POLYNUCLEOTIDES COMPRISING N1-METHYL-PSEUDOURIDINE AND METHODS FOR PREPARING THE SAME

POLYNUCLEOTIDES COMPRENANT DU N1-METHYL-PSEUDOURACILS ET METHODES POUR LES PREPARER

English Abstract

The invention relates to compositions and methods for the preparation, manufacture and therapeutic use of polynucleotides, primary transcripts and mmRNA molecules.

French Abstract

L'invention concerne des compositions et des procédés de préparation, de fabrication et d'utilisation thérapeutique de polynucléotides, de transcrits primaires et de molécules d'ARNm modifiées.

Claims

Claims
We claim:
1. An isolated polynucleotide comprising;
(a) a first region of linked nucleosides, said first region encoding a
polypeptide of interest, said polypeptide of interest selected from the group
consisting of SEQ ID NOs 769-1392;
(b) a first flanking region located at the 5' terminus of said first region
comprising;
(i) a sequence of linked nucleosides selected from the group consisting of
the native 5' UTR of any of the nucleic acids that encode any of SEQ ID NOs
769-1392, SEQ ID NOs: 1-4 and functional variants thereof; and
(ii) at least one 5' terminal cap;
(c) a second flanking region located at the 3' terminus of said first region
comprising;
(i) a sequence of linked nucleosides selected from the group consisting of
the native 3' UTR of any of the nucleic acids that encode any of SEQ ID NOs
769-1392, SEQ ID NOs 5-21 and functional variants thereof; and
(ii') a 3' tailing sequence of linked nucleosides.
2. The isolated polynucleotide of claim 1 wherein the first region of
linked
nucleosides comprises at least an open reading frame of a nucleic acid
sequence, wherein
the nucleic acid sequence selected from the group consisting of SEQ ID NOs:
1393-
21423, 21435-21438, 21442-21443, 21449, 21451-21473, 21521-21542, 21639-21642,

21671-21679, 21681, 21682, 21684, 21686, 21687, 21724-21726, 21728 and 21730-
21738.
3. The isolated polynucleotide of claim 1, wherein the 3'tailing sequence
of linked
nucleosides is selected from the group consisting of a poly-A tail of
approximately 160
nucleotides and a polyA-G quartet.
742

4. The isolated polynucleotide of any one of claims 1-3 which is purified.
5. The isolated polynucleotide of any one of claims 1-4, wherein the at
least one 5'
terminal cap is selected from the group consisting of Cap0, Cap1, ARCA,
inosine, N1-
methyl-guanosine, 2'fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-
amino-
guanosine, LNA-guanosine, and 2-azido-guanosine.
6. The isolated polynucleotide of any preceding claim, wherein at least one
of said
linked nucleosides comprises at least one modification as compared to the
chemical
structure of an A, G, U or C ribonucleotide.
7. The isolated polynucleotide of claim 6, wherein at least one said
modification is
located in a nucleoside base and/or sugar portion.
8. The isolated polynucleotide of any one of claims 1-7, wherein said first
region
comprises n number of linked nucleosides having Formula (Ia):
Image (Ia), or a pharmaceutically acceptable salt or
stereoisomer thereof,
wherein
U is O, S, N(R U)nu, or C(R U)nu, wherein nu is an integer from 0 to 2 and
each R U
is, independently, H, halo, or optionally substituted alkyl;
Image is a single or double bond;
- - - is a single bond or absent;
each of R1', R2', R1", R2", R3, R4, and R5 is, independently, H, halo,
hydroxy, thiol,
optionally substituted alkyl, optionally substituted alkoxy, optionally
substituted
743

alkenyloxy, optionally substituted alkynyloxy, optionally substituted
aminoalkoxy,
optionally substituted alkoxyalkoxy, optionally substituted hydroxyalkoxy,
optionally
substituted amino, azido, optionally substituted aryl, optionally substituted
aminoalkyl, or
absent; wherein the combination of R3 with one or more of R1', R1", R2', R2",
or R5 can
join together to form optionally substituted alkylene or optionally
substituted
heteroalkylene and, taken together with the carbons to which they are
attached, provide
an optionally substituted heterocyclyl; wherein the combination of R5 with one
or more
of R1', R1", R2', or R2" can join together to form optionally substituted
alkylene or
optionally substituted heteroalkylene and, taken together with the carbons to
which they
are attached, provide an optionally substituted heterocyclyl; and wherein the
combination
of R4 and one or more of R1', R1", R2', R2", R3, or R5 can join together to
form optionally
substituted alkylene or optionally substituted heteroalkylene and, taken
together with the
carbons to which they are attached, provide an optionally substituted
heterocyclyl;
each of Y1, Y2, and Y3, is, independently, O, S, -NR N1-, optionally
substituted
alkylene, or optionally substituted heteroalkylene, wherein R N1 is H,
optionally
substituted alkyl, optionally substituted alkenyl, optionally substituted
alkynyl, optionally
substituted aryl, or absent;
each Y4 is, independently, H, hydroxy, thiol, optionally substituted alkyl,
optionally substituted alkenyl, optionally substituted alkynyl, optionally
substituted
alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy,
optionally
substituted thioalkoxy, optionally substituted alkoxyalkoxy, or optionally
substituted
amino;
each Y5 is, independently, O, S, optionally substituted alkylene, or
optionally
substituted heteroalkylene;
n is an integer from 1 to 100,000; and
B is a nucleobase, wherein the combination of B and R1', the combination of B
and R2', the combination of B and R1", or the combination of B and R2" can,
taken
together with the carbons to which they are attached, optionally form a
bicyclic group or
wherein the combination of B, R1", and R3 or the combination of B, R2", and R3
can
optionally form a tricyclic or tetracyclic group.
744

9. The isolated polynucleotide of claim 8, wherein B is not pseudouridine
(.PSI.) or 5-
methyl-cytidine (m5C).
10. The isolated polynucleotide of any one of claims 8-9, wherein
U is O or C(R U)nu, wherein nu is an integer from 1 to 2 and each R U is,
independently, H, halo, or optionally substituted alkyl;
each of R1, R1', R1", R2, R2', and R2", if present, is, independently, H,
halo,
hydroxy, optionally substituted alkyl, optionally substituted alkoxy,
optionally substituted
alkenyloxy, optionally substituted alkynyloxy, optionally substituted
aminoalkoxy,
optionally substituted alkoxyalkoxy, optionally substituted amino, azido,
optionally
substituted aryl, or optionally substituted aminoalkyl;
each of R3 and R4 is, independently, H, halo, hydroxy, optionally substituted
alkyl, or optionally substituted alkoxyalkoxy;
each of Y1, Y2, and Y3, is, independently, O, S, -NR N1-, optionally
substituted
alkylene, or optionally substituted heteroalkylene, wherein R N1 is H,
optionally
substituted alkyl, optionally substituted alkenyl, or optionally substituted
alkynyl;
each Y4 is, independently, H, hydroxy, thiol, optionally substituted alkyl,
optionally substituted alkenyl, optionally substituted alkynyl, optionally
substituted
alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy,
optionally
substituted thioalkoxy, or optionally substituted amino;
each Y5 is, independently, O or optionally substituted alkylene; and
n is an integer from 10 to 10,000.
11. The isolated polynucleotide of claim 10, wherein each of R1, R1', and
R1", if
present, is H.
12. The isolated polynucleotide of claim 11, wherein each of R2, R2', and
R2", if
present, is, independently, H, halo, hydroxy, optionally substituted alkoxy,
or optionally
substituted alkoxyalkoxy.
745

13. The isolated polynucleotide of claim 10, wherein each of R2, R2', and
R2", if
present, is H.
14. The isolated polynucleotide of claim 13, wherein each of R1, R1', and
R1", if
present, is, independently, H, halo, hydroxy, optionally substituted alkoxy,
or optionally
substituted alkoxyalkoxy.
15. The isolated polynucleotide of claim 8, wherein said first region
comprises n
number of linked nucleotides having Formula (IIa):
Image (IIa), or a pharmaceutically acceptable salt or
stereoisomer
thereof.
16. isolated The polypeptide of claim 15, wherein said first region
comprises n
number of linked nucleosides having Formula (IIb) or (IIc), or a
pharmaceutically
acceptable salt thereof.
17. The isolated polynucleotide of claim 8, wherein said first region
comprises n
number of linked nucleosides having Formula (IId):
Image (IId), or a pharmaceutically acceptable salt or
stereoisomer
thereof.
746

18. The isolated polypeptide of claim 17, wherein said first region
comprises n
number of linked nucleosides having Formula (IIe) or (IIf), or a
pharmaceutically
acceptable salt thereof.
19. The isolated polynucleotide of claim 8, wherein said first region
comprises n
number of linked nucleotides, each of said linked nucleotides independently
having one
of Formulas (IIg)-(IIj):
Image
pharmaceutically acceptable salt or stereoisomer thereof
20. The isolated polynucleotide of claim 8, wherein said first region
comprises n number
of linked nucleosides having Formula (IIk):
Image (IIk) , or a pharmaceutically acceptable salt or
stereoisomer
thereof.
747




21. The isolated polynucleotide of claim 20, wherein said first region
comprises n
number of linked nucleosides having Formula (IIl):
Image (II1), or a pharmaceutically acceptable salt or
stereoisomer
thereof
22. The isolated polynucleotide of claim 20, wherein said first region
comprises n
number of linked nucleosides having Formula (IIm):
Image (IIm), or a pharmaceutically acceptable salt or
stereoisomer
thereof,
wherein
each of R1', R1", R2', and R2" is, independently, H, halo, hydroxy, optionally

substituted alkyl, optionally substituted alkoxy, optionally substituted
alkenyloxy,
optionally substituted alkynyloxy, optionally substituted aminoalkoxy,
optionally
substituted alkoxyalkoxy, or absent; and wherein the combination of R2' and R3
or the
combination of R2" and R3 can be taken together to form optionally substituted
alkylene
or optionally substituted heteroalkylene.
23. The isolated polynucleotide of any one of claims 11-22, wherein
U is O or C(R U)nu, wherein nu is an integer from 1 to 2 and each R U is,
independently, H, halo, or optionally substituted alkyl;
748




each of R1 and R2 is, independently, H, halo, hydroxy, optionally substituted
alkyl, optionally substituted alkoxy, optionally substituted alkenyloxy,
optionally
substituted alkynyloxy, optionally substituted aminoalkoxy, optionally
substituted
alkoxyalkoxy, optionally substituted amino, azido, optionally substituted
aryl, or
optionally substituted aminoalkyl;
each of R3 and R4 is, independently, H or optionally substituted alkyl;
each of Y1, Y2, and Y3, is, independently, O, S, -NR N1-, optionally
substituted
alkylene, or optionally substituted heteroalkylene, wherein R N1 is H,
optionally
substituted alkyl, optionally substituted alkenyl, or optionally substituted
alkynyl;
each Y4 is, independently, H, hydroxy, thiol, optionally substituted alkyl,
optionally substituted alkenyl, optionally substituted alkynyl, optionally
substituted
alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy,
optionally
substituted thioalkoxy, or optionally substituted amino;
each Y5 is, independently, O or optionally substituted alkylene; and
n is an integer from 10 to 10,000.
24. The isolated polynucleotide of claim 8, wherein said first region
comprises n
number of linked nucleosides having Formula (IIn):
Image
(IIn), or a pharmaceutically acceptable salt or stereoisomer
thereof,
wherein
U is O or C(R U)nu, wherein nu is an integer from 1 to 2 and each R U is,
independently, H, halo, or optionally substituted alkyl;
each of R1 and R4 is, independently, H, halo, hydroxy, optionally substituted
alkyl, optionally substituted alkoxy, optionally substituted alkenyloxy,
optionally
substituted alkynyloxy, optionally substituted aminoalkoxy, optionally
substituted
749




alkoxyalkoxy, optionally substituted amino, azido, optionally substituted
aryl, or
optionally substituted aminoalkyl;
R3' is O, S, or -NR N1-, wherein R N1 is H, optionally substituted alkyl,
optionally
substituted alkenyl, optionally substituted alkynyl, or optionally substituted
aryl;
R3" is optionally substituted alkylene or optionally substituted
heteroalkylene;
each of Y1, Y2, and Y3, is, independently, O, S, -NR N1-, optionally
substituted
alkylene, or optionally substituted heteroalkylene, wherein R N1 is H,
optionally
substituted alkyl, optionally substituted alkenyl, or optionally substituted
alkynyl;
each Y4 is, independently, H, hydroxy, thiol, optionally substituted alkyl,
optionally substituted alkenyl, optionally substituted alkynyl, optionally
substituted
alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy,
optionally
substituted thioalkoxy, or optionally substituted amino;
each Y5 is, independently, O, S, optionally substituted alkylene (e.g.,
methylene),
or optionally substituted heteroalkylene; and
n is an integer from 10 to 10,000.
25. The
isolated polynucleotide of any one of claims 8-24, wherein in said n number
of B has, each B independently has a formula selected from Formula (b1)-(b5):
or Image (b5), or
a pharmaceutically acceptable salt or stereoisomer thereof,
wherein
Image connecting V1 and V2 in formula (b1) is a single or double bond;
750




each of T1', T1", T2', and T2" is, independently, H, optionally substituted
alkyl,
optionally substituted alkoxy, or optionally substituted thioalkoxy, or the
combination of
T1' and T1" or the combination of T2' and T2" join together to form O (oxo), S
(thio), or Se
(seleno);
each of V1 and V2 is, independently, O, S, N(R Vb), or C(R Vb), wherein nv is
an
integer from 0 to 2 and each R Vb is, independently, H, halo, optionally
substituted amino
acid, optionally substituted alkyl, optionally substituted alkenyl, optionally
substituted
alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, or
optionally
substituted alkynyloxy;
R10 is H, halo, optionally substituted amino acid, hydroxy, optionally
substituted
alkyl, optionally substituted alkenyl, optionally substituted alkynyl,
optionally substituted
aminoalkyl, optionally substituted alkoxy, optionally substituted
alkoxycarbonylalkyl,
optionally substituted alkoxycarbonylalkoxy, optionally substituted
carboxyalkoxy,
optionally substituted carboxyalkyl, or optionally substituted carbamoylalkyl;
R11 is H or optionally substituted alkyl;
R12a is H, optionally substituted alkyl, or optionally substituted aminoalkyl;
and
R12c is H, halo, optionally substituted alkyl, optionally substituted alkoxy,
optionally substituted thioalkoxy, optionally substituted amino, or optionally
substituted
aminoalkyl.
26. The isolated polynucleotide of any one of claims 8-25, wherein n number
of B has
Formula (b6)-(b9):
Image
(b9), or a pharmaceutically acceptable salt or stereoisomer thereof,
wherein
is a single or double bond;
751




each of T1', T1", T2', and T2" is, independently, H, optionally substituted
alkyl,
optionally substituted alkoxy, or optionally substituted thioalkoxy, or the
combination of
T1' and T1" or the combination of T2' and T2" join together to form O (oxo), S
(thio), or Se
(seleno);
each of W1 and W2 is, independently, N(R Wa)nw or c(R Wa)nw, wherein nw is an
integer from 0 to 2 and each R Wa is, independently, H, optionally substituted
alkyl, or
optionally substituted alkoxy;
each V3 is, independently, O, S, N(R Va)nv, or C(R Va), wherein nv is an
integer
from 0 to 2 and each R Va is, independently, H, halo, optionally substituted
amino acid,
optionally substituted alkyl, optionally substituted alkenyl, optionally
substituted alkynyl,
optionally substituted heterocyclyl, optionally substituted alkheterocyclyl,
optionally
substituted alkoxy, optionally substituted alkenyloxy, or optionally
substituted
alkynyloxy, and wherein R Va and R12C taken together with the carbon atoms to
which they
are attached can form optionally substituted cycloalkyl, optionally
substituted aryl, or
optionally substituted heterocyclyl;
R12a is H, optionally substituted alkyl, optionally substituted aminoalkyl, or
absent;
R12b is H, optionally substituted alkyl, optionally substituted alkenyl,
optionally
substituted alkynyl, optionally substituted alkaryl, optionally substituted
heterocyclyl,
optionally substituted alkheterocyclyl, or optionally substituted amino acid,
wherein the
combination of R12b and T1' or the combination of R12b and R12C can join
together to form
optionally substituted heterocyclyl; and
R12c is H, halo, optionally substituted alkyl, optionally substituted alkoxy,
optionally substituted thioalkoxy, optionally substituted amino, or optionally
substituted
aminoalkyl.
27. The isolated polynucleotide of claim 26, wherein R12a, R12b, R12c, or R
Va is
substituted with -(CH2)s2(OCH2CH2)s1(CH2)OR', wherein s1 is an integer from 1
to 10,
each of s2 and s3, independently, is an integer from 0 to 10, and R' is H or
C1-20 alkyl); or
-NR N1(CH2)s2(CH2CH2O)s1(CH2)s3NR N1, wherein s1 is an integer from 1 to 10,
each of s2
752

and s3, independently, is an integer from 0 to 10, and each R N1 is,
independently,
hydrogen or optionally substituted C1-6 alkyl.
28. The isolated polynucleotide of any one of claims 8-27, wherein n number
of B has
Formula (b 10)-(b 14):
Image , or a pharmaceutically acceptable salt
or stereoisomer thereof,
wherein
each of T3' and T3" is, independently, H, optionally substituted alkyl,
optionally
substituted alkoxy, or optionally substituted thioalkoxy, or the combination
of T3' and T3"
join together to form O (oxo), S (thio), or Se (seleno);
each V4 is, independently, O, S, N(R Vc)nv, or C(R Vc)nv, wherein nv is an
integer
from 0 to 2 and each R Vc is, independently, H, halo, optionally substituted
amino acid,
optionally substituted alkyl, optionally substituted alkenyl, optionally
substituted alkynyl,
optionally substituted alkoxy, optionally substituted alkenyloxy, optionally
substituted
heterocyclyl, optionally substituted alkheterocyclyl, or optionally
substituted alkynyloxy,
wherein the combination of R13b and R Vc can be taken together to form
optionally
substituted heterocyclyl;
each of R13a and R13b is, independently, H, optionally substituted acyl,
optionally
substituted alkyl, or optionally substituted alkoxy, wherein the combination
of R13b and
R14 can be taken together to form optionally substituted heterocyclyl;
753


each R14 is, independently, H, halo, hydroxy, thiol, optionally substituted
acyl,
optionally substituted amino acid, optionally substituted alkyl, optionally
substituted
alkenyl, optionally substituted alkynyl, optionally substituted hydroxyalkyl,
optionally
substituted alkoxy, optionally substituted alkenyloxy, optionally substituted
alkynyloxy,
optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy,
optionally
substituted amino, azido, optionally substituted aryl, optionally substituted
heterocyclyl,
optionally substituted alkheterocyclyl, or optionally substituted aminoalkyl;
and
each of R15 and R16 is, independently, H, optionally substituted alkyl,
optionally
substituted alkenyl, or optionally substituted alkynyl.
29. The isolated polynucleotide of any one of claims 7-27, wherein n number
of B has
Formula (b15)-(b17):
Image , or a pharmaceutically acceptable salt or stereoisomer
thereof,
wherein
each of T4', T4", T5', T5", T6', and T6" is, independently, H, optionally
substituted
alkyl, or optionally substituted alkoxy, and wherein the combination of T4'
and T4" or the
combination of T5' and T5" or the combination of T6' and T6" together form O,
S, or Se;
each of V5 and V6 is, independently, O, S, N(R Vd), or C(R Vd), wherein nv is
an
integer from 0 to 2 and each R Vd is, independently, H, halo, optionally
substituted amino
acid, cyano, amidine, optionally substituted aminoalkyl, optionally
substituted alkyl,
optionally substituted alkenyl, optionally substituted alkynyl, optionally
substituted
alkoxy, optionally substituted alkenyloxy, or optionally substituted
alkynyloxy; and
754

each of R17, R18, R19a R19b, R21, R22, R23, and R24 is, independently, H,
optionally
substituted alkyl, optionally substituted alkenyl, optionally substituted
alkynyl, or
optionally substituted amino acid.
30. The polynucleotide of any one of claims 8-29, wherein n number of B has
Formula (b18)-(b20):
Image (b20), or a pharmaceutically acceptable salt or
stereoisomer
thereof,
wherein
each V7 is, independently, O, S, N(R Ve)nv, or C(R Ve)nv, wherein nv is an
integer
from 0 to 2 and each R Ve is, independently, H, halo, optionally substituted
amino acid,
optionally substituted alkyl, optionally substituted alkenyl, optionally
substituted alkynyl,
optionally substituted alkoxy, optionally substituted alkenyloxy, or
optionally substituted
alkynyloxy;
each R25 is, independently, H, optionally substituted alkyl, optionally
substituted
alkenyl, or optionally substituted alkynyl;
each of R26a and R26b is, independently, H, optionally substituted acyl,
optionally
substituted amino acid, optionally substituted carbamoylalkyl, optionally
substituted
alkyl, optionally substituted alkenyl, optionally substituted alkynyl,
optionally substituted
hydroxyalkyl, optionally substituted hydroxyalkenyl, or optionally substituted
alkoxy;
each R27 is, independently, H, optionally substituted alkyl, optionally
substituted
alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, or
optionally
substituted amino;
755

each R28 is, independently, H, optionally substituted alkyl, optionally
substituted
alkenyl, or optionally substituted alkynyl; and
each R29 is, independently, H, optionally substituted acyl, optionally
substituted
amino acid, optionally substituted carbamoylalkyl, optionally substituted
alkyl, optionally
substituted alkenyl, optionally substituted alkynyl, optionally substituted
hydroxyalkyl,
optionally substituted hydroxyalkenyl, optionally substituted alkoxy, or
optionally
substituted amino.
31. The isolated polynucleotide of claim 30, wherein R26a, R26b, or R29 is
substituted
with -(CH2)s2(OCH2CH2)s1(CH2)s3OR', wherein s1 is an integer from 1 to 10,
each of s2
and s3, independently, is an integer from 0 to 10, and R' is H or C1-20
alkyl); or -
NR Ni(CH2)s2(CH2CH2O)s1(CH2)s3NR N1, wherein s1 is an integer from 1 to 10,
each of s2
and s3, independently, is an integer from 0 to 10, and each R N1 is,
independently,
hydrogen or optionally substituted C1-6 alkyl.
32. The isolated polynucleotide of any one of claims 8-3 1, wherein n
number of B has
Formula (b21):
Image (b21), or a pharmaceutically acceptable salt or stereoisomer
thereof,
756

wherein X12 is, independently, O, S, optionally substituted alkylene, or
optionally
substituted heteroalkylene; xa is an integer from 0 to 3; R12a is H,
optionally substituted
alkyl, optionally substituted aminoalkyl, or absent; and T2 is O, S, or Se.
33. The isolated polynucleotide of any one of claims 8-32, wherein n number
of B has
Formula (b22):
Image (b22), or a pharmaceutically acceptable salt or
stereoisomer thereof, wherein R10' is, independently, optionally substituted
alkyl,
optionally substituted alkenyl, optionally substituted alkynyl, optionally
substituted aryl,
optionally substituted heterocyclyl, optionally substituted aminoalkyl,
optionally
substituted alkoxy, optionally substituted alkoxycarbonylalkyl, optionally
substituted
alkoxycarbonylalkoxy, optionally substituted carboxyalkoxy, optionally
substituted
carboxyalkyl, or optionally substituted carbamoylalkyl; R11 is H or optionally
substituted
alkyl; R12a is H, optionally substituted alkyl, optionally substituted
aminoalkyl, or absent;
and each of T1 and T2 is, independently, O, S, or Se.
34. The isolated polynucleotide of any one of claims 8-33, wherein n number
of B has
Formula (b23):
Image , wherein R10 is optionally substituted heterocyclyl or
optionally
substituted aryl; R11 is H or optionally substituted alkyl; R12a is H,
optionally substituted
alkyl, optionally substituted aminoalkyl, or absent; and each of T1 and T2 is,

independently, O, S, or Se.
757


35. The isolated polynucleotide of any one of claims 8-34, wherein n number
of B has
Formula (b24):
Image
T3 is O, S, or Se;
each of R13a and R13b is, independently, H, optionally substituted acyl,
optionally
substituted alkyl, or optionally substituted alkoxy, wherein the combination
of R13b and
R14 can be taken together to form optionally substituted heterocyclyl;
R14' is, independently, optionally substituted alkyl, optionally substituted
alkenyl,
optionally substituted alkynyl, optionally substituted aryl, optionally
substituted
heterocyclyl, optionally substituted alkheterocyclyl, optionally substituted
alkaryl,
optionally substituted aminoalkyl, optionally substituted alkoxy, optionally
substituted
alkoxycarbonylalkyl, optionally substituted alkoxycarbonylalkoxy, optionally
substituted
carboxyalkoxy, optionally substituted carboxyalkyl, or optionally substituted
carbamoylalkyl; and
each R15 is, independently, H, optionally substituted alkyl, optionally
substituted
alkenyl, or optionally substituted alkynyl.
36. The isolated polynucleotide of any one of claims 1-35, further
comprising a
targeting moiety, wherein said targeting moiety is covalently bound to said
polynucleotide.
37. The isolated polynucleotide of claim 36, wherein said targeting moiety
is an
antibody, thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein
A, Mucin
carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-
galactosamine, N-
acetyl-gulucosamine multivalent mannose, multivalent fucose, glycosylated
polyaminoacids, multivalent galactose, transferrin, bisphosphonate,
polyglutamate,

758


polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin
B12, biotin, an RGD
peptide, an RGD peptide mimetic, or an aptamer.
38. A pharmaceutical composition comprising the isolated polynucleotide of
any one
of claims 1-37.
39. A pharmaceutical composition comprising the isolated polynucleotide of
any one
of claims 1-37 and a pharmaceutically acceptable excipient.
40. The pharmaceutical composition of claim 39, wherein the excipient is
selected
from a solvent, aqueous solvent, non-aqueous solvent, dispersion media,
diluent,
dispersion, suspension aid, surface active agent, isotonic agent, thickening
or emulsifying
agent, preservative, lipid, lipidoids liposome, lipid nanoparticle, core-shell
nanoparticles,
polymer, lipoplexe peptide, protein, cell, hyaluronidase, and mixtures
thereof.
41. The pharmaceutical composition of claim 40, where the pharmaceutical
composition comprises a lipid and wherein said lipid is selected from DLin-
DMA, DLin-
K-DMA, DLin-KC2-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DODMA, DSDMA,
DLenDMA, reLNPs, PLGA and PEGylated lipids and mixtures thereof.
42. A method of producing a polypeptide of interest in a mammalian cell,
tissue or
organism comprising administering to said cell, tissue or organism the
isolated
polynucleotide of any one of claims 1-37 or the pharmaceutical composition of
any of
claims 38-41.
43. The method of claim 42, wherein the isolated polynucleotide is
formulated.
44. The method of claim 43, wherein the formulation comprises a lipid which
is
selected from one of DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, 98N12-5, C12-200,
DLin-MC3-DMA, DODMA, DSDMA, DLenDMA, reLNPs, PLGA, PEGylated lipids
and mixtures or combinations thereof.

759


45. The method of claim 44, wherein the isolated polynucleotide is
administered at a
total daily dose of between 1 ug and 150 ug.
46. The method of claim 45, wheriein administration is by injection.
47. The method of claim 46, wherein administration is intradermal or
subcutaneous or
intramuscular or intravitreal.
48. The method of claim 45, wherein levels of the polypeptide of interest
in the serum
of the mammal are at least 50 pg/mL at least two hours after administration.
49. The method of claim 45, wherein the levels of the polypeptide of
interest in the
serum of the mammal remain above 50 pg/mL for at least 72 hours after
administration.
50. The method of claim 49, wherein the levels of the polypeptide of
interest in the
serum of the mammal remain above 60 pg/mL for at least 72 hours after
administration.
51. The method of claim 44, wherein the resulting polynucleotide
formulation has a
mean particle size of 80 nm-160 nm, a PDI of between 0.02 and 0.20 and a lipid
to
polnucleotide ratio (wt/wt) of between 10-20.
52. A method for producing an increased level of a polypeptide of interest
selected
from the group consisting of SEQ ID NOs 769-1392 in a mammalian cell, tissue
or
organism, comprising administering to said cell, tissue or organism a total
daily dose of
the isolated polynucleotide of any one of claims 4-37 or the pharmaceutical
composition
of any one of claims 38-41 in two or more equal or unequal split doses.
53. The method of claim 52, wherein the level of the polypeptide produced
in
response to said administration is greater than the levels produced by
administering the
same total daily dose of the isolated polynucleotide or pharmaceutical
composition as a

760


single administration.
54. The method of claim 52, wherein the mammalian organism is a human
patient in
need of an increased level of the polypeptide of interest.
55. The method of claim 54, wherein the increased level of the polypeptide
of interest
is detectable in a bodily fluid of said patient.
56. The method of claim 55, wherein the bodily fluid is selected from the
group
consisting of peripheral blood, serum, plasma, ascites, urine, cerebrospinal
fluid (CSF),
sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid,
cerumen,
breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper's
fluid or pre-
ejaculatory fluid, sweat, fecal matter, hair, tears, cyst fluid, pleural and
peritoneal fluid,
pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus,
sebum, vomit,
vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage
fluids from
sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, and
umbilical cord
blood.
57. The method of claim 56, wherein the bodily fluid is serum and the
polpeptide per
unit drug (PUD) is greater than 1.
58. The method of claim 57, wherein the dose splitting factor (DSF) is
greater than 4.
59. The method of claim 55, wherein administration is transdermal.
60. The method of claim 59, wherein transdermal administration comprises
utilization
of one or more members selected from the group consisting of a patch, cream,
ointment,
mechanical device, needle, sponge, depot and fabric.
61. The method of claim 59, wherein administration is according to a dosing
regimen
which occurs over the course of hours, days, weeks, months, or years.

761


62. The method of claim 52, wherein said two or more split doses comprise a
first
dose of the polynucleotide or pharmaceutical composition administered at a
time T1
followed by a second dose of the polynucleotide or pharmaceutical composition
administered at a time T2, wherein said time T1 and said time T2 are separated
by no
more than 1 minute and wherein said first dose and said second dose are
administered in
amounts that result in higher levels of the polypeptide of interest in said
subject than if
the amounts of polynucleotide or pharmaceutical composition were administered
together
in a single unit dose.
63. The method of claim 62, further comprising administering a plurality of
doses of
said polynucleotide or pharmaceutical composition, Nx at times Tn, wherein x
and n are
independently selected from 3 to about 1000 and where the time between Tn and
Tn+1 is
separated by increments of no more than 10 seconds.
64. The method of claim 63, wherein administration occurs by direct
injection.
65. The method of claim 64, wherein direct injection is selected from the
group
consisting of intravenous, intradermal, subcutaneous, intramuscular and
intravitreal.
66. The method of claim 64, wherein said first dose is administered
proximal to said
second or plurality of doses.
67. The method of claim 64, wherein said first dose is administered distal
to said
second or plurality of doses.
68. The method of claim 64, wherein the distance between the site of
injection of said
first dose and the site of injection of any second or plurality of doses is
from about 1mm
to about 10 cm.

762


69. The method of claim 64, wherein injection is made at a depth of from
0.1mm to
about 1cm.
70. The method of claim 65, wherein direct injection is achieved by using
one or
more devices selected from multineedle injection systems, catheter or lumen
systems, and
ultrasound, electrical or radiation based systems.
71. The method of claim 63, wherein the amount polynucleotide or
pharmaceutical
composition administered in any dose is substantially equal.
72. The method of claim 63, wherein time T1 and time T2 are separated by no
more
than 30 seconds.
73. The method of claim 63, wherein time T1 and time T2 are separated by no
more
than 10 seconds.
74. The method of claim 63, wherein the first dose, the second dose and any
of a
plurality of doses are administered at substantially the same time.
75. The method of claim 63, wherein the single unit dose is between about
10 mg/kg
and about 500 mg/kg.
76. The method of claim 63, wherein the single unit dose is between about
1.0 mg/kg
and about 10 mg/kg.
77. The method of claim 63, wherein the single unit dose is between about
0.001
mg/kg and about 1.0 mg/kg.
78. A method of preparing a lipid nanopartical formulation of a
polynucleotide
encoding a polypeptide of interest comprising rapidly injecting a first
ethanolic solution
into a second aqueous solution wherein,

763


(a) said first ethanolic solution comprises a mixture of lipid:DSPC:
Cholesterol:
PEG-c-DOMG to yield a molar ratio of 50:10:38.5:1.5 and having a final lipid
concentration of approximately 25mM, and
(b) said second aqueous solution comprises a sodium citrate buffered solution
of the polynucleotide encoding the polypeptide of interest having a
concentration of 1-2 mg/mL and a pH of approximately 3,
wherein the rapid injection results in a suspension containing 33% ethanol and
a total lipid
to polynucleotide weight ratio of at least 10:1.
79. The method of claim 78, wherein the rapid injection is performed either
manually
(MI) or by the aid of a syringe pump (SP).
80. The method of claim 79, further comprising dialyzing the resultant
suspension
against phosphate buffered saline (PBS) at pH 7.4.
81. The method of claim 80, wherein dialysis is performed more than once.
82. The method of claim 81, further comprising filtering the dialyzed
suspension
through a 0.2 µm sterile filter.
83. The method of any of claims 78-82, wherein the lipid is selected from
the group
consisting of DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, 98N12-5, C12-200, DLin-
MC3-DMA, DODMA, DSDMA, DLenDMA, reLNPs, PLGA and PEGylated lipids.
84. The method of any of claims 78-83, wherein the polynucleotide is
selected from
the polynucleotide of any of claims 1-37.
85. A lipid nanopartical formulation of a polynucleotide encoding a
polypeptide of
interest produced by the method of any of claims 78-84 and having a particle
size of 80
nm-160 nm, a PDI of between 0.02 and 0.20 and a lipid to polnucleotide ratio
(wt/wt) of
between 10-30.

764


86. A lipid nanopartical formulation of claim 85, wherein the
polynucleotide is
selected from the polynucleotide of any of claims 1-37.
87. A reLNP formulation of a polynucleotide said polynucleotide encoding a
polypeptide of interest.
88. A reLNP formulation of claim 87, wherein the polynucleotide is selected
from the
polynucleotide of any of claims 1-36.
89. A sustained release formulation of a polynucleotide said polynucleotide
encoding
a polypeptide of interest.
90. A sustained release formulation of claim 89, wherein the polynucleotide
is
selected from the polynucleotide of any of claims 1-36.
91. A polynucleotide encoding a fusion protein said fusion protein
comprising a first
polypeptide and a second polypeptide.
92. The polynucleotide of claim 91 where the first polypeptide is selected
from the
group consisting of Fc receptor, Fab fragment, Fab' fragment, F(ab')2
fragment, Fv
fragment, IgA domain, IgD domain, IgE domain, IgD domain, IgM domain, IgV
domain,
IgC1 domain, IgC2 domain and IgI domain and the second polypeptide is a
polypeptide
of interest.

765

Description

DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.
CECI EST LE TOME 1 DE 3
CONTENANT LES PAGES 1 A 339
NOTE : Pour les tomes additionels, veuillez contacter le Bureau canadien des
brevets
JUMBO APPLICATIONS/PATENTS
THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE THAN ONE
VOLUME
THIS IS VOLUME 1 OF 3
CONTAINING PAGES 1 TO 339
NOTE: For additional volumes, please contact the Canadian Patent Office
NOM DU FICHIER / FILE NAME:
NOTE POUR LE TOME / VOLUME NOTE:

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MODIFIED POLYNUCLEOTIDES FOR THE PRODUCTION OF BIOLOGICS
AND PROTEINS ASSOCIATED WITH HUMAN DISEASE
REFERENCE TO SEQUENCE LISTING
[0001] The present application is being filed along with a Sequence Listing
in
electronic format. The Sequence Listing file entitled M300PCTSQLST.txt, was
created
on March 9, 2013 and is 49,417,315 bytes in size. The information in
electronic format
of the Sequence Listing is incorporated herein by reference in its entirety.
CROSS REFERENCE TO RELATED APPLICATIONS
[0002] This application claims priority to U.S. Provisional Patent
Application No
61/681,742, filed, August 10, 2012, entitled Modified Polynucleotides for the
Production
of Oncology-Related Proteins and Peptides, U.S. Provisional Patent Application
No
61/737,224, filed December 14, 2012, entitled Terminally Optimized Modified
RNAs,
International Application No PCT/U52012/069610, filed December 14, 2012,
entitled
Modified Nucleoside, Nucleotide, and Nucleic Acid Compositions, U.S.
Provisional
Patent Application No 61/618,862, filed April 2, 2012, entitled Modified
Polynucleotides
for the Production of Biologics, U.S. Provisional Patent Application No
61/681,645, filed
August 10, 2012, entitled Modified Polynucleotides for the Production of
Biologics, U.S.
Provisional Patent Application No 61/737,130, filed December 14, 2012,
entitled
Modified Polynucleotides for the Production of Biologics, U.S. Provisional
Patent
Application No 61/618,866, filed April 2, 2012, entitled Modified
Polynucleotides for the
Production of Antibodies, U.S. Provisional Patent Application No 61/681,647,
filed
August 10, 2012, entitled Modified Polynucleotides for the Production of
Antibodies,
U.S. Provisional Patent Application No 61/737,134, filed December 14, 2012,
entitled
Modified Polynucleotides for the Production of Antibodies, U.S. Provisional
Patent
Application No 61/618,868, filed April 2, 2012, entitled Modified
Polynucleotides for the
Production of Vaccines, U.S. Provisional Patent Application No 61/681,648,
filed August
10, 2012, entitled Modified Polynucleotides for the Production of Vaccines,
U.S.
Provisional Patent Application No 61/737,135, filed December 14, 2012,
entitled
Modified Polynucleotides for the Production of Vaccines, U.S. Provisional
Patent
Application No 61/618,870, filed April 2, 2012, entitled Modified
Polynucleotides for the
Production of Therapeutic Proteins and Peptides, U.S. Provisional Patent
Application No
1

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PCT/US2013/030062
61/681,649, filed August 10, 2012, entitled Modified Polynucleotides for the
Production
of Therapeutic Proteins and Peptides, U.S. Provisional Patent Application No
61/737,139, filed December 14, 2012, Modified Polynucleotides for the
Production of
Therapeutic Proteins and Peptides, U.S. Provisional Patent Application No
61/618,873,
filed April 2, 2012, entitled Modified Polynucleotides for the Production of
Secreted
Proteins, U.S. Provisional Patent Application No 61/681,650, filed August 10,
2012,
entitled Modified Polynucleotides for the Production of Secreted Proteins,
U.S.
Provisional Patent Application No 61/737,147, filed December 14, 2012,
entitled
Modified Polynucleotides for the Production of Secreted Proteins, U.S.
Provisional
Patent Application No 61/618,878, filed April 2, 2012, entitled Modified
Polynucleotides
for the Production of Plasma Membrane Proteins, U.S. Provisional Patent
Application No
61/681,654, filed August 10, 2012, entitled Modified Polynucleotides for the
Production
of Plasma Membrane Proteins, U.S. Provisional Patent Application No
61/737,152, filed
December 14, 2012, entitled Modified Polynucleotides for the Production of
Plasma
Membrane Proteins, U.S. Provisional Patent Application No 61/618,885, filed
April 2,
2012, entitled Modified Polynucleotides for the Production of Cytoplasmic and
Cytoskeletal Proteins, U.S. Provisional Patent Application No 61/681,658,
filed August
10, 2012, entitled Modified Polynucleotides for the Production of Cytoplasmic
and
Cytoskeletal Proteins, U.S. Provisional Patent Application No 61/737,155,
filed
December 14, 2012, entitled Modified Polynucleotides for the Production of
Cytoplasmic
and Cytoskeletal Proteins, U.S. Provisional Patent Application No 61/618,896,
filed April
2, 2012, entitled Modified Polynucleotides for the Production of Intracellular
Membrane
Bound Proteins, U.S. Provisional Patent Application No 61/668,157, filed July
5, 2012,
entitled Modified Polynucleotides for the Production of Intracellular Membrane
Bound
Proteins, U.S. Provisional Patent Application No 61/681,661, filed August 10,
2012,
entitled Modified Polynucleotides for the Production of Intracellular Membrane
Bound
Proteins, U.S. Provisional Patent Application No 61/737,160, filed December
14, 2012,
entitled Modified Polynucleotides for the Production of Intracellular Membrane
Bound
Proteins, U.S. Provisional Patent Application No 61/618,911, filed April 2,
2012, entitled
Modified Polynucleotides for the Production of Nuclear Proteins, U.S.
Provisional Patent
Application No 61/681,667, filed August 10, 2012, entitled Modified
Polynucleotides for
2

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the Production of Nuclear Proteins, U.S. Provisional Patent Application No
61/737,168,
filed December 14, 2012, entitled Modified Polynucleotides for the Production
of
Nuclear Proteins, U.S. Provisional Patent Application No 61/618,922, filed
April 2, 2012,
entitled Modified Polynucleotides for the Production of Proteins, U.S.
Provisional Patent
Application No 61/681,675, filed August 10, 2012, entitled Modified
Polynucleotides for
the Production of Proteins, U.S. Provisional Patent Application No 61/737,174,
filed
December 14, 2012, entitled Modified Polynucleotides for the Production of
Proteins,
U.S. Provisional Patent Application No 61/618,935, filed April 2, 2012,
entitled Modified
Polynucleotides for the Production of Proteins Associated with Human Disease,
U.S.
Provisional Patent Application No 61/681,687, filed August 10, 2012, entitled
Modified
Polynucleotides for the Production of Proteins Associated with Human Disease,
U.S.
Provisional Patent Application No 61/737,184, filed December 14, 2012,
entitled
Modified Polynucleotides for the Production of Proteins Associated with Human
Disease,
U.S. Provisional Patent Application No 61/618,945, filed April 2, 2012,
entitled Modified
Polynucleotides for the Production of Proteins Associated with Human Disease,
U.S.
Provisional Patent Application No 61/681,696, filed August 10, 2012, entitled
Modified
Polynucleotides for the Production of Proteins Associated with Human Disease,
U.S.
Provisional Patent Application No 61/737,191, filed December 14, 2012,
entitled
Modified Polynucleotides for the Production of Proteins Associated with Human
Disease,
U.S. Provisional Patent Application No 61/618,953, filed April 2, 2012,
entitled Modified
Polynucleotides for the Production of Proteins Associated with Human Disease,
U.S.
Provisional Patent Application No 61/681,704, filed August 10, 2012, entitled
Modified
Polynucleotides for the Production of Proteins Associated with Human Disease,
U.S.
Provisional Patent Application No 61/737,203, filed December 14, 2012,
entitled
Modified Polynucleotides for the Production of Proteins Associated with Human
Disease,
U.S. Provisional Patent Application No 61/618,961, filed April 2, 2012,
entitled Dosing
Methods for Modified mRNA, U.S. Provisional Patent Application No 61/648,286,
filed
May 17, 2012, entitled Dosing Methods for Modified mRNA, the contents of each
of
which are herein incorporated by reference in its entirety.
[0003] This application is also related to International Publication No.
PCT/US2012/58519, filed October 3, 2012, entitled Modified Nucleosides,
Nucleotides,
3

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and Nucleic Acids, and Uses Thereof and International Publication No.
PCT/US2012/69610, filed December 14, 2012, entitled Modified Nucleoside,
Nucleotide,
and Nucleic Acid Compositions.
[0004] The
instant application is also related to co-pending applications, each filed
concurrently herewith on March 9, 2013 and having Attorney Docket Number
M301.20,
(PCT/US13/ ) entitled Modified Polynucleotides; Attorney Docket Number
M304.20 (PCT/US13/ ),
entitled Modified Polynucleotides for the Production of
Secreted Proteins; Attorney Docket Number M305.20 (PCT/U513/)0000(), entitled
Modified Polynucleotides for the Production of Membrane Proteins; Attorney
Docket
Number M306.20 (PCT/U513/ ), entitled Modified Polynucleotides for the
Production of Cytoplasmic and Cytoskeletal Proteins; Attorney Docket Number
M308.20
(PCT/U513/ ), entitled Modified Polynucleotides for the Production of
Nuclear
Proteins; Attorney Docket Number M309.20 (PCT/U513/ ),
entitled Modified
Polynucleotides for the Production of Proteins; Attorney Docket Number M310.20
(PCT/US13/ ), entitled Modified Polynucleotides for the Production of
Proteins
Associated with Human Disease; Attorney Docket Number MNC1.20
(PCT/US13/ ), entitled Modified Polynucleotides for the Production of
Cosmetic
Proteins and Peptides and Attorney Docket Number MNC2.20 (PCT/U513/ ),
entitled Modified Polynucleotides for the Production of Oncology-Related
Proteins and
Peptides, the contents of each of which are herein incorporated by reference
in its
entirety.
FIELD OF THE INVENTION
[0005] The
invention relates to compositions, methods, processes, kits and devices
for the design, preparation, manufacture and/or formulation of
polynucleotides, primary
constructs and modified mRNA molecules (mmRNA).
BACKGROUND OF THE INVENTION
[0006] There are multiple problems with prior methodologies of effecting
protein
expression. For example, introduced DNA can integrate into host cell genomic
DNA at
some frequency, resulting in alterations and/or damage to the host cell
genomic DNA.
Alternatively, the heterologous deoxyribonucleic acid (DNA) introduced into a
cell can
be inherited by daughter cells (whether or not the heterologous DNA has
integrated into
4

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the chromosome) or by offspring. In addition, assuming proper delivery and
no
damage or integration into the host genome, there are multiple steps which
must occur
before the encoded protein is made. Once inside the cell, DNA must be
transported into
the nucleus where it is transcribed into RNA. The RNA transcribed from DNA
must then
enter the cytoplasm where it is translated into protein. Not only do the
multiple
processing steps from administered DNA to protein create lag times before the
generation
of the functional protein, each step represents an opportunity for error and
damage to the
cell. Further, it is known to be difficult to obtain DNA expression in cells
as DNA
frequently enters a cell but is not expressed or not expressed at reasonable
rates or
concentrations. This can be a particular problem when DNA is introduced into
primary
cells or modified cell lines.
[0007] In the early 1990's Bloom and colleagues successfully rescued
vasopressin-
deficient rats by injecting in vitro-transcribed vasopressin mRNA into the
hypothalamus
(Science 255: 996-998; 1992). However, the low levels of translation and the
immunogenicity of the molecules hampered the development of mRNA as a
therapeutic
and efforts have since focused on alternative applications that could instead
exploit these
pitfalls, i.e. immunization with mRNAs coding for cancer antigens.
[0008] Others have investigated the use of mRNA to deliver a polypeptide of
interest
and shown that certain chemical modifications of mRNA molecules, particularly
pseudouridine and 5-methyl-cytosine, have reduced immunostimulatory effect.
[0009] These studies are disclosed in, for example, Ribostem Limited in
United
Kingdom patent application serial number 0316089.2 filed on July 9, 2003 now
abandoned, PCT application number PCT/GB2004/002981 filed on July 9, 2004
published as W02005005622, United States patent application national phase
entry serial
number 10/563,897 filed on June 8, 2006 published as US20060247195 now
abandoned,
and European patent application national phase entry serial number
EP2004743322 filed
on July 9, 2004 published as EP1646714 now withdrawn; Novozymes, Inc. in PCT
application number PCT/U52007/88060 filed on December 19, 2007 published as
W02008140615, United States patent application national phase entry serial
number
12/520,072 filed on July 2, 2009 published as US20100028943 and European
patent
application national phase entry serial number EP2007874376 filed on July 7,
2009

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PCT/US2013/030062
published as EP2104739; University of Rochester in PCT application number
PCT/US2006/46120 filed on December 4, 2006 published as W02007064952 and
United
States patent application serial number 11/606,995 filed on December 1, 2006
published
as U520070141030; BioNTech AG in European patent application serial number
EP2007024312 filed December 14, 2007 now abandoned, PCT application number
PCT/EP2008/01059 filed on December 12, 2008 published as W02009077134,
European
patent application national phase entry serial number EP2008861423 filed on
June 2,
2010 published as EP2240572, United States patent application national phase
entry
serial number 12/,735,060 filed November 24, 2010 published as US20110065103,
German patent application serial number DE 10 2005 046 490 filed September 28,
2005,
PCT application PCT/EP2006/0448 filed September 28, 2006 published as
W02007036366, national phase European patent EP1934345 published March, 21,
2012
and national phase US patent application serial number 11/992,638 filed August
14,
2009 published as 20100129877; Immune Disease Institute Inc. in United States
patent
application serial number 13/088,009 filed April 15, 2011 published as
US20120046346
and PCT application PCT/US2011/32679 filed April 15, 2011 published as
W020110130624; Shire Human Genetic Therapeutics in United States patent
application
serial number 12/957,340 filed on November 20, 2010 published as
US20110244026;
Sequitur Inc. in PCT application PCT/U51998/019492 filed on September 18, 1998

published as W01999014346; The Scripps Research Institute in PCT application
number
PCT/U52010/00567 filed on February 24, 2010 published as W02010098861, and
United States patent application national phase entry serial number 13/203,229
filed
November 3, 2011 published as US20120053333; Ludwig-Maximillians University in

PCT application number PCT/EP2010/004681 filed on July 30, 2010 published as
W02011012316; Cellscript Inc. in United States patent number 8,039,214 filed
June 30,
2008 and granted October 18, 2011, United States patent application serial
numbers
12/962,498 filed on December 7, 2010 published as US20110143436, 12/962,468
filed
on December 7, 2010 published as US20110143397, 13/237,451 filed on September
20,
2011 published as US20120009649, and PCT applications PCT/U52010/59305 filed
December 7, 2010 published as W02011071931 and PCT/U52010/59317 filed on
December 7, 2010 published as W02011071936; The Trustees of the University of
6

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Pennsylvania in PCT application number PCT/US2006/32372 filed on August 21,
2006
published as W02007024708, and United States patent application national phase
entry
serial number 11/990,646 filed on March 27, 2009 published as U520090286852;
Curevac GMBH in German patent application serial numbers DE10 2001 027 283.9
filed
June 5,2001, DE10 2001 062 480.8 filed December 19, 2001, and DE 20 2006 051
516
filed October 31, 2006 all abandoned, European patent numbers EP1392341
granted
March 30, 2005 and EP1458410 granted January 2, 2008, PCT application numbers
PCT/EP2002/06180 filed June 5, 2002 published as W02002098443,
PCT/EP2002/14577 filed on December 19, 2002 published as W02003051401,
PCT/EP2007/09469 filed on December 31, 2007 published as W02008052770,
PCT/EP2008/03033 filed on April 16, 2008 published as W02009127230,
PCT/EP2006/004784 filed on May 19, 2005 published as W02006122828,
PCT/EP2008/00081 filed on January 9, 2007 published as W02008083949, and
United
States patent application serial numbers 10/729,830 filed on December 5, 2003
published
as U520050032730, 10/870,110 filed on June 18, 2004 published as
U520050059624,
11/914,945 filed on July 7, 2008 published as U520080267873, 12/446,912 filed
on
October 27, 2009 published as U52010047261 now abandoned, 12/522,214 filed on
January 4,2010 published as U520100189729, 12/787,566 filed on May 26, 2010
published as US20110077287, 12/787,755 filed on May 26, 2010 published as
U520100239608, 13/185,119 filed on July 18, 2011 published as US20110269950,
and
13/106,548 filed on May 12, 2011 published as US20110311472 all of which are
herein
incorporated by reference in their entirety.
[00010] Notwithstanding these reports which are limited to a selection of
chemical
modifications including pseudouridine and 5-methyl-cytosine, there remains a
need in the
art for therapeutic modalities to address the myriad of barriers surrounding
the efficacious
modulation of intracellular translation and processing of nucleic acids
encoding
polypeptides or fragments thereof
[00011] To this end, the inventors have shown that certain modified mRNA
sequences
have the potential as therapeutics with benefits beyond just evading, avoiding
or
diminishing the immune response. Such studies are detailed in published co-
pending
applications International Application PCT/U52011/046861 filed August 5, 2011
and
7

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PCT/US2011/054636 filed October 3, 2011, International Application number
PCT/US2011/054617 filed October 3, 2011, the contents of which are
incorporated
herein by reference in their entirety.
[00012] The present invention addresses this need by providing nucleic acid
based
compounds or polynucleotides which encode a polypeptide of interest (e.g.,
modified
mRNA or mmRNA) and which have structural and/or chemical features that avoid
one or
more of the problems in the art, for example, features which are useful for
optimizing
formulation and delivery of nucleic acid-based therapeutics while retaining
structural and
functional integrity, overcoming the threshold of expression, improving
expression rates,
half life and/or protein concentrations, optimizing protein localization, and
avoiding
deleterious bio-responses such as the immune response and/or degradation
pathways.
SUMMARY OF THE INVENTION
[00013] Described herein are compositions, methods, processes, kits and
devices for
the design, preparation, manufacture and/or formulation of modified mRNA
(mmRNA)
molecules.
[00014] The details of various embodiments of the invention are set forth in
the
description below. Other features, objects, and advantages of the invention
will be
apparent from the description and the drawings, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[00015] The foregoing and other objects, features and advantages will be
apparent
from the following description of particular embodiments of the invention, as
illustrated
in the accompanying drawings in which like reference characters refer to the
same parts
throughout the different views. The drawings are not necessarily to scale,
emphasis
instead being placed upon illustrating the principles of various embodiments
of the
invention.
[00016] FIG. 1 is a schematic of a primary construct of the present invention.
[00017] FIG. 2 illustrates lipid structures in the prior art useful in the
present
invention. Shown are the structures for 98N12-5 (TETA5-LAP), DLin-DMA, DLin-K-
DMA (2,2-Dilinoley1-4-dimethylaminomethyl-[1,3]-dioxolane), DLin-KC2-DMA,
DLin-MC3-DMA and C12-200.
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[00018] FIG. 3 is a representative plasmid useful in the IVT reactions taught
herein.
The plasmid contains Insert 64818, designed by the instant inventors.
[00019] FIG. 4 is a gel profile of modified mRNA encapsulated in PLGA
micro spheres.
[00020] FIG. 5 is a histogram of Factor IX protein production PLGA formulation

Factor IX modified mRNA.
[00021] FIG. 6 is a histogram showing VEGF protein production in human
keratinocyte cells after transfection of modified mRNA at a range of doses.
Figure 6A
shows protein production after transfection of modified mRNA comprising
natural
nucleoside triphosphate (NTP). Figure 6B shows protein production after
transfection of
modified mRNA fully modified with pseudouridine (Pseudo-U) and 5-
methylcytosine
(5mC). Figure 6C shows protein production after transfection of modified mRNA
fully
modified with Nl-methyl-pseudouridine (N 1-methyl-Pseudo-U)and 5-
methylcytosine
(5mC).
[00022] FIG. 7 is a histogram of VEGF protein production in HEK293 cells.
[00023] FIG. 8 is a histogram of VEGF expression and IFN-alpha induction after

transfection of VEGF modified mRNA in peripheral blood mononuclear cells
(PBMC).
Figure 8A shows VEGF expression. Figure 8B shows IFN-alpha induction.
[00024] FIG. 9 is a histogram of VEGF protein production in HeLa cells from
VEGF
modified mRNA.
[00025] FIG. 10 is a histogram of VEGF protein production from lipoplexed VEGF

modified mRNA in mice.
[00026] FIG. 11 is a histogram of G-CSF protein production in HeLa cells from
G-
CSF modified mRNA.
[00027] FIG. 12 is a histogram of G-CSF protein production in mice from
lipoplexedG-CSF modified mRNA.
[00028] FIG. 13 is a histogram of Factor IX protein production in HeLa cell
supernatant from Factor IX modified mRNA.
[00029] FIG. 14 is a histogram of AP0A1 protein production in HeLa cells from
AP0A1 wild-type modified mRNA, AP0A1 Milano modified mRNA or AP0A1 Paris
modified mRNA.
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[00030] FIG. 15 is a gel profile of AP0A1 protein from AP0A1 wild-type
modified
mRNA.
[00031] FIG. 16 is a gel profile of AP0A1 protein from AP0A1 Paris modified
mRNA.
[00032] FIG. 17 is a gel profile of AP0A1 protein from AP0A1 Milano modified
mRNA.
[00033] FIG. 18 is a gel profile of Fibrinogen alpha (FGA) protein from FGA
modified mRNA.
[00034] FIG. 19 is a histogram of Plasminogen protein production in HeLa cell
supernatant from Plasminogen modified mRNA.
[00035] FIG. 20 is a gel profile of Plasminogen protein from Plasminogen
modified
mRNA.
[00036] FIG. 21 is a gel profile of galactose- 1 -phosphate
uridylyltransferase (GALT)
protein from GALT modified mRNA.
[00037] FIG. 22 is a gel profile of argininosuccinate lyase (ASL) protein from
ASL
modified mRNA.
[00038] FIG. 23 is a gel profile of tyrosine aminotransferase (TAT) protein
from TAT
modified mRNA.
[00039] FIG. 24 is a gel profile of glucan (1,4-alpha-), branching enzyme 1
(GBE1)
protein from GBE1 modified mRNA.
[00040] FIG. 25 is a histogram of Prothrombin protein production in HeLa cell
supernatant from Prothrombin modified mRNA.
[00041] FIG. 26 is a histogram of Prothrombin protein production in HeLa cell
supernatant from Prothrombin modified mRNA.
[00042] FIG. 27 is a gel profile of ceruloplasmin (CP or CLP) protein from CP
modified mRNA.
[00043] FIG. 28 is a histogram of transforming growth factor beta 1 (TGF-
betal)
protein production in HeLa cell supernatant from TGF-betal modified mRNA.
[00044] FIG. 29 is a gel profile of ornithine carbamoyltransferase (OTC)
protein from
OTC modified mRNA.

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[00045] FIG. 30 is a flow cytometry plot of low density lipoprotein receptor
(LDLR)
modified mRNA.
[00046] FIG. 31 is a gel profile of UDP glucuronosyltransferase 1 family,
polypeptide
Al (UGT1A1) protein from UGT1A1 modified mRNA.
[00047] FIG. 32 is a histogram of Factor XI protein production in HEK293
cells.
[00048] FIG. 33 is a gel profile of Aquaporin-5 protein from Aquaporin-5
modified
mRNA.
[00049] FIG. 34 is a histogram of Factor VII protein production in HeLa cells
from
Factor VII modified mRNA.
[00050] FIG. 35 is a histogram of Insulin Glargine protein production in HeLa
cells
from Insulin Glargine modified mRNA.
[00051] FIG. 36 is a histogram of Tissue Factor protein production in HeLa
cells from
Tissue Factor modified mRNA.
[00052] FIG. 37 is a histogram of Factor XI protein production in HeLa cells
from
Factor XI modified mRNA.
[00053] FIG. 38 is a histogram of Factor XI protein production in HeLa cell
supernatant from Factor XI modified mRNA.
[00054] FIG. 39 is a histogram of Insulin Aspart protein production in HeLa
cells from
Insulin Aspart modified mRNA.
[00055] FIG. 40 is a histogram of Insulin Lispro protein production in HeLa
cells from
Insulin Lispro modified mRNA.
[00056] FIG. 41 is a histogram of Insulin Glulisine protein production in HeLa
cells
from Insulin Glulisine modified mRNA.
[00057] FIG. 42 is a histogram of human growth hormone protein production in
HeLa
cells from human growth hormone modified mRNA.
[00058] FIG. 43 is a gel profile of tumor protein 53 (p53) protein from p53
modified
mRNA. Figure 43A shows the expected size of p53. Figure 43B shows the expected
size
of p53.
[00059] FIG. 44 is a gel profile of tuftelin (TUFT1) protein from TUFT1
modified
mRNA.
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[00060] FIG. 45 is a gel profile of galactokinase 1 (GALK1) protein from GALK1
modified mRNA. Figure 45A shows the expected size of GALK1. Figure 45B shows
the expected size of GALK1.
[00061] FIG. 46 is a gel profile of defensin, beta 103A (DEFB103A) protein
from
DEFB103A modified mRNA.
[00062] FIG. 47 is a flow cytometry plot of LDLR modified mRNA.
[00063] FIG. 48 is a histogram of vascular endothelial growth factor
expression in
HeLa.
[00064] FIG. 49 is a histogram of the cell vitality of HeLa cells transfected
with
vascular endothelial growth factor mRNA.
[00065] FIG. 50 is a histogram of Insulin Aspart protein expression.
[00066] FIG. 51 is a histogram of Insulin Glargine protein expression.
[00067] FIG. 52 is a histogram of Insulin Glulisine protein expression.
[00068] FIG. 53 is a histogram of Interleukin 7 (IL-7) protein expression.
[00069] FIG. 54 is a histogram of Erythropoietin (EPO) protein expression.
[00070] FIG. 55 is a gel profile of Lysosomal Acid Lipase protein from
Lysosomal
Acid Lipase modified mRNA.
[00071] FIG. 56 is a gel profile of Glucocerebrosidase protein from
Glucocerebrosidase modified mRNA.
[00072] FIG. 57 is a gel profile of Iduronate 2-Sulfatase protein from
Iduronate 2-
Sulfatase modified mRNA.
[00073] FIG. 58 is a gel profile of Luciferase protein from Luciferase
modified
mRNA.
[00074] FIG. 59 is a histogram of IgG concentration after administration with
formulated Herceptin modified mRNA in mammals.
[00075] FIG. 60 is a histogram of IgG concentration (in ng/ml) after
transfection with
Herceptin modified mRNA.
[00076] FIG. 61 is a gel profile of Herceptin protein from Herceptin modified
mRNA.
[00077] FIG. 62 is a histogram of Glucocerebrosidase enzyme activity.
[00078] FIG. 63 is a histogram of Lysosomal Acid Lipase enzyme activity.
[00079] FIG. 64 a is histogram of Factor VIII protein expression.
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[00080] FIG. 65 is a histogram of Factor VIII Chromogenic Activity.
[00081] FIG. 66 is a graph of LDLR Expression. Figure 66A shows LDL Receptor
Expression of cells compared to LDLR mRNA added. Figure 66B shows LDL Receptor

Expression of cells post transfection. Figure 66C shows the saturation of
BODIPYO
labeled LRL. Figure 66D shows the binding affinity of BODIPY-LDL to cells.
[00082] FIG. 67 is a graph showing the percent positive cells for UGT1A1
Expression.
[00083] FIG. 68 is a graph showing UGT1A1 protein accumulation.
[00084] FIG. 69 is a gel profile of UGT1A1 protein and OTC from eith UGT1A1 or

OTC modified mRNA.
[00085] FIG. 70 is a flow cytometry plot of HEK293 cells transfected with PAh
or
UGT1A1.
[00086] FIG. 71 is a gel profile of UGT1A1 protein from UGT1A1 modified mRNA.
[00087] FIG. 72 is a gel profile of microsomal extracts of mice treated with
LNPs
containing UGT1A1.
DETAILED DESCRIPTION
[00088] It is
of great interest in the fields of therapeutics, diagnostics, reagents and for
biological assays to be able to deliver a nucleic acid, e.g., a ribonucleic
acid (RNA) inside
a cell, whether in vitro, in vivo, in situ or ex vivo, such as to cause
intracellular translation
of the nucleic acid and production of an encoded polypeptide of interest. Of
particular
importance is the delivery and function of a non-integrative polynucleotide.
[00089] Described herein are compositions (including pharmaceutical
compositions)
and methods for the design, preparation, manufacture and/or formulation of
polynucleotides encoding one or more polypeptides of interest. Also provided
are
systems, processes, devices and kits for the selection, design and/or
utilization of the
polynucleotides encoding the polypeptides of interest described herein.
[00090] According to the present invention, these polynucleotides are
preferably
modified as to avoid the deficiencies of other polypeptide-encoding molecules
of the art.
Hence these polynucleotides are referred to as modified mRNA or mmRNA.
[00091] The use of modified polynucleotides in the fields of antibodies,
viruses,
veterinary applications and a variety of in vivo settings has been explored by
the
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inventors and these studies are disclosed in for example, co-pending and co-
owned
United States provisional patent application serial numbers 61/470,451 filed
March 31,
2011 teaching in vivo applications of mmRNA; 61/517,784 filed on April 26,
2011
teaching engineered nucleic acids for the production of antibody polypeptides;

61/519,158 filed May 17, 2011 teaching veterinary applications of mmRNA
technology;
61/533, 537 filed on September 12, 2011 teaching antimicrobial applications of
mmRNA
technology; 61/533,554 filed on September 12, 2011 teaching viral applications
of
mmRNA technology, 61/542,533 filed on October 3, 2011 teaching various
chemical
modifications for use in mmRNA technology; 61/570,690 filed on December 14,
2011
teaching mobile devices for use in making or using mmRNA technology;
61/570,708
filed on December 14, 2011 teaching the use of mmRNA in acute care situations;

61/576,651 filed on December 16, 2011 teaching terminal modification
architecture for
mmRNA; 61/576,705 filed on December 16, 2011 teaching delivery methods using
lipidoids for mmRNA; 61/578,271 filed on December 21, 2011 teaching methods to

increase the viability of organs or tissues using mmRNA; 61/581,322 filed on
December
29, 2011 teaching mmRNA encoding cell penetrating peptides; 61/581,352 filed
on
December 29, 2011 teaching the incorporation of cytotoxic nucleosides in mmRNA
and
61/631,729 filed on January 10, 2012 teaching methods of using mmRNA for
crossing
the blood brain barrier; all of which are herein incorporated by reference in
their entirety.
[00092] Provided herein, in part, are polynucleotides, primary constructs
and/or
mmRNA encoding polypeptides of interest which have been designed to improve
one or
more of the stability and/or clearance in tissues, receptor uptake and/or
kinetics, cellular
access by the compositions, engagement with translational machinery, mRNA half-
life,
translation efficiency, immune evasion, protein production capacity, secretion
efficiency
(when applicable), accessibility to circulation, protein half-life and/or
modulation of a
cell's status, function and/or activity.
I. Compositions of the Invention (mmRNA)
[00093] The present invention provides nucleic acid molecules, specifically
polynucleotides, primary constructs and/or mmRNA which encode one or more
polypeptides of interest. The term "nucleic acid," in its broadest sense,
includes any
compound and/or substance that comprise a polymer of nucleotides. These
polymers are
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often referred to as polynucleotides. Exemplary nucleic acids or
polynucleotides of the
invention include, but are not limited to, ribonucleic acids (RNAs),
deoxyribonucleic
acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs),
peptide
nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a 0- D-
ribo
configuration, a-LNA having an a-L-ribo configuration (a diastereomer of LNA),
2'-
amino-LNA having a 2'-amino functionalization, and 2'-amino- a-LNA having a 2'-

amino functionalization) or hybrids thereof
[00094] In preferred embodiments, the nucleic acid molecule is a messenger RNA

(mRNA). As used herein, the term "messenger RNA" (mRNA) refers to any
polynucleotide which encodes a polypeptide of interest and which is capable of
being
translated to produce the encoded polypeptide of interest in vitro, in vivo,
in situ or ex
vivo.
[00095] Traditionally, the basic components of an mRNA molecule include at
least a
coding region, a 5'UTR, a 3'UTR, a 5' cap and a poly-A tail. Building on this
wild type
modular structure, the present invention expands the scope of functionality of
traditional
mRNA molecules by providing polynucleotides or primary RNA constructs which
maintain a modular organization, but which comprise one or more structural
and/or
chemical modifications or alterations which impart useful properties to the
polynucleotide including, in some embodiments, the lack of a substantial
induction of the
innate immune response of a cell into which the polynucleotide is introduced.
As such,
modified mRNA molecules of the present invention are termed "mmRNA." As used
herein, a "structural" feature or modification is one in which two or more
linked
nucleotides are inserted, deleted, duplicated, inverted or randomized in a
polynucleotide,
primary construct or mmRNA without significant chemical modification to the
nucleotides themselves. Because chemical bonds will necessarily be broken and
reformed
to effect a structural modification, structural modifications are of a
chemical nature and
hence are chemical modifications. However, structural modifications will
result in a
different sequence of nucleotides. For example, the polynucleotide "ATCG" may
be
chemically modified to "AT-5meC-G". The same polynucleotide may be
structurally
modified from "ATCG" to "ATCCCG". Here, the dinucleotide "CC" has been
inserted,
resulting in a structural modification to the polynucleotide.

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mmRNA Architecture
[00096] The mmRNA of the present invention are distinguished from wild type
mRNA in their functional and/or structural design features which serve to, as
evidenced
herein, overcome existing problems of effective polypeptide production using
nucleic
acid-based therapeutics.
[00097] Figure 1 shows a representative polynucleotide primary construct
100 of the
present invention. As used herein, the term "primary construct" or "primary
mRNA
construct" refers to a polynucleotide transcript which encodes one or more
polypeptides
of interest and which retains sufficient structural and/or chemical features
to allow the
polypeptide of interest encoded therein to be translated. Primary constructs
may be
polynucleotides of the invention. When structurally or chemically modified,
the primary
construct may be referred to as an mmRNA.
[00098] Returning to FIG. 1, the primary construct 100 here contains a first
region of
linked nucleotides 102 that is flanked by a first flanking region 104 and a
second flaking
region 106. As used herein, the "first region" may be referred to as a "coding
region" or
"region encoding" or simply the "first region." This first region may include,
but is not
limited to, the encoded polypeptide of interest. The polypeptide of interest
may comprise
at its 5' terminus one or more signal sequences encoded by a signal sequence
region 103.
The flanking region 104 may comprise a region of linked nucleotides comprising
one or
more complete or incomplete 5' UTRs sequences. The flanking region 104 may
also
comprise a 5' terminal cap 108. The second flanking region 106 may comprise a
region
of linked nucleotides comprising one or more complete or incomplete 3' UTRs.
The
flanking region 106 may also comprise a 3' tailing sequence 110.
[00099] Bridging the 5' terminus of the first region 102 and the first
flanking region
104 is a first operational region 105. Traditionally this operational region
comprises a
Start codon. The operational region may alternatively comprise any translation
initiation
sequence or signal including a Start codon.
[000100] Bridging the 3' terminus of the first region 102 and the second
flanking region
106 is a second operational region 107. Traditionally this operational region
comprises a
Stop codon. The operational region may alternatively comprise any translation
initiation
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sequence or signal including a Stop codon. According to the present invention,
multiple
serial stop codons may also be used.
[000101] Generally, the shortest length of the first region of the primary
construct of the
present invention can be the length of a nucleic acid sequence that is
sufficient to encode
for a dipeptide, a tripeptide, a tetrapeptide, a pentapeptide, a hexapeptide,
a heptapeptide,
an octapeptide, a nonapeptide, or a decapeptide. In another embodiment, the
length may
be sufficient to encode a peptide of 2-30 amino acids, e.g. 5-30, 10-30, 2-25,
5-25, 10-25,
or 10-20 amino acids. The length may be sufficient to encode for a peptide of
at least 11,
12, 13, 14, 15, 17, 20, 25 or 30 amino acids, or a peptide that is no longer
than 40 amino
acids, e.g. no longer than 35, 30, 25, 20, 17, 15, 14, 13, 12, 11 or 10 amino
acids.
Examples of dipeptides that the polynucleotide sequences can encode or
include, but are
not limited to, carnosine and anserine.
[000102] Generally, the length of the first region encoding the polypeptide of
interest of
the present invention is greater than about 30 nucleotides in length (e.g., at
least or
greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160,
180, 200, 250,
300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300,
1,400, 1,500,
1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000, 4,000, 5,000, 6,000,
7,000, 8,000,
9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000
or up to
and including 100,000 nucleotides). As used herein, the "first region" may be
referred to
as a "coding region" or "region encoding" or simply the "first region."
[000103] In some embodiments, the polynucleotide, primary construct, or mmRNA
includes from about 30 to about 100,000 nucleotides (e.g., from 30 to 50, from
30 to 100,
from 30 to 250, from 30 to 500, from 30 to 1,000, from 30 to 1,500, from 30 to
3,000,
from 30 to 5,000, from 30 to 7,000, from 30 to 10,000, from 30 to 25,000, from
30 to
50,000, from 30 to 70,000, from 100 to 250, from 100 to 500, from 100 to
1,000, from
100 to 1,500, from 100 to 3,000, from 100 to 5,000, from 100 to 7,000, from
100 to
10,000, from 100 to 25,000, from 100 to 50,000, from 100 to 70,000, from 100
to
100,000, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to
3,000,
from 500 to 5,000, from 500 to 7,000, from 500 to 10,000, from 500 to 25,000,
from 500
to 50,000, from 500 to 70,000, from 500 to 100,000, from 1,000 to 1,500, from
1,000 to
2,000, from 1,000 to 3,000, from 1,000 to 5,000, from 1,000 to 7,000, from
1,000 to
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10,000, from 1,000 to 25,000, from 1,000 to 50,000, from 1,000 to 70,000, from
1,000 to
100,000, from 1,500 to 3,000, from 1,500 to 5,000, from 1,500 to 7,000, from
1,500 to
10,000, from 1,500 to 25,000, from 1,500 to 50,000, from 1,500 to 70,000, from
1,500 to
100,000, from 2,000 to 3,000, from 2,000 to 5,000, from 2,000 to 7,000, from
2,000 to
10,000, from 2,000 to 25,000, from 2,000 to 50,000, from 2,000 to 70,000, and
from
2,000 to 100,000).
[000104] According to the present invention, the first and second flanking
regions may
range independently from 15-1,000 nucleotides in length (e.g., greater than
30, 40, 45,
50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450,
500, 600,
700, 800, and 900 nucleotides or at least 30, 40, 45, 50, 55, 60, 70, 80, 90,
100, 120, 140,
160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, and 1,000
nucleotides).
[000105] According to the present invention, the tailing sequence may range
from
absent to 500 nucleotides in length (e.g., at least 60, 70, 80, 90, 120, 140,
160, 180, 200,
250, 300, 350, 400, 450, or 500 nucleotides). Where the tailing region is a
polyA tail, the
length may be determined in units of or as a function of polyA Binding Protein
binding.
In this embodiment, the polyA tail is long enough to bind at least 4 monomers
of PolyA
Binding Protein. PolyA Binding Protein monomers bind to stretches of
approximately 38
nucleotides. As such, it has been observed that polyA tails of about 80
nucleotides and
160 nucleotides are functional.
[000106] According to the present invention, the capping region may comprise a
single
cap or a series of nucleotides forming the cap. In this embodiment the capping
region
may be from 1 to 10, e.g. 2-9, 3-8, 4-7, 1-5, 5-10, or at least 2, or 10 or
fewer nucleotides
in length. In some embodiments, the cap is absent.
[000107] According to the present invention, the first and second operational
regions
may range from 3 to 40, e.g., 5-30, 10-20, 15, or at least 4, or 30 or fewer
nucleotides in
length and may comprise, in addition to a Start and/or Stop codon, one or more
signal
and/or restriction sequences.
Cyclic mmRNA
[000108] According to the present invention, a primary construct or mmRNA may
be
cyclized, or concatemerized, to generate a translation competent molecule to
assist
interactions between poly-A binding proteins and 5'-end binding proteins. The
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mechanism of cyclization or concatemerization may occur through at least 3
different
routes: 1) chemical, 2) enzymatic, and 3) ribozyme catalyzed. The newly formed
5'-/3'-
linkage may be intramolecular or intermolecular.
[000109] In the first route, the 5'-end and the 3'-end of the nucleic acid
contain
chemically reactive groups that, when close together, form a new covalent
linkage
between the 5'-end and the 3'-end of the molecule. The 5'-end may contain an
NHS-ester
reactive group and the 3'-end may contain a 3'-amino-terminated nucleotide
such that in
an organic solvent the 3'-amino-terminated nucleotide on the 3'-end of a
synthetic mRNA
molecule will undergo a nucleophilic attack on the 5'-NHS-ester moiety forming
a new
5'-/3'-amide bond.
[000110] In the second route, T4 RNA ligase may be used to enzymatically link
a 5'-
phosphorylated nucleic acid molecule to the 3'-hydroxyl group of a nucleic
acid forming
a new phosphorodiester linkage. In an example reaction, liug of a nucleic acid
molecule
is incubated at 37 C for 1 hour with 1-10 units of T4 RNA ligase (New England
Biolabs,
Ipswich, MA) according to the manufacturer's protocol. The ligation reaction
may occur
in the presence of a split oligonucleotide capable of base-pairing with both
the 5'- and 3'-
region in juxtaposition to assist the enzymatic ligation reaction.
[000111] In the third route, either the 5'-or 3'-end of the cDNA template
encodes a
ligase ribozyme sequence such that during in vitro transcription, the
resultant nucleic acid
molecule can contain an active ribozyme sequence capable of ligating the 5'-
end of a
nucleic acid molecule to the 3'-end of a nucleic acid molecule. The ligase
ribozyme may
be derived from the Group I Intron, Group I Intron, Hepatitis Delta Virus,
Hairpin
ribozyme or may be selected by SELEX (systematic evolution of ligands by
exponential
enrichment). The ribozyme ligase reaction may take 1 to 24 hours at
temperatures
between 0 and 37 C.
mmRNA Multimers
[000112] According to the present invention, multiple distinct
polynucleotides, primary
constructs or mmRNA may be linked together through the 3'-end using
nucleotides which
are modified at the 3'-terminus. Chemical conjugation may be used to control
the
stoichiometry of delivery into cells. For example, the glyoxylate cycle
enzymes,
isocitrate lyase and malate synthase, may be supplied into HepG2 cells at a
1:1 ratio to
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alter cellular fatty acid metabolism. This ratio may be controlled by
chemically linking
polynucleotides, primary constructs or mmRNA using a 3'-azido terminated
nucleotide
on one polynucleotide, primary construct or mmRNA species and a C5-ethynyl or
alkynyl-containing nucleotide on the opposite polynucleotide, primary
construct or
mmRNA species. The modified nucleotide is added post-transcriptionally using
terminal
transferase (New England Biolabs, Ipswich, MA) according to the manufacturer's

protocol. After the addition of the 3'-modified nucleotide, the two
polynucleotide,
primary construct or mmRNA species may be combined in an aqueous solution, in
the
presence or absence of copper, to form a new covalent linkage via a click
chemistry
mechanism as described in the literature.
[000113] In another example, more than two polynucleotides may be linked
together
using a functionalized linker molecule. For example, a functionalized
saccharide
molecule may be chemically modified to contain multiple chemical reactive
groups (SH-,
NH2-, N3, etc...) to react with the cognate moiety on a 3'-functionalized mRNA
molecule
(i.e., a 3'-maleimide ester, 3'-NHS-ester, alkynyl). The number of reactive
groups on the
modified saccharide can be controlled in a stoichiometric fashion to directly
control the
stoichiometric ratio of conjugated polynucleotide, primary construct or mmRNA.

mmRNA Conjugates and Combinations
[000114] In order to further enhance protein production, primary constructs or
mmRNA
of the present invention can be designed to be conjugated to other
polynucleotides, dyes,
intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene,
mitomycin C),
porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons
(e.g.,
phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), alkylating
agents,
phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino,
alkyl,
substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin),
transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid),
synthetic
ribonucleases, proteins, e.g., glycoproteins, or peptides, e.g., molecules
having a specific
affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a
specified cell type
such as a cancer cell, endothelial cell, or bone cell, hormones and hormone
receptors,
non-peptidic species, such as lipids, lectins, carbohydrates, vitamins,
cofactors, or a drug.

