Note: Descriptions are shown in the official language in which they were submitted.
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TRANSPOSON SYSTEM AND METHODS OF USE
RELATED APPLICATIONS
[01] This application claims priority to U.S. Patent Application No.
62/300,387, filed
February 26, 2016, the contents of which are herein incorporated by reference
in their
entirety.
INCORPORATION OF SEQUENCE LISTING
[02] The contents of the text file named "POTH-007001W0 SeqList.txt", which
was
created on February 24, 2017 and is 26 KB in size, are hereby incorporated by
reference in
their entirety.
FIELD OF THE DISCLOSURE
[03] The present invention is directed to compositions and methods for
targeted gene
modification.
BACKGROUND
[04] Ex vivo genetic modification of non-transformed primary human T
lymphocytes using
non-viral vector-based gene transfer delivery systems has been extremely
difficult. As a
result, most groups have generally used viral vector-based transduction such
as retrovirus,
including lentivirus. A number of non-viral methods have been tested and
include antibody-
targeted liposomes, nanoparticles, aptamer siRNA chimeras, electroporation,
nucleofection,
lipofection, and peptide transduction. Overall, these approaches have resulted
in poor
transfection efficiency, direct cell toxicity, or a lack of experimental
throughput.
[05] The use of plasmid vectors for genetic modification of human lymphocytes
has been
limited by low efficiency using currently available plasmid transfection
systems and by the
toxicity that many plasmid transfection reagents have on these cells. There is
a long-felt and
unmet need for a method of nonviral gene modification in immune cells.
SUMMARY
[06] When compared with viral transduction of immune cells, such as T
lymphocytes,
delivery of transgenes via DNA transposons, such as piggyBac and Sleeping
Beauty, offers
significant advantages in ease of use, ability to delivery much larger cargo,
speed to clinic
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and cost of production. The piggyBac DNA transposon, in particular, offers
additional
advantages in giving long-term, high-level and stable expression of
transgenes, and in being
significantly less mutagenic than a retrovirus, being non-oncogenic and being
fully
reversible. Previous attempts to use DNA transposons to deliver transgenes to
T cells have
been unsuccessful at generating commercially viable products or manufacturing
methods
because the previous methods have been inefficient. For example, the poor
efficiency
demonstrated by previous methods of using DNA transposons to deliver
transgenes to T cells
has resulted in the need for prolonged expansion ex vivo. Previous
unsuccessful attempts by
others to solve this problem have all focused on increasing the amount of DNA
transposon
delivered to the immune cell, which has been a strategy that worked well for
non-immune
cells. This disclosure demonstrates that increasing the amount of DNA
transposon makes the
efficiency problem worse in immune cells by increasing DNA-mediated toxicity.
To solve
this problem, counterintuitively, the methods of the disclosure decrease the
amount of DNA
delivered to the immune cell. Using the methods of the disclosure, the data
provided herein
demonstrate not only that decreasing the amount of DNA transposon introduced
into the cell
increased viability but also that this method increased the percentage of
cells that harbored a
transposition event, resulting in a viable commercial process and a viable
commercial
product. Thus, the methods of the disclosure demonstrate success where others
have failed.
[07] The disclosure provides a nonviral method for the ex-vivo genetic
modification of an
immune cell comprising delivering to the immune cell, (a) a nucleic acid or
amino acid
sequence comprising a sequence encoding a transposase enzyme and (b) a
recombinant and
non-naturally occurring DNA sequence comprising a DNA sequence encoding a
transposon.
In certain embodiments, the method further comprises the step of stimulating
the immune cell
with one or more cytokine(s).
[08] In certain embodiments of the methods of the disclosure, the sequence
encoding a
transposase enzyme is an mRNA sequence. The mRNA sequence encoding a
transposase
enzyme may be produced in vitro.
[09] In certain embodiments of the methods of the disclosure, the sequence
encoding a
transposase enzyme is a DNA sequence. The DNA sequence encoding a transposase
enzyme
may be produced in vitro. The DNA sequence may be a cDNA sequence.
[010] In certain embodiments of the methods of the disclosure, the sequence
encoding a
transposase enzyme is an amino acid sequence. The amino acid sequence encoding
a
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transposase enzyme may be produced in vitro. A protein sPBo may be delivered
following
pre-incubation with transposon DNA.
10111 In certain embodiments of the methods of the disclosure, the delivering
step
comprises electroporation or nucleofection of the immune cell.
[012] In certain embodiments of the methods of the disclosure, the step of
stimulating the
immune cell with one or more cytokine(s) occurs following the delivering step.
Alternatively,
or in addition, in certain embodiments, the step of stimulating the immune
cell with one or
more cytokine(s) occurs prior to the delivering step. In certain embodiments,
the one or more
cytokine(s) comprise(s) IL-2, IL-21, IL-7 and/or IL-15.
[013] In certain embodiments of the methods of the disclosure, the immune cell
is an
autologous immune cell. The immune cell may be a human immune cell and/or an
autologous
immune cell. The immune cell may be derived from a non-autologous source,
including, but
not limited to a primary cell, a cultured cell or cell line, an embryonic or
adult stem cell, an
induced pluripotent stem cell or a transdifferentiated cell. The immune cell
may have been
previously genetically modified or derived from a cell or cell line that has
been genetically
modified. The immune cell may be modified or may be derived from a cell or
cell line that
has been modified to suppress one or more apoptotic pathways. The immune cell
may be
modified or may be derived from a cell or cell line that has been modified to
be "universally"
allogenic by a majority of recipients in the context, for example, of a
therapy involving an
adoptive cell transfer.
[014] In certain embodiments of the methods of the disclosure, the immune cell
is an
activated immune cell.
[015] In certain embodiments of the methods of the disclosure, the immune cell
is an
resting immune cell.
[016] In certain embodiments of the methods of the disclosure, the immune cell
is a T-
lymphocyte. In certain embodiments, the T-lymphocyte is an activated T-
lymphocyte. In
certain embodiments, the T-lymphocyte is a resting T-lymphocyte.
[017] In certain embodiments of the methods of the disclosure, the immune cell
is a
Natural Killer (NK) cell.
[018] In certain embodiments of the methods of the disclosure, the immune cell
is a
Cytokine-induced Killer (CIK) cell.
[019] In certain embodiments of the methods of the disclosure, the immune cell
is a
Natural Killer T (NKT) cell.
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[020] In certain embodiments of the methods of the disclosure, the immune cell
is isolated
or derived from a human.
[021] In certain embodiments of the methods of the disclosure, the immune cell
is isolated
or derived from a non-human mammal. In certain embodiments, the non-human
mammal is a
rodent, a rabbit, a cat, a dog, a pig, a horse, a cow, or a camel. In certain
embodiments, the
immune cell is isolated or derived from a non-human primate.
[022] In certain embodiments of the methods of the disclosure, the transposase
enzyme is a
Super piggyBacTM (sPBo) transposase enzyme. The Super piggyBac (PB)
transposase
enzyme may comprise or consist of an amino acid sequence at least 75%
identical to:
MGS S LDDEHIL S ALLQ SDDELVGED SD SEV SDHV SEDDVQ SDTEEAFIDEVHEVQP TS
S GSEILDEQNVIEQPGS SLASNRILTLPQRTIRGKNKHCWSTSKSTRRSRVSALNIVRS
QRGPTRMCRNIYDPLLCFKLFFTDEIISEIVKWTNAEISLKRRESMTSATFRDTNEDEI
YAFFGILVMTAVRKDNHMSTDDLFDRSL SMVYVSVMSRDRFDFLIRCLRMDDKSIR
P TLRENDVF TPVRKIWDLF IHQ CI QNYTP GAHLTIDEQLL GFRGRCPF RVYIPNKP S KY
GIKILMMCDS GTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGS CRNITCDN
WFTSIPLAKNLLQEPYKLTIVGTVRSNKREIPEVLKNSRSRPVGTSMFCFDGPLTLVSY
KPKPAKMVYLLS S CDEDASINESTGKPQMVMYYNQTKGGVDTLDQMCSVMTCSRK
TNRWPMALLYGMINIACINSFIIYSHNVS S KGEKV Q S RKKF MRNLYM S LT S SFMRKR
LEAPTLKRYLRDNISNILPKEVPGTSDDSTEEPVMKKRTYCTYCPSKIRRKANAS CKK
CKKVICREHNIDMCQSCF (SEQ ID NO: 1).
[023] In certain embodiments of the methods of the disclosure, the transposase
enzyme is a
Sleeping Beauty transposase enzyme (see, for example, US Patent No. 9,228,180,
the
contents of which are incorporated herein in their entirety). In certain
embodiments, the
Sleeping Beauty transposase is a hyperactive Sleeping Beauty SB100X
transposase. In
certain embodiments, the Sleeping Beauty transposase enzyme comprises an amino
acid
sequence at least 75% identical to:
MGKS KEI S QDLRKKIVDLHKS GS SLGAISKRLKVPRS SVQTIVRKYKHHGTTQP SYRS
GRRRYL S P RDERTLVRKV QINPRTTAKDLVKMLEETGTKV S I S TVKRVLYRHNLKGR
SARKKPLLQNRHKKARLRFATAHGDKDRTFWRNVLWSDETKIELFGHNDHRYVWR
KKGEACKPKNTIPTVKHGGGSIMLWGCFAAGGTGALHKIDGIMRKENYVDILKQHL
KTSVRKLKLGRKWVFQMDNDPKHTSKVVAKWLKDNKVKVLEWPSQSPDLNPIENL
WAELKKRVRARRPTNLTQLHQLC QEEWAKIHPTYC GKLVEGYPKRLTQVKQF KGN
ATKY (SEQ ID NO: 2). In certain embodiments, including those wherein the
Sleeping
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Beauty transposase is a hyperactive Sleeping Beauty SB100X transposase, the
Sleeping
Beauty transposase enzyme comprises an amino acid sequence at least 75%
identical to:
MGKSKEISQDLRKRIVDLHKSGSSLGAISKRLAVPRSSVQTIVRKYKHHGTTQPSYRS
GRRRYLSPRDERTLVRKVQINPRTTAKDLVKMLEETGTKVSISTVKRVLYRHNLKGH
SARKKPLLQNRHKKARLRFATAHGDKDRTFWRNVLWSDETKIELFGHNDHRYVWR
KKGEACKPKNTIPTVKHGGGSIMLWGCFAAGGTGALHKIDGIMDAVQYVDILKQHL
KTSVRKLKLGRKWVFQHDNDPKHTSKVVAKWLKDNKVKVLEWPSQSPDLNPIENL
WAELKKRVRARRPTNLTQLHQLCQEEWAKIHPNYCGKLVEGYPKRLTQVKQFKGN
ATKY (SEQ ID NO: 3).
[024] In certain embodiments of the methods of the disclosure, the recombinant
and non-
naturally occurring DNA sequence comprising a DNA sequence encoding a
transposon may
be circular. As a nonlimiting example, the DNA sequence encoding a transposon
may be a
plasmid vector. As a nonlimiting example, the DNA sequence encoding a
transposon may be
a minicircle DNA vector.
[025] In certain embodiments of the methods of the disclosure, the recombinant
and non-
naturally occurring DNA sequence encoding a transposon may be linear. The
linear
recombinant and non-naturally occurring DNA sequence encoding a transposon may
be
produced in vitro. Linear recombinant and non-naturally occurring DNA
sequences of the
disclosure may be a product of a restriction digest of a circular DNA. In
certain
embodiments, the circular DNA is a plasmid vector or a minicircle DNA vector.
Linear
recombinant and non-naturally occurring DNA sequences of the disclosure may be
a product
of a polymerase chain reaction (PCR). Linear recombinant and non-naturally
occurring DNA
sequences of the disclosure may be a double-stranded doggyboneTM DNA sequence.
DoggyboneTM DNA sequences of the disclosure may be produced by an enzymatic
process
that solely encodes an antigen expression cassette, comprising antigen,
promoter, poly-A tail
and telomeric ends.
[026] In certain embodiments of the methods of the disclosure, the recombinant
and non-
naturally occurring DNA sequence encoding a transposon further comprises a
sequence
encoding a chimeric antigen receptor or a portion thereof Chimeric antigen
receptors (CARs)
of the disclosure may comprise (a) an ectodomain comprising an antigen
recognition region,
(b) a transmembrane domain, and (c) an endodomain comprising at least one
costimulatory
domain. In certain embodiments, the ectodomain may further comprise a signal
peptide.
Alternatively, or in addition, in certain embodiments, the ectodomain may
further comprise a
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hinge between the antigen recognition region and the transmembrane domain. In
certain
embodiments of the CARs of the disclosure, the signal peptide may comprise a
sequence
encoding a human CD2, CD36, CD3E, CD3y, CDK CD4, CD8a, CD19, CD28, 4-1BB or
GM-CSFR signal peptide. In certain embodiments of the CARs of the disclosure,
the signal
peptide may comprise a sequence encoding a human CD8a signal peptide. In
certain
embodiments, the transmembrane domain may comprise a sequence encoding a human
CD2,
CD36, CD3E, CD3y, CDK CD4, CD8a, CD19, CD28, 4-1BB or GM-CSFR transmembrane
domain. In certain embodiments of the CARs of the disclosure, the
transmembrane domain
may comprise a sequence encoding a human CD8a transmembrane domain. In certain
embodiments of the CARs of the disclosure, the endodomain may comprise a human
CD3
endodomain. In certain embodiments of the CARs of the disclosure, the at least
one
costimulatory domain may comprise a human 4-1BB, CD28, CD40, ICOS, MyD88, OX-
40
intracellular segment, or any combination thereof In certain embodiments of
the CARs of the
disclosure, the at least one costimulatory domain may comprise a CD28 and/or a
4-1BB
costimulatory domain. In certain embodiments of the CARs of the disclosure,
the hinge may
comprise a sequence derived from a human CD8a, IgG4, and/or CD4 sequence. In
certain
embodiments of the CARs of the disclosure, the hinge may comprise a sequence
derived
from a human CD8a sequence.
[027] In certain embodiments of the methods of the disclosure, the recombinant
and non-
naturally occurring DNA sequence encoding a transposon further comprises a
sequence
encoding a chimeric antigen receptor or a portion thereof The portion of the
sequence
encoding a chimeric antigen receptor may encode an antigen recognition region.
The antigen
recognition region may comprise one or more complementarity determining
region(s). The
antigen recognition region may comprise an antibody, an antibody mimetic, a
protein scaffold
or a fragment thereof In certain embodiments, the antibody is a chimeric
antibody, a
recombinant antibody, a humanized antibody or a human antibody. In certain
embodiments,
the antibody is affinity-tuned. Nonlimiting examples of antibodies of the
disclosure include a
single-chain variable fragment (scFv), a VHH, a single domain antibody (sdAB),
a small
modular immunopharmaceutical (SMIP) molecule, or a nanobody. In certain
embodiments,
the VHH is camelid. Alternatively, or in addition, in certain embodiments, the
VHH is
humanized. Nonlimiting examples of antibody fragments of the disclosure
include a
complementary determining region, a variable region, a heavy chain, a light
chain, or any
combination thereof Nonlimiting examples of antibody mimetics of the
disclosure include an
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affibody, an afflilin, an affimer, an affitin, an alphabody, an anticalin, and
avimer, a DARPin,
a Fynomer, a Kunitz domain peptide, or a monobody. Nonlimiting examples of
protein
scaffolds of the disclosure include a Centyrin.
[028] In certain embodiments of the methods of the disclosure, the nucleic
acid sequence
encoding the transposase enzyme is a DNA sequence, and an amount of the DNA
sequence
encoding the transposase enzyme and an amount of the DNA sequence encoding the
transposon is equal to or less than 10.0 lig per 100 uL of an electroporation
or nucleofection
reaction. In certain embodiments, a concentration of the amount of the DNA
sequence
encoding the transposase enzyme and an amount of the DNA sequence encoding the
transposon in the electroporation or nucleofection reaction is equal to or
less than 100 ug/mL.
[029] In certain embodiments of the methods of the disclosure, the nucleic
acid sequence
encoding the transposase enzyme is a DNA sequence, and an amount of the DNA
sequence
encoding the transposase enzyme and an amount of the DNA sequence encoding the
transposon is equal to or less than 7.5 lig per 100 uL of an electroporation
or nucleofection
reaction. In certain embodiments, a concentration of the amount of the DNA
sequence
encoding the transposase enzyme and an amount of the DNA sequence encoding the
transposon in the electroporation or nucleofection reaction is equal to or
less than 75 ug/mL.
[030] In certain embodiments of the methods of the disclosure, the nucleic
acid sequence
encoding the transposase enzyme is a DNA sequence, and an amount of the DNA
sequence
encoding the transposase enzyme and an amount of the DNA sequence encoding the
transposon is equal to or less than 6.0 lig per 100 uL of an electroporation
or nucleofection
reaction. In certain embodiments, a concentration of the amount of the DNA
sequence
encoding the transposase enzyme and an amount of the DNA sequence encoding the
transposon in the electroporation or nucleofection reaction is equal to or
less than 60 ug/mL.
In certain embodiments, the transposase is a Sleeping Beauty transposase. In
certain
embodiments, the Sleeping Beauty transposase is a Sleeping Beauty 100X
(SB100X)
transposase.
[031] In certain embodiments of the methods of the disclosure, the nucleic
acid sequence
encoding the transposase enzyme is a DNA sequence, and an amount of the DNA
sequence
encoding the transposase enzyme and an amount of the DNA sequence encoding the
transposon is equal to or less than 5.0 lig per 100 uL of an electroporation
or nucleofection
reaction. In certain embodiments, a concentration of the amount of the DNA
sequence
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encoding the transposase enzyme and an amount of the DNA sequence encoding the
transposon in the electroporation or nucleofection reaction is equal to or
less than 50 pg/mL.
[032] In certain embodiments of the methods of the disclosure, the nucleic
acid sequence
encoding the transposase enzyme is a DNA sequence, and an amount of the DNA
sequence
encoding the transposase enzyme and an amount of the DNA sequence encoding the
transposon is equal to or less than 2.5 pg per 100 pL of an electroporation or
nucleofection
reaction. In certain embodiments, a concentration of the amount of the DNA
sequence
encoding the transposase enzyme and an amount of the DNA sequence encoding the
transposon in the electroporation or nucleofection reaction is equal to or
less than 25 pg/mL.
[033] In certain embodiments of the methods of the disclosure, the nucleic
acid sequence
encoding the transposase enzyme is a DNA sequence, and an amount of the DNA
sequence
encoding the transposase enzyme and an amount of the DNA sequence encoding the
transposon is equal to or less than 1.67 pg per 100 pL of an electroporation
or nucleofection
reaction. In certain embodiments, a concentration of the amount of the DNA
sequence
encoding the transposase enzyme and an amount of the DNA sequence encoding the
transposon in the electroporation or nucleofection reaction is equal to or
less than 16.7
pg/mL. In certain embodiments, the transposase is a Super piggyBac (PB)
transposase.
[034] In certain embodiments of the methods of the disclosure, the nucleic
acid sequence
encoding the transposase enzyme is a DNA sequence, and an amount of the DNA
sequence
encoding the transposase enzyme and an amount of the DNA sequence encoding the
transposon is equal to or less than 0.55 pg per 100 pL of an electroporation
or nucleofection
reaction. In certain embodiments, a concentration of the amount of the DNA
sequence
encoding the transposase enzyme and an amount of the DNA sequence encoding the
transposon in the electroporation or nucleofection reaction is equal to or
less than 5.5 pg/mL.
[035] In certain embodiments of the methods of the disclosure, the nucleic
acid sequence
encoding the transposase enzyme is a DNA sequence, and an amount of the DNA
sequence
encoding the transposase enzyme and an amount of the DNA sequence encoding the
transposon is equal to or less than 0.19 pg per 100 pL of an electroporation
or nucleofection
reaction. In certain embodiments, a concentration of the amount of the DNA
sequence
encoding the transposase enzyme and an amount of the DNA sequence encoding the
transposon in the electroporation or nucleofection reaction is equal to or
less than 1.9 pg/mL.
[036] In certain embodiments of the methods of the disclosure, the nucleic
acid sequence
encoding the transposase enzyme is a DNA sequence, and an amount of the DNA
sequence
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encoding the transposase enzyme and an amount of the DNA sequence encoding the
transposon is equal to or less than 0.10 pg per 100 pL of an electroporation
or nucleofection
reaction. In certain embodiments, a concentration of the amount of the DNA
sequence
encoding the transposase enzyme and an amount of the DNA sequence encoding the
transposon in the electroporation or nucleofection reaction is equal to or
less than 1.0 pg/mL.
[037] In certain embodiments of the methods of the disclosure, the nucleic
acid sequence
encoding the transposase enzyme is a RNA sequence, and an amount of the DNA
sequence
encoding the transposon is equal to or less than 10.0 pg per 100 pL of an
electroporation or
nucleofection reaction. In certain embodiments, a concentration of the amount
of the DNA
sequence encoding the transposon in the electroporation or nucleofection
reaction is equal to
or less than 100 pg/mL.
[038] In certain embodiments of the methods of the disclosure, the nucleic
acid sequence
encoding the transposase enzyme is a RNA sequence, and an amount of the DNA
sequence
encoding the transposon is equal to or less than 7.5 pg per 100 pL of an
electroporation or
nucleofection reaction. In certain embodiments, a concentration of the amount
of the DNA
sequence encoding the transposon in the electroporation or nucleofection
reaction is equal to
or less than 75 pg/mL.
[039] In certain embodiments of the methods of the disclosure, the nucleic
acid sequence
encoding the transposase enzyme is a RNA sequence, and an amount of the DNA
sequence
encoding the transposon is equal to or less than 6.0 pg per 100 pL of an
electroporation or
nucleofection reaction. In certain embodiments, a concentration of the amount
of the DNA
sequence encoding the transposon in the electroporation or nucleofection
reaction is equal to
or less than 60 pg/mL. In certain embodiments, the transposase is a Sleeping
Beauty
transposase. In certain embodiments, the Sleeping Beauty transposase is a
Sleeping Beauty
100X (SB100X) transposase.
[040] In certain embodiments of the methods of the disclosure, the nucleic
acid sequence
encoding the transposase enzyme is a RNA sequence, and an amount of the DNA
sequence
encoding the transposon is equal to or less than 5.0 pg per 100 pL of an
electroporation or
nucleofection reaction. In certain embodiments, a concentration of the amount
of the DNA
sequence encoding the transposon in the electroporation or nucleofection
reaction is equal to
or less than 50 pg/mL.
[041] In certain embodiments of the methods of the disclosure, the nucleic
acid sequence
encoding the transposase enzyme is a RNA sequence, and an amount of the DNA
sequence
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encoding the transposon is equal to or less than 2.5 pg per 100 pL of an
electroporation or
nucleofection reaction. In certain embodiments, a concentration of the amount
of the DNA
sequence encoding the transposon in the electroporation or nucleofection
reaction is equal to
or less than 25 pg/mL.
[042] In certain embodiments of the methods of the disclosure, the nucleic
acid sequence
encoding the transposase enzyme is a RNA sequence, and an amount of the DNA
sequence
encoding the transposon is equal to or less than 1.67 pg per 100 pL of an
electroporation or
nucleofection reaction. In certain embodiments, a concentration of the amount
of the DNA
sequence encoding the transposon in the electroporation or nucleofection
reaction is equal to
or less than 16.7 pg/mL. In certain embodiments, the transposase is a Super
piggyBac (PB)
transposase.
[043] In certain embodiments of the methods of the disclosure, the nucleic
acid sequence
encoding the transposase enzyme is a RNA sequence, and an amount of the DNA
sequence
encoding the transposon is equal to or less than 0.55 pg per 100 pL of an
electroporation or
nucleofection reaction. In certain embodiments, a concentration of the amount
of the DNA
sequence encoding the transposon in the electroporation or nucleofection
reaction is equal to
or less than 5.5 pg/mL.
[044] In certain embodiments of the methods of the disclosure, the nucleic
acid sequence
encoding the transposase enzyme is a RNA sequence, and an amount of the DNA
sequence
encoding the transposon is equal to or less than 0.19 pg per 100 pL of an
electroporation or
nucleofection reaction. In certain embodiments, a concentration of the amount
of the DNA
sequence encoding the transposon in the electroporation or nucleofection
reaction is equal to
or less than 1.9 pg/mL.
[045] In certain embodiments of the methods of the disclosure, the nucleic
acid sequence
encoding the transposase enzyme is a RNA sequence, and an amount of the DNA
sequence
encoding the transposon is equal to or less than 1.0 pg per 100 pL of an
electroporation or
nucleofection reaction. In certain embodiments, a concentration of the amount
of the DNA
sequence encoding the transposon in the electroporation or nucleofection
reaction is equal to
or less than 1.0 pg/mL.
[046] The disclosure provides an immune cell modified according to the method
of the
disclosure. The immune cell may be a T-lymphocyte, a Natural Killer (NK) cell,
a Cytokine-
induced Killer (CIK) cell or a Natural Killer T (NKT) cell. The immune cell
may be further
modified by a second gene editing tool, including, but not limited to those
gene editing tools
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comprising an endonuclease operably-linked to either a Cas9 or a TALE
sequence. In certain
embodiments of the second gene editing tool, the endonuclease is operably-
linked to either a
Cas9 or a TALE sequence covalently. In certain embodiments of the second gene
editing
tool, the endonuclease is operably-linked to either a Cas9 or a TALE sequence
non-
covalently. In certain embodiments, the Cas9 is an inactivated Cas9 (dCas9).
In certain
embodiments, the inactivated Cas9 comprises D1 OA and N580A within the
catalytic site. In
certain embodiments, the Cas9 is a small and inactivated Cas9 (dSaCas9). In
certain
embodiments, the dSaCas9 comprises the amino acid sequence of
1 mkrnyilglA igitsvgygi idyetrdvid agvrlfkean vennegrrsk rgarrlkrrr
61 rhriqrvkkl lfdynlltdh selsginpye arvkglsqkl seeefsaall hlakrrgvhn
121 vneveedtgn elstkeqisr nskaleekyv aelqlerlkk dgevrgsinr fktsdyvkea
181 kqllkvqkay hqldqsfidt yidlletrrt yyegpgegsp fgwkdikewy emlmghctyf
241 peelrsvkya ynadlynaln dlnnlvitrd enekleyyek fqiienvfkq kkkptlkqia
301 keilvneedi kgyrvtstgk peftnlkvyh dikditarke iienaelldq iakiltiyqs
361 sediqeeltn lnseltqeei eqisnlkgyt gthnlslkai nlildelwht ndnqiaifnr
421 lklvpkkvd1 sqqkeipttl vddfilspvv krsfiqsikv inaiikkygl pndiiielar
481 eknskdaqkm inemqkrnrq tnerieeiir ttgkenakyl iekiklhdmq egkclyslea
541 ipledllnnp fnyevdhiip rsysfdnsfn nkvlvkqeeA skkgnrtpfq ylsssdskis
601 yetfkkhiln lakgkgrisk tkkeylleer dinrfsvqkd finrnlvdtr yatrglmnll
661 rsyfrvnnld vkvksinggf tsflrrkwkf kkernkgykh haedaliian adfifkewkk
721 ldkakkvmen qmfeekqaes mpeieteqey keifitphqi khikdfkdyk yshrvdkkpn
781 relindtlys trkddkgntl ivnnlnglyd kdndklkkli nkspekllmy hhdpqtyqkl
841 klimeqygde knplykyyee tgnyltkysk kdngpvikki kyygnklnah lditddypns
901 rnkvvklslk pyrfdvyldn gvykfvtvkn ldvikkenyy evnskcyeea kklkkisnqa
961 efiasfynnd likingelyr vigvnndlln rievnmidit yreylenmnd krppriikti
1021 asktqsikky stdilgnlye vkskkhpqii kkg(SEX)IIMD:4).
[047] The disclosure provides an immune cell modified according to the method
of the
disclosure. The immune cell may be a T-lymphocyte, a Natural Killer (NK) cell,
a Cytokine-
induced Killer (CIK) cell or a Natural Killer T (NKT) cell. The immune cell
may be further
modified by a second gene editing tool, including, but not limited to those
gene editing tools
comprising an endonuclease operably-linked to either a Cas9 or a TALE
sequence.
Alternatively or in addition, the second gene editing tool may include an
excision-only
piggyBac transposase to re-excise the inserted sequences or any portion
thereof For example,
the excision-only piggyBac transposase may be used to "re-excise" the
transposon.
[048] The disclosure provides a composition comprising the immune cell of the
disclosure.
[049] The disclosure provides a use of a composition comprising the immune
cell of the
disclosure for the treatment of a disease or disorder in a subject in need
thereof In certain
embodiments, the disease or disorder is a cancer. In certain embodiments, the
disease or
disorder is an infectious disease. For example, the infectious disease may be
caused by a
virus, bacterium, yeast, microbe or any combination thereof In certain
embodiments, the
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immune cell of the composition is autologous. In certain embodiments, the
immune cell of
the composition is allogeneic.
[050] The disclosure provides a culture media for enhancing viability of a
modified immune
cell comprising IL-2, IL-21, IL-7, IL-15 or any combination thereof The
modified immune
cell may be a T-lymphocyte, a Natural Killer (NK) cell, a Cytokine-induced
Killer (CIK) cell
or a Natural Killer T (NKT) cell. The modified immune cell may contain one or
more
exogenous DNA sequences. The modified immune cell may contain one or more
exogenous
RNA sequences. The modified immune cell may have been electroporated or
nucleofected.
BRIEF DESCRIPTION OF THE DRAWINGS
[051] Figure 1 is a series of graphs depicting transfection efficiency and
cell viability
following plasmid DNA nucleofection in primary human T lymphocytes.
[052] Figure 2 is a series of graphs depicting DNA cytotoxicity to T cells.
[053] Figure 3 is a series of graphs showing that DNA-mediated cytotoxicity in
T cells is
dose dependent.
[054] Figure 4 is a series of graphs showing that extracellular plasmid DNA is
not
cytotoxic.
[055] Figure 5 is a series of graphs depicting efficient transposition using
sPBo mRNA in
Jurkat cells.
[056] Figure 6 is a series of graphs depicting efficient transposition in T
lymphocytes using
sPBo mRNA
[057] Figure 7 is a series of graphs depicting efficient delivery of
linearized DNA
transposon products.
[058] Figure 8 is a series of graphs showing that addition of that IL-7 and IL-
15 and
immediate stimulation of T cells post-nucleofection enhances cell viability.
[059] Figure 9 is a series of graphs showing that IL-7 and IL-15 rescue T
cells from DNA
mediated toxicity
[060] Figure 10 is a series of graphs showing that immediate stimulation of T
cells post-
nucleofection enhances cell viability.
[061] Figure 11A-C is a series of graphs depicting T cell transposition with
varying
amounts of DNA. Primary human pan T cells were nucleofected with varying
amounts of
DNA using piggyBacTM. T cells were nucleofected with the indicated amounts of
transposon
and 5 [ig sPBo mRNA. Cells were then stimulated on day 2 post-nucleofection
through CD3
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and CD28. As expected, T cells nucleofected with high amounts of DNA exhibited
high
episomal expression at day 1 post nucleofection whereas almost no episomal
expression was
observed at low DNA doses. In contrast, following expansion at day 21 post
nucleofection
the greatest percentage of transgene positive cells were observed in lower DNA
amounts
peaking at 1.67 [ig for this transposon. (A) Flow analysis for transgene
positive cells at day 1
and 21. (B) Percentage of transgene positive T cells. (C) Percentage of viable
T cells at day 1
and 21. For all graphs shown in this figure, the Y-axis ranges from 0 to 100%
in increments
of 20% and the X-axis ranges from 0 to 105 by powers of 10.
[062] Figure 12A-B is a series of graphs depicting T cell transposition with
low DNA
amounts using the Sleeping BeautyTM 100X (SB100X) transposase. Primary human
pan T
cells were nucleofected with GFP plasmids encoding either the piggyBacTM (PB)
or Sleeping
BeautyTM (SB) ITRs. (A) Cells were nucleofected with the indicated amounts of
SB
transposon and 1 [ig SB transposase mRNA. (B) Cells were nucleofected with the
indicated
amounts of SB transposase and 0.75 [ig SB transposon. Flow analysis was
performed on day
14 post nucleofection for all samples. For all graphs shown in this figure,
the Y-axis ranges
from 0 to 250K in increments of 50K and the X-axis ranges from 0 to 105 by
powers of 10.
DETAILED DESCRIPTION
[063] Disclosed are compositions and methods for the ex-vivo genetic
modification of an
immune cell comprising delivering to the immune cell, (a) a nucleic acid or
amino acid
sequence comprising a sequence encoding a transposase enzyme and (b) a
recombinant and
non-naturally occurring DNA sequence comprising a DNA sequence encoding a
transposon.
In certain embodiments, the method further comprises the step of stimulating
the immune cell
with one or more cytokine(s).
[064] Centyrins of the disclosure may comprise a protein scaffold, wherein the
scaffold is
capable of specifically binding an antigen. Centyrins of the disclosure may
comprise a protein
scaffold comprising a consensus sequence of at least one fibronectin type III
(FN3) domain,
wherein the scaffold is capable of specifically binding an antigen. The at
least one fibronectin
type III (FN3) domain may be derived from a human protein. The human protein
may be
Tenascin-C. The consensus sequence may comprise
LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDL
TGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT (SEQ ID NO: 5) or
MLPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYD
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LTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT (SEQ ID NO: 6). The consensus
sequence may comprise an amino sequence at least 74% identical to
LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDL
TGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT (SEQ ID NO: 5) or
MLPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYD
LTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT (SEQ ID NO: 6). The consensus
sequence may encoded by a nucleic acid sequence comprising
atgctgcctgcaccaaagaacctggtggtgtctcatgtgacagaggatagtgccagactgtcatggactgctcccgacg
cagccttcg
ataglittatcatcgtgtaccgggagaacatcgaaaccggcgaggccattgtcctgacagtgccagggtccgaacgctc
ttatgacctg
acagatctgaagcccggaactgagtactatgtgcagatcgccggcgtcaaaggaggcaatatcagcttccctctgtccg
caatcttcac
caca (SEQ ID NO: 7). The consensus sequence may be modified at one or more
positions
within (a) a A-B loop comprising or consisting of the amino acid residues TEDS
(SEQ ID
NO: 8) at positions 13-16 of the consensus sequence; (b) a B-C loop comprising
or consisting
of the amino acid residues TAPDAAF (SEQ ID NO: 9) at positions 22-28 of the
consensus
sequence; (c) a C-D loop comprising or consisting of the amino acid residues
SEKVGE (SEQ
ID NO: 10) at positions 38-43 of the consensus sequence; (d) a D-E loop
comprising or
consisting of the amino acid residues GSER (SEQ ID NO: 11) at positions 51-54
of the
consensus sequence; (e) a E-F loop comprising or consisting of the amino acid
residues
GLKPG (SEQ ID NO: 12) at positions 60-64 of the consensus sequence; (0 a F-G
loop
comprising or consisting of the amino acid residues KGGHRSN (SEQ ID NO: 13) at
positions 75-81 of the consensus sequence; or (g) any combination of (a)-(f).
Centyrins of the
disclosure may comprise a consensus sequence of at least 5 fibronectin type
III (FN3)
domains, at least 10 fibronectin type III (FN3) domains or at least 15
fibronectin type III
(FN3) domains. The scaffold may bind an antigen with at least one affinity
selected from a
KD of less than or equal to 10-9M, less than or equal to 10-1 M, less than or
equal to 10-11M,
less than or equal to 10-12m, less than or equal to 10-13M, less than or equal
to 10-14M, and
less than or equal to 10-15M. The KD may be determined by surface plasmon
resonance.
[065] The term "antibody mimetic" is intended to describe an organic compound
that
specifically binds a target sequence and has a structure distinct from a
naturally-occurring
antibody. Antibody mimetics may comprise a protein, a nucleic acid, or a small
molecule.
The target sequence to which an antibody mimetic of the disclosure
specifically binds may be
an antigen. Antibody mimetics may provide superior properties over antibodies
including, but
not limited to, superior solubility, tissue penetration, stability towards
heat and enzymes (e.g.
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resistance to enzymatic degradation), and lower production costs. Exemplary
antibody
mimetics include, but are not limited to, an affibody, an afflilin, an
affimer, an affitin, an
alphabody, an anticalin, and avimer (also known as avidity multimer), a DARPin
(Designed
Ankyrin Repeat Protein), a Fynomer, a Kunitz domain peptide, and a monobody.
[066] Affibody molecules of the disclosure comprise a protein scaffold
comprising or
consisting of one or more alpha helix without any disulfide bridges.
Preferably, affibody
molecules of the disclosure comprise or consist of three alpha helices. For
example, an
affibody molecule of the disclosure may comprise an immunoglobulin binding
domain. An
affibody molecule of the disclosure may comprise the Z domain of protein A.
[067] Affilin molecules of the disclosure comprise a protein scaffold produced
by
modification of exposed amino acids of, for example, either gamma-B crystallin
or ubiquitin.
Affilin molecules functionally mimic an antibody's affinity to antigen, but do
not structurally
mimic an antibody. In any protein scaffold used to make an affilin, those
amino acids that are
accessible to solvent or possible binding partners in a properly-folded
protein molecule are
considered exposed amino acids. Any one or more of these exposed amino acids
may be
modified to specifically bind to a target sequence or antigen.
[068] Affimer molecules of the disclosure comprise a protein scaffold
comprising a highly
stable protein engineered to display peptide loops that provide a high
affinity binding site for
a specific target sequence. Exemplary affimer molecules of the disclosure
comprise a protein
scaffold based upon a cystatin protein or tertiary structure thereof Exemplary
affimer
molecules of the disclosure may share a common tertiary structure of
comprising an alpha-
helix lying on top of an anti-parallel beta-sheet.
[069] Affitin molecules of the disclosure comprise an artificial protein
scaffold, the
structure of which may be derived, for example, from a DNA binding protein
(e.g. the DNA
binding protein Sac7d). Affitins of the disclosure selectively bind a target
sequence, which
may be the entirety or part of an antigen. Exemplary affitins of the
disclosure are
manufactured by randomizing one or more amino acid sequences on the binding
surface of a
DNA binding protein and subjecting the resultant protein to ribosome display
and selection.
Target sequences of affitins of the disclosure may be found, for example, in
the genome or on
the surface of a peptide, protein, virus, or bacteria. In certain embodiments
of the disclosure,
an affitin molecule may be used as a specific inhibitor of an enzyme. Affitin
molecules of the
disclosure may include heat-resistant proteins or derivatives thereof
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[070] Alphabody molecules of the disclosure may also be referred to as Cell-
Penetrating
Alphabodies (CPAB). Alphabody molecules of the disclosure comprise small
proteins
(typically of less than 10 kDa) that bind to a variety of target sequences
(including antigens).
Alphabody molecules are capable of reaching and binding to intracellular
target sequences.
Structurally, alphabody molecules of the disclosure comprise an artificial
sequence forming
single chain alpha helix (similar to naturally occurring coiled-coil
structures). Alphabody
molecules of the disclosure may comprise a protein scaffold comprising one or
more amino
acids that are modified to specifically bind target proteins. Regardless of
the binding
specificity of the molecule, alphabody molecules of the disclosure maintain
correct folding
and thermostability.
[071] Anticalin molecules of the disclosure comprise artificial proteins
that bind to target
sequences or sites in either proteins or small molecules. Anticalin molecules
of the disclosure
may comprise an artificial protein derived from a human lipocalin. Anticalin
molecules of
the disclosure may be used in place of, for example, monoclonal antibodies or
fragments
thereof Anticalin molecules may demonstrate superior tissue penetration and
thermostability
than monoclonal antibodies or fragments thereof Exemplary anticalin molecules
of the
disclosure may comprise about 180 amino acids, having a mass of approximately
20 kDa.
Structurally, anticalin molecules of the disclosure comprise a barrel
structure comprising
antiparallel beta-strands pairwise connected by loops and an attached alpha
helix. In preferred
embodiments, anticalin molecules of the disclosure comprise a barrel structure
comprising
eight antiparallel beta-strands pairwise connected by loops and an attached
alpha helix.
[072] Avimer molecules of the disclosure comprise an artificial protein
that specifically
binds to a target sequence (which may also be an antigen). Avimers of the
disclosure may
recognize multiple binding sites within the same target or within distinct
targets. When an
avimer of the disclosure recognize more than one target, the avimer mimics
function of a bi-
specific antibody. The artificial protein avimer may comprise two or more
peptide sequences
of approximately 30-35 amino acids each. These peptides may be connected via
one or more
linker peptides. Amino acid sequences of one or more of the peptides of the
avimer may be
derived from an A domain of a membrane receptor. Avimers have a rigid
structure that may
optionally comprise disulfide bonds and/or calcium. Avimers of the disclosure
may
demonstrate greater heat stability compared to an antibody.
[073] DARPins (Designed Ankyrin Repeat Proteins) of the disclosure comprise
genetically-engineered, recombinant, or chimeric proteins having high
specificity and high
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affinity for a target sequence. In certain embodiments, DARPins of the
disclosure are derived
from ankyrin proteins and, optionally, comprise at least three repeat motifs
(also referred to
as repetitive structural units) of the ankyrin protein. Ankyrin proteins
mediate high-affinity
protein-protein interactions. DARPins of the disclosure comprise a large
target interaction
surface.
[074] Fynomers of the disclosure comprise small binding proteins (about 7 kDa)
derived
from the human Fyn SH3 domain and engineered to bind to target sequences and
molecules
with equal affinity and equal specificity as an antibody.
[075] Kunitz domain peptides of the disclosure comprise a protein scaffold
comprising a
Kunitz domain. Kunitz domains comprise an active site for inhibiting protease
activity.
Structurally, Kunitz domains of the disclosure comprise a disulfide-rich
alpha+beta fold. This
structure is exemplified by the bovine pancreatic trypsin inhibitor. Kunitz
domain peptides
recognize specific protein structures and serve as competitive protease
inhibitors. Kunitz
domains of the disclosure may comprise Ecallantide (derived from a human
lipoprotein-
associated coagulation inhibitor (LAC)).
[076] Monobodies of the disclosure are small proteins (comprising about 94
amino acids
and having a mass of about 10 kDa) comparable in size to a single chain
antibody. These
genetically engineered proteins specifically bind target sequences including
antigens.
Monobodies of the disclosure may specifically target one or more distinct
proteins or target
sequences. In preferred embodiments, monobodies of the disclosure comprise a
protein
scaffold mimicking the structure of human fibronectin, and more preferably,
mimicking the
structure of the tenth extracellular type III domain of fibronectin. The tenth
extracellular type
III domain of fibronectin, as well as a monobody mimetic thereof, contains
seven beta sheets
forming a barrel and three exposed loops on each side corresponding to the
three
complementarity determining regions (CDRs) of an antibody. In contrast to the
structure of
the variable domain of an antibody, a monobody lacks any binding site for
metal ions as well
as a central disulfide bond. Multispecific monobodies may be optimized by
modifying the
loops BC and FG. Monobodies of the disclosure may comprise an adnectin.
[077] As used throughout the disclosure, the singular forms "a," "and," and
"the" include
plural referents unless the context clearly dictates otherwise. Thus, for
example, reference to
"a method" includes a plurality of such methods and reference to "a dose"
includes reference
to one or more doses and equivalents thereof known to those skilled in the
art, and so forth.
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[078] The term "about" or "approximately" means within an acceptable error
range for the
particular value as determined by one of ordinary skill in the art, which will
depend in part on
how the value is measured or determined, e.g., the limitations of the
measurement system.
For example, "about" can mean within 1 or more standard deviations.
Alternatively, "about"
can mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a
given value.
Alternatively, particularly with respect to biological systems or processes,
the term can mean
within an order of magnitude, preferably within 5-fold, and more preferably
within 2-fold, of
a value. Where particular values are described in the application and claims,
unless otherwise
stated the term "about" meaning within an acceptable error range for the
particular value
should be assumed.
[079] The disclosure provides isolated or substantially purified
polynucleotide or protein
compositions. An "isolated" or "purified" polynucleotide or protein, or
biologically active
portion thereof, is substantially or essentially free from components that
normally accompany
or interact with the polynucleotide or protein as found in its naturally
occurring environment.
Thus, an isolated or purified polynucleotide or protein is substantially free
of other cellular
material or culture medium when produced by recombinant techniques, or
substantially free
of chemical precursors or other chemicals when chemically synthesized.
Optimally, an
"isolated" polynucleotide is free of sequences (optimally protein encoding
sequences) that
naturally flank the polynucleotide (i.e., sequences located at the 5' and 3'
ends of the
polynucleotide) in the genomic DNA of the organism from which the
polynucleotide is
derived. For example, in various embodiments, the isolated polynucleotide can
contain less
than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide
sequence that naturally
flank the polynucleotide in genomic DNA of the cell from which the
polynucleotide is
derived. A protein that is substantially free of cellular material includes
preparations of
protein having less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of
contaminating
protein. When the protein of the invention or biologically active portion
thereof is
recombinantly produced, optimally culture medium represents less than about
30%, 20%,
10%, 5%, or 1% (by dry weight) of chemical precursors or non-protein-of-
interest chemicals.
[080] The disclosure provides fragments and variants of the disclosed DNA
sequences and
proteins encoded by these DNA sequences. As used throughