Note: Descriptions are shown in the official language in which they were submitted.
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
ANTISENSE COMPOUNDS AND METHODS FOR TARGETING CUG REPEATS
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Application
Serial Nos: 63/213,900,
filed on June 23, 2021; 63/239,847, filed on September 1, 2021; 63/290,892,
filed on December
17, 2021; 63/305,071, filed on January 31, 2022; 63/314,369, filed on February
26, 2022;
63/316,634, filed on March 4, 2022; 63/317,856, filed on March 8, 2022;
63/326,201, filed on
March 31, 2022; 63/327,179, filed on April 4, 2022; 63/339,250, filed on May
6, 2022; 63/362,295,
filed on March 31, 2022; 63/239,671, filed on September 1, 2021; 63/290,960,
filed on December
17, 2021; 63/298,565, filed on January 11, 2022; and 63/268,577, filed on
February 25, 2022.
FIELD
[0002] The present disclosure relates to compounds, compositions, and methods
for modulating
the activity and/or levels of genes that include expanded nucleotide repeats,
in particular expanded
CTG=CUG repeats. The compounds and compositions containing the same may be
used to treat
diseases associated with genes that include an expanded nucleotide repeat, in
particular expanded
CTG=CUG repeats.
INTRODUCTION
[0003] Several diseases are associated with genes have expanded nucleotide
repeats, that is, a
greater number of nucleotide repeats than is observed in a healthy phenotype.
The expanded repeat
may cause aggregation and/or nucleation of the expanded repeat containing
transcript and/or cause
nucleation of proteins that bind to expanded repeat containing transcript. The
expanded repeats
may result in some proteins, such as pre-mRNA processing proteins, being
sequestered on the
repeat, thus inhibiting the proteins from performing their normal functions,
such as processing pre-
mRNA transcripts of other genes that do not contain the expanded repeat.
[0004] There are several diseases associated with genes having expanded
CTG=CUG trinucleotide
repeats (CTG refers to the DNA repeat and CUG refers to the corresponding RNA
repeat that
occurs upon transcription). Diseases associated with genes having expanded
CTG=CUG
trinucleotide repeats include, but are not limited to, myotonic dystrophy type
1 (DM1),
1
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
Spinocerebellar Ataxia-8 (SCA8), Huntington's disease like-2 (HDL2), and
Fuchs' endothelial
corneal dystrophy (FECD).
[0005] Myotonic dystrophy type 1 (DM1), the most common cause of muscular
dystrophy in
adults, affecting 1 in 8500 individuals worldwide, is associated with a gene
that has an expanded
trinucleotide repeat (Lee and Cooper. (2009) "Pathogenic mechanisms of
myotonic dystrophy,"
Biochem Soc Trans. 37(06): 10.1042/B5T0371281). DM1 is a disorder that affects
skeletal and
smooth muscle, as well as the eye, heart, endocrine system, and central
nervous system. DM1 is
caused by abnormal expansion of a CTG-trinucleotide repeat in the non-coding
region of the gene
encoding Dystrophia Myotonica Protein Kinase (DMPK). The CTG expansion lies
within a region
corresponding to the 3' untranslated region (3'-UTR) of the DMPK mRNA. Whereas
the DMPK
gene in healthy individuals contains between 5 and 40 CTG trinucleotide
repeats, patients with
DM1 have from 50 and up to several thousand CTG trinucleotide repeats. CTG-
trinucleotide
repeat expansion results in global deregulation of gene expression in affected
individuals due to
nucleation of some regulatory RNA-binding proteins in the CUG-expansion in the
3' untranslated
region (3'-UTR), rendering the RNA-binding proteins, such as muscleblind-like
protein (MBNL1-
3) unable to perform their normal cellular function. The nucleated RNA-binding
proteins are not
available to bind and affect translation of other mRNA transcripts. These CUG-
expanded mRNA¨
protein aggregates form distinct nuclear foci. The activity of additional
splicing factors, such as
CUGBP Elav-like family member 1 (CELF1), is also disrupted, leading to the mis-
splicing of a
large number of downstream gene transcripts associated with symptoms of DM1.
Disease severity
increases and age of onset decreases with an increasing number of repeats
(Pettersson etal. (2015)
"Molecular mechanisms in DM1 ¨ a focus on foci." Nucleic Acids Res. 43(4):2433-
2441).
[0006] The CUG-trinucleotide repeats in the 3' untranslated region of DMPK
mRNA form
imperfect stable hairpin structures that accumulate in the cell nucleus in
small ribonuclear
complexes or microscopically visible inclusions, and impair the function of
proteins implicated in
transcription, splicing or RNA export. Although DMPK genes with CUG repeats
are transcribed
into mRNA, the mutant transcripts are sequestered in the nucleus as aggregates
(foci), which
results in a decrease in cytoplasmic DMPK mRNA levels. These aggregations lead
to the
deregulation of the alternative splicing of many different transcripts due to
sequestration of two
RNA-binding proteins: MBNL1 (muscleblind-like 1) and CUGBP1 (CUG-binding
protein 1),
2
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
resulting in loss-of-function of MBNL1 and upregulation of CUGBP1 (Lee and
Cooper. (2009)
"Pathogenic mechanisms of myotonic dystrophy," Biochem Soc Trans. 37(06):
10.1042/B5T0371281). MBNL1 and CUGBP-ETR-3 like factor 1 (CELF1) are
developmental
regulators of splicing events during fetal to adult transition and
modification of their activities in
DM1 leads to expression of a fetal splicing pattern in adult tissues. The
downstream impact of
decreased MBNL1 and increased CELF1 levels includes disruption of alternative
splicing, mRNA
translation and mRNA decay in proteins such as cardiac troponin T (cTNT),
insulin receptor
(INSR), muscle-specific chloride ion channel (CLCN1) and
sarcoplasmic/endoplasmic reticulum
calcium ATPase 1 (ATP2A1) transcripts, in addition to 1V1BNL1 (Konieczny et
al. (2017)
"Myotonic dystrophy: candidate small molecule therapeutics." Drug Discov
Today. 22(11):1740-
174).
[0007] Possible therapeutic approaches to treat DM1, or other diseases
associated with expanded
CTG=CUG repeats, include the use of therapeutic oligonucleotide containing
compounds.
However, a major problem associated with the use of oligonucleotide compounds
in therapeutics
is their limited ability to gain access to the intracellular compartment when
administered
systemically. Intracellular delivery of oligonucleotide compounds can be
facilitated by use of
carrier systems such as polymers, cationic liposomes or by chemical
modification of the construct,
for example by the covalent attachment of cholesterol molecules. However,
intracellular delivery
efficiency of oligonucleotide compounds remains low. Improved delivery systems
are still
required to increase the potency of these compounds.
[0008] There is an unmet need for effective compositions to deliver
therapeutic oligonucleotide
compounds to intracellular compartments to treat diseases that are caused by
expanded CTG=CUG
repeats, such as DM1.
SUMMARY
[0009] Compounds, compositions, and methods for treating a disease associated
with an expanded
CTG=CUG repeat are described herein. In embodiments, this disclosure relates
to compounds that
include an antisense compound (AC) and a cyclic peptide, such as a cyclic cell
penetrating peptide
(cCPP). In embodiments, the AC binds to a gene or gene transcript comprising
an expanded CUG
repeat. In embodiments, the cyclic peptide facilitates intracellular
localization of the AC. The
3
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
compounds may comprise an endosomal escape vehicle (EEV). The EEV may comprise
the cyclic
peptide and an exocyclic peptide.
[0010] In embodiments, provided herein is a compound comprising: (a) at least
one cyclic peptide
and (b) an antisense compound (AC) that is complementary to a target
nucleotide. In
embodiments, the target nucleotide comprises at least one expanded CUG or CTG
repeat. In
embodiments, the target nucleotide is a gene that comprises at least one
expanded CTG repeat. In
embodiments, the target nucleotide is RNA that comprises at least one expanded
CUG repeat. In
embodiments, the RNA that comprises at least one expanded CUG repeat is a pre-
mRNA
sequence. In embodiments, the expanded CUG repeat corresponds to an expanded
CTG repeat in
a gene from which the pre-mRNA is transcribed. In embodiments, the antisense
compound binds
to the expanded CTG repeat or the expanded CUG repeat. In embodiments, the AC
comprises 5-
40 CAG repeats (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26,
27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 repeats). In
embodiments, the AC comprises
a sequence of a nucleotide listed in Table 2, Table 10, or Table 11. In
embodiments, the AC
comprises a sequence of a nucleotide listed in Table 2.
[0011] In embodiments, the AC comprises at least one modified nucleotide or
nucleic acid selected
from a phosphorothioate (PS) nucleotide, a phosphorodiamidate morpholino (PMO)
nucleotide, a
locked nucleic acid (LNA), a peptide nucleic acid (PNA), a nucleotide
comprising a 2' -0-methyl
(2'-0Me) modified backbone, a 2'0-methoxy-ethyl (2'-M0E) nucleotide, a 2,4'
constrained ethyl
(cEt) nucleotide, a 2'-deoxy-2'-fluoro-beta-D-arabinonucleic acid (2'F-ANA),
and combinations
thereof. In embodiments, the AC comprises a PMO nucleotide.
[0012] In embodiments, compounds are provided that include a cyclic peptide
having 6 to 12
amino acids, wherein at least two amino acids of the cyclic peptide are
charged amino acids, at
least two amino acids of the cyclic peptide are aromatic hydrophobic amino
acids and at least
two amino acids of the cyclic peptide are uncharged, non-aromatic amino acids.
In embodiments,
the anti sense compound (AC) is complementary to at least a portion of an
expanded CUG repeat
in a target mRNA sequence. In embodiments, the AC is a phosphorodiamidate
morpholino
(PMO) nucleotide.
[0013] In embodiments, at least two charged amino acids of the cyclic peptide
are arginine. In
embodiments, at least two aromatic, hydrophobic amino acids of the cyclic
peptide are
4
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
phenylalanine, naphthylalanine (3-Naphth-2-yl-alanine), or a combination
thereof. In
embodiments, at least two uncharged, non-aromatic amino acids of the cyclic
peptide are
citrulline, glycine, or a combination thereof. In embodiments, the compound is
a cyclic peptide
having 6 to 12 amino acids wherein two amino acids of the cyclic peptide are
arginine, at least
two amino acids are aromatic, hydrophobic amino acids selected from
phenylalanine,
naphthylalanine, and combinations thereof, and at least two amino acids are
uncharged, non-
aromatic amino acids selected from citrulline, glycine, and combinations
thereof.
[0014] In embodiments, the compound comprises an endosomal escape vehicle
comprising a
cyclic peptide and an exocyclic peptide (EP). In embodiments, the EP is
conjugated to a linker
at an amino group. The linker may be a linker as described herein. In
embodiments, the EP is
conjugated to the cyclic peptide via the linker. In embodiments, the EP is
conjugated to the AC
via the linker. In embodiments, the EP is conjugated to the linker that
conjugates the AC to the
cyclic peptide.
[0015] In embodiments, the EP comprises from 2 to 10 amino acids. In
embodiments, the EP
comprises from 4 to 8 amino acid residues. In embodiments, the EP comprises 1
or 2 amino acids
comprising a side chain comprising a guanidine group, or a protonated form
thereof In
embodiments, the EP comprises 1, 2, 3, or 4 lysine residues. In embodiments,
the amino group on
the side chain of each lysine residue is substituted with a trifluoroacetyl (-
COCF3) group,
allyloxycarbonyl (Alloc), 1-(4,4-dimethy1-2,6-dioxocyclohexylidene)ethyl
(Dde), or (4,4-
dimethy1-2,6-dioxocyclohex-1-ylidene-3)-methylbutyl (ivDde) group. In
embodiments, EP
comprises at least 2 amino acid residues with a hydrophobic side chain. In
embodiments, the amino
acid residue with a hydrophobic side chain is selected from valine, proline,
alanine, leucine,
isoleucine, and methionine. In embodiments, the exocyclic peptide comprises
one of the following
sequences: PKKKRKV; KR; RR; KKK; KGK; KBK; KBR; KRK; KRR; RKK; RRR; KKKK;
KKRK; KRKK; KRRK; RKKR; RRRR; KGKK; KKGK; KKKKK; KKKRK; KBKBK;
KKKRKV; PGKKRKV; PKGKRKV; PKKGRKV; PKKKGKV; PKKKRGV; or PKKKRKG. In
embodiments, the exocyclic peptide consists of one of the following sequences:
PKKKRKV; KR;
RR; KKK; KGK; KBK; KBR; KRK; KRR; RKK; RRR; KKKK; KKRK; KRKK; KRRK; RKKR;
RRRR; KGKK; KKGK; KKKKK; KKKRK; KBKBK; KKKRKV; PGKKRKV; PKGKRKV;
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
PKKGRKV; PKKKGKV; PKKKRGV; or PKKKRKG. In embodiments, the exocyclic peptide
has the structure: Ac-P-K-K-K-R-K-V-.
[0016] In embodiments, the cyclic peptide comprises 4 to 12 amino acids. In
embodiments, the
cyclic peptide comprises 6 to 12 amino acids. In embodiments, at least two
amino acids of the
cyclic peptide are charged amino acids, at least two amino acids of the cyclic
peptide are
aromatic hydrophobic amino acids and at least two amino acids of the cyclic
peptide are
uncharged, non-aromatic amino acids. In embodiments, at least two charged
amino acids of the
cyclic peptide are arginine, at least two aromatic hydrophobic amino acids of
the cyclic peptide
are phenylalanine, napthylalanine, or combinations thereof, and at least two
uncharged, non-
aromatic amino acids are citrulline, glycine, or combinations thereof.
[0017] In embodiments, the cyclic peptide has 4 to 12 amino acids, wherein at
least two amino
acids are arginine and at least two amino acids comprise a hydrophobic side
chain, provided that
the cyclic peptide is not a cyclic peptide having a sequence of SEQ ID NO: 89-
117. In
embodiments, the cyclic peptide is not a cyclic peptide having a sequence of
SEQ ID NO: 89-
117.
CPP sequences and SEQ ID NOs
FORRRQ 89 RRFRORQ 99 FORRRRQK 109
FORRRC 90 FRRRROQ 100 FORRRRQC 110
FORRRU 91 rRFRORQ 101 fORrRrRQ 111
RRROFQ 92 RROFRRQ 102 FORRRRRQ 112
RRRIt(toF 93 CRRRRFWQ 103 RRRROFDS2C 113
FORRRR 94 FfORrRrQ 104 FORRR 114
FORrRq 95 FFORRRRQ 105 FWRRR 115
FORIRQ 96 RFRFRORQ 106 RRROF 116
FORRRRQ 97 URRRRFWQ 107 RRRWF 117
fORrRrQ 98 CRRRRFWQ 108
where F is L-phenylalanine, f is D-phenylalanine,(1) is L-3-(2-naphthyl)-
alanine,(1) is D-3-(2-
naphthyl)-alanine, R is L-arginine, r is D-arginine, Q is L-glutamine, q is D-
glutamine, C is L-
cysteine, U is L-selenocysteine, W is L-tryptophan, K is L-lysine, D is L-
aspartic acid, and S2 is
L-norleucine.
[0018] In embodiments, the cyclic peptide has the following structure:
6
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
Rg R2
0
AAsc
HN R3
NH
0)(
1:-)4
R7 NH
ONN
R6 0
0 R5 (A), or
a protonated form thereof,
wherein:
R1, R2, and R3 are each independently H or an aromatic or heteroaromatic side
chain of an
amino acid;
at least one of R1, R2, and R3 is an aromatic or heteroaromatic side chain of
an amino acid;
R4, R5, R6, R7 are independently H or an amino acid side chain;
at least one of R4, R5, R6, R7 is the side chain of 3-guanidino-2-
aminopropionic acid, 4-
guanidino-2-aminobutanoic acid, arginine, homoarginine, N-methylarginine, N,N-
dimethylarginine, 2,3-diaminopropionic acid, 2,4-diaminobutanoic acid, ly
sine, N-
methylly sine, N,N-dimethyllysine, N-ethyllysine,
N,N,N-trimethyllysine, 4-
guanidinophenylalanine, citrulline, N,N-dimethyllysine,
P-homoarginine, 3-(1-
piperidinyl)alanine;
AAsc is an amino acid side chain to which the antisense compound is
conjugated; and
q is 1, 2, 3 or 4.
[0019] In embodiments, at least one of R4, R5, R6, R7 are independently a
uncharged, non-
aromatic side chain of an amino acid. In embodiments, at least one of R4, R5,
R6, R7 are
independently H or a side chain of citrulline.
[0020] In embodiments, the cyclic peptide has the structure of Formula I:
7
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
R g R2
0
H¨Cr
AAsc
HN R3
NH
01/
HI\k)
/ VCNH
wfµi R4 q
NH 0 N --
H2N
R7 0
NH
qm
NH
NH
H2N (I)
or a protonated form thereof,
wherein:
R1, R2, and R3 are each independently H or an amino acid residue having a side
chain
comprising an aromatic group;
at least one of R1, R2, and R3 is an aromatic or heteroaromatic side chain of
an amino acid;
R4 and R7 are independently H or an amino acid side chain;
AAsc is an amino acid side chain to which the antisense compound is
conjugated;
q is 1, 2, 3 or 4; and
each m is independently an integer of 0, 1, 2, or 3.
[0021] In embodiments, the cyclic peptide of Formula (I) has one of the
following structures:
Ri 0
R2
AAsc
NH HN
0 NH
H2N¨I(Nm HNX XR3
m NH
NH FHN 0
R4
0
H2N_1NH
NH (I-a),
8
CA 03222824 2023-12-07
WO 2022/271818
PCT/US2022/034517
Ri 0
R2
AAsc41/415\--Ho
NH
oTNH
HN
H2N¨NN--NL X- R3
H kN)ni NH
K_ HN 0
O
NH
,s4
0
rni
H2N_\(NH
NH (I-b), or
protonated form thereof
[0022] In embodiments, the cyclic peptide of Formula (I) has one of the
following structures:
0 0
AAõy\---N
NH HN----\r0
0 NH
H2N-AN--)c+.T HN
H m NH
Ok_NH 0 fik
0)ni
H2N-1NH
NH
sc
NH 0
NH
A 0
I-12¨N
HN
¨ N--N
H k-)m- NH
Ok_ HN 0
NH
0
0 r m
H2N_INH
NH (I-2),
9
CA 03222824 2023-12-07
WO 2022/271818
PCT/US2022/034517
NH2
HN\
N HN
HN
0 N,oe
NH H
HN
NH 0
0 HN
NH
HN
0
z
0
(1-3),
AAsc
H2N 0
NH µ'.?-11 HN
HN 0
oy NH
HN
NH
C
NH Ed =
HN 0
C 0/
NH
H2N-A
NH
(I-4),
HN
NH2
H AAR,
J
0 I HN H14L=e"
= %,
H2N IN1H
0 -4
Mr*
H a 0
, .NH 0f
,
NHfI
(1-5),
CA 03222824 2023-12-07
WO 2022/271818
PCT/US2022/034517
NH2
="' IN -" ''''...
O. tolka H
HN . , -- ==,--'"" HN ,.õ.1 --i, %,µ
,
.,?--NH
1-12N .
NH HN
\ i
(
NH CC;c>TI¨N'irS
0)
%.µ 11---= NH I s 1
1.11---. =õ....,:-.:.:õ.
(I-6), or
a protonated form thereof
[0023] In embodiments, the cyclic peptide of Formula (I) has one of the
following structures:
11
CA 03222824 2023-12-07
WO 2022/271818
PCT/US2022/034517
0 0
AAõ,y--N
HN¨
NH \r0
11 0y NH
H2N¨\N
H I\47.T' NH MN
NH Ni-_(HN
0),71
NH
H2N-1
NH
0
AAscYLHNN-----co.
NH 0 NH
H2NAN--N HN
H 111.. NH
NH 0
0
0 rm
H2N_1NH
NH (I-2), or
a protonated form thereof
[0024] In embodiments, the cyclic peptide of Formula (I) has the following
structure:
0 0
AAscYLHN H
NH N¨Nr0
0, HN
H2N¨\N
H 1\417--NH
Ok_
HN
NH 0
NH
H2N-1
NH (I-1), or
a protonated form thereof
12
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
[0025] In embodiments, the compound has a structure of Formula C:
H 0 Cargo
4'
0 (81114
NH
'L 0 P
Nrf iks¨it R2
1/4\y" 11N-4
NH
0, .14H
H Hi;s\tot
lit,\?=\`'µo
cro\ t 1
N
Rol
\
lm
1144,õ(NH
NH
(C), or
a protonated form or salt thereof,
wherein:
R1, R2, and R3 are each independently H or a side chain comprising an aryl or
heteroaryl
group, wherein at least one of R1, R2, and R3 is a side chain comprising an
aryl or
heteroaryl group;
R4 and R7 are independently H or an amino acid side chain;
EP is the exocyclic peptide;
each m is independently an integer from 0-3;
n is an integer from 0-2;
x' is an integer from 1-23;
y is an integer from 1-5;
q is an integer from 1-4;
z' is an integer from 1-23, and
Cargo is the antisense compound._
13
CA 03222824 2023-12-07
WO 2022/271818
PCT/US2022/034517
[0026] In embodiments, the compound has one or the following structures:
H (ill oligonucleotide
EP 0
0 - H
(aH2)
4
NH
0
yLN
NH HN---\r.0
0 NH
HN
NH
HN'O
O
NH H
0
NH
NH (C-1),
H Nit oligonucleotide
0 - H
(oH2)
4
NH
z-4)
T N
0
NH
0 NH
HN
H2NAN__\,,T
NH
HN
H H N
0
0
NH
NH (C-2),
14
CA 03222824 2023-12-07
WO 2022/271818
PCT/US2022/034517
0 11 oligonucleotide
EP
. N
Thi
NNC)0t\LA
H -__. , H
0 (cH2)4
I
HN 0
H2N
N
HN
Oz NH 0
HN
,...4.:,1
NH
HN 0 4/
NH H
._.......7 N
0 0
NH
H2N¨
NH (C-3),
0 0
ET:, H
NC)(:)-(NAN O)Loligonuoleotide
H - H '11
0
N
0 H
0 Z.40
H2N / __ \
)---NH
HN 0
oy NH
c HN
X%
NH
H HN 0 4Ik
C 0/ AO
NH
H2N¨
I.
NH
(C-4)
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
or a protonated form or salt thereof,
wherein EP is the exocyclic peptide, and
oligonucleotide is the antisense compound.
[0027] In embodiments, the oligonucleotide of the compound of Formula (C-1),
(C-2), (C-3), or
(C-4) comprises the following sequence: 5'-CAG CAG CAG CAG CAG CAG CAG-3'.
[0028] In embodiments, the EP of the compound of Formula (C-1), (C-2), (C-3),
or (C-4)
comprises the following sequence: PKKKRKV.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1 is a schematic showing multiple strategies for targeting CUG
repeats in mRNA.
[0030] FIG. 2 shows modified nucleotides used in antisense oligonucleotides
described herein.
Structures 1-3 (1 = Phosphorothioate; 2 = (Sc5-RP)-a,f3-CAN; 3 = PMO) are
phosphate backbone
modifications; 4 (2-thio-dT) is a base modification; 5-8 (5 = 2'-0Me-RNA; 6 =
2'0-M0E-RNA;
7 = 2'F-RNA; 8 = 2'F-ANA) are 2' sugar modifications; 9-11 are constrained
nucleotides; 12-14
(9 = LNA; 10 = (S)-cET; 11 = tcDNA; 12 = FHNA; 13 = (S)5'-C-methyl; 14 = UNA)
are additional
sugar modification; and 15-18 (15= E-VP; 16 = Methyl phosphonate; 17 = 5'
phosphorothioate;
18 = (S)-5'-C-methyl with phosphate) are 5' phosphate stabilization
modifications; 19 is a
morpholino sugar. Reformatted from Khvorova, A., et al., Nat, Biotechnol. 2017
Mar; 35(3): 238---
248.
[0031] FIGS. 3A-3D illustrate conjugation chemistries for connecting an AC to
a cyclic cell
penetrating peptide. FIG. 3A shows the amide bond formation between peptides
with a carboxylic
acid group or with TFP activated ester and primary amine residues at the 5'
end of an AC. FIG.
3B shows the conjugation of secondary amine or primary amine modified AC at 3'
and peptide-
TFP ester through amide bond formation. FIG. 3C shows the conjugation of a
peptide-azide to the
5' cyclooctyne modified AC via copper-free azide-alkyne cycloaddition. FIG. 3D
demonstrates
another exemplary conjugation between a 3' modified cyclooctyne ACs or 3'
modified azide ACs
and CPP containing linker-azide or linker-alkyne/cyclooctyne moiety, via a
copper-free azide-
alkyne cycloaddition or cupper catalyzed azide-alkyne cycloaddition,
respectively (click reaction).
[0032] FIG. 4 shows the conjugation chemistry for connecting an AC and CPP
with an additional
linker modality containing a polyethylene glycol (PEG) moiety.
16
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
[0033] FIGS. 5A-5D provide structures of the adenine (5A), cytosine (5B),
guanine (5C), and
thymine (5D) morpholino subunit monomers that may be used to synthesize
phosphorodiamidate-
linked morpholino oligomers (PM0s).
[0034] FIG. 6A-6F show RT-PCR analysis of alternative RNA splicing events
(e.g., exon
inclusion or exclusion) of MBNL1 (exon 5; FIG. 6A, 6B, 6E) and CLASP1 (exon
19; FIG. 6B,
6D, 6F) 24 hours (6A-6B) and 48 hours (6C-6D) after HeLa-48 cells were treated
with 1 tM, 3
or 10 tM of various PM0s or PMO-EEV compounds using the Endo-Porter
transfection
agent (6A-6C) or without the Endo-Porter agent (6E-6F). The parental HeLa cell
line and the
HeLa-480 cell line treated with (6A-6D) or without (6E-6F) the Endo-Porter
agent were included
as controls.
[0035] FIG. 7A-7B show RT-PCR analysis of alternative RNA splicing events
(e.g., exon
inclusion or exclusion) of MBNL1 (exon 5; FIG. 7A) and CLASP1 (exon 19; FIG.
7B) 48 hours
after DM1 myoblasts were treated with 1 1..LM of various PM0 or PMO-EEV
compounds without
the Endo-Porter transfection reagent. Two controls, DM-04 without endo-porter
treated and DM-
05 without endo-porter were included as controls.
[0036] FIG. 8A-8D show RT-PCR analysis of the alternative RNA splicing events
(e.g., exon
inclusion or exclusion) of Atp2a1 (exon 22; FIG. 8A), Nfix (exon 7; FIG. 8B),
Clcn1 (exon 7a;
FIG. 8C) and Mbnll (exon 5; FIG. 8D) from gastrocnemius muscle tissues one
week after HSA-
LR (DM1-mouse model) mice were treated with a PM0, 20 mpk PMO-EEV 221-1106, or
40 mpk
PMO-EEV 221-1106. FVB/NJ (wild type inbred mouse) and HSA-LR (without
treatment) mice
were included as control groups.
[0037] FIG. 9A-9D show RT-PCR analysis of the alternative RNA splicing events
(e.g., exon
inclusion or exclusion) of Atp2a1 (exon 22; FIG. 9A), Nfix (exon 7; FIG. 9B),
Clcn1 (exon 7a;
FIG. 9C) and Mbnll (exon 5; FIG. 9D) from quadricep muscle tissues one week
after HSA-LR
(DM1-mouse model) mice were treated with a PM0, 20 mpk PMO-EEV 221-1106, or 40
mpk
PMO-EEV 221-1106. FVB/NJ (wild type inbred mouse) and HSA-LR (without
treatment) mice
were included as control groups.
[0038] FIG. 10A-10D show RT-PCR analysis of the alternative RNA splicing
events (e.g., exon
inclusion or exclusion) of Atp2a1 (exon 22; FIG. 10A), Nfix (exon 7; FIG.
10B), Clcn1 (exon 7a;
FIG. 10C) and Mbnl 1 (exon 5; FIG. 10D) from tibialis anterior muscle tissues
one week after
17
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
HSA-LR (DM1-mouse model) mice were treated with a PM0, 20 mpk PMO-EEV 221-
1106, or
40 mpk PMO-EEV 221-1106. FVB/NJ (wild type inbred mouse) and HSA-LR (without
treatment)
mice were included as control groups.
[0039] FIG. 11A-11F show RT-PCR analysis of alternative RNA splicing events
(e.g., exon
inclusion or exclusion) of MBNL1 (exon 5, FIG. 11A), SOS1 (exon 25, FIG. 11B),
IR (exon 11,
FIG. 11C), DMD (exon 78, FIG. 11D), BIN1 (exon 11, FIG. 11E) and LDB3 (exon
11, FIG.
11F) after DM1 patient derived muscle cells were treated with different
concentrations (101.tm,
3 p.m, 1pm, 0.3 [tm) of DMPK CUG-targeting EEV-PM0s (CUGexP 197-777 and CUG"P
221-
1106). Muscle cells from two groups, healthy people (negative control) and DM1
patients (positive
control), were tested for the alternative RNA splicing events as control. All
data was collected
from three individual experiments (n=3). T- test of treated versus untreated
DM1 myotubes was
conducted; * p <0.05; ** p <0.01; *** p< 0.001.
[0040] FIG. 12A-12F shows RT-PCR analysis of the alternative RNA splicing
events (e.g., exon
inclusion or exclusion) of MBL1 (exon 5, FIG. 12A), SOS1 (exon 25, FIG. 12B),
INSR (exon 11,
FIG. 12C), DMD (exon 78, FIG. 12D), BIN1 (exon 11, FIG. 12E) and LDB3 (exon
11, FIG.
12F) after patient derived DM1 myoblasts and myotubes were treated with
101.tm, 3 p.m, or li.tm
of D1VIPK CUG-targeting EEV-PMO 197-777. Healthy patient cells and DM1 cells
were used as
controls. All data was collected from three individual experiments (n=3). T-
test of treated versus
untreated DM1 myotubes was conducted; * p <0.05; ** p < 0.01; *** p< 0.001.
[0041] FIG. 13A-13B show the relative levels of mRNA after HSA-LR mice were
treated with
various concentrations of PMO-EEV 221-1120. FIG. 13A shows the relative mRNA
level for the
gastrocnemius, triceps, tibialis anterior, and diaphragm. FIG. 13B shows the
relative mRNA levels
in the diaphragm.
[0042] FIG. 14A-14C show the relative levels of mRNA in the quadricep (14A),
gastrocnemius
(14B), tricep (14C) and tibialis anterior (14D) tissues after HSA-LR mice were
treated with various
concentrations of PMO-EEV 221-1120.
[0043] FIG. 15A-15D show the mouse DM1 splicing index (mDSI) for various genes
in quadricep
(FIG. 15A), gastrocnemius (FIG. 15B), tricep (FIG. 15C), and tibialis anterior
(FIG. 15D) tissues
after HSA-LR mice were treated with various concentrations of PMO-EEV 221-
1120.
18
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
[0044] FIGS. 16A-16C show the prevalence of RNA foci in the tibialis anterior
muscle after HSA-
LR mice were either untreated or treated with EEV-PMO 221-1120 (EEV-PMO-DM1-3;
DM1-
3). FIG. 16A-16B show images of tibialis anterior muscle tissue stained for
RNA CUG foci (red)
and nuclei (blue). FIG. 16C is a plot quantifying the percent of nuclei that
have a CUG foci from
data associated with the images in FIG. 16A-16B.
[0045] FIG. 17A-17F are plots showing a dose-dependent response for drug
levels in the
quadricep (17A), tricep (17B), heart (17C), gastrocnemius (17D), tibialis
anterior (17E),
diaphragm (17F), brain (1711), liver (17I), and kidney (17J) tissues after HSA-
LR mice were
treated with various concentrations of EEV-PMO-DM1-3. FIG. 17K shows drug
exposure of
various tissues at a 60 mpk dosage level.
[0046] FIG. 18 shows a dose dependent myotonia reduction in HSA-LR mice 7 days
after
treatment with EEV-PMO-DM1-3 at 15, 30, 60 and 90 mpk.
[0047] FIG. 19A-19D are plots show the results of a principal component
analysis comparing
gene expression in un-diseased mice (WT), DM1 mice (HSA-LR), and HSA-LR mice
treated with
PMO-EEV 221-1120. FIG. 19A and 19C are plots showing three principal
components and FIG.
19B and 19D are plots showing two principal components.
[0048] FIG. 20A-20B show heatmaps of differentially expressed genes between un-
diseased mice
(WT), DM1 mice (HSA-LR), and HSA-LR mice treated with 60 mpk PMO-EEV 221-1120.
FIG.
20A is a clustered heatmap showing 513 differentially expressed genes. FIG.
20B is a clustered
heatmap showing 40 genes that are known to have CTG=CUG repeats.
[0049] FIG. 21 is a volcano plot showing the global transcriptional change
across the untreated
HSA-LR mice and mice treated with PMO-EEV 221-1120.
[0050] FIG. 22A-22E are plots show the result of a principal component
analysis for the Scube2
(22A), Grebl (22B), Ttc7 (22C), Tx1nb(CUG)9 (22D), and Ndrg3 (22E) genes from
undiseased
mice, HSA-LR mice, and HSA-LR mice treated with PMO-EEV 221-1120.
[0051] FIG. 23A-23D show RNA sequencing (RNAseq) data for Atp2a1 (23A; exon 22
is boxed),
Clcn1 (23B; exon 7a is boxed), Nfix (23C; exon 7 is boxed), and Mbnl (23D;
exon 5 is boxed)
for un-diseased mice (WT-saline), HSA-LR mice (HSA-LR saline), and HSA-LR mice
treated
with PMO-EEV 221-1120. Two reads are shown for each treatment group.
19
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
[0052] FIG. 24 shows the percent spliced index (PSI) of individual exons for
various genes of
interest for undiseased mice (WT-saline), HSA-LR mice (HSA-LR saline), and HSA-
LR mice
treated with PMO-EEV 221-1120.
[0053] FIG. 25A-25D shows the drug levels in HSA-LR mice treated with 80 mpk
(60 mpk oligo,
80 mpk whole drug) EEV-PMO-DM1-3 after 1 week to 4 weeks in the tibialis
anterior (25A),
gastrocnemius (25B), triceps (25C) and quadricep (25D) tissues.
[0054] FIG. 26A-26D show the drug levels in mice after HSA-LR mice were
treated with a single
80 mpk dose of EEV-PMO-DM1-3. FIG. 26A-26B show the drug levels in the liver
from 1 week
to 12 weeks post treatment. FIG. 26C-26D show the drug levels in the kidney
from 1 week to 12
weeks post treatment.
[0055] FIG. 27A-27C are plots showing the level of exon inclusion for MBNL1
(exon 5; 26A),
SOS1 (exon 25; 26B), and NFIX (exon 7; 26C) after DM1 patient derived muscle
cells were
treated with 30 i.tM EEV-PMO-DM1-3.
[0056] FIG. 28A-28C shows that EEV-PMO-DM1-3 reduces CUG nuclear foci (green)
in the
nucleus (blue) in DM1 patient-derived muscle cells. FIG. 28A-28B are images of
DM1 patient
derived muscle cells that are either untreated or treated with EEV-PMO-DM1-3
or untreated. FIG.
28C is the quantification of the number of CUG foci per nucleus for data
associated with the
images in FIG. 28A.
[0057] FIG. 29A-29B show the raw data (29A) and the normalized data (29B) of a
CELLTITER-
GLO luminescent viability assay where RPTEC cells were treated with various
concentrations of
PMO-DM1 or EEV-PMO-DM1-3. Melittin was used as a positive control.
[0058] FIGS. 30A-30C show images depicting RNA CUG repeat foci in DM1 patient-
derived
cells (30A) and DM1 patient-derived cells treated with an EEV-PMO 221-1113
(30B). Cells were
stained for nuclei (blue; Hoechst) and RNA CUG foci (green). FIG. 30C is a
plot of the CUG
RNA foci per nuclear area for data associated with the images of FIG. 30A-30B.
[0059] FIG. 31A-31B show the prevalence of RNA CUG7 foci in HeLa, untreated
HeLa480 cells,
and HeLa480 cells treated with EEV-PMO 221-1113. FIG. 31A shows images of
cells stained for
RNA CUG7 foci (green) and nuclei (blue). FIG. 31B is a plot quantifying the
CUG7 foci per
nuclear are for data associated with the images in FIG. 31A.
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
[0060] FIGS. 32A-32C are plots showing the percent inclusion of exon 5 in
MBNL1 (32A), exon
25 in SOS1 (32B), and exon 7 in NFIX (32C) after DM1 patient-derived cells
were treated with
30 i.tM of EEV-PMO 221-1113.
[0061] FIGS. 33A-33E show RT-PCR analysis of the alternative RNA splicing
events (e.g., exon
inclusion) of MBNL1 (exon 5; 33A), SO S1 (exon 25; 33B), CLASP1 (exon 19,
33C), NFIX (exon
7, 33D), and INSR (exon 11, 33E) after DM1 patient derived muscle cells were
treated with
various concentrations of PMO-EEV 221-1113. T- test was used to determine
significance; * p <
0.05; ** p < 0.01; *** p< 0.001.
[0062] FIGS. 34A-34D show RT-PCR analysis of the alternative RNA splicing
events (e.g., exon
inclusion) Atp2a1 (exon 22, 34A), Nfix (exon 7, 34B), Clcn1 (exon 7a, 34C),
and Mbnll (exon 5,
34D) in the gastrocnemius tissue of mice treated with various concentrations
of PM0 221 or EEV-
PMO 221-1106.
[0063] FIGS. 35A-35C show RT-PCR analysis of the alternative RNA splicing
events (e.g., exon
inclusion or exclusion) of Mbnll (exon 5, 35A), Nfix (exon 7, 35B), and Atp2a1
(exon 22, 35C)
in the tibialis anterior tissue of HSA-LR mice treated with either PMO-EEV
0221-1121 (21-mer)
or PMO-EEV 0325-1121 (24-mer).
[0064] FIGS. 36A-36C show RT-PCR analysis of the alternative RNA splicing
events (e.g., exon
inclusion) of Mbnll (exon 5, 36A), Nfix (exon 7, 36B), and Atp2a1 (exon 22,
36C) in the
gastrocnemius tissue of HSA-LR mice treated with either PMO-EEV 0221-1121 (21-
mer) or
PMO-EEV 0325-1121 (24-mer).
[0065] FIGS. 37 shows PM0-0221a, the major metabolite of PMO-EEV 220-1120
detected in
vivo.
[0066] FIGS. 38A-38B show the percent exon inclusion in the tibialis anterior
(38A) and the
gastrocnemius (38B) for MBNL1 (exon 5) after Hela480 cells were treated with
various
concentration of EEV-PMO 221-1120.
[0067] FIGS. 39A-39B show the percent exon inclusion in the tibialis anterior
(39A) and the
gastrocnemius (39B) for NFIX (exon 7) after Hela480 cells were treated with
various
concentration of EEV-PMO 221-1120.
21
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
[0068] FIGS. 40A-40B show the percent exon inclusion in the tibialis anterior
(40A) and the
gastrocnemius (40B) for Atp2a1 (exon 22) after Hela480 cells were treated with
various
concentration of EEV-PMO 221-1120.
[0069] FIG. 41 show images (41A) depicting RNA CUG repeat foci in Hela480
cells after
treatment with various concentrations of EEV-PMO 221-1120. FIG. 41B is a plot
of the RNA foci
per nuclear area for data associated with the images of FIG. 41A.
[0070] FIGS. 42A-42D show the relative r(CUG480) repeat mRNA levels, (42A),
the relative
DMPK mRNA levels (42B), percent exon 5 inclusion of MBNL1 (42C), and percent
exon 25
inclusion in SOS1 (42D) in HeLa480 cells after treatment with various
concentrations of EEV-
PMO 221-1120.
[0071] FIG. 43 is a bar chart showing examples of genes expressed in muscle
tissue that are known
to have CTG=CUG repeats.
[0072] FIGS. 44A-44D show phenotypic myotonia reduction in HSA-LR mouse model
treated
with 20 mpk PMO-EEV 221-1106. FIGS. 44A and 44C show plots of relaxation. FIG.
44B shows
an example raw force trace. FIG. 44D shows representative electromyography
traces.
DETAILED DESCRIPTION
Compounds
[0073] In embodiments, compounds are provided that modulate the level and/or
activity of a gene
transcript having an expanded CUG trinucleotide repeat. In embodiments, the
compounds of the
present disclosure include at least one cyclic cell penetrating peptide (cCPP)
and a therapeutic
moiety (TM). The cCPP faciliates entry of the TM into the cell. In
embodiments, the compound
includes an ensomal escape vehicle (EEV) that comprises the cCPP and an
exocyclic peptide (EP).
The cCPP or the EEV may permit the TM to enter the cytosol or a cellular
compartment to interact
with the target transcritpt.
Therapeutic Moieties
[0074] Generally, the TM is the effector moitey that elicites a response. In
embodimetns, the TM
elicites a response by modulating the expression, activity, and/or level of a
target transcript and/or
22
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
a target protein. In embodiments, the traget transcript includes an expanded
CUG trinucelotide
repeat. In embodiments, the TM modulates the levels or a target transcript
and/or target protein
within a cell. In embodimetns, the TM decreases the level of the target
transcript and/or target
protein within a cell.
[0075] In embodiments, the TM modulates the activity of the target transcript
by reducing the
affinity between the target transcript and one or more proteins that bind to
the target transcript. By
reducing the affinity between the target transcript and the one or more
proteins, the TM may
effectively modulate the activity of the one or more proteins that would
otherwise be associated
with the target transcript. For example, if the one or more proteins are not
bound to the target
transcript, they are available to carry out their functions on other
molecules. For example, if the
one or more proteins are involved in pre-mRNA processing, reducing the
affinity of the one or
more proteins for a transcript comprising an expanded CUG repeat may allow the
one or more
proteins to process pre-mRNA transcripts that do not comprise expanded CUG
repeats. As such,
the TM may modulate the activity, expression, and/or levels of the downstream
genes (genes that
do not contain the expanded CTG repeat) that are regulated by the one or more
proteins whose
interaction with the target transcript is disrupted by the TM.
[0076] In embodiments, the TM comprises an oligonucleotide, a peptide, an
antibody, and/or a
small molecule. The class and identity of the TM depends on the mechanism
being used to
modulate the level and/or activity of the target transcript that includes an
expanded CUG
trinucleotide repeat.
Antisense compound
[0077] In various embodiments, the compounds disclosed herein comprise a cell
penetrating
peptide (CPP) conjugated to an antisense compound (AC).
[0078] The term "antisense compound" refers to an oligonucleotide sequence
that is
complementary, or at least partially complementary, to a target nucleotide
sequence. An AC is an
oligonucleotide that includes natural DNA bases, modified DNA bases, natural
RNA bases,
modified RNA bases, natural RNA sugars, modified RNA sugars, natural DNA
sugars, modified
DNA sugars, natural internucleoside linkages, modified internucleoside
linkages, or any
combination thereof ACs include, but are not limited to, antisense
oligonucleotides RNAi,
microRNA, antagomirs, aptamers, ribozymes, immunostimulatory oligonucleotides,
decoy
23
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
oligonucleotides, supermir, miRNA mimics, miRNA inhibitors, Ul adapters, and
combinations
thereof.
[0079] In embodiments, the AC includes a nucleotide sequence that is at least
partially
complementary to a target transcript that has an expanded CUG trinucleotide
repeat. In
embodiments, the AC includes a nucleotide sequence that is at least partially
complementary to an
expanded CUG trinucleotide repeat in a target mRNA sequence. Several diseases
are associated
with expanded CUG trinucleotide repeats, for example, myotonic dystrophy type
1 (DM1), Fuchs'
Endothelial Corneal Dystrophy (FECD), Spinocerebellar Ataxia-8 (SCA8), and
Huntington's
Disease-Like (HDL2). Table 1 provides examples of nucleotide repeat disorders,
and
characteristics of genes with expanded nucleotide repeats associated with such
disorders. The
following document describes exemplary oligonucleotides for treating tandem
repeat diseases and
is incorporated by reference herein in its entirety: Zain et al.
Neurotherapeutics. 2019; 16(2): 248-
262; Zarouchlioti et al. Am J Hum Genet. 2018;102(4):528-539; Fautsch et al.
Prog Retin Eye Res.
2021; 81:100883.
Table /: Diseases associated with expanded CUG trinucleotide repeats
Normal Expanded
Disease Gene Repeat Location
Gene repeat repeat
(abbreviation) length length product sequence of Repeat
Dystrophia
DM1 D1VIPK 5-35 > 50 myotonicaCTG=CAG 3' UTR
protein
kinase
FECD TCF4 <30 >40 Transcription CTG=CAG Intron 3
factor 4
ATXN8OS A ta xi n 8 and
SCA8 and/or 15-50 > 50 ataxin 8 CTG=CAG 3' UTR
ATXN8 opposite
strand
HDL2 JPH3 6-27 >40 JunctophilinCTG=CAG 3' UTR
3
[0080] In embodiments, the AC includes a nucleotide sequence that is at least
partially
complementary to a nucleotide sequence that is within a target mRNA transcript
that includes an
expanded CTG=CUG trinucleotide repeat. In embodiments, the AC includes a
nucleotide sequence
24
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
that is at least partially complementary to an expanded CTG=CUG trinucleotide
repeat in a target
mRNA transcript.
[0081] In embodiments, the AC includes a nucleotide sequence that is at least
partially
complementary to a nucleotide sequence that is within a DMPK1 target
transcript that includes an
expanded CTG=CUG trinucleotide repeat. In embodiments, the AC includes a
nucleotide sequence
that is at least partially complementary to a nucleotide sequence that is
within a TCF4 target
transcript that includes an expanded CTG=CUG trinucleotide repeat. In
embodiments, the AC
includes a nucleotide sequence that is at least partially complementary to a
nucleotide sequence
that is within a ATXN80S/ATXN8 target transcript that includes an expanded
CTG=CUG
trinucleotide repeat. In embodiments, the AC includes a nucleotide sequence
that is at least
partially complementary to a nucleotide sequence that is within a JPH3 target
transcript that
includes an expanded CTG=CUG trinucleotide repeat.
[0082] In embodiments, the AC includes a nucleotide sequence that is at least
partially
complementary to a trinucleotide repeat in a 3'UTR of a target mRNA
transcript. In embodiments,
the AC includes a nucleotide sequence that is at least partially complementary
to an expanded
CTG=CUG trinucleotide repeat in a 3'UTR of a DMPK1 target transcript. In
embodiments, the AC
includes a nucleotide sequence that is at least partially complementary to an
expanded CTG=CUG
trinucleotide repeat in a 3 'UTR of a ATXN80S/ATXN8 target transcript. In
embodiments, the AC
includes a nucleotide sequence that is at least partially complementary to an
expanded CTG=CUG
trinucleotide repeat in a 3'UTR of a JPH3 target transcript.
[0083] In embodiments, the AC includes a nucleotide sequence that is at least
partially
complementary to trinucleotide repeats, such as CTG=CUG repeats. In
embodiments, the target
nucleotide sequence comprises at least one expanded trinucleotide repeat
(e.g., CTG=CUG
repeats). In embodiments the target nucleotide sequence comprises at least 40,
at least 45, at least
50, at least 60, at least 70, at least 80, at least 90, at least 100, at least
150, at least 200, at least 300,
at least 400, at least 500, at least 600, at least 700, at least 800, at least
900, at least 1000, or at
least 2000 CTG=CUG trinucleotide repeats. In embodiments, the expanded
trinucleotide repeat is
in the 3'UTR of the target nucleotide sequence.
[0084] In embodiments, the AC includes a nucleotide sequence that is at least
partially
complementary to, and may hybridize with, at least a portion of the contiguous
expanded
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
trinucleotide repeats present in the target transcript. In embodiments, the AC
includes a nucleotide
sequence that is at least partially complementary to, and may hybridize with,
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, at
least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at
least 19, at least 20, and up to
50,up to 100, up to 150, up to 200, up to 300, up to 400, up to 500, up to
600, up to 700, up to 800,
up to 900, up to 1000, or up to 2000 trinucleotide repeats in a target
transcript. In embodiments,
the AC includes a nucleotide sequence that is at least partially complementary
to, and may
hybridize with, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19
or 20 trinucleotide repeats
in a target transcript. In embodiments, the AC includes a nucleotide sequence
that is at least
partially complementary to, and may hybridize with, 5 to 10 trinucleotide
repeats in a target
transcript. In embodiments, the AC includes a nucleotide sequence that is at
least partially
complementary to, and may hybridize with, 5 to 9 trinucleotide repeats in a
target transcript. In
embodiments, the AC includes a nucleotide sequence that is at least partially
complementary to,
and may hybridize with, 5 to 8 trinucleotide repeats in a target transcript.
In embodiments, the AC
includes a nucleotide sequence that is at least partially complementary to,
and may hybridize with,
to 7 trinucleotide repeats in a target transcript. In embodiments, the AC
includes a nucleotide
sequence that is at least partially complementary to, and may hybridize with,
5 to 6 trinucleotide
repeats in a target transcript. In embodiments, the AC includes a nucleotide
sequence that is at
least partially complementary to, and may hybridize with, 5 trinucleotide
repeats in a target
transcript. In embodiments, the AC includes a nucleotide sequence that is at
least partially
complementary to, and may hybridize with, 6 trinucleotide repeats in a target
transcript. In
embodiments, the AC includes a nucleotide sequence that is at least partially
complementary to,
and may hybridize with, 7 trinucleotide repeats in a target transcript. In
embodiments, the AC
includes a nucleotide sequence that is at least partially complementary to,
and may hybridize with,
8 trinucleotide repeats in a target transcript. In embodiments, the AC
includes a nucleotide
sequence that is complementary at least partially to, and may hybridize with,
9 trinucleotide repeats
in a target transcript. In embodiments, the AC includes a nucleotide sequence
that
[0085] In embodiments, the AC may include a nucleotide sequence that is at
least partially
complementary to, and my hybridize with, at least a portion of the contiguous
expanded
trinucleotide repeats present at any location in a target transcript. In
embodiments, the AC includes
26
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
a nucleotide sequence that is at least partially complementary to, and may
hybridize with, at least
a portion of the contiguous expanded trinucleotide repeats present in the 3'
UTR of a target
transcript. In embodiments, the AC includes a nucleotide sequence that is at
least partially
complementary to, and may hybridize with, at least a portion of the contiguous
expanded
trinucleotide repeats present in the 3' UTR of DMPK1, SCA8, and/or HDL2 target
transcript. In
embodiments, the AC includes a nucleotide sequence that is at least partially
complementary to,
and may hybridize with, at least a portion of the contiguous expanded
trinucleotide repeats present
an intron of a target transcript. In embodiments, the AC includes a nucleotide
sequence that is at
least partially complementary to, and may hybridize with, at least a portion
of the contiguous
expanded trinucleotide repeats present in the intron 3 of a TCF4 target
transcript. In embodiments,
the AC includes a nucleotide sequence that is at least partially complementary
to, and may
hybridize with, at least a portion of the contiguous expanded trinucleotide
repeats present in the
CTG18.1 locus of the TCF4 transcript. In embodiments, the AC includes a
nucleotide sequence
that is at least partially complementary to, and may hybridize with, at least
a portion of the
contiguous expanded trinucleotide repeats present an exon of a target
transcript.
[0086] In embodiments, the AC is 5 or more, 10 or more, 15 or more, 20 or
more, 25 or more, 30
or more, 35 or more, 40 or more, or 45 or more nucleic acids in length. In
embodiments, the AC
is 50 or less, 45 or less, 40 or less, 35 or less, 30 or less, 25 or less, 20
or less, 15 or less, or 10 or
less nucleic acids in length. In embodiments, the AC is 5 to 50, 5 to 45, 5 to
40, 5 to 35, 5 to 30, 5
to 25, 5 to 20, 5 to 15, or 5 to 10 nucleic acids in length. In embodiments,
the AC is 10 to 50, 10
to 45, 10 to 40, 10 to 35, 10 to 30, 10 to 25, 10 to 20, or 10 to 15 nucleic
acids in length. In
embodiments, the AC is 15 to 50, 15 to 45, 15 to 40, 15 to 35, 15 to 30, 15 to
25, or 15 to 20
nucleic acids in length. In embodiments, the AC is 20 to 50, 20 to 45, 20 to
40, 20 to 35, 20 to 30,
or 20 to 25 nucleic acids in length. In embodiments, the AC is 25 to 50, 25 to
45, 25 to 40, 25 to
35, or 25 to 30 nucleic acids in length. In embodiments, the AC is 30 to 50,
30 to 45, 30 to 40, or
30 to 35 nucleic acids in length. In embodiments, the AC is 35 to 50, 35 to
45, or 35 to 40 nucleic
acids in length. In embodiments, the AC is 40 to 50 or 40 to 45 nucleic acids
in length. In
embodiments, the AC is 45 to 50 nucleic acids in length. In embodiments, the
AC is 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 31, 32, 33, 34, 35,
36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleic acids in
length.
27
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
[0087] In embodiments, the AC has 100% complementarity to a target nucleotide
sequence. In
embodiments, the AC does not have 100% complementarity to a target nucleotide
sequence. As
used herein, the term "percent complementarity" refers to the number of
nucleobases (e.g., natural
nucleobase or modified nucleobase) of an AC that have nucleobase
complementarity with a
corresponding nucleobase of an oligomeric compound or nucleic acid (e.g., a
target nucleotide
sequence) divided by the total length (number of nucleobases) of the AC. One
skilled in the art
recognizes that the inclusion of mismatches is possible without eliminating
the activity of the
anti sense compound.
[0088] In embodiments, the AC includes 20% or less, 15% or less, 10% or less,
5% or less, or zero
mismatches to the target nucleotide sequence. In some embodiments, the AC
includes 5% or more,
10% or more, or 15% or more mismatched. In embodiments, the AC includes zero
to 5%, zero to
10%, zero to 15%, or zero to 20% mismatches to the target nucleotide sequence.
In embodiments,
the AC includes 5% to 10%, 5% to 15%, or 5% to 20% mismatches to the target
nucleotide
sequence. In embodiments, the AC includes 10% to 15% or 10% to 20% mismatches
to the target
nucleotide sequence. In embodiments, the AC includes 10% to 20% mismatches to
the target
nucleotide sequence.
[0089] In embodiments, the AC has 80% or greater, 85% or greater, 90% or
greater, 95% or
greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater
complementarity to a
target nucleotide sequence. In embodiments, the AC has 100% or less, 99% or
less, 98% or less,
97% or less 96% or less 95% or less, 90% or less, 85% or less complementarity
to a target
nucleotide sequence. In embodiments, the AC has 80% to 100%, 80% to 99%, 80%
to 98%, 80%
to 97% 80% to 96%, 80% to 95%, 80% to 90% or 80% to 85% complementarity to a
target
nucleotide sequence. In embodiments, the AC has 85% to 100%, 85% to 99%, 85%
to 98%, 85%
to 97% 85% to 96%, 85% to 95%, or 85% to 90% complementarity to a target
nucleotide sequence.
In embodiments, the AC has 90% to 100%, 90% to 99%, 90% to 98%, 90% to 97%,
90% to 96%,
or 90% to 95% complementarity to a target nucleotide sequence. In embodiments,
the AC has 95%
to 100%, 95% to 99%, 95% to 98%, 95% to 97%, or 95% to 96% complementarity to
a target
nucleotide sequence. In embodiments, the AC has 96% to 100%, 96% to 99%, 96%
to 98%, or
96% to 97% complementarity to a target nucleotide sequence. In embodiments,
the AC has 97%
to 100%, 97% to 99%, or 97% to 98% complementarity to a target nucleotide
sequence. In
28
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
embodiments, the AC has 98% to 100% or 98% to 99% complementarity to a target
nucleotide
sequence. In embodiments, the AC has 99% to 100% complementarity to a target
nucleotide
sequence.
[0090] In embodiments, incorporation of nucleotide affinity modifications
allows for a greater
number of mismatches compared to an unmodified compound. Similarly, certain
oligonucleotide
sequences may be more tolerant to mismatches than other oligonucleotide
sequences. One of
ordinary skill in the art is capable of determining an appropriate number of
mismatches between
an AC and a target nucleotide sequence, such as by determining the thermal
melting temperature
(Tm). Tm or ATm can be calculated by techniques that are familiar to one of
ordinary skill in the
art. For example, techniques described in Freier et al. (Nucleic Acids
Research, 1997, 25, 22: 4429-
4443) allow one of ordinary skill in the art to evaluate nucleotide
modifications for their ability to
increase the melting temperature of an RNA:DNA duplex.
[0091] In embodiments, the AC includes a nucleotide sequence that in itself is
a trinucleotide
repeat, that is, a CAG trinucleotide repeat. The reverse complement of a 5'-
CAG-3' has 100%
complementarity and may hybridize with a 5'-CUG-3' trinucleotide repeat. In
embodiments, the
AC includes one to 50 CAG repeats. In embodiments, the CAG repeats are
contiguous. In
embodiments, the CAG repeats are not contiguous. In embodiments, the AC
includes a nucleotide
sequence that includes incomplete CAG repeats on either the 5' or 3' end. For
example, in
embodiments, the AC includes a sequence such as AG(CAG),, G(CAG),, (CAG)AG, or
(CAG)A
where n is an integer from 1 to 50. In embodiments, the AC includes a
nucleotide sequence that
includes one or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7
or more, 8 or more,
9 or more, 10 or more, 20 or more, 30 or more, or 50 or more CAG repeats. In
embodiments, the
AC includes a nucleotide sequence that includes 50 or less, 40 or less, 30 or
less, 20 or less, 10 or
less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or
less, or 2 or less CAG repeats.
In embodiments, the AC includes a nucleotide sequence that includes 2 to 50, 2
to 20, 2 to 10, 4
to 10, 5 to 10, 6 to 10, 6 to 9, 6 to 8, or 6 to 7 CAG repeats. In
embodiments, the AC includes any
one of the nucleotide sequences in Table 2 (SEQ ID NO: 151-291).
Table 2: CAG repeat AC nucleotide sequences
AC sequence (5' to 3')
SEQ ID NO:
CAG NA
29
CA 03222824 2023-12-07
WO 2022/271818
PCT/US2022/034517
CAG-CAG NA
CAG-CAG-CAG NA
CAG-CAG-CAG-CAG 151
CAG-CAG-CAG-CAG-CAG 152
CAG-CAG-CAG-CAG-CAG-CAG 153
CAG-CAG-CAG-CAG-CAG-CAG-CAG 154
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 155
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 156
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 157
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 158
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 159
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 160
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG 161
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG 162
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG 163
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG 164
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG 165
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG 166
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 167
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 168
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 169
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 170
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 171
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 172
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG 173
CA 03222824 2023-12-07
WO 2022/271818
PCT/US2022/034517
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG 174
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG 175
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG 176
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG 177
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG 178
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 179
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 180
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 181
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 182
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 183
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 184
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG 185
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG 186
31
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG 187
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG 188
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG 189
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG 190
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 191
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 192
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 193
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 194
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 195
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 196
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-
CAG-CAG 197
32
CA 03222824 2023-12-07
WO 2022/271818
PCT/US2022/034517
AGC NA
AGCAGC NA
AGCAGCAGC NA
AGCAGCAGCAGC 198
AGCAGCAGCAGCAGC 199
AGCAGCAGCAGCAGCAGC 200
AGCAGCAGCAGCAGCAGCAGC 201
AGCAGCAGCAGCAGCAGCAGCAGC 202
AGCAGCAGCAGCAGCAGCAGCAGCAGC 203
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 204
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 205
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 206
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 207
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 208
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 209
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGC 210
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGC 211
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGCAGC 212
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGCAGCAGC 213
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGCAGCAGCAGC 214
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGCAGCAGCAGCAGC 215
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGCAGCAGCAGCAGCAGC 216
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGCAGCAGCAGCAGCAGCAGC 217
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGCAGCAGCAGCAGCAGCAGCAGC 218
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 219
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 220
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 221
33
CA 03222824 2023-12-07
WO 2022/271818
PCT/US2022/034517
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 222
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 223
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 224
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGC 225
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGC 226
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGCAGC 227
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGCAGCAGC 228
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGCAGCAGCAGC 229
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGCAGCAGCAGCAGC 230
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGCAGCAGCAGCAGCAGC 231
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGCAGCAGCAGCAGCAGCAGC 232
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGCAGCAGCAGCAGCAGCAGCAGC 233
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 234
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 235
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 236
34
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 237
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 238
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 239
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGC 240
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGC 241
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGCAGC 242
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGCAGCAGC 243
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGCAGCAGCAGC 244
GCA NA
GCAGCA NA
GCAGCAGCA NA
GCAGCAGCAGCA 245
GCAGCAGCAGCAGCA 246
GCAGCAGCAGCAGCAGCA 247
GCAGCAGCAGCAGCAGCAGCA 248
GCAGCAGCAGCAGCAGCAGCAGCA 249
GCAGCAGCAGCAGCAGCAGCAGCAGCA 250
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 251
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 252
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 253
CA 03222824 2023-12-07
WO 2022/271818
PCT/US2022/034517
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 254
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 255
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 256
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCA 257
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCA 258
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCA 259
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCAGCA 260
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCAGCAGCA 261
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCAGCAGCAGCA 262
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCAGCAGCAGCAGCA 263
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCAGCAGCAGCAGCAGCA 264
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCAGCAGCAGCAGCAGCAGCA 265
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 266
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 267
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 268
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 269
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 270
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 271
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCA 272
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCA 273
36
CA 03222824 2023-12-07
WO 2022/271818
PCT/US2022/034517
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCA 274
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCAGCA 275
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCAGCAGCA 276
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCAGCAGCAGCA 277
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCAGCAGCAGCAGCA 278
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCAGCAGCAGCAGCAGCA 279
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCAGCAGCAGCAGCAGCAGCA 280
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 281
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 282
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 283
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 284
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 285
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 286
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCA 287
37
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCA 288
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCA 289
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCAGCA 290
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCAGCAGCA 291
[0092] In embodiments, an AC that has a nucleotide that includes CAG repeats
may include
additional nucleotide sequences on the 5' end, 3' end, or both, of the CAG
repeat. In embodiments,
the additional nucleotide sequences may have 80% to 100% or 95% to 100%
complementarity to
portions of the target transcript to which they hybridize. The additional
nucleotide sequences may
be added to a CAG repeat nucleotide sequence in order to increase the
selectivity of the AC for
hybridizing to a specific target transcript.
[0093] In embodiments, the AC includes a nucleotide sequence that includes 1
to 50 CAG repeats
and that is a gapmer. Gapmers are oligonucleotides that are a DNA/RNA hybrid
and that induce
RNase decay mechanism. For example, gapmers may have a central DNA or DNA
mimic segment
that is flanked by an RNA or RNA mimic segment on both the 5' and 3' ends of
the DNA or DNA
mimetic segment. In embodiments, the AC includes a gapmer that includes a
nucleotide sequence
that hybridizes with a target nucleic acid sequence of the target transcript
that is separate from the
expanded CUG repeat of the target transcript.
[0094] In embodiments, an AC of the disclosure is a gapmer oligonucleotide as
disclosed in U.S.
Patent No. 9,550,988, the disclosure of which is incorporated by reference
herein.
[0095] In embodiments, an AC of the disclosure comprises the sequence and/or
structure of any
one of the ACs targeting DMPK disclosed in U.S. Patent Publication No.
2017/0260524, the
disclosure of which is incorporated by reference herein.
38
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
[0096] In embodiments, an AC of the disclosure comprises the sequence and/or
structure of any
one of the ACs or oligonucleotides disclosed in U.S. Patent Publications
US20030235845A1,
US20060099616A1, US 2013/0072671 Al, US 2014/0275212 Al, US 2009/0312532 Al,
US20100125099A1, US 2010/0125099 Al, US 2009/0269755 Al, US 2011/0294753 Al,
US
2012/0022134 Al, US 2011/0263682 Al, US 2014/0128592 Al, US 2015/0073037 Al,
and
US20120059042A1, the contents of each of which are incorporated herein in
their entirety for all
purposes.
[0097] When using an AC to target and/or hybridize to an expanded CTG=CUG
repeat, care must
be taken to avoid off-target effects where the AC unintentionally binds to off-
target transcripts that
include CTG=CUG repeats (e.g., a transcript that includes CTG=CUG repats that
are not an
expanded CTG=CUG repeat). An in-silico analysis of the human genome reveals
that in total, 63
human genes have CTG=CUG repeats (Uhlen, et. al., Science 2015
347(6220):1260419)). The 63
genes can be ranked by the expression of mRNA plus the amount of protein
expressed in total
muscle (cardiac, skeletal, and smooth muscle). The expression level can be
quantified using RPM
(reads per million) mRNA expression is FPKM (Fragments per kilo base of
transcript per million
mapped fragments) and protein expression is pTPM (transcripts per million
protein coding genes),
using greater than 10 RPM as the cutoff for non-insignificant expression. FIG.
43 shows the results
of such an in-silico analysis. Thirty-six genes show an expression level of >
10 RPM. Of the 36
genes, only three genes (besides D1VIPK) had > 10 CTG=CUG repeats. Genes with
10 CTG=CUG
repeats represent the lowest risk for off-target binding and toxicity. The
number of CTG=CUG
repeats (11-24) in these 3 genes (TCF4, CASK, MAP3k4) is nonetheless
significantly lower than
that seen in classic and congenital DM1 patients. For example, late-onset DM1
patients have 100-
600 CTG=CUG repeats on DMPK, classical DM1 patients have 250-750 CTG=CUG
repeats on
DMPK, and congenital DM1 patients have 750-1,400 CTG=CUG repeats on DMPK. The
same
in-silico analysis may be performed for the liver and kidney. CASK is the only
significant gene
with > 10 CTG=CUG repeats in the kidney. No genes with > 10 CTG=CUG repeats
were significant
in the liver.
[0098] The ACs described herein may contain one or more asymmetric centers and
thus give rise
to enantiomers, diastereomers, and other stereoisomeric configurations that
may be defined, in
terms of absolute stereochemistry, as (R) or (S); a or 13; or as (D) or (L).
Included in the anti sense
39
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
compounds provided herein are all such possible isomers, as well as their
racemic and optically
pure forms.
[0099] The efficacy of the ACs may be assessed by evaluating the antisense
activity effected by
their administration. As used herein, the term "antisense activity" refers to
any detectable and/or
measurable activity attributable to the hybridization of an antisense compound
to its target
nucleotide sequence. Such detection and/or measuring may be direct or
indirect. In embodiments,
antisense activity is assessed by detecting and or measuring the amount of the
protein expressed
from the transcript of interest. In embodiments, antisense activity is
assessed by detecting and/or
measuring the amount of the transcript of interest. In embodiments, antisense
activity is assessed
by detecting and/or measuring the amount of alternatively spliced RNA and/or
the amount of
protein isoforms translated from the target transcript.
AC mechanisms of modulation
[0100] In embodiments, the AC may modulate the activity and/or level of the
target transcript
within the cell. FIG. 1 shows exemplary mechanisms of how an AC can modulate
level and/or
activity of a target transcript.
[0101] In embodiments, the AC may modulate the level of the target transcript
within a cell. For
example, in embodiments where the AC is a gamper, binding of the AC to the
target transcript
induces the degradation of the target transcript via RNase H pathways (FIG. 1,
arrows A and B).
In embodiments, the gapmer hybridizes to a portion of the target transcript
that is distinct from the
expanded CUG trinucleotide repeat and thereby induces degradation of the
target transcript via
RNase H pathways (FIG. 1, Arrow A). In embodiments, the gapmer hybridizes to
at least a portion
of the expanded CUG repeat withing the target transcript and thereby induces
degradation of the
target transcript via RNase H pathways (FIG. 1, Arrow B).
[0102] In embodiments, the AC may modulate the activity of the target
transcript. Modulation of
activity may include increasing or decreasing the ability of the target
transcript to bind with a
binding partner. In embodiments, the AC may modulate the activity of the
target transcript by
decreasing the ability of the target transcript to bind with one or more
proteins that may associate
with the transcript, particularly proteins that associate with at least a
portion of the expanded CUG
repeat of a target transcript (FIG. 1, arrow C). In embodiments, decreasing
the ability of the target
transcript to bind with one or more proteins includes decreasing the affinity
of the target transcript
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
for the one or more proteins. In embodiments, decreasing the ability of the
target transcript to bind
with one or more proteins includes partial or full steric blocking of the
target transcript from
binding the one or more proteins. For example, the AC may occupy at least a
portion of the binding
site that may be occupied by the one or more proteins if not sterically
blocked. In embodiments,
the binding site of the one or more proteins to the target transcript includes
at least a portion of the
expanded trinucleotide repeat of the target transcript. As such, in
embodiments, the AC may
occupy at least a portion of the expanded trinucleotide repeat (e.g., expanded
CUG repeat) that
may be occupied by the one or more proteins if not sterically blocked. Partial
steric blocking of
the target transcript may result in decreased affinity between the target
transcript and the one or
more proteins. For example, in embodiments, the AC binds to at least a portion
of the expanded
CUG repeat of the target transcript thereby sterically blocking and/or
decreasing the affinity of
target transcript for a protein that may bind to the expanded CUG repeat (FIG.
1, arrow C). The
following review article describes additional applications for steric blocking
antisense
oligonucleotides and is incorporated by reference herein in its entirety:
Roberts et al. Nature
Reviews Drug Discovery (2020) 19: 673-694.
[0103] The CUG repeats of an expanded CUG repeat may form a double stranded
hairpin
structure. In the disease state, proteins bind to the double stranded hairpin
structure and become
sequestered and unable to perform other functions. In embodiments, the AC
binds to at least a
portion of the double stranded hairpin structure thereby sterically blocking
and/or decreasing the
affinity of the double stranded hairpin structure for a protein binding
partner. In embodiments, the
AC binds to at least a portion of a single stranded expanded CUG repeat
thereby inhibiting the
formation of the double stranded hairpin structure, and thus, inhibiting one
or more protein from
binding to the double stranded hairpin structure. In embodiments,
hybridization of the AC to the
double hairpin structure sterically blocks and/or decreases the affinity for
one or more proteins for
binding to the double hairpin structure. In embodiments, hybridization of the
AC to at least a
portion of a single stranded region of the expanded trinucleotide repeat,
inhibits the formation of
a double stranded hairpin structure.
[0104] Decreasing the ability of the target transcript to bind with the one or
more proteins, may
allow the one or more proteins to carry out other functions such as, for
example, regulating splicing
of downstream transcripts (transcripts that do not contain an expanded CUG
repeat). As such, in
41
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
embodiments, decreasing the ability of the target transcript to bind with the
one or more proteins,
may increase the level of the one or more proteins within the cell that are
available to provide other
functions or function on other transcripts. In embodiments, decreasing the
ability of the target
transcript to bind with the one or more proteins, may increase the cytosolic
level of the one or more
proteins within the cell that are available to provide other functions or
function on other transcripts.
Therefore, in embodiments, binding of the AC to the target transcript may
result in the modulation
of the level and/or activity of the one or more proteins that interact with
the target transcript.
[0105] In embodiments, hybridization of the AC to at least a portion of the
expanded CUG repeat,
decreases the affinity and or sterically blocks the binding of MNBL1 to the
target transcript.
MNBL1 is a splicing factor that regulates the splicing of downstream gene
transcripts. In a DM1
disease phenotype, MNBL1 binds to the expanded CUG repeat of a target
transcript. While bound
to target transcript, MNBL1 is sequestered in the nucleus and is not able to
regulate the splicing
of downstream gene transcripts (transcripts that do not contain an expanded
CUG repeat). In
embodiments, hybridization of the AC to at least a portion of the expanded CUG
repeat in target
transcript, sterically blocks and/or decreases the affinity of MNBL1 for the
target transcript,
thereby allowing it to regulate splicing of downstream gene transcripts. In
embodiments,
hybridization of the AC to at least a portion of the expanded CUG repeat in
the target transcript,
sterically blocks and/or decreases the affinity of MNBL1 for the target
transcript, thereby
increasing the amount of free (e.g., not bound to a transcript having a CUG
repeat) MNBL1. In
embodiments, hybridization of the AC to at least a portion of the expanded CUG
repeat in the
target transcript, sterically blocks and/or decreases the affinity of MNBL1
for the target transcript,
thereby decreasing the amount of MBNL1 bound to and sequestered by the target
transcript.
[0106] In embodiments where the target transcript is DMPK, hybridization of
the AC to at least a
portion of the expanded CUG repeat, decreases the affinity and or sterically
blocks the binding of
MNBL1 to the target transcript. MNBL1 is a splicing factor that regulates the
splicing of
downstream gene transcripts. In a DM1 disease phenotype, MNBL1 binds to the
expanded CUG
repeat of DMPK1. While bound to DMPK1, MNBL1 is sequestered in the nucleus and
is not able
to regulate the splicing of downstream gene transcripts (transcripts that do
not contain an expanded
CUG repeat). In embodiments, hybridization of the AC to at least a portion of
the expanded CUG
repeat in DMPK, sterically blocks and/or decreases the affinity of MNBL1 for
the DMPK
42
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
transcript, thereby allowing it to regulate splicing of downstream gene
transcripts. In embodiments,
hybridization of the AC to at least a portion of the expanded CUG repeat in
D1VIPK, sterically
blocks and/or decreases the affinity of MNBL1 for the D1VIPK transcript,
thereby increasing the
amount of free (e.g., not bound to a transcript having a CUG repeat) 1\4NBL1.
In embodiments,
hybridization of the AC to at least a portion of the expanded CUG repeat in
D1VIPK, sterically
blocks and/or decreases the affinity of MNBL1 for the D1VIPK transcript,
thereby decreasing the
amount of MBNL1 bound to and sequestered by the D1VIPK1 transcript.
[0107] In embodiments where the target transcript is D1VIPK, hybridization of
the AC to at least a
portion of the expanded CUG repeat, results in the decrease of CUGBP1 levels.
In the DM1 disease
state, the level of free MBNL1 (able to function) decreases while the level of
free (able to function)
of CUGBP1 increases. An increase in CUGBP1 levels is associated with the
disease state. As such,
in embodiments, hybridization of the AC to at least a portion of the expanded
CUG repeat results
in increased levels of free (able to function) 1V1BNL1 and/or a decrease in
free (able to function)
CUGBP1 levels.
[0108] Decreasing the ability of the target transcript to bind with the one or
more proteins, may
reduce, or inhibit the formation of CUG repeat foci. Transcripts that include
expanded nucleotide
repeats (e.g., expanded CUG repeats) may be transcribed and then sequestered
in the nucleus.
Within the nucleus, the sequestered transcripts may form aggregates. Proteins
that bind to the
transcript may then being to nucleate on sequestered transcript and/or
sequester transcript
aggregate thereby forming expanded nucleotide repeat (e.g., CUG repeat) foci.
The CUG repeat
foci may be visible using microscopy. In embodiments, decreasing the ability
of the target
transcript to bind with the one or more proteins, may reduce, or inhibit the
formation of aggregates
that include the target transcript. In embodiments, decreasing the ability of
the target transcript to
bind with the one or more proteins, may reduce, or inhibit the nucleation of
the one or more
proteins on the target transcript, on a double stranded hairpin region of a
transcript, or on an
aggregate of target transcripts. In embodiments where the target transcript is
D1VIPK, decreasing
the ability of the DMPK1 target transcript to bind with MNBL1 may reduce, or
inhibit the
nucleation MNBL1 on a DMPK1 target transcript or DMPK1 target transcript
aggregate. In
embodiments, hybridization of the AC to the target transcript may result in
inhibition or reduction
in formation of CUG repeat nuclear foci. In embodiments, hybridization of the
AC to the target
43
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
transcript may result in inhibition or reduction in formation of CUG repeat
nuclear foci formed
from a DMPK, TCF4, JPH3, and/or ATXN80S/ATXN8 target transcript.
[0109] In embodiments, hybridization of the AC to the target transcript may
result in the
modulation of the level, expression, and/or activity of one or more downstream
genes. For
example, hybridization of the AC to the target transcript may be used to
induce target transcript
degradation or sterically block or decreases the affinity of the target
transcript for one or more
proteins, thereby allowing the one or more proteins that were sequestered by
the target transcript
to regulate the expression, level, and/or activity of downstream genes. For
example, in
embodiments, the one or more proteins may include a protein that is involved
in regulating the
splicing of one or more downstream transcripts (transcripts that do not
contain an expanded CUG
repeat). In embodiments, the splicing of downstream transcripts is altered
when the protein
involved in splicing is bound and sequestered on the target transcript. For
example, alteration of
splicing may include the exclusion of one or more exons or the inclusion of
one or more introns in
a transcript thereby leading to the expression of various protein isoforms. In
embodiments, the
alteration of splicing may result in the inclusion of an exon and/or intron
that includes a premature
stop codon thereby resulting in a truncated isoform that may have no or
deleterious activity.
Alteration of downstream gene transcript splicing may lead to a change in
level, folding, and/or
activity of the downstream gene product which may be associated with a disease
phenotype. When
not bound to the target transcript comprising the expanded CUG repeat, the
protein involved in
splicing is free to regulate splicing which may results in a correction (or
rescue) of the splicing of
the downstream gene transcript, thereby at least partially restoring the
protein level, folding, and/or
activity of the downstream gene product associated with a healthy phenotype.
[0110] In embodiments, AC hybridization to the target transcript may result in
the modulation of
the splicing of downstream gene transcripts that are regulated by proteins
that are sequestered by
the target transcript in a disease state associated with expanded nucleotide
repeats (e.g., expanded
trinucleotide repeats). In disease associated with expanded trinucleotide
repeats, downstream gene
transcripts are often mis-processed, for example, mis-spliced. The mis-
splicing leads of the
downstream gene transcripts may lead to gene products that are destroyed
before translation or
translated into proteins that have aberrant structure and/or function. For
example, sequestration of
proteins that regulate the processing of downstream gene transcripts may lead
to the inclusion of
44
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
exons and/or introns with premature stop codons, the inclusion of introns, the
exclusion of exons,
and/or the inclusion of alternative exons, which may lead to a transcript and
or gene product that
is destroyed prior to translation or that is translated into an gene product
with aberrant function.
The change in levels of the downstream gene transcript and/or gene product
and/or the aberrant
structure and/or function of the downstream gene products are associated with
expanded
trinucleotide disease phenotypes. In embodiments, AC hybridization to the
target transcript may
result in the modulation of exon inclusion, exon exclusion, intron inclusion,
and/or intron
exclusion in downstream transcripts whose splicing is regulated by proteins
that are sequestered
to the target transcript during a disease state. As such, hybridization of the
AC to the target
transcript may result in the upregulation of downstream protein isomers and/or
transcripts
associated with a healthy phenotype. Similarly, hybridization of the AC to the
target transcript
may result in the downregulation (e.g., suppression) of downstream transcripts
and/or protein
isomers associated with a disease phenotype.
[0111] In embodiments where the target transcript is DMPK, AC hybridization to
the target
transcript may result in the modulation of the splicing of downstream gene
transcripts that are
regulated by proteins that are sequestered by the DMPK target transcript
during a disease state. In
DM1, several downstream gene transcripts are mis-spliced leading. The mis-
spliced genes are
associated with the disease phenotype. As such, modulation of gene splicing
may include
correcting (e.g., rescuing) the splicing of genes to result in gene products
of downstream genes
that are associated with a healthy phenotype. In embodiments where the target
transcript is DMPK,
AC hybridization to the target transcript may result in the modulation of the
splicing of
downstream gene transcripts that are regulated by MNBL1, a splicing regulator
that is sequestered
by the DMPK target transcript during a disease state. In embodiments where the
target transcript
is DMPK, AC hybridization to the target transcript may result in the
modulation of the splicing of
downstream gene transcripts that are regulated by CUGBP1, a protein whose
activity is affected
by expanded CUG repeats. In embodiments where the target transcript if DMPK,
AC hybridization
to the target transcript may result in the correct processing (e.g., splicing)
of downstream genes
that are regulated by MNBL1 and/or CUGBP1. In embodiments where the target
transcript is
D1VIPK, AC hybridization to the target transcript may result in the modulation
of splicing of
downstream genes including, but not limited to, 4833439L19Rik, Abcc9, Atp2a1,
Arhgef10,
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
Arhgap28, Armcx6, Angell, Best3, Binl, Brd2, Cacnals, Cacna2d1, Cpd, Cpeb3,
Ccpgl, Claspl,
C1C-1, Clcnl, Clk4, Cpeb2, Camk2g, Capzb, Copz2, Coch, cTNT, Ctu2, Cyp2s1,
Dctn4, Dnmll,
Eya4, Efna3, Efna2, Fbxo31, Fbxo21, Frem2, Fgd4, Fucal, Fnl, Gogla4, Gpr3711,
Grebl, Hegl,
Insr, Impdh2, IR, Itgav, Jag2, Kid, Kcan6, Kif13a, Ldb3, Lrrfip2, Mapt, Macfl,
Map3k4,
Mapkapl, Mbnll, Mllt3, Mbn12, Mef2c, Mpdz, Mrpll, Mxra7, Mybpcl, Myo9a,
Ncapd3, Ngfr,
Ndrg3, Ndufv3, Neb, Nfix, Numal, Opal, Pacsin2, Pcolce, Pdlim3, Pla2g15,
Phactr4, Phkal,
Phtf2, Ppplrl2b, Ppp3cc, Ppplcc, Ramp2, Rapgefl, Run, Ryrl, Sorcs2, Spsb4,
Scube2,
Sema6c, Sfc8a3, Slain2, Sorbsl, Spag9, Tmem28, Taccl, Tacc2, Ttc7, Tnik,
Tnfrsf22, Tnfrsf25,
Trappc9, Trim55, Ttn, Txn14a, Txlnb, Ube2d3, Vsp39, or any combination thereof
[0112] Mis-splicing of many of the above-mentioned downstream gene transcripts
results in
specific DM1 disease phenotypes. For example, MNBL1 is a splicing factor with
loss of function
in DM1 due to exon 5 inclusion. MNBL1 is sequestered by D1VIPK CUG expansion
and forms
RNA nuclear foci. Additionally, SOS1 promotes Ras activation to positively
regulate RAS/MAPK
signaling pathway. In DM1, exon 25 of SOS1 is excluded leading to inhibition
of muscle
hypertrophy pathways. In DM1, IR/INSR has exon 11 exclusion, which results in
higher levels of
low-signaling non-muscle isoform and decreased metabolic response to insulin
in DM1 (insulin
resistance). Similarly, exon 78 exclusion of DMD is observed in DM. Exon 78
exclusion results
in out-of-frame transcript at C-terminal domain. This mutated protein is
expressed in DM1 patients
and associated with a mechanism responsible for muscle wasting in patients.
BIN1 is required for
proper muscle T-tubule formation (EC coupling). Exon 11 exclusion produces
inactive isoform
and is found in DM1 patients. LDB3 interacts with a-actinin at the Z-disc in
striated muscle and
maintains muscle structure. Exon 11 inclusion of LDB3 detected in DM1 results
in reduction of
affinity for Protein kinase C (PKC). Consequently, PKC becomes hyperactive in
DM1. In
embodiments modulation of one or more downstream genes results in the
correction, or rescue, of
transcript splicing that is associated with a healthy phenotype. As such, in
embodiments,
hybridization of the AC to the D1VIPK target transcript, results in the rescue
of mis-splicing of
downstream genes/transcripts, thereby, reducing the level of downstream
genes/transcripts
associated with a disease phenotype. As such, in embodiments, hybridization of
the AC to the
DMPK target transcript, results in the rescue of mis-splicing of downstream
genes/transcripts,
thereby, increasing the level of downstream genes/transcripts associated with
a healthy phenotype.
46
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
[0113] In embodiments, the AC inhibits expression of the target transcript. In
embodiments, the
AC inhibits expression of the target transcript by blocking the pre-mRNA
processing machinery
and/or translation machinery from accessing and/or completing translation
and/or pre-mRNA
processing. In embodiments, the AC inhibits expression of the target
transcript by inducing
degradation of the target transcript, for example, through RNase H pathways.
AC structure
[0114] The AC includes an oligonucleotide and/or an oligonucleoside.
Oligonucleotides and/or
oligonucleotides are nucleotides or nucleosides linked through internucleoside
linkages.
Nucleosides include a pentose sugar (e.g., ribose or deoxyribose) and a
nitrogenous base
covalently attached to sugar. The naturally occurring (or traditional basses)
bases found in DNA
and/or RNA are adenine (A), guanine (G), thymine (T), cytosine (C), and uracil
(U). The naturally
occurring sugars (or traditional sugars) found in DNA and/or RNA deoxyribose
(DNA) and ribose
(RNA). The naturally occurring nucleoside linkage (or traditional
internucleoside linkage) is a
phosphodiester bond. In embodiments, the ACs of the present disclosure may
have all natural
sugars, bases, and internucleoside linkages.
[0115] Chemically modified nucleosides are routinely used for incorporation
into antisense
compounds to enhance one or more properties, such as nuclease resistance,
pharmacokinetics, or
affinity for a target RNA. In embodiments, the ACs of the present disclosure
may have one or
more modified nucleosides. In embodiments, the ACs of the present disclosure
may have one or
more modified sugars. In embodiments, the ACs of the present disclosure may
have one or more
modified bases. In embodiments, the ACs of the present disclosure may have one
or more modified
internucleoside linkages.
[0116] In general, a nucleobase is any group that contains one or more atom or
groups of atoms
capable of hydrogen bonding to a base of another nucleic acid. In addition to
"unmodified" or
"natural" nucleobases (A, G, T, C, and U) many modified nucleobases or
nucleobase mimetics are
known to those skilled in the art are amenable with the compounds described
herein Generally a
modified nucleobase refers to a nucleobase that is fairly similar in structure
to the parent
nucleobase, such as for example a 7-deaza purine, a 5-methyl cytosine, 2-thio-
dT (FIG. 2) or a G-
clamp. Generally, a nucleobase mimetic is a nucleobase that includes a
structure that is more
complicated than a modified nucleobase, such as for example a tricyclic
phenoxazine nucleobase
47
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
mimetic. Methods for preparation of the above noted modified nucleobases are
well known to
those skilled in the art.
[0117] In embodiments, the AC may include one or more nucleosides having a
modified sugar
moiety. In embodiments, the furanosyl sugar of a natural nucleoside may have a
2' modification,
modifications to make a constrained nucleoside, and others (see FIG. 2). For
example, in
embodiments, the furanosyl sugar ring of a natural nucleoside can be modified
in a number of
ways including, but not limited to, addition of a substituent group, bridging
of two non-geminal
ring atoms to form a bicyclic nucleic acid (BNA) or a locked nucleic acid;
exchanging the oxygen
of the furanosyl ring with C or N; and/or substitution of an atom or group
such (see FIG. 2).
Modified sugars are well known and can be used to increase or decrease the
affinity of the AC for
its target nucleotide sequence. Modified sugars may also be used increase AC
resistance to
nucleases. Sugars can also be replaced with sugar mimetic groups among others.
In embodiments,
one or more sugars of the nucleosides of the AC is replaced with a
methylenemorpholine ring as
shown as 19 in FIG. 2.
[0118] In embodiments, the AC includes one or more nucleosides that include a
bicyclic modified
sugar (BNA; sometimes called bridged nucleic acids). Examples of BNAs include,
but are not
limited to LNA (4'-(CH2)-0-2' bridge), 2'-thio-LNA (4'-(CH2)-S-2' bridge), 2'-
amino-LNA (4'-
(CH2)-NR-2' bridge), ENA (4'-(CH2)2-0-2' bridge), 4'-(CH2)3-2' bridged BNA, 4'-
(CH2CH(CH3))-
2' bridged BNA" cEt (4'-(CH(CH3)-0-2' bridge), and cM0E BNAs (4'-(CH(CH2OCH3)-
0-2'
bridge). BNA's have been prepared and disclosed in the patent literature as
well as in scientific
literature (Srivastava, et al. J. Am. Chem. Soc. (2007), ACS Advanced online
publication,
10.1021/ja071106y; Albaek et al. J. Org. Chem. (2006), 71, 7731 -7740;
Fluiter, et al.
Chembiochem (2005), 6, 1104-1109; Singh et al., Chem. Commun. (1998), 4, 455-
456; Koshkin
et al., Tetrahedron (1998), 54, 3607-3630; Wahlestedt et al., Proc. Natl.
Acad. Sci. U.S.A. (2000),
97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett. (1998), 8, 2219-2222; WO
94/14226; WO
2005/021570; Singh et al., J. Org. Chem. (1998), 63, 10035-10039, WO
2007/090071; U.S. Patent
Nos. 7,053,207; 6,268,490; 6,770,748; 6,794,499; 7,034,133; and 6,525,191; and
U.S. Pre-Grant
Publication Nos. 2004-0171570; 2004-0219565; 2004-0014959; 2003-0207841; 2004-
0143114;
and 20030082807).
48
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
[0119] In embodiments, the AC includes one or more nucleosides that include a
locked nucleic
acid (LNA). In LNAs the 2'-hydroxyl group of the ribosyl sugar ring is linked
to the 4' carbon
atom of the sugar ring thereby forming a 2'-C,4'-C-oxymethylene linkage to
form the bicyclic sugar
moiety (reviewed in Elayadi et al., Curr. Opinion Invens. Drugs (2001), 2, 558-
561; Braasch et al.,
Chem. Biol. (2001), 8 1-7; and Orum et al., Curr. Opinion Mol. Ther. (2001),
3, 239-243; see also
U.S. Patents: 6,268,490 and 6,670,461). The linkage can be a methylene (-CH2-)
group bridging
the 2' oxygen atom and the 4' carbon atom, for which the term LNA is used for
the bicyclic moiety;
in the case of an ethylene group in this position, the term ENATM is used
(Singh et al., Chem.
Commun. (1998), 4, 455-456; ENATM; Morita et al., Bioorganic Medicinal
Chemistry (2003), 11,
2211-2226). LNA and other bicyclic sugar analogs display very high duplex
thermal stabilities
with complementary DNA and RNA (Tm = +3 to +10 C), stability towards 3'-
exonucleolytic
degradation and good solubility properties. Potent and nontoxic antisense
oligonucleotides
containing LNAs have been described (Wahlestedt et al., Proc. Natl. Acad. Sci.
U.S.A. (2000), 97,
5633-5638).
[0120] An isomer of LNA that has also been studied is alpha-L-LNA which has
been shown to
have superior stability against a 3'-exonuclease. The alpha-L-LNA's were
incorporated into
antisense gapmers and chimeras that showed potent antisense activity (Frieden
et al., Nucleic
Acids Research (2003), 21, 6365-6372).
[0121] The synthesis and preparation of the LNA monomers adenine, cytosine,
guanine, 5-methyl-
cytosine, thymine and uracil, along with their oligomerization, and nucleic
acid recognition
properties have been described (Koshkin et al., Tetrahedron, 1998, 54, 3607-
3630). LNAs and
preparation thereof are also described in WO 98/39352 and WO 99/14226.
[0122] Analogs of LNA, phosphorothioate-LNA and 2'-thio-LNAs, have also been
prepared
(Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). Preparation of
LNAanalogs
containing oligodeoxyribonucleotide duplexes as substrates for nucleic acid
polymerases has also
been described (Wengel et al., WO 99/14226). Furthermore, synthesis of 2'-
amino-LNA, a
conformationally restricted high-affinity oligonucleotide analog has been
described (Singh et al.,
J. Org. Chem. (1998), 63, 10035-10039). In addition, 2'-amino- and 2'-
methylamino-LNA's have
been prepared and the thermal stability of their duplexes with complementary
RNA and DNA
strands has been previously reported.
49
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
[0123] Methods for the preparations of modified sugars are well known to those
skilled in the art.
Some representative patents and publications that teach the preparation of
such modified sugars
include, but are not limited to, U.S. Patents: 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;
5,792,747;
5,700,920; and 6,600,032; and WO 2005/121371.
Internucleoside Linkages
[0124] Described herein are internucleoside linking groups that link the
nucleosides or otherwise
modified nucleoside monomer units together thereby forming an oligonucleotide
and/or an
oligonucleotide containing AC. The ACs may include naturally occurring
internucleoside
linkages, unnatural internucleoside linkages, or both.
[0125] In naturally occurring DNA and RNA, the internucleoside linking group
is a
phosphodiester that covalently links adjacent nucleosides to one another to
form a linear polymeric
compound. In naturally occurring DNA and RNA, phosphodiester is linked to the
2', 3' or 5'
hydroxyl moiety of the sugar. Within oligonucleotides, the phosphate groups
are commonly
referred to as forming the internucleoside backbone of the oligonucleotide. In
naturally occurring
DNA and RNA, the linkage or backbone of RNA and DNA, is a 3' to 5'
phosphodiester linkage.
In embodiments, the internucleoside linking groups of the ACs are
phosphodiesters. In
embodiments, the internucleoside linking groups of the ACs are 3' to 5'
phosphodiester linkages.
[0126] The two main classes of unnatural internucleoside linking groups are
defined by the
presence or absence of a phosphorus atom. Representative phosphorus containing
internucleoside
linkages include, but are not limited to, phosphotriesters,
methylphosphonates, phosphoramidate,
and phosphorothioates. Representative non-phosphorus containing
internucleoside linking groups
include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-0-CH2-),
thiodiester (-0-
C(0)-S-), thionocarbamate (-0-C(0)(NH)-S-); siloxane (-0-Si(H2-0-); and N,N'-
dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). ACs having phosphorus internucleoside
linking
groups are referred to as oligonucleotides. Antisense compounds having non-
phosphorus
internucleoside linking groups are referred to as oligonucleosides. Modified
internucleoside
linkages, compared to natural phosphodiester linkages, can be used to alter,
typically increase,
nuclease resistance of the antisense compound. Internucleoside linkages having
a chiral atom can
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
be prepared as racemic, chiral, or as a mixture. Representative chiral
internucleoside linkages
include, but are not limited to, alkylphosphonates and phosphorothioates.
Methods of preparation
of phosphorous-containing and non-phosphorous-containing linkages are well
known to those
skilled in the art.
[0127] In embodiments, two or more nucleosides having modified sugars and/or
modified
nucleobases may be joined using a phosphoramidate. In embodiments, two or more
nucleosides
having a methyl en OM orp h line ring may be connected through a pliosphoram
id a te internucleoside
linkage.
[01281 Antisense compounds that include nucleobases with a methylenemorpholine
ring that are
linked through ph osphorami date internucleosi de linkage may be referred to
as phosp horarn i date
morpholino olig,omers (PM0s).
Conjugate Groups
[0129] In embodiments, ACs are modified by covalent attachment of one or more
conjugate
groups. In general, conjugate groups modify one or more properties of the
attached AC including
but not limited to pharmacodynamic, pharmacokinetic, binding, absorption,
cellular distribution,
cellular uptake, charge and clearance. Conjugate groups are routinely used in
the chemical arts and
are linked directly or via an optional linking moiety or linking group to a
parent compound such
as an AC. Conjugate groups include without limitation, intercalators, reporter
molecules,
polyamines, polyamides, polyethylene glycols, thioethers, polyethers,
cholesterols,
thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, biotin,
phenazine,
phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines,
coumarins and
dyes. In embodiments, the conjugate group is a polyethylene glycol (PEG), and
the PEG is
conjugated to either the AC or the CPP (CPP discussed elsewhere herein).
[0130] In embodiments, conjugate groups include lipid moieties such as a
cholesterol moiety
(Letsinger et al., Proc. Natl. Acad. Sci. USA (1989), 86, 6553); cholic acid
(Manoharan et al.,
Bioorg. Med. Chem. Lett. (1994), 4, 1053); a thioether, e.g., hexyl-S-
tritylthiol (Manoharan et al.,
Ann. N.Y. Acad. Sci. (1992), 660, 306; Manoharan et al., Bioorg. Med. Chem.
Let. (1993), 3,
2765); a thiocholesterol (Oberhauser et al., Nucl. Acids Res. (1992), 20,
533); an aliphatic chain,
e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J.
(1991), 10, 111;
Kabanov et al., FEBS Lett. (1990), 259, 327; Svinarchuk et al., Biochimie
(1993), 75, 49); a
51
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium-1,2-di-O-
hexadecyl-rac-
glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett. (1995), 36, 3651;
Shea et al., Nucl.
Acids Res. (1990), 18, 3777); a polyamine or a polyethylene glycol chain
(Manoharan et al.,
Nucleosides & Nucleotides (1995), 14, 969); adamantane acetic acid (Manoharan
et al.,
Tetrahedron Lett. (1995), 36, 3651); a palmityl moiety (Mishra et al.,
Biochim. Biophys. Acta.
(1995), 1264, 229); or an octadecylamine or hexylamino-carbonyl-oxycholesterol
moiety (Crooke
et al., J. Pharmacol. Exp. Ther. (1996) ,277,923).
Types of Antisense Compounds
[0131] Various types of AC may be used for example, including an antisense
oligonucleotide,
siRNA, microRNA, antagomir, aptamer, ribozyme, supermir, miRNA mimic, miRNA
inhibitor,
or combinations thereof.
Antisense Oligonucleotides
[0132] In various embodiments, the antisense compound (AC) is an antisense
oligonucleotide
(ASO) that is complementary to a target nucleotide sequence. The term
"antisense oligonucleotide
(ASO)" or simply "antisense" is meant to include oligonucleotides that are
complementary to a
target nucleotide sequence. The term also encompasses ASOs that may not be
fully complementary
to the desired target nucleotide sequence. ASOs include single strands of DNA
and/or RNA that
are complementary to a chosen target nucleotide sequence or a target gene.
ASOs may include one
or more modified DNA and/or RNA bases, modified sugars, and/or unnatural
internucleoside
linkages. In embodiments, the ASOs may include one or more phosphoramidate
internucleoside
linkages. In embodiments, the ASO is phosphoramidate morpholino oligomers
(PM0s). ASOs
may have any characteristic, be any length, bind to any target nucleotide
sequence and/or sequence
element, and effect any mechanism as described relative to an AC.
[0133] Antisense oligonucleotides have been demonstrated to be effective as
targeted inhibitors
of protein synthesis, and, consequently, can be used to specifically inhibit
protein synthesis by a
targeted gene. The efficacy of ASO for inhibiting protein synthesis is well
established. To date,
these compounds have shown promise in several in vitro and in vivo models,
including models of
inflammatory disease, cancer, and HIV (Agrawal, Trends in Biotech. (1996),
14:376-387).
Antisense can also affect cellular activity by hybridizing specifically with
chromosomal DNA.
52
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
[0134] Methods of producing antisense oligonucleotides are known in the art
and can be readily
adapted to produce an antisense oligonucleotide that targets any
polynucleotide sequence.
Selection of antisense oligonucleotide sequences specific for a given target
sequence is based upon
analysis of the chosen target sequence and determination of secondary
structure, Tm, binding
energy, and relative stability. Antisense oligonucleotides may be selected
based upon their relative
inability to form dimers, hairpins, or other secondary structures that would
reduce or prohibit
specific binding to the target mRNA in a host cell. Target regions of the mRNA
include those
regions at or near the AUG translation initiation codon and those sequences
that are substantially
complementary to 5' regions of the mRNA. These secondary structure analyses
and target site
selection considerations can be performed, for example, using v.4 of the OLIGO
primer analysis
software (Molecular Biology Insights) and/or the BLASTN 2Ø5 algorithm
software (Altschul et
ai, Nucleic Acids Res. 1997, 25(17):3389-402).
RNA Interference
[0135] In embodiments, the AC includes a molecule that mediates RNA
interference (RNAi). As
used herein, the phrase "mediates RNAi" refers to the ability to silence, in a
sequence specific
manner, a target transcript. While not wishing to be bound by theory, it is
believed that silencing
uses the RNAi machinery or process and a guide RNA, e.g., an siRNA compound of
from about
21 to about 23 nucleotides. In embodiments, the AC targets the target
transcript for degradation.
As such, in embodiments, RNAi molecule may be used to disrupt the expression
of a gene or
polynucleotide of interest. In embodiments, RNAi molecule is used to induce
degradation of the
target transcript, such as a pre-mRNA or a mature mRNA.
[0136] In embodiments, the AC includes a small interfering RNA (siRNA) that
elicits an RNAi
response.
[0137] Small interfering RNAs (siRNAs) are nucleic acid duplexes normally from
about 16 to
about 30 nucleotides long that can associate with a cytoplasmic multi-protein
complex known as
RNAi-induced silencing complex (RISC). RISC loaded with siRNA mediates the
degradation of
homologous transcripts, therefore siRNA can be designed to knock down protein
expression with
high specificity. Unlike other antisense technologies, siRNA function through
a natural mechanism
evolved to control gene expression through non-coding RNA. A variety of RNAi
reagents,
53
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
including siRNAs targeting clinically relevant targets, are currently under
pharmaceutical
development, as described, e.g., in de Fougerolles, A. et al., Nature Reviews
(2007) 6:443-453.
[0138] While the first described RNAi molecules were RNA:RNA hybrids that
include both an
RNA sense and an RNA antisense strand, it has now been demonstrated that DNA
sense:RNA
antisense hybrids, RNA sense:DNA antisense hybrids, and DNA:DNA hybrids are
capable of
mediating RNAi (Lamberton, J. S . and Christian, A.T., Molecular Biotechnology
(2003), 24:111-
119). In embodiments, RNAi molecules are used that include any of these
different types of
double-stranded molecules. In addition, it is understood that RNAi molecules
may be used and
introduced to cells in a variety of forms. Accordingly, as used herein, RNAi
molecules
encompasses any and all molecules capable of mediating an RNAi in cells,
including, but not
limited to, double-stranded oligonucleotides that include two separate
strands, i.e. a sense strand
and an antisense strand, e.g., small interfering RNA (siRNA); double-stranded
oligonucleotide that
includes two separate strands that are linked together by non-nucleotidyl
linker; oligonucleotides
that include a hairpin loop of complementary sequences, which forms a double-
stranded region,
e.g., shRNAi molecules, and expression vectors that express one or more
polynucleotides capable
of forming a double-stranded polynucleotide alone or in combination with
another polynucleotide.
[0139] A "single strand siRNA compound" as used herein, is an siRNA compound
which is made
up of a single molecule. It may include a duplexed region, formed by intra-
strand pairing, e.g., it
may be, or include, a hairpin or pan-handle structure. Single strand siRNA
compounds may be
antisense with regard to the target molecule.
[0140] A single strand siRNA compound may be sufficiently long that it can
enter the RISC and
participate in RISC mediated cleavage of a target mRNA. A single strand siRNA
compound is at
least about 14, at least about 15, at least about 20, at least about 25, at
least about 30, at least about
35, at least about 40, or up to about 50 nucleotides in length. In certain
embodiments, the single
strand siRNA is less than about 200, about 100, or about 60 nucleotides in
length.
[0141] Hairpin siRNA compounds may have a duplex region equal to or at least
about 17, about
18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25
nucleotide pairs. The
duplex region may be equal to or less than about 200, about 100, or about 50
nucleotide pairs in
length. In certain embodiments, ranges for the duplex region are from about 15
to about 30, from
about 17 to about 23, from about 19 to about 23, and from about 19 to about 21
nucleotides pairs
54
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
in length. The hairpin may have a single strand overhang or terminal unpaired
region. In certain
embodiments, the overhangs are from about 2 to about 3 nucleotides in length.
In embodiments,
the overhang is at the same side of the hairpin and in embodiments on the
antisense side of the
hairpin.
[0142] A "double stranded siRNA compound" as used herein, is an siRNA compound
which
includes more than one, and in some cases two, strands in which interchain
hybridization can form
a region of duplex structure.
[0143] The antisense strand of a double stranded siRNA compound may be equal
to or at least
about 14, about 15, about 16 about 17, about 18, about 19, about 20, about 25,
about 30, about 40,
or about 60 nucleotides in length. It may be equal to or less than about 200,
about 100, or about 50
nucleotides in length. Ranges may be from about 17 to about 25, from about 19
to about 23, and
from about 19 to about 21 nucleotides in length. As used herein, term
"antisense strand" means the
strand of an siRNA compound that is sufficiently complementary to a target
molecule, e.g., the
target nucleotide sequence of a target transcript.
[0144] The sense strand of a double stranded siRNA compound may be equal to or
at least about
14, about 15, about 16, about 17, about 18, about 19, about 20, about 25,
about 30, about 40, or
about 60 nucleotides in length. It may be equal to or less than about 200,
about 100, or about 50,
nucleotides in length. Ranges may be from about 17 to about 25, from about 19
to about 23, and
from about 19 to about 21 nucleotides in length.
[0145] The double strand portion of a double stranded siRNA compound may be
equal to or at
least about 14, about 15, about 16, about 17, about 18, about 19, about 20,
about 21, about 22,
about 23, about 24, about 25, about 30, about 40, or about 60 nucleotide pairs
in length. It may be
equal to or less than about 200, about 100, or about 50, nucleotides pairs in
length. Ranges may be
from about 15 to about 30, from about 17 to about 23, from about 19 to about
23, and from about
19 to about 21 nucleotides pairs in length.
[0146] In embodiments, the siRNA compound is sufficiently large that it can be
cleaved by an
endogenous molecule, e.g., by Dicer, to produce smaller siRNA compounds, e.g.,
siRNAs agents.
[0147] The sense and antisense strands may be chosen such that the double-
stranded siRNA
compound includes a single strand or unpaired region at one or both ends of
the molecule. Thus, a
double-stranded siRNA compound may contain sense and antisense strands, paired
to contain an
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
overhang, e.g., one or two 5' or 3' overhangs, or a 3' overhang of 1 to 3
nucleotides. The overhangs
can be the result of one strand being longer than the other, or the result of
two strands of the same
length being staggered. Some embodiments will have at least one 3' overhang.
In embodiments,
both ends of an siRNA molecule will have a 3' overhang. In embodiments, the
overhang is 2
nucleotides.
[0148] In embodiments, the length for the duplexed region is from about 15 to
about 30, or about
18, about 19, about 20, about 21, about 22, or about 23 nucleotides in length,
e.g., in the ssiRNA
(siRNA with sticky overhangs) compound range discussed above. ssiRNA compounds
can
resemble in length and structure the natural Dicer processed products from
long dsiRNAs.
Embodiments in which the two strands of the ssiRNA compound are linked, e.g.,
covalently linked
are also included. In embodiments, hairpin, or other single strand structures
which provide a double
stranded region, and a 3' over hangs are included.
[0149] The siRNA compounds described herein, including double-stranded siRNA
compounds
and single- stranded siRNA compounds can mediate silencing of a target RNA,
e.g., mRNA, e.g.,
a transcript of a gene that encodes a protein. For convenience, such mRNA is
also referred to
herein as mRNA to be silenced. Such a gene is also referred to as a target
gene. In general, the
RNA to be silenced is an endogenous gene.
[0150] In embodiments, an siRNA compound is "sufficiently complementary" to a
target
transcript, such that the siRNA compound silences production of protein
encoded by the target
mRNA. In embodiments, the siRNA compound is "sufficiently complementary" to at
least a
portion of a target transcript, such that the siRNA compound silences
production of the gene
product encoded by the target transcript. In another embodiment, the siRNA
compound is "exactly
complementary" to a target nucleotide sequence (e.g., a portion of a target
transcript) such that the
target nucleotide sequence and the siRNA compound anneal, for example to form
a hybrid made
exclusively of Watson-Crick base pairs in the region of exact complementarity.
A "sufficiently
complementary" to a target nucleotide sequence can include an internal region
(e.g., of at least
about 10 nucleotides) that is exactly complementary to a target nucleotide
sequence. Moreover, in
certain embodiments, the siRNA compound specifically discriminates a single-
nucleotide
difference. In this case, the siRNA compound only mediates RNAi if exact
complementary is
found in the region (e.g., within 7 nucleotides of) the single-nucleotide
difference.
56
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
[0151] The therapeutic applications of RNAi are extremely broad, since siRNA
and miRNA
constructs can be synthesized with any nucleotide sequence directed against a
target protein. To
date, siRNA constructs have shown the ability to specifically down-regulate
target proteins in both
in vitro and in vivo models, as well as in clinical studies
MicroRNAs
[0152] In embodiments, the AC includes a microRNA molecule. MicroRNAs (miRNAs)
are a
highly conserved class of small RNA molecules that are transcribed from DNA in
the genomes of
plants and animals but are not translated into protein. Processed miRNAs are
single stranded 17-
25 nucleotide RNA molecules that become incorporated into the RNA-induced
silencing complex
(RISC) and have been identified as key regulators of development, cell
proliferation, apoptosis
and differentiation. They are believed to play a role in regulation of gene
expression by binding to
the 3 '-untranslated region of specific mRNAs. RISC mediates down-regulation
of gene expression
through translational inhibition, transcript cleavage, or both. RISC is also
implicated in
transcriptional silencing in the nucleus of a wide range of eukaryotes.
Antagomirs
[0153] In embodiments, the AC is an antagomir. Antagomirs are RNA-like
oligonucleotides that
harbor various modifications for RNAse protection and pharmacologic
properties, such as
enhanced tissue and cellular uptake. They differ from normal RNA by, for
example, complete 2'-
0-methylation of sugar, phosphorothioate backbone and, for example, a
cholesterol-moiety at 3'-
end. Antagomirs may be used to efficiently silence endogenous miRNAs by
forming duplexes that
include the antagomir and endogenous miRNA, thereby preventing miRNA-induced
gene
silencing. An example of antagomir-mediated miRNA silencing is the silencing
of miR-122,
described in Krutzfeldt et al., Nature (2005), 438: 685-689, which is
expressly incorporated by
reference herein in its entirety. Antagomir RNAs may be synthesized using
standard solid phase
oligonucleotide synthesis protocols (U.S. Patent Application Ser. Nos.
11/502,158 and
11/657,341; the disclosure of each of which are incorporated herein by
reference).
[0154] An antagomir can include ligand-conjugated monomer subunits and
monomers for
oligonucleotide synthesis. Monomers are described in U.S. Application No.
10/916,185. An
antagomir can have a ZXY structure, such as is described in PCT Application
No.
57
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
PCT/US2004/07070. An antagomir can be complexed with an amphipathic moiety.
Amphipathic
moieties for use with oligonucleotide agents are described in PCT Application
No.
PCT/US2004/07070.
Aptamers
[0155] In embodiments, the AC includes an aptamer. Aptamers are nucleic acid
or peptide
molecules that bind to a particular molecule of interest with high affinity
and specificity (Tuerk
and Gold, Science 249:505 (1990); Ellington and Szostak, Nature 346:818
(1990)). DNA or RNA
aptamers have been successfully produced which bind many different entities
from large proteins
to small organic molecules (Eaton, Curr. Opin. Chem. Biol. (1997), 1: 10-16;
Famulok, Curr. Opin.
Struct. Biol. (1999), 9:324-9; and Hermann and Patel, Science (2000), 287:820-
5). Aptamers may
be RNA or DNA based and may include a riboswitch. A riboswitch is a part of an
mRNA molecule
that can directly bind a small target molecule, and whose binding of the
target affects the gene's
activity. Thus, an mRNA that contains a riboswitch is directly involved in
regulating its own
activity, depending on the presence or absence of its target molecule.
Generally, aptamers are
engineered through repeated rounds of in vitro selection or equivalently,
SELEX (systematic
evolution of ligands by exponential enrichment) to bind to various molecular
targets such as small
molecules, proteins, nucleic acids, and even cells, tissues and organisms. The
aptamer may be
prepared by any known method, including synthetic, recombinant, and
purification methods, and
may be used alone or in combination with other aptamers specific for the same
target. Further, the
term "aptamer" also includes "secondary aptamers" containing a consensus
sequence derived from
comparing two or more known aptamers to a given target. In embodiments, the
aptamer is an
"intracellular aptamer", or "intramer", which specifically recognize
intracellular targets (Famulok
et al., Chem Biol. (2001),8(10):931-939; Yoon and Rossi, Adv. Drug Deliv. Rev.
(2018), 134:22-
35; each incorporated by reference herein).
Ribozymes
[0156] In embodiments, the AC is a ribozyme. Ribozymes are RNA molecules
complexes having
specific catalytic domains that possess endonuclease activity (Kim and Cech,
Proc. Natl. Acad.
Sci. USA (1987),84(24):8788-92; Forster and Symons, Cell (1987) 24, 49(2):211-
20). For
example, a large number of ribozymes accelerate phosphoester transfer
reactions with a high
58
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
degree of specificity, often cleaving only one of several phosphoesters in an
oligonucleotide
substrate (Cech et al., Cell (1981), 27(3 Pt 2):487-96; Michel and Westhof, J.
Mol. Biol. (1990),
5, 216(3):585-610; Reinhold-Hurek and Shub, Nature (1992), 14, 357(6374): 173-
6). This
specificity has been attributed to the requirement that the substrate bind via
specific base-pairing
interactions to the internal guide sequence (IGS) of the ribozyme prior to
chemical reaction.
[0157] At least six basic varieties of naturally occurring enzymatic RNAs are
known presently.
Each can catalyze the hydrolysis of RNA phosphodiester bonds in trans (and
thus can cleave other
RNA molecules) under physiological conditions, In general, enzymatic nucleic
acids act by first
binding to a target RNA. Such binding occurs through the target binding
portion of an enzymatic
nucleic acid which is held in close proximity to an enzymatic portion of the
molecule that acts to
cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and
then binds a target
RNA through complementary base-pairing, and once bound to the correct site,
acts enzymatically
to cut the target RNA. Strategic cleavage of such a target RNA will destroy
its ability to direct
synthesis of an encoded protein. After an enzymatic nucleic acid has bound and
cleaved its RNA
target, it is released from that RNA to search for another target and can
repeatedly bind and cleave
new targets.
[0158] The enzymatic nucleic acid molecule may be formed in a hammerhead,
hairpin, a hepatitis
6 virus, group I intron or RNaseP RNA (in association with an RNA guide
sequence) or
Neurospora VS RNA motif, for example. Specific examples of hammerhead motifs
are described
by Rossi et al. Nucleic Acids Res. (1992), 20(17):4559-65. Examples of hairpin
motifs are
described by Hampel et al. (Eur. Pat. Appl. Publ. No. EP 0360257), Hampel and
Tritz,
Biochemistry (1989), 28(12):4929- 33; Hampel et al, Nucleic Acids Res.
(1990),18(2):299-304
and U. S. Patent 5,631,359. An example of the hepatitis virus motif is
described by Perrotta and
Been, Biochemistry (1992), 31(47): 11843-52; an example of the RNaseP motif is
described by
Guerrier-Takada et al., Cell (1983), 35(3 Pt 2):849-57; Neurospora VS RNA
ribozyme motif is
described by Collins (Saville and Collins, Cell (1990), 61(4):685-96; Saville
and Collins, Proc.
Natl. Acad. Sci. USA (1991),88(19):8826-30; Collins and Olive, Biochemistry
(1993),32(l
1):2795-9); and an example of the Group I intron is described in U. S. Patent
4,987,071. In
embodiments, enzymatic nucleic acid molecules have a specific substrate
binding site which is
complementary to one or more of the target gene DNA or RNA regions, and that
they have
59
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
nucleotide sequences within or surrounding that substrate binding site which
impart an RNA
cleaving activity to the molecule. Thus, the ribozyme constructs need not be
limited to specific
motifs mentioned herein.
[0159] Ribozymes may be designed as described in Int. Pat. Appl. Publ. No. WO
93/23569 and
Int. Pat. Appl. Publ. No. WO 94/02595, each specifically incorporated herein
by reference, and
synthesized to be tested in vitro and in vivo, as described therein. In
embodiments, the ribozyme
is targeted to a target nucleotide sequence of a target transcript.
[0160] Ribozyme activity can be increased by altering the length of the
ribozyme binding arms or
chemically synthesizing ribozymes with modifications that prevent their
degradation by serum
ribonucleases (see e.g. , Int. Pat. Appl. Publ. No. WO 92/07065; Int. Pat.
Appl. Publ. No. WO
93/15187; Int. Pat. Appl. Publ. No. WO 91/03162; Eur. Pat. Appl. Publ. No.
92110298.4; U. S.
Patent 5,334,711; and Int. Pat. Appl. Publ. No. WO 94/13688, which describe
various chemical
modifications that can be made to the sugar moieties of enzymatic RNA
molecules), modifications
which enhance their efficacy in cells, and removal of stem H bases to shorten
RNA synthesis times
and reduce chemical requirements.
Supermir
[0161] In embodiments, the AC is a supermir. A supermir refers to a single
stranded, double
stranded, or partially double stranded oligomer or polymer of RNA, polymer of
DNA, or both, or
modifications thereof, which has a nucleotide sequence that is substantially
identical to an miRNA
and that is antisense with respect to its target, This term includes
oligonucleotides composed of
naturally-occurring nucleobases, sugars and covalent internucleoside
(backbone) linkages and
which contain at least one non-naturally- occurring portion which functions
similarly. Such
modified or substituted oligonucleotides have desirable properties such as,
for example, enhanced
cellular uptake, enhanced affinity for nucleic acid target and increased
stability in the presence of
nucleases. In embodiments, the supermir does not include a sense strand, and
in another
embodiment, the supermir does not self-hybridize to a significant extent. A
supermir can have
secondary structure, but it is substantially single-stranded under
physiological conditions. A
supermir that is substantially single-stranded is single-stranded to the
extent that less than about
50% (e.g., less than about 40%, about 30%, about 20%, about 10%, or about 5%)
of the supermir
is duplexed with itself. The supermir can include a hairpin segment, e.g.,
sequence, for example,
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
at the 3' end can self-hybridize and form a duplex region, e.g., a duplex
region of at least about 1,
about 2, about 3, or about 4 or less than about 8, about 7, about 6, or about
5 nucleotides, or about
nucleotides. The duplexed region can be connected by a linker, e.g., a
nucleotide linker, e.g.,
about 3, about 4, about 5, or about 6 dTs, e.g., modified dTs. In another
embodiment the supermir
is duplexed with a shorter oligo, e.g., of about 5, about 6, about 7, about 8,
about 9, or about 10
nucleotides in length, e.g., at one or both of the 3' and 5' end or at one end
and in the non-terminal
or middle of the supermir.
miRNA mimics
[0162] In embodiments, the AC is a miRNA mimic. miRNA mimics represent a class
of molecules
that can be used to imitate the gene silencing ability of one or more miRNAs.
Thus, the term
"microRNA mimic" refers to synthetic non-coding RNAs (i.e., the miRNA is not
obtained by
purification from a source of the endogenous miRNA) that are capable of
entering the RNAi
pathway and regulating gene expression. miRNA mimics can be designed as mature
molecules
(e.g., single stranded) or mimic precursors (e.g., pri- or pre-miRNAs). miRNA
mimics can include
nucleic acid (modified or modified nucleic acids) including oligonucleotides
that include, without
limitation, RNA, modified RNA, DNA, modified DNA, locked nucleic acids, or 2'-
0,4'-C-
ethylene-bridged nucleic acids (ENA), or any combination of the above
(including DNA-RNA
hybrids). In addition, miRNA mimics can include conjugates that can affect
delivery, intracellular
compartmentalization, stability, specificity, functionality, strand usage,
and/or potency. In one
design, miRNA mimics are double stranded molecules (e.g., with a duplex region
of between about
16 and about 31 nucleotides in length) and contain one or more sequences that
have identity with
the mature strand of a given miRNA. Modifications can include 2' modifications
(including 2'-0
methyl modifications and 2' F modifications) on one or both strands of the
molecule and
internucleoside modifications (e.g., phosphorothioate modifications) that
enhance nucleic acid
stability and/or specificity. In addition, miRNA mimics can include overhangs.
The overhangs can
include from about 1 to about 6 nucleotides on either the 3' or 5' end of
either strand and can be
modified to enhance stability or functionality. In embodiments, a miRNA mimic
includes a duplex
region of from about 16 to about 31 nucleotides and one or more of the
following chemical
modification patterns: the sense strand contains 2'-0-methyl modifications of
nucleotides 1 and 2
(counting from the 5' end of the sense oligonucleotide), and all of the Cs and
Us; the antisense
61
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
strand modifications can include 2' F modification of all of the Cs and Us,
phosphorylation of the
5' end of the oligonucleotide, and stabilized internucleoside linkages
associated with a 2 nucleotide
3 'overhang.
miRNA inhibitor
[0163] In embodiments, the AC is a miRNA inhibitor. The terms "antimir"
"microRNA inhibitor",
"miR inhibitor", or "miRNA inhibitor" are synonymous and refer to
oligonucleotides or modified
oligonucleotides that interfere with the ability of specific miRNAs. In
general, the inhibitors are
nucleic acid or modified nucleic acids in nature including oligonucleotides
that include RNA,
modified RNA, DNA, modified DNA, locked nucleic acids (LNAs), or any
combination of the
above.
[0164] Modifications include 2' modifications (including 2'-0 alkyl
modifications and 2' F
modifications) and internucleoside modifications (e.g., phosphorothioate
modifications) that can
affect delivery, stability, specificity, intracellular compartmentalization,
or potency. In addition,
miRNA inhibitors can include conjugates that can affect delivery,
intracellular
compartmentalization, stability, and/or potency. Inhibitors can adopt a
variety of configurations
including single stranded, double stranded (RNA/RNA or RNA/DNA duplexes), and
hairpin
designs, in general, microRNA inhibitors include contain one or more sequences
or portions of
sequences that are complementary or partially complementary with the mature
strand (or strands)
of the miRNA to be targeted. In addition, the miRNA inhibitor may also include
additional
sequences located 5' and 3' to the sequence that is the reverse complement of
the mature miRNA.
The additional sequences may be the reverse complements of the sequences that
are adjacent to
the mature miRNA in the pri-miRNA from which the mature miRNA is derived, or
the additional
sequences may be arbitrary sequences (having a mixture of A, G, C, or U). In
embodiments, one
or both of the additional sequences are arbitrary sequences capable of forming
hairpins. Thus, in
embodiments, the sequence that is the reverse complement of the miRNA is
flanked on the 5' side
and on the 3' side by hairpin structures. Micro-RNA inhibitors, when double
stranded, may include
mismatches between nucleotides on opposite strands. Furthermore, micro-RNA
inhibitors may be
linked to conjugate moieties in order to facilitate uptake of the inhibitor
into a cell. For example,
a micro-RNA inhibitor may be linked to cholesteryl 5-(bis(4-
methoxyphenyl)(phenyl)methoxy)-
3 hydroxypentylcarbamate) which allows passive uptake of a micro-RNA inhibitor
into a cell.
62
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
Micro-RNA inhibitors, including hairpin miRNA inhibitors, are described in
detail in Vermeulen
et al., RNA 13: 723- 730 (2007) and in W02007/095387 and WO 2008/036825 each
of which is
incorporated herein by reference in its entirety. A person of ordinary skill
in the art can select a
sequence from the database for a desired miRNA and design an inhibitor useful
for the methods
disclosed herein.
[0165] Linking groups or bifunctional linking moieties such as those known in
the art are
amenable to the compounds provided herein. Linking groups are useful for
attachment of chemical
functional groups, conjugate groups, reporter groups and other groups to
selective sites in a parent
compound such as for example an AC. In general, a bifunctional linking moiety
includes a
hydrocarbyl moiety having two functional groups. One of the functional groups
is selected to bind
to a parent molecule or compound of interest and the other is selected to bind
essentially any
selected group such as chemical functional group or a conjugate group. Any of
the linkers
described here may be used. In embodiments, the linker includes a chain
structure or an oligomer
of repeating units such as ethylene glycol or amino acid units. Examples of
functional groups that
are routinely used in a bifunctional linking moiety include, but are not
limited to, electrophiles for
reacting with nucleophilic groups and nucleophiles for reacting with
electrophilic groups. In
embodiments, bifunctional linking moieties include amino, hydroxyl, carboxylic
acid, thiol,
unsaturations (e.g., double or triple bonds), and the like. Some nonlimiting
examples of
bifunctional linking moieties include 8-amino-3,6-dioxaoctanoic acid (ADO),
succinimidyl 4-(N-
maleimidomethyl) cyclohexane-l-carboxylate (SMCC) and 6-aminohexanoic acid
(AHEX or
AHA). Other linking groups include, but are not limited to, substituted Cl-C10
alkyl, substituted
or unsubstituted C2-C10 alkenyl or substituted or unsubstituted C2-C10
alkynyl, wherein a
nonlimiting list of sub stituent groups includes hydroxyl, amino, alkoxy,
carboxy, benzyl, phenyl,
nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.
[0166] In embodiments, AC includes nucleotide modification designed to not
support RNase H
activity. Nucleotide modifications of antisense compounds that do not support
RNase H activity
are known and include, but are not limited to, 2'-0-methoxy
ethyl/phosphorothioate (MOE)
modifications. Advantageously, AC with MOE modifications have increased
affinity for target
RNA and increase nuclease stability.
63
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
Immunostimulatory Oligonucleotides
[0167] In embodiments, the therapeutic moiety is an immunostimulatory
oligonucleotide.
Immunostimulatory oligonucleotides (ISS; single-or double- stranded) are
capable of inducing an
immune response when administered to a patient, which may be a mammal or other
patient. ISS
include, e.g., certain palindromes leading to hairpin secondary structures
(see Yamamoto S., et al.
(1992) J. Immunol. 148: 4072-4076), or CpG motifs, as well as other known ISS
features (such as
multi-G domains, see WO 96/11266).
[0168] The immune response may be an innate or an adaptive immune response.
The immune
system is divided into a more innate immune system, and acquired adaptive
immune system of
vertebrates, the latter of which is further divided into humoral cellular
components. In particular
embodiments, the immune response may be mucosal.
[0169] Immunostimulatory nucleic acids are considered to be non-sequence
specific when it is not
required that they specifically bind to and reduce the expression of a target
polynucleotide in order
to provoke an immune response. Thus, certain immunostimulatory nucleic acids
may include a
sequence corresponding to a region of a naturally occurring gene or mRNA, but
they may still be
considered non-sequence specific immunostimulatory nucleic acids.
[0170] In embodiments, the immunostimulatory nucleic acid or oligonucleotide
includes at least
one CpG dinucleotide. The oligonucleotide or CpG dinucleotide may be
unmethylated or
methylated. In another embodiment, the immunostimulatory nucleic acid includes
at least one CpG
dinucleotide having a methylated cytosine. In embodiments, the nucleic acid
includes a single CpG
dinucleotide, wherein the cytosine in said CpG dinucleotide is methylated. In
a specific
embodiment, the nucleic acid includes the sequence 5' TAACGTTGAGGG' CAT 3'
(SEQ ID NO:
369). In an alternative embodiment, the nucleic acid includes at least two CpG
dinucleotides,
wherein at least one cytosine in the CpG dinucleotides is methylated. In a
further embodiment,
each cytosine in the CpG dinucleotides present in the sequence is methylated.
In another
embodiment, the nucleic acid includes a plurality of CpG dinucleotides,
wherein at least one of
said CpG dinucleotides includes a methylated cytosine.
[0171] Additional specific nucleic acid sequences of oligonucleotides (ODNs)
suitable for use in
the compositions and methods are described in Raney et al, Journal of
Pharmacology and
Experimental Therapeutics, 298:1185-1192 (2001). In certain embodiments, ODNs
used in the
64
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
compositions and methods have a phosphodiester("PO") backbone or a
phosphorothioate ("PS")
backbone, and/or at least one methylated cytosine residue in a CpG motif.
Decoy Oligonucleotides
[0172] In embodiments, the therapeutic moiety is a decoy oligonucleotide.
Because transcription
factors recognize their relatively short binding sequences, even in the
absence of surrounding
genomic DNA, short oligonucleotides bearing the consensus binding sequence of
a specific
transcription factor can be used as tools for manipulating gene expression in
living cells. This
strategy involves the intracellular delivery of such "decoy oligonucleotides",
which are then
recognized and bound by the target factor. Occupation of the transcription
factor's DNA-binding
site by the decoy renders the transcription factor incapable of subsequently
binding to the promoter
regions of target genes. Decoys can be used as therapeutic agents, either to
inhibit the expression
of genes that are activated by a transcription factor, or to upregulate genes
that are suppressed by
the binding of a transcription factor. Examples of the utilization of decoy
oligonucleotides may be
found in Mann et al., J. Clin. Invest, 2000, 106: 1071-1075, which is
expressly incorporated by
reference herein, in its entirety.
U] adaptor
[0173] In some embodiments, the therapeutic moiety is a Ul adaptor. Ul
adaptors inhibit polyA
sites and are bifunctional oligonucleotides with a target domain complementary
to a site in the
target gene's terminal exon and a 'Ul domain' that binds to the Ul smaller
nuclear RNA component
of the Ul snRNP (Goraczniak, et al., 2008, Nature Biotechnology, 27(3), 257-
263, which is
expressly incorporated by reference herein, in its entirety). Ul snRNP is a
ribonucleoprotein
complex that functions primarily to direct early steps in spliceosome
formation by binding to the
pre-mRNA exon- intron boundary (Brown and Simpson, 1998, Annu Rev Plant
Physiol Plant Mol
Biol 49:77-95). Nucleotides 2-11 of the 5'end of Ul snRNA base pair bind with
the 5'ss of the pre
mRNA. In one embodiment, oligonucleotides are Ul adaptors. In one embodiment,
the Ul adaptor
can be administered in combination with at least one other iRNA agent.
(CRISPR) Gene-Editing Machinery
[0174] In embodiments, the compounds disclosed herein include one or more CPP
(or cCPP)
conjugated to CRISPR gene-editing machinery. As used herein, "CRISPR gene-
editing
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
machinery" refers to protein, nucleic acids, or combinations thereof, which
may be used to edit a
genome. Non-limiting examples of gene-editing machinery include gRNAs,
nucleases, nuclease
inhibitors, and combinations and complexes thereof The following patent
documents describe
CRISPR gene-editing machinery: U.S. Pat. No. 8,697,359, U.S. Pat. No.
8,771,945, U.S. Pat. No.
8,795,965, U.S. Pat. No. 8,865,406, U.S. Pat. No. 8,871,445, U.S. Pat. No.
8,889,356, U.S. Pat.
No. 8,895,308, U.S. Pat. No. 8,906,616, U.S. Pat. No. 8,932,814, U.S. Pat. No.
8,945,839, U.S.
Pat. No. 8,993,233, U.S. Pat. No. 8,999,641, U.S. Pat. App. No. 14/704,551,
and U.S. Pat. App.
No. 13/842,859. Each of the aforementioned patent documents is incorporated by
reference herein
in its entirety.
[0175] In embodiments, a linker conjugates the cCPP to the CRISPR gene-editing
machinery. Any
linker described in this disclosure or that is known to a person of skill in
the art may be utilized.
gRNA
[0176] In embodiments, the compounds include the CPP (or cCPP) is conjugated
to a gRNA. A
gRNA targets a genomic loci in a prokaryotic or eukaryotic cell.
[0177] In embodiments, the gRNA is a single-molecule guide RNA (sgRNA). A
sgRNA includes
a spacer sequence and a scaffold sequence. A spacer sequence is a short
nucleic acid sequence
used to target a nuclease (e.g., a Cas9 nuclease) to a specific nucleotide
region of interest (e.g., a
genomic DNA sequence to be cleaved). In embodiments, the spacer may be about
17-24 bases in
length, such as about 20 bases in length. In embodiments, the spacer may be
about 15, about 16,
about 17, about 18, about 19, about 20, about 21, about 22, about 23, about
24, about 25, about 26,
about 27, about 28, about 29, or about 30 bases in length. In embodiments, the
spacer may be at
least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at
least 21, at least 22, at least
23, at least 24, at least 25, at least 26, at least 27, at least 28, at least
29, or at least 30 bases in
length. In embodiments, the spacer may be about 15, about 16, about 17, about
18, about 19, about
20, about 21, about 22, about 23, about 24, about 25, about 26, about 27,
about 28, about 29, or
about 30 bases in length. In embodiments, the spacer sequence has between
about 40% to about
80% GC content.
[0178] In embodiments, the spacer targets a site that immediately precedes a
5' protospacer
adjacent motif (PAM). The PAM sequence may be selected based on the desired
nuclease. For
example, the PAM sequence may be any one of the PAM sequences shown in Table 3
below,
66
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
wherein N refers to any nucleic acid, R refers to A or G, Y refers to C or T,
W refers to A or T,
and V refers to A or C or G.
Table 3: Exemplary Nucleases and PAM sequences
PAM sequence (5' to 3') Nuclease Isolated from
NGG SpCas9 Streptococcus pyogenes
NGRRT or NGRRN SaCas9 Staphylococcus aureus
NNNNGAT T NmeCas9 Neisseria meningitidis
NNNNRYAC Cj Cas9 Campylobacter jejuni
NNAGAAW StCas9 Streptococcus thermophiles
TTTV LbCpfl Lachnospiraceae bacterium
TTTV AsCpfl Acidaminococcus sp.
[0179] In embodiments, a spacer may target a sequence of a mammalian gene,
such as a human
gene. In embodiments, the spacer may target a mutant gene. In embodiments, the
spacer may target
a coding sequence. In embodiments, the spacer may target an exonic sequence.
In embodiments,
the spacer may target a polyadenylation site (PS). In embodiments, the spacer
may target a
sequence element of a PS. In embodiments, the spacer may target a
polyadenylation signal (PAS),
an intervening sequence (IS), a cleavage site (CS), a downstream element
(DES), or a portion or
combination thereof. In embodiments, a spacer may target a splicing element
(SE) or a cis-splicing
regulatory element (SRE).
[0180] The scaffold sequence is the sequence within the sgRNA that is
responsible for nuclease
(e.g., Cas9) binding. The scaffold sequence does not include the
spacer/targeting sequence. In
embodiments, the scaffold may be about 1 to about 10, about 10 to about 20,
about 20 to about 30,
about 30 to about 40, about 40 to about 50, about 50 to about 60, about 60 to
about 70, about 70
to about 80, about 80 to about 90, about 90 to about 100, about 100 to about
110, about 110 to
about 120, or about 120 to about 130 nucleotides in length. In embodiments,
the scaffold may be
about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about
9, about 10, about 11,
about 12, about 13, about 14, about 15, about 16, about 17, about 18, about
19, about 20, about 21,
about 22, about 23, about 24, about 25, about 26, about 27, about 28, about
29, about 30, about 31,
about 32, about 33, about 34, about 35, about 36, about 37, about 38, about
39, about 40, about 41,
67
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
about 42, about 43, about 44, about 45, about 46, about 47, about 48, about
49, about 50, about 51,
about 52, about 53, about 54, about 55, about 56, about 57, about 58, about
59, about 60,about 60,
about 61, about 62, about 63, about 64, about 65, about 66, about 67, about
68, about 69, about 70,
about 71, about 72, about 73, about 74, about 75, about 76, about 77, about
78, about 79, about 80,
about 81, about 82, about 83, about 84, about 85, about 86, about 87, about
88, about 89, about 90,
about 91, about 92, about 93, about 94, about 95, about 96, about 97, about
98, about 99, about
100, about 101, about 102, about 103, about 104, about 105, about 106, about
107, about 108,
about 109, about 110, about 111, about 112, about 113, about 114, about 115,
about 116, about
117, about 118, about 119, about 120, about 121, about 122, about 123, about
124, or about 125
nucleotides in length. In embodiments, the scaffold may be at least 10, at
least 20, at least 30, at
least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at
least 100, at least 110, at least
120, or at least 125 nucleotides in length.
[0181] In embodiments, the gRNA is a dual-molecule guide RNA, e.g, crRNA and
tracrRNA. In
embodiments, the gRNA may further include a poly(A) tail.
[0182] In embodiments, a compound that includes a CPP is conjugated to a
nucleic acid that
includes a gRNA. In embodiments, the nucleic acid includes about 1, about 2,
about 3, about 4,
about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12,
about 13, about 14, about
15, about 16, about 17, about 18, about 19, or about 20 gRNAs. In embodiments,
the gRNAs
recognize the same target. In embodiments, the gRNAs recognize different
targets. In
embodiments, the nucleic acid that includes a gRNA includes a sequence
encoding a promoter,
wherein the promoter drives expression of the gRNA.
Nuclease
[0183] In embodiments, the compounds include a cell penetrating peptide
conjugated to a
nuclease. In embodiments, the nuclease is a Type II, Type V-A, Type V-B, Type
VC, Type V-U,
Type VI-B nuclease. In embodiments, the nuclease is a transcription, activator-
like effector
nuclease (TALEN), a meganuclease, or a zinc-finger nuclease. In embodiments,
the nuclease is a
Cas9, Cas12a (Cpfl), Cas12b, Cas12c, Tnp-B like, Cas13a (C2c2), Cas13b, or
Cas14 nuclease.
For example, in some embodiments, the nuclease is a Cas9 nuclease or a Cpfl
nuclease.
[0184] In embodiments, the nuclease is a modified form or variant of a Cas9,
Cas12a (Cpfl),
Cas12b, Cas12c, Tnp-B like, Cas13a (C2c2), Cas13b, or Cas14 nuclease. In
embodiments, the
68
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
nuclease is a modified form or variant of a TAL nuclease, a meganuclease, or a
zinc-finger
nuclease. A "modified" or "variant" nuclease is one that is, for example,
truncated, fused to another
protein (such as another nuclease), catalytically inactivated, etc. In
embodiments, the nuclease may
have at least about 80%, at least about 85%, at least about 90%, at least
about 95%, at least about
98%, at least about 99%, or about 100% sequence identity to a naturally
occurring Cas9, Cas12a
(Cpfl), Cas12b, Cas12c, Tnp-B like, Cas13a (C2c2), Cas13b, Cas14 nuclease, or
a TALEN,
meganuclease, or zinc-finger nuclease. In embodiments, the nuclease is a Cas9
nuclease derived
from S. pyogenes (SpCas9). In embodiments, a nuclease has at least about 90%,
at least about 95%,
at least about 96%, at least about 97%, at least about 98%, or at least about
99% sequence identity
to a Cas9 nuclease derived from S. pyogenes (SpCas9). In embodiments, the
nuclease is a Cas9
derived from S. aureus (SaCas9). In embodiments, the nuclease has at least
about 90%, at least
about 95%, at least about 96%, at least about 97%, at least about 98%, or at
least about 99%
sequence identity to a Cas9 derived from S. aureus (SaCas9). In embodiments,
the Cpfl is a Cpfl
enzyme from Acidaminococcus (species BV3L6, UniProt Accession No. U2UMQ6). In
embodiments, the nuclease has at least about 90%, at least about 95%, at least
about 96%, at least
about 97%, at least about 98%, or at least about 99% sequence identity to a
Cpfl enzyme from
Acidaminococcus (species BV3L6, UniProt Accession No. U2UMQ6).
[0185] In embodiments, the Cpfl is a Cpfl enzyme from Lachnospiraceae (species
ND2006,
UniProt Accession No. A0A182DWE3). In embodiments, the nuclease has at least
about 90%, at
least about 95%, at least about 96%, at least about 97%, at least about 98%,
or at least about 99%
sequence identity to a Cpfl enzyme from Lachnospiraceae. In embodiments, a
sequence encoding
the nuclease is codon optimized for expression in mammalian cells. In
embodiments, the sequence
encoding the nuclease is codon optimized for expression in human cells or
mouse cells.
[0186] In embodiments, a compound that includes a CPP is conjugated to a
nuclease. In
embodiments, the nuclease is a soluble protein.
[0187] In embodiments, a compound that includes a CPP is conjugated to a
nucleic acid encoding
a nuclease. In embodiments, the nucleic acid encoding a nuclease includes a
sequence encoding a
promoter, wherein the promoter drives expression of the nuclease.
69
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
gRNA and Nuclease Combinations
[0188] In embodiments, the compounds include one or more CPP (or cCPP)
conjugated to a gRNA
and a nuclease. In embodiments, the one or more CPP (or cCPP) are conjugated
to a nucleic acid
encoding a gRNA and/or a nuclease. In embodiments, the nucleic acid encoding a
nuclease and a
gRNA includes a sequence encoding a promoter, wherein the promoter drives
expression of the
nuclease and the gRNA. In embodiments, the nucleic acid encoding a nuclease
and a gRNA
includes two promoters, wherein a first promoter controls expression of the
nuclease and a second
promoter controls expression of the gRNA. In embodiments, the nucleic acid
encoding a gRNA
and a nuclease encodes from about 1 to about 20 gRNAs, or from about 1, about
2, about 3, about
4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12,
about 13, about 14,
about 15, about 16, about 17, about 18, or about 19, and up to about 20 gRNAs.
In embodiments,
the gRNAs recognize different targets. In embodiments, the gRNAs recognize the
same target.
[0189] In embodiments, the compounds include a cell penetrating peptide (or
cCPP) conjugated
to a ribonucleoprotein (RNP) that includes a gRNA and a nuclease.
[0190] In embodiments, a composition that includes: (a) a CPP conjugated to a
gRNA and (b) a
nuclease is delivered to a cell. In embodiments, a composition that includes:
(a) a CPP conjugated
to a nuclease and (b) an gRNA is delivered to a cell.
[0191] In embodiments, a composition that includes: (a) a first CPP conjugated
to a gRNA and (b)
a second CPP conjugated to a nuclease is delivered to a cell. In embodiments,
the first CPP and
second CPP are the same. In embodiments, the first CPP and second CPP are
different.
Genetic Element of Interest
[0192] In embodiments, the compounds disclosed herein include a cell
penetrating peptide
conjugated to a genetic element of interest. In embodiments, a genetic element
of interest replaces
a genomic DNA sequence cleaved by a nuclease. Non-limiting examples of genetic
elements of
interest include genes, a single nucleotide polymorphism, promoter, or
terminators.
Nuclease Inhibitors
[0193] In embodiments, the compounds disclosed herein include a cell
penetrating peptide
conjugated to an inhibitor of a nuclease (e.g., Cas9). A limitation of gene
editing is potential off-
target editing. The delivery of a nuclease inhibitor will limit off-target
editing. In embodiments,
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
the nuclease inhibitor is a polypeptide, polynucleotide, or small molecule.
Exemplary nuclease
inhibitors are described in U.S. Publication No. 2020/087354, International
Publication No.
2018/085288, U.S. Publication No. 2018/0382741, International Publication No.
2019/089761,
International Publication No. 2020/068304, International Publication No.
2020/041384, and
International Publication No. 2019/076651, each of which is incorporated by
reference herein in
its entirety.
Therapeutic polyp eptides
[0194] In embodiments, the therapeutic moiety includes a polypeptide. In
embodiments, the
therapeutic moiety includes a protein or a fragment thereof In embodiments,
the therapeutic
moiety includes an RNA binding protein or an RNA binding fragment thereof. In
embodiments,
the therapeutic moiety includes an enzyme. In embodiments, the therapeutic
moiety includes an
RNA-cleaving enzyme or an active fragment thereof.
Conjugate Groups
[0195] In embodiments, ACs are modified by covalent attachment of one or more
conjugate
groups. In general, conjugate groups modify one or more properties of the
attached AC including
but not limited to pharmacodynamic, pharmacokinetic, binding, absorption,
cellular distribution,
cellular uptake, charge and clearance. Conjugate groups are routinely used in
the chemical arts and
are linked directly or via an optional linking moiety or linking group to a
parent compound such
as an AC. Conjugate groups include without limitation, intercalators, reporter
molecules,
polyamines, polyamides, polyethylene glycols, thioethers, polyethers,
cholesterols,
thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, biotin,
phenazine,
phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines,
coumarins and
dyes. In embodiments, the conjugate group is a polyethylene glycol (PEG), and
the PEG is
conjugated to either the AC or the CPP.
[0196] Conjugate groups include lipid moieties such as a cholesterol moiety
(Letsinger et al., Proc.
Natl. Acad. Sci. USA, 1989, 86, 6553); cholic acid (Manoharan et al., Bioorg.
Med. Chem. Lett.,
1994, 4, 1053); a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann.
N.Y. Acad. Sci., 1992,
660, 306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765); a
thiocholesterol
(Oberhauser et al., Nucl. Acids Res., 1992, 20, 533); an aliphatic chain,
e.g., dodecandiol or
71
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 111; Kabanov et
al., FEB S Lett.,
1990, 259, 327; Svinarchuk et al., Biochimie, 1993, 75, 49); a phospholipid,
e.g., di-hexadecyl-
rac-glycerol or triethylammonium-1,2-di-O-hexadecyl-rac-glycero-3-H-
phosphonate (Manoharan
et al., Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acids Res.,
1990, 18, 3777); a
polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides &
Nucleotides, 1995,
14, 969); adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995,
36, 3651); a palmityl
moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229); or an
octadecylamine or
hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp.
Ther.,
1996,277,923).
[0197] Linking groups or bifunctional linking moieties such as those known in
the art are
amenable to the compounds provided herein. Linking groups are useful for
attachment of chemical
functional groups, conjugate groups, reporter groups and other groups to
selective sites in a parent
compound such as for example an AC. In general, a bifunctional linking moiety
comprises a
hydrocarbyl moiety having two functional groups. One of the functional groups
is selected to bind
to a parent molecule or compound of interest and the other is selected to bind
essentially any
selected group such as chemical functional group or a conjugate group. Any of
the linkers
described here may be used. In embodiments, the linker comprises a chain
structure or an oligomer
of repeating units such as ethylene glycol or amino acid units. Examples of
functional groups that
are routinely used in a bifunctional linking moiety include, but are not
limited to, electrophiles for
reacting with nucleophilic groups and nucleophiles for reacting with
electrophilic groups. In
embodiments, bifunctional linking moieties include amino, hydroxyl, carboxylic
acid, thiol,
unsaturations (e.g., double or triple bonds), and the like. Some nonlimiting
examples of
bifunctional linking moieties include 8-amino-3,6-dioxaoctanoic acid (ADO),
succinimidyl 4-(N-
maleimidomethyl) cyclohexane-l-carboxylate (SMCC) and 6-aminohexanoic acid
(AHEX or
AHA). Other linking groups include, but are not limited to, substituted Cl-C10
alkyl, substituted
or unsubstituted C2-C10 alkenyl or substituted or unsubstituted C2-C10
alkynyl, wherein a
nonlimiting list of sub stituent groups includes hydroxyl, amino, alkoxy,
carboxy, benzyl, phenyl,
nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.
[0198] In embodiments, the AC may be linked to a 10 arginine-serine dipeptide
repeat. ACs linked
to 10 arginine-serine dipeptide repeats for the artificial recruitment of
splicing enhancer factors
72
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
have been applied in vitro to induce inclusion of mutated BRCA1 and SMN2 exons
that otherwise
would be skipped. See Cartegni and Krainer 2003, incorporated by reference
herein.
Endosomal Escape Vehicles (EEVs)
[0199] An endosomal escape vehicle (EEV) can be used to transport a cargo
across a cellular
membrane, for example, to deliver the cargo to the cytosol or nucleus of a
cell. Cargo can
include a therapeutic moiety (TM). The EEV can comprise a cell penetrating
peptide (CPP), for
example, a cyclic cell penetrating peptide (cCPP). In embodiments, the EEV
comprises a cCPP,
which is conjugated to an exocyclic peptide (EP). The EP can be referred to
interchangeably as a
modulatory peptide (MP). The EP can comprise a sequence of a nuclear
localization signal
(NLS). The EP can be coupled to the cargo. The EP can be coupled to the cCPP.
The EP can be
coupled to the cargo and the cCPP. Coupling between the EP, cargo, cCPP, or
combinations
thereof, may be non-covalent or covalent. The EP can be attached through a
peptide bond to the
N-terminus of the cCPP. The EP can be attached through a peptide bond to the C-
terminus of the
cCPP. The EP can be attached to the cCPP through a side chain of an amino acid
in the cCPP.
The EP can be attached to the cCPP through a side chain of a lysine which can
be conjugated to
the side chain of a glutamine in the cCPP. The EP can be conjugated to the 5'
or 3' end of an
oligonucleotide cargo. The EP can be coupled to a linker. The exocyclic
peptide can be
conjugated to an amino group of the linker. The EP can be coupled to a linker
via the C-terminus
of an EP and a cCPP through a side chain on the cCPP and/or EP. For example,
an EP may
comprise a terminal lysine which can then be coupled to a cCPP containing a
glutamine through
an amide bond. When the EP contains a terminal lysine, and the side chain of
the lysine can be
used to attach the cCPP, the C- or N-terminus may be attached to a linker on
the cargo.
Exocyclic Peptides
[0200] The exocyclic peptide (EP) can comprise from 2 to 10 amino acid
residues e.g., 2, 3, 4, 5,
6, 7, 8, 9, or 10 amino acid residues, inclusive of all ranges and values
therebetween. The EP can
comprise 6 to 9 amino acid residues. The EP can comprise from 4 to 8 amino
acid residues.
[0201] Each amino acid in the exocyclic peptide may be a natural or non-
natural amino acid. The
term "non-natural amino acid" refers to an organic compound that is a congener
of a natural
amino acid in that it has a structure similar to a natural amino acid so that
it mimics the structure
73
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
and reactivity of a natural amino acid. The non-natural amino acid can be a
modified amino acid,
and/or amino acid analog, that is not one of the 20 common naturally occurring
amino acids or
the rare natural amino acids selenocysteine or pyrrolysine. Non-natural amino
acids can also be
the D-isomer of the natural amino acids. Examples of suitable amino acids
include, but are not
limited to, alanine, allosoleucine, arginine, citrulline, asparagine, aspartic
acid, cysteine,
glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine,
methionine,
napthylalanine, phenylalanine, proline, pyroglutamic acid, serine, threonine,
tryptophan,
tyrosine, valine, a derivative thereof, or combinations thereof. These, and
others amino acids, are
listed in the Table 4 along with their abbreviations used herein. For example,
the amino acids
can be A, G, P, K, R, V, F, H, Nal, or citrulline.
[0202] The EP can comprise at least one positively charged amino acid residue,
e.g., at least one
lysine residue and/or at least one amine acid residue comprising a side chain
comprising a
guanidine group, or a protonated form thereof The EP can comprise 1 or 2 amino
acid residues
comprising a side chain comprising a guanidine group, or a protonated form
thereof. The amino
acid residue comprising a side chain comprising a guanidine group can be an
arginine residue.
Protonated forms can mean salt thereof throughout the disclosure.
[0203] The EP can comprise at least two, at least three or at least four or
more lysine residues.
The EP can comprise 2, 3, or 4 lysine residues. The amino group on the side
chain of each lysine
residue can be substituted with a protecting group, including, for example,
trifluoroacetyl (-
COCF3), allyloxycarbonyl (Alloc), 1-(4,4-dimethy1-2,6-
dioxocyclohexylidene)ethyl (Dde), or
(4,4-dimethy1-2,6-dioxocyclohex-1-ylidene-3)-methylbutyl (ivDde) group. The
amino group on
the side chain of each lysine residue can be substituted with a
trifluoroacetyl (-COCF 3) group.
The protecting group can be included to enable amide conjugation. The
protecting group can be
removed after the EP is conjugated to a cCPP.
[0204] The EP can comprise at least 2 amino acid residues with a hydrophobic
side chain. The
amino acid residue with a hydrophobic side chain can be selected from valine,
proline, alanine,
leucine, isoleucine, and methionine. The amino acid residue with a hydrophobic
side chain can
be valine or proline.
74
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
[0205] The EP can comprise at least one positively charged amino acid residue,
e.g., at least one
lysine residue and/or at least one arginine residue. The EP can comprise at
least two, at least
three or at least four or more lysine residues and/or arginine residues.
[0206] The EP can comprise KK, KR, RR, HH, HK, HR, RH, KKK, KGK, KBK, KBR,
KRK,
KRR, RKK, RRR, KKH, KHK, HKK, HRR, HRH, HHR, HBH, HHH, HHHH (SEQ ID NO:1),
KHKK (SEQ ID NO:2), KKHK (SEQ ID NO:3), KKKH (SEQ ID NO:4), KHKH (SEQ ID
NO:5), HKHK (SEQ ID NO:6), KKKK (SEQ ID NO:7), KKRK (SEQ ID NO:8), KRKK (SEQ
ID NO:9), KRRK (SEQ ID NO:10), RKKR (SEQ ID NO:11), RRRR (SEQ ID NO:12), KGKK
(SEQ ID NO:13), KKGK (SEQ ID NO:14), HBHBH (SEQ ID NO:15), HBKBH (SEQ ID
NO:16), RRRRR (SEQ ID NO:17), KKKKK (SEQ ID NO:18), KKKRK (SEQ ID NO:19),
RKKKK (SEQ ID NO:20), KRKKK (SEQ ID NO:21), KKRKK (SEQ ID NO:22), KKKKR
(SEQ ID NO:23), KBKBK (SEQ ID NO:24), RKKKKG (SEQ ID NO:25), KRKKKG (SEQ ID
NO:26), KKRKKG (SEQ ID NO:27), KKKKRG (SEQ ID NO:28), RKKKKB (SEQ ID NO:29),
KRKKKB (SEQ ID NO:30), KKRKKB (SEQ ID NO:31), KKKKRB (SEQ ID NO:32),
KKKRKV (SEQ ID NO:33), RRRRRR (SEQ ID NO:34), HEIHHHH (SEQ ID NO:35),
RHRHRH (SEQ ID NO:36), HRHRHR (SEQ ID NO:37), KRKRKR (SEQ ID NO:38),
RKRKRK (SEQ ID NO:39), RBRBRB (SEQ ID NO:40), KBKBKB (SEQ ID NO:41),
PKKKRKV (SEQ ID NO:42), PGKKRKV (SEQ ID NO:43), PKGKRKV (SEQ ID NO:44),
PKKGRKV (SEQ ID NO:45), PKKKGKV (SEQ ID NO:46), PKKKRGV (SEQ ID NO:47), or
PKKKRKG (SEQ ID NO:48), wherein B is beta-alanine. The amino acids in the EP
can have D
or L stereochemistry.
[0207] The EP can comprise KK, KR, RR, KKK, KGK, KBK, KBR, KRK, KRR, RKK, RRR,
KKKK (SEQ ID NO:7), KKRK (SEQ ID NO:8), KRKK (SEQ ID NO:9), KRRK (SEQ ID
NO:10), RKKR (SEQ ID NO:11), RRRR (SEQ ID NO:12), KGKK (SEQ ID NO:13), KKGK
(SEQ ID NO:14), KKKKK (SEQ ID NO: 18), KKKRK (SEQ ID NO:19), KBKBK (SEQ ID
NO:24), KKKRKV (SEQ ID NO:33), PKKKRKV (SEQ ID NO:42), PGKKRKV (SEQ ID
NO:43), PKGKRKV (SEQ ID NO:44), PKKGRKV (SEQ ID NO:45), PKKKGKV (SEQ ID
NO:46), PKKKRGV (SEQ ID NO:47), or PKKKRKG (SEQ ID NO:48). The EP can comprise
PKKKRKV (SEQ ID NO:42), RR, RRR, RHR, RBR, RBRBR (SEQ ID NO:49), RBHBR (SEQ
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
ID NO:50), or HBRBH (SEQ ID NO:51), wherein B is beta-alanine. The amino acids
in the EP
can have D or L stereochemistry.
[0208] The EP can consist of KK, KR, RR, KKK, KGK, KBK, KBR, KRK, KRR, RKK,
RRR,
KKKK (SEQ ID NO:7), KKRK (SEQ ID NO:8), KRKK (SEQ ID NO:9), KRRK (SEQ ID
NO:10), RKKR (SEQ ID NO:11), RRRR (SEQ ID NO:12), KGKK (SEQ ID NO:13), KKGK
(SEQ ID NO:14), KKKKK (SEQ ID NO:18), KKKRK (SEQ ID NO:19), KBKBK (SEQ ID
NO:24), KKKRKV (SEQ ID NO:33), PKKKRKV (SEQ ID NO:42), PGKKRKV (SEQ ID
NO:Z43), PKGKRKV (SEQ ID NO:Z44), PKKGRKV (SEQ ID NO:Z45), PKKKGKV (SEQ ID
NO:46), PKKKRGV (SEQ ID NO:47), or PKKKRKG (SEQ ID NO:48). The EP can consist
of
PKKKRKV (SEQ ID NO:42), RR, RRR, RHR, RBR, RBRBR (SEQ ID NO:49), RBHBR (SEQ
ID NO:50), or HBRBH (SEQ ID NO:51), wherein B is beta-alanine. The amino acids
in the EP
can have D or L stereochemistry.
[0209] The EP can comprise an amino acid sequence identified in the art as a
nuclear
localization sequence (NLS). The EP can consist of an amino acid sequence
identified in the art
as a nuclear localization sequence (NLS). The EP can comprise an NLS
comprising the amino
acid sequence PKKKRKV (SEQ ID NO:42). The EP can consist of an NLS comprising
the
amino acid sequence PKKKRKV (SEQ ID NO:42). The EP can comprise an NLS
comprising an
amino acid sequence selected from NLSKRPAAIKKAGQAKKKK (SEQ ID NO:52),
PAAKRVKLD (SEQ ID NO:53), RQRRNELKRSF (SEQ ID NO:54),
RMRKFKNKGKDTAELRRRRVEVSVELR (SEQ ID NO:Z55), KAKKDEQILKRRNV (SEQ
ID NO:56), VSRKRPRP (SEQ ID NO:57), PPKKARED (SEQ ID NO:58), PQPKKKPL (SEQ
ID NO:59), SALIKKKKKMAP (SEQ ID NO:60), DRLRR (SEQ ID NO:61), PKQKKRK (SEQ
ID NO:62), RKLKKKIKKL (SEQ ID NO:63), REKKKFLKRR (SEQ ID NO:64),
KRKGDEVDGVDEVAKKKSKK (SEQ ID NO:65), and RKCLQAGMNLEARKTKK (SEQ ID
NO:66). The EP can consist of an NLS comprising an amino acid sequence
selected from
NLSKRPAAIKKAGQAKKKK (SEQ ID NO:52), PAAKRVKLD (SEQ ID NO:53),
RQRRNELKRSF (SEQ ID NO:54), RMRKFKNKGKDTAELRRRRVEVSVELR (SEQ ID
NO:55), KAKKDEQILKRRNV (SEQ ID NO:56), VSRKRPRP (SEQ ID NO:57), PPKKARED
(SEQ ID NO:58), PQPKKKPL (SEQ ID NO:59), SALIKKKKKMAP (SEQ ID NO:60), DRLRR
(SEQ ID NO:61), PKQKKRK (SEQ ID NO:62), RKLKKKIKKL (SEQ ID NO:63),
76
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
REKKKFLKRR (SEQ ID NO:64), KRKGDEVDGVDEVAKKKSKK (SEQ ID NO:65), and
RKCLQAGMNLEARKTKK (SEQ ID NO:66).
[0210] All exocyclic sequences can also contain an N-terminal acetyl group.
Hence, for
example, the EP can have the structure: Ac-PKKKRKV (SEQ ID NO:42).
Cell Penetrating Peptides (CPP)
[0211] The cell penetrating peptide (CPP) can comprise 6 to 20 amino acid
residues. The cell
penetrating peptide can be a cyclic cell penetrating peptide (cCPP). The cCPP
is capable of
penetrating a cell membrane. An exocyclic peptide (EP) can be conjugated to
the cCPP, and the
resulting construct can be referred to as an endosomal escape vehicle (EEV).
The cCPP can
direct a cargo (e.g., a therapeutic moiety (TM) such as an oligonucleotide,
peptide or small
molecule) to penetrate the membrane of a cell. The cCPP can deliver the cargo
to the cytosol of
the cell. The cCPP can deliver the cargo to a cellular location where a target
(e.g., pre-mRNA) is
located. To conjugate the cCPP to a cargo (e.g., peptide, oligonucleotide, or
small molecule), at
least one bond or lone pair of electrons on the cCPP can be replaced.
[0212] The total number of amino acid residues in the cCPP is in the range of
from 6 to 20 amino
acid residues, e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20
amino acid residues,
inclusive of all ranges and subranges therebetween. The cCPP can comprise 6 to
13 amino acid
residues. The cCPP disclosed herein can comprise 6 to 10 amino acids. By way
of example, cCPP
comprising 6-10 amino acid residues can have a structure according to any of
Formula I-A to I-E:
77
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
AA,I
AA1,AA2 AA8¨AA1 AA9
.Aiki, AA7 / \ / \AA2
AA AA, 2 / \ AA7 AA2 AA8 AA3
i I AA6 AA3 I I I I
AA5 \ / / AA6 AA3 AA7 /
AA4
\ \
N V AA3
AA4 AA6¨AA4 AA6¨AA4 AA6¨AA5
1-A I-B I-C T-D
or
, , , ,
z AA1 0 ¨AN
\
AA9 AA2
/ \
AA8 AA3
\ /
AA7 AA4
/
AA6¨AA5
I-E
, wherein AA', AA2, AA3, AA4, AA5, AA6, AA7, AAg, AA9, and AAio
are amino acid residues.
[0213] The cCPP can comprise 6 to 8 amino acids. The cCPP can comprise 8 amino
acids.
[0214] Each amino acid in the cCPP may be a natural or non-natural amino acid.
The term "non-
natural amino acid" refers to an organic compound that is a congener of a
natural amino acid in
that it has a structure similar to a natural amino acid so that it mimics the
structure and reactivity
of a natural amino acid. The non-natural amino acid can be a modified amino
acid, and/or amino
acid analog, that is not one of the 20 common naturally occurring amino acids
or the rare natural
amino acids selenocysteine or pyrrolysine. Non-natural amino acids can also be
a D-isomer of a
natural amino acid. Examples of suitable amino acids include, but are not
limited to, alanine,
allosoleucine, arginine, citrulline, asparagine, aspartic acid, cysteine,
glutamine, glutamic acid,
glycine, histidine, isoleucine, leucine, lysine, methionine, napthylalanine,
phenylalanine, proline,
pyroglutamic acid, serine, threonine, tryptophan, tyrosine, valine, a
derivative thereof, or
combinations thereof These, and others amino acids, are listed in the Table 5
along with their
abbreviations used herein.
Table 4. Amino Acid Abbreviations
Amino Acid Abbreviations* Abbreviations*
L-amino acid D-amino acid
2-[2-[2-aminoethoxy]ethoxy]acetic acid AEEA, miniPEG, PEG2 NA
Alanine Ala (A) ala (a)
78
CA 03222824 2023-12-07
WO 2022/271818
PCT/US2022/034517
Amino Acid Abbreviations* Abbreviations*
L-amino acid D-amino acid
Allo-isoleucine Aile Aile
Arginine Arg (R) arg (r)
Asparagine Asn (N) asn (n)
aspartic acid Asp (D) asp (d)
Cy steine Cys (C) cys (c)
Citrulline Cit Cit
Cycl ohexyl al anine Cha cha
2,3-diaminopropionic acid Dap dap
4-fluorophenylalanine Fpa (/) pfa
glutamic acid Glu (E) glu (e)
glutamine Gin (Q) gin (q)
glycine Gly (G) gly (g)
hi sti dine His (H) his (h)
Homoproline (aka pipecolic acid) Pip (0) Pip (e)
isoleucine Ile (I) ile (i)
leucine Leu (L) leu (1)
lysine Lys (K) lys (k)
methionine Met (M) met (m)
3 -(2-naphthyl)-alanine Nal (4:1)) nal (4))
3 -(1 -naphthyl)-al anine 1-Nal 1-nal
norleucine Nle (2) nle
phenylalanine Phe (F) phe (f)
phenylglycine Phg (1ll) phg
4-(phosphonodifluoromethyl)phenylalanine F2Pmp (A) f2pmp
proline Pro (P) pro (p)
sarcosine Sar (E) sar
selenocysteine Sec (U) sec (u)
serine Ser (S) ser (s)
threonine Thr (T) thr (y)
tyrosine Tyr (Y) tyr (y)
tryptophan Trp (W) trp (w)
valine Val (V) val (v)
Tert-butyl-alanine Tie tie
Penicillamine Pen Pen
Homoargi nine HomoArg homoarg
Nicotinyl-lysine Lys(NIC) lys(NIC)
Triflouroacetyl -lysine Lys(TFA) lys(TFA)
Methyl -leucine MeLeu meLeu
3 -(3 -benzothieny1)-al anine Bta bta
* single letter abbreviations: capital letters indicate the L-amino acid form,
lower
case letter indicate the D-amino acid form.
79
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
[0215] As used herein, "polyethylene glycol" and "PEG" are used
interchangeably. "PEGm," and
"PEG.," are, or are derived from, a molecule of the formula HO(C0)-(CH2),-
(OCH2CH2).-
NH2 where n is any integer from 1 to 5 and m is any integer from 1 to 23. In
embodiments, n is 1
or 2. In embodiments, n is 1. In embodiments, n is 2. In embodiments, n is 1
and m is 2. In
embodiments, n is 2 and m is 2. In embodiments, n is 1 and m is 4. In
embodiments, n is 2 and m
is 4. In embodiments, n is 1 and m is 12. In embodiments, n is 2 and m is 12.
[0216] As used herein, "miniPEGm" or "miniPEG." are, or are derived from, a
molecule of the
formula HO(C0)-(CH2).-(OCH2CH2).4'4H2 where n is 1 and m is any integer from 1
to 23. For
example, "miniPEG2" or "miniPEG2" is, or is derived from, (2-[2-[2-
aminoethoxy]ethoxy]acetic
acid), and "miniPEG4" or "miniPEG4" is, or is derived from, HO(C0)-(CH2),-
(OCH2CH2).-
NH2 where n is 1 and m is 4.
[0217] The cCPP can comprise 4 to 20 amino acids, wherein: (i) at least one
amino acid has a
side chain comprising a guanidine group, or a protonated form thereof (ii) at
least one amino
0 NH 0
H2NANA' H2NA N)y N N-
acid has no side chain or a side chain comprising H H H
\.
N N¨ N,4f
, or a protonated form thereof; and (iii) at least two amino acids
independently have a side chain comprising an aromatic or heteroaromatic
group.
0
H2NAN
[0218] At least two amino acids can have no side chain or a side chain
comprising
NH 0
H2NAN)Y N- N(N)aµ FiNO I
H H H ,
or a protonated form thereof As
used herein, when no side chain is present, the amino acid has two hydrogen
atoms on the carbon
atom(s) (e.g., -CH2-) linking the amine and carboxylic acid.
[0219] The amino acid having no side chain can be glycine or 13-alanine.
[0220] The cCPP can comprise from 6 to 20 amino acid residues which form the
cCPP, wherein:
(i) at least one amino acid can be glycine, 13-alanine, or 4-aminobutyric acid
residues; (ii) at least
one amino acid can have a side chain comprising an aryl or heteroaryl group;
and (iii) at least
CA 03222824 2023-12-07
WO 2022/271818
PCT/US2022/034517
0 NH 0
H2NANN H2NAN)Y
one amino acid has a side chain comprising a guanidine group,
N 1\n= N N
H H , or a protonated form thereof
[0221] The cCPP can comprise from 6 to 20 amino acid residues which form the
cCPP, wherein:
(i) at least two amino acid can independently be glycine, 13-alanine, or 4-
aminobutyric acid
residues; (ii) at least one amino acid can have a side chain comprising an
aryl or heteroaryl
group; and (iii) at least one amino acid has a side chain comprising a
guanidine group,
0 NH 0 CN H2NANN H2NA N i N HNOI
N N
H HH , or
a protonated
form thereof.
[0222] The cCPP can comprise from 6 to 20 amino acid residues which form the
cCPP, wherein:
(i) at least three amino acids can independently be glycine, 13-alanine, or 4-
aminobutyric acid
residues; (ii) at least one amino acid can have a side chain comprising an
aromatic or
heteroaromatic group; and (iii) at least one amino acid can have a side chain
comprising a
0 NH 0 H2NANN H2NAN)Y NLN)k N)LN HNOIN. NN,/
guanidine group, H H H
or a protonated form thereof
Glycine and Related Amino Acid Residues
[0223] The cCPP can comprise (i) 1, 2, 3, 4, 5, or 6 glycine, 13-alanine, 4-
aminobutyric acid
residues, or combinations thereof The cCPP can comprise (i) 2 glycine, 13-
alanine, 4-
aminobutyric acid residues, or combinations thereof. The cCPP can comprise (i)
3 glycine, 13-
alanine, 4-aminobutyric acid residues, or combinations thereof. The cCPP can
comprise (i) 4
glycine, 13-alanine, 4-aminobutyric acid residues, or combinations thereof.
The cCPP can
comprise (i) 5 glycine, 13-alanine, 4-aminobutyric acid residues, or
combinations thereof The
cCPP can comprise (i) 6 glycine, 13-alanine, 4-aminobutyric acid residues, or
combinations
thereof. The cCPP can comprise (i) 3, 4, or 5 glycine, 13-alanine, 4-
aminobutyric acid residues, or
81
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
combinations thereof The cCPP can comprise (i) 3 or 4 glycine, 13-alanine, 4-
aminobutyric acid
residues, or combinations thereof
[0224] The cCPP can comprise (i) 1, 2, 3, 4, 5, or 6 glycine residues. The
cCPP can comprise (i)
2 glycine residues. The cCPP can comprise (i) 3 glycine residues. The cCPP can
comprise (i) 4
glycine residues. The cCPP can comprise (i) 5 glycine residues. The cCPP can
comprise (i) 6
glycine residues. The cCPP can comprise (i) 3, 4, or 5 glycine residues. The
cCPP can comprise
(i) 3 or 4 glycine residues. The cCPP can comprise (i) 2 or 3 glycine
residues. The cCPP can
comprise (i) 1 or 2 glycine residues.
[0225] The cCPP can comprise (i) 3, 4, 5, or 6 glycine, 13-alanine, 4-
aminobutyric acid residues,
or combinations thereof. The cCPP can comprise (i) 3 glycine, 13-alanine, 4-
aminobutyric acid
residues, or combinations thereof The cCPP can comprise (i) 4 glycine, 13-
alanine, 4-
aminobutyric acid residues, or combinations thereof. The cCPP can comprise (i)
5 glycine, 0-
alanine, 4-aminobutyric acid residues, or combinations thereof. The cCPP can
comprise (i) 6
glycine, 13-alanine, 4-aminobutyric acid residues, or combinations thereof.
The cCPP can
comprise (i) 3, 4, or 5 glycine, 13-alanine, 4-aminobutyric acid residues, or
combinations thereof.
The cCPP can comprise (i) 3 or 4 glycine, 13-alanine, 4-aminobutyric acid
residues, or
combinations thereof
[0226] The cCPP can comprise at least three glycine residues. The cCPP can
comprise (i) 3, 4, 5,
or 6 glycine residues. The cCPP can comprise (i) 3 glycine residues. The cCPP
can comprise (i)
4 glycine residues. The cCPP can comprise (i) 5 glycine residues. The cCPP can
comprise (i) 6
glycine residues. The cCPP can comprise (i) 3, 4, or 5 glycine residues. The
cCPP can comprise
(i) 3 or 4 glycine residues
[0227] In embodiments, none of the glycine, 13-alanine, or 4-aminobutyric acid
residues in the
cCPP are contiguous. Two or three glycine, 13-alanine, 4-or aminobutyric acid
residues can be
contiguous. Two glycine, 13-alanine, or 4-aminobutyric acid residues can be
contiguous.
[0228] In embodiments, none of the glycine residues in the cCPP are
contiguous. Each glycine
residues in the cCPP can be separated by an amino acid residue that cannot be
glycine. Two or
three glycine residues can be contiguous. Two glycine residues can be
contiguous
82
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
Amino Acid Side Chains with an Aromatic or Heteroaromatic Group
[0229] The cCPP can comprise (ii) 2, 3, 4, 5 or 6 amino acid residues
independently having a
side chain comprising an aromatic or heteroaromatic group. The cCPP can
comprise (ii) 2 amino
acid residues independently having a side chain comprising an aromatic or
heteroaromatic group.
The cCPP can comprise (ii) 3 amino acid residues independently having a side
chain comprising
an aromatic or heteroaromatic group. The cCPP can comprise (ii) 4 amino acid
residues
independently having a side chain comprising an aromatic or heteroaromatic
group. The cCPP
can comprise (ii) 5 amino acid residues independently having a side chain
comprising an
aromatic or heteroaromatic group. The cCPP can comprise (ii) 6 amino acid
residues
independently having a side chain comprising an aromatic or heteroaromatic
group. The cCPP
can comprise (ii) 2, 3, or 4 amino acid residues independently having a side
chain comprising an
aromatic or heteroaromatic group. The cCPP can comprise (ii) 2 or 3 amino acid
residues
independently having a side chain comprising an aromatic or heteroaromatic
group.
[0230] The cCPP can comprise (ii) 2, 3, 4, 5 or 6 amino acid residues
independently having a
side chain comprising an aromatic group. The cCPP can comprise (ii) 2 amino
acid residues
independently having a side chain comprising an aromatic group. The cCPP can
comprise (ii) 3
amino acid residues independently having a side chain comprising an aromatic
group. The cCPP
can comprise (ii) 4 amino acid residues independently having a side chain
comprising an
aromatic group. The cCPP can comprise (ii) 5 amino acid residues independently
having a side
chain comprising an aromatic group. The cCPP can comprise (ii) 6 amino acid
residues
independently having a side chain comprising an aromatic group. The cCPP can
comprise (ii) 2,
3, or 4 amino acid residues independently having a side chain comprising an
aromatic group. The
cCPP can comprise (ii) 2 or 3 amino acid residues independently having a side
chain comprising
an aromatic group.
[0231] The aromatic group can be a 6- to 14-membered aryl. Aryl can be phenyl,
naphthyl or
anthracenyl, each of which is optionally substituted. Aryl can be phenyl or
naphthyl, each of
which is optionally substituted. The heteroaromatic group can be a 6- to 14-
membered heteroaryl
having 1, 2, or 3 heteroatoms selected from N, 0, and S. Heteroaryl can be
pyridyl, quinolyl, or
isoquinolyl.
83
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
102321 The amino acid residue having a side chain comprising an aromatic or
heteroaromatic
group can each independently be bis(homonaphthylalanine), homonaphthylalanine,
naphthylalanine, phenylglycine, bis(homophenylalanine), homophenylalanine,
phenylalanine,
tryptophan, 3-(3-benzothieny1)-alanine, 3-(2-quinoly1)-alanine, 0-
benzylserine, 3-(4-
(benzyloxy)pheny1)-alanine, S-(4-methylbenzyl)cy steine, N-(naphthalen-2-
yl)glutamine, 3 -(1, 1 '-
bipheny1-4-y1)-alanine, 3-(3-benzothieny1)-alanine or tyrosine, each of which
is optionally
substituted with one or more substituents. The amino acid having a side chain
comprising an
aromatic or heteroaromatic group can each independently be selected from:
0 140:1
0
H2N CO2H H2N CO2H H2N CO2H
3 -(2 -quinoly1)-a lanine 0-benzylserine 3 -(4-(benzyloxy)p heny1)-al an
ine
N
H2N CO2H H2NCO2H H2N CO2H
S-(4-methylbenzyl)cysteine N5-(n aph th al en-2-yl)gl utamine 3-(1,1'-bipheny1-
4-y1)-a1anine
and
H2N CO2H
3 -(3 -benzothieny1)-alanine
, wherein the H on the N-terminus and/or the H on the C-
terminus are replaced by a peptide bond.
102331 The amino acid residue having a side chain comprising an aromatic or
heteroaromatic
group can each be independently a residue of phenylalanine, naphthylalanine,
phenylglycine,
homophenylalanine, homonaphthylalanine, bis(homophenylalanine), bis-
(homonaphthylalanine),
tryptophan, or tyrosine, each of which is optionally substituted with one or
more substituents.
84
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
The amino acid residue having a side chain comprising an aromatic group can
each
independently be a residue of tyrosine, phenylalanine, 1-naphthylalanine, 2-
naphthylalanine,
tryptophan, 3-benzothienylalanine, 4-phenylphenylalanine, 3,4-
difluorophenylalanine, 4-
trifluoromethylphenylalanine, 2,3,4,5,6-pentafluorophenylalanine,
homophenylalanine, f3-
homophenylalanine, 4-tert-butyl-phenylalanine, 4-pyridinylalanine, 3-
pyridinylalanine, 4-
methylphenylalanine, 4-fluorophenylalanine, 4-chlorophenylalanine, 3-(9-
anthry1)-alanine. The
amino acid residue having a side chain comprising an aromatic group can each
independently be
a residue of phenylalanine, naphthylalanine, phenylglycine, homophenylalanine,
or
homonaphthylalanine, each of which is optionally substituted with one or more
substituents. The
amino acid residue having a side chain comprising an aromatic group can each
be independently
a residue of phenylalanine, naphthylalanine, homophenylalanine,
homonaphthylalanine,
bis(homonaphthylalanine), or bis(homonaphthylalanine), each of which is
optionally substituted
with one or more substituents. The amino acid residue having a side chain
comprising an
aromatic group can each be independently a residue of phenylalanine or
naphthylalanine, each of
which is optionally substituted with one or more substituents. At least one
amino acid residue
having a side chain comprising an aromatic group can be a residue of
phenylalanine. At least two
amino acid residues having a side chain comprising an aromatic group can be
residues of
phenylalanine. Each amino acid residue having a side chain comprising an
aromatic group can be
a residue of phenylalanine.
[0234] In embodiments, none of the amino acids having the side chain
comprising the aromatic
or heteroaromatic group are contiguous. Two amino acids having the side chain
comprising the
aromatic or heteroaromatic group can be contiguous. Two contiguous amino acids
can have
opposite stereochemistry. The two contiguous amino acids can have the same
stereochemistry.
Three amino acids having the side chain comprising the aromatic or
heteroaromatic group can be
contiguous. Three contiguous amino acids can have the same stereochemistry.
Three contiguous
amino acids can have alternating stereochemistry.
[0235] The amino acid residues comprising aromatic or heteroaromatic groups
can be L-amino
acids. The amino acid residues comprising aromatic or heteroaromatic groups
can be D-amino
acids. The amino acid residues comprising aromatic or heteroaromatic groups
can be a mixture
of D- and L-amino acids.
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
[0236] The optional substituent can be any atom or group which does not
significantly reduce
(e.g., by more than 50%) the cytosolic delivery efficiency of the cCPP, e.g.,
compared to an
otherwise identical sequence which does not have the substituent. The optional
substituent can be
a hydrophobic substituent or a hydrophilic substituent. The optional
substituent can be a
hydrophobic substituent. The substituent can increase the solvent-accessible
surface area (as
defined herein) of the hydrophobic amino acid. The substituent can be halogen,
alkyl, alkenyl,
alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, aryl,
heteroaryl, alkoxy, aryloxy,
acyl, alkylcarbamoyl, alkylcarboxamidyl, alkoxycarbonyl, alkylthio, or
arylthio. The substituent
can be halogen.
[0237] While not wishing to be bound by theory, it is believed that amino
acids having an
aromatic or heteroaromatic group having higher hydrophobicity values (i.e.,
amino acids having
side chains comprising aromatic or heteroaromatic groups) can improve
cytosolic delivery
efficiency of a cCPP relative to amino acids having a lower hydrophobicity
value. Each
hydrophobic amino acid can independently have a hydrophobicity value greater
than that of
glycine. Each hydrophobic amino acid can independently be a hydrophobic amino
acid having a
hydrophobicity value greater than that of alanine. Each hydrophobic amino acid
can
independently have a hydrophobicity value greater or equal to phenylalanine.
Hydrophobicity
may be measured using hydrophobicity scales known in the art. Table 5 lists
hydrophobicity
values for various amino acids as reported by Eisenberg and Weiss (Proc. Natl.
Acad. Sci. U. S.
A. 1984;81(1):140-144), Engleman, et al. (Ann. Rev. of Biophys. Biophys. Chem.
1986;1986(15):321-53), Kyte and Doolittle (J. Mol. Biol. 1982;157(1):105-132),
Hoop and
Woods (Proc. Natl. Acad. Sci. U. S. A. 1981;78(6):3824-3828), and Janin
(Nature. 1979;277(5696):491-492), the entirety of each of which is herein
incorporated by
reference. Hydrophobicity can be measured using the hydrophobicity scale
reported in
Engleman, et al.
Table 5. Amino Acid Hydrophobicity
Amino Eisenberg Engleman Kyrie and Hoop and
Group Janin
Acid and Weiss et al. Doolittle Woods
Ile Nonpolar 0.73 3.1 4.5 -1.8 0.7
Phe Nonpolar 0.61 3.7 2.8 -2.5 0.5
Val Nonpolar 0.54 2.6 4.2 -1.5 0.6
Leu Nonpolar 0.53 2.8 3.8 -1.8 0.5
86
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
Amino Eisenberg Engleman Kyrie and Hoop and
Group Janin
Acid and Weiss et at. Doolittle Woods
Trp Nonpolar 0.37 1.9 -0.9 -3.4 0.3
Met Nonpolar 0.26 3.4 1.9 -1.3 0.4
Ala Nonpolar 0.25 1.6 1.8 -0.5 0.3
Gly Nonpolar 0.16 1.0 -0.4 0.0 0.3
Cys Unch/Polar 0.04 2.0 2.5 -1.0 0.9
Tyr Unch/Polar 0.02 -0.7 -1.3 -2.3 -0.4
Pro Nonpolar -0.07 -0.2 -1.6 0.0 -0.3
Thr Unch/Polar -0.18 1.2 -0.7 -0.4 -0.2
Ser Unch/Polar -0.26 0.6 -0.8 0.3 -0.1
His Charged -0.40 -3.0 -3.2 -0.5 -0.1
Glu Charged -0.62 -8.2 -3.5 3.0 -0.7
Asn Unch/Polar -0.64 -4.8 -3.5 0.2 -0.5
Gln Unch/Polar -0.69 -4.1 -3.5 0.2 -0.7
Asp Charged -0.72 -9.2 -3.5 3.0 -0.6
Lys Charged -1.10 -8.8 -3.9 3.0 -1.8
Arg Charged -1.80 -12.3 -4.5 3.0 -1.4
[0238] The size of the aromatic or heteroaromatic groups may be selected to
improve cytosolic
delivery efficiency of the cCPP. While not wishing to be bound by theory, it
is believed that a
larger aromatic or heteroaromatic group on the side chain of amino acid may
improve cytosolic
delivery efficiency compared to an otherwise identical sequence having a
smaller hydrophobic
amino acid. The size of the hydrophobic amino acid can be measured in terms of
molecular
weight of the hydrophobic amino acid, the steric effects of the hydrophobic
amino acid, the
solvent-accessible surface area (SASA) of the side chain, or combinations
thereof The size of
the hydrophobic amino acid can be measured in terms of the molecular weight of
the
hydrophobic amino acid, and the larger hydrophobic amino acid has a side chain
with a
molecular weight of at least about 90 g/mol, or at least about 130 g/mol, or
at least about 141
g/mol. The size of the amino acid can be measured in terms of the SASA of the
hydrophobic side
chain. The hydrophobic amino acid can have a side chain with a SASA of greater
than or equal
to alanine, or greater than or equal to glycine. Larger hydrophobic amino
acids can have a side
chain with a SASA greater than alanine, or greater than glycine. The
hydrophobic amino acid
can have an aromatic or heteroaromatic group with a SASA greater than or equal
to about
piperidine-2-carboxylic acid, greater than or equal to about tryptophan,
greater than or equal to
about phenylalanine, or greater than or equal to about naphthylalanine. A
first hydrophobic
87
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
amino acid (AAFH) can have a side chain with a SASA of at least about 200 A2,
at least about
210 A2, at least about 220 A2, at least about 240 A2, at least about 250 A2,
at least about 260 A2,
at least about 270 A2, at least about 280 A2, at least about 290 A2, at least
about 300 A2, at least
about 310 A2, at least about 320 A2, or at least about 330 A2. A second
hydrophobic amino acid
(AAH2) can have a side chain with a SASA of at least about 200 A2, at least
about 210 A2, at least
about 220 A2, at least about 240 A2, at least about 250 A2, at least about 260
A2, at least about
270 A2, at least about 280 A2, at least about 290 A2, at least about 300 A2,
at least about 310 A2,
at least about 320 A2, or at least about 330 A2. The side chains of AAFH and
AAH2 can have a
combined SASA of at least about 350 A2, at least about 360 A2, at least about
370 A2, at least
about 380 A2, at least about 390 A2, at least about 400 A2, at least about 410
A2, at least about
420 A2, at least about 430 A2, at least about 440 A2, at least about 450 A2,
at least about 460 A2,
at least about 470 A2, at least about 480 A2, at least about 490 A2, greater
than about 500 A2, at
least about 510 A2, at least about 520 A2, at least about 530 A2, at least
about 540 A2, at least
about 550 A2, at least about 560 A2, at least about 570 A2, at least about 580
A2, at least about
590 A2, at least about 600 A2, at least about 610 A2, at least about 620 A2,
at least about 630 A2,
at least about 640 A2, greater than about 650 A2, at least about 660 A2, at
least about 670 A2, at
least about 680 A2, at least about 690 A2, or at least about 700 A2. AAH2 can
be a hydrophobic
amino acid residue with a side chain having a SASA that is less than or equal
to the SASA of the
hydrophobic side chain of AAFH. By way of example, and not by limitation, a
cCPP having a
Nal-Arg motif may exhibit improved cytosolic delivery efficiency compared to
an otherwise
identical cCPP having a Phe-Arg motif; a cCPP having a Phe-Nal-Arg motif may
exhibit
improved cytosolic delivery efficiency compared to an otherwise identical cCPP
having a Nal-
Phe-Arg motif; and a phe-Nal-Arg motif may exhibit improved cytosolic delivery
efficiency
compared to an otherwise identical cCPP having a nal-Phe-Arg motif
[0239] As used herein, "hydrophobic surface area" or "SASA" refers to the
surface area
(reported as square Angstroms; A2) of an amino acid side chain that is
accessible to a solvent.,
SASA can be calculated using the 'rolling ball' algorithm developed by Shrake
& Rupley Plot
Biol. 79 (2): 351-71), which is herein incorporated by reference in its
entirety for all purposes.
This algorithm uses a "sphere" of solvent of a particular radius to probe the
surface of the
88
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
molecule. A typical value of the sphere is 1.4 A, which approximates to the
radius of a water
molecule.
[0240] SASA values for certain side chains are shown below in Table 6. The
SASA values
described herein are based on the theoretical values listed in Table 6 below,
as reported by Tien,
et al. (PLOS ONE 8(11): e80635, available at
doi.org/10.1371/journal.pone.0080635), which is
herein incorporated by reference in its entirety for all purposes.
Table 6. Amino Acid SASA Values
Residue Theoretical Empirical Miller et al. (1987) Rose et al.
(1985)
Alanine 129.0 121.0 113.0 118.1
Arginine 274.0 265.0 241.0 256.0
Asparagine 195.0 187.0 158.0 165.5
Aspartate 193.0 187.0 151.0 158.7
Cysteine 167.0 148.0 140.0 146.1
Glutamate 223.0 214.0 183.0 186.2
Glutamine 225.0 214.0 189.0 193.2
Glycine 104.0 97.0 85.0 88.1
Histidine 224.0 216.0 194.0 202.5
Isoleucine 197.0 195.0 182.0 181.0
Leucine 201.0 191.0 180.0 193.1
Lysine 236.0 230.0 211.0 225.8
Methionine 224.0 203.0 204.0 203.4
Phenylalanine 240.0 228.0 218.0 222.8
Proline 159.0 154.0 143.0 146.8
Serine 155.0 143.0 122.0 129.8
Threonine 172.0 163.0 146.0 152.5
Tryptophan 285.0 264.0 259.0 266.3
Tyrosine 263.0 255.0 229.0 236.8
Valine 174.0 165.0 160.0 164.5
Amino Acid Residues Having a Side Chain Comprising a Guanidine Group,
Guanidine
Replacement Group, or Protonated Form Thereof
[0241] As used herein, guanidine refers to the structure:
NH2
HN N
H
=
[0242] As used herein, a protonated form of guanidine refers to the structure:
89
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
NH2
C)
H2N
H
[0243] Guanidine replacement groups refer to functional groups on the side
chain of amino acids
that will be positively charged at or above physiological pH or those that can
recapitulate the
hydrogen bond donating and accepting activity of guanidinium groups.
[0244] The guanidine replacement groups facilitate cell penetration and
delivery of therapeutic
agents while reducing toxicity associated with guanidine groups or protonated
forms thereof The
cCPP can comprise at least one amino acid having a side chain comprising a
guanidine or
guanidinium replacement group. The cCPP can comprise at least two amino acids
having a side
chain comprising a guanidine or guanidinium replacement group. The cCPP can
comprise at
least three amino acids having a side chain comprising a guanidine or
guanidinium replacement
group
[0245] The guanidine or guanidinium group can be an isostere of guanidine or
guanidinium. The
guanidine or guanidinium replacement group can be less basic than guanidine.
0 NH 0
H2NANN H2NAN)Y
[0246] As used herein, a guanidine replacement group refers to
,N Niai
N N-= N N H N4
H H , or a protonated form thereof
[0247] The disclosure relates to a cCPP comprising from 4 to 20 amino acids
residues, wherein:
(i) at least one amino acid has a side chain comprising a guanidine group, or
a protonated form
thereof; (ii) at least one amino acid residue has no side chain or a side
chain comprising
0 NH 0 A l IL JIL
H2NANN H2N N)L,õ N J N H NaNv
H H , or a
protonated
form thereof, and (iii) at least two amino acids residues independently have a
side chain
comprising an aromatic or heteroaromatic group.
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
[0248] At least two amino acids residues can have no side chain or a side
chain comprising
0 NH 0 N r N
A a µ \ F.INOI I
H2NANN H2N N N N- N N N- N NN,
H H / H H H , , i , or a
protonated
form thereof. As used herein, when no side chain is present, the amino acid
residue have two
hydrogen atoms on the carbon atom(s) (e.g., -CH2-) linking the amine and
carboxylic acid.
[0249] The cCPP can comprise at least one amino acid having a side chain
comprising one of the
0 NH 0 C N CN HNOI
:( )
H2NANN H2NA N , N NN N. NN
following moieties: H H r H H H ,
I
N v
1 , or a protonated form thereof
[0250] The cCPP can comprise at least two amino acids each independently
having one of the
0 NH 0 N N H Na/ 1
A )y a i, N
H2NANN H2N N N N'N, r jµ N N N
following moieties H H H H H
or a protonated form thereof At least two amino acids can have a side chain
comprising the same
C 0 NH 0 N r,N H Nal/
H2NANN H2NA N / N'' N\ r.it N N-Nµ
moiety selected from: H H H H H
I
N
1 , or a protonated form thereof At least one amino acid can have a side chain
comprising
0
H2NANN
H , or a protonated form thereof At least two amino acids can have a
side chain
0
H2NANN
comprising H , or a protonated form thereof One, two, three, or four amino
acids can
0
H2NANN
have a side chain comprising H , or a protonated form thereof. One amino
acid can
91
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
0
H2NANN
have a side chain comprising H , or a protonated form thereof. Two amino
acids can
0 0
H2NANN
H2NANN
have a side chain comprising H , or a protonated form thereof.
NH 0
Cri
H2NAN)Y N¨NN HNOof
H H H ,
or a protonated form thereof, can
0
H2NAN
be attached to the terminus of the amino acid side chain. H can be
attached to the
terminus of the amino acid side chain.
[0251] The cCPP can comprise (iii) 2, 3, 4, 5 or 6 amino acid residues
independently having a
side chain comprising a guanidine group, guanidine replacement group, or a
protonated form
thereof. The cCPP can comprise (iii) 2 amino acid residues independently
having a side chain
comprising a guanidine group, guanidine replacement group, or a protonated
form thereof. The
cCPP can comprise (iii) 3 amino acid residues independently having a side
chain comprising a
guanidine group, guanidine replacement group, or a protonated form thereof.
The cCPP can
comprise (iii) 4 amino acid residues independently having a side chain
comprising a guanidine
group, guanidine replacement group, or a protonated form thereof. The cCPP can
comprise (iii) 5
amino acid residues independently having a side chain comprising a guanidine
group, guanidine
replacement group, or a protonated form thereof. The cCPP can comprise (iii) 6
amino acid
residues independently having a side chain comprising a guanidine group,
guanidine replacement
group, or a protonated form thereof The cCPP can comprise (iii) 2, 3, 4, or 5
amino acid
residues independently having a side chain comprising a guanidine group,
guanidine replacement
group, or a protonated form thereof The cCPP can comprise (iii) 2, 3, or 4
amino acid residues
independently having a side chain comprising a guanidine group, guanidine
replacement group,
or a protonated form thereof The cCPP can comprise (iii) 2 or 3 amino acid
residues
independently having a side chain comprising a guanidine group, guanidine
replacement group,
or a protonated form thereof The cCPP can comprise (iii) at least one amino
acid residue having
a side chain comprising a guanidine group or protonated form thereof. The cCPP
can comprise
92
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
(iii) two amino acid residues having a side chain comprising a guanidine group
or protonated
form thereof. The cCPP can comprise (iii) three amino acid residues having a
side chain
comprising a guanidine group or protonated form thereof
[0252] The amino acid residues can independently have the side chain
comprising the guanidine
group, guanidine replacement group, or the protonated form thereof that are
not contiguous. Two
amino acid residues can independently have the side chain comprising the
guanidine group,
guanidine replacement group, or the protonated form thereof can be contiguous.
Three amino
acid residues can independently have the side chain comprising the guanidine
group, guanidine
replacement group, or the protonated form thereof can be contiguous. Four
amino acid residues
can independently have the side chain comprising the guanidine group,
guanidine replacement
group, or the protonated form thereof can be contiguous. The contiguous amino
acid residues can
have the same stereochemistry. The contiguous amino acids can have alternating
stereochemistry.
[0253] The amino acid residues independently having the side chain comprising
the guanidine
group, guanidine replacement group, or the protonated form thereof, can be L-
amino acids. The
amino acid residues independently having the side chain comprising the
guanidine group,
guanidine replacement group, or the protonated form thereof, can be D-amino
acids. The amino
acid residues independently having the side chain comprising the guanidine
group, guanidine
replacement group, or the protonated form thereof, can be a mixture of L- or D-
amino acids.
[0254] Each amino acid residue having the side chain comprising the guanidine
group, or the
protonated form thereof, can independently be a residue of arginine,
homoarginine, 2-amino-3-
propionic acid, 2-amino-4-guanidinobutyric acid or a protonated form thereof.
Each amino acid
residue having the side chain comprising the guanidine group, or the
protonated form thereof,
can independently be a residue of arginine or a protonated form thereof.
102551 Each amino acid having the side chain comprising a guanidine
replacement group, or
0 NH 0
H2NANAL H2NA N)y N
protonated form thereof, can independently be H H H
N N
, or a protonated form thereof
93
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
[0256] Without being bound by theory, it is hypothesized that guanidine
replacement groups
have reduced basicity, relative to arginine and in some cases are uncharged at
physiological pH
(e.g., a -N(H)C(0)), and are capable of maintaining the bidentate hydrogen
bonding interactions
with phospholipids on the plasma membrane that is believed to facilitate
effective membrane
association and subsequent internalization. The removal of positive charge is
also believed to
reduce toxicity of the cCPP.
[0257] Those skilled in the art will appreciate that the N- and/or C-termini
of the above non-
natural aromatic hydrophobic amino acids, upon incorporation into the peptides
disclosed herein,
form amide bonds.
[0258] The cCPP can comprise a first amino acid having a side chain comprising
an aromatic or
heteroaromatic group and a second amino acid having a side chain comprising an
aromatic or
heteroaromatic group, wherein an N-terminus of a first glycine forms a peptide
bond with the
first amino acid having the side chain comprising the aromatic or
heteroaromatic group, and a C-
terminus of the first glycine forms a peptide bond with the second amino acid
having the side
chain comprising the aromatic or heteroaromatic group. Although by convention,
the term "first
amino acid" often refers to the N-terminal amino acid of a peptide sequence,
as used herein "first
amino acid" is used to distinguish the referent amino acid from another amino
acid (e.g., a
"second amino acid") in the cCPP such that the term "first amino acid" may or
may refer to an
amino acid located at the N-terminus of the peptide sequence.
[0259] The cCPP can comprise an N-terminus of a second glycine forms a peptide
bond with an
amino acid having a side chain comprising an aromatic or heteroaromatic group,
and a C-
terminus of the second glycine forms a peptide bond with an amino acid having
a side chain
comprising a guanidine group, or a protonated form thereof.
[0260] The cCPP can comprise a first amino acid having a side chain comprising
a guanidine
group, or a protonated form thereof, and a second amino acid having a side
chain comprising a
guanidine group, or a protonated form thereof, wherein an N-terminus of a
third glycine forms a
peptide bond with a first amino acid having a side chain comprising a
guanidine group, or a
protonated form thereof, and a C-terminus of the third glycine forms a peptide
bond with a
second amino acid having a side chain comprising a guanidine group, or a
protonated form
thereof.
94
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
[0261] The cCPP can comprise a residue of asparagine, aspartic acid,
glutamine, glutamic acid,
or homoglutamine. The cCPP can comprise a residue of asparagine. The cCPP can
comprise a
residue of glutamine.
[0262] The cCPP can comprise a residue of tyrosine, phenylalanine, 1-
naphthylalanine, 2-
naphthylalanine, tryptophan, 3-benzothienylalanine, 4-phenylphenylalanine, 3,4-
difluorophenylalanine, 4-trifluoromethylphenylalanine, 2,3,4,5,6-
pentafluorophenylalanine,
homophenylalanine, P-homophenylalanine, 4-tert-butyl-phenylalanine, 4-
pyridinylalanine, 3-
pyridinylalanine, 4-methylphenylalanine, 4-fluorophenylalanine, 4-
chlorophenylalanine, 3-(9-
anthry1)-alanine.
[0263] While not wishing to be bound by theory, it is believed that the
chirality of the amino
acids in the cCPPs may impact cytosolic uptake efficiency. The cCPP can
comprise at least one
D amino acid. The cCPP can comprise one to fifteen D amino acids. The cCPP can
comprise one
to ten D amino acids. The cCPP can comprise 1, 2, 3, or 4 D amino acids. The
cCPP can
comprise 2, 3, 4, 5, 6, 7, or 8 contiguous amino acids having alternating D
and L chirality. The
cCPP can comprise three contiguous amino acids having the same chirality. The
cCPP can
comprise two contiguous amino acids having the same chirality. At least two of
the amino acids
can have the opposite chirality. The at least two amino acids having the
opposite chirality can be
adjacent to each other. At least three amino acids can have alternating
stereochemistry relative to
each other. The at least three amino acids having the alternating chirality
relative to each other
can be adjacent to each other. At least four amino acids have alternating
stereochemistry relative
to each other. The at least four amino acids having the alternating chirality
relative to each other
can be adjacent to each other. At least two of the amino acids can have the
same chirality. At
least two amino acids having the same chirality can be adjacent to each other.
At least two amino
acids have the same chirality and at least two amino acids have the opposite
chirality. The at
least two amino acids having the opposite chirality can be adjacent to the at
least two amino
acids having the same chirality. Accordingly, adjacent amino acids in the cCPP
can have any of
the following sequences: D-L; L-D; D-L-L-D; L-D-D-L; L-D-L-L-D; D-L-D-D-L; D-L-
L-D-L;
or L-D-D-L-D. The amino acid residues that form the cCPP can all be L-amino
acids. The amino
acid residues that form the cCPP can all be D-amino acids.
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
[0264] At least two of the amino acids can have a different chirality. At
least two amino acids
having a different chirality can be adjacent to each other. At least three
amino acids can have
different chirality relative to an adjacent amino acid. At least four amino
acids can have different
chirality relative to an adjacent amino acid. At least two amino acids have
the same chirality and
at least two amino acids have a different chirality. One or more amino acid
residues that form the
cCPP can be achiral. The cCPP can comprise a motif of 3, 4, or 5 amino acids,
wherein two
amino acids having the same chirality can be separated by an achiral amino
acid. The cCPPs can
comprise the following sequences: D-X-D; D-X-D-X; D-X-D-X-D; L-X-L; L-X-L-X;
or L-X-L-
X-L, wherein X is an achiral amino acid. The achiral amino acid can be
glycine.
[0265] An amino acid having a side chain comprising:
0 ANH N,HN
H2NANN H2N N N N N
H H H , or a
protonated form thereof, can be adjacent to an amino acid having a side chain
comprising an
0
H2NAN
aromatic or heteroaromatic group. An amino acid having a side chain
comprising:
NH 0 H2NAN)Y NLNA.= HNO I
H H H NI/
f , or a protonated form thereof,
can be adjacent to at least one amino acid having a side chain comprising a
guanidine or
protonated form thereof An amino acid having a side chain comprising a
guanidine or
protonated form thereof can be adjacent to an amino acid having a side chain
comprising an
aromatic or heteroaromatic group. Two amino acids having a side chain
comprising:
0 NH 0 N
A )IL JIL
H2NANN H2N Ny P- J N __ N N H
H H or protonated
forms thereof, can be adjacent to each other. Two amino acids having a side
chain comprising a
guanidine or protonated form thereof are adjacent to each other. The cCPPs can
comprise at least
two contiguous amino acids having a side chain can comprise an aromatic or
heteroaromatic
96
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
0
H2NANN
group and at least two non-adjacent amino acids having a side chain
comprising:
NH 0 H2NAN)Y NLNA.= HNO I
H H H NNI/
, or a protonated form thereof
The cCPPs can comprise at least two contiguous amino acids having a side chain
comprising an
aromatic or heteroaromatic group and at least two non-adjacent amino acids
having a side chain
0
H2N
comprising H , or a protonated form thereof. The adjacent amino acids can
have the
same chirality. The adjacent amino acids can have the opposite chirality.
Other combinations of
amino acids can have any arrangement of D and L amino acids, e.g., any of the
sequences
described in the preceding paragraph.
[0266] At least two amino acids having a side chain comprising:
0 NH 0 N HNil
IL IL
H2NANN H2NA P- J N N N J Na
H H H , or a
protonated form thereof, are alternating with at least two amino acids having
a side chain
comprising a guanidine group or protonated form thereof
[0267] The cCPP can comprise the structure of Formula (A):
Rg R2
AAsc
HN R3
NH
HN
R7 NH
ON
HN R4 q
0 FN5 (A)
or a protonated form thereof,
wherein:
R1, R2, and R3 are each independently H or an aromatic or heteroaromatic side
97
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
chain of an amino acid;
at least one of Ri, R2, and R3 is an aromatic or heteroaromatic side chain of
an amino
acid;
R4, R5, R6, R7 are independently H or an amino acid side chain;
at least one of R4, R5, R6, R7 is the side chain of 3-guanidino-2-
aminopropionic acid, 4-
guanidino-2-aminobutanoic acid, arginine, homoarginine, N-methylarginine, N,N-
dimethylarginine, 2,3-diaminopropionic acid, 2,4-diaminobutanoic acid, lysine,
N-methyllysine,
N,N-dimethyllysine, N-ethyllysine, N,N,N-trimethyllysine, 4-
guanidinophenylalanine, citrulline,
N,N-dimethyllysineõ P-homoarginine, 3-(1-piperidinyl)alanine;
AAsc is an amino acid side chain; and
q is 1, 2, 3 or 4.
[0268] In embodiments, at least one of R4, R5, R6, R7 are independently an
uncharged, non-
aromatic side chain of an amino acid. In embodiments, at least one of R4, R5,
R6, R7 are
independently H or a side chain of citrulline.
[0269] In embodiments, compounds are provided that include a cyclic peptide
having 6 to 12
amino acids, wherein at least two amino acids of the cyclic peptide are
charged amino acids, at
least two amino acids of the cyclic peptide are aromatic hydrophobic amino
acids and at least
two amino acids of the cyclic peptide are uncharged, non-aromatic amino acids.
In embodiments,
at least two charged amino acids of the cyclic peptide are arginine. In
embodiments, at least two
aromatic, hydrophobic amino acids of the cyclic peptide are phenylalanine or
naphthylalanine. In
embodiments, at least two uncharged, non-aromatic amino acids of the cyclic
peptide are
citrulline or glycine.
[0270] In embodiments, the cyclic peptide of Formula (A) is not selected from
a cyclic peptide
having a sequence of SEQ ID NO: 89-117.
[0271] In embodiments, the cyclic peptide of Formula (A) is selected from a
cyclic peptide
having a sequence of SEQ ID NO: 89-117.
CPP sequences and SEQ ID NOs
FORRRQ 89 RRFRORQ 99 FORRRRQK 109
FORRRC 90 FRRRROQ 100 FORRRRQC 110
FORRRU 91 rRFRORQ 101 fORrRrRQ 111
RRROFQ 92 RROFRRQ 102 FORRRRRQ 112
98
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
RRRR0F 93 CRRRRFWQ 103 RRRROFDS2C 113
FORRRR 94 FfORrRrQ 104 FORRR 114
FckrRrRq 95 FFORRRRQ 105 FWRRR 115
FORIRQ 96 RFRFRORQ 106 RRROF 116
FORRRRQ 97 URRRRFWQ 107 RRRWF 117
fORrRrQ 98 CRRRRFWQ 108
0 = L-naphthylalanine; (I) = D-naphthylalanine; = L-norleucine
[0272] The cCPP can comprise the structure of Formula (I):
Rg R2
AAsc
HN R3
NH
/CNN
km R4 q
NH 0 N
R7 0
NH
qm
NH
NH
H2N (I)
or a protonated form thereof,
wherein:
Ri, R2, and R3 can each independently be H or an amino acid residue having a
side chain comprising an aromatic group;
at least one of Ri, R2, and R3 is an aromatic or heteroaromatic side chain of
an amino
acid;
R4 and R7 are independently H or an amino acid side chain;
AAsc is an amino acid side chain;
q is 1, 2, 3 or 4; and
each m is independently an integer of 0, 1, 2, or 3.
[0273] Ri, R2, and R3 can each independently be H, -alkylene-aryl, or -
alkylene-heteroaryl. Ri,
R2, and R3 can each independently be H, -Ci_3alkylene-aryl, or -Ci_3alkylene-
heteroaryl. Ri, R2,
and R3 can each independently be H or -alkylene-aryl. Ri, R2, and R3 can each
independently be
99
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
H or -Ci_3alkylene-aryl. Ci_3alkylene can be methylene. Aryl can be a 6- to 14-
membered aryl.
Heteroaryl can be a 6- to 14-membered heteroaryl having one or more
heteroatoms selected from
N, 0, and S. Aryl can be selected from phenyl, naphthyl, or anthracenyl. Aryl
can be phenyl or
naphthyl. Aryl can be phenyl. Heteroaryl can be pyridyl, quinolyl, and
isoquinolyl. R1, R2, and
R3 can each independently be H, -Ci_3alkylene-Ph or -Ci_3alkylene-Naphthyl.
R1, R2, and R3 can
each independently be H, -CH2Ph, or -CH2Naphthyl. R1, R2, and R3 can each
independently be H
or -CH2Ph.
[0274] R1, R2, and R3 can each independently be the side chain of tyrosine,
phenylalanine, 1-
naphthylalanine, 2-naphthylalanine, tryptophan, 3-benzothienylalanine, 4-
phenylphenylalanine,
3,4-difluorophenylalanine, 4-trifluoromethylphenylalanine, 2,3,4,5,6-
pentafluorophenylalanine,
homophenylalanine, 3-homophenylalanine, 4-tert-butyl-phenylalanine, 4-
pyridinylalanine, 3-
pyridinylalanine, 4-methylphenylalanine, 4-fluorophenylalanine, 4-
chlorophenylalanine, 3-(9-
anthry1)-alanine.
[0275] Ri can be the side chain of tyrosine. Ri can be the side chain of
phenylalanine. Ri can be
the side chain of 1-naphthylalanine. Ri can be the side chain of 2-
naphthylalanine. Ri can be the
side chain of tryptophan. Ri can be the side chain of 3-benzothienylalanine.
Ri can be the side
chain of 4-phenylphenylalanine. Ri can be the side chain of 3,4-
difluorophenylalanine. Ri can be
the side chain of 4-trifluoromethylphenylalanine. Ri can be the side chain of
2,3,4,5,6-
pentafluorophenylalanine. Ri can be the side chain of homophenylalanine. Ri
can be the side
chain of 3-homophenylalanine. Ri can be the side chain of 4-tert-butyl-
phenylalanine. Ri can be
the side chain of 4-pyridinylalanine. Ri can be the side chain of 3-
pyridinylalanine. Ri can be the
side chain of 4-methylphenylalanine. Ri can be the side chain of 4-
fluorophenylalanine. Ri can
be the side chain of 4-chlorophenylalanine. Ri can be the side chain of 3-(9-
anthry1)-alanine.
[0276] R2 can be the side chain of tyrosine. R2 can be the side chain of
phenylalanine. R2 can be
the side chain of 1-naphthylalanine. Ri can be the side chain of 2-
naphthylalanine. R2 can be the
side chain of tryptophan. R2 can be the side chain of 3-benzothienylalanine.
R2 can be the side
chain of 4-phenylphenylalanine. R2 can be the side chain of 3,4-
difluorophenylalanine. R2 can be
the side chain of 4-trifluoromethylphenylalanine. R2 can be the side chain of
2,3,4,5,6-
pentafluorophenylalanine. R2 can be the side chain of homophenylalanine. R2
can be the side
chain of 3-homophenylalanine. R2 can be the side chain of 4-tert-butyl-
phenylalanine. R2 can be
100
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
the side chain of 4-pyridinylalanine. R2 can be the side chain of 3-
pyridinylalanine. R2 can be the
side chain of 4-methylphenylalanine. R2 can be the side chain of 4-
fluorophenylalanine. R2 can
be the side chain of 4-chlorophenylalanine. R2 can be the side chain of 3-(9-
anthry1)-alanine.
[0277] R3 can be the side chain of tyrosine. R3 can be the side chain of
phenylalanine. R3 can be
the side chain of 1-naphthylalanine. R3 can be the side chain of 2-
naphthylalanine. R3 can be the
side chain of tryptophan. R3 can be the side chain of 3-benzothienylalanine.
R3 can be the side
chain of 4-phenylphenylalanine. R3 can be the side chain of 3,4-
difluorophenylalanine. R3 can be
the side chain of 4-trifluoromethylphenylalanine. R3 can be the side chain of
2,3,4,5,6-
pentafluorophenylalanine. R3 can be the side chain of homophenylalanine. R3
can be the side
chain of P-homophenylalanine. R3 can be the side chain of 4-tert-butyl-
phenylalanine. R3 can be
the side chain of 4-pyridinylalanine. R3 can be the side chain of 3-
pyridinylalanine. R3 can be the
side chain of 4-methylphenylalanine. R3 can be the side chain of 4-
fluorophenylalanine. R3 can
be the side chain of 4-chlorophenylalanine. R3 can be the side chain of 3-(9-
anthry1)-alanine.
[0278] R4 can be H, -alkylene-aryl, -alkylene-heteroaryl. R4 can be H, -
C1_3alkylene-aryl, or -Ci-
3alkylene-heteroaryl. R4 can be H or -alkylene-aryl. R4 can be H or -
C1_3alkylene-aryl. C
3alkylene can be a methylene. Aryl can be a 6- to 14-membered aryl. Heteroaryl
can be a 6- to
14-membered heteroaryl haying one or more heteroatoms selected from N, 0, and
S. Aryl can be
selected from phenyl, naphthyl, or anthracenyl. Aryl can be phenyl or
naphthyl. Aryl can phenyl.
Heteroaryl can be pyridyl, quinolyl, and isoquinolyl. R4 can be H, -
Ci_3alkylene-Ph or -
C1_3alkylene-Naphthyl. R4 can be H or the side chain of an amino acid in Table
4 or Table 6. R4
can be H or an amino acid residue haying a side chain comprising an aromatic
group. R4 can be
H, -CH2Ph, or -CH2Naphthyl. R4 can be H or -CH2Ph.
[0279] R5 can be H, -alkylene-aryl, -alkylene-heteroaryl. R5 can be H, -
C1_3alkylene-aryl, or -
3a1ky1ene-heteroaryl. R5 can be H or -alkylene-aryl. R5 can be H or -
C1_3alkylene-aryl. C
3a1ky1ene can be a methylene. Aryl can be a 6- to 14-membered aryl. Heteroaryl
can be a 6- to
14-membered heteroaryl haying one or more heteroatoms selected from N, 0, and
S. Aryl can be
selected from phenyl, naphthyl, or anthracenyl. Aryl can be phenyl or
naphthyl. Aryl can phenyl.
Heteroaryl can be pyridyl, quinolyl, and isoquinolyl. R5 can be H, -
Ci_3alkylene-Ph or -
C1_3alkylene-Naphthyl. R5 can be H or the side chain of an amino acid in Table
4 or Table 6. R4
101
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
can be H or an amino acid residue haying a side chain comprising an aromatic
group. R5 can be
H, -CH2Ph, or -CH2Naphthyl. R4 can be H or -CH2Ph.
[0280] R6 can be H, -alkylene-aryl, -alkylene-heteroaryl. R6 can be H, -
C1_3alkylene-aryl, or -Ci-
3alkylene-heteroaryl. R6 can be H or -alkylene-aryl. R6 can be H or -
C1_3alkylene-aryl. C
3alkylene can be a methylene. Aryl can be a 6- to 14-membered aryl. Heteroaryl
can be a 6- to
14-membered heteroaryl haying one or more heteroatoms selected from N, 0, and
S. Aryl can be
selected from phenyl, naphthyl, or anthracenyl. Aryl can be phenyl or
naphthyl. Aryl can phenyl.
Heteroaryl can be pyridyl, quinolyl, and isoquinolyl. R6 can be H, -
Ci_3alkylene-Ph or -
C1_3alkylene-Naphthyl. R6 can be H or the side chain of an amino acid in Table
4 or Table 6. R6
can be H or an amino acid residue haying a side chain comprising an aromatic
group. R6 can be
H, -CH2Ph, or -CH2Naphthyl. R6 can be H or -CH2Ph.
[0281] R7 can be H, -alkylene-aryl, -alkylene-heteroaryl. R7 can be H, -
C1_3alkylene-aryl, or -
3a1ky1ene-heteroaryl. R7 can be H or -alkylene-aryl. R7 can be H or -
C1_3alkylene-aryl. C
3a1ky1ene can be a methylene. Aryl can be a 6- to 14-membered aryl. Heteroaryl
can be a 6- to
14-membered heteroaryl haying one or more heteroatoms selected from N, 0, and
S. Aryl can be
selected from phenyl, naphthyl, or anthracenyl. Aryl can be phenyl or
naphthyl. Aryl can phenyl.
Heteroaryl can be pyridyl, quinolyl, and isoquinolyl. R7 can be H, -
Ci_3alkylene-Ph or -
C1_3alkylene-Naphthyl. R7 can be H or the side chain of an amino acid in Table
4 or Table 6. R7
can be H or an amino acid residue haying a side chain comprising an aromatic
group. R7 can be
H, -CH2Ph, or -CH2Naphthyl. R7 can be H or -CH2Ph.
[0282] One, two or three of Ri, R2, R3, R4, Rs, R6, and R7 can be -CH2Ph. One
of Ri, R2, R3, R4,
R5, R6, and R7 can be -CH2Ph. Two of Ri, R2, R3, R4, Rs, R6, and R7 can be -
CH2Ph. Three of R1,
R2, R3, R4, R5, R6, and R7 can be -CH2Ph. At least one of R1, R2, R3, R4, R5,
R6, and R7 can be -
CH2Ph. No more than four of Ri, R2, R3, R4, Rs, R6, and R7 can be -CH2Ph.
[0283] One, two or three of R1, R2, R3, and R4 are -CH2Ph. One of R1, R2, R3,
and R4 is -CH2Ph.
Two of R1, R2, R3, and R4 are -CH2Ph. Three of R1, R2, R3, and R4 are -CH2Ph.
At least one of
Ri, R2, R3, and R4 is -CH2Ph.
[0284] One, two or three of Ri, R2, R3, R4, Rs, R6, and R7 can be H. One of
R1, R2, R3, R4, R5,
R6, and R7 can be H. Two of Ri, R2, R3, R4, Rs, R6, and R7 are H. Three of R1,
R2, R3, R5, R6, and
102
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
R7 can be H. At least one of R1, R2, R3, R4, R5, R6, and R7 can be H. No more
than three of R1,
R2, R3, R4, R5, R6, and R7 can be -CH2Ph.
[0285] One, two or three of R1, R2, R3, and R4 are H. One of R1, R2, R3, and
R4 is H. Two of R1,
R2, R3, and R4 are H. Three of Ri, R2, R3, and R4 are H. At least one of R1,
R2, R3, and R4 is H.
[0286] At least one of R4, R5, R6, and R7 can be side chain of 3-guanidino-2-
aminopropionic
acid. At least one of R4, R5, R6, and R7 can be side chain of 4-guanidino-2-
aminobutanoic acid.
At least one of R4, Rs, R6, and R7 can be side chain of arginine. At least one
of R4, Rs, R6, and R7
can be side chain of homoarginine. At least one of R4, R5, R6, and R7 can be
side chain of N-
methylarginine. At least one of R4, R5, R6, and R7 can be side chain of N,N-
dimethylarginine. At
least one of R4, R5, R6, and R7 can be side chain of 2,3-diaminopropionic
acid. At least one of R4,
R5, R6, and R7 can be side chain of 2,4-diaminobutanoic acid, lysine. At least
one of R4, R5, R6,
and R7 can be side chain of N-methyllysine. At least one of R4, Rs, R6, and R7
can be side chain
of N,N-dimethyllysine. At least one of R4, R5, R6, and R7 can be side chain of
N-ethyllysine. At
least one of R4, Rs, R6, and R7 can be side chain of N,N,N-trimethyllysine, 4-
guanidinophenylalanine. At least one of R4, Rs, R6, and R7 can be side chain
of citrulline. At least
one of R4, Rs, R6, and R7 can be side chain of N,N-dimethyllysineõ 3-
homoarginine. At least one
of R4, R5, R6, and R7 can be side chain of 3-(1-piperidinyl)alanine.
[0287] At least two of R4, R5, R6, and R7 can be side chain of 3-guanidino-2-
aminopropionic
acid. At least two of R4, R5, R6, and R7 can be side chain of 4-guanidino-2-
aminobutanoic acid.
At least two of R4, Rs, R6, and R7 can be side chain of arginine. At least two
of R4, R5, R6, and R7
can be side chain of homoarginine. At least two of R4, Rs, R6, and R7 can be
side chain of N-
methylarginine. At least two of R4, R5, R6, and R7 can be side chain of N,N-
dimethylarginine. At
least two of R4, R5, R6, and R7 can be side chain of 2,3-diaminopropionic
acid. At least two of R4,
R5, R6, and R7 can be side chain of 2,4-diaminobutanoic acid, lysine. At least
two of R4, R5, R6,
and R7 can be side chain of N-methyllysine. At least two of R4, R5, R6, and R7
can be side chain
of N,N-dimethyllysine. At least two of R4, R5, R6, and R7 can be side chain of
N-ethyllysine. At
least two of R4, R5, R6, and R7 can be side chain of N,N,N-trimethyllysine, 4-
guanidinophenylalanine. At least two of R4, R5, R6, and R7 can be side chain
of citrulline. At
least two of R4, R5, R6, and R7 can be side chain of N,N-dimethyllysineõ 3-
homoarginine. At
least two of R4, R5, R6, and R7 can be side chain of 3-(1-piperidinyl)alanine.
103
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
[0288] At least three of R4, Rs, R6, and R7 can be side chain of 3-guanidino-2-
aminopropionic
acid. At least three of R4, R5, R6, and R7 can be side chain of 4-guanidino-2-
aminobutanoic acid.
At least three of R4, R5, R6, and R7 can be side chain of arginine. At least
three of R4, R5, R6, and
R7 can be side chain of homoarginine. At least three of R4, Rs, R6, and R7 can
be side chain of N-
methylarginine. At least three of R4, Rs, R6, and R7 can be side chain of N,N-
dimethylarginine.
At least three of R4, R5, R6, and R7 can be side chain of 2,3-diaminopropionic
acid. At least three
of R4, R5, R6, and R7 can be side chain of 2,4-diaminobutanoic acid, lysine.
At least three of R4,
R5, R6, and R7 can be side chain of N-methyllysine. At least three of R4, R5,
R6, and R7 can be
side chain of N,N-dimethyllysine. At least three of R4, R5, R6, and R7 can be
side chain of N-
ethyllysine. At least three of R4, Rs, R6, and R7 can be side chain of N,N,N-
trimethyllysine, 4-
guanidinophenylalanine. At least three of R4, Rs, R6, and R7 can be side chain
of citrulline,. At
least three of R4, R5, R6, and R7 can be side chain of N,N-dimethyllysineõ P-
homoarginine. At
least three of R4, R5, R6, and R7 can be side chain of 3-(1-
piperidinyl)alanine.
[0289] AAsc can be a side chain of a residue of asparagine, glutamine, or
homoglutamine. AAsc
can be a side chain of a residue of glutamine. The cCPP can further comprise a
linker conjugated
the AAsc, e.g., the residue of asparagine, glutamine, or homoglutamine. Hence,
the cCPP can
further comprise a linker conjugated to the asparagine, glutamine, or
homoglutamine residue.
The cCPP can further comprise a linker conjugated to the glutamine residue.
[0290] q can be 1,2, or 3. q can 1 or 2. q can be 1. q can be 2. q can be 3. q
can be 4.
[0291] m can be 1-3. m can be 1 or 2. m can be 0. m can be 1. m can be 2. m
can be 3.
104
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
[0292] The cCPP of Formula (A) can comprise the structure of Formula (I)
N}i 0 ,44 \pc-0
A =,..õ4õ.
HN
4 '
tr:21c H.
,A 0
R4 -,,
",i¨NH .14,1, .
Ft, > ' µ
- <1 (1)4
H2N¨õeNH
NH
(I) or protonated form thereof, wherein AAsc, R1,
R2, R3, R4, R7, m, and q are as defined herein.
[0293] The cCPP of Formula (A) can comprise the structure of Formula (I-a) or
Formula (I-b):
Ri AAõ....7 o R1 a
41/415_
R2
N HN----cr
R2 AAõ N HN
H H
NH 0 NH 0
ii _TNH B 0,NH
HN HN
H2N--\N X- R3 H2N--\N--\ M\ i
H NNH X- R3
H m NH
Ok__
NH EdHN 0
HN 0
NH
R4
e--- 0
t--)111 0 0 r m
NH NH
H2N--1 H2N--1
NH (I-a), NH (I-
b),
or protonated form thereof, wherein AAsc, Ri, R2, R3, R4, and m are as defined
herein.
[0294] The cCPP of Formula (A) can comprise the structure of Formula (I-1), (I-
2), (I-3), or (I-
4):
105
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
0 sc /_40 0.
0
0 0
AA 46?\---N HN*._
H
r
H HN-\r NH
0 0 NH
rThr,04...TNH H m---I( HN
H,N-N
HN -2- N T
H ni NH
Ok_ HN 0
0*___
NH H
HN'. NH
e--- 0 (0
rn
NH NH
H2N-1 H2N-1
NH (I-1), NH (1-2),
NH2
HN\
HN
N
H
HN 0 AAsc 0
H2N
0 N"-----foe / __ \ )----( .
)--NH 1,,,.?\-- HN
HN ,1 m HN 0
oyNH
NH 0
C HN
1.0
0 HN 0
HN
NH
NH HN 1
____\--"AAsc =
OK_ H
0 ,õ= NH ENI
C) -
11 0
H2N¨µNal
NH
0-3), (1-4)
or protonated form thereof, wherein AAsc and m are as defined herein.
[0295] The cCPP of Formula (A) can comprise the structure of Formula (1-5) or
(1-6):
106
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
NH2
NH? HN'''Cs
N
':.
-1\ AA M
' 0
g AAs,
1 ,
i..04 ,,,,,,, o\,.....N1,1 H
3-IN .. ? \ H 1114µ ---/FM:::;--;;', µ=-=-NH =*-
--i: ,\zz=zzO \''''''../
\..----NH \.-4 , ,..,õ =
..tzzzO '' H?N' NH HNµ
H214 kii HN I
; .,---_,' NH H HN-40
..=
.....N..,,....
, 0
NH ce--..."
0 I NH r
b---.
\:.-r-ri
...-N.
H2Nt, NH
(I-5), or
(I-6)
or protonated form thereof, wherein AAsc is as defined herein.
[0296] The cCPP of Formula (A) can comprise the structure of Formula (I-1):
it
o
o
AAscYLN NH HN--\ro
H
0 NH
H2N-I(NmX HN
H m NH
Ok__NH HHN 0 O
N
C?---)ni 0
H2N...1NH
NH (I-1), or a protonated form thereof,
wherein AAsc and m are as defined herein.
[0297] The cCPP of Formula (A) can comprise the structure of Formula (I-2):
107
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
0
AAõy\---NHN
NH 0
0 NH
1--1 -2-
H ot. HNo ,; NH
NH
C7/ (())111
NH
NH (1-2), or a protonated form thereof,
wherein AAsc and m are as defined herein.
[0298] The cCPP of Formula (A) can comprise the structure of Formula (1-3):
NH2
HNN HN
)-NH2
HN
NH
0
HIJ
HN m
NH 0
0 HN
NH
HN
0
=
(1-3), or a protonated form thereof,
wherein AAsc and m are as defined herein.
[0299] The cCPP of Formula (A) can comprise the structure of Formula (1-4):
108
CA 03222824 2023-12-07
WO 2022/271818
PCT/US2022/034517
AAsc 0
H2N u )-4
HN 0
HN
NH
OK_NH Ed HN
C OZ
NH
H2N¨µ
NH
(I-4), or a protonated form thereof,
wherein AAsc and m are as defined herein.
[0300] The cCPP of Formula (A) can comprise the structure of Formula (1-5):
tfl
\ 0
HN r
Hick.seof¨'N';>'
=
\.>-- NH ,
H2N Htsi
0
.;.==
' N\H
H2N,,,(NH 0
0
d =
(1-5), or a protonated form thereof,
wherein AAsc and m are as defined herein.
[0301] The cCPP of Formula (A) can comprise the structure of Formula (1-6):
109
CA 03222824 2023-12-07
WO 2022/271818 PC T/US2022/034517
_P1H2
AAso
H 0
cit ..1,e
\
HP'NH k
HN
=).
NH
\*=¨=4 Azz==0
14X
HN
NH H HN
, N ,
NH Cl;' fr
NH
HN
H2N 'NH
(1-6), or a protonated form thereof, wherein AAsc
and m are as defined herein.
[0302] The cCPP can comprise one of the following sequences: FGFGRGR (SEQ ID
NO:68);
GfFGrGr (SEQ ID NO:69), FfitoGRGR (SEQ ID NO:70); FfFGRGR (SEQ ID NO:71); or
FfitoGrGr (SEQ ID NO:72). The cCPP can have one of the following sequences:
FGF (SEQ ID
NO:73); GfFGrGrQ (SEQ ID NO:74), FfitoGRGRQ (SEQ ID NO:75); FfFGRGRQ (SEQ ID
NO:76); or FfitoGrGrQ (SEQ ID NO:77).
[0303] The disclosure also relates to a cCPP having the structure of Formula
(II):
R2b
R2a 0
H N 0
N H R2d
H N/0
rN H
Ria
0 N H N Msc
0
Rib )nu
n" (II)
wherein:
AAsc is an amino acid side chain;
Rib,
and Ric are each independently a 6- to 14-membered aryl or a 6- to 14-
membered heteroaryl;
110
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
R2a, R2b, R2c and R2d
are independently an amino acid side chain;
0 NH 0 CN
N- 1X
H2NANN H2NAN)Y N
at least one of R2a, R2b, R2c and R2d is H H H H ,
CI N FiNOI 1
N N N
H , , f , or a protonated form thereof
at least one of R2a, R2b, R2c and R2d is guanidine or a protonated form
thereof
each n" is independently an integer 0, 1, 2, 3, 4, or 5;
each n' is independently an integer from 0, 1, 2, or3; and
if n' is 0 then R2a, R2b, R2b or R2d is absent.
0 NH 0 CN
NX
H2NAN H2NAN)Y N - 1
[0304] At least two of R2a, R2b, R2c and R2d can be H H H H
r ,
N
JIL X I
N N- % FiNOI N
NI/
, , H f , or a protonated form thereof Two or three of R2a, R2b, R2c
and
H2NANN H2NAN)y CN'.N,
0 NH 0 N N HNaii 1
), r), N- 1
N µ N x 1 N
. y
R2d can be H H , H H , H , , f ,
or a
0 NH 0
H2NANN H2NAN)Y
protonated form thereof One of R2a, R2b, R2 and R2d can be H H ,
rs-N Nr rNN- x
HNail 1
N µ i
N µ N N
H H H
i , , , or a protonated form thereof. At
least one of R2a,
0
H2NAN
KN
-r%2b,
R2c and R2d can be H , or a protonated form thereof, and the
remaining of R2a,
R2b,
R2 and R2d can be guanidine or a protonated form thereof. At least two of R2a,
R2b, R2c and
0
H2NANN
R2d can be H , or
a protonated form thereof, and the remaining of R2a, R2b, R2c and
R2d can be guanidine, or a protonated form thereof
111
CA 03222824 2023-12-07
WO 2022/271818
PCT/US2022/034517
0 NH 0
H2NANN H2NAN)Y N)LN\
[0305] All of R2a, R2b, R2c and =-=2d
can be H H H
FiN0/N N
, or a protonated form thereof At least of R2a, R2b, R2c and Rat
0
H2NANN
can be H , or a protonated form thereof, and the remaining of R2a,
R2b, R2c and Rat
can be guaninide or a protonated form thereof At least two R2a, R2b, R2c and .-
µ2d
groups can be
0
H2NAN \
H , or a protonated form thereof, and the remaining of R2a, R2b, R2c
and Rat are
guanidine, or a protonated form thereof.
[0306] Each of R2a, R2b, R2c and 2d
can independently be 2,3-diaminopropionic acid, 2,4-
diaminobutyric acid, the side chains of ornithine, lysine, methyllysine,
dimethyllysine,
trimethyllysine, homo-lysine, serine, homo-serine, threonine, allo-threonine,
histidine, 1-
methylhistidine, 2-aminobutanedioic acid, aspartic acid, glutamic acid, or
homo-glutamic acid.
cssspr N H2 ,scsprco2H
[0307] AAsc can be " t
or t ,
wherein t can be an integer from 0 to 5.
,scspi-0O2H
t
AAsc can be ,wherein t can be an integer from 0 to 5. t can be 1 to 5.
t is 2 or 3. t can
be 2. t can be 3.
[0308] Ria, Rib, and Ric can each independently be 6- to 14-membered aryl.
Ria, Rib, and Ric can
be each independently a 6- to 14-membered heteroaryl having one or more
heteroatoms selected
from N, 0, or S. Ria, Rib, and Ric can each be independently selected from
phenyl, naphthyl,
anthracenyl, pyridyl, quinolyl, or isoquinolyl. Ria, Rib, and Ric can each be
independently
selected from phenyl, naphthyl, or anthracenyl. Ria, Rib, and Ric can each be
independently
phenyl or naphthyl. Ria, Rib, and Ric can each be independently selected
pyridyl, quinolyl, or
isoquinolyl.
112
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
[0309] Each n' can independently be 1 or 2. Each n' can be 1. Each n' can be
2. At least one n'
can be 0. At least one n' can be 1. At least one n' can be 2. At least one n'
can be 3. At least one
n' can be 4. At least one n' can be 5.
[0310] Each n" can independently be an integer from 1 to 3. Each n" can
independently be 2 or
3. Each n" can be 2. Each n" can be 3. At least one n" can be 0. At least one
n" can be 1. At least
one n" can be 2. At least one n" can be 3.
[0311] Each n" can independently be 1 or 2 and each n' can independently be 2
or 3. Each n"
can be 1 and each n' can independently be 2 or 3. Each n" can be 1 and each n'
can be 2. Each n"
is 1 and each n' is 3.
[0312] The cCPP of Formula (II) can have the structure of Formula (II-1):
fin, R2c
R2a\
*tiii7 NH N4
HN /-1 n!
NH 7sR2d
01/
0
NH HN
Rla s
0 N HN c
0
Rib in" r(S. Ric
n" (II-1),
wherein lea, RR, R2a, R2b, R2c, R2d, AAsc, n' and n" are as defined herein.
[0313] The cCPP of Formula (II) can have the structure of Formula (Ha):
R2b¨Hn' 0 R2c
R21
*ti'l7 NH N nn
NH HN
0 pR2d
0
NH HN
0
0
(Ha),
wherein lea, R1b, Ric, R2a, R2b, R2c, R2d, AAsc and n' are as defined herein.
113
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
[0314] The cCPP of formula (II) can have the structure of Formula (IIb):
H2N-fNH
HN . 0
R2a\ R2c
( NH N n 0
, NH
N
0 H
HN (JJ
0
HN
NH
HNsc
0
0
(llb),
wherein R2a, R2b, AAsc, and n' are as defined herein.
[0315] The cCPP can have the structure of Formula (IIc):
H2N.,fNH
0
0
HN
H
)L NNH c---\
H2N 4.?-NH h140
, NH
n'NH 0 HNNH
(41
0
HN
NH
HN AAsc
0
0
(IIc), or a protonated form thereof,
wherein:
AAsc and n' are as defined herein.
[0316] The cCPP of Formula (Ha) has one of the following structures:
114
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
H2N--..f0
0 H2N -__o
HN _
NH2
---NH2 7- 0 HN-...t...1 0 HN--.1...1 n
NH 0
k 1. NH
)NH )---%,,cn
H2N , y--NH H----0 H2N , )NH "N fl' 0
l NH t H
n'
NH HN.,in' A n'NH HN.
NH2 0
BnN1' INN
H H 2
0 0
NH HN
NH HN
CAA HN4.".AAsc \FIHN4Asc
1110 0
Si 0
IP IP
H2N--,fNH
0 H2N-__o
HN ---NH2 o____NH2
0 -1--,) , 0
.szjictNH HN 0
HNI---).f/j4. (,.-NH
NH
H2N , \.4...1),, NH IN n 0 H2N ,
\.4.1)\-NH H n 0
t H , 0 t , 0
n'NH HN ,HI1 A n'NH HN f 41 jt
0 H NH2 0
, N-NH2
0 0 H
NH HN NH HN
0\--FNI HN4-"I HN AAsc AAso
0----11 --\<1."'s'
101 0
101 0
IP ,or
wherein AAsc and n are as defined herein.
[0317] The cCPP of Formula (Ha) has one of the following structures:
H2N-NH ¨f 0 H2N-..fo
HN
---NH2 .--NH2
0 0 0 HN) , 0
HN) '-NH2
_c\IH
NH 14 n 0 H2N ,p4...?\-NH El n 0
t H , NH l , NH
n'NH HN ,Bn A n'NH HN
0 IF1 NH2 0 hi NH2
0 0
NH HN NH HN
0-FN-1 HN___\441P\As0 ________ 0\--Ed HN ?-41AAso
Oa so
115
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
H2N-fNH
0 H2N--f
HN
HN
HN¨..f...1 n ---NH2 HN --NH2
k 1./' " "-
NH HN ¨N , 0
_i(n NH
)Lt NH O\ )---L --NH 0
H2N , \.4.1), __ NH 11 / H---i. ,0 __ H2N t ,Lq NH
HN 11'0
0 , 0
n'NH HN n )( n'
NH HN 1,111 1
O H NH2 0
rN- 'NH2
H
O 0
HN HN
NH NH
AAsc
0\---FN-1 HNi 0___H HN4""VkAsc
O 0
So
SI 0
111 ,or
wherein AAsc and n are as defined herein
[0318] The cCPP of Formula (ha) has one of the following structures:
H2N....f NH H2N-....f0
HN HN
--NH2
0 HN) , 0 --NH2 HN HN) , 0
NH NH
)\---NH .)--An ,, n
H2N ,y¨NH ,N 0 H2N
t H , NH l , NH
n'NH HNn'NH HN.
O I-VNH2 0 KnN)(NH2
O H
NH HN NH HN 0
0-H H 4-""AAsc 0d 4sc
N --F HN
O 0
Oa
Oa
H2NNH 0 H2NfNH
HN
HN HN-1--
---NH2 HN HN-14 .--NH2
, 110 0
-mtNHNH
)LNH
H2N , \.4...1),µ NH Pi n 0 H2N
,y¨NN H n 0
t H , NH
n'NH HN. n'NH HN
O KnN).(NH 0 KnN)(NH
2 2
O H
0 H
NH HN NH HN
A
0 ----H HN--\<LsiAsc HN
?-41AA sc
0\¨FNI
O 0
0 0 0 0
111 , or
116
CA 03222824 2023-12-07
WO 2022/271818
PCT/US2022/034517
wherein AAsc and n are as defined herein.
[0319] The cCPP of Formula (II) can have the structure:
H2N--fNH
H2N
NH 0
NH2
(---- HN
()N 0 ' _____
H y.___Nr4 ___(r. )
H HN
0
0 NH
HN NH2
Inµ \----\ i
NH N----NH
Ok_ H HN 0 H
* NH
0 0
0
. OH
[0320] The cCPP of Formula (II) can have the structure:
NH H2N--fNH
H2N---.f I NH
1[1 (NH N
...- ,..
C-----. õ \
N¨
NH '-'1=7)---N ' HN
N HN
H 0 NH
0 NH HN
HN x, N.......N
!µ1H2
1zH2
NH N--k=
NH H NH
HN 0
Ok___
HN 0 H NH
NH N
H---eµVr0 0 0
0 0
410 OH
or it OH
,
[0321] The cCPP can have the structure of Formula (III):
117
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
pp2b
' R2c
R2aõ\___O
( HN
NH HN (õ),r1'
0.y R2d
HN/0
( in-NH
Rla
0 NHNSC
0
R1 b )nõ
( RI'
n" (III),
wherein:
AAsc is an amino acid side chain;
Rib,
and Ric are each independently a 6- to 14-membered aryl or a 6- to 14-
membered heteroaryl;
0 NH 0 N
JIL
H2NAN C
)µ H2NAN)Y N N-
R2a and R2c are each independently H, H H H
J HNO/
IL
N N¨ Nv
, or a protonated form thereof;
R2b and R2d are each independently guanidine or a protonated form thereof;
each n" is independently an integer from 1 to 3;
each n' is independently an integer from 1 to 5; and
each p' is independently an integer from 0 to 5.
[0322] The cCPP of Formula (III) can have the structure of Formula (III-1):
R2b
+4._11111 R2c
R2a
1**0-17
Or):NH PR2d
(R) 0
n" NH HN
Ria sc
0 N (RpN
0 ( Ric
n" (III-1),
118
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
wherein:
AAsc, R,RR, R2a, R2c, R2b, R2d n,, n,,, and p' are as defined herein.
[0323] The cCPP of Formula (III) can have the structure of Formula (Ma):
ppab_f
')
pµ's R2c
R2a NEP\ >-1(11
i*Itzr 0
HN
NH
0 k PR2d
(S) NH HN 0
siNAAsc
(y
0
0
(Ma),
wherein:
AAsc, R2a, R2c, R2b, R2d n,, and p' are as defined herein.
[0324] In Formulas (III), (III-1), and (Ma), Ra and RC can be H. Ra and Itc
can be H and Rb and
Rd can each independently be guanidine or protonated form thereof. Ra can be
H. Rb can be H. p'
can be 0. Ra and Itc can be H and each p' can be 0.
[0325] In Formulas (III), (III-1), and (Ma), Ra and Itc can be H, Rb and Rd
can each
independently be guanidine or protonated form thereof, n" can be 2 or 3, and
each p' can be 0.
[0326] p' can 0. p' can 1. p' can 2. p' can 3. p' can 4. p' can be 5.
[0327] The cCPP can have the structure:
119
CA 03222824 2023-12-07
WO 2022/271818
PCT/US2022/034517
H2N---fNH
NH
0
HNN
--Nro
0 NH H
HN
NH2
NH
HN 0 H NH
NH kl.__\(c
0 0
O OH
[0328] The cCPP of Formula (A) can be selected from:
CPP Sequence SEQ ID NO:
(FKoRrRrQ) 78
(FRICit-r-Cit-rQ) 79
(Ff(toGrGrQ) 80
(FfFGRGRQ) 81
(FGFGRGRQ) 82
(GfFGrGrQ) 83
(FGFGRRRQ) 84
(FGFRRRRQ) 85
[0329] The cCPP of Formula (A) can be selected from:
CPP Sequence SEQ ID NO:
F(toRRRRQ 86
f(toltrRrQ 87
Ff(toltrRrQ 78
Ff(toCit-r-Cit-rQ 79
Ff(toGrGrQ 80
Ff(toRGRGQ 88
FfFGRGRQ 81
FGFGRGRQ 82
GfFGrGrQ 83
FGFGRRRQ 84
FGFRRRRQ 85
[0330] In embodiments, the cCPP is selected from:
120
CA 03222824 2023-12-07
WO 2022/271818
PCT/US2022/034517
CPP sequences and SEQ ID NOs
FORRRQ 89 RRFRORQ 99 FORRRRQK 109
FORRRC 90 FRRRROQ 100 FORRRRQC 110
FORRRU 91 rRFRORQ 101 fORrRrRQ 111
RRROFQ 92 RROFRRQ 102 FORRRRRQ 112
RRRR0F 93 CRRRRFWQ 103 RRRROFDS/C 113
FORRRR 94 FfORrRrQ 104 FORRR 114
FckrRrRq 95 FFORRRRQ 105 FWRRR 115
FORIRQ 96 RFRFRORQ 106 RRROF 116
FORRRRQ 97 URRRRFWQ 107 RRRWF 117
fORrRrQ 98 CRRRRFWQ 108
Where 0 = L-naphthylalanine; (I) = D-naphthylalanine; S2 = L-norleucine
[0331] In embodiments, the cCPP is not selected from:
CPP sequences and SEQ ID NOs
FORRRQ 89 RRFRORQ 99 FORRRRQK 109
FORRRC 90 FRRRROQ 100 FORRRRQC 110
FORRRU 91 rRFRORQ 101 fORrRrRQ 111
RRROFQ 92 RROFRRQ 102 FORRRRRQ 112
RRRR0F 93 CRRRRFWQ 103 RRRROFDS/C 113
FORRRR 94 FfORrRrQ 104 FORRR 114
FckrRrRq 95 FFORRRRQ 105 FWRRR 115
FORIRQ 96 RFRFRORQ 106 RRROF 116
FORRRRQ 97 URRRRFWQ 107 RRRWF 117
fORrRrQ 98 CRRRRFWQ 108
Where 0 = L-naphthylalanine; (I) = D-naphthylalanine; S2 = L-norleucine
121
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
[0332] The cCPP can comprise the structure of Formula (D)
NH
H2N11(NH
H2N--fNH
R ,6 0
im NH Nr0
HNAAsc
0
o/Y
R4
NH
NH FN-I
R3
(D)
or a protonated form thereof, wherein:
R1, R2, and R3 can each independently be H or an amino acid residue haying a
side chain
comprising an aromatic group;
at least one of R1, R2, and R3 is an aromatic or heteroaromatic side chain of
an amino
acid;
R4 and R6 are independently H or an amino acid side chain;
0
HN)L1
0
H2N
H2N
0
N NH2
m NH
Y is 0 m H
0
HNJLI
N
) H2N
Jwv
0+)-S.N>L
NH2 n m H =
, or
AAsc is an amino acid side chain;
122
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
q is 1, 2, 3 or 4;
each m is independently an integer 0, 1, 2, or 3, and
each n is independently an integer 0, 1, 2, or 3.
[0333] The cCPP of Formula (D) can have the structure of Formula (D-I):
NH
H2N-1(NH
H
HN 0
0
HNTAAsc
i\uNH
0
R4 0/
NH
o NH EN-11
(QR1
R3
of 1R2
(D-I)
or a protonated form thereof,
wherein:
R1, R2, and R3 can each independently be H or an amino acid residue having a
side chain
comprising an aromatic group;
at least one of R1, R2, and R3 is an aromatic or heteroaromatic side chain of
an amino
acid;
R4 and R6 are independently H or an amino acid side chain;
AAsc is an amino acid side chain;
q is 1, 2, 3 or 4;
each m is independently an integer 0, 1, 2, or 3, and
0
)=
HN z
i,NThrNH2
Y is 0
123
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
[0334] The cCPP of Formula (D) can have the structure of Formula (D-II):
NH
H2N¨I(NH
H2N---fNH
HN 0
0
HNTAAse
\nNH
0
R4 0/
NH
NH FN-1.....\(L Ri
R3
1R2
or a protonated form thereof,
wherein:
R1, R2, and R3 can each independently be H or an amino acid residue having a
side chain
comprising an aromatic group;
at least one of R1, R2, and R3 is an aromatic or heteroaromatic side chain of
an amino
acid;
R4 and R6 are independently H or an amino acid side chain;
AAsc is an amino acid side chain;
q is 1, 2, 3 or 4;
each m is independently an integer 0, 1, 2, or 3,
each m is independently an integer 0, 1, 2, or 3, and
=y0
HN ),, H2N
Y is m H
[0335] The cCPP of Formula (D) can have the structure of Formula
124
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
NH
H2N1(NH
H2N--fNH
R5_2(
HN 0
0
HNTAAsc
0
R4 0/
NH
o NH EN-11
(QR1
R3
0" 1R2
(D-III)
or a protonated form thereof,
wherein:
Ri, R2, and R3 can each independently be H or an amino acid residue having a
side chain
comprising an aromatic group;
at least one of Ri, R2, and R3 is an aromatic or heteroaromatic side chain of
an amino
acid;
R4 and R6 are independently H or an amino acid side chain;
AAsc is an amino acid side chain;
q is 1, 2, 3 or 4;
each m is independently an integer 0, 1, 2, or 3,
each n is independently an integer 0, 1, 2, or 3, and
0
H2N
0
m NH
Y is
[0336] The cCPP of Formula (D) can have the structure of Formula (D-IV):
125
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
NH
H2N1(NH
H
R5_2(
HN 0
0
HNTAAsc
0
R4 0/
NH
o NH EN-11
(QR1
R3
0" 1R2
(D-IV)
or a protonated form thereof,
wherein:
Ri, R2, and R3 can each independently be H or an amino acid residue having a
side chain
comprising an aromatic group;
at least one of Ri, R2, and R3 is an aromatic or heteroaromatic side chain of
an amino
acid;
R4 and R6 are independently H or an amino acid side chain;
AAsc is an amino acid side chain;
q is 1, 2, 3 or 4;
each m is independently an integer 0, 1, 2, or 3, and
0
HNAI
)n
Ns
NN)rn
Y is NH2
[0337] The cCPP of Formula (D) can have the structure of Formula (D-V):
126
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
NH
H2N1(NH
H2N--..fNH
HN 0
0
HNTAAsc
0
R4 0/
NH
o NH EN-11
(LR1
of 1R2
(D-V)
or a protonated form thereof,
wherein:
Ri, R2, and R3 can each independently be H or an amino acid residue having a
side chain
comprising an aromatic group;
at least one of Ri, R2, and R3 is an aromatic or heteroaromatic side chain of
an amino
acid;
R4 and R6 are independently H or an amino acid side chain;
AAsc is an amino acid side chain;
q is 1, 2, 3 or 4;
each m is independently an integer 0, 1, 2, or 3, and
H2N
nS
Y is ni H
[0338] The AAsc can be conjugated to a linker.
Linker
[0339] The cCPP of the disclosure can be conjugated to a linker. The linker
can link a cargo to
the cCPP. The linker can be attached to the side chain of an amino acid of the
cCPP, and the
cargo can be attached at a suitable position on linker.
127
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
[0340] The linker can be any appropriate moiety which can conjugate a cCPP to
one or more
additional moieties, e.g., an exocyclic peptide (EP) and/or a cargo. Prior to
conjugation to the
cCPP and one or more additional moieties, the linker has two or more
functional groups, each of
which are independently capable of forming a covalent bond to the cCPP and one
or more
additional moieties. If the cargo is an oligonucleotide, the linker can be
covalently bound to the
5' end of the cargo or the 3' end of the cargo. The linker can be covalently
bound to the 5' end of
the cargo. The linker can be covalently bound to the 3' end of the cargo. If
the cargo is a peptide,
the linker can be covalently bound to the N-terminus or the C-terminus of the
cargo. The linker
can be covalently bound to the backbone of the oligonucleotide or peptide
cargo. The linker can
be any appropriate moiety which conjugates a cCPP described herein to a cargo
such as an
oligonucleotide, peptide or small molecule.
[0341] The linker can comprise hydrocarbon linker.
[0342] The linker can comprise a cleavage site. The cleavage site can be a
disulfide, or caspase-
cleavage site (e.g, Val-Cit-PABC).
[0343] The linker can comprise: (i) one or more D or L amino acids, each of
which is optionally
substituted; (ii) optionally substituted alkylene; (iii) optionally
substituted alkenylene; (iv)
optionally substituted alkynylene; (v) optionally substituted carbocyclyl;
(vi) optionally
substituted heterocyclyl; (vii) one or more -(R1-J-R2)z"- subunits, wherein
each of Wand R2, at
each instance, are independently selected from alkylene, alkenylene,
alkynylene, carbocyclyl,
and heterocyclyl, each J is independently C, NR3, -NR3C(0)-, S, and 0, wherein
R3 is
independently selected from H, alkyl, alkenyl, alkynyl, carbocyclyl, and
heterocyclyl, each of
which is optionally substituted, and z" is an integer from 1 to 50; (viii) -
(R1-J)z"- or
wherein each of R1, at each instance, is independently alkylene, alkenylene,
alkynylene,
carbocyclyl, or heterocyclyl, each J is independently C, NR3, -NR3C(0)-, S, or
0, wherein R3 is
H, alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl, each of which is
optionally substituted,
and z" is an integer from 1 to 50; or (ix) the linker can comprise one or more
of (i) through (x).
[0344] The linker can comprise one or more D or L amino acids and/or -(R1-J-
R2)z"-, wherein
each of Wand R2, at each instance, are independently alkylene, each J is
independently C, NR3, -
NR3C(0)-, S, and 0, wherein R4 is independently selected from H and alkyl, and
z" is an integer
from 1 to 50; or combinations thereof.
128
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
[0345] The linker can comprise a -(OCH2CH2),- (e.g., as a spacer), wherein z'
is an integer from
1 to 23, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, or 23. "-
(OCH2CH2) z' can also be referred to as polyethylene glycol (PEG).
[0346] The linker can comprise one or more amino acids. The linker can
comprise a peptide. The
linker can comprise a -(OCH2CH2)z,-, wherein z' is an integer from 1 to 23,
and a peptide. The
peptide can comprise from 2 to 10 amino acids. The linker can further comprise
a functional
group (FG) capable of reacting through click chemistry. FG can be an azide or
alkyne, and a
triazole is formed when the cargo is conjugated to the linker.
[0347] The linker can comprises (i) a 0 alanine residue and lysine residue;
(ii) -(J-R1)z"; or (iii)
a combination thereof. Each R1 can independently be alkylene, alkenylene,
alkynylene,
carbocyclyl, or heterocyclyl, each J is independently C, NR3, -NR3C(0)-, S, or
0, wherein R3 is
H, alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl, each of which is
optionally substituted,
and z" can be an integer from 1 to 50. Each R1 can be alkylene and each J can
be 0.
[0348] The linker can comprise (i) residues of 13-alanine, glycine, lysine, 4-
aminobutyric acid, 5-
aminopentanoic acid, 6-aminohexanoic acid or combinations thereof; and (ii) -
(R1-J)z"- or -(J-
R1)z". Each R1 can independently be alkylene, alkenylene, alkynylene,
carbocyclyl, or
heterocyclyl, each J is independently C, NR3, -NR3C(0)-, S, or 0, wherein R3
is H, alkyl,
alkenyl, alkynyl, carbocyclyl, or heterocyclyl, each of which is optionally
substituted, and z" can
be an integer from 1 to 50. Each R1 can be alkylene and each J can be 0. The
linker can
comprise glycine, beta-alanine, 4-aminobutyric acid, 5-aminopentanoic acid, 6-
aminohexanoic
acid, or a combination thereof.
wuv
[0349] The linker can be a trivalent linker. The linker can have the
structure: 0
Juv ' Juw
7'
Ai
ZHN(C1 B1NOZ
ZHNH.roz
0 0 , or 0 wherein Ai, Bi, and Ci, can
independently
be a hydrocarbon linker (e.g., NRH-(CH2),-000H), a PEG linker (e.g., NRH-
(CH20),-000H,
wherein R is H, methyl or ethyl) or one or more amino acid residue, and Z is
independently a
129
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
protecting group. The linker can also incorporate a cleavage site, including a
disulfide [NH2-
(CH20),-S-S-(CH20),-COOH], or caspase-cleavage site (Val-Cit-PABC).
[0350] The hydrocarbon can be a residue of glycine or beta-alanine.
[0351] The linker can be bivalent and link the cCPP to a cargo. The linker can
be bivalent and
link the cCPP to an exocyclic peptide (EP).
[0352] The linker can be trivalent and link the cCPP to a cargo and to an EP.
[0353] The linker can be a bivalent or trivalent C i-050 alkylene, wherein 1-
25 methylene groups
are optionally and independently replaced by -N(H)-, -N(Ci-C4 alkyl)-, -
N(cycloalkyl)-, -0-, -
C(0)-, -C(0)0-, -S-, -S(0)-, -S(0)2-, -S(0)2N(Ci-C4 alkyl)-, -
S(0)2N(cycloalkyl)-, -N(H)C(0)-,
-N(Ci-C4 alkyl)C(0)-, -N(cycloalkyl)C(0)-, -C(0)N(H)-, -C(0)N(Ci-C4 alkyl), -
C(0)N(cycloalkyl), aryl, heterocyclyl, heteroaryl, cycloalkyl, or
cycloalkenyl. The linker can be
a bivalent or trivalent C i-050 alkylene, wherein 1-25 methylene groups are
optionally and
independently replaced by -N(H)-, -0-, -C(0)N(H)-, or a combination thereof.
[0354] The linker can have the structure:
H0
FM*N
AA
" (614
, wherein: each AA is independently an amino acid residue; *
is the point of attachment to the AAsc, and AAsc is side chain of an amino
acid residue of the
cCPP; x is an integer from 1-10; y is an integer from 1-5; and z is an integer
from 1-10. x can be
an integer from 1-5. x can be an integer from 1-3. x can be 1. y can be an
integer from 2-4. y can
be 4. z can be an integer from 1-5. z can be an integer from 1-3. z can be 1.
Each AA can
independently be selected from glycine, 13-alanine, 4-aminobutyric acid, 5-
aminopentanoic acid,
and 6-aminohexanoic acid.
[0355] The cCPP can be attached to the cargo through a linker ("L"). The
linker can be
conjugated to the cargo through a bonding group ("M").
[0356] The linker can have the structure:
130
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
/.4 0
, H ,õJf _AAt
(cHz)
, wherein: x is an integer from 1-10; y is an integer from 1-
5; z is an integer from 1-10; each AA is independently an amino acid residue;
* is the point of
attachment to the AAsc, and AAsc is side chain of an amino acid residue of the
cCPP; and M is a
bonding group defined herein.
[0357] The linker can have the structure:
0
ck, N N N M
/ 0
X' z H
0 (CH2)y z'
vw
wherein: x' is an integer from 1-23; y is an integer from 1-5; z' is an
integer from 1-23; *
is the point of attachment to the AAsc, and AAsc is a side chain of an amino
acid residue of the
cCPP; and M is a bonding group defined herein.
[0358] The linker can have the structure:
0 OH
40 N .)=L N
/ 0
= H
0 (CH2)y z'
TA'
wherein: x' is an integer from 1-23; y is an integer from 1-5; and z' is an
integer from 1-
23; * is the point of attachment to the AAsc, and AAsc is a side chain of an
amino acid residue of
the cCPP.
[0359] x can be an integer from 1-10, e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9, or 10,
inclusive of all ranges
and subranges therebetween.
[0360] x' can be an integer from 1-23, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, or 23, inclusive of all ranges and subranges therebetween.
x' can be an integer
from 5-15. x' can be an integer from 9-13. x' can be an integer from 1-5. x'
can be 1.
131
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
[0361] y can be an integer from 1-5, e.g., 1, 2, 3, 4, or 5, inclusive of all
ranges and subranges
therebetween. y can be an integer from 2-5. y can be an integer from 3-5. y
can be 3 or 4. y can
be 4 or 5. y can be 3. y can be 4. y can be 5.
[0362] z can be an integer from 1-10, e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9, or 10,
inclusive of all ranges
and subranges therebetween.
[0363] z' can be an integer from 1-23, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, or 23, inclusive of all ranges and subranges therebetween.
z' can be an integer
from 5-15. z' can be an integer from 9-13. z' can be 11.
[0364] As discussed above, the linker or M (wherein M is part of the linker)
can be covalently
bound to cargo at any suitable location on the cargo. The linker or M (wherein
M is part of the
linker) can be covalently bound to the 3' end of oligonucleotide cargo or the
5' end of an
oligonucleotide cargo. The linker or M (wherein M is part of the linker) can
be covalently bound
to the N-terminus or the C-terminus of a peptide cargo. The linker or M
(wherein M is part of the
linker) can be covalently bound to the backbone of an oligonucleotide or a
peptide cargo.
[0365] The linker can be bound to the side chain of aspartic acid, glutamic
acid, glutamine,
asparagine, or lysine, or a modified side chain of glutamine or asparagine
(e.g., a reduced side
chain having an amino group), on the cCPP. The linker can be bound to the side
chain of lysine
on the cCPP.
[0366] The linker can be bound to the side chain of aspartic acid, glutamic
acid, glutamine,
asparagine, or lysine, or a modified side chain of glutamine or asparagine
(e.g., a reduced side
chain having an amino group), on a peptide cargo. The linker can be bound to
the side chain of
lysine on the peptide cargo.
[0367] The linker can have a structure:
HIV-My
A)0
HAAs-(Mx)p NH2
0
wherein
M is a group that conjugates L to a cargo, for example, an oligonucleotide;
AA, is a side chain or terminus of an amino acid on the cCPP;
132
CA 03222824 2023-12-07
WO 2022/271818
PCT/US2022/034517
each AA x is independently an amino acid residue;
o is an integer from 0 to 10; and
p is an integer from 0 to 5.
[0368] The linker can have a structure:
NW 7
0 9 r
, .1õNH,
0
wherein
M is a group that conjugates L to a cargo, for example, an oligonucleotide;
AA, is a side chain or terminus of an amino acid on the cCPP;
each AA x is independently an amino acid residue;
o is an integer from 0 to 10; and
p is an integer from 0 to 5.
[0369] M can comprise an alkylene, alkenylene, alkynylene, carbocyclyl, or
heterocyclyl, each
of which is optionally substituted. M can be selected from:
0
1-17-1
SH 0
sH
0 H
O
,s
0 R srsrvsg 0 R HS
,ecr S
µsDr
2-61- N N
NH
133
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
I N>`
N 0 HN.,..,t0
R , wherein R is alkyl, alkenyl, alkynyl, carbocyclyl, or
heterocyclyl.
[0370] M can be selected from:
0 0 0
R1
SA, o S' cos AN)LA AN).L/Sy
0 0 ,
N..-.1
pN--).....
Nr'N*N-1 ill /-
0 µ =-=¨==i k¨C) 6r0
rkN)-S,Ri) x
H Oi
0 ,
-r
0
liL0j 0 0H
.0 \ N
NA,
(:)F'' 0
HO' A H
uN)HiN
H
0 ,and
--r-
0
B
Ipl N Lr0
---1 0 0
.0 \ NN,......,..õ,NNANN
(:)
HO'A H H
)L7iN
H
0
wherein:
Aoic/
10 is alkylene, cycloalkyl, or a , wherein a is 0 to 10.
134
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
0
S'
[0371] M can be 0 , Iti can be a ,
and a is 0 to 10. M can be .
0
[0372] M can be a heterobifunctional crosslinker, e.g., 0 , which is
disclosed in Williams et al. Curr. Protoc Nucleic Acid Chem. 2010, 42, 4.41.1-
4.41.20,
incorporated herein by reference its entirety.
[0373] M can be -C(0)-.
[0374] AA, can be a side chain or terminus of an amino acid on the cCPP. Non-
limiting
examples of AA, include aspartic acid, glutamic acid, glutamine, asparagine,
or lysine, or a
modified side chain of glutamine or asparagine (e.g., a reduced side chain
having an amino
group). AA, can be an AAsc as defined herein.
[0375] Each AA x is independently a natural or non-natural amino acid. One or
more AA x can be
a natural amino acid. One or more AA x can be a non-natural amino acid. One or
more AA x can
be a I3-amino acid. The I3-amino acid can be I3-alanine.
[0376] o can be an integer from 0 to 10, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,
and 10. o can be 0, 1, 2, or
3. o can be O. o can be 1. o can be 2. o can be 3.
[0377] p can be 0 to 5, e.g., 0, 1, 2, 3, 4, or 5. p can be 0. p can be 1. p
can be 2. p can be 3. p can
be 4. p can be 5.
[0378] The linker can have the structure:
0
H NAR1¨J
o " z" H2NO 0
1¨AAs 2 , NH
NMI
0 or 0 = z"
wherein M, AA,, each -(10-J-R2)z"-, o and z" are defined herein; r can be 0 or
1.
[0379] r can be 0. r can be 1.
[0380] The linker can have the structure:
135
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
0 .
N-MA
0)()13\ - H
z" H2NO
AAsNH
ThrNH2 P
H0 or 0 = -z" 0
wherein each of M, AA, o, p, q, r and z" can be as defined herein.
[0381] z" can be an integer from 1 to 50, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,
36, 37, 38, 39, 40, 41, 42,
43, 44, 45, 46, 47, 48, 49, and 50, inclusive of all ranges and values
therebetween. z" can be an
integer from 5-20. z" can be an integer from 10-15.
[0382] The linker can have the structure:
HN-My
,(J)0
HAAs .iNH 2
0
wherein:
M, AA s and o are as defined herein.
[0383] Other non-limiting examples of suitable linkers include:
0
HN)Hrml 0 0
0 AAs A
N M AAsNC)N)Hrikil
1 2 0
AAs-....N NH2
0
0
HNIVI)/ 0 0
0 AAs )(mA
2 H
0
AAs--...N NH2
0
136
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
H
AAs AAs
N N rA
No,r my
- ,..,-....,...õ,0
-(N'i)/
H H
0 0 ,
0
0 0
MX
AAs
NN()NOC)NON()0()NONO
H H H
0 MA'
AAsNNC)0C)0)LNO
H H
AAs.
N,0
H H
0 0
AAs. N 0.oc)ro
AAs, N
_.õ. õ,..,,.....-0.,.,,.(0õ , 24 MX, H
H 0 M
, f ,
AAs
\ 0
HN---\_..? )rvi A
HN
H 0
)
HN
M¨I AAs,,
NH2 NNIF12
0 , H
0
0
M
H NM A, HN Y
)
0
H
AAs NC)r N 04)LNr NH2 AAs....N NH2
H H H
0 0 0
,
and
137
CA 03222824 2023-12-07
WO 2022/271818
PCT/US2022/034517
0 0
HN'
>la
L..., 0
r 1 -
0
[0384] wherein M and AA s are as defined herein.
[0385] Provided herein is a compound comprising a cCPP and an AC that is
complementary to a
target in a pre-mRNA sequence further comprising L, wherein the linker is
conjugated to the AC
0
FN
Ri
0 S y
through a bonding group (M), wherein M is .
[0386] Provided herein is a compound comprising a cCPP and a cargo that
comprises an
antisense compound (AC), for example, an antisense oligonucleotide, that is
complementary to a
target in a pre-mRNA sequence, wherein the compound further comprises L,
wherein the linker
is conjugated to the AC through a bonding group (M), wherein M is selected
from:
0 0 0
0
i¨Nei I-N I-N 0 0
SA ,R1
S y N AH)LA AN)LS1
0
N=N
0
0 kig-01¨
, 6(0
R1
H Oi
0
Nz--_-N
N--zN
1
\ N)se
0:1µ511 0
6r N
___________________________ 0
rrYcss
0 , and ...AL ; wherein: le is alkylene, cycloalkyl, or
,
138
CA 03222824 2023-12-07
WO 2022/271818
PCT/US2022/034517
wherein t' is 0 to 10 wherein each R is independently an alkyl, alkenyl,
alkynyl, carbocyclyl, or
0
csss1)-Lcss,
heterocyclyl, wherein R1 is t , and t' is 2.
[0387] The linker can have the structure:
"ii.
0::;z;4,,,,
\s.
./
.,,.L r... .
.õ,k, =
1-,
I
,-
-LI ms,t4ii , = - At'sh
.
. 0
,
wherein AA, is as defined herein, and m' is 0-10.
[0388] The linker can be of the formula:
,sk NH
0
H H
H 1 1
0 0 .
0
Base N 0,,N 0
(k) " 11 H
[0389] The linker can be of the formula: X,
, wherein
"base" is a nucleobase at the 3' end of a cargo phosphorodiamidate morpholino
oligomer.
[0390] The linker can be of the formula:
139
CA 03222824 2023-12-07
WO 2022/271818
PCT/US2022/034517
Base
)s ) N 0
(2)
0
0
= H
H2N 0
,wherein
"base" corresponds to a nucleobase at the 3' end of a cargo phosphorodiamidate
morpholino
oligomer.
[0391] The linker can be of the formula:
Base
o
)ss,)Ny0
o
0
0
LCD$
Th,µNH
(s)
H2N 0
, wherein
"base" is a nucleobase at the 3' end of a cargo phosphorodiamidate morpholino
oligomer.
0
Base N)
0 .11
[0392] The linker can be of the formula: ,
wherein
"base" is a nucleobase at the 3' end of a cargo phosphorodiamidate morpholino
oligomer.
[0393] The linker can be of the formula:
140
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
Base) \ 9
O N-4(
_______________ 0/\_
l3µ ______________________ \
0
/ 0,µ
, N ______________________________________ \
CAT-,2N
N H2N \-0
0 \¨\
HN¨(
0
=
[0394] The linker can be covalently bound to a cargo at any suitable location
on the cargo. The
linker is covalently bound to the 3' end of cargo or the 5' end of an
oligonucleotide cargo The
linker can be covalently bound to the backbone of a cargo.
[0395] The linker can be bound to the side chain of aspartic acid, glutamic
acid, glutamine,
asparagine, or lysine, or a modified side chain of glutamine or asparagine
(e.g., a reduced side
chain having an amino group), on the cCPP. The linker can be bound to the side
chain of lysine
on the cCPP.
cCPP-linker conjugates
[0396] The cCPP can be conjugated to a linker defined herein. The linker can
be conjugated to
an AAsc of the cCPP as defined herein.
[0397] The linker can comprise a -(OCH2CH2)z¨ subunit (e.g., as a spacer),
wherein z' is an
integer from 1 to 23, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22
or 23. "-(OCH2CH2)z' is also referred to as PEG. The cCPP-linker conjugate can
have a structure
selected from Table 7:
Table 7: cCPP-linker conjugates and SEQ ID NOs
cyc/o(FRI3-4gp-r-4gp-rQ)-PEG4-K-Nth cyclo(SEQ ID NO:118)-PEG4-K-NH2
cyc/o(FRI3-Cit-r-Cit-rQ)-PEG4-K-Nth cyclo(SEQ ID NO:119)-PEG4-K-NH2
cyc/o(FRI3-Pia-r-Pia-rQ)-PEG4-K-Nth cyclo(SEQ ID NO:120)-PEG4-K-NH2
cyc/o(FRI3-Dml-r-Dml -rQ)-PEG4-K-NH2 cyclo(SEQ ID NO:121)-PEG4-K-NH2
cyc/o(FRI3-Cit-r-Cit-rQ)-PEG12-0H cyclo(SEQ ID NO:122)-PEG12-0H
141
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
cyc/o(fOR-Cit-R-Cit-Q)-PEG12-0H cyclo(SEQ ID NO:123)-PEG12-0H
[0398] The linker can comprise a -(OCH2CH2)f- subunit, wherein z' is an
integer from 1 to 23,
and a peptide subunit. The peptide subunit can comprise from 2 to 10 amino
acids. The cCPP-
linker conjugate can have a structure selected from Table 8:
Table 8: cCPP-linker conjugate and SEQ ID NOs
Ac-PKKKRKV-Lys(cyclo[FfO-R-r-Cit-rQ])- Ac-SEQ ID NO:42-Lys(cyclo[SEQ ID
PEG12-K(N3)-NH2 NO:127])-PEG12-K(N3)-NH2
Ac-PKKKRKV-Lys(cyclo[Ff0-Cit-r-R-rQ])- Ac- SEQ ID NO:42-Lys(cyclo[SEQ ID
PEG12-K(N3)-NH2 NO:128])-PEG12-K(N3)-NH2
Ac-PKKKRKV-K(cyclo(FfOR-cit-R-cit-Q))- Ac- SEQ ID NO:42-K(cyclo(SEQ ID
PEG12-K(N3)-NH2 NO:129))-PEG12-K(N3)-NH2
Ac-PKKKRKV-PEG2-Lys(cyclo[Ff0-Cit-r-Cit- Ac- SEQ ID NO:42-PEG2-Lys(cyclo[SEQ
rQ])-B-k(N3)-NH2 ID NO:130])-B-k(N3)-NH2
Ac-PKKKRKV-PEG2-Lys(cyclo[Ff0-Cit-r-Cit- Ac- SEQ ID NO:42-PEG2-Lys(cyclo[SEQ
rQ])-PEG2-k(N3)-NH2 ID NO:130])-PEG2-k(N3)-NH2
Ac-PKKKRKV-PEG2-Lys(cyclo[Ff0-Cit-r-Cit- Ac- SEQ ID NO:42-PEG2-Lys(cyclo[SEQ
rQ])-PEG4-k(N3)-NH2 ID NO:130])-PEG4-k(N3)-NH2
Ac-PKKKRKV-Lys(cyclo[Ff0-Cit-r-Cit-rQ])- Ac- SEQ ID NO:42-Lys(cyclo[SEQ ID
PEG12-k(N3)-NH2 NO:130D-PEG12-k(N3)-NH2
Ac-pkkkrkv-PEG2-Lys(cyclo[Ff0-Cit-r-Cit-rQ])- Ac- SEQ ID NO:131-PEG2-
Lys(cyclo[SEQ
PEG12-k(N3)-NH2 ID NO:130D-PEG12-k(N3)-NH2
Ac-rrv-PEG2-Lys(cyclo[Ff0-Cit-r-Cit-rQ])- Ac-rrv-PEG2-Lys(cyclo[SEQ ID
NO:130])-
PEG12-0H PEG12-0H
Ac-PKKKRKV-PEG2-Lys(cyclo[Ff0-Cit-r-Cit-r- Ac- SEQ ID NO:42-PEG2-Lys(cyclo[SEQ
Q])-PEG12-k(N3)-NH2 ID NO:130])-PEG12-k(N3)-NH2
Ac-PKKK-Cit-KV-PEG2-Lys(cyclo[Ff0-Cit-r- Ac- SEQ ID NO:126-PEG2-Lys(cyclo[SEQ
Cit-r-Q])-PEG12-k(N3)-NH2 ID NO:130])-PEG12-k(N3)-NH2
Ac-PKKKRKV-PEG2-Lys(cyclo[Ff0-Cit-r-Cit-r- Ac- SEQ ID NO:42-PEG2-Lys(cyclo[SEQ
Q]EG12-K(N3)-NH2 ID NO:130]-PEG12-K(N3)-NH2
[0399] EEVs comprising a cyclic cell penetrating peptide (cCPP), linker and
exocyclic peptide
(EP) are provided. An EEV can comprise the structure of Formula (B):
142
CA 03222824 2023-12-07
WO 2022/271818
PCT/US2022/034517
, P
9 ,s4 OH
N
H 1= H ' =
(OH2).
NH
F=1
07' 1 -
82
r H
NH 1
NH
\ HN
r
H NH
H
1
0,3
= s = 4 N.
ii
b
Li I)) \
,m
NH
H.2 N
NH (B), or a protonated form thereof,
wherein:
R1, R2, and R3 are each independently H or an aromatic or heteroaromatic side
chain of
an amino acid;
R4 and R7 are independently H or an amino acid side chain;
EP is an exocyclic peptide as defined herein;
each m is independently an integer from 0-3;
n is an integer from 0-2;
x' is an integer from 1-20;
y is an integer from 1-5;
q is 1-4; and
z' is an integer from 1-23.
[0400] Ri, R2, R3, R4, R7, EP, m, q, y, x', z' are as described herein.
[0401] n can be 0. n can be 1. n can be 2.
[0402] The EEV can comprise the structure of Formula (B-a) or (B-b):
143
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
1.4 0 OH
ER - .0 = N 10
""¨.0
6
2 4
NH
wk, 0 ""/5-)
: 1?.
-1) AN---/ =
NH \F-ii
0-
N
Hrn,
171
õ
µs)
NH
;\
NH (B-a),
H OH
EP- == 0 N =
'`=-= 'y- 1"0'.'"="0
0 =,:." 11
NH
1! a;f-1-,,, 9s, :
NH
HN
- N." = \
k= s qn = NH
HN = A)
F./ ----- =:*
a f...; 0
y
NH
NH (B-
b), or a protonated form thereof, wherein
EP, RI-, R2, R3, R4, m and z' are as defined above in Formula (B).
[0403] The EEV can comprises the structure of Formula (B-c):
144
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
C)
EP-k4 YL(' AA*M-I
(e1-12)
I Y
HN
2
HN
NH
0 MN
I-12N AN.---\\Nõ)...T HN
/tR3
NH
HN 0
H2N-1NH
NH (B-c),
or a protonated form thereof, wherein EP, Rl, R2, R3, R4, and m are as defined
above in
Formula (B); AA is an amino acid as defined herein; M is as defined herein; n
is an integer from
0-2; x is an integer from 1-10; y is an integer from 1-5; and z is an integer
from 1-10.
[0404] The EEV can have the structure of Formula (B-1), (B-2), (B-3), or (B-
4):
0 OH
- H
(oH2)
I 4
NH
0
y\--N
HN
NH
0 NH
HN
NH
HN 0
0
H2N-NH1
NH (B-1),
145
CA 03222824 2023-12-07
WO 2022/271818
PCT/US2022/034517
H 9 OH
.--,õõ0õõ--,,o
OOO
HN
0 = H
(CH2),
NH
0 0
NH
(:)/NH
HN
H2NAN
NH
HN
0
NH
NH (B-2),
H 0 OH
EP, NI N
H
0
NH2
HN
HThj
r.. NH H HN
0.
NH HN
NH H HNOh
N
H2N-1H 0 0
NH
(B-3),
146
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
0 0
EP H
N0.Ø)L0H
z H 11
NH
OZ40
0
H2N
)--NH HN
HN o NH
NH
4110
OK_NH HNO
C0
NH
H2N4
NH
(B-4)or a protonated form thereof,
wherein EP is as defined above in Formula (B).
[0405] The EEV can comprise Formula (B) and can have the structure: Ac-
PKKKRKVAEEA-
K(cyclo[FGFGRGRQ])-PEG12-0H (Ac-SEQ ID NO:132- K(cyclo[SEQ ID NO:82])-PEG12-
0H)
or Ac-PK-KKR-KV-AEEA-K(cyclo[GIFGrGrQ])-PEG12-0H (Ac- SEQ ID NO:133-
K(cyc/o[SEQ ID NO:83])-PEG-12-0H).
[0406] The EEV can comprise a cCPP of formula:
NH2
HN(
N HN
)--N H2
HN
0 N
I-1
HN
(s)
NH 0
0 HN
(s)
(3) 0
NH
0 OH
41111 0
147
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
[0407] The EEV can comprise formula: Ac-PKKKRKV-miniPEG2-Lys(cyclo(FfFGRGRQ)-
PEG2-K(N3) (Ac-SEQ ID NO:42-miniPEG2-Lys(cyclo(SEQ ID NO:81)-PEG2-K3)).
[0408] The EEV can be:
NH2
H2N NH
y
HN
(s) , 0ONH2
j urF1 ()rH 0 'J;rH 0
¨ '0 N&L Nks.)A
N ,(0,i-0/AN/,N3
H 0 K H 0 H 0 : H /11 H
H (oH2)4
\ 0
N NH2 NH2 NH2 H¨I___,µ *
p
H2N 0 H N (.3)
--NH \---N H
HN HNyµ =
NH /0
() HN
NH
_ l'1)
, 1,1 H N ,...,
- u
H2N H .._.)
--N
HN
[0409] The EEV can be
0 0 F 0 F
HN)LF-'F HNF HN)L'F
F F
CEI 0\rH \ H H 0 0
H
Q)N N'A, N N: N 1\1')L¨: NOh-rN`:AN,0AOH
1 FIO'I-1 O F1 0/- 2 : H s il
r HyJ 0 ,
(eH2)4
\ 0
N
H¨I___µ p
HNIOF H2NNH 4*
H2N 0 N 0
F F --1\1H rNH H
HN \......./"
HI\1,0 *
NH 0
0 HN
NH
0 H HN _
M-N U
-
H2N H j 0
---N
HN
148
CA 03222824 2023-12-07
WO 2022/271818
PCT/US2022/034517
[0410] The EEV can be Ac-P-K(Tfa)-K(Tfa)-K(Tfa)-R-K(Tfa)-V-miniPEG2-K(cyc/o(Ff-
Nal-
GrGrQ)-PEG12-0H (Ac-SEQ ID NO:134-miniPEG2-K(cyc/o(SEQ ID NO:135)-PEG12-0H).
[0411] The EEV can be
NH2 NH2 NH2
0\r ICI Ei F& F
r 0 0 0
NN )"OH
Q\)1,____H 0 H,c,Ho' 01,20 H
11N, N ,1p
=
HN)
(oH2)4
\ 0
*
NH2 H2NNEI
H2N 0 N 0
HN ?--NH H
NH
HN
NH
0Nr-/N
H 0
H2N H
HN
[0412] The EEV can be Ac-P-K-K-K-R-K-V-miniPEG2-K(cyc/o(Ff-Na1-GrGrQ)-PEG12-0H
(Ac- SEQ ID NO:42-miniPEG2-K(cyc/o(SEQ ID NO:135)-PEG12-0H).
[0413] The EEV can be
149
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
0 0 0 p
HN HN
.,F JF HN)-'F
F F F
0
iFi 0\r H \ H H 0
H
Q
Nj=i\j NN
. N 1All 0 H 0 H 0 t '2 " : H
0 HN) 0 =
(OH2)4
\ 0
O HN 0 I HN--1----
___kCIN 0 F H2N NH
H2N ( F F H \/...rNH H
N HN
NH 0
0 HN
NH
H HN ip,
0,...? 0
H2N H
---N
HN
[0414] The EEV can be
NH2 NH2 NH2
0 0
Cril 9\rH H j ___ kii [11
N2.c NN N
12 n
o E F-1
(O-H2)4
c5 HN) \ 0
O NH2 H2N NH HN-Ic----
,__koN 0
H2N
--NH ,--NH H
HN \/"'( HI\1
NH /0
0 HN
NH
H HN
OThr\II--/ 0 *,
H2N H)
--N
HN
The EEV can be
150
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
H2N1,1\1H
NH2 I
HN
fly cr 0 or
H IirH 91 H 9
-4(DHN N2=C N XirNC
,(0i.o.AN
H (-) H 0 H 0 i H 111 H
(oH2)4
\ 0
_____ j
NH2 NH2 NH2 *
H
H2N N.-: % 0
¨NH ,---NH H
HN Ly"'( HINJ.,µ .
NH /0
H C
HN NV-)N HN
1
NH2 okli HN 0
H2N H3
---N NH
HN =NH
H2N
[0415] The EEV can be
H N1,1\1H
NH2 2 f
HN
flly0 cNi n
H 0 0
0 H
-40 HN)L / \
. N . N . N -VO 1\1)= .0-f. jL
_
- H 0 H 0 H 0 nr _
" H
0 =
(H2)4
\NIL, y * -
-\
NH2 NH2 NH2 H
H2N 0 )LN 0
--NH --NH H
HN V/1" HN 0 to N3
NH 0
H HN
HN.,N--- NH
1
NH2 okli HN
)0i 0
.:
H2N H .._...)
--N NH
HN =NH
H2N
[0416] The EEV can be
151
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
N3
NH2 NH2 NH2
0\r1-1 ?I H H 0 0
N....),
,,,ri 0/ 0 H 0 i
HN) 2 0 E H
H 0
(oH2)4
\ 0
N
H-c___,0 =
NH2 H2NNFI
H N 0 N 0
H
HN FIN.,µ tit
NH
0 HN
O W
NH
H Ni 0 1p
HN H HN
0
---N
HN
[0417] The EEV can be
NH2
H2N NH
Y
fil,r0
FJ A
(: HN
0
NI-1,, C)rN NH, 11 r\rNILNO,:))LOH
H 0
r7
H = H : H \ /11
0 =
(OH2)4
\ 0
NH2 NH2 NH2 H
H2N c N f
HN \/"( HFIN.,µ .
NH /0
0 HN
NH
u
Id H 0
- )1---/N
H2N H ....._) 0
---N
HN
[0418] The EEV can be:
152
CA 03222824 2023-12-07
WO 2022/271818
PCT/US2022/034517
0 0 0
).Q.- F m F ).LF.F
H F
HN HN N
F F F
0 0 H 0 0 0 0
H H H
(s) N (s) N iNi-LsAN (s) N N
0(:)-r N s).(N 0(1)/\)LOH
H = H z H = H = H 11
0 0 0
#
0 HN )
r
HN 0 NH
H2N LI\IH 0
XF
F F
0 Z40
H2N /--\
)--NH
HN
QT
NH
c HN
l"
NH
HNt 0 4Ik
0 NH \fi H (S)
µ,. N
C '`)/
NH
H2N--µ
NH
[0419] The EEV can be
NH2 NH2 NH2
o o H 0 0 0 0
H H H
(s) N.Z.)..,N (s)
- N
H = H z H z H = H 11
N 0 0 0 0
0 HN
17----
)
r
NH2 0 NH
1-121\1LNH
0 Z.40
H2N /---\ )¨ NH 1,,
N (s) '
HN
oy NH
c HN
(R)..:µ
NH
HN 'O =
0*Rj_ H (5)
C C?---/ Os
NH
H2N¨
NH
[0420] The EEV can be
153
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
0 0 0
).Q.= F ).LF..F ).Q..F
HN HN HN
F F F
0 0 H 0 0 0
H H H
N,LSA N =,(S)LNOcy.iN,S.ANO(y-yH
H = H = H = H 11
N 0 0 0 0 0
0 )1.--
HN)
r
HN 0 NH
XF F, H2N NH
F OL
NH2
HNN 0
H (s) [\11 (S) HN (s) II
ty\--
0 NH FIN
H2NyNH NH HN
0 (S) NH N
H (S)
HN
NH
H2N¨(
NH =
[0421] The EEV can be
NH2 NH2 NH2
o o ).(Fi 0 0 0 0
N (s) . N (s) N'e)..'. N 0=1N4*.SJANC)0")''''.'".---
'1LOH
H = H = H = H = H \ 11
N 0 0 0 0
0 17---
HN)
r
NH2 NH
H2 NH 0
L
NH2
1
HNN C)
)
HNN
(5) 0
(s)
0 NH HN
I
H2Ny NH
NH HN n
s-'
(5)
0 (5)
HN NH H N
NH
H2N¨
NH
154
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
[0422] The EEV can be
0 0 F 0
F
F F 1-11\1)F HN"
OrH \H 9 H 0 0
0 =
(oH2)4
HN \ 0
_10
HNICF) H2NNH
H2N 0 N 0
F F
HN I-
11\k
NH
HNO
r1D,NH
H HN
0 4111
111r/
H2NI NH 0
NH NH
NH NH
H2N-4NH H2N
[0423] The EEV can be selected from
Ac-rr-miniPEG2-Dap(cyclo[SEQ
Ac-rr-miniPEG2-Dap(cyc/o[FK0-Cit-r-Cit-rQ])-PEG12-0H ID NO:136])-PEG12-0H
Ac-frr-PEG2-Dap(cyclo[SEQ ID
Ac-frr-PEG2-Dap(cyc/o[Ff40-Cit-r-Cit-rQ])-PEG12-0H NO:136])-PEG12-0H
Ac-rfr-PEG2-Dap(cyclo[SEQ ID
Ac-rfr-PEG2-Dap(cyc/o[Ff40-Cit-r-Cit-rQ])-PEG12-0H NO:136])-PEG12-0H
Ac-SEQ ID NO:137-PEG2-
Dap(cyc/o[SEQ ID NO:136])-
Ac-rbfbr-PEG2-Dap(cyc/o[FK0-Cit-r-Cit-rQ])-PEG12-0H PEG12-0H
Ac-rrr-PEG2-Dap(cyclo[SEQ ID
Ac-rrr-PEG2-Dap(cyc/o[Ff(D-Cit-r-Cit-rQ])-PEG12-0H NO:136])-PEG12-0H
Ac-rbr-PEG2-Dap(cyclo[SEQ ID
Ac-rbr-PEG2-Dap(cyc/o[Ff40-Cit-r-Cit-rQ])-PEG12-0H NO:136])-PEG12-0H
Ac-SEQ ID NO:138-PEG2-
Dap(cyc/o[SEQ ID NO:136])-
Ac-rbrbr-PEG2-Dap(cyc/o[FK0-Cit-r-Cit-rQ])-PEG12-0H PEG12-0H
Ac-hh-PEG2-Dap(cyclo[SEQ ID
Ac-hh-PEG2-Dap(cyc/o[Ff40-Cit-r-Cit-rQ])-PEG12-0H NO:136])-PEG12-0H
155
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
Ac-hbh-PEG2-Dap(cyclo[SEQ ID
Ac-hbh-PEG2-Dap(cyc/o[FRD-Cit-r-Cit-rQ])-PEG12-0H NO:136D-PEG12-0H
Ac-SEQ IN NO: 139-PEG2-
Dap(cyc/o[SEQ ID NO:136])-
Ac-hbhbh-PEG2-Dap(cyc/o[FK0-Cit-r-Cit-rQ])-PEG12-0H PEG12-0H
Ac- SEQ ID NO: 140-PEG2-
Dap(cyc/o[SEQ ID NO:136])-
Ac-rbhbh-PEG2-Dap(cyc/o[Ff40-Cit-r-Cit-rQ])-PEG12-0H PEG12-0H
Ac-SEQ ID NO: 141-PEG2-
Dap(cyc/o[SEQ ID NO:136])-
Ac-hbrbh-PEG2-Dap(cyc/o[Ff40-Cit-r-Cit-rQ])-PEG12-0H PEG12-0H
Ac-rr-Dap(cyclo[SEQ ID
Ac-rr-Dap(cyclo[Ff(D-Cit-r-Cit-rQ])-b-OH NO:136])-b-OH
Ac-frr-Dap(cyclo[SEQ ID
Ac-frr-Dap(cyclo[Ff(D-Cit-r-Cit-rQ])-b-OH NO:136])-b-OH
Ac-rfr-Dap(cyclo[SEQ ID
Ac-rfr-Dap(cyclo[Ff(D-Cit-r-Cit-rQ])-b-OH NO:136])-b-OH
Ac- SEQ ID NO:137-
Dap(cyc/o[SEQ ID NO:136])-b-
Ac-rbfbr-Dap(cyc/o[Ff40-Cit-r-Cit-rQ])-b-OH OH
Ac-rrr-Dap(cyclo[SEQ ID
Ac-m-Dap(cyclo[Ff40-Cit-r-Cit-rQ])-b-OH NO:136])-b-OH
Ac-rbr-Dap(cyclo[SEQ ID
Ac-rbr-Dap(cyclo[Ff(D-Cit-r-Cit-rQ])-b-OH NO:136])-b-OH
Ac- SEQ ID NO:138-
Dap(cyc/o[SEQ ID NO:136])-b-
Ac-rbrbr-Dap(cyclo[Ff(D-Cit-r-Cit-rQ])-b-OH OH
Ac-hh-Dap(cyclo[SEQ ID
Ac-hh-Dap(cyclo[Ff(D-Cit-r-Cit-rQ])-b-OH NO:136])-b-OH
Ac-hbh-Dap(cyclo[SEQ ID
Ac-hbh-Dap(cyclo[Ff(D-Cit-r-Cit-rQ])-b-OH NO:136])-b-OH
Ac-SEQ IN NO: 139-
Dap(cyc/o[SEQ ID NO:136])-b-
Ac-hbhbh-Dap(cyclo[Ff(D-Cit-r-Cit-rQ])-b-OH OH
Ac-SEQ ID NO: 140-
Dap(cyc/o[SEQ ID NO:136])-b-
Ac-rbhbh-Dap(cyc/o[Ff40-Cit-r-Cit-rQ])-b-OH OH
Ac- SEQ ID NO:141-
Dap(cyc/o[SEQ ID NO:136])-b-
Ac-hbrbh-Dap(cyc/o[Ff40-Cit-r-Cit-rQ])-b-OH OH
Ac- SEQ ID NO:7-miniPEG2-
Ac-KKKK-miniPEG2-Lys(cyclo[FK0GrGrQ])-miniPEG2- Lys(cyclo[SEQ ID NO:Z80])-
K(N3)-NH2 miniPEG2-K(N3)-NH2
156
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
Ac- SEQ ID NO:13-miniPEG2-
Ac-KGKK-miniPEG2-Lys(cyc/o[FK0GrGrQ])-miniPEG2- Lys(cyclo[SEQ ID NO:Z80])-
K(N3)-NH2 miniPEG2-K(N3)-NH2
Ac- SEQ ID NO:14-miniPEG2-
Ac-KKGK-miniPEG2-Lys(cyc/o[MoGrGrQ])-miniPEG2- Lys(cyclo[SEQ ID NO:Z80])-
K(N3)-NH2 miniPEG2-K(N3)-NH2
Ac-KKK-miniPEG2-
Ac-KKK-miniPEG2-Lys(cyclo[FDI0GrGrQ])-miniPEG2- Lys(cyclo[SEQ ID NO:80])-
K(N3)-NH2 miniPEG2-K(N3)-NH2
Ac-KK-miniPEG2-Lys(cyclo[FRI)GrGrQ])-miniPEG2- Ac-KK-miniPEG2-Lys(cyclo[SEQ
K(N3)-NH2 ID NO:80])-miniPEG2-K(N3)-NH2
Ac-KGK-miniPEG2-
Ac-KGK-miniPEG2-Lys(cyc/o[FK0GrGrQ])-miniPEG2- Lys(cyclo[SEQ ID NO:80])-
K(N3)-NH2 miniPEG2-K(N3)-NH2
Ac-KBK-miniPEG2-
Ac-KBK-miniPEG2-Lys(cyc/o[FK0GrGrQ])-miniPEG2- Lys(cyclo[SEQ ID NO:80])-
K(N3)-NH2 miniPEG2-K(N3)-NH2
Ac- SEQ ID NO:24-miniPEG2-
Ac-KBKBK-miniPEG2-Lys(cyc/o[MoGrGrQ])-miniPEG2- Lys(cyclo[SEQ ID NO:80])-
K(N3)-NH2 miniPEG2-K(N3)-NH2
Ac-KR-miniPEG2-Lys(cyclo[MoGrGrQ])-miniPEG2- Ac-KR-miniPEG2-Lys(cyclo[SEQ
K(N3)-NH2 ID NO:80])-miniPEG2-K(N3)-NH2
Ac-KBR-miniPEG2-
Ac-KBR-miniPEG2-Lys(cyc/o[FK0GrGrQ])-miniPEG2- Lys(cyclo[SEQ ID NO:80])-
K(N3)-NH2 miniPEG2-K(N3)-NH2
Ac- SEQ ID NO:42-miniPEG2-
Ac-PKKKRKV-miniPEG2-Lys(cyclo[FK0GrGrQ])- Lys(cyclo[SEQ ID NO:80])-
miniPEG2-K(N3)-NH2 miniPEG2-K(N3)-NH2
Ac- SEQ ID NO:42-miniPEG2-
Ac-PKKKRKV-miniPEG2-Lys(cyclo[FK0GrGrQ])- Lys(cyclo[SEQ ID NO:80])-
miniPEG2-K(N3)-NH2 miniPEG2-K(N3)-NH2
Ac- SEQ ID NO:43-miniPEG2-
Ac-PGKKRKV-miniPEG2-Lys(cyclo[FK0GrGrQ])- Lys(cyclo[SEQ ID NO:80])-
miniPEG2-K(N3)-NH2 miniPEG2-K(N3)-NH2
Ac- SEQ ID NO:44-miniPEG2-
Ac-PKGKRKV-miniPEG2-Lys(cyclo[FK0GrGrQ])- Lys(cyclo[SEQ ID NO:80])-
miniPEG2-K(N3)-NH2 miniPEG2-K(N3)-NH2
Ac- SEQ ID NO:45-miniPEG2-
Ac-PKKGRKV-miniPEG2-Lys(cyclo[FK0GrGrQ])- Lys(cyclo[SEQ ID NO:80])-
miniPEG2-K(N3)-NH2 miniPEG2-K(N3)-NH2
Ac- SEQ ID NO:46-miniPEG2-
Ac-PKKKGKV-miniPEG2-Lys(cyclo[FK0GrGrQ])- Lys(cyclo[SEQ ID NO:80])-
miniPEG2-K(N3)-NH2 miniPEG2-K(N3)-NH2
157
CA 03222824 2023-12-07
WO 2022/271818
PCT/US2022/034517
Ac- SEQ ID NO:47-miniPEG2-
Ac-PKKKRGV-miniPEG2-Lys(cyclo[Ff(1)GrGrQ])- Lys(cyclo[SEQ ID NO:80])-
miniPEG2-K(N3)-NH2 miniPEG2-K(N3)-NH2
Ac- SEQ ID NO:48-miniPEG2-
Ac-PKKKRKG-miniPEG2-Lys(cyclo[Ff(1)GrGrQ])- Lys(cyclo[SEQ ID NO:80])-
miniPEG2-K(N3)-NH2 miniPEG2-K(N3)-NH2
Ac- SEQ ID NO:19-miniPEG2-
Ac-KKKRK-miniPEG2-Lys(cyclo[Ff(1)GrGrQ])- Lys(cyclo[SEQ ID NO:80])-
miniPEG2-K(N3)-NH2 miniPEG2-K(N3)-NH2
Ac- SEQ ID NO:8-miniPEG2-
Ac-KKRK-miniPEG2-Lys(cyclo[Ff(1)GrGrQ])-miniPEG2- Lys(cyclo[SEQ ID NO:80])-
K(N3)-NH2 and miniPEG2-K(N3)-NH2 and
Ac-KRK-miniPEG2-
Ac-KRK-miniPEG2-Lys(cyclo[Ff(1)GrGrQ])-miniPEG2- Lys(cyclo[SEQ ID NO:80])-
K(N3)-NH2. miniPEG2-K(N3)-NH2.
[0424] The EEV can be selected from:
Ac-PKKKRKV-Lys(cyclo[FRI)GrGrQ])-PEG12-K(N3)-NH2
(Ac- SEQ ID NO:42-Lys(cyc/o[SEQ ID NO:80])-PEG12-K(N3)-NH2)
Ac-PKKKRKV-miniPEG2-Lys(cyclo[Ff(toGrGrQ])-miniPEG2-K(N3)-NH2
(Ac- SEQ ID NO:42-miniPEG2-Lys(cyc/o[SEQ ID NO:80])-miniPEG2-K(N3)-NH2)
Ac-PKKKRKV-miniPEG2-Lys(cyclo[FGFGRGRQ])-miniPEG2-K(N3)-NH2
(Ac- SEQ ID NO:42-miniPEG2-Lys(cyc/o[SEQ ID NO:82])-miniPEG2-K(N3)-NH2)
Ac-KR-PEG2-K(cyc/o[FGFGRGRQ])-PEG2-K(N3)-NH2
(Ac-KR-PEG2-K(cyclo[SEQ ID NO:82])-PEG2-K(N3)-NH2)
Ac-PKKKGKV-PEG2-K(cyc/o[FGFGRGRQ])-PEG2-K(N3)-NH2
(Ac- SEQ ID NO:46-PEG2-K(cyc/o[SEQ ID NO:82])-PEG2-K(N3)-NH2)
Ac-PKKKRKG-PEG2-K(cyc/o[FGFGRGRQ])-PEG2-K(N3)-NH2
(Ac- SEQ ID NO:48-PEG2-K(cyc/o[SEQ ID NO:82])-PEG2-K(N3)-NH2)
Ac-KKKRK-PEG2-K(cyc/o[FGFGRGRQ])-PEG2-K(N3)-NH2
(Ac- SEQ ID NO:19-PEG2-K(cyc/o[SEQ ID NO:82])-PEG2-K(N3)-NH2)
Ac-PKKKRKV-miniPEG2-Lys(cyclo[FF(DGRGRQ])-miniPEG2-K(N3)-NH2
(Ac- SEQ ID NO:42-miniPEG2-Lys(cyc/o[SEQ ID NO:80])-miniPEG2-K(N3)-NH2)
Ac-PKKKRKV-miniPEG2-Lys(cyclo[f3hFDI0GrGrQ])-miniPEG2-K(N3)-NH2
158
CA 03222824 2023-12-07
WO 2022/271818
PCT/US2022/034517
(Ac- SEQ ID NO:42-miniPEG2-Lys(cyc/o[SEQ ID NO:142])-miniPEG2-K(N3)-NH2)
Ac-PKKKRKV-miniPEG2-Lys(cyclo[Ff(toSrSrQ] )-miniPEG2-K(N3)-NH2
(Ac- SEQ ID NO:42-miniPEG2-Lys(cyc/o[SEQ ID NO:143])-miniPEG2-K(N3)-NH2).
[0425] The EEV can be selected from:
Ac-PKKKRKV-miniPEG2-Lys(cyclo(GfFGrGrQD-PEG12-0H
(Ac- SEQ ID NO:42-miniPEG2-Lys(cyc/o(SEQ ID NO:133D-PEG12-0H)
Ac-PKKKRKV-miniPEG2-Lys(cyclo[FGFKRKRQ] )-PEG12-0H
(Ac- SEQ ID NO: ID NO:144])-PEG12-0H)
Ac-PKKKRKV-miniPEG2-Lys(cyclo[FGFRGRGQ] )-PEG12-0H
(Ac- SEQ ID NO:42-miniPEG2-Lys(cyc/o[SEQ ID NO:145])-PEG12-0H)
Ac-PKKKRKV-miniPEG2-Lys(cyclo[FGFGRGRGRQ])-PEG12-0H
(Ac- SEQ ID NO:42-miniPEG2-Lys(cyc/o[SEQ ID NO:146])-PEG12-0H)
Ac-PKKKRKV-miniPEG2-Lys(cyclo[FGFGRIRQ] )-PEG12-0H
(Ac- SEQ ID NO:42-miniPEG2-Lys(cyc/o[SEQ ID NO:147])-PEG12-0H)
Ac-PKKKRKV-miniPEG2-Lys(cyclo[FGFGRRRQ] )-PEG12-0H
(Ac- SEQ ID NO: ID NO:84])-PEG12-0H)and
Ac-PKKKRKV-miniPEG2-Lys(cyclo[FGFRRRRQ] )-PEG12-0H
(Ac- SEQ ID NO:42-miniPEG2-Lys(cyc/o[SEQ ID NO:85])-PEG12-0H).
[0426] The EEV can be selected from:
Ac-K-K-K-R-K-G-miniPEG2-K(cyc/o[FGFGRGRQ])-PEG12-0H
(Ac-SEQ ID NO:148-miniPEG2-K(cyc/o[SEQ ID NO:82])-PEG12-0H)
Ac-K-K-K-R-K-miniPEG2-K(cyc/o[FGFGRGRQ])-PEG12-0H
(Ac- SEQ ID NO:19-miniPEG2-K(cyc/o[SEQ ID NO:82])-PEG12-0H)
Ac-K-K-R-K-K-PEG4-K(cyc/o[FGFGRGRQ] )-PEG12-0H
(Ac- SEQ ID NO:22-PEG4-K(cyc/o[SEQ ID NO:82])-PEG12-0H)
Ac-K-R-K-K-K-PEG4-K(cyc/o[FGFGRGRQ] )-PEG12-0H
(Ac- SEQ ID NO:21-PEG4-K(cyc/o[SEQ ID NO:82])-PEG12-0H)
Ac-K-K-K-K-R-PEG4-K(cyc/o[FGFGRGRQ] )-PEG12-0H
(Ac- SEQ ID NO:23-PEG4-K(cyc/o[SEQ ID NO:82])-PEG12-0H)
Ac-R-K-K-K-K-PEG4-K(cyc/o[FGFGRGRQ] )-PEG12-0H
159
CA 03222824 2023-12-07
WO 2022/271818
PCT/US2022/034517
(Ac- SEQ ID NO:20-PEG4-K(cyc/o[SEQ ID NO:82])-PEG12-0H) and
Ac-K-K-K-R-K-PEG4-K(cyc/o[FGFGRGRQ] )-PEG12-0H
(Ac- SEQ ID NO:19-PEG4-K(cyc/o[SEQ ID NO:82])-PEG12-0H).
[0427] The EEV can be selected from:
Ac-PKKKRKV-PEG,-K(cyc/o[FGFGRGRQ] )-PEG2-K(N3)-NH2
(Ac- SEQ ID NO:42-PEG2-K(cyc/o[SEQ ID NO:82])-PEG2-K(N3)-NH2)
Ac-PKKKRKV-PEG,-K(cyc/o[FGFGRGRQ] )-PEG12-0H
(Ac- SEQ ID NO:42-PEG2-K(cyc/o[SEQ ID NO:82])-PEG12-0H)
Ac-PKKKRKV-PEG,-K(cyc/o[GfFGrGrQ] )-PEG2-K(N3)-NH2
(Ac- SEQ ID NO:42-PEG2-K(cyc/o[SEQ ID NO:133])-PEG2-K(N3)-NH2) and
Ac- PKKKRKV-PEG2-K(cyc/o[GfFGrGrQ] )-PEG12-0H
(Ac- SEQ ID NO:42-PEG2-K(cyc/o[SEQ ID NO:133])-PEG12-0H).
[0428] The cargo can be an AC and the EEV can be selected from:
Ac-PKKKRKV-PEG,-K(cyc/o[Ff(toGrGrQ])-PEG12-0H
(Ac- SEQ ID NO:42-PEG2-K(cyc/o[SEQ ID NO:80])-PEG12-0H)
Ac-PKKKRKV-PEG,-K(cyc/o[Ff(1)Cit-r-Cit-rQ] )-PEG12-0H
(Ac- SEQ ID NO:42-PEG2-K(cyc/o[SEQ ID NO:79])-PEG12-0H)
Ac-PKKKRKV-PEG,-K(cyc/o[FfFGRGRQ] )-PEG12-0H
(Ac- SEQ ID NO:42-PEG2-K(cyc/o[SEQ ID NO:81])-PEG12-0H)
Ac-PKKKRKV-PEG,-K(cyc/o[FGFGRGRQ] )-PEG12-0H
(Ac- SEQ ID NO:42-PEG2-K(cyc/o[SEQ ID NO:82])-PEG12-0H)
Ac-PKKKRKV-PEG,-K(cyc/o[GfFGrGrQ] )-PEG12-0H
(Ac- SEQ ID NO:42-PEG2-K(cyc/o[SEQ ID NO:133])-PEG12-0H)
Ac-PKKKRKV-PEG,-K(cyc/o[FGFGRRRQ])-PEG12-0H
(Ac- SEQ ID NO:42-PEG2-K(cyc/o[SEQ ID NO:84])-PEG12-0H)
Ac-PKKKRKV-PEG,-K(cyc/o[FGFRRRRQ])-PEG12-0H
(Ac- SEQ ID NO:42-PEG2-K(cyc/o[SEQ ID NO:85])-PEG12-0H)
Ac-rr-PEG,-K(cyc/o[Ff(toGrGrQ] )-PEG12-0H
(Ac-rr-PEG,-K(cyc/o[SEQ ID NO :80]
Ac-rr-PEG,-K(cyc/o[Ff(toCit-r-Cit-rQ] )-PEG12-0H
160
CA 03222824 2023-12-07
WO 2022/271818
PCT/US2022/034517
(Ac-rr-PEG,-K(cyc/o[SEQ ID NO :79]
Ac-rr-PEG,-K(cyc/o[FfF-GRGRQ] )-PEG12-0H
(Ac-rr-PEG,-K(cyc/o[SEQ ID NO :81]
Ac-rr-PEG,-K(cyc/o[FGFGRGRQ])-PEG12-0H
(Ac-rr-PEG,-K(cyc/o[SEQ ID NO :82]
Ac-rr-PEG2-K(cyc/o[GfFGrGrQ] )-PEG12-0H
(Ac-rr-PEG,-K(cyc/o[ SEQ ID NO :133] )-PEG12-0H)
Ac-rr-PEG,-K(cyc/o[FGFGRRRQ] )-PEG12-0H
(Ac-rr-PEG,-K(cyc/o[SEQ ID NO :84]
Ac-rr-PEG,-K(cyc/o[FGFRRRRQ] )-PEG12-0H
(Ac-rr-PEG,-K(cyc/o[SEQ ID NO :85]
Ac-rrr-PEG,-K(cyc/o[MoGrGrQ] )-PEG12-0H
(Ac-rrr-PEG,-K(cyc/o[SEQ ID NO:80] )-PEG12-0H)
Ac-rrr-PEG,-K(cyc/o[MoCit-r-Cit-rQ] )-PEG12-0H
(Ac-rrr-PEG,-K(cyc/o[SEQ ID NO:79] )-PEG12-0H)
Ac-rrr-PEG,-K(cyc/o[FfFGRGRQ])-PEG12-0H
(Ac-rrr-PEG,-K(cyc/o[ SEQ ID NO : 81] )-PEG12-0H)
Ac-rrr-PEG,-K(cyc/o[FGFGRGRQ] )-PEG12-0H
(Ac-rrr-PEG,-K(cyc/o[SEQ ID NO:82] )-PEG12-0H)
Ac-rrr-PEG,-K(cyc/o[GfFGrGrQ] )-PEG12-0H
(Ac-rrr-PEG,-K(cyc/o[SEQ ID NO:133] )-PEG12-0H)
Ac-rrr-PEG,-K(cyc/o[FGFGRRRQ] )-PEG12-0H
(Ac-rrr-PEG,-K(cyc/o[SEQ ID NO:84] )-PEG12-0H)
Ac-rrr-PEG,-K(cyc/o[FGFRRRRQ] )-PEG12-0H
(Ac-rrr-PEG,-K(cyc/o[SEQ ID NO:85] )-PEG12-0H)
Ac-rhr-PEG,-K(cyc/o[Ff40GrGrQ] )-PEG12-0H
(Ac-rhr-PEG,-K(cyc/o[SEQ ID NO :80]
Ac-rhr-PEG,-K(cyc/o[FDICit-r-Cit-rQ])-PEG12-0H
161
CA 03222824 2023-12-07
WO 2022/271818
PCT/US2022/034517
(Ac-rhr-PEG,-K(cyc/o[SEQ ID NO :79] )-PEG12-0H)
Ac-rhr-PEG,-K(cyc/o[FfFGRGRQ] )-PEG12-0H
(Ac-rhr-PEG,-K(cyc/o[ SEQ ID NO:8 1 ] )-PEG12-0H)
Ac-rhr-PEG,-K(cyc/o[FGFGRGRQ] )-PEG12-0H
(Ac-rhr-PEG,-K(cyc/o[SEQ ID NO :82] )-PEG12-0H)
Ac-rhr-PEG2-K(cyc/o[GfFGrGrQ])-PEG12-0H
(Ac-rhr-PEG,-K(cyc/o[SEQ ID NO:133] )-PEG12-0H)
Ac-rhr-PEG,-K(cyc/o[FGFGRRRQ] )-PEG12-0H
(Ac-rhr-PEG,-K(cyc/o[SEQ ID NO :84] )-PEG12-0H)
Ac-rhr-PEG,-K(cyc/o[FGFRRRRQ] )-PEG12-0H
(Ac-rhr-PEG,-K(cyc/o[SEQ ID NO :85] )-PEG12-0H)
Ac-rbr-PEG,-K(cyc/o[Ff40GrGrQ] )-PEG12-0H
(Ac-rbr-PEG2-K(cyc/o[SEQ ID NO :80] )-PEG12-0H)
Ac-rbr-PEG,-K(cyc/o[FDICit-r-Cit-rQ])-PEG12-0H
(Ac-rbr-PEG,-K(cyc/o[SEQ ID NO :79] )-PEG12-0H)
Ac-rbr-PEG,-K(cyc/o[FfFGRGRQ] )-PEG12-0H
(Ac-rbr-PEG2-K(cyc/o[ SEQ ID NO:8 1 ] )-PEG12-0H)
Ac-rbr-PEG,-K(cyc/o[FGFGRGRQ] )-PEG12-0H
(Ac-rbr-PEG2-K(cyc/o[SEQ ID NO :82] )-PEG12-0H)
Ac-rbr-PEG2-K(cyc/o[GfFGrGrQ])-PEG12-0H
(Ac-rbr-PEG,-K(cyc/o[SEQ ID NO:133] )-PEG12-0H)
Ac-rbr-PEG,-K(cyc/o[FGFGRRRQ] )-PEG12-0H
(Ac-rbr-PEG,-K(cyc/o[SEQ ID NO :84] )-PEG12-0H)
Ac-rbr-PEG,-K(cyc/o[FGFRRRRQ] )-PEG12-0H
(Ac-rbr-PEG,-K(cyc/o[SEQ ID NO :85] )-PEG12-0H)
Ac-rbrbr-PEG,-K(cyc/o[FDI0GrGrQ] )-PEG12-0H
(Ac-SEQ ID NO:138-PEG2-K(cyc/o[SEQ ID NO:80])-PEG12-0H)
Ac-rbrbr-PEG,-K(cyc/o[Ff(1)Cit-r-Cit-rQ] )-PEG12-0H
162
CA 03222824 2023-12-07
WO 2022/271818
PCT/US2022/034517
(Ac- SEQ ID NO:138-PEG2-K(cyc/o[SEQ ID NO:79])-PEG12-0H)
Ac-rbrbr-PEG,-K(cyc/o[FfFGRGRQ])-PEG12-0H
(Ac- SEQ ID NO:138-PEG2-K(cyc/o[SEQ ID NO:81])-PEG12-0H)
Ac-rbrbr-PEG,-K(cyc/o[FGFGRGRQ] )-PEG12-0H
(Ac- SEQ ID NO:138-PEG2-K(cyc/o[SEQ ID NO:82])-PEG12-0H)
Ac-rbrbr-PEG,-K(cyc/o[GfFGrGrQ])-PEG12-0H
(Ac- SEQ ID NO:138-PEG2-K(cyc/o[SEQ ID NO:133])-PEG12-0H)
Ac-rbrbr-PEG,-K(cyc/o[FGFGRRRQ])-PEG12-0H
(Ac- SEQ ID NO:138-PEG2-K(cyc/o[SEQ ID NO:84])-PEG12-0H)
Ac-rbrbr-PEG2-K(cyc/o[FGFRRRRQ])-PEG12-0H
(Ac- SEQ ID NO:138-PEG2-K(cyc/o[SEQ ID NO:85])-PEG12-0H)
Ac-rbhbr-PEG,-K(cyc/o[Ff40GrGrQ] )-PEG12-0H
(Ac- SEQ ID NO:149-PEG2-K(cyc/o[SEQ ID NO:80])-PEG12-0H)
Ac-rbhbr-PEG,-K(cyc/o[FDICit-r-Cit-rQ] )-PEG12-0H
(Ac- SEQ ID NO:149-PEG2-K(cyc/o[SEQ ID NO:79])-PEG12-0H)
Ac-rbhbr-PEG,-K(cyc/o[FfFGRGRQ] )-PEG12-0H
(Ac- SEQ ID NO:149-PEG2-K(cyc/o[SEQ ID NO:81])-PEG12-0H)
Ac-rbhbr-PEG,-K(cyc/o[FGFGRGRQ])-PEG12-0H
(Ac- SEQ ID NO:149-PEG2-K(cyc/o[SEQ ID NO:82])-PEG12-0H)
Ac-rbhbr-PEG,-K(cyc/o[GfFGrGrQ] )-PEG12-0H
(Ac- SEQ ID NO:149-PEG2-K(cyc/o[SEQ ID NO:133])-PEG12-0H)
Ac-rbhbr-PEG,-K(cyc/o[FGFGRRRQ] )-PEG12-0H
(Ac- SEQ ID NO:149-PEG2-K(cyc/o[SEQ ID NO:84])-PEG12-0H)
Ac-rbhbr-PEG,-K(cyc/o[FGFRRRRQ] )-PEG12-0H
(Ac- SEQ ID NO:149-PEG2-K(cyc/o[SEQ ID NO:85])-PEG12-0H)
Ac-hbrbh-PEG,-K(cyc/o[FDI0GrGrQ] )-PEG12-0H
(Ac- SEQ ID NO:141-PEG2-K(cyc/o[SEQ ID NO:80])-PEG12-0H)
Ac-hbrbh-PEG,-K(cyc/o[Ff(toCit-r-Cit-rQ] )-PEG12-0H
163
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
(Ac- SEQ ID NO:141-PEG2-K(cyc/o[SEQ ID NO:79])-PEG12-0H)
Ac-hbrbh-PEG2-K(cyc/o[FfFGRGRQ])-PEG12-0H
(Ac- SEQ ID NO:141-PEG2-K(cyc/o[ SEQ ID NO:8 1 ])-PEG12-0H)
Ac-hbrbh-PEG2-K(cyc/o[FGFGRGRQ])-PEG12-0H
(Ac- SEQ ID NO:141-PEG2-K(cyc/o[SEQ ID NO:82])-PEG12-0H)
Ac-hbrbh-PEG2-K(cyc/o[GfFGrGrQ])-PEG12-0H
(Ac- SEQ ID NO:141-PEG2-K(cyc/o[SEQ ID NO:133])-PEG12-0H)
Ac-hbrbh-PEG2-K(cyc/o[FGFGRRRQ])-PEG12-0H
(Ac- SEQ ID NO:141-PEG2-K(cyc/o[SEQ ID NO:84])-PEG-12-0H)
Ac- hbrbh -PEG2-K(cyc/o[FGFRRRRQ])-PEG12-0H
(Ac- SEQ ID NO:141-PEG2-K(cyc/o[SEQ ID NO:85])-PEG-12-0H),
wherein b is beta-alanine, and the exocyclic sequence can be D or L
stereochemistry.
Cargo
[0429] The cell penetrating peptide (CPP), such as a cyclic cell penetrating
peptide (e.g., cCPP),
can be conjugated to a cargo. As used herein, "cargo" is a compound or moiety
for which delivery
into a cell is desired. The cargo can be conjugated to a terminal carbonyl
group of a linker. At
least one atom of the cyclic peptide can be replaced by a cargo or at least
one lone pair can form a
bond to a cargo. The cargo can be conjugated to the cCPP by a linker. The
cargo can be conjugated
to an AAsc by a linker. At least one atom of the cCPP can be replaced by the
cargoty or at least
one lone pair of the cCPP forms a bond to the cargo. A hydroxyl group on an
amino acid side chain
of the cCPP can be replaced by a bond to the cargo. A hydroxyl group on a
glutamine side chain
of the cCPP can be replaced by a bond to the cargo. The cargo can be
conjugated to the cCPP by
a linker. The cargo can be conjugated to an AAsc by a linker.
[0430] In embodiments, the amino acid side chain comprises a chemically
reactive group to which
the linker or cargo is conjugated. The chemically reactive group can comprise
an amine group, a
carboxylic acid, an amide, a hydroxyl group, a sulfhydryl group, a guanidinyl
group, a phenolic
group, a thioether group, an imidazolyl group, or an indolyl group. In
embodiments, the amino
acid of the cCPP to which the cargo is conjugated comprises lysine, arginine,
aspartic acid,
glutamic acid, asparagine, glutamine, homoglutamine, serine, threonine,
tyrosine, cysteine,
arginine, tyrosine, methionine, histidine or tryptophan.
164
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
[0431] The cargo can comprise one or more detectable moieties, one or more
therapeutic
moieties (TMs), one or more targeting moieties, or any combination thereof In
embodiments,
the cargo comprises a TM. In embodiments, the cargo comprises an AC.
Cyclic cell penetrating peptides (cCPPs) conjugated to a cargo moiety
[0432] The cyclic cell penetrating peptide (cCPP) can be conjugated to a cargo
moiety.
[0433] The cargo moiety can be conjugated to the linker at the terminal
carbonyl group to
provide the following structure:
EP N
-\,(0),0Cargo / 0 N
H
X' Q (aH2)Juuv
, wherein:
EP is an exocyclic peptide and M, AAsc, Cargo, x', y, and z' are as defined
above, * is
the point of attachment to the AAsc.. x' can be 1. y can be 4. z' can be 11. -
(OCH2CH2)x,- and/or
-(OCH2CH2)f- can be independently replaced with one or more amino acids,
including, for
example, glycine, beta-alanine, 4-aminobutyric acid, 5-aminopentanoic acid, 6-
aminohexanoic
acid, or combinations thereof.
[0434] An endosomal escape vehicle (EEV) can comprise a cyclic cell
penetrating peptide
(cCPP), an exocyclic peptide (EP) and linker, and can be conjugated to a cargo
to form an EEV-
conjugate comprising the structure of Formula (C):
165
CA 03222824 2023-12-07
WO 2022/271818
PCT/US2022/034517
Eftõ (0 \xµ Cago
=
= y 'sr 0
= (cH2)
NH
. -Jv
az> 11)n R2
titsk¨e,
=11,NA. HN
r
H st.'1W)v
NH
==. ,N /14 A
= le .= =
*
i$e $
NH
(C)
or a protonated form thereof,
wherein:
R1, R2, and R3 can each independently be H or an amino acid residue having a
side chain comprising an aromatic group;
R4 is H or an amino acid side chain;
EP is an exocyclic peptide as defined herein;
Cargo is a moiety as defined herein;
each m is independently an integer from 0-3;
n is an integer from 0-2;
x' is an integer from 2-20;
y is an integer from 1-5;
q is an integer from 1-4; and
z' is an integer from 2-20.
[0435] R1, R2, R3, R4, EP, cargo, m, n, x', y, q, and z' are as defined
herein.
166
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
[0436] The EEV can be conjugated to a cargo and the EEV-conjugate can comprise
the structure
of Formula (C-a) or (C-b):
H 9 Cargo
'N= --' '/1- 10 0
0 H
(c1-12.)4
R1 0
0 õ
0." = õ
HN
NH
0, NH
,$
= µ1 HN
H r =
,NH
HN"
\
' ---- H
cc 0
----
V L.
.1M
NH
NH (C-a),
0 =Cargo
0
(c1-12j 4
NH
R4 0
- 0'
sss,
R2
H HN"--< õ
NH
0., ..AH
H 1-1N,
-N- \ .
H 1m NH
HN.
H
µR4
(5!' )
NH (C-b), or a protonated form
167
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
thereof, wherein EP, m and z are as defined above in Formula (C).
[0437] The EEV can be conjugated to a cargo and the EEV-conjugate can comprise
the structure
of Formula (C-c):
0
N
E x ,AA17Cargo
(oH2)
I Y
HN
n
R2
YLHN
NH 0
0 NH
HN
1-R3
NH
HN 0
NH
N R4
NH
NH (C-c),
or a protonated form thereof, wherein EP, 10, R2, R3, R4, and m are as defined
above in
Formula (III); AA can be an amino acid as defined herein; n can be an integer
from 0-2; x can be
an integer from 1-10; y can be an integer from 1-5; and z can be an integer
from 1-10.
[0438] The EEV can be conjugated to an oligonucleotide cargo and the EEV-
oligonucleotide
conjugate can comprises a structure of Formula (C-1), (C-2), (C-3), or (C-4):
168
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
0 oligonucleotide
EPO Fd)k 110
. N
0 - H
(CH2)
4
NH
0
T N
0
NH
NH
HN
H2NANõ...-NX
NH
HN
NH
0
NH
NH (C-1),
H 0 oligonucleotide
0 H
(CH2)
4
NH
T N
0
NH
o NH
HN
NH
HN '0 çi
- 0
0 j
NH
NH (C-2),
169
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
0 11 oligonucleotide
EP
NN II . N µ
/ 0 0
H -__. , H
0 (cH2)4
i
HN 0
HN
N
HN
Oz NH 0
HN
,...4.:,1
NH
HN 0 4/
NH H
0 0
NH
H2N---i
NH (C-3),
0 0
EP H
N N C)c)-r :AN (:)
.,,,. .
0 oligonucleotide
H - H = 11
0
NH
0
0 Z.40
HN 7---\
HN 0
oyNH
c HN
X%
NH
0*....
H HN 0 .
s. NH N
Ns -----Y
C 0 110
NH
H2N-4
I. (C-4)
NH
170
CA 03222824 2023-12-07
WO 2022/271818
PCT/US2022/034517
[0439] The EEV can be conjugated to an oligonucleotide cargo and the EEV-
conjugate can
comprise the structure:
NH Npf
Crd
Ho, rz)<'"Nsi N
L.N
N a) rs''1,NLO
...,o
N112 NFO NO
NNH2
0=P-N/ 11 IrLiN 0=P-N - CL-N
6, 'µ N re 0=P-K N
(z)1-1'.:11
.6, ' I N,L,
-0,( ....0 0 N N
'N' N112 NH2 0=P-N.-
.7,:m N NH2
1AN N
a I o i l-Nzz,(INI-1:::,
()=-N-.,--
*Orsi N
0 O I )
N N
NoeH
,/ NrEf
0=P¨N C''''=N o=rsi=l-N'' 11
41 rs'l / ersl
O \PH ., ' 0=1-N \ 0 1
i.....0,N 0 O N 0
0 ,C)/ 0 , rsr-np
N 'N 0 n/
0=P-Nc(Z)N1f-1(4LNH I
OIT
s-NC-4L,71., N NH
0, N N-Sil'eNH2 0 N '0 0=F1'-NZ))))
0 I
,
0
04-4
0=rs'11.-N
(:0 /MNI(Z) NH
rsil /
;k-rILNH
(:) 0, j 1 \
0 N N (z) NH2
-oy N,.,.?H
I NH2
0' N- --
0 ....
- 'N) Ali
N N
O 0
0=1:1;-N,-.7(4111:::,
*07/rsi
*Cy 0
N , N / .,. ,I,
0=P-N (z)NXII'NH
arV'NH2 ti o NXI0 --
r
O" \ m
siLrsi Yrs.iri
..,o
`I-No
N
0=1. __ 0
0=1. __ 0 0 rs
N =P-N CCN
/ \
NH NH NH /N
C:../ 01)y 0 4 0 4 0
isiz,11.1(s) 0 sioj HI,INH2
A o
NH NH
NH H2N--INH 0 NH
0 4
0 NA
rHN(S) 00
NH MN
NH H iiiNl
C)
0 ' '0
H1,1.1.11-) ON IS
0
NH2
Cytosolic Delivery Efficiency
[0440] Modifications to a cyclic cell penetrating peptide (cCPP) may improve
cytosolic delivery
efficiency. Improved cytosolic uptake efficiency can be measured by comparing
the cytosolic
delivery efficiency of a cCPP having a modified sequence to a control
sequence. The control
171
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
sequence does not include a particular replacement amino acid residue in the
modified sequence
(including, but not limited to arginine, phenylalanine, and/or glycine), but
is otherwise identical.
[0441] As used herein cytosolic delivery efficiency refers to the ability of a
cCPP to traverse a
cell membrane and enter the cytosol of a cell. Cytosolic delivery efficiency
of the cCPP is not
necessarily dependent on a receptor or a cell type. Cytosolic delivery
efficiency can refer to
absolute cytosolic delivery efficiency or relative cytosolic delivery
efficiency.
[0442] Absolute cytosolic delivery efficiency is the ratio of cytosolic
concentration of a cCPP
(or a cCPP-cargo conjugate) over the concentration of the cCPP (or the cCPP-
cargo conjugate) in
the growth medium. Relative cytosolic delivery efficiency refers to the
concentration of a cCPP
in the cytosol compared to the concentration of a control cCPP in the cytosol.
Quantification can
be achieved by fluorescently labeling the cCPP (e.g., with a FITC dye) and
measuring the
fluorescence intensity using techniques well-known in the art.
[0443] Relative cytosolic delivery efficiency is determined by comparing (i)
the amount of a
cCPP of the invention internalized by a cell type (e.g., HeLa cells) to (ii)
the amount of a control
cCPP internalized by the same cell type. To measure relative cytosolic
delivery efficiency, the
cell type may be incubated in the presence of a cCPP for a specified period of
time (e.g., 30
minutes, 1 hour, 2 hours, etc.) after which the amount of the cCPP
internalized by the cell is
quantified using methods known in the art, e.g., fluorescence microscopy.
Separately, the same
concentration of the control cCPP is incubated in the presence of the cell
type over the same
period of time, and the amount of the control cCPP internalized by the cell is
quantified.
[0444] Relative cytosolic delivery efficiency can be determined by measuring
the IC50 of a cCPP
having a modified sequence for an intracellular target and comparing the IC50
of the cCPP
having the modified sequence to a control sequence (as described herein).
[0445] The relative cytosolic delivery efficiency of the cCPPs can be in the
range of from about
50% to about 450% compared to cyclo(FfORrRrQ, SEQ ID NO:150), e.g., about 60%,
about
70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%,
about 140%,
about 150%, about 160%, about 170%, about 180%, about 190%, about 200%, about
210%,
about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about
280%,
about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about
350%,
about 360%, about 370%, about 380%, about 390%, about 400%, about 410%, about
420%,
172
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
about 430%, about 440%, about 450%, about 460%, about 470%, about 480%, about
490%,
about 500%, about 510%, about 520%, about 530%, about 540%, about 550%, about
560%,
about 570%, about 580%, or about 590%, inclusive of all values and subranges
therebetween.
The relative cytosolic delivery efficiency of the cCPPs can be improved by
greater than about
600% compared to a cyclic peptide comprising cyclo(FfORrRrQ, SEQ ID NO:150).
[0446] The absolute cytosolic delivery efficacy of from about 40% to about
100%, e.g., about
45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about
80%, about
85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about
96%, about
97%, about 98%, about 99%, inclusive of all values and subranges therebetween.
[0447] The cCPPs of the present disclosure can improve the cytosolic delivery
efficiency by
about 1.1 fold to about 30 fold, compared to an otherwise identical sequence,
e.g., about 1.2,
about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9,
about 2.0, about 2.5,
about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0,
about 6.5, about 7.0,
about 7.5, about 8.0, about 8.5, about 9.0, about 10, about 10.5, about 11.0,
about 11.5, about
12.0, about 12.5, about 13.0, about 13.5, about 14.0, about 14.5, about 15.0,
about 15.5, about
16.0, about 16.5, about 17.0, about 17.5, about 18.0, about 18.5, about 19.0,
about 19.5, about
20, about 20.5, about 21.0, about 21.5, about 22.0, about 22.5, about 23.0,
about 23.5, about
24.0, about 24.5, about 25.0, about 25.5, about 26.0, about 26.5, about 27.0,
about 27.5, about
28.0, about 28.5, about 29.0, or about 29.5 fold, inclusive of all values and
subranges
therebetween.
Detectable moiety
[0448] In embodiments, the compound disclosed herein includes a detectable
moiety. In
embodiments, the detectible moiety is attached to the cell penetrating peptide
at the amino group,
the carboxylate group, or the side chain of any of the amino acids of the cell
penetrating peptide
moiety (e.g., at the amino group, the carboxylate group, or the side chain of
any amino acid in the
CPP). In embodiments, the therapeutic moiety includes a detectable moiety. The
detectable moiety
can include any detectable label. Examples of suitable detectable labels
include, but are not limited
to, a UV-Vis label, a near-infrared label, a luminescent group, a
phosphorescent group, a magnetic
spin resonance label, a photosensitizer, a photocleavable moiety, a chelating
center, a heavy atom,
a radioactive isotope, an isotope detectable spin resonance label, a
paramagnetic moiety, a
173
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
chromophore, or any combination thereof. In embodiments, the label is
detectable without the
addition of further reagents.
[0449] In embodiments, the detectable moiety is a biocompatible detectable
moiety, such that the
compounds can be suitable for use in a variety of biological applications.
"Biocompatible" and
"biologically compatible", as used herein, generally refer to compounds that
are, along with any
metabolites or degradation products thereof, generally non-toxic to cells and
tissues, and which do
not cause any significant adverse effects to cells and tissues when cells and
tissues are incubated
(e.g., cultured) in their presence.
[0450] The detectable moiety can contain a luminophore such as a fluorescent
label or near-
infrared label. Examples of suitable luminophores include, but are not limited
to, metal porphyrins;
benzoporphyrins; azabenzoporphyrine; napthoporphyrin; phthalocyanine;
polycyclic aromatic
hydrocarbons such as perylene diimine, pyrenes; azo dyes; xanthene dyes; boron
dipyoromethene,
aza-boron dipyoromethene, cyanine dyes, metal-ligand complex such as
bipyridine, bipyridyls,
phenanthroline, coumarin, and acetylacetonates of ruthenium and iridium;
acridine, oxazine
derivatives such as benzophenoxazine; aza-annulene, squaraine; 8-
hydroxyquinoline,
polymethines, luminescent producing nanoparticle, such as quantum dots,
nanocrystals;
carbostyril; terbium complex; inorganic phosphor; ionophore such as crown
ethers affiliated or
derivatized dyes; or combinations thereof. Specific examples of suitable
luminophores include,
but are not limited to, Pd (II) octaethylporphyrin; Pt (II)-
octaethylporphyrin; Pd (II)
tetraphenylporphyrin; Pt (II) tetraphenylporphyrin; Pd (II) meso-
tetraphenylporphyrin
tetrabenzoporphine; Pt (II) meso-tetraphenyl metrylbenzoporphyrin; Pd (II)
octaethylporphyrin
ketone; Pt (II) octaethylporphyrin ketone; Pd (II) meso-
tetra(pentafluorophenyl)porphyrin; Pt (II)
meso-tetra (pentafluorophenyl) porphyrin; Ru (II) tris(4,7-dipheny1-1,10-
phenanthroline) (Ru
(dpp)3); Ru (II) tris(1,10-phenanthroline) (Ru(phen)3), tris(2,2'-
bipyridine)rutheniurn (II) chloride
hexahydrate (Ru(bpy)3); erythrosine B; fluorescein; fluorescein isothiocyanate
(FITC); eosin;
iridium (III)
((N-m ethyl-b enzimi dazol-2-y1)-7-(di ethyl amino)-c oumarin)); 1 74
enzothiazol e)
((b enzothi az o1-2-y1)-7- (di ethyl amino)-
coumarin))-2-(ac etyl acetonate); Lumogen dyes;
Macroflex fluorescent red; Macrolex fluorescent yellow; Texas Red; rhodamine
B; rhodamine 6G;
sulfur rhodamine; m-cresol; thymol blue; xylenol blue; cresol red;
chlorophenol blue; bromocresol
green; bromcresol red; bromothymol blue; Cy2; a Cy3; a Cy5; a Cy5.5; Cy7; 4-
nitirophenol;
174
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
alizarin; phenolphthalein; o-cresolphthalein; chlorophenol red; calmagite;
bromo-xylenol; phenol
red; neutral red; nitrazine; 3,4,5,6-tetrabromphenolphtalein; congo red;
fluor' Sc' in; eosin; 2',7'-
dichlorofluorescein; 5(6)-carboxy-fluorecsein; carboxynaphthofluorescein; 8-
hydroxypyrene-
1,3,6-trisulfonic acid; semi-naphthorhodafluor; semi-naphthofluorescein; tris
(4,7-dipheny1-1,10-
phenanthroline) ruthenium (II) dichloride; (4,7-dipheny1-1,10-phenanthroline)
ruthenium (II)
tetraphenylboron; platinum (II) octaethylporphyin;
dialkylcarbocyanine;
dioctadecylcycloxacarbocyanine;
fluorenylmethyloxycarbonyl chloride; 7-amino-4-
methylcourmarin (Amc); green fluorescent protein (GFP); and derivatives or
combinations
thereof.
[0451] In some examples, the detectable moiety can include Rhodamine B (Rho),
fluorescein
isothiocyanate (FITC), 7-amino-4-methylcourmarin (Amc), green fluorescent
protein (GFP), or
derivatives or combinations thereof.
Methods of Making
[0452] The compounds described herein can be prepared in a variety of ways
known to one
skilled in the art of organic synthesis or variations thereon as appreciated
by those skilled in the
art. The compounds described herein can be prepared from readily available
starting materials.
Optimum reaction conditions can vary with the particular reactants or solvents
used, but such
conditions can be determined by one skilled in the art.
[0453] Variations on the compounds described herein include the addition,
subtraction, or
movement of the various constituents as described for each compound.
Similarly, when one or
more chiral centers are present in a molecule, the chirality of the molecule
can be changed.
Additionally, compound synthesis can involve the protection and deprotection
of various
chemical groups. The use of protection and deprotection, and the selection of
appropriate
protecting groups can be determined by one skilled in the art. The chemistry
of protecting groups
can be found, for example, in Wuts and Greene, Protective Groups in Organic
Synthesis, 4th Ed.,
Wiley & Sons, 2006, which is incorporated herein by reference in its entirety.
[0454] The starting materials and reagents used in preparing the disclosed
compounds and
compositions are either available from commercial suppliers such as Aldrich
Chemical Co.,
(Milwaukee, WI), Acros Organics (Morris Plains, NJ), Fisher Scientific
(Pittsburgh, PA), Sigma
(St. Louis, MO), Pfizer (New York, NY), GlaxoSmithKline (Raleigh, NC), Merck
(Whitehouse
175
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
Station, NJ), Johnson & Johnson (New Brunswick, NJ), Aventis (Bridgewater,
NJ), AstraZeneca
(Wilmington, DE), Novartis (Basel, Switzerland), Wyeth (Madison, NJ), Bristol-
Myers-Squibb
(New York, NY), Roche (Basel, Switzerland), Lilly (Indianapolis, IN), Abbott
(Abbott Park, IL),
Schering Plough (Kenilworth, NJ), or Boehringer Ingelheim (Ingelheim,
Germany), or are
prepared by methods known to those skilled in the art following procedures set
forth in
references such as Fieser and Fieser's Reagents for Organic Synthesis, Volumes
1-17 (John
Wiley and Sons, 1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and
Supplementals (Elsevier Science Publishers, 1989); Organic Reactions, Volumes
1-40 (John
Wiley and Sons, 1991); March's Advanced Organic Chemistry, (John Wiley and
Sons, 4th
Edition); and Larock's Comprehensive Organic Transformations (VCH Publishers
Inc., 1989).
Other materials, such as the pharmaceutical carriers disclosed herein can be
obtained from
commercial sources.
[0455] Reactions to produce the compounds described herein can be carried out
in solvents,
which can be selected by one of skill in the art of organic synthesis.
Solvents can be substantially
nonreactive with the starting materials (reactants), the intermediates, or
products under the
conditions at which the reactions are carried out, i.e., temperature and
pressure. Reactions can be
carried out in one solvent or a mixture of more than one solvent. Product or
intermediate
formation 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., 'H or '3C) infrared spectroscopy, spectrophotometry (e.g.,
UV-visible), or
mass spectrometry, or by chromatography such as high-performance liquid
chromatography
(HPLC) or thin layer chromatography.
[0456] The disclosed compounds can be prepared by solid phase peptide
synthesis wherein the
amino acid a-N-terminus is protected by an acid or base protecting group. Such
protecting
groups should have the properties of being stable to the conditions of peptide
linkage formation
while being readily removable without destruction of the growing peptide chain
or racemization
of any of the chiral centers contained therein. Suitable protecting groups are
9-
fluorenylmethyloxycarbonyl (Fmoc), t-butyloxycarbonyl (Boc), benzyloxycarbonyl
(Cbz),
biphenylisopropyloxycarbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl, a,a-
dimethy1-3,5-
dimethoxybenzyloxycarbonyl, o-nitrophenylsulfenyl, 2-cyano-t-butyloxycarbonyl,
and the like.
176
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
The 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group is particularly
preferred for the
synthesis of the disclosed compounds. Other preferred side chain protecting
groups are, for side
chain amino groups like lysine and arginine, 2,2,5,7,8-pentamethylchroman-6-
sulfonyl (pmc),
nitro, p-toluenesulfonyl, 4-methoxybenzene- sulfonyl, Cbz, Boc, and
adamantyloxycarbonyl; for
tyrosine, benzyl, o-bromobenzyloxy-carbonyl, 2,6-dichlorobenzyl, isopropyl, t-
butyl (t-Bu),
cyclohexyl, cyclopenyl and acetyl (Ac); for serine, t-butyl, benzyl and
tetrahydropyranyl; for
histidine, trityl, benzyl, Cbz, p-toluenesulfonyl and 2,4-dinitrophenyl; for
tryptophan, formyl; for
asparticacid and glutamic acid, benzyl and t-butyl and for cysteine,
triphenylmethyl (trityl).
[0457] In the solid phase peptide synthesis method, the a-C-terminal amino
acid is attached to a
suitable solid support or resin. Suitable solid supports useful for the above
synthesis are those
materials which are inert to the reagents and reaction conditions of the
stepwise condensation-
deprotection reactions, as well as being insoluble in the media used. Solid
supports for synthesis
of a-C-terminal carboxy peptides is 4-hydroxymethylphenoxymethyl-
copoly(styrene-1%
divinylbenzene) or 4-(2',4'-dimethoxyphenyl-Fmoc-
aminomethyl)phenoxyacetamidoethyl resin
available from Applied Biosystems (Foster City, Calif.). The a-C-terminal
amino acid is coupled
to the resin by means of N,N'-dicyclohexylcarbodiimide (DCC), N,N'-
diisopropylcarbodiimide
(DIC) or 0-benzotriazol-1-yl-N,N,N',N'-tetramethyluroniumhexafluorophosphate
(HBTU), with
or without 4-dimethylaminopyridine (DMAP), 1-hydroxybenzotriazole (HOBT),
benzotriazol-1-
yloxy-tris(dimethylamino)phosphoniumhexafluorophosphate (BOP) or bis(2-oxo-3-
oxazolidinyl)phosphine chloride (BOPC1), mediated coupling for from about 1 to
about 24 hours
at a temperature of between 10 C and 50 C in a solvent such as dichloromethane
or DMF. When
the solid support is 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy-
acetamidoethyl resin,
the Fmoc group is cleaved with a secondary amine, preferably piperidine, prior
to coupling with
the a-C-terminal amino acid as described above. One method for coupling to the
deprotected 4
(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy-acetamidoethyl resin is 0-
benzotriazol-1-
yl-N,N,N',N'-tetramethyluroniumhexafluorophosphate (HBTU, 1 equiv.) and 1-
hydroxybenzotriazole (HOBT, 1 equiv.) in DMF. The coupling of successive
protected amino
acids can be carried out in an automatic polypeptide synthesizer. In one
example, the a-N-
terminus in the amino acids of the growing peptide chain are protected with
Fmoc. The removal
of the Fmoc protecting group from the a-N-terminal side of the growing peptide
is accomplished
177
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
by treatment with a secondary amine, preferably piperidine. Each protected
amino acid is then
introduced in about 3-fold molar excess, and the coupling is preferably
carried out in DMF. The
coupling agent can be 0-benzotriazol-1-yl-N,N,N',N'-
tetramethyluroniumhexafluorophosphate
(HBTU, 1 equiv.) and 1-hydroxybenzotriazole (HOBT, 1 equiv.). At the end of
the solid phase
synthesis, the polypeptide is removed from the resin and deprotected, either
successively or in a
single operation. Removal of the polypeptide and deprotection can be
accomplished in a single
operation by treating the resin-bound polypeptide with a cleavage reagent
comprising thianisole,
water, ethanedithiol and trifluoroacetic acid. In cases wherein the a-C-
terminal of the
polypeptide is an alkylamide, the resin is cleaved by aminolysis with an
alkylamine.
Alternatively, the peptide can be removed by transesterification, e.g. with
methanol, followed by
aminolysis or by direct transamidation. The protected peptide can be purified
at this point or
taken to the next step directly. The removal of the side chain protecting
groups can be
accomplished using the cleavage cocktail described above. The fully
deprotected peptide can be
purified by a sequence of chromatographic steps employing any or all of the
following types: ion
exchange on a weakly basic resin (acetate form); hydrophobic adsorption
chromatography on
underivitized polystyrene-divinylbenzene (for example, Amberlite XAD); silica
gel adsorption
chromatography; ion exchange chromatography on carboxymethylcellulose;
partition
chromatography, e.g. on Sephadex G-25, LH-20 or countercurrent distribution;
high performance
liquid chromatography (HPLC), especially reverse-phase HPLC on octyl- or
octadecylsilyl-silica
bonded phase column packing.
[0458] The above polymers, such as PEG groups, can be attached to an
oligonucleotide, such as
an AC, under any suitable conditions. Any means known in the art can be used,
including via
acylation, reductive alkylation, Michael addition, thiol alkylation or other
chemoselective
conjugation/ligation methods through a reactive group on the PEG moiety (e.g.,
an aldehyde,
amino, ester, thiol, a-haloacetyl, maleimido or hydrazino group) to a reactive
group on the AC
(e.g., an aldehyde, amino, ester, thiol, a-haloacetyl, maleimido or hydrazino
group). Activating
groups which can be used to link the water soluble polymer to one or more
proteins include without
limitation sulfone, maleimide, sulfhydryl, thiol, triflate, tresylate,
azidirine, oxirane, 5-pyridyl, and
alpha-halogenated acyl group (e.g., a-iodo acetic acid, a-bromoacetic acid, a-
chloroacetic acid).
If attached to the AC by reductive alkylation, the polymer selected should
have a single reactive
178
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
aldehyde so that the degree of polymerization is controlled. See, for example,
Kinstler et al., Adv.
Drug. Delivery Rev. (2002), 54: 477-485; Roberts et al., Adv. Drug Delivery
Rev. (2002), 54: 459-
476; and Zalipsky et al., Adv. Drug Delivery Rev. (1995), 16: 157-182.
[0459] In order to direct covalently link the AC or linker to the CPP,
appropriate amino acid
residues of the CPP may be reacted with an organic derivatizing agent that is
capable of reacting
with a selected side chain or the N- or C-termini of an amino acids. Reactive
groups on the peptide
or conjugate moiety include, e.g., an aldehyde, amino, ester, thiol, a-
haloacetyl, maleimido or
hydrazino group. Derivatizing agents include, for example, maleimidobenzoyl
sulfosuccinimide
ester (conjugation through cysteine residues), N-hydroxysuccinimide (through
lysine residues),
glutaraldehyde, succinic anhydride or other agents known in the art.
[0460] Methods of making AC and conjugating AC to linear CPP are generally
described in US
Pub. No. 2018/0298383, which is herein incorporated by reference for all
purposes. The methods
may be applied to the cyclic CPPs disclosed herein.
[0461] Synthetic schemes are provided in FIG. 3A-3D and FIG. 4.
[0462] Non-limiting examples of compounds that include a CPPs and a reactive
group useful for
conjugation to an AC are shown in Table 9. Example linker groups are also
shown. Example
reactive groups include tetrafluorophenyl ester (TFP), free carboxylic acid
(COOH), and azide
(N3). In Table 9, n is an integer from 0 to 20; Pipa6 is
AcRXRRBRRXRYQFLIRXRBRXRB
wherein B is 13-Alanine and X is aminohexanoic acid; Dap is 2,3-
diaminopropionic acid; NLS is a
nuclear localization sequence; (3A is beta alanine; -ss- is a disulfide; PABC
is poly(A) binding
protein C-terminal domain; Cx where x is a number is an alkyl chain of length
x; and BCN is
bicyclo [6.1.0]nonyne.
Table 9: Compounds that include a CPPs and a reactive group
TFP-PEGn-K(CPP)
TFP-PEGn-K(CPP)-PEGn-Dap(palmitoyl)
TFP-PEGn-K(CPP)-PEGn-Dap(CPP)
TFP-Pip6a
CPP-PEGn-TFP
CPP-PEGn-K(CPP)-PEGn-TFP
CPP-PEGn-Lys(N3)
CPP-K(CPP)-PEGn-K(N3)
179
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
CPP-PEG1-K(PEG1-CPP)-PEG1-K(N3)
CPP-PEGn-K(PEGn-CPP)-PEGn-K(N3)
CPP-K(CPP)-K(CPP)-PEG11-K(N3)
CPP-PEGn-K(PEGn-CPP)-K(PEGn-CPP)-PEGn-K(N3)
CPP-PEGn-K(PEGn-CPP)-K(PEGn-CPP)-PEGn-K(N3)
Ac-NLS-Lys(CPP)-PEGn-K(N3)
K(N3)- PEGn-NLS-ss-PEGn-CPP
BCN-NLS-ss-CPP
CPP-PEG11-Val-Cit-PABC-K(N3)
CPP-PEG11-Cys-ss-Cys-K(N3)
CPP-PEG11-Cys-ss-Cys-K(N3)
CPP-PEGn-TFP
CPP-PEG11-Lys(N3)
CPP-PEG11-Cys-prodisulfide-K(N3)
CPP-PEG11-K(N3)
CPP-K(CPP)-PEG11-K(N3)
CPP-PEGn-K(CPP)-PEGn-TFP
CPP-C6-TFP
CPP-PEGn-K(PEGn-CPP)PEGn-K(N3)
Ac-T9-PEG11-Lys(CPP-PEG11)-K(N3)
Ac-MSP-PEG11-K(CPP-PEG11)-K(N3)
CPP-PEGn-TFP (ENTRD 802)
CPP-C6-TFP (ENTRD 696)
CPP-PEGn-K(CPP)-PEGn-TFP (ENTRD-344)
CPP-PEGn-COOH
CPP-C12-TFP (ENTD-695)
palmitoyl-PEGn-K(CPP)-PEGn-TFP (ENTD-343)
CPP-PEG11-K(N3) (ENTRD-617)
Ac-T9-PEG11-K(CPP)-K(N3) (ENTRD 673)
Ac-MSP-PEG11-K(CPP-PEG11)-K(N3) (ENTRD 675)
Ac-NLS-K(CPP)-PEG11-K(N3) (ENTRD 684)
K(N3)-PEG11-NLS-ss-PEG11-CPP (ETRD-681)
K(N3)-PEGn-NLS-K-13A-13A-CPP (ETRD-682)
[0463] In embodiments, the CPPs have free carboxylic acid groups that may be
utilized for
conjugation to an AC. In embodiments, the EEVs have free carboxylic acid
groups that may be
utilized for conjugation to an AC.
180
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
[0464] The structure below is a 3' cyclooctyne modified PM0 used for a click
reaction with a
\ PM
0 0 N 0
o
0- '0
compound that includes an azide:
[0465] An example scheme of conjugation of a CPP and linker to the 3' end of
an AC via an amide
bond is shown below.
181
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
H2NTO H2NO
N
1 1
041"¨NMe2 0=P¨NMe2
1
0 0
HCB 0 B
N)
N
1 1
04¨NMe2 0=1:1)¨NMe2
0 0
CPP-Linker-COOH
LOB ________________ LOB
PYAOP. DIPEA
1\1 DMF, r.t N
0=171)¨NMe2 0=1¨NMe2
0 0
OB 0 B
NI
¨1¨ .-1¨
CY=P¨NMe2 GEP¨NMe2
O (!)
OB 0 B
1\1 N
1 1
R1 R2
0 0
R1=H,
0 R2: 71NH2 Linker¨CPP 0 N)LLinker¨CPP
1 1 H
m=1-4 m=1-4
[0466] An example scheme of conjugation of a CPP and linker to a 3 ' -
cyclooctyne modified PM0
via strain-promoted azide-alkyne cycloaddition is shown below:
182
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
H2NO H2NTO
-..N..--
N
I
0=1'¨NMe2 0= 1: i'¨NMe2
0 0
N
(0B ) cOxB
N
0=F¨NMe2 0=1,¨NMe2
0 0
oCi CPP-Linker-N3
cr B ________________________ cOxB
N)
Nuclease-free water
1-10 mM, r.t N
0=I¨NMe2 0=P¨NMe2
o O
(0( B
N) (OxB
N
--1-- --1--
OP¨NMe2 OP¨NMe2
0 0
c(Dr B
N) (OxB
,N,
N N=NLinker-CPP
N
00C)---C¨) 00()---?--
_
Mixture of regioisomers
[0467] An example of the conjugation chemistry used to connect an AC and CPP
with an
additional linker containing a polyethylene glycol moiety is shown below:
183
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
H2NT0 H2NT0
Y N
i
0=c'¨NMe2 0=P¨NMe2
0 0
cOyB cOTB
m)
7 Y
0=P¨NMe2 0=P¨NMe2
0 0
CPP-Linker-N3
crDB cr:)TB
N)y _____________________ a
Nuclease-free water
1-10 mM, r.t N
¨1¨= ¨1¨=
0=P¨NMe2 0=P¨NMe2
0 ,
0
cN
Oy B cOTB )
N
--1¨ --1-
0=P¨NMe2 0=P¨NMe2
i
0 0
cOyB cOTB
m)
7 N
R3 14
R3: R4:
,
,N
NJ' N.vLinker-CPP
=-*--", -",õ=-- -. /III
0 0 0 0
Mixture of regioisomers
0
07
N'eC)N).L.0" 0
H 1 H T
endo 0
1=0-12 / H 1 H
1= 0-12 ____________________________________________________________________
/ N,Linker-CPP
N=N
Mixture of regioisomers
[0468] An example of conjugation of a CPP-linker to a 5'-cyclooctyne modified
PM0 via strain-
promoted azide-alkyne cycloaddition (click chemistry) is shown below:
184
H I C H I 0
0,0.....---õ....--,01,...N,,,,a...^.õ0-,,N,,N,,,,..0)1.N...---õeNH2 0 II
0-.,,,Nõ,
0
Y
0 N..õ.....N 1 8 CPP¨LinkeNyN, ,N 0
N.õ,.....N 1 8
1 N I
N
0
(N) (I\I
k...)
0
1 N
1
k...)
0=F,,¨Nme2 0=P-NMe2
k...)
0 6
k....)
--4
ccoy B Lx0y B
I,
N) N)
00
I-,
CPP-Unker-N3
00
1 1
C=P-NMe, _________________________ I 0=P-NMe.,
6 Nuclease-free water 6
1_10 mM, r.t
I=x0y 6 1,..,(0),B
N) N
-1- -1-=
0=P-NMe2 0=P-
NMe2
6 6
(tOyB IxOTB
N.) N
--1-=
0=P-NMe2 0-NA/le2
0 0
ccay B 1,..,(0y
B P
N) N)
o
L.
Iv
H H
"
Iv
0
Iv
o.
Iv
o
Iv
L.
1
r
Iv
O
...1
IV
n
cp
k....)
c,
k....)
k....)
c,
c...,
4=.
(A
1-,
--1
H I 0 H I 0
0
II Y o ii 1
0 N.....õ....N I 0 CPP¨Linker^,N,N,N 0 NN 1 g
1 1
N N
0
0
0=1:,,¨NMe; OP¨NMe.2
k.4
,
0 0 k.4
CPP-Linker-N3
00
I¨,
i _____________________________________ r 7
00
OP¨NMe, Nuclease-frea water 0=P¨N Mei:
I 1-10 mM.r t O
0
1....,(0,),B 1....õ(0),B
N) N
¨1¨ ¨1-
0=P¨NMe2 0=Fr ¨NMe,
0 0
1....(0,7,B
N) N
¨1¨= ¨1-
0.F;,¨NMe2 OP¨NMe2
1
0 0
N
6 1,,(0),BH2
N.)
P
o
Iv
0=S=0 0=S=0
Iv
Iv
a)
0 8
Iv
o.
035 0,5
Iv
o
Iv
0 0
1
1,,
2 c ) 1-.
,
.
-4
IV
n
cp
k....)
k....)
k....)
c...,
4=,
(../1
I¨,
--1
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
[0469] Methods of synthesizing oligomeric antisense compounds are known in the
art. The present
disclosure is not limited by the method of synthesizing the AC. In
embodiments, provided herein
are compounds having reactive phosphorus groups useful for forming
internucleoside linkages
including for example phosphodiester and phosphorothioate internucleoside
linkages. Methods of
preparation and/or purification of precursors or antisense compounds are not a
limitation of the
compositions or methods provided herein. Methods for synthesis and
purification of DNA, RNA,
and the antisense compounds are well known to those skilled in the art.
[0470] Oligomerization of modified and unmodified nucleosides can be routinely
performed
according to literature procedures for DNA (Protocols for Oligonucleotides and
Analogs, Ed.
Agrawal (1993), Humana Press) and/or RNA (Scaringe, Methods (2001), 23, 206-
217. Gait et al.,
Applications of Chemically synthesized RNA in RNA: Protein Interactions, Ed.
Smith (1998), 1-
36. Gallo et al., Tetrahedron (2001), 57, 5707-5713).
[0471] Antisense compounds provided herein can be conveniently and routinely
made through the
well-known technique of solid phase synthesis. Equipment for such synthesis is
sold by several
vendors including, for example, Applied Biosystems (Foster City, CA). Any
other means for such
synthesis known in the art may additionally or alternatively be employed. It
is well known to use
similar techniques to prepare oligonucleotides such as the phosphorothioates
and alkylated
derivatives. The invention is not limited by the method of antisense compound
synthesis.
[0472] Methods of oligonucleotide purification and analysis are known to those
skilled in the art.
Analysis methods include capillary electrophoresis (CE) and electrospray-mass
spectroscopy.
Such synthesis and analysis methods can be performed in multi-well plates. The
method of the
invention is not limited by the method of oligomer purification.
[0473] In the compounds disclosed herein, the AC is coupled to the CPP (e.g.,
cyclic peptide). As
used herein, "coupled" can refer to a covalent or non-covalent association
between the CPP to the
AC, including fusion of the CPP to the AC and chemical conjugation of the CPP
(e.g., cyclic
peptide) to the AC. A non-limiting example of a means to non-covalently attach
the CPP to the
AC is through the streptavidin/biotin interaction, e.g., by conjugating biotin
to CPP and fusing AC
to streptavidin. In the resulting compound, the CPP is coupled to the AC via
non-covalent
association between biotin and streptavidin.
[0474] In embodiments, the CPP (e.g., cyclic peptide) is conjugated, directly
or indirectly, to the
AC to thereby form a CPP-AC conjugate. Conjugation of the AC to the CPP may
occur at any
187
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
appropriate site on these moieties. For example, In embodiments, the 5' or the
3' end of the AC
may be conjugated to the C-terminus, the N-terminus, or a side chain of an
amino acid in the CPP.
[0475] In embodiments, the AC is covalently linked to the CPP (e.g., cyclic
peptide). Covalent
linkage, as used herein, refer to constructs where a CPP moiety is covalently
linked to the 5' and/or
3' end of the AC moiety. Such conjugates may alternatively be described as
having a CPP moiety
(e.g., cyclic peptide moiety) and an oligonucleotide moiety. A covalently-
linked AC-CPP or CPP-
AC conjugate, in accordance with certain embodiments, includes the AC
component and the CPP
component associated with one another by a linker described herein.
[0476] In embodiments, the AC may be conjugated to the CPP (e.g. cyclic
peptide) through a side
chain of an amino acid on the CPP. Any amino acid side chain on the CPP which
is capable of
forming a covalent bond, or which may be so modified, can be used to link AC
to the CPP. The
amino acid on the CPP can be a natural or non-natural amino acid. In
embodiments, the amino
acid on the CPP used to conjugate the AC is aspartic acid, glutamic acid,
glutamine, asparagine,
lysine, ornithine, 2,3-diaminopropionic acid, or analogs thereof, wherein the
side chain is
substituted with a bond to the AC or linker. In embodiments, the amino acid is
lysine, or an analog
thereof. In embodiments, the amino acid is glutamic acid, or an analog
thereof. In embodiments,
the amino acid is aspartic acid, or an analog thereof.
[0477] In embodiments, the CPP is cyclic. There are numerous possible
configurations for the
compounds disclosed herein. In embodiments, the compounds of the disclosure
include
compounds wherein AC is conjugated to the side chain of an amino acid in the
cyclic peptide. In
embodiments, the compounds disclosed herein have a structure (i.e., exocyclic)
according to
Formula I-A:
CPP-L-AC
(I-A) ,
wherein the linker is covalently bound to the side chain of an amino acid on
the CPP and to the 5'
end of the AC, the backbone of the AC, or the 3' end of the AC.
Diseases and Target Genes
[0478] In embodiments, compounds and methods are provided for treating a
disease or disorder
associated with one or more genes having an expanded nucleotide repeats (e.g.,
expanded
trinucleotide repeats such as expanded trinucleotide repeats). In embodiments,
compounds and
methods are provided for treating a disease or disorder associated with one or
more genes having
188
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
an expanded CTG=CUG trinucleotide repeat. In embodiments, compounds and
methods are
provided for treating a disease or disorder associated with one or more genes
having an expanded
CTG=CUG trinucleotide repeat in the 3'-UTR of the gene. In embodiments,
compounds and
methods are provided for treating a disease or disorder associated with a gene
that has an expanded
CTG=CUG in the 3' UTR such as DMPK, ATXN8OS ATXN8, and/or JPH3. In
embodiments,
compounds and methods are provided for treating a disease or disorder
associated with one or
more genes having an expanded CTG=CUG trinucleotide repeat in the intron of a
gene. In
embodiments, compounds and methods are provided for treating a disease or
disorder associated
with an expanded CTG=CUG trinucleotide repeat in an intron of TCF4. In
embodiments,
compounds and methods are provided for treating myotonic dystrophy type 1
(DM1), Fuchs'
Endothelial Corneal Dystrophy (FECD), Spinocerebellar Ataxia-8 (SCA8), and/or
Huntington's
Disease-Like (HDL2).
Myotonic dystrophy type 1 (DM1)
[0479] In embodiments, compounds, compositions, and methods are provided to
treat Myotonic
dystrophy (DM1 or Steinert's disease). DM1 is a multisystemic disorder often
characterized by
muscle degeneration and myotonia or delayed muscle relaxation due to
repetitive action potentials
in myofibers. Myotonic dystrophy type 1 (DM1) is the most common form of
muscular dystrophy,
affecting about 1 in 8000 people. DM1 is a paradigm for genetic disorders
caused by CTG=CUG
expansions. DM1 is a neuromuscular disorder caused by a CTG=CUG repeat
expansion in the 3'-
untranslated region (UTR) of the dystrophia myotonia protein kinase (DMPK)
gene. At the RNA
level, the DMPK transcript (e.g., the expanded CUG repeat) sequesters splicing
regulator proteins,
for example, muscleblind-like (MBNL) protein, which results in incorrect
splicing of a number of
downstream pre-mRNAs (pre-mRNAs that do not contain an expanded CUG repeat)
that are
regulated by MBNL1. This gain-of-function is the cause of DM1.
[0480] The excessive number of CUG repeats impart toxic activity, referred to
as a toxic gain-of-
function. Multiple key proteins are misprocessed, and this contributes to the
multi systemic nature
of the disease, which includes generalized limb weakness, respiratory muscle
impairment, cardiac
abnormalities, fatigue, gastrointestinal complications, cataracts,
incontinence, and excessive
daytime sleepiness.
[0481] DM1 patients with CTG=CUG expansions within the 3'-untranslated region
of DMPK gene
are at increased risk for FECD and form CUGexp-MBNL1 foci in corneal
endothelium. (Mootha
189
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
etal., Investigative ophthalmology &visual science, 2017; 58, 4579-4585).
Association of MBNL1
with mutant RNA affects the cellular pool of free 1V1BNL1 and triggers mis-
splicing of some
1V1BNL1 target genes (e.g., regulated by MBNL1) in affected brain, muscle, and
heart tissues (Jiang
et al. Hum Mol Genet. 2004; 13: 3079-3088). Gattey et al. (Cornea. 2014; 33:
96-98) reported
FECD in four DM1 subjects including a mother¨daughter pair. Thus, the
association between DM1
and FECD is likely to be present (FECD is described in more detail elsewhere
herein).
[0482] Without being bound by theory, there are at least two hypotheses
proposed to explain the
pathogenesis of DM1. One is that the expanded CTG=CUG repeats inhibit DMPK
mRNA or
protein production, resulting in D1VIPK haploinsufficiency. This was supported
by studies
demonstrating decreased expression of D1VIPK mRNA and protein in DM1 muscle
(Fu, Y.H.et al.
(1993) Decreased expression of myotonin-protein kinase messenger RNA and
protein in adult
form of myotonic dystrophy. Science 260, 235-238). In embodiments, the
compounds and
methods described herein ameliorate D1VIPK haploinsufficiency. Another RNA
gain-of-function
hypothesis proposes that the mutant RNA transcribed from the expanded allele
is sufficient to
induce symptoms of the disease. This was suggested by observations: (i) the
expanded CTG
repeats are transcribed into CUG repeats that accumulate in discrete nuclear
foci, (ii) expression
of only the DMPK 3'-UTR with 200 CTG repeats is sufficient to inhibit
myogenesis (Davis, B.M.,
et al. (1997) Expansion of a CUG trinucleotide repeat in the 31 untranslated
region of myotonic
dystrophy protein kinase transcripts results in nuclear retention of
transcripts. Proc. Natl. Acad.
Sci. U.S.A. 94, 7388-7393; Amack, J.D. et al., (1999) Cis and trans effects of
the myotonic
dystrophy (DM) mutation in a cell culture model. Hum. Mol. Genet. 8, 1975-
1984). In
embodiments, the compounds and methods described herein reduce transcription
of mutant RNA
which are associated with the expanded allele.
[0483] The expanded CTG=CUG trinucleotide repeats in the 3' untranslated
region of D1VIPK
mRNA form imperfect stable hairpin structures that accumulate in the cell
nucleus in small
ribonuclear complexes or microscopically visible inclusions, and impair the
function of proteins
implicated in transcription, splicing or RNA export. Although D1VIPK genes
with CUG repeats are
transcribed into mRNA, the mutant transcripts are sequestered in the nucleus
as aggregates (foci),
which results in a decrease in cytoplasmic DMPK mRNA levels. These
aggregations lead to the
deregulation of the alternative splicing of many different transcripts due to
sequestration of two
RNA-binding proteins: MBNL1 (muscleblind-like 1) and CUGBP1 (CUG-binding
protein 1),
190
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
resulting in loss-of-function of MBNL1 and upregulation of CUGBP1 (Lee and
Cooper. (2009)
"Pathogenic mechanisms of myotonic dystrophy," Biochem Soc Trans. 37(06): 1281-
1286).
[0484] In DM1, the RNA-binding protein MBNL1, is sequestered to the double-
stranded hairpin
structure formed by CUG repeats, depleting it from the nucleoplasm. Then, the
CUG repeats to
which MBNL1 bound stimulate Protein Kinase C (PKC) activation through an
unknown
mechanism, which induces CUGBP1 hyperphosphorylation and stabilization. The
downstream
effects include disruption of alternative splicing, mRNA translation and mRNA
decay of
downstream genes. An important molecular feature of DM1 is the misregulation
of alternative
splicing due to sequestration of MBNL1 to CUG repeats with double-stranded
hairpin structure.
Among more than two dozen splicing events mis-regulated in DM1, the abnormal
splicing of the
skeletal muscle-specific C1C-1 (chloride channel 1) is known to be one of the
causes for myotonia.
Increased inclusion of exons containing premature stop codons result in down-
regulation of C1C-
1 mRNA and protein, which is sufficient to cause myotonia (Charlet-B et al.
(2002) Loss of the
muscle-specific chloride channel in type 1 myotonic dystrophy due to
misregulated alternative
splicing. Mol. Cell 10, 45-53; Mankodi, A.et al. (2002) Expanded CUG repeats
trigger aberrant
splicing of C1C-1 chloride channel pre-mRNA and hyperexcitability of skeletal
muscle in
myotonic dystrophy. Mol. Cell 10, 35-44). In embodiments, the compounds and
methods
described herein ameliorate the downstream effects, including disruption of
alternative splicing,
mRNA translation, and mRNA decay of downstream genes. In embodiments, the
compounds and
methods described herein reduce the number of splicing events mis-regulated in
DM1 compared
to a subject with DM1 that is not treated with compounds or methods of the
disclosure. For
example, in some embodiments, the compounds and methods described herein may
reduce the
number of mis-regulated splicing events in one or more downstream genes such
as
4833439L19Rik, Abcc9, Atp2a1, Arhgef10, Arhgap28, Armcx6, Angell, Best3, Binl,
Brd2,
Cacnals, Cacna2d1, Cpd, Cpeb3, Ccpgl, Claspl, Clcnl, Clk4, Cpeb2, Camk2g,
Capzb, Copz2,
Coch, cTNT, Ctu2, Cyp2s1, Dctn4, Dnmll, Eya4, Efna3, Efna2, Fbxo31, Fbxo21,
Frem2, Fgd4,
Fucal, Fnl, Gogla4, Gpr3711, Grebl, Hegl, Insr, Impdh2, IR, Itgav, Jag2, Klcl,
Kcan6, Kif13a,
Ldb3, Lrrfip2, Mapt, Macfl, Map3k4, Mapkapl, Mbnll, Mllt3, Mbn12, Mef2c, Mpdz,
Mrpll,
Mxra7, Mybpcl, Myo9a, Ncapd3, Ngfr, Ndrg3, Ndufv3, Neb, Nfix, Numal, Opal,
Pacsin2,
Pcolce, Pdlim3, Pla2g15, Phactr4, Phkal, Phtf2, Ppplrl2b, Ppp3cc, Ppplcc,
Ramp2, Rapgefl,
Run, Ryrl, 5orcs2, 5psb4, 5cube2, 5ema6c, 5fc8a3, 51ain2, Sorbsl, 5pag9,
Tmem28, Taccl,
191
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
Tacc2, Ttc7, Tnik, Tnfrsf22, Tnfrsf25, Trappc9, Trim55, Ttn, Txn14a, Txlnb,
Ube2d3, or Vsp39.
In embodiments, the compounds and methods described herein reduce the number
of exons
containing premature stop codons which result in down-regulation of C1C-1 mRNA
compared to
a subject with DM1 that is not treated with compounds or methods of the
disclosure.
[0485] The levels of MBNL1 and CUGBP1 in the nucleus control a subset of
developmentally
regulated splicing events that are reversed in DM1. In the embryonic stage,
MBNL1 nuclear levels
are low and CUGBP1 levels are high. During development, 1V1BNL1 nuclear levels
increase while
CUGBP1 levels decrease, inducing an embryonic-to-adult transition of
downstream splice targets
(including IR exon 11, CC-1 exons containing stop codons and cTNT exon 5).
However, in DM1,
MBNL1 is sequestered to CUG repeats, resulting in a decrease of functional
MBNL1, while
CUGBP1 levels are increased due to phosphorylation and stabilization. This
simulates the
embryonic condition and enhances expression of embryonic isoforms in adults,
resulting in
multiple disease symptoms (Lee and Cooper.; 2009). In embodiments, the
compounds and
methods described herein reduce amount of MBNL1 sequestered, increase the
amount of
functional MBNL1, decrease CUGBP1 levels compared to a subject with DM1 that
is not treated
with compounds or methods of the disclosure.
[0486] 1V1BNL1 and CELF1 (also referred to as "CUGBP1") are developmental
regulators of
splicing events during fetal to adult transition and modification of their
activities in DM1 leads to
expression of a fetal splicing pattern in adult tissues. The downstream impact
of low MBNL1 and
high CELF1 includes disruption of alternative splicing, mRNA translation and
mRNA decay in
proteins such as cardiac troponin T (cTNT), insulin receptor (INSR), muscle-
specific chloride ion
channel (CLCN1) and sarcoplasmic/endoplasmic reticulum calcium ATPase 1
(ATP2A1)
transcripts, in addition to MBNL1. Konieczny et al. (2017) "Myotonic
dystrophy: candidate small
molecule therapeutics," Drug Discovery Today. 22(11):1740-1748.
[0487] Compounds and methods for treating myotonic dystrophy using antisense
oligomers
targeting polyCUG repeats in the 3'-UTR of DMPK gene are described in
US10106796B2,
US10111962B2, US20150080311A1, each of which is herein incorporated by
reference in its
entirety for all purposes. However, such PM0s or PPM0s targeting CUG repeats
to treat DM1
might have limitations in oligo delivery to muscles, which is the disease
affected tissues.
[0488] In embodiments, the present disclosure teaches use of diverse cell
penetrating peptide
(CPP) to deliver the AC (e.g., PM0 or ASO) and a degradation sequence
described herein e.g., in
192
CA 03222824 2023-12-07
WO 2022/271818
PCT/US2022/034517
Tables 2 and 10, to the cytosol of the cell. In embodiments, the CPP or EEV
conjugated with the
AC delivers the AC of interest to the cellular location where the target
sequence on pre-mRNA is
located.
[0489] In embodiments, the disease is a form of myotonic dystrophy (e.g.,
myotonic dystrophy
type 1 or myotonic dystrophy type 2). In embodiments, the target gene is the
DMPK gene, which
encodes myotonic-protein kinase. In embodiments, the compounds provided herein
comprise an
AC (e.g., ASO) that targets DMPK (e.g., the 3'-untranslated
region/polyadenylation of DMPK
gene) to degrade DMPK gene. Exemplary oligonucleotides that target DMPK for
degradation are
provided in Table 10. The degradation sequence may be used in combination with
an AC sequence
comprising from 10-40 CAG repeats, including but not limited to the AC
provided in Table 2.
Table 10. Oligonucleotides (AC) targeting DMPK for degradation.
Oligo (AC) ID Sequence (5'-3') SEQ ID
Target
NO:
5'-CAG CAG CAG CAG CAG CAG CAG-3'-click- DMPK
K-PEG12-Lys(CPP12)-NLS-AC
(all PM0 monomers)
D1VIPK-A-17 GGGCCTTTTATTCGCGAGGGTCGGG 151
DMPK
D1VIPK-A-18 GAGGGCCTTTTATTCGCGAGGGTCG 152
DMPK
DMPK-A-19 TGGAGGGCCTTTTATTCGCGAGGGT 153
DMPK
DMPK-A-20 GATGGAGGGCCTTTTATTCGCGAGG 154
DMPK
DMPK-A-21 CAGATGGAGGGCCTTTTATTCGCGA 155
DMPK
DMPK-A-22 GGCAGATGGAGGGCCTTTTATTCGC 156
DMPK
DMPK-A-23 TGGGCAGATGGAGGGCCTTTTATTC 157
DMPK
DMPK-A-24 TTTGGGCAGATGGAGGGCCTTTTAT 158
DMPK
DMPK-A-25 GCTTTGGGCAGATGGAGGGCCTTTT 159
DMPK
DMPK-A-26 GAGCTTTGGGCAGATGGAGGGCCTT 160
DMPK
DMPK-A-27 CAGAGCTTTGGGCAGATGGAGGGCC 161
DMPK
DMPK-A-28 TCCAGAGCTTTGGGCAGATGGAGGG 162
DMPK
DMPK-A-29 AGTCCAGAGCTTTGGGCAGATGGAG 163
DMPK
DMPK-A-30 GGAGTCCAGAGCTTTGGGCAGATGG 164
DMPK
DMPK-A-31 GT GGAGT CCAGAGC T TT GGGC AGAT 165 DMPK
DMPK-A-32 CTGTGGAGTCCAGAGCTTTGGGCAG 166
DMPK
DMPK-A-33 CACTGTGGAGTCCAGAGCTTTGGGC 177
DMPK
DMPK-A-34 GACACTGTGGAGTCCAGAGCTTTGG 178
DMPK
DMPK-A-35 CGGACACTGTGGAGTCCAGAGCTTT 179
DMPK
DMPK-A-36 CGCGGACACTGTGGAGTCCAGAGCT 180
DMPK
DMPK-A-37 ACCGCGGACACTGTGGAGTCCAGAG 181
DMPK
DMPK-A-38 AAACCGCGGACACTGTGGAGTCCAG 182
DMPK
DMPK-A-39 GCAAACCGCGGACACTGTGGAGTCC 183
DMPK
DMPK-A-40 ACGCAAACCGCGGACACTGTGGAGT 184
DMPK
193
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
D1VIPK-A-41 CAACGCAAACCGCGGACACTGTGGA 185 D1VIP K
Spinocerebellar Ataxia-8 (SCA8)
[0490] In embodiments, compounds, compositions, and methods are provided to
treat
Spinocerebellar Ataxia-8 (SCA8). SCA8 is an inherited neurodegenerative
condition that is
characterized by slowly progressing ataxia. Symptoms normally emerge during
the third to fifth
decades of life. Symptoms include eye movement abnormalities, sensory
neuropathy, dysphagia,
cerebellar ataxia, and cognitive impairment.
[0491] SCA8 is associated with heterozygous abnormal expanded CTG=CUG repeat
in the 3' UTR
of two overlapping genes ATXN8OS and ATXN8. Healthy individuals generally have
between
15 and 50 CTG=CUG repeats in the ATXN8OS and ATXN8 genes. Patients with SCA8
have
greater than 50 CTG=CUG repeats, sometimes as many as 240 CTG=CUG repeats in
the ATXN8OS
and ATXN8 genes.
Huntington's disease like-2 (HDL2)
[0492] In embodiments, compounds, compositions and methods are provided to
treat Huntington's
disease like-2 (HDL2) disease. HDL2 is an autosomal dominant neurodegenerative
disorder that
is phenotypically related to Huntington's disease. HDL2 is characterized by
symptoms that include
chorea, dystonia, rigidity, bradykinesia, and psychiatric symptoms such as
dementia. Symptoms
of HDL2 typically occur in mid-life and may lead to a premature death by about
10-15 years.
[0493] HDL2 is associated with an expanded CTG=CUG in the 3' UTR of the
junctophilin 3
(JPH3) gene (16q24.3). Healthy individuals generally have between 6 and 27
CTG=CUG repeats
in the JPH3 gene. Patients with HDL2 have greater than 40 CTG=CUG repeats,
sometimes as many
as 60 or more CTG=CUG repeats in the JPH3 gene.
Fuchs' Endothelial Corneal Dystrophy
[0494] In embodiments, compounds, compositions, and methods are provided to
treat Fuchs'
Endothelial Corneal Dystrophy (FECD). FECD (MINI 136800) is an age-related
degenerative
disorder of the corneal endothelium. FECD is characterized by progressive loss
of corneal
endothelial cells, thickening of Descement' s membrane, and deposition of
extracellular matrix in
the form of guttae. When the number of endothelial cells becomes critically
low, the cornea swells
and causes loss of vision (Elhalis et al. Ocul Surf. 2010; 8(4):173-184).
194
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
[0495] FECD can be inherited as an autosomal dominant trait with genetic
heterogeneity. Rare
heterozygous mutations in collagen, type VIII, alpha 2 gene (COL8A2, MINI
120252) can give
rise to an early-onset corneal endothelial dystrophy. Other genes such as
solute carrier family 4,
sodium borate transporter, member 11 (SLC4A11, MINI 610206), transcription
factor 8 (TCF8,
MINI 189909), lipoxygenase homology domains 1 (LOXHD1, MINI 613267), and
ATP/GTP
binding protein-like 1 (AGBL1, MINI 615523) are collectively associated with a
small fraction of
adult-onset FECD cases. The genome-wide association studies of adult-onset
FECD have
suggested that transcription factor 4 (TCF4, MIM 602272) and more recently KN
motif¨ and
ankyrin repeat domain¨containing protein 4 (KANK4, MINI 614612), laminin gamma-
1 (LAMC1,
MIM150290), Na/ K+ transporting ATPase, and beta-1 polypeptide (ATP1B1, MINI
182330),
with the TCF4 locus noted have a predominant effect on FECD (Mootha et al.,
Investigative
ophthalmology &visual science, 2017; 58, 4579-4585).
[0496] Expanded trinucleotide repeats at the CTG18.1 locus in intron 2 of TCF4
are associated
with FECD (Wieben et al., PLoS One. 2012; 7(11):e49083). Each copy of the
expanded CTG18.1
allele of more than 40 CTG=CUG trinucleotide repeats leads to significant risk
for development of
FECD (Mootha et al., Invest Ophthalmol Vis Sci. 2014; 55: 33-42). RNA nuclear
foci, a hallmark
of toxic gain of function RNA, has been reported in neurodegenerative
disorders caused by simple
repeat expansions. Expanded CUG repeat RNA accumulate as nuclear foci in the
corneal
endothelium of FECD subjects with the CTG18.1 triplet repeat expansion while
absent in control
samples lacking the triplet expansion (Mootha et al., Invest Ophthalmol Vis
Sci. 2015;56(3):2003-
2011). Expanded CUG repeat RNA colocalize with mRNA-splicing factor,
muscleblind-like 1
(MBNL1), in nuclear foci in endothelium as a molecular hallmark. The triplet
repeat expansion at
the CTG18.1 locus may mediate endothelial dysfunction via aberrant gene
splicing as a result of
the mutant CUG RNA transcripts sequestering the 1VIBNL1 (Du et al., J Biol
Chem. 2015; 290:
5979-5990). Thus, two distinct triplet repeats converge on RNA foci and FECD,
and it is likely
that the foci may play a causal role for FECD.
[0497] In embodiments, compounds and methods useful in the treatment of Fuchs'
Endothelial
Corneal Dystrophy (FECD) that reduce expanded CUG repeat RNA with antisense
oligonucleotides are described in W02018165541A1, US10760076B2, each of which
is herein
incorporated by reference in its entirety for all purposes. However, such
phosphorodiamidate
morpholino oligomers (PM0s) or peptide-conjugated PM0s (PPM0s) targeting CUG
repeats such
195
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
as those described in the prior art, to treat DM1 might have limitations in
oligo delivery to the
target tissue (e.g. endothelial layers in cornea), which is the disease
affected tissues.
[0498] In embodiments, the present disclosure teaches use of diverse cell
penetrating peptide
(CPP) or endosomal escape vehicle (EEV) to deliver the AC (e.g. PM0 or ASO)
described herein,
e.g., in Table 6 to the cytosol of the cell. In embodiments, the CPP or EEV
conjugated to the AC
delivers the AC of interest to the cellular location where the target sequence
on pre-mRNA is
located.
[0499] In embodiments, the disease is Fuchs' Endothelial Corneal Dystrophy
(FECD). In
embodiments, the target gene is TCF4, which encodes transcription factor 4
(TCF-4), which is also
known as immunoglobulin transcription factor 2 (ITF-2). In embodiments, the
compounds
provided herein comprise an antisense oligonucleotide that targets TCF4.
Exemplary
oligonucleotides that may be used to target TCF4 are provided in Table 2 and
Table 11.
Table 11. Exemplary Oligonucleotides targeting the expanded triplet repeat of
TCF4
Oligo Design Target
chemistry
21-mer 5'-CAG CAG CAG CAG CAG CAG CAG -3' TCF4
PM0 (all PM0 monomers; SEQ ID NO:146) (CUG)n
25-mer 5'-CAG CAG CAG CAG CAG CAG CAG CAG C-3' TCF4
PM0 (all PM0 monomers: SEQ ID NO:147) (CUG)n
21-mer EEV-NLS PMO CAG 21 mer TCF4
EEV-NLS- (conjugation from SEQ ID NO:146) (CUG)n
PM0
30-mer 5'-CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG-3' TCF4
PM0 (all PM0 monomers; SEQ ID NO:148) (CUG)n
30-mer 5' -AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC-3' TCF4
PM0 (all PM0 monomers; SEQ ID NO:149) (CUG)n
30-mer 5' -GCA GCA GCA GCA GCA GCA GCA GCA GCA GCA-3' TCF4
PM0 (all PM0 monomers; SEQ ID NO:150) (CUG)n
PMO: Phosphorodiamidate Morpholino Oligomer
[0500] Mootha et al. (2017) reported that DM1 and FECD originate from
noncoding CTG
expansions even though both are not identical diseases. The DMPK expansion in
DM1 results in
a multiorgan disease that involves various tissues in the eye including lens,
retina, and corneal
endothelium. In contrast, the TCF4 repeat expansion appears to affect the
corneal endothelium
without any clinically apparent sequela to other ocular tissues or bodily
organs. Mutant expansions
in DMPK and TCF4 share important similarities, such as (i) nuclear foci that
contain expanded
CUG repeats, (ii) association of foci with MBNL1 protein, and (iii) an ability
to cause FECD. It is
196
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
suggested that the triplet expansions in both D1VIPK and TCF4 may cause the
same corneal
endothelial tissue phenotype of FECD through shared molecular mechanisms.
[0501] See U.S. Patent No. 10760076B2, International Application Publication
No.
W02018165541A1, U.S. Pat. Appl. Publ. No. 2016/0355796 and U.S. Pat. Appl.
Publ. No.
2018/0344817, each of which is incorporated by reference herein, and which
discloses diseases
and corresponding genes prone to forming and/or expanding tandem nucleotide
repeats.
Compositions and Methods of Administration
[0502] The compounds of the present disclosure may be formulated into
compositions suitable for
in vivo applications. The compounds and/or compositions may be administered to
a patient that
has, or is suspected of having, a disease associated with an expanded
trinucleotide repeat.
[0503] In vivo application of the disclosed compounds, and compositions
containing them, can be
accomplished by any suitable method and technique presently or prospectively
known to those
skilled in the art. For example, the disclosed compounds can be formulated in
a physiologically-
or pharmaceutically-acceptable composition and administered by any suitable
route known in the
art including, for example, oral and parenteral routes of administration. As
used herein, the term
parenteral includes subcutaneous, intradermal, intravenous, intramuscular,
intraperitoneal,
intrasternal, and intrathecal administration, such as by injection.
Administration of the disclosed
compounds or compositions can be a single administration, or at continuous or
distinct intervals
as can be readily determined by a person skilled in the art.
[0504] The compounds disclosed herein, and compositions comprising them, can
also be
administered utilizing liposome technology, slow-release capsules, implantable
pumps, and
biodegradable containers. These delivery methods can, advantageously, provide
a uniform dosage
over an extended period of time. The compounds can also be administered in
their salt derivative
forms or crystalline forms.
[0505] The compounds disclosed herein can be formulated into pharmaceutical
compositions
according to known methods for preparing pharmaceutically acceptable
compositions.
Formulations are described in detail in a number of sources which are well
known and readily
available to those skilled in the art. For example, Remington 's
Pharmaceutical Science by E.W.
Martin (1995) describes formulations that can be used in connection with the
disclosed methods.
In general, the compounds disclosed herein can be formulated such that an
effective amount of the
compound is combined with a suitable carrier in order to facilitate effective
administration of the
197
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
compound. The compositions used can also be in a variety of forms. These
include, for example,
solid, semi-solid, and liquid dosage forms, such as tablets, pills, powders,
liquid solutions or
suspension, suppositories, injectable and infusible solutions, and sprays. The
form depends on the
intended mode of administration and therapeutic application. The compositions
also include
conventional pharmaceutically acceptable carriers and diluents which are known
to those skilled
in the art. Examples of carriers or diluents for use with the compounds
include ethanol, dimethyl
sulfoxide, glycerol, alumina, starch, saline, and equivalent carriers and
diluents. To provide for the
administration of such dosages for the desired therapeutic treatment,
compositions disclosed herein
can advantageously comprise between about 0.1% and 100% by weight of the total
of one or more
of the subject compounds based on the weight of the total composition
including carrier or diluent.
[0506] Formulations suitable for administration include, for example, aqueous
sterile injection
solutions, which can contain antioxidants, buffers, bacteriostats, and solutes
that render the
formulation isotonic with the blood of the intended recipient; and aqueous and
nonaqueous sterile
suspensions, which can include suspending agents and thickening agents. The
formulations can be
presented in unit-dose or multi-dose containers, for example sealed ampoules
and vials, and can
be stored in a freeze dried (lyophilized) condition requiring only the
condition of the sterile liquid
carrier, for example, water for injections, prior to use. Extemporaneous
injection solutions and
suspensions can be prepared from sterile powder, granules, tablets, etc. It
should be understood
that in addition to the ingredients particularly mentioned above, the
compositions disclosed herein
can include other agents conventional in the art having regard to the type of
formulation in
question.
[0507] Compounds disclosed herein, and compositions comprising them, can be
delivered to a cell
either through direct contact with the cell or via a carrier means. Carrier
means for delivering
compounds and compositions to cells are known in the art and include, for
example, encapsulating
the composition in a liposome moiety. Another means for delivery of compounds
and
compositions disclosed herein to a cell comprises attaching the compounds to a
protein or nucleic
acid that is targeted for delivery to the target cell. U.S. Patent No.
6,960,648 and U.S. Application
Publication Nos. 20030032594 and 20020120100 disclose amino acid sequences
that can be
coupled to another composition and that allows the composition to be
translocated across
biological membranes. U.S. Application Publication No. 20020035243 also
describes
compositions for transporting biological moieties across cell membranes for
intracellular delivery.
198
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
Compounds can also be incorporated into polymers, examples of which include
poly (D-L lactide-
co-glycolide) polymer for intracranial tumors; poly[bis(p-carboxyphenoxy)
propane:sebacic acid]
in a 20:80 molar ratio (as used in GLIADEL); chondroitin; chitin; and
chitosan.
[0508] Compounds and compositions disclosed herein, including pharmaceutically
acceptable
salts or prodrugs thereof, can be administered intravenously, intramuscularly,
or intraperitoneally
by infusion or injection. Solutions of the active agent or its salts can be
prepared in water,
optionally mixed with a nontoxic surfactant. Dispersions can also be prepared
in glycerol, liquid
polyethylene glycols, triacetin, and mixtures thereof and in oils. Under
ordinary conditions of
storage and use, these preparations can contain a preservative to prevent the
growth of
microorganisms.
[0509] The pharmaceutical dosage forms suitable for injection or infusion can
include sterile
aqueous solutions or dispersions or sterile powders comprising the active
ingredient, which are
adapted for the extemporaneous preparation of sterile injectable or infusible
solutions or
dispersions, optionally encapsulated in liposomes. The ultimate dosage form
should be sterile,
fluid and stable under the conditions of manufacture and storage. The liquid
carrier or vehicle can
be a solvent or liquid dispersion medium comprising, for example, water,
ethanol, a polyol (for
example, glycerol, propylene glycol, liquid polyethylene glycols, and the
like), vegetable oils,
nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity
can be maintained, for
example, by the formation of liposomes, by the maintenance of the required
particle size in the
case of dispersions or by the use of surfactants. Optionally, the prevention
of the action of
microorganisms can be brought about by various other antibacterial and
antifungal agents, for
example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the
like. In many cases,
isotonic agents may be included, for example, sugars, buffers or sodium
chloride. Prolonged
absorption of the injectable compositions can be brought about by the
inclusion of agents that
delay absorption, for example, aluminum monostearate and gelatin.
[0510] Sterile injectable solutions are prepared by incorporating a compound
and/or agent
disclosed herein in the required amount in the appropriate solvent with
various other ingredients
enumerated above, as required, followed by filter sterilization. In the case
of sterile powders for
the preparation of sterile injectable solutions, methods of preparation
include vacuum drying and
the freeze-drying techniques, which yield a powder of the active ingredient
plus any additional
desired ingredient present in the previously sterile-filtered solutions.
199
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
[0511] Useful dosages of the compounds and agents and pharmaceutical
compositions disclosed
herein can be determined by comparing their in vitro activity, and in vivo
activity in animal models.
Methods for the extrapolation of effective dosages in mice, and other animals,
to humans are
known to the art.
[0512] The dosage ranges for the administration of the compositions are those
large enough to
produce the desired effect in which the symptoms or disorder are affected. The
dosage should not
be so large as to cause adverse side effects, such as unwanted cross-
reactions, anaphylactic
reactions, and the like. Generally, the dosage will vary with the age,
condition, sex and extent of
the disease in the patient and can be determined by one of skill in the art.
The dosage can be
adjusted by the individual physician in the event of any counterindications.
Dosage can vary, and
can be administered in one or more dose administrations daily, for one or
several days.
[0513] Also disclosed are pharmaceutical compositions that comprise a compound
disclosed
herein in combination with a pharmaceutically acceptable carrier.
Pharmaceutical compositions
adapted for oral, topical or parenteral administration, comprising an amount
of a compound are
disclosed herein. The dose administered to a patient, particularly a human,
should be sufficient to
achieve a therapeutic response in the patient over a reasonable time frame,
without lethal toxicity,
and causing no more than an acceptable level of side effects or morbidity. One
skilled in the art
will recognize that dosage will depend upon a variety of factors including the
condition (health)
of the subject, the body weight of the subject, kind of concurrent treatment,
if any, frequency of
treatment, therapeutic ratio, as well as the severity and stage of the
pathological condition.
[0514] Also disclosed are kits that comprise a compound disclosed herein
and/or pharmaceutical
compositions containing the same, in one or more containers. The disclosed
kits can optionally
include pharmaceutically acceptable carriers and/or diluents. In one
embodiment, a kit includes
one or more other components, adjuncts, or adjuvants as described herein. In
one embodiment, a
kit includes instructions or packaging materials that describe how to
administer a compound or
composition of the kit. Containers of the kit can be of any suitable material,
e.g., glass, plastic,
metal, etc., and of any suitable size, shape, or configuration. In one
embodiment, a compound
and/or agent disclosed herein is provided in the kit as a solid, such as a
tablet, pill, or powder form.
In another embodiment, a compound and/or agent disclosed herein is provided in
the kit as a liquid
or solution. In one embodiment, the kit comprises an ampoule or syringe
containing a compound
and/or agent disclosed herein in liquid or solution form.
200
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
[0515] In embodiments, the compound and or composition of the of the
disclosure is administered
to a patient diagnosed with a disease associated with a nucleotide repeat
expansion at a dose of
between about 0.1 mg/kg and about 1000 mg/kg, for example, about 0.1 mg/kg,
about 0.2 mg/kg,
about 0.3 mg/kg, about 0.4 mg/kg, about 0.5 mg/kg, about 0.6 mg/kg, about 0.7
mg/kg, about 0.8
mg/kg, about 0.9 mg/kg, about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4
mg/kg, about 5
mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10
mg/kg, about 11
mg/kg, about 12 mg/kg, about 13 mg/kg, about 14 mg/kg, about 15 mg/kg, about
16 mg/kg, about
17 mg/kg, about 18 mg/kg, about 19 mg/kg, about 20 mg/kg, about 21 mg/kg,
about 22 mg/kg,
about 23 mg/kg, about 24 mg/kg, about 25 mg/kg, about 26 mg/kg, about 27
mg/kg, about 28
mg/kg, about 29 mg/kg, about 30 mg/kg, about 31 mg/kg, about 32 mg/kg, about
33 mg/kg, about
34 mg/kg, about 35 mg/kg, about 36 mg/kg, about 37 mg/kg, about 38 mg/kg,
about 39 mg/kg,
about 40 mg/kg, about 41 mg/kg, about 42 mg/kg, about 43 mg/kg, about 44
mg/kg, about 45
mg/kg, about 46 mg/kg, about 47 mg/kg, about 48 mg/kg, about 49 mg/kg, about
50 mg/kg, about
51 mg/kg, about 52 mg/kg, about 53 mg/kg, about 54 mg/kg, about 55 mg/kg,
about 56 mg/kg,
about 57 mg/kg, about 58 mg/kg, about 59 mg/kg, about 60 mg/kg, about 61
mg/kg, about 62
mg/kg, about 63 mg/kg, about 64 mg/kg, about 65 mg/kg, about 66 mg/kg, about
67 mg/kg, about
68 mg/kg, about 69 mg/kg, about 70 mg/kg, about 71 mg/kg, about 72 mg/kg,
about 73 mg/kg,
about 74 mg/kg, about 75 mg/kg, about 76 mg/kg, about 77 mg/kg, about 78
mg/kg, about 79
mg/kg, about 80 mg/kg, about 81 mg/kg, about 82 mg/kg, about 83 mg/kg, about
84 mg/kg, about
85 mg/kg, about 86 mg/kg, about 87 mg/kg, about 88 mg/kg, about 89 mg/kg,
about 90 mg/kg,
about 91 mg/kg, about 92 mg/kg, about 93 mg/kg, about 94 mg/kg, about 95
mg/kg, about 96
mg/kg, about 97 mg/kg, about 98 mg/kg, about 99 mg/kg, about 100 mg/kg, about
110 mg/kg,
about 120 mg/kg, about 130 mg/kg, about 140 mg/kg, about 150 mg/kg, about 160
mg/kg, about
170 mg/kg, about 180 mg/kg, about 190 mg/kg, about 200 mg/kg, about 210 mg/kg,
about 220
mg/kg, about 230 mg/kg, about 240 mg/kg, about 250 mg/kg, about 260 mg/kg,
about 270 mg/kg,
about 280 mg/kg, about 290 mg/kg, about 300 mg/kg, about 310 mg/kg, about 320
mg/kg, about
330 mg/kg, about 340 mg/kg, about 350 mg/kg, about 360 mg/kg, about 370 mg/kg,
about 380
mg/kg, about 390 mg/kg, about 400 mg/kg, about 410 mg/kg, about 420 mg/kg,
about 430 mg/kg,
about 440 mg/kg, about 450 mg/kg, about 460 mg/kg, about 470 mg/kg, about 480
mg/kg, about
490 mg/kg, about 500 mg/kg, about 510 mg/kg, about 520 mg/kg, about 530 mg/kg,
about 540
mg/kg, about 550 mg/kg, about 560 mg/kg, about 570 mg/kg, about 580 mg/kg,
about 590 mg/kg,
201
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
about 600 mg/kg, about 610 mg/kg, about 620 mg/kg, about 630 mg/kg, about 640
mg/kg, about
650 mg/kg, about 660 mg/kg, about 670 mg/kg, about 680 mg/kg, about 690 mg/kg,
about 700
mg/kg, about 710 mg/kg, about 720 mg/kg, about 730 mg/kg, about 740 mg/kg,
about 750 mg/kg,
about 760 mg/kg, about 770 mg/kg, about 780 mg/kg, about 790 mg/kg, about 800
mg/kg, about
810 mg/kg, about 820 mg/kg, about 830 mg/kg, about 840 mg/kg, about 850 mg/kg,
about 860
mg/kg, about 870 mg/kg, about 880 mg/kg, about 890 mg/kg, about 900 mg/kg,
about 910 mg/kg,
about 920 mg/kg, about 930 mg/kg, about 940 mg/kg, about 950 mg/kg, about 960
mg/kg, about
970 mg/kg, about 980 mg/kg, about 990 mg/kg, or about 1000 mg/kg, including
all values and
ranges therein and in between.
Methods of Treatment
[0516] The present disclosure provides a method of treating disease in a
subject in need thereof,
comprising administering a compound and/or composition containing the compound
disclosed
herein. In embodiments, the disease is any of the diseases provided in the
present disclosure. In
embodiments, the target gene or gene transcript is any of the target genes or
gene transcripts
provided in the present disclosure.
[0517] In embodiments, the patient is identified as having, or at risk of
having, any disease as
described herein. In embodiments, a method is provided for treating a disease
associated with a
CTG.CUG repeat in a 3' untranslated region of a gene/transcript. In
embodiments, a method is
provided for treating myotonic dystrophy. In embodiments, a method is provided
for treating
myotonic dystrophy type 1 (DM1). In embodiments, a method is provided for
treating SCA8. In
embodiments, a method is provided for treating HDL2. In embodiments, a method
is provided for
treating FECD.
[0518] In embodiments, treatment refers to partial or complete alleviation,
amelioration, relief,
inhibition, delaying onset, reducing severity and/or incidence of one or more
symptoms in a
subj ect.
[0519] Treatment of the disease and/or symptoms of the disease may occur
through a variety of
molecular mechanisms such as those described herein.
[0520] In embodiments, a method is provided for altering the expression and/or
activity of a target
gene in a subject in need thereof, comprising administering a compound
disclosed herein. In
embodiments, the treatment results in the lowered expression of a target
protein from a target
transcript. In embodiments, treatment results in the lowered levels of a
target transcript. In
202
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
embodiments, treatment results in the modulation of splicing of downstream
gene transcripts that
are regulated by the target transcript and/or proteins that bind to the target
transcript. In
embodiments, modulation of splicing of downstream gene transcripts results in
an increase in
downstream transcripts and/or downstream proteins isoforms that are associated
with healthy
phenotypes. In embodiments, the alternative splicing results in a decrease in
downstream
transcripts and/or downstream proteins isoforms that are associated with
disease phenotypes.
[0521] In embodiments, a method is provided for treating DM1 by reducing
sequestration of at
least one RNA-binding protein to a pre-mRNA comprising at least one expanded
CUG repeat. In
embodiments, a method is provided for treating DM1 by reducing accumulation of
a pre-mRNA
comprising at least one expanded CUG repeat. In embodiments, a method is
provided for treating
DM1 by correcting splicing defects of downstream gene transcripts.
[0522] In embodiments, treatment according to the present disclosure results
in a decreased level
of the target transcript and/or expression of the target transcript (e.g.,
DMPK, TCF4, JPH3,
ATXN8OS and/or ATXN8) genes by more than about 5%, about 10%, about 15%, about
20%,
about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%,
about 60%,
about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%,
and about
100%, as compared to the average level of the protein in the subject before
the treatment or of one
or more control individuals with similar disease without treatment, or
compared to treatment with
an AC not conjugated to a cyclic CPP disclosed herein. In embodiments,
treatment according to
the present disclosure results in a decreased level of the target transcript
(e.g., DMPK, TCF4, JPH3,
ATXN8OS and/or ATXN8) and/or expression of the target transcript by about 5%
to about 100%,
about 10% to about 100%, about 20% to about 100%, about 50% to about 100%,
about 70% to
about 100%, about 80% to about 100%, about 90% to about 100%, about 95% to
about 100%,
about 40% to about 95%, about 50% to about 95%, about 70% to about 95%, or
about 90% to
about 95% as compared to the average level of the transcript and/or protein in
the subject before
the treatment or of one or more control individuals with similar disease
without treatment, or
compared to treatment with an AC not conjugated to a cyclic CPP disclosed
herein.
[0523] In embodiments, treatment according to the present disclosure results
in a decreased
number of CUG repeat RNA nuclear foci of a target gene (e.g., DMPK, TCF4,
JPH3, ATXN8OS
and/or ATXN8) by more than about 5%, e.g., about 5%, about 10%, about 15%,
about 20%, about
25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about
60%, about
203
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, and
about 100%, as
compared to the average level foci in the subject before the treatment or of
one or more control
individuals with similar disease without treatment, or compared to treatment
with an AC not
conjugated to a cyclic CPP disclosed herein. In embodiments, treatment
according to the present
disclosure results in a decreased number of CUG repeat RNA nuclear foci of a
target gene (e.g.,
DMPK, TCF4, JPH3, ATXN8OS and/or ATXN8) by about 5% to about 100%, about 10%
to about
100%, about 20% to about 100%, about 50% to about 100%, about 70% to about
100%, about
80% to about 100%, about 90% to about 100%, about 95% to about 100%, about 40%
to about
95%, about 50% to about 95%, about 70% to about 95%, or about 90% to about 95%
as compared
to the average level of foci in the subject before the treatment or of one or
more control individuals
with similar disease without treatment, or compared to treatment with an AC
not conjugated to a
cyclic CPP disclosed herein.
[0524] In embodiments, treatment according to the present disclosure results
in a decreased level
of downstream transcript and/or expression of a downstream gene product that
is associated with
a disease phenotype by more than about 5%, e.g., about 5%, about 10%, about
15%, about 20%,
about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%,
about 60%,
about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%,
and about
100%, as compared to the average level of the protein in the subject before
the treatment or of one
or more control individuals with similar disease without treatment, or
compared to treatment with
an AC not conjugated to a cyclic CPP disclosed herein. In embodiments,
treatment according to
the present disclosure results in a decreased level of downstream transcript
and/or expression of a
downstream gene product that is associated with a disease phenotype by about
5% to about 100%,
about 10% to about 100%, about 20% to about 100%, about 50% to about 100%,
about 70% to
about 100%, about 80% to about 100%, about 90% to about 100%, about 95% to
about 100%,
about 40% to about 95%, about 50% to about 95%, about 70% to about 95%, or
about 90% to
about 95% as compared to the average level of the protein in the subject
before the treatment or of
one or more control individuals with similar disease without treatment, or
compared to treatment
with an AC not conjugated to a cyclic CPP disclosed herein.
[0525] In embodiments, treatment according to the present disclosure results
in an increased level
of downstream transcript and/or expression of a downstream gene product that
is associated with
a healthy phenotype by more than about 5%, e.g., about 5%, about 10%, about
15%, about 20%,
204
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%,
about 60%,
about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%,
and about
100%, as compared to the average level of the protein in the subject before
the treatment or of one
or more control individuals with similar disease without treatment, or
compared to treatment with
an AC not conjugated to a cyclic CPP disclosed herein. In embodiments,
treatment according to
the present disclosure results in an increased level of downstream transcript
and/or expression of
a downstream gene product that is associated with a healthy phenotype by about
5% to about
100%, about 10% to about 100%, about 20% to about 100%, about 50% to about
100%, about
70% to about 100%, about 80% to about 100%, about 90% to about 100%, about 95%
to about
100%, about 40% to about 95%, about 50% to about 95%, about 70% to about 95%,
or about 90%
to about 95% as compared to the average level of the protein in the subject
before the treatment or
of one or more control individuals with similar disease without treatment, or
compared to treatment
with an AC not conjugated to a cyclic CPP disclosed herein.
[0526] In embodiments, treatment according to the present disclosure results
in decreased
expression of a protein isoform associated with a disease phenotype in a
subject's corneal tissue,
muscle tissue, diaphragm tissue, quadriceps, triceps, tibialis anterior,
gastrocnemius, or heart by
more than about 5%, e.g., about 5%, about 10%, about 15%, about 20%, about
25%, about 30%,
about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%,
about 70%,
about 75%, about 80%, about 85%, about 90%, about 95%, and about 100%, as
compared to the
average level of the protein in the subject's corneal tissue, muscle tissue,
diaphragm tissue,
quadriceps, triceps, tibialis anterior, gastrocnemius, or heart before the
treatment, compared to one
or more control individuals with similar disease without treatment, or
compared to treatment with
an AC not conjugated to a cyclic CPP disclosed herein. In embodiments,
treatment according to
the present disclosure results in decreased expression of a protein isoform
associated with a disease
phenotype in a subject's corneal tissue, muscle tissue, diaphragm tissue,
quadriceps, triceps,
tibialis anterior, gastrocnemius, or heart by more than about by about 5% to
about 100%, about
10% to about 100%, about 20% to about 100%, about 50% to about 100%, about 70%
to about
100%, about 80% to about 100%, about 90% to about 100%, about 95% to about
100%, about
40% to about 95%, about 50% to about 95%, about 70% to about 95%, or about 90%
to about 95%
as compared to the average level of the protein in the subject's corneal
tissue, muscle tissue,
diaphragm tissue, quadriceps, triceps, tibialis anterior, gastrocnemius, or
heart before the
205
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
treatment, compared to one or more control individuals with similar disease
without treatment, or
compared to treatment with an AC not conjugated to a cyclic CPP disclosed
herein.
[0527] In embodiments, treatment according to the present disclosure results
in increased
expression of an alternately spliced downstream protein in a subject's corneal
tissue, muscle tissue,
diaphragm tissue, quadriceps, or heart by more than about 5%, e.g., about 5%,
about 10%, about
15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about
50%, about
55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about
90%, about
95%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%,
about 400%,
about 450%, about 500%, about 550%, about 600%, about 650%, about 700%, about
750%, about
800, about 850%, about 900%, about 950%, or about 1000% or more, as compared
to the average
level of the downstream protein in the subject's corneal tissue, muscle
tissue, diaphragm tissue,
quadriceps, or heart before the treatment, compared to one or more control
individuals with similar
disease without treatment, or compared to treatment with an AC not conjugated
to a cyclic CPP
disclosed herein.
[0528] In embodiments, treatment according to the present disclosure results
in increased or
decreased expression of a wild type protein isomer in a subject's corneal
tissue, muscle tissue,
diaphragm tissue, quadriceps, or heart by more than about 5%, e.g., about 5%,
about 10%, about
15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about
50%, about
55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about
90%, about
95%, and about 100%, as compared to the average level of the wild type protein
isomer in the
subject's corneal tissue, muscle tissue, diaphragm tissue, quadriceps, or
heart before the treatment,
compared to one or more control individuals with similar disease without
treatment, or compared
to treatment with an AC not conjugated to a cyclic CPP disclosed herein.
[0529] In embodiments, treatment according to the present disclosure results
in decreased
expression of a protein in a subject's tissue of interest by more than about
5%, e.g., about 5%,
about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%,
about 45%,
about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%,
about 85%,
about 90%, about 95%, and about 100%, as compared to the average level of the
protein in the
subject's tissue of interest before the treatment, compared to one or more
control individuals with
similar disease without treatment, or compared to treatment with an AC not
conjugated to a cyclic
CPP disclosed herein.
206
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
[0530] In embodiments, treatment according to the present disclosure results
in increased
expression of an alternately spliced downstream protein in a subject's tissue
of interest by more
than about 5%, e.g., about 5%, about 10%, about 15%, about 20%, about 25%,
about 30%, about
35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about
70%, about
75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 150%, about
200%, about
250%, about 300%, about 350%, about 400%, about 450%, about 500%, about 550%,
about 600%,
about 650%, about 700%, about 750%, about 800, about 850%, about 900%, about
950%, or about
1000% or more, as compared to the average level of the downstream protein in
the subject's tissue
of interest before the treatment, compared to one or more control individuals
with similar disease
without treatment, or compared to treatment with an AC not conjugated to a
cyclic CPP disclosed
herein.
[0531] In embodiments, treatment according to the present disclosure results
in increased or
decreased expression of a wild type downstream protein isomer in a subject's
tissue of interest by
more than about 5%, e.g., about 5%, about 10%, about 15%, about 20%, about
25%, about 30%,
about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%,
about 70%,
about 75%, about 80%, about 85%, about 90%, about 95%, and about 100%, as
compared to the
average level of the downsteam protein in the subject's tissue of interest
before the treatment,
compared to one or more control individuals with similar disease without
treatment, or compared
to treatment with an AC not conjugated to a cyclic CPP disclosed herein.
[0532] In embodiments, the subject's tissue of interest is corneal tissue or
muscle tissue.
[0533] The terms, "improve," "increase," "reduce," "decrease," and the like,
as used herein,
indicate values that are relative to a control. In embodiments, a suitable
control is a baseline
measurement, such as a measurement in the same individual prior to initiation
of the treatment
described herein, or a measurement in a control individual (or multiple
control individuals) in the
absence of the treatment described herein. A "control individual" is an
individual afflicted with
the same disease, who is about the same age and/or gender as the individual
being treated (to ensure
that the stages of the disease in the treated individual and the control
individual(s) are comparable).
[0534] The individual (also referred to as "patient" or "subject") being
treated is an individual
(fetus, infant, child, adolescent, or adult human) having a disease or having
the potential to develop
a disease. The individual may have a disease mediated by aberrant gene
expression or aberrant
gene splicing. In various embodiments, the individual having the disease may
have downstream
207
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
protein expression or activity levels that are less than about 1-99% of normal
wild type protein
expression or activity levels in an individual not afflicted with the disease.
In embodiments, the
range includes, but is not limited to less than about 80-99%, less than about
65-80%, less than
about 50-65%, less than about 30-50%, less than about 25-30%, less than about
20-25%, less than
about 15-20%, less than about 10-15%, less than about 5-10%, less than about 1-
5% of normal
wild type protein expression or activity levels. In embodiments, the
individual may have
downstream protein expression or activity levels that are 1-500% higher than
normal wild type
target protein expression or activity levels in an individual not afflicted
with the disease. In
embodiments, the range includes, but is not limited to, greater than about 1-
10%, about 10-50%,
about 50-100%, about 100-200%, about 200-300%, about 300-400%, about 400-500%,
or about
500-1000% of normal wild type target protein expression or activity levels.
[0535] In embodiments, the individual is an individual who has been recently
diagnosed with the
disease. Typically, early treatment (treatment commencing as soon as possible
after diagnosis) is
important to minimize the effects of the disease and to maximize the benefits
of treatment.
Certain Definitions
[0536] As used in the description and the appended claims, the singular forms
"a," "an," and "the"
include plural referents unless the context clearly dictates otherwise. Thus,
for example, reference
to "a composition" includes mixtures of two or more such compositions,
reference to "an agent"
includes mixtures of two or more such agents, reference to "the component"
includes mixtures of
two or more such components, and the like.
[0537] The term "about" when immediately preceding a numerical value means a
range (e.g., plus
or minus 20%, 10%, or 5% of that value). For example, "about 50" can mean 45
to 55, "about
25,000" can mean 22,500 to 27,500, etc., unless the context of the disclosure
indicates otherwise,
or is inconsistent with such an interpretation. For example, in a list of
numerical values such as
"about 49, about 50, about 55, ...", "about 50" means a range extending to
less than half the
interval(s) between the preceding and subsequent values, e.g., more than 49.5
to less than 52.5.
Furthermore, the phrases "less than about" a value or "greater than about" a
value should be
understood in view of the definition of the term "about" provided herein.
Similarly, the term
"about" when preceding a series of numerical values or a range of values
(e.g., "about 10, 20, 30"
or "about 10-30") refers, respectively to all values in the series, or the
endpoints of the range.
208
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
[0538] As used herein, "cell penetrating peptide" or "CPP" refers to a peptide
that facilitates
delivery of a cargo, e.g., a therapeutic moiety (TM) into a cell. In
embodiments, the CPP is cyclic,
and is represented as "cCPP". In embodiments, the cCPP is capable of directing
a therapeutic
moiety to penetrate the membrane of a cell. In embodiments, the cCPP delivers
the therapeutic
moiety to the cytosol of the cell. In embodiments, the cCPP delivers an
antisense compound (AC)
to a cellular location where a pre-mRNA is located.
[0539] As used herein, the term "endosomal escape vehicle" (EEV) refers to a
cCPP that is
conjugated by a chemical linkage (i.e., a covalent bond or non-covalent
interaction) to a linker
and/or an exocyclic peptide (EP). The EEV can be an EEV of Formula (B).
[0540] As used herein, the term "EEV-conjugate" refers to an endosomal escape
vehicle defined
herein conjugated by a chemical linkage (i.e., a covalent bond or non-covalent
interaction) to a
cargo. The cargo can be a therapeutic moiety (e.g., an oligonucleotide,
peptide, or small molecule)
that can be delivered into a cell by the EEV. The EEV-conjugate can be an EEV-
conjugate of
Formula (C).
[0541] As used herein, the term "exocyclic peptide" (EP) and "modulatory
peptide" (MP) may be
used interchangeably to refer to two or more amino acid residues linked by a
peptide bond that can
be conjugated to a cyclic cell penetrating peptide (cCPP) disclosed herein.
The EP, when
conjugated to a cyclic peptide disclosed herein, may alter the tissue
distribution and/or retention
of the compound. Typically, the EP comprises at least one positively charged
amino acid residue,
e.g., at least one lysine residue and/or at least one arginine residue. Non-
limiting examples of EP
are described herein. The EP can be a peptide that has been identified in the
art as a "nuclear
localization sequence" (NLS). Non-limiting examples of nuclear localization
sequences include
the nuclear localization sequence of the SV40 virus large T-antigen, the
minimal functional unit
of which is the seven amino acid sequence PKKKRKV (SEQ ID NO:42), the
nucleoplasmin
bipartite NLS with the sequence NLSKRPAAIKKAGQAKKKK(SEQ ID NO:52), the c-myc
nuclear localization sequence having the amino acid sequence PAAKRVKLD (SEQ ID
NO:53)
or RQRRNELKR SF (SEQ ID NO:54), the
sequence
RMRKFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO:50) of the IBB
domain from importin-alpha, the sequences VSRKRPRP (SEQ ID NO:57) and PPKKARED
(SEQ ID NO:58)of the myoma T protein, the sequence PQPKKKPL (SEQ ID NO:59) of
human
p53, the sequence SALIKKKKKMAP (SEQ ID NO:60) of mouse c-abl IV, the sequences
DRLRR
209
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
(SEQ ID NO:61) and PKQKKRK (SEQ ID NO:62) of the influenza virus NS1, the
sequence
RKLKKKIKKL (SEQ ID NO:63) of the Hepatitis virus delta antigen, the sequence
REKKKFLKRR (SEQ ID NO:64) of the mouse Mxl protein, the sequence
KRKGDEVDGVDEVAKKKSKK (SEQ ID NO:65) of the human poly(ADP-ribose) polymerase,
and the sequence RKCLQAGMNLEARKTKK (SEQ ID NO:66) of the steroid hormone
receptors
(human) glucocorticoid. International Publication No. 2001/038547 describes
additional examples
of NLSs and is incorporated by reference herein in its entirety.
[0542] As used herein, "linker" or "L" refers to a moiety that covalently
bonds one or more
moieties (e.g., an exocyclic peptide (EP) and a cargo, e.g., an
oligonucleotide, peptide or small
molecule) to the cyclic cell penetrating peptide (cCPP). The linker can
comprise a natural or non-
natural amino acid or polypeptide. The linker can be a synthetic compound
containing two or more
appropriate functional groups suitable to bind the cCPP to a cargo moiety, to
thereby form the
compounds disclosed herein. The linker can comprise a polyethylene glycol
(PEG) moiety. The
linker can comprise one or more amino acids. The cCPP may be covalently bound
to a cargo via a
linker.
[0543] The terms "peptide," "protein," and "polypeptide" are used
interchangeably to refer to a
natural or synthetic molecule comprising two or more amino acids linked by the
carboxyl group
of one amino acid to the alpha amino group of another. Two or more amino acid
residues can be
linked by the carboxyl group of one amino acid to the alpha amino group. Two
or more amino
acids of the polypeptide can be joined by a peptide bond. The polypeptide can
include a peptide
backbone modification in which two or more amino acids are covalently attached
by a bond other
than a peptide bond. The polypeptide can include one or more non-natural amino
acids, amino acid
analogs, or other synthetic molecules that are capable of integrating into a
polypeptide. The term
polypeptide includes naturally occurring and artificially occurring amino
acids. The term
polypeptide includes peptides, for example, that include from about 2 to about
100 amino acid
residues as well as proteins, that include more than about 100 amino acid
residues, or more than
about 1000 amino acid residues, including, but not limited to therapeutic
proteins such as
antibodies, enzymes, receptors, soluble proteins, and the like.
[0544] As used herein, the term "contiguous" refers to two amino acids, which
are connected by
a covalent bond. For example, in the context of a representative cyclic cell
penetrating peptide
210
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
AA5 \AA2
AA4
(cCPP) such as AA3 , AAi/AA2, AA2/AA3, AA3/AA4, and AA5/AA1 exemplify
pairs of
contiguous amino acids.
[0545] A residue of a chemical species, as used herein, refers to a derivative
of the chemical
species that is present in a particular product. To form the product, at least
one atom of the species
is replaced by a bond to another moiety, such that the product contains a
derivative, or residue, of
the chemical species. For example, the cyclic cell penetrating peptides (cCPP)
described herein
have amino acids (e.g., arginine) incorporated therein through formation of
one or more peptide
bonds. The amino acids incorporated into the cCPP may be referred to residues,
or simply as an
NH2
HNN
y
zNH
amino acid. For example, arginine or an arginine residue refers to
[0546] The term "protonated form thereof' refers to a protonated form of an
amino acid or side
chain. For example, the guanidine group on the side chain of arginine may be
protonated to form
NH2
0
H2N
y
zNH
a guanidinium group. The structure of a protonated form of arginine is
[0547] As used herein, the term "chirality" refers to a molecule that has more
than one
stereoisomer that differs in the three-dimensional spatial arrangement of
atoms, in which one
stereoisomer is a non-superimposable mirror image of the other. Amino acids,
except for glycine,
have a chiral carbon atom adjacent to the carboxyl group. The term
"enantiomer" refers to
stereoisomers that are chiral. The chiral molecule can be an amino acid
residue having a "D" and
"L" enantiomer. Molecules without a chiral center, such as glycine, can be
referred to as "achiral."
[0548] As used herein, the term "hydrophobic" refers to a moiety that is not
soluble in water or
has minimal solubility in water. Generally, neutral moieties and/or non-polar
moieties, or moieties
211
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
that are predominately neutral and/or non-polar are hydrophobic.
Hydrophobicity can be measured
by one of the methods disclosed herein.
[0549] As used herein "aromatic" refers to an unsaturated cyclic molecule
having 4n + 2 it
electrons, wherein n is any integer. "Heteroaromatic," defined below, is a
subset of aromatic.
Examples of aromatic amino acids include phenylalanine and napthylalinine. The
term "non-
aromatic" refers to any molecule that does not fall within the definition of
aromatic. For example,
any linear, branched or cyclic molecule which does not fall within the
definition of aromatic is
non-aromatic. Examples of non-aromatic amino acids include, but are not
limited to, glycine and
citrulline.
[0550] "Alkyl", "alkyl chain" or "alkyl group" refer to a fully saturated,
straight or branched
hydrocarbon chain radical having from one to forty carbon atoms, and which is
attached to the rest
of the molecule by a single bond. Alkyls comprising any number of carbon atoms
from 1 to 40 are
included. An alkyl comprising up to 40 carbon atoms is a Ci-C40 alkyl, an
alkyl comprising up to
1 0 carbon atoms is a Ci-C10 alkyl, an alkyl comprising up to 6 carbon atoms
is a Ci-C6 alkyl and
an alkyl comprising up to 5 carbon atoms is a C i-05 alkyl. A C i-05 alkyl
includes C5 alkyls, C4
alkyls, C3 alkyls, C2 alkyls and Ci alkyl (i.e., methyl). A C1-C6 alkyl
includes all moieties described
above for C1-05 alkyls but also includes C6 alkyls. A C1-C10 alkyl includes
all moieties described
above for C1-05 alkyls and Ci-C6 alkyls, but also includes C7, Cg, C9 and Cio
alkyls. Similarly, a
C i-C12 alkyl includes all the foregoing moieties, but also includes C11 and
C12 alkyls. Non-limiting
examples of CI-Cu alkyl include methyl, ethyl, n-propyl, i-propyl, sec-propyl,
n-butyl, i-butyl,
sec-butyl, t-butyl, n-pentyl, t-amyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-
decyl, n-undecyl, and n-
dodecyl. Unless stated otherwise specifically in the specification, an alkyl
group can be optionally
substituted.
[0551] "Alkylene", "alkylene chain" or "alkylene group" refers to a fully
saturated, straight or
branched divalent hydrocarbon chain radical, having from one to forty carbon
atoms. Non-limiting
examples of C2-C40 alkylene include ethylene, propylene, n-butylene,
ethenylene, propenylene,
n-butenylene, propynylene, n-butynylene, and the like. Unless stated otherwise
specifically in the
specification, an alkylene chain can be optionally substituted.
[0552] "Alkenyl", "alkenyl chain" or "alkenyl group" refers to a straight or
branched hydrocarbon
chain radical having from two to forty carbon atoms and having one or more
carbon-carbon double
bonds. Each alkenyl group is attached to the rest of the molecule by a single
bond. Alkenyl groups
212
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
comprising any number of carbon atoms from 2 to 40 are included. An alkenyl
group comprising
up to 40 carbon atoms is a C2-C40 alkenyl, an alkenyl comprising up to 10
carbon atoms is a C2-
Cio an alkenyl group comprising up to 6 carbon atoms is a C2-C6 alkenyl
and an alkenyl
comprising up to 5 carbon atoms is a C2-05 alkenyl. A C2-05 alkenyl includes
C5 alkenyls, C4
alkenyls, C3 alkenyls, and C2 alkenyls. A C2-C6 alkenyl includes all moieties
described above for
C2-05 alkenyls but also includes C6 alkenyls. A C2-C10 alkenyl includes all
moieties described
above for C2-05 alkenyls and C2-C6 alkenyls, but also includes C7, C8, C9 and
Cio alkenyls.
Similarly, a C2-C12 alkenyl includes all the foregoing moieties, but also
includes C11 and C12
alkenyls. Non-limiting examples of C2-C12 alkenyl include ethenyl (vinyl), 1 -
propenyl, 2-propenyl
(allyl), iso-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-
pentenyl, 2-
pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl,
5-hexenyl, 1-
heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5-heptenyl, 6-heptenyl, 1 -
octenyl, 2-octenyl, 3-
octenyl, 4-octenyl, 5-octenyl, 6-octenyl, 7-octenyl, 1 -nonenyl, 2-nonenyl, 3-
nonenyl, 4-nonenyl,
5-nonenyl, 6-nonenyl, 7-nonenyl, 8-nonenyl, 1-decenyl, 2-decenyl, 3-decenyl, 4-
decenyl, 5-
decenyl, 6-decenyl, 7-decenyl, 8-decenyl, 9-decenyl, 1-undecenyl, 2-undecenyl,
3-undecenyl, 4-
undecenyl, 5-undecenyl, 6-undecenyl, 7-undecenyl, 8-undecenyl, 9-undecenyl, 1
0-undecenyl, 1-
dodecenyl, 2-dodecenyl, 3-dodecenyl, 4-dodecenyl, 5-dodecenyl, 6-dodecenyl, 7-
dodecenyl, 8-
dodecenyl, 9-dodecenyl, 1 0-dodecenyl, and 1 1-dodecenyl. Unless stated
otherwise specifically in
the specification, an alkyl group can be optionally substituted.
[0553] "Alkenylene", "alkenylene chain" or "alkenylene group" refers to a
straight or branched
divalent hydrocarbon chain radical, having from two to forty carbon atoms, and
having one or
more carbon-carbon double bonds. Non-limiting examples of C2-C40 alkenylene
include ethene,
propene, butene, and the like. Unless stated otherwise specifically in the
specification, an
alkenylene chain can be optionally.
[0554] "Alkoxy" or "alkoxy group" refers to the group -OR, where R is alkyl,
alkenyl, alkynyl,
cycloalkyl, or heterocyclyl as defined herein. Unless stated otherwise
specifically in the
specification, an alkoxy group can be optionally substituted.
[0555] "Acyl" or "acyl group" refers to groups -C(0)R, where R is hydrogen,
alkyl, alkenyl,
alkynyl, carbocyclyl, or heterocyclyl, as defined herein. Unless stated
otherwise specifically in the
specification, acyl can be optionally substituted.
213
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
[0556] "Alkylcarbamoyl" or "alkylcarbamoyl group" refers to the group -0-
C(0)4'4IRaRb, where
Ra and Rb are the same or different and are independently an alkyl, alkenyl,
alkynyl, aryl,
heteroaryl, as defined herein, or RaRb can be taken together to form a
cycloalkyl group or
heterocyclyl group, as defined herein. Unless stated otherwise specifically in
the specification, an
alkylcarbamoyl group can be optionally substituted.
[0557] "Alkylcarboxamidyl" or "alkylcarboxamidyl group" refers to the group
¨C(0)-NRaRb,
where Ra and Rb are the same or different and are independently an alkyl,
alkenyl, alkynyl, aryl,
heteroaryl, cycloalkyl, cycloalkenyl, cycloalkynyl, or heterocyclyl group, as
defined herein, or
RaRb can be taken together to form a cycloalkyl group, as defined herein.
Unless stated otherwise
specifically in the specification, an alkylcarboxamidyl group can be
optionally substituted.
[0558] "Aryl" refers to a hydrocarbon ring system radical comprising hydrogen,
6 to 18 carbon
atoms and at least one aromatic ring. For purposes of this invention, the aryl
radical can be a
monocyclic, bicyclic, tricyclic or tetracyclic ring system, which can include
fused or bridged ring
systems. Aryl radicals include, but are not limited to, aryl radicals derived
from aceanthrylene,
acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene,
fluoranthene,
fluorene, as-indacene, s-indacene, indane, indene, naphthalene, phenalene,
phenanthrene,
pleiadene, pyrene, and triphenylene. Unless stated otherwise specifically in
the specification, the
term "aryl" is meant to include aryl radicals that are optionally substituted.
[0559] "Heteroaryl" refers to a 5- to 20-membered ring system radical
comprising hydrogen
atoms, one to thirteen carbon atoms, one to six heteroatoms selected from
nitrogen, oxygen and
sulfur, and at least one aromatic ring. For purposes of this invention, the
heteroaryl radical can be
a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which can
include fused or bridged ring
systems; and the nitrogen, carbon or sulfur atoms in the heteroaryl radical
can be optionally
oxidized; the nitrogen atom can be optionally quaternized. Examples include,
but are not limited
to, azepinyl, acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl,
benzodioxolyl, benzofuranyl,
benzooxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, 1,4-
benzodioxanyl,
benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl,
benzopyranonyl, benzofuranyl, benzofuranonyl, b enzothienyl (benzothiophenyl),
benzotriazolyl,
benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl, dibenzofuranyl,
dibenzothiophenyl,
furanyl, furanonyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl,
isoindolyl, indolinyl,
isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, naphthyridinyl,
oxadiazolyl, 2-oxoazepinyl,
214
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
oxazolyl, oxiranyl, 1-oxidopyridinyl, 1-oxidopyrimidinyl, 1-oxidopyrazinyl, 1-
oxidopyridazinyl,
1-pheny1-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl,
pteridinyl, purinyl,
pyrrolyl, pyrazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl,
quinazolinyl, quinoxalinyl,
quinolinyl, quinuclidinyl, isoquinolinyl, tetrahydroquinolinyl, thiazolyl,
thiadiazolyl, triazolyl,
tetrazolyl, triazinyl, and thiophenyl (i.e. thienyl). Unless stated otherwise
specifically in the
specification, a heteroaryl group can be optionally substituted.
[0560] The term "substituted" used herein means any of the above groups (i.e.,
alkyl, alkenyl,
alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, aryl,
heteroaryl, alkoxy, aryloxy,
acyl, alkylcarbamoyl, alkylcarboxamidyl, alkoxycarbonyl, alkylthio, or
arylthio) wherein at least
one atom is replaced by a non-hydrogen atoms such as, but not limited to: a
halogen atom such as
F, Cl, Br, and I; an oxygen atom in groups such as hydroxyl groups, alkoxy
groups, and ester
groups; a sulfur atom in groups such as thiol groups, thioalkyl groups,
sulfone groups, sulfonyl
groups, and sulfoxide groups; a nitrogen atom in groups such as amines,
amides, alkylamines,
dialkylamines, arylamines, alkylarylamines, diarylamines, N-oxides, imides,
and enamines; a
silicon atom in groups such as trialkylsilyl groups, dialkylarylsilyl groups,
alkyldiarylsilyl groups,
and triarylsilyl groups; and other heteroatoms in various other groups.
"Substituted" also means
any of the above groups in which one or more atoms are replaced by a higher-
order bond (e.g., a
double- or triple-bond) to a heteroatom such as oxygen in oxo, carbonyl,
carboxyl, and ester
groups; and nitrogen in groups such as imines, oximes, hydrazones, and
nitriles. For example,
"substituted" includes any of the above groups in which one or more atoms are
replaced
with -NRgRh, -NRgC(=0)Rh, -NRgC(=0)NRgRh, 4'4RgC(=0)0Rh, -NRgS02Rh, -
0C(=0)NRgRh, -
ORg, -SRg, -SORg, -SO2Rg, -0S02Rg, -S020Rg, =NSO2Rg, and -SO2NRgRh.
"Substituted also
means any of the above groups in which one or more hydrogen atoms are replaced
with -C(=0)Rg, -C(=0)0Rg, -C(=0)NRgRh, -CH2S02Rg, -CH2S02NRgRh. In the
foregoing, Rg and
Rh are the same or different and independently hydrogen, alkyl, alkenyl,
alkynyl, alkoxy,
alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, cycloalkynyl,
cycloalkylalkyl,
haloalkyl, haloalkenyl, haloalkynyl, heterocyclyl, N-heterocyclyl,
heterocyclylalkyl, heteroaryl,
N-heteroaryl and/or heteroarylalkyl. "Substituted" further means any of the
above groups in which
one or more atoms are replaced by an amino, cyano, hydroxyl, imino, nitro,
oxo, thioxo, halo,
alkyl, alkenyl, alkynyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl,
cycloalkyl, cycloalkenyl,
cycloalkynyl, cycloalkylalkyl, haloalkyl, haloalkenyl, haloalkynyl,
heterocyclyl, N-heterocyclyl,
215
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
heterocyclylalkyl, heteroaryl, N-heteroaryl and/or heteroarylalkyl group.
"Substituted" can also
mean an amino acid in which one or more atoms on the side chain are replaced
by alkyl, alkenyl,
alkynyl, acyl, alkylcarboxamidyl, alkoxycarbonyl, carbocyclyl, heterocyclyl,
aryl, or heteroaryl.
In addition, each of the foregoing substituents can also be optionally
substituted with one or more
of the above substituents.
[0561] As used herein, the symbol "
" (hereinafter can be referred to as "a point of
attachment bond") denotes a bond that is a point of attachment between two
chemical entities, one
of which is depicted as being attached to the point of attachment bond and the
other of which is
XY-1- not depicted as being attached to the point of attachment bond. For
example," õindicates
that the chemical entity "XY" is bonded to another chemical entity via the
point of attachment
bond. Furthermore, the specific point of attachment to the non-depicted
chemical entity can be
XY-1- specified by inference. For example, the compound CH3-R3, wherein R3 is
H or" t õinfers
that when R3 is "XY", the point of attachment bond is the same bond as the
bond by which R3 is
depicted as being bonded to CH3.
[0562] As used herein, by a "subject" is meant an individual. Thus, the
"subject" can include
domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle,
horses, pigs, sheep, goats, etc.),
laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.), and birds.
"Subject" can also include
a mammal, such as a primate or a human. Thus, the subject can be a human or
veterinary patient.
The term "patient" refers to a subject under the treatment of a clinician,
e.g., physician.
[0563] The terms "inhibit", "inhibiting" or "inhibition" refer to a decrease
in an activity,
expression, function or other biological parameter and can include, but does
not require complete
ablation of the activity, expression, function or other biological parameter.
Inhibition can include,
for example, at least about a 10% reduction in the activity, response,
condition, or disease as
compared to a control. In embodiments, expression, activity or function of a
gene or protein is
decreased by a statistically significant amount. In embodiments, activity or
function is decreased
by at least about 10%, about 20%, about 30%, about 40%, about 50%, and up to
about 60%, about
70%, about 80%õ. about 90% or about 100%.
216
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
[0564] By "reduce" or other forms of the word, such as "reducing" or
"reduction," is meant
lowering of an event or characteristic (e.g., tumor growth). It is understood
that this is typically in
relation to some standard or expected value, in other words it is relative,
but that it is not always
necessary for the standard or relative value to be referred to. For example,
"reduces tumor growth"
means reducing the rate of growth of a tumor relative to a standard or a
control (e.g., an untreated
tumor).
[0565] As used herein, "treat," "treating," "treatment" and variants thereof,
refers to any
administration of the disclosed compounds that partially or completely
alleviates, ameliorates,
relieves, inhibits, delays onset of, reduces severity of, and/or reduces
incidence of one or more
symptoms or features of a disease as described herein. In reference to a
patient, the term
"treatment" refers to the medical management of a patient with the intent to
cure, ameliorate,
stabilize, or prevent a disease, pathological condition, or disorder. This
term includes active
treatment, that is, treatment directed specifically toward the improvement of
a disease, pathological
condition, or disorder, and also includes causal treatment, that is, treatment
directed toward
removal of the cause of the associated disease, pathological condition, or
disorder. In addition, this
term includes palliative treatment, that is, treatment designed for the relief
of symptoms rather than
the curing of the disease, pathological condition, or disorder; preventative
treatment, that is,
treatment directed to minimizing or partially or completely inhibiting the
development of the
associated disease, pathological condition, or disorder; and supportive
treatment, that is, treatment
employed to supplement another specific therapy directed toward the
improvement of the
associated disease, pathological condition, or disorder.
[0566] The term "therapeutically effective" refers to the amount of the
disclosed compound and/or
composition used is of sufficient quantity to ameliorate one or more causes or
symptoms of a
disease or disorder. Such amelioration only requires a reduction or
alteration, not necessarily
elimination.
[0567] The term "pharmaceutically acceptable" refers to those compounds,
materials,
compositions, and/or dosage forms which are, within the scope of sound medical
judgment,
suitable for use in contact with the tissues of human beings and/or animals
without excessive
toxicity, irritation, allergic response, or other problems or complications
commensurate with a
reasonable benefit/risk ratio.
217
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
[0568] The term "carrier" means a compound, composition, substance, or
structure that, when in
combination with a compound or composition of the present disclosure, aids or
facilitates
preparation, storage, administration, delivery, effectiveness, selectivity, or
any other feature of the
compound or composition for its intended use or purpose, or combinations
thereof. For example,
a carrier can be selected to minimize any degradation of the active ingredient
and to minimize any
adverse side effects in the subject.
[0569] As used herein, the term "pharmaceutically acceptable carrier" refers
to a carrier suitable
for administration to a patient. A pharmaceutical carrier may be a substance
that aids or facilitates
preparation, storage, administration, delivery, effectiveness, selectivity, or
any other feature of the
compound or composition of the present disclosure for its intended use or
purpose, or combinations
thereof. For example, a carrier can be selected to reduce degradation of the
compound or to reduce
adverse side effects in the patient. In embodiments, a pharmaceutically
acceptable carrier can be
a sterile aqueous or nonaqueous solutions, dispersions, suspensions or
emulsions, as well as sterile
powders for reconstitution into sterile injectable solutions or dispersions
just prior to use.
Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or
vehicles include
water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene
glycol and the like),
carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as
olive oil) and
injectable organic esters such as ethyl oleate. Proper fluidity can be
maintained, for example, by
the use of coating materials such as lecithin, by the maintenance of the
required particle size in the
case of dispersions and by the use of surfactants. These compositions can also
contain adjuvants
such as preservatives, wetting agents, emulsifying agents and dispersing
agents. Prevention of the
action of microorganisms can be ensured by the inclusion of various
antibacterial and antifungal
agents such as paraben, chlorobutanol, phenol, sorbic acid, and the like. It
can also be desirable to
include isotonic agents such as sugars, sodium chloride, and the like. The
injectable formulations
can be sterilized, for example, by filtration through a bacterial-retaining
filter 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 media just prior to use. Suitable
inert carriers can include
sugars such as lactose.
[0570] The term "pharmaceutically acceptable salts" include those obtained by
reacting the active
compound functioning as a base, with an inorganic or organic acid to form a
salt, for example,
salts of hydrochloric acid, sulfuric acid, phosphoric acid, methanesulfonic
acid, camphorsulfonic
218
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
acid, oxalic acid, maleic acid, succinic acid, citric acid, formic acid,
hydrobromic acid, benzoic
acid, tartaric acid, fumaric acid, salicylic acid, mandelic acid, carbonic
acid, etc. Those skilled in
the art will further recognize that acid addition salts may be prepared by
reaction of the compounds
with the appropriate inorganic or organic acid via any of a number of known
methods. The term
"pharmaceutically acceptable salts" also includes those obtained by reacting
the active compound
functioning as an acid, with an inorganic or organic base to form a salt, for
example salts of
ethylenediamine, N-methyl-glucamine, lysine, arginine, ornithine, choline,
N,N'-
dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-
benzylphenethylamine,
diethylamine, piperazine, tris-(hydroxymethyl)-aminomethane,
tetramethylammonium hydroxide,
triethylamine, dibenzylamine, ephenamine, dehydroabietylamine, N-
ethylpiperidine,
benzylamine, tetramethylammonium, tetraethylammonium, methylamine,
dimethylamine,
trimethylamine, ethylamine, basic amino acids, and the like. Non limiting
examples of inorganic
or metal salts include lithium, sodium, calcium, potassium, magnesium salts,
and the like.
[0571] As used herein, the term "parenteral administration," refers to
administration through
injection or infusion. Parenteral administration includes, but is not limited
to, subcutaneous
administration, intravenous administration, or intramuscular administration.
[0572] As used herein, the term "subcutaneous administration" refers to
administration just below
the skin. "Intravenous administration" means administration into a vein.
[0573] As used herein, the term "dose" refers to a specified quantity of a
pharmaceutical agent
provided in a single administration. In embodiments, a dose may be
administered in two or more
boluses, tablets, or injections. In embodiments, where subcutaneous
administration is desired, the
desired dose requires a volume not easily accommodated by a single injection.
In such
embodiments, two or more injections may be used to achieve the desired dose.
In embodiments, a
dose may be administered in two or more injections to reduce injection site
reaction in a patient.
[0574] As used herein, the term "dosage unit" refers to a form in which a
pharmaceutical agent is
provided. In embodiments, a dosage unit is a vial that includes lyophilized
compounds or
compositions described herein. In embodiments, a dosage unit is a vial that
includes reconstituted
compounds or compositions described herein.
[0575] The term "therapeutic moiety" (TM) refers to a compound that can be
used for treating, at
least one symptom of a disease or disorder and can include, but is not limited
to, therapeutic
polypeptides, oligonucleotides, small molecules and other agents that can be
used to treat at least
219
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
one symptom of a disease or disorder. In embodiments, the TM modulates the
activity, expression,
and/or levels of a target transcript. In embodiments, the TM decreases the
levels of the target
transcript through decay mechanisms. In embodiments the activity is the
ability of the target
transcript to bind to (e.g., sequester) one or more proteins. In embodiments,
the TM modulates the
activity of the target transcript by reducing the affinity between the target
transcript and one or
more proteins that bind to the target transcript. As a result of reducing the
affinity between the
target transcript and the one or more proteins the activity of the one or more
proteins may be
modulated. For example, if the one or more proteins are not bound to the
target transcript, they are
available to carry out their functions, such as, for example, facilitating the
splicing, alternative
splicing, and/or exon skipping of other transcripts. As a result of the
function of a TM, the activity,
expression, and/or levels of the downstream genes that are regulated by the
one or more proteins
whose interaction with the target transcript is disrupted by the TM may be
modulated.
[0576] The terms "modulate", "modulating" and "modulation" refer to a
perturbation of
expression, function or activity when compared to the level of expression,
function or activity prior
to modulation. Modulation can include an increase (stimulation or induction)
or a decrease
(inhibition or reduction) in expression, function, or activity. In
embodiments, the activity of a
target transcript is modulated. In embodiments, modulating the activity of the
target transcript
includes decreasing the ability of the target transcript to bind to one or
more proteins. In
embodiments, decreasing the affinity between the target transcript and the one
or more proteins
results in the modulation of the activity of the one or more proteins that
interact with the target
transcript. For example, if the one or more proteins are not bound to the
target transcript, they are
available to carry out their functions, such as, for example, facilitating the
splicing, alternative
splicing, and/or exon skipping of other transcripts (e.g., downstream
transcripts). As such,
modulating the activity of the target transcript may result in the modulation
of the activity,
expression, and/or levels of the downstream genes that are regulated by the
one or more proteins
whose interaction with the target transcript may be disrupted.
[0577] "Amino acid" refers to an organic compound that includes an amino group
and a carboxylic
Z4,14 ............................. C, .. COOH
acid group and has the general formula
H where R can be any organic group. An
amino acid may be a naturally occurring amino acid or non-naturally occurring
amino acid. An
220
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
amino acid may be a proteogenic amino acid or a non-proteogenic amino acid. An
amino acid can
be an L-amino acid or a D- amino acid. The term "amino acid side chain" or
"side chain" refers to
the characterizing substituent ("R") bound to the a-carbon of a natural or non-
natural a-amino
acid. An amino acid may be incorporated into a polypeptide via a peptide bond.
[0578] As used herein, an "uncharged" amino acid is an amino acid having a
side chain that has a
net neutral charge at pH 7.35 to 7.45. Examples of uncharged amino acids
include, but are not
limited to, glycine and citrulline.
[0579] As used herein, a "charged" amino acid is an amino acid having a side
chain having a net
charge at a pH of 7.35 to 7.45. An example of a charged amino acid is
arginine.
[0580] As used herein, the term "sequence identity" refers to the percentage
of nucleic acids or
amino acids between two oligonucleotide or polypeptide sequences,
respectively, that are the same
and in the same relative position. As such, one sequence has a certain
percentage of sequence
identity compared to another sequence. For sequence comparison, typically one
sequence acts as
a reference sequence, to which test sequences are compared. Those of ordinary
skill in the art will
appreciate that two sequences are generally considered to be "substantially
identical" if they
contain identical residues in corresponding positions. In embodiments, the
sequence identity
between sequences may be determined using the Needleman-Wunsch algorithm
(Needleman and
Wunsch, 1970, 1 Mol. Biol. 48: 443-453) as implemented in the Needle program
of the EMBOSS
package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et
al., Trends
Genet.(2000), 16: 276-277), in the version that exists as of the date of
filing. The parameters used
are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62
(EMBOSS version
of BLOSUM62) substitution matrix. The output of Needle labeled "longest
identity" (obtained
using the ¨nobrief option) is used as the percent identity and is calculated
as follows: (Identical
Residues x 100)/(Length of Alignment¨Total Number of Gaps in Alignment)
[0581] In other embodiments, sequence identity may be determined using the
Smith-Waterman
algorithm, in the version that exists as of the date of filing.
[0582] As used herein, "sequence homology" refers to the percentage of amino
acids between two
polypeptide sequences that are homologous and in the same relative position.
As such one
polypeptide sequence has a certain percentage of sequence homology compared to
another
polypeptide sequence. As will be appreciated by those of ordinary skill in the
art, two sequences
are generally considered to be "substantially homologous" if they contain
homologous residues in
221
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
corresponding positions. Homologous residues may be identical residues.
Alternatively,
homologous residues may be non-identical residues with appropriately similar
structural and/or
functional characteristics. For example, as is well known by those of ordinary
skill in the art,
certain amino acids are typically classified as "hydrophobic" or "hydrophilic"
amino acids, and/or
as having "polar" or "non-polar" side chains, and substitution of one amino
acid for another of the
same type may often be considered a "homologous" substitution.
[0583] As is well known in this art, amino acid sequences may be compared
using any of a variety
of algorithms, including those available in commercial computer programs such
as BLASTP,
gapped BLAST, and PSI-BLAST, in existence as of the date of filing. Such
programs are described
in Altschul, et al., J. Mol. Biol., (1990),215(3): 403-410; Altschul, et al.,
Nucleic Acids Res.
(1997), 25:3389-3402; Baxevanis et al., Bioinformatics A Practical Guide to
the Analysis of Genes
and Proteins, Wiley, 1998; and Misener, et al., (eds.), Bioinformatics Methods
and Protocols
(Methods in Molecular Biology, Vol. 132), Humana Press, 1999. In addition to
identifying
homologous sequences, the programs mentioned above typically provide an
indication of the
degree of homology.
[0584] As used herein, "cell targeting moiety" refers to a molecule or
macromolecule that
specifically binds to a molecule, such as a receptor, on the surface of a
target cell. In embodiments,
the cell surface molecule is expressed only on the surface of a target cell.
In embodiments, the cell
surface molecule is also present on the surface of one or more non-target
cells, but the amount of
cell surface molecule expression is higher on the surface of the target cells.
Examples of a cell
targeting moiety include, but are not limited to, an antibody, a peptide, a
protein, an aptamer, or a
small molecule.
[0585] As used herein, the terms "antisense compound" and "AC" are used
interchangeably to
refer to a polymeric nucleic acid structure which is at least partially
complementary to a target
nucleic acid molecule to which it (the AC) hybridizes. The AC may be a short
(in embodiments,
less than 50 bases) polynucleotide or polynucleotide homologue that includes
at least a portion of
a sequence complimentary to a target sequence. In embodiments, the AC is a
polynucleotide or
polynucleotide homologue that includes a portion that has a sequence
complimentary to a target
sequence in a target pre-mRNA strand. The AC may be formed of natural nucleic
acids, synthetic
nucleic acids, nucleic acid homologues, or any combination thereof In
embodiments, the AC
includes oligonucleosides. In embodiments, AC includes antisense
oligonucleotides. In
222
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
embodiments, the AC includes conjugate groups. Nonlimiting examples of ACs
include, but are
not limited to, primers, probes, antisense oligonucleotides, external guide
sequence (EGS)
oligonucleotides, siRNAs, oligonucleotides, oligonucleosides, oligonucleotide
analogs,
oligonucleotide mimetics, and chimeric combinations of these. As such, these
compounds can be
introduced in the form of single-stranded, double-stranded, circular, branched
or hairpins and can
contain structural elements such as internal or terminal bulges or loops.
Oligomeric double-
stranded compounds can be two strands hybridized to form double-stranded
compounds or a single
strand with sufficient self-complementarity to allow for hybridization and
formation of a fully or
partially double-stranded compound. In embodiments, an AC modulates
(increases, decreases, or
changes) the expression, levels, and/or activity of a target transcript (e.g.,
target nucleic acid). In
embodiments, the AC decreases the level of the target transcript through
inducing decay
mechanisms. In embodiments, the AC modulates the activity of the target
transcript. In
embodiments, the AC modulates the activity of the target transcript by
decreasing the ability of
the target transcript to bind one or more proteins. In embodiments, decreasing
the affinity between
the target transcript and the one or more proteins may result in the
modulation of the activity of
the one or more proteins. For example, if the one or more proteins are not
bound to the target
transcript, they are available to carry out their functions, such as, for
example, facilitating the
splicing, alternative splicing, and/or exon skipping of other transcripts
(downstream transcripts).
As such, AC mediated modulation of the activity of the target transcript may
result in modulation
of the activity, expression, and/or levels of the downstream genes that are
regulated by the one or
more proteins whose interaction with the target transcript may be disrupted.
[0586] As used herein, the terms "targeting" or "targeted to" refer to the
association of a
therapeutic moiety, for example, an antisense compound with a target nucleic
acid molecule or a
region of a target nucleic acid molecule. In embodiments, the therapeutic
moiety includes an
antisense compound that is capable of hybridizing to a target nucleic acid
under physiological
conditions. 111 embodiments, the antisense cornpound targets a spedti c
portion or site within the
target nucleic acid, for example, a portion of the target nucleic acid having
at least one identifiable
structure, function, or characteristic such as a particular eXOfl or intron,
or selected nucleobases or
motifs within an exon or introit
[0587] As used herein, the terms "target nucleic acid sequence," "target
nucleotide sequence," and
"target sequence" refer to the nucleic acid sequence or the nucleotide
sequence to which a
223
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
therapeutic moiety, such as an anti sense compound, binds or hybridizes.
Target nucleic acids
include, but are not limited, to a portion of a target transcript, target RNA
(including, but not
limited to pre-mRNA and mRNA or portions thereof), a portion of target cDNA
derived from such
RNA, as well as a portion of target non-translated RNA, such as miRNA. For
example, in
embodiments, a target nucleic acid can be a portion of a target cellular gene
(or mRNA transcribed
from such gene) whose expression or transcription is associated with a
particular disorder or
disease state. The term "portion" refers to a defined number of contiguous
(i.e., linked) nucleotides
of a nucleic acid.
[0588] As used herein, the term "transcript" or "gene transcript" refers to an
RNA molecule
transcribed from DNA and includes, but is not limited to mRNA, pre-mRNA, and
partially
processed RNA.
[0589] The terms "target transcript" and "target RNA" refer to the pre-mRNA or
mRNA transcript
that is bound by the therapeutic moiety. The target transcript may include a
target nucleotide
sequence. In embodiments, the target transcript includes a target nucleotide
sequence that includes
an expanded CUG trinucleotide repeat.
[0590] The term "target gene" and "gene of interest" refer to the gene of
which modulation of the
expression and/or activity is desired or intended. The target gene may be
transcribed into a target
transcript that includes a target nucleotide sequence. The target transcript
may be translated into a
protein of interest.
[0591] The term "target protein" refers to the polypeptide or protein encoded
by the target
transcript (e.g., target mRNA).
[0592] As used herein, the term "mRNA" refers to an RNA molecule that encodes
a protein and
includes pre-mRNA and mature mRNA. "Pre-mRNA" refers to a newly synthesized
eukaryotic
mRNA molecule directly after DNA transcription. In embodiments, a pre-mRNA is
capped with
a 5' cap, modified with a 3' poly-A tail, and/or spliced to produce a mature
mRNA sequence. In
embodiments, pre-mRNA includes one or more introns. In embodiments, the pre-
mRNA
undergoes a process known as splicing to remove introns and join exons. In
embodiments, pre-
mRNA includes one or more splicing elements or splice regulatory elements. In
embodiments,
pre-mRNA includes a polyadenylation site.
[0593] As used herein, the term "expression," "gene expression," "expression
of a gene," or the
like refers to all the functions and steps by which information encoded in a
gene is converted into
224
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
a functional gene product, such as a polypeptide or a non-coding RNA, in a
cell. Examples of non-
coding RNA include transfer RNA (tRNA) and ribosomal RNA. Gene expression of a
polypeptide
includes transcription of the gene to form a pre-mRNA, processing of the pre-
mRNA to form a
mature mRNA, translocating the mature mRNA from the nucleus to the cytoplasm,
translation of
the mature mRNA into the polypeptide, and assembly of the encoded polypeptide.
Expression
includes partial expression. For example, expression of a gene may be referred
to as generation of
a gene transcript. Translation of a mature mRNA may be referred to as
expression of the mature
mRNA.
[0594] As used herein, "modulation of gene expression" or the like refers to
modulation of one or
more of the processes associated with gene expression. For example,
modification of gene
expression may include modification of one or more of gene transcription, RNA
processing, RNA
translocation from the nucleus to the cytoplasm, and translation of mRNA into
a protein.
[0595] As used herein, the term "gene" refers to a nucleic acid sequence that
encompasses a 5'
promoter region associated with the expression of the gene product, and any
intron and exon
regions and 3 untransiated regions ("UM") associated with the expression of
the gene product.
[0596] The term "immune cell" refers to a cell of hematopoietic origin and
that plays a role in the
immune response. Immune cells include, but are not limited to, lymphocytes
(e.g., B cells and T
cells), natural killer (NK) cells, and myeloid cells. The term "myeloid cells"
includes monocytes,
macrophages and granulocytes (e.g., basophils, neutrophils, eosinophils and
mast cells).
Monocytes are lymphocytes that circulate through the blood for 1-3 days, after
which time, they
either migrate into tissues and differentiate into macrophages or inflammatory
dendritic cells or
die. The term "macrophage" as used herein includes fetal-derived macrophages
(which also can
be referred to as resident tissue macrophages) and macrophages derived from
monocytes that have
migrated from the bloodstream into a tissue in the body (which can be referred
to as monocyte-
derived macrophages). Depending on which tissue the macrophage is located, it
be referred to as
a Kupffer cell (liver), an intraglomular mesangial cell (kidney), an alveolar
macrophage (lungs), a
sinus histiocyte (lymph nodes), a hofbauer cell (placenta), microglia (brain
and spinal cord), or
langerhans (skin), among others.
[0597] As used herein, the term "oligonucleotide" refers to an oligomeric
compound comprising
a plurality of linked nucleotides or nucleosides. One or more nucleotides of
an oligonucleotide can
be modified. An oligonucleotide can comprise ribonucleic acid (RNA) or
deoxyribonucleic acid
225
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
(DNA). Oligonucleotides can be composed of natural and/or modified
nucleobases, sugars and
covalent internucleoside linkages, and can further include non-nucleic acid
conjugates.
[0598] As used herein, the term "nucleoside" refers to a glycosylamine that
includes a nucleobase
and a sugar. Nucleosides include, but are not limited to, natural nucleosides,
abasic nucleosides,
modified nucleosides, and nucleosides having mimetic bases and/or sugar
groups. A "natural
nucleoside" or "unmodified nucleoside" is a nucleoside that includes a natural
nucleobase and a
natural sugar. Natural nucleosides include RNA and DNA nucleosides.
[0599] As used herein, the term "natural sugar" refers to a sugar of a
nucleoside that is unmodified
from its naturally occurring form in RNA (2'-OH) or DNA (2'-H).
[0600] As used herein, the term "nucleotide" refers to a nucleoside having a
phosphate group
covalently linked to the sugar. Nucleotides may be modified with any of a
variety of substituents.
[0601] As used herein, the term "nucleobase" refers to the base portion of a
nucleoside or
nucleotide. A nucleobase may include any atom or group of atoms capable of
hydrogen bonding
to a base of another nucleic acid. A natural nucleobase is a nucleobase that
is unmodified from its
naturally occurring form in RNA or DNA.
[0602] As used herein, the term "heterocyclic base moiety" refers to a
nucleobase that includes a
heterocycle.
[0603] As used herein "internucleoside linkage" refers to a covalent linkage
between adjacent
nucleosides.
[0604] As used herein "natural internucleoside linkage" refers to a 3' to 5'
phosphodiester linkage.
[0605] As used herein, the term "modified internucleoside linkage" refers to
any linkage between
nucleosides or nucleotides other than a naturally occurring internucleoside
linkage.
[0606] As used herein "oligonucleoside" refers to an oligonucleotide in which
the internucleoside
linkages do not contain a phosphorus atom.
[0607] As used herein the term "chimeric antisense compound" refers to an
antisense compound,
having at least one sugar, nucleobase, and/or internucleoside linkage that is
differentially modified
as compared to the other sugars, nucleobases, and internucleoside linkages
within the same
oligomeric compound. The remainder of the sugars, nucleobases, and
internucleoside linkages can
be independently modified or unmodified. In general, a chimeric oligomeric
compound will have
modified nucleosides that can be in isolated positions or grouped together in
regions that will
226
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
define a particular motif. Any combination of modifications and or mimetic
groups can include a
chimeric oligomeric compound as described herein.
[0608] As used herein, the term "mixed-backbone antisense oligonucleotide"
refers to an antisense
oligonucleotide wherein at least one internucleoside linkage of the antisense
oligonucleotide is
different from at least one other internucleoside linkage of the antisense
oligonucleotide.
[0609] As used herein, the term "nucleobase complementarity" refers to a
nucleobase that is
capable of base pairing with another nucleobase. For example, in DNA, adenine
(A) is
complementary to thymine (T). For example, in RNA, adenine (A) is
complementary to uracil (U).
In embodiments, complementary nucleobase refers to a nucleobase of an
antisense compound that
is capable of base pairing with a nucleobase of its target nucleic acid. For
example, if a nucleobase
at a certain position of an antisense compound is capable of hydrogen bonding
with a nucleobase
at a certain position of a target nucleic acid, then the position of hydrogen
bonding between the
oligonucleotide and the target nucleic acid is considered to be complementary
at that nucleobase
pair.
[0610] As used herein, the term "non-complementary nucleobase" refers to a
pair of nucleobases
that do not form hydrogen bonds with one another or otherwise support
hybridization.
[0611] As used herein, the term "complementary" refers to the capacity of an
oligomeric
compound to hybridize to another oligomeric compound or nucleic acid through
nucleobase
complementarity. In embodiments, an antisense compound and its target are
complementary to
each other when a sufficient number of corresponding positions in each
molecule are occupied by
nucleobases that can bond with each other to allow stable association between
the antisense
compound and the target. One skilled in the art recognizes that the inclusion
of mismatches is
possible without eliminating the ability of the oligomeric compounds to remain
in association.
Therefore, described herein are antisense compounds that may include up to
about 20%
nucleotides that are mismatched (i.e., are not nucleobase complementary to the
corresponding
nucleotides of the target). In embodiments, the antisense compounds contain no
more than about
15%, for example, not more than about 10%, for example, not more than 5%, or
no mismatches.
The remaining nucleotides are nucleobase complementary or otherwise do not
disrupt
hybridization (e.g., universal bases). One of ordinary skill in the art would
recognize the
compounds provided herein are at least 80%, at least 85%, at least 90%, at
least 95%, at least 96%,
227
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
at least 97%, at least 98%, at least 99%, or 100% nucleobase complementary to
a target nucleic
acid.
[0612] As used herein, "hybridization" means the pairing of complementary
oligomeric
compounds (e.g., an antisense compound and its target nucleic acid). While not
limited to a
particular mechanism, the most common mechanism of pairing involves hydrogen
bonding, which
may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between
complementary nucleoside or nucleotide bases (nucleobases). For example, the
natural base
adenine is nucleobase complementary to the natural nucleobases thymine and
uracil which pair
through the formation of hydrogen bonds. The natural base guanine is
nucleobase complementary
to the natural bases cytosine and 5-methyl cytosine. Hybridization can occur
under varying
circumstances.
[0613] As used herein, the term "specifically hybridizes" refers to the
ability of an oligomeric
compound to hybridize to one nucleic acid site with greater affinity than it
hybridizes to another
nucleic acid site. In embodiments, an antisense oligonucleotide specifically
hybridizes to more
than one target site. In embodiments, an oligomeric compound specifically
hybridizes with its
target under stringent hybridization conditions.
[0614] "Stringent hybridization conditions" and "stringent hybridization wash
conditions" in the
context of nucleic acid hybridization are sequence dependent and are different
under different
environmental parameters. An extensive guide to the hybridization of nucleic
acids is found in
Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology-
Hybridization with
Nucleic Acid Probes part I chapter 2 "Overview of principles of hybridization
and the strategy of
nucleic acid probe assays" Elsevier, New York (1993). Generally, highly
stringent hybridization
and wash conditions are selected to be about 5 C lower than the thermal
melting point (Tm) for
the specific sequence at a defined ionic strength and pH. The Tm is the
temperature (under defined
ionic strength and pH) at which 50% of the target sequence hybridizes to a
perfectly matched
probe. Very stringent conditions are selected to be equal to the Tm for a
particular probe. An
example of stringent hybridization conditions for hybridization of
complementary nucleotide
sequences which have more than 100 complementary residues on a filter in a
Southern or Northern
blot is 50% formamide with 1 mg of heparin at 42 C, with the hybridization
being carried out
overnight. An example of highly stringent wash conditions is 0.15M NaCl at 72
C for about 15
minutes. An example of stringent wash conditions is a 0.2x SSC wash at 65 C
for 15 minutes (see,
228
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
Sambrook and Russel, Molecular Cloning: A laboratory Manual, 3rd ed., Cold
Spring Harbor
Laboratory Press, 2001 for a description of SSC buffer). Often, a high
stringency wash is preceded
by a low stringency wash to remove background probe signal. An example of a
medium stringency
wash for a duplex of, e.g., more than 100 nucleotides, is lx SSC at 45 C for
15 minutes. An
example of a low stringency wash for a duplex of, e.g., more than 100
nucleotides, is 4-6x SSC at
40 C for 15 minutes. For short probes (e.g., about 10 to 50 nucleotides),
stringent conditions
typically involve salt concentrations of less than about 1.0 M Na ion,
typically about 0.01 to 1.0
M Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature
is typically at least
about 30 C. Stringent conditions can also be achieved with the addition of
destabilizing agents
such as formamide.
[0615] As used herein, the term "2'-modified" or "2'-substituted" means a
sugar that includes
substituent at the 2' position other than H or OH. 2'-modified monomers,
include, but are not
limited to, BNA's and monomers (e.g., nucleosides and nucleotides) with 2'-
substituents, such as
allyl, amino, azido, thio, 0-allyl, 0-C1-C10 alkyl, -0CF3, 0-(CH2)2-0-CH3, 2'-
0(CH2)25CH3,
0-(CH2)2-0-N(Rm)(Rn), or 0-CH2-C(=0)-N(Rm)(Rn), where each Rm and Rn is,
independently, H or substituted or unsubstituted C1-C10 alkyl.
[0616] As used herein, the term "MOE" refers to a 2'-0-methoxyethyl
substituent.
[0617] As used herein, the term "high-affinity modified nucleotide" refers to
a nucleotide having
at least one modified nucleobase, internucleoside linkage or sugar moiety,
such that the
modification increases the affinity of an antisense compound that includes the
modified nucleotide
to a target nucleic acid. High-affinity modifications include, but are not
limited to, BNAs, LNAs
and 2'-M0E.
[0618] As used herein the term "mimetic" refers to groups that are substituted
for a sugar, a
nucleobase, and/ or internucleoside linkage in an AC. Generally, a mimetic is
used in place of the
sugar or sugar-internucleoside linkage combination, and the nucleobase is
maintained for
hybridization to a selected target. Representative examples of a sugar mimetic
include, but are not
limited to, cyclohexenyl or morpholino. Representative examples of a mimetic
for a sugar-
internucleoside linkage combination include, but are not limited to, peptide
nucleic acids (PNA)
and morpholino groups linked by uncharged achiral linkages. In some instances,
a mimetic is used
in place of the nucleobase. Representative nucleobase mimetics are well known
in the art and
include, but are not limited to, tricyclic phenoxazine analogs and universal
bases (Berger et al.,
229
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
Nuc Acid Res. 2000, 28:2911-14, incorporated herein by reference). Methods of
synthesis of
sugar, nucleoside, and nucleobase mimetics are well known to those skilled in
the art.
[0619] As used herein, the term "bicyclic nucleoside" or "BNA" refers to a
nucleoside wherein the
furanose portion of the nucleoside includes a bridge connecting two atoms on
the furanose ring,
thereby forming a bicyclic ring system. BNAs include, but are not limited to,
a-L-LNA, f3-D-LNA,
ENA, Oxyamino BNA (2'-0-N(CH3)-CH2-4') and Aminooxy BNA (2'-N(CH3)-0-CH2-4').
[0620] As used herein, the term "4' to 2' bicyclic nucleoside" refers to a BNA
wherein the bridge
connecting two atoms of the furanose ring bridges the 4' carbon atom and the
2' carbon atom of
the furanose ring, thereby forming a bicyclic ring system.
[0621] As used herein, a "locked nucleic acid" or "LNA" refers to a nucleotide
modified such that
the 2'-hydroxyl group of the ribosyl sugar ring is linked to the 4' carbon
atom of the sugar ring via
a methylene group, thereby forming a 2'-C,4'-C-oxymethylene linkage. LNAs
include, but are not
limited to, a-L-LNA, and f3-D-LNA.
[0622] As used herein, the term "cap structure" or "terminal cap moiety"
refers to chemical
modifications, which have been incorporated at either end of an AC.The term
"therapeutic
polypeptide" refers to a naturally occurring or recombinantly produced
macromolecule that
includes two or more amino acids and has therapeutic, prophylactic or other
biological activity.
[0623] The term "small molecule" refers to an organic compound with
pharmacological activity
and a molecular weight of less than about 2000 Daltons, or less than about
1000 Daltons, or less
than about 500 Daltons. Small molecule therapeutics are typically manufactured
by chemical
synthesis.
[0624] "Wild type target protein" refers to a native, functional protein
isomer produced by a wild
type, normal, or unmutated version of the target gene. The wild type target
protein also refers to a
protein resulting from a target pre-mRNA that has been re-spliced.
[0625] A "re-spliced target protein", as used herein, refers to the protein
encoded by the mRNA
resulting from the splicing of the target pre-mRNA to which the AC hybridizes.
Re-spliced target
protein may be identical to a wild type target protein, may be homologous to a
wild type target
protein, may be a functional variant of a wild type target protein, may be an
isoform of a wild type
target protein, or may be an active fragment of a wild type target protein.
[0626] As used herein, an "expanded trinucleotide repeat," such as an
"expanded" CUG or and
"expanded" CTG repeat, means a gene containing or encoding the trinucleotide
repeat contains a
230
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
number of repeated consecutive trinucleotides that is greater than present in
a wild type gene.
Expanded nucleotide repeats may be written as XXX=NNN or (XXX=NNN) where XXX
refers to
the DNA repeat and NNN refers to the RNA repeat that is transcribed from the
DNA repeat. For
example, the CTG=CUG repeat, refers to a gene having a CTG DNA repeat from
which a RNA
having a CUG repeat is transcribed. In embodiments, the number of repeats in
an expanded
trinucleotide repeat is 5 or more, 10 or more 15, or more or 20 or more than
the wild type gene. In
embodiments, the expanded trinucleotide repeat includes 2x, 3x, 4x, 5x, 10x,
20x, 50x or more
trinucleotide repeats than the wild type gene. The expanded trinucleotide
repeat may result in a
disease in a subject having a gene that contains the expanded trinucleotide
repeat. For example, a
subject having an expanded CTG repeat in a gene may suffer from DM1 or FECD.
In DM1, the
DPMK gene contains an expanded CTG repeat. Subjects that suffer from DM1 may
have 50 or
more CTG repeats in the 3' untranslated region (UTR) of the DPMK gene, while
non-disease
subjects typically have 5 to 34 CTG repeats in the 3' UTR of the DPMK gene. In
FECD, the TCF4
gene contains an expanded CTG repeat. Subjects that suffer from FECD may have
40 or more
CTG repeats in a CTG18.1 locus of the TCF4 gene, while non-disease subjects
typically have 30
or less CTG repeats in the CTG18.1 locus of the TCF4 gene. mRNA transcribed
from a gene
having an expanded CTG repeat will have an expanded CUG repeat.
[0627] The term "downstream" in the present disclosure, as it relates to a
gene, mRNA, or protein,
refers to a gene, mRNA, or protein that is affected by binding of AC to the
target nucleotide (e.g.,
target transcript) but is not the gene, mRNA, or protein corresponding to the
target nucleotide.
Binding of the AC to the target nucleotide may reduce aggregation or
sequestration of RNA
binding protein such as MBNL1 or CUGBP1 on accumulated mRNA having CUG
repeats, which
may make available such RNA binding proteins for proper transcription, RNA
processing, and/or
expression of downstream gene products.
[0628] As used herein, "functional fragment" or "active fragment" refers to a
portion of a
eukaryotic wild type target protein that exhibits an activity, such as one or
more activities of a full-
length wild type target protein, or that possesses another activity. In
embodiments, a re-spliced
target protein that shares at least one biological activity of wild type
target protein is considered to
be an active fragment of the wild type target protein. Activity can be any
percentage of activity
(i.e., more or less) of the full-length wild type target protein, including
but not limited to, about
1% of the activity, about 2%, about 3%, about 4%, about 5%, about 10%, about
20%, about 30%,
231
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%,
about 96%,
about 97%, about 98%, about 99%, about 100%, about 200%, about 300%, about
400%, about
500%, or more (including all values and ranges in between these values)
activity compared to the
wild type target protein. Thus, in embodiments, the active fragment may retain
at least a portion
of one or more biological activities of wild type target protein. In
embodiments, the active fragment
may enhance one or more biological activities of wild type target protein.
[0629] "Wild type target protein" refers to a native, functional protein
isomer produced by a wild
type, normal, or unmutated version of the target gene. The wild type target
protein also refers to
the protein resulting from a target pre-mRNA that has been properly spliced.
[0630] As used herein, the terms "splicing" and "processing" refer to the
modification of a pre-
mRNA following transcription, in which introns are removed and exons are
joined. Splicing occurs
in a series of reactions that are catalyzed by a large RNA-protein complex
composed of five small
nuclear ribonucleoproteins (snRNPs) referred to as a spliceosome. Within an
intron, a 3' splice
site, a 5' splice site, and a branch site are required for splicing. The RNA
components of snRNPs
interact with the intron and may be involved in catalysis.
[0631] As used herein, al Lernative splicing refers to the splicing of
different combinations of exons
present in a gene, which rest d ts in the generation of different rn RNA
transcripts from a single gene.
[0632] A "re-spliced target protein", as used herein, refers to the protein
encoded by the mRNA
resulting from the splicing of the target pre-mRNA to which the AC hybridizes.
Re-spliced target
protein may be identical to a wild type target protein, may be homologous to a
wild type target
protein, may be a functional variant of a wild type target protein, or may be
an active fragment of
a wild type target protein.
[0633] All publications, patents and patent applications mentioned in the
specification are
indicative of the level of skill of those skilled in the art to which this
invention pertains. All
publications, patents and patent applications are herein incorporated by
reference to the same
extent as if each individual publication or patent application was
specifically and individually
indicated to be incorporated by reference.
232
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
EXAMPLES
Example 1. Assessment of PM0s and PMO-EEV compounds for impacts on RNA foci
formation and splicing rescue in DM1-related cell lines
[0634] The effect of a (CUG)7 repeat PM0 and a (CUG)7 repeat PMO-EEV compound
(A and D
in Table 12) on RNA foci formation and splicing rescue was evaluated in DM1-
related cell lines.
A PMO-EEV compound having a mismatched PM0 sequence and a PM0 having a
scrambled
sequence were also included (B and C in Table 12).
[0635] Experimental
[0636] Cells. The PM0s and PM0-EEVs were evaluated in a DM1 HeLa cell model
(HeLa-480),
which is a stable cell line with high CUG repeat load and downstream splicing
defects, and DM1
myoblasts derived from a DM1 patient with about 2600 CTG repeats and
downstream splicing
defects. HeLa-480 recapitulates pathogenic hallmarks of DM1, including CUG
ribonuclear foci
and mis-splicing of pre-mRNA targets of the muscleblind (MBNL) alternative
splicing factors. It
was noted that DM1 myoblasts grow quite slowly (doubling time of about 7 days)
and do not
transfect well. Control HeLa and HeLa-480 cells were treated with ENDOPORTER
without
additional compounds. HeLa-480 cells represent the disease state and HeLa
cells represent the un-
diseased state.
[0637] RNA foci analysis. HeLa-480 cells or DM1 myoblasts were treated with 1
tM, 3 tM, or
i.tM of compounds A-D (Table 12). All compounds were transfected without a
transfection
reagent or using the ENDOPORTER (available from GENETOOLS LLC in Philomath,
Oregon)
transfection agent designed to deliver naturally charged PM0s into cells.
Cells were incubated for
24 hours and then fixed for qualitative RNA foci analysis via microscopy. Upon
qualitative visual
inspection of the treated Hela-480 cells, compound A (PMO-EEV) showed the
least amount of
RNA foci. No conclusion was drawn from the DM1 myoblasts.
[0638] Splicing analysis and RT-PCR. HeLa-480 cells treated as described above
were harvested
after 24 or 48 hours of the incubation and the total RNA was extracted. RT-PCR
was performed
to measure and/or quantify splicing patterns of DM-affected exons in target
RNAs such as MBNL1
and CLASP1. The percentage of exon inclusion of interest was evaluated.
Table 12: PM0s and PM0-EEVs tested in Example 1.
ID Sequence (all PMO) Purity Solvent
233
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
CAGCAGCAGCAGCAGCAGCAG-click-K-PEG12-
K(Ff(WrRrQ)-PKKKRKV-Ac
A 91 Saline (0.9%
NaCl)
(SEQ ID NO: 154)-click-K-PEG12-(SEQ ID NO:78)-(SEQ ID
NO: 42)-Ac
GTAACTGTATTTGGTACTTCC-C3-NH2-PEG4-COT-
PEG12-PKKKRKV-(Ff(toRrRrQ)
99 PBSX1
(SEQ ID NO: 317)-C3-NH2-PEG4-COT-PEG12-(SEQ ID NO:
78)-(SEQ ID NO:42)-Ac
C AGCCAGAGCACCGCAACCGGACGAG (SEQ ID NO: 318) 81 Saline (0.9%
NaCl)
D CAGCAGCAGCAGCAGCAGCAG (SEQ ID NO: 154) 75 Saline (0.9%
NaCl)
[0639] Results. FIG. 6A-6D show the RT-PCR results of the alternative splicing
events of
MBNL1 (6A and 6C, exon 5 inclusion) and CLASP1 (6B and 6D, exon 19 inclusion)
24 hours
(6A and 6B) and 48 hours (6C and 6D) after HeLa-480 cells were treated with
compounds A-D
and the ENDOPORTER transfection agent. A reduction of exon 5 inclusion in
MBNL1 was
observed for cells treated with each of compounds A-D at both the 24- and 48-
hour time points
(FIG. 6A and 6C). Cells treated with compound A showed the largest rescue
(decrease in exon 5
inclusion). An increase in exon 19 inclusion in CLASP1 was observed for cells
treated with each
of compounds A-D at both the 24- and 48-hour time points (FIG. 6B and 6D).
Cells treated with
compound A showed the largest rescue (increase in exon 19 inclusion). In
contrast, when HeLa-
480 cells were treated with A-D without the ENDOPORTER transfection reagent no
change in the
splicing events of MBNL1 (FIG. 6E) or CLASP1 (FIG. 6F) were observed, except
for compound
A.
[0640] FIG. 7A-7B show the RT-PCR results of alternative splicing events of
MBNL1 and
CLASP1 after DM1 myoblasts were treated with compound A-D, the negative
control DM-04, or
the positive control DM-05. Treatment with all the compounds resulted in the
rescue of the splicing
events of MBNL1 (FIG. 7A) and CLASP1 (FIG. 7B).
Example 2. Assessment of EEV-PMO 221-1106 for impacts on splicing rescue in
DM1-
mouse model
[0641] The effect of a PM0 only and a PMO-EEV (Table 13) on splicing rescue
was evaluated
in vivo using an HSA-LR DM1-mouse model.
234
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
[0642] Experimental.
[0643] Mouse Model. HSA-LR is a transgenic mouse model having expanded long
CUG repeats
(LR) in the 3'-UTR of a human skeletal actin (HSA) transgene and expresses
CUGexp RNA (e.g.,
expanded CUG RNA) at high levels in skeletal muscle (Mankodi et al., Science
2000,
289(5485):1769-1773). The HSA-LR mouse shows myotonic phenotype along with
splicing
defects. A Friend Virus B NIH Jackson (FVB/NJ) mouse model was used as a
control and to make
the HSA-LR transgenic mice.
[0644] Experimental design. Compounds A-D were administered to the mice via
Retro-orbital
injection or intravenous (IV) injection at a single dose of 100 tL compound
solution per 20 g body
weight. The scale was proportional to body weight of each mouse (e.g., 150 tL
per 30 g body
weight).
Table 13: PM0s and PM0-EEVs tested in Example 2.
ID Compound Sequence (all PM0s)
Con.Solvent
(mg/ml)
Saline
(A) Saline Vehicle
control NA (0.9%
NaCl)
5'-CAG CAG CAG CAG CAG CAG CAG- Saline
6 (0.9%
(B) 221
3' (SEQ ID NO: 154)
_________________________________________________________________ NaC1)
5'-CAG CAG CAG CAG CAG CAG CAG-3'-
PEG4COT -click-K-miniPEG2-
221-1106 Lys(FfFGRGRE)-miniPEG2-VKRKKKP-Ac
Saline
(C) 4 (0.9%
at 20 mpk
(SEQ ID NO: 154)-PEG4COT-click-K- NaCl)
miniPEG2-K(SEQ ID NO: 76)-miniPEG2-
(SEQ ID NO: 42)-Ac
5'-CAG CAG CAG CAG CAG CAG CAG-3'-
PEG4COT-click-K-miniPEG2-
221 - 1106 Lys(FfFGRGRE)-miniPEG2-VKRKKKP-AC
Saline
(D) 8 (0.9%
at 40 mpk
(SEQ ID NO: 154)-PEG4COT-click-K- NaCl)
miniPEG2-K(SEQ ID NO: 76)-miniPEG2-
(SEQ ID NO: 42)-Ac
235
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
[0645] Animals were age matched and assigned into six treatment groups. Two
control groups
were used; Group 1: FVB/NJ mice (FVB/NJ; un-diseased control) and Group 2: HSA-
LR
(diseased control) mice, each of which were injected with saline. Four
treatment groups (Groups
A-D) were used; HSA-LR mice injected with compounds A, B, C, or D. The FVB/NJ
mice were
weeks and 4 days old when injected, while the HSA-LR mice were 6 weeks and 1
or 2 days old
when injected. Four mice (two males and two females) per group were utilized
for this experiment.
Mice were sacrificed 1 week post treatment. Tissues (Gastrocnemius,
Quadriceps, Tibialis
Anterior (TA)) were harvested and flash frozen in liquid nitrogen and stored
at -80 C for further
evaluation of splicing rescue analysis.
[0646] Total RNA was extracted from tissue samples and analyzed by RT-PCR to
assess AC-
induced alternative RNA splicing rescue events on (i) Atp2a1 exon 22, (ii)
Nfix exon 7, (iii) Clcn1
exon 7a, and (iv) Mbnll exon 5. The percentage of exon inclusion of interest
was evaluated.
[0647] Results. Prior to treatment all HSA-LR mice had myotonia. After
injection of compounds,
all mice were disoriented. Mice injected with compounds A and B (Groups A and
B) recovered
within 15 minutes, while mice injected with compounds C and D (Groups C and D)
took several
hours to recover. All treated mice completely recovered by the next day. At
time of sacrifice,
Groups A and B had myotonia; however, Groups C and D clearly did not have
myotonia.
[0648] FIGs. 8A-10D show the RNA splicing measurements for Atp2a1 (for exon 22
inclusion;
FIGs. 8A, 9A, and 10A), Nfix (for exon 7 inclusion; FIG. 8B, 9B, and 10B),
Clcn1 (for exon 7a
inclusion; FIG. 8C, 9C, and 10C) and Mbnll (for exon 5 inclusion; FIG. 8D, 9D,
and 10D) in
gastrocnemius (FIGs. 8A-8D), quadricep (FIG. 9A-9D), and tibialis anterior
(FIG. 10A-10D)
muscle tissue of treated mice. Mice treated with compounds C and D (PMO-EEV)
showed a rescue
of Atp2a1 and Nfix splicing events in gastrocnemius, quadriceps, and tibialis
anterior tissue while
the PM0 and saline groups did not show rescue of splicing events (FIG. 8A, 8B,
9A, 9B, 10A,
and 10B. Similarly, in the gastrocnemius and quadricep muscle tissue, mice
treated with
compounds C and D showed a rescue of Clcn1 (FIG. 8C and 9C) and Mbnll (FIG. 8D
and 9D)
splicing events while the PM0 and saline groups did not show rescue of
splicing events. Regarding
the splicing of Clcn1 and Mbnll in the tibialis tissue (FIG. 10C-10D), no
alternative splicing
defects were detected in the control mouse line (FVB/NJ) and the DM1 mouse
model (HSA-LR),
As such, treatment with compounds A-D did not result in splicing rescue in the
Clcn1 and Mbnll
genes in the tibialis tissue.
236
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
[0649] These results demonstrate that positive impacts of PMO-EEV treatment on
splicing rescue
in vivo study using DM1 mouse model as well as potential use of PMO-EEV
compounds to treat
myotonic dystrophy (DM).
Example 3. Assessment of various PMO-EEV compounds for correcting mis-splicing
events
in Immortalized Myoblasts from DM1 patients
[0650] The effect of two DMPK CUG-targeting PM0-EEVs (197-777 and 221-1106;
Table 14)
on splicing rescue of DM1-related genes was evaluated in vitro using DM1
patient derived muscle
myoblasts and myotubes.
[0651] Experimental.
[0652] Cell culture. Immortalized myoblasts from DM1 patients (ASA308DM1), and
unaffected
individuals (KM1421; AB1190) were obtained. DM1 patient myoblasts harbor 2600
CTG repeats
in the 3'-UTR of DMPK. Myoblasts were cultured in a growth medium of Skeletal
Muscle Cell
Growth Medium (available from PromoCell in Heidelberg, Germany), 2% horse
serum (available
from Gibco in Bristol, RI), 1% chick embryo extract (available from USB Corp
in Cleveland, OH),
and 0.5 mg/mL penicillin/streptomycin (Gibco). For myogenic differentiation,
confluent cultures
were switched to differentiation medium of DMEM supplemented with 2% horse
serum and
cultured for four days.
Table 14: Compounds tested in Example 3
Compound EEV Sequence (N to C) PM0 sequence PM0 Conjugation
ID (5'-3') modifications
Chemistry
CAGCAGCA
GCAGCAGC
AGCAG
PM0 only NA 5' OH NA
(SEQ ID NO:
154)
Ac-PKKKRKV-
CAGCAGCA
Lys(cyclo[Ff-Nal-RrRrQ])-
GCAGCAGC
PEG12-K(N3)-NH2 5' -sarcosine
197-777 AGCAG
(DM1-1) amide; 3'-C4- Click
Ac-(SEQ ID NO: 42)-- cyclooctyne
(SEQ ID NO:
Lys(SEQ ID NO: 78)-
154)
PEG12-K(N3)-NH2
Ac-PKKKRKV-miniPEG2- CAGCAGCA
5'-OH; 3'- ?Click with
221-1106 Lys(cyclo[FfFGRGRQ]- GCAGCAGC
secondary amine a PEG4-
(3M1-2) miniPEG2-K(N3)-NH2 AGCAG
morpholino COT linker
237
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
Ac-(SEQ ID NO: 42)- (SEQ ID NO:
miniPEG2-Lys(SEQ ID 154)
NO: 76)-miniPEG2-
K (N3 )NH2
[0653] Treatment. DM1 patient muscle cells were treated with 10 p.m, 3 p.m, 1
p.m, or 0.3 p.m of
the compounds using two different treatment conditions. In the first
condition, myoblasts were
plated at 75-80% confluence, the compounds were serially diluted in growth
medium, and cells
were bathed for 24 hours to allow for free-uptake of compound. The compound-
containing media
was removed, myoblasts washed with lx DPBS (Gibco), and differentiated for
four days prior to
harvest. For the second condition run in parallel, myoblasts were
differentiated three days prior to
treatment, compounds were serially diluted in differentiation medium, and
myotubes were
harvested for analysis 24 hours later.
[0654] RNA isolation and PCR. Total RNA was isolated with the RNEASY Mini Kit
(available
from Qiagen in Germantown, MD) according to the manufacturer's instructions.
For exon
inclusion, 100 ng RNA was reverse transcribed and used for PCR (OneStep RT-PCR
Kit, Qiagen).
Samples were analyzed by LabChip (available from PerkinElmer in Waltham, MD)
with the HT
DNA High Sensitivity Assay Kit.
[0655] Results. DMPK CUG targeting PM0 (not conjugated with EEV) improved mis-
splicing
of 1V1BNL1 exon 5 (data not shown). FIG. 11A-11F shows mixed rescue of
splicing defect of
1V1BNL1 (FIG. 11A) and its targets (SOS1, IR, DMD, BIN1, LDB3; FIG. 11B-11F)
in DM1
patient derived muscle cells treated with various concentrations of DMPK CUG-
targeting EEV-
PM0s (CUGexP 197-777 and CUGexP 221-1106). EEV-PMO 197-777 elicited moderate
correction
of mis-splicing events in DM1 patient muscle cells. MBNL1 and SOS1 showed the
best response
of mis-splicing correction. EEV-PMO 197-777 chosen as tool compound for follow-
up
experiments described below.
[0656] DM1 patient derived myoblasts and myotubes were treated with 10 p.m, 3
p.m, and 1 p.m
of DMPK CUG-targeting EEV-PMO 197-777 using methods similar to those described
above.
Rescue of alternative RNA splicing events of MNBL1 and 1V1NBL1 targets was
evaluated. Various
extendts of splicing correction was observed for 1V1NBL1 (exon 5 exclusion;
FIG. 12A), SOS1
(exon 25 inclusion; FIG. 12B), INSR (exon 11 inclusion; FIG. 12C), DMD (exon
78 inclusion,
238
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
FIG. 12D), BIN1(exon 11 inclusion; FIG. 12E), and LDB3 (exon 11 exclusion;
FIG. 12F) after
myoblasts and myotubes were treated with EEV-PMO.
[0657] FIGS. 44A-44D show the reversal of myotonia phenotypes in HSA-LR mice
treated with
20 mpk 221-1106 quantified by muscle relaxation assay. FIG. 44A and 44C show
plots of
relaxation time to 80% of peak isometric force and FIG. 44B shows the force
trace raw data. FIG.
44D shows the reversal of myotonia phenotypes in HSA-LR mice treated with 20
mpk 221-1106
quantified by representative electromyography (EMG) traces.
Example 4. Evaluation of PMO-EEV 221-1120 in a DM1 mouse model
[0658] A DM1 mouse model was done to study the effect of EEV-PMO 221-1120
(also referred
to as EEV-PMO- DM1-3 or DM1-3; PM0 221 = 5'-CAG CAG CAG CAG CAG CAG CAG-3'
(SEQ ID NO: 154; all PM0 monomers); EEV 1120 = Ac-PKKKRKV-AEEA-
Lys(cyclo[FGFGRGRQ]-PEG12-0H (Ac-(SEQ ID NO: 42)-AEEA-Lys(SEQ ID NO: 82)-
PEG12-0H)) on the splicing and mRNA levels of downstream genes in the same HSA-
LR
transgenic mouse model as described in Example 2. The PM0 and EEV were
conjugated using
amide chemistry.
[0659] Experimental. There were two general treatment groups: 1) wild-type
mice; and 2) HSA-
LR mice (DM1 disease model). Within the HSA-LR treatment group there were two
sub-treatment
groups: 1) HSA-LR treated with saline (control); and 2) HSA-LR + EEV-PMO 221-
1120. Mice
were treated with 15 mpk, 30 mpk, 60 mpk, or 90 mpk (based on the PMO) of the
PMO-EEV or
saline via tail intravenous injection. Seven days after treatment, mice were
sacrificed, and tissues
were collected for analysis.
[0660] RT-PCR assays (correction of splicing). Tissues were homogenized by
OMNI BEAD
MILL HOMOGENIZER and the RNA was extracted by QIACUBEQ. RT-PCR assays were
performed using one-step RT-PCT kit (Qiagen) following the manufacturer's
protocols with 35
PCR cycles of 94 C for 30 seconds; 60 C for 30 seconds and 72 C for 30
seconds. Sequence
of gene specific primers are as follows: Clcn1 exon 7a inclusion Forward
primer = 5'-
TTCACATCGCCAGCATCTGTGC-3' (SEQ ID NO: 319), Reverse primer = 5' -
CACGGAACACAAAGGCACTGAATGT-3' (SEQ ID NO: 320); Mbnl 1 exon5 inclusion
forward primer = 5'-GCTGCCCAATACCAGGTCAAC-3' (SEQ ID NO: 321), reverse primer
=
5'-TGGTGGGAGAAATGCTGTATGC-3' (SEQ ID NO: 322);
239
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
Atp2a1 exon 22 inclusion forward primer = 5' -GCTCATGGTCCTCAAGATCTCAC-3' (SEQ
ID NO: 323), reverse primer: 5' -GGGTCAGTGCCTCAGCTTTG-3' (SEQ ID NO: 324);
Nfix exon 7 inclusion forward primer = 5' -TCGACGACAGTGAGATGGAG-3' (SEQ ID NO:
325), reverse primer 5' CAAACTCCTTCAGCGAGTCC-3' (SEQ ID NO: 326). Primers for
Clcnl, Mbnll, and Atp2a1 were from Klein et al., The Journal of Clinical
Investigation. 2019,
129 (11), pg. 4739; and primers for Nfix were from Chen et al., Scientific
Reports. 2016, 6(1),
pg. 1. The cDNA products were separated on 2% agarose E-gel with SYBR SAFE
dye. The
percentage exon inclusion was calculated by the ratio of the un-skipped
band/(un-skipped band +
skipped band).
[0661] Mouse DM1 splicing index (mDSI) calculation. The mDSI was calculated
following the
literature protocol from Tanner et. al. (Nucleic acids research. 2021, 49 (4),
pg. 2240-54). For each
sample i, normalized splicing values were calculated for each splice event j
as (PSI, j ¨
PSIwildtypej)/(PSIHSALR,j ¨PSIwildtypej), where PSIwildtypej is the average
PSI for event j across the
wildtype mice, and PSIHsALR,j is the average PSI for event j across the HSALR
mice. mDSI is then
calculated as the mean of all normalized splicing values, which are Atp2a1,
Nfix, Mbnll and Clcn1
in the studies.
[0662] qRT-PCR assays (HSA mRNA knockdown) . Reverse transcription was
performed using the
High-Capacity cDNA Reverse Transcription Kit from Life Technologies
Corporation following
the manufacturer's protocols. Quantitative real-time PCR were performed using
Bio-Rad SyBr
Green Supermix and QuantStudio3 qPCR machine with gene specific primers: HSA
mRNA
forward primer = 5' -TTCCATCGTCCACCGCAAAT-3' (SEQ ID NO: 327), reverse primer
= 5' -
AGTTTACGATGGCAGCAACG-3' (SEQ ID NO; 328), both primers from Klein et al., The
Journal of Clinical Investigation. 2019, 129 (11), pg. 4739; and mouse GAPDH
forward primer =
5' -AGGTCGGTGTGAACGGATTTG-3' (SEQ ID NO: 329), reverse primer = 5' -
TGTAGACCATGTAGTTGAGGTCA-3' (SEQ ID NO: 330).
[0663] RNAseq. PolyA RNAseq using Next Generation Sequencing was done for
transcriptome
profiling. The Z-score for each gene was calculated as the (sample value ¨ the
mean)/(the standard
deviation). Differential splicing analysis was done on the RNAseq data to
calculate the percent
spliced (PSI) of individual exons for each gene. The PSI is a ratio of
normalized read counts
indicating the inclusion of a transcript element over the total normalized
reads for that event
240
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
(inclusion and exclusion reads). For example, if an exon is included in the
reads 100% of the time,
the PSI is 1. Additionally, if an exon is excluded from the reads 100% of the
time, the PSI is 0.
[0664] Twenty-two genes of interest known to be predictive of DM1 were
analyzed. Additionally,
the genes studied in Wagner et al. (PLOS Gen 2016 (47)) and Tanner et al. (NAR
2021 (48), 4,
2240-2254) were analyzed. The mouse exons were mapped to the human location.
In some cases,
the boundary of the exons in mice and/or the human genome was not completely
known. As such,
the data was analyzed using different boundaries. The correct boundaries were
verified using the
RNAseq data.
[0665] RNA CUG Foci analysis. Tibialis anterior muscle sections were stained
for CUG foci
(FISH, red) and nuclei (Hoechst, blue). TA muscle sections were imaged and the
number of nuclei
having a CUG RNA foci were quantified.
[0666] Results
[0667] HSA mRNA knockdown. The diaphragm only expressed 5-10% of the HSA mRNA
levels
compared to quadriceps, tibialis anterior, and triceps (FIG. 13A). EEV-PMO
treatment did not
seem to change the HSA mRNA levels in the diaphragm (FIG. 13B). The expression
level of the
HSA 220 CUG repeats may not be sufficient for the mis-splicing phenotype in
DM1 in the
diaphragm.
[0668] The EEV-PMO knock downed HSA mRNA in a dose-dependent manner,
confirming
target engagement in quadricep (FIG. 14A), gastrocnemius (FIG. 14B), tricep
(FIG. 14C), and
tibialis anterior (FIG. 14D) tissue. Additionally, the Ct (cycle threshold)
value of HSA mRNA is
similar to the level of mouse GAPDH (-15), suggesting high expression of HSA
transgene in
HSA-LR mice in the quadriceps.
[0669] mDSI (correction of splicing). The mouse DM1 splicing index (mDSI) for
the quadriceps,
the gastrocnemius, the triceps, and the tibialis anterior are shown in FIG.
15A-15D. Treatment
with EEV-PMO corrected DM1 relevant splicing defects (Atp2a1 exon 22, Nfix
exon 7, Clcn1
exon 7a, Mbnl 1 exon 5) at 1-week post injection in the quadriceps (FIG. 15A),
gastrocnemius
(FIG. 15B), triceps (FIG. 15C), and tibialis anterior (FIG. 15D) in a dose
dependent manner with
higher doses approaching or equivalent to wild type (full correction).
Approximately 50%-60%
human skeletal actin RNA knockdown in HSA-LR mice was achieved at drug
concentrations that
achieve near complete splicing correction.
241
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
[0670] FIGS. 16A-B show images of tibialis anterior tissue of HSA-LR mice
(FIG. 16A) and
HSA-LR mice treated with EEV-PMO (FIG. 16B) stained for CUG foci (red) and
nuclei (blue).
Qualitative and quantitative assessment (FIG. 16C) showed that EEV-PMO
treatment reduced
number of nuclei had CUG foci.
[0671] Drug Exposure. Drug exposure was studied using LC-MS. FIG. 17A-17D show
a dose
dependent response for PMO-EEV exposure in the quadriceps (FIG. 17A), triceps
(FIG. 17B),
heart (FIG. 17C), gastrocnemius (FIG. 17D), tibialis anterior (TA; FIG. 17F),
liver (FIG. 171),
and kidney (FIG. 17J). No dose-dependent response was observed in the
diaphragm (FIG. 17G).
The EEV-PMO was not detected in the brain except at the 60 mpk and 90 mpk
dosage levels.
FIG. 17K shows drug exposure of various tissues at the 60 mpk dosage level.
[0672] Myotonia Response: A dose dependent myotonia reduction in HSA-LR mice 7
days after
treatment with EEV-PMO-DM1-3at 15, 30, 60 and 90 mpk was observed (FIG. 18A).
Myotonia
is likely ameliorated one week after treatment with EEV-PMO-DM1-3. HSA-LR mice
treated with
a single dose of 90 mpk EEV-PMO-DM1-3 did not exhibit obvious signs of hind
limb myotonia
after induction.
[0673] RNAseq Data Analysis. FIG. 19A-19D show the results of a principal
component analysis.
Principal component analysis can be used to reveal the similarity between
samples based on the
distance matrix. This type of plot is useful for visualizing the overall
effect of experimental
covariates and batch effects. The x-axis is the direction that explains the
most variance and the y-
axis is the second most. The percentage of the total variance per direction is
shown as PCA. The
wild type and HSA-LR mice are in distinct groups. Gene expression in the
gastrocnemius muscle
of HSA-LR mice treated with PMO-EEV was shifted toward that of wild type mice.
[0674] FIG. 20A is a heatmap showing differentially expressed genes (by Z-
score) from three
treatment groups: 1) WT mice; 2) HSA-LR mice; and 3) HSA-LR + EEV-PMO 221-1120
(60
mpk). A total of 956 (p < 0.5) genes differentially expressed between the
treatment groups,
indicating a difference between the wild type mice (WT) and the disease model
mice (HSA-LR).
Treatment with EEV-PMO (HSA-LR (+,+)) resulted in global gene expression
correction, shifting
away from a disease profile (in red, HSA-LR (-,-)) and toward that of wild
type mice (WT above).
[0675] FIG. 20B is a heatmap used to visualize the expression profile of 40 of
the 43 genes found
to have more than 7 CTG repeats from a BLAST analysis. Three of the CTG repeat
gene found in
242
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
the Blast analysis (Crb2, Hsd3b6, and Inhbe) were not included due to low
reads number. This
analysis is useful to identify co-regulated genes across the treatment
conditions.
[0676] FIG. 21 shows a volcano plot of the global transcriptional change
across the EEV-PMO
treated group and the HSA-LA group. Each data point in the scatter plot
represents a gene. The
fold change of each gene is represented on the x-axis and the log10 of its
adjusted p-value is on
the y-axis. Genes with an adjusted p-value less than 0.05 and a fold change
greater than 2 are
indicated by red dots. These represent up-regulated genes. Genes with an
adjusted p-value less
than 0.05 and a fold change less than -2 are indicated by blue dots. These
represent down-regulated
genes. Three genes were found to be significantly downregulated (Txlnb, Scube2
and Grebl) and
one gene was significantly upregulated (Txlnb). Most transcripts containing at
least (CUG)7 were
not significantly influenced. PCA Analysis of these genes showed that Scube2
(FIG. 22A), Grebl
(FIG. 22B), Ttc7 (FIG. 22C), Tx1nb(CUG)9, and Ndrg3 (FIG. 22E) showed
correction when
treated with EEV-PMO. Txlnb is overcorrected by treatment (FIG. 22D).
[0677] FIGS. 23A-23D show the transcriptome data for various genes and various
treatment
groups. The HSA-LR+ EEV-PMO 221-1120 treatment group showed correction of the
inclusion
of exon 22 of Atp2a1 (FIG. 23A), exclusion of exon 7 of Clcnl(FIG. 23B),
exclusion of exon 7
of Nfix (FIG. 23C), and the exclusion of exon 7 of Mbnll (FIG. 23D).
[0678] FIGS. 24 show the percent spliced (PSI) of individual exons for various
genes. The genes
are MBNL-1 responsive splicing biomarkers (e.g., downstream genes). The choice
of MBNL-1
dependent biomarkers was selected based on the dynamic range between the
wildtype and disease
groups as described in the literature. The HSA-LR+EEV-PM0 221-1120 treatment
group showed
correction for exon inclusion/exclusion for all of the 20 genes of interest
including Mbnll, Nfix,
Atp2a1, Ldb3, Camk2g, Trim55, Fbox31, 51c8a3, Map3k4, Dctn4, Cacnals, Ryrl,
51ain2, Phkal,
Ppp3cc, Ttn, Neb, 1rrfip2, Rapgefl, and Vsp39.
Example 5. Evaluation of PMO-EEV 221-1120 in a DM1 mouse model second DM1
mouse model
[0679] PMO-EEV DM1-3 (221-1120; see Example 4 for the sequence) was evaluated
in a second
DM1 mouse model using method similar to those described in Example 4.
[0680] Experimental. Seven-week-old HSA-LR mice were administered a single
dose 80 mpk of
EEV-PMO-DM1-3 or a 20 mpk dose EEV-PMO-DM1-3 every other week for six weeks
(total of
80 mpk over 4 doses) intravenously and tissues were harvested after 1 week to
12 weeks post the
243
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
single dose or two weeks after the final dose. RT-PCR was used to determine
alternative splicing
for specific genes (Atp2a1, Clcnl, Nfix, MBNL1). Q-PCR was used to determine
the reduction of
mRNA level of actin-HSA after treatment. LC-mass was used to determine drug
level in
quadricep, gastrocnemius, tibialis anterior, triceps, diaphragm, heart,
kidney, liver, brain, and
plasma. Myotonia reduction was recorded 7 days after treatment with the EEV-
PMO-DM1-3
compound.
[0681] Results. Similar trends to those observed in Example 4 were observed
for the rescue of
splicing for Atp2a1, Clcnl, Nfix, and MBNL1 in the tibialis anterior,
gastrocnemius, tricep, and
quadricep tissues (data not shown). Additionally, similar trends to those
observed in Example 4
were observed for the HSA mRNA knockdown tibialis anterior, gastrocnemius,
tricep, and
quadricep tissues both 1-week and 4-weeks post treatment (data not shown).
[0682] FIG. 25A-25D are plots showing a decrease in drug level with 80 mpk EEV-
PMO-DM1-
3 after 1 week to 8 weeks in the tibialis anterior (FIG. 25A), gastrocnemius
(FIG. 25B), triceps
(FIG. 25C), and quadricep (FIG. 25D) tissues. EEV-PMO-DM1-3 (60 mpk oligo, 80
mpk whole
drug) fully correct mis-splicing in gastrocnemius, triceps, tibialis anterior
and quadricep post 1
week treatment. FIG. 26A-26B are plots showing a decrease in drug levels was
observed with the
single 80 mpk dose of EEV-PMO-DM1-3 after 1 week to 4 weeks, to 8 weeks, and
to 12 weeks in
the liver. A relatively higher amount of EEV-PMO-DM1-3 in the liver was
observed 2 weeks post
the last dose of the 6-week dosing regime when compared to 4 weeks post the
single dose regime.
FIG. 26C-26D shows a decrease in drug levels was observed with the single 80
mpk dose of EEV-
PMO-DM1-3 after 1 week to 4 weeks, to 8 weeks, and to 12 weeks in the kidney.
A relatively low
amount of EEV-PMO-DM1-3 in the kidney was also observed from 2 weeks post the
last dose of
the 6-week dosing regime when compared to 4 weeks post the single dose regime.
At 12 weeks
post the single dose of EEV-PMO-DM1-3, the drug was still present in the
kidney but not in the
liver.
[0683] Subjective myotonia observations were made and shown in Table 15. The
multi-dosing
regime (Q2W) showed no signs of myotonia rescue after two weeks post
treatment. There was a
mixed effect on myotonia in mice treated with the single 80 mpk dose that
disappeared by 12
weeks post treatment.
244
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
Table 15. Myotonia Observations
Pre- 1 week post 4-week 8-week 12-week
Group dosing dose post dose
post dose post dose
Gender F M F M F MFM F
FVB 0 0 0 0 0
0 0 0 0 0
HSA-LR ++ ++ ++
++ ++ ++ ++ ++ ++ ++
221-112 80- mpk ++ ++ + + + 0 + ++ ++
221-1120 20 mpk
Q2W ++ ++ NA
NA NA NA ++ ++ NA NA
F = female; M= male; 0 = no mice displayed myotonia; + = mixed myotonia, some
mice displayed reduced myotonia; ++ = all mice displayed myotonia
[0684] A similar experiment was performed to evaluate intravenous
administration of EEV-PMO-
DM1-3 for a longer duration and at a higher dose. Eight-week-old HSA-LR mice
were treated with
40 mpk, 60 mpk, 80 mpk, or 120 mpk of EEV-PMO-DM1-3 intravenously and tissues
were
harvested after 4 weeks to 12 weeks. RT-PCR was used to determine alternative
splicing for
specific genes (Atp2a1, Clcnl, Nfix, MBNL1). Myotonia reduction was recorded 7
days after
treatment with EEV-PMO-DM1-3. Similar trends to those observed in Example 4
were observed
for the rescue of splicing for Atp2a1, Clcnl, Nfix, and MBNL1 in the tibialis
anterior
gastrocnemius tissues (data not shown).
[0685] Subjective myotonia observations were made and shown in Table 16.
Females displayed
more myotonia than males. There are no signs of myotonia in both male and
female mice dosed
with 120 mpk after 8 weeks post treatment.
Table 16. Myotonia Observations
Pre- 1 week post 4-week 8-week 12-week
Group dosing dose post dose
post dose post dose
Gender F M F M F MFM F
FVB 0 0 0 0 0
0 0 0 0 0
HSA-LR ++ ++ ++
++ ++ ++ ++ ++ ++ ++
221-1120 40 mpk ++ ++ + ++ ++ ++
NA NA NA NA
221-1120 60 mpk ++ ++ + 0 + + NA NA
NA NA
221-1120 80 mpk ++ ++ 0 0 + + + 0 0
221-1120 120
mpk ++ ++ 0 0 0 0 0 0
0 0
F = female; M= male; 0 = no mice displayed myotonia; + = mixed myotonia, some
mice displayed reduced myotonia; ++ = all mice displayed myotonia
245
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
Example 6. Treatment of Patient derived DM1 cells with EEV-PMO-DM1-3
[0686] PMO-EEV DM1-3 (221-1120; see Example 4 for the sequence) was evaluated
in DM1
patient derived myoblasts.
[0687] Experimental. Patient myoblasts were treated with 30 micromolar of DM1-
3 throughout
four days of differentiation. Splicing correction was assessed by one-step RT-
PCR and Labpchip
(Plotted mean SD; n=4). HCR-FISH and sequestered 1V1BNL1 protein detection
assays were used
to detect RNA CUG foci. Results: EEV-PMO-DM1-3promotes significant biomarker
splicing
correction and a reduction in nuclear foci in DM1 patient-derived muscle
cells.
[0688] Results. FIG. 27A-27C are plots showing that EEV-PMO-DM1-3 promotes
significant
biomarker splicing correction (MBLN1, SOS1, and NFIX) in DM1 patient-derived
muscle cells.
Additionally, treatment with DM1-3 resulted in the reduction of nuclear foci
in DM1 patient-
derived muscle cells (FIG. 28A-28C).
Example 7. Cytotoxicity Screening of EEV-PMO-DM1-3 in Renal Cells
[0689] PMO-EEV DM1-3 (221-1120; see Example 4 for the sequence) was evaluated
in human
renal cells.
[0690] Experimental. Human Primary Renal Proximal Tubular Epithelial Cells
(RPTECs) were
exposed to varying concentrations (1:2 serial dilution in saline with a final
dilution factor of 4x
from about 6 1.1.M to about 800 l.M) of PMO-DM1 and EEV-PMO-DM1-3 for 24 hours
and
screened for viability using a CELLTITER-GLO luminescent viability assay.
Melittin was used as
a positive control at 16.6 04.
[0691] Results. FIG. 29A-29B show that PMO-DM1 or its conjugated EEV-PMO-DM1-3
did not
show any toxicity even with the highest concentration of 817 tM or 79704,
respectively.
Example 8. Assessment of PMO-EEV 221-1113 for ability to correct mis-splicing
events
and downstream splicing in immortalized cells DM1 patients and HeLa-480 cells
[0692] Immortalized DM1 patient-derived (2,600 CUG repeats) muscle cells and
HeLa-480 (DM1
model cell line, see Example 1) were treated with the EEV-PMO construct 221-
1113 and analyzed
for correction of aberrant splicing and foci quantification. EEV 1113 is Ac-
PKKKRKV-miniPEG-
K(cyc/o(Ff-Nal-GrGrQ)-PEG12-0H (Ac-(SEQ ID NO: 42)-miniPEG-K(cyclo(SEQ ID NO:
80)-
PEG12-0H). EEV-PMO 221-1113 is EEV 1113 conjugated to PM0 sequence 221 (5' -
CAG CAG
CAG CAG CAG CAG CAG-3' (SEQ ID NO: 154; all PM0 monomers) via amide bond
chemistry.
[0693] Experimental. Methods similar to those described in Example 1 and
Example 3 were used.
246
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
[0694] Results.
[0695] RNA CUG Foci analysis. Cells were stained for nuclei (Hoeschet, blue)
and for RNA CUG
repeat foci (green) and imaged. A reduction in RNA CUG foci was observed
between untreated
DM1 patient cells and EEV-PMO treated DM1 cells (FIG. 30A-30C). Similarly, a
reduction in
RNA CUG foci was observed between untreated HeLa-480 cells and EEV-PMO HeLa-
480 cells
(FIG. 31A-31B).
[0696] Correction of downstream splicing. PMO-EEV treated DM1 patient derived
cells and
HeLa-480 cells were analyzed for percent exon 5 inclusion for MBLN1, percent
exon 25 inclusion
for SOS1, and percent inclusion of exon 7 for NFIX. Treatment with EEV-PMO
resulted in a
rescue of splicing events for Mbnll (FIG. 32A), Sosl (FIG. 32B), and NFIX
(FIG. 32C).
[0697] Additionally, HeLa-480 cell treated EEV-PMO showed a dose dependent
correction of
MBNL1 (FIG. 33A) splicing and the downstream missplicings of SOS1 (FIG. 33B),
CLASP1
(FIG. 33C), NFIX (FIG. 33D), and INSR (FIG. 33E) in a dose dependent manner.
Example 9. Evaluation of EEV-PMO 221-1106 in a second DM1 mouse model
[0698] A DM1 mouse model was done to study the effect of EEV-PMO 221-1106 on
the splicing
and mRNA levels of downstream genes.
[0699] Experimental. Human skeletal actin long repeat (HSA-LR) transgenic mice
were used as
the DM1 disease model. Similar methods to those described in Example 5 were
used.
[0700] Results. FIGS. 34A-34D show a dose dependent correction of the
inclusion of exon 22 in
Atp2a1 (FIG. 34A), exon 7 in Nfix (FIG. 34B), exon 7A in Clcn1 (FIG. 34C), and
Mbnll (FIG.
34D) in the gastrocnemius of mice treated with various concentrations of EEV-
PMO 221-1106.
Treatment with PM0 221 alone did not result in correction of splicing.
Example 10. DM1 mouse model to study effect of different lengths of CUG
repeats in
PM0s
[0701] A DM1 mouse model was done to study the effect of PMO-EEV 221-1121 (PM0
has7
CAG repeats, 21-mer) and PMO-EEV 0325-1121 (PM0 has 8 CAG repeats, 24-mer) on
the
splicing and mRNA levels of downstream genes. PMO-EEV 221-1121 is PM0 221 (5' -
CAG CAG
CAG CAG CAG CAG CAG-3'; SEQ ID NO: 154; all PM0 monomers) conjugated to EEV
1121
(Ac-PKKKRKV-miniPEG2-Lys(cyclo[GfFGrGrQ])-PEG12-0H; Ac-(SEQ ID NO: 42)-
miniPEG2-Lys(SEQ ID NO:74)-PEG12-0H ) via amide chemistry. PMO-EEV 0325-1121
is
247
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
PM0 0325 (5'-CAG CAG CAG CAG CAG CAG CAG-CAG-3'; SEQ ID NO: 155; all PM0
monomers) conjugated to EEV 1121 via amide chemistry.
[0702] Experimental. Human skeletal actin long repeat (HSA-LR) transgenic mice
were used as
the DM1 disease model. Briefly, HSA-LR mice were dosed with 20 mpk, 40 mpk, or
60 mpk of
either 0221-1121 or 0325-1121 via intravenous injection into the tail vein.
One week post
injection, mice were sacrificed, and tissue was collected. Other experimental
methods are similar
to those described in Example 5 were used.
[0703] Results. 0221-1121 (21-mer) was more effective in correcting exon
splicing in Mbnll
(FIG. 35A), Nfix (FIG. 35B), and Atp2a1 (FIG. 35C) than 0325-1121 (24-mer) in
the tibialis
anterior tissue. This result was unexpected. It was expected that the 24-mer
would be more
effective as it would have a higher hybridization efficiency and higher
thermal melting
temperature. In the gastrocnemius tissue, the differences were less pronounced
as shown in FIGS.
36A-36C. Subjective myotonia observations were made using the male mice (Table
17). Mixed
myotonia was observed at 1 week post treatment for the 21-mer at 40 mpk,
similar to the results
in Table 11.
Table 17: Myotonia Observations
1 week post 4 week post
Pre-dosing
Group dose dose
Gender
FVB 0 0 NA
HSA-LR ++ ++ NA
221-112 20 mpk + + + + NA
221-112040
mpk ++
325-1120 20
mpk ++ ++ NA
325-1120 40
mpk ++ ++
M= male; 0 = no mice displayed myotonia; + = mixed myotonia,
some mice displayed reduced myotonia ; ++ = all mice displayed
myotonia
Example 11. Pharmacokinetic studies of the EEV-PMO 221-1120 in CD! mice
[0704] A CD1 mouse model was used to study the plasma, kidney, and tibialis
anterior drug
exposure (AUC) to the EEV-PMO construct 221-1120 (see Example 4 for the
sequence) and PM0-
0221a, the major metabolite of 221-1120 (see FIG. 37) was also measured.
248
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
[0705] Experimental. Five- to seven-week-old CD1 mice were treated with 80 mpk
of the EEV-
PM0 construct 221-1120 via intravenous injection. Mice were bled and/or
scarified at various time
points.
[0706] Results. Table 18, Table 19, and Table 20 show the pharmacokinetic
properties observed
in the plasma, kidney, and tibialis anterior, respectively. For the tables:
AUCIast = area under the
curve from zero to last quantifiable concentration; D = dose; C. = maximum
serum or plasma
concentration; T. = time to reach C.; CL = total plasma, serum, or blood
clearance; t1/2 =
elimination half-life; Vss = apparent volume of distribution at equilibrium;
Qh = hepatic blood flow
(ml/min/kg).
[0707] The AUC values for the metabolite is ¨1000-fold lower in the tibias
anterior compared to
the kidney. The metabolite mean residence time (MRT) values in plasma may be
directly related
to tissue MRT values as a result of moving from tissues to plasma before
urinary excretion.
Table 18: Plasma pharmacokinetic properties
221-1120 PM0-0221a
AUCiasoM*hr) 9290 953
AUCiast/D 1121 115
Cmax(nM) 16217 12
CmaxiD 1956 1.4
T(hr) 0.1 24
CL (mL/min/kg) 15
0h(%) 16
t1/4(hr) 19 68
MRTiast(hr) 1.2 60.0
Vss(mL/kg) 1325
Table 19: Kidney pharmacokinetic properties
221-1120 PM0-0221a
AUCiast(pmol/g*hr) 51609 10113709
AUCiast/D 6225 1219865
Cmax(pmol/g) 8754 105053
CmaxiD 1056 12671
T(hr) 4 24
t1/4(hr) 5 87
MRTiast(hr) 8 68
249
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
Table 20: Tibialis anterior pharmacokinetic properties
PM0-
221-1120 0221a
AUCiast(pmolig*hr) 9140
AUCiast/D 1102
Cmax(pmol/g) 34 162
Cmax/D 4 20
T(hr) 4 24
t1/4(hr) 48
MRTiast(hr) 50
Example 12. Evaluation of PMO-EEV 221-1120 in a third DM1 mouse model
[0708] A DM1 mouse model study similar to Examples 5 and 9 was conducted to
evaluate the
effect of various doses of PMO-EEV 221-220 (see Example 4 for the sequence) in
HSA-LR mice.
[0709] Experimental. Eight-week-old HSA-LR mice were administered 40, 60, 80
or 120 mpk
of PMO-EEV 221-1120 intravenously and tissues were harvested after 4 to 12
weeks. RT-PCR
was used to determine alternative splicing for specific genes (Atp2a1, Clcnl,
Nfix, MBNL1). LC-
mass was used to determine drug level in Quad, gastro, TA, Triceps, diaphragm,
heart, kidney,
liver, brain, plasma. RNA-seq was used to determine the transcription level
change between a
treated disease model, an untreated disease model and wild-type. Q-PCR was
used to determine
the reduction of mRNA level of actin-HSA after treatment.
[0710] Results. Fluorescence imaging was used to determine RNA Foci reduction
after treatment
with the EEV-oligo compound (data no shown). Myotonia reduction was recorded 7
days after
treatment with the EEV-oligo compound (data not shown). The results of these
experiments show
similar trends to the mice treated with PMO-EEV 221-1120 in Example 5 and
Example 14.
[0711] 1V1BNL1, NFIX, and ATP2A1 splicing correction was observed in the
tibialis anterior
(FIGS. 38A, 39A, 40A) and gastrocnemius (FIGS. 38B, 39B, 40B) at various doses
of the EEV-
PMO. MBNL1, NFIX, and ATP2A1splicing correction was observed in both the
tibialis anterior
and gastrocnemius 12 weeks post treatment with 120 mpk EEV-PMO.
Example 13. Evaluation of EEV-PMO 221-1120 in HeLa480 cells
[0712] HeLa480 cells were treated with various concentrations of EEV-PMO 221-
1120 (see
Example 4 for the sequence) and analyzed for CUG repeat foci, selective r(CUG)
reduction, and
downstream splicing correction of MBNL1 and SOS1.
250
CA 03222824 2023-12-07
WO 2022/271818 PCT/US2022/034517
[0713] Experimental. Hela480 cells were constructed as described in earlier
Examples. RT-PCR
and foci staining was performed similar to other Examples described herein.
[0714] Results. FIG. 41A-41B shows example images of control cells (untreated)
and cells treated
with 5 tM, 10 p,M, 20 p,M, 50 p,M, and 100 1..LM of EEV-PMO 221-1120. There is
a reduction in
CUG foci (green) in the treated HeLa480 group when compared to the untreated
HeLa480 cells.
FIG. 41B is a plot quantifying the foci per nuclear area. FIG.41A-41B show
that EEV-PMO 221-
1120 can reduce nuclear CUG RNA foci. Almost a complete reduction is observed
in the 5 1..LM
dose.
[0715] FIG. 42A-42B indicate the EEV-PMO 221-1120 treatment can selectively
knockdown
repeat expansion-containing DMPK transcript in the HeLa480 cell line.
[0716] FIG. 42C-42D show that treatment with EEV-PMO 221-1120 corrected MBNL1
(FIG.
42C) and SOCS1 (FIG. 42D) splicing in a dose dependent manner.
[0717] A number of embodiments of the invention have been described.
Nevertheless, it will be
understood that various modifications may be made without departing from the
spirit and scope of
the invention. Accordingly, other embodiments are within the scope of the
following claims.
251