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[000115] Conjugation may result in increased stability and/or half life and
may be
particularly useful in targeting the polynucleotides, primary constructs or
mmRNA to
specific sites in the cell, tissue or organism.
[000116] According to the present invention, the mmRNA or primary constructs
may be
administered with, or further encode one or more of RNAi agents, siRNAs,
shRNAs,
miRNAs, miRNA binding sites, antisense RNAs, ribozymes, catalytic DNA, tRNA,
RNAs that induce triple helix formation, aptamers or vectors, and the like.
Bifunctional mmRNA
[000117] In one embodiment of the invention are bifunctional polynucleotides
(e.g.,
bifunctional primary constructs or bifunctional mmRNA). As the name implies,
bifunctional polynucleotides are those having or capable of at least two
functions. These
molecules may also by convention be referred to as multi-functional.
[000118] The multiple functionalities of bifunctional polynucleotides may be
encoded
by the RNA (the function may not manifest until the encoded product is
translated) or
may be a property of the polynucleotide itself. It may be structural or
chemical.
Bifunctional modified polynucleotides may comprise a function that is
covalently or
electrostatically associated with the polynucleotides. Further, the two
functions may be
provided in the context of a complex of a mmRNA and another molecule.
[000119] Bifunctional polynucleotides may encode peptides which are anti-
proliferative. These peptides may be linear, cyclic, constrained or random
coil. They
may function as aptamers, signaling molecules, ligands or mimics or mimetics
thereof
Anti-proliferative peptides may, as translated, be from 3 to 50 amino acids in
length.
They may be 5-40, 10-30, or approximately 15 amino acids long. They may be
single
chain, multichain or branched and may form complexes, aggregates or any multi-
unit
structure once translated.
Noncoding polynucleotides and primary constructs
[000120] As described herein, provided are polynucleotides and primary
constructs
having sequences that are partially or substantially not translatable, e.g.,
having a
noncoding region. Such noncoding region may be the "first region" of the
primary
construct. Alternatively, the noncoding region may be a region other than the
first region.
Such molecules are generally not translated, but can exert an effect on
protein production
21

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by one or more of binding to and sequestering one or more translational
machinery
components such as a ribosomal protein or a transfer RNA (tRNA), thereby
effectively
reducing protein expression in the cell or modulating one or more pathways or
cascades
in a cell which in turn alters protein levels. The polynucleotide or primary
construct may
contain or encode one or more long noncoding RNA (lncRNA, or lincRNA) or
portion
thereof, a small nucleolar RNA (sno-RNA), micro RNA (miRNA), small interfering

RNA (siRNA) or Piwi-interacting RNA (piRNA).
Polypeptides of interest
[000121] According to the present invention, the primary construct is designed
to
encode one or more polypeptides of interest or fragments thereof. A
polypeptide of
interest may include, but is not limited to, whole polypeptides, a plurality
of polypeptides
or fragments of polypeptides, which independently may be encoded by one or
more
nucleic acids, a plurality of nucleic acids, fragments of nucleic acids or
variants of any of
the aforementioned. As used herein, the term "polypeptides of interest" refer
to any
polypeptide which is selected to be encoded in the primary construct of the
present
invention. As used herein, "polypeptide" means a polymer of amino acid
residues
(natural or unnatural) linked together most often by peptide bonds. The term,
as used
herein, refers to proteins, polypeptides, and peptides of any size, structure,
or function. In
some instances the polypeptide encoded is smaller than about 50 amino acids
and the
polypeptide is then termed a peptide. If the polypeptide is a peptide, it will
be at least
about 2, 3, 4, or at least 5 amino acid residues long. Thus, polypeptides
include gene
products, naturally occurring polypeptides, synthetic polypeptides, homologs,
orthologs,
paralogs, fragments and other equivalents, variants, and analogs of the
foregoing. A
polypeptide may be a single molecule or may be a multi-molecular complex such
as a
dimer, trimer or tetramer. They may also comprise single chain or multichain
polypeptides such as antibodies or insulin and may be associated or linked.
Most
commonly disulfide linkages are found in multichain polypeptides. The term
polypeptide
may also apply to amino acid polymers in which one or more amino acid residues
are an
artificial chemical analogue of a corresponding naturally occurring amino
acid.
[000122] The term "polypeptide variant" refers to molecules which differ in
their amino
acid sequence from a native or reference sequence. The amino acid sequence
variants
22

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may possess substitutions, deletions, and/or insertions at certain positions
within the
amino acid sequence, as compared to a native or reference sequence.
Ordinarily, variants
will possess at least about 50% identity (homology) to a native or reference
sequence,
and preferably, they will be at least about 80%, more preferably at least
about 90%
identical (homologous) to a native or reference sequence.
[000123] In some embodiments "variant mimics" are provided. As used herein,
the term
"variant mimic" is one which contains one or more amino acids which would
mimic an
activated sequence. For example, glutamate may serve as a mimic for phosphoro-
threonine and/or phosphoro-serine. Alternatively, variant mimics may result in

deactivation or in an inactivated product containing the mimic, e.g.,
phenylalanine may
act as an inactivating substitution for tyrosine; or alanine may act as an
inactivating
substitution for serine.
[000124] "Homology" as it applies to amino acid sequences is defined as the
percentage
of residues in the candidate amino acid sequence that are identical with the
residues in the
amino acid sequence of a second sequence after aligning the sequences and
introducing
gaps, if necessary, to achieve the maximum percent homology. Methods and
computer
programs for the alignment are well known in the art. It is understood that
homology
depends on a calculation of percent identity but may differ in value due to
gaps and
penalties introduced in the calculation.
[000125] By "homologs" as it applies to polypeptide sequences means the
corresponding sequence of other species having substantial identity to a
second sequence
of a second species.
[000126] "Analogs" is meant to include polypeptide variants which differ by
one or
more amino acid alterations, e.g., substitutions, additions or deletions of
amino acid
residues that still maintain one or more of the properties of the parent or
starting
polypeptide.
[000127] The present invention contemplates several types of compositions
which are
polypeptide based including variants and derivatives. These include
substitutional,
insertional, deletion and covalent variants and derivatives. The term
"derivative" is used
synonymously with the term "variant" but generally refers to a molecule that
has been
modified and/or changed in any way relative to a reference molecule or
starting molecule.
23

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[000128] As such, mmRNA encoding polypeptides containing substitutions,
insertions
and/or additions, deletions and covalent modifications with respect to
reference
sequences, in particular the polypeptide sequences disclosed herein, are
included within
the scope of this invention. For example, sequence tags or amino acids, such
as one or
more lysines, can be added to the peptide sequences of the invention (e.g., at
the N-
terminal or C-terminal ends). Sequence tags can be used for peptide
purification or
localization. Lysines can be used to increase peptide solubility or to allow
for
biotinylation. Alternatively, amino acid residues located at the carboxy and
amino
terminal regions of the amino acid sequence of a peptide or protein may
optionally be
deleted providing for truncated sequences. Certain amino acids (e.g., C-
terminal or N-
terminal residues) may alternatively be deleted depending on the use of the
sequence, as
for example, expression of the sequence as part of a larger sequence which is
soluble, or
linked to a solid support.
[000129] "Substitutional variants" when referring to polypeptides are those
that have at
least one amino acid residue in a native or starting sequence removed and a
different
amino acid inserted in its place at the same position. The substitutions may
be single,
where only one amino acid in the molecule has been substituted, or they may be
multiple,
where two or more amino acids have been substituted in the same molecule.
[000130] As used herein the term "conservative amino acid substitution" refers
to the
substitution of an amino acid that is normally present in the sequence with a
different
amino acid of similar size, charge, or polarity. Examples of conservative
substitutions
include the substitution of a non-polar (hydrophobic) residue such as
isoleucine, valine
and leucine for another non-polar residue. Likewise, examples of conservative
substitutions include the substitution of one polar (hydrophilic) residue for
another such
as between arginine and lysine, between glutamine and asparagine, and between
glycine
and serine. Additionally, the substitution of a basic residue such as lysine,
arginine or
histidine for another, or the substitution of one acidic residue such as
aspartic acid or
glutamic acid for another acidic residue are additional examples of
conservative
substitutions. Examples of non-conservative substitutions include the
substitution of a
non-polar (hydrophobic) amino acid residue such as isoleucine, valine,
leucine, alanine,
24

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methionine for a polar (hydrophilic) residue such as cysteine, glutamine,
glutamic acid or
lysine and/or a polar residue for a non-polar residue.
[000131] "Insertional variants" when referring to polypeptides are those with
one or
more amino acids inserted immediately adjacent to an amino acid at a
particular position
in a native or starting sequence. "Immediately adjacent" to an amino acid
means
connected to either the alpha-carboxy or alpha-amino functional group of the
amino acid.
[000132] "Deletional variants" when referring to polypeptides are those with
one or
more amino acids in the native or starting amino acid sequence removed.
Ordinarily,
deletional variants will have one or more amino acids deleted in a particular
region of the
molecule.
[000133] "Covalent derivatives" when referring to polypeptides include
modifications
of a native or starting protein with an organic proteinaceous or non-
proteinaceous
derivatizing agent, and/or post-translational modifications. Covalent
modifications are
traditionally introduced by reacting targeted amino acid residues of the
protein with an
organic derivatizing agent that is capable of reacting with selected side-
chains or terminal
residues, or by harnessing mechanisms of post-translational modifications that
function in
selected recombinant host cells. The resultant covalent derivatives are useful
in programs
directed at identifying residues important for biological activity, for
immunoassays, or for
the preparation of anti-protein antibodies for immunoaffinity purification of
the
recombinant glycoprotein. Such modifications are within the ordinary skill in
the art and
are performed without undue experimentation.
[000134] Certain post-translational modifications are the result of the action
of
recombinant host cells on the expressed polypeptide. Glutaminyl and
asparaginyl
residues are frequently post-translationally deamidated to the corresponding
glutamyl and
aspartyl residues. Alternatively, these residues are deamidated under mildly
acidic
conditions. Either form of these residues may be present in the polypeptides
produced in
accordance with the present invention.
[000135] Other post-translational modifications include hydroxylation of
proline and
lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues,
methylation of
the alpha-amino groups of lysine, arginine, and histidine side chains (T. E.
Creighton,

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Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San
Francisco, pp.
79-86 (1983)).
[000136] "Features" when referring to polypeptides are defined as distinct
amino acid
sequence-based components of a molecule. Features of the polypeptides encoded
by the
mmRNA of the present invention include surface manifestations, local
conformational
shape, folds, loops, half-loops, domains, half-domains, sites, termini or any
combination
thereof
[000137] As used herein when referring to polypeptides the term "surface
manifestation" refers to a polypeptide based component of a protein appearing
on an
outermost surface.
[000138] As used herein when referring to polypeptides the term "local
conformational
shape" means a polypeptide based structural manifestation of a protein which
is located
within a definable space of the protein.
[000139] As used herein when referring to polypeptides the term "fold" refers
to the
resultant conformation of an amino acid sequence upon energy minimization. A
fold may
occur at the secondary or tertiary level of the folding process. Examples of
secondary
level folds include beta sheets and alpha helices. Examples of tertiary folds
include
domains and regions formed due to aggregation or separation of energetic
forces.
Regions formed in this way include hydrophobic and hydrophilic pockets, and
the like.
[000140] As used herein the term "turn" as it relates to protein conformation
means a
bend which alters the direction of the backbone of a peptide or polypeptide
and may
involve one, two, three or more amino acid residues.
[000141] As used herein when referring to polypeptides the term "loop" refers
to a
structural feature of a polypeptide which may serve to reverse the direction
of the
backbone of a peptide or polypeptide. Where the loop is found in a polypeptide
and only
alters the direction of the backbone, it may comprise four or more amino acid
residues.
Oliva et al. have identified at least 5 classes of protein loops (J. Mol Biol
266 (4): 814-
830; 1997). Loops may be open or closed. Closed loops or "cyclic" loops may
comprise
2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids between the bridging moieties.
Such bridging
moieties may comprise a cysteine-cysteine bridge (Cys-Cys) typical in
polypeptides
26

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having disulfide bridges or alternatively bridging moieties may be non-protein
based such
as the dibromozylyl agents used herein.
[000142] As used herein when referring to polypeptides the term "half-loop"
refers to a
portion of an identified loop having at least half the number of amino acid
resides as the
loop from which it is derived. It is understood that loops may not always
contain an even
number of amino acid residues. Therefore, in those cases where a loop contains
or is
identified to comprise an odd number of amino acids, a half-loop of the odd-
numbered
loop will comprise the whole number portion or next whole number portion of
the loop
(number of amino acids of the loop/2+/-0.5 amino acids). For example, a loop
identified
as a 7 amino acid loop could produce half-loops of 3 amino acids or 4 amino
acids
(7/2=3.5+/-0.5 being 3 or 4).
[000143] As used herein when referring to polypeptides the term "domain"
refers to a
motif of a polypeptide having one or more identifiable structural or
functional
characteristics or properties (e.g., binding capacity, serving as a site for
protein-protein
interactions).
[000144] As used herein when referring to polypeptides the term "half-domain"
means
a portion of an identified domain having at least half the number of amino
acid resides as
the domain from which it is derived. It is understood that domains may not
always
contain an even number of amino acid residues. Therefore, in those cases where
a domain
contains or is identified to comprise an odd number of amino acids, a half-
domain of the
odd-numbered domain will comprise the whole number portion or next whole
number
portion of the domain (number of amino acids of the domain/2+/-0.5 amino
acids). For
example, a domain identified as a 7 amino acid domain could produce half-
domains of 3
amino acids or 4 amino acids (7/2=3.5+/-0.5 being 3 or 4). It is also
understood that sub-
domains may be identified within domains or half-domains, these subdomains
possessing
less than all of the structural or functional properties identified in the
domains or half
domains from which they were derived. It is also understood that the amino
acids that
comprise any of the domain types herein need not be contiguous along the
backbone of
the polypeptide (i.e., nonadjacent amino acids may fold structurally to
produce a domain,
half-domain or subdomain).
27

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[000145] As used herein when referring to polypeptides the terms "site" as it
pertains to
amino acid based embodiments is used synonymously with "amino acid residue"
and
"amino acid side chain." A site represents a position within a peptide or
polypeptide that
may be modified, manipulated, altered, derivatized or varied within the
polypeptide based
molecules of the present invention.
[000146] As used herein the terms "termini" or "terminus" when referring to
polypeptides refers to an extremity of a peptide or polypeptide. Such
extremity is not
limited only to the first or final site of the peptide or polypeptide but may
include
additional amino acids in the terminal regions. The polypeptide based
molecules of the
present invention may be characterized as having both an N-terminus
(terminated by an
amino acid with a free amino group (NH2)) and a C-terminus (terminated by an
amino
acid with a free carboxyl group (COOH)). Proteins of the invention are in some
cases
made up of multiple polypeptide chains brought together by disulfide bonds or
by non-
covalent forces (multimers, oligomers). These sorts of proteins will have
multiple N- and
C-termini. Alternatively, the termini of the polypeptides may be modified such
that they
begin or end, as the case may be, with a non-polypeptide based moiety such as
an organic
conjugate.
[000147] Once any of the features have been identified or defined as a desired

component of a polypeptide to be encoded by the primary construct or mmRNA of
the
invention, any of several manipulations and/or modifications of these features
may be
performed by moving, swapping, inverting, deleting, randomizing or
duplicating.
Furthermore, it is understood that manipulation of features may result in the
same
outcome as a modification to the molecules of the invention. For example, a
manipulation
which involved deleting a domain would result in the alteration of the length
of a
molecule just as modification of a nucleic acid to encode less than a full
length molecule
would.
[000148] Modifications and manipulations can be accomplished by methods known
in
the art such as, but not limited to, site directed mutagenesis. The resulting
modified
molecules may then be tested for activity using in vitro or in vivo assays
such as those
described herein or any other suitable screening assay known in the art.
28

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[000149] According to the present invention, the polypeptides may comprise a
consensus sequence which is discovered through rounds of experimentation. As
used
herein a "consensus" sequence is a single sequence which represents a
collective
population of sequences allowing for variability at one or more sites.
[000150] As recognized by those skilled in the art, protein fragments,
functional protein
domains, and homologous proteins are also considered to be within the scope of

polypeptides of interest of this invention. For example, provided herein is
any protein
fragment (meaning a polypeptide sequence at least one amino acid residue
shorter than a
reference polypeptide sequence but otherwise identical) of a reference protein
10, 20, 30,
40, 50, 60, 70, 80, 90, 100 or greater than 100 amino acids in length. In
another example,
any protein that includes a stretch of about 20, about 30, about 40, about 50,
or about 100
amino acids which are about 40%, about 50%, about 60%, about 70%, about 80%,
about
90%, about 95%, or about 100% identical to any of the sequences described
herein can be
utilized in accordance with the invention. In certain embodiments, a
polypeptide to be
utilized in accordance with the invention includes 2, 3, 4, 5, 6, 7, 8, 9, 10,
or more
mutations as shown in any of the sequences provided or referenced herein.
Encoded Polypeptides
[000151] The primary constructs or mmRNA of the present invention may be
designed
to encode polypeptides of interest selected from any of several target
categories
including, but not limited to, biologics, antibodies, vaccines, therapeutic
proteins or
peptides, cell penetrating peptides, secreted proteins, plasma membrane
proteins,
cytoplasmic or cytoskeletal proteins, intracellular membrane bound proteins,
nuclear
proteins, proteins associated with human disease, targeting moieties or those
proteins
encoded by the human genome for which no therapeutic indication has been
identified
but which nonetheless have utility in areas of research and discovery.
[000152] In one embodiment primary constructs or mmRNA may encode variant
polypeptides which have a certain identity with a reference polypeptide
sequence. As
used herein, a "reference polypeptide sequence" refers to a starting
polypeptide sequence.
Reference sequences may be wild type sequences or any sequence to which
reference is
made in the design of another sequence. A "reference polypeptide sequence"
may, e.g.,
be any one of SEQ ID NOs: 769-1392as disclosed herein, e.g., any of SEQ ID NOs
769,
29

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770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784,
785, 786, 787,
788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802,
803, 804, 805,
806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820,
821, 822, 823,
824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838,
839, 840, 841,
842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856,
857, 858, 859,
860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874,
875, 876, 877,
878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892,
893, 894, 895,
896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910,
911, 912, 913,
914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928,
929, 930, 931,
932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946,
947, 948, 949,
950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964,
965, 966, 967,
968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982,
983, 984, 985,
986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, 1000,
1001, 1002,
1003, 1004, 1005, 1006, 1007, 1008, 1009, 1010, 1011, 1012, 1013, 1014, 1015,
1016,
1017, 1018, 1019, 1020, 1021, 1022, 1023, 1024, 1025, 1026, 1027, 1028, 1029,
1030,
1031, 1032, 1033, 1034, 1035, 1036, 1037, 1038, 1039, 1040, 1041, 1042, 1043,
1044,
1045, 1046, 1047, 1048, 1049, 1050, 1051, 1052, 1053, 1054, 1055, 1056, 1057,
1058,
1059, 1060, 1061, 1062, 1063, 1064, 1065, 1066, 1067, 1068, 1069, 1070, 1071,
1072,
1073, 1074, 1075, 1076, 1077, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1085,
1086,
1087, 1088, 1089, 1090, 1091, 1092, 1093, 1094, 1095, 1096, 1097, 1098, 1099,
1100,
1101, 1102, 1103, 1104, 1105, 1106, 1107, 1108, 1109, 1110, 1111, 1112, 1113,
1114,
1115, 1116, 1117, 1118, 1119, 1120, 1121, 1122, 1123, 1124, 1125, 1126, 1127,
1128,
1129, 1130, 1131, 1132, 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141,
1142,
1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, 1151, 1152, 1153, 1154, 1155,
1156,
1157, 1158, 1159, 1160, 1161, 1162, 1163, 1164, 1165, 1166, 1167, 1168, 1169,
1170,
1171, 1172, 1173, 1174, 1175, 1176, 1177, 1178, 1179, 1180, 1181, 1182, 1183,
1184,
1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197,
1198,
1199, 1200, 1201, 1202, 1203, 1204, 1205, 1206, 1207, 1208, 1209, 1210, 1211,
1212,
1213, 1214, 1215, 1216, 1217, 1218, 1219, 1220, 1221, 1222, 1223, 1224, 1225,
1226,
1227, 1228, 1229, 1230, 1231, 1232, 1233, 1234, 1235, 1236, 1237, 1238, 1239,
1240,
1241, 1242, 1243, 1244, 1245, 1246, 1247, 1248, 1249, 1250, 1251, 1252, 1253,
1254,

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1255, 1256, 1257, 1258, 1259, 1260, 1261, 1262, 1263, 1264, 1265, 1266, 1267,
1268,
1269, 1270, 1271, 1272, 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281,
1282,
1283, 1284, 1285, 1286, 1287, 1288, 1289, 1290, 1291, 1292, 1293, 1294, 1295,
1296,
1297, 1298, 1299, 1300, 1301, 1302, 1303, 1304, 1305, 1306, 1307, 1308, 1309,
1310,
1311, 1312, 1313, 1314, 1315, 1316, 1317, 1318, 1319, 1320, 1321, 1322, 1323,
1324,
1325, 1326, 1327, 1328, 1329, 1330, 1331, 1332, 1333, 1334, 1335, 1336, 1337,
1338,
1339, 1340, 1341, 1342, 1343, 1344, 1345, 1346, 1347, 1348, 1349, 1350, 1351,
1352,
1353, 1354, 1355, 1356, 1357, 1358, 1359, 1360, 1361, 1362, 1363, 1364, 1365,
1366,
1367, 1368, 1369, 1370, 1371, 1372, 1373, 1374, 1375, 1376, 1377, 1378, 1379,
1380,
1381, 1382, 1383, 1384, 1385, 1386, 1387, 1388, 1389, 1390, 1391, 1392.
[000153] The term "identity" as known in the art, refers to a relationship
between the
sequences of two or more peptides, as determined by comparing the sequences.
In the art,
identity also means the degree of sequence relatedness between peptides, as
determined
by the number of matches between strings of two or more amino acid residues.
Identity
measures the percent of identical matches between the smaller of two or more
sequences
with gap alignments (if any) addressed by a particular mathematical model or
computer
program (i.e., "algorithms"). Identity of related peptides can be readily
calculated by
known methods. Such methods include, but are not limited to, those described
in
Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press,
New York,
1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,
Academic
Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A.
M., and
Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in
Molecular
Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer,
Gribskov, M.
and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carillo et al.,
SIAM J.
Applied Math. 48, 1073 (1988).
[000154] In some embodiments, the polypeptide variant may have the same or a
similar
activity as the reference polypeptide. Alternatively, the variant may have an
altered
activity (e.g., increased or decreased) relative to a reference polypeptide.
Generally,
variants of a particular polynucleotide or polypeptide of the invention will
have at least
about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity to that
particular
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reference polynucleotide or polypeptide as determined by sequence alignment
programs
and parameters described herein and known to those skilled in the art. Such
tools for
alignment include those of the BLAST suite (Stephen F. Altschul, Thomas L.
Madden,
Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J.
Lipman
(1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database
search
programs", Nucleic Acids Res. 25:3389-3402.) Other tools are described herein,

specifically in the definition of "Identity."
[000155] Default parameters in the BLAST algorithm include, for example, an
expect
threshold of 10, Word size of 28, Match/Mismatch Scores 1, -2, Gap costs
Linear. Any
filter can be applied as well as a selection for species specific repeats,
e.g., Homo sapiens.
Biologics
[000156] The polynucleotides, primary constructs or mmRNA disclosed herein,
may
encode one or more biologics. As used herein, a "biologic" is a polypeptide-
based
molecule produced by the methods provided herein and which may be used to
treat, cure,
mitigate, prevent, or diagnose a serious or life-threatening disease or
medical condition.
Biologics, according to the present invention include, but are not limited to,
allergenic
extracts (e.g. for allergy shots and tests), blood components, gene therapy
products,
human tissue or cellular products used in transplantation, vaccines,
monoclonal
antibodies, cytokines, growth factors, enzymes, thrombolytics, and
immunomodulators,
among others.
[000157] According to the present invention, one or more biologics currently
being
marketed or in development may be encoded by the polynucleotides, primary
constructs
or mmRNA of the present invention. While not wishing to be bound by theory, it
is
believed that incorporation of the encoding polynucleotides of a known
biologic into the
primary constructs or mmRNA of the invention will result in improved
therapeutic
efficacy due at least in part to the specificity, purity and/or selectivity of
the construct
designs.
Antibodies
[000158] The primary constructs or mmRNA disclosed herein, may encode one or
more
antibodies or fragments thereof The term "antibody" includes monoclonal
antibodies
(including full length antibodies which have an immunoglobulin Fc region),
antibody
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compositions with polyepitopic specificity, multispecific antibodies (e.g.,
bispecific
antibodies, diabodies, and single-chain molecules), as well as antibody
fragments. The
term "immunoglobulin" (Ig) is used interchangeably with "antibody" herein. As
used
herein, the term "monoclonal antibody" refers to an antibody obtained from a
population
of substantially homogeneous antibodies, i.e., the individual antibodies
comprising the
population are identical except for possible naturally occurring mutations
and/or post-
translation modifications (e.g., isomerizations, amidations) that may be
present in minor
amounts. Monoclonal antibodies are highly specific, being directed against a
single
antigenic site.
[000159] The monoclonal antibodies herein specifically include "chimeric"
antibodies
(immunoglobulins) in which a portion of the heavy and/or light chain is
identical with or
homologous to corresponding sequences in antibodies derived from a particular
species
or belonging to a particular antibody class or subclass, while the remainder
of the
chain(s) is(are) identical with or homologous to corresponding sequences in
antibodies
derived from another species or belonging to another antibody class or
subclass, as well
as fragments of such antibodies, so long as they exhibit the desired
biological activity.
Chimeric antibodies of interest herein include, but are not limited to,
"primatized"
antibodies comprising variable domain antigen-binding sequences derived from a
non-
human primate (e.g., Old World Monkey, Ape etc.) and human constant region
sequences.
[000160] An "antibody fragment" comprises a portion of an intact antibody,
preferably
the antigen binding and/or the variable region of the intact antibody.
Examples of
antibody fragments include Fab, Fab', F(ab')2 and Fv fragments; diabodies;
linear
antibodies; nanobodies; single-chain antibody molecules and multispecific
antibodies
formed from antibody fragments.
[000161] Any of the five classes of immunoglobulins, IgA, IgD, IgE, IgG and
IgM, may
be encoded by the mmRNA of the invention, including the heavy chains
designated
alpha, delta, epsilon, gamma and mu, respectively. Also included are
polynucleotide
sequences encoding the subclasses, gamma and mu. Hence any of the subclasses
of
antibodies may be encoded in part or in whole and include the following
subclasses:
IgGl, IgG2, IgG3, IgG4, IgAl and IgA2.
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[000162] According to the present invention, one or more antibodies or
fragments
currently being marketed or in development may be encoded by the
polynucleotides,
primary constructs or mmRNA of the present invention. While not wishing to be
bound
by theory, it is believed that incorporation into the primary constructs of
the invention
will result in improved therapeutic efficacy due at least in part to the
specificity, purity
and selectivity of the mmRNA designs.
[000163] Antibodies encoded in the polynucleotides, primary constructs or
mmRNA of
the invention may be utilized to treat conditions or diseases in many
therapeutic areas
such as, but not limited to, blood, cardiovascular, CNS, poisoning (including
antivenoms), dermatology, endocrinology, gastrointestinal, medical imaging,
musculoskeletal, oncology, immunology, respiratory, sensory and anti-
infective.
[000164] In one embodiment, primary constructs or mmRNA disclosed herein may
encode monoclonal antibodies and/or variants thereof. Variants of antibodies
may also
include, but are not limited to, substitutional variants, conservative amino
acid
substitution, insertional variants, deletional variants and/or covalent
derivatives. In one
embodiment, the primary construct and/or mmRNA disclosed herein may encode an
immunoglobulin Fc region. In another embodiment, the primary constructs and/or

mmRNA may encode a variant immunoglobulin Fc region. As a non-limiting
example,
the primary constructs and/or mmRNA may encode an antibody having a variant
immunoglobulin Fc region as described in U.S. Pat. No. 8,217,147 herein
incorporated by
reference in its entirety.
Vaccines
[000165] The primary constructs or mmRNA disclosed herein, may encode one or
more
vaccines. As used herein, a "vaccine" is a biological preparation that
improves immunity
to a particular disease or infectious agent. According to the present
invention, one or
more vaccines currently being marketed or in development may be encoded by the

polynucleotides, primary constructs or mmRNA of the present invention. While
not
wishing to be bound by theory, it is believed that incorporation into the
primary
constructs or mmRNA of the invention will result in improved therapeutic
efficacy due at
least in part to the specificity, purity and selectivity of the construct
designs.
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[000166] Vaccines encoded in the polynucleotides, primary constructs or mmRNA
of
the invention may be utilized to treat conditions or diseases in many
therapeutic areas
such as, but not limited to, cardiovascular, CNS, dermatology, endocrinology,
oncology,
immunology, respiratory, and anti-infective.
Therapeutic proteins or peptides
[000167] The primary constructs or mmRNA disclosed herein, may encode one or
more
validated or "in testing" therapeutic proteins or peptides.
[000168] According to the present invention, one or more therapeutic proteins
or
peptides currently being marketed or in development may be encoded by the
polynucleotides, primary constructs or mmRNA of the present invention. While
not
wishing to be bound by theory, it is believed that incorporation into the
primary
constructs or mmRNA of the invention will result in improved therapeutic
efficacy due at
least in part to the specificity, purity and selectivity of the construct
designs.
[000169] Therapeutic proteins and peptides encoded in the polynucleotides,
primary
constructs or mmRNA of the invention may be utilized to treat conditions or
diseases in
many therapeutic areas such as, but not limited to, blood, cardiovascular,
CNS, poisoning
(including antivenoms), dermatology, endocrinology, genetic, genitourinary,
gastrointestinal, musculoskeletal, oncology, and immunology, respiratory,
sensory and
anti-infective.
Cell-Penetrating Polypeptides
[000170] The primary constructs or mmRNA disclosed herein, may encode one or
more
cell-penetrating polypeptides. As used herein, "cell-penetrating polypeptide"
or CPP
refers to a polypeptide which may facilitate the cellular uptake of molecules.
A cell-
penetrating polypeptide of the present invention may contain one or more
detectable
labels. The polypeptides may be partially labeled or completely labeled
throughout. The
polynucleotide, primary construct or mmRNA may encode the detectable label
completely, partially or not at all. The cell-penetrating peptide may also
include a signal
sequence. As used herein, a "signal sequence" refers to a sequence of amino
acid
residues bound at the amino terminus of a nascent protein during protein
translation. The
signal sequence may be used to signal the secretion of the cell-penetrating
polypeptide.

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[000171] In one embodiment, the polynucleotides, primary constructs or mmRNA
may
also encode a fusion protein. The fusion protein may be created by operably
linking a
charged protein to a therapeutic protein. As used herein, "operably linked"
refers to the
therapeutic protein and the charged protein being connected in such a way to
permit the
expression of the complex when introduced into the cell. As used herein,
"charged
protein" refers to a protein that carries a positive, negative or overall
neutral electrical
charge. Preferably, the therapeutic protein may be covalently linked to the
charged
protein in the formation of the fusion protein. The ratio of surface charge to
total or
surface amino acids may be approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,
0.8 or 0.9.
[000172] The cell-penetrating polypeptide encoded by the polynucleotides,
primary
constructs or mmRNA may form a complex after being translated. The complex may

comprise a charged protein linked, e.g. covalently linked, to the cell-
penetrating
polypeptide. "Therapeutic protein" refers to a protein that, when administered
to a cell
has a therapeutic, diagnostic, and/or prophylactic effect and/or elicits a
desired biological
and/or pharmacological effect.
[000173] In one embodiment, the cell-penetrating polypeptide may comprise a
first
domain and a second domain. The first domain may comprise a supercharged
polypeptide. The second domain may comprise a protein-binding partner. As used

herein, "protein-binding partner" includes, but is not limited to, antibodies
and functional
fragments thereof, scaffold proteins, or peptides. The cell-penetrating
polypeptide may
further comprise an intracellular binding partner for the protein-binding
partner. The
cell-penetrating polypeptide may be capable of being secreted from a cell
where the
polynucleotide, primary construct or mmRNA may be introduced. The cell-
penetrating
polypeptide may also be capable of penetrating the first cell.
[000174] In a further embodiment, the cell-penetrating polypeptide is capable
of
penetrating a second cell. The second cell may be from the same area as the
first cell, or
it may be from a different area. The area may include, but is not limited to,
tissues and
organs. The second cell may also be proximal or distal to the first cell.
[000175] In one embodiment, the polynucleotides, primary constructs or mmRNA
may
encode a cell-penetrating polypeptide which may comprise a protein-binding
partner.
The protein binding partner may include, but is not limited to, an antibody, a
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supercharged antibody or a functional fragment. The polynucleotides, primary
constructs
or mmRNA may be introduced into the cell where a cell-penetrating polypeptide
comprising the protein-binding partner is introduced.
Secreted proteins
[000176] Human and other eukaryotic cells are subdivided by membranes into
many
functionally distinct compartments. Each membrane-bounded compartment, or
organelle,
contains different proteins essential for the function of the organelle. The
cell uses
"sorting signals," which are amino acid motifs located within the protein, to
target
proteins to particular cellular organelles.
[000177] One type of sorting signal, called a signal sequence, a signal
peptide, or a
leader sequence, directs a class of proteins to an organelle called the
endoplasmic
reticulum (ER).
[000178] Proteins targeted to the ER by a signal sequence can be released into
the
extracellular space as a secreted protein. Similarly, proteins residing on the
cell
membrane can also be secreted into the extracellular space by proteolytic
cleavage of a
"linker" holding the protein to the membrane. While not wishing to be bound by
theory,
the molecules of the present invention may be used to exploit the cellular
trafficking
described above. As such, in some embodiments of the invention,
polynucleotides,
primary constructs or mmRNA are provided to express a secreted protein. The
secreted
proteins may be selected from those described herein or those in US Patent
Publication,
20100255574, the contents of which are incorporated herein by reference in
their entirety.
[000179] In one embodiment, these may be used in the manufacture of large
quantities
of valuable human gene products.
Plasma membrane proteins
[000180] In some embodiments of the invention, polynucleotides, primary
constructs or
mmRNA are provided to express a protein of the plasma membrane.
Cytoplasmic or cytoskeletal proteins
[000181] In some embodiments of the invention, polynucleotides, primary
constructs or
mmRNA are provided to express a cytoplasmic or cytoskeletal protein.
Intracellular membrane bound proteins
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[000182] In some embodiments of the invention, polynucleotides, primary
constructs or
mmRNA are provided to express an intracellular membrane bound protein.
Nuclear proteins
[000183] In some embodiments of the invention, polynucleotides, primary
constructs or
mmRNA are provided to express a nuclear protein.
Proteins associated with human disease
[000184] In some embodiments of the invention, polynucleotides, primary
constructs or
mmRNA are provided to express a protein associated with human disease.
Miscellaneous proteins
[000185] In some embodiments of the invention, polynucleotides, primary
constructs or
mmRNA are provided to express a protein with a presently unknown therapeutic
function.
Targeting Moieties
[000186] In some embodiments of the invention, polynucleotides, primary
constructs or
mmRNA are provided to express a targeting moiety. These include a protein-
binding
partner or a receptor on the surface of the cell, which functions to target
the cell to a
specific tissue space or to interact with a specific moiety, either in vivo or
in vitro.
Suitable protein-binding partners include, but are not limited to, antibodies
and functional
fragments thereof, scaffold proteins, or peptides. Additionally,
polynucleotide, primary
construct or mmRNA can be employed to direct the synthesis and extracellular
localization of lipids, carbohydrates, or other biological moieties or
biomolecules.
Polyp eptide Libraries
[000187] In one embodiment, the polynucleotides, primary constructs or mmRNA
may
be used to produce polypeptide libraries. These libraries may arise from the
production of
a population of polynucleotides, primary constructs or mmRNA, each containing
various
structural or chemical modification designs. In this embodiment, a population
of
polynucleotides, primary constructs or mmRNA may comprise a plurality of
encoded
polypeptides, including but not limited to, an antibody or antibody fragment,
protein
binding partner, scaffold protein, and other polypeptides taught herein or
known in the
art. In a preferred embodiment, the polynucleotides are primary constructs of
the present
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invention, including mmRNA which may be suitable for direct introduction into
a target
cell or culture which in turn may synthesize the encoded polypeptides.
[000188] In certain embodiments, multiple variants of a protein, each with
different
amino acid modification(s), may be produced and tested to determine the best
variant in
terms of pharmacokinetics, stability, biocompatibility, and/or biological
activity, or a
biophysical property such as expression level. Such a library may contain 10,
102, 103,
104, 105, 106, 107, 108, 109, or over 109 possible variants (including, but
not limited to,
substitutions, deletions of one or more residues, and insertion of one or more
residues).
Anti-Microbial and Anti-viral Polypeptides
[000189] The polynucleotides, primary constructs and mmRNA of the present
invention
may be designed to encode on or more antimicrobial peptides (AMP) or antiviral
peptides
(AVP). AMPs and AVPs have been isolated and described from a wide range of
animals
such as, but not limited to, microorganisms, invertebrates, plants,
amphibians, birds, fish,
and mammals (Wang et al., Nucleic Acids Res. 2009; 37 (Database issue):D933-
7). For
example, anti-microbial polypeptides are described in Antimicrobial Peptide
Database
(http://aps.unmc.edu/AP/main.php; Wang et al., Nucleic Acids Res. 2009; 37
(Database
issue):D933-7), CAMP: Collection of Anti-Microbial Peptides
(http://www.bicnirrh.res.in/antimicrobial/); Thomas et al., Nucleic Acids Res.
2010; 38
(Database issue):D774-80), US 5221732, US 5447914, US 5519115, US 5607914, US
5714577, US 5734015, US 5798336, US 5821224, US 5849490, US 5856127, US
5905187, US 5994308, US 5998374, US 6107460, US 6191254, US 6211148, US
6300489, US 6329504, US 6399370, US 6476189, US 6478825, US 6492328, US
6514701, US 6573361, US 6573361, US 6576755, US 6605698, US 6624140, US
6638531, US 6642203, US 6653280, US 6696238, US 6727066, US 6730659, US
6743598, US 6743769, US 6747007, US 6790833, US 6794490, US 6818407, US
6835536, US 6835713, US 6838435, US 6872705, US 6875907, US 6884776, US
6887847, US 6906035, US 6911524, US 6936432, US 7001924, US 7071293, US
7078380, US 7091185, US 7094759, US 7166769, US 7244710, US 7314858, and US
7582301, the contents of which are incorporated by reference in their
entirety.
[000190] The anti-microbial polypeptides described herein may block cell
fusion and/or
viral entry by one or more enveloped viruses (e.g., HIV, HCV). For example,
the anti-
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microbial polypeptide can comprise or consist of a synthetic peptide
corresponding to a
region, e.g., a consecutive sequence of at least about 5, 10, 15, 20, 25, 30,
35, 40, 45, 50,
55, or 60 amino acids of the transmembrane subunit of a viral envelope
protein, e.g.,
HIV-1 gp120 or gp41. The amino acid and nucleotide sequences of HIV-1 gp120 or

gp41 are described in, e.g., Kuiken et at., (2008). "HIV Sequence Compendium,"
Los
Alamos National Laboratory.
[000191] In some embodiments, the anti-microbial polypeptide may have at least
about
75%, 80%, 85%, 90%, 95%, 100% sequence homology to the corresponding viral
protein
sequence. In some embodiments, the anti-microbial polypeptide may have at
least about
75%, 80%, 85%, 90%, 95%, or 100% sequence homology to the corresponding viral
protein sequence.
[000192] In other embodiments, the anti-microbial polypeptide may comprise or
consist
of a synthetic peptide corresponding to a region, e.g., a consecutive sequence
of at least
about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 amino acids of the
binding domain of
a capsid binding protein. In some embodiments, the anti-microbial polypeptide
may have
at least about 75%, 80%, 85%, 90%, 95%, or 100% sequence homology to the
corresponding sequence of the capsid binding protein.
[000193] The anti-microbial polypeptides described herein may block protease
dimerization and inhibit cleavage of viral proproteins (e.g., HIV Gag-pol
processing) into
functional proteins thereby preventing release of one or more enveloped
viruses (e.g.,
HIV, HCV). In some embodiments, the anti-microbial polypeptide may have at
least
about 75%, 80%, 85%, 90%, 95%, 100% sequence homology to the corresponding
viral
protein sequence.
[000194] In other embodiments, the anti-microbial polypeptide can comprise or
consist
of a synthetic peptide corresponding to a region, e.g., a consecutive sequence
of at least
about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 amino acids of the
binding domain of
a protease binding protein. In some embodiments, the anti-microbial
polypeptide may
have at least about 75%, 80%, 85%, 90%, 95%, 100% sequence homology to the
corresponding sequence of the protease binding protein.
[000195] The anti-microbial polypeptides described herein can include an in
vitro-
evolved polypeptide directed against a viral pathogen.

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Anti-Microbial Polypeptides
[000196] Anti-microbial polypeptides (AMPs) are small peptides of variable
length,
sequence and structure with broad spectrum activity against a wide range of
microorganisms including, but not limited to, bacteria, viruses, fungi,
protozoa, parasites,
prions, and tumor/cancer cells. (See, e.g., Zaiou, J Mol Med, 2007; 85:317;
herein
incorporated by reference in its entirety). It has been shown that AMPs have
broad-
spectrum of rapid onset of killing activities, with potentially low levels of
induced
resistance and concomitant broad anti-inflammatory effects.
[000197] In some embodiments, the anti-microbial polypeptide (e.g., an anti-
bacterial
polypeptide) may be under 10kDa, e.g., under 8kDa, 6kDa, 4kDa, 2kDa, or lkDa.
In
some embodiments, the anti-microbial polypeptide (e.g., an anti-bacterial
polypeptide)
consists of from about 6 to about 100 amino acids, e.g., from about 6 to about
75 amino
acids, about 6 to about 50 amino acids, about 6 to about 25 amino acids, about
25 to
about 100 amino acids, about 50 to about 100 amino acids, or about 75 to about
100
amino acids. In certain embodiments, the anti-microbial polypeptide (e.g., an
anti-
bacterial polypeptide) may consist of from about 15 to about 45 amino acids.
In some
embodiments, the anti-microbial polypeptide (e.g., an anti-bacterial
polypeptide) is
substantially cationic.
[000198] In some embodiments, the anti-microbial polypeptide (e.g., an anti-
bacterial
polypeptide) may be substantially amphipathic. In certain embodiments, the
anti-
microbial polypeptide (e.g., an anti-bacterial polypeptide) may be
substantially cationic
and amphipathic. In some embodiments, the anti-microbial polypeptide (e.g., an
anti-
bacterial polypeptide) may be cytostatic to a Gram-positive bacterium. In some

embodiments, the anti-microbial polypeptide (e.g., an anti-bacterial
polypeptide) may be
cytotoxic to a Gram-positive bacterium. In some embodiments, the anti-
microbial
polypeptide (e.g., an anti-bacterial polypeptide) may be cytostatic and
cytotoxic to a
Gram-positive bacterium. In some embodiments, the anti-microbial polypeptide
(e.g., an
anti-bacterial polypeptide) may be cytostatic to a Gram-negative bacterium. In
some
embodiments, the anti-microbial polypeptide (e.g., an anti-bacterial
polypeptide) may be
cytotoxic to a Gram-negative bacterium. In some embodiments, the anti-
microbial
polypeptide (e.g., an anti-bacterial polypeptide) may be cytostatic and
cytotoxic to a
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Gram-positive bacterium. In some embodiments, the anti-microbial polypeptide
may be
cytostatic to a virus, fungus, protozoan, parasite, prion, or a combination
thereof In
some embodiments, the anti-microbial polypeptide may be cytotoxic to a virus,
fungus,
protozoan, parasite, prion, or a combination thereof. In certain embodiments,
the anti-
microbial polypeptide may be cytostatic and cytotoxic to a virus, fungus,
protozoan,
parasite, prion, or a combination thereof In some embodiments, the anti-
microbial
polypeptide may be cytotoxic to a tumor or cancer cell (e.g., a human tumor
and/or
cancer cell). In some embodiments, the anti-microbial polypeptide may be
cytostatic to a
tumor or cancer cell (e.g., a human tumor and/or cancer cell). In certain
embodiments,
the anti-microbial polypeptide may be cytotoxic and cytostatic to a tumor or
cancer cell
(e.g., a human tumor or cancer cell). In some embodiments, the anti-microbial
polypeptide (e.g., an anti-bacterial polypeptide) may be a secreted
polypeptide.
[000199] In some embodiments, the anti-microbial polypeptide comprises or
consists of
a defensin. Exemplary defensins include, but are not limited to, a-defensins
(e.g.,
neutrophil defensin 1, defensin alpha 1, neutrophil defensin 3, neutrophil
defensin 4,
defensin 5, defensin 6), 13-defensins (e.g., beta-defensin 1, beta-defensin 2,
beta-defensin
103, beta-defensin 107, beta-defensin 110, beta-defensin 136), and 0-
defensins. In other
embodiments, the anti-microbial polypeptide comprises or consists of a
cathelicidin (e.g.,
hCAP18).
Anti-Viral Polypeptides
[000200] Anti-viral polypeptides (AVPs) are small peptides of variable length,

sequence and structure with broad spectrum activity against a wide range of
viruses. See,
e.g., Zaiou, J Mol Med, 2007; 85:317. It has been shown that AVPs have a broad-

spectrum of rapid onset of killing activities, with potentially low levels of
induced
resistance and concomitant broad anti-inflammatory effects. In some
embodiments, the
anti-viral polypeptide is under 10kDa, e.g., under 8kDa, 6kDa, 4kDa, 2kDa, or
lkDa. In
some embodiments, the anti-viral polypeptide comprises or consists of from
about 6 to
about 100 amino acids, e.g., from about 6 to about 75 amino acids, about 6 to
about 50
amino acids, about 6 to about 25 amino acids, about 25 to about 100 amino
acids, about
50 to about 100 amino acids, or about 75 to about 100 amino acids. In certain
embodiments, the anti-viral polypeptide comprises or consists of from about 15
to about
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45 amino acids. In some embodiments, the anti-viral polypeptide is
substantially
cationic. In some embodiments, the anti-viral polypeptide is substantially
amphipathic.
In certain embodiments, the anti-viral polypeptide is substantially cationic
and
amphipathic. In some embodiments, the anti-viral polypeptide is cytostatic to
a virus. In
some embodiments, the anti-viral polypeptide is cytotoxic to a virus. In some
embodiments, the anti-viral polypeptide is cytostatic and cytotoxic to a
virus. In some
embodiments, the anti-viral polypeptide is cytostatic to a bacterium, fungus,
protozoan,
parasite, prion, or a combination thereof In some embodiments, the anti-viral
polypeptide is cytotoxic to a bacterium, fungus, protozoan, parasite, prion or
a
combination thereof In certain embodiments, the anti-viral polypeptide is
cytostatic and
cytotoxic to a bacterium, fungus, protozoan, parasite, prion, or a combination
thereof. In
some embodiments, the anti-viral polypeptide is cytotoxic to a tumor or cancer
cell (e.g.,
a human cancer cell). In some embodiments, the anti-viral polypeptide is
cytostatic to a
tumor or cancer cell (e.g., a human cancer cell). In certain embodiments, the
anti-viral
polypeptide is cytotoxic and cytostatic to a tumor or cancer cell (e.g., a
human cancer
cell). In some embodiments, the anti-viral polypeptide is a secreted
polypeptide.
Cytotoxic Nucleosides
[000201] In one embodiment, the polynucleotides, primary constructs or mmRNA
of
the present invention may incorporate one or more cytotoxic nucleosides. For
example,
cytotoxic nucleosides may be incorporated into polynucleotides, primary
constructs or
mmRNA such as bifunctional modified RNAs or mRNAs. Cytotoxic nucleoside anti-
cancer agents include, but are not limited to, adenosine arabinoside,
cytarabine, cytosine
arabinoside, 5-fluorouracil, fludarabine, floxuridine, FTORAFURO (a
combination of
tegafur and uracil), tegafur ((RS)-5-fluoro-1-(tetrahydrofuran-2-yl)pyrimidine-

2,4(1H,3H)-dione), and 6-mercaptopurine.
[000202] A number of cytotoxic nucleoside analogues are in clinical use, or
have been
the subject of clinical trials, as anticancer agents. Examples of such
analogues include,
but are not limited to, cytarabine, gemcitabine, troxacitabine, decitabine,
tezacitabine, 2'-
deoxy-2'-methylidenecytidine (DMDC), cladribine, clofarabine, 5-azacytidine,
4'-thio-
aracytidine, cyclopentenylcytosine and 1-(2-C-cyano-2-deoxy-beta-D-arabino-
pentofuranosyl)-cytosine. Another example of such a compound is fludarabine
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phosphate. These compounds may be administered systemically and may have side
effects which are typical of cytotoxic agents such as, but not limited to,
little or no
specificity for tumor cells over proliferating normal cells.
[000203] A number of prodrugs of cytotoxic nucleoside analogues are also
reported in
the art. Examples include, but are not limited to, N4-behenoy1-1-beta-D-
arabinofuranosylcytosine, N4-octadecy1-1-beta-D-arabinofuranosylcytosine, N4-
palmitoy1-1-(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl) cytosine, and P-
4055
(cytarabine 5'-elaidic acid ester). In general, these prodrugs may be
converted into the
active drugs mainly in the liver and systemic circulation and display little
or no selective
release of active drug in the tumor tissue. For example, capecitabine, a
prodrug of 5'-
deoxy-5-fluorocytidine (and eventually of 5-fluorouracil), is metabolized both
in the liver
and in the tumor tissue. A series of capecitabine analogues containing "an
easily
hydrolysable radical under physiological conditions" has been claimed by Fujiu
et al.
(U.S. Pat. No. 4,966,891) and is herein incorporated by reference. The series
described by
Fujiu includes N4 alkyl and aralkyl carbamates of 5'-deoxy-5-fluorocytidine
and the
implication that these compounds will be activated by hydrolysis under normal
physiological conditions to provide 5'-deoxy-5-fluorocytidine.
[000204] A series of cytarabine N4-carbamates has been by reported by Fadl et
al
(Pharmazie. 1995, 50, 382-7, herein incorporated by reference) in which
compounds
were designed to convert into cytarabine in the liver and plasma. WO
2004/041203,
herein incorporated by reference, discloses prodrugs of gemcitabine, where
some of the
prodrugs are N4-carbamates. These compounds were designed to overcome the
gastrointestinal toxicity of gemcitabine and were intended to provide
gemcitabine by
hydrolytic release in the liver and plasma after absorption of the intact
prodrug from the
gastrointestinal tract. Nomura et al (Bioorg Med. Chem. 2003, 11, 2453-61,
herein
incorporated by reference) have described acetal derivatives of 1-(3-C-ethyny1-
13-D-ribo-
pentofaranosyl) cytosine which, on bioreduction, produced an intermediate that
required
further hydrolysis under acidic conditions to produce a cytotoxic nucleoside
compound.
[000205] Cytotoxic nucleotides which may be chemotherapeutic also include, but
are
not limited to, pyrazolo [3,4-D]-pyrimidines, allopurinol, azathioprine,
capecitabine,
cytosine arabinoside, fluorouracil, mercaptopurine, 6-thioguanine, acyclovir,
ara-
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adenosine, ribavirin, 7-deaza-adenosine, 7-deaza-guanosine, 6-aza-uracil, 6-
aza-cytidine,
thymidine ribonucleotide, 5-bromodeoxyuridine, 2-chloro-purine, and inosine,
or
combinations thereof
Flanking Regions: Untranslated Regions (UTRs)
[000206] Untranslated regions (UTRs) of a gene are transcribed but not
translated. The
5'UTR starts at the transcription start site and continues to the start codon
but does not
include the start codon; whereas, the 3'UTR starts immediately following the
stop codon
and continues until the transcriptional termination signal. There is growing
body of
evidence about the regulatory roles played by the UTRs in terms of stability
of the
nucleic acid molecule and translation. The regulatory features of a UTR can be

incorporated into the polynucleotides, primary constructs and/or mmRNA of the
present
invention to enhance the stability of the molecule. The specific features can
also be
incorporated to ensure controlled down-regulation of the transcript in case
they are
misdirected to undesired organs sites.
5' UTR and Translation Initiation
[000207] Natural 5'UTRs bear features which play roles in for translation
initiation.
They harbor signatures like Kozak sequences which are commonly known to be
involved
in the process by which the ribosome initiates translation of many genes.
Kozak
sequences have the consensus CCR(A/G)CCAUGG, where R is a purine (adenine or
guanine) three bases upstream of the start codon (AUG), which is followed by
another
'G'. 5'UTR also have been known to form secondary structures which are
involved in
elongation factor binding.
[000208] By engineering the features typically found in abundantly expressed
genes of
specific target organs, one can enhance the stability and protein production
of the
polynucleotides, primary constructs or mmRNA of the invention. For example,
introduction of 5' UTR of liver-expressed mRNA, such as albumin, serum amyloid
A,
Apolipoprotein A/B/E, transferrin, alpha fetoprotein, erythropoietin, or
Factor VIII, could
be used to enhance expression of a nucleic acid molecule, such as a mmRNA, in
hepatic
cell lines or liver. Likewise, use of 5' UTR from other tissue-specific mRNA
to improve
expression in that tissue is possible for muscle (MyoD, Myosin, Myoglobin,
Myogenin,
Herculin), for endothelial cells (Tie-1, CD36), for myeloid cells (C/EBP,
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GM-CSF, CD1 lb, MSR, Fr-1, i-NOS), for leukocytes (CD45, CD18), for adipose
tissue
(CD36, GLUT4, ACRP30, adiponectin) and for lung epithelial cells (SP-A/B/C/D).

[000209] Other non-UTR sequences may be incorporated into the 5' (or 3' UTR)
UTRs.
For example, introns or portions of introns sequences may be incorporated into
the
flanking regions of the polynucleotides, primary constructs or mmRNA of the
invention.
Incorporation of intronic sequences may increase protein production as well as
mRNA
levels.
3' UTR and the AU Rich Elements
[000210] 3' UTRs are known to have stretches of Adenosines and Uridines
embedded in
them. These AU rich signatures are particularly prevalent in genes with high
rates of
turnover. Based on their sequence features and functional properties, the AU
rich
elements (AREs) can be separated into three classes (Chen et al, 1995): Class
I AREs
contain several dispersed copies of an AUUUA motif within U-rich regions. C-
Myc and
MyoD contain class I AREs. Class II AREs possess two or more overlapping
UUAUUUA(U/A)(U/A) nonamers. Molecules containing this type of AREs include
GM-CSF and TNF-a. Class III ARES are less well defined. These U rich regions
do not
contain an AUUUA motif. c-Jun and Myogenin are two well-studied examples of
this
class. Most proteins binding to the AREs are known to destabilize the
messenger,
whereas members of the ELAV family, most notably HuR, have been documented to
increase the stability of mRNA. HuR binds to AREs of all the three classes.
Engineering
the HuR specific binding sites into the 3' UTR of nucleic acid molecules will
lead to HuR
binding and thus, stabilization of the message in vivo.
[000211] Introduction, removal or modification of 3' UTR AU rich elements
(AREs)
can be used to modulate the stability of polynucleotides, primary constructs
or mmRNA
of the invention. When engineering specific polynucleotides, primary
constructs or
mmRNA, one or more copies of an ARE can be introduced to make polynucleotides,

primary constructs or mmRNA of the invention less stable and thereby curtail
translation
and decrease production of the resultant protein. Likewise, AREs can be
identified and
removed or mutated to increase the intracellular stability and thus increase
translation and
production of the resultant protein. Transfection experiments can be conducted
in
relevant cell lines, using polynucleotides, primary constructs or mmRNA of the
invention
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and protein production can be assayed at various time points post-
transfection. For
example, cells can be transfected with different ARE-engineering molecules and
by using
an ELISA kit to the relevant protein and assaying protein produced at 6 hour,
12 hour, 24
hour, 48 hour, and 7 days post-transfection.
Incorporating microRNA Binding Sites
[000212] microRNAs (or miRNA) are 19-25 nucleotide long noncoding RNAs that
bind
to the 3'UTR of nucleic acid molecules and down-regulate gene expression
either by
reducing nucleic acid molecule stability or by inhibiting translation. The
polynucleotides, primary constructs or mmRNA of the invention may comprise one
or
more microRNA target sequences, microRNA seqences, or microRNA seeds. Such
sequences may correspond to any known microRNA such as those taught in US
Publication U52005/0261218 and US Publication U52005/0059005, the contents of
which are incorporated herein by reference in their entirety.
[000213] A microRNA sequence comprises a "seed" region, i.e., a sequence in
the
region of positions 2-8 of the mature microRNA, which sequence has perfect
Watson-
Crick complementarity to the miRNA target sequence. A microRNA seed may
comprise
positions 2-8 or 2-7 of the mature microRNA. In some embodiments, a microRNA
seed
may comprise 7 nucleotides (e.g., nucleotides 2-8 of the mature microRNA),
wherein the
seed-complementary site in the corresponding miRNA target is flanked by an
adenine (A)
opposed to microRNA position 1. In some embodiments, a microRNA seed may
comprise 6 nucleotides (e.g., nucleotides 2-7 of the mature microRNA), wherein
the
seed-complementary site in the corresponding miRNA target is flanked byan
adenine (A)
opposed to microRNA position 1. See for example, Grimson A, Farh KK, Johnston
WK,
Garrett-Engele P, Lim LP, Bartel DP; Mol Cell. 2007 Jul 6;27(1):91-105; each
of which
is herein incorporated by reference in their entirety. The bases of the
microRNA seed
have complete complementarity with the target sequence. By engineering
microRNA
target sequences into the 3'UTR of polynucleotides, primary constructs or
mmRNA of
the invention one can target the molecule for degradation or reduced
translation, provided
the microRNA in question is available. This process will reduce the hazard of
off target
effects upon nucleic acid molecule delivery. Identification of microRNA,
microRNA
target regions, and their expression patterns and role in biology have been
reported
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(Bonauer et al., Curr Drug Targets 2010 11:943-949; Anand and Cheresh Curr
Opin
Hematol 201118:171-176; Contreras and Rao Leukemia 2012 26:404-413 (2011 Dec
20.
doi: 10.1038/1eu.2011.356); Bartel Cell 2009 136:215-233; Landgraf et al,
Cell, 2007
129:1401-1414; each of which is herein incorporated by reference in its
entirety).
[000214] For example, if the nucleic acid molecule is an mRNA and is not
intended to
be delivered to the liver but ends up there, then miR-122, a microRNA abundant
in liver,
can inhibit the expression of the gene of interest if one or multiple target
sites of miR-122
are engineered into the 3' UTR of the polynucleotides, primary constructs or
mmRNA.
Introduction of one or multiple binding sites for different microRNA can be
engineered
to further decrease the longevity, stability, and protein translation of a
polynucleotides,
primary constructs or mmRNA.
[000215] As used herein, the term "microRNA site" refers to a microRNA target
site or
a microRNA recognition site, or any nucleotide sequence to which a microRNA
binds or
associates. It should be understood that "binding" may follow traditional
Watson-Crick
hybridization rules or may reflect any stable association of the microRNA with
the target
sequence at or adjacent to the microRNA site.
[000216] Conversely, for the purposes of the polynucleotides, primary
constructs or
mmRNA of the present invention, microRNA binding sites can be engineered out
of (i.e.
removed from) sequences in which they naturally occur in order to increase
protein
expression in specific tissues. For example, miR-122 binding sites may be
removed to
improve protein expression in the liver. Regulation of expression in multiple
tissues can
be accomplished through introduction or removal or one or several microRNA
binding
sites.
[000217] Examples of tissues where microRNA are known to regulate mRNA, and
thereby protein expression, include, but are not limited to, liver (miR-122),
muscle (miR-
133, miR-206, miR-208), endothelial cells (miR-17-92, miR-126), myeloid cells
(miR-
142-3p, miR-142-5p, miR-16, miR-21, miR-223, miR-24, miR-27), adipose tissue
(let-7,
miR-30c), heart (miR-1d, miR-149), kidney (miR-192, miR-194, miR-204), and
lung
epithelial cells (let-7, miR-133, miR-126). MicroRNA can also regulate complex

biological processes such as angiogenesis (miR-132) (Anand and Cheresh Curr
Opin
Hematol 201118:171-176; herein incorporated by reference in its entirety). In
the
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polynucleotides, primary constructs or mmRNA of the present invention, binding
sites
for microRNAs that are involved in such processes may be removed or
introduced, in
order to tailor the expression of the polynucleotides, primary constructs or
mmRNA
expression to biologically relevant cell types or to the context of relevant
biological
processes. A listing of MicroRNA, miR sequences and miR binding sites is
listed in
Table 9 of U.S. Provisional Application No. 61/753,661 filed January 17, 2013,
in Table
9 of U.S. Provisional Application No. 61/754,159 filed January 18, 2013, and
in Table 7
of U.S. Provisional Application No. 61/758,921 filed January 31, 2013, each of
which are
herein incorporated by reference in their entireties.
[000218] Lastly, through an understanding of the expression patterns of
microRNA in
different cell types, polynucleotides, primary constructs or mmRNA can be
engineered
for more targeted expression in specific cell types or only under specific
biological
conditions. Through introduction of tissue-specific microRNA binding sites,
polynucleotides, primary constructs or mmRNA could be designed that would be
optimal
for protein expression in a tissue or in the context of a biological
condition. Examples of
use of microRNA to drive tissue or disease-specific gene expression are listed
(Getner
and Naldini, Tissue Antigens. 2012, 80:393-403; herein incoroporated by
reference in its
entirety). In addition, microRNA seed sites can be incorporated into mRNA to
decrease
expression in certain cells which results in a biological improvement. An
example of this
is incorporation of miR-142 sites into a UGT1A1-expressing lentiviral vector.
The
presence of miR-142 seed sites reduced expression in hematopoietic cells, and
as a
consequence reduced expression in antigen-presentating cells, leading to the
absence of
an immune response against the virally expressed UGT1A1 (Schmitt et al.,
Gastroenterology 2010; 139:999-1007; Gonzalez-Asequinolaza et al.
Gastroenterology
2010, 139:726-729; both herein incorporated by reference in its entirety) .
Incorporation
of miR-142 sites into modified mRNA could not only reduce expression of the
encoded
protein in hematopoietic cells, but could also reduce or abolish immune
responses to the
mRNA-encoded protein. Incorporation of miR-142 seed sites (one or multiple)
into
mRNA would be important in the case of treatment of patients with complete
protein
deficiencies (UGT1A1 type I, LDLR-deficient patients, CRIM-negative Pompe
patients,
etc.) .
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[000219] Transfection experiments can be conducted in relevant cell lines,
using
engineered polynucleotides, primary constructs or mmRNA and protein production
can
be assayed at various time points post-transfection. For example, cells can be
transfected
with different microRNA binding site-engineering polynucleotides, primary
constructs or
mmRNA and by using an ELISA kit to the relevant protein and assaying protein
produced at 6 hour, 12 hour, 24 hour, 48 hour, 72 hour and 7 days post-
transfection. In
vivo experiments can also be conducted using microRNA-binding site-engineered
molecules to examine changes in tissue-specific expression of formulated
polynucleotides, primary constructs or mmRNA.
5' Capping
[000220] The 5' cap structure of an mRNA is involved in nuclear export,
increasing
mRNA stability and binds the mRNA Cap Binding Protein (CBP), which is
responsibile
for mRNA stability in the cell and translation competency through the
association of CBP
with poly(A) binding protein to form the mature cyclic mRNA species. The cap
further
assists the removal of 5' proximal introns removal during mRNA splicing.
[000221] Endogenous mRNA molecules may be 5'-end capped generating a 5'-ppp-5'-

triphosphate linkage between a terminal guanosine cap residue and the 5'-
terminal
transcribed sense nucleotide of the mRNA molecule. This 5'-guanylate cap may
then be
methylated to generate an N7-methyl-guanylate residue. The ribose sugars of
the
terminal and/or anteterminal transcribed nucleotides of the 5' end of the mRNA
may
optionally also be 2'-0-methylated. 5'-decapping through hydrolysis and
cleavage of the
guanylate cap structure may target a nucleic acid molecule, such as an mRNA
molecule,
for degradation.
[000222] Modifications to the polynucleotides, primary constructs, and mmRNA
of the
present invention may generate a non-hydrolyzable cap structure preventing
decapping
and thus increasing mRNA half-life. Because cap structure hydrolysis requires
cleavage
of 5'-ppp-5' phosphorodiester linkages, modified nucleotides may be used
during the
capping reaction. For example, a Vaccinia Capping Enzyme from New England
Biolabs
(Ipswich, MA) may be used with a-thio-guanosine nucleotides according to the
manufacturer's instructions to create a phosphorothioate linkage in the 5'-ppp-
5' cap.

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Additional modified guanosine nucleotides may be used such as a-methyl-
phosphonate
and seleno-phosphate nucleotides.
[000223] Additional modifications include, but are not limited to, 2'-0-
methylation of
the ribose sugars of 5'-terminal and/or 5'-anteterminal nucleotides of the
mRNA (as
mentioned above) on the 2'-hydroxyl group of the sugar ring. Multiple distinct
5'-cap
structures can be used to generate the 5'-cap of a nucleic acid molecule, such
as an
mRNA molecule.
[000224] Cap analogs, which herein are also referred to as synthetic cap
analogs,
chemical caps, chemical cap analogs, or structural or functional cap analogs,
differ from
natural (i.e. endogenous, wild-type or physiological) 5'-caps in their
chemical structure,
while retaining cap function. Cap analogs may be chemically (i.e. non-
enzymatically) or
enzymatically synthesized and/or linked to a nucleic acid molecule.
[000225] For example, the Anti-Reverse Cap Analog (ARCA) cap contains two
guanines linked by a 5'-5'-triphosphate group, wherein one guanine contains an
N7
methyl group as well as a 3'-0-methyl group (i.e., N7,3'-0-dimethyl-guanosine-
5'-
triphosphate-5'-guanosine (m7G-3'mppp-G; which may equivaliently be designated
3' 0-
Me-m7G(5')ppp(5')G). The 3'-0 atom of the other, unmodified, guanine becomes
linked
to the 5'-terminal nucleotide of the capped nucleic acid molecule (e.g. an
mRNA or
mmRNA). The N7- and 3'-0-methlyated guanine provides the terminal moiety of
the
capped nucleic acid molecule (e.g. mRNA or mmRNA).
[000226] Another exemplary cap is mCAP, which is similar to ARCA but has a 2'-
0-
methyl group on guanosine (i.e., N7,2'-0-dimethyl-guanosine-5'-triphosphate-5'-

guanosine, m7Gm-ppp-G).
[000227] While cap analogs allow for the concomitant capping of a nucleic acid

molecule in an in vitro transcription reaction, up to 20% of transcripts can
remain
uncapped. This, as well as the structural differences of a cap analog from an
endogenous
5'-cap structures of nucleic acids produced by the endogenous, cellular
transcription
machinery, may lead to reduced translational competency and reduced cellular
stability.
[000228] Polynucleotides, primary constructs and mmRNA of the invention may
also
be capped post-transcriptionally, using enzymes, in order to generate more
authentic 5'-
cap structures. As used herein, the phrase "more authentic" refers to a
feature that
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closely mirrors or mimics, either structurally or functionally, an endogenous
or wild type
feature. That is, a "more authentic" feature is better representative of an
endogenous,
wild-type, natural or physiological cellular function and/or structure as
compared to
synthetic features or analogs, etc., of the prior art, or which outperforms
the
corresponding endogenous, wild-type, natural or physiological feature in one
or more
respects. Non-limiting examples of more authentic 5'cap structures of the
present
invention are those which, among other things, have enhanced binding of cap
binding
proteins, increased half life, reduced susceptibility to 5' endonucleases
and/or reduced
5'decapping, as compared to synthetic 5'cap structures known in the art (or to
a wild-type,
natural or physiological 5'cap structure). For example, recombinant Vaccinia
Virus
Capping Enzyme and recombinant 2'-0-methyltransferase enzyme can create a
canonical
5'-5'-triphosphate linkage between the 5'-terminal nucleotide of an mRNA and a
guanine
cap nucleotide wherein the cap guanine contains an N7 methylation and the 5'-
terminal
nucleotide of the mRNA contains a 2'-0-methyl. Such a structure is termed the
Capl
structure. This cap results in a higher translational-competency and cellular
stability and
a reduced activation of cellular pro-inflammatory cytokines, as compared,
e.g., to other
5'cap analog structures known in the art. Cap structures include, but are not
limited to,
7mG(5')ppp(5')N,pN2p (cap 0), 7mG(5')ppp(5')NlmpNp (cap 1), and 7mG(5')-
ppp(5')NlmpN2mp (cap 2).
[000229] Because the polynucleotides, primary constructs or mmRNA may be
capped
post-transcriptionally, and because this process is more efficient, nearly
100% of the
polynucleotides, primary constructs or mmRNA may be capped. This is in
contrast to
¨80% when a cap analog is linked to an mRNA in the course of an in vitro
transcription
reaction.
[000230] According to the present invention, 5' terminal caps may include
endogenous
caps or cap analogs. According to the present invention, a 5' terminal cap may
comprise
a guanine analog. Useful guanine analogs include, but are not limited to,
inosine, N1-
methyl-guanosine, 2'fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-
amino-
guanosine, LNA-guanosine, and 2-azido-guanosine.
Viral Sequences
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[000231] Additional viral sequences such as, but not limited to, the
translation enhancer
sequence of the barley yellow dwarf virus (BYDV-PAV), the Jaagsiekte sheep
retrovirus
(JSRV) and/or the Enzootic nasal tumor virus (See e.g., International Pub. No.

W02012129648; herein incorporated by reference in its entirety) can be
engineered and
inserted in the 3' UTR of the polynucleotides, primary constructs or mmRNA of
the
invention and can stimulate the translation of the construct in vitro and in
vivo.
Transfection experiments can be conducted in relevant cell lines at and
protein
production can be assayed by ELISA at 12hr, 24hr, 48hr, 72 hr and day 7 post-
transfection.
IRES Sequences
[000232] Further, provided are polynucleotides, primary constructs or mmRNA
which
may contain an internal ribosome entry site (IRES). First identified as a
feature Picorna
virus RNA, IRES plays an important role in initiating protein synthesis in
absence of the
5' cap structure. An IRES may act as the sole ribosome binding site, or may
serve as one
of multiple ribosome binding sites of an mRNA. Polynucleotides, primary
constructs or
mmRNA containing more than one functional ribosome binding site may encode
several
peptides or polypeptides that are translated independently by the ribosomes
("multicistronic nucleic acid molecules"). When polynucleotides, primary
constructs or
mmRNA are provided with an IRES, further optionally provided is a second
translatable
region. Examples of IRES sequences that can be used according to the invention
include
without limitation, those from picornaviruses (e.g. FMDV), pest viruses
(CFFV), polio
viruses (PV), encephalomyocarditis viruses (ECMV), foot-and-mouth disease
viruses
(FMDV), hepatitis C viruses (HCV), classical swine fever viruses (CSFV),
murine
leukemia virus (MLV), simian immune deficiency viruses (SIV) or cricket
paralysis
viruses (CrPV).
Poly-A tails
[000233] During RNA processing, a long chain of adenine nucleotides (poly-A
tail)
may be added to a polynucleotide such as an mRNA molecules in order to
increase
stability. Immediately after transcription, the 3' end of the transcript may
be cleaved to
free a 3' hydroxyl. Then poly-A polymerase adds a chain of adenine nucleotides
to the
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RNA. The process, called polyadenylation, adds a poly-A tail that can be
between, for
example, approximately 100 and 250 residues long.
[000234] It has been discovered that unique poly-A tail lengths provide
certain
advantages to the polynucleotides, primary constructs or mmRNA of the present
invention.
[000235] Generally, the length of a poly-A tail of the present invention is
greater than
30 nucleotides in length. In another embodiment, the poly-A tail is greater
than 35
nucleotides in length (e.g., at least or greater than about 35, 40, 45, 50,
55, 60, 70, 80, 90,
100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800,
900, 1,000,
1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500,
and 3,000
nucleotides). In some embodiments, the polynucleotide, primary construct, or
mmRNA
includes from about 30 to about 3,000 nucleotides (e.g., from 30 to 50, from
30 to 100,
from 30 to 250, from 30 to 500, from 30 to 750, from 30 to 1,000, from 30 to
1,500, from
30 to 2,000, from 30 to 2,500, from 50 to 100, from 50 to 250, from 50 to 500,
from 50 to
750, from 50 to 1,000, from 50 to 1,500, from 50 to 2,000, from 50 to 2,500,
from 50 to
3,000, from 100 to 500, from 100 to 750, from 100 to 1,000, from 100 to 1,500,
from 100
to 2,000, from 100 to 2,500, from 100 to 3,000, from 500 to 750, from 500 to
1,000, from
500 to 1,500, from 500 to 2,000, from 500 to 2,500, from 500 to 3,000, from
1,000 to
1,500, from 1,000 to 2,000, from 1,000 to 2,500, from 1,000 to 3,000, from
1,500 to
2,000, from 1,500 to 2,500, from 1,500 to 3,000, from 2,000 to 3,000, from
2,000 to
2,500, and from 2,500 to 3,000).
[000236] In one embodiment, the poly-A tail is designed relative to the length
of the
overall polynucleotides, primary constructs or mmRNA. This design may be based
on the
length of the coding region, the length of a particular feature or region
(such as the first or
flanking regions), or based on the length of the ultimate product expressed
from the
polynucleotides, primary constructs or mmRNA.
[000237] In this context the poly-A tail may be 10, 20, 30, 40, 50, 60, 70,
80, 90, or
100% greater in length than the polynucleotides, primary constructs or mmRNA
or
feature thereof The poly-A tail may also be designed as a fraction of
polynucleotides,
primary constructs or mmRNA to which it belongs. In this context, the poly-A
tail may
be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the total length of the
construct or the
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total length of the construct minus the poly-A tail. Further, engineered
binding sites and
conjugation of polynucleotides, primary constructs or mmRNA for Poly-A binding

protein may enhance expression.
[000238] Additionally, multiple distinct polynucleotides, primary constructs
or
mmRNA may be linked together to the PABP (Poly-A binding protein) through the
3'-
end using modified nucleotides at the 3'-terminus of the poly-A tail.
Transfection
experiments can be conducted in relevant cell lines at and protein production
can be
assayed by ELISA at 12hr, 24hr, 48hr, 72 hr and day 7 post-transfection.
[000239] In one embodiment, the polynucleotide primary constructs of the
present
invention are designed to include a polyA-G Quartet. The G-quartet is a cyclic
hydrogen
bonded array of four guanine nucleotides that can be formed by G-rich
sequences in both
DNA and RNA. In this embodiment, the G-quartet is incorporated at the end of
the poly-
A tail. The resultant mmRNA construct is assayed for stability, protein
production and
other parameters including half-life at various time points. It has been
discovered that the
polyA-G quartet results in protein production equivalent to at least 75% of
that seen using
a poly-A tail of 120 nucleotides alone.
Quantification
[000240] In one embodiment, the polynucleotides, primary constructs or mmRNA
of
the present invention may be quantified in exosomes derived from one or more
bodily
fluid. As used herein "bodily fluids" include peripheral blood, serum, plasma,
ascites,
urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid,
aqueous
humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid,
semen,
prostatic fluid, cowper's fluid or pre-ejaculatory fluid, sweat, fecal matter,
hair, tears, cyst
fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle,
bile, interstitial
fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool
water,
pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary
aspirates, blastocyl
cavity fluid, and umbilical cord blood. Alternatively, exosomes may be
retrieved from an
organ selected from the group consisting of lung, heart, pancreas, stomach,
intestine,
bladder, kidney, ovary, testis, skin, colon, breast, prostate, brain,
esophagus, liver, and
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[000241] In the quantification method, a sample of not more than 2mL is
obtained from
the subject and the exosomes isolated by size exclusion chromatography,
density gradient
centrifugation, differential centrifugation, nanomembrane ultrafiltration,
immunoabsorbent capture, affinity purification, microfluidic separation, or
combinations
thereof In the analysis, the level or concentration of a polynucleotide,
primary construct
or mmRNA may be an expression level, presence, absence, truncation or
alteration of the
administered construct. It is advantageous to correlate the level with one or
more clinical
phenotypes or with an assay for a human disease biomarker. The assay may be
performed
using construct specific probes, cytometry, qRT-PCR, real-time PCR, PCR, flow
cytometry, electrophoresis, mass spectrometry, or combinations thereof while
the
exosomes may be isolated using immunohistochemical methods such as enzyme
linked
immunosorbent assay (ELISA) methods. Exosomes may also be isolated by size
exclusion chromatography, density gradient centrifugation, differential
centrifugation,
nanomembrane ultrafiltration, immunoabsorbent capture, affinity purification,
microfluidic separation, or combinations thereof
[000242] These methods afford the investigator the ability to monitor, in real
time, the
level of polynucleotides, primary constructs or mmRNA remaining or delivered.
This is
possible because the polynucleotides, primary constructs or mmRNA of the
present
invention differ from the endogenous forms due to the structural or chemical
modifications.
II. Design and synthesis of mmRNA
[000243] Polynucleotides, primary constructs or mmRNA for use in accordance
with
the invention may be prepared according to any available technique including,
but not
limited to chemical synthesis, enzymatic synthesis, which is generally termed
in vitro
transcription (IVT) or enzymatic or chemical cleavage of a longer precursor,
etc.
Methods of synthesizing RNAs are known in the art (see, e.g., Gait, M.J. (ed.)

Oligonucleotide synthesis: a practical approach, Oxford [Oxfordshire],
Washington, DC:
IRL Press, 1984; and Herdewijn, P. (ed.) Oligonucleotide synthesis: methods
and
applications, Methods in Molecular Biology, v. 288 (Clifton, N.J.) Totowa,
N.J.: Humana
Press, 2005; both of which are incorporated herein by reference).
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[000244] The process of design and synthesis of the primary constructs of the
invention
generally includes the steps of gene construction, mRNA production (either
with or
without modifications) and purification. In the enzymatic synthesis method, a
target
polynucleotide sequence encoding the polypeptide of interest is first selected
for
incorporation into a vector which will be amplified to produce a cDNA
template.
Optionally, the target polynucleotide sequence and/or any flanking sequences
may be
codon optimized. The cDNA template is then used to produce mRNA through in
vitro
transcription (IVT). After production, the mRNA may undergo purification and
clean-up
processes. The steps of which are provided in more detail below.
Gene Construction
[000245] The step of gene construction may include, but is not limited to gene

synthesis, vector amplification, plasmid purification, plasmid linearization
and clean-up,
and cDNA template synthesis and clean-up.
Gene Synthesis
[000246] Once a polypeptide of interest, or target, is selected for
production, a primary
construct is designed. Within the primary construct, a first region of linked
nucleosides
encoding the polypeptide of interest may be constructed using an open reading
frame
(ORF) of a selected nucleic acid (DNA or RNA) transcript. The ORF may comprise
the
wild type ORF, an isoform, variant or a fragment thereof As used herein, an
"open
reading frame" or "ORF" is meant to refer to a nucleic acid sequence (DNA or
RNA)
which is capable of encoding a polypeptide of interest. ORFs often begin with
the start
codon, ATG and end with a nonsense or termination codon or signal.
[000247] Further, the nucleotide sequence of the first region may be codon
optimized.
Codon optimization methods are known in the art and may be useful in efforts
to achieve
one or more of several goals. These goals include to match codon frequencies
in target
and host organisms to ensure proper folding, bias GC content to increase mRNA
stability
or reduce secondary structures, minimize tandem repeat codons or base runs
that may
impair gene construction or expression, customize transcriptional and
translational
control regions, insert or remove protein trafficking sequences, remove/add
post
translation modification sites in encoded protein (e.g. glycosylation sites),
add, remove or
shuffle protein domains, insert or delete restriction sites, modify ribosome
binding sites
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and mRNA degradation sites, to adjust translational rates to allow the various
domains of
the protein to fold properly, or to reduce or eliminate problem secondary
structures within
the mRNA. Codon optimization tools, algorithms and services are known in the
art, non-
limiting examples include services from GeneArt (Life Technologies), DNA2.0
(Menlo
Park CA) and/or proprietary methods. In one embodiment, the ORF sequence is
optimized using optimization algorithms. Codon options for each amino acid are
given in
Table 1.
Table 1. Codon Options
Amino Acid Single Letter Code Codon Options
Isoleucine I ATT, ATC, ATA
Leucine L CTT, CTC, CTA, CTG, TTA, TTG
Valine V GTT, GTC, GTA, GTG
Phenylalanine F TTT, TTC
Methionine M ATG
Cysteine C TGT, TGC
Alanine A GCT, GCC, GCA, GCG
Glycine G GGT, GGC, GGA, GGG
Proline P CCT, CCC, CCA, CCG
Threonine T ACT, ACC, ACA, ACG
Serine S TCT, TCC, TCA, TCG, AGT, AGC
Tyrosine Y TAT, TAC
Tryptophan W TGG
Glutamine Q CAA, CAG
Asparagine N AAT, AAC
Histidine H CAT, CAC
Glutamic acid E GAA, GAG
Aspartic acid D GAT, GAC
Lysine K AAA, AAG
Arginine R CGT, CGC, CGA, CGG, AGA, AGG
Selenocysteine Sec UGA in mRNA in presence of Selenocystein
insertion element (SECIS)
Stop codons Stop TAA, TAG, TGA
[000248] Features, which may be considered beneficial in some embodiments of
the
present invention, may be encoded by the primary construct and may flank the
ORF as a
first or second flanking region. The flanking regions may be incorporated into
the
primary construct before and/or after optimization of the ORF. It is not
required that a
primary construct contain both a 5' and 3' flanking region. Examples of such
features
include, but are not limited to, untranslated regions (UTRs), Kozak sequences,
an
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oligo(dT) sequence, and detectable tags and may include multiple cloning sites
which
may have XbaI recognition.
[000249] In some embodiments, a 5' UTR and/or a 3' UTR may be provided as
flanking
regions. Multiple 5' or 3' UTRs may be included in the flanking regions and
may be the
same or of different sequences. Any portion of the flanking regions, including
none, may
be codon optimized and any may independently contain one or more different
structural
or chemical modifications, before and/or after codon optimization.
Combinations of
features may be included in the first and second flanking regions and may be
contained
within other features. For example, the ORF may be flanked by a 5' UTR which
may
contain a strong Kozak translational initiation signal and/or a 3' UTR which
may include
an oligo(dT) sequence for templated addition of a poly-A tail. 5'UTR may
comprise a
first polynucleotide fragment and a second polynucleotide fragment from the
same and/or
different genes such as the 5'UTRs described in US Patent Application
Publication No.
20100293625, herein incorporated by reference in its entirety.
[000250] Tables 2 and 3 provide a listing of exemplary UTRs which may be
utilized in
the primary construct of the present invention as flanking regions. Shown in
Table 2 is a
listing of a 5'-untranslated region of the invention. Variants of 5' UTRs may
be utilized
wherein one or more nucleotides are added or removed to the termini, including
A, T, C
or G.
Table 2. 5'-Untranslated Regions
5' UTR Name/Desc SEQ ID
Sequence
Identifier ription NO.
Upstream GGGAAATAAGAGAGAAAAGAAGAGTA
5UTR-001 1
UTR AGAAGAAATATAAGAGCCACC
Upstream GGGAGATCAGAGAGAAAAGAAGAGTA
5UTR-002 2
UTR AGAAGAAATATAAGAGCCACC
GGAATAAAAGTCTCAACACAACATATA
CAAAACAAACGAATCTCAAGCAATCAA
Upstream GCATTCTACTTCTATTGCAGCAATTTAA
5UTR-003 3
UTR ATCATTTCTTTTAAAGCAAAAGCAATTT
TCTGAAAATTTTCACCATTTACGAACGA
TAGCAAC
5UTR 004 Upstream GGGAGACAAGCUUGGCAUUCCGGUAC 4
-
UTR UGUUGGUAAAGCCACC
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[000251] Shown in Table 3 is a representative listing of 3'-untranslated
regions of the
invention. Variants of 3' UTRs may be utilized wherein one or more nucleotides
are
added or removed to the termini, including A, T, C or G.
Table 3. 3'-Untranslated Regions
3' UTR Name/Desc SEQ
Sequence ID
Identifier ription
NO.
GCGCCTGCCCACCTGCCACCGACTGCTGGA
ACCCAGCCAGTGGGAGGGCCTGGCCCACC
AGAGTCCTGCTCCCTCACTCCTCGCCCCGC
CCCCTGTCCCAGAGTCCCACCTGGGGGCTC
TCTCCACCCTTCTCAGAGTTCCAGTTTCAAC
C reatine CAGAGTTCCAACCAATGGGCTCCATCCTCT
3UTR-001 GGATTCTGGCCAATGAAATATCTCCCTGGC 5
Kinase
AGGGTCCTCTTCTTTTCCCAGAGCTCCACCC
CAACCAGGAGCTCTAGTTAATGGAGAGCTC
CCAGCACACTCGGAGCTTGTGCTTTGTCTC
CACGCAAAGCGATAAATAAAAGCATTGGT
GGCCTTTGGTCTTTGAATAAAGCCTGAGTA
GGAAGTCTAGA
GCCCCTGCCGCTCCCACCCCCACCCATCTG
GGCCCCGGGTTCAAGAGAGAGCGGGGTCT
GATCTCGTGTAGCCATATAGAGTTTGCTTC
TGAGTGTCTGCTTTGTTTAGTAGAGGTGGG
CAGGAGGAGCTGAGGGGCTGGGGCTGGGG
TGTTGAAGTTGGCTTTGCATGCCCAGCGAT
GCGCCTCCCTGTGGGATGTCATCACCCTGG
GAACCGGGAGTGGCCCTTGGCTCACTGTGT
TCTGCATGGTTTGGATCTGAATTAATTGTCC
3UTR-002 Myoglobin TTTCTTCTAAATCCCAACCGAACTTCTTCCA 6
ACCTCCAAACTGGCTGTAACCCCAAATCCA
AGCCATTAACTACACCTGACAGTAGCAATT
GTCTGATTAATCACTGGCCCCTTGAAGACA
GCAGAATGTCCCTTTGCAATGAGGAGGAG
ATCTGGGCTGGGCGGGCCAGCTGGGGAAG
CATTTGACTATCTGGAACTTGTGTGTGCCTC
CTCAGGTATGGCAGTGACTCACCTGGTTTT
AATAAAACAACCTGCAACATCTCATGGTCT
TTGAATAAAGCCTGAGTAGGAAGTCTAGA
ACACACTCCACCTCCAGCACGCGACTTCTC
AGGACGACGAATCTTCTCAATGGGGGGGC
GGCTGAGCTCCAGCCACCCCGCAGTCACTT
C. T TTTGTAACAACTTCCGTTGCTGCCATCGT
3UTR-003 ct-actmAAACTGACACAGTGTTTATAACGTGTACAT 7
ACATTAACTTATTACCTCATTTTGTTATTTT
TCGAAACAAAGCCCTGTGGAAGAAAATGG
AAAACTTGAAGAAGCATTAAAGTCATTCTG
TTAAGCTGCGTAAATGGTCTTTGAATAAAG

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CCTGAGTAGGAAGTCTAGA
CATCACATTTAAAAGCATCTCAGCCTACCA
TGAGAATAAGAGAAAGAAAATGAAGATCA
AAAGCTTATTCATCTGTTTTTCTTTTTCGTT
GGTGTAAAGCCAACACCCTGTCTAAAAAAC
ATAAATTTCTTTAATCATTTTGCCTCTTTTC
Alb umm C. T TGTGCTTCAATTAATAAAAAATGGAAAG
3UTR-004 AATCTAATAGAGTGGTACAGCACTGTTATT 8
TTTCAAAGATGTGTTGCTATCCTGAAAATT
CTGTAGGTTCTGTGGAAGTTCCAGTGTTCT
CTCTTATTCCACTTCGGTAGAGGATTTCTAG
TTTCTTGTGGGCTAATTAAATAAATCATTA
ATACTCTTCTAATGGTCTTTGAATAAAGCC
TGAGTAGGAAGTCTAGA
GCTGCCTTCTGCGGGGCTTGCCTTCTGGCC
a-globin ATGCCCTTCTTCTCTCCCTTGCACCTGTACC
3UTR-005 9
TCTTGGTCTTTGAATAAAGCCTGAGTAGGA
AGGCGGCCGCTCGAGCATGCATCTAGA
GCCAAGCCCTCCCCATCCCATGTATTTATCT
CTATTTAATATTTATGTCTATTTAAGCCTCA
TATTTAAAGACAGGGAAGAGCAGAACGGA
GCCCCAGGCCTCTGTGTCCTTCCCTGCATTT
CTGAGTTTCATTCTCCTGCCTGTAGCAGTG
AGAAAAAGCTCCTGTCCTCCCATCCCCTGG
ACTGGGAGGTAGATAGGTAAATACCAAGT
ATTTATTACTATGACTGCTCCCCAGCCCTG
GCTCTGCAATGGGCACTGGGATGAGCCGCT
GTGAGCCCCTGGTCCTGAGGGTCCCCACCT
GGGACCCTTGAGAGTATCAGGTCTCCCACG
TGGGAGACAAGAAATCCCTGTTTAATATTT
AAACAGCAGTGTTCCCCATCTGGGTCCTTG
CACCCCTCACTCTGGCCTCAGCCGACTGCA
CAGCGGCCCCTGCATCCCCTTGGCTGTGAG
3UTR-006 G-C SF GCCCCTGGACAAGCAGAGGTGGCCAGAGC 10
TGGGAGGCATGGCCCTGGGGTCCCACGAAT
TTGCTGGGGAATCTCGTTTTTCTTCTTAAGA
CTTTTGGGACATGGTTTGACTCCCGAACAT
CACCGACGCGTCTCCTGTTTTTCTGGGTGG
CCTCGGGACACCTGCCCTGCCCCCACGAGG
GTCAGGACTGTGACTCTTTTTAGGGCCAGG
CAGGTGCCTGGACATTTGCCTTGCTGGACG
GGGACTGGGGATGTGGGAGGGAGCAGACA
GGAGGAATCATGTCAGGCCTGTGTGTGAAA
GGAAGCTCCACTGTCACCCTCCACCTCTTC
ACCCCCCACTCACCAGTGTCCCCTCCACTG
TCACATTGTAACTGAACTTCAGGATAATAA
AGTGTTTGCCTCCATGGTCTTTGAATAAAG
CCTGAGTAGGAAGGCGGCCGCTCGAGCAT
GCATCTAGA
3UTR-007 Col 1 a2; ACTCAATCTAAATTAAAAAAGAAAGAAAT 11
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collagen, TTGAAAAAACTTTCTCTTTGCCATTTCTTCT
type I, alpha TCTTCTTTTTTAACTGAAAGCTGAATCCTTC
2 CATTTCTTCTGCACATCTACTTGCTTAAATT
GTGGGCAAAAGAGAAAAAGAAGGATTGAT
CAGAGCATTGTGCAATACAGTTTCATTAAC
TCCTTCCCCCGCTCCCCCAAAAATTTGAATT
TTTTTTTCAACACTCTTACACCTGTTATGGA
AAATGTCAACCTTTGTAAGAAAACCAAAAT
AAAAATTGAAAAATAAAAACCATAAACAT
TTGCACCACTTGTGGCTTTTGAATATCTTCC
ACAGAGGGAAGTTTAAAACCCAAACTTCC
AAAGGTTTAAACTACCTCAAAACACTTTCC
CATGAGTGTGATCCACATTGTTAGGTGCTG
ACCTAGACAGAGATGAACTGAGGTCCTTGT
TTTGTTTTGTTCATAATACAAAGGTGCTAAT
TAATAGTATTTCAGATACTTGAAGAATGTT
GATGGTGCTAGAAGAATTTGAGAAGAAAT
ACTCCTGTATTGAGTTGTATCGTGTGGTGT
ATTTTTTAAAAAATTTGATTTAGCATTCATA
TTTTCCATCTTATTCCCAATTAAAAGTATGC
AGATTATTTGCCCAAATCTTCTTCAGATTCA
GCATTTGTTCTTTGCCAGTCTCATTTTCATC
TTCTTCCATGGTTCCACAGAAGCTTTGTTTC
TTGGGCAAGCAGAAAAATTAAATTGTACCT
ATTTTGTATATGTGAGATGTTTAAATAAAT
TGTGAAAAAAATGAAATAAAGCATGTTTG
GTTTTCCAAAAGAACATAT
CGCCGCCGCCCGGGCCCCGCAGTCGAGGGT
CGTGAGCCCACCCCGTCCATGGTGCTAAGC
GGGCCCGGGTCCCACACGGCCAGCACCGCT
Co16a2; GCTCACTCGGACGACGCCCTGGGCCTGCAC
collagen, CTCTCCAGCTCCTCCCACGGGGTCCCCGTA
3UTR-008 12
type VI, GCCCCGGCCCCCGCCCAGCCCCAGGTCTCC
alpha 2 CCAGGCCCTCCGCAGGCTGCCCGGCCTCCC
TCCCCCTGCAGCCATCCCAAGGCTCCTGAC
CTACCTGGCCCCTGAGCTCTGGAGCAAGCC
CTGACCCAATAAAGGCTTTGAACCCAT
GGGGCTAGAGCCCTCTCCGCACAGCGTGGA
GACGGGGCAAGGAGGGGGGTTATTAGGAT
TGGTGGTTTTGTTTTGCTTTGTTTAAAGCCG
TGGGAAAATGGCACAACTTTACCTCTGTGG
GAGATGCAACACTGAGAGCCAAGGGGTGG
GAGTTGGGATAATTTTTATATAAAAGAAGT
RPN1; TTTTCCACTTTGAATTGCTAAAAGTGGCATT
3UTR-009 13
rthophorin I TTTCCTATGTGCAGTCACTCCTCTCATTTCT
AAAATAGGGACGTGGCCAGGCACGGTGGC
TCATGCCTGTAATCCCAGCACTTTGGGAGG
CCGAGGCAGGCGGCTCACGAGGTCAGGAG
ATCGAGACTATCCTGGCTAACACGGTAAAA
CCCTGTCTCTACTAAAAGTACAAAAAATTA
GCTGGGCGTGGTGGTGGGCACCTGTAGTCC
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CAGCTACTCGGGAGGCTGAGGCAGGAGAA
AGGCATGAATCCAAGAGGCAGAGCTTGCA
GTGAGCTGAGATCACGCCATTGCACTCCAG
CCTGGGCAACAGTGTTAAGACTCTGTCTCA
AATATAAATAAATAAATAAATAAATAAAT
AAATAAATAAAAATAAAGCGAGATGTTGC
CCTCAAA
GGCCCTGCCCCGTCGGACTGCCCCCAGAAA
GCCTCCTGCCCCCTGCCAGTGAAGTCCTTC
AGTGAGCCCCTCCCCAGCCAGCCCTTCCCT
GGCCCCGCCGGATGTATAAATGTAAAAATG
AAGGAATTACATTTTATATGTGAGCGAGCA
AGCCGGCAAGCGAGCACAGTATTATTTCTC
CATCCCCTCCCTGCCTGCTCCTTGGCACCCC
CATGCTGCCTTCAGGGAGACAGGCAGGGA
GGGCTTGGGGCTGCACCTCCTACCCTCCCA
CCAGAACGCACCCCACTGGGAGAGCTGGT
LRP1; l ow GGTGCAGCCTTCCCCTCCCTGTATAAGACA
CTTTGCCAAGGCTCTCCCCTCTCGCCCCATC
density. CCTGCTTGCCCGCTCCCACAGCTTCCTGAG
lm
3UTR-010 ipoproteGGCTAATTCTGGGAAGGGAGAGTTCTTTGC 14
receptor-
TGCCCCTGTCTGGAAGACGTGGCTCTGGGT
related
GAGGTAGGCGGGAAAGGATGGAGTGTTTT
protein 1
AGTTCTTGGGGGAGGCCACCCCAAACCCCA
GCCCCAACTCCAGGGGCACCTATGAGATGG
CCATGCTCAACCCCCCTCCCAGACAGGCCC
TCCCTGTCTCCAGGGCCCCCACCGAGGTTC
CCAGGGCTGGAGACTTCCTCTGGTAAACAT
TCCTCCAGCCTCCCCTCCCCTGGGGACGCC
AAGGAGGTGGGCCACACCCAGGAAGGGAA
AGCGGGCAGCCCCGTTTTGGGGACGTGAAC
GTTTTAATAATTTTTGCTGAATTCCTTTACA
ACTAAATAACACAGATATTGTTATAAATAA
AATTGT
ATATTAAGGATCAAGCTGTTAGCTAATAAT
GCCACCTCTGCAGTTTTGGGAACAGGCAAA
TAAAGTATCAGTATACATGGTGATGTACAT
CTGTAGCAAAGCTCTTGGAGAAAATGAAG
ACTGAAGAAAGCAAAGCAAAAACTGTATA
GAGAGATTTTTCAAAAGCAGTAATCCCTCA
Nntl; ATTTTAAAAAAGGATTGAAAATTCTAAATG
cardiotrophi TCTTTCTGTGCATATTTTTTGTGTTAGGAAT
3UTR-011 n-like CAAAAGTATTTTATAAAAGGAGAAAGAAC 15
cytokine AGCCTCATTTTAGATGTAGTCCTGTTGGATT
factor 1 TTTTATGCCTCCTCAGTAACCAGAAATGTTT
TAAAAAACTAAGTGTTTAGGATTTCAAGAC
AACATTATACATGGCTCTGAAATATCTGAC
ACAATGTAAACATTGCAGGCACCTGCATTT
TATGTTTTTTTTTTCAACAAATGTGACTAAT
TTGAAACTTTTATGAACTTCTGAGCTGTCCC
CTTGCAATTCAACCGCAGTTTGAATTAATC
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ATATCAAATCAGTTTTAATTTTTTAAATTGT
ACTTCAGAGTCTATATTTCAAGGGCACATT
TTCTCACTACTATTTTAATACATTAAAGGA
CTAAATAATCTTTCAGAGATGCTGGAAACA
AATCATTTGCTTTATATGTTTCATTAGAATA
CCAATGAAACATACAACTTGAAAATTAGTA
ATAGTATTTTTGAAGATCCCATTTCTAATTG
GAGATCTCTTTAATTTCGATCAACTTATAAT
GTGTAGTACTATATTAAGTGCACTTGAGTG
GAATTCAACATTTGACTAATAAAATGAGTT
CATCATGTTGGCAAGTGATGTGGCAATTAT
CTCTGGTGACAAAAGAGTAAAATCAAATAT
TTCTGCCTGTTACAAATATCAAGGAAGACC
TGCTACTATGAAATAGATGACATTAATCTG
TCTTCACTGTTTATAATACGGATGGATTTTT
TTTCAAATCAGTGTGTGTTTTGAGGTCTTAT
GTAATTGATGACATTTGAGAGAAATGGTGG
CTTTTTTTAGCTACCTCTTTGTTCATTTAAG
CACCAGTAAAGATCATGTCTTTTTATAGAA
GTGTAGATTTTCTTTGTGACTTTGCTATCGT
GCCTAAAGCTCTAAATATAGGTGAATGTGT
GATGAATACTCAGATTATTTGTCTCTCTATA
TAATTAGTTTGGTACTAAGTTTCTCAAAAA
ATTATTAACACATGAAAGACAATCTCTAAA
CCAGAAAAAGAAGTAGTACAAATTTTGTTA
CTGTAATGCTCGCGTTTAGTGAGTTTAAAA
CACACAGTATCTTTTGGTTTTATAATCAGTT
TCTATTTTGCTGTGCCTGAGATTAAGATCTG
TGTATGTGTGTGTGTGTGTGTGTGCGTTTGT
GTGTTAAAGCAGAAAAGACTTTTTTAAAAG
TTTTAAGTGATAAATGCAATTTGTTAATTG
ATCTTAGATCACTAGTAAACTCAGGGCTGA
ATTATACCATGTATATTCTATTAGAAGAAA
GTAAACACCATCTTTATTCCTGCCCTTTTTC
TTCTCTCAAAGTAGTTGTAGTTATATCTAG
AAAGAAGCAATTTTGATTTCTTGAAAAGGT
AGTTCCTGCACTCAGTTTAAACTAAAAATA
ATCATACTTGGATTTTATTTATTTTTGTCAT
AGTAAAAATTTTAATTTATATATATTTTTAT
TTAGTATTATCTTATTCTTTGCTATTTGCCA
ATCCTTTGTCATCAATTGTGTTAAATGAATT
GAAAATTCATGCCCTGTTCATTTTATTTTAC
TTTATTGGTTAGGATATTTAAAGGATTTTTG
TATATATAATTTCTTAAATTAATATTCCAAA
AGGTTAGTGGACTTAGATTATAAATTATGG
CAAAAATCTAAAAACAACAAAAATGATTTT
TATACATTCTATTTCATTATTCCTCTTTTTCC
AATAAGTCATACAATTGGTAGATATGACTT
ATTTTATTTTTGTATTATTCACTATATCTTT
ATGATATTTAAGTATAAATAATTAAAAAAA
TTTATTGTACCTTATAGTCTGTCACCAAAA
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AAAAAAAATTATCTGTAGGTAGTGAAATGC
TAATGTTGATTTGTCTTTAAGGGCTTGTTAA
CTATCCTTTATTTTCTCATTTGTCTTAAATT
AGGAGTTTGTGTTTAAATTACTCATCTAAG
CAAAAAATGTATATAAATCCCATTACTGGG
TATATACCCAAAGGATTATAAATCATGCTG
CTATAAAGACACATGCACACGTATGTTTAT
TGCAGCACTATTCACAATAGCAAAGACTTG
GAACCAACCCAAATGTCCATCAATGATAGA
CTTGATTAAGAAAATGTGCACATATACACC
ATGGAATACTATGCAGCCATAAAAAAGGA
TGAGTTCATGTCCTTTGTAGGGACATGGAT
AAAGCTGGAAACCATCATTCTGAGCAAACT
ATTGCAAGGACAGAAAACCAAACACTGCA
TGTTCTCACTCATAGGTGGGAATTGAACAA
TGAGAACACTTGGACACAAGGTGGGGAAC
ACCACACACCAGGGCCTGTCATGGGGTGG
GGGGAGTGGGGAGGGATAGCATTAGGAGA
TATACCTAATGTAAATGATGAGTTAATGGG
TGCAGCACACCAACATGGCACATGTATACA
TATGTAGCAAACCTGCACGTTGTGCACATG
TACCCTAGAACTTAAAGTATAATTAAAAAA
AAAAAGAAAACAGAAGCTATTTATAAAGA
AGTTATTTGCTGAAATAAATGTGATCTTTC
CCATTAAAAAAATAAAGAAATTTTGGGGTA
AAAAAACACAATATATTGTATTCTTGAAAA
ATTCTAAGAGAGTGGATGTGAAGTGTTCTC
ACCACAAAAGTGATAACTAATTGAGGTAAT
GCACATATTAATTAGAAAGATTTTGTCATT
CCACAATGTATATATACTTAAAAATATGTT
ATACACAATAAATACATACATTAAAAAATA
AGTAAATGTA
CCCACCCTGCACGCCGGCACCAAACCCTGT
CCTCCCACCCCTCCCCACTCATCACTAAAC
AGAGTAAAATGTGATGCGAATTTTCCCGAC
CAACCTGATTCGCTAGATTTTTTTTAAGGA
AAAGCTTGGAAAGCCAGGACACAACGCTG
CTGCCTGCTTTGTGCAGGGTCCTCCGGGGC
TCAGCCCTGAGTTGGCATCACCTGCGCAGG
C o16 a 1. GCCCTCTGGGGCTCAGCCCTGAGCTAGTGT
,
CACCTGCACAGGGCCCTCTGAGGCTCAGCC
3UTR-012 collagen,
CTGAGCTGGCGTCACCTGTGCAGGGCCCTC 16
type VI,
TGGGGCTCAGCCCTGAGCTGGCCTCACCTG
alpha 1
GGTTCCCCACCCCGGGCTCTCCTGCCCTGC
CCTCCTGCCCGCCCTCCCTCCTGCCTGCGCA
GCTCCTTCCCTAGGCACCTCTGTGCTGCATC
CCACCAGCCTGAGCAAGACGCCCTCTCGGG
GCCTGTGCCGCACTAGCCTCCCTCTCCTCTG
TCCCCATAGCTGGTTTTTCCCACCAATCCTC
ACCTAACAGTTACTTTACAATTAAACTCAA
AGCAAGCTCTTCTCCTCAGCTTGGGGCAGC

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CATTGGCCTCTGTCTCGTTTTGGGAAACCA
AGGTCAGGAGGCCGTTGCAGACATAAATCT
CGGCGACTCGGCCCCGTCTCCTGAGGGTCC
TGCTGGTGACCGGCCTGGACCTTGGCCCTA
CAGCCCTGGAGGCCGCTGCTGACCAGCACT
GACCCCGACCTCAGAGAGTACTCGCAGGG
GCGCTGGCTGCACTCAAGACCCTCGAGATT
AACGGTGCTAACCCCGTCTGCTCCTCCCTC
CCGCAGAGACTGGGGCCTGGACTGGACAT
GAGAGCCCCTTGGTGCCACAGAGGGCTGTG
TCTTACTAGAAACAACGCAAACCTCTCCTT
CCTCAGAATAGTGATGTGTTCGACGTTTTA
TCAAAGGCCCCCTTTCTATGTTCATGTTAGT
TTTGCTCCTTCTGTGTTTTTTTCTGAACCAT
ATCCATGTTGCTGACTTTTCCAAATAAAGG
TTTTCACTCCTCTC
AGAGGCCTGCCTCCAGGGCTGGACTGAGG
CCTGAGCGCTCCTGCCGCAGAGCTGGCCGC
GCCAAATAATGTCTCTGTGAGACTCGAGAA
CTTTCATTTTTTTCCAGGCTGGTTCGGATTT
GGGGTGGATTTTGGTTTTGTTCCCCTCCTCC
ACTCTCCCCCACCCCCTCCCCGCCCTTTTTT
TTTTTTTTTTTTAAACTGGTATTTTATCTTTG
ATTCTCCTTCAGCCCTCACCCCTGGTTCTCA
TCTTTCTTGATCAACATCTTTTCTTGCCTCT
Calr; GTCCCCTTCTCTCATCTCTTAGCTCCCCTCC
3UTR-013 17
calreticulin AACCTGGGGGGCAGTGGTGTGGAGAAGCC
ACAGGCCTGAGATTTCATCTGCTCTCCTTCC
TGGAGCCCAGAGGAGGGCAGCAGAAGGGG
GTGGTGTCTCCAACCCCCCAGCACTGAGGA
AGAACGGGGCTCTTCTCATTTCACCCCTCC
CTTTCTCCCCTGCCCCCAGGACTGGGCCAC
TTCTGGGTGGGGCAGTGGGTCCCAGATTGG
CTCACACTGAGAATGTAAGAACTACAAAC
AAAATTTCTATTAAATTAAATTTTGTGTCTC
C
CTCCCTCCATCCCAACCTGGCTCCCTCCCAC
CCAACCAACTTTCCCCCCAACCCGGAAACA
GACAAGCAACCCAAACTGAACCCCCTCAA
AAGCCAAAAAATGGGAGACAATTTCACAT
GGACTTTGGAAAATATTTTTTTCCTTTGCAT
C oll a 1 = TCATCTCTCAAACTTAGTTTTTATCTTTGAC
' CAACCGAACATGACCAAAAACCAAAAGTG
3UTR-014 collagen,
CATTCAACCTTACCAAAAAAAAAAAAAAA 18
type I, alpha
AAAAGAATAAATAAATAACTTTTTAAAAA
1
AGGAAGCTTGGTCCACTTGCTTGAAGACCC
ATGCGGGGGTAAGTCCCTTTCTGCCCGTTG
GGCTTATGAAACCCCAATGCTGCCCTTTCT
GCTCCTTTCTCCACACCCCCCTTGGGGCCTC
CCCTCCACTCCTTCCCAAATCTGTCTCCCCA
GAAGACACAGGAAACAATGTATTGTCTGCC
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CAGCAATCAAAGGCAATGCTCAAACACCC
AAGTGGCCCCCACCCTCAGCCCGCTCCTGC
CCGCCCAGCACCCCCAGGCCCTGGGGGACC
TGGGGTTCTCAGACTGCCAAAGAAGCCTTG
CCATCTGGCGCTCCCATGGCTCTTGCAACA
TCTCCCCTTCGTTTTTGAGGGGGTCATGCCG
GGGGAGCCACCAGCCCCTCACTGGGTTCGG
AGGAGAGTCAGGAAGGGCCACGACAAAGC
AGAAACATCGGATTTGGGGAACGCGTGTC
AATCCCTTGTGCCGCAGGGCTGGGCGGGAG
AGACTGTTCTGTTCCTTGTGTAACTGTGTTG
CTGAAAGACTACCTCGTTCTTGTCTTGATGT
GTCACCGGGGCAACTGCCTGGGGGCGGGG
ATGGGGGCAGGGTGGAAGCGGCTCCCCAT
TTTATACCAAAGGTGCTACATCTATGTGAT
GGGTGGGGTGGGGAGGGAATCACTGGTGC
TATAGAAATTGAGATGCCCCCCCAGGCCAG
CAAATGTTCCTTTTTGTTCAAAGTCTATTTT
TATTCCTTGATATTTTTCTTTTTTTTTTTTTT
TTTTTGTGGATGGGGACTTGTGAATTTTTCT
AAAGGTGCTATTTAACATGGGAGGAGAGC
GTGTGCGGCTCCAGCCCAGCCCGCTGCTCA
CTTTCCACCCTCTCTCCACCTGCCTCTGGCT
TCTCAGGCCTCTGCTCTCCGACCTCTCTCCT
CTGAAACCCTCCTCCACAGCTGCAGCCCAT
CCTCCCGGCTCCCTCCTAGTCTGTCCTGCGT
CCTCTGTCCCCGGGTTTCAGAGACAACTTC
CCAAAGCACAAAGCAGTTTTTCCCCCTAGG
GGTGGGAGGAAGCAAAAGACTCTGTACCT
ATTTTGTATGTGTATAATAATTTGAGATGTT
TTTAATTATTTTGATTGCTGGAATAAAGCA
TGTGGAAATGACCCAAACATAATCCGCAGT
GGCCTCCTAATTTCCTTCTTTGGAGTTGGGG
GAGGGGTAGACATGGGGAAGGGGCTTTGG
GGTGATGGGCTTGCCTTCCATTCCTGCCCTT
TCCCTCCCCACTATTCTCTTCTAGATCCCTC
CATAACCCCACTCCCCTTTCTCTCACCCTTC
TTATACCGCAAACCTTTCTACTTCCTCTTTC
ATTTTCTATTCTTGCAATTTCCTTGCACCTT
TTCCAAATCCTCTTCTCCCCTGCAATACCAT
ACAGGCAATCCACGTGCACAACACACACA
CACACTCTTCACATCTGGGGTTGTCCAAAC
CTCATACCCACTCCCCTTCAAGCCCATCCA
CTCTCCACCCCCTGGATGCCCTGCACTTGG
TGGCGGTGGGATGCTCATGGATACTGGGAG
GGTGAGGGGAGTGGAACCCGTGAGGAGGA
CCTGGGGGCCTCTCCTTGAACTGACATGAA
GGGTCATCTGGCCTCTGCTCCCTTCTCACCC
ACGCTGACCTCCTGCCGAAGGAGCAACGC
AACAGGAGAGGGGTCTGCTGAGCCTGGCG
AGGGTCTGGGAGGGACCAGGAGGAAGGCG
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TGCTCCCTGCTCGCTGTCCTGGCCCTGGGG
GAGTGAGGGAGACAGACACCTGGGAGAGC
TGTGGGGAAGGCACTCGCACCGTGCTCTTG
GGAAGGAAGGAGACCTGGCCCTGCTCACC
ACGGACTGGGTGCCTCGACCTCCTGAATCC
CCAGAACACAACCCCCCTGGGCTGGGGTG
GTCTGGGGAACCATCGTGCCCCCGCCTCCC
GCCTACTCCTTTTTAAGCTT
TTGGCCAGGCCTGACCCTCTTGGACCTTTCT
TCTTTGCCGACAACCACTGCCCAGCAGCCT
CTGGGACCTCGGGGTCCCAGGGAACCCAGT
CCAGCCTCCTGGCTGTTGACTTCCCATTGCT
CTTGGAGCCACCAATCAAAGAGATTCAAA
GAGATTCCTGCAGGCCAGAGGCGGAACAC
ACCTTTATGGCTGGGGCTCTCCGTGGTGTT
CTGGACCCAGCCCCTGGAGACACCATTCAC
TTTTACTGCTTTGTAGTGACTCGTGCTCTCC
Plod 1; AACCTGTCTTCCTGAAAAACCAAGGCCCCC
procollagen- TTCCCCCACCTCTTCCATGGGGTGAGACTT
lysine, 2- GAGCAGAACAGGGGCTTCCCCAAGTTGCCC
3UTR-015 oxoglutarate AGAAAGACTGTCTGGGTGAGAAGCCATGG 19
5-
CCAGAGCTTCTCCCAGGCACAGGTGTTGCA
dioxygenase CCAGGGACTTCTGCTTCAAGTTTTGGGGTA
1 AAGACACCTGGATCAGACTCCAAGGGCTG
CCCTGAGTCTGGGACTTCTGCCTCCATGGC
TGGTCATGAGAGCAAACCGTAGTCCCCTGG
AGACAGCGACTCCAGAGAACCTCTTGGGA
GACAGAAGAGGCATCTGTGCACAGCTCGA
TCTTCTACTTGCCTGTGGGGAGGGGAGTGA
CAGGTCCACACACCACACTGGGTCACCCTG
TCCTGGATGCCTCTGAAGAGAGGGACAGA
CCGTCAGAAACTGGAGAGTTTCTATTAAAG
GTCATTTAAACCA
TCCTCCGGGACCCCAGCCCTCAGGATTCCT
GATGCTCCAAGGCGACTGATGGGCGCTGG
ATGAAGTGGCACAGTCAGCTTCCCTGGGGG
CTGGTGTCATGTTGGGCTCCTGGGGCGGGG
GCACGGCCTGGCATTTCACGCATTGCTGCC
ACCCCAGGTCCACCTGTCTCCACTTTCACA
GCCTCCAAGTCTGTGGCTCTTCCCTTCTGTC
CTCCGAGGGGCTTGCCTTCTCTCGTGTCCA
Nucbl.
. ' . GTGAGGTGCTCAGTGATCGGCTTAACTTAG
3UTR-016 nucleobmdt 20
AGAAGCCCGCCCCCTCCCCTTCTCCGTCTG
n 1
TCCCAAGAGGGTCTGCTCTGAGCCTGCGTT
CCTAGGTGGCTCGGCCTCAGCTGCCTGGGT
TGTGGCCGCCCTAGCATCCTGTATGCCCAC
AGCTACTGGAATCCCCGCTGCTGCTCCGGG
CCAAGCTTCTGGTTGATTAATGAGGGCATG
GGGTGGTCCCTCAAGACCTTCCCCTACCTT
TTGTGGAACCAGTGATGCCTCAAAGACAGT
GTCCCCTCCACAGCTGGGTGCCAGGGGCAG
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GGGATCCTCAGTATAGCCGGTGAACCCTGA
TACCAGGAGCCTGGGCCTCCCTGAACCCCT
GGCTTCCAGCCATCTCATCGCCAGCCTCCT
CCTGGACCTCTTGGCCCCCAGCCCCTTCCC
CACACAGCCCCAGAAGGGTCCCAGAGCTG
ACCCCACTCCAGGACCTAGGCCCAGCCCCT
CAGCCTCATCTGGAGCCCCTGAAGACCAGT
CCCACCCACCTTTCTGGCCTCATCTGACACT
GCTCCGCATCCTGCTGTGTGTCCTGTTCCAT
GTTCCGGTTCCATCCAAATACACTTTCTGG
AACAAA
GCTGGAGCCTCGGTGGCCATGCTTCTTGCC
3 UTR- 01 a-globin CCTTGGGCCTCCCCCCAGCCCCTCCTCCCCT 21
7
TCCTGCACCCGTACCCCCGTGGTCTTTGAA
TAAAGTCTGAGTGGGCGGC
[000252] It should be understood that those listed in the previous tables are
examples
and that any UTR from any gene may be incorporated into the respective first
or second
flanking region of the primary construct. Furthermore, multiple wild-type UTRs
of any
known gene may be utilized. It is also within the scope of the present
invention to
provide artificial UTRs which are not variants of wild type genes. These UTRs
or
portions thereof may be placed in the same orientation as in the transcript
from which
they were selected or may be altered in orientation or location. Hence a 5' or
3' UTR may
be inverted, shortened, lengthened, made chimeric with one or more other 5'
UTRs or 3'
UTRs. As used herein, the term "altered" as it relates to a UTR sequence,
means that the
UTR has been changed in some way in relation to a reference sequence. For
example, a 3'
or 5' UTR may be altered relative to a wild type or native UTR by the change
in
orientation or location as taught above or may be altered by the inclusion of
additional
nucleotides, deletion of nucleotides, swapping or transposition of
nucleotides. Any of
these changes producing an "altered" UTR (whether 3' or 5') comprise a variant
UTR.
[000253] In one embodiment, a double, triple or quadruple UTR such as a 5' or
3' UTR
may be used. As used herein, a "double" UTR is one in which two copies of the
same
UTR are encoded either in series or substantially in series. For example, a
double beta-
globin 3' UTR may be used as described in US Patent publication 20100129877,
the
contents of which are incorporated herein by reference in its entirety.
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[000254] It is also within the scope of the present invention to have
patterned UTRs. As
used herein "patterned UTRs" are those UTRs which reflect a repeating or
alternating
pattern, such as ABABAB or AABBAABBAABB or ABCABCABC or variants thereof
repeated once, twice, or more than 3 times. In these patterns, each letter, A,
B, or C
represent a different UTR at the nucleotide level.
[000255] In one embodiment, flanking regions are selected from a family of
transcripts
whose proteins share a common function, structure, feature of property. For
example,
polypeptides of interest may belong to a family of proteins which are
expressed in a
particular cell, tissue or at some time during development. The UTRs from any
of these
genes may be swapped for any other UTR of the same or different family of
proteins to
create a new chimeric primary transcript. As used herein, a "family of
proteins" is used in
the broadest sense to refer to a group of two or more polypeptides of interest
which share
at least one function, structure, feature, localization, origin, or expression
pattern.
[000256] After optimization (if desired), the primary construct components are

reconstituted and transformed into a vector such as, but not limited to,
plasmids, viruses,
cosmids, and artificial chromosomes. For example, the optimized construct may
be
reconstituted and transformed into chemically competent E. coli, yeast,
neurospora,
maize, drosophila, etc. where high copy plasmid-like or chromosome structures
occur by
methods described herein.
[000257] The untranslated region may also include translation enhancer
elements
(TEE). As a non-limiting example, the TEE may include those described in US
Application No. 20090226470, herein incorporated by reference in its entirety,
and those
known in the art.
Stop Codons
[000258] In one embodiment, the primary constructs of the present invention
may
include at least two stop codons before the 3' untranslated region (UTR). The
stop codon
may be selected from TGA, TAA and TAG. In one embodiment, the primary
constructs
of the present invention include the stop codon TGA and one additional stop
codon. In a
further embodiment the addition stop codon may be TAA. In another embodiment,
the
primary constructs of the present invention include three stop codons.
Vector Amplification

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[000259] The vector containing the primary construct is then amplified and the
plasmid
isolated and purified using methods known in the art such as, but not limited
to, a maxi
prep using the Invitrogen PURELNKTM HiPure Maxiprep Kit (Carlsbad, CA).
Plasmid Linearization
[000260] The plasmid may then be linearized using methods known in the art
such as,
but not limited to, the use of restriction enzymes and buffers. The
linearization reaction
may be purified using methods including, for example Invitrogen's PURELINKTM
PCR
Micro Kit (Carlsbad, CA), and HPLC based purification methods such as, but not
limited
to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC
(RP-
HPLC), and hydrophobic interaction HPLC (HIC-HPLC) and Invitrogen's standard
PURELINKTM PCR Kit (Carlsbad, CA). The purification method may be modified
depending on the size of the linearization reaction which was conducted. The
linearized
plasmid is then used to generate cDNA for in vitro transcription (IVT)
reactions.
cDNA Template Synthesis
[000261] A cDNA template may be synthesized by having a linearized plasmid
undergo
polymerase chain reaction (PCR). Table 4 is a listing of primers and probes
that may be
usefully in the PCR reactions of the present invention. It should be
understood that the
listing is not exhaustive and that primer-probe design for any amplification
is within the
skill of those in the art. Probes may also contain chemically modified bases
to increase
base-pairing fidelity to the target molecule and base-pairing strength. Such
modifications
may include 5-methyl-Cytidine, 2, 6-di-amino-purine, 2'-fluoro, phosphoro-
thioate, or
locked nucleic acids.
Table 4. Primers and Probes
Primer/SEQ
Hybridization
Probe Sequence (5'-3') ID
Identifier target NO.
TTGGACCCTCGTACAGAAGCTAA
UFP TACG cDNA Template 22
URP TxmoCTTCCTACTCAGGCTTTATTC
C Templ 23
ate
AAAGACCA
CCTTGACCTTCTGGAACTTC Acid
GBA1 24
glucocerebrosidase
CCAAGCACTGAAACGGATAT Acid
GBA2 25
glucocerebrosidase
GATGAAAAGTGCTCCAAGGA
LUC1 Luciferase 26
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AACCGTGATGAAAAGGTACC
LUC2 Luciferase 27
TCATGCAGATTGGAAAGGTC
LUC3 Luciferase 28
CTTCTTGGACTGTCCAGAGG
GCSF1 G-C SF 29
GCAGTCCCTGATACAAGAAC
GCSF2 G-C SF 30
GATTGAAGGTGGCTCGCTAC
GCSF3 G-C SF 31
*UFP is universal forward primer; URP is universal reverse primer.
[000262] In one embodiment, the cDNA may be submitted for sequencing analysis
before undergoing transcription.
mRNA Production
[000263] The process of mRNA or mmRNA production may include, but is not
limited
to, in vitro transcription, cDNA template removal and RNA clean-up, and mRNA
capping and/or tailing reactions.
In Vitro Transcription
[000264] The cDNA produced in the previous step may be transcribed using an in
vitro
transcription (IVT) system. The system typically comprises a transcription
buffer,
nucleotide triphosphates (NTPs), an RNase inhibitor and a polymerase. The NTPs
may
be manufactured in house, may be selected from a supplier, or may be
synthesized as
described herein. The NTPs may be selected from, but are not limited to, those
described
herein including natural and unnatural (modified) NTPs. The polymerase may be
selected
from, but is not limited to, T7 RNA polymerase, T3 RNA polymerase and mutant
polymerases such as, but not limited to, polymerases able to incorporate
modified nucleic
acids.
RNA Polymerases
[000265] Any number of RNA polymerases or variants may be used in the design
of the
primary constructs of the present invention.
[000266] RNA polymerases may be modified by inserting or deleting amino acids
of
the RNA polymerase sequence. As a non-limiting example, the RNA polymerase may
be
modified to exhibit an increased ability to incorporate a 2'-modified
nucleotide
triphosphate compared to an unmodified RNA polymerase (see International
Publication
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W02008078180 and U.S. Patent 8,101,385; herein incorporated by reference in
their
entireties).
[000267] Variants may be obtained by evolving an RNA polymerase, optimizing
the
RNA polymerase amino acid and/or nucleic acid sequence and/or by using other
methods
known in the art. As a non-limiting example, T7 RNA polymerase variants may be

evolved using the continuous directed evolution system set out by Esvelt et
at. (Nature
(2011) 472(7344):499-503; herein incorporated by reference in its entirety)
where clones
of T7 RNA polymerase may encode at least one mutation such as, but not limited
to,
lysine at position 93 substituted for threonine (K93T), I4M, A7T, E63V, V64D,
A65E,
D66Y, T76N, C125R, 5128R, A136T, N1655, G175R, H176L, Y178H, F182L, L196F,
G198V, D208Y, E222K, 5228A, Q239R, T243N, G259D, M267I, G280C, H300R,
D351A, A3545, E356D, L360P, A383V, Y385C, D388Y, 5397R, M401T, N4105,
K450R, P45 1T, G452V, E484A, H523L, H524N, G542V, E565K, K577E, K577M,
N6015, 5684Y, L699I, K713E, N748D, Q754R, E775K, A827V, D851N or L864F. As
another non-limiting example, T7 RNA polymerase variants may encode at least
mutation as described in U.S. Pub. Nos. 20100120024 and 20070117112; herein
incorporated by reference in their entireties. Variants of RNA polymerase may
also
include, but are not limited to, substitutional variants, conservative amino
acid
substitution, insertional variants, deletional variants and/or covalent
derivatives.
[000268] In one embodiment, the primary construct may be designed to be
recognized
by the wild type or variant RNA polymerases. In doing so, the primary
construct may be
modified to contain sites or regions of sequence changes from the wild type or
parent
primary construct.
[000269] In one embodiment, the primary construct may be designed to include
at least
one substitution and/or insertion upstream of an RNA polymerase binding or
recognition
site, downstream of the RNA polymerase binding or recognition site, upstream
of the
TATA box sequence, downstream of the TATA box sequence of the primary
construct
but upstream of the coding region of the primary construct, within the 5'UTR,
before the
5'UTR and/or after the 5'UTR.
[000270] In one embodiment, the 5'UTR of the primary construct may be replaced
by
the insertion of at least one region and/or string of nucleotides of the same
base. The
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region and/or string of nucleotides may include, but is not limited to, at
least 3, at least 4,
at least 5, at least 6, at least 7 or at least 8 nucleotides and the
nucleotides may be natural
and/or unnatural. As a non-limiting example, the group of nucleotides may
include 5-8
adenine, cytosine, thymine, a string of any of the other nucleotides disclosed
herein
and/or combinations thereof.
[000271] In one embodiment, the 5'UTR of the primary construct may be replaced
by
the insertion of at least two regions and/or strings of nucleotides of two
different bases
such as, but not limited to, adenine, cytosine, thymine, any of the other
nucleotides
disclosed herein and/or combinations thereof For example, the 5'UTR may be
replaced
by inserting 5-8 adenine bases followed by the insertion of 5-8 cytosine
bases. In another
example, the 5'UTR may be replaced by inserting 5-8 cytosine bases followed by
the
insertion of 5-8 adenine bases.
[000272] In one embodiment, the primary construct may include at least one
substitution and/or insertion downstream of the transcription start site which
may be
recognized by an RNA polymerase. As a non-limiting example, at least one
substitution
and/or insertion may occur downstream the transcription start site by
substituting at least
one nucleic acid in the region just downstream of the transcription start site
(such as, but
not limited to, +1 to +6). Changes to region of nucleotides just downstream of
the
transcription start site may affect initiation rates, increase apparent
nucleotide
triphosphate (NTP) reaction constant values, and increase the dissociation of
short
transcripts from the transcription complex curing initial transcription
(Brieba et al,
Biochemistry (2002) 41: 5144-5149; herein incorporated by reference in its
entirety).
The modification, substitution and/or insertion of at least one nucleic acid
may cause a
silent mutation of the nucleic acid sequence or may cause a mutation in the
amino acid
sequence.
[000273] In one embodiment, the primary construct may include the substitution
of at
least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least
7, at least 8, at least 9,
at least 10, at least 11, at least 12 or at least 13 guanine bases downstream
of the
transcription start site.
[000274] In one embodiment, the primary construct may include the substitution
of at
least 1, at least 2, at least 3, at least 4, at least 5 or at least 6 guanine
bases in the region
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just downstream of the transcription start site. As a non-limiting example, if
the
nucleotides in the region are GGGAGA the guanine bases may be substituted by
at least
1, at least 2, at least 3 or at least 4 adenine nucleotides. In another non-
limiting example,
if the nucleotides in the region are GGGAGA the guanine bases may be
substituted by at
least 1, at least 2, at least 3 or at least 4 cytosine bases. In another non-
limiting example,
if the nucleotides in the region are GGGAGA the guanine bases may be
substituted by at
least 1, at least 2, at least 3 or at least 4 thymine, and/or any of the
nucleotides described
herein.
[000275] In one embodiment, the primary construct may include at least one
substitution and/or insertion upstream of the start codon. For the purpose of
clarity, one
of skill in the art would appreciate that the start codon is the first codon
of the protein
coding region whereas the transcription start site is the site where
transcription begins.
The primary construct may include, but is not limited to, at least 1, at least
2, at least 3, at
least 4, at least 5, at least 6, at least 7 or at least 8 substitutions and/or
insertions of
nucleotide bases. The nucleotide bases may be inserted or substituted at 1, at
least 1, at
least 2, at least 3, at least 4 or at least 5 locations upstream of the start
codon. The
nucleotides inserted and/or substituted may be the same base (e.g., all A or
all C or all T
or all G), two different bases (e.g., A and C, A and T, or C and T), three
different bases
(e.g., A, C and T or A, C and T) or at least four different bases. As a non-
limiting
example, the guanine base upstream of the coding region in the primary
construct may be
substituted with adenine, cytosine, thymine, or any of the nucleotides
described herein.
In another non-limiting example the substitution of guanine bases in the
primary
construct may be designed so as to leave one guanine base in the region
downstream of
the transcription start site and before the start codon (see Esvelt et at.
Nature (2011)
472(7344):499-503; herein incorporated by reference in its entirety). As a non-
limiting
example, at least 5 nucleotides may be inserted at 1 location downstream of
the
transcription start site but upstream of the start codon and the at least 5
nucleotides may
be the same base type.
cDNA Template Removal and Clean-Up
[000276] The cDNA template may be removed using methods known in the art such
as,
but not limited to, treatment with Deoxyribonuclease I (DNase I). RNA clean-up
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also include a purification method such as, but not limited to, AGENCOURTO
CLEANSEQO system from Beckman Coulter (Danvers, MA), HPLC based purification
methods such as, but not limited to, strong anion exchange HPLC, weak anion
exchange
HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-
HPLC) .
Capping and/or Tailing Reactions
[000277] The primary construct or mmRNA may also undergo capping and/or
tailing
reactions. A capping reaction may be performed by methods known in the art to
add a 5'
cap to the 5' end of the primary construct. Methods for capping include, but
are not
limited to, using a Vaccinia Capping enzyme (New England Biolabs, Ipswich,
MA).
[000278] A poly-A tailing reaction may be performed by methods known in the
art,
such as, but not limited to, 2' 0-methyltransferase and by methods as
described herein. If
the primary construct generated from cDNA does not include a poly-T, it may be

beneficial to perform the poly-A-tailing reaction before the primary construct
is cleaned.
mRNA Purification
[000279] Primary construct or mmRNA purification may include, but is not
limited to,
mRNA or mmRNA clean-up, quality assurance and quality control. mRNA or mmRNA
clean-up may be performed by methods known in the arts such as, but not
limited to,
AGENCOURTO beads (Beckman Coulter Genomics, Danvers, MA), poly-T beads,
LNATM oligo-T capture probes (EXIQONO Inc, Vedbaek, Denmark) or HPLC based
purification methods such as, but not limited to, strong anion exchange HPLC,
weak
anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction

HPLC (HIC-HPLC). The term "purified" when used in relation to a polynucleotide
such
as a "purified mRNA or mmRNA" refers to one that is separated from at least
one
contaminant. As used herein, a "contaminant" is any substance which makes
another
unfit, impure or inferior. Thus, a purified polynucleotide (e.g., DNA and RNA)
is present
in a form or setting different from that in which it is found in nature, or a
form or setting
different from that which existed prior to subjecting it to a treatment or
purification
method.
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[000280] A quality assurance and/or quality control check may be conducted
using
methods such as, but not limited to, gel electrophoresis, UV absorbance, or
analytical
HPLC.
[000281] In another embodiment, the mRNA or mmRNA may be sequenced by
methods including, but not limited to reverse-transcriptase-PCR.
[000282] In one embodiment, the mRNA or mmRNA may be quantified using methods
such as, but not limited to, ultraviolet visible spectroscopy (UVNis). A non-
limiting
example of a UVNis spectrometer is a NANODROPO spectrometer (ThermoFisher,
Waltham, MA). The quantified mRNA or mmRNA may be analyzed in order to
determine if the mRNA or mmRNA may be of proper size, check that no
degradation of
the mRNA or mmRNA has occurred. Degradation of the mRNA and/or mmRNA may be
checked by methods such as, but not limited to, agarose gel electrophoresis,
HPLC based
purification methods such as, but not limited to, strong anion exchange HPLC,
weak
anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction

HPLC (HIC-HPLC), liquid chromatography-mass spectrometry (LCMS), capillary
electrophoresis (CE) and capillary gel electrophoresis (CGE).
Signal Sequences
[000283] The primary constructs or mmRNA may also encode additional features
which facilitate trafficking of the polypeptides to therapeutically relevant
sites. One such
feature which aids in protein trafficking is the signal sequence. As used
herein, a "signal
sequence" or "signal peptide" is a polynucleotide or polypeptide,
respectively, which is
from about 9 to 200 nucleotides (3-60 amino acids) in length which is
incorporated at the
5' (or N-terminus) of the coding region or polypeptide encoded, respectively.
Addition of
these sequences result in trafficking of the encoded polypeptide to the
endoplasmic
reticulum through one or more secretory pathways. Some signal peptides are
cleaved
from the protein by signal peptidase after the proteins are transported.
[000284] Table 5 is a representative listing of protein signal sequences which
may be
incorporated for encoding by the polynucleotides, primary constructs or mmRNA
of the
invention.
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Table 5. Signal Sequences
ID Description NUCLEOTIDE SEQUENCE SEQ ENCODED SEQ ID
(5'-3') ID PEPTIDE NO.
NO.
SS-001 a-1- ATGATGCCATCCTCAGTCTCA 32 MMPSSVSWGI 94
antitrypsin TGGGGTATTTTGCTCTTGGCG LLAGLCCLVP
GGTCTGTGCTGTCTCGTGCCG VSLA
GTGTCGCTCGCA
SS-002 G-CSF ATGGCCGGACCGGCGACTCAG 33 MAGPATQSPM 95
TCGCCCATGAAACTCATGGCC KLMALQLLLW
CTGCAGTTGTTGCTTTGGCAC HSALWTVQEA
TCAGCCCTCTGGACCGTCCAA
GAGGCG
SS-003 Factor IX ATGCAGAGAGTGAACATGATT 34 MQRVNMIMAE 96
ATGGCCGAGTCCCCATCGCTC SP SLITICLLGY
ATCACAATCTGCCTGCTTGGT LLSAECTVFLD
ACCTGCTTTCCGCCGAATGCA HENANKILNRP
CTGTCTTTCTGGATCACGAGA KR
ATGCGAATAAGATCTTGAACC
GACCCAAACGG
SS-004 Prolactin ATGAAAGGATCATTGCTGTTG 35 MKGSLLLLLV 97
CTCCTCGTGTCGAACCTTCTG SNLLLCQSVAP
CTTTGCCAGTCCGTAGCCCCC
SS-005 Albumin ATGAAATGGGTGACGTTCATC 36 MKWVTFISLLF 98
TCACTGTTGTTTTTGTTCTCGT LFSSAYSRG
CCGCCTACTCCAGGGGAGTAT VFRR
TCCGCCGA
SS-006 HMMSP38 ATGTGGTGGCGGCTCTGGTGG 37 MWWRLWWLL 99
CTGCTCCTGTTGCTCCTCTTGC LLLLLLPMWA
TGTGGCCCATGGTGTGGGCA
MLS- ornithine TGCTCTTTAACCTCCGCATCCT 38 MLFNLRILLNN 100
001 carbamoyltr GTTGAATAACGCTGCGTTCCG AAFRNGHNFM
ansferase AAATGGGCATAACTTCATGGT VRNFRCGQPL
ACGCAACTTCAGATGCGGCCA Q
GCCACTCCAG
MLS- Cytochrome ATGTCCGTCTTGACACCCCTG 39 MSVLTPLLLRG 101
002 C Oxidase CTCTTGAGAGGGCTGACGGGG LTGSARRLPVP
subunit 8A TCCGCTAGACGCCTGCCGGTA RAKIHSL
CCGCGAGCGAAGATCCACTCC
CTG
MLS- Cytochrome ATGAGCGTGCTCACTCCGTTG 40 MSVLTPLLLRG 102
003 C Oxidase CTTCTTCGAGGGCTTACGGGA LTGSARRLPVP
subunit 8A TCGGCTCGGAGGTTGCCCGTC RAKIHSL
CCGAGAGCGAAGATCCATTCG
TTG
SS-007 Type III, TGACAAAAATAACTTTATCTC 41 MVTKITLSPQN 103
bacterial CCCAGAATTTTAGAATCCAAA FRIQKQETTLL
AACAGGAAACCACACTACTA KEKSTEKNSLA
AAAGAAAAATCAACCGAGAA KSILAVKNHFI
AAATTCTTTAGCAAAAAGTAT ELRSKLSERFIS
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TCTCGCAGTAAAAATCACTTC HKNT
ATCGAATTAAGGTCAAAATTA
TCGGAACGTTTTATTTCGCAT
AAGAACACT
SS-008 Viral ATGCTGAGCTTTGTGGATACC 42 MLSFVDTRTLL 104
CGCACCCTGCTGCTGCTGGCG LLAVTSCLATC
GTGACCAGCTGCCTGGCGACC Q
TGCCAG
SS-009 viral ATGGGCAGCAGCCAGGCGCC 43 MGS SQAPRMG 105
GCGCATGGGCAGCGTGGGCG SVGGHGLMAL
GCCATGGCCTGATGGCGCTGC LMAGLILPGIL
TGATGGCGGGCCTGATTCTGC A
CGGGCATTCTGGCG
SS-010 Viral ATGGCGGGCATTTTTTATTTTC 44 MAGIFYFLF SF 106
TGTTTAGCTTTCTGTTTGGCAT LFGICD
TTGCGAT
SS-011 Viral ATGGAAAACCGCCTGCTGCGC 45 MENRLLRVFL 107
GTGTTTCTGGTGTGGGCGGCG VWAALTMDG
CTGACCATGGATGGCGCGAGC ASA
GCG
SS-012 Viral ATGGCGCGCCAGGGCTGCTTT 46 MARQGCFGSY 108
GGCAGCTATCAGGTGATTAGC QVISLFTFAIGV
CTGTTTACCTTTGCGATTGGC NLCLG
GTGAACCTGTGCCTGGGC
SS-013 Bacillus ATGAGCCGCCTGCCGGTGCTG 47 MSRLPVLLLLQ 109
CTGCTGCTGCAGCTGCTGGTG LLVRPGLQ
CGCCCGGGCCTGCAG
SS-014 Bacillus ATGAAACAGCAGAAACGCCT 48 MKQQKRLYAR 110
GTATGCGCGCCTGCTGACCCT LLTLLFALIFLL
GCTGTTTGCGCTGATTTTTCTG PHS SASA
CTGCCGCATAGCAGCGCGAGC
GCG
SS-015 Secretion ATGGCGACGCCGCTGCCTCCG 49 MATPLPPPSPR 111
signal CCCTCCCCGCGGCACCTGCGG HLRLLRLLL SG
CTGCTGCGGCTGCTGCTCTCC
GCCCTCGTCCTCGGC
SS-016 Secretion ATGAAGGCTCCGGGTCGGCTC 50 MKAPGRLVLII 112
signal GTGCTCATCATCCTGTGCTCC LCSVVFS
GTGGTCTTCTCT
SS-017 Secretion ATGCTTCAGCTTTGGAAACTT 51 MLQLWKLLCG 113
signal GTTCTCCTGTGCGGCGTGCTC VLT
ACT
SS-018 Secretion ATGCTTTATCTCCAGGGTTGG 52 MLYLQGWSM 114
signal AGCATGCCTGCTGTGGCA PAVA
SS-019 Secretion ATGGATAACGTGCAGCCGAA 53 MDNVQPKIKH 115
signal AATAAAACATCGCCCCTTCTG RPFCFSVKGHV
CTTCAGTGTGAAAGGCCACGT KMLRLDIINSL
GAAGATGCTGCGGCTGGATAT VTTVFMLIVSV
TATCAACTCACTGGTAACAAC LALIP
AGTATTCATGCTCATCGTATC
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TGTGTTGGCACTGATACCA
SS-020 Secretion ATGCCCTGCCTAGACCAACAG 54 MPCLDQQLTV 116
signal CTCACTGTTCATGCCCTACCCT HALPCPAQPS S
GCCCTGCCCAGCCCTCCTCTC LAFCQVGFLT
TGGCCTTCTGCCAAGTGGGGT A
TCTTAACAGCA
SS-021 Secretion ATGAAAACCTTGTTCAATCCA 55 MKTLFNPAPAI 117
signal GCCCCTGCCATTGCTGACCTG ADLDPQFYTLS
GATCCCCAGTTCTACACCCTC DVFCCNESEAE
TCAGATGTGTTCTGCTGCAAT ILTGLTVGSAA
GAAAGTGAGGCTGAGATTTTA DA
ACTGGCCTCACGGTGGGCAGC
GCTGCAGATGCT
SS-022 Secretion ATGAAGCCTCTCCTTGTTGTG 56 MKPLLVVFVF 118
signal TTTGTCTTTCTTTTCCTTTGGG LFLWDPVLA
ATCCAGTGCTGGCA
SS-023 Secretion ATGTCCTGTTCCCTAAAGTTT 57 MSCSLKFTLIVI 119
signal ACTTTGATTGTAATTTTTTTTT FFTCTLS SS
ACTGTTGGCTTTCATCCAGC
SS-024 Secretion ATGGTTCTTACTAAACCTCTTC 58 MVLTKPLQRN 120
signal AAAGAAATGGCAGCATGATG GSMMSFENVK
AGCTTTGAAAATGTGAAAGAA EKSREGGPHA
AAGAGCAGAGAAGGAGGGCC HTPEEELCFVV
CCATGCACACACACCCGAAGA THTPQVQTTL
AGAATTGTGTTTCGTGGTAAC NLFFHIFKVLT
ACACTACCCTCAGGTTCAGAC QPLSLLWG
CACACTCAACCTGTTTTTCCAT
ATATTCAAGGTTCTTACTCAA
CCACTTTCCCTTCTGTGGGGT
SS-025 Secretion ATGGCCACCCCGCCATTCCGG 59 MATPPFRLIRK 121
signal CTGATAAGGAAGATGTTTTCC MFSFKVSRWM
TTCAAGGTGAGCAGATGGATG GLACFRSLAAS
GGGCTTGCCTGCTTCCGGTCC
CTGGCGGCATCC
SS-026 Secretion ATGAGCTTTTTCCAACTCCTG 60 MSFFQLLMKR 122
signal ATGAAAAGGAAGGAACTCAT KELIPLVVFMT
TCCCTTGGTGGTGTTCATGAC VAAGGASS
TGTGGCGGCGGGTGGAGCCTC
ATCT
SS-027 Secretion ATGGTCTCAGCTCTGCGGGGA 61 MVSALRGAPLI 123
signal GCACCCCTGATCAGGGTGCAC RVHS SPVS SPS
TCAAGCCCTGTTTCTTCTCCTT VSGPAALVSCL
CTGTGAGTGGACCACGGAGGC SSQSSALS
TGGTGAGCTGCCTGTCATCCC
AAAGCTCAGCTCTGAGC
SS-028 Secretion ATGATGGGGTCCCCAGTGAGT 62 MMGSPVSHLL 124
signal CATCTGCTGGCCGGCTTCTGT AGFCVWVVLG
GTGTGGGTCGTCTTGGGC
SS-029 Secretion ATGGCAAGCATGGCTGCCGTG 63 MASMAAVLT 125
signal CTCACCTGGGCTCTGGCTCTT WALALLSAFS

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CTTTCAGCGTTTTCGGCCACC ATQA
CAGGCA
SS-030 Secretion ATGGTGCTCATGTGGACCAGT 64 MVLMWTSGD 126
signal GGTGACGCCTTCAAGACGGCC AFKTAYFLLK
TACTTCCTGCTGAAGGGTGCC GAPLQFSVCGL
CCTCTGCAGTTCTCCGTGTGC LQVLVDLAILG
GGCCTGCTGCAGGTGCTGGTG QATA
GACCTGGCCATCCTGGGGCAG
GCCTACGCC
SS-031 Secretion ATGGATTTTGTCGCTGGAGCC 65 MDFVAGAIGG 127
signal ATCGGAGGCGTCTGCGGTGTT VCGVAVGYPL
GCTGTGGGCTACCCCCTGGAC DTVKVRIQTEP
ACGGTGAAGGTCAGGATCCA LYTGIWHCVR
GACGGAGCCAAAGTACACAG DTYHRERVWG
GCATCTGGCACTGCGTCCGGG FYRGLSLPVCT
ATACGTATCACCGAGAGCGCG VSLVS S
TGTGGG
GCTTCTACCGGGGCCTCTCGC
TGCCCGTGTGCACGGTGTCCC
TGGTATCTTCC
SS-032 Secretion ATGGAGAAGCCCCTCTTCCCA 66 MEKPLFPLVPL 128
signal TTAGTGCCTTTGCATTGGTTTG HWFGFGYTAL
GCTTTGGCTACACAGCACTGG VVSGGIVGYV
TTGTTTCTGGTGGGATCGTTG KTGSVPSLAA
GCTATGTAAAAACAGGCAGC GLLFGSLA
GTGCCGTCCCTGGCTGCAGGG
CTGCTCTTCGGCAGTCTAGCC
SS-033 Secretion ATGGGTCTGCTCCTTCCCCTG 67 MGLLLPLALCI 129
signal GCACTCTGCATCCTAGTCCTG LVLC
TGC
SS-034 Secretion ATGGGGATCCAGACGAGCCCC 68 MGIQTSPVLLA 130
signal GTCCTGCTGGCCTCCCTGGGG SLGVGLVTLL
GTGGGGCTGGTCACTCTGCTC GLAVG
GGCCTGGCTGTGGGC
SS-035 Secretion ATGTCGGACCTGCTACTACTG 69 MSDLLLLGLIG 131
signal GGCCTGATTGGGGGCCTGACT GLTLLLLLTLL
CTCTTACTGCTGCTGACGCTG AFA
CTAGCCTTTGCC
SS-036 Secretion ATGGAGACTGTGGTGATTGTT 70 METVVIVAIGV 132
signal GCCATAGGTGTGCTGGCCACC LATIFLAS FAA
ATGTTTCTGGCTTCGTTTGCAG LVLVCRQ
CCTTGGTGCTGGTTTGCAGGC
AG
SS-037 Secretion ATGCGCGGCTCTGTGGAGTGC 71 MAGSVECTWG 133
signal ACCTGGGGTTGGGGGCACTGT WGHCAPSPLL
GCCCCCAGCCCCCTGCTCCTT LWTLLLFAAPF
TGGACTCTACTTCTGTTTGCA GLLG
GCCCCATTTGGCCTGCTGGGG
SS-038 Secretion ATGATGCCGTCCCGTACCAAC 72 MMPSRTNLAT 134
signal CTGGCTACTGGAATCCCCAGT GIPS SKVKYSR
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AGTAAAGTGAAATATTCAAGG LSSTDDGYIDL
CTCTCCAGCACAGACGATGGC QFKKTPPKIPY
TACATTGACCTTCAGTTTAAG KAIALATVLFL
AAAACCCCTCCTAAGATCCCT IGA
TATAAGGCCATCGCACTTGCC
ACTGTGCTGTTTTTGATTGGC
GCC
SS-039 Secretion ATGGCCCTGCCCCAGATGTGT 73 MALPQMCDGS 135
signal GACGGGAGCCACTTGGCCTCC HLASTLRYCM
ACCCTCCGCTATTGCATGACA TVSGTVVLVA
GTCAGCGGCACAGTGGTTCTG GTLCFA
GTGGCCGGGACGCTCTGCTTC
GCT
SS-041 Vrg-6 TGAAAAAGTGGTTCGTTGCTG 74
MKKWFVAAGI 136
CCGGCATCGGCGCTGCCGGAC GAGLLML S SA
TCATGCTCTCCAGCGCCGCCA A
SS-042 PhoA ATGAAACAGAGCACCATTGCG 75
MKQSTIALALL 137
CTGGCGCTGCTGCCGCTGCTG PLLFTPVTKA
TTTACCCCGGTGACCAAAGCG
SS-043 OmpA ATGAAAAAAACCGCGATTGC 76 MKKTAIAIAV 138
GATTGCGGTGGCGCTGGCGGG ALAGFATVAQ
CTTTGCGACCGTGGCGCAGGC A
G
SS-044 STI ATGAAAAAACTGATGCTGGCG 77 MKKLMLAIFFS 139
ATTTTTTTTAGCGTGCTGAGCT VLSFPSFSQS
TTCCGAGCTTTAGCCAGAGC
SS-045 STII ATGAAAAAAAACATTGCGTTT 78 MKKNIAFLLAS 140
CTGCTGGCGAGCATGTTTGTG MFVFSIATNAY
TTTAGCATTGCGACCAACGCG A
TATGCG
SS-046 Amylase ATGTTTGCGAAACGCTTTAAA 79 MFAKRFKTSL 141
ACCAGCCTGCTGCCGCTGTTT LPLFAGFLLLF
GCGGGCTTTCTGCTGCTGTTTC HLVLAGPAAA
ATCTGGTGCTGGCGGGCCCGG S
CGGCGGCGAGC
SS-047 Alpha ATGCGCTTTCCGAGCATTTTT 80
MRFPSIFTAVL 142
Factor ACCGCGGTGCTGTTTGCGGCG FAAS SALA
AGCAGCGCGCTGGCG
SS-048 Alpha ATGCGCTTTCCGAGCATTTTT 81
MRFPSIFTTVL 143
Factor ACCACCGTGCTGTTTGCGGCG FAAS SALA
AGCAGCGCGCTGGCG
SS-049 Alpha ATGCGCTTTCCGAGCATTTTT 82
MRFPSIFTSVLF 144
Factor ACCAGCGTGCTGTTTGCGGCG AAS SALA
AGCAGCGCGCTGGCG
SS-050 Alpha ATGCGCTTTCCGAGCATTTTT 83
MRFPSIFTHVL 145
Factor ACCCATGTGCTGTTTGCGGCG FAAS SALA
AGCAGCGCGCTGGCG
SS-051 Alpha ATGCGCTTTCCGAGCATTTTT 84
MRFPSIFTIVLF 146
Factor ACCATTGTGCTGTTTGCGGCG AAS SALA
AGCAGCGCGCTGGCG
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SS-052 Alpha ATGCGCTTTCCGAGCATTTTT 85 MRFPSIFTFVLF 147
Factor ACCTTTGTGCTGTTTGCGGCG AASSALA
AGCAGCGCGCTGGCG
SS-053 Alpha ATGCGCTTTCCGAGCATTTTT 86 MRFPSIFTEVL 148
Factor ACCGAAGTGCTGTTTGCGGCG FAAS SALA
AGCAGCGCGCTGGCG
SS-054 Alpha ATGCGCTTTCCGAGCATTTTT 87 MRFPSIFTGVL 149
Factor ACCGGCGTGCTGTTTGCGGCG FAAS SALA
AGCAGCGCGCTGGCG
SS-055 Endoglucan ATGCGTTCCTCCCCCCTCCTCC 88 MRSSPLLRSAV 150
ase V GCTCCGCCGTTGTGGCCGCCC VAALPVLALA
TGCCGGTGTTGGCCCTTGCC
SS-056 Secretion ATGGGCGCGGCGGCCGTGCGC 89 MGAAAVRWH 151
signal TGGCACTTGTGCGTGCTGCTG LCVLLALGTR
GCCCTGGGCACACGCGGGCG GRL
GCTG
SS-057 Fungal ATGAGGAGCTCCCTTGTGCTG 90 MRSSLVLFFVS 152
TTCTTTGTCTCTGCGTGGACG AWTALA
GCCTTGGCCAG
SS-058 Fibronectin ATGCTCAGGGGTCCGGGACCC 91 MLRGPGPGRL 153
GGGCGGCTGCTGCTGCTAGCA LLLAVLCLGTS
GTCCTGTGCCTGGGGACATCG VRCTETGKSK
GTGCGCTGCACCGAAACCGGG R
AAGAGCAAGAGG
SS-059 Fibronectin ATGCTTAGGGGTCCGGGGCCC 92 MLRGPGPGLL 154
GGGCTGCTGCTGCTGGCCGTC LLAVQCLGTA
CAGCTGGGGACAGCGGTGCCC VPSTGA
TCCACG
SS-060 Fibronectin ATGCGCCGGGGGGCCCTGACC 93 MRRGALTGLL 155
GGGCTGCTCCTGGTCCTGTGC LVLCLSVVLR
CTGAGTGTTGTGCTACGTGCA AAPSATSKKR
GCCCCCTCTGCAACAAGCAAG R
AAGCGCAGG
[000285] In the table, SS is secretion signal and MLS is mitochondrial leader
signal.
The primary constructs or mmRNA of the present invention may be designed to
encode
any of the signal sequences of SEQ ID NOs 94-155, or fragments or variants
thereof
These sequences may be included at the beginning of the polypeptide coding
region, in
the middle or at the terminus or alternatively into a flanking region.
Further, any of the
polynucleotide primary constructs of the present invention may also comprise
one or
more of the sequences defined by SEQ ID NOs 32-93. These may be in the first
region
or either flanking region.
[000286] Additional signal sequences which may be utilized in the present
invention
include those taught in, for example, databases such as those found at
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http://www.signalpeptide.de/ or http://proline.bic.nus.edu.sg/spdb/. Those
described in
US Patents 8,124,379; 7,413,875 and 7,385,034 are also within the scope of the
invention
and the contents of each are incorporated herein by reference in their
entirety.
Target Selection
[000287] According to the present invention, the primary constructs comprise
at least a
first region of linked nucleosides encoding at least one polypeptide of
interest. The
polypeptides of interest or "Targets" of the present invention are listed in
Table 6. Shown
in Table 6, in addition to the name and description of the gene encoding the
polypeptide
of interest are the ENSEMBL Transcript ID (ENST), the ENSEMBL Protein ID
(ENSP)
and when available the optimized transcript sequence ID (Optim Trans SEQ ID)
or
optimized open reading frame sequence ID (Optim ORF SEQ ID). For any
particular
gene there may exist one or more variants or isoforms. Where these exist, they
are shown
in the table as well. It will be appreciated by those of skill in the art that
disclosed in the
Table are potential flanking regions. These are encoded in each ENST
transcript either to
the 5' (upstream) or 3' (downstream) of the ORF or coding region. The coding
region is
definitively and specifically disclosed by teaching the ENSP sequence.
Consequently,
the sequences taught flanking that encoding the protein are considered
flanking regions.
It is also possible to further characterize the 5' and 3' flanking regions by
utilizing one or
more available databases or algorithms. Databases have annotated the features
contained
in the flanking regions of the ENST transcripts and these are available in the
art.
Table 6. Targets
Target Target ENST Trans ENSP Peptide Optim ORF Optim
No. Description SEQ SEQ ID SEQ ID Trans
ID NO NO SEQ ID
NO
1 4- 289004 156 289004 769 1673, 2293,
hydroxyphenylpyr 2913, 3533,
uvate dioxygenase 4153,5433-
5519
2 4- 543163 157 441677 770 1674,2294,
hydroxyphenylpyr 2914, 3534,
uvate dioxygenase 4154
3 4- 545969 158 437419 771 1675,2295,
hydroxyphenylpyr 2915, 3535,
uvate dioxygenase 4155
4 6- 280362 159 280362 772 1676,2296,
1609
pyruvoyltetrahydr 2916, 3536,
opterin synthase 4156, 4773,
5103,5520-
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5606
A disintegrin and 563979 160 456186 773 1677, 2297,
1609
metalloproteinase 2917, 3537,
with 4157, 4774,
thrombospondin 5104, 5607-
motifs 13 isoform 5693
1 preproprotein
6 activity-dependent 349014 161 342905 774 1678, 2298,
neuroprotector 2918, 3538,
homeobox 4158, 4775,
5105, 5694-
5780
7 activity-dependent 371602 162 360662 775 1679, 2299,
neuroprotector 2919, 3539,
homeobox 4159, 4776,
5106
8 activity-dependent 396029 163 379346 776 1680, 2300,
neuroprotector 2920, 3540,
homeobox 4160, 4777,
5107
9 activity-dependent 396032 164 379349 777 1681, 2301,
neuroprotector 2921, 3541,
homeobox 4161, 4778,
5108
activity-dependent 534467 165 436181 778 1682, 2302,
neuroprotector 2922, 3542,
homeobox 4162, 4779,
5109
11 ADAM 338351 166 345120 779 1683,2303,
metallopeptidase 2923, 3543,
with 4163, 4780,
thrombospondin 5110
type 1 motif, 13
12 ADAM 355699 167 347927 780 1393, 1684,
metallopeptidase 2304, 2924,
with 3544, 4164,
thrombospondin 4781, 5111
type 1 motif, 13
13 ADAM 356589 168 348997 781 1394, 1685,
metallopeptidase 2305, 2925,
with 3545, 4165,
thrombospondin 4782, 5112
type 1 motif, 13
14 ADAM 371910 169 360978 782 1395, 1686,
metallopeptidase 2306, 2926,
with 3546, 4166,
thrombospondin 4783, 5113
type 1 motif, 13
ADAM 371911 170 360979 783 1396, 1687,
metallopeptidase 2307, 2927,
with 3547, 4167,
thrombospondin 4784, 5114

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type 1 motif, 13
16 ADAM 371916 171 360984 784 1397, 1688,
metallopeptidase 2308, 2928,
with 3548, 4168,
thrombospondin 4785, 5115
type 1 motif, 13
17 ADAM 371929 172 360997 785 1398, 1689, 1610
metallopeptidase 2309, 2929,
with 3549, 4169,
thrombospondin 4786, 5116
type 1 motif, 13
18 ADAM 536611 173 444504 786 1399, 1690,
metallopeptidase 2310, 2930,
with 3550, 4170,
thrombospondin 4787, 5117
type 1 motif, 13
19 AIF sh
20 Albiglutide;
albiglutide
21 aldolase A 563060 174 455800 787 1691,2311,
fructose- 2931, 3551,
bisphosphate 4171
22 aldolase A 564546 175 455917 788 1692,2312,
fructose- 2932, 3552,
bisphosphate 4172
23 aldolase A 564595 176 457468 789 1693, 2313,
fructose- 2933, 3553,
bisphosphate 4173
24 aldolase A, 338110 177 336927 790 1694, 2314,
fructose- 2934, 3554,
bisphosphate 4174
25 aldolase A, 395240 178 378661 791 1695,2315,
fructose- 2935, 3555,
bisphosphate 4175
26 aldolase A, 395248 179 378669 792 1696,2316,
fructose- 2936, 3556,
bisphosphate 4176, 5781-
5867
27 aldolase A, 412304 180 400452 793 1697, 2317,
fructose- 2937, 3557,
bisphosphate 4177
28 ameloblastin 322937 181 313809 794 1698,2318,
1611
(enamel matrix 2938, 3558,
protein) 4178, 4788,
5118, 5868-
5954
29 ameloblastin 449493 182 391234 795 1699,2319,
1611
(enamel matrix 2939, 3559,
protein) 4179, 4789,
5119
30 ameloblastin 538728 183 445605 796 1700, 2320,
1611
(enamel matrix 2940, 3560,
protein) 4180, 4790,
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5120
31 amelogenin, X- 348912 184 335312 797 1701,2321,
linked 2941, 3561,
4181, 4791,
5121
32 amelogenin, X- 380712 185 370088 798 1702,2322,
linked 2942, 3562,
4182, 4792,
5122, 5955-
6041
33 amelogenin, X- 380714 186 370090 799 1703, 2323,
linked 2943, 3563,
4183, 4793,
5123
34 amelogenin, Y- 215479 187 215479 800 1704, 2324,
1612
linked 2944, 3564,
4184
35 amelogenin, Y- 383036 188 372505 801 1705,2325,
1612
linked 2945, 3565,
4185
36 amelogenin, Y- 383037 189 372506 802 1706, 2326,
1612
linked 2946, 3566,
4186
37 amylo-alpha-1, 6- 294724 190 294724 803 1707, 2327,
glucosidase, 4- 2947, 3567,
alpha- 4187
glucanotransferase
38 amylo-alpha-1, 6- 361302 191 354971 804 1708,2328,
glucosidase, 4- 2948, 3568,
alpha- 4188
glucanotransferase
39 amylo-alpha-1, 6- 361522 192 354635 805 1709,2329,
glucosidase, 4- 2949, 3569,
alpha- 4189
glucanotransferase
40 amylo-alpha-1, 6- 361915 193 355106 806 1710,2330,
glucosidase, 4- 2950, 3570,
alpha- 4190, 6042-
glucanotransferase 6128
41 amylo-alpha-1, 6- 370161 194 359180 807 1711,2331,
glucosidase, 4- 2951, 3571,
alpha- 4191
glucanotransferase
42 amylo-alpha-1, 6- 370163 195 359182 808 1712,2332,
glucosidase, 4- 2952, 3572,
alpha- 4192
glucanotransferase
43 amylo-alpha-1, 6- 370165 196 359184 809 1713,2333,
glucosidase, 4- 2953, 3573,
alpha- 4193
glucanotransferase
44 amyloid P 255040 197 255040 810 1714,2334,
component, serum 2954, 3574,
87

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4194, 6129-
6215
45 angiotensin I 252519 198 252519 811 1715,2335,
converting enzyme 2955, 3575,
(peptidyl- 4195, 6216-
dipeptidase A) 2 6302
46 angiotensin I 427411 199 389326 812 1716, 2336,
converting enzyme 2956, 3576,
(peptidyl- 4196
dipeptidase A) 2
47 Anti-TNFalpha
(TNFRFusion;
Enbrel)
48 AP0A1 Milano; 813 1400, 1717, 1613
apo A- 2337, 2957,
I(R173C)Milano , 3577, 4197,
ETC-216 4794, 5124
49 AP0A1 Paris; apo 814 1401, 1718,
A-I(R151C)Paris 2338, 2958,
3578, 4198,
4795, 5125
50 ApoA-a optimized 1614
mRNA
51 apolipoprotein A-I 236850 200 236850 815 1402,
1719, 1615
2339, 2959,
3579, 4199,
4796, 5126,
6303-5363
52 apolipoprotein A-I 359492 201 352471 816 1720,
2340, 1615
2960, 3580,
4200, 4797,
5127
53 apolipoprotein A-I 375320 202 364469 817 1721,
2341, 1615
2961, 3581,
4201, 4798,
5128
54 apolipoprotein A-I 375323 203 364472 818 1722,
2342, 1615
2962, 3582,
4202, 4799,
5129
55 apolipoprotein B 233242 204 233242 819 1723, 2343,
(including Ag(x) 2963, 3583,
antigen) 4203
56 apolipoprotein B 535079 205 439731 820 1724,2344,
(including Ag(x) 2964, 3584,
antigen) 4204
57 aquaporin 5 293599 206 293599 821 1725,2345, 1616
2965,3585,
4205,4800,
5130,6564-
6650
58 arginase, liver 356962 207 349446 822 1726,2346,
2966,3586,
4206,
88

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59 arginase, liver 368087 208 357066 823 1727,2347,
2967,3587,
4207,6651-
6737
60 arginase, liver 476845 209 417694 824 1728, 2348,
2968, 3588,
4208
61 argininosuccinate 304874 210 307188 825 1729, 2349,
lyase 2969, 3589,
4209, 6738-
6876
62 argininosuccinate 380839 211 370219 826 1730, 2350,
lyase 2970, 3590,
4210
63 argininosuccinate 395331 212 378740 827 1731, 2351,
lyase 2971, 3591,
4211
64 argininosuccinate 395332 213 378741 828 1732, 2352,
lyase 2972, 3592,
4212
65 argininosuccinate 502022 214 441778 829 1733, 2353,
lyase 2973, 3593,
4213
66 argininosuccinate 334909 215 361470 830 1403, 1734,
synthase 1 2354, 2974,
3594, 4214
67 argininosuccinate 352480 216 253004 831 1735, 2355,
synthase 1 2975, 3595,
4215, 6877-
6963
68 argininosuccinate 372386 217 361461 832 1404, 1736,
synthase 1 2356, 2976,
3596, 4216
69 argininosuccinate 372393 218 361469 833 1737, 2357,
synthase 1 2977, 3597,
4217
70 argininosuccinate 372394 219 361471 834 1738, 2358,
synthase 1 2978, 3598,
4218
71 argininosuccinate 422569 220 394212 835 1405, 1739,
synthase 1 2359, 2979,
3599, 4219
72 argininosuccinate 443588 221 397785 836 1406, 1740,
synthase 1 2360, 2980,
3600, 4220
73 artemin 372354 222 361429 837 1741, 2361,
2981, 3601,
4221, 4801,
5131, 6964-
7050
74 artemin 372359 223 361434 838 1742, 2362,
2982, 3602,
4222, 4802,
5132
89

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75 artemin 414809 224 387435 839 1743, 2363,
2983, 3603,
4223, 4803,
5133
76 artemin 471394 225 435804 840 1744, 2364,
2984, 3604,
4224, 4804,
5134
77 artemin 474592 226 434856 841 1745, 2365,
2985, 3605,
4225, 4805,
5135
78 artemin 477048 227 434784 842 1746, 2366,
2986, 3606,
4226, 4806,
5136
79 artemin 491846 228 436149 843 1747, 2367,
2987, 3607,
4227, 4807,
5137
80 artemin 498139 229 436727 844 1748, 2368,
2988, 3608,
4228, 4808,
5138
81 arylsulfatase B 264914 230 264914 845 1749,2369,
1617
2989,3609,
4229,4809,
5139,7051-
7137
82 arylsulfatase B 264914 231 264914 846 1750, 2370,
1617
2990, 3610,
4230, 4810,
5140
83 arylsulfatase B 396151 232 379455 847 1751,2371,
1617
2991,3611,
4231,4811,
5141
84 arylsulfatase B 521117 233 428611 848 1752,2372,
1617
2992,3612,
4232,4812,
5142
85 asparaginase 299234 234 299234 849 1753, 2373,
homolog (S. 2993, 3613,
cerevisiae) 4233, 4813,
5143
86 asparaginase 455920 235 389003 850 1754, 2374,
homolog (S. 2994, 3614,
cerevisiae) 4234, 4814,
5144
87 asparaginase 550583 236 446856 851 1755, 2375,
homolog (S. 2995, 3615,
cerevisiae) 4235, 4815,
5145

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88 asparaginase 551177 237 450040 852 1756, 2376,
homolog (S. 2996, 3616,
cerevisiae) 4236, 4816,
5146
89 aspartoacylase 263080 238 263080 853 1757, 2377,
2997, 3617,
4237
90 aspartoacylase 456349 239 409976 854 1758, 2378,
2998, 3618,
4238, 7138-
7224
91 ATPase, 283684 240 283684 855 1407, 1759,
aminophospholipi 2379, 2999,
d transporter, class 3619, 4239,
I, type 8B, 7225-7398
member 1
92 ATPase, 536015 241 445359 856 1760, 2380,
aminophospholipi 3000, 3620,
d transporter, class 4240
I, type 8B,
member 1
93 ATPase, Cu++ 242839 242 242839 857 1761,2381,
1618
transporting, beta 3001, 3621,
polypeptide 4241, 4817,
5147, 7399-
7485
94 ATPase, Cu++ 344297 243 342559 858 1762,2382,
1618
transporting, beta 3002, 3622,
polypeptide 4242, 4818,
5148
95 ATPase, Cu++ 400366 244 383217 859 1763,2383,
1618
transporting, beta 3003, 3623,
polypeptide 4243, 4819,
5149
96 ATPase, Cu++ 400370 245 383221 860 1764,2384,
1618
transporting, beta 3004, 3624,
polypeptide 4244, 4820,
5150
97 ATPase, Cu++ 417240 246 390360 861 1765,2385,
transporting, beta 3005, 3625,
polypeptide 4245, 4821,
5151
98 ATPase, Cu++ 418097 247 393343 862 1766,2386,
1618
transporting, beta 3006, 3626,
polypeptide 4246, 4822,
5152
99 ATPase, Cu++ 448424 248 416738 863 1767,2387,
1618
transporting, beta 3007, 3627,
polypeptide 4247, 4823,
5153
100 ATPase, Cu++ 542656 249 443128 864 1768, 2388,
transporting, beta 3008, 3628,
polypeptide 4248, 4824,
5154
91

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101 ATP-binding 263817 250 263817 865 1408, 1769,
cassette, sub- 2389, 3009,
family B 3629, 4249
(MDR/TAP),
member 11
102 ATP-binding 265723 251 265723 866 1409, 1770,
cassette, sub- 2390, 3010,
family B 3630, 4250,
(MDR/TAP), 7486-7659
member 4
103 ATP-binding 358400 252 351172 867 1410, 1771,
cassette, sub- 2391, 3011,
family B 3631, 4251
(MDR/TAP),
member 4
104 ATP-binding 359206 253 352135 868 1411, 1772,
cassette, sub- 2392, 3012,
family B 3632, 4252
(MDR/TAP),
member 4
105 ATP-binding 417608 254 394511 869 1412, 1773,
cassette, sub- 2393, 3013,
family B 3633, 4253
(MDR/TAP),
member 4
106 ATP-binding 453593 255 392983 870 1774, 2394,
cassette, sub- 3014, 3634,
family B 4254
(MDR/TAP),
member 4
107 ATP-binding 545634 256 437465 871 1775, 2395,
cassette, sub- 3015, 3635,
family B 4255
(MDR/TAP),
member 4
108 bactericidal/perme 262865 257 262865 872 1776, 2396,
ability-increasing 3016, 3636,
protein 4256, 7660-
7746
109 basic helix-loop- 242728 258 242728 873 1413, 1777,
helix family, 2397, 3017,
member e41 3637, 4257,
7747-7833
110 basic helix-loop- 540731 259 437369 874 1778, 2398,
helix family, 3018, 3638,
member e41 4258
111 bone 378827 260 368104 875 1414, 1779,
morphogenetic 2399, 3019,
protein 2 3639, 4259,
7834-7920
112 bone 395863 261 379204 876 1415, 1780,
morphogenetic 2400, 3020,
protein 7 3640, 4260,
4825, 5155,
7921-8007
92

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113 bone 395864 262 379205 877 1416, 1781,
morphogenetic 2401, 3021,
protein 7 3641, 4261,
4826, 5156
114 Cap18
115 carbamoyl- 233072 263 233072 878 1417,1782,
phosphate 2402,3022,
synthase 1, 3642,4262
mitochondrial
116 carbamoyl- 417946 264 388496 879 1418, 1783,
phosphate 2403, 3023,
synthase 1, 3643, 4263
mitochondrial
117 carbamoyl- 430249 265 402608 880 1419, 1784,
phosphate 2404, 3024,
synthase 1, 3644, 4264,
mitochondrial 8008-8146
118 carbamoyl- 451903 266 406136 881 1420, 1785,
phosphate 2405, 3025,
synthase 1, 3645, 4265
mitochondrial
119 carbamoyl- 518043 267 430697 882 1786, 2406,
phosphate 3026, 3646,
synthase 1, 4266,
mitochondrial
120 carbamoyl- 536125 268 445539 883 1787,2407,
phosphate 3027,3647,
synthase 1, 4267
mitochondrial
121 carbamoyl- 539150 269 444139 884 1788, 2408,
phosphate 3028, 3648,
synthase 1, 4268
mitochondrial
122 carbamoyl- 544169 270 442790 885 1789,2409,
phosphate 3029,3649,
synthase 1, 4269
mitochondrial
123 cardiotrophin 1 279804 271 279804 886 1790, 2410,
3030, 3650,
4270, 8147-
8233
124 cardiotrophin 1 395019 272 378465 887 1791,2411,
3031,3651,
4271
125 cathelicidin 296435 273 296435 888 1792,2412,
1619
antimicrobial 3032, 3652,
peptide 4272, 4827,
5157, 8234-
8407
126 cathelicidin 576243 274 458149 889 1793,2413,
antimicrobial 3033, 3653,
peptide 4273, 4828,
5158
93

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127 Cbp/p300- 372638 275 361721 890 1421, 1794,
interacting 2414, 3034,
transactivator, 3654, 4274,
with Glu/Asp-rich 8408-8494
carboxy-terminal
domain, 4
128 ceruloplasmin 264613 276 264613 891 1422, 1795,
1620
(ferroxidase) 2415, 3035,
3655, 4275,
4829, 5159,
8495-8581
129 Cheetah Program
2; hyaluronidase
(human); insulin
lispro
130 ciliary 361987 277 355370 892 1796, 2416,
neurotrophic 3036, 3656,
factor 4276
131 coagulation factor 311907 278 308541 893 1423, 1797,
II (thrombin) 2417, 3037,
3657, 4277,
4830, 5160,
8582-8668
132 coagulation factor 446804 279 406403 894 1798, 2418,
II (thrombin) 3038, 3658,
4278, 4831,
5161
133 coagulation factor 530231 280 433907 895 1424, 1799,
II (thrombin) 2419, 3039,
3659, 4279,
4832, 5162
134 coagulation factor 334047 281 334145 896 1425, 1800,
III 2420, 3040,
(thromboplastin, 3660, 4280,
tissue factor) 8669-8755
135 coagulation factor 370207 282 359226 897 1426, 1801,
III 2421, 3041,
(thromboplastin, 3661, 4281,
tissue factor) 8756-8842
136 coagulation factor 218099 283 218099 898 1427,
1802, 1621
IX 2422, 3042,
3662, 4282,
8843-9016
137 coagulation factor 394090 284 377650 899 1428,
1803, 1621
IX 2423, 3043,
3663, 4283,
4833, 5163
138 coagulation factor 367796 285 356770 900 1429, 1804,
V (proaccelerin, 2424, 3044,
labile factor) 3664, 4284
139 coagulation factor 367797 286 356771 901 1430, 1805,
V (proaccelerin, 2425, 3045,
labile factor) 3665, 4285,
9017-9155
94

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140 coagulation factor 546081 287 439664 902 1431, 1806,
V (proaccelerin, 2426, 3046,
labile factor) 3666, 4286
141 coagulation factor 346342 288 329546 903 1432,
1807, 1622
VII (serum 2427, 3047,
prothrombin 3667, 4287,
conversion 4834, 5164
accelerator)
142 coagulation factor 375581 289 364731 904 1433,
1808, 1622
VII (serum 2428, 3048,
prothrombin 3668, 4288,
conversion 4835, 5165,
accelerator) 9156-9242
143 coagulation factor 541084 290 442051 905 1434,
1809, 1622
VII (serum 2429, 3049,
prothrombin 3669, 4289,
conversion 4836, 5166
accelerator)
144 coagulation factor 330287 291 327895 906 1810, 2430,
VIII procoagulant 3050, 3670,
component 4290, 4837,
5167
145 coagulation factor 360256 292 353393 907 1435,
1811, 1623
VIII, procoagulant 2431, 3051,
component 3671, 4291,
4838, 5168
146 coagulation factor 375551 293 364701 908 1436, 1812,
X 2432, 3052,
3672, 4292,
4839, 5169
147 coagulation factor 375559 294 364709 909 1437, 1813,
X 2433, 3053,
3673, 4293,
4840, 5170,
9243-9381
148 coagulation factor 409306 295 387092 910 1438, 1814,
X 2434, 3054,
3674, 4294,
4841, 5171
149 coagulation factor 264692 296 264692 911 1439,
1815, 1624
XI 2435, 3055,
3675, 4295,
4842, 5172
150 coagulation factor 403665 297 384957 912 1440,
1816, 1624
XI 2436, 3056,
3676, 4296,
4843, 5173,
9382-9468
151 coagulation factor 452239 298 397401 913 1441, 1817,
XI 2437, 3057,
3677, 4297,
4844, 5174,
152 coagulation factor 264870 299 264870 914 1442, 1818,
XIII, Al 2438, 3058,
polypeptide 3678, 4298,

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9469-9555
153 coagulation factor 414279 300 413334 915 1443, 1819,
XIII, Al 2439, 3059,
polypeptide 3679, 4299
154 coagulation factor 441301 301 416127 916 1820, 2440,
XIII, Al 3060, 3680,
polypeptide 4300
155 collagen, type VII, 328333 302 332371 917
1821,2441, 1625
alpha 1 3061,3681,
4301, 4845,
5175, 9556-
9642
156 collagen, type VII, 454817 303 412569 918 1822, 2442,
alpha 1 3062, 3682,
4302, 4846,
5176,
157 colony stimulating 296871 304 296871 919 1823,
2443, 1626
factor 2 3063, 3683,
(granulocyte- 4303, 4847,
macrophage) 5177, 9643-
9781
158 colony stimulating 225474 305 225474 920 1824, 2444,
1627
factor 3 3064, 3684,
(granulocyte) 4304, 9782-
9868
159 colony stimulating 331769 306 327766 921 1825, 2445,
factor 3 3065, 3685,
(granulocyte) 4305, 4848,
5178, 9869-
9955
160 colony stimulating 394148 307 377704 922 1826,
2446, 1627
factor 3 3066, 3686,
(granulocyte) 4306, 4849,
5179,
161 colony stimulating 394149 308 377705 923 1827,
2447, 1627
factor 3 3067, 3687,
(granulocyte) 4307, 4850,
5180, 9956-
10042
162 complement factor 367429 309 356399 924 1444, 1828,
1627
H 2448, 3068,
3688, 4308,
4851, 5181
163 complement factor 391986 310 375846 925 1829, 2449,
H 3069, 3689,
4309, 4852,
5182
164 complement factor 439155 311 402656 926 1445, 1830,
H 2450, 3070,
3690, 4310,
4853, 5183
165 copper metabolism 311832 312 308236 927 1446, 1831,
(Murrl) domain 2451, 3071,
96

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containing 1 3691, 4311,
10043-10129
166 copper metabolism 427417 313 413207 928 1447, 1832,
(Murrl) domain 2452, 3072,
containing 1 3692, 4312
167 copper metabolism 444166 314 410050 929 1448, 1833,
(Murrl) domain 2453, 3073,
containing 1 3693, 4313
168 copper metabolism 458337 315 401236 930 1834, 2454,
(Murrl) domain 3074, 3694,
containing 1 4314
169 copper metabolism 538736 316 438961 931 1449, 1835,
(Murrl) domain 2455, 3075,
containing 1 3695, 4315
170 corticotropin 276571 317 276571 932 1836, 2456,
releasing hormone 3076, 3696,
4316, 4854,
5184
171 CTLA4-Ig
(Orencia)
172 cystic fibrosis 3084 318 3084 933 1450, 1837, 1628
transmembrane 2457, 3077,
conductance 3697, 4317,
regulator (ATP- 4855, 5185,
binding cassette 10130-10216
sub-family C,
member 7)
173 cystic fibrosis 426809 319 389119 934 1451, 1838,
transmembrane 2458, 3078,
conductance 3698, 4318,
regulator (ATP- 4856, 5186
binding cassette
sub-family C,
member 7)
174 cystic fibrosis 454343 320 403677 935 1452, 1839,
1628
transmembrane 2459, 3079,
conductance 3699, 4319,
regulator (ATP- 4857, 5187
binding cassette
sub-family C,
member 7)
175 cystic fibrosis 468795 321 419254 936 1453, 1840,
transmembrane 2460, 3080,
conductance 3700, 4320,
regulator (ATP- 4858, 5188
binding cassette
sub-family C,
member 7)
176 cystinosin 381870 322 371294 937 1841,2461,
lysosomal cystine 3081, 3701,
transporter 4321
97

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177 cystinosin, 46640 323 46640 938 1454, 1842,
lysosomal cystine 2462, 3082,
transporter 3702, 4322,
4859, 5189,
10217-10355
178 cystinosin, 414524 324 395471 939 1455, 1843,
lysosomal cystine 2463, 3083,
transporter 3703, 4323
179 cystinosin, 441220 325 411465 940 1456, 1844,
lysosomal cystine 2464, 3084,
transporter 3704, 4324
180 cystinosin, 452111 326 408652 941 1457, 1845,
lysosomal cystine 2465, 3085,
transporter 3705, 4325
181 defensin, beta 314357 327 320951 942 1846,2466,
1629
103A 3086,3706,
4326,4860,
5190
182 defensin, beta 318124 328 324633 943 1847,2467,
1630
103B 3087,3707,
4327,4861,
5191,10356-
10442
183 defensin, beta 4A 302247 329 303532 944 1848,
2468, 1631
3088, 3708,
4328, 4862,
5192, 10443-
10529
184 defensin, beta 4B 318157 330 424598 945 1849,
2469, 1632
3089, 3709,
4329, 4863,
5193
185 DegludecPlus; insulin aspart; insulin
degludec
186 deoxyribonuclease 246949 331 246949 946 1850, 2470,
1633
3090, 3710,
4330, 4864,
5194, 10530-
10616
187 deoxyribonuclease 407479 332 385905 947 1851,
2471, 1633
3091, 3711,
4331, 4865,
5195
188 deoxyribonuclease 414110 333 416699 948 1852, 2472,
3092, 3712,
4332, 4866,
5196
189 dysferlin, limb 258104 334 258104 949 1458, 1853,
girdle muscular 2473, 3093,
dystrophy 2B 3713, 4333,
(autosomal 4867, 5197
recessive)
98

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190 dysferlin, limb 394120 335 377678 950 1459, 1854,
1634
girdle muscular 2474, 3094,
dystrophy 2B 3714, 4334,
(autosomal 4868, 5198,
recessive) 10617-10703
191 dysferlin, limb 409366 336 386512 951 1460, 1855,
girdle muscular 2475, 3095,
dystrophy 2B 3715,4335,
(autosomal 4869, 5199
recessive)
192 dysferlin, limb 409582 337 386547 952 1461, 1856,
girdle muscular 2476, 3096,
dystrophy 2B 3716, 4336,
(autosomal 4870, 5200
recessive)
193 dysferlin, limb 409651 338 386683 953 1462, 1857,
girdle muscular 2477, 3097,
dystrophy 2B 3717, 4337,
(autosomal 4871, 5201
recessive)
194 dysferlin, limb 409744 339 386285 954 1463, 1858,
girdle muscular 2478, 3098,
dystrophy 2B 3718, 4338,
(autosomal 4872, 5202
recessive)
195 dysferlin, limb 409762 340 387137 955 1464, 1859,
girdle muscular 2479, 3099,
dystrophy 2B 3719, 4339,
(autosomal 4873, 5203
recessive)
196 dysferlin, limb 410020 341 386881 956 1465,1860,
girdle muscular 2480, 3100,
dystrophy 2B 3720, 4340,
(autosomal 4874, 5204
recessive)
197 dysferlin, limb 410041 342 386617 957 1466,1861,
girdle muscular 2481, 3101,
dystrophy 2B 3721, 4341,
(autosomal 4875, 5205
recessive)
198 dysferlin, limb 413539 343 407046 958 1467, 1862,
girdle muscular 2482, 3102,
dystrophy 2B 3722, 4342,
(autosomal 4876, 5206
recessive)
199 dysferlin, limb 429174 344 398305 959 1468, 1863,
girdle muscular 2483, 3103,
dystrophy 2B 3723, 4343,
(autosomal 4877, 5207
recessive)
200 ectodysplasin A 338901 345 340611 960 1864, 2484,
3104, 3724,
4344, 4878,
5208
99

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201 ectodysplasin A 374552 346 363680 961 1865, 2485,
3105, 3725,
4345, 4879,
5209
202 ectodysplasin A 374553 347 363681 962 1866, 2486,
1635
3106, 3726,
4346, 4880,
5210, 10704-
10790
203 ectodysplasin A 525810 348 434195 963 1867, 2487,
3107, 3727,
4347, 4881,
5211
204 ectodysplasin A 524573 349 432585 964 1868, 2488,
3108, 3728,
4348, 4882,
5212
205 ectodysplasin A 527388 350 434861 965 1869, 2489,
3109, 3729,
4349, 4883,
5213
206 enamelin 396073 351 379383 966 1870, 2490,
1636
3110, 3730,
4350, 4884,
5214
207 erythropoietin 252723 352 252723 967 1871,2491,
1637
3111,3731,
4351,4885,
5215,10791-
11051
208 Exenafide 968 1872,2492,
3112,3732,
4352,4886,
5216
209 family with 252530 353 252530 969 1873,2493,
sequence 3113,3733,
similarity 98, 4353
member C
210 family with 343358 354 340348 970 1874,2494,
sequence 3114,3734,
similarity 98, 4354
member C
211 fibrinogen alpha 302053 355 306361 971 1469, 1875,
chain 2495,3115,
3735, 4355,
11052-11190
212 fibrinogen alpha 403106 356 385981 972 1470, 1876,
chain 2496,3116,
3736, 4356
213 fibrinogen alpha 457487 357 407891 973 1877, 2497,
chain 3117, 3737,
4357
214 fibrinogen beta 302068 358 306099 974 1471,1878,
chain 2498,3118,
3738,4358,
100

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11191-11329
215 fibrinogen beta 537843 359 437727 975 1879,
2499,
chain 3119, 3739,
4359
216 fibrinogen gamma 336098 360 336829 976 1472,
1880,
chain 2500,3120,
3740, 4360,
11330-11468
217 fibrinogen gamma 404648 361 384860 977 1473,
1881,
chain 2501,3121,
3741, 4361
218 fibroblast growth 222157 362 222157 978 1474,
1882,
factor 21 2502, 3122,
3742, 4362
219 fibroblast growth 593756 363 471477 979 1883,2503,
factor 21 3123,3743,
4363, 11469-
11555
220 fibronectin type III 373470 364 362569 980 1884,
2504,
domain containing 3124, 3744,
4364,
221 fibronectin type III 373471 365 362570 981 1885,2505,
domain containing 3125, 3745,
5 4365, 11556-
11642
222 follicle stimulating 254122 366 254122 982 1886,
2506,
hormone, beta 3126, 3746,
polypeptide 4366, 4887,
5217, 11643-
11729
223 follicle stimulating 417547 367 416606 983 1887,
2507,
hormone, beta 3127, 3747,
polypeptide 4367, 4888,
5218
224 follicle stimulating 533718 368 433424 984 1888,2508,
hormone, beta 3128, 3748,
polypeptide 4368, 4889,
5219
225 fumarylacetoacetat 261755 369 261755 985 1475,
1889,
e hydrolase 2509, 3129,
(fumarylacetoaceta 3749, 4369
se)
226 fumarylacetoacetat 407106 370 385080 986 1890,
2510,
e hydrolase 3130, 3750,
(fumarylacetoaceta 4370, 11730-
se) 11868
227 fumarylacetoacetat 561421 371 453347 987 1891,
2511,
e hydrolase 3131,3751,
(fumarylacetoaceta 4371
se)
228 galactokinase 1 225614 372 225614 988 1892,2512,
3132,3752,
4372
101

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229 galactokinase 1 375188 373 364334 989 1893,2513,
3133,3753,
4373
230 galactokinase 1 437911 374 406305 990 1894,2514,
3134,3754,
4374
231 galactokinase 1 588479 375 465930 991 1895,
2515,
3135, 3755,
4375, 11869-
11955
232 galactosamine (N- 268695 376 268695 992 1476,
1896, 1638
acetyl)-6-sulfate 2516, 3136,
sulfatase 3756, 4376,
4890, 5220,
11956-12042
233 galactosamine (N- 439266 377 402127 993 1897,
2517,
acetyl)-6-sulfate 3137, 3757,
sulfatase 4377, 4891,
5221
234 galactosamine (N- 542788 378 438197 994 1477,
1898,
acetyl)-6-sulfate 2518, 3138,
sulfatase 3758, 4378,
4892, 5222
235 galactose-1- 378842 379 368119 995 1899,2519,
phosphate 3139,3759,
uridylyltransferase 4379, 12043-
12181
236 galactose-1- 450095 380 401956 996 1900,
2520,
phosphate 3140, 3760,
uridylyltransferase 4380
237 galactose-1- 554550 381 451435 997 1901,2521,
phosphate 3141,3761,
uridylyltransferase 4381
238 galactosidase, 218516 382 218516 998 1902,
2522, 1639
alpha 3142, 3762,
4382, 12182-
12268
239 Galectin 3 383 999 1640
inhibitor (A9)
240 Galectin 3 384 1000 1641
inhibitor (C12)
241 glial cell derived 326524 385 317145 1001 1903,
2523,
neurotrophic 3143, 3763,
factor 4383, 4893,
5223
242 glial cell derived 344622 386 339703 1002 1904,
2524,
neurotrophic 3144, 3764,
factor 4384, 4894,
5224
243 glial cell derived 427982 387 409007 1003 1905,
2525, 1642
neurotrophic 3145, 3765,
factor 4385, 4895,
5225, 12269-
12355
102

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244 glial cell derived 502572 388 423557 1004
1906,2526,
neurotrophic 3146, 3766,
factor 4386, 4896,
5226
245 glial cell derived 510177 389 424592 1005 1907,
2527,
neurotrophic 3147, 3767,
factor 4387, 4897,
5227
246 glial cell derived 515058 390 425928 1006 1908,
2528,
neurotrophic 3148, 3768,
factor 4388, 4898,
5228
247 glial cell derived 381826 391 371248 1007 1909,
2529,
neurotrophic 3149, 3769,
factor 4389, 4899,
5229
248 GLP-1 Fc;
dulaglutide
249 glucagon 375497 392 364647 1008 1910, 2530,
1643
3150, 3770,
4390, 4900,
5230
250 glucagon 418842 393 387662 1009 1911, 2531,
1643
3151, 3771,
4391, 4901,
5231, 12356-
12703
251 glucan (1,4-alpha- 264326 394 264326 1010
1912,2532,
), branching 3152, 3772,
enzyme 1 4392
252 glucan (1,4-alpha- 429644 395 410833 1011 1478,
1913,
), branching 2533, 3153,
enzyme 1 3773, 4393,
12704-12842
253 glucan (1,4-alpha- 536832 396 445365 1012
1914,2534,
), branching 3154, 3774,
enzyme 1 4394
254 glucose-6- 253801 397 253801 1013 1915, 2535,
phosphatase, 3155, 3775,
catalytic subunit 4395, 12843-
12981
255 glucosidase, alpha; 302262 398 305692 1014 1916,
2536, 1644
acid 3156, 3776,
4396, 4902,
5232, 12982-
13155
256 glucosidase, alpha; 390015 399 374665 1015 1917,
2537, 1644
acid 3157, 3777,
4397, 4903,
5233
257 glucosidase, beta, 327247 400 314508 1016
1918,2538, 1645
acid 3158, 3778,
4398, 4904,
5234
103

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258 glucosidase, beta, 368373 401 357357 1017 1919,2539,
1645
acid 3159, 3779,
4399, 4905,
5235
259 glucosidase, beta, 402928 402 385813 1018 1920,
2540, 1645
acid 3160, 3780,
4400, 4906,
5236
260 glucosidase, beta, 427500 403 402577 1019 1921,2541,
1645
acid 3161, 3781,
4401, 4907,
5237
261 glucosidase, beta, 428024 404 397986 1020 1922,
2542, 1645
acid 3162, 3782,
4402, 4908,
5238
262 glucosidase, beta, 536555 405 446457 1021 1923,2543,
1645
acid 3163, 3783,
4403, 4909,
5239
263 glucosidase, beta, 536770 406 445560 1022 1924,
2544, 1645
acid 3164, 3784,
4404, 4910,
5240
264 glycogen synthase 261195 407 261195 1023 1925,
2545,
2 (liver) 3165, 3785,
4405, 13156-
13242
265 glycoprotein 369582 408 358595 1024 1926,2546,
1646
hormones, alpha 3166, 3786,
polypeptide 4406, 4911,
5241, 13243-
13329
266 GP2013;
rituximab
267 Granulysin 9 kDa 1479
(non-secreted)
268 Granulysin 9 kDa 1480
(secreted)
269 growth hormone 1 323322 409 312673 1025 1927,2547,
1647
3167, 3787,
4407, 4912,
5242, 13330-
13416
270 growth hormone 1 342364 410 339278 1026 1928,2548,
1647
3168, 3788,
4408, 4913,
5243
271 growth hormone 1 351388 411 343791 1027 1929,2549,
1647
3169, 3789,
4409, 4914,
5244
272 growth hormone 1 458650 412 408486 1028 1930,2550,
1647
3170, 3790,
4410, 4915,
104

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5245
273 growthhormone 2 332800 413 333157 1029 1931,2551,
3171,3791,
4411,4916,
5246
274 growth hormone 2 423893 414 409294 1030 1932,2552,
3172,3792,
4412,4917,
5247
275 growthhormone 2 449787 415 410618 1031 1933,2553,
3173,3793,
4413,4918,
5248
276 growthhormone 2 456543 416 394122 1032 1934,2554,
3174,3794,
4414,4919,
5249
277 (111) 395514 417 378890 1033 1935,2555, 1648
cyclohydrolase 1 3175,3795,
4415,4920,
5250,13417-
13503
278 (111) 395524 418 378895 1034 1936,2556,
cyclohydrolase 1 3176,3796,
4416,4921,
5251
279 (111) 491895 419 419045 1035 1937,2557, 1649
cyclohydrolase 1 3177,3797,
4417,4922,
5252
280 (111) 536224 420 445246 1036 1938,2558,
cyclohydrolase 1 3178,3798,
4418,4923,
5253
281 (111) 543643 421 444011 1037 1939,2559,
cyclohydrolase 1 3179,3799,
4419,4924,
5254
282 hemochromatosis 309234 422 311698 1038 1940,2560,
3180,3800,
4420
283 hemochromatosis 317896 423 313776 1039 1941,2561,
3181,3801,
4421
284 hemochromatosis 336625 424 337819 1040 1942,2562,
3182,3802,
4422
285 hemochromatosis 349999 425 259699 1041 1943,2563,
3183,3803,
4423
286 hemochromatosis 352392 426 315936 1042 1944,2564,
3184,3804,
4424
105

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287 hemochromatosis 353147 427 312342 1043 1945,
2565,
3185, 3805,
4425
288 hemochromatosis 357618 428 417404 1044 1946,
2566,
3186, 3806,
4426
289 hemochromatosis 397022 429 380217 1045 1947,
2567,
3187, 3807,
4427
290 hemochromatosis 461397 430 420802 1046 1948,
2568,
3188, 3808,
4428
291 hemochromatosis 470149 431 419725 1047 1949,
2569,
3189, 3809,
4429
292 hemochromatosis 488199 432 420559 1048 1950,
2570,
3190, 3810,
4430
293 hemochromatosis 535098 433 445872 1049 1951,
2571,
3191, 3811,
4431
294 hemochromatosis 539147 434 445098 1050 1952,
2572,
3192, 3812,
4432
295 hemochromatosis 336751 435 337014 1051 1481,
1953,
type 2 (juvenile) 2573, 3193,
3813, 4433,
4925, 5255,
13504-13590
296 hemochromatosis 357836 436 350495 1052 1482,
1954,
type 2 (juvenile) 2574, 3194,
3814, 4434,
4926, 5256
297 hemochromatosis 421822 437 411863 1053 1483,
1955,
type 2 (juvenile) 2575, 3195,
3815, 4435,
4927, 5257
298 hemochromatosis 475797 438 425716 1054 1484,
1956,
type 2 (juvenile) 2576, 3196,
3816, 4436,
4928, 5258
299 hemochromatosis 497365 439 421820 1055 1957,
2577,
type 2 (juvenile) 3197, 3817,
4437, 4929,
5259
300 hemochromatosis 577520 440 463276 1056 1958,
2578,
type 2 (juvenile) 3198, 3818,
4438
301 hemochromatosis 580693 441 464413 1057 1959,
2579,
type 2 (juvenile) 3199, 3819,
4439
302 hepatocyte growth 222390 442 222390 1058 1960,
2580,
factor (hepapoietin 3200, 3820,
A; scatter factor) 4440, 13591-
13677
106

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303 hepatocyte growth 354224 443 346164 1059 1961,
2581,
factor (hepapoietin 3201, 3821,
A; scatter factor) 4441
304 hepatocyte growth 394769 444 378250 1060 1962,
2582,
factor (hepapoietin 3202, 3822,
A; scatter factor) 4442
305 hepatocyte growth 412881 445 396307 1061 1963,
2583,
factor (hepapoietin 3203, 3823,
A; scatter factor) 4443
306 hepatocyte growth 421558 446 388592 1062 1964,
2584,
factor (hepapoietin 3204, 3824,
A; scatter factor) 4444
307 hepatocyte growth 423064 447 413829 1063 1965,
2585,
factor (hepapoietin 3205, 3825,
A; scatter factor) 4445
308 hepatocyte growth 444829 448 389854 1064 1966,
2586,
factor (hepapoietin 3206, 3826,
A; scatter factor) 4446
309 hepatocyte growth 453411 449 408270 1065 1967,
2587,
factor (hepapoietin 3207, 3827,
A; scatter factor) 4447
310 hepatocyte growth 457544 450 391238 1066 1968,
2588,
factor (hepapoietin 3208, 3828,
A; scatter factor) 4448
311 hepcidin 222304 451 222304 1067 14851969,
antimicrobial 2589, 3209,
peptide 3829, 4449
312 hepcidin 598398 452 471894 1068 1970, 2590,
antimicrobial 3210, 3830,
peptide 4450, 13678-
13764
313 Herceptin Heavy 1069 1971, 2591,
Chain 3211, 3831,
4451, 4930,
5260
314 Herceptin Light 1070 1972, 2592,
Chain 3212, 3832,
4452, 4931,
5261
315 hypoxanthine 298556 453 298556 1071 1486,
1973, 1650
phosphoribosyltra 2593, 3213,
nsferase 1 3833, 4453,
4932, 5262,
13765-13851
316 hypoxanthine 370796 454 359832 1072
1974,2594,
phosphoribosyltra 3214, 3834,
nsferase 1 4454, 4933,
5263
317 IDegLira; insulin
degludec;
liraglutide
318 iduronate 2- 340855 455 339801 1073 1975,
2595, 1651
sulfatase 3215, 3835,
4455, 4934,
5264
107

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319 kluronate2- 370441 456 359470 1074 1976,2596, 1651
sulfalase 3216,3836,
4456,4935,
5265
320 kluronate2- 370443 457 359472 1075 1977,2597, 1651
sulfalase 3217,3837,
4457,4936,
5266
321 kluronate2- 428056 458 390241 1076 1978,2598, 1651
sulfalase 3218,3838,
4458,4937,
5267
322 kluronate2- 521702 459 429745 1077 1979,2599, 1651
sulfalase 3219,3839,
4459,4938,
5268
323 kluronate2- 537071 460 440324 1078 1980,2600, 1651
sulfalase 3220,3840,
4460,4939,
5269
324 kluronate2- 541269 461 441261 1079 1981,2601, 1651
sulfalase 3221,3841,
4461,4940,
5270
325 iduronidase, alpha- 247933 462 247933 1080 1982,2602,
1652
L, 3222,3842,
4462,4941,
5271,13852-
13938
326 iduronidase, alpha- 453894 463 396458 1081 1983,2603,
1652
L, 3223,3843,
4463,4942,
5272
327 IgIVI Heavy
328 IgNnight
329 1NS-IGF2 397270 464 380440 1082 1984,2604,
readthrough 3224,3844,
4464,4943,
5273
330 insulin 250971 465 250971 1083 1985,2605, 1653
3225,3845,
4465,4944,
5274,13939-
14286
331 insulin 381330 466 370731 1084 1986,2606, 1653
3226,3846,
4466,4945,
5275
332 insulin 397262 467 380432 1085 1987,2607, 1653
3227,3847,
4467,4946,
5276,14287-
14373
108

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333 insulin 512523 468 424008 1086 1988, 2608,
3228, 3848,
4468, 4947,
5277
334 Insulin Aspart 1087 1989, 2609,
3229, 3849,
4469, 4948,
5278
335 Insulin Glargine 1088 1990,2610,
3230, 3850,
4470, 4949,
5279
336 Insulin Glulisine 1089 1991,2611,
3231, 3851,
4471, 4950,
5280
337 Insulin Lispro 1090 1992, 2612,
3232, 3852,
4472, 4951,
5281
338 interferon, alpha 357374 469 369566 1091 1993,2613,
3233, 3853,
4473, 4952,
5282
339 interferon, alpha 449498 470 394494 1092 1994,
2614,
13 3234, 3854,
4474, 4953,
5283
340 interferon, alpha 380222 471 369571 1093 1995,
2615,
14 3235, 3855,
4475, 4954,
5284
341 interferon, alpha 380216 472 369564 1094 1996,
2616,
16 3236, 3856,
4476, 4955,
5285
342 interferon, alpha 413767 473 411940 1095 1997,
2617,
17 3237, 3857,
4477, 4956,
5286
343 interferon, alpha 2 380206 474 369554 1096 1998,
2618, 1654
3238, 3858,
4478, 4957,
5287, 14374-
14460
344 interferon, alpha 380225 475 369574 1097 1999,
2619,
21 3239, 3859,
4479, 4958,
5288
345 interferon, alpha 4 421715 476 412897 1098 2000,
2620,
3240, 3860,
4480, 4959,
5289
109

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346 interferon, alpha 5 259555 477 259555 1099 2001,
2621,
3241, 3861,
4481, 4960,
5290
347 interferon, alpha 6 380210 478 369558 1100 2002,
2622,
3242, 3862,
4482, 4961,
5291
348 interferon, alpha 7 239347 479 239347 1101 2003,
2623,
3243, 3863,
4483, 4962,
5292
349 interferon, alpha 8 380205 480 369553 1102 2004,
2624,
3244, 3864,
4484, 4963,
5293
350 interferon, beta 1, 380232 481 369581 1103 2005,2625,
1655
fibroblast 3245, 3865,
4485, 4964,
5294, 14461-
14547
351 interleukin 10 423557 482 412237 1104 1487,2006,
2626,3246,
3866,4486,
14548-14634
352 interleukin 21 264497 483 264497 1105 2007,2627,
3247,3867,
4487,14635-
14721
353 interleukin 7 263851 484 263851 1106 1488,2008,
1656
2628,3248,
3868,4488,
4965,5295,
14722-14860
354 interleukin 7 379113 485 368408 1107 1489,2009,
1656
2629,3249,
3869,4489,
4966,5296
355 interleukin 7 379114 486 368409 1108 2010,2630,
3250, 3870,
4490, 4967,
5297
356 interleukin 7 518982 487 430272 1109 2011,2631,
3251,3871,
4491,4968,
5298
357 interleukin 7 520215 488 428364 1110 1490,2012,
2632,3252,
3872,4492,
4969,5299
358 interleukin 7 520269 489 427750 1111 1491,2013,
2633,3253,
3873,4493,
4970,5300
110

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359 interleukin 7 520317 490 427800 1112 2014,2634,
3254,3874,
4494,4971,
5301
360 interleukin 7 541183 491 438922 1113 1492,
2015,
2635, 3255,
3875, 4495,
4972, 5302
361 KIT ligand 228280 492 228280 1114 2016, 2636,
3256, 3876,
4496, 14861-
14947
362 KIT ligand 347404 493 54216 1115 2017, 2637,
3257, 3877,
4497
363 KIT ligand 537835 494 438889 1116 2018,2638,
3258, 3878,
4498
364 klotho 380099 495 369442 1117 1493, 2019,
2639, 3259,
3879, 4499,
14948-15347
365 klotho 426690 496 399513 1118 14942020,
2640, 3260,
3880, 4500
366 Kruppel-like 374672 497 363804 1119 2021,
2641,
factor 4 (gut) 3261,3881,
4501
367 Kruppel-like 411706 498 399921 1120 2022,
2642,
factor 4 (gut) 3262, 3882,
4502
368 Kruppel-like 420475 499 404922 1121 2023,
2643,
factor 4 (gut) 3263, 3883,
4503
369 Kruppel-like 439281 500 396294 1122 2024,
2644,
factor 4 (gut) 3264, 3884,
4504
370 lecithin- 264005 501 264005 1123 1495, 2025,
cholesterol 2645, 3265,
acyltransferase 3885, 4505,
15348-15434
371 lectin, galactoside- 254301 502 254301 1124 2026,
2646,
binding, soluble, 3 3266, 3886,
4506, 15435-
15521
372 lin-28 homolog A 254231 503 254231 1125 2027,
2647,
(C. elegans) 3267, 3887,
4507
373 lin-28 homolog A 326279 504 363314 1126 2028,2648,
(C. elegans) 3268, 3888,
4508, 15522-
15608
374 lipase A, 282673 505 282673 1127 1496 , 2029,
lysosomal acid, 2649, 3269,
cholesterol 3889, 4509,
1 1 1

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esterase 4973, 5303
375 lipase A, 336233 506 337354 1128 1497,2030, 1657
lysosomal acid, 2650, 3270,
cholesterol 3890, 4510,
esterase 4974, 5304,
15609-15782
376 lipase A, 354621 507 360895 1129 2031,2651,
lysosomal acid, 3271, 3891,
cholesterol 4511, 4975,
esterase 5305
377 lipase A, 371829 508 360894 1130 2032,2652, 1657
lysosomal acid, 3272, 3892,
cholesterol 4512, 4976,
esterase 5306
378 lipase A, 371837 509 360903 1131 1498,2033,
lysosomal acid, 2653, 3273,
cholesterol 3893, 4513,
esterase 4977, 5307
379 lipase A, 425287 510 392037 1132 1499,2034,
lysosomal acid, 2654, 3274,
cholesterol 3894, 4514,
esterase 4978, 5308
380 lipase A, 428800 511 388415 1133 1500,2035, 1657
lysosomal acid, 2655, 3275,
cholesterol 3895, 4515,
esterase 4979, 5309
381 lipase A, 456827 512 413019 1134 2036,2656, 1657
lysosomal acid, 3276, 3896,
cholesterol 4516, 4980,
esterase 5310
382 lipase A, 540050 513 439727 1135 2037,2657,
lysosomal acid, 3277, 3897,
cholesterol 4517, 4981,
esterase 5311
383 lipase A, 541980 514 438127 1136 2038,2658, 1657
lysosomal acid, 3278, 3898,
cholesterol 4518, 4982,
esterase 5312
384 lipase A, 542307 515 437564 1137 1501,2039, 1657
lysosomal acid, 2659, 3279,
cholesterol 3899, 4519,
esterase 4983, 5313
385 lipoprotein lipase 311322 516 309757 1138 1502,2040,
2660, 3280,
3900, 4520,
4984, 5314,
15783-15869
386 lipoprotein lipase 520959 517 428496 1139 1503, 2041,
2661, 3281,
3901, 4521,
4985, 5315
387 lipoprotein lipase 522701 518 428557 1140 1504, 2042,
2662, 3282,
112

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3902, 4522,
4986, 5316
388 lipoprotein lipase 524029 519 428237 1141 1505,2043,
2663, 3283,
3903, 4523,
4987, 5317
389 lipoprotein lipase 535763 520 438606 1142 2044, 2664,
3284, 3904,
4524, 4988,
5318
390 lipoprotein lipase 538071 521 439407 1143 2045, 2665,
3285, 3905,
4525, 4989,
5319
391 liraglutide native 1506
7-37
392 liraglutide native 1507
7-37-A8G
393 liraglutide native 1508
7-37-A8G-10X
394 liraglutide; GLP1 ; 1509
Victoza
395 LL-37; 1144 1620
Cathelicidin anti-
microbial peptide
396 low density 455727 522 397829 1145 1510, 2046,
lipoprotein 2666, 3286,
receptor 3906, 4526
397 low density 535915 523 440520 1146 1511, 2047,
lipoprotein 2667, 3287,
receptor 3907, 4527
398 low density 545707 524 437639 1147 1512, 2048,
lipoprotein 2668, 3288,
receptor 3908, 4528
399 low density 558013 525 453346 1148 1513,2049,
lipoprotein 2669, 3289,
receptor 3909, 4529
400 low density 558518 526 454071 1149 1514 , 2050,
lipoprotein 2670, 3290,
receptor 3910, 4530,
15870-16478
401 low density 561343 527 454147 1150 2051,2671,
lipoprotein 3291, 3911,
receptor 4531
402 low density 252444 528 252444 1151 2052, 2672,
lipoprotein 3292, 3912,
receptor 4532
403 luteinizing 221421 529 221421 1152 2053, 2673,
hormone beta 3293, 3913,
polypeptide 4533
404 luteinizing 391870 530 375743 1153 2054, 2674,
hormone beta 3294, 3914,
polypeptide 4534, 4990,
5320,
113

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405 LY2963016;
insulin glargine
406 Ly6/neurotoxin 1 317543 531 319846 1154 2055,2675,
3295, 3915,
4535
407 MabThera SC;
rituximab
408 mannosidase alpha 221363 532 221363 1155 2056,2676,
class 2B member 1 3296, 3916,
4536
409 mannosidase, 433513 533 390583 1156 1515, 2057,
alpha, class 2B, 2677, 3297,
member 1 3917, 4537
410 mannosidase, 456935 534 395473 1157 1516, 2058,
alpha, class 2B, 2678, 3298,
member 1 3918, 4538,
16479-16565
411 mannosidase, 536796 535 438266 1158 2059, 2679,
alpha, class 2B, 3299, 3919,
member 1 4539
412 meteorin glial cell 568223 536 455068 1159 2060, 2680,
differentiation 3300, 3920,
regulator 4540, 16566-
16652
413 meteorin, glial cell 219542 537 219542 1160 2061,2681,
differentiation 3301, 3921,
regulator 4541
414 methylmalonyl 274813 538 274813 1161 1517,2062,
1658
CoAnnAase 2682,3302,
3922,4542,
4991,5321,
16653-16739
415 methylmalonyl 540138 539 445535 1162 2063,2683,
CoAnnAase 3303,3923,
4543,4992,
5322,
416 microsomal 265517 540 265517 1163 1518,2064,
higlycelide 2684,3304,
transfer protein 3924, 4544,
16740-16878
417 microsomal 457717 541 400821 1164 2065,2685,
h*glyceride 3305,3925,
transfer protein 4545
418 microsomal 506883 542 426755 1165 1519,2066,
higlycelide 2686,3306,
transfer protein 3926, 4546
419 microsomal 538053 543 437358 1166 2067,2687,
triglyceride 3307, 3927,
transfer protein 4547
420 N- 299314 544 299314 1167 1520, 2068,
acetylglucos amine 2688, 3308,
-1-phosphate 3928, 4548,
transferase, alpha 16879-17017
and beta subunits
114

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421 N- 549940 545 449150 1168 1521, 2069,
acetylglucos amine 2689, 3309,
-1-phosphate 3929, 4549
transferase, alpha
and beta subunits
422 N-acetylglutamate 293404 546 293404 1169 2070, 2690,
synthase 3310, 3930,
4550, 17018-
17156
423 N-acetylglutamate 541745 547 441262 1170 2071, 2691,
synthase 3311, 3931,
4551
424 N-acylsphingosine 262097 548 262097 1171 1522, 2072,
amidohydrolase 2692, 3312,
(acid ceramidase) 3932,
1 455217157-
17295
425 N-acylsphingosine 314146 549 326970 1172 1523, 2073,
amidohydrolase 2693, 3313,
(acid ceramidase) 3933, 4553
1
426 N-acylsphingosine 381733 550 371152 1173 1524 , 2074,
amidohydrolase 2694, 3314,
(acid ceramidase) 3934, 4554
1
427 N-acylsphingosine 417108 551 394125 1174 1525 , 2075,
amidohydrolase 2695, 3315,
(acid ceramidase) 3935, 4555
1
428 N-acylsphingosine 520781 552 427751 1175 1526, 2076,
amidohydrolase 2696, 3316,
(acid ceramidase) 3936, 4556
1
429 natriuretic peptide 376468 553 365651 1176 2077, 2697,
B 3317, 3937,
4557, 4993,
5323,
430 nerve growth 369512 554 358525 1177 2078, 2698,
factor (beta 3318, 3938,
polypeptide) 4558, 4994,
5324, 17296-
17382
431 neuregulin 1 287840 555 287840 1178 2079,2699,
3319, 3939,
4559
432 neuregulin 1 287842 556 287842 1179 2080, 2700,
3320, 3940,
4560, 17383-
17469
433 neuregulin 1 287845 557 287845 1180 2081,2701,
3321, 3941,
4561
434 neuregulin 1 338921 558 343395 1181 2082,2702,
3322, 3942,
4562
115

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435 neuregulin 1 341377 559 340497 1182 2083,2703,
3323, 3943,
4563
436 neuregulin 1 356819 560 349275 1183 2084,2704,
3324, 3944,
4564
437 neuregulin 1 405005 561 384620 1184 2085, 2705,
3325, 3945,
4565
438 neuregulin 1 519301 562 429582 1185 2086, 2706,
3326, 3946,
4566
439 neuregulin 1 520407 563 434640 1186 2087, 2707,
3327, 3947,
4567, 17470-
17556
440 neuregulin 1 520502 564 433289 1187 2088, 2708,
3328, 3948,
4568
441 neuregulin 1 521670 565 428828 1188 2089, 2709,
3329, 3949,
4569
442 neuregulin 1 539990 566 439276 1189 2090, 2710,
3330, 3950,
4570
443 neuregulin 1 523079 567 430120 1190 2091, 2711,
3331, 3951,
4571
444 OMOMYC 1191 1527, 17557-
17643
445 OMOMYC90 1528, 17644-
(90AA truncated 17784
domain)
446 Orencia S.C.;
abatacept
447 ornithine 39007 568 39007 1192 1529,2092, 1659
carbamoyltransfer 2712, 3332,
ase 3952, 4572,
4995, 5325,
17785-17871
448 parathyroid 282091 569 282091 1193 2093,2713,
hormone 3333,3953,
4573,4996,
5326
449 parathyroid 529816 570 433208 1194 2094, 2714,
hormone 3334, 3954,
4574, 4997,
5327
450 PBO-326;
rituximab
451 phenylalanine 546844 571 446658 1195 1530, 2095,
hydroxylase 2715, 3335,
3955, 4575,
4998, 5328
116

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452 phenylalanine 551337 572 447620 1196 1531,
2096,
hydroxylase 2716, 3336,
3956, 4576,
4999, 5329
453 phenylalanine 553106 573 448059 1197 1532,
2097, 1660
hydroxylase 2717, 3337,
3957, 4577,
5000, 5330,
17872-17958
454 phosphorylase 566044 574 456729 1198 2098,
2718,
kinase beta 3338, 3958,
4578
455 phosphorylase 563588 575 455607 1199
2099,2719,
kinase gamma 2 3339, 3959,
(testis) 4579, 17959-
18045
456 phosphorylase 379942 576 369274 1200 2100,
2720,
kinase, alpha 2 3340, 3960,
(liver) 4580, 18046-
18132
457 phosphorylase 299167 577 299167 1201 2101,
2721,
kinase, beta 3341, 3961,
4581
458 phosphorylase 323584 578 313504 1202 2102,
2722,
kinase, beta 3342, 3962,
4582, 18133-
18219
459 phosphorylase 455779 579 414345 1203 2103,
2723,
kinase, beta 3343, 3963,
4583
460 phosphorylase 328273 580 329968 1204 2104,
2724,
kinase, gamma 2 3344, 3964,
(testis) 4584
461 phosphorylase 424889 581 388571 1205 2105,
2725,
kinase, gamma 2 3345, 3965,
(testis) 4585
462 phosphorylase, 216392 582 216392 1206 2106,
2726,
glycogen, liver 3346, 3966,
4586
463 phosphorylase, 544180 583 443787 1207
2107,2727,
glycogen, liver 3347, 3967,
4587
464 plasminogen 308192 584 308938 1208 1533,2108,
2728,3348,
3968,4588,
18220-18271
465 plasminogen 316325 585 321466 1209 2109,2729,
3349,3969,
4589
466 plasminogen 366924 586 355891 1210 1534, 2110,
2730, 3350,
3970, 4590
467 plasminogen 220809 587 220809 1211 1535,2111,
1661
activator, tissue 2731, 3351,
3971, 4591,
117

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18272-18358
468 plasminogen 270189 588 270189 1212 1536,2112, 1661
activator, tissue 2732, 3352,
3972, 4592,
5001, 5331
469 plasminogen 352041 589 270188 1213 1537,2113, 1661
activator, tissue 2733, 3353,
3973, 4593,
5002, 5332
470 plasminogen 429089 590 392045 1214 2114,2734, 1661
activator, tissue 3354, 3974,
4594, 5003,
5333
471 plasminogen 429710 591 407861 1215 1538,2115,
activator, tissue 2735, 3355,
3975, 4595,
5004, 5334
472 plasminogen 519510 592 428886 1216 1539,2116,
activator, tissue 2736, 3356,
3976, 4596,
5005, 5335
473 plasminogen 520523 593 428797 1217 1540, 2117,
1661
activator, tissue 2737, 3357,
3977, 4597,
5006, 5336
474 plasminogen 521694 594 429801 1218 1541,2118, 1661
activator, tissue 2738, 3358,
3978, 4598,
5007, 5337
475 plasminogen 524009 595 429401 1219 1542,2119,
activator, tissue 2739, 3359,
3979, 4599,
5008, 5338
476 POU class 5 259915 596 259915 1220 2120, 2740,
homeobox 1 3360, 3980,
4600
477 POU class 5 376243 597 365419 1221 2121, 2741,
homeobox 1 3361, 3981,
4601
478 POU class 5 383524 598 373016 1222 2122, 2742,
homeobox 1 3362, 3982,
4602
479 POU class 5 412166 599 387646 1223 2123, 2743,
homeobox 1 3363, 3983,
4603
480 POU class 5 419095 600 413622 1224 2124, 2744,
homeobox 1 3364, 3984,
4604
481 POU class 5 429603 601 392877 1225 2125, 2745,
homeobox 1 3365, 3985,
4605
482 POU class 5 433063 602 405041 1226 2126, 2746,
homeobox 1 3366, 3986,
118

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4606
483 POU class 5 433348 603 412665 1227 2127, 2747,
homeobox 1 3367, 3987,
4607
484 POU class 5 434616 604 388842 1228 2128, 2748,
homeobox 1 3368, 3988,
4608, 18359-
18445
485 POU class 5 448657 605 416165 1229 2129, 2749,
homeobox 1 3369, 3989,
4609
486 POU class 5 451077 606 391507 1230 2130, 2750,
homeobox 1 3370, 3990,
4610
487 POU class 5 454714 607 400047 1231 2131, 2751,
homeobox 1 3371, 3991,
4611
488 POU class 5 546505 608 448154 1232 2132, 2752,
homeobox 1 3372, 3992,
4612
489 POU class 5 547234 609 449442 1233 2133, 2753,
homeobox 1 3373, 3993,
4613
490 POU class 5 547658 610 446962 1234 2134, 2754,
homeobox 1 3374, 3994,
4614
491 POU class 5 548682 611 446815 1235 2135, 2755,
homeobox 1 3375, 3995,
4615
492 POU class 5 550059 612 447874 1236 2136, 2756,
homeobox 1 3376, 3996,
4616
493 POU class 5 550521 613 447969 1237 2137, 2757,
homeobox 1 3377, 3997,
4617
494 POU class 5 550572 614 448254 1238 2138, 2758,
homeobox 1 3378, 3998,
4618
495 POU class 5 553069 615 448231 1239 2139, 2759,
homeobox 1 3379, 3999,
4619
496 POU class 5 553206 616 446757 1240 2140, 2760,
homeobox 1 3380, 4000,
4620
497 proprotein 302118 617 303208 1241 2141,2761,
convertase 3381,4001,
subtilisin/kexin 4621
type 9
498 proprotein 452118 618 401598 1242 2142,2762,
convertase 3382,4002,
subtilisin/kexin 4622
type 9
119

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499 proprotein 543384 619 441859 1243 2143,2763,
convertase 3383,4003,
subtilisin/kexin 4623
type 9
500 protein C 234071 620 234071 1244 2144,2764,
(inactivator of 3384, 4004,
coagulation factors 4624
Va and Villa)
501 protein C 409048 621 386679 1245 2145,2765,
(inactivator of 3385, 4005,
coagulation factors 4625, 5009,
Va and Villa) 5339
502 protein C 422777 622 409543 1246 2146,2766,
(inactivator of 3386, 4006,
coagulation factors 4626, 5010,
Va and VIIIa) 5340
503 protein C 427769 623 406295 1247 2147,2767,
(inactivator of 3387, 4007,
coagulation factors 4627, 5011,
Va and VIIIa) 5341
504 protein C 429925 624 412697 1248 2148,2768,
(inactivator of 3388, 4008,
coagulation factors 4628, 5012,
Va and VIIIa) 5342
505 protein C 453608 625 404030 1249 2149,2769,
(inactivator of 3389, 4009,
coagulation factors 4629, 5013,
Va and VIIIa) 5343
506 protein C 537436 626 442106 1250 2150,2770,
(inactivator of 3390, 4010,
coagulation factors 4630, 5014,
Va and VIIIa) 5344
507 relaxin 2 308420 627 308018 1251 2151,2771,
3391,4011,
4631
508 Raxh12 381627 628 371040 1252 2152,2772,
3392,4012,
4632,18446-
18532
509 Reverse Caspase 3 1543
(cleavable)
510 Reverse Caspase 3 1544
(non-cleavable)
511 Reverse Caspase 6 1545
512 rhodopsin 296271 629 296271 1253 2153,2773,
3393,4013,
4633,5015,
5345,18533-
18619
513 Rituximab;
rituximab
514 serpin peptidase 355814 630 348068 1254 2154,
2774, 1662
inhibitor, clade A 3394, 4014,
(alpha-1 4634, 5016,
antiproteinase, 5346, 18620-
120

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antitrypsin), 18706
member 1
515 serpin peptidase 393087 631 376802 1255 2155,
2775, 1662
inhibitor, clade A 3395, 4015,
(alpha-1 4635, 5017,
antiproteinase, 5347
antitrypsin),
member 1
516 serpin peptidase 393088 632 376803 1256 2156,
2776, 1662
inhibitor, clade A 3396, 4016,
(alpha-1 4636, 5018,
antiproteinase, 5348
antitrypsin),
member 1
517 serpin peptidase 402629 633 386094 1257 2157,
2777,
inhibitor, clade A 3397, 4017,
(alpha-1 4637, 5019,
antiproteinase, 5349
antitrypsin),
member 1
518 serpin peptidase 404814 634 385960 1258 2158,
2778, 1662
inhibitor, clade A 3398, 4018,
(alpha-1 4638, 5020,
antiproteinase, 5350
antitrypsin),
member 1
519 serpin peptidase 437397 635 408474 1259 2159,
2779, 1662
inhibitor, clade A 3399, 4019,
(alpha-1 4639, 5021,
antiproteinase, 5351
antitrypsin),
member 1
520 serpin peptidase 440909 636 390299 1260 2160,
2780, 1662
inhibitor, clade A 3400, 4020,
(alpha-1 4640, 5022,
antiproteinase, 5352
antitrypsin),
member 1
521 serpin peptidase 448921 637 416066 1261 2161,2781,
1662
inhibitor, clade A 3401, 4021,
(alpha-1 4641, 5023,
antiproteinase, 5353
antitrypsin),
member 1
522 serpin peptidase 449399 638 416354 1262 2162,
2782, 1662
inhibitor, clade A 3402, 4022,
(alpha-1 4642, 5024,
antiproteinase, 5354
antitrypsin),
member 1
121

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523 serpin peptidase 553327 639 452480 1263 2163, 2783,
inhibitor, clade A 3403, 4023,
(alpha-1 4643, 5025,
antiproteinase, 5355, 18707-
antitrypsin), 18793
member 1
524 serpin peptidase 556091 640 452169 1264 2164, 2784,
inhibitor, clade A 3404, 4024,
(alpha-1 4644, 5026,
antiproteinase, 5356
antitrypsin),
member 1
525 serpin peptidase 556955 641 451098 1265 2165, 2785,
inhibitor, clade A 3405, 4025,
(alpha-1 4645, 5027,
antiproteinase, 5357
antitrypsin),
member 1
526 serpin peptidase 557118 642 451826 1266 2166, 2786,
inhibitor, clade A 3406, 4026,
(alpha-1 4646, 5028,
antiproteinase, 5358
antitrypsin),
member 1
527 serpin peptidase 557492 643 452452 1267 2167, 2787,
inhibitor, clade A 3407, 4027,
(alpha-1 4647, 5029,
antiproteinase, 5359
antitrypsin),
member 1
528 serpin peptidase 351522 644 307953 1268 2168, 2788,
inhibitor, clade C 3408, 4028,
(antithrombin), 4648, 5030,
member 1 5360
529 serpin peptidase 367698 645 356671 1269 1546, 2169,
inhibitor, clade C 2789, 3409,
(antithrombin), 4029, 4649,
member 1 5031, 5361,
18794-18932
530 serpin peptidase 324015 646 321853 1270 1547, 2170,
inhibitor, clade F 2790, 3410,
(alpha-2 4030, 4650
antiplasmin,
pigment
epithelium derived
factor), member 2
531 serpin peptidase 382061 647 371493 1271 2171, 2791,
inhibitor, clade F 3411, 4031,
(alpha-2 4651, 18933-
antiplasmin, 19071
pigment
epithelium derived
factor), member 2
532 serpin peptidase 450523 648 403877 1272 1548, 2172,
inhibitor, clade F 2792, 3412,
122

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(alpha-2 4032, 4652
antiplasmin,
pigment
epithelium derived
factor), member 2
533 serpin peptidase 453066 649 402286 1273 1549 , 2173,
inhibitor, clade F 2793, 3413,
(alpha-2 4033, 4653
antiplasmin,
pigment
epithelium derived
factor), member 2
534 serpin peptidase 278407 650 278407 1274 1550, 2174,
inhibitor, clade G 2794, 3414,
(Cl inhibitor), 4034, 4654,
member 1 5032, 5362,
19072-19210
535 serpin peptidase 378323 651 367574 1275 1551 ,2175,
inhibitor, clade G 2795, 3415,
(Cl inhibitor), 4035, 4655,
member 1 5033, 5363
536 serpin peptidase 378324 652 367575 1276 1552, 2176,
inhibitor, clade G 2796, 3416,
(Cl inhibitor), 4036, 4656,
member 1 5034, 5364
537 serpin peptidase 405496 653 384561 1277 1553, 2177,
inhibitor, clade G 2797, 3417,
(Cl inhibitor), 4037, 4657,
member 1 5035, 5365
538 serpin peptidase 433668 654 399800 1278 2178, 2798,
inhibitor, clade G 3418, 4038,
(Cl inhibitor), 4658, 5036,
member 1 5366
539 sirtuin 1 212015 655 212015 1279 1554, 2179, 1663
2799, 3419,
4039, 4659,
19211-19297
540 sirtuin 1 403579 656 384063 1280 1555,2180,
2800, 3420,
4040, 4660,
5037, 5367
541 sirtuin 1 406900 657 384508 1281 2181,2801,
3421, 4041,
4661, 5038,
5368
542 sirtuin 1 432464 658 409208 1282 1556,2182, 1664
2802, 3422,
4042, 4662,
5039, 5369
543 sirtuin 6 305232 659 305310 1283 1557, 2183,
2803, 3423,
4043, 4663,
5040, 5370
123

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544 sirtuin 6 337491 660 337332 1284 1558,2184, 1665
2804,3424,
4044,4664,
5041,5371,
19298-19384
545 sh-tuin6 381935 661 371360 1285 1559,2185,
2805,3425,
4045,4665,
5042,5372
546 solute carrier 314251 662 323568 1286 1560,2186,
family 2 2806, 3426,
(facilitated glucose 4046, 4666,
transporter), 19385-19523
member 2
547 solute carrier 382808 663 372258 1287 1561,2187,
family 2 2807, 3427,
(facilitated glucose 4047, 4667
transporter),
member 2
548 solute carrier 577093 664 461344 1288 2188,2808,
family 37 3428, 4048,
(glucose-6- 4668õ 19524-
phosphate 19610
transporter)
member 4
549 solute carrier 590663 665 464769 1289 2189, 2809,
family 37 3429, 4049,
(glucose-6- 4669
phosphate
transporter)
member 4
550 solute carrier 261024 666 261024 1290 2190,2810,
family 40 (iron- 3430, 4050,
regulated 4670
transporter),
member 1
551 solute carrier 427241 667 390005 1291 2191,2811,
family 40 (iron- 3431, 4051,
regulated 4671
transporter),
member 1
552 solute carrier 427419 668 392730 1292 2192,2812,
family 40 (iron- 3432, 4052,
regulated 4672
transporter),
member 1
553 solute carrier 440626 669 396134 1293 2193,2813,
family 40 (iron- 3433, 4053,
regulated 4673
transporter),
member 1
554 solute carrier 455320 670 413549 1294 2194,2814,
family 40 (iron- 3434, 4054,
regulated 4674
transporter),
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member 1
555 solute carrier 481497 671 446069 1295 2195,2815,
family 40 (iron- 3435, 4055,
regulated 4675
transporter),
member 1
556 solute carrier 544056 672 444582 1296 2196,2816,
family 40 (iron- 3436, 4056,
regulated 4676
transporter),
member 1
557 sortilin 1 256637 673 256637 1297 1562,2197,
2817,3437,
4057,4677,
5043,5373
558 sortilhll 538502 674 438597 1298 1563,2198,
2818,3438,
4058,4678,
5044,5374,
19611-19697
559 sperm adhesion 223028 675 223028 1299 2199,2819,
molecule 1 (PH-20 3439, 4059,
hyaluronidase, 4679
zona pellucida
binding)
560 sperm adhesion 340011 676 345849 1300 2200,2820,
molecule 1 (PH-20 3440, 4060,
hyaluronidase, 4680, 5045,
zona pellucida 5375
binding)
561 sperm adhesion 402183 677 386028 1301 2201,
2821,
molecule 1 (PH-20 3441, 4061,
hyaluronidase, 4681, 5046,
zona pellucida 5376
binding)
562 sperm adhesion 413927 678 391491 1302 2202,2822,
molecule 1 (PH-20 3442, 4062,
hyaluronidase, 4682, 5047,
zona pellucida 5377
binding)
563 sperm adhesion 439500 679 402123 1303 2203,2823,
molecule 1 (PH-20 3443, 4063,
hyaluronidase, 4683, 5048,
zona pellucida 5378
binding)
564 sperm adhesion 460182 680 417934 1304 2204,
2824,
molecule 1 (PH-20 3444, 4064,
hyaluronidase, 4684, 5049,
zona pellucida 5379
binding)
565 sphingomyelin 299397 681 299397 1305 1564,2205,
phosphodiesterase 2825, 3445,
1, acid lysosomal 4065, 4685,
125

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5050, 5380,
566 sphingomyelin 342245 682 340409 1306 1565, 2206,
1666
phosphodiesterase 2826, 3446,
1, acid lysosomal 4066, 4686,
5051, 5381,
19698-19784
567 sphingomyelin 356761 683 349203 1307 1566, 2207,
phosphodiesterase 2827, 3447,
1, acid lysosomal 4067, 4687,
5052, 5382
568 sphingomyelin 527275 684 435350 1308 1567, 2208,
phosphodiesterase 2828, 3448,
1, acid lysosomal 4068, 4688,
5053, 5383
569 sphingomyelin 533123 685 435950 1309 1568,2209,
phosphodiesterase 2829, 3449,
1, acid lysosomal 4069, 4689,
5054, 5384
570 sphingomyelin 534405 686 434353 1310 1569, 2210,
phosphodiesterase 2830, 3450,
1, acid lysosomal 4070, 4690,
5055, 5385
571 SRY (sex 325404 687 323588 1311 2211,2831,
determining region 3451, 4071,
Y)-box 2 4691
572 SRY (sex 431565 688 439111 1312 2212,2832,
determining region 3452, 4072,
Y)-box 2 4692, 19785-
19871
573 Subcutaneous
Herceptin;
trastuzumab
574 surfactant protein 393822 689 377409 1313 2213, 2833,
B 3453, 4073,
4693
575 surfactant protein 409383 690 386346 1314 2214, 2834,
B 3454, 4074,
4694
576 surfactant protein 441838 691 395757 1315 2215,2835,
B 3455, 4075,
4695
577 surfactant protein 519937 692 428719 1316 2216, 2836,
B 3456, 4076,
4696
578 Synagis
579 thrombomodulin 377103 693 366307 1317 1570, 2217,
2837, 3457,
4077, 4697,
19872-19958
580 thrombomodulin 503590 694 440119 1318 2218, 2838,
3458, 4078,
4698
126

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581 thrombopoietin 204615 695 204615 1319 1571,2219,
1667
2839,3459,
4079,4699,
5056,5386,
19959-20045
582 thrombopoietin 353488 696 341335 1320 2220,
2840,
3460, 4080,
4700, 5057,
5387
583 thrombopoietin 421442 697 411704 1321 1572,2221,
2841,3461,
4081,4701,
5058,5388
584 thrombopoietin 445696 698 410763 1322 1573,
2222,
2842,3462,
4082,4702,
5059,5389
585 TL011; rituximab
586 transfenin 223051 699 223051 1323 2223, 2843,
receptor 2 3463, 4083,
4703
587 transfenin 431692 700 413905 1324 2224, 2844,
receptor 2 3464, 4084,
4704
588 transfenin 462107 701 420525 1325 2225, 2845,
receptor 2 3465, 4085,
4705
589 transfenin 544242 702 443656 1326 2226, 2846,
receptor 2 3466, 4086,
4706
590 transforming 221930 703 221930 1327 2227,2847,
1668
growth factor, beta 3467, 4087,
1 4707 , 20046-
20132
591 transforming 366929 704 355896 1328 2228,
2848,
growth factor, beta 3468, 4088,
2 4708
592 transforming 366930 705 355897 1329 2229,2849,
growth factor, beta 3469, 4089,
2 4709
593 transforming 238682 706 238682 1330 2230,
2850,
growth factor, beta 3470, 4090,
3 4710, 5060,
5390, 20133-
20219
594 transforming 556285 707 451110 1331 2231,
2851,
growth factor, beta 3471, 4091,
3 4711, 5061,
5391
595 transthyretin 237014 708 237014 1332 2232,2852,
3472,4092,
4712,20220-
20358
127

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596 transthyretin 432547 709 396762 1333 2233,2853,
3473,4093,
4713
597 transthyretin 541025 710 442134 1334 2234,2854,
3474,4094,
4714
598 tufMinl 353024 711 343781 1335 2235,2855, 1669
3475,4095,
4715,5062,
5392
599 tufMinl 368848 712 357841 1336 2236,2856, 1669
3476,4096,
4716,5063,
5393
600 tufUhll 368849 713 357842 1337 2237,2857, 1669
3477,4097,
4717,5064,
5394,20359-
20445
601 tufUhll 507671 714 443643 1338 2238,2858, 1669
3478,4098,
4718,5065,
5395
602 tufUhll 538902 715 437997 1339 2239,2859,
3479,4099,
4719,5066,
5396
603 tufUh11 544350 716 441557 1340 2240,2860, 1669
3480,4100,
4720,5067,
5397
604 tumorproteinp53 269305 717 269305 1341 1574,2241, 1670
2861,3481,
4101,4721,
5068,5398,
20446-20532
605 tumorproteinp53 359597 718 352610 1342 1575,2242,
2862,3482,
4102,4722,
5069,5399
606 tumorproteinp53 396473 719 379735 1343 2243,2863,
3483,4103,
4723,5070,
5400
607 tumorproteinp53 413465 720 410739 1344 1576,2244,
2864,3484,
4104,4724,
5071,5401
608 tumorproteinp53 414315 721 394195 1345 2245,2865,
3485,4105,
4725,5072,
5402
609 tumorproteinp53 419024 722 402130 1346 2246,2866,
3486,4106,
4726,5073,
128

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5403
610 tumor protein p53 420246 723 391127 1347 1577,
2247,
2867, 3487,
4107, 4727,
5074, 5404
611 tumor protein p53 445888 724 391478 1348 2248,
2868, 1670
3488, 4108,
4728, 5075,
5405
612 tumor protein p53 455263 725 398846 1349 1578,
2249,
2869, 3489,
4109, 4729,
5076, 5406
613 tumor protein p53 503591 726 426252 1350 1579 ,
2250,
2870, 3490,
4110, 4730,
5077, 5407
614 tumor protein p53 508793 727 424104 1351 1580,
2251,
2871, 3491,
4111, 4731,
5078, 5408
615 tumor protein p53 509690 728 425104 1352 1581õ
2252,
2872, 3492,
4112, 4732,
5079, 5409
616 tumor protein p53 514944 729 423862 1353 1582,
2253,
2873, 3493,
4113, 4733,
5080, 5410
617 tumor protein p53 545858 730 437792 1354 2254,
2874,
3494, 4114,
4734, 5081,
5411
618 tyrosinase 263321 731 263321 1355 2255,2875,
(oculocutaneous 3495, 4115,
albinism IA) 4735õ , 20533-
20619
619 tyrosine 355962 732 348234 1356 1583, 2256,
aminotransferase 2876, 3496,
4116, 4736õ ,
20620-20758
620 UDP 305208 733 304845 1357 1584, 2257,
glucuronosyltransf 2877, 3497,
erase 1 family, 4117, 4737õ ,
polypeptide Al 20759-20845
621 UDP 360418 734 353593 1358 1585, 2258,
glucuronosyltransf 2878, 3498,
erase 1 family, 4118, 4738
polypeptide Al
622 UDP 344644 735 343838
1359 2259,2879,
glucuronosyltransf 3499, 4119,
erase 1 family, 4739
polypeptide A10
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623 UDP 482026 736 418532 1360 2260, 2880,
glucuronosyltransf 3500, 4120,
erase 1 family, 4740
polypeptide A3
624 UDP 513300 737 427404 1361 2261, 2881,
glycosyltransferas 3501, 4121,
e 3 family 4741, 5082,
polypeptide A2 5412
625 UDP 282507 738 282507
1362 2262,2882, 1671
glycosyltransferas 3502, 4122,
e 3 family, 4742, 5083,
polypeptide A2 5413
626 UDP 515131 739 420865 1363 2263,2883,
glycosyltransferas 3503, 4123,
e 3 family, 4743, 5084,
polypeptide A2 5414
627 UDP 545528 740 445367
1364 2264,2884,
glycosyltransferas 3504, 4124,
e 3 family, 4744, 5085,
polypeptide A2 5415
628 vascular 230480 741 230480 1365 1586,2265,
endothelial growth 2885, 3505,
factor A 4125, 4745
629 vascular 324450 742 317598 1366 1587,2266,
endothelial growth 2886, 3506,
factor A 4126, 4746,
5086, 5416
630 vascular 372055 743 361125 1367 1588,2267,
endothelial growth 2887, 3507,
factor A 4127, 4747,
5087, 5417
631 vascular 372064 744 361134 1368 1589, 2268,
endothelial growth 2888, 3508,
factor A 4128, 4748,
5088, 5418
632 vascular 372067 745 361137 1369 1590, 2269,
1672
endothelial growth 2889, 3509,
factor A 4129, 4749,
5089, 5419,
20846-20932
633 vascular 372077 746 361148 1370 1591,2270,
endothelial growth 2890, 3510,
factor A 4130, 4750,
5090, 5420
634 vascular 413642 747 389864 1371 1592, 2271,
endothelial growth 2891, 3511,
factor A 4131,4751,
5091, 5421
635 vascular 417285 748 388663 1372 1593,2272,
endothelial growth 2892, 3512,
factor A 4132, 4752,
5092, 5422
636 vascular 425836 749 388465 1373 1594,2273,
endothelial growth 2893, 3513,
factor A 4133, 4753,
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5093, 5423
637 vascular 457104 750 409911 1374 1595,2274,
endothelial growth 2894, 3514,
factor A 4134, 4754,
5094, 5424
638 vascular 482630 751 421561 1375 1596,2275,
endothelial growth 2895, 3515,
factor A 4135, 4755,
5095, 5425
639 vascular 518689 752 430829 1376 1597, 2276,
endothelial growth 2896, 3516,
factor A 4136, 4756,
5096, 5426
640 vascular 518824 753 430002 1377 1598,2277,
endothelial growth 2897, 3517,
factor A 4137, 4757,
5097, 5427
641 vascular 519767 754 430594 1378 1599, 2278,
endothelial growth 2898, 3518,
factor A 4138, 4758,
5098, 5428
642 vascular 520948 755 428321 1379 1600, 2279,
endothelial growth 2899, 3519,
factor A 4139, 4759,
5099, 5429
643 vascular 523125 756 429008 1380 1601,2280,
endothelial growth 2900, 3520,
factor A 4140, 4760,
5100, 5430
644 vascular 523873 757 430479 1381 1602, 2281,
endothelial growth 2901, 3521,
factor A 4141,4761,
5101, 5431
645 vascular 523950 758 429643 1382 1603,2282,
endothelial growth 2902, 3522,
factor A 4142, 4762,
5102, 5432
646 vascular 280193 759 280193 1383 1604,2283,
endothelial growth 2903, 3523,
factor C 4143, 4763,
20933-21019
647 vasoactive 367243 760 356212 1384 1605,2284,
intestinal peptide 2904, 3524,
4144, 4764
648 vasoactive 367244 761 356213 1385 1606,2285,
intestinal peptide 2905, 3525,
4145, 4765,
21020-21106
649 vasoactive 431366 762 410356 1386 1607, 2286,
intestinal peptide 2906, 3526,
4146, 4766
650 v-myc 259523 763 259523
1387 2287,2907,
myelocytomatosis 3527, 4147,
131

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viral oncogene 4767
homolog (avian)
651 v-myc 377970 764 367207 1388 2288, 2908,
myelocytomatosis 3528, 4148,
viral oncogene 4768, 21107-
homolog (avian) 21110
652 v-myc 454617 765 405312 1389 2289, 2909,
myelocytomatosis 3529, 4149,
viral oncogene 4769
homolog (avian)
653 v-myc 524013 766 430235 1390 2290, 2910,
myelocytomatosis 3530, 4150,
viral oncogene 4770,21111-
homolog (avian) 21284
654 von Willebrand 261405 767 261405 1391 1608,2291,
factor 2911,3531,
4151, 4771,
21285-21423
655 zinc finger, 403903 768 384434 1392 2292,2912,
GATA-like 3532, 4152,
protein 1 4772
Protein Cleavage Signals and Sites
[000288] In one embodiment, the polypeptides of the present invention may
include at
least one protein cleavage signal containing at least one protein cleavage
site. The
protein cleavage site may be located at the N-terminus, the C-terminus, at any
space
between the N- and the C- termini such as, but not limited to, half-way
between the N-
and C-termini, between the N-terminus and the half way point, between the half
way
point and the C-terminus, and combinations thereof.
[000289] The polypeptides of the present invention may include, but is not
limited to, a
proprotein convertase (or prohormone convertase), thrombin or Factor Xa
protein
cleavage signal. Proprotein convertases are a family of nine proteinases,
comprising
seven basic amino acid-specific subtilisin-like serine proteinases related to
yeast kexin,
known as prohormone convertase 1/3 (PC1/3), PC2, furin, PC4, PC5/6, paired
basic
amino-acid cleaving enzyme 4 (PACE4) and PC7, and two other subtilases that
cleave at
non-basic residues, called subtilisin kexin isozyme 1 (SKI-1) and proprotein
convertase
subtilisin kexin 9 (PCSK9). Non-limiting examples of protein cleavage signal
amino
acid sequences are listing in Table 7. In Table 7, "X" refers to any amino
acid, "n" may
be 0, 2, 4 or 6 amino acids and "*" refers to the protein cleavage site. In
Table 7, SEQ ID
NO: 21426 refers to when n=4 and SEQ ID NO: 21427 refers to when n=6.
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Table 7. Protein Cleavage Site Sequences
Protein Cleavage Signal Amino Acid Cleavage Sequence SEQ ID NO
Proprotein convertase R-X-X-R* 21424
R-X-K/R-R* 21425
K/R-Xn-K/R* 21426 or 21427
Thrombin L-V-P-R*-G-S 21428
L-V-P-R* 21429
A/F/G/I/L/TN/M-A/F/G/I/L/TN/W-P-R* 21430
Factor Xa I-E-G-R* 21431
I-D-G-R* 21432
A-E-G-R* 21433
A/F/G/I/L/TN/M-D/E-G-R* 21434
[000290] In one embodiment, the primary constructs and the mmRNA of the
present
invention may be engineered such that the primary construct or mmRNA contains
at least
one encoded protein cleavage signal. The encoded protein cleavage signal may
be
located before the start codon, after the start codon, before the coding
region, within the
coding region such as, but not limited to, half way in the coding region,
between the start
codon and the half way point, between the half way point and the stop codon,
after the
coding region, before the stop codon, between two stop codons, after the stop
codon and
combinations thereof
[000291] In one embodiment, the primary constructs or mmRNA of the present
invention may include at least one encoded protein cleavage signal containing
at least one
protein cleavage site. The encoded protein cleavage signal may include, but is
not
limited to, a proprotein convertase (or prohormone convertase), thrombin
and/or Factor
Xa protein cleavage signal. One of skill in the art may use Table 1 above or
other known
methods to determine the appropriate encoded protein cleavage signal to
include in the
primary constructs or mmRNA of the present invention. For example, starting
with the
signal of Table 7 and considering the codons of Table 1 one can design a
signal for the
primary construct which can produce a protein signal in the resulting
polypeptide.
[000292] In one embodiment, the polypeptides of the present invention include
at least
one protein cleavage signal and/or site.
133

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[000293] As a non-limiting example, U.S. Pat. No. 7,374,930 and U.S. Pub. No.
20090227660, herein incorporated by reference in their entireties, use a furin
cleavage
site to cleave the N-terminal methionine of GLP-1 in the expression product
from the
Golgi apparatus of the cells. In one embodiment, the polypeptides of the
present
invention include at least one protein cleavage signal and/or site with the
proviso that the
polypeptide is not GLP-1.
[000294] In one embodiment, the primary constructs or mmRNA of the present
invention includes at least one encoded protein cleavage signal and/or site.
[000295] In one embodiment, the primary constructs or mmRNA of the present
invention includes at least one encoded protein cleavage signal and/or site
with the
proviso that the primary construct or mmRNA does not encode GLP-1.
[000296] In one embodiment, the primary constructs or mmRNA of the present
invention may include more than one coding region. Where multiple coding
regions are
present in the primary construct or mmRNA of the present invention, the
multiple coding
regions may be separated by encoded protein cleavage sites. As a non-limiting
example,
the primary construct or mmRNA may be signed in an ordered pattern. On such
pattern
follows AXBY form where A and B are coding regions which may be the same or
different coding regions and/or may encode the same or different polypeptides,
and X and
Y are encoded protein cleavage signals which may encode the same or different
protein
cleavage signals. A second such pattern follows the form AXYBZ where A and B
are
coding regions which may be the same or different coding regions and/or may
encode the
same or different polypeptides, and X, Y and Z are encoded protein cleavage
signals
which may encode the same or different protein cleavage signals. A third
pattern follows
the form ABXCY where A, B and C are coding regions which may be the same or
different coding regions and/or may encode the same or different polypeptides,
and X and
Y are encoded protein cleavage signals which may encode the same or different
protein
cleavage signals.
[000297] In one embodiment, the polypeptides, primary constructs and mmRNA can

also contain sequences that encode protein cleavage sites so that the
polypeptides,
primary constructs and mmRNA can be released from a carrier region or a fusion
partner
by treatment with a specific protease for said protein cleavage site.
134

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[000298] In one embodiment, the polypeptides, primary constructs and mmRNA of
the
present invention may include a sequence encoding the 2A peptide. In one
embodiment,
this sequence may be used to separate the coding region of two or more
polypeptides of
interest. As a non-limiting example, the sequence encoding the 2A peptide may
be
between coding region A and coding region B (A-2Apep-B). The presence of the
2A
peptide would result in the cleavage of one long protein into protein A,
protein B and the
2A peptide. Protein A and protein B may be the same or different polypeptides
of
interest. In another embodiment, the 2A peptide may be used in the
polynucleotides,
primary constructs and/or mmRNA of the present invention to produce two,
three, four,
five, six, seven, eight, nine, ten or more proteins.
Incorporating Post Transcriptional Control Modulators
[000299] In one embodiment, the polynucleotides, primary constructs and/or
mmRNA
of the present invention may include at least one post transcriptional control
modulator.
These post transcriptional control modulators may be, but are not limited to,
small
molecules, compounds and regulatory sequences. As a non-limiting example, post

transcriptional control may be achieved using small molecules identified by
PTC
Therapeutics Inc. (South Plainfield, NJ) using their GEMSTm (Gene Expression
Modulation by Small-Moleclues) screening technology.
[000300] The post transcriptional control modulator may be a gene expression
modulator which is screened by the method detailed in or a gene expression
modulator
described in International Publication No. W02006022712, herein incorporated
by
reference in its entirety. Methods identifying RNA regulatory sequences
involved in
translational control are described in International Publication No.
W02004067728,
herein incorporated by reference in its entirety; methods identifying
compounds that
modulate untranslated region dependent expression of a gene are described in
International Publication No. W02004065561, herein incorporated by reference
in its
entirety.
[000301] In one embodiment, the polynucleotides, primary constructs and/or
mmRNA
of the present invention may include at least one post transcriptional control
modulator is
located in the 5' and/or the 3' untranslated region of the polynucleotides,
primary
constructs and/or mmRNA of the present invention
135

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[000302] In another embodiment, the polynucleotides, primary constructs and/or

mmRNA of the present invention may include at least one post transcription
control
modulator to modulate premature translation termination. The post
transcription control
modulators may be compounds described in or a compound found by methods
outlined in
International Publication Nso. W02004010106, W02006044456, W02006044682,
W02006044503 and W02006044505, each of which is herein incorporated by
reference
in its entirety. As a non-limiting example, the compound may bind to a region
of the 28S
ribosomal RNA in order to modulate premature translation termination (See
e.g.,
W02004010106, herein incorporated by reference in its entirety).
[000303] In one embodiment, polynucleotides, primary constructs and/or mmRNA
of
the present invention may include at least one post transcription control
modulator to
alter protein expression. As a non-limiting example, the expression of VEGF
may be
regulated using the compounds described in or a compound found by the methods
described in International Publication Nos. W02005118857, W02006065480,
W02006065479 and W02006058088, each of which is herein incorporated by
reference
in its entirety.
[000304] The polynucleotides, primary constructs and/or mmRNA of the present
invention may include at least one post transcription control modulator to
control
translation. In one embodiment, the post transcription control modulator may
be a RNA
regulatory sequence. As a non-limiting example, the RNA regulatory sequence
may be
identified by the methods described in International Publication No.
W02006071903,
herein incorporated by reference in its entirety.
III. Modifications
[000305] Herein, in a polynucleotide (such as a primary construct or an mRNA
molecule), the terms "modification" or, as appropriate, "modified" refer to
modification
with respect to A, G, U or C ribonucleotides. Generally, herein, these terms
are not
intended to refer to the ribonucleotide modifications in naturally occurring
5'-terminal
mRNA cap moieties. In a polypeptide, the term "modification" refers to a
modification
as compared to the canonical set of 20 amino acids, moiety)
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[000306] The modifications may be various distinct modifications. In some
embodiments, the coding region, the flanking regions and/or the terminal
regions may
contain one, two, or more (optionally different) nucleoside or nucleotide
modifications.
In some embodiments, a modified polynucleotide, primary construct, or mmRNA
introduced to a cell may exhibit reduced degradation in the cell, as compared
to an
unmodified polynucleotide, primary construct, or mmRNA.
[000307] The polynucleotides, primary constructs, and mmRNA can include any
useful
modification, such as to the sugar, the nucleobase, or the internucleoside
linkage (e.g. to a
linking phosphate / to a phosphodiester linkage / to the phosphodiester
backbone). One
or more atoms of a pyrimidine nucleobase may be replaced or substituted with
optionally
substituted amino, optionally substituted thiol, optionally substituted alkyl
(e.g., methyl
or ethyl), or halo (e.g., chloro or fluoro). In certain embodiments,
modifications (e.g.,
one or more modifications) are present in each of the sugar and the
internucleoside
linkage. Modifications according to the present invention may be modifications
of
ribonucleic acids (RNAs) to deoxyribonucleic acids (DNAs), threose nucleic
acids
(TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked
nucleic acids
(LNAs) or hybrids thereof). Additional modifications are described herein.
[000308] As described herein, the polynucleotides, primary constructs, and
mmRNA of
the invention do not substantially induce an innate immune response of a cell
into which
the mRNA is introduced. Featues of an induced innate immune response include
1)
increased expression of pro-inflammatory cytokines, 2) activation of
intracellular PRRs
(RIG-I, MDA5, etc, and/or 3) termination or reduction in protein translation.
[000309] In certain embodiments, it may desirable to intracellularly degrade a
modified
nucleic acid molecule introduced into the cell. For example, degradation of a
modified
nucleic acid molecule may be preferable if precise timing of protein
production is
desired. Thus, in some embodiments, the invention provides a modified nucleic
acid
molecule containing a degradation domain, which is capable of being acted on
in a
directed manner within a cell. In another aspect, the present disclosure
provides
polynucleotides comprising a nucleoside or nucleotide that can disrupt the
binding of a
major groove interacting, e.g. binding, partner with the polynucleotide (e.g.,
where the
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modified nucleotide has decreased binding affinity to major groove interacting
partner, as
compared to an unmodified nucleotide).
[000310] The polynucleotides, primary constructs, and mmRNA can optionally
include
other agents (e.g., RNAi-inducing agents, RNAi agents, siRNAs, shRNAs, miRNAs,

antisense RNAs, ribozymes, catalytic DNA, tRNA, RNAs that induce triple helix
formation, aptamers, vectors, etc.). In some embodiments, the polynucleotides,
primary
constructs, or mmRNA may include one or more messenger RNAs (mRNAs) and one or

more modified nucleoside or nucleotides (e.g., mmRNA molecules). Details for
these
polynucleotides, primary constructs, and mmRNA follow.
Polynucleotides and Primary Constructs
[000311] The polynucleotides, primary constructs, and mmRNA of the invention
includes a first region of linked nucleosides encoding a polypeptide of
interest, a first
flanking region located at the 5' terminus of the first region, and a second
flanking region
located at the 3' terminus of the first region.
[000312] In some embodiments, the polynucleotide, primary construct, or mmRNA
(e.g., the first region, first flanking region, or second flanking region)
includes n number
of linked nucleosides having Formula (Ia) or Formula (Ia-1):
__ yl y5 I B ________________ yl _y5
R3 L.L4,R4 /
% R4
R1),rt
R5 ; (1, j R54-1 \4r
mil mil
y2 y2 \ m,
111
Y3=131 __________________________ Y3=131 ______________
4 4
_ (Ia) (Ia-1) or a
pharmaceutically acceptable salt or stereoisomer thereof,
[000313] wherein
[000314] U is 0, S, N(Ru)õu, or C(Ru)õu, wherein nu is an integer from 0 to 2
and each
RU is, independently, H, halo, or optionally substituted alkyl;
[000315] - -- is a single bond or absent;
[000316] each of R1', R2', R1", R2", Rl, R2, R3, R4, and R5 is, independently,
if present,
H, halo, hydroxy, thiol, optionally substituted alkyl, optionally substituted
alkoxy,
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optionally substituted alkenyloxy, optionally substituted alkynyloxy,
optionally
substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally
substituted
hydroxyalkoxy, optionally substituted amino, azido, optionally substituted
aryl,
optionally substituted aminoalkyl, optionally substituted aminoalkenyl,
optionally
substituted aminoalkynyl,or absent; wherein the combination of R3 with one or
more of
R1', R1", R2', R2", or R5 (e.g., the combination of R1' and R3, the
combination of Rl"
and R3, the combination of R2' and R3, the combination of R2" and R3, or the
combination of R5 and R3) can join together to form optionally substituted
alkylene or
optionally substituted heteroalkylene and, taken together with the carbons to
which they
are attached, provide an optionally substituted heterocyclyl (e.g., a
bicyclic, tricyclic, or
tetracyclic heterocyclyl); wherein the combination of R5 with one or more of
R1', R1",
R2', or R2" (e.g., the combination of R1' and R5, the combination of Rl" and
R5, the
combination of R2' and R5, or the combination of R2" and R5) can join together
to form
optionally substituted alkylene or optionally substituted heteroalkylene and,
taken
together with the carbons to which they are attached, provide an optionally
substituted
heterocyclyl (e.g., a bicyclic, tricyclic, or tetracyclic heterocyclyl); and
wherein the
combination of R4 and one or more of R1' , R3, or R5 can join together to
form optionally substituted alkylene or optionally substituted heteroalkylene
and, taken
together with the carbons to which they are attached, provide an optionally
substituted
heterocyclyl (e.g., a bicyclic, tricyclic, or tetracyclic heterocycly1);each
of m' and m" is,
independently, an integer from 0 to 3 (e.g., from 0 to 2, from 0 to 1, from 1
to 3, or from
1 to 2);
[000317] each of Yl, Y2, and Y3, is, independently, 0, S, Se, -NRN1-,
optionally
substituted alkylene, or optionally substituted heteroalkylene, wherein RN1 is
H,
optionally substituted alkyl, optionally substituted alkenyl, optionally
substituted alkynyl,
optionally substituted aryl, or absent;
[000318] each Y4 is, independently, H, hydroxy, thiol, boranyl, optionally
substituted
alkyl, optionally substituted alkenyl, optionally substituted alkynyl,
optionally substituted
alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy,
optionally
substituted thioalkoxy, optionally substituted alkoxyalkoxy, or optionally
substituted
amino;
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[000319] each Y5 is, independently, 0, S, Se, optionally substituted alkylene
(e.g.,
methylene), or optionally substituted heteroalkylene;
[000320] n is an integer from 1 to 100,000; and
[000321] B is a nucleobase (e.g., a purine, a pyrimidine, or derivatives
thereof), wherein
the combination of B and Ry, the combination of B and R2', the combination of
B and
or the combination of B and R2" can, taken together with the carbons to which
they
are attached, optionally form a bicyclic group (e.g., a bicyclic heterocycly1)
or wherein
the combination of B, Ri", and R3 or the combination of B, R2", and R3 can
optionally
form a tricyclic or tetracyclic group (e.g., a tricyclic or tetracyclic
heterocyclyl, such as in
Formula (IIo)-(IIp) herein),In some embodiments, the polynucleotide, primary
construct,
or mmRNA includes a modified ribose. In some embodiments, the polynucleotide,
primary construct, or mmRNA (e.g., the first region, the first flanking
region, or the
second flanking region) includes n number of linked nucleosides having Formula
(Ia-2)-
(Ia-5) or a pharmaceutically acceptable salt or stereoisomer thereof
__ yl v5 __________________ y1 y5
' U v-U
IV' 1R4
y2
R3 -C2/R4 R R\
y2 \R2
'
y3=I? ______________________ y3I m
=I? ___________________________________________
y4
Y4
¨ (ia-2) ¨ ¨ (Ia-3)
_____ 15u0 y1 y5
IR4
4 R5/ iroR)'
Y2 rnI u
y-2 k m
\ nn'
Y3=I? ______________________ Y3=1? ___________
1
y4
Y4
¨ ¨ ¨ (Ia-5).
[000322] In some embodiments, the polynucleotide, primary construct, or mmRNA
(e.g., the first region, the first flanking region, or the second flanking
region) includes n
number of linked nucleosides having Formula (Ib) or Formula (lb-1):
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R3" u B U B
1iyR3.¨<1 y . R
RI3N1--- R4 \I--- R4
R5 Y2 R5 Y2
1 1
Y3=I? _________________ y=1? __
3
I 4 yI4
Y
_ ¨ (Ib), _ _ (lb-1) or a pharmaceutically
acceptable salt or stereoisomer thereof,
[000323] wherein
[000324] U is 0, S, N(Ru)õu, or C(Ru)õu, wherein nu is an integer from 0 to 2
and each
Ru is, independently, H, halo, or optionally substituted alkyl;
[000325] - - - is a single bond or absent;
[000326] each of Rl, R3', R3", and R4 is, independently, H, halo, hydroxy,
optionally
substituted alkyl, optionally substituted alkoxy, optionally substituted
alkenyloxy,
optionally substituted alkynyloxy, optionally substituted aminoalkoxy,
optionally
substituted alkoxyalkoxy, optionally substituted hydroxyalkoxy, optionally
substituted
amino, azido, optionally substituted aryl, optionally substituted aminoalkyl,
optionally
substituted aminoalkenyl, optionally substituted aminoalkynyl, or absent; and
wherein the
combination of Rl and R3' or the combination of Rl and R3" can be taken
together to form
optionally substituted alkylene or optionally substituted heteroalkylene
(e.g., to produce a
locked nucleic acid);
[000327] each R5 is, independently, H, halo, hydroxy, optionally substituted
alkyl,
optionally substituted alkoxy, optionally substituted alkenyloxy, optionally
substituted
alkynyloxy, optionally substituted aminoalkoxy, optionally substituted
alkoxyalkoxy, or
absent;
[000328] each of Yl, Y2, and Y3 is, independently, 0, S, Se, -NRN1-,
optionally
substituted alkylene, or optionally substituted heteroalkylene, wherein RN1 is
H,
optionally substituted alkyl, optionally substituted alkenyl, optionally
substituted alkynyl,
or optionally substituted aryl;
[000329] each Y4 is, independently, H, hydroxy, thiol, boranyl, optionally
substituted
alkyl, optionally substituted alkenyl, optionally substituted alkynyl,
optionally substituted
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alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy,
optionally
substituted alkoxyalkoxy, or optionally substituted amino;
[000330] n is an integer from 1 to 100,000; and
[000331] B is a nucleobase.
[000332] In some embodiments, the polynucleotide, primary construct, or mmRNA
(e.g., the first region, first flanking region, or second flanking region)
includes n number
of linked nucleosides having Formula (Ic):
__ 1 5
Y ¨Y B3
q _.---- U Rb3
R- 2
B1 B
R5.1' ' 4, - "K
Y2 I b1 Rb2
i R
y3=p _______________
I
Y4
¨ ¨ (Ic), or a pharmaceutically acceptable salt or
stereoisomer
thereof,
[000333] wherein
[000334] U is 0, S, N(Ru)õu, or C(Ru)õu, wherein nu is an integer from 0 to 2
and each
RU is, independently, H, halo, or optionally substituted alkyl;
[000335] - - - is a single bond or absent;
[000336] each of B', B2, and B3 is, independently, a nucleobase (e.g., a
purine, a
pyrimidine, or derivatives thereof, as described herein), H, halo, hydroxy,
thiol,
optionally substituted alkyl, optionally substituted alkoxy, optionally
substituted
alkenyloxy, optionally substituted alkynyloxy, optionally substituted
aminoalkoxy,
optionally substituted alkoxyalkoxy, optionally substituted hydroxyalkoxy,
optionally
substituted amino, azido, optionally substituted aryl, optionally substituted
aminoalkyl,
optionally substituted aminoalkenyl, or optionally substituted aminoalkynyl,
wherein one
and only one of Bl, B2, and B3 is a nucleobase;
[000337] each of Rbl, Rb2, Rb3, R3, and R5 is, independently, H, halo,
hydroxy, thiol,
optionally substituted alkyl, optionally substituted alkoxy, optionally
substituted
alkenyloxy, optionally substituted alkynyloxy, optionally substituted
aminoalkoxy,
optionally substituted alkoxyalkoxy, optionally substituted hydroxyalkoxy,
optionally
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substituted amino, azido, optionally substituted aryl, optionally substituted
aminoalkyl,
optionally substituted aminoalkenyl or optionally substituted aminoalkynyl;
[000338] each of Yl, Y2, and Y3, is, independently, 0, S, Se, -NRN1-,
optionally
substituted alkylene, or optionally substituted heteroalkylene, wherein RN1 is
H,
optionally substituted alkyl, optionally substituted alkenyl, optionally
substituted alkynyl,
or optionally substituted aryl;
[000339] each Y4 is, independently, H, hydroxy, thiol, boranyl, optionally
substituted
alkyl, optionally substituted alkenyl, optionally substituted alkynyl,
optionally substituted
alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy,
optionally
substituted thioalkoxy, optionally substituted alkoxyalkoxy, or optionally
substituted
amino;
[000340] each Y5 is, independently, 0, S, Se, optionally substituted alkylene
(e.g.,
methylene), or optionally substituted heteroalkylene;
[000341] n is an integer from 1 to 100,000; and
[000342] wherein the ring including U can include one or more double bonds.
[000343] In particular embodiments, the ring including U does not have a
double bond
between U-CB3Rb3 or between CB3Rb3_cB2Rb2.
[000344] In some embodiments, the polynucleotide, primary construct, or mmRNA
(e.g., the first region, first flanking region, or second flanking region)
includes n number
of linked nucleosides having Formula (Id):
, B
__ yl y., 1
-J
R3
y2
y3I=R _______
I
Y4
¨ (Id), or a pharmaceutically acceptable salt or stereoisomer thereof,
[000345] wherein
[000346] U is 0, S, N(Ru)õu, or C(Ru)õu, wherein nu is an integer from 0 to 2
and each
Ru is, independently, H, halo, or optionally substituted alkyl;
[000347] each R3 is, independently, H, halo, hydroxy, thiol, optionally
substituted alkyl,
optionally substituted alkoxy, optionally substituted alkenyloxy, optionally
substituted
alkynyloxy, optionally substituted aminoalkoxy, optionally substituted
alkoxyalkoxy,
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optionally substituted hydroxyalkoxy, optionally substituted amino, azido,
optionally
substituted aryl, optionally substituted aminoalkyl, optionally substituted
aminoalkenyl,
or optionally substituted aminoalkynyl;
[000348] each of Yl, Y2, and Y3, is, independently, 0, S, Se, -NRN1-,
optionally
substituted alkylene, or optionally substituted heteroalkylene, wherein RN1 is
H,
optionally substituted alkyl, optionally substituted alkenyl, optionally
substituted alkynyl,
or optionally substituted aryl;
[000349] each Y4 is, independently, H, hydroxy, thiol, boranyl, optionally
substituted
alkyl, optionally substituted alkenyl, optionally substituted alkynyl,
optionally substituted
alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy,
optionally
substituted thioalkoxy, optionally substituted alkoxyalkoxy, or optionally
substituted
amino;
[000350] each Y5 is, independently, 0, S, optionally substituted alkylene
(e.g.,
methylene), or optionally substituted heteroalkylene;
[000351] n is an integer from 1 to 100,000; and
[000352] B is a nucleobase (e.g., a purine, a pyrimidine, or derivatives
thereof).
[000353] In some embodiments, the polynucleotide, primary construct, or mmRNA
(e.g., the first region, first flanking region, or second flanking region)
includes n number
of linked nucleosides having Formula (le):
N
yu"
R6 N __________
¨ (le), or a pharmaceutically acceptable salt or stereoisomer
thereof,
[000354] wherein
[000355] each of U' and U" is, independently, 0, S, N(Ru)õu, or C(Ru)õu,
wherein nu is
an integer from 0 to 2 and each Ru is, independently, H, halo, or optionally
substituted
alkyl;
[000356] each R6 is, independently, H, halo, hydroxy, thiol, optionally
substituted alkyl,
optionally substituted alkoxy, optionally substituted alkenyloxy, optionally
substituted
alkynyloxy, optionally substituted aminoalkoxy, optionally substituted
alkoxyalkoxy,
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optionally substituted hydroxyalkoxy, optionally substituted amino, azido,
optionally
substituted aryl, optionally substituted aminoalkyl, optionally substituted
aminoalkenyl,
or optionally substituted aminoalkynyl;
[000357] each Y5' is, independently, 0, S, optionally substituted alkylene
(e.g.,
methylene or ethylene), or optionally substituted heteroalkylene;
[000358] n is an integer from 1 to 100,000; and
[000359] B is a nucleobase (e.g., a purine, a pyrimidine, or derivatives
thereof).
[000360] In some embodiments, the polynucleotide, primary construct, or mmRNA
(e.g., the first region, first flanking region, or second flanking region)
includes n number
of linked nucleosides having Formula (If) or (If-1):
__ y1 y5 g ¨ ¨
R3LrUt yl R1y5 g
-R4 )õ_,:-.....Ut
-R4
õ -
R2/ 11j R2" R2' y R2"
y2 y2
1 1
Y3=P ___________________________ Y3=P ______
I (zi
y4
_ _ (If), ¨ ¨ (If-1), or a pharmaceutically
acceptable salt or stereoisomer thereof,
[000361] wherein
[000362] each of U' and U" is, independently, 0, S, N, N(Ru)õu, or C(Ru)õu,
wherein nu
is an integer from 0 to 2 and each RU is, independently, H, halo, or
optionally substituted
alkyl (e.g., U' is 0 and U" is N);
[000363] - - - is a single bond or absent;
[000364] each of R1', R2', Ri", R2", R3, and R4 is, independently, H, halo,
hydroxy, thiol,
optionally substituted alkyl, optionally substituted alkoxy, optionally
substituted
alkenyloxy, optionally substituted alkynyloxy, optionally substituted
aminoalkoxy,
optionally substituted alkoxyalkoxy, optionally substituted hydroxyalkoxy,
optionally
substituted amino, azido, optionally substituted aryl, optionally substituted
aminoalkyl,
optionally substituted aminoalkenyl, optionally substituted aminoalkynyl, or
absent; and
wherein the combination of R1' and R3, the combination of Ri" and R3, the
combination
of R2' and R3, or the combination of R2" and R3 can be taken together to form
optionally
substituted alkylene or optionally substituted heteroalkylene (e.g., to
produce a locked
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nucleic acid);each of m' and m" is, independently, an integer from 0 to 3
(e.g., from 0 to
2, from 0 to 1, from 1 to 3, or from 1 to 2);
[000365] each of Yl, Y2, and Y3, is, independently, 0, S, Se, -NRN1-,
optionally
substituted alkylene, or optionally substituted heteroalkylene, wherein RN1 is
H,
optionally substituted alkyl, optionally substituted alkenyl, optionally
substituted alkynyl,
optionally substituted aryl, or absent;
[000366] each Y4 is, independently, H, hydroxy, thiol, boranyl, optionally
substituted
alkyl, optionally substituted alkenyl, optionally substituted alkynyl,
optionally substituted
alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy,
optionally
substituted thioalkoxy, optionally substituted alkoxyalkoxy, or optionally
substituted
amino;
[000367] each Y5 is, independently, 0, S, Se, optionally substituted alkylene
(e.g.,
methylene), or optionally substituted heteroalkylene;
[000368] n is an integer from 1 to 100,000; and
[000369] B is a nucleobase (e.g., a purine, a pyrimidine, or derivatives
thereof).
[000370] In some embodiments of the polynucleotides, primary constructs, or
mmRNA
(e.g., Formulas (Ia), (Ia-1)-(Ia-3), (Ib)-(If), and (IIa)-(IIp)), the ring
including U has one
or two double bonds.
[000371] In some embodiments of the polynucleotides, primary constructs, or
mmRNA
(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (llb-2), (IIc-
1)-(IIc-2), (IIn-1),
(IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), each of Rl, R1', and Ri", if present,
is H. In further
embodiments, each of R2, R2', and R2", if present, is, independently, H, halo
(e.g., fluoro),
hydroxy, optionally substituted alkoxy (e.g., methoxy or ethoxy), or
optionally
substituted alkoxyalkoxy. In particular embodiments, alkoxyalkoxy is -
(CH2)s2(0CH2CH2)s1(CH2)s30R', wherein sl is an integer from 1 to 10 (e.g.,
from 1 to 6
or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10
(e.g., from 0
to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R' is H or
C1_20 alkyl). In
some embodiments, s2 is 0, sl is 1 or 2, s3 is 0 or 1, and R' is Ci_6 alkyl.
[000372] In some embodiments of the polynucleotides, primary constructs, or
mmRNA
(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (llb-2), (IIc-
1)-(IIc-2), (IIn-1),
(IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), each of R2, R2', and R2", if present,
is H. In further
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embodiments, each of Rl, Ry, and Ri", if present, is, independently, H, halo
(e.g., fluoro),
hydroxy, optionally substituted alkoxy (e.g., methoxy or ethoxy), or
optionally
substituted alkoxyalkoxy. In particular embodiments, alkoxyalkoxy is -
(CH2)s2(OCH2CH2)si(CH2)s3OR', wherein sl is an integer from 1 to 10 (e.g.,
from 1 to 6
or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10
(e.g., from 0
to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R' is H or
Ci_20 alkyl). In
some embodiments, s2 is 0, sl is 1 or 2, s3 is 0 or 1, and R' is Ci_6 alkyl.
[000373] In some embodiments of the polynucleotides, primary constructs, or
mmRNA
(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (llb-2), (IIc-
1)-(IIc-2), (IIn-1),
(IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), each of R3, R4, and R5 is,
independently, H, halo
(e.g., fluoro), hydroxy, optionally substituted alkyl, optionally substituted
alkoxy (e.g.,
methoxy or ethoxy), or optionally substituted alkoxyalkoxy. In particular
embodiments,
R3 is H, R4 is H, R5 is H, or R3, R4, and R5 are all H. In particular
embodiments, R3 is C1_
6 alkyl, R4 is Ci_6 alkyl, R5 is Ci_6 alkyl, or R3, R4, and R5 are all Ci_6
alkyl. In particular
embodiments, R3 and R4 are both H, and R5 is Ci_6 alkyl.
[000374] In some embodiments of the polynucleotides, primary constructs, or
mmRNA
(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (llb-2), (IIc-
1)-(IIc-2), (IIn-1),
(IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), R3 and R5 join together to form
optionally
substituted alkylene or optionally substituted heteroalkylene and, taken
together with the
carbons to which they are attached, provide an optionally substituted
heterocyclyl (e.g., a
bicyclic, tricyclic, or tetracyclic heterocyclyl, such as trans-3',4' analogs,
wherein R3 and
R5 join together to form heteroalkylene (e.g., -(CH2)biO(CH2)b20(CH2)b3-,
wherein each
of bl, b2, and b3 are, independently, an integer from 0 to 3).
[000375] In some embodiments of the polynucleotides, primary constructs, or
mmRNA
(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (llb-2), (IIc-
1)-(IIc-2), (IIn-1),
(IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), R3 and one or more of R1' , or R5
join
together to form optionally substituted alkylene or optionally substituted
heteroalkylene
and, taken together with the carbons to which they are attached, provide an
optionally
substituted heterocyclyl (e.g., a bicyclic, tricyclic, or tetracyclic
heterocyclyl, R3 and one
or more of R1' , or R5 join together to form heteroalkylene (e.g., -
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(CH2)1,10(CH2)b20(CH2)b3-, wherein each of bl, b2, and b3 are, independently,
an integer
from 0 to 3).
[000376] In some embodiments of the polynucleotides, primary constructs, or
mmRNA
(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (llb-2), (IIc-
1)-(IIc-2), (IIn-1),
(IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), R5 and one or more of Ry, Ri", R2', or
R2" join
together to form optionally substituted alkylene or optionally substituted
heteroalkylene
and, taken together with the carbons to which they are attached, provide an
optionally
substituted heterocyclyl (e.g., a bicyclic, tricyclic, or tetracyclic
heterocyclyl, R5 and one
or more of Ry, Ri", R2', or R2" join together to form heteroalkylene (e.g., -
(CH2)biO(CH2)b20(CH2)b3-, wherein each of bl, b2, and b3 are, independently,
an integer
from 0 to 3).
[000377] In some embodiments of the polynucleotides, primary constructs, or
mmRNA
(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (llb-2), (IIc-
1)-(IIc-2), (IIn-1),
(IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), each Y2 is, independently, 0, S, or -
NRN1-, wherein
RN1 is H, optionally substituted alkyl, optionally substituted alkenyl,
optionally
substituted alkynyl, or optionally substituted aryl. In particular
embodiments, Y2 is
NR -, wherein RN1 is H or optionally substituted alkyl (e.g., C1-6 alkyl, such
as methyl,
ethyl, isopropyl, or n-propyl).
[000378] In some embodiments of the polynucleotides, primary constructs, or
mmRNA
(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (llb-2), (IIc-
1)-(IIc-2), (IIn-1),
(IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), each Y3 is, independently, 0 or S.
[000379] In some embodiments of the polynucleotides, primary constructs, or
mmRNA
(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (llb-2), (IIc-
1)-(IIc-2), (IIn-1),
(IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), Rl is H; each R2 is, independently, H,
halo (e.g.,
fluoro), hydroxy, optionally substituted alkoxy (e.g., methoxy or ethoxy), or
optionally
substituted alkoxyalkoxy (e.g., -(CH2)s2(0CH2CH2),1(CH2),30R', wherein sl is
an integer
from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,
independently, is an
integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to
6, or from 1 to
10), and R' is H or C1_20 alkyl, such as wherein s2 is 0, sl is 1 or 2, s3 is
0 or 1, and R' is
C1_6 alkyl); each Y2 is, independently, 0 or -NRN1-, wherein RN1 is H,
optionally
substituted alkyl, optionally substituted alkenyl, optionally substituted
alkynyl, or
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optionally substituted aryl (e.g., wherein RN1 is H or optionally substituted
alkyl (e.g., C1-
6 alkyl, such as methyl, ethyl, isopropyl, or n-propyl)); and each Y3 is,
independently, 0
or S (e.g., S). In further embodiments, R3 is H, halo (e.g., fluoro), hydroxy,
optionally
substituted alkyl, optionally substituted alkoxy (e.g., methoxy or ethoxy), or
optionally
substituted alkoxyalkoxy. In yet further embodiments, each Y1 is ,
independently, 0 or -
NRN1-, wherein RN1 is H, optionally substituted alkyl, optionally substituted
alkenyl,
optionally substituted alkynyl, or optionally substituted aryl (e.g., wherein
RN1 is H or
optionally substituted alkyl (e.g., Ci_6 alkyl, such as methyl, ethyl,
isopropyl, or n-
propy1)); and each Y4 is, independently, H, hydroxy, thiol, optionally
substituted alkyl,
optionally substituted alkoxy, optionally substituted thioalkoxy, optionally
substituted
alkoxyalkoxy, or optionally substituted amino.
[000380] In some embodiments of the polynucleotides, primary constructs, or
mmRNA
(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (llb-2), (IIc-
1)-(IIc-2), (IIn-1),
(IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), each R1 is, independently, H, halo
(e.g., fluoro),
hydroxy, optionally substituted alkoxy (e.g., methoxy or ethoxy), or
optionally
substituted alkoxyalkoxy (e.g., -(CH2)s2(0CH2CH2),1(CH2),30R', wherein sl is
an integer
from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,
independently, is an
integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to
6, or from 1 to
10), and R' is H or C1_20 alkyl, such as wherein s2 is 0, sl is 1 or 2, s3 is
0 or 1, and R' is
C1_6 alkyl); R2 is H; each Y2 is, independently, 0 or -NRN1-, wherein RN1 is
H, optionally
substituted alkyl, optionally substituted alkenyl, optionally substituted
alkynyl, or
optionally substituted aryl (e.g., wherein RN1 is H or optionally substituted
alkyl (e.g., Cl
-
6 alkyl, such as methyl, ethyl, isopropyl, or n-propyl)); and each Y3 is,
independently, 0
or S (e.g., S). In further embodiments, R3 is H, halo (e.g., fluoro), hydroxy,
optionally
substituted alkyl, optionally substituted alkoxy (e.g., methoxy or ethoxy), or
optionally
substituted alkoxyalkoxy. In yet further embodiments, each Y1 is ,
independently, 0 or -
NRN1-, wherein RN1 is H, optionally substituted alkyl, optionally substituted
alkenyl,
optionally substituted alkynyl, or optionally substituted aryl (e.g., wherein
RN1 is H or
optionally substituted alkyl (e.g., Ci_6 alkyl, such as methyl, ethyl,
isopropyl, or n-
propy1)); and each Y4 is, independently, H, hydroxy, thiol, optionally
substituted alkyl,
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optionally substituted alkoxy, optionally substituted thioalkoxy, optionally
substituted
alkoxyalkoxy, or optionally substituted amino.
[000381] In some embodiments of the polynucleotides, primary constructs, or
mmRNA
(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (llb-2), (IIc-
1)-(IIc-2), (IIn-1),
(IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), the ring including U is in the I3-D
(e.g., I3-D-ribo)
configuration.
[000382] In some embodiments of the polynucleotides, primary constructs, or
mmRNA
(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (llb-2), (IIc-
1)-(IIc-2), (IIn-1),
(IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), the ring including U is in the a-L
(e.g., a-L-ribo)
configuration.
[000383] In some embodiments of the polynucleotides, primary constructs, or
mmRNA
(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (llb-2), (IIc-
1)-(IIc-2), (IIn-1),
(IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), one or more B is not pseudouridine (y)
or 5-
methyl-cytidine (m5C). In some embodiments, about 10% to about 100% of n
number of
B nucleobases is not y or m5C (e.g., from 10% to 20%, from 10% to 35%, from
10% to
50%, from 10% to 60%, from 10% to 75%, from 10% to 90%, from 10% to 95%, from
10% to 98%, from 10% to 99%, from 20% to 35%, from 20% to 50%, from 20% to
60%,
from 20% to 75%, from 20% to 90%, from 20% to 95%, from 20% to 98%, from 20%
to
99%, from 20% to 100%, from 50% to 60%, from 50% to 75%, from 50% to 90%, from

50% to 95%, from 50% to 98%, from 50% to 99%, from 50% to 100%, from 75% to
90%, from 75% to 95%, from 75% to 98%, from 75% to 99%, and from 75% to 100%
of
n number of B is not y or m5C). In some embodiments, B is not y or m5C.
[000384] In some embodiments of the polynucleotides, primary constructs, or
mmRNA
(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (llb-2), (IIc-
1)-(IIc-2), (IIn-1),
(IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), when B is an unmodified nucleobase
selected from
cytosine, guanine, uracil and adenine, then at least one of Yl, Y2, or Y3 is
not 0.
[000385] In some embodiments, the polynucleotide, primary construct, or mmRNA
includes a modified ribose. In some embodiments, the polynucleotide, primary
construct,
or mmRNA (e.g., the first region, the first flanking region, or the second
flanking region)
includes n number of linked nucleosides having Formula (IIa)-(IIc):
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________ y1 y5
B ____yl_y5 B
\1U,RI
U
1
R3 __ 02 R4 R4
R2
y2
Y2 Fµ
y3=pI 1
Y3=1? ______________________________________
I I
Y4 y4
¨ ¨ (Ha), _ _ (IIb), or
_______________ yi y5 u g
R3 ____________ R4
R2
y2
I
Y3=Pi ____________
- - (lie), or a pharmaceutically acceptable salt or
stereoisomer
thereof In particular embodiments, U is 0 or C(Ru)nu, wherein nu is an integer
from 0 to
2 and each Ru is, independently, H, halo, or optionally substituted alkyl
(e.g., U is ¨CH2¨
or ¨CH¨). In other embodiments, each of Rl, R2, R3, R4, and R5 is,
independently, H,
halo, hydroxy, thiol, optionally substituted alkyl, optionally substituted
alkoxy, optionally
substituted alkenyloxy, optionally substituted alkynyloxy, optionally
substituted
aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted
hydroxyalkoxy,
optionally substituted amino, azido, optionally substituted aryl, optionally
substituted
aminoalkyl, optionally substituted aminoalkenyl, optionally substituted
aminoalkynyl, or
absent (e.g., each Rl and R2 is, independently, H, halo, hydroxy, optionally
substituted
alkyl, or optionally substituted alkoxy; each R3 and R4 is, independently, H
or optionally
substituted alkyl; and R5 is H or hydroxy), and --- is a single bond or double
bond.
[000386] In particular embodiments, the polynucleotidesor mmRNA includes n
number
of linked nucleosides having Formula (IIb- 1 )-(IIb -2) :
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_
_y1_y5 u g _y 1 _y5 B
R2
y2 R2' y2
I 1
y3=1? _________________________ Y3=I? _____
yi4 yi4
- - (IIb-1) or ¨ ¨ (IIb-2) or a pharmaceutically
acceptable salt or stereoisomer thereof In some embodiments, U is 0 or
C(Ru)õõ,
wherein nu is an integer from 0 to 2 and each Ru is, independently, H, halo,
or optionally
substituted alkyl (e.g., U is ¨CH2¨ or ¨CH¨). In other embodiments, each of Rl
and R2 is,
independently, H, halo, hydroxy, thiol, optionally substituted alkyl,
optionally substituted
alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy,
optionally
substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally
substituted
hydroxyalkoxy, optionally substituted amino, azido, optionally substituted
aryl,
optionally substituted aminoalkyl, optionally substituted aminoalkenyl,
optionally
substituted aminoalkynyl, or absent (e.g., each Rl and R2 is, independently,
H, halo,
hydroxy, optionally substituted alkyl, or optionally substituted alkoxy, e.g.,
H, halo,
hydroxy, alkyl, or alkoxy). In particular embodiments, R2 is hydroxy or
optionally
substituted alkoxy (e.g., methoxy, ethoxy, or any described herein).
[000387] In particular embodiments, the polynucleotide, primary construct, or
mmRNA
includes n number of linked nucleosides having Formula (IIc-1)-(IIc-4):
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_______ Y1 Y5
Y1 Y5 0
11 11
n2 n2
Y2 rµ Y2 rµ
y3=pI 3_ I
y _p __
Y4 Y4
¨ (lie-1), ¨ (IIc-2),
_______ Y1 Y5
Y1 Y5 0
111
R3 __
n2
Y2 rµ Y2 rµ
y3=pI 3_ I
y _p __________________________________________
Y4 Y4
¨ (IIc-3), or ¨ (IIc-4), or a
pharmaceutically acceptable salt or stereoisomer thereof In some embodiments,
U is 0
or C(Ru)nu, wherein nu is an integer from 0 to 2 and each Ru is,
independently, H, halo,
or optionally substituted alkyl (e.g., U is ¨CH2¨ or ¨CH¨). In some
embodiments, each
of Rl, R2, and R3 is, independently, H, halo, hydroxy, thiol, optionally
substituted alkyl,
optionally substituted alkoxy, optionally substituted alkenyloxy, optionally
substituted
alkynyloxy, optionally substituted aminoalkoxy, optionally substituted
alkoxyalkoxy,
optionally substituted hydroxyalkoxy, optionally substituted amino, azido,
optionally
substituted aryl, optionally substituted aminoalkyl, optionally substituted
aminoalkenyl,
optionally substituted aminoalkynyl, or absent (e.g., each Rl and R2 is,
independently, H,
halo, hydroxy, optionally substituted alkyl, or optionally substituted alkoxy,
e.g., H, halo,
hydroxy, alkyl, or alkoxy; and each R3 is, independently, H or optionally
substituted
alkyl)). In particular embodiments, R2 is optionally substituted alkoxy (e.g.,
methoxy or
ethoxy, or any described herein). In particular embodiments, Rl is optionally
substituted
alkyl, and R2 is hydroxy. In other embodiments, Rl is hydroxy, and R2 is
optionally
substituted alkyl. In further embodiments, R3 is optionally substituted alkyl.
[000388] In some embodiments, the polynucleotide, primary construct, or mmRNA
includes an acyclic modified ribose. In some embodiments, the polynucleotide,
primary
construct, or mmRNA (e.g., the first region, the first flanking region, or the
second
flanking region) includes n number of linked nucleosides having Formula (IId)-
(IIf):
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___ Y1 Y5
B 4 _______ Y1 Y5
B 4 __________________________________________________ Y1 Y5
B 4
C r
R31Lifp U R R1 lUV
R3
R5 R5 R5 m.2 n2 Cm.2
Y2 rµ Y2 rµ Y2 rµ
I I I 4
Y4 Y4 Y
¨ ¨ (lid), ¨ ¨ (He), or ¨ ¨
(Ili), or a pharmaceutically acceptable salt or stereoisomer thereof
[000389] In some embodiments, the polynucleotide, primary construct, or mmRNA
includes an acyclic modified hexitol. In some embodiments, the polynucleotide,
primary
construct, or mmRNA (e.g., the first region, the first flanking region, or the
second
flanking region) includes n number of linked nucleosides Formula (IIg)-(IID:
__ y1 y5 B _____________ y1 y5 B
R3'ij "'WI R3:riji "R4
R5 i .., .IR'' R1" R5 R'i ' R1"
y2 12 y2 12'
1
y31=1? _______________________ y3=P1 ____________
1 4 1 Y 4
Y
_ _ (hg), _ _ (IIh),
_
__ y1 y5 B3 ______________ y1 y5 B3
R3'lj '''Rb3 R32rUliiRb3
R5 B1 B2 R5 B1 B2
"Rb2 - iRb2
y2 ib1 y2 : b1
R
I I
y3=1? _________________________ y3=1? ___________
I 4 I
Y Y4
_ _ (Iii), or _ _ (IIj), or a
pharmaceutically acceptable salt or stereoisomer thereof
[000390] In some embodiments, the polynucleotide, primary construct, or mmRNA
includes a sugar moiety having a contracted or an expanded ribose ring. In
some
embodiments, the polynucleotide, primary construct, or mmRNA (e.g., the first
region,
the first flanking region, or the second flanking region) includes n number of
linked
nucleosides having Formula
(IIk)-(IIm):
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_______________ y1_y5 u g __ yl_y5
U D
R5 '1
R3 R4 R3 R4
2
y2 m y2
y3=1? __________________________ y3=P1 _____
Y4 y4
- (Ilk), ¨ (Ill), or
__ y1 y5
R3% '" R4
R5> R1"
= R2"
y2
Y3=1? _______________
4
(IIm),or a pharmaceutically acceptable salt or stereoisomer
thereof, wherein each of R1', Ri", R2', and R2" is, independently, H, halo,
hydroxy,
optionally substituted alkyl, optionally substituted alkoxy, optionally
substituted
alkenyloxy, optionally substituted alkynyloxy, optionally substituted
aminoalkoxy,
optionally substituted alkoxyalkoxy, or absent; and wherein the combination of
R2' and
R3 or the combination of R2" and R3 can be taken together to form optionally
substituted
alkylene or optionally substituted heteroalkylene.
[000391] In some embodiments, the polynucleotide, primary construct, or mmRNA
includes a locked modified ribose. In some embodiments, the polynucleotide,
primary
construct, or mmRNA (e.g., the first region, the first flanking region, or the
second
flanking region) includes n number of linked nucleosides having Formula (In):
__ y 1 y5
11
R3\ ____________ R4
y3=pI
¨ (IIn), or a pharmaceutically acceptable salt or stereoisomer
thereof, wherein R3' is 0, S, or -NRN1-, wherein RN1 is H, optionally
substituted alkyl,
optionally substituted alkenyl, optionally substituted alkynyl, or optionally
substituted
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aryl and R3" is optionally substituted alkylene (e.g., -CH2-, -CH2CH2-, or -
CH2CH2CH2-)
or optionally substituted heteroalkylene (e.g., -CH2NH-, -CH2CH2NH-, -CH2OCH2-
, or -
CH2CH2OCH2-)(e.g., R3' is 0 and R3" is optionally substituted alkylene (e.g., -
CH2-, -
CH2CH2-, or -CH2CH2CH2-)).
[000392] In some embodiments, the polynucleotide, primary construct, or mmRNA
includes n number of linked nucleosides having Formula (IIn-1)-(II-n2):
__ yi y5 u B1 5
_______________________________ Y -Y
U B
R __________________________________ 3\
R ---
-2-R-'
Y Y2 0
y3=pI 3_ I
y _p __
I I
Y4 Y4
¨ ¨ (IIn-1) or ¨ ¨ (IIn-2), or a
pharmaceutically acceptable salt or stereoisomer thereof, wherein R3' is 0, S,
or
wherein RN1 is H, optionally substituted alkyl, optionally substituted
alkenyl, optionally
substituted alkynyl, or optionally substituted aryl and R3" is optionally
substituted
alkylene (e.g., -CH2-, -CH2CH2-, or -CH2CH2CH2-) or optionally substituted
heteroalkylene (e.g., -CH2NH-, -CH2CH2NH-, -CH2OCH2-, or -CH2CH2OCH2-) (e.g.,
R3'
is 0 and R3" is optionally substituted alkylene (e.g., -CH2-, -CH2CH2-, or -
CH2CH2CH2-
)).
[000393] In some embodiments, the polynucleotide, primary construct, or mmRNA
includes a locked modified ribose that forms a tetracyclic heterocyclyl. In
some
embodiments, the polynucleotide, primary construct, or mmRNA (e.g., the first
region,
the first flanking region, or the second flanking region) includes n number of
linked
nucleosides having Formula (Ho):
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___ Y1 Y5 ______________________________ Y1 Y5
UR4 -r2T' 2õ U R4 72' "
X 7K T2
R3" ______ ( \N--N ¨R12a R3 .,,. N---"\N_R12a
v1+1-1"
v 12c
R
T1'
y2 y2
y3=pI 3_ I
y _p ______________________________________________________
I I
Y4 Y4
¨ ¨ (Ho) or ¨ ¨ (Hp), or a
pharmaceutically acceptable salt or stereoisomer thereof, wherein R12a5 R12c5
T1', Tr5T2',
T2", V1, and V3 are as described herein.
[000394] Any of the formulas for the polynucleotides, primary constructs, or
mmRNA
can include one or more nucleobases described herein (e.g., Formulas (b1)-
(b43)).
[000395] In one embodiment, the present invention provides methods of
preparing a
polynucleotide, primary construct, or mmRNA, wherein the polynucleotide
comprises n
number of nucleosides having Formula (Ia), as defined herein:
_______________________ y1 y5 B
R3.V-U
R5,,,( R) RI
11
..n,,
y2 152'
1 ' x MI
Y3=P1 _________
1 4
Y
¨ ¨ (Ia),
the method comprising reacting a compound of Formula (Ma), as defined herein:
(
y3 \
y6 __________________________ II:i y1 y5 B
IiLl / RR5 >--"U ,õR4
...
.. .7,4 71i 1,,, R)
M
/ y \
R 2) R m.
I
y3=1? ________________________________ y7
\ /
/ a (Ma),
with an RNA polymerase, and a cDNA template.
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[000396] In a further embodiment, the present invention provides methods of
amplifying a polynucleotide, primary construct, or mmRNA comprising at least
one
nucleotide (e.g., mmRNA molecule), the method comprising: reacting a compound
of
Formula (Ma), as defined herein, with a primer, a cDNA template, and an RNA
polymerase.
[000397] In one embodiment, the present invention provides methods of
preparing a
polynucleotide, primary construct, or mmRNA comprising at least one nucleotide
(e.g.,
mmRNA molecule), wherein the polynucleotide comprises n number of nucleosides
having Formula (Ia), as defined herein:
_______________________ yl y5 B
U,,,,R4
-----;= . 1 .
(Ri,
R5 s 1,K 2..
: \R õ
y2 ikq m, M
I
y3=1? ______________________________________
1 4
Y
_ _ (Ia-1),
the method comprising reacting a compound of Formula (IIIa-1), as defined
herein:
I I
y6 ____________________ p y 1 __ y5 B
\I /
y4 r
R5/ R =) R)'
.....-- .1.,õ
7 mõ
Y3=1? _____________________________ Y7
\ ir,4/ /
a (IIIa-1),
with an RNA polymerase, and a cDNA template.
[000398] In a further embodiment, the present invention provides methods of
amplifying a polynucleotide, primary construct, or mmRNA comprising at least
one
nucleotide (e.g., mmRNA molecule), the method comprising:
[000399] reacting a compound of Formula (IIIa-1), as defined herein, with a
primer, a
cDNA template, and an RNA polymerase.
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[000400] In one embodiment, the present invention provides methods of
preparing a
modified mRNA comprising at least one nucleotide (e.g., mmRNA molecule),
wherein
the polynucleotide comprises n number of nucleosides having Formula (Ia-2), as
defined
herein:
_________________________ yl_y5 B
U
5c.44
R3 -
2
y2 mi
I
Y3=131 ____________________________________
Niii4
- - (Ia-2),
the method comprising reacting a compound of Formula (IIIa-2), as defined
herein:
u
y6 _____________________ p yl y5 B
I U
\ y4 / 4
4
R3 -
2
7 y2 \ m
I µ
y3=Pi __ y7
\ itzt /
/ a (IIIa-2),
with an RNA polymerase, and a cDNA template.
[000401] In a further embodiment, the present invention provides methods of
amplifying a modified mRNA comprising at least one nucleotide (e.g., mmRNA
molecule), the method comprising:
[000402] reacting a compound of Formula (IIIa-2), as defined herein, with a
primer, a
cDNA template, and an RNA polymerase.
[000403] In some embodiments, the reaction may be repeated from 1 to about
7,000
times. In any of the embodiments herein, B may be a nucleobase of Formula (b1)-
(b43).
[000404] The polynucleotides, primary constructs, and mmRNA can optionally
include
5' and/or 3' flanking regions, which are described herein.
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Modified RNA (mmRNA) Molecules
[000405] The present invention also includes building blocks, e.g., modified
ribonucleosides, modified ribonucleotides, of modified RNA (mmRNA) molecules.
For
example, these building blocks can be useful for preparing the
polynucleotides, primary
constructs, or mmRNA of the invention.
In some embodiments, the building block molecule has Formula (Ma) or (IIIa-1):
/ y3 \
y6 __ p yl __ y5 y6 __ p yl __ y5
H B
yl4 /
"R4 \!4. 1
Pk_ R1,
" R5( Ri)
R2,, ====\ R /mi,
y2 \ rn y2 62,
rµ rµ
Y3=I? Y7 ____ Y3=I? Y7
(Ma),
\ y4 /
/ q (IIIa-1) or a
pharmaceutically acceptable salt or stereoisomer thereof, wherein the
substituents are as
described herein (e.g., for Formula (Ia) and (Ia-1)), and wherein when B is an
unmodified
nucleobase selected from cytosine, guanine, uracil and adenine, then at least
one of Yl,
Y2, or Y3 is not 0.
[000406] In some embodiments, the building block molecule, which may be
incorporated into a polynucleotide, primary construct, or mmRNA, has Formula
(IVa)-
(IVb):
\i(3
y6 __ Fi y5 B
7y3
xi 1y 5
4 T
\ Y
0,0 r p
(IVa) or HO OH (IVb), or a pharmaceutically
acceptable salt or stereoisomer thereof, wherein B is as described herein
(e.g., any one of
(b1)-(b43)). In particular embodiments, Formula (IVa) or (IVb) is combined
with a
modified uracil (e.g., any one of formulas (b1)-(b9), (b21)-(b23), and (b28)-
(b31), such
as formula ()1), (b8), (b28), (b29), or (b30)). In particular embodiments,
Formula (IVa)
or (IVb) is combined with a modified cytosine (e.g., any one of formulas (b10)-
(b14),
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(b24), (b25), and (b32)-(b36), such as formula (b10) or (b32)). In particular
embodiments, Formula (IVa) or (IVb) is combined with a modified guanine (e.g.,
any
one of formulas (b15)-(b17) and (b37)-(b40)). In particular embodiments,
Formula (IVa)
or (IVb) is combined with a modified adenine (e.g., any one of formulas (b18)-
(b20) and
(b41)-(b43)).
[000407] In some embodiments, the building block molecule, which may be
incorporated into a polynucleotide, primary construct, or mmRNA, has Formula
(IVc)-
(IVk):
II I I
y6 __ p y _____ 5 y6 __ p vl .
1 ' t-----Y`' n
\ 4 r R3µµ ' Y'vU R1 \ y4 /r *
HO I-2 (IVO, HO 12(ivd.),
/ y3 \ (3\
ii y6 __ A y1,_
y6 __ p y1 5,....._ 5
1
\''4 ir ,\y4 / r
HO R2(IVe), HO R2(ivf),
7 y3 \
(y3\
I I
y6 __ p yl,...... 5 y6 __ A y1 5
1
\y4 1 ir Y,,u13 y4 / 3µµ('v r U /3
R ____ R1
HO R2
m(IVg), Ho bCH3(IVh),
7 y3 \ (3\
i I
y6 __ p yip__ 5 y6 __ A yl 5
1 1
\4 / r 3YvU /3RI y4 iR3µµ r Yvuy
Rµµ R1
Ho F (Ivo, Ha bCH3(Ivi),
161

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7 y3 \ 7 y3 \
y6 __ p NI/I5 y6 __ p y 1 5
\ yi 4 / V ' r Y.,u13 i I U
\ y4 i
R R ' __________________________________ / rR3õ - Ri
Ho "CI (IVk), or Ha i (IV1), or a
pharmaceutically acceptable salt or stereoisomer thereof, wherein B is as
described herein
(e.g., any one of (b1)-(b43)). In particular embodiments, one of Formulas
(IVc)-(IVk) is
combined with a modified uracil (e.g., any one of formulas (b1)-(b9), (b21)-
(b23), and
(b28)-(b31), such as formula (bl), (b8), (b28), (b29), or (b30)). In
particular
embodiments, one of Formulas (IVc)-(IVk) is combined with a modified cytosine
(e.g.,
any one of formulas (b10)-(b14), (b24), (b25), and (b32)-(b36), such as
formula (b10) or
(b32)). In particular embodiments, one of Formulas (IVc)-(IVk) is combined
with a
modified guanine (e.g., any one of formulas (b15)-(b17) and (b37)-(b40)). In
particular
embodiments, one of Formulas (IVc)-(IVk) is combined with a modified adenine
(e.g.,
any one of formulas (b18)-(b20) and (b41)-(b43)).
[000408] In other embodiments, the building block molecule, which may be
incorporated into a polynucleotide, primary construct, or mmRNA, has Formula
(Va) or
(Vb):
R29
/3 \/ ____---1\1, 27
I I y3 \ V7 \ ----1:Z
y6 __ Fr yl ________ D N
y6 ________________________________ ig yl I N
\ y4 4 R3y,1,, , ili 1 4 U
.7.- -(-:. r =
R3
rn (Va) or y-7 -1:z2 (Vb),
or a
pharmaceutically acceptable salt or stereoisomer thereof, wherein B is as
described herein
(e.g., any one of (b1)-(b43)).
[000409] In other embodiments, the building block molecule, which may be
incorporated into a polynucleotide, primary construct, or mmRNA, has Formula
(IXa)-
(IXd):
162

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l y3 \ 7 Y3
II
y6 ___________ p ylr_____ 5 __ y6 ig yl 5
1
\(4 Jr VI \i4Jr V/3
HO F (IXa), Ho Br (IXb),
y6 __ ig yl 5 V/
yoa ____________________________ II 4
1
.... __
HO al (IXc), or H6 1 (IXd), or a
pharmaceutically
acceptable salt or stereoisomer thereof, wherein B is as described herein
(e.g., any one of
(b1)-(b43)). In particular embodiments, one of Formulas (IXa)-(IXd) is
combined with a
modified uracil (e.g., any one of formulas (b1)-(b9), (b21)-(b23), and (b28)-
(b31), such
as formula ()1), (b8), (b28), (b29), or (b30)). In particular embodiments, one
of
Formulas (IXa)-(IXd) is combined with a modified cytosine (e.g., any one of
formulas
(b10)-(b14), (b24), (b25), and (b32)-(b36), such as formula (b10) or (b32)).
In particular
embodiments, one of Formulas (IXa)-(IXd) is combined with a modified guanine
(e.g.,
any one of formulas (b15)-(b17) and (b37)-(b40)). In particular embodiments,
one of
Formulas (IXa)-(IXd) is combined with a modified adenine (e.g., any one of
formulas
(b18)-(b20) and (b41)-(b43)).
[000410] In other embodiments, the building block molecule, which may be
incorporated into a polynucleotide, primary construct, or mmRNA, has Formula
(IXe)-
(IXg):
163

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y 3 \ y 3 \
I I I I
y6 __ p k 5 y6 __ p y1, 5
k I I
BH2 ir V/31 \H2 /r V/3
- _
Ha R.- 2 (IXe), HO 2(IXf), or
S
lie
y6 __ p yl, 5
NI(4
r V/3
Ha R2 (IXg), or a pharmaceutically acceptable salt or
stereoisomer
thereof, wherein B is as described herein (e.g., any one of (b1)-(b43)). In
particular
embodiments, one of Formulas (IXe)-(IXg) is combined with a modified uracil
(e.g., any
one of formulas (b1)-(b9), (b21)-(b23), and (b28)-(b31), such as formula ()1),
(b8),
(b28), (b29), or (b30)). In particular embodiments, one of Formulas (IXe)-
(IXg) is
combined with a modified cytosine (e.g., any one of formulas (b10)-(b14),
(b24), (b25),
and (b32)-(b36), such as formula (b10) or (b32)). In particular embodiments,
one of
Formulas (IXe)-(IXg) is combined with a modified guanine (e.g., any one of
formulas
(b15)-(b17) and (b37)-(b40)). In particular embodiments, one of Formulas (IXe)-
(IXg) is
combined with a modified adenine (e.g., any one of formulas (b18)-(b20) and
(b41)-
(b43)).
[000411] In other embodiments, the building block molecule, which may be
incorporated into a polynucleotide, primary construct, or mmRNA, has Formula
(IXh)-
(Da):
164

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II II
___ p yl 5 y6
y6
R1 \
1 (L1 jr 0/3 \ y4 /Y

(.0
OH
.. __________________________________________ ..
HO 0 (IXh), Ho t H 3 (IM),
/ y3 7 y3
y6 __ IlLy1 5 y6 __ ig yl 5
1 I VO
\43,
Y r 'Y,,__, \y4 r
%.,n3
H3C ... ..
Ho OH (IXj), or Ho OH (Da), or a
pharmaceutically acceptable salt or stereoisomer thereof, wherein B is as
described herein
(e.g., any one of (b1)-(b43)). In particular embodiments, one of Formulas
(IXh)-(IXk) is
combined with a modified uracil (e.g., any one of formulas (b1)-(b9), (b21)-
(b23), and
(b28)-(b31), such as formula (bl), (b8), (b28), (b29), or (b30)). In
particular
embodiments, one of Formulas (IXh)-(IXk) is combined with a modified cytosine
(e.g.,
any one of formulas (b10)-(b14), (b24), (b25), and (b32)-(b36), such as
formula (b10) or
(b32)). In particular embodiments, one of Formulas (IXh)-(IXk) is combined
with a
modified guanine (e.g., any one of formulas (b15)-(b17) and (b37)-(b40)). In
particular
embodiments, one of Formulas (IXh)-(IXk) is combined with a modified adenine
(e.g.,
any one of formulas (b18)-(b20) and (b41)-(b43)).
[000412] In other embodiments, the building block molecule, which may be
incorporated into a polynucleotide, primary construct, or mmRNA, has Formula
(IX1)-
(IXr):
165

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/0 \ /079 \/
HO __ P 0 ___ P ¨o .-.L B HO PO ________ PO B
/r2
I I I I
\OH \OH / r2 \ \ CH3 rl09
/ \ /
õ
Ha bH(IX1), HO bH(IXm),
/0 \ /Se \ /0
HO __ 1¨O ___ l¨o B HO-- IJ-0 B
I
\OH j
/ r2\ 0H A09
/ r \OH Jr 0)
HO OH(IXn), HO -.F (Ixo),
/so II
/0 \ /0
HO-H-0 HO-HP-0 B H04-0 B
p1 B I
r
r
.. _
- ____________________________________ _
¨
Ha al (IXp), HO br(IXq), or Ha 0CH3
(IXr) or a pharmaceutically acceptable salt or stereoisomer thereof, wherein
each rl and
r2 is, independently, an integer from 0 to 5 (e.g., from 0 to 3, from 1 to 3,
or from 1 to 5)
and B is as described herein (e.g., any one of (b1)-(b43)). In particular
embodiments, one
of Formulas (IX1)-(IXr) is combined with a modified uracil (e.g., any one of
formulas
(b1)-(b9), (b21)-(b23), and (b28)-(b31), such as formula (bl), (b8), (b28),
(b29), or
(b30)). In particular embodiments, one of Formulas (IX1)-(IXr) is combined
with a
modified cytosine (e.g., any one of formulas (b10)-(b14), (b24), (b25), and
(b32)-(b36),
such as formula (b10) or (b32)). In particular embodiments, one of Formulas
(IX1)-(IXr)
is combined with a modified guanine (e.g., any one of formulas (b15)-(b17) and
(b37)-
(b40)). In particular embodiments, one of Formulas (IX1)-(IXr) is combined
with a
modified adenine (e.g., any one of formulas (b18)-(b20) and (b41)-(b43)).
[000413] In some embodiments, the building block molecule, which may be
incorporated into a polynucleotide, primary construct, or mmRNA, can be
selected from
the group consisting of:
166

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NN2
NH N
(:
N,...)--...N /0 \ N 3 I 1 N ----N-
4-1
Ho H0L0 0
P-0-yi N NH2
I I r /
H ir \OH
Ha OH (BB- 1), Ha -OH (BB- 2),
NH CI
NN/L N--.../L.N
/9 \ I 0
NN- NN
HO-P-0 HO-(11L0
I O/
\OH iA I r i
OH A
Ho OH (BB- 3), Ho OH (BB- 4),
NH 0
N) .CH3 1\1)*L
)1 /0 \ I NH
H0+0 N.'"i\r NN
0 H04-0
\OH /A i I r /
\OH A
Ho OH (BB- 5), Ho b1-1 (BB- 6),
H3C0
NH2
N,.../N
(--)1N /0 \ I
/9 \ N'. II N ----". N":;-1-- N
HO-T-O-LA .0i N NO-H3-0A04 H2
I
\OH ir QH ir
_ _____________________________________________ /
Ha OH (BB- 7), Ho OH (BB- 8),
0 0
N)LN=CH2 FI).L
/0 \ I
,1/4,----..., 4:2) \ oN 1 r
HO-P-0 " N NH2 õ,,,..., .....;-.A.,
I Ao/ HO-ILO " N NH2
\OH /r I AO'
\OH ir
Ha OH (BB- 9), Ho OH
167

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CI
N--......./L.N
II
HO'-FrO-O+ NH2
\OH ir
(BB- 10), Ha OH (BB- 11), and
0
II
HO-1?-0 N----NNH2
\OH /-r r.-\ '
Ha OH (BB- 12), or a pharmaceutically acceptable salt or
stereoisomer thereof, wherein each r is, independently, an integer from 0 to 5
(e.g., from
0 to 3, from 1 to 3, or from 1 to 5).
[000414] In some embodiments, the building block molecule, which may be
incorporated into a polynucleotide, primary construct, or mmRNA, can be
selected from
the group consisting of:
168

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H2N
c, rN-...N, _NI
N N
/0 \ N
/0 \ I 1
N----õ,-,
p
1-1044-0-Noy - " HO-ILO
, I oq
1
\OH /1. H /r
... ___________________________________________ (.
HO OH (BB- 13), HO -OH (BB- 14),
e 9
o s
\ I\(),)
N N N N
/0 \ /0
NNH04-11=1)-0-yi N
\OH /r H01-P-0-y/
I
Ho OH (BB- 15), Ho OH (BB- 16),
NH2 e
1\1-__N-0
N-----r\j'
H01-0-yi
\OH ir
Ho OH (BB- 17),
0
H'NOhNNH2 /0 il(NH
si
N-..../LN HOi-\0Pi -HO r "0
NN
HO-T-0 r 0"
',)F1
Ha bil (BB- 18), (BB- 19),
0
/0 (NH
---µ
HO¨ILO N 0
I
\OH
r _______________
õ
and Ha OH (BB- 20), or a pharmaceutically acceptable salt
or
169

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stereoisomer thereof, wherein each r is, independently, an integer from 0 to 5
(e.g., from
0 to 3, from 1 to 3, or from 1 to 5) and sl is as described herein.
[000415] In some embodiments, the building block molecule, which may be
incorporated into a nucleic acid (e.g., RNA, mRNA, polynucleotide, primary
construct, or
mmRNA), is a modified uridine (e.g., selected from the group consisting of:
0 0
H3CA HOJ.L
Y3 1 li / y3 1 11H
(
y6 __ ilLy1 N 0 y6 __ p yl N 0
I I
y4r 0) \ y4 0)
r
Fib OH (BB- 21), HO OH (BB- 22),
0
I ).(
/ y3 \ I X
II
y6 __ p_y1 N 0
I
\ y4
r ___________ )
Ha OH (BB- 23),
0
0
H2N.L NH
7Y3 \ \ ANN
/Y3
II NLO
y6 __ p yl y6 __ p yl N 0
I I
\y4 -iA0) \Y4 ---"'s."0
r _____________________________________ r __ )
HO OH (BB- 24), Ho OH (BB- 25),
0 S
A
/y3\ )-NH / y3 I X
y6 __ ig_y1 NLID y6 _ilLy1 N 0
I
\(L1 ----'sV) \ y4 0)
r ______________________________ \ r __
Ha OH (BB- 26), Ho OH (BB- 27),
170

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0
0
/ y3 H)Arti /y3\ ?L NH
N,N=L0
y6 _p_yl
y6_yl
(L1- 1
0 \ y4 -----":\õ0õ)
\ r __
Ha OH (BB- 28), Ho OH (BB- 29),
0
0
HNAN/CH3
H3C,NANH Y3 \
/y3 \ y6 __ ig yl
ii ro
y6 __ p _y1
(
\ yl 4 ---4-:\,0 zi
Ho OH (BB- 30), Ho OH (BB- 31),
0 0 0
F3CA )c70LL
0CH3
/y3 \ t XI HN 1
y6 __ pli_y1 N 0 i\i
Y ¨Pi -Y)1 s-' n -
\i4 i 0) (Lt 0)
r r
Ho OH (BB- 32), HO OH (BB-
0
0
)-7.y0CH3 HNNH2
\ HN
( 3 \ j
y3 \ j
Y I -Y1 ON 0
6
+4 ii7r0) Y
Y 171 Y/I-A1 CI Oi
33), Ha OH (BB- 34), Ho OH (BB-
171

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0 0 0
HN)-1-.....7-,..NCF3
7
Y3 \ Y3 \ HN 1 NH2
ii 0 N H 7 j
ii SN
y6_p_y1 y6_p_y1
1 1
\ y4 /7::\r0)
35), Ho OH (BB- 36), H6 OH
0 0
)=
HN 1 N C F3
y3 ),,1 H
y ¨ig¨yi S- -1\1
6(
)!/4 0)
r
(BB- 37), Ha OH (BB- 38),
0
CH3
H)\1!Vri 0
Y3 HN
1 NCH3
y6(ig_yi 0 N y Y3 \ i
/1 0) ilLyi 0 N 0 CF3
1
1-7 \,0)
- --__
HO OH (BB- 39), Ho OH (BB-
0
1-11\1N--Thr
6(I(I 3N OCF3C)
Y Pi ¨Y1
N;f4 0)
r
),_
40), Ha OH (BB- 41),
0 0
HN)-N.r0H
HN)-N.r0H
/ y3 (:)
),,\ N j H
0 / v3 ) S
H
0
ii " - III "-N
µ,61_0¨µ,1 µ,61_0¨µ,1
' 'I ' ' 'I '
\ y4 o \ y4 0
r r
_ ___________ ..
Ha OH (BB- 42), Ha OH
172

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0
N ...r0H
HN)CV
Y3 \ 0 N 0
y _ilLyi
j
)1(4 )
/ r ______ H
6(
(BB- 43), Ha 0CH3 (BB- 44),
0
HN)c,Nr0Fmoc
_1_13 \ 1 H 0
y yl
0 N
Nir4 /AO)
6( i
Ha OH (BB- 45),
0
)czN .r0Fmoc
, HN - - N
Y6( 14-3 Y/17.4\r\i S j H
0
- -
HO OH (BB-46),
0
HN )czN.r0Fmoc
_ir \ H
i
y _Nit
6( 0 Nj
NI(4 'Ai 0)
/ r
0
y6 il t_L
r Ho 6cNH3 (BB- 47),
o
A 7L
coN2F:Fmoc
3 oc
I -Y1 N 0
y4 0)
- ____________ _
HO OH (BB- 48),
173

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0 CO2H
).L
/e N NH2
\ t
y6 _y1
\ -
I
\ y4 irT-V))
Ha ol-I (BB- 49),
0 0 0 0
),,O0Fmoc HN)70-LOH
/ y3 ),s\rHI j
y3 \ j
I I 0 N
0 N
y6_ p_yl Y
r
Ho OH (BB- 50), Ho OH
0 OFmoc
OFmoc
\ HNri
_YA3 \i ON" 0
y _y
6(
NI(4 -AO)
/ r
z s_
(BB- 51), HO OH (BB- 52),
0 OCOCF3
0 OH
OH HN)yy0Me
(\ HN)YY y3 i 0
r
3 \ i
y _Nit
6( 0 N
0
NI(4 ;TAO) y _ilLy1 N
(LI 0)
HO OH (BB- 53), Ho OH (BB-
174

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0 OH 0
OMe ),y0Me
, HN).VY , HN 1
/ Y3 \ ONi 0 Y3 \ ON 0
ii
Y6 _ p _y1 y _IlLy1
\ 2r7V)) ilLi ;AO)
54), 1-16 OH (BB- 55), Ha OH
0
).r0Me
, HN 1
7 Y3 \
ONi 0
y6 _ilLy1
1
\ y4 / 1,0)
(BB-56), H6 0- CH3 (BB-57),
0 0
)cv.r0Me
FINN--CH3
HN 1
7 Y3 ), \ Y3 H
\
ii S N 0 7 II 0 N j
p_y 1 y6 _ p _y
Y6 _Y)
1 1
\ y4 0) y4 /1,C))
r
Ha OH (BB- 58), Ha OH
175

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0
HNI N-CH3
H
y6_1Ly1 S N
\ !r4 0)
(BB- 59), HO OH (BB- 60),
0
HN)CN -CH
3
Y3 /
y _Ig_yi Se N I H
6(
(L1 0)
r
Ha OH (BB-61),
0
NH2
HN).r
7 y3 I 0
yo
,, I I 1 0 N
1
\ y4 0)
r
Ha OH (BB- 62),
0
HN
7 y3 \ ACcN H2
I I 0 N
y6_p_yl
1
\ y4 /1-7-\/0)
Ho oCH3 (BB- 63),
176

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0 CO2Fmoc
H3CNANNHFmoc
Y3 \
6
v ( ig yl 0
1 1
Ho OH (BB- 64),
0 CO2H
H3C,NANNH2
Y3 \
6
v ( ig yl 0
1 1
Ho OH (BB- 65),
0
)czy0H
HN 1
II 0 N
y6_p_yl
NI(4 0)
r
Ho OH (BB- 66),
0
yF3 ))yr0 MOC
0
HN 1
y ig_yi 0 N
6(
(41 0)
r
Ho OH (BB- 67),
177

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0
H N )C7 N
7 y3
y6F, j H
1 1
)
ryi 0 N
\ y4 0
r
Ho OH (BB-68),
0
H N
7 y3 j 11
y6 yi S N
\ yl 4 0)
r
Ho OH (BB- 69),
0
H N
7 y3 ! il
y6 il:Lyi - (-) N
\ (zi 0)
r
Ho oCH3 (BB- 70),
178

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0
0
HNAN
H3C,,,AN-CH3
7 Y3
/y3\ IN
, ,,
y6 __ ilLy1 o yo_Fry 1
(LI -(:) \ y4 0
\ r
r ____________
Ha OH (BB- 71), Ho OH (BB- 72),
0 0
6(
\ HNAN/ \ HNAN
y3 \
y ¨_yl
NI4 0
/ r Y3 \
ii
Y _ry1
(
y4 --'---4\r
ir
Ha OH (BB- 73), Ha OH (BB-
0 0
HNAN HNAN''
/y3\ /y3\
y6HLy1 0 y6_,g_y1 0
r r
74), Ho OH (BB- 75), Ho OH
0
µ HNAN
ye(y3 \
ig_y1
I
(BB- 76), Ha OH (BB- 77),
179

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0
0
HN A NrOH HN)C71
Y3 / y3 ), i
, 1 1
y6(_y).._.C:lri yo_p_y1 0 N
1
y4 0 \ y4 r 0)
r
Ha OH (BB- 78), Ho OH (BB-
0
HNI o).1
), i HN)I
Y3 \
7 y3 0 N i
y6_ig_yl
1 y _ig 0 N
\ y4 r 0)Ty)
79), Ho OH (BB- 80), Ha OH
0
).c7\
7 y3 HN j
II 0 N
y6_p_y
41
l
\ ( 0)
r
),,
(BB- 81), Ho OH (BB- 82),
0 0
HN HNOH
)
/ y3 \ j 0 N / y3 \ j
µ,6_ig_µ,1 µ,6_0_µ,1 0
1 I 1 1 'T '
HO OH (BB- 83), Ha OH (BB-
180

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0
0
)*LI ).NH
/ y3 NH / \I(13 t
I
v6 __________ ig yi N .L0 y6 __ p yi N 0
1 I0
\4 ONJ \f4
r H
r
iCH3
84), Ho OH (BB- 85), HO tH3 (BB- 86),
O o
INH A, NH
7 Y 3 / Y3 \ &
v6 _______ ig yi N .LO v6 __ ig y1 N 0
1 I A I I
r ,-
H3d ____________
Ho OH (BB- 87), H(57:6 (BB- 88),
O 0
7 3
INH tAyH
Y y3
y6 _______ ig yi N y6( A yi NO
I,
\
(zi 0)
r r
Ha -I (BB- 89), Ho CI (BB- 90),
o o
AI NH AI NH
/ y3 /y3\
y6 _______ ig yi NO y6 __ ig yi N
I I
\(4 0) \(4 0)
r r
H6 Br (BB- 91), Ho -I (BB- 92),
181

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0 0
A ANH
/y3\ t 11F1
/ y3 \ t
I I
y6 __ ig yl 'N 'O y6 __ p y1 N 0
I
\ y4r 0) \y4
Ha tH3 (BB- 93), HO Ocl--13 (BB- 94),
0 0
A ,cH3 1-13c0
1 N
7 Y3 7 NH
y3 t
y6 __ ig y I NO v6 __ i¨yl N 0
I I
\ y-rA I r 0) \ y4 r 0)
Ho OH (BB- 95), HO OH (BB- 96),
H3C 0 s
ANH
/ y3 t yH HN
y6 __ ig yi N 0 y6 __ ir yl r0
1
\ y-r,
r 0) (z1 r 0
Ho OH (BB- 97), HO OH (BB- 98),
0 0
HNANH H3C,NANH
Y3 \
CI3

v6 \ )rS v6 __ ig yl S
D yl
"1 1 1
y4 0 y4 0
Ho OH (BB- 99), Ho OH (BB- 100),
182

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S
H3CNANH
7Y3 \
y6 __ ig y1
\)r0
1
y4 0
Ho OH (BB-1O1),
0
HN
AN N V\ SO3H
-
7 i(13 H
)
y6_p_y1
1
\ y4 r 0
Ho OH (BB-102),
0
HNAN/\ N SO3Fmoc
/ y3 H
y6 ii:Ly1
1
\ y4 0
r
Ho OH (BB-103),
0
...11-...\ ,...------........õ.....S03H
HN 1 N
O N H
y6_,g_y1
\ (41 0)
r
Ha OH (BB-104),
183

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0
)-7\ SO3FMOC
HN N
/ y3 ), N j H
y6 __y1 0
\ y4 0
r
Ho OH (BB-105),
0
)'cZ\
HN N
SO3H
1
y3 H
y6(11:Ly1 S N
(zi 0)
r
Ho OH (BB-106),
0
HN)=cz N SO3Fmoc
/ y3 ) S
j H
II N
y6 _p_yl
\r
Ha OH (BB-107),
0
\I(3 \ Th\10
y6 p y1
( 1
yl4
)T' )
HO OH (BB- 108),
184

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0 0
)..L
HN OCH3
y3 j
Y6(11'1-Y1 0 N
Izt 0)
r
Ho -OH (BB-109),
0
).c
HN 1z NH2
y3
y6(11:Lyi 0 N
r
Ho OH (BB-11O),
0
0 0 0
Y3
H3Cj-L
1 Ir
HN
),_
)C)Cj OC H 3
y6 ( A yl NS y3
yi 4 y _I
0 ilLy1 O N
)
\A 0)
r r
Ha OH (BB-111), Ha OH
9 9
HN (
)c2OI ICH 3
7 y3 ),_
y6 ON O N 0
\ (zt 0)
r
(BB- 112), Ha OH (BB- 113),
185

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0
0
/ y3 \ AI NH
ANCH3
/ y3 \
y6 __ py1
y6 __ A _y N'O
A
\ \!/4
\ \I /17-4\,0)
H
HO OCH3 (BB- 114), a OCH3 (BB- 115),
O 0
HN
A
NH A
HN NH
y3 \ y3 \
4
6 __ ( " 1 LL

L0 y6 ( ig y1 r)
y P 1(,4,,
yl
yl 0
0 4
r
Ho F (BB-116), Ho ol (BB-117),
O 0
HNA NH HNA NH
7 __ NI(13) 0 6 1(3 rLO
N/ ( igy
,),..
i4 1 1
0 y 4
\y
y6 p yL'I- r __
Ha OCH3 (BB-118), HO 1 (BB-119),
O 0
HNANH HNANH
/
y6 __ p-yl 0 y6 I

\
\ y4 Ni(3 6 ( " 1 )rL0
P y,i_.
yl4
1 0
...I.-4NC)
Ha tH3 (BB- 120), Ha OCH3 (BB- 121),
186

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0 0
A
HN NH HNANH
/Y3 \ Y3 \
II 0 y6 __ 1g y 1 cr 0
y6 __ p yl
1
\
______________________ CH3
Ho OH (BB- 122), HO OcH3(BB- 123),
0 0
7 y3 )1NANH / y3 HNA NH
ii )r=L II r
y6 __ p yl 0 y6 __ p yl 0
1 1
\ y4 r 0 \ y4 r 0
: =---_
Ho OH (BB- 124), and HO 0 (BB-
125), or a pharmaceutically acceptable salt or stereoisomer thereof, wherein
Yl, Y3, Y4,
Y6, and r are as described herein (e.g., each r is, independently, an integer
from 0 to 5,
such as from 0 to 3, from 1 to 3, or from 1 to 5)).
[000416] In some embodiments, the building block molecule, which may be
incorporated into a polynucleotide, primary construct, or mmRNA, is a modified
cytidine
(e.g., selected from the group consisting of:
187

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NH2 NH2
I Id3CN
7 Y3 \ Y3 \ 1
y6 __ A yi NO y6 __ A y......õ\1 1\1LQ
yi4
\ yi 4 AO
HO OH (BB-126), HO OH (BB-127),
NH2 NH2
),
rN
(3\ HN - N
0
\ _____________________________________ y 4 Ao N 0
v6 D v I
"1 1
r ________________________________________________ )
.. ______________ _
Ha old (BB- 128), HO OH (BB- 129),
H3C
NH2 / NH
y3 \ 1 1
A yl N - S
(
f.4 -VONJ
Y3
y6 1
y6 ____________________________________ A 1 NO
I Y ()NI
y4
r __________________________________________________ /
.. ______________ :.
Ha old (BB-130), Ho OH (BB-131),
,CH3
NH HN
)-L ,CH3
1 N
Y3 I Y3 1 I
y6 7 A yi NO y6 ( yl N 0
N!izt 0) NI(4 ()
\ r ________________________________ r __
Ho OH (BB-132), Ha OH (BB-133),
188

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,CH3
HN H3C,N,CH3
/y3 t 11
/y3 t 11
y6 __ A y 1 N 0 y6 __ A y 1 N 0
1
\y4 0)0)
r __________________________________ \ r __
HO oCH3 (BB-134), Ho OH (BB-135),
NH2
Fi3C,N,CH3
7y3 HO
y3 \ 1 N
NO
\ 11
1
11
y6 __ p _y1 N 0 y6 __ :i_y1
1 11
\Y4 0) y4 A. 0)
HO oCH3 (BB- 136), Ho OH ( BB-
137),
NHAc NH2
Ac0 N TBDMS,oN
1
\
/y3 \ 1\1LC) y3 \ N 0
11
y6 __ p y1 y6 __ ig _y 1
\
y1 A.
\ 4 yi 4 A
r 0) 0)
Ha OH (BB-138), Ho OH (BB-139),
N H2
NH 2
F3C N
.....,...õ--'L
/ X3 t / y3 t 11
y6 ____________________________________ A y1 N 0
y6 __ 15 y1 NO 1
y14y4 0
0)
\ r __
HO OH (BB-140), HO bl--13 (BB-141),
189

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NH2 NH2
)1 N
/y3 \ 1 I I
Y6( \IIDIC3 yl r\i'Lci
y6 __ ig_y1 1\1 0
I
y14 0)
\ Y4 A-o/CH3
H30' _______________________________________________
HO OH (BB- 142), Ha OH (BB- 143),
NH2 NH2
((),,\)
Y3 1 Y3 1
Y ¨11"¨Y1 N-10 y/¨_yi N-10
I
y14 0) y4 0
HO 0 (BB- 144), HO 0 (BB- 145),
NH2 NH2
/y3 1 y3 1 N
y6 __ A yl NOy6 ________________ A y1 \ NO
)\(L1 y14
0)
r r*
HO Br (BB- 146), HO OH (BB- 147),
NHAc
NH2
)N
NO
\i3 1 I / (13
y6 __ p y1 N 0 v6 __ D v:)s.1, )
/c
IA "I I
\y-r 0) \Y4 0
r _________________________________________ r ____
HO tH3 (BB- 148), Ho OH (BB- 149),
190

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NHAc NH
OHCN
1 1 1
/y3 N 0 y3 \ N 0
y6 __ II:i yl ) y6
1
\ y4 0 y4 0
r ______________
Ha OCH3 (BB-150), HO OH
(BB-151),
NH2 S
OHCJ
I 11 H3C,NAN
7Y3 \ N 0
/ y3 \
NH2
y6 __ ilLy1 y6py1
I 1
\Y4 ---A, 0) \(4 A5,
r
Ha OCH3 (BB- 152), HO OH (BB- 153),
NH2 NH2
Br y Br
7 y3 \ t
y6 __ A yl N 0 y6 __ A yl 1\10
I
\ y14 AO)
\ r _____________________ \ y4
r 0)
Ho -OH (BB- 154), Ho OH (BB- 155),
NH2 H3C NH2
HO N 1\1
t tN/0
/y3 \ k N 0 43 \ 1
II 0
y6_p_e_yi...,...\/ ) y6,_p_y_1..,..\/ )
1
\ +4 0 \ y4 r 0
r __________
Ho 01-1 (BB- 156), Ho OH (BB- 157),
191

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NH
/ y3 \ ). N CO2Fmoc
II I JL
y6y1 N N NHFmoc
I H
\Y4 AO
r )
Ho OH (BB- 158), and
NH
7
).N CO2H
Y3 I *
y6 __ ilLy1 N N NH2
itzl- 0) H
\ r __
Ha OH (BB- 159), or a pharmaceutically
acceptable salt or stereoisomer thereof, wherein Yl, Y3, Y4, Y6, and r are as
described
herein (e.g., each r is, independently, an integer from 0 to 5, such as from 0
to 3, from 1
to 3, or from 1 to 5)). For example, the building block molecule, which may be

incorporated into a polynucleotide, primary construct, or mmRNA, can be:
0 0
H3CI 1111-i)
/9
H
ii )......
HO-H=1-0 N 0 H0,¨P-0 N 0
1
\O r
H 0) \OH 0)
r
\:
- ______________________________________________ _
HO OH (BB- 160) or Ho OH (BB- 161), or a
pharmaceutically acceptable salt or stereoisomer thereof, wherein each r is,
independently, an integer from 0 to 5 (e.g., from 0 to 3, from 1 to 3, or from
1 to 5).
[000417] In some embodiments, the building block molecule, which may be
incorporated into a polynucleotide, primary construct, or mmRNA, is a modified

adenosine (e.g., selected from the group consisting of:
192

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NH2 NH2
/y3 \ e p N N
/y3 \ I II
, 1 1
yo __ p _y1 1\1---- N y6Ly1 N ----- N
1
\Y4 7\O \Y4 AO
r _________________ /CH3 r __ /OH
HO OH (BB- 162), Ha al-13 (BB- 163),
NH2 NH2
N,)m N,)N
r 1 1 1/3
H3d _____________________________________________________ 1
y6 __ p y 1 N-----N- y6 ( A y 1 N-----N
/y3 \

y 4 0)
r ,, r ,,
_ .).
) ____________________________________________________
:----
Hici OH (BB- 164), HO b (BB-
NH2 NH2
7 NN N,)N
Y3 1 Y3 1
y6 ______ 11:i yl N-----N- y6 __ A y 1 N----N-
1
\Y4 fLI
0) 0)
r ________________________________________________________
r __________________
165), Ha F (BB-166), Ha bl
NH2
NN
/y3 1
y6 __________ 11:i yl N-----N
I
\Y4 0)
r _______________________
(BB- 167), Hici Br (BB- 168),
193

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N
NH2 H2
7 3 NN /X3 1
/1
p yl N N
y6 __ p yl N--"N y6 __
y4
10)
0) \Y4 r _____
r ______________
Ha 1 (BB- 169), Ho tH3 (BB-
NH2
/y3 \ N-....)N
1
I I
y6 ______ p y1 N---N
VI Ao
r )
170), Ha OCH3 (BB-171),
N
NH2 H2
/y3 \ ,)L
N......)m
/y3 N..... m :.Q
.1
µ,6 _\/1 n
ii Ni, 4
wo_4
N N
N N I '
T 'T '
y4 0)
\Y4 7o
\ r
Ha OH (BB- 172), HO OH (BB-
NH2N H2
N......)
/y3 \
N1-...,N
1 /y3 \ </ ---I N
ii
ii
y-
Ni6_o_yl N---N 1 v "i
6_1 N N OCH3
\Y4 .A0)
" 1
\ y4 AO
r _______________ )
173), Ha OH (BB- 174), Ho OH
(BB- 175),
194

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OH
NH2
HN
/y3 \ /1\1-,./IN
N....,)
< I / y3 \ i N
< I
y6Ly1
r )N---"kr SCH 3 II
y6_,_p_yl
1,.4 A0)1\1----N SCH3
I
\Y4 AO
\ r
Ha OH (BB- 176), Ha OH
OH
HN
N....,)
/y3
I N
y6 N
yl ----\ %L
1,4 0)I N OCH3
\ r __
(BB- 177), HO OH (BB- 178),
OH NH2
N,)
HN /y3 \ H3C- I N
1
II
/y3 \ N...1 N y6 __ p yl NN
y6 ____ p_y1 N."---Nr \\ y4
I
\ y4 Arj)
-O)
1.-
Ha OH (BB- 179), Ho OH (BB-
NH2 NH2
------L
/y3 \ e 1 )\1
11 y3 ( _____ eN
y6 ______ p yl N----N y6 NN
___________________________________________________ yl N----N
\ fzi.fzi.
0) 0)
r _______________________________ r __
180), Ha OH (BB- 181), Ha OH
195

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NH2
(y3 e,N
y6 __ p yl
NN
y4 r 0)
(BB- 182), Ho OH (BB- 183),
NH2 NH2
(Y p113 1 _11\1 /Y113 Br¨e_INI
Y6( yl N---"N y6 __ p yl N----N
fz1 0) \ y14 0)
r ____________________________________________ r __
Ha OH (BB- 184), Ha OH (BB-
NH2 NH2
N,)
6'13 \ Cl¨e 1 _11\1 Yll3 1¨ 1 _11\1
v6 _______ wl ni y6 __
' 'T '
y14
\Y4
r ________________________________________________________
185), Ha OH (BB-186), Ha OH
NH2
/Y113 FIS- N1 NI
y6 ___________ p yl N.---
1 N
\Y4 0)
r ________________________
(BB- 187), Ha OH (BB- 188),
196

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NH2 NH2
7 Yi 1 3 \S¨e NI N,/L
(
Y 04 113 )s_ 1 1
y6 ______ p y 1 N ---
1 N y6 ________ yi N---N
\ y4 0) 0)
r ____________________________________________ r ___
Ha old (BB- 189), Ha OH (BB-
NH2 ------
NH2
7 Y3 S¨e,INI /Y113 S¨e 1 NI
II ,
yo ___________ p y 1 N----N y6

I 1 N
\Y4 !r ) \Y4 0)
r ________________________________________________________
190), Ho OH (BB-191), Ha OH
\
NH2
I I
yoa p yl N"--N!
\Y4 AO
r ________________________ )
(BB- 192), Ho OH (BB- 193),
NH2 NH
4113 H N-NI\II /y3 \ NN
y i': yl 2 N---- \ I
6 __
I N y6 _______ ilj) y1 N N CH3
---..., ---
\Y4 0) \Y4 TA )0, 1
r _______________________________________ i r __ 7
Ho ohi (BB- 194), Ho OH (BB-
197

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HN
7y3 I I
NN
y6 _____ A yl N-..--tN
I
\Y4 0)
r __________________
195), HO OH (BB-196),
HN
N,/L
' N
(
YE3 1
Y6( __ E.) yl N----N,
yI4 0)
r ______________
Ha old (BB-197),
HN
N,/L
' N
IY13 ), 1
Y6( F., yl N--\j
(
Th,
yI4 0)
r ______________
Ha old (BB-198),
HN
N,)
i N
Y3 \ I
y6( ig yl N-----N
yI4
A- )
Ha OH (BB- 199), and
198

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HNONH2
N,)
N
(
,(23 ), 1
y6 __ !Jr yl N----\N,
yi4 0) 5
r ______________
Ho OH (BB- 200) or a
pharmaceutically acceptable salt or stereoisomer thereof, wherein Yl, Y3, Y4,
Y6, and r
are as described herein (e.g., each r is, independently, an integer from 0 to
5, such as from
0 to 3, from 1 to 3, or from 1 to 5)).
[000418] In some embodiments, the building block molecule, which may be
incorporated into a polynucleotide, primary construct, or mmRNA, is a modified

guanosine (e.g., selected from the group consisting of:
0
0
(
y6 p_yl
_ N __ õA NH
jic3 \ ( I
(.4 Ao N---"N NH2 7 y3 N.....,A
1 r
y6_y1 N-----NNH2
14 0
\ Y
/ r/OH CH3
r ___________
Ha -OH (BB- 201), Ha tH3
0
/y3 ),, NNH
y6 _p_yl
N----N N H2
1
\y4 0)
r .-
H3d :... .:
(BB- 202), HO OH (BB- 203),
0 0
/y3 ): NNH /y3
1 NH
ii I 1
y6 _p_yl
N---- kr N H2 y6Ly1
N----NLN H2
1
\y4 0) \(.4 r 0)
r
_
HO 0 (BB- 204), HO
199

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0
/y3 ________ \ ,N -......,--11-, NH
II _________ I
y6 _________ p _y 1
I
\ y4 0)
\ A-
(BB- 205), Ha CI (BB- 206),
0
0
/y3 \
N NH /y3 N 1 NH
/I
1 y6 N N H2
--- L
y614-yA1 oil ---- Nr NH2 0)
1\1
\ r ________________________ \ r __
.7 s
¨
HO Br (BB- 207), Ha CI (BB-
208),
0
0
N,ANH
43 \ J,N NH /y3 \ < I
ii K I
y6 p)_y1
1\1-NN H2
v6_1_ o _v1 N----Nr NH2 I
' 'T '
\y4 Ao) \Y4 AO)
r r
Ha tH3 (BB- 209), Ho oCH3
0
N,ANH
/y3\ I
Ni_µ,1
NN N
1 I 1 ,0) H
r ____________________
(BB-210), Ho OH (BB-211),
0 0
43 \i
N "")Li NH
H < I /y3 \
11 I
N/6 __ n_v1 N N N y6 __ p y1 NNN
\Y4
' if ' I
.AO) H \Y4 -;:\,o)
H
r _________
Ho OH (BB- 212), Ha OH (BB-
200

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(:)
OCH3
N
1 N
43 \
4
NN 3 \
II \ I
y6_yl N N NH2
y6_Hp_yl
1\1---N NH I
\Nk ....-'N\1 2 \Y4 A. 0)
r
213), Ha old (BB- 214), Ha el
o\/
N) /y3 \ // I N
\ I
II
y6_1.1,-_: -y1 N---- NNH2
\Y4
i0) r V
(BB-215), Ha OH (BB-216),
0
0
N )LNH
/y3 \
N) /y3 \
y6_y1 ,,, N NH 1 H3C- I .L
I NI II
ii ....- 1 N-----N NH2
'A
.1(4 cl,1 2 v6_1_o_v \4
Ha OH (BB- 217), Ha ol-1 (BB-
0
/y3 \\ ______________________ e-).(1 NH
---1
y6ziTyl a IN -N NH2
\ r .. 7
218), Ho OH (BB-219),
0
I
NjLNH
/y3 \( __ < N).cH /y3 I
y6_yl N----NL NH2
\,6_4_\,1 N----1\1 NH2 I
" I 1 )
\Y4 AO
\y4 "r\'
r __________ )
Ha OH (BB- 220), Ha OH (BB-
201

CA 02868391 2014-09-24
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0
N,A
7 y3 \ F_ 1 NH
il
y6 _____ p_y1 N--- NNH 2
14 Acd
\ Y r __ 1
-___
221), HO OH (BB- 222),
0
0
N,A NH N,)(NH
43 Cl- I / 3 \ //
X Br-\ I
.---
y6 __ ig _y1 N - N NH2 y6_p_p_yl
N---"N NH
I IONI 2
\y4 0)
r \ / r \ /
. _
_
Ho OH (BB- 223), HO OH (BB-
0
0
N,A NH
/Y3 \
N,A NH 43 \ HS I
1¨ Iii - -6 _i_11:i _s =1
Y ifzi Y,,\ ,cd1\1---NNH2
-6-_-1
Y 1--1:4 1r,,\ .0, iii--NLNEI2
\ ir v __ 7 \Y
v
_ __ 1
224), Ho OH (BB- 225), HO OH
0
/ _ \ \ N......}..
NH
i ri N s¨ I
,6_,_,g_õ1 N N NH2
\Y4 -
1 1 1

N\,0
r __ )
(BB- 226), Ho OH (BB- 227),
0 (1:311
r ____________
N,A
/y s_ I I] Fl y3 \ s_N NH
II
y6 __ ilLy1
N N NH2 v/6 ig_wi N-----
1 (zi. M A N NH2
\
I
yl 0)
/ r _________________________________________________
_ 7
Ha OH (BB- 228), Ha OH
202

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V
/y3 s_e NH
ii ---1
y6_y1 N -N NH2
I
\y4 0)
r
(BB- 229), Ha OH (BB- 230),
\
W 0
N NH /y3 \
N.....)LNH
/y3 \s¨
\ I
II
L
y6 y1
N------ N N H2 y6_Nit',1A m,
......i, ......
¨ N N
y4 AO) \Y4 0) H
\ r r
.. ________________________________________________ ._
Ho OH (BB- 231), Ho ol-I (BB-
0 S
/X3
N 43 \ -.....A NH N.,...)L NH
I <
ii I
y6_y1 N----,..N%L.N..--' y6_y1
1\1----N NH
I
\Y4 0) I (.4 VICI) 2
\
r ___________________________________________________ / r \
232), Ha OH (BB- 233), Ho OH
S
/y3 \
I
ii
y6_y1 N N NH2
1
\Y4 AO)
- ____________________ ..
(BB- 234), Ha OH (BB- 235),
0
N-õA ,CH3 0
/ii3 I 11 /Y3 \ H N-N.----)1NH
y6 __ p_y1 ii
0)N----N NH2 2 _____,....
1.1,1,....
I y6_Hp_yl
N N NH2
\ yzi
\ NI(4 -AO)
r r _
Ho OH (BB- 236), and Ha ol-I
(BB- 237), or a pharmaceutically acceptable salt or stereoisomer thereof,
wherein Yl, Y3,
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Y4, Y6, and r are as described herein (e.g., each r is, independently, an
integer from 0 to 5,
such as from 0 to 3, from 1 to 3, or from 1 to 5)).
[000419] In some embodiments, the chemical modification can include
replacement of
C group at C-5 of the ring (e.g., for a pyrimidine nucleoside, such as
cytosine or uracil)
with N (e.g., replacement of the >CH group at C-5 with >NRN1 group, wherein
RN1 is H
or optionally substituted alkyl). For example, the building block molecule,
which may be
incorporated into a polynucleotide, primary construct, or mmRNA, can be:
0 0
HNANH H3C,NANH
/0 /0 \
II
HO¨A-0 cr0 HO¨P-0 c)r.L0
I I
\OH r \OH /A
r
Ha OH (BB- 238) or Ha OH (BB-
239)
0
0 H3Ci A CH3
HO _________ 0
CH3 \IN-
HNAN / 0
/ 0 II
õ ro HO
PI \\A
\ OH 0 OH O
\
/ r ______________________________________________ r
Or Ho OH (BB- 240) or Ho OH
(BB- 241), or a pharmaceutically acceptable salt or stereoisomer thereof,
wherein each r
is, independently, an integer from 0 to 5 (e.g., from 0 to 3, from 1 to 3, or
from 1 to 5).
[000420] In another embodiment, the chemical modification can include
replacement of
the hydrogen at C-5 of cytosine with halo (e.g., Br, Cl, F, or I) or
optionally substituted
alkyl (e.g., methyl). For example, the building block molecule, which may be
incorporated into a polynucleotide, primary construct, or mmRNA, can be:
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NH2 NH2
IN H3CN
/0 \tNc) /9 t
ii
HO¨P-0 HO-nP-0 N 0
I , I
\OH -70 \OH 0)
r ) r
Ha OH (BB- 242) or HO OH (BB- 243)
NH2 NHAc
TBDMS,oN AcON
0
(
NO i 0
HO II:' 0) ) I
OH 0 \
HO
I o
OH At
/ r
Or Ha OH (BB- 244) or Ha OH (BB-
245), or a pharmaceutically acceptable salt or stereoisomer thereof, wherein
each r is,
independently, an integer from 0 to 5 (e.g., from 0 to 3, from 1 to 3, or from
1 to 5).
[000421] In yet a further embodiment, the chemical modification can include a
fused
ring that is formed by the NH2 at the C-4 position and the carbon atom at the
C-5
position. For example, the building block molecule, which may be incorporated
into a
polynucleotide, primary construct, or mmRNA, can be:
H3C
/ NH
/0 I
HO-4-0 N1 0
I
\OH ()
r
: __ s
Ha OH (BB- 246), or a pharmaceutically acceptable salt or
stereoisomer thereof, wherein each r is, independently, an integer from 0 to 5
(e.g., from
0 to 3, from 1 to 3, or from 1 to 5).
Modifications on the Sugar
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[000422] The modified nucleosides and nucleotides (e.g., building block
molecules),
which may be incorporated into a polynucleotide, primary construct, or mmRNA
(e.g.,
RNA or mRNA, as described herein), can be modified on the sugar of the
ribonucleic
acid. For example, the 2' hydroxyl group (OH) can be modified or replaced with
a
number of different substituents. Exemplary substitutions at the 2'-position
include, but
are not limited to, H, halo, optionally substituted C1-6 alkyl; optionally
substituted C1-6
alkoxy; optionally substituted C6_10 aryloxy; optionally substituted C3-8
cycloalkyl;
optionally substituted C3-8 cycloalkoxy; optionally substituted C6_10 aryloxy;
optionally
substituted C6_10 aryl-C1_6 alkoxy, optionally substituted C1_12
(heterocyclyl)oxy; a sugar
(e.g., ribose, pentose, or any described herein); a polyethyleneglycol (PEG), -

0(CH2CH20)õCH2CH2OR, where R is H or optionally substituted alkyl, and n is an

integer from 0 to 20 (e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to
16, from 1 to
4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4, from 2
to 8, from 2
to 10, from 2 to 16, from 2 to 20, from 4 to 8, from 4 to 10, from 4 to 16,
and from 4 to
20); "locked" nucleic acids (LNA) in which the 2'-hydroxyl is connected by a
C1_6
alkylene or C1_6 heteroalkylene bridge to the 4'-carbon of the same ribose
sugar, where
exemplary bridges included methylene, propylene, ether, or amino bridges;
aminoalkyl,
as defined herein; aminoalkoxy, as defined herein; amino as defined herein;
and amino
acid, as defined herein
Generally, RNA includes the sugar group ribose, which is a 5-membered ring
having an
oxygen. Exemplary, non-limiting modified nucleotides include replacement of
the
oxygen in ribose (e.g., with S, Se, or alkylene, such as methylene or
ethylene); addition
of a double bond (e.g., to replace ribose with cyclopentenyl or cyclohexenyl);
ring
contraction of ribose (e.g., to form a 4-membered ring of cyclobutane or
oxetane); ring
expansion of ribose (e.g., to form a 6- or 7-membered ring having an
additional carbon or
heteroatom, such as for anhydrohexitol, altritol, mannitol, cyclohexanyl,
cyclohexenyl,
and morpholino that also has a phosphoramidate backbone); multicyclic forms
(e.g.,
tricyclo; and "unlocked" forms, such as glycol nucleic acid (GNA) (e.g., R-GNA
or S-
GNA, where ribose is replaced by glycol units attached to phosphodiester
bonds), threose
nucleic acid (TNA, where ribose is replace with a-L-threofuranosyl-(3'¨>2)) ,
and
peptide nucleic acid (PNA, where 2-amino-ethyl-glycine linkages replace the
ribose and
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phosphodiester backbone). The sugar group can also contain one or more carbons
that
possess the opposite stereochemical configuration than that of the
corresponding carbon
in ribose. Thus, a polynucleotide, primary construct, or mmRNA molecule can
include
nucleotides containing, e.g., arabinose, as the sugar.
Modifications on the Nucleobase
[000423] The present disclosure provides for modified nucleosides and
nucleotides. As
described herein "nucleoside" is defined as a compound containing a sugar
molecule
(e.g., a pentose or ribose) or a derivative thereof in combination with an
organic base
(e.g., a purine or pyrimidine) or a derivative thereof (also referred to
herein as
"nucleobase"). As described herein, "nucleotide" is defined as a nucleoside
including a
phosphate group. The modified nucleotides may by synthesized by any useful
method, as
described herein (e.g., chemically, enzymatically, or recombinantly to include
one or
more modified or non-natural nucleosides).
[000424] The modified nucleotide base pairing encompasses not only the
standard
adenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs, but
also base
pairs formed between nucleotides and/or modified nucleotides comprising non-
standard
or modified bases, wherein the arrangement of hydrogen bond donors and
hydrogen bond
acceptors permits hydrogen bonding between a non-standard base and a standard
base or
between two complementary non-standard base structures. One example of such
non-
standard base pairing is the base pairing between the modified nucleotide
inosine and
adenine, cytosine or uracil.
[000425] The modified nucleosides and nucleotides can include a modified
nucleobase.
Examples of nucleobases found in RNA include, but are not limited to, adenine,
guanine,
cytosine, and uracil. Examples of nucleobase found in DNA include, but are not
limited
to, adenine, guanine, cytosine, and thymine. These nucleobases can be modified
or
wholly replaced to provide polynucleotides, primary constructs, or mmRNA
molecules
having enhanced properties, e.g., resistance to nucleases through disruption
of the
binding of a major groove binding partner. Table 8 below identifies the
chemical faces of
each canonical nucleotide. Circles identify the atoms comprising the
respective chemical
regions.
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Table 8
Watson-Crick
Major Groove Minor Groove Base-pairing
Face Face Face
I-
0
Cyt Wine; 0P01 )l
.4 .)0
. 0470
&E(SE4 H
Pyrimidines OHO OHO
I./
r,----mi 0 1 f,:
0 o...., _ 0- :.I
=5
,I <
Uridinel 0-P-I -4 " (3.4!õ..,..:-. -,-, <õ0 c''' 3
s'y
---1.
oi-ioii OtiOil (MON
$012 N
.-,> .4
Adenosine: 0-P7P-W:- `N- 031:0
Purines tict-i. 01-04 01-c=H
0 0 n
110.1:
/ 1 .
GUanasine: 0--P-.0, '.:Thi*iz
ol-t6H 01-0N 01-8)H
[000426] In some embodiments, B is a modified uracil. Exemplary modified
uracils
include those having Formula (b1)-(b5):
Tv\ /Tv' R12c R12c
R12c
)c R12a
V1
N NIA
Rio N Rio
m 12a
, N
I
I :2
I
/\ 2"
V...., ....... T2" N
N Rii c) Rii N T N '0
' T2' 1 T2' 1
' (bl), n't (b2),rvvvv,
' (b3),,vs.A.,-,
' (b4),
0
io
R.....,õ.õ.õ...,... _Ri2c
N
I
N,
- N 0
,,,,,L,,,
Or (b5), or a pharmaceutically acceptable salt or stereoisomer
thereof,
[000427] wherein
[000428] is a single or double bond;
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[000429] each of T", Ti", T2', and T2" is, independently, H, optionally
substituted alkyl,
optionally substituted alkoxy, or optionally substituted thioalkoxy, or the
combination of
Tr and Ti" or the combination of T2' and T2" join together (e.g., as in T2) to
form 0 (oxo),
S (thio), or Se (seleno);
[000430] each of Vl and V2 is, independently, 0, S, N(R), or C(Rvb), wherein
nv
is an integer from 0 to 2 and each Rvb is, independently, H, halo, optionally
substituted
amino acid, optionally substituted alkyl, optionally substituted haloalkyl,
optionally
substituted alkenyl, optionally substituted alkynyl, optionally substituted
alkoxy,
optionally substituted alkenyloxy, optionally substituted alkynyloxy,
optionally
substituted hydroxyalkyl, optionally substituted hydroxyalkenyl, optionally
substituted
hydroxyalkynyl, optionally substituted aminoalkyl (e.g., substituted with an N-
protecting
group, such as any described herein, e.g., trifluoroacetyl), optionally
substituted
aminoalkenyl, optionally substituted aminoalkynyl, optionally substituted
acylaminoalkyl
(e.g., substituted with an N-protecting group, such as any described herein,
e.g.,
trifluoroacetyl), optionally substituted alkoxycarbonylalkyl, optionally
substituted
alkoxycarbonylalkenyl, optionally substituted alkoxycarbonylalkynyl, or
optionally
substituted alkynyloxy (e.g., optionally substituted with any substituent
described herein,
such as those selected from (1)-(21) for alkyl);
[000431] Rm is H, halo, optionally substituted amino acid, hydroxy, optionally

substituted alkyl, optionally substituted alkenyl, optionally substituted
alkynyl, optionally
substituted aminoalkyl, optionally substituted hydroxyalkyl, optionally
substituted
hydroxyalkenyl, optionally substituted hydroxyalkynyl, optionally substituted
aminoalkenyl, optionally substituted aminoalkynyl, optionally substituted
alkoxy,
optionally substituted alkoxycarbonylalkyl, optionally substituted
alkoxycarbonylalkenyl,
optionally substituted alkoxycarbonylalkynyl, optionally substituted
alkoxycarbonylalkoxy, optionally substituted carboxyalkoxy, optionally
substituted
carboxyalkyl, or optionally substituted carbamoylalkyl;
[000432] R" is H or optionally substituted alkyl;
[000433] R12a is H, optionally substituted alkyl, optionally substituted
hydroxyalkyl,
optionally substituted hydroxyalkenyl, optionally substituted hydroxyalkynyl,
optionally
substituted aminoalkyl, optionally substituted aminoalkenyl, or optionally
substituted
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aminoalkynyl, optionally substituted carboxyalkyl (e.g., optionally
substituted with
hydroxy), optionally substituted carboxyalkoxy, optionally substituted
carboxyaminoalkyl, or optionally substituted carbamoylalkyl; and
[000434] R12c is H, halo, optionally substituted alkyl, optionally substituted
alkoxy,
optionally substituted thioalkoxy, optionally substituted amino, optionally
substituted
hydroxyalkyl, optionally substituted hydroxyalkenyl, optionally substituted
hydroxyalkynyl, optionally substituted aminoalkyl, optionally substituted
aminoalkenyl,
or optionally substituted aminoalkynyl.
[000435] Other exemplary modified uracils include those having Formula (b6)-
(b9):
Ri2c R12c
T1 T1 R12c
R12b X IR12a F.D12b
....., ,......--...,
N N N N
1:1 ill
W-1m2 \---Tf W` 2T2
T '2'
W
NVIAP T 'SNAP
-7-- (b6), ---r" (b7), I (b8), or I
(b9), or a pharmaceutically acceptable salt or stereoisomer thereof,
[000436] wherein
[000437] is a single or double bond;
[000438] each of Ty, Tr, T2', and T2" is, independently, H, optionally
substituted alkyl,
optionally substituted alkoxy, or optionally substituted thioalkoxy, or the
combination of
T1' and T" join together (e.g., as in T1) or the combination of T2' and T2"
join together
(e.g., as in T2) to form 0 (oxo), S (thio), or Se (seleno), or each Tl and T2
is,
independently, 0 (oxo), S (thio), or Se (seleno);
[000439] each of Wl and W2 is, independently, N(Rwa)õ, or C(Rwa), wherein nw
is an
integer from 0 to 2 and each ea is, independently, H, optionally substituted
alkyl, or
optionally substituted alkoxy;
[000440] each V3 is, independently, 0, S, N(R), or C(Rva), wherein nv is an
integer from 0 to 2 and each Rva is, independently, H, halo, optionally
substituted amino
acid, optionally substituted alkyl, optionally substituted hydroxyalkyl,
optionally
substituted hydroxyalkenyl, optionally substituted hydroxyalkynyl, optionally
substituted
alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl,
optionally
substituted alkheterocyclyl, optionally substituted alkoxy, optionally
substituted
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alkenyloxy, or optionally substituted alkynyloxy , optionally substituted
aminoalkyl (e.g.,
substituted with an N-protecting group, such as any described herein, e.g.,
trifluoroacetyl,
or sulfoalkyl), optionally substituted aminoalkenyl, optionally substituted
aminoalkynyl,
optionally substituted acylaminoalkyl (e.g., substituted with an N-protecting
group, such
as any described herein, e.g., trifluoroacetyl), optionally substituted
alkoxycarbonylalkyl,
optionally substituted alkoxycarbonylalkenyl, optionally substituted
alkoxycarbonylalkynyl, optionally substituted alkoxycarbonylacyl, optionally
substituted
alkoxycarbonylalkoxy, optionally substituted carboxyalkyl (e.g., optionally
substituted
with hydroxy and/or an 0-protecting group), optionally substituted
carboxyalkoxy,
optionally substituted carboxyaminoalkyl, or optionally substituted
carbamoylalkyl (e.g.,
optionally substituted with any substituent described herein, such as those
selected from
(1)-(21) for alkyl), and wherein Rva and R12c taken together with the carbon
atoms to
which they are attached can form optionally substituted cycloalkyl, optionally
substituted
aryl, or optionally substituted heterocyclyl (e.g., a 5- or 6-membered ring);
[000441] R12a is H, optionally substituted alkyl, optionally substituted
hydroxyalkyl,
optionally substituted hydroxyalkenyl, optionally substituted hydroxyalkynyl,
optionally
substituted aminoalkyl, optionally substituted aminoalkenyl, optionally
substituted
aminoalkynyl, optionally substituted carboxyalkyl (e.g., optionally
substituted with
hydroxy and/or an 0-protecting group), optionally substituted carboxyalkoxy,
optionally
substituted carboxyaminoalkyl, optionally substituted carbamoylalkyl, or
absent;
[000442] Rub is H, optionally substituted alkyl, optionally substituted
alkenyl,
optionally substituted alkynyl, optionally substituted hydroxyalkyl,
optionally substituted
hydroxyalkenyl, optionally substituted hydroxyalkynyl, optionally substituted
aminoalkyl, optionally substituted aminoalkenyl, optionally substituted
aminoalkynyl,
optionally substituted alkaryl, optionally substituted heterocyclyl,
optionally substituted
alkheterocyclyl, optionally substituted amino acid, optionally substituted
alkoxycarbonylacyl, optionally substituted alkoxycarbonylalkoxy, optionally
substituted
alkoxycarbonylalkyl, optionally substituted alkoxycarbonylalkenyl, optionally
substituted
alkoxycarbonylalkynyl, optionally substituted alkoxycarbonylalkoxy, optionally

substituted carboxyalkyl (e.g., optionally substituted with hydroxy and/or an
0-protecting
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group), optionally substituted carboxyalkoxy, optionally substituted
carboxyaminoalkyl,
or optionally substituted carbamoylalkyl,
[000443] wherein the combination of R12b and Ty or the combination of R12b and
R12c
can join together to form optionally substituted heterocyclyl; and
[000444] R12c is H, halo, optionally substituted alkyl, optionally substituted
alkoxy,
optionally substituted thioalkoxy, optionally substituted amino, optionally
substituted
aminoalkyl, optionally substituted aminoalkenyl, or optionally substituted
aminoalkynyl.
[000445] Further exemplary modified uracils include those having Formula (b28)-

(b31):
T1 T1 Ti
Vb 12a
R',......................õ,õ.......õ N .õ, R m,, Vb'
IA ....................-----.., , R12a R12b R12a
1 N N N
1 I
RVb"/\ k , T2
IN T2 N -F2
(b28), (b29), '-n-r`. (b30), or
T1
Rvio. ,R 12a
N
\ NT2
(b31), or a pharmaceutically acceptable salt or stereoisomer thereof,
[000446] wherein
[000447] each of T1 and T2 is, independently, 0 (oxo), S (thio), or Se
(seleno);
[000448] each Rvb' and Rvb" is, independently, H, halo, optionally substituted
amino
acid, optionally substituted alkyl, optionally substituted haloalkyl,
optionally substituted
hydroxyalkyl, optionally substituted hydroxyalkenyl, optionally substituted
hydroxyalkynyl, optionally substituted alkenyl, optionally substituted
alkynyl, optionally
substituted alkoxy, optionally substituted alkenyloxy, optionally substituted
alkynyloxy,
optionally substituted aminoalkyl (e.g., substituted with an N-protecting
group, such as
any described herein, e.g., trifluoroacetyl, or sulfoalkyl), optionally
substituted
aminoalkenyl, optionally substituted aminoalkynyl, optionally substituted
acylaminoalkyl
(e.g., substituted with an N-protecting group, such as any described herein,
e.g.,
trifluoroacetyl), optionally substituted alkoxycarbonylalkyl, optionally
substituted
alkoxycarbonylalkenyl, optionally substituted alkoxycarbonylalkynyl,
optionally
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substituted alkoxycarbonylacyl, optionally substituted alkoxycarbonylalkoxy,
optionally
substituted carboxyalkyl (e.g., optionally substituted with hydroxy and/or an
0-protecting
group), optionally substituted carboxyalkoxy, optionally substituted
carboxyaminoalkyl,
or optionally substituted carbamoylalkyl (e.g., optionally substituted with
any substituent
described herein, such as those selected from (1)-(21) for alkyl) (e.g., Rvb'
is optionally
substituted alkyl, optionally substituted alkenyl, or optionally substituted
aminoalkyl,
e.g., substituted with an N-protecting group, such as any described herein,
e.g.,
trifluoroacetyl, or sulfoalkyl);
[000449] R12a is H, optionally substituted alkyl, optionally substituted
carboxyaminoalkyl, optionally substituted aminoalkyl (e.g., e.g., substituted
with an N-
protecting group, such as any described herein, e.g., trifluoroacetyl, or
sulfoalkyl),
optionally substituted aminoalkenyl, or optionally substituted aminoalkynyl;
and
[000450] Rub is H, optionally substituted alkyl, optionally substituted
alkenyl,
optionally substituted alkynyl, optionally substituted hydroxyalkyl,
optionally substituted
hydroxyalkenyl, optionally substituted hydroxyalkynyl, optionally substituted
aminoalkyl, optionally substituted aminoalkenyl, optionally substituted
aminoalkynyl
(e.g., e.g., substituted with an N-protecting group, such as any described
herein, e.g.,
trifluoroacetyl, or sulfoalkyl),
[000451] optionally substituted alkoxycarbonylacyl, optionally substituted
alkoxycarbonylalkoxy, optionally substituted alkoxycarbonylalkyl, optionally
substituted
alkoxycarbonylalkenyl, optionally substituted alkoxycarbonylalkynyl,
optionally
substituted alkoxycarbonylalkoxy, optionally substituted carboxyalkoxy,
optionally
substituted carboxyalkyl, or optionally substituted carbamoylalkyl.
[000452] In particular embodiments, Tl is 0 (oxo), and T2 is S (thio) or Se
(seleno). In
other embodiments, Tl is S (thio), and T2 is 0 (oxo) or Se (seleno). In some
embodiments, Rvb' is H, optionally substituted alkyl, or optionally
substituted alkoxy.
[000453] In other embodiments, each R12a and R12b is, independently, H,
optionally
substituted alkyl, optionally substituted alkenyl, optionally substituted
alkynyl, or
optionally substituted hydroxyalkyl. In particular embodiments, R12a is H. In
other
embodiments, both R12a and R12b are H.
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[000454] In some embodiments, each Rvb' of Rub =s5
1 independently, optionally
substituted aminoalkyl (e.g., substituted with an N-protecting group, such as
any
described herein, e.g., trifluoroacetyl, or sulfoalkyl), optionally
substituted aminoalkenyl,
optionally substituted aminoalkynyl, or optionally substituted acylaminoalkyl
(e.g.,
substituted with an N-protecting group, such as any described herein, e.g.,
trifluoroacetyl). In some embodiments, the amino and/or alkyl of the
optionally
substituted aminoalkyl is substituted with one or more of optionally
substituted alkyl,
optionally substituted alkenyl, optionally substituted sulfoalkyl, optionally
substituted
carboxy (e.g., substituted with an 0-protecting group), optionally substituted
hydroxy
(e.g., substituted with an 0-protecting group), optionally substituted
carboxyalkyl (e.g.,
substituted with an 0-protecting group), optionally substituted
alkoxycarbonylalkyl (e.g.,
substituted with an 0-protecting group), or N-protecting group. In some
embodiments,
optionally substituted aminoalkyl is substituted with an optionally
substituted sulfoalkyl
or optionally substituted alkenyl. In particular embodiments, R12a and Rvb"
are both H.
In particular embodiments, Tl is 0 (oxo), and T2 is S (thio) or Se (seleno).
[000455] In some embodiments, Rvb' is optionally substituted
alkoxycarbonylalkyl or
optionally substituted carbamoylalkyl.
[000456] In particular embodiments, the optional substituent for R12a5 R12b5
R12c5 or RVa
is a polyethylene glycol group (e.g., -(CH2)s2(0CH2CH2)s1(CH2)s30R', wherein
sl is an
integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,
independently, is
an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1
to 6, or from 1
to 10), and R' is H or C1_20 alkyl); or an amino-polyethylene glycol group
(e.g., -
NRNi(CH2)s2(CH2CH20)si(CH2)s3NRN1, wherein sl is an integer from 1 to 10
(e.g., from
1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0
to 10 (e.g.,
from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each
RN1 is,
independently, hydrogen or optionally substituted Ci_6 alkyl).
[000457] In some embodiments, B is a modified cytosine. Exemplary modified
cytosines include compounds of Formula (b10)-(b14):
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R13a ,R13b R13b R1 3aN,R13b R1 3aN,R13b
,
,R16
R14
N
N N V4 N
i 5
3"
R15
R15 R15 R 3.
Ti T'5 T
(b10), (b11), (b12),
V4N
RI5
T-.
(b13), or (b14), or a pharmaceutically acceptable salt or
stereoisomer
thereof,
wherein
[000458] each of T3' and T3" is, independently, H, optionally substituted
alkyl,
optionally substituted alkoxy, or optionally substituted thioalkoxy, or the
combination of
T3' and T3" join together (e.g., as in T3) to form 0 (oxo), S (thio), or Se
(seleno);
[000459] each V4 is, independently, 0, S, N(Rvc)õ,,, or C(Rvc)õ,,, wherein nv
is an
integer from 0 to 2 and each Rvc is, independently, H, halo, optionally
substituted amino
acid, optionally substituted alkyl, optionally substituted alkenyl, optionally
substituted
alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy,
optionally
substituted heterocyclyl, optionally substituted alkheterocyclyl, or
optionally substituted
alkynyloxy (e.g., optionally substituted with any substituent described
herein, such as
those selected from (1)-(21) for alkyl), wherein the combination of R13b and
Rvc can be
taken together to form optionally substituted heterocyclyl;
[000460] each V5 is, independently, N(R), or C(Rvd), wherein nv is an integer
from 0 to 2 and each Rvd is, independently, H, halo, optionally substituted
amino acid,
optionally substituted alkyl, optionally substituted alkenyl, optionally
substituted alkynyl,
optionally substituted alkoxy, optionally substituted alkenyloxy, optionally
substituted
heterocyclyl, optionally substituted alkheterocyclyl, or optionally
substituted alkynyloxy
(e.g., optionally substituted with any substituent described herein, such as
those selected
from (1)-(21) for alkyl) (e.g., V5 is ¨CH or N);
[000461] each of R13a and R13b is, independently, H, optionally substituted
acyl,
optionally substituted acyloxyalkyl, optionally substituted alkyl, or
optionally substituted
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alkoxy, wherein the combination of R13b and R14 can be taken together to form
optionally
substituted heterocyclyl;
[000462] each R14 is, independently, H, halo, hydroxy, thiol, optionally
substituted acyl,
optionally substituted amino acid, optionally substituted alkyl, optionally
substituted
haloalkyl, optionally substituted alkenyl, optionally substituted alkynyl,
optionally
substituted hydroxyalkyl (e.g., substituted with an 0-protecting group),
optionally
substituted hydroxyalkenyl, optionally substituted hydroxyalkynyl, optionally
substituted
alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy,
optionally
substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally
substituted
acyloxyalkyl, optionally substituted amino (e.g., -NHR, wherein R is H, alkyl,
aryl, or
phosphoryl), azido, optionally substituted aryl, optionally substituted
heterocyclyl,
optionally substituted alkheterocyclyl, optionally substituted aminoalkyl,
optionally
substituted aminoalkenyl, or optionally substituted aminoalkyl; and
[000463] each of R15 and R16 is, independently, H, optionally substituted
alkyl,
optionally substituted alkenyl, or optionally substituted alkynyl.
[000464] Further exemplary modified cytosines include those having Formula
(b32)-
(b35):
R13a ,R13b
,R13b
N
N Ti
I
R14N R14 ,R16 R14 A
1 N N N
I I
R15 N 3 R15 N T I 3 N-R13a
T
I
NW, I
NW, I 3b
, (b32), , (b33), ' Ri (b34), or
R13aN,R13b
R14
1
R15 \N/
(b35), or a pharmaceutically acceptable salt or stereoisomer thereof,
[000465] wherein
[000466] each of T1 and T3 is, independently, 0 (oxo), S (thio), or Se
(seleno);
[000467] each of R13' and R13b is, independently, H, optionally substituted
acyl,
optionally substituted acyloxyalkyl, optionally substituted alkyl, or
optionally substituted
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alkoxy, wherein the combination of R13b and R14 can be taken together to form
optionally
substituted heterocyclyl;
[000468] each R14 is, independently, H, halo, hydroxy, thiol, optionally
substituted acyl,
optionally substituted amino acid, optionally substituted alkyl, optionally
substituted
haloalkyl, optionally substituted alkenyl, optionally substituted alkynyl,
optionally
substituted hydroxyalkyl (e.g., substituted with an 0-protecting group),
optionally
substituted hydroxyalkenyl, optionally substituted hydroxyalkynyl, optionally
substituted
alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy,
optionally
substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally
substituted
acyloxyalkyl, optionally substituted amino (e.g., -NHR, wherein R is H, alkyl,
aryl, or
phosphoryl), azido, optionally substituted aryl, optionally substituted
heterocyclyl,
optionally substituted alkheterocyclyl, optionally substituted aminoalkyl
(e.g.,
hydroxyalkyl, alkyl, alkenyl, or alkynyl), optionally substituted
aminoalkenyl, or
optionally substituted aminoalkynyl; and
[000469] each of R15 and R16 is, independently, H, optionally substituted
alkyl,
optionally substituted alkenyl, or optionally substituted alkynyl (e.g., R15
is H, and R16 is
H or optionally substituted alkyl).
[000470] In some embodiments, R15 is H, and R16 is H or optionally substituted
alkyl.
In particular embodiments, R14 is H, acyl, or hydroxyalkyl. In some
embodiments, R14 is
halo. In some embodiments, both R14 and R15 are H. In some embodiments, both
R15
and R16 are H. In some embodiments, each of R14 and R15 and R16 is H. In
further
embodiments, each of R13a and R13b is independently, H or optionally
substituted alkyl.
[000471] Further non-limiting examples of modified cytosines include compounds
of
Formula (b36):
_Rub
N
R14a I
N
1
R14b
?
NIIIIIP
i (b36) or a pharmaceutically acceptable salt or stereoisomer
thereof,
[000472] wherein
[000473] each R13b is, independently, H, optionally substituted acyl,
optionally
substituted acyloxyalkyl, optionally substituted alkyl, or optionally
substituted alkoxy,
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wherein the combination of Ri3b and Ri4b can be taken together to form
optionally
substituted heterocyclyl;
[000474] each Rma and R14b is, independently, H, halo, hydroxy, thiol,
optionally
substituted acyl, optionally substituted amino acid, optionally substituted
alkyl, optionally
substituted haloalkyl, optionally substituted alkenyl, optionally substituted
alkynyl,
optionally substituted hydroxyalkyl (e.g., substituted with an 0-protecting
group),
optionally substituted hydroxyalkenyl, optionally substituted alkoxy,
optionally
substituted alkenyloxy, optionally substituted alkynyloxy, optionally
substituted
aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted
acyloxyalkyl,
optionally substituted amino (e.g., -NHR, wherein R is H, alkyl, aryl,
phosphoryl,
optionally substituted aminoalkyl, or optionally substituted
carboxyaminoalkyl), azido,
optionally substituted aryl, optionally substituted heterocyclyl, optionally
substituted
alkheterocyclyl, optionally substituted aminoalkyl, optionally substituted
aminoalkenyl,
or optionally substituted aminoalkynyl; and
[000475] each of le is, independently, H, optionally substituted alkyl,
optionally
substituted alkenyl, or optionally substituted alkynyl.
[000476] In particular embodiments, R14b is an optionally substituted amino
acid (e.g.,
optionally substituted lysine). In some embodiments, Rma is H.
[000477] In some embodiments, B is a modified guanine. Exemplary modified
guanines include compounds of Formula (b15)-(b17):
"
R18 R23
m
R21 " R24
\ N ,R19a
N N
I N N1913 I
R (b15), , R22
(b16), or
T5 T5"
R18
R17flN
6"
N T.
N
R22
(b17), or a pharmaceutically acceptable salt or stereoisomer
thereof,
[000478] wherein
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[000479] each of T4', T4", T5', T5", T6', and T6" is, independently, H,
optionally
substituted alkyl, or optionally substituted alkoxy, and wherein the
combination of T4'
and T4" (e.g., as in T4) or the combination of T5' and T5" (e.g., as in T5) or
the combination
of T6' and T6" (e.g., as in T6) join together form 0 (oxo), S (thio), or Se
(seleno);
[000480] each of V5 and V6 is, independently, 0, S, N(R), or C(Rvd)õv, wherein
nv
is an integer from 0 to 2 and each Rvd is, independently, H, halo, thiol,
optionally
substituted amino acid, cyano, amidine, optionally substituted aminoalkyl,
optionally
substituted aminoalkenyl, optionally substituted aminoalkynyl, optionally
substituted
alkyl, optionally substituted alkenyl, optionally substituted alkynyl,
optionally substituted
alkoxy, optionally substituted alkenyloxy, or optionally substituted
alkynyloxy (e.g.,
optionally substituted with any substituent described herein, such as those
selected from
(1)-(21) for alkyl) , optionally substituted thioalkoxy, or optionally
substituted amino;
and
[000481] each of R17, R18, R19a, R191), R21, R22, R235 and R24 is,
independently, H, halo,
thiol, optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted
alkynyl, optionally substituted thioalkoxy, optionally substituted amino, or
optionally
substituted amino acid.
[000482] Exemplary modified guanosines include compounds of Formula (b37)-
(b40):
T4 T4' T4
N-_,./IcN.R18
//N N
-----i N ,R18
1 \ 1 &C
N N.R19a
I 19b 1 N N
19b
R 1 N N
, R (b37), ' (b38), ' R19b
(b39),
T4
Nõ....>c N .R18
R21 __
1
N---\ %\ N .R19a
I N
=I.n.P.Nl I
I
or R19b (b40), or a pharmaceutically acceptable salt or
stereoisomer thereof,
[000483] wherein
[000484] each of T4' is, independently, H, optionally substituted alkyl, or
optionally
substituted alkoxy, and each T4 is, independently, 0 (oxo), S (thio), or Se
(seleno);
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[000485] each of R185 R19a, R191)5 and R21 is, independently, H, halo, thiol,
optionally
substituted alkyl, optionally substituted alkenyl, optionally substituted
alkynyl, optionally
substituted thioalkoxy, optionally substituted amino, or optionally
substituted amino acid.
[000486] In some embodiments, R18 is H or optionally substituted alkyl. In
further
embodiments, T4 is oxo. In some embodiments, each of R19a and R19b is,
independently,
H or optionally substituted alkyl.
[000487] In some embodiments, B is a modified adenine. Exemplary modified
adenines include compounds of Formula (b18)-(b20):
R26a R2613 R2613
N1
_R28
1
R25 __________________________ R25
R27
'NAM
(b18), (b19), or
R29
VN
R25
N R27
(b20), or a pharmaceutically acceptable salt or stereoisomer
thereof,
[000488] wherein
[000489] each V7 is, independently, 0, S, N(R), or C(Rve), wherein nv is an
integer from 0 to 2 and each RVe is, independently, H, halo, optionally
substituted amino
acid, optionally substituted alkyl, optionally substituted alkenyl, optionally
substituted
alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, or
optionally
substituted alkynyloxy (e.g., optionally substituted with any substituent
described herein,
such as those selected from (1)-(21) for alkyl);
[000490] each R25 is, independently, H, halo, thiol, optionally substituted
alkyl,
optionally substituted alkenyl, optionally substituted alkynyl, optionally
substituted
thioalkoxy, or optionally substituted amino;
[000491] each of R26a and R26b is, independently, H, optionally substituted
acyl,
optionally substituted amino acid, optionally substituted carbamoylalkyl,
optionally
substituted alkyl, optionally substituted alkenyl, optionally substituted
alkynyl, optionally
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substituted hydroxyalkyl, optionally substituted hydroxyalkenyl, optionally
substituted
hydroxyalkynyl, optionally substituted alkoxy, or polyethylene glycol group
(e.g., -
(CH2)s2(OCH2CH2)s1(CH2)s3OR', wherein sl is an integer from 1 to 10 (e.g.,
from 1 to 6
or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10
(e.g., from 0
to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R' is H or
C1_20 alkyl); or
an amino-polyethylene glycol group (e.g., -NRNi(CH2)s2(CH2CH20)si(CH2)s3NRN1,
wherein sl is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each
of s2 and s3,
independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6,
from 1 to 4, from
1 to 6, or from 1 to 10), and each RN1 is, independently, hydrogen or
optionally
substituted C1-6 alkyl);
[000492] each R27 is, independently, H, optionally substituted alkyl,
optionally
substituted alkenyl, optionally substituted alkynyl, optionally substituted
alkoxy,
optionally substituted thioalkoxy or optionally substituted amino;
[000493] each R28 is, independently, H, optionally substituted alkyl,
optionally
substituted alkenyl, or optionally substituted alkynyl; and
[000494] each R29 is, independently, H, optionally substituted acyl,
optionally
substituted amino acid, optionally substituted carbamoylalkyl, optionally
substituted
alkyl, optionally substituted alkenyl, optionally substituted alkynyl,
optionally substituted
hydroxyalkyl, optionally substituted hydroxyalkenyl, optionally substituted
alkoxy, or
optionally substituted amino.
[000495] Exemplary modified adenines include compounds of Formula (b41)-(b43):
R26a R2 6b R26a R26b R26a R26b
N N N
N....,....../N N-.........N N-..,...,./N
1 R25 ____ 1 i 1 j
N -
NR2 7 N---e N"---N
[000496] , (b41), , (b42), or ,
(b43), or a pharmaceutically acceptable salt or stereoisomer thereof,
[000497] wherein
[000498] each R25 is, independently, H, halo, thiol, optionally substituted
alkyl,
optionally substituted alkenyl, optionally substituted alkynyl, optionally
substituted
thioalkoxy, or optionally substituted amino;
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[000499] each of R26a and R26b is, independently, H, optionally substituted
acyl,
optionally substituted amino acid, optionally substituted carbamoylalkyl,
optionally
substituted alkyl, optionally substituted alkenyl, optionally substituted
alkynyl, optionally
substituted hydroxyalkyl, optionally substituted hydroxyalkenyl, optionally
substituted
hydroxyalkynyl, optionally substituted alkoxy, or polyethylene glycol group
(e.g., -
(CH2)s2(OCH2CH2)s1(CH2)s3OR', wherein sl is an integer from 1 to 10 (e.g.,
from 1 to 6
or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10
(e.g., from 0
to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R' is H or
C1_20 alkyl); or
an amino-polyethylene glycol group (e.g., -NRNi(CH2)s2(CH2CH20)si(CH2)s3NRN1,
wherein sl is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each
of s2 and s3,
independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6,
from 1 to 4, from
1 to 6, or from 1 to 10), and each RN1 is, independently, hydrogen or
optionally
substituted C1-6 alkyl); and
[000500] each R27 is, independently, H, optionally substituted alkyl,
optionally
substituted alkenyl, optionally substituted alkynyl, optionally substituted
alkoxy,
optionally substituted thioalkoxy, or optionally substituted amino.
[000501] In some embodiments, R26a is H, and R26b is optionally substituted
alkyl. In
some embodiments, each of R26a and R26b is, independently, optionally
substituted alkyl.
In particular embodiments, R27 is optionally substituted alkyl, optionally
substituted
alkoxy, or optionally substituted thioalkoxy. In other embodiments, R25 is
optionally
substituted alkyl, optionally substituted alkoxy, or optionally substituted
thioalkoxy.
[000502] In particular embodiments, the optional substituent for R26a, R26b,
or

R29 is a
polyethylene glycol group (e.g., -(CH2)s2(OCH2CH2)s1(CH2)s3OR', wherein sl is
an
integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,
independently, is
an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1
to 6, or from 1
to 10), and R' is H or C1_20 alkyl); or an amino-polyethylene glycol group
(e.g., -
NRNi(CH2)s2(CH2CH20)si(CH2)s3NRN1, wherein sl is an integer from 1 to 10
(e.g., from
1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0
to 10 (e.g.,
from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each
RN1 is,
independently, hydrogen or optionally substituted Ci_6 alkyl).
[000503] In some embodiments, B may have Formula (b21):
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ex12
R12a
(b21), wherein X'2 is, independently, 0, S, optionally substituted
alkylene (e.g., methylene), or optionally substituted heteroalkylene, xa is an
integer from
0 to 3, and R12a and T2 are as described herein.
[000504] In some embodiments, B may have Formula (b22):
0 T1
R10 it ,R12a
N
H I
R N T2
'NNW
(b22), wherein R1 ' is, independently, optionally substituted
alkyl, optionally substituted alkenyl, optionally substituted alkynyl,
optionally substituted
aryl, optionally substituted heterocyclyl, optionally substituted aminoalkyl,
optionally
substituted aminoalkenyl, optionally substituted aminoalkynyl, optionally
substituted
alkoxy, optionally substituted alkoxycarbonylalkyl, optionally substituted
alkoxycarbonylalkenyl, optionally substituted alkoxycarbonylalkynyl,
optionally
substituted alkoxycarbonylalkoxy, optionally substituted carboxyalkoxy,
optionally
substituted carboxyalkyl, or optionally substituted carbamoylalkyl, and R11,
R12a5 T15 and
T2 are as described herein.
[000505] In some embodiments, B may have Formula (b23):
T1
IR10N R1 2a
R 2
N T
fvvvv,
(b23), wherein Rlo is optionally substituted heterocyclyl (e.g.,
optionally substituted furyl, optionally substitued thienyl, or optionally
substitued
pyrrolyl), optionally substituted aryl (e.g., optionally substituted phenyl or
optionally
substituted naphthyl), or any substituent described herein (e.g., for R1 )
;and wherein R"
(e.g., H or any substituent described herein), R12a (e.g., H or any
substituent described
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herein), Tl (e.g., oxo or any substituent described herein), and T2 (e.g., oxo
or any
substituent described herein) are as described herein.
In some embodiments, B may have Formula (b24):
Ri 3aN, R13b
0
R14' )1 N
R15 3
N T
(b24), wherein R14' is, independently, optionally substituted alkyl,
optionally substituted alkenyl, optionally substituted alkynyl, optionally
substituted aryl,
optionally substituted heterocyclyl, optionally substituted alkaryl,
optionally substituted
alkheterocyclyl, optionally substituted aminoalkyl, optionally substituted
aminoalkenyl,
optionally substituted aminoalkynyl, optionally substituted alkoxy, optionally
substituted
alkoxycarbonylalkenyl, optionally substituted alkoxycarbonylalkynyl,
optionally
substituted alkoxycarbonylalkyl, optionally substituted alkoxycarbonylalkoxy,
optionally
substituted carboxyalkoxy, optionally substituted carboxyalkyl, or optionally
substituted
carbamoylalkyl, and R13a, R13b, R15, and T3 are as described herein.
[000506] In some embodiments, B may have Formula (b25):
R13a R13b
0
R14' N N
R15 N-r3
(b25), wherein R14' is optionally substituted heterocyclyl (e.g.,
optionally substituted furyl, optionally substitued thienyl, or optionally
substitued
pyrrolyl), optionally substituted aryl (e.g., optionally substituted phenyl or
optionally
substituted naphthyl), or any substituent described herein (e.g., for R14 or
R14'); and
wherein R13a (e.g., H or any substituent described herein), R13b (e.g., H or
any substituent
described herein), R15 (e.g., H or any substituent described herein), and T3
(e.g., oxo or
any substituent described herein) are as described herein.
[000507] In some embodiments, B is a nucleobase selected from the group
consisting of
cytosine, guanine, adenine, and uracil. In some embodiments, B may be:
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N
'N NH2 e
N N
N N N
(b26) or + (b27).
[000508] In some embodiments, the modified nucleobase is a modified uracil.
Exemplary nucleobases and nucleosides having a modified uracil include
pseudouridine
(y), pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-
uridine, 2-
thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-
pseudouridine, 5-
hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-
uridine or 5-
bromo-uridine), 3-methyl-uridine (m3U), 5-methoxy-uridine (mo5U), uridine 5-
oxyacetic
acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-
uridine
(cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U),
5-
carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-
uridine (mcm5U), 5-methoxycarbonylmethy1-2-thio-uridine (mcm5s2U), 5-
aminomethy1-
2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 5-
methylaminomethy1-
2-thio-uridine (mnm5s2U), 5-methylaminomethy1-2-seleno-uridine (mnm5se2U), 5-
carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U),
5-
carboxymethylaminomethy1-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1-
propynyl-
pseudouridine, 5-taurinomethyl-uridine (Tm5U), 1-taurinomethyl-pseudouridine,
5-
taurinomethy1-2-thio-uridine('rm5s2U), 1-taurinomethy1-4-thio-pseudouridine, 5-
methyl-
uridine (m5U, i.e., having the nucleobase deoxythymine), 1-methylpseudouridine
(mly),
5-methy1-2-thio-uridine (m5s2U), 1-methy1-4-thio-pseudouridine (m1s4lif), 4-
thio-1-
methyl-pseudouridine, 3-methyl-pseudouridine (m3y), 2-thio-1-methyl-
pseudouridine, 1-
methyl-1 -deaza-pseudouridine, 2-thio- 1 -methyl- 1 -de aza-ps eudouridine,
dihydrouridine
(D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D),
2-thio-
dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-
thio-
uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-
pseudouridine (also known as 1-methylpseudouridine (mly)), 3-(3-amino-3-
carboxypropyl)uridine (acp3U), 1-methy1-3-(3-amino-3-
carboxypropyl)pseudouridine
(acp3 kv), 5-(isopentenylaminomethyl)uridine (inm5U), 5-
(isopentenylaminomethyl)-2-
thio-uridine (inm5s2U), a-thio-uridine, 2'-0-methyl-uridine (Um), 5,2'-0-
dimethyl-
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uridine (m5Um), 2'-0-methyl-pseudouridine (wm), 2-thio-2'-0-methyl-uridine
(s2Um), 5-
methoxycarbonylmethy1-2'-0-methyl-uridine (mcm5Um), 5-carbamoylmethy1-2'-0-
methyl-uridine (ncm5Um), 5-carboxymethylaminomethy1-2'-0-methyl-uridine
(cmnm5Um), 3,2'-0-dimethyl-uridine (m3Um), 5-(isopentenylaminomethyl)-2'-0-
methyl-uridine (inm5Um), 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-
F-uridine,
2'-0H-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, and 5-[3-(1-E-
propenylamino)uridine.
[000509] In some embodiments, the modified nucleobase is a modified cytosine.
Exemplary nucleobases and nucleosides haying a modified cytosine include 5-aza-

cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m3C), N4-
acetyl-cytidine
(ac4C), 5-formyl-cytidine (f5C), N4-methyl-cytidine (m4C), 5-methyl-cytidine
(m5C), 5-
halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-
methyl-
pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-
cytidine (s2C), 2-
thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-
pseudoisocytidine, 4-
thio- 1-methyl-1 -deaza-pseudoisocytidine, 1 -methyl- 1 -deaza-
pseudoisocytidine,
zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-
thio-
zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-
pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysidine (k2C), a-
thio-
cytidine, 2'-0-methyl-cytidine (Cm), 5,2'-0-dimethyl-cytidine (m5Cm), N4-
acety1-2'-0-
methyl-cytidine (ac4Cm), N4,2'-0-dimethyl-cytidine (m4Cm), 5-formy1-2'-0-
methyl-
cytidine (f5Cm), N4,N4,2'-0-trimethyl-cytidine (m42Cm), 1-thio-cytidine, 2'-F-
ara-
cytidine, 2'-F-cytidine, and 2'-0H-ara-cytidine.
[000510] In some embodiments, the modified nucleobase is a modified adenine.
Exemplary nucleobases and nucleosides having a modified adenine include 2-
amino-
purine, 2, 6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-
purine), 6-
halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-
adenosine, 7-
deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-
amino-
purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl-
adenosine (m1A), 2-methyl-adenine (m2A), N6-methyl-adenosine (m6A), 2-
methylthio-
N6-methyl-adenosine (ms2m6A), N6-isopentenyl-adenosine (i6A), 2-methylthio-N6-
isopentenyl-adenosine (ms2i6A), N6-(cis-hydroxyisopentenyl)adenosine (io6A), 2-

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methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms2io6A), N6-
glycinylcarbamoyl-
adenosine (g6A), N6-threonylcarbamoyl-adenosine (t6A), N6-methyl-N6-
threonylcarbamoyl-adenosine (m6t6A), 2-methylthio-N6-threonylcarbamoyl-
adenosine
(ms2g6A), N6,N6-dimethyl-adenosine (m62A), N6-hydroxynorvalylcarbamoyl-
adenosine
(hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms2hn6A), N6-
acetyl-
adenosine (ac6A), 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, a-
thio-
adenosine, 2'-0-methyl-adenosine (Am), N6,2'-0-dimethyl-adenosine (m6Am),
N6,N6,2'-0-trimethyl-adenosine (m62Am), 1,2'-0-dimethyl-adenosine (mlAm), 2'-0-

ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-
adenosine, 8-
azido-adenosine, 2'-F-ara-adenosine, 2'-F-adenosine, 2'-OH-ara-adenosine, and
N6-(19-
amino-pentaoxanonadecy1)-adenosine.
[000511] In some embodiments, the modified nucleobase is a modified guanine.
Exemplary nucleobases and nucleosides having a modified guanine include
inosine (I), 1-
methyl-inosine (mil), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine
(imG-14), isowyosine (imG2), wybutosine (yW), peroxywybutosine (o2yW),
hydroxywybutosine (OHyW), undermodified hydroxywybutosine (OHyW*), 7-deaza-
guanosine, queuosine (Q), epoxyqueuosine (oQ), galactosyl-queuosine (galQ),
mannosyl-
queuosine (manQ), 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethy1-7-deaza-
guanosine (preQi), archaeosine (G d), 7-deaza-8-aza-guanosine, 6-thio-
guanosine, 6-thio-
7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m7G), 6-
thio-
7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine
(m' G), N2-methyl-guanosine (m2G), N2,N2-dimethyl-guanosine (m22G), N2,7-
dimethyl-
guanosine (m2'7G), N2, N2,7-dimethyl-guanosine (m2'2'7G), 8-oxo-guanosine, 7-
methyl-
8-oxo-guanosine, 1-methy1-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-
dimethy1-6-thio-guanosine, a-thio-guanosine, 2'-0-methyl-guanosine (Gm), N2-
methy1-
2'-0-methyl-guanosine (m2Gm), N2,N2-dimethy1-2'-0-methyl-guanosine (m22Gm), 1-
methy1-2'-0-methyl-guanosine (m' Gm), N2,7-dimethy1-2'-0-methyl-guanosine
(m2'7Gm), 2'-0-methyl-inosine (Im), 1,2'-0-dimethyl-inosine (m1Im), and 2'-0-
ribosylguanosine (phosphate) (Gr(p)).
[000512] The nucleobase of the nucleotide can be independently selected from a
purine,
a pyrimidine, a purine or pyrimidine analog. For example, the nucleobase can
each be
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independently selected from adenine, cytosine, guanine, uracil, or
hypoxanthine. In
another embodiment, the nucleobase can also include, for example, naturally-
occurring
and synthetic derivatives of a base, including pyrazolo[3,4-d]pyrimidines, 5-
methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-
aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-
propyl and
other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine
and 2-
thiocytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and
thymine, 5-uracil
(pseudouracil), 4-thiouracil, 8-halo (e.g., 8-bromo), 8-amino, 8-thiol, 8-
thioalkyl, 8-
hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-
bromo, 5-
trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine
and 7-
methyladenine, 8-azaguanine and 8-azaadenine, deazaguanine, 7-deazaguanine, 3-
deazaguanine, deazaadenine, 7-deazaadenine, 3-deazaadenine, pyrazolo[3,4-
d]pyrimidine, imidazo[1,5-a]1,3,5 triazinones, 9-deazapurines, imidazo[4,5-
d]pyrazines,
thiazolo[4,5-d]pyrimidines, pyrazin-2-ones, 1,2,4-triazine, pyridazine; and
1,3,5 triazine.
When the nucleotides are depicted using the shorthand A, G, C, T or U, each
letter refers
to the representative base and/or derivatives thereof, e.g., A includes
adenine or adenine
analogs, e.g., 7-deaza adenine).
Modifications on the Internucleoside Linkage
[000513] The modified nucleotides, which may be incorporated into a
polynucleotide,
primary construct, or mmRNA molecule, can be modified on the internucleoside
linkage
(e.g., phosphate backbone). Herein, in the context of the polynucleotide
backbone, the
phrases "phosphate" and "phosphodiester" are used interchangeably. Backbone
phosphate groups can be modified by replacing one or more of the oxygen atoms
with a
different substituent. Further, the modified nucleosides and nucleotides can
include the
wholesale replacement of an unmodified phosphate moiety with another
internucleoside
linkage as described herein. Examples of modified phosphate groups include,
but are not
limited to, phosphorothioate, phosphoroselenates,
boranophosphates,boranophosphate
esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or
aryl
phosphonates, and phosphotriesters. Phosphorodithioates have both non-linking
oxygens
replaced by sulfur. The phosphate linker can also be modified by the
replacement of a
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linking oxygen with nitrogen (bridged phosphoramidates), sulfur (bridged
phosphorothioates), and carbon (bridged methylene-phosphonates).
[000514] The a-thio substituted phosphate moiety is provided to confer
stability to
RNA and DNA polymers through the unnatural phosphorothioate backbone linkages.

Phosphorothioate DNA and RNA have increased nuclease resistance and
subsequently a
longer half-life in a cellular environment. Phosphorothioate linked
polynucleotides,
primary constructs, or mmRNA molecules are expected to also reduce the innate
immune
response through weaker binding/activation of cellular innate immune
molecules.
[000515] In specific embodiments, a modified nucleoside includes an alpha-thio-

nucleoside (e.g., 5'-0-(1-thiophosphate)-adenosine, 5'-0-(1-thiophosphate)-
cytidine (a-
thio-cytidine), 5'-0-(1-thiophosphate)-guanosine, 5'-0-(1-thiophosphate)-
uridine, or 5'-0-
(1-thiophosphate)-pseudouridine).
[000516] Other internucleoside linkages that may be employed according to the
present
invention, including internucleoside linkages which do not contain a
phosphorous atom,
are described herein below.
Combinations of Modified Sugars, Nucleobases, and Internucleoside Linkages
[000517] The polynucleotides, primary constructs, and mmRNA of the invention
can
include a combination of modifications to the sugar, the nucleobase, and/or
the
internucleoside linkage. These combinations can include any one or more
modifications
described herein. For examples, any of the nucleotides described herein in
Formulas (Ia),
(Ia-1)-(Ia-3), (Ib)-(If), (IIa)-(IIp), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-
1), (IIn-2), (IVa)-
(IV1), and (IXa)-(IXr) can be combined with any of the nucleobases described
herein
(e.g., in Formulas (b1)-(b43) or any other described herein).
Synthesis of Polypeptides, Primary Constructs, and mmRNA Molecules
[000518] The polypeptides, primary constructs, and mmRNA molecules for use in
accordance with the invention may be prepared according to any useful
technique, as
described herein. The modified nucleosides and nucleotides used in the
synthesis of
polynucleotides, primary constructs, and mmRNA molecules disclosed herein can
be
prepared from readily available starting materials using the following general
methods
and procedures. Where typical or preferred process conditions (e.g., reaction
temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are
provided, a
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skilled artisan would be able to optimize and develop additional process
conditions.
Optimum reaction conditions may vary with the particular reactants or solvent
used, but
such conditions can be determined by one skilled in the art by routine
optimization
procedures.
[000519] The processes described herein can be monitored according to any
suitable
method known in the art. For example, product formation can be monitored by
spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., 1H
or 13C)
infrared spectroscopy, spectrophotometry (e.g., UV-visible), or mass
spectrometry, or by
chromatography such as high performance liquid chromatography (HPLC) or thin
layer
chromatography.
[000520] Preparation of polypeptides, primary constructs, and mmRNA molecules
of
the present invention can involve the protection and deprotection of various
chemical
groups. The need for protection and deprotection, and the selection of
appropriate
protecting groups can be readily determined by one skilled in the art. The
chemistry of
protecting groups can be found, for example, in Greene, et al., Protective
Groups in
Organic Synthesis, 2d. Ed., Wiley & Sons, 1991, which is incorporated herein
by
reference in its entirety.
[000521] The reactions of the processes described herein can be carried out in
suitable
solvents, which can be readily selected by one of skill in the art of organic
synthesis.
Suitable solvents can be substantially nonreactive with the starting materials
(reactants),
the intermediates, or products at the temperatures at which the reactions are
carried out,
i.e., temperatures which can range from the solvent's freezing temperature to
the
solvent's boiling temperature. A given reaction can be carried out in one
solvent or a
mixture of more than one solvent. Depending on the particular reaction step,
suitable
solvents for a particular reaction step can be selected.
[000522] Resolution of racemic mixtures of modified nucleosides and
nucleotides can
be carried out by any of numerous methods known in the art. An example method
includes fractional recrystallization using a "chiral resolving acid" which is
an optically
active, salt-forming organic acid. Suitable resolving agents for fractional
recrystallization
methods are, for example, optically active acids, such as the D and L forms of
tartaric
acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic
acid, lactic acid or
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the various optically active camphorsulfonic acids. Resolution of racemic
mixtures can
also be carried out by elution on a column packed with an optically active
resolving agent
(e.g., dinitrobenzoylphenylglycine). Suitable elution solvent composition can
be
determined by one skilled in the art.
Modified nucleosides and nucleotides (e.g., building block molecules) can be
prepared
according to the synthetic methods described in Ogata et al., J. Org. Chem.
74:2585-2588
(2009); Purmal et al., Nucl. Acids Res. 22(1): 72-78, (1994); Fukuhara et al.,

Biochemistry, 1(4): 563-568 (1962); and Xu et al., Tetrahedron, 48(9): 1729-
1740
(1992), each of which are incorporated by reference in their entirety.
[000523] The polypeptides, primary constructs, and mmRNA of the invention may
or
may not be uniformly modified along the entire length of the molecule. For
example, one
or more or all types of nucleotide (e.g., purine or pyrimidine, or any one or
more or all of
A, G, U, C) may or may not be uniformly modified in a polynucleotide of the
invention,
or in a given predetermined sequence region thereof (e.g. one or more of the
sequence
regions represented in Figure 1). In some embodiments, all nucleotides X in a
polynucleotide of the invention (or in a given sequence region thereof) are
modified,
wherein X may any one of nucleotides A, G, U, C, or any one of the
combinations A+G,
A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C or A+G+C.
[000524] Different sugar modifications, nucleotide modifications, and/or
internucleoside linkages (e.g., backbone structures) may exist at various
positions in the
polynucleotide, primary construct, or mmRNA. One of ordinary skill in the art
will
appreciate that the nucleotide analogs or other modification(s) may be located
at any
position(s) of a polynucleotide, primary construct, or mmRNA such that the
function of
the polynucleotide, primary construct, or mmRNA is not substantially
decreased. A
modification may also be a 5' or 3' terminal modification. The polynucleotide,
primary
construct, or mmRNA may contain from about 1% to about 100% modified
nucleotides
(either in relation to overall nucleotide content, or in relation to one or
more types of
nucleotide, i.e. any one or more of A, G, U or C) or any intervening
percentage (e.g.,
from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to
70%,
from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to
25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from
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10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to
50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from
20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to
80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from

70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to
95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to
100%).
[000525] In some embodiments, the polynucleotide, primary construct, or mmRNA
includes a modified pyrimidine (e.g., a modified uracil/uridine/U or modified
cytosine/cytidine/C). In some embodiments, the uracil or uridine (generally:
U) in the
polynucleotide, primary construct, or mmRNA molecule may be replaced with from

about 1% to about 100% of a modified uracil or modified uridine (e.g., from 1%
to 20%,
from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to
80%,
from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to
50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from
10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to
60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from
20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to
90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from

70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to
100%, from 90% to 95%, from 90% to 100%, and from 95% to 100% of a modified
uracil or modified uridine). The modified uracil or uridine can be replaced by
a
compound having a single unique structure or by a plurality of compounds
having
different structures (e.g., 2, 3, 4 or more unique structures, as described
herein). In some
embodiments, the cytosine or cytidine (generally: C) in the polynucleotide,
primary
construct, or mmRNA molecule may be replaced with from about 1% to about 100%
of a
modified cytosine or modified cytidine (e.g., from 1% to 20%, from 1% to 25%,
from 1%
to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from
1%
to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%,
from
10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to
100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from
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20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to
60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from
50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to
100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%,
from
90% to 100%, and from 95% to 100% of a modified cytosine or modified
cytidine). The
modified cytosine or cytidine can be replaced by a compound having a single
unique
structure or by a plurality of compounds having different structures (e.g., 2,
3, 4 or more
unique structures, as described herein).
In some embodiments, the present disclosure provides methods of synthesizing a
polynucleotide, primary construct, or mmRNA (e.g., the first region, first
flanking region,
or second flanking region) including n number of linked nucleosides having
Formula (Ia-
1):
___ Y1 Y5
R-
R1-(RR2
Y"m
1
Y3=P _____________
I 4
- (Ia-1), comprising:
a) reacting a nucleotide of Formula (IV-1):
B
3 =
RR4 \
(tk;r2 P2 m (IV-1),
with a phosphoramidite compound of Formula (V-1):
P -Y-Y5 Ui
R31_
R5 9 2)
3 Y m
P\ 1
O-P\ _(
(V-1),
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wherein Y9 is H, hydroxy, phosphoryl, pyrophosphate, sulfate, amino, thiol,
optionally
substituted amino acid, or a peptide (e.g., including from 2 to 12 amino
acids); and each
Pl, P2, and P3 is, independently, a suitable protecting group; and 0 denotes a
solid
support;
to provide a polynucleotide, primary construct, or mmRNA of Formula (VI-1):
R31-U
R5v2 Y9-P2)m
P\3 I
O-P
vi
B
R3 1-Lj
R5
cr2Y9-4m
(VI-1), and
b) oxidizing or sulfurizing the polynucleotide, primary construct, or mmRNA of
Formula
(V) to yield a polynucleotide, primary construct, or mmRNA of Formula (V11-1):
R3 1-Lji
R5 2 (Y9-P2im
p3 Y
' \ I 3
y5
R31-U/
R5
Cr2 \Y9-P)m
(VII-1), and
c) removing the protecting groups to yield the polynucleotide, primary
construct, or
mmRNA of Formula (Ia).
[000526] In some embodiments, steps a) and b) are repeated from 1 to about
10,000
times. In some embodiments, the methods further comprise a nucleotide (e.g.,
mmRNA
molecule) selected from the group consisting of A, C, G and U adenosine,
cytosine,
guanosine, and uracil. In some embodiments, the nucleobase may be a pyrimidine
or
derivative thereof In some embodiments, the polynucleotide, primary construct,
or
mmRNA is translatable.
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[000527] Other components of polynucleotides, primary constructs, and mmRNA
are
optional, and are beneficial in some embodiments. For example, a 5'
untranslated region
(UTR) and/or a 3'UTR are provided, wherein either or both may independently
contain
one or more different nucleotide modifications. In such embodiments,
nucleotide
modifications may also be present in the translatable region. Also provided
are
polynucleotides, primary constructs, and mmRNA containing a Kozak sequence.
[000528] Exemplary syntheses of modified nucleotides, which are incorporated
into a
modified nucleic acid or mmRNA, e.g., RNA or mRNA, are provided below in
Scheme 1
through Scheme 11. Scheme 1 provides a general method for phosphorylation of
nucleosides, including modified nucleosides.
Scheme 1
/1.1¨/ /1.1¨/
------(cL-- --------N ------(Ct--------N
Z \ N)(N \ )
ii
N 1) POCI3 0 0 0
HO _________________________ IP e0¨P¨O¨P¨O¨P-0
)0 2) Pyrophosphate 1 1 1
0 o e oe ..----o----?
OH OH OH OH
[000529] Various protecting groups may be used to control the reaction. For
example,
Scheme 2 provides the use of multiple protecting and deprotecting steps to
promote
phosphorylation at the 5' position of the sugar, rather than the 2' and 3'
hydroxyl groups.
Scheme 2
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H2N H2N
_"-------N _----------N
\ ) Acetone/H+ HO /k1
HO N \ N)
N
0-..,..\j
OH OH 00
Ac20
H
H2N 2N
_---"---------N
_---"-------N N \ N)
N \ N)
Ac0
Ac0 Dowex Ii+ N
______________________________________________ (
OH OH 0
( 0
ph3c.
H2N
1, H2N
I) OH-
_--- ----N
_------------N rij \ )
N \
Ac0 N) 0 0 0
2) POCI3 N
%1:,p//
N 3) Pyrophosphate 8 0 0/ 0
....õ...-0.-____ 4) H+
e e e ____
OH OH
0 0
Ph3C/ CPh3
[000530] Modified nucleotides can be synthesized in any useful manner. Schemes
3, 4,
and 7 provide exemplary methods for synthesizing modified nucleotides having a

modified purine nucleobase; and Schemes 5 and 6 provide exemplary methods for
synthesizing modified nucleotides having a modified pseudouridine or
pseudoisocytidine,
respectively.
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Scheme 3
o
o
N----
N........,NCH3
NH
< 1 < 1
N"-
N"----NNH2 N NH2
CH3I/heat HO
HO
___________________________________ 0/1 c0
<L1
OH OH
OH OH
1) POCI3
2) Pyrophosphate
V
0
Ne0H3
0 0 0 ( 1
N.......-........ ,.."-..,
II II II N NH2
0 0--o--o¨i-0
I ,
01 e 0 e 01e <LI
OH OH
Scheme 4
e
o
e
l
o e
\
< 1 0
ll 0
ll 0
11 Nr-----.'"eL'NH2
o 0¨P¨O¨P¨O¨P-0.
N"----'-N-----.'"NF12 I) POCI3 I
HO ol e O e o e c _
c0 2) Pyrophosphate
OH OH
OH OH
Scheme 5
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0 0
HN/\NH N NH
RBr/Heat
R = alkyl, alkenyl,
HO _________________ ally!, and benzyl __ HO c0
CcL>
OH OH OH OH
1) POCI3
2) Pyrophosphate
0
N NH
0 0 0 0
8 0¨P ¨0¨P ¨0¨P-0
0
oe oe oe
OH OH
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Scheme 6
NH2 NH2
R...N..k.õ.N
HNN
0 RBr/Heat
0
R = alkyl, alkenyl,
HO _____________________________ allyl, and benzyl HO
c0 (cLI
OH OH OH OH
1) POCI3
2) Pyrophosphate
V
NH2
R
N ' N
===.1 %. ..,....õ....-..õ
0 0 0 0
II II II
eo¨P¨O¨P¨O¨P-0-
1 1 1 0
Oe oe oe
OH OH
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Scheme 7
CI
NHCH3
N-................,N
< 1 N.......,..õ..-
"'\\...N
N-------NI.---NH2 < 1 ,
N,...----,,,
HO CH3N H2/ Heat N NH2
---õ,..
HO
00. ====,....
c0
OH OH
OH OH
1) POCI3
,1
2) Pyrophosphate
NHCH3
N.................N
< 1
0 0 0
II II 11
8 0-P-O-P-O-P----0
O I I 0
8 e e
OH OH
[000531] Schemes 8 and 9 provide exemplary syntheses of modified nucleotides.
Scheme 10 provides a non-limiting biocatalytic method for producing
nucleotides.
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Scheme 8
AcOOH cc Ph Pd(0) 3 Ac0Cr OAc
Ac20
0
Enzymatic
Hydrolysis
(/ INN Ph3Pd(0)
H0.0õ,...N--\<
Hahn4.0Ac
Uracil
(1) 0504
(2) Acetone,
Ts0H/......,
ID
(/ INF! 01 )
(/ NH
H016.24=N--\( (1) (Et0)2POCH2C7Ts 0...2.00AN-i
0 0
_õ,_
0,0 00
(2)TMSil
(1) DCC, Morpholine
(2) Pyrophosphate
-0 P
0
- ,
0 *
(/ NH
- 0...20N--c(
0
00
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Scheme 9
HO
----H2
zzlo rstz-NH2 Fin \.......
Ph3P(Pd) _N ......
).. _
\
I CH2COCH3 HO OH
HO OH
COCH3
1 1) H-
2) -OH, heat
0 Frs1 H
H __________
1 ¨N
\Z 1) POCI3 HO/----
sON 2) Pyrophosphate HO OH
pO¨OH
H
0
(:)=1:(-0H
b
P:¨OH
0
HO,p'
0' %
OH
Scheme 10
HO -0,1,0
B enzyme, ATP P B
_0_?1 yeast enzymes,
B
, -4
OH OH OH OH 1-27
OH OH
[000532] Scheme 11 provides an exemplary synthesis of a modified uracil, where
the
Ni position is modified with Rim, as provided elsewhere, and the 5'-position
of ribose is
phosphorylated. Tl, T2, R12a, R12b, and r are as provided herein. This
synthesis, as well
as optimized versions thereof, can be used to modify other pyrimidine
nucleobases and
purine nucleobases (see e.g., Formulas (b1)-(b43)) and/or to install one or
more
phosphate groups (e.g., at the 5' position of the sugar). This alkylating
reaction can also
be used to include one or more optionally substituted alkyl group at any
reactive group
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(e.g., amino group) in any nucleobase described herein (e.g., the amino groups
in the
Watson-Crick base-pairing face for cytosine, uracil, adenine, and guanine).
Scheme 11
Ti Ti Ti
,R12a R12b/R12a R12b,, ,R12a
HN N N N
T2
R12bX/ileat 7 0 \
1) POC13
(X is halo) HO-
2) Pyi ()phosphate HO
c0 0
\ 01H /r
OH OH OH OH OH OH
Combinations of Nucleotides in mmRNA
[000533] Further examples of modified nucleotides and modified nucleotide
combinations are provided below in Table 9. These combinations of modified
nucleotides can be used to form the polypeptides, primary constructs, or mmRNA
of the
invention. Unless otherwise noted, the modified nucleotides may be completely
substituted for the natural nucleotides of the modified nucleic acids or mmRNA
of the
invention. As a non-limiting example, the natural nucleotide uridine may be
substituted
with a modified nucleoside described herein. In another non-limiting example,
the
natural nucleotide uridine may be partially substituted (e.g., about 0.1%, 1%,
5%, 10%,
15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95% or 99.9%) with at least one of the modified nucleoside disclosed
herein.
Table 9
Modified Nucleotide Modified Nucleotide Combination
a-thio-cytidine a-thio-cytidine/5-iodo-uridine
a-thio-cytidine/Nl-methyl-pseudouridine
a-thio-cytidine/a-thio-uridine
a-thio-cytidine/5-methy1-uridine
a-thio-cytidine/pseudo-uridine
about 50% of the cytosines are a-thio-cytidine
pseudoisocytidine pseudoisocytidine/5-iodo-uridine
pseudoisocytidine/Nl-methyl-pseudouridine
pseudoisocytidine/a-thio-uridine
pseudoisocytidine/5-methyl-uridine
pseudoisocytidine/pseudouridine
about 25% of cytosines are pseudoisocytidine
pseudoisocytidine/about 50% of uridines are N1-methyl-
pseudouridine and about 50% of uridines are
pseudouridine
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pseudoisocytidine/about 25% of uridines are N1-methyl-
pseudouridine and about 25% of uridines are
pseudouridine
pyrrolo-cytidine pyrrolo-cytidine/5-iodo-uridine
pyrrolo-cytidine/Nl-methyl-pseudouridine
pyrrolo-cytidine/a-thio-uridine
pyrrolo-cytidine/5-methyl-uridine
pyrrolo-cytidine/pseudouridine
about 50% of the cytosines are pyrrolo-cytidine
5-methyl-cytidine 5-methyl-cytidine/5-iodo-uridine
5-methyl-cytidine/N1-methyl-pseudouridine
5-methyl-cytidine/a-thio-uridine
5-methyl-cytidine/5-methyl-uridine
5-methyl-cytidine/pseudouridine
about 25% of cytosines are 5-methyl-cytidine
about 50% of cytosines are 5-methyl-cytidine
5-methyl-cytidine/5-methoxy-uridine
5-methyl-cytidine/5-bromo-uridine
5-methyl-cytidine/2-thio-uridine
5-methyl-cytidine/about 50% of uridines are 2-thio-
uridine
about 50% of uridines are 5-methyl-cytidine/ about 50%
of uridines are 2-thio-uridine
N4-acetyl-cytidine N4-acetyl-cytidine /5-iodo-uridine
N4-acetyl-cytidine /Nl-methyl-pseudouridine
N4-acetyl-cytidine /a-thio-uridine
N4-acetyl-cytidine /5-methyl-uridine
N4-acetyl-cytidine /pseudouridine
about 50% of cytosines are N4-acetyl-cytidine
about 25% of cytosines are N4-acetyl-cytidine
N4-acetyl-cytidine /5-methoxy-uridine
N4-acetyl-cytidine /5-bromo-uridine
N4-acetyl-cytidine /2-thio-uridine
about 50% of cytosines are N4-acetyl-cytidine/ about
50% of uridines are 2-thio-uridine
[000534] Further examples of modified nucleotide combinations are provided
below in
Table 10. These combinations of modified nucleotides can be used to form the
polypeptides, primary constructs, or mmRNA of the invention.
Table 10
Modified Nucleotide __ Modified Nucleotide Combination
_
modified cytidine modified cytidine with (b10)/pseudouridine
having one or more modified cytidine with (b10)/N1-methyl-pseudouridine
nucleobases of Formula modified cytidine with (b10)/5-methoxy-uridine
(b10) modified cytidine with (b10)/5-methyl-uridine
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modified cytidine with (b10)/5-bromo-uridine
modified cytidine with (b10)/2-thio-uridine
about 50% of cytidine substituted with modified cytidine (b10)/
about 50% of uridines are 2-thio-uridine
modified cytidine modified cytidine with (b32)/pseudouridine
having one or more modified cytidine with (b32)/N1-methyl-pseudouridine
nucleobases of Formula modified cytidine with (b32)/5-methoxy-uridine
(b32) modified cytidine with (b32)/5-methyl-uridine
modified cytidine with (b32)/5-bromo-uridine
modified cytidine with (b32)/2-thio-uridine
about 50% of cytidine substituted with modified cytidine (b32)/
about 50% of uridines are 2-thio-uridine
modified uridine modified uridine with (b 1)! N4-acetyl-cytidine
having one or more modified uridine with (b 1)! 5-methyl-cytidine
nucleobases of Formula
(b 1)
modified uridine modified uridine with (b8)/ N4-acetyl-cytidine
having one or more modified uridine with (b8)/ 5-methyl-cytidine
nucleobases of Formula
(b8)
modified uridine modified uridine with (b28)/ N4-acetyl-cytidine
having one or more modified uridine with (b28)/ 5-methyl-cytidine
nucleobases of Formula
(b28)
modified uridine modified uridine with (b29)/ N4-acetyl-cytidine
having one or more modified uridine with (b29)/ 5-methyl-cytidine
nucleobases of Formula
(b29)
modified uridine modified uridine with (b30)/ N4-acetyl-cytidine
having one or more modified uridine with (b30)/ 5-methyl-cytidine
nucleobases of Formula
(b30)
[000535] In some embodiments, at least 25% of the cytosines are replaced by a
compound of Formula (b10)-(b14) (e.g., at least about 30%, at least about 35%,
at least
about 40%, at least about 45%, at least about 50%, at least about 55%, at
least about 60%,
at least about 65%, at least about 70%, at least about 75%, at least about
80%, at least
about 85%, at least about 90%, at least about 95%, or about 100%).
[000536] In some embodiments, at least 25% of the uracils are replaced by a
compound
of Formula (b1)-(b9) (e.g., at least about 30%, at least about 35%, at least
about 40%, at
least about 45%, at least about 50%, at least about 55%, at least about 60%,
at least about
65%, at least about 70%, at least about 75%, at least about 80%, at least
about 85%, at
least about 90%, at least about 95%, or about 100%).
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[000537] In some embodiments, at least 25% of the cytosines are replaced by a
compound of Formula (b10)-(b14), and at least 25% of the uracils are replaced
by a
compound of Formula (b1)-(b9) (e.g., at least about 30%, at least about 35%,
at least
about 40%, at least about 45%, at least about 50%, at least about 55%, at
least about 60%,
at least about 65%, at least about 70%, at least about 75%, at least about
80%, at least
about 85%, at least about 90%, at least about 95%, or about 100%).
IV. Pharmaceutical Compositions
Formulation, Administration, Delivery and Dosing
[000538] The present invention provides polynucleotides, primary constructs
and
mmRNA compositions and complexes in combination with one or more
pharmaceutically
acceptable excipients. Pharmaceutical compositions may optionally comprise one
or
more additional active substances, e.g. therapeutically and/or
prophylactically active
substances. General considerations in the formulation and/or manufacture of
pharmaceutical agents may be found, for example, in Remington: The Science and

Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005
(incorporated herein
by reference).
[000539] In some embodiments, compositions are administered to humans, human
patients or subjects. For the purposes of the present disclosure, the phrase
"active
ingredient" generally refers to polynucleotides, primary constructs and mmRNA
to be
delivered as described herein.
[000540] Although the descriptions of pharmaceutical compositions provided
herein are
principally directed to pharmaceutical compositions which are suitable for
administration
to humans, it will be understood by the skilled artisan that such compositions
are
generally suitable for administration to any other animal, e.g., to non-human
animals, e.g.
non-human mammals. Modification of pharmaceutical compositions suitable for
administration to humans in order to render the compositions suitable for
administration
to various animals is well understood, and the ordinarily skilled veterinary
pharmacologist can design and/or perform such modification with merely
ordinary, if
any, experimentation. Subjects to which administration of the pharmaceutical
compositions is contemplated include, but are not limited to, humans and/or
other
primates; mammals, including commercially relevant mammals such as cattle,
pigs,
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horses, sheep, cats, dogs, mice, and/or rats; and/or birds, including
commercially relevant
birds such as poultry, chickens, ducks, geese, and/or turkeys.
[000541] Formulations of the pharmaceutical compositions described herein may
be
prepared by any method known or hereafter developed in the art of
pharmacology. In
general, such preparatory methods include the step of bringing the active
ingredient into
association with an excipient and/or one or more other accessory ingredients,
and then, if
necessary and/or desirable, dividing, shaping and/or packaging the product
into a desired
single- or multi-dose unit.
[000542] A pharmaceutical composition in accordance with the invention may be
prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a
plurality of
single unit doses. As used herein, a "unit dose" is discrete amount of the
pharmaceutical
composition comprising a predetermined amount of the active ingredient. The
amount of
the active ingredient is generally equal to the dosage of the active
ingredient which would
be administered to a subject and/or a convenient fraction of such a dosage
such as, for
example, one-half or one-third of such a dosage.
[000543] Relative amounts of the active ingredient, the pharmaceutically
acceptable
excipient, and/or any additional ingredients in a pharmaceutical composition
in
accordance with the invention will vary, depending upon the identity, size,
and/or
condition of the subject treated and further depending upon the route by which
the
composition is to be administered. By way of example, the composition may
comprise
between 0.1% and 100%, e.g., between .5 and 50%, between 1-30%, between 5-80%,
at
least 80% (w/w) active ingredient.
Formulations
[000544] The polynucleotide, primary construct, and mmRNA of the invention can
be
formulated using one or more excipients to: (1) increase stability; (2)
increase cell
transfection; (3) permit the sustained or delayed release (e.g., from a depot
formulation of
the polynucleotide, primary construct, or mmRNA); (4) alter the
biodistribution (e.g.,
target the polynucleotide, primary construct, or mmRNA to specific tissues or
cell types);
(5) increase the translation of encoded protein in vivo; and/or (6) alter the
release profile
of encoded protein in vivo. In addition to traditional excipients such as any
and all
solvents, dispersion media, diluents, or other liquid vehicles, dispersion or
suspension
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aids, surface active agents, isotonic agents, thickening or emulsifying
agents,
preservatives, excipients of the present invention can include, without
limitation,
lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell
nanoparticles,
peptides, proteins, cells transfected with polynucleotide, primary construct,
or mmRNA
(e.g., for transplantation into a subject), hyaluronidase, nanoparticle mimics
and
combinations thereof. Accordingly, the formulations of the invention can
include one or
more excipients, each in an amount that together increases the stability of
the
polynucleotide, primary construct, or mmRNA, increases cell transfection by
the
polynucleotide, primary construct, or mmRNA, increases the expression of
polynucleotide, primary construct, or mmRNA encoded protein, and/or alters the
release
profile of polynucleotide, primary construct, or mmRNA encoded proteins.
Further, the
primary construct and mmRNA of the present invention may be formulated using
self-
assembled nucleic acid nanoparticles.
[000545] Formulations of the pharmaceutical compositions described herein may
be
prepared by any method known or hereafter developed in the art of
pharmacology. In
general, such preparatory methods include the step of associating the active
ingredient
with an excipient and/or one or more other accessory ingredients.
[000546] A pharmaceutical composition in accordance with the present
disclosure may
be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a
plurality of
single unit doses. As used herein, a "unit dose" refers to a discrete amount
of the
pharmaceutical composition comprising a predetermined amount of the active
ingredient.
The amount of the active ingredient may generally be equal to the dosage of
the active
ingredient which would be administered to a subject and/or a convenient
fraction of such
a dosage including, but not limited to, one-half or one-third of such a
dosage.
[000547] Relative amounts of the active ingredient, the pharmaceutically
acceptable
excipient, and/or any additional ingredients in a pharmaceutical composition
in
accordance with the present disclosure may vary, depending upon the identity,
size,
and/or condition of the subject being treated and further depending upon the
route by
which the composition is to be administered. For example, the composition may
comprise
between 0.1% and 99% (w/w) of the active ingredient.
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[000548] In some embodiments, the formulations described herein may contain at
least
one mmRNA. As a non-limiting example, the formulations may contain 1, 2, 3, 4
or 5
mmRNA. In one embodiment the formulation may contain modified mRNA encoding
proteins selected from categories such as, but not limited to, human proteins,
veterinary
proteins, bacterial proteins, biological proteins, antibodies, immunogenic
proteins,
therapeutic peptides and proteins, secreted proteins, plasma membrane
proteins,
cytoplasmic and cytoskeletal proteins, intrancellular membrane bound proteins,
nuclear
proteins, proteins associated with human disease and/or proteins associated
with non-
human diseases. In one embodiment, the formulation contains at least three
modified
mRNA encoding proteins. In one embodiment, the formulation contains at least
five
modified mRNA encoding proteins.
[000549] Pharmaceutical formulations may additionally comprise a
pharmaceutically
acceptable excipient, which, as used herein, includes, but is not limited to,
any and all
solvents, dispersion media, diluents, or other liquid vehicles, dispersion or
suspension
aids, surface active agents, isotonic agents, thickening or emulsifying
agents,
preservatives, and the like, as suited to the particular dosage form desired.
Various
excipients for formulating pharmaceutical compositions and techniques for
preparing the
composition are known in the art (see Remington: The Science and Practice of
Pharmacy,
21st Edition, A. R. Gennaro, Lippincott, Williams & Wilkins, Baltimore, MD,
2006;
incorporated herein by reference in its entirety). The use of a conventional
excipient
medium may be contemplated within the scope of the present disclosure, except
insofar
as any conventional excipient medium may be incompatible with a substance or
its
derivatives, such as by producing any undesirable biological effect or
otherwise
interacting in a deleterious manner with any other component(s) of the
pharmaceutical
composition.
[000550] In some embodiments, the particle size of the lipid nanoparticle may
be
increased and/or decreased. The change in particle size may be able to help
counter
biological reaction such as, but not limited to, inflammation or may increase
the
biological effect of the modified mRNA delivered to mammals.
[000551] Pharmaceutically acceptable excipients used in the manufacture of
pharmaceutical compositions include, but are not limited to, inert diluents,
surface active
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agents and/or emulsifiers, preservatives, buffering agents, lubricating
agents, and/or oils.
Such excipients may optionally be included in the pharmaceutical formulations
of the
invention.
Lipidoids
[000552] The synthesis of lipidoids has been extensively described and
formulations
containing these compounds are particularly suited for delivery of
polynucleotides,
primary constructs or mmRNA (see Mahon et al., Bioconjug Chem. 2010 21:1448-
1454;
Schroeder et al., J Intern Med. 2010 267:9-21; Akinc et al., Nat Biotechnol.
2008 26:561-
569; Love et al., Proc Natl Acad Sci U S A. 2010 107:1864-1869; Siegwart et
al., Proc
Natl Acad Sci U S A. 2011108:12996-3001; all of which are incorporated herein
in their
entireties).
[000553] While these lipidoids have been used to effectively deliver double
stranded
small interfering RNA molecules in rodents and non-human primates (see Akinc
et al.,
Nat Biotechnol. 2008 26:561-569; Frank-Kamenetsky et al., Proc Natl Acad Sci U
S A.
2008 105:11915-11920; Akinc et al., Mol Ther. 2009 17:872-879; Love et al.,
Proc Natl
Acad Sci USA. 2010 107:1864-1869; Leuschner et al., Nat Biotechnol. 2011
29:1005-
1010; all of which is incorporated herein in their entirety), the present
disclosure
describes their formulation and use in delivering single stranded
polynucleotides, primary
constructs, or mmRNA. Complexes, micelles, liposomes or particles can be
prepared
containing these lipidoids and therefore, can result in an effective delivery
of the
polynucleotide, primary construct, or mmRNA, as judged by the production of an

encoded protein, following the injection of a lipidoid formulation via
localized and/or
systemic routes of administration. Lipidoid complexes of polynucleotides,
primary
constructs, or mmRNA can be administered by various means including, but not
limited
to, intravenous, intramuscular, or subcutaneous routes.
[000554] In vivo delivery of nucleic acids may be affected by many parameters,

including, but not limited to, the formulation composition, nature of particle
PEGylation,
degree of loading, oligonucleotide to lipid ratio, and biophysical parameters
such as, but
not limited to, particle size (Akinc et al., Mol Ther. 2009 17:872-879; herein
incorporated
by reference in its entirety). As an example, small changes in the anchor
chain length of
poly(ethylene glycol) (PEG) lipids may result in significant effects on in
vivo efficacy.
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Formulations with the different lipidoids, including, but not limited to
penta[3-(1-
laurylaminopropionyl)]-triethylenetetramine hydrochloride (TETA-5LAP; aka
98N12-5,
see Murugaiah et al., Analytical Biochemistry, 401:61(2010); herein
incorporated by
reference in its entirety), C12-200 (including derivatives and variants), and
MD1, can be
tested for in vivo activity.
[000555] The lipidoid referred to herein as "98N12-5" is disclosed by Akinc et
al., Mol
Ther. 2009 17:872-879 and is incorporated by reference in its entirety. (See
Figure 2)
[000556] The lipidoid referred to herein as "C12-200" is disclosed by Love et
al., Proc
Natl Acad Sci U S A. 2010 107:1864-1869 (see Figure 2) and Liu and Huang,
Molecular
Therapy. 2010 669-670 (see Figure 2); both of which are herein incorporated by

reference in their entirety. The lipidoid formulations can include particles
comprising
either 3 or 4 or more components in addition to polynucleotide, primary
construct, or
mmRNA. As an example, formulations with certain lipidoids, include, but are
not limited
to, 98N12-5 and may contain 42% lipidoid, 48% cholesterol and 10% PEG (C14
alkyl
chain length). As another example, formulations with certain lipidoids,
include, but are
not limited to, C12-200 and may contain 50% lipidoid, 10%
disteroylphosphatidyl
choline, 38.5% cholesterol, and 1.5% PEG-DMG.
[000557] In one embodiment, a polynucleotide, primary construct, or mmRNA
formulated with a lipidoid for systemic intravenous administration can target
the liver.
For example, a final optimized intravenous formulation using polynucleotide,
primary
construct, or mmRNA, and comprising a lipid molar composition of 42% 98N12-5,
48%
cholesterol, and 10% PEG-lipid with a final weight ratio of about 7.5 to 1
total lipid to
polynucleotide, primary construct, or mmRNA, and a C14 alkyl chain length on
the PEG
lipid, with a mean particle size of roughly 50-60 nm, can result in the
distribution of the
formulation to be greater than 90% to the liver.(see, Akinc et al., Mol Ther.
2009 17:872-
879; herein incorporated by reference in its entirety). In another example, an
intravenous
formulation using a C12-200 (see US provisional application 61/175,770 and
published
international application W02010129709, each of which is herein incorporated
by
reference in their entirety) lipidoid may have a molar ratio of 50/10/38.5/1.5
of C12-
200/disteroylphosphatidyl choline/cholesterol/PEG-DMG, with a weight ratio of
7 to 1
total lipid to polynucleotide, primary construct, or mmRNA, and a mean
particle size of
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80 nm may be effective to deliver polynucleotide, primary construct, or mmRNA
to
hepatocytes (see, Love et al., Proc Natl Acad Sci U S A. 2010 107:1864-1869
herein
incorporated by reference in its entirety). In another embodiment, an MD1
lipidoid-
containing formulation may be used to effectively deliver polynucleotide,
primary
construct, or mmRNA to hepatocytes in vivo. The characteristics of optimized
lipidoid
formulations for intramuscular or subcutaneous routes may vary significantly
depending
on the target cell type and the ability of formulations to diffuse through the
extracellular
matrix into the blood stream. While a particle size of less than 150 nm may be
desired for
effective hepatocyte delivery due to the size of the endothelial fenestrae
(see, Akinc et al.,
Mol Ther. 2009 17:872-879 herein incorporated by reference in its entirety),
use of a
lipidoid-formulated polynucleotide, primary construct, or mmRNA to deliver the

formulation to other cells types including, but not limited to, endothelial
cells, myeloid
cells, and muscle cells may not be similarly size-limited. Use of lipidoid
formulations to
deliver siRNA in vivo to other non-hepatocyte cells such as myeloid cells and
endothelium has been reported (see Akinc et al., Nat Biotechnol. 2008 26:561-
569;
Leuschner et al., Nat Biotechnol. 2011 29:1005-1010; Cho et al. Adv. Funct.
Mater. 2009
19:3112-3118; 8th International Judah Folkman Conference, Cambridge, MA
October 8-
9, 2010; each of which is herein incorporated by reference in its entirety).
Effective
delivery to myeloid cells, such as monocytes, lipidoid formulations may have a
similar
component molar ratio. Different ratios of lipidoids and other components
including, but
not limited to, disteroylphosphatidyl choline, cholesterol and PEG-DMG, may be
used to
optimize the formulation of the polynucleotide, primary construct, or mmRNA
for
delivery to different cell types including, but not limited to, hepatocytes,
myeloid cells,
muscle cells, etc. For example, the component molar ratio may include, but is
not limited
to, 50% C12-200, 10% disteroylphosphatidyl choline, 38.5% cholesterol, and
%1.5 PEG-
DMG (see Leuschner et al., Nat Biotechnol 2011 29:1005-1010; herein
incorporated by
reference in its entirety). The use of lipidoid formulations for the localized
delivery of
nucleic acids to cells (such as, but not limited to, adipose cells and muscle
cells) via
either subcutaneous or intramuscular delivery, may not require all of the
formulation
components desired for systemic delivery, and as such may comprise only the
lipidoid
and the polynucleotide, primary construct, or mmRNA.
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[000558] Combinations of different lipidoids may be used to improve the
efficacy of
polynucleotide, primary construct, or mmRNA directed protein production as the

lipidoids may be able to increase cell transfection by the polynucleotide,
primary
construct, or mmRNA; and/or increase the translation of encoded protein (see
Whitehead
et al., Mol. Ther. 2011, 19:1688-1694, herein incorporated by reference in its
entirety).
Liposomes, Lipoplexes, and Lipid Nanoparticles
[000559] The polynucleotide, primary construct, and mmRNA of the invention can
be
formulated using one or more liposomes, lipoplexes, or lipid nanoparticles. In
one
embodiment, pharmaceutical compositions of polynucleotide, primary construct,
or
mmRNA include liposomes. Liposomes are artificially-prepared vesicles which
may
primarily be composed of a lipid bilayer and may be used as a delivery vehicle
for the
administration of nutrients and pharmaceutical formulations. Liposomes can be
of
different sizes such as, but not limited to, a multilamellar vesicle (MLV)
which may be
hundreds of nanometers in diameter and may contain a series of concentric
bilayers
separated by narrow aqueous compartments, a small unicellular vesicle (SUV)
which
may be smaller than 50 nm in diameter, and a large unilamellar vesicle (LUV)
which may
be between 50 and 500 nm in diameter. Liposome design may include, but is not
limited
to, opsonins or ligands in order to improve the attachment of liposomes to
unhealthy
tissue or to activate events such as, but not limited to, endocytosis.
Liposomes may
contain a low or a high pH in order to improve the delivery of the
pharmaceutical
formulations.
[000560] The formation of liposomes may depend on the physicochemical
characteristics such as, but not limited to, the pharmaceutical formulation
entrapped and
the liposomal ingredients , the nature of the medium in which the lipid
vesicles are
dispersed, the effective concentration of the entrapped substance and its
potential
toxicity, any additional processes involved during the application and/or
delivery of the
vesicles, the optimization size, polydispersity and the shelf-life of the
vesicles for the
intended application, and the batch-to-batch reproducibility and possibility
of large-scale
production of safe and efficient liposomal products.
[000561] In one embodiment, pharmaceutical compositions described herein may
include, without limitation, liposomes such as those formed from 1,2-
dioleyloxy-N,N-
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dimethylaminopropane (DODMA) liposomes, DiLa2 liposomes from Marina Biotech
(Bothell, WA), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA), 2,2-
dilinoley1-
4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), and MC3
(US20100324120; herein incorporated by reference in its entirety) and
liposomes which
may deliver small molecule drugs such as, but not limited to, DOXILO from
Janssen
Biotech, Inc. (Horsham, PA).
[000562] In one embodiment, pharmaceutical compositions described herein may
include, without limitation, liposomes such as those formed from the synthesis
of
stabilized plasmid-lipid particles (SPLP) or stabilized nucleic acid lipid
particle (SNALP)
that have been previously described and shown to be suitable for
oligonucleotide delivery
in vitro and in vivo (see Wheeler et al. Gene Therapy. 1999 6:271-281; Zhang
et al. Gene
Therapy. 1999 6:1438-1447; Jeffs et al. Pharm Res. 2005 22:362-372; Morrissey
et al.,
Nat Biotechnol. 2005 2:1002-1007; Zimmermann et al., Nature. 2006 441:111-114;

Heyes et al. J Contr Rel. 2005 107:276-287; Semple et al. Nature Biotech. 2010
28:172-
176; Judge et al. J Clin Invest. 2009 119:661-673; deFougerolles Hum Gene
Ther. 2008
19:125-132; all of which are incorporated herein in their entireties). The
original
manufacture method by Wheeler et al. was a detergent dialysis method, which
was later
improved by Jeffs et al. and is referred to as the spontaneous vesicle
formation method.
The liposome formulations are composed of 3 to 4 lipid components in addition
to the
polynucleotide, primary construct, or mmRNA. As an example a liposome can
contain,
but is not limited to, 55% cholesterol, 20% disteroylphosphatidyl choline
(DSPC), 10%
PEG-S-DSG, and 15% 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), as
described by Jeffs et al. As another example, certain liposome formulations
may contain,
but are not limited to, 48% cholesterol, 20% DSPC, 2% PEG-c-DMA, and 30%
cationic
lipid, where the cationic lipid can be 1,2-distearloxy-/V,N-
dimethylaminopropane
(DSDMA), DODMA, DLin-DMA, or 1,2-dilinolenyloxy-3-dimethylaminopropane
(DLenDMA), as described by Heyes et al.
[000563] In one embodiment, pharmaceutical compositions may include liposomes
which may be formed to deliver mmRNA which may encode at least one immunogen.
The mmRNA may be encapsulated by the liposome and/or it may be contained in an

aqueous core which may then be encapsulated by the liposome (see International
Pub.
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Nos. W02012031046, W02012031043, W02012030901 and W02012006378; each of
which is herein incorporated by reference in their entirety). In another
embodiment, the
mmRNA which may encode an immunogen may be formulated in a cationic oil-in-
water
emulsion where the emulsion particle comprises an oil core and a cationic
lipid which can
interact with the mmRNA anchoring the molecule to the emulsion particle (see
International Pub. No. W02012006380; herein incorporated by reference in its
entirety).
In yet another embodiment, the lipid formulation may include at least cationic
lipid, a
lipid which may enhance transfection and a least one lipid which contains a
hydrophilic
head group linked to a lipid moiety (International Pub. No. W02011076807 and
U.S.
Pub. No. 20110200582; each of which is herein incorporated by reference in
their
entirety). In another embodiment, the polynucleotides, primary constructs
and/or
mmRNA encoding an immunogen may be formulated in a lipid vesicle which may
have
crosslinks between functionalized lipid bilayers (see U.S. Pub. No.
20120177724, herein
incorporated by reference in its entirety).
[000564] In one embodiment, the polynucleotides, primary constructs and/or
mmRNA
may be formulated in a lipid vesicle which may have crosslinks between
functionalized
lipid bilayers.
[000565] In one embodiment, the polynucleotides, primary constructs and/or
mmRNA
may be formulated in a liposome comprising a cationic lipid. The liposome may
have a
molar ratio of nitrogen atoms in the cationic lipid to the phophates in the
RNA (N:P ratio)
of between 1:1 and 20:1 as described in International Publication No.
W02013006825,
herein incorporated by reference in its entirety. In another embodiment, the
liposome
may have a N:P ratio of greater than 20:1 or less than 1:1.
[000566] In one embodiment, the polynucleotides, primary constructs and/or
mmRNA
may be formulated in a lipid-polycation complex. The formation of the lipid-
polycation
complex may be accomplished by methods known in the art and/or as described in
U.S.
Pub. No. 20120178702, herein incorporated by reference in its entirety. As a
non-
limiting example, the polycation may include a cationic peptide or a
polypeptide such as,
but not limited to, polylysine, polyornithine and/or polyarginine and the
cationic peptides
described in International Pub. No. W02012013326; herein incorporated by
reference in
its entirety. In another embodiment, the polynucleotides, primary constructs
and/or
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mmRNA may be formulated in a lipid-polycation complex which may further
include a
neutral lipid such as, but not limited to, cholesterol or dioleoyl
phosphatidylethanolamine
(DOPE).
[000567] The liposome formulation may be influenced by, but not limited to,
the
selection of the cationic lipid component, the degree of cationic lipid
saturation, the
nature of the PEGylation, ratio of all components and biophysical parameters
such as
size. In one example by Semple et al. (Semple et al. Nature Biotech. 2010
28:172-176;
herein incorporated by reference in its entirety), the liposome formulation
was composed
of 57.1 % cationic lipid, 7.1% dipalmitoylphosphatidylcholine, 34.3 %
cholesterol, and
1.4% PEG-c-DMA. As another example, changing the composition of the cationic
lipid
could more effectively deliver siRNA to various antigen presenting cells
(Basha et al.
Mol Ther. 201119:2186-2200; herein incorporated by reference in its entirety).
[000568] In some embodiments, the ratio of PEG in the lipid nanoparticle (LNP)

formulations may be increased or decreased and/or the carbon chain length of
the PEG
lipid may be modified from C14 to C18 to alter the pharmacokinetics and/or
biodistribution of the LNP formulations. As a non-limiting example, LNP
formulations
may contain 1-5% of the lipid molar ratio of PEG-c-DOMG as compared to the
cationic
lipid, DSPC and cholesterol. In another embodiment the PEG-c-DOMG may be
replaced
with a PEG lipid such as, but not limited to, PEG- DSG (1,2-Distearoyl-sn-
glycerol,
methoxypolyethylene glycol) or PEG-DPG (1,2-Dipalmitoyl-sn-glycerol,
methoxypolyethylene glycol). The cationic lipid may be selected from any lipid
known
in the art such as, but not limited to, DLin-MC3-DMA, DLin-DMA, C12-200 and
DLin-
KC2-DMA.
[000569] In one embodiment, the polynucleotides, primary constructs or mmRNA
may
be formulated in a lipid nanoparticle such as those described in International
Publication
No. W02012170930, herein incorporated by reference in its entirety.
[000570] In one embodiment, the cationic lipid may be selected from, but not
limited to,
a cationic lipid described in International Publication Nos. W02012040184,
W02011153120, W02011149733, W02011090965, W02011043913, W02011022460,
W02012061259, W02012054365, W02012044638, W02010080724, W0201021865
and W02008103276, US Patent Nos. 7,893,302, 7,404,969 and 8,283,333 and US
Patent
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Publication No. US20100036115 and US20120202871; each of which is herein
incorporated by reference in their entirety. In another embodiment, the
cationic lipid may
be selected from, but not limited to, formula A described in International
Publication
Nos. W02012040184, W02011153120, W02011149733, W02011090965,
W02011043913, W02011022460, W02012061259, W02012054365 and
W02012044638; each of which is herein incorporated by reference in their
entirety. In
yet another embodiment, the cationic lipid may be selected from, but not
limited to,
formula CLI-CL)(XIX of International Publication No. W02008103276, formula CLI-

CL)(XIX of US Patent No. 7,893,302, formula CLI-CLXXXXII of US Patent No.
7,404,969 and formula 1-VI of US Patent Publication No. US20100036115; each of

which is herein incorporated by reference in their entirety. As a non-limiting
example,
the cationic lipid may be selected from (20Z,23Z)-N,N-dimethylnonacosa-20,23-
dien-10-
amine, (17Z,20Z)-N,N-dimemylhexacosa-17,20-dien-9-amine, (1Z,19Z)-N5N-
dimethylpentacosa-16, 19-dien-8-amine, (13Z,16Z)-N,N-dimethyldocosa-13,16-dien-
5-
amine, (12Z,15Z)-N,N-dimethylhenicosa-12,15-dien-4-amine, (14Z,17Z)-N,N-
dimethyltricosa-14,17-dien-6-amine, (15Z,18Z)-N,N-dimethyltetracosa-15,18-dien-
7-
amine, (18Z,21Z)-N,N-dimethylheptacosa-18,21-dien-10-amine, (15Z,18Z)-N,N-
dimethyltetracosa-15,18-dien-5-amine, (14Z,17Z)-N,N-dimethyltricosa-14,17-dien-
4-
amine, (19Z,22Z)-N,N-dimeihyloctacosa-19,22-dien-9-amine, (18Z,21 Z)-N,N-
dimethylheptacosa- 18 ,21 -dien-8 ¨amine, (17Z,20Z)-N,N-dimethylhexacosa-
17,20-
dien-7-amine, (16Z,19Z)-N,N-dimethylpentacosa-16,19-dien-6-amine, (22Z,25Z)-
N,N-
dimethylhentriaconta-22,25-dien-10-amine, (21 Z ,24Z)-N,N-dimethyltriaconta-
21,24-
dien-9-amine, (18Z)-N,N-dimetylheptacos-18-en-10-amine, (17Z)-N,N-
dimethylhexacos-
17-en-9-amine, (19Z,22Z)-N,N-dimethyloctacosa-19,22-dien-7-amine, N,N-
dimethylheptacosan-10-amine, (20Z,23Z)-N-ethyl-N-methylnonacosa-20,23-dien-10-
amine, 1-[(11Z,14Z)-1-nonylicosa-11,14-dien-l-yl] pyrrolidine, (20Z)-N,N-
dimethylheptacos-20-en-10-amine, (15Z)-N,N-dimethyl eptacos-15-en-10-amine,
(14Z)-
N,N-dimethylnonacos-14-en-10-amine, (17Z)-N,N-dimethylnonacos-17-en-10-amine,
(24Z)-N,N-dimethyltritriacont-24-en-10-amine, (20Z)-N,N-dimethylnonacos-20-en-
10-
amine, (22Z)-N,N-dimethylhentriacont-22-en-10-amine, (16Z)-N,N-
dimethylpentacos-16-
en-8-amine, (12Z,15Z)-N,N-dimethy1-2-nonylhenicosa-12,15-dien-1¨amine,
(13Z,16Z)-
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N,N-dimethy1-3-nonyldocosa-13,16-dien-1-amine, N,N-dimethy1-1-[(1S,2R)-2-
octylcyclopropyl] eptadecan-8-amine, 1- [(1S,2R)-2-hexylcyclopropyl]-N,N-
dimethylnonadecan-10-amine, N,N-dimethy1-1 - [(1S ,2R)-2-o
ctylcyclopropyl]nonadecan-
10-amine, N,N-dimethy1-21-[(1S,2R)-2-octylcyclopropyl]henicosan-10-amine,N,N-
dimethyl-1-[(1S,25)-2- { [(1R,2R)-2-pentylcycIopropyl]methyl}
cyclopropyl]nonadecan-
10-amine,N,N-dimethy1-1-[(1S,2R)-2-octylcyclopropyl]hexadecan-8-amine, N,N-
dimethyl-[(1R,2 S)-2-undecyIcyclopropyl]tetradecan-5 -amine, N,N-dimethy1-3- {
7-
[(1 S ,2R)-2-octylcyclopropyl]heptyl} dodecan-l-amine, 1- [(1R,2S)-2-hepty
lcyclopropy1]-N,N-dimethyloctadecan-9-amine, 1- [(1S,2R)-2-decylcyclopropy1]-
N,N-
dimethylpentadecan-6-amine, N,N-dimethy1-1- [(1S ,2R)-2-
octylcyclopropyl]pentadecan-8 -
amine, R-N,N-dimethy1-1 - [(9Z,12Z)-octadeca-9,12-dien-1-yloxy] -3 -
(octyloxy)propan-2-
amine, S-N,N-dimethy1-1 - [(9Z,12Z)-o ctadeca-9,12-dien-1 -yloxy]-3 -
(octyloxy)propan-2-
amine, 1- {2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-
Roctyloxy)methyllethyl}pyrrolidine, (2 S)-N,N-dimethy1-1 -[(9Z,12Z)-octadeca-
9,12-
dien-1 -yloxy] -3- [(5Z)-oct-5 -en-1 -yloxy]propan-2-amine, 1- {2-[(9Z,12Z)-
octadeca-9,12-
dien-1 -yloxy]-1 -[(octyloxy)methyl] ethyl} azetidine, (25)-i -(hexyloxy)-N,N-
dimethy1-3 -
[(9Z,12Z)-octadeca-9,12-dien-1 -yloxy]propan-2-amine, (2 S)-1 -(heptyloxy)-N,N-

dimethy1-3 - [(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, N,N-dimethy1-
1-
(nonyloxy)-3-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-2-amine, N,N-dimethy1-
1-
[(9Z)-octadec-9-en-l-yloxy]-3-(octyloxy)propan-2-amine; (2 S)-N,N-dimethy1-1 -

[(6Z,9Z,12Z)-octadeca-6,9,12-trien-1 -ylo xy]-3 -(octyloxy)propan-2-amine, (2
S)-1 -
[(11Z,14Z)-icosa-11,14-dien-1 -yloxy]-N,N-dimethy1-3 -(pentyloxy)propan-2-
amine, (2S)-
1 -(hexylo xy)-3 - [(11Z,14Z)-icosa-11,14-dien-l-yloxy]-N,N-dimethylpropan-2 -
amine, 1 -
[(11Z,14Z)-icosa-11,14-dien-1 -yloxy]-N,N-dimethyl -3 -(octyloxy)propan-2-
amine, 1 -
[(13Z,16Z)-docosa-13 ,16-dien-l-yloxy]-N,N-dimethy1-3-(octyloxy)propan-2-
amine, (2 S)-
1 - [(13Z,16Z)-docosa-13 ,16-dien-1 -yloxy] -3 -(hexyloxy)-N,N-dimethylpropan-
2-amine,
(2 S)-1 -[(13Z)-docos-13-en-1 -yloxy]-3 -(hexyloxy)-N,N-dimethylpropan-2-
amine, 1 -
[(13Z)-docos-13 -en-1 -yloxy]-N,N-dimethy1-3 -(octyloxy)propan-2-amine, 1-
[(9Z)-
hexadec-9-en-1-yloxy]-N,N-dimethy1-3-(octyloxy)propan-2-amine, (2R)-N,N-
dimethyl-
H(1 -metoylo ctyl)oxy]-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,
(2R)-1-
[(3 ,7-dimethyloctyl)oxy]-N,N-dimethy1-3- [(9Z,12Z)-octadeca-9,12-dien-1 -
yloxy]propan-
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2-amine, N,N-dimethy1-1-(octyloxy)-3-({8-[(1S,2S)-2-{[(1R,2R)-2-
pentylcyclopropyl]methyl} cyc lopropyl] o ctyl} oxy)propan-2-amine, N,N-
dimethy1-1- { [8-
(2-oc1ylcyclopropyl)octyl]oxy} -3-(octyloxy)propan-2-amine and (11E,20Z,23Z)-
N,N-
dimethylnonacosa-11,20,2-trien-10-amine or a pharmaceutically acceptable salt
or
stereoisomer thereof.
[000571] In one embodiment, the lipid may be a cleavable lipid such as those
described
in International Publication No. W02012170889, herein incorporated by
reference in its
entirety.
[000572] In one embodiment, the cationic lipid may be synthesized by methods
known
in the art and/or as described in International Publication Nos. W02012040184,

W02011153120, W02011149733, W02011090965, W02011043913, W02011022460,
W02012061259, W02012054365, W02012044638, W02010080724 and
W0201021865; each of which is herein incorporated by reference in their
entirety.
[000573] In one embodiment, the LNP formulations of the polynucleotides,
primary
constructs and/or mmRNA may contain PEG-c-DOMG at 3% lipid molar ratio. In
another embodiment, the LNP formulations polynucleotides, primary constructs
and/or
mmRNA may contain PEG-c-DOMG at 1.5% lipid molar ratio.
[000574] In one embodiment, the pharmaceutical compositions of the
polynucleotides,
primary constructs and/or mmRNA may include at least one of the PEGylated
lipids
described in International Publication No. 2012099755, herein incorporated by
reference.
[000575] In one embodiment, the LNP formulation may contain PEG-DMG 2000 (1,2-
dimyristoyl-sn-glycero-3-phophoethanolamine-N-[methoxy(polyethylene glycol)-
2000).
In one embodiment, the LNP formulation may contain PEG-DMG 2000, a cationic
lipid
known in the art and at least one other component. In another embodiment, the
LNP
formulation may contain PEG-DMG 2000, a cationic lipid known in the art, DSPC
and
cholesterol. As a non-limiting example, the LNP formulation may contain PEG-
DMG
2000, DLin-DMA, DSPC and cholesterol. As another non-limiting example the LNP
formulation may contain PEG-DMG 2000, DLin-DMA, DSPC and cholesterol in a
molar
ratio of 2:40:10:48 (see e.g., Geall et al., Nonviral delivery of self-
amplifying RNA
vaccines, PNAS 2012; PMID: 22908294; herein incorporated by reference in its
entirety).
. As another non-limiting example, modified RNA described herein may be
formulated
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in a nanoparticle to be delivered by a parenteral route as described in U.S.
Pub. No.
20120207845; herein incorporated by reference in its entirety.
In one embodiment, the LNP formulation may be formulated by the methods
described in
International Publication Nos. W02011127255 or W02008103276, each of which is
herein incorporated by reference in their entirety. As a non-limiting example,
modified
RNA described herein may be encapsulated in LNP formulations as described in
W02011127255 and/or W02008103276; each of which is herein incorporated by
reference in their entirety.
In one embodiment, LNP formulations described herein may comprise a
polycationic
composition. As a non-limiting example, the polycationic composition may be
selected
from formula 1-60 of US Patent Publication No. U520050222064; herein
incorporated by
reference in its entirety. In another embodiment, the LNP formulations
comprising a
polycationic composition may be used for the delivery of the modified RNA
described
herein in vivo and/or in vitro.
[000576] In one embodiment, the LNP formulations described herein may
additionally
comprise a permeability enhancer molecule. Non-limiting permeability enhancer
molecules are described in US Patent Publication No. U52005 0222064; herein
incorporated by reference in its entirety.
[000577] In one embodiment, the pharmaceutical compositions may be formulated
in
liposomes such as, but not limited to, DiLa2 liposomes (Marina Biotech,
Bothell, WA),
SMARTICLESO (Marina Biotech, Bothell, WA), neutral DOPC (1,2-dioleoyl-sn-
glycero-3-phosphocholine) based liposomes (e.g., siRNA delivery for ovarian
cancer
(Landen et al. Cancer Biology & Therapy 2006 5(12)1708-1713); herein
incorporated by
reference in its entirety) and hyaluronan-coated liposomes (Quiet
Therapeutics, Israel).
[000578] The nanoparticle formulations may be a carbohydrate nanoparticle
comprising
a carbohydrate carrier and a modified nucleic acid molecule (e.g., mmRNA). As
a non-
limiting example, the carbohydrate carrier may include, but is not limited to,
an
anhydride-modified phytoglycogen or glycogen-type material, phtoglycogen
octenyl
succinate, phytoglycogen beta-dextrin, anhydride-modified phytoglycogen beta-
dextrin.
(See e.g., International Publication No. W02012109121; herein incorporated by
reference in its entirety).
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[000579] Lipid nanoparticle formulations may be improved by replacing the
cationic
lipid with a biodegradable cationic lipid which is known as a rapidly
eliminated lipid
nanoparticle (reLNP). Ionizable cationic lipids, such as, but not limited to,
DLinDMA,
DLin-KC2-DMA, and DLin-MC3-DMA, have been shown to accumulate in plasma and
tissues over time and may be a potential source of toxicity. The rapid
metabolism of the
rapidly eliminated lipids can improve the tolerability and therapeutic index
of the lipid
nanoparticles by an order of magnitude from a 1 mg/kg dose to a 10 mg/kg dose
in rat.
Inclusion of an enzymatically degraded ester linkage can improve the
degradation and
metabolism profile of the cationic component, while still maintaining the
activity of the
reLNP formulation. The ester linkage can be internally located within the
lipid chain or it
may be terminally located at the terminal end of the lipid chain. The internal
ester
linkage may replace any carbon in the lipid chain.
[000580] In one embodiment, the internal ester linkage may be located on
either side of
the saturated carbon. Non-limiting examples of reLNPs include,
0
9
1-
0
and
[000581] In one embodiment, an immune response may be elicited by delivering a
lipid
nanoparticle which may include a nanospecies, a polymer and an immunogen.
(U.S.
Publication No. 20120189700 and International Publication No. W02012099805;
each of
which is herein incorporated by reference in their entirety). The polymer may
encapsulate the nanospecies or partially encapsulate the nanospecies. The
immunogen
may be a recombinant protein, a modified RNA and/or a primary construct
described
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herein. In one embodiment, the lipid nanoparticle may be formulated for use in
a vaccine
such as, but not limited to, against a pathogen.
[000582] Lipid nanoparticles may be engineered to alter the surface properties
of
particles so the lipid nanoparticles may penetrate the mucosal barrier. Mucus
is located
on mucosal tissue such as, but not limted to, oral (e.g., the buccal and
esophageal
membranes and tonsil tissue), ophthalmic, gastrointestinal (e.g., stomach,
small intestine,
large intestine, colon, rectum), nasal, respiratory (e.g., nasal, pharyngeal,
tracheal and
bronchial membranes), genital (e.g., vaginal, cervical and urethral
membranes).
Nanoparticles larger than 10-200 nm which are preferred for higher drug
encapsulation
efficiency and the ability to provide the sustained delivery of a wide array
of drugs have
been thought to be too large to rapidly diffuse through mucosal barriers.
Mucus is
continuously secreted, shed, discarded or digested and recycled so most of the
trapped
particles may be removed from the mucosla tissue within seconds or within a
few hours.
Large polymeric nanoparticles (200nm -500nm in diameter) which have been
coated
densely with a low molecular weight polyethylene glycol (PEG) diffused through
mucus
only 4 to 6-fold lower than the same particles diffusing in water (Lai et al.
PNAS 2007
104(5):1482-487; Lai et al. Adv Drug Deliv Rev. 2009 61(2): 158-171; each of
which is
herein incorporated by reference in their entirety). The transport of
nanoparticles may be
determined using rates of permeation and/or fluorescent microscopy techniques
including, but not limited to, fluorescence recovery after photobleaching
(FRAP) and
high resolution multiple particle tracking (MPT). As a non-limiting example,
compositions which can penetrate a mucosal barrier may be made as described in
U.S.
Pat. No. 8,241,670, herein incorporated by reference in its entirety.
[000583] The lipid nanoparticle engineered to penetrate mucus may comprise a
polymeric material (i.e. a polymeric core) and/or a polymer-vitamin conjugate
and/or a
tri-block co-polymer. The polymeric material may include, but is not limited
to,
polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas,
polycarbonates, poly(styrenes), polyimides, polysulfones, polyurethanes,
polyacetylenes,
polyethylenes, polyethyeneimines, polyisocyanates, polyacrylates,
polymethacrylates,
polyacrylonitriles, and polyarylates. The polymeric material may be
biodegradable and/or
biocompatible. The polymeric material may additionally be irradiated. As a non-
limiting
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example, the polymeric material may be gamma irradiated (See e.g.,
International App.
No. W0201282165, herein incorporated by reference in its entirety). Non-
limiting
examples of specific polymers include poly(caprolactone) (PCL), ethylene vinyl
acetate
polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA),
poly(glycolic acid)
(PGA), poly(lactic acid-co-glycolic acid) (PLGA), poly(L-lactic acid-co-
glycolic acid)
(PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide-co-

caprolactone), poly(D,L-lactide-co-caprolactone-co-glycolide), poly(D,L-
lactide-co-
PEO-co-D,L-lactide), poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl
cyanoacralate,
polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA),
polyethyleneglycol, poly-L-glutamic acid, poly(hydroxy acids), polyanhydrides,

polyorthoesters, poly(ester amides), polyamides, poly(ester ethers),
polycarbonates,
polyalkylenes such as polyethylene and polypropylene, polyalkylene glycols
such as
poly(ethylene glycol) (PEG), polyalkylene oxides (PEO), polyalkylene
terephthalates
such as poly(ethylene terephthalate), polyvinyl alcohols (PVA), polyvinyl
ethers,
polyvinyl esters such as poly(vinyl acetate), polyvinyl halides such as
poly(vinyl
chloride) (PVC), polyvinylpyrrolidone, polysiloxanes, polystyrene (PS),
polyurethanes,
derivatized celluloses such as alkyl celluloses, hydroxyalkyl celluloses,
cellulose ethers,
cellulose esters, nitro celluloses, hydroxypropylcellulose,
carboxymethylcellulose,
polymers of acrylic acids, such as poly(methyl(meth)acrylate) (PMMA),
poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate),
poly(isobutyl(meth)acrylate),
poly(hexyl(meth)acrylate), poly(isodecyl(meth)acrylate),
poly(lauryl(meth)acrylate),
poly(phenyl(meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate),
poly(isobutyl acrylate), poly(octadecyl acrylate) and copolymers and mixtures
thereof,
polydioxanone and its copolymers, polyhydroxyalkanoates, polypropylene
fumarate,
polyoxymethylene, poloxamers, poly(ortho)esters, poly(butyric acid),
poly(valeric acid),
poly(lactide-co-caprolactone), and trimethylene carbonate,
polyvinylpyrrolidone .The
lipid nanoparticle may be coated or associated with a co-polymer such as, but
not limited
to, a block co-polymer (such as a branched polyether-polyamide block copolymer

described in International Publication No. W02013012476, herein incorporated
by
reference in its entirety), and (poly(ethylene glycol))-(poly(propylene
oxide))-
(poly(ethylene glycol)) triblock copolymer (see e.g., US Publication
20120121718 and
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US Publication 20100003337 and U.S. Pat. No. 8,263,665; each of which is
herein
incorporated by reference in their entirety). The co-polymer may be a polymer
that is
generally regarded as safe (GRAS) and the formation of the lipid nanoparticle
may be in
such a way that no new chemical entities are created. For example, the lipid
nanoparticle
may comprise poloxamers coating PLGA nanoparticles without forming new
chemical
entities which are still able to rapidly penetrate human mucus (Yang et al.
Angew. Chem.
Int. Ed. 2011 50:2597-2600; herein incorporated by reference in its entirety).
[000584] The vitamin of the polymer-vitamin conjugate may be vitamin E. The
vitamin
portion of the conjugate may be substituted with other suitable components
such as, but
not limited to, vitamin A, vitamin E, other vitamins, cholesterol, a
hydrophobic moiety,
or a hydrophobic component of other surfactants (e.g., sterol chains, fatty
acids,
hydrocarbon chains and alkylene oxide chains).
[000585] The lipid nanoparticle engineered to penetrate mucus may include
surface
altering agents such as, but not limited to, mmRNA, anionic proteins (e.g.,
bovine serum
albumin), surfactants (e.g., cationic surfactants such as for example
dimethyldioctadecyl-
ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrin), nucleic
acids,
polymers (e.g., heparin, polyethylene glycol and poloxamer), mucolytic agents
(e.g., N-
acetylcysteine, mugwort, bromelain, papain, clerodendrum, acetylcysteine,
bromhexine,
carbocisteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letosteine,
stepronin,
tiopronin, gelsolin, thymosin P4 dornase alfa, neltenexine, erdosteine) and
various
DNases including rhDNase.. The surface altering agent may be embedded or
enmeshed in
the particle's surface or disposed (e.g., by coating, adsorption, covalent
linkage, or other
process) on the surface of the lipid nanoparticle. (see e.g., US Publication
20100215580
and US Publication 20080166414; each of which is herein incorporated by
reference in
their entirety).
[000586] The mucus penetrating lipid nanoparticles may comprise at least one
mmRNA
described herein. The mmRNA may be encapsulated in the lipid nanoparticle
and/or
disposed on the surface of the paricle. The mmRNA may be covalently coupled to
the
lipid nanoparticle. Formulations of mucus penetrating lipid nanoparticles may
comprise
a plurality of nanoparticles. Further, the formulations may contain particles
which may
interact with the mucus and alter the structural and/or adhesive properties of
the
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surrounding mucus to decrease mucoadhesion which may increase the delivery of
the
mucus penetrating lipid nanoparticles to the mucosal tissue.
[000587] In one embodiment, the polynucleotide, primary construct, or mmRNA is

formulated as a lipoplex, such as, without limitation, the ATUPLEXTm system,
the
DACC system, the DBTC system and other siRNA-lipoplex technology from Silence
Therapeutics (London, United Kingdom), STEMFECTTm from STEMGENTO
(Cambridge, MA), and polyethylenimine (PEI) or protamine-based targeted and
non-
targeted delivery of nucleic acids acids (Aleku et al. Cancer Res. 2008
68:9788-9798;
Strumberg et al. Int J Clin Pharmacol Ther 2012 50:76-78; Santel et al., Gene
Ther 2006
13:1222-1234; Santel et al., Gene Ther 2006 13:1360-1370; Gutbier et al., Pulm

Pharmacol. Ther. 2010 23:334-344; Kaufmann et al. Microvasc Res 2010 80:286-
293Weide et al. J Immunother. 2009 32:498-507; Weide et al. J Immunother. 2008

31:180-188; Pascolo Expert Opin. Biol. Ther. 4:1285-1294; Fotin-Mleczek et
al., 2011 J.
Immunother. 34:1-15; Song et al., Nature Biotechnol. 2005, 23:709-717; Peer et
al., Proc
Natl Acad Sci U S A. 2007 6;104:4095-4100; deFougerolles Hum Gene Ther. 2008
19:125-132; all of which are incorporated herein by reference in its
entirety).
[000588] In one embodiment such formulations may also be constructed or
compositions altered such that they passively or actively are directed to
different cell
types in vivo, including but not limited to hepatocytes, immune cells, tumor
cells,
endothelial cells, antigen presenting cells, and leukocytes (Akinc et al. Mol
Ther. 2010
18:1357-1364; Song et al., Nat Biotechnol. 2005 23:709-717; Judge et al., J
Clin Invest.
2009 119:661-673; Kaufmann et al., Microvasc Res 2010 80:286-293; Santel et
al., Gene
Ther 2006 13:1222-1234; Santel et al., Gene Ther 2006 13:1360-1370; Gutbier et
al.,
Pulm Pharmacol. Ther. 2010 23:334-344; Basha et al., Mol. Ther. 201119:2186-
2200;
Fenske and Cullis, Expert Opin Drug Deliv. 2008 5:25-44; Peer et al., Science.
2008
319:627-630; Peer and Lieberman, Gene Ther. 201118:1127-1133; all of which are

incorporated herein by reference in its entirety). One example of passive
targeting of
formulations to liver cells includes the DLin-DMA, DLin-KC2-DMA and DLin-MC3-
DMA-based lipid nanoparticle formulations which have been shown to bind to
apolipoprotein E and promote binding and uptake of these formulations into
hepatocytes
in vivo (Akinc et al. Mol Ther. 2010 18:1357-1364; herein incorporated by
reference in
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its entirety). Formulations can also be selectively targeted through
expression of
different ligands on their surface as exemplified by, but not limited by,
folate, transferrin,
N-acetylgalactosamine (GalNAc), and antibody targeted approaches (Kolhatkar et
al.,
Curr Drug Discov Technol. 2011 8:197-206; Musacchio and Torchilin, Front
Biosci.
2011 16:1388-1412; Yu et al., Mol Membr Biol. 2010 27:286-298; Patil et al.,
Crit Rev
Ther Drug Carrier Syst. 2008 25:1-61; Benoit et al., Biomacromolecules. 2011
12:2708-
2714; Zhao et al., Expert Opin Drug Deliv. 2008 5:309-319; Akinc et al., Mol
Ther. 2010
18:1357-1364; Srinivasan et al., Methods Mol Biol. 2012 820:105-116; Ben-Arie
et al.,
Methods Mol Biol. 2012 757:497-507; Peer 2010 J Control Release. 20:63-68;
Peer et al.,
Proc Natl Acad Sci U S A. 2007 104:4095-4100; Kim et al., Methods Mol Biol.
2011
721:339-353; Subramanya et al., Mol Ther. 2010 18:2028-2037; Song et al., Nat
Biotechnol. 2005 23:709-717; Peer et al., Science. 2008 319:627-630; Peer and
Lieberman, Gene Ther. 201118:1127-1133; all of which are incorporated herein
by
reference in its entirety)..
[000589] In one embodiment, the polynucleotide, primary construct, or mmRNA is

formulated as a solid lipid nanoparticle. A solid lipid nanoparticle (SLN) may
be
spherical with an average diameter between 10 to 1000 nm. SLN possess a solid
lipid
core matrix that can solubilize lipophilic molecules and may be stabilized
with
surfactants and/or emulsifiers. In a further embodiment, the lipid
nanoparticle may be a
self-assembly lipid-polymer nanoparticle (see Zhang et al., ACS Nano, 2008, 2
(8), pp
1696-1702; herein incorporated by reference in its entirety).
[000590] Liposomes, lipoplexes, or lipid nanoparticles may be used to improve
the
efficacy of polynucleotide, primary construct, or mmRNA directed protein
production as
these formulations may be able to increase cell transfection by the
polynucleotide,
primary construct, or mmRNA; and/or increase the translation of encoded
protein. One
such example involves the use of lipid encapsulation to enable the effective
systemic
delivery of polyplex plasmid DNA (Heyes et al., Mol Ther. 2007 15:713-720;
herein
incorporated by reference in its entirety). The liposomes, lipoplexes, or
lipid
nanoparticles may also be used to increase the stability of the
polynucleotide, primary
construct, or mmRNA.
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[000591] In one embodiment, the the polynucleotides, primary constructs,
and/or the
mmRNA of the present invention can be formulated for controlled release and/or
targeted
delivery. As used herein, "controlled release" refers to a pharmaceutical
composition or
compound release profile that conforms to a particular pattern of release to
effect a
therapeutic outcome. In one embodiment, the polynucleotides, primary
constructs or the
mmRNA may be encapsulated into a delivery agent described herein and/or known
in the
art for controlled release and/or targeted delivery. As used herein, the term
"encapsulate"
means to enclose, surround or encase. As it relates to the formulation of the
compounds
of the invention, encapsulation may be substantial, complete or partial. The
term
"substitantially encapsulated" means that at least greater than 50, 60, 70,
80, 85, 90, 95,
96, 97, 98, 99, 99.9, 99.9 or greater than 99.999% of the pharmaceutical
composition or
compound of the invention may be enclosed, surrounded or encased within the
delivery
agent. "Partially encapsulation" means that less than 10, 10, 20, 30, 40 50 or
less of the
pharmaceutical composition or compound of the invention may be enclosed,
surrounded
or encased within the delivery agent. Advantageously, encapsulation may be
determined
by measuring the escape or the activity of the pharmaceutical composition or
compound
of the invention using fluorescence and/or electron micrograph. For example,
at least 1,
5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.99 or
greater than
99.99% of the pharmaceutical composition or compound of the invention are
encapsulated in the delivery agent.
[000592] In one embodiment, the controlled release formulation may include,
but is not
limited to, tri-block co-polymers. As a non-limiting example, the formulation
may
include two different types of tri-block co-polymers (International Pub. No.
W02012131104 and W02012131106; each of which is herein incorporated by
reference
in its entirety).
[000593] In another embodiment, the the polynucleotides, primary constructs,
or the
mmRNA may be encapsulated into a lipid nanoparticle or a rapidly eliminated
lipid
nanoparticle and the lipid nanoparticles or a rapidly eliminated lipid
nanoparticle may
then be encapsulated into a polymer, hydrogel and/or surgical sealant
described herein
and/or known in the art. As a non-limiting example, the polymer, hydrogel or
surgical
sealant may be PLGA, ethylene vinyl acetate (EVAc), poloxamer, GELSITEO
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(Nanotherapeutics, Inc. Alachua, FL), HYLENEXO (Halozyme Therapeutics, San
Diego
CA), surgical sealants such as fibrinogen polymers (Ethicon Inc. Cornelia,
GA),
TISSELLO (Baxter International, Inc Deerfield, IL), PEG-based sealants, and
COSEALO (Baxter International, Inc Deerfield, IL).
[000594] In another embodiment, the lipid nanoparticle may be encapsulated
into any
polymer known in the art which may form a gel when injected into a subject. As
another
non-limiting example, the lipid nanoparticle may be encapsulated into a
polymer matrix
which may be biodegradable.
[000595] In one embodiment, the the polynucleotide, primary construct, or
mmRNA
formulation for controlled release and/or targeted delivery may also include
at least one
controlled release coating. Controlled release coatings include, but are not
limited to,
OPADRYO, polyvinylpyrrolidone/vinyl acetate copolymer, polyvinylpyrrolidone,
hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxyethyl
cellulose,
EUDRAGIT RLO, EUDRAGIT RS and cellulose derivatives such as ethylcellulose
aqueous dispersions (AQUACOATO and SURELEASEO).
[000596] In one embodiment, the controlled release and/or targeted delivery
formulation may comprise at least one degradable polyester which may contain
polycationic side chains. Degradeable polyesters include, but are not limited
to,
poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline
ester), and
combinations thereof. In another embodiment, the degradable polyesters may
include a
PEG conjugation to form a PEGylated polymer.
[000597] In one embodiment, the the polynucleotides, primary constructs,
and/or the
mmRNA of the present invention may be encapsulated in a therapeutic
nanoparticle.
Therapeutic nanoparticles may be formulated by methods described herein and
known in
the art such as, but not limited to, International Pub Nos. W02010005740,
W02010030763, W02010005721, W02010005723, W02012054923, US Pub. Nos.
U520110262491, U520100104645, U520100087337, U520100068285,
U520110274759, U520100068286 and U520120288541 and US Pat No. 8,206,747,
8,293,276, 8,318,208 and 8,318,211 each of which is herein incorporated by
reference in
their entirety. In another embodiment, therapeutic polymer nanoparticles may
be
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identified by the methods described in US Pub No. US20120140790, herein
incorporated
by reference in its entirety.
[000598] In one embodiment, the therapeutic nanoparticle may be formulated for

sustained release. As used herein, "sustained release" refers to a
pharmaceutical
composition or compound that conforms to a release rate over a specific period
of time.
The period of time may include, but is not limited to, hours, days, weeks,
months and
years. As a non-limiting example, the sustained release nanoparticle may
comprise a
polymer and a therapeutic agent such as, but not limited to, the the
polynucleotides,
primary constructs, and mmRNA of the present invention (see International Pub
No.
2010075072 and US Pub No. U520100216804, US20110217377 and U520120201859,
each of which is herein incorporated by reference in their entirety).
[000599] In one embodiment, the therapeutic nanoparticles may be formulated to
be
target specific. As a non-limiting example, the thereapeutic nanoparticles may
include a
corticosteroid (see International Pub. No. W02011084518; herein incorporated
by
reference in its entirety). In one embodiment, the therapeutic nanoparticles
may be
formulated to be cancer specific. As a non-limiting example, the therapeutic
nanoparticles may be formulated in nanoparticles described in International
Pub No.
W02008121949, W02010005726, W02010005725, W02011084521 and US Pub No.
U520100069426, U520120004293 and U520100104655, each of which is herein
incorporated by reference in their entirety.
[000600] In one embodiment, the nanoparticles of the present invention may
comprise a
polymeric matrix. As a non-limiting example, the nanoparticle may comprise two
or
more polymers such as, but not limited to, polyethylenes, polycarbonates,
polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones,
polyamides,
polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates,
polyvinyl
alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates,
polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysine,
poly(ethylene
imine), poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-
proline ester)
or combinations thereof.
[000601] In one embodiment, the therapeutic nanoparticle comprises a diblock
copolymer. In one embodiment, the diblock copolymer may include PEG in
combination
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with a polymer such as, but not limited to, polyethylenes, polycarbonates,
polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones,
polyamides,
polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates,
polyvinyl
alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates,
polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysine,
poly(ethylene
imine), poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-
proline ester)
or combinations thereof
[000602] As a non-limiting example the therapeutic nanoparticle comprises a
PLGA-
PEG block copolymer (see US Pub. No. US20120004293 and US Pat No. 8,236,330,
each of which is herein incorporated by reference in their entirety). In
another non-
limiting example, the therapeutic nanoparticle is a stealth nanoparticle
comprising a
diblock copolymer of PEG and PLA or PEG and PLGA (see US Pat No 8,246,968 and
International Publication No. W02012166923, each of which is herein
incorporated by
reference in its entirety).
[000603] In one embodiment, the therapeutic nanoparticle may comprise a
multiblock
copolymer (See e.g., U.S. Pat. No. 8,263,665 and 8,287,910; each of which is
herein
incorporated by reference in its entirety).
[000604] In one embodiment, the block copolymers described herein may be
included
in a polyion complex comprising a non-polymeric micelle and the block
copolymer. (See
e.g., U.S. Pub. No. 20120076836; herein incorporated by reference in its
entirety).
[000605] In one embodiment, the therapeutic nanoparticle may comprise at least
one
acrylic polymer. Acrylic polymers include but are not limited to, acrylic
acid,
methacrylic acid, acrylic acid and methacrylic acid copolymers, methyl
methacrylate
copolymers, ethoxyethyl methacrylates, cyanoethyl methacrylate, amino alkyl
methacrylate copolymer, poly(acrylic acid), poly(methacrylic acid),
polycyanoacrylates
and combinations thereof
[000606] In one embodiment, the therapeutic nanoparticles may comprise at
least one
cationic polymer described herein and/or known in the art.
[000607] In one embodiment, the therapeutic nanoparticles may comprise at
least one
amine-containing polymer such as, but not limited to polylysine, polyethylene
imine,
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poly(amidoamine) dendrimers, poly(beta-amino esters) (See e.g., U.S. Pat. No.
8,287,849; herein incorporated by reference in its entirety) and combinations
thereof.
[000608] In one embodiment, the therapeutic nanoparticles may comprise at
least one
degradable polyester which may contain polycationic side chains. Degradeable
polyesters include, but are not limited to, poly(serine ester), poly(L-lactide-
co-L-lysine),
poly(4-hydroxy-L-proline ester), and combinations thereof In another
embodiment, the
degradable polyesters may include a PEG conjugation to form a PEGylated
polymer.
[000609] In another embodiment, the therapeutic nanoparticle may include a
conjugation of at least one targeting ligand. The targeting ligand may be any
ligand
known in the art such as, but not limited to, a monoclonal antibody. (Kirpotin
et al,
Cancer Res. 2006 66:6732-6740; herein incorporated by reference in its
entirety).
[000610] In one embodiment, the therapeutic nanoparticle may be formulated in
an
aqueous solution which may be used to target cancer (see International Pub No.

W02011084513 and US Pub No. US20110294717, each of which is herein
incorporated
by reference in their entirety).
[000611] In one embodiment, the polynucleotides, primary constructs, or mmRNA
may
be encapsulated in, linked to and/or associated with synthetic nanocarriers. .
Synthetic
nanocarriers include, but are not limited to, those described in International
Pub. Nos.
W02010005740, W02010030763, W0201213501, W02012149252, W02012149255,
W02012149259, W02012149265, W02012149268, W02012149282, W02012149301,
W02012149393, W02012149405, W02012149411, W02012149454 and
W02013019669, and US Pub. Nos. US20110262491, U520100104645, U520100087337
and US20120244222, each of which is herein incorporated by reference in their
entirety.
The synthetic nanocarriers may be formulated using methods known in the art
and/or
described herein. As a non-limiting example, the synthetic nanocarriers may be

formulated by the methods described in International Pub Nos. W02010005740,
W02010030763 and W0201213501and US Pub. Nos. US20110262491,
U520100104645, U520100087337 and U52012024422, each of which is herein
incorporated by reference in their entirety. In another embodiment, the
synthetic
nanocarrier formulations may be lyophilized by methods described in
International Pub.
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No. W02011072218 and US Pat No. 8,211,473; each of which is herein
incorporated by
reference in their entirety.
[000612] In one embodiment, the synthetic nanocarriers may contain reactive
groups to
release the polynucleotides, primary constructs and/or mmRNA described herein
(see
International Pub. No. W020120952552 and US Pub No. U520120171229, each of
which is herein incorporated by reference in their entirety).
[000613] In one embodiment, the synthetic nanocarriers may contain an
immunostimulatory agent to enhance the immune response from delivery of the
synthetic
nanocarrier. As a non-limiting example, the synthetic nanocarrier may comprise
a Thl
immunostimulatory agent which may enhance a Thl-based response of the immune
system (see International Pub No. W02010123569 and US Pub. No. US20110223201,
each of which is herein incorporated by reference in its entirety).
[000614] In one embodiment, the synthetic nanocarriers may be formulated for
targeted
release. In one embodiment, the synthetic nanocarrier is formulated to release
the
polynucleotides, primary constructs and/or mmRNA at a specified pH and/or
after a
desired time interval. As a non-limiting example, the synthetic nanoparticle
may be
formulated to release the polynucleotides, primary constructs and/or mmRNA
after 24
hours and/or at a pH of 4.5 (see International Pub. Nos. W02010138193 and
W02010138194 and US Pub Nos. U520110020388 and U520110027217, each of which
is herein incorporated by reference in their entireties).
[000615] In one embodiment, the synthetic nanocarriers may be formulated for
controlled and/or sustained release of the polynucleotides, primary constructs
and/or
mmRNA described herein. As a non-limiting example, the synthetic nanocarriers
for
sustained release may be formulated by methods known in the art, described
herein
and/or as described in International Pub No. W02010138192 and US Pub No.
20100303850, each of which is herein incorporated by reference in their
entirety.
[000616] In one embodiment, the synthetic nanocarrier may be formulated for
use as a
vaccine. In one embodiment, the synthetic nanocarrier may encapsulate at least
one
polynucleotide, primary construct and/or mmRNA which encode at least one
antigen. As
a non-limiting example, the synthetic nanocarrier may include at least one
antigen and an
excipient for a vaccine dosage form (see International Pub No. W02011150264
and US
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Pub No. US20110293723, each of which is herein incorporated by reference in
their
entirety). As another non-limiting example, a vaccine dosage form may include
at least
two synthetic nanocarriers with the same or different antigens and an
excipient (see
International Pub No. W02011150249 and US Pub No. US20110293701, each of which

is herein incorporated by reference in their entirety). The vaccine dosage
form may be
selected by methods described herein, known in the art and/or described in
International
Pub No. W02011150258 and US Pub No. US20120027806, each of which is herein
incorporated by reference in their entirety).
[000617] In one embodiment, the synthetic nanocarrier may comprise at least
one
polynucleotide, primary construct and/or mmRNA which encodes at least one
adjuvant.
As non-limiting example, the adjuvant may comprise dimethyldioctadecylammonium-

bromide, dimethyldioctadecylammonium-chloride, dimethyldioctadecylammonium-
phosphate or dimethyldioctadecylammonium-acetate (DDA) and an apolar fraction
or
part of said apolar fraction of a total lipid extract of a mycobacterium (See
e.g, U.S. Pat.
No. 8,241,610; herein incorporated by reference in its entirety). In another
embodiment,
the synthetic nanocarrier may comprise at least one polynucleotide, primary
construct
and/or mmRNA and an adjuvant. As a non-limiting example, the synthetic
nanocarrier
comprising and adjuvant may be formulated by the methods described in
International
Pub No. W02011150240 and US Pub No. US20110293700, each of which is herein
incorporated by reference in its entirety.
[000618] In one embodiment, the synthetic nanocarrier may encapsulate at least
one
polynucleotide, primary construct and/or mmRNA which encodes a peptide,
fragment or
region from a virus. As a non-limiting example, the synthetic nanocarrier may
include,
but is not limited to, the nanocarriers described in International Pub No.
W02012024621,
W0201202629, W02012024632 and US Pub No. US20120064110, U520120058153
and U520120058154, each of which is herein incorporated by reference in their
entirety.
[000619] In one embodiment, the synthetic nanocarrier may be coupled to a
polynucleotide, primary construct or mmRNA which may be able to trigger a
humoral
and/or cytotoxic T lymphocyte (CTL) response (See e.g., International
Publication No.
W02013019669, herein incorporated by reference in its entirety).
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[000620] In one embodiment, the nanoparticle may be optimized for oral
administration. The nanoparticle may comprise at least one cationic biopolymer
such as,
but not limited to, chitosan or a derivative thereof As a non-limiting
example, the
nanoparticle may be formulated by the methods described in U.S. Pub. No.
20120282343; herein incorporated by reference in its entirety.
Polymers, Biodegradable Nanoparticles, and Core-Shell Nanoparticles
[000621] The polynucleotide, primary construct, and mmRNA of the invention can
be
formulated using natural and/or synthetic polymers. Non-limiting examples of
polymers
which may be used for delivery include, but are not limited to, DYNAMIC
POLYCONJUGATEO (Arrowhead Reasearch Corp., Pasadena, CA) formulations from
MIRUSO Bio (Madison, WI) and Roche Madison (Madison, WI), PHASERXTM polymer
formulations such as, without limitation, SMARTT POLYMER TECHNOLOGYTm
(PHASERXO, Seattle, WA), DMRI/DOPE, poloxamer, VAXFECTINO adjuvant from
Vical (San Diego, CA), chitosan, cyclodextrin from Calando Pharmaceuticals
(Pasadena,
CA), dendrimers and poly(lactic-co-glycolic acid) (PLGA) polymers. RONDELTM
(RNAi/Oligonucleotide Nanoparticle Delivery) polymers (Arrowhead Research
Corporation, Pasadena, CA) and pH responsive co-block polymers such as, but
not
limited to, PHASERXO (Seattle, WA).
[000622] A non-limiting example of chitosan formulation includes a core of
positively
charged chitosan and an outer portion of negatively charged substrate (U.S.
Pub. No.
20120258176; herein incorporated by reference in its entirety). Chitosan
includes, but is
not limited to N-trimethyl chitosan, mono-N-carboxymethyl chitosan (MCC), N-
palmitoyl chitosan (NPCS), EDTA-chitosan, low molecular weight chitosan,
chitosan
derivatives, or combinations thereof
[000623] In one embodiment, the polymers used in the present invention have
undergone processing to reduce and/or inhibit the attachement of unwanted
substances
such as, but not limited to, bacteria, to the surface of the polymer. The
polymer may be
processed by methods known and/or described in the art and/or described in
International
Pub. No. W02012150467, herein incorporated by reference in its entirety.
[000624] A non-limiting example of PLGA formulations include, but are not
limited to,
PLGA injectable depots (e.g., ELIGARDO which is formed by dissolving PLGA in
66%
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N-methyl-2-pyrrolidone (NMP) and the remainder being aqueous solvent and
leuprolide.
Once injected, the PLGA and leuprolide peptide precipitates into the
subcutaneous
space).
[000625] Many of these polymer approaches have demonstrated efficacy in
delivering
oligonucleotides in vivo into the cell cytoplasm (reviewed in deFougerolles
Hum Gene
Ther. 2008 19:125-132; herein incorporated by reference in its entirety). Two
polymer
approaches that have yielded robust in vivo delivery of nucleic acids, in this
case with
small interfering RNA (siRNA), are dynamic polyconjugates and cyclodextrin-
based
nanoparticles. The first of these delivery approaches uses dynamic
polyconjugates and
has been shown in vivo in mice to effectively deliver siRNA and silence
endogenous
target mRNA in hepatocytes (Rozema et al., Proc Natl Acad Sci U S A. 2007
104:12982-
12887; herein incorporated by reference in its entirety). This particular
approach is a
multicomponent polymer system whose key features include a membrane-active
polymer
to which nucleic acid, in this case siRNA, is covalently coupled via a
disulfide bond and
where both PEG (for charge masking) and N-acetylgalactosamine (for hepatocyte
targeting) groups are linked via pH-sensitive bonds (Rozema et al., Proc Natl
Acad Sci U
S A. 2007 104:12982-12887; herein incorporated by reference in its entirety).
On binding
to the hepatocyte and entry into the endosome, the polymer complex
disassembles in the
low-pH environment, with the polymer exposing its positive charge, leading to
endosomal escape and cytoplasmic release of the siRNA from the polymer.
Through
replacement of the N-acetylgalactosamine group with a mannose group, it was
shown one
could alter targeting from asialoglycoprotein receptor-expressing hepatocytes
to
sinusoidal endothelium and Kupffer cells. Another polymer approach involves
using
transferrin-targeted cyclodextrin-containing polycation nanoparticles. These
nanoparticles have demonstrated targeted silencing of the EWS-FLI1 gene
product in
transferrin receptor-expressing Ewing's sarcoma tumor cells (Hu-Lieskovan et
at.,
Cancer Res.2005 65: 8984-8982; herein incorporated by reference in its
entirety) and
siRNA formulated in these nanoparticles was well tolerated in non-human
primates
(Heidel et at., Proc Natl Acad Sci USA 2007 104:5715-21; herein incorporated
by
reference in its entirety). Both of these delivery strategies incorporate
rational approaches
using both targeted delivery and endosomal escape mechanisms.
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[000626] The polymer formulation can permit the sustained or delayed release
of
polynucleotide, primary construct, or mmRNA (e.g., following intramuscular or
subcutaneous injection). The altered release profile for the polynucleotide,
primary
construct, or mmRNA can result in, for example, translation of an encoded
protein over
an extended period of time. The polymer formulation may also be used to
increase the
stability of the polynucleotide, primary construct, or mmRNA. Biodegradable
polymers
have been previously used to protect nucleic acids other than mmRNA from
degradation
and been shown to result in sustained release of payloads in vivo (Rozema et
al., Proc
Natl Acad Sci U S A. 2007 104:12982-12887; Sullivan et al., Expert Opin Drug
Deliv.
2010 7:1433-1446; Convertine et al., Biomacromolecules. 2010 Oct 1; Chu et
al., Acc
Chem Res. 2012 Jan 13; Manganiello et al., Biomaterials. 2012 33:2301-2309;
Benoit et
al., Biomacromolecules. 201112:2708-2714; Singha et al., Nucleic Acid Ther.
2011
2:133-147; deFougerolles Hum Gene Ther. 2008 19:125-132; Schaffert and Wagner,

Gene Ther. 2008 16:1131-1138; Chaturvedi et al., Expert Opin Drug Deliv. 2011
8:1455-
1468; Davis, Mol Pharm. 2009 6:659-668; Davis, Nature 2010 464:1067-1070; each
of
which is herein incorporated by reference in its entirety).
[000627] In one embodiment, the pharmaceutical compositions may be sustained
release formulations. In a further embodiment, the sustained release
formulations may be
for subcutaneous delivery. Sustained release formulations may include, but are
not
limited to, PLGA microspheres, ethylene vinyl acetate (EVAc), poloxamer,
GELSITEO
(Nanotherapeutics, Inc. Alachua, FL), HYLENEXO (Halozyme Therapeutics, San
Diego
CA), surgical sealants such as fibrinogen polymers (Ethicon Inc. Cornelia,
GA),
TISSELLO (Baxter International, Inc Deerfield, IL), PEG-based sealants, and
COSEALO (Baxter International, Inc Deerfield, IL).
[000628] As a non-limiting example modified mRNA may be formulated in PLGA
microspheres by preparing the PLGA microspheres with tunable release rates
(e.g., days
and weeks) and encapsulating the modified mRNA in the PLGA microspheres while
maintaining the integrity of the modified mRNA during the encapsulation
process.
EVAc are non-biodegradeable, biocompatible polymers which are used extensively
in
pre-clinical sustained release implant applications (e.g., extended release
products
Ocusert a pilocarpine ophthalmic insert for glaucoma or progestasert a
sustained release
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progesterone intrauterine deivce; transdermal delivery systems Testoderm,
Duragesic and
Selegiline; catheters). Poloxamer F-407 NF is a hydrophilic, non-ionic
surfactant
triblock copolymer of polyoxyethylene-polyoxypropylene-polyoxyethylene having
a low
viscosity at temperatures less than 5 C and forms a solid gel at temperatures
greater than
15 C. PEG-based surgical sealants comprise two synthetic PEG components mixed
in a
delivery device which can be prepared in one minute, seals in 3 minutes and is

reabsorbed within 30 days. GELSITEO and natural polymers are capable of in-
situ
gelation at the site of administration. They have been shown to interact with
protein and
peptide therapeutic candidates through ionic ineraction to provide a
stabilizing effect.
[000629] Polymer formulations can also be selectively targeted through
expression of
different ligands as exemplified by, but not limited by, folate, transferrin,
and N-
acetylgalactosamine (GalNAc) (Benoit et al., Biomacromolecules. 201112:2708-
2714;
Rozema et al., Proc Natl Acad Sci U S A. 2007 104:12982-12887; Davis, Mol
Pharm.
2009 6:659-668; Davis, Nature 2010 464:1067-1070; each of which is herein
incorporated by reference in its entirety).
[000630] The modified nucleic acid, and mmRNA of the invention may be
formulated
with or in a polymeric compound. The polymer may include at least one polymer
such
as, but not limited to, polyethenes, polyethylene glycol (PEG), poly(1-
lysine)(PLL), PEG
grafted to PLL, cationic lipopolymer, biodegradable cationic lipopolymer,
polyethyleneimine (PEI), cross-linked branched poly(alkylene imines), a
polyamine
derivative, a modified poloxamer, a biodegradable polymer, elastic
biodegradable
polymer, biodegradable block copolymer, biodegradable random copolymer,
biodegradable polyester copolymer, biodegradable polyester block copolymer,
biodegradable polyester block random copolymer, multiblock copolymers, linear
biodegradable copolymer, poly[a-(4-aminobuty1)-L-glycolic acid) (PAGA),
biodegradable cross-linked cationic multi-block copolymers, polycarbonates,
polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones,
polyamides,
polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates,
polyvinyl
alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates,
polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysine,
poly(ethylene
imine), poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-
proline ester),
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acrylic polymers, amine-containing polymers, dextran polymers, dextran polymer

derivatives or or combinations thereof.
[000631] As a non-limiting example, the modified nucleic acid or mmRNA of the
invention may be formulated with the polymeric compound of PEG grafted with
PLL as
described in U.S. Pat. No. 6,177,274; herein incorporated by reference in its
entirety.
The formulation may be used for transfecting cells in vitro or for in vivo
delivery of the
modified nucleic acid and mmRNA. In another example, the modified nucleic acid
and
mmRNA may be suspended in a solution or medium with a cationic polymer, in a
dry
pharmaceutical composition or in a solution that is capable of being dried as
described in
U.S. Pub. Nos. 20090042829 and 20090042825; each of which are herein
incorporated
by reference in their entireties.
[000632] As another non-limiting example the polynucleotides, primary
constructs or
mmRNA of the invention may be formulated with a PLGA-PEG block copolymer (see
US Pub. No. US20120004293 and US Pat No. 8,236,330, herein incorporated by
reference in their entireties) or PLGA-PEG-PLGA block copolymers (See U.S.
Pat. No.
6,004,573, herein incorporated by reference in its entirety). As a non-
limiting example,
the polynucleotides, primary constructs or mmRNA of the invention may be
formulated
with a diblock copolymer of PEG and PLA or PEG and PLGA (see US Pat No
8,246,968,
herein incorporated by reference in its entirety).
[000633] A polyamine derivative may be used to deliver nucleic acids or to
treat and/or
prevent a disease or to be included in an implantable or injectable device
(U.S. Pub. No.
20100260817 herein incorporated by reference in its entirety). As a non-
limiting
example, a pharmaceutical composition may include the modified nucleic acids
and
mmRNA and the polyamine derivative described in U.S. Pub. No. 20100260817 (the

contents of which are incorporated herein by reference in its entirety. As a
non-limiting
example the polynucleotides, primary constructs and mmRNA of the present
invention
may be delivered using a polyaminde polymer such as, but not limited to, a
polymer
comprising a 1,3-dipolar addition polymer prepared by combining a carbohydrate
diazide
monomer with a dilkyne unite comprising oligoamines (U.S. Pat. No. 8,236,280;
herein
incorporated by reference in its entirety).
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[000634] In one embodiment, the polynucleotides, primary constructs or mmRNA
of
the present invention may be formulated with at least one polymer and/or
derivatives
thereof described in International Publication Nos. W02011115862, W02012082574
and
W02012068187 and U.S. Pub. No. 20120283427, each of which are herein
incorporated
by reference in their entireties. In another embodiment, the modified nucleic
acid or
mmRNA of the present invention may be formulated with a polymer of formula Z
as
described in W02011115862, herein incorporated by reference in its entirety.
In yet
another embodiment, the modified nucleic acid or mmRNA may be formulated with
a
polymer of formula Z, Z' or Z" as described in International Pub. Nos.
W02012082574
or W02012068187 and U.S. Pub. No. 2012028342, each of which are herein
incorporated by reference in their entireties. The polymers formulated with
the modified
RNA of the present invention may be synthesized by the methods described in
International Pub. Nos. W02012082574 or W02012068187, each of which are herein

incorporated by reference in their entireties.
[000635] The polynucleotides, primary constructs or mmRNA of the invention may
be
formulated with at least one acrylic polymer. Acrylic polymers include but are
not
limited to, acrylic acid, methacrylic acid, acrylic acid and methacrylic acid
copolymers,
methyl methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl
methacrylate,
amino alkyl methacrylate copolymer, poly(acrylic acid), poly(methacrylic
acid),
polycyanoacrylates and combinations thereof
[000636] Formulations of polynucleotides, primary constructs or mmRNA of the
invention may include at least one amine-containing polymer such as, but not
limited to
polylysine, polyethylene imine, poly(amidoamine) dendrimers or combinations
thereof
[000637] For example, the modified nucleic acid or mmRNA of the invention may
be
formulated in a pharmaceutical compound including a poly(alkylene imine), a
biodegradable cationic lipopolymer, a biodegradable block copolymer, a
biodegradable
polymer, or a biodegradable random copolymer, a biodegradable polyester block
copolymer, a biodegradable polyester polymer, a biodegradable polyester random

copolymer, a linear biodegradable copolymer, PAGA, a biodegradable cross-
linked
cationic multi-block copolymer or combinations thereof The biodegradable
cationic
lipopolymer may be made by methods known in the art and/or described in U.S.
Pat. No.
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6,696,038, U.S. App. Nos. 20030073619 and 20040142474 each of which is herein
incorporated by reference in their entireties. The poly(alkylene imine) may be
made using
methods known in the art and/or as described in U.S. Pub. No. 20100004315,
herein
incorporated by reference in its entirety. The biodegradabale polymer,
biodegradable
block copolymer, the biodegradable random copolymer, biodegradable polyester
block
copolymer, biodegradable polyester polymer, or biodegradable polyester random
copolymer may be made using methods known in the art and/or as described in
U.S. Pat.
Nos. 6,517,869 and 6,267,987, the contents of which are each incorporated
herein by
reference in their entirety. The linear biodegradable copolymer may be made
using
methods known in the art and/or as described in U.S. Pat. No. 6,652,886. The
PAGA
polymer may be made using methods known in the art and/or as described in U.S.
Pat.
No. 6,217,912 herein incorporated by reference in its entirety. The PAGA
polymer may
be copolymerized to form a copolymer or block copolymer with polymers such as
but not
limited to, poly-L-lysine, polyargine, polyornithine, histones, avidin,
protamines,
polylactides and poly(lactide-co-glycolides). The biodegradable cross-linked
cationic
multi-block copolymers may be made my methods known in the art and/or as
described
in U.S. Pat. No. 8,057,821 or U.S. Pub. No. 2012009145 each of which are
herein
incorporated by reference in their entireties. For example, the multi-block
copolymers
may be synthesized using linear polyethyleneimine (LPEI) blocks which have
distinct
patterns as compared to branched polyethyleneimines. Further, the composition
or
pharmaceutical composition may be made by the methods known in the art,
described
herein, or as described in U.S. Pub. No. 20100004315 or U.S. Pat. Nos.
6,267,987 and
6,217,912 each of which are herein incorporated by reference in their
entireties.
[000638] The polynucleotides, primary constructs, and mmRNA of the invention
may
be formulated with at least one degradable polyester which may contain
polycationic side
chains. Degradeable polyesters include, but are not limited to, poly(serine
ester), poly(L-
lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), and combinations
thereof In
another embodiment, the degradable polyesters may include a PEG conjugation to
form a
PEGylated polymer.
[000639] The polynucleotides, primary construct, mmRNA of the invention may be

formulated with at least one crosslinkable polyester. Crosslinkable polyesters
include
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those known in the art and described in US Pub. No. 20120269761, herein
incorporated
by reference in its entirety.
[000640] In one embodiment, the polymers described herein may be conjugated to
a
lipid-terminating PEG. As a non-limiting example, PLGA may be conjugated to a
lipid-
terminating PEG forming PLGA-DSPE-PEG. As another non-limiting example, PEG
conjugates for use with the present invention are described in International
Publication
No. W02008103276, herein incorporated by reference in its entirety. The
polymers may
be conjugated using a ligand conjugate such as, but not limited to, the
conjugates
described in U.S. Pat. No. 8,273,363, herein incorporated by reference in its
entirety.
[000641] In one embodiment, the modified RNA described herein may be
conjugated
with another compound. Non-limiting examples of conjugates are described in US
Patent
Nos. 7,964,578 and 7,833,992, each of which are herein incorporated by
reference in
their entireties. In another embodiment, modified RNA of the present invention
may be
conjugated with conjugates of formula 1-122 as described in US Patent Nos.
7,964,578
and 7,833,992, each of which are herein incorporated by reference in their
entireties. The
polynucleotides, primary constructs and/or mmRNA described herein may be
conjugated
with a metal such as, but not limited to, gold. (See e.g., Giljohann et al.
Journ. Amer.
Chem. Soc. 2009 131(6): 2072-2073; herein incorporated by reference in its
entirety). In
another embodiment, the polynucleotides, primary constructs and/or mmRNA
described
herein may be conjugated and/or encapsulated in gold-nanoparticles.
(Interantional Pub.
No. W0201216269 and U.S. Pub. No. 20120302940; each of which is herein
incorporated by reference in its entirety).
[000642] As described in U.S. Pub. No. 20100004313, herein incorporated by
reference
in its entirety, a gene delivery composition may include a nucleotide sequence
and a
poloxamer. For example, the modified nucleic acid and mmRNA of the present
inveition
may be used in a gene delivery composition with the poloxamer described in
U.S. Pub.
No. 20100004313.
[000643] In one embodiment, the polymer formulation of the present invention
may be
stabilized by contacting the polymer formulation, which may include a cationic
carrier,
with a cationic lipopolymer which may be covalently linked to cholesterol and
polyethylene glycol groups. The polymer formulation may be contacted with a
cationic
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lipopolymer using the methods described in U.S. Pub. No. 20090042829 herein
incorporated by reference in its entirety. The cationic carrier may include,
but is not
limited to, polyethylenimine, poly(trimethylenimine),
poly(tetramethylenimine),
polypropylenimine, aminoglycoside-polyamine, dideoxy-diamino-b-cyclodextrin,
spermine, spermidine, poly(2-dimethylamino)ethyl methacrylate, poly(lysine),
poly(histidine), poly(arginine), cationized gelatin, dendrimers, chitosan, 1,2-
Dioleoy1-3-
Trimethylammonium-Propane(DOTAP), N-[1-(2,3-dioleoyloxy)propyll-N,N,N-
trimethylammonium chloride (DOTMA), 142-(oleoyloxy)ethy1]-2-oley1-3-(2-
hydroxyethyl)imidazolinium chloride (DOTIM), 2,3-dioleyloxy-N-
[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate
(DOSPA), 3B-[N¨(N',N'-Dimethylaminoethane)-carbamoyl]Cholesterol Hydrochloride

(DC-Cholesterol HC1) diheptadecylamidoglycyl spermidine (DOGS), N,N-distearyl-
N,N-
dimethylammonium bromide (DDAB), N-(1,2-dimyristyloxyprop-3-y1)-N,N-dimethyl-N-

hydroxyethyl ammonium bromide (DMRIE), N,N-dioleyl-N,N-dimethylammonium
chloride DODAC) and combinations thereof.
[000644] The polynucleotides, primary constructs and/or mmRNA of the invention
may
be formulated in a polyplex of one or more polymers (U.S. Pub. No. 20120237565
and
20120270927; each of which is herein incorporated by reference in its
entirety). In one
embodiment, the polyplex comprises two or more cationic polymers. The
catioinic
polymer may comprise a poly(ethylene imine) (PEI) such as linear PEI.
[000645] The polynucleotide, primary construct, and mmRNA of the invention can
also
be formulated as a nanoparticle using a combination of polymers, lipids,
and/or other
biodegradable agents, such as, but not limited to, calcium phosphate.
Components may
be combined in a core-shell, hybrid, and/or layer-by-layer architecture, to
allow for fine-
tuning of the nanoparticle so to delivery of the polynucleotide, primary
construct and
mmRNA may be enhanced (Wang et al., Nat Mater. 2006 5:791-796; Fuller et al.,
Biomaterials. 2008 29:1526-1532; DeKoker et al., Adv Drug Deliv Rev. 2011
63:748-
761; Endres et al., Biomaterials. 2011 32:7721-7731; Su et al., Mol Pharm.
2011 Jun
6;8(3):774-87; herein incorporated by reference in its entirety). As a non-
limiting
example, the nanoparticle may comprise a plurality of polymers such as, but
not limited
to hydrophilic-hydrophobic polymers (e.g., PEG-PLGA), hydrophobic polymers
(e.g.,
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PEG) and/or hydrophilic polymers (International Pub. No. W020120225129; herein

incorporated by reference in its entirety).
[000646] Biodegradable calcium phosphate nanoparticles in combination with
lipids
and/or polymers have been shown to deliver polynucleotides, primary constructs
and
mmRNA in vivo. In one embodiment, a lipid coated calcium phosphate
nanoparticle,
which may also contain a targeting ligand such as anisamide, may be used to
deliver the
polynucleotide, primary construct and mmRNA of the present invention. For
example, to
effectively deliver siRNA in a mouse metastatic lung model a lipid coated
calcium
phosphate nanoparticle was used (Li et al., J Contr Rel. 2010 142: 416-421; Li
et al., J
Contr Rel. 2012 158:108-114; Yang et al., Mol Ther. 2012 20:609-615; herein
incorporated by reference in its entirety). This delivery system combines both
a targeted
nanoparticle and a component to enhance the endosomal escape, calcium
phosphate, in
order to improve delivery of the siRNA.
[000647] In one embodiment, calcium phosphate with a PEG-polyanion block
copolymer may be used to delivery polynucleotides, primary constructs and
mmRNA
(Kazikawa et al., J Contr Rel. 2004 97:345-356; Kazikawa et al., J Contr Rel.
2006
111:368-370; herein incorporated by reference in its entirety).
[000648] In one embodiment, a PEG-charge-conversional polymer (Pitella et al.,

Biomaterials. 2011 32:3106-3114) may be used to form a nanoparticle to deliver
the
polynucleotides, primary constructs and mmRNA of the present invention. The
PEG-
charge-conversional polymer may improve upon the PEG-polyanion block
copolymers
by being cleaved into a polycation at acidic pH, thus enhancing endosomal
escape.
[000649] The use of core-shell nanoparticles has additionally focused on a
high-
throughput approach to synthesize cationic cross-linked nanogel cores and
various shells
(Siegwart et al., Proc Natl Acad Sci U S A. 2011108:12996-13001). The
complexation,
delivery, and internalization of the polymeric nanoparticles can be precisely
controlled by
altering the chemical composition in both the core and shell components of the

nanoparticle. For example, the core-shell nanoparticles may efficiently
deliver siRNA to
mouse hepatocytes after they covalently attach cholesterol to the
nanoparticle.
[000650] In one embodiment, a hollow lipid core comprising a middle PLGA layer
and
an outer neutral lipid layer containg PEG may be used to delivery of the
polynucleotide,
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primary construct and mmRNA of the present invention. As a non-limiting
example, in
mice bearing a luciferease-expressing tumor, it was determined that the lipid-
polymer-
lipid hybrid nanoparticle significantly suppressed luciferase expression, as
compared to a
conventional lipoplex (Shi et al, Angew Chem Int Ed. 2011 50:7027-7031; herein

incorporated by reference in its entirety).
[000651] In one embodiment, the lipid nanoparticles may comprise a core of the
modified nucleic acid molecules disclosed herein and a polymer shell. The
polymer shell
may be any of the polymers described herein and are known in the art. In an
additional
embodiment, the polymer shell may be used to protect the modified nucleic
acids in the
core.
[000652] Core¨shell nanoparticles for use with the modified nucleic acid
molecules of
the present invention are described and may be formed by the methods described
in U.S.
Pat. No. 8,313,777 herein incorporated by reference in its entirety.
[000653] In one embodiment, the core-shell nanoparticles may comprise a core
of the
modified nucleic acid molecules disclosed herein and a polymer shell. The
polymer shell
may be any of the polymers described herein and are known in the art. In an
additional
embodiment, the polymer shell may be used to protect the modified nucleic acid

molecules in the core. As a non-limiting example, the core-shell nanoparticle
may be
used to treat an eye disease or disorder (See e.g. US Publication No.
20120321719, herein
incorporated by reference in its entirety).
[000654] In one embodiment, the polymer used with the formulations described
herein
may be a modified polymer (such as, but not limited to, a modified polyacetal)
as
described in International Publication No. W02011120053, herein incorporated
by
reference in its entirety.
Peptides and Proteins
[000655] The polynucleotide, primary construct, and mmRNA of the invention can
be
formulated with peptides and/or proteins in order to increase transfection of
cells by the
polynucleotide, primary construct, or mmRNA. In one embodiment, peptides such
as, but
not limited to, cell penetrating peptides and proteins and peptides that
enable intracellular
delivery may be used to deliver pharmaceutical formulations. A non-limiting
example of
a cell penetrating peptide which may be used with the pharmaceutical
formulations of the
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present invention includes a cell-penetrating peptide sequence attached to
polycations
that facilitates delivery to the intracellular space, e.g., HIV-derived TAT
peptide,
penetratins, transportans, or hCT derived cell-penetrating peptides (see,
e.g., Caron et al.,
Mol. Ther. 3(3):310-8 (2001); Langel, Cell-Penetrating Peptides: Processes and

Applications (CRC Press, Boca Raton FL, 2002); El-Andaloussi et al., Curr.
Pharm. Des.
11(28):3597-611 (2003); and Deshayes et al., Cell. Mol. Life Sci. 62(16):1839-
49 (2005),
all of which are incorporated herein by reference in their entirety). The
compositions can
also be formulated to include a cell penetrating agent, e.g., liposomes, which
enhance
delivery of the compositions to the intracellular space. Polynucleotides,
primary
constructs, and mmRNA of the invention may be complexed to peptides and/or
proteins
such as, but not limited to, peptides and/or proteins from Aileron
Therapeutics
(Cambridge, MA) and Permeon Biologics (Cambridge, MA) in order to enable
intracellular delivery (Cronican et al., ACS Chem. Biol. 2010 5:747-752;
McNaughton et
al., Proc. Natl. Acad. Sci. USA 2009 106:6111-6116; Sawyer, Chem Biol Drug
Des.
2009 73:3-6; Verdine and Hilinski, Methods Enzymol. 2012;503:3-33; all of
which are
herein incorporated by reference in its entirety).
[000656] In one embodiment, the cell-penetrating polypeptide may comprise a
first
domain and a second domain. The first domain may comprise a supercharged
polypeptide. The second domain may comprise a protein-binding partner. As used
herein,
"protein-binding partner" includes, but are not limited to, antibodies and
functional
fragments thereof, scaffold proteins, or peptides. The cell-penetrating
polypeptide may
further comprise an intracellular binding partner for the protein-binding
partner. The cell-
penetrating polypeptide may be capable of being secreted from a cell where the

polynucleotide, primary construct, or mmRNA may be introduced.
[000657] Formulations of the including peptides or proteins may be used to
increase
cell transfection by the polynucleotide, primary construct, or mmRNA, alter
the
biodistribution of the polynucleotide, primary construct, or mmRNA (e.g., by
targeting
specific tissues or cell types), and/or increase the translation of encoded
protein. (See e.g.,
International Pub. No. W02012110636; herein incorporated by reference in its
entirety).
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Cells
[000658] The polynucleotide, primary construct, and mmRNA of the invention can
be
transfected ex vivo into cells, which are subsequently transplanted into a
subject. As non-
limiting examples, the pharmaceutical compositions may include red blood cells
to
deliver modified RNA to liver and myeloid cells, virosomes to deliver modified
RNA in
virus-like particles (VLPs), and electroporated cells such as, but not limited
to, from
MAXCYTEO (Gaithersburg, MD) and from ERYTECHO (Lyon, France) to deliver
modified RNA. Examples of use of red blood cells, viral particles and
electroporated
cells to deliver payloads other than mmRNA have been documented (Godfrin et
al.,
Expert Opin Biol Ther. 2012 12:127-133; Fang et al., Expert Opin Biol Ther.
2012
12:385-389; Hu et al., Proc Natl Acad Sci U S A. 2011 108:10980-10985; Lund et
al.,
Pharm Res. 2010 27:400-420; Huckriede et al., J Liposome Res. 2007;17:39-47;
Cusi,
Hum Vaccin. 2006 2:1-7; de Jonge et al., Gene Ther. 2006 13:400-411; all of
which are
herein incorporated by reference in its entirety).
[000659] The polynucleotides, primary constructs and mmRNA may be delivered in

synthetic VLPs synthesized by the methods described in International Pub No.
W02011085231 and US Pub No. 20110171248, each of which are herein incorporated

by reference in their entireties.
[000660] Cell-based formulations of the polynucleotide, primary construct, and

mmRNA of the invention may be used to ensure cell transfection (e.g., in the
cellular
carrier), alter the biodistribution of the polynucleotide, primary construct,
or mmRNA
(e.g., by targeting the cell carrier to specific tissues or cell types),
and/or increase the
translation of encoded protein.
[000661] A variety of methods are known in the art and suitable for
introduction of
nucleic acid into a cell, including viral and non-viral mediated techniques.
Examples of
typical non-viral mediated techniques include, but are not limited to,
electroporation,
calcium phosphate mediated transfer, nucleofection, sonoporation, heat shock,
magnetofection, liposome mediated transfer, microinjection, microprojectile
mediated
transfer (nanoparticles), cationic polymer mediated transfer (DEAE-dextran,
polyethylenimine, polyethylene glycol (PEG) and the like) or cell fusion.
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[000662] The technique of sonoporation, or cellular sonication, is the use of
sound (e.g.,
ultrasonic frequencies) for modifying the permeability of the cell plasma
membrane.
Sonoporation methods are known to those in the art and are used to deliver
nucleic acids
in vivo (Yoon and Park, Expert Opin Drug Deliv. 2010 7:321-330; Postema and
Gilja,
Curr Pharm Biotechnol. 2007 8:355-361; Newman and Bettinger, Gene Ther. 2007
14:465-475; all herein incorporated by reference in their entirety).
Sonoporation methods
are known in the art and are also taught for example as it relates to bacteria
in US Patent
Publication 20100196983 and as it relates to other cell types in, for example,
US Patent
Publication 20100009424, each of which are incorporated herein by reference in
their
entirety.
[000663] Electroporation techniques are also well known in the art and are
used to
deliver nucleic acids in vivo and clinically (Andre et al., Curr Gene Ther.
2010 10:267-
280; Chiarella et al., Curr Gene Ther. 2010 10:281-286; Hojman, Curr Gene
Ther. 2010
10:128-138; all herein incorporated by reference in their entirety). In one
embodiment,
polynucleotides, primary constructs or mmRNA may be delivered by
electroporation as
described in Example 8.
Hyaluronidase
[000664] The intramuscular or subcutaneous localized injection of
polynucleotide,
primary construct, or mmRNA of the invention can include hyaluronidase, which
catalyzes the hydrolysis of hyaluronan. By catalyzing the hydrolysis of
hyaluronan, a
constituent of the interstitial barrier, hyaluronidase lowers the viscosity of
hyaluronan,
thereby increasing tissue permeability (Frost, Expert Opin. Drug Deliv. (2007)
4:427-
440; herein incorporated by reference in its entirety). It is useful to speed
their dispersion
and systemic distribution of encoded proteins produced by transfected cells.
Alternatively, the hyaluronidase can be used to increase the number of cells
exposed to a
polynucleotide, primary construct, or mmRNA of the invention administered
intramuscularly or subcutaneously.
Nanoparticle Mimics
[000665] The polynucleotide, primary construct or mmRNA of the invention may
be
encapsulated within and/or absorbed to a nanoparticle mimic. A nanoparticle
mimic can
mimic the delivery function organisms or particles such as, but not limited
to, pathogens,
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viruses, bacteria, fungus, parasites, prions and cells. As a non-limiting
example the
polynucleotide, primary construct or mmRNA of the invention may be
encapsulated in a
non-viron particle which can mimic the delivery function of a virus (see
International
Pub. No. W02012006376 herein incorporated by reference in its entirety).
Nanotubes
[000666] The polynucleotides, primary constructs or mmRNA of the invention can
be
attached or otherwise bound to at least one nanotube such as, but not limited
to, rosette
nanotubes, rosette nanotubes having twin bases with a linker, carbon nanotubes
and/or
single-walled carbon nanotubes, The polynucleotides, primary constructs or
mmRNA
may be bound to the nanotubes through forces such as, but not limited to,
steric, ionic,
covalent and/or other forces.
[000667] In one embodiment, the nanotube can release one or more
polynucleotides,
primary constructs or mmRNA into cells. The size and/or the surface structure
of at least
one nanotube may be altered so as to govern the interaction of the nanotubes
within the
body and/or to attach or bind to the polynucleotides, primary constructs or
mmRNA
disclosed herein. In one embodiment, the building block and/or the functional
groups
attached to the building block of the at least one nanotube may be altered to
adjust the
dimensions and/or properties of the nanotube. As a non-limiting example, the
length of
the nanotubes may be altered to hinder the nanotubes from passing through the
holes in
the walls of normal blood vessels but still small enough to pass through the
larger holes
in the blood vessels of tumor tissue.
[000668] In one embodiment, at least one nanotube may also be coated with
delivery
enhancing compounds including polymers, such as, but not limited to,
polyethylene
glycol. In another embodiment, at least one nanotube and/or the
polynucleotides,
primary constructs or mmRNA may be mixed with pharmaceutically acceptable
excipients and/or delivery vehicles.
[000669] In one embodiment, the polynucleotides, primary constructs or mmRNA
are
attached and/or otherwise bound to at least one rosette nanotube. The rosette
nanotubes
may be formed by a process known in the art and/or by the process described in

International Publication No. W02012094304, herein incorporated by reference
in its
entirety. At least one polynucleotide, primary construct and/or mmRNA may be
attached
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and/or otherwise bound to at least one rosette nanotube by a process as
described in
International Publication No. W02012094304, herein incorporated by reference
in its
entirety, where rosette nanotubes or modules forming rosette nanotubes are
mixed in
aqueous media with at least one polynucleotide, primary construct and/or mmRNA
under
conditions which may cause at least one polynucleotide, primary construct or
mmRNA to
attach or otherwise bind to the rosette nanotubes.
[000670] In one embodiment, the polynucleotides, primary constructs or mmRNA
may
be attached to and/or otherwise bound to at least one carbon nanotube. As a
non-limiting
example, the polynucleotides, primary constructs or mmRNA may be bound to a
linking
agent and the linked agent may be bound to the carbon nanotube (See e.g., U.S.
Pat No.
8,246,995; herein incorporated by reference in its entirety). The carbon
nanotube may be
a single-walled nanotube (See e.g., U.S. Pat No. 8,246,995; herein
incorporated by
reference in its entirety).
Conjugates
[000671] The polynucleotides, primary constructs, and mmRNA of the invention
include conjugates, such as a polynucleotide, primary construct, or mmRNA
covalently
linked to a carrier or targeting group, or including two encoding regions that
together
produce a fusion protein (e.g., bearing a targeting group and therapeutic
protein or
peptide).
[000672] The conjugates of the invention include a naturally occurring
substance, such
as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL),
high-
density lipoprotein (HDL), or globulin); an carbohydrate (e.g., a dextran,
pullulan, chitin,
chitosan, inulin, cyclodextrin or hyaluronic acid); or a lipid. The ligand may
also be a
recombinant or synthetic molecule, such as a synthetic polymer, e.g., a
synthetic
polyamino acid, an oligonucleotide (e.g. an aptamer). Examples of polyamino
acids
include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-
glutamic acid,
styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied)
copolymer,
divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide
copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA),
polyurethane,
poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or
polyphosphazine.
Example of polyamines include: polyethylenimine, polylysine (PLL), spermine,
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spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine,
dendrimer
polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin,
quaternary
salt of a polyamine, or an alpha helical peptide.
[000673] Representative U.S. patents that teach the preparation of
polynucleotide
conjugates, particularly to RNA, include, but are not limited to, U.S. Pat.
Nos. 4,828,979;
4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717,
5,580,731;
5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439;
5,578,718;
5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941;
4,835,263;
4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830;
5,112,963;
5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873;
5,317,098;
5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785;
5,565,552;
5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923;
5,599,928
and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297;
7,037,646; each
of which is herein incorporated by reference in their entireties.
[000674] In one embodiment, the conjugate of the present invention may
function as a
carrier for the modified nucleic acids and mmRNA of the present invention. The

conjugate may comprise a cationic polymer such as, but not limited to,
polyamine,
polylysine, polyalkylenimine, and polyethylenimine which may be grafted to
with
poly(ethylene glycol). As a non-limiting example, the conjugate may be similar
to the
polymeric conjugate and the method of synthesizing the polymeric conjugate
described in
U.S. Pat. No. 6,586,524 herein incorporated by reference in its entirety.
[000675] The conjugates can also include targeting groups, e.g., a cell or
tissue
targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an
antibody, that binds to
a specified cell type such as a kidney cell. A targeting group can be a
thyrotropin,
melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate,
multivalent
lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine
multivalent mannose, multivalent fucose, glycosylated polyaminoacids,
multivalent
galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid,
cholesterol,
a steroid, bile acid, folate, vitamin B12, biotin, an RGD peptide, an RGD
peptide mimetic
or an aptamer.
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[000676] Targeting groups can be proteins, e.g., glycoproteins, or peptides,
e.g.,
molecules having a specific affinity for a co-ligand, or antibodies e.g., an
antibody, that
binds to a specified cell type such as a cancer cell, endothelial cell, or
bone cell.
Targeting groups may also include hormones and hormone receptors. They can
also
include non-peptidic species, such as lipids, lectins, carbohydrates,
vitamins, cofactors,
multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-
gulucosamine multivalent mannose, multivalent fucose, or aptamers. The ligand
can be,
for example, a lipopolysaccharide, or an activator of p38 MAP kinase.
[000677] The targeting group can be any ligand that is capable of targeting a
specific
receptor. Examples include, without limitation, folate, GalNAc, galactose,
mannose,
mannose-6P, apatamers, integrin receptor ligands, chemokine receptor ligands,
transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII,
somatostatin,
LDL, and HDL ligands. In particular embodiments, the targeting group is an
aptamer.
The aptamer can be unmodified or have any combination of modifications
disclosed
herein.
[000678] In one embodiment, pharmaceutical compositions of the present
invention
may include chemical modifications such as, but not limited to, modifications
similar to
locked nucleic acids.
[000679] Representative U.S. Patents that teach the preparation of locked
nucleic acid
(LNA) such as those from Santaris, include, but are not limited to, the
following: U.S.
Pat. Nos. 6,268,490; 6,670,461; 6,794,499; 6,998,484; 7,053,207; 7,084,125;
and
7,399,845, each of which is herein incorporated by reference in its entirety.
[000680] Representative U.S. patents that teach the preparation of PNA
compounds
include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and
5,719,262, each
of which is herein incorporated by reference. Further teaching of PNA
compounds can be
found, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.
[000681] Some embodiments featured in the invention include polynucleotides,
primary
constructs or mmRNA with phosphorothioate backbones and oligonucleosides with
other
modified backbones, and in particular --CH2--NH¨CH2--, --CH2--N(CH3)--0--CH2--
[known as a methylene (methylimino) or MMI backbone], --CH2-0--N(CH3)--CH2--, -
-
CH2--N(CH3)--N(CH3)--CH2-- and --N(CH3)--CH2--CH2-4wherein the native
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phosphodiester backbone is represented as --0¨P(0)2-0--CH2--] of the above-
referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-
referenced
U.S. Pat. No. 5,602,240. In some embodiments, the polynucletotides featured
herein have
morpholino backbone structures of the above-referenced U.S. Pat. No.
5,034,506.
[000682] Modifications at the 2' position may also aid in delivery.
Preferably,
modifications at the 2' position are not located in a polypeptide-coding
sequence, i.e., not
in a translatable region. Modifications at the 2' position may be located in a
5'UTR, a
3'UTR and/or a tailing region. Modifications at the 2' position can include
one of the
following at the 2' position: H (i.e., 2'-deoxy); F; 0-, S-, or N-alkyl; 0-, S-
, or N-alkenyl;
0-, S- or N-alkynyl; or 0-alkyl-0-alkyl, wherein the alkyl, alkenyl and
alkynyl may be
substituted or unsubstituted Ci to Cio alkyl or C2 to C10 alkenyl and alkynyl.
Exemplary
suitable modifications include O[(CH2)õ0] mCH3, 0(CH2).õOCH3, 0(CH2)õNH2,
0(CH2)
õCH3, 0(CH2)õONH2, and 0(CH2)õONRCH2)õCH3)]2, where n and m are from 1 to
about
10. In other embodiments, the polynucleotides, primary constructs or mmRNA
include
one of the following at the 2' position: Ci to C10 lower alkyl, substituted
lower alkyl,
alkaryl, aralkyl, 0-alkaryl or 0-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3,
OCF3,
SOCH3, 502CH3, 0NO2, NO2, N35 NH25 heterocycloalkyl, heterocycloalkaryl,
aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a
reporter
group, an intercalator, a group for improving the pharmacokinetic properties,
or a group
for improving the pharmacodynamic properties, and other substituents having
similar
properties. In some embodiments, the modification includes a 2'-methoxyethoxy
(2'-0--
CH2CH2OCH3, also known as 2'-0-(2-methoxyethyl) or 2'-M0E) (Martin et at.,
Hely.
Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary
modification is 2'-dimethylaminooxyethoxy, i.e., a 0(CH2)20N(CH3)2 group, also
known
as 2'-DMA0E, as described in examples herein below, and 2'-
dimethylaminoethoxyethoxy (also known in the art as 2'-0-
dimethylaminoethoxyethyl or
2'-DMAEOE), i.e., 2' -0--CH2-0--CH2--N(CH2)2, also described in examples
herein
below. Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'-
OCH2CH2CH2NH2) and 2'-fluoro (2'-F). Similar modifications may also be made at
other
positions, particularly the 3' position of the sugar on the 3' terminal
nucleotide or in 2'-5'
linked dsRNAs and the 5' position of 5' terminal nucleotide. Polynucleotides
of the
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invention may also have sugar mimetics such as cyclobutyl moieties in place of
the
pentofuranosyl sugar. Representative U.S. patents that teach the preparation
of such
modified sugar structures include, but are not limited to, U.S. Pat. Nos.
4,981,957;
5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785;
5,519,134;
5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;
5,646,265;
5,658,873; 5,670,633; and 5,700,920 and each of which is herein incorporated
by
reference.
[000683] In still other embodiments, the polynucleotide, primary construct, or
mmRNA
is covalently conjugated to a cell penetrating polypeptide. The cell-
penetrating peptide
may also include a signal sequence. The conjugates of the invention can be
designed to
have increased stability; increased cell transfection; and/or altered the
biodistribution
(e.g., targeted to specific tissues or cell types).
[000684] In one embodiment, the polynucleotides, primary constructs or mmRNA
may
be conjugated to an agent to enhance delivery. As a non-limiting example, the
agent may
be a monomer or polymer such as a targeting monomer or a polymer having
targeting
blocks as described in International Publication No. W02011062965, herein
incorporated
by reference in its entirety. In another non-limiting example, the agent may
be a
transport agent covalently coupled to the polynucleotides, primary constructs
or mmRNA
of the present invention (See e.g., U.S. Pat. Nos. 6,835.393 and 7,374,778,
each of which
is herein incorporated by reference in its entirety). In yet another non-
limiting example,
the agent may be a membrane barrier transport enhancing agent such as those
described
in U.S. Pat. Nos. 7,737,108 and 8,003,129, each of which is herein
incorporated by
reference in its entirety.
[000685] In another embodiment, polynucleotides, primary constructs or mmRNA
may
be conjugated to SMARTT POLYMER TECHNOLOGY (PHASERXO, Inc. Seattle,
WA).
Self-Assembled Nanoparticles
Nucleic Acid Self-Assembled Nanoparticles
[000686] Self-assembled nanoparticles have a well-defined size which may be
precisely
controlled as the nucleic acid strands may be easily reprogrammable. For
example, the
optimal particle size for a cancer-targeting nanodelivery carrier is 20-100 nm
as a
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diameter greater than 20 nm avoids renal clearance and enhances delivery to
certain
tumors through enhanced permeability and retention effect. Using self-
assembled
nucleic acid nanoparticles a single uniform population in size and shape
having a
precisely controlled spatial orientation and density of cancer-targeting
ligands for
enhanced delivery. As a non-limiting example, oligonucleotide nanoparticles
were
prepared using programmable self-assembly of short DNA fragments and
therapeutic
siRNAs. These nanoparticles are molecularly identical with controllable
particle size and
target ligand location and density. The DNA fragments and siRNAs self-
assembled into
a one-step reaction to generate DNA/siRNA tetrahedral nanoparticles for
targeted in vivo
delivery. (Lee et al., Nature Nanotechnology 2012 7:389-393; herein
incorporated by
reference in its entirety).
[000687] In one embodiment, the polynucleotides, primary constructs and/or
mmRNA
disclosed herein may be formulated as self-assembled nanoparticles. As a non-
limiting
example, nucleic acids may be used to make nanoparticles which may be used in
a
delivery system for the polynucleotides, primary constructs and/or mmRNA of
the
present invention (See e.g., International Pub. No. W02012125987; herein
incorporated
by reference in its entirety).
[000688] In one embodiment, the nucleic acid self-assembled nanoparticles may
comprise a core of the polynucleotides, primary constructs or mmRNA disclosed
herein
and a polymer shell. The polymer shell may be any of the polymers described
herein and
are known in the art. In an additional embodiment, the polymer shell may be
used to
protect the polynucleotides, primary contructs and mmRNA in the core.
Polymer-Based Self-Assembled Nanoparticles
[000689] Polymers may be used to form sheets which self-assembled into
nanoparticles.
These nanoparticles may be used to deliver the polynucleotides, primary
constructs and
mmRNA of the present invention. In one embodiment, these self-assembled
nanoparticles may be microsponges formed of long polymers of RNA hairpins
which
form into crystalline 'pleated' sheets before self-assembling into
microsponges. These
microsponges are densely-packed sponge like microparticles which may function
as an
efficient carrier and may be able to deliver cargo to a cell. The microsponges
may be
from lum to 300 nm in diameter. The microsponges may be complexed with other
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agents known in the art to form larger microsponges. As a non-limiting
example, the
microsponge may be complexed with an agent to form an outer layer to promote
cellular
uptake such as polycation polyethyleneime (PEI). This complex can form a 250-
nm
diameter particle that can remain stable at high temperatures (150 C) (Grabow
and
Jaegar, Nature Materials 2012, 11:269-269; herein incorporated by reference in
its
entirety). Additionally these microsponges may be able to exhibit an
extraordinary
degree of protection from degradation by ribonucleases.
[000690] In another embodiment, the polymer-based self-assembled nanoparticles
such
as, but not limited to, microsponges, may be fully programmable nanoparticles.
The
geometry, size and stoichiometry of the nanoparticle may be precisely
controlled to
create the optimal nanoparticle for delivery of cargo such as, but not limited
to,
polynucleotides, primary constructs and/or mmRNA.
[000691] In one embodiment, the polymer based nanoparticles may comprise a
core of
the polynucleotides, primary constructs and/or mmRNA disclosed herein and a
polymer
shell. The polymer shell may be any of the polymers described herein and are
known in
the art. In an additional embodiment, the polymer shell may be used to protect
the
polynucleotides, primary construct and/or mmRNA in the core.
[000692] In yet another embodiment, the polymer based nanoparticle may
comprise a
non-nucleic acid polymer comprising a plurality of heterogenous monomers such
as those
described in Interantional Publication No. W02013009736, herein incorporated
by
reference in its entirety.
Inorganic Nanoparticles
[000693] The polynucleotides, primary constructs and/or mmRNAs of the present
invention may be formulated in inorganic nanoparticles (U.S. Pat. No.
8,257,745, herein
incorporated by reference in its entirety). The inorganic nanoparticles may
include, but
are not limited to, clay substances that are water swellable. As a non-
limiting example,
the inorganic nanoparticle may include synthetic smectite clays which are made
from
simple silicates (See e.g., U.S. Pat. No. 5,585,108 and 8,257,745 each of
which are herein
incorporated by reference in their entirety).
[000694] In one embodiment, the inorganic nanoparticles may comprise a core of
the
modified nucleic acids disclosed herein and a polymer shell. The polymer shell
may be
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any of the polymers described herein and are known in the art. In an
additional
embodiment, the polymer shell may be used to protect the modified nucleic
acids in the
core.
Semi-conductive and Metallic Nanoparticles
[000695] The polynucleotides, primary constructs and/or mmRNAs of the present
invention may be formulated in water-dispersible nanoparticle comprising a
semiconductive or metallic material (U.S. Pub. No. 20120228565; herein
incorporated by
reference in its entirety) or formed in a magnetic nanoparticle (U.S. Pub. No.

20120265001 and 20120283503; each of which is herein incorporated by reference
in its
entirety). The water-dispersible nanoparticles may be hydrophobic
nanoparticles or
hydrophilic nanoparticles.
[000696] In one embodiment, the semi-conductive and/or metallic nanoparticles
may
comprise a core of the polynucleotides, primary constructs and/or mmRNA
disclosed
herein and a polymer shell. The polymer shell may be any of the polymers
described
herein and are known in the art. In an additional embodiment, the polymer
shell may be
used to protect the polynucleotides, primary constructs and/or mmRNA in the
core.
Gels and Hydrogels
[000697] In one embodiment, the polynucleotides, primary constructs and/or
mmRNA
disclosed herein may be encapsulated into any hydrogel known in the art which
may form
a gel when injected into a subject. Hydrogels are a network of polymer chains
that are
hydrophilic, and are sometimes found as a colloidal gel in which water is the
dispersion
medium. Hydrogels are highly absorbent (they can contain over 99% water)
natural or
synthetic polymers. Hydrogels also possess a degree of flexibility very
similar to natural
tissue, due to their significant water content. The hydrogel described herein
may used to
encapsulate lipid nanoparticles which are biocompatible, biodegradable and/or
porous.
[000698] As a non-limiting example, the hydrogel may be an aptamer-
functionalized
hydrogel. The aptamer-functionalized hydrogel may be programmed to release one
or
more polynucleotides, primary constructs and/or mmRNA using nucleic acid
hybridization. (Battig et al., J. Am. Chem. Society. 2012 134:12410-12413;
herein
incorporated by reference in its entirety).
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[000699] As another non-limiting example, the hydrogel may be a shaped as an
inverted
opal.
The opal hydrogels exhibit higher swelling ratios and the swelling kinetics is
an order of
magnitude faster as well. Methods of producing opal hydrogels and description
of opal
hydrogels are described in International Pub. No. W02012148684, herein
incorporated
by reference in its entirety.
[000700] In yet another non-limiting example, the hydrogel may be an
antibacterial
hydrogel. The antibacterial hydrogel may comprise a pharmaceutical acceptable
salt or
organic material such as, but not limited to pharmaceutical grade and/or
medical grade
silver salt and aloe vera gel or extract. (International Pub. No.
W02012151438, herein
incorporated by reference in its entirety).
[000701] In one embodiment, the modified mRNA may be encapsulated in a lipid
nanoparticle and then the lipid nanoparticle may be encapsulated into a
hyrdogel.
[000702] In one embodiment, the polynucleotides, primary constructs and/or
mmRNA
disclosed herein may be encapsulated into any gel known in the art. As a non-
limiting
example the gel may be a fluorouracil injectable gel or a fluorouracil
injectable gel
containing a chemical compound and/or drug known in the art. As another
example, the
polynucleotides, primary constructs and/or mmRNA may be encapsulated in a
fluorouracil gel containing epinephrine (See e.g., Smith et al. Cancer
Chemotherapty and
Pharmacology, 1999 44(4):267-274; herein incorporated by reference in its
entirety).
[000703] In one embodiment, the polynucleotides, primary constructs and/or
mmRNA
disclosed herein may be encapsulated into a fibrin gel, fibrin hydrogel or
fibrin glue. In
another embodiment, the polynucleotides, primary constructs and/or mmRNA may
be
formulated in a lipid nanoparticle or a rapidly eliminated lipid nanoparticle
prior to being
encapsulated into a fibrin gel, fibrin hydrogel or a fibrin glue. In yet
another
embodiment, the polynucleotides, primary constructs and/or mmRNA may be
formulated
as a lipoplex prior to being encapsulated into a fibrin gel, hydrogel or a
fibrin glue.
Fibrin gels, hydrogels and glues comprise two components, a fibrinogen
solution and a
thrombin solution which is rich in calcium (See e.g., Spicer and Mikos,
Journal of
Controlled Release 2010. 148: 49-55; Kidd et al. Journal of Controlled Release
2012.
157:80-85; each of which is herein incorporated by reference in its entirety).
The
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concentration of the components of the fibrin gel, hydrogel and/or glue can be
altered to
change the characteristics, the network mesh size, and/or the degradation
characteristics
of the gel, hydrogel and/or glue such as, but not limited to changing the
release
characteristics of the fibrin gel, hydrogel and/or glue. (See e.g., Spicer and
Mikos,
Journal of Controlled Release 2010. 148: 49-55; Kidd et al. Journal of
Controlled Release
2012. 157:80-85; Catelas et al. Tissue Engineering 2008. 14:119-128; each of
which is
herein incorporated by reference in its entirety). This feature may be
advantageous when
used to deliver the modified mRNA disclosed herein. (See e.g., Kidd et al.
Journal of
Controlled Release 2012. 157:80-85; Catelas et al. Tissue Engineering 2008.
14:119-128;
each of which is herein incorporated by reference in its entirety).
Cations and Anions
[000704] Formulations of polynucleotides, primary constructs and/or mmRNA
disclosed herein may include cations or anions. In one embodiment, the
formulations
include metal cations such as, but not limited to, Zn2+, Ca2+, Cu2+, Mg+ and
combinations thereof As a non-limiting example, formulations may include
polymers
and a polynucleotides, primary constructs and/or mmRNA complexed with a metal
cation
(See e.g., U.S. Pat. Nos. 6,265,389 and 6,555,525, each of which is herein
incorporated
by reference in its entirety).
Molded Nanoparticles and Microparticles
[000705] The polynucleotides, primary constructs and/or mmRNA disclosed herein

may be formulated in nanoparticles and/or microparticles. These nanoparticles
and/or
microparticles may be molded into any size shape and chemistry. As an example,
the
nanoparticles and/or microparticles may be made using the PRINT technology by

LIQUIDA TECHNOLOGIES (Morrisville, NC) (See e.g., International Pub. No.
W02007024323; herein incorporated by reference in its entirety).
[000706] In one embodiment, the molded nanoparticles may comprise a core of
the
polynucleotides, primary constructs and/or mmRNA disclosed herein and a
polymer
shell. The polymer shell may be any of the polymers described herein and are
known in
the art. In an additional embodiment, the polymer shell may be used to protect
the
polynucleotides, primary construct and/or mmRNA in the core.
NanoJackets and NanoLiposomes
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[000707] The polynucleotides, primary constructs and/or mmRNA disclosed herein

may be formulated in NanoJackets and NanoLiposomes by Keystone Nano (State
College, PA). NanoJackets are made of compounds that are naturally found in
the body
including calcium, phosphate and may also include a small amount of silicates.

Nanojackets may range in size from 5 to 50 nm and may be used to deliver
hydrophilic
and hydrophobic compounds such as, but not limited to, polynucleotides,
primary
constructs and/or mmRNA.
[000708] NanoLiposomes are made of lipids such as, but not limited to, lipids
which
naturally occur in the body. NanoLiposomes may range in size from 60-80 nm and
may
be used to deliver hydrophilic and hydrophobic compounds such as, but not
limited to,
polynucleotides, primary constructs and/or mmRNA. In one aspect, the
polynucleotides,
primary constructs and/or mmRNA disclosed herein are formulated in a
NanoLiposome
such as, but not limited to, Ceramide NanoLiposomes.
Excipients
[000709] Pharmaceutical formulations may additionally comprise a
pharmaceutically
acceptable excipient, which, as used herein, includes any and all solvents,
dispersion
media, diluents, or other liquid vehicles, dispersion or suspension aids,
surface active
agents, isotonic agents, thickening or emulsifying agents, preservatives,
solid binders,
lubricants and the like, as suited to the particular dosage form desired.
Remington's The
Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro (Lippincott,
Williams &
Wilkins, Baltimore, MD, 2006; incorporated herein by reference in its
entirety) discloses
various excipients used in formulating pharmaceutical compositions and known
techniques for the preparation thereof Except insofar as any conventional
excipient
medium is incompatible with a substance or its derivatives, such as by
producing any
undesirable biological effect or otherwise interacting in a deleterious manner
with any
other component(s) of the pharmaceutical composition, its use is contemplated
to be
within the scope of this invention.
[000710] In some embodiments, a pharmaceutically acceptable excipient is at
least
95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In
some
embodiments, an excipient is approved for use in humans and for veterinary
use. In some
embodiments, an excipient is approved by United States Food and Drug
Administration.
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In some embodiments, an excipient is pharmaceutical grade. In some
embodiments, an
excipient meets the standards of the United States Pharmacopoeia (USP), the
European
Pharmacopoeia (EP), the British Pharmacopoeia, and/or the International
Pharmacopoeia.
[000711] Pharmaceutically acceptable excipients used in the manufacture of
pharmaceutical compositions include, but are not limited to, inert diluents,
dispersing
and/or granulating agents, surface active agents and/or emulsifiers,
disintegrating agents,
binding agents, preservatives, buffering agents, lubricating agents, and/or
oils. Such
excipients may optionally be included in pharmaceutical compositions.
[000712] Exemplary diluents include, but are not limited to, calcium
carbonate, sodium
carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium
hydrogen
phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline
cellulose,
kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch,
powdered
sugar, etc., and/or combinations thereof.
[000713] Exemplary granulating and/or dispersing agents include, but are not
limited to,
potato starch, corn starch, tapioca starch, sodium starch glycolate, clays,
alginic acid,
guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural
sponge,
cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-
linked
poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium
starch
glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl
cellulose
(croscarmellose), methylcellulose, pregelatinized starch (starch 1500),
microcrystalline
starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium
aluminum
silicate (VEEGUM8), sodium lauryl sulfate, quaternary ammonium compounds,
etc.,
and/or combinations thereof.
[000714] Exemplary surface active agents and/or emulsifiers include, but are
not
limited to, natural emulsifiers (e.g. acacia, agar, alginic acid, sodium
alginate, tragacanth,
chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat,
cholesterol,
wax, and lecithin), colloidal clays (e.g. bentonite [aluminum silicate] and
VEEGUM
[magnesium aluminum silicate]), long chain amino acid derivatives, high
molecular
weight alcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin
monostearate,
ethylene glycol distearate, glyceryl monostearate, and propylene glycol
monostearate,
polyvinyl alcohol), carbomers (e.g. carboxy polymethylene, polyacrylic acid,
acrylic acid
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polymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g.
carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose,
hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose),
sorbitan fatty
acid esters (e.g. polyoxyethylene sorbitan monolaurate [TWEEN 20],
polyoxyethylene
sorbitan [TWEENn 60], polyoxyethylene sorbitan monooleate [TWEEN 80], sorbitan

monopalmitate [SPAN 40], sorbitan monostearate [SPAN 60], sorbitan tristearate

[SPAN 65], glyceryl monooleate, sorbitan monooleate [SPAN 80]),
polyoxyethylene
esters (e.g. polyoxyethylene monostearate [MYRJ 45], polyoxyethylene
hydrogenated
castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and
SOLUTOL8),
sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g.
CREMOPHOR ),
polyoxyethylene ethers, (e.g. polyoxyethylene lauryl ether [BRIJ 30]),
poly(vinyl-
pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium
oleate,
potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl
sulfate,
PLUORNC F 68, POLOXAMER 188, cetrimonium bromide, cetylpyridinium chloride,
benzalkonium chloride, docusate sodium, etc. and/or combinations thereof.
[000715] Exemplary binding agents include, but are not limited to, starch
(e.g.
cornstarch and starch paste); gelatin; sugars (e.g. sucrose, glucose,
dextrose, dextrin,
molasses, lactose, lactitol, mannitol,); natural and synthetic gums (e.g.
acacia, sodium
alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol
husks,
carboxymethylcellulose, methylcellulose, ethylcellulose,
hydroxyethylcellulose,
hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline
cellulose,
cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate
(Veegum ), and
larch arabogalactan); alginates; polyethylene oxide; polyethylene glycol;
inorganic
calcium salts; silicic acid; polymethacrylates; waxes; water; alcohol; etc.;
and
combinations thereof
[000716] Exemplary preservatives may include, but are not limited to,
antioxidants,
chelating agents, antimicrobial preservatives, antifungal preservatives,
alcohol
preservatives, acidic preservatives, and/or other preservatives. Exemplary
antioxidants
include, but are not limited to, alpha tocopherol, ascorbic acid, acorbyl
palmitate,
butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol,
potassium
metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium
bisulfite, sodium
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metabisulfite, and/or sodium sulfite. Exemplary chelating agents include
ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium
edetate,
dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid,
sodium
edetate, tartaric acid, and/or trisodium edetate. Exemplary antimicrobial
preservatives
include, but are not limited to, benzalkonium chloride, benzethonium chloride,
benzyl
alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine,
chlorobutanol,
chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine,
imidurea, phenol,
phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol,
and/or
thimerosal. Exemplary antifungal preservatives include, but are not limited
to, butyl
paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid,
hydroxybenzoic
acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium
propionate, and/or
sorbic acid. Exemplary alcohol preservatives include, but are not limited to,
ethanol,
polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol,
hydroxybenzoate, and/or phenylethyl alcohol. Exemplary acidic preservatives
include,
but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric
acid, acetic
acid, dehydroacetic acid, ascorbic acid, sorbic acid, and/or phytic acid.
Other
preservatives include, but are not limited to, tocopherol, tocopherol acetate,
deteroxime
mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened
(BHT),
ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate
(SLES),
sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium
metabisulfite,
GLYDANT PLUS , PHENONIP , methylparaben, GERMALL 115, GERMABEN H,
NEOLONETM, KATHONTm, and/or EUXYL .
[000717] Exemplary buffering agents include, but are not limited to, citrate
buffer
solutions, acetate buffer solutions, phosphate buffer solutions, ammonium
chloride,
calcium carbonate, calcium chloride, calcium citrate, calcium glubionate,
calcium
gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate,
calcium
lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium
phosphate,
phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate,
potassium
acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic
potassium
phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium

acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate,
dibasic
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sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures,
tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-
free
water, isotonic saline, Ringer's solution, ethyl alcohol, etc., and/or
combinations thereof
[000718] Exemplary lubricating agents include, but are not limited to,
magnesium
stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl
behanate, hydrogenated
vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium
chloride,
leucine, magnesium lauryl sulfate, sodium lauryl sulfate, etc., and
combinations thereof
[000719] Exemplary oils include, but are not limited to, almond, apricot
kernel,
avocado, babassu, bergamot, black current seed, borage, cade, camomile,
canola,
caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee,
corn,
cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol,
gourd, grape
seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin,
lavender, lemon,
litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink,
nutmeg,
olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy
seed,
pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana,
savoury,
sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree,
thistle, tsubaki,
vetiver, walnut, and wheat germ oils. Exemplary oils include, but are not
limited to,
butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone,
diethyl sebacate,
dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl
alcohol, silicone
oil, and/or combinations thereof.
[000720] Excipients such as cocoa butter and suppository waxes, coloring
agents,
coating agents, sweetening, flavoring, and/or perfuming agents can be present
in the
composition, according to the judgment of the formulator.
Delivery
[000721] The present disclosure encompasses the delivery of polynucleotides,
primary
constructs or mmRNA for any of therapeutic, pharmaceutical, diagnostic or
imaging by
any appropriate route taking into consideration likely advances in the
sciences of drug
delivery. Delivery may be naked or formulated.
Naked Delivery
[000722] The polynucleotides, primary constructs or mmRNA of the present
invention
may be delivered to a cell naked. As used herein in, "naked" refers to
delivering
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polynucleotides, primary constructs or mmRNA free from agents which promote
transfection. For example, the polynucleotides, primary constructs or mmRNA
delivered
to the cell may contain no modifications. The naked polynucleotides, primary
constructs
or mmRNA may be delivered to the cell using routes of administration known in
the art
and described herein.
Formulated Delivery
[000723] The polynucleotides, primary constructs or mmRNA of the present
invention
may be formulated, using the methods described herein. The formulations may
contain
polynucleotides, primary constructs or mmRNA which may be modified and/or
unmodified. The formulations may further include, but are not limited to, cell
penetration
agents, a pharmaceutically acceptable carrier, a delivery agent, a bioerodible
or
biocompatible polymer, a solvent, and a sustained-release delivery depot. The
formulated polynucleotides, primary constructs or mmRNA may be delivered to
the cell
using routes of administration known in the art and described herein.
[000724] The compositions may also be formulated for direct delivery to an
organ or
tissue in any of several ways in the art including, but not limited to, direct
soaking or
bathing, via a catheter, by gels, powder, ointments, creams, gels, lotions,
and/or drops, by
using substrates such as fabric or biodegradable materials coated or
impregnated with the
compositions, and the like.
Administration
[000725] The polynucleotides, primary constructs or mmRNA of the present
invention
may be administered by any route which results in a therapeutically effective
outcome.
These include, but are not limited to enteral, gastroenteral, epidural, oral,
transdermal,
epidural (peridural), intracerebral (into the cerebrum),
intracerebroventricular (into the
cerebral ventricles), epicutaneous (application onto the skin), intradermal,
(into the skin
itself), subcutaneous (under the skin), nasal administration (through the
nose),
intravenous (into a vein), intraarterial (into an artery), intramuscular (into
a muscle),
intracardiac (into the heart), intraosseous infusion (into the bone marrow),
intrathecal
(into the spinal canal), intraperitoneal, (infusion or injection into the
peritoneum),
intravesical infusion, intravitreal, (through the eye), intracavernous
injection, ( into the
base of the penis), intravaginal administration, intrauterine, extra-amniotic
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administration, transdermal (diffusion through the intact skin for systemic
distribution),
transmucosal (diffusion through a mucous membrane), insufflation (snorting),
sublingual,
sublabial, enema, eye drops (onto the conjunctiva), or in ear drops. In
specific
embodiments, compositions may be administered in a way which allows them cross
the
blood-brain barrier, vascular barrier, or other epithelial barrier.Non-
limiting routes of
administration for the polynucleotides, primary constructs or mmRNA of the
present
invention are described below.
Parenteral and Injecfible Administration
[000726] Liquid dosage forms for parenteral administration include, but are
not limited
to, pharmaceutically acceptable emulsions, microemulsions, solutions,
suspensions,
syrups, and/or elixirs. In addition to active ingredients, liquid dosage forms
may
comprise inert diluents commonly used in the art such as, for example, water
or other
solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl
alcohol,
ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene
glycol, 1,3-
butylene glycol, dimethylformamide, oils (in particular, cottonseed,
groundnut, corn,
germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol,
polyethylene
glycols and fatty acid esters of sorbitan, and mixtures thereof Besides inert
diluents, oral
compositions can include adjuvants such as wetting agents, emulsifying and
suspending
agents, sweetening, flavoring, and/or perfuming agents. In certain embodiments
for
parenteral administration, compositions are mixed with solubilizing agents
such as
CREMOPHOR , alcohols, oils, modified oils, glycols, polysorbates,
cyclodextrins,
polymers, and/or combinations thereof.
[000727] Injectable preparations, for example, sterile injectable aqueous or
oleaginous
suspensions may be formulated according to the known art using suitable
dispersing
agents, wetting agents, and/or suspending agents. Sterile injectable
preparations may be
sterile injectable solutions, suspensions, and/or emulsions in nontoxic
parenterally
acceptable diluents and/or solvents, for example, as a solution in 1,3-
butanediol. Among
the acceptable vehicles and solvents that may be employed are water, Ringer's
solution,
U.S.P., and isotonic sodium chloride solution. Sterile, fixed oils are
conventionally
employed as a solvent or suspending medium. For this purpose any bland fixed
oil can
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be employed including synthetic mono- or diglycerides. Fatty acids such as
oleic acid
can be used in the preparation of injectables.
[000728] Injectable formulations can be sterilized, for example, by filtration
through a
bacterial-retaining filter, and/or by incorporating sterilizing agents in the
form of sterile
solid compositions which can be dissolved or dispersed in sterile water or
other sterile
injectable medium prior to use.
[000729] In order to prolong the effect of an active ingredient, it is often
desirable to
slow the absorption of the active ingredient from subcutaneous or
intramuscular injection.
This may be accomplished by the use of a liquid suspension of crystalline or
amorphous
material with poor water solubility. The rate of absorption of the drug then
depends upon
its rate of dissolution which, in turn, may depend upon crystal size and
crystalline form.
Alternatively, delayed absorption of a parenterally administered drug form is
accomplished by dissolving or suspending the drug in an oil vehicle.
Injectable depot
forms are made by forming microencapsule matrices of the drug in biodegradable

polymers such as polylactide-polyglycolide. Depending upon the ratio of drug
to
polymer and the nature of the particular polymer employed, the rate of drug
release can
be controlled. Examples of other biodegradable polymers include
poly(orthoesters) and
poly(anhydrides). Depot injectable formulations are prepared by entrapping the
drug in
liposomes or microemulsions which are compatible with body tissues.
Rectal and Vaginal Administration
[000730] Compositions for rectal or vaginal administration are typically
suppositories
which can be prepared by mixing compositions with suitable non-irritating
excipients
such as cocoa butter, polyethylene glycol or a suppository wax which are solid
at ambient
temperature but liquid at body temperature and therefore melt in the rectum or
vaginal
cavity and release the active ingredient.
Oral Administration
[000731] Liquid dosage forms for oral administration include, but are not
limited to,
pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions,
syrups,
and/or elixirs. In addition to active ingredients, liquid dosage forms may
comprise inert
diluents commonly used in the art such as, for example, water or other
solvents,
solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol,
ethyl
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carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,
1,3-butylene
glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn,
germ, olive,
castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene
glycols and
fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents,
oral
compositions can include adjuvants such as wetting agents, emulsifying and
suspending
agents, sweetening, flavoring, and/or perfuming agents. In certain embodiments
for
parenteral administration, compositions are mixed with solubilizing agents
such as
CREMOPHOR , alcohols, oils, modified oils, glycols, polysorbates,
cyclodextrins,
polymers, and/or combinations thereof.
[000732] Solid dosage forms for oral administration include capsules, tablets,
pills,
powders, and granules. In such solid dosage forms, an active ingredient is
mixed with at
least one inert, pharmaceutically acceptable excipient such as sodium citrate
or dicalcium
phosphate and/or fillers or extenders (e.g. starches, lactose, sucrose,
glucose, mannitol,
and silicic acid), binders (e.g. carboxymethylcellulose, alginates, gelatin,
polyvinylpyrrolidinone, sucrose, and acacia), humectants (e.g. glycerol),
disintegrating
agents (e.g. agar, calcium carbonate, potato or tapioca starch, alginic acid,
certain
silicates, and sodium carbonate), solution retarding agents (e.g. paraffin),
absorption
accelerators (e.g. quaternary ammonium compounds), wetting agents (e.g. cetyl
alcohol
and glycerol monostearate), absorbents (e.g. kaolin and bentonite clay), and
lubricants
(e.g. talc, calcium stearate, magnesium stearate, solid polyethylene glycols,
sodium lauryl
sulfate), and mixtures thereof. In the case of capsules, tablets and pills,
the dosage form
may comprise buffering agents.
Topical or Transdermal Administration
[000733] As described herein, compositions containing the polynucleotides,
primary
constructs or mmRNA of the invention may be formulated for administration
topically.
The skin may be an ideal target site for delivery as it is readily accessible.
Gene
expression may be restricted not only to the skin, potentially avoiding
nonspecific
toxicity, but also to specific layers and cell types within the skin.
[000734] The site of cutaneous expression of the delivered compositions will
depend on
the route of nucleic acid delivery. Three routes are commonly considered to
deliver
polynucleotides, primary constructs or mmRNA to the skin: (i) topical
application (e.g.
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for local/regional treatment and/or cosmetic applications); (ii) intradermal
injection (e.g.
for local/regional treatment and/or cosmetic applications); and (iii) systemic
delivery (e.g.
for treatment of dermatologic diseases that affect both cutaneous and
extracutaneous
regions). Polynucleotides, primary constructs or mmRNA can be delivered to the
skin by
several different approaches known in the art. Most topical delivery
approaches have
been shown to work for delivery of DNA, such as but not limited to, topical
application
of non-cationic liposome¨DNA complex, cationic liposome¨DNA complex, particle-
mediated (gene gun), puncture-mediated gene transfections, and viral delivery
approaches. After delivery of the nucleic acid, gene products have been
detected in a
number of different skin cell types, including, but not limited to, basal
keratinocytes,
sebaceous gland cells, dermal fibroblasts and dermal macrophages.
[000735] In one embodiment, the invention provides for a variety of dressings
(e.g.,
wound dressings) or bandages (e.g., adhesive bandages) for conveniently and/or

effectively carrying out methods of the present invention. Typically dressing
or bandages
may comprise sufficient amounts of pharmaceutical compositions and/or
polynucleotides,
primary constructs or mmRNA described herein to allow a user to perform
multiple
treatments of a subject(s).
[000736] In one embodiment, the invention provides for the polynucleotides,
primary
constructs or mmRNA compositions to be delivered in more than one injection.
[000737] In one embodiment, before topical and/or transdermal administration
at least
one area of tissue, such as skin, may be subjected to a device and/or solution
which may
increase permeability. In one embodiment, the tissue may be subjected to an
abrasion
device to increase the permeability of the skin (see U.S. Patent Publication
No.
20080275468, herein incorporated by reference in its entirety). In another
embodiment,
the tissue may be subjected to an ultrasound enhancement device. An ultrasound

enhancement device may include, but is not limited to, the devices described
in U.S.
Publication No. 20040236268 and U.S. Patent Nos. 6,491,657 and 6,234,990; each
of
which are herein incorporated by reference in their entireties. Methods of
enhancing the
permeability of tissue are described in U.S. Publication Nos. 20040171980 and
20040236268 and U.S. Pat. No. 6,190,315; each of which are herein incorporated
by
reference in their entireties.
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[000738] In one embodiment, a device may be used to increase permeability of
tissue
before delivering formulations of modified mRNA described herein. The
permeability of
skin may be measured by methods known in the art and/or described in U.S.
Patent No.
6,190,315, herein incorporated by reference in its entirety. As a non-limiting
example, a
modified mRNA formulation may be delivered by the drug delivery methods
described in
U.S. Patent No. 6,190,315, herein incorporated by reference in its entirety.
[000739] In another non-limiting example tissue may be treated with a eutectic
mixture
of local anesthetics (EMLA) cream before, during and/or after the tissue may
be
subjected to a device which may increase permeability. Katz et al. (Anesth
Analg
(2004); 98:371-76; herein incorporated by reference in its entirety) showed
that using the
EMLA cream in combination with a low energy, an onset of superficial cutaneous

analgesia was seen as fast as 5 minutes after a pretreatment with a low energy
ultrasound.
[000740] In one embodiment, enhancers may be applied to the tissue before,
during,
and/or after the tissue has been treated to increase permeability. Enhancers
include, but
are not limited to, transport enhancers, physical enhancers, and cavitation
enhancers.
Non-limiting examples of enhancers are described in U.S. Patent No. 6,190,315,
herein
incorporated by reference in its entirety.
[000741] In one embodiment, a device may be used to increase permeability of
tissue
before delivering formulations of modified mRNA described herein, which may
further
contain a substance that invokes an immune response. In another non-limiting
example,
a formulation containing a substance to invoke an immune response may be
delivered by
the methods described in U.S. Publication Nos. 20040171980 and 20040236268;
each of
which are herein incorporated by reference in their entireties.
[000742] Dosage forms for topical and/or transdermal administration of a
composition
may include ointments, pastes, creams, lotions, gels, powders, solutions,
sprays, inhalants
and/or patches. Generally, an active ingredient is admixed under sterile
conditions with a
pharmaceutically acceptable excipient and/or any needed preservatives and/or
buffers as
may be required.
[000743] Additionally, the present invention contemplates the use of
transdermal
patches, which often have the added advantage of providing controlled delivery
of a
compound to the body. Such dosage forms may be prepared, for example, by
dissolving
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and/or dispensing the compound in the proper medium. Alternatively or
additionally,
rate may be controlled by either providing a rate controlling membrane and/or
by
dispersing the compound in a polymer matrix and/or gel.
[000744] Formulations suitable for topical administration include, but are not
limited to,
liquid and/or semi liquid preparations such as liniments, lotions, oil in
water and/or water
in oil emulsions such as creams, ointments and/or pastes, and/or solutions
and/or
suspensions.
[000745] Topically-administrable formulations may, for example, comprise from
about
0.1% to about 10% (w/w) active ingredient, although the concentration of
active
ingredient may be as high as the solubility limit of the active ingredient in
the solvent.
Formulations for topical administration may further comprise one or more of
the
additional ingredients described herein.
Depot Administration
[000746] As described herein, in some embodiments, the composition is
formulated in
depots for extended release. Generally, a specific organ or tissue (a "target
tissue") is
targeted for administration.
[000747] In some aspects of the invention, the polynucleotides, primary
constructs or
mmRNA are spatially retained within or proximal to a target tissue. Provided
are method
of providing a composition to a target tissue of a mammalian subject by
contacting the
target tissue (which contains one or more target cells) with the composition
under
conditions such that the composition, in particular the nucleic acid
component(s) of the
composition, is substantially retained in the target tissue, meaning that at
least 10, 20, 30,
40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.99 or greater than
99.99% of the
composition is retained in the target tissue. Advantageously, retention is
determined by
measuring the amount of the nucleic acid present in the composition that
enters one or
more target cells. For example, at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80,
85, 90, 95, 96,
97, 98, 99, 99.9, 99.99 or greater than 99.99% of the nucleic acids
administered to the
subject are present intracellularly at a period of time following
administration. For
example, intramuscular injection to a mammalian subject is performed using an
aqueous
composition containing a ribonucleic acid and a transfection reagent, and
retention of the
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composition is determined by measuring the amount of the ribonucleic acid
present in the
muscle cells.
[000748] Aspects of the invention are directed to methods of providing a
composition to
a target tissue of a mammalian subject, by contacting the target tissue
(containing one or
more target cells) with the composition under conditions such that the
composition is
substantially retained in the target tissue. The composition contains an
effective amount
of a polynucleotides, primary constructs or mmRNA such that the polypeptide of
interest
is produced in at least one target cell. The compositions generally contain a
cell
penetration agent, although "naked" nucleic acid (such as nucleic acids
without a cell
penetration agent or other agent) is also contemplated, and a pharmaceutically
acceptable
carrier.
[000749] In some circumstances, the amount of a protein produced by cells in a
tissue is
desirably increased. Preferably, this increase in protein production is
spatially restricted
to cells within the target tissue. Thus, provided are methods of increasing
production of a
protein of interest in a tissue of a mammalian subject. A composition is
provided that
contains polynucleotides, primary constructs or mmRNA characterized in that a
unit
quantity of composition has been determined to produce the polypeptide of
interest in a
substantial percentage of cells contained within a predetermined volume of the
target
tissue.
[000750] In some embodiments, the composition includes a plurality of
different
polynucleotides, primary constructs or mmRNA, where one or more than one of
the
polynucleotides, primary constructs or mmRNA encodes a polypeptide of
interest.
Optionally, the composition also contains a cell penetration agent to assist
in the
intracellular delivery of the composition. A determination is made of the dose
of the
composition required to produce the polypeptide of interest in a substantial
percentage of
cells contained within the predetermined volume of the target tissue
(generally, without
inducing significant production of the polypeptide of interest in tissue
adjacent to the
predetermined volume, or distally to the target tissue). Subsequent to this
determination,
the determined dose is introduced directly into the tissue of the mammalian
subject.
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[000751] In one embodiment, the invention provides for the polynucleotides,
primary
constructs or mmRNA to be delivered in more than one injection or by split
dose
injections.
[000752] In one embodiment, the invention may be retained near target tissue
using a
small disposable drug reservoir, patch pump or osmotic pump. Non-limiting
examples of
patch pumps include those manufactured and/or sold by BD (Franklin Lakes,
NJ),
Insulet Corporation (Bedford, MA), SteadyMed Therapeutics (San Francisco, CA),

Medtronic (Minneapolis, MN) (e.g., MiniMed), UniLife (York, PA), Valeritas
(Bridgewater, NJ), and SpringLeaf Therapeutics (Boston, MA). A non-limiting
example
of an osmotic pump include those manufactured by DURECTO (Cupertino, CA)
(e.g.,
DUROSO and ALZET 0).
Pulmonary Administration
[000753] A pharmaceutical composition may be prepared, packaged, and/or sold
in a
formulation suitable for pulmonary administration via the buccal cavity. Such
a
formulation may comprise dry particles which comprise the active ingredient
and which
have a diameter in the range from about 0.5 nm to about 7 nm or from about 1
nm to
about 6 nm. Such compositions are suitably in the form of dry powders for
administration using a device comprising a dry powder reservoir to which a
stream of
propellant may be directed to disperse the powder and/or using a self
propelling
solvent/powder dispensing container such as a device comprising the active
ingredient
dissolved and/or suspended in a low-boiling propellant in a sealed container.
Such
powders comprise particles wherein at least 98% of the particles by weight
have a
diameter greater than 0.5 nm and at least 95% of the particles by number have
a diameter
less than 7 nm. Alternatively, at least 95% of the particles by weight have a
diameter
greater than 1 nm and at least 90% of the particles by number have a diameter
less than 6
nm. Dry powder compositions may include a solid fine powder diluent such as
sugar and
are conveniently provided in a unit dose form.
[000754] Low boiling propellants generally include liquid propellants having a
boiling
point of below 65 F at atmospheric pressure. Generally the propellant may
constitute
50% to 99.9% (w/w) of the composition, and active ingredient may constitute
0.1% to
20% (w/w) of the composition. A propellant may further comprise additional
ingredients
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such as a liquid non-ionic and/or solid anionic surfactant and/or a solid
diluent (which
may have a particle size of the same order as particles comprising the active
ingredient).
[000755] As a non-limiting example, the polynucleotides, primary constructs
and/or
mmRNA described herein may be formulated for pulmonary delivery by the methods

described in U.S. Pat. No. 8,257,685; herein incorporated by reference in its
entirety.
[000756] Pharmaceutical compositions formulated for pulmonary delivery may
provide
an active ingredient in the form of droplets of a solution and/or suspension.
Such
formulations may be prepared, packaged, and/or sold as aqueous and/or dilute
alcoholic
solutions and/or suspensions, optionally sterile, comprising active
ingredient, and may
conveniently be administered using any nebulization and/or atomization device.
Such
formulations may further comprise one or more additional ingredients
including, but not
limited to, a flavoring agent such as saccharin sodium, a volatile oil, a
buffering agent, a
surface active agent, and/or a preservative such as methylhydroxybenzoate.
Droplets
provided by this route of administration may have an average diameter in the
range from
about 0.1 nm to about 200 nm.
Intranasal, nasal and buccal Administration
[000757] Formulations described herein as being useful for pulmonary delivery
are
useful for intranasal delivery of a pharmaceutical composition. Another
formulation
suitable for intranasal administration is a coarse powder comprising the
active ingredient
and having an average particle from about 0.2 um to 500 um. Such a formulation
is
administered in the manner in which snuff is taken, i.e. by rapid inhalation
through the
nasal passage from a container of the powder held close to the nose.
[000758] Formulations suitable for nasal administration may, for example,
comprise
from about as little as 0.1% (w/w) and as much as 100% (w/w) of active
ingredient, and
may comprise one or more of the additional ingredients described herein. A
pharmaceutical composition may be prepared, packaged, and/or sold in a
formulation
suitable for buccal administration. Such formulations may, for example, be in
the form
of tablets and/or lozenges made using conventional methods, and may, for
example, 0.1%
to 20% (w/w) active ingredient, the balance comprising an orally dissolvable
and/or
degradable composition and, optionally, one or more of the additional
ingredients
described herein. Alternately, formulations suitable for buccal administration
may
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comprise a powder and/or an aerosolized and/or atomized solution and/or
suspension
comprising active ingredient. Such powdered, aerosolized, and/or aerosolized
formulations, when dispersed, may have an average particle and/or droplet size
in the
range from about 0.1 nm to about 200 nm, and may further comprise one or more
of any
additional ingredients described herein.
Ophthalmic Administration
[000759] A pharmaceutical composition may be prepared, packaged, and/or sold
in a
formulation suitable for ophthalmic administration. Such formulations may, for
example,
be in the form of eye drops including, for example, a 0.1/1.0% (w/w) solution
and/or
suspension of the active ingredient in an aqueous or oily liquid excipient.
Such drops
may further comprise buffering agents, salts, and/or one or more other of any
additional
ingredients described herein. Other ophthalmically-administrable formulations
which are
useful include those which comprise the active ingredient in microcrystalline
form and/or
in a liposomal preparation. Ear drops and/or eye drops are contemplated as
being within
the scope of this invention. A multilayer thin film device may be prepared to
contain a
pharmaceutical composition for delivery to the eye and/or surrounding tissue.
Payload Administration: Detectable Agents and Therapeutic Agents
[000760] The polynucleotides, primary constructs or mmRNA described herein can
be
used in a number of different scenarios in which delivery of a substance (the
"payload")
to a biological target is desired, for example delivery of detectable
substances for
detection of the target, or delivery of a therapeutic agent. Detection methods
can include,
but are not limited to, both imaging in vitro and in vivo imaging methods,
e.g.,
immunohistochemistry, bioluminescence imaging (BLI), Magnetic Resonance
Imaging
(MRI), positron emission tomography (PET), electron microscopy, X-ray computed

tomography, Raman imaging, optical coherence tomography, absorption imaging,
thermal imaging, fluorescence reflectance imaging, fluorescence microscopy,
fluorescence molecular tomographic imaging, nuclear magnetic resonance
imaging, X-
ray imaging, ultrasound imaging, photoacoustic imaging, lab assays, or in any
situation
where tagging/staining/imaging is required.
[000761] The polynucleotides, primary constructs or mmRNA can be designed to
include both a linker and a payload in any useful orientation. For example, a
linker
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having two ends is used to attach one end to the payload and the other end to
the
nucleobase, such as at the C-7 or C-8 positions of the deaza-adenosine or
deaza-
guanosine or to the N-3 or C-5 positions of cytosine or uracil. The
polynucleotide of the
invention can include more than one payload (e.g., a label and a transcription
inhibitor),
as well as a cleavable linker. In one embodiment, the modified nucleotide is a
modified
7-deaza-adenosine triphosphate, where one end of a cleavable linker is
attached to the C7
position of 7-deaza-adenine, the other end of the linker is attached to an
inhibitor (e.g., to
the C5 position of the nucleobase on a cytidine), and a label (e.g., Cy5) is
attached to the
center of the linker (see, e.g., compound 1 of A*pCp C5 Parg Capless in Fig. 5
and
columns 9 and 10 of U.S. Pat. No. 7,994,304, incorporated herein by
reference). Upon
incorporation of the modified 7-deaza-adenosine triphosphate to an encoding
region, the
resulting polynucleotide having a cleavable linker attached to a label and an
inhibitor
(e.g., a polymerase inhibitor). Upon cleavage of the linker (e.g., with
reductive
conditions to reduce a linker having a cleavable disulfide moiety), the label
and inhibitor
are released. Additional linkers and payloads (e.g., therapeutic agents,
detectable labels,
and cell penetrating payloads) are described herein.
[000762] Scheme 12 below depicts an exemplary modified nucleotide wherein the
nucleobase, adenine, is attached to a linker at the C-7 carbon of 7-deaza
adenine. In
addition, Scheme 12 depicts the modified nucleotide with the linker and
payload, e.g., a
detectable agent, incorporated onto the 3' end of the mRNA. Disulfide cleavage
and 1,2-
addition of the thiol group onto the propargyl ester releases the detectable
agent. The
remaining structure (depicted, for example, as pApC5Parg in Scheme 12) is the
inhibitor.
The rationale for the structure of the modified nucleotides is that the
tethered inhibitor
sterically interferes with the ability of the polymerase to incorporate a
second base. Thus,
it is critical that the tether be long enough to affect this function and that
the inhibiter be
in a stereochemical orientation that inhibits or prohibits second and follow
on nucleotides
into the growing polynucleotide strand.
Scheme 12
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03s so, so3H
, ...- ...- ---
Ne N lir
0
HN 0
1N H2
NV \ 0 8
I
Llit.111H2
N N
A Capless pCpC5 Parg 1 11
0
N 0
II II II-OPOPOPes-q
0- -
0 0 -
OH OH
0o ,0
,P
-0 \
0-
1
incorporation Cy5
''.0
NH2 HN 0
I\ V H
^-^i I \ 131-S-SrI
RNA^^- ..õ. m
I N N 0 N L430
0
VL-i2
0
./ N.
( N
I
OH OH N 0
Cleavage of S-S bond
-0. 5)
P.
-o, 0
NH2
0
N.---)....--=, 0 r 0
..'
1-rSH P'0
RNA-1n ,....
I N N 0
0 (3)/
OH OH I
NH2
NV i \ = OH
RNA-1-1 .._, 0
I N N
0
_________ 0) + S\)
/
OH OH
[000763] For example, the polynucleotides, primary constructs or mmRNA
described
herein can be used in reprogramming induced pluripotent stem cells (iPS
cells), which
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can directly track cells that are transfected compared to total cells in the
cluster. In
another example, a drug that may be attached to the polynucleotides, primary
constructs
or mmRNA via a linker and may be fluorescently labeled can be used to track
the drug in
vivo, e.g. intracellularly. Other examples include, but are not limited to,
the use of a
polynucleotides, primary constructs or mmRNA in reversible drug delivery into
cells.
[000764] The polynucleotides, primary constructs or mmRNA described herein can
be
used in intracellular targeting of a payload, e.g., detectable or therapeutic
agent, to
specific organelle. Exemplary intracellular targets can include, but are not
limited to, the
nuclear localization for advanced mRNA processing, or a nuclear localization
sequence
(NLS) linked to the mRNA containing an inhibitor.
[000765] In addition, the polynucleotides, primary constructs or mmRNA
described
herein can be used to deliver therapeutic agents to cells or tissues, e.g., in
living animals.
For example, the polynucleotides, primary constructs or mmRNA described herein
can be
used to deliver highly polar chemotherapeutics agents to kill cancer cells.
The
polynucleotides, primary constructs or mmRNA attached to the therapeutic agent
through
a linker can facilitate member permeation allowing the therapeutic agent to
travel into a
cell to reach an intracellular target.
[000766] In one example, the linker is attached at the 2'-position of the
ribose ring
and/or at the 3' and/or 5' positionof the polynucleotides, primary constructs
mmRNA
(See e.g., International Pub. No. W02012030683, herein incorporated by
reference in its
entirety). The linker may be any linker disclosed herein, known in the art
and/or
disclosed in International Pub. No. W02012030683, herein incorporated by
reference in
its entirety.
[000767] In another example, the polynucleotides, primary constructs or mmRNA
can
be attached to the polynucleotides, primary constructs or mmRNA a viral
inhibitory
peptide (VIP) through a cleavable linker. The cleavable linker can release the
VIP and
dye into the cell. In another example, the polynucleotides, primary constructs
or
mmRNA can be attached through the linker to an ADP-ribosylate, which is
responsible
for the actions of some bacterial toxins, such as cholera toxin, diphtheria
toxin, and
pertussis toxin. These toxin proteins are ADP-ribosyltransferases that modify
target
proteins in human cells. For example, cholera toxin ADP-ribosylates G proteins
modifies
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human cells by causing massive fluid secretion from the lining of the small
intestine,
which results in life-threatening diarrhea.
[000768] In some embodiments, the payload may be a therapeutic agent such as a

cytotoxin, radioactive ion, chemotherapeutic, or other therapeutic agent. A
cytotoxin or
cytotoxic agent includes any agent that may be detrimental to cells. Examples
include,
but are not limited to, taxol, cytochalasin B, gramicidin D, ethidium bromide,
emetine,
mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine,
doxorubicin,
daunorubicin, dihydroxyanthracinedione, mitoxantrone, mithramycin, actinomycin
D, 1-
dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,
propranolol,
puromycin, maytansinoids, e.g., maytansinol (see U.S. Pat. No. 5,208,020
incorporated
herein in its entirety), rachelmycin (CC-1065, see U.S. Pat. Nos. 5,475,092,
5,585,499,
and 5,846,545, all of which are incorporated herein by reference), and analogs
or
homologs thereof Radioactive ions include, but are not limited to iodine
(e.g., iodine
125 or iodine 131), strontium 89, phosphorous, palladium, cesium, iridium,
phosphate,
cobalt, yttrium 90, samarium 153, and praseodymium. Other therapeutic agents
include,
but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine,
6-
thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,

mechlorethamine, thiotepa chlorambucil, rachelmycin (CC-1065), melphalan,
carmustine
(BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol,
streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)
cisplatin),
anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin),
antibiotics
(e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and
anthramycin
(AMC)), and anti-mitotic agents (e.g., vincristine, vinblastine, taxol and
maytansinoids).
[000769] In some embodiments, the payload may be a detectable agent, such as
various organic small molecules, inorganic compounds, nanoparticles, enzymes
or
enzyme substrates, fluorescent materials, luminescent materials (e.g.,
luminol),
bioluminescent materials (e.g., luciferase, luciferin, and aequorin),
chemiluminescent
materials, radioactive materials (e.g., 18F5 67Ga, 8imKr5 82Rb, 111In5 12315
133xe5 201115 12515
35, 14-5
3H, or 99mTc (e.g., as pertechnetate (technetate(VII), Tc04-)), and contrast
agents
(e.g., gold (e.g., gold nanoparticles), gadolinium (e.g., chelated Gd), iron
oxides (e.g.,
superparamagnetic iron oxide (SPIO), monocrystalline iron oxide nanoparticles
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(MIONs), and ultrasmall superparamagnetic iron oxide (USPIO)), manganese
chelates
(e.g., Mn-DPDP), barium sulfate, iodinated contrast media (iohexol),
microbubbles, or
perfluorocarbons). Such optically-detectable labels include for example,
without
limitation, 4-acetamido-4'-isothiocyanatostilbene-2,2'disulfonic acid;
acridine and
derivatives (e.g., acridine and acridine isothiocyanate); 5-(2'-
aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS); 4-amino-N-[3-
vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate; N-(4-anilino-l-
naphthyl)maleimide;
anthranilamide; BODIPY; Brilliant Yellow; coumarin and derivatives (e.g.,
coumarin, 7-
amino-4-methylcoumarin (AMC, Coumarin 120), and 7-amino-4-
trifluoromethylcoumarin (Coumarin 151)); cyanine dyes; cyanosine; 4',6-
diaminidino-2-
phenylindole (DAPI); 5' 5"-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol
Red);
7-diethylamino-3-(4'-isothiocyanatopheny1)-4-methylcoumarin;
diethylenetriamine
pentaacetate; 4,4'-diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid; 4,4'-

diisothiocyanatostilbene-2,2'-disulfonic acid; 5-[dimethylamino]-naphthalene-1-
sulfonyl
chloride (DNS, dansylchloride); 4-dimethylaminophenylazopheny1-4'-
isothiocyanate
(DABITC); eosin and derivatives (e.g., eosin and eosin isothiocyanate);
erythrosin and
derivatives (e.g., erythrosin B and erythrosin isothiocyanate); ethidium;
fluorescein and
derivatives (e.g., 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2-
yl)aminofluorescein (DTAF), 2',7'-dimethoxy-4'5'-dichloro-6-
carboxyfluorescein,
fluorescein, fluorescein isothiocyanate, X-rhodamine-5-(and-6)-isothiocyanate
(QFITC
or XRITC), and fluorescamine); 2-[2-[3-[[1,3-dihydro-1,1-dimethy1-3-(3-
sulfopropy1)-
2H-benz[e]indo1-2-ylidene]ethylidene]-2-[4-(ethoxycarbony1)-1-piperazinyl]-1-
cyclopenten-1-yl]etheny1]-1,1-dimethyl-3-(3-sulforpropy1)-1H-benz[e]indolium
hydroxide, inner salt, compound with n,n-diethylethanamine(1:1) (IR144); 5-
chloro-2-[2-
[3-[(5-chloro-3-ethy1-2(3H)-benzothiazol- ylidene)ethylidene]-2-
(diphenylamino)-1-
cyclopenten-1-yl]etheny1]-3-ethyl benzothiazolium perchlorate (IR140);
Malachite Green
isothiocyanate; 4-methylumbelliferone orthocresolphthalein; nitrotyrosine;
pararosaniline; Phenol Red; B-phycoerythrin; o-phthaldialdehyde; pyrene and
derivatives(e.g., pyrene, pyrene butyrate, and succinimidyl 1-pyrene);
butyrate quantum
dots; Reactive Red 4 (CIBACRONTM Brilliant Red 3B-A); rhodamine and
derivatives
(e.g., 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine
rhodamine
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B sulfonyl chloride rhodarnine (Rhod), rhodamine B, rhodamine 123, rhodamine X

isothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloride
derivative of
sulforhodamine 101 (Texas Red), N,N,N ',N 'tetramethyl-6-carboxyrhodamine
(TAMRA)
tetramethyl rhodamine, and tetramethyl rhodamine isothiocyanate (TRITC));
riboflavin;
rosolic acid; terbium chelate derivatives; Cyanine-3 (Cy3); Cyanine-5 (Cy5);
cyanine-5.5
(Cy5.5), Cyanine-7 (Cy7); IRD 700; IRD 800; Alexa 647; La Jolta Blue; phthalo
cyanine; and naphthalo cyanine.
[000770] In some embodiments, the detectable agent may be a non-detectable pre-

cursor that becomes detectable upon activation (e.g., fluorogenic tetrazine-
fluorophore
constructs (e.g., tetrazine-BODIPY FL, tetrazine-Oregon Green 488, or
tetrazine-
BODIPY TMR-X) or enzyme activatable fluorogenic agents (e.g., PROSENSEO (VisEn

Medical))). In vitro assays in which the enzyme labeled compositions can be
used
include, but are not limited to, enzyme linked immunosorbent assays (ELISAs),
immunoprecipitation assays, immunofluorescence, enzyme immunoassays (EIA),
radioimmunoassays (RIA), and Western blot analysis.
Combinations
[000771] The polynucleotides, primary constructs or mmRNA may be used in
combination with one or more other therapeutic, prophylactic, diagnostic, or
imaging
agents. By "in combination with," it is not intended to imply that the agents
must be
administered at the same time and/or formulated for delivery together,
although these
methods of delivery are within the scope of the present disclosure.
Compositions can be
administered concurrently with, prior to, or subsequent to, one or more other
desired
therapeutics or medical procedures. In general, each agent will be
administered at a dose
and/or on a time schedule determined for that agent. In some embodiments, the
present
disclosure encompasses the delivery of pharmaceutical, prophylactic,
diagnostic, or
imaging compositions in combination with agents that may improve their
bioavailability,
reduce and/or modify their metabolism, inhibit their excretion, and/or modify
their
distribution within the body. As a non-limiting example, the nucleic acids or
mmRNA
may be used in combination with a pharmaceutical agent for the treatment of
cancer or to
control hyperproliferative cells. In U.S. Pat. No. 7,964,571, herein
incorporated by
reference in its entirety, a combination therapy for the treatment of solid
primary or
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metastasized tumor is described using a pharmaceutical composition including a
DNA
plasmid encoding for interleukin-12 with a lipopolymer and also administering
at least
one anticancer agent or chemotherapeutic. Further, the nucleic acids and mmRNA
of the
present invention that encodes anti-proliferative molecules may be in a
pharmaceutical
composition with a lipopolymer (see e.g., U.S. Pub. No. 20110218231, herein
incorporated by reference in its entirety, claiming a pharmaceutical
composition
comprising a DNA plasmid encoding an anti-proliferative molecule and a
lipopolymer)
which may be administered with at least one chemotherapeutic or anticancer
agent.
[000772] It will further be appreciated that therapeutically,
prophylactically,
diagnostically, or imaging active agents utilized in combination may be
administered
together in a single composition or administered separately in different
compositions. In
general, it is expected that agents utilized in combination with be utilized
at levels that do
not exceed the levels at which they are utilized individually. In some
embodiments, the
levels utilized in combination will be lower than those utilized individually.
In one
embodiment, the combinations, each or together may be administered according
to the
split dosing regimens described herein.
Dosing
[000773] The present invention provides methods comprising administering
modified
mRNAs and their encoded proteins or complexes in accordance with the invention
to a
subject in need thereof. Nucleic acids, proteins or complexes, or
pharmaceutical,
imaging, diagnostic, or prophylactic compositions thereof, may be administered
to a
subject using any amount and any route of administration effective for
preventing,
treating, diagnosing, or imaging a disease, disorder, and/or condition (e.g.,
a disease,
disorder, and/or condition relating to working memory deficits). The exact
amount
required will vary from subject to subject, depending on the species, age, and
general
condition of the subject, the severity of the disease, the particular
composition, its mode
of administration, its mode of activity, and the like. Compositions in
accordance with the
invention are typically formulated in dosage unit form for ease of
administration and
uniformity of dosage. It will be understood, however, that the total daily
usage of the
compositions of the present invention may be decided by the attending
physician within
the scope of sound medical judgment. The specific therapeutically effective,
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prophylactically effective, or appropriate imaging dose level for any
particular patient
will depend upon a variety of factors including the disorder being treated and
the severity
of the disorder; the activity of the specific compound employed; the specific
composition
employed; the age, body weight, general health, sex and diet of the patient;
the time of
administration, route of administration, and rate of excretion of the specific
compound
employed; the duration of the treatment; drugs used in combination or
coincidental with
the specific compound employed; and like factors well known in the medical
arts.
[000774] In certain embodiments, compositions in accordance with the present
invention may be administered at dosage levels sufficient to deliver from
about 0.0001
mg/kg to about 100 mg/kg, from about 0.001 mg/kg to about 0.05 mg/kg, from
about
0.005 mg/kg to about 0.05 mg/kg, from about 0.001 mg/kg to about 0.005 mg/kg,
from
about 0.05 mg/kg to about 0.5 mg/kg, from about 0.01 mg/kg to about 50 mg/kg,
from
about 0.1 mg/kg to about 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg,
from about
0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or from
about 1
mg/kg to about 25 mg/kg, of subject body weight per day, one or more times a
day, to
obtain the desired therapeutic, diagnostic, prophylactic, or imaging effect.
The desired
dosage may be delivered three times a day, two times a day, once a day, every
other day,
every third day, every week, every two weeks, every three weeks, or every four
weeks.
In certain embodiments, the desired dosage may be delivered using multiple
administrations (e.g., two, three, four, five, six, seven, eight, nine, ten,
eleven, twelve,
thirteen, fourteen, or more administrations). When multiple administrations
are
employed, split dosing regimens such as those described herein may be used.
[000775] According to the present invention, it has been discovered that
administration
of mmRNA in split-dose regimens produce higher levels of proteins in mammalian

subjects. As used herein, a "split dose" is the division of single unit dose
or total daily
dose into two or more doses, e.g, two or more administrations of the single
unit dose. As
used herein, a "single unit dose" is a dose of any therapeutic administed in
one dose/at
one time/single route/single point of contact, i.e., single administration
event. As used
herein, a "total daily dose" is an amount given or prescribed in 24 hr period.
It may be
administered as a single unit dose. In one embodiment, the mmRNA of the
present
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invention are administed to a subject in split doses. The mmRNA may be
formulated in
buffer only or in a formulation described herein.
Dosage Forms
[000776] A pharmaceutical composition described herein can be formulated into
a
dosage form described herein, such as a topical, intranasal, intratracheal, or
injectable
(e.g., intravenous, intraocular, intravitreal, intramuscular, intracardiac,
intraperitoneal,
subcutaneous).
Liquid dosage forms
[000777] Liquid dosage forms for parenteral administration include, but are
not limited
to, pharmaceutically acceptable emulsions, microemulsions, solutions,
suspensions,
syrups, and/or elixirs. In addition to active ingredients, liquid dosage forms
may comprise
inert diluents commonly used in the art including, but not limited to, water
or other
solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl
alcohol,
ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene
glycol, 1,3-
butylene glycol, dimethylformamide, oils (in particular, cottonseed,
groundnut, corn,
germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol,
polyethylene
glycols and fatty acid esters of sorbitan, and mixtures thereof In certain
embodiments for
parenteral administration, compositions may be mixed with solubilizing agents
such as
CREMOPHOR , alcohols, oils, modified oils, glycols, polysorbates,
cyclodextrins,
polymers, and/or combinations thereof.
Injectable
[000778] Injectable preparations, for example, sterile injectable aqueous or
oleaginous
suspensions may be formulated according to the known art and may include
suitable
dispersing agents, wetting agents, and/or suspending agents. Sterile
injectable
preparations may be sterile injectable solutions, suspensions, and/or
emulsions in
nontoxic parenterally acceptable diluents and/or solvents, for example, a
solution in 1,3-
butanediol. Among the acceptable vehicles and solvents that may be employed
include,
but are not limited to, water, Ringer's solution, U.S.P., and isotonic sodium
chloride
solution. Sterile, fixed oils are conventionally employed as a solvent or
suspending
medium. For this purpose any bland fixed oil can be employed including
synthetic mono-
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or diglycerides. Fatty acids such as oleic acid can be used in the preparation
of
injectables.
Injectable formulations can be sterilized, for example, by filtration through
a bacterial-
retaining filter, and/or by incorporating sterilizing agents in the form of
sterile solid
compositions which can be dissolved or dispersed in sterile water or other
sterile
injectable medium prior to use.
In order to prolong the effect of an active ingredient, it may be desirable to
slow the
absorption of the active ingredient from subcutaneous or intramuscular
injection. This
may be accomplished by the use of a liquid suspension of crystalline or
amorphous
material with poor water solubility. The rate of absorption of the
polynucleotide, primary
construct or mmRNA then depends upon its rate of dissolution which, in turn,
may
depend upon crystal size and crystalline form. Alternatively, delayed
absorption of a
parenterally administered polynucleotide, primary construct or mmRNA may be
accomplished by dissolving or suspending the polynucleotide, primary construct
or
mmRNA in an oil vehicle. Injectable depot forms are made by forming
microencapsule
matrices of the polynucleotide, primary construct or mmRNA in biodegradable
polymers
such as polylactide-polyglycolide. Depending upon the ratio of polynucleotide,
primary
construct or mmRNA to polymer and the nature of the particular polymer
employed, the
rate of polynucleotide, primary construct or mmRNA release can be controlled.
Examples
of other biodegradable polymers include, but are not limited to,
poly(orthoesters) and
poly(anhydrides). Depot injectable formulations may be prepared by entrapping
the
polynucleotide, primary construct or mmRNA in liposomes or microemulsions
which are
compatible with body tissues.
Pulmonary
[000779] Formulations described herein as being useful for pulmonary delivery
may
also be used for intranasal delivery of a pharmaceutical composition. Another
formulation suitable for intranasal administration may be a coarse powder
comprising the
active ingredient and having an average particle from about 0.2 ilm to 500 pm.
Such a
formulation may be administered in the manner in which snuff is taken, i.e. by
rapid
inhalation through the nasal passage from a container of the powder held close
to the
nose.
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[000780] Formulations suitable for nasal administration may, for example,
comprise
from about as little as 0.1% (w/w) and as much as 100% (w/w) of active
ingredient, and
may comprise one or more of the additional ingredients described herein. A
pharmaceutical composition may be prepared, packaged, and/or sold in a
formulation
suitable for buccal administration. Such formulations may, for example, be in
the form of
tablets and/or lozenges made using conventional methods, and may, for example,
contain
about 0.1% to 20% (w/w) active ingredient, where the balance may comprise an
orally
dissolvable and/or degradable composition and, optionally, one or more of the
additional
ingredients described herein. Alternately, formulations suitable for buccal
administration
may comprise a powder and/or an aerosolized and/or atomized solution and/or
suspension comprising active ingredient. Such powdered, aerosolized, and/or
aerosolized
formulations, when dispersed, may have an average particle and/or droplet size
in the
range from about 0.1 nm to about 200 nm, and may further comprise one or more
of any
additional ingredients described herein.
[000781] General considerations in the formulation and/or manufacture of
pharmaceutical agents may be found, for example, in Remington: The Science and

Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005
(incorporated herein
by reference in its entirety).
Coatings or Shells
[000782] Solid dosage forms of tablets, dragees, capsules, pills, and granules
can be
prepared with coatings and shells such as enteric coatings and other coatings
well known
in the pharmaceutical formulating art. They may optionally comprise opacifying
agents
and can be of a composition that they release the active ingredient(s) only,
or
preferentially, in a certain part of the intestinal tract, optionally, in a
delayed manner.
Examples of embedding compositions which can be used include polymeric
substances
and waxes. Solid compositions of a similar type may be employed as fillers in
soft and
hard-filled gelatin capsules using such excipients as lactose or milk sugar as
well as high
molecular weight polyethylene glycols and the like.
Properties of Pharmaceutical Compositions
[000783] The pharmaceutical compositions described herein can be characterized
by
one or more of bioavailability, therapeutic window and/or volume of
distribution.
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Bioavailability
[000784] The polynucleotides, primary constructs or mmRNA, when formulated
into a
composition with a delivery agent as described herein, can exhibit an increase
in
bioavailability as compared to a composition lacking a delivery agent as
described herein.
As used herein, the term "bioavailability" refers to the systemic availability
of a given
amount of polynucleotides, primary constructs or mmRNA administered to a
mammal.
Bioavailability can be assessed by measuring the area under the curve (AUC) or
the
maximum serum or plasma concentration (C.) of the unchanged form of a compound

following administration of the compound to a mammal. AUC is a determination
of the
area under the curve plotting the serum or plasma concentration of a compound
along the
ordinate (Y-axis) against time along the abscissa (X-axis). Generally, the AUC
for a
particular compound can be calculated using methods known to those of ordinary
skill in
the art and as described in G. S. Banker, Modern Pharmaceutics, Drugs and the
Pharmaceutical Sciences, v. 72, Marcel Dekker, New York, Inc., 1996, herein
incorporated by reference in its entirety.
[000785] The C. value is the maximum concentration of the compound achieved in

the serum or plasma of a mammal following administration of the compound to
the
mammal. The C. value of a particular compound can be measured using methods
known to those of ordinary skill in the art. The phrases "increasing
bioavailability" or
"improving the pharmacokinetics," as used herein mean that the systemic
availability of a
first polynucleotide, primary construct or mmRNA, measured as AUC, Cmax, Or
Cmin in a
mammal is greater, when co-administered with a delivery agent as described
herein, than
when such co-administration does not take place. In some embodiments, the
bioavailability of the polynucleotide, primary construct or mmRNA can increase
by at
least about 2%, at least about 5%, at least about 10%, at least about 15%, at
least about
20%, at least about 25%, at least about 30%, at least about 35%, at least
about 40%, at
least about 45%, at least about 50%, at least about 55%, at least about 60%,
at least about
65%, at least about 70%, at least about 75%, at least about 80%, at least
about 85%, at
least about 90%, at least about 95%, or about 100%.
Therapeutic Window
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[000786] The polynucleotides, primary constructs or mmRNA, when formulated
into a
composition with a delivery agent as described herein, can exhibit an increase
in the
therapeutic window of the administered polynucleotide, primary construct or
mmRNA
composition as compared to the therapeutic window of the administered
polynucleotide,
primary construct or mmRNA composition lacking a delivery agent as described
herein.
As used herein "therapeutic window" refers to the range of plasma
concentrations, or the
range of levels of therapeutically active substance at the site of action,
with a high
probability of eliciting a therapeutic effect. In some embodiments, the
therapeutic
window of the polynucleotide, primary construct or mmRNA when co-administered
with
a delivery agent as described herein can increase by at least about 2%, at
least about 5%,
at least about 10%, at least about 15%, at least about 20%, at least about
25%, at least
about 30%, at least about 35%, at least about 40%, at least about 45%, at
least about 50%,
at least about 55%, at least about 60%, at least about 65%, at least about
70%, at least
about 75%, at least about 80%, at least about 85%, at least about 90%, at
least about 95%,
or about 100%.
Volume of Distribution
[000787] The polynucleotides, primary constructs or mmRNA, when formulated
into a
composition with a delivery agent as described herein, can exhibit an improved
volume
of distribution (Vdist), e.g., reduced or targeted, relative to a composition
lacking a
delivery agent as described herein. The volume of distribution (Vdist) relates
the amount
of the drug in the body to the concentration of the drug in the blood or
plasma. As used
herein, the term "volume of distribution" refers to the fluid volume that
would be
required to contain the total amount of the drug in the body at the same
concentration as
in the blood or plasma: Vdist equals the amount of drug in the
body/concentration of drug
in blood or plasma. For example, for a 10 mg dose and a plasma concentration
of 10
mg/L, the volume of distribution would be 1 liter. The volume of distribution
reflects the
extent to which the drug is present in the extravascular tissue. A large
volume of
distribution reflects the tendency of a compound to bind to the tissue
components
compared with plasma protein binding. In a clinical setting, Vdist can be used
to
determine a loading dose to achieve a steady state concentration. In some
embodiments,
the volume of distribution of the polynucleotide, primary construct or mmRNA
when co-
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administered with a delivery agent as described herein can decrease at least
about 2%, at
least about 5%, at least about 10%, at least about 15%, at least about 20%, at
least about
25%, at least about 30%, at least about 35%, at least about 40%, at least
about 45%, at
least about 50%, at least about 55%, at least about 60%, at least about 65%,
at least about
70%.
Biological Effect
[000788] In one embodiment, the biological effect of the modified mRNA
delivered to
the animals may be categorized by analyzing the protein expression in the
animals. The
protein expression may be determined from analyzing a biological sample
collected from
a mammal administered the modified mRNA of the present invention. In one
embodiment, the expression protein encoded by the modified mRNA administered
to the
mammal of at least 50 pg/ml may be preferred. For example, a protein
expression of 50-
200 pg/ml for the protein encoded by the modified mRNA delivered to the mammal
may
be seen as a therapeutically effective amount of protein in the mammal.
Detection of Modified Nucleic Acids by Mass Spectrometry
[000789] Mass spectrometry (MS) is an analytical technique that can provide
structural
and molecular mass/concentration information on molecules after their
conversion to
ions. The molecules are first ionized to acquire positive or negative charges
and then
they travel through the mass analyzer to arrive at different areas of the
detector according
to their mass/charge (m/z) ratio.
[000790] Mass spectrometry is performed using a mass spectrometer which
includes an
ion source for ionizing the fractionated sample and creating charged molecules
for further
analysis. For example ionization of the sample may be performed by
electrospray
ionization (ESI), atmospheric pressure chemical ionization (APCI),
photoionization,
electron ionization, fast atom bombardment (FAB)/liquid secondary ionization
(LSIMS),
matrix assisted laser desorption/ionization (MALDI), field ionization, field
desorption,
thermospray/plasmaspray ionization, and particle beam ionization. The skilled
artisan
will understand that the choice of ionization method can be determined based
on the
analyte to be measured, type of sample, the type of detector, the choice of
positive versus
negative mode, etc.
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[000791] After the sample has been ionized, the positively charged or
negatively
charged ions thereby created may be analyzed to determine a mass-to-charge
ratio (i.e.,
m/z). Suitable analyzers for determining mass-to-charge ratios include
quadropole
analyzers, ion traps analyzers, and time-of-flight analyzers. The ions may be
detected
using several detection modes. For example, selected ions may be detected
(i.e., using a
selective ion monitoring mode (SIM)), or alternatively, ions may be detected
using a
scanning mode, e.g., multiple reaction monitoring (MRM) or selected reaction
monitoring (SRM).
[000792] Liquid chromatography-multiple reaction monitoring (LC-MS/MRM)
coupled
with stable isotope labeled dilution of peptide standards has been shown to be
an
effective method for protein verification (e.g., Keshishian et al., Mol Cell
Proteomics
2009 8: 2339-2349; Kuhn et al., Clin Chem 2009 55:1108-1117; Lopez et al.,
Clin Chem
2010 56:281-290; each of which are herein incorporated by reference in its
entirety).
Unlike untargeted mass spectrometry frequently used in biomarker discovery
studies,
targeted MS methods are peptide sequence¨based modes of MS that focus the full

analytical capacity of the instrument on tens to hundreds of selected peptides
in a
complex mixture. By restricting detection and fragmentation to only those
peptides
derived from proteins of interest, sensitivity and reproducibility are
improved
dramatically compared to discovery-mode MS methods. This method of mass
spectrometry-based multiple reaction monitoring (MRM) quantitation of proteins
can
dramatically impact the discovery and quantitation of biomarkers via rapid,
targeted,
multiplexed protein expression profiling of clinical samples.
[000793] In one embodiment, a biological sample which may contain at least one

protein encoded by at least one modified mRNA of the present invention may be
analyzed by the method of MRM-MS. The quantification of the biological sample
may
further include, but is not limited to, isotopically labeled peptides or
proteins as internal
standards.
[000794] According to the present invention, the biological sample, once
obtained from
the subject, may be subjected to enzyme digestion. As used herein, the term
"digest"
means to break apart into shorter peptides. As used herein, the phrase
"treating a sample
to digest proteins" means manipulating a sample in such a way as to break down
proteins
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in a sample. These enzymes include, but are not limited to, trypsin,
endoproteinase Glu-
C and chymotrypsin. In one embodiment, a biological sample which may contain
at least
one protein encoded by at least one modified mRNA of the present invention may
be
digested using enzymes.
[000795] In one embodiment, a biological sample which may contain protein
encoded
by modified mRNA of the present invention may be analyzed for protein using
electrospray ionization. Electrospray ionization (ESI) mass spectrometry
(ESIMS) uses
electrical energy to aid in the transfer of ions from the solution to the
gaseous phase
before they are analyzed by mass spectrometry. Samples may be analyzed using
methods
known in the art (e.g., Ho et al., Clin Biochem Rev. 2003 24(1):3-12; herein
incorporated
by reference in its entirety). The ionic species contained in solution may be
transferred
into the gas phase by dispersing a fine spray of charge droplets, evaporating
the solvent
and ejecting the ions from the charged droplets to generate a mist of highly
charged
droplets. The mist of highly charged droplets may be analyzed using at least
1, at least 2,
at least 3 or at least 4 mass analyzers such as, but not limited to, a
quadropole mass
analyzer. Further, the mass spectrometry method may include a purification
step. As a
non-limiting example, the first quadrapole may be set to select a single m/z
ratio so it
may filter out other molecular ions having a different m/z ratio which may
eliminate
complicated and time-consuming sample purification procedures prior to MS
analysis.
[000796] In one embodiment, a biological sample which may contain protein
encoded
by modified mRNA of the present invention may be analyzed for protein in a
tandem
ESIMS system (e.g., MS/MS). As non-limiting examples, the droplets may be
analyzed
using a product scan (or daughter scan) a precursor scan (parent scan) a
neutral loss or a
multiple reaction monitoring.
[000797] In one embodiment, a biological sample which may contain protein
encoded
by modified mRNA of the present invention may be analyzed using matrix-
assisted laser
desorption/ionization (MALDI) mass spectrometry (MALDIMS). MALDI provides for
the nondestructive vaporization and ionization of both large and small
molecules, such as
proteins. In MALDI analysis, the analyte is first co-crystallized with a large
molar excess
of a matrix compound, which may also include, but is not limited to, an
ultraviolet
absorbing weak organic acid. Non-limiting examples of matrices used in MALDI
are a-
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cyano-4-hydroxycinnamic acid, 3,5-dimethoxy-4-hydroxycinnamic acid and 2,5-
dihydroxybenzoic acid. Laser radiation of the analyte-matrix mixture may
result in the
vaporization of the matrix and the analyte. The laser induced desorption
provides high
ion yields of the intact analyte and allows for measurement of compounds with
high
accuracy. Samples may be analyzed using methods known in the art (e.g., Lewis,
Wei
and Siuzdak, Encyclopedia of Analytical Chemistry 2000:5880-5894; herein
incorporated
by reference in its entirety). As non-limiting examples, mass analyzers used
in the
MALDI analysis may include a linear time-of-flight (TOF), a TOF reflectron or
a Fourier
transform mass analyzer.
[000798] In one embodiment, the analyte-matrix mixture may be formed using the

dried-droplet method. A biologic sample is mixed with a matrix to create a
saturated
matrix solution where the matrix-to-sample ratio is approximately 5000:1. An
aliquot
(approximately 0.5-2.0 uL) of the saturated matrix solution is then allowed to
dry to form
the analyte-matrix mixture.
[000799] In one embodiment, the analyte-matrix mixture may be formed using the

thin-layer method. A matrix homogeneous film is first formed and then the
sample is
then applied and may be absorbed by the matrix to form the analyte-matrix
mixture.
[000800] In one embodiment, the analyte-matrix mixture may be formed using the

thick-layer method. A matrix homogeneous film is formed with a nitro-cellulose
matrix
additive. Once the uniform nitro-cellulose matrix layer is obtained the sample
is applied
and absorbed into the matrix to form the analyte-matrix mixture.
[000801] In one embodiment, the analyte-matrix mixture may be formed using the

sandwich method. A thin layer of matrix crystals is prepared as in the thin-
layer method
followed by the addition of droplets of aqueous trifluoroacetic acid, the
sample and
matrix. The sample is then absorbed into the matrix to form the analyte-matrix
mixture.
V. Uses of Polynucleotides, primary constructs and mmRNA of the Invention
[000802] The polynucleotides, primary constructs and mmRNA of the present
invention
are designed, in preferred embodiments, to provide for avoidance or evasion of

deleterious bio-responses such as the immune response and/or degradation
pathways,
overcoming the threshold of expression and/or improving protein production
capacity,
improved expression rates or translation efficiency, improved drug or protein
half life
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and/or protein concentrations, optimized protein localization, to improve one
or more of
the stability and/or clearance in tissues, receptor uptake and/or kinetics,
cellular access by
the compositions, engagement with translational machinery, secretion
efficiency (when
applicable), accessibility to circulation, and/or modulation of a cell's
status, function
and/or activity.
Therapeutics
Therapeutic Agents
[000803] The polynucleotides, primary constructs or mmRNA of the present
invention,
such as modified nucleic acids and modified RNAs, and the proteins translated
from them
described herein can be used as therapeutic or prophylactic agents. They are
provided for
use in medicine. For example, a polynucleotide, primary construct or mmRNA
described
herein can be administered to a subject, wherein the polynucleotide, primary
construct or
mmRNA is translated in vivo to produce a therapeutic or prophylactic
polypeptide in the
subject. Provided are compositions, methods, kits, and reagents for diagnosis,
treatment
or prevention of a disease or condition in humans and other mammals. The
active
therapeutic agents of the invention include polynucleotides, primary
constructs or
mmRNA, cells containing polynucleotides, primary constructs or mmRNA or
polypeptides translated from the polynucleotides, primary constructs or mmRNA.

[000804] In certain embodiments, provided herein are combination therapeutics
containing one or more polynucleotide, primary construct or mmRNA containing
translatable regions that encode for a protein or proteins that boost a
mammalian
subject's immunity along with a protein that induces antibody-dependent
cellular
toxicity. For example, provided herein are therapeutics containing one or more
nucleic
acids that encode trastuzumab and granulocyte-colony stimulating factor (G-
CSF). In
particular, such combination therapeutics are useful in Her2+ breast cancer
patients who
develop induced resistance to trastuzumab. (See, e.g., Albrecht,
Immunotherapy.
2(6):795-8 (2010)).
[000805] Provided herein are methods of inducing translation of a recombinant
polypeptide in a cell population using the polynucleotide, primary construct
or mmRNA
described herein. Such translation can be in vivo, ex vivo, in culture, or in
vitro. The cell
population is contacted with an effective amount of a composition containing a
nucleic
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acid that has at least one nucleoside modification, and a translatable region
encoding the
recombinant polypeptide. The population is contacted under conditions such
that the
nucleic acid is localized into one or more cells of the cell population and
the recombinant
polypeptide is translated in the cell from the nucleic acid.
[000806] An "effective amount" of the composition is provided based, at least
in part,
on the target tissue, target cell type, means of administration, physical
characteristics of
the nucleic acid (e.g., size, and extent of modified nucleosides), and other
determinants.
In general, an effective amount of the composition provides efficient protein
production
in the cell, preferably more efficient than a composition containing a
corresponding
unmodified nucleic acid. Increased efficiency may be demonstrated by increased
cell
transfection (i.e., the percentage of cells transfected with the nucleic
acid), increased
protein translation from the nucleic acid, decreased nucleic acid degradation
(as
demonstrated, e.g., by increased duration of protein translation from a
modified nucleic
acid), or reduced innate immune response of the host cell.
[000807] Aspects of the invention are directed to methods of inducing in vivo
translation of a recombinant polypeptide in a mammalian subject in need
thereof.
Therein, an effective amount of a composition containing a nucleic acid that
has at least
one structural or chemical modification and a translatable region encoding the

recombinant polypeptide is administered to the subject using the delivery
methods
described herein. The nucleic acid is provided in an amount and under other
conditions
such that the nucleic acid is localized into a cell of the subject and the
recombinant
polypeptide is translated in the cell from the nucleic acid. The cell in which
the nucleic
acid is localized, or the tissue in which the cell is present, may be targeted
with one or
more than one rounds of nucleic acid administration.
[000808] In certain embodiments, the administered polynucleotide, primary
construct or
mmRNA directs production of one or more recombinant polypeptides that provide
a
functional activity which is substantially absent in the cell, tissue or
organism in which
the recombinant polypeptide is translated. For example, the missing functional
activity
may be enzymatic, structural, or gene regulatory in nature. In related
embodiments, the
administered polynucleotide, primary construct or mmRNA directs production of
one or
more recombinant polypeptides that increases (e.g., synergistically) a
functional activity
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which is present but substantially deficient in the cell in which the
recombinant
polypeptide is translated.
[000809] In other embodiments, the administered polynucleotide, primary
construct or
mmRNA directs production of one or more recombinant polypeptides that replace
a
polypeptide (or multiple polypeptides) that is substantially absent in the
cell in which the
recombinant polypeptide is translated. Such absence may be due to genetic
mutation of
the encoding gene or regulatory pathway thereof In some embodiments, the
recombinant
polypeptide increases the level of an endogenous protein in the cell to a
desirable level;
such an increase may bring the level of the endogenous protein from a
subnormal level to
a normal level or from a normal level to a super-normal level.
[000810] Alternatively, the recombinant polypeptide functions to antagonize
the
activity of an endogenous protein present in, on the surface of, or secreted
from the cell.
Usually, the activity of the endogenous protein is deleterious to the subject;
for example,
due to mutation of the endogenous protein resulting in altered activity or
localization.
Additionally, the recombinant polypeptide antagonizes, directly or indirectly,
the activity
of a biological moiety present in, on the surface of, or secreted from the
cell. Examples
of antagonized biological moieties include lipids (e.g., cholesterol), a
lipoprotein (e.g.,
low density lipoprotein), a nucleic acid, a carbohydrate, a protein toxin such
as shiga and
tetanus toxins, or a small molecule toxin such as botulinum, cholera, and
diphtheria
toxins. Additionally, the antagonized biological molecule may be an endogenous
protein
that exhibits an undesirable activity, such as a cytotoxic or cytostatic
activity.
[000811] The recombinant proteins described herein may be engineered for
localization
within the cell, potentially within a specific compartment such as the
nucleus, or are
engineered for secretion from the cell or translocation to the plasma membrane
of the
cell.
[000812] In some embodiments, modified mRNAs and their encoded polypeptides in

accordance with the present invention may be used for treatment of any of a
variety of
diseases, disorders, and/or conditions, including but not limited to one or
more of the
following: autoimmune disorders (e.g. diabetes, lupus, multiple sclerosis,
psoriasis,
rheumatoid arthritis); inflammatory disorders (e.g. arthritis, pelvic
inflammatory disease);
infectious diseases (e.g. viral infections (e.g., HIV, HCV, RSV), bacterial
infections,
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fungal infections, sepsis); neurological disorders (e.g. Alzheimer's disease,
Huntington's
disease; autism; Duchenne muscular dystrophy); cardiovascular disorders (e.g.
atherosclerosis, hypercholesterolemia, thrombosis, clotting disorders,
angiogenic
disorders such as macular degeneration); proliferative disorders (e.g. cancer,
benign
neoplasms); respiratory disorders (e.g. chronic obstructive pulmonary
disease); digestive
disorders (e.g. inflammatory bowel disease, ulcers); musculoskeletal disorders
(e.g.
fibromyalgia, arthritis); endocrine, metabolic, and nutritional disorders
(e.g. diabetes,
osteoporosis); urological disorders (e.g. renal disease); psychological
disorders (e.g.
depression, schizophrenia); skin disorders (e.g. wounds, eczema); blood and
lymphatic
disorders (e.g. anemia, hemophilia); etc.
[000813] Diseases characterized by dysfunctional or aberrant protein activity
include
cystic fibrosis, sickle cell anemia, epidermolysis bullosa, amyotrophic
lateral sclerosis,
and glucose-6-phosphate dehydrogenase deficiency. The present invention
provides a
method for treating such conditions or diseases in a subject by introducing
nucleic acid or
cell-based therapeutics containing the polynucleotide, primary construct or
mmRNA
provided herein, wherein the polynucleotide, primary construct or mmRNA encode
for a
protein that antagonizes or otherwise overcomes the aberrant protein activity
present in
the cell of the subject. Specific examples of a dysfunctional protein are the
missense
mutation variants of the cystic fibrosis transmembrane conductance regulator
(CFTR)
gene, which produce a dysfunctional protein variant of CFTR protein, which
causes
cystic fibrosis.
[000814] Diseases characterized by missing (or substantially diminished such
that
proper (normal or physiological protein function does not occur) protein
activity include
cystic fibrosis, Niemann-Pick type C, 0 thalassemia major, Duchenne muscular
dystrophy, Hurler Syndrome, Hunter Syndrome, and Hemophilia A. Such proteins
may
not be present, or are essentially non-functional. The present invention
provides a
method for treating such conditions or diseases in a subject by introducing
nucleic acid or
cell-based therapeutics containing the polynucleotide, primary construct or
mmRNA
provided herein, wherein the polynucleotide, primary construct or mmRNA encode
for a
protein that replaces the protein activity missing from the target cells of
the subject.
Specific examples of a dysfunctional protein are the nonsense mutation
variants of the
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cystic fibrosis transmembrane conductance regulator (CFTR) gene, which produce
a
nonfunctional protein variant of CFTR protein, which causes cystic fibrosis.
[000815] Thus, provided are methods of treating cystic fibrosis in a mammalian
subject
by contacting a cell of the subject with a polynucleotide, primary construct
or mmRNA
having a translatable region that encodes a functional CFTR polypeptide, under

conditions such that an effective amount of the CTFR polypeptide is present in
the cell.
Preferred target cells are epithelial, endothelial and mesothelial cells, such
as the lung,
and methods of administration are determined in view of the target tissue;
i.e., for lung
delivery, the RNA molecules are formulated for administration by inhalation.
[000816] In another embodiment, the present invention provides a method for
treating
hyperlipidemia in a subject, by introducing into a cell population of the
subject with a
modified mRNA molecule encoding Sortilin, a protein recently characterized by
genomic
studies, thereby ameliorating the hyperlipidemia in a subject. The SORT] gene
encodes a
trans-Golgi network (TGN) transmembrane protein called Sortilin. Genetic
studies have
shown that one of five individuals has a single nucleotide polymorphism,
rs12740374, in
the 1p13 locus of the SORT1 gene that predisposes them to having low levels of
low-
density lipoprotein (LDL) and very-low-density lipoprotein (VLDL). Each copy
of the
minor allele, present in about 30% of people, alters LDL cholesterol by 8
mg/dL, while
two copies of the minor allele, present in about 5% of the population, lowers
LDL
cholesterol 16 mg/dL. Carriers of the minor allele have also been shown to
have a 40%
decreased risk of myocardial infarction. Functional in vivo studies in mice
describes that
overexpression of SORT] in mouse liver tissue led to significantly lower LDL-
cholesterol
levels, as much as 80% lower, and that silencing SORT1 increased LDL
cholesterol
approximately 200% (Musunuru K et al. From noncoding variant to phenotype via
SORT] at the 1p13 cholesterol locus. Nature 2010; 466: 714-721).
[000817] In another embodiment, the present invention provides a method for
treating
hematopoietic disorders, cardiovascular disease, oncology, diabetes, cystic
fibrosis,
neurological diseases, inborn errors of metabolism, skin and systemic
disorders, and
blindness. The identity of molecular targets to treat these specific diseases
has been
described (Templeton ed., Gene and Cell Therapy: Therapeutic Mechanisms and
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Strategies, 3rd Edition, Bota Raton, FL:CRC Press; herein incorporated by
reference in its
entirety).
[000818] Provided herein, are methods to prevent infection and/or sepsis in a
subject at
risk of developing infection and/or sepsis, the method comprising
administering to a
subject in need of such prevention a composition comprising a polynucleotide,
primary
construct or mmRNA precursor encoding an anti-microbial polypeptide (e.g., an
anti-
bacterial polypeptide), or a partially or fully processed form thereof in an
amount
sufficient to prevent infection and/or sepsis. In certain embodiments, the
subject at risk
of developing infection and/or sepsis may be a cancer patient. In certain
embodiments,
the cancer patient may have undergone a conditioning regimen. In some
embodiments,
the conditioning regiment may include, but is not limited to, chemotherapy,
radiation
therapy, or both. As a non-limiting example, a polynucleotide, primary
construct or
mmRNA can encode Protein C, its zymogen or prepro-protein, the activated form
of
Protein C (APC) or variants of Protein C which are known in the art. The
polynucleotides, primary constructs or mmRNA may be chemically modified and
delivered to cells. Non-limiting examples of polypeptides which may be encoded
within
the chemically modified mRNAs of the present invention include those taught in
US
Patents 7,226,999; 7,498,305; 6,630,138 each of which is incorporated herein
by
reference in its entirety. These patents teach Protein C like molecules,
variants and
derivatives, any of which may be encoded within the chemically modified
molecules of
the present invention.
[000819] Further provided herein, are methods to treat infection and/or sepsis
in a
subject, the method comprising administering to a subject in need of such
treatment a
composition comprising a polynucleotide, primary construct or mmRNA precursor
encoding an anti-microbial polypeptide (e.g., an anti-bacterial polypeptide),
e.g., an anti-
microbial polypeptide described herein, or a partially or fully processed form
thereof in
an amount sufficient to treat an infection and/or sepsis. In certain
embodiments, the
subject in need of treatment is a cancer patient. In certain embodiments, the
cancer
patient has undergone a conditioning regimen. In some embodiments, the
conditioning
regiment may include, but is not limited to, chemotherapy, radiation therapy,
or both.
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[000820] In certain embodiments, the subject may exhibits acute or chronic
microbial
infections (e.g., bacterial infections). In certain embodiments, the subject
may have
received or may be receiving a therapy. In certain embodiments, the therapy
may
include, but is not limited to, radiotherapy, chemotherapy, steroids,
ultraviolet radiation,
or a combination thereof In certain embodiments, the patient may suffer from a

microvascular disorder. In some embodiments, the microvascular disorder may be

diabetes. In certain embodiments, the patient may have a wound. In some
embodiments,
the wound may be an ulcer. In a specific embodiment, the wound may be a
diabetic foot
ulcer. In certain embodiments, the subject may have one or more burn wounds.
In
certain embodiments, the administration may be local or systemic. In certain
embodiments, the administration may be subcutaneous. In certain embodiments,
the
administration may be intravenous. In certain embodiments, the administration
may be
oral. In certain embodiments, the administration may be topical. In certain
embodiments, the administration may be by inhalation. In certain embodiments,
the
administration may be rectal. In certain embodiments, the administration may
be vaginal.
[000821] Other aspects of the present disclosure relate to transplantation of
cells
containing polynucleotide, primary construct, or mmRNA to a mammalian subject.

Administration of cells to mammalian subjects is known to those of ordinary
skill in the
art, and include, but is not limited to, local implantation (e.g., topical or
subcutaneous
administration), organ delivery or systemic injection (e.g., intravenous
injection or
inhalation), and the formulation of cells in pharmaceutically acceptable
carrier. Such
compositions containing polynucleotide, primary construct, or mmRNA can be
formulated for administration intramuscularly, transarterially,
intraperitoneally,
intravenously, intranasally, subcutaneously, endoscopically, transdermally, or

intrathecally. In some embodiments, the composition may be formulated for
extended
release.
[000822] The subject to whom the therapeutic agent may be administered suffers
from
or may be at risk of developing a disease, disorder, or deleterious condition.
Provided are
methods of identifying, diagnosing, and classifying subjects on these bases,
which may
include clinical diagnosis, biomarker levels, genome-wide association studies
(GWAS),
and other methods known in the art.
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Rare Liver Diseases or Disorders
Progressive Familial Intrahepatic Cholestasis (PFIC)
[000823] In one embodiment, the rare liver disease or disorder polynucleotide,
primary
construct, or mmRNA of the present invention may be used to treat progressive
familial
intrahepatic cholestasis (PFIC). The term "progressive familial intrahepatic
cholestasis"
or "PFIC" as used herein refers to a liver disorder that can lead to liver
failure. PFIC is
characterized by autosomal recessive defects in bile formation and
hepatocellular
cholestasis. As used herein, "hepatocellular" is a term used to describe
something that
affects or pertains to liver cells (also referred to as hepatocytes). As used
herein, the term
"cholestasis" refers to a condition characterized by slowed or disrupted flow
of bile from
the liver. As used herein, the term "bile" refers to a liquid substance
produced by the liver
comprising water, bile salts, mucus, fats inorganic salts and cholesterol,
which aids in the
emulsification and digestion of dietary fats.
[000824] There are three known types of PFIC (PFIC-1, PFIC-2 and PFIC-3), and
they
have been traced to mutations in genes encoding proteins involved in the
hepatocellular
transport system and in the formation of bile.
[000825] PFIC-1 and PFIC-2 are typically diagnosed in infancy but diagnosis
may
occur during the prenatal period or the neonatal period. As used herein, the
term
"prenatal" refers to the period of time in the life of an organism that occurs
before birth.
As used herein, the term "neonatal" refers to a period of time in the life of
an organism
that occurs after birth. In humans, the neonatal period may include a period
of time from
birth to about 1 month, to about 3 months or to about 6 months after birth. As
used
herein, the term "infancy" refers to a period in the life of an organism that
occurs
between birth and childhood. In humans, infancy may include the period of time
from
birth to about 1 year of life, to about 2 years of life, to about 3 years of
life or to about 4
years of life.
[000826] PFIC-3 may be diagnosed during the prenatal period, during the
neonatal
period or during infancy. In some cases, PFIC-3 may be able evade diagnosis
until
childhood or young adulthood. As used herein, the term "childhood" refers to
the period
of time in the life of an organism that occurs after infancy and before young
adulthood. In
humans, childhood may include the period of time from about 2 years of age to
about 10
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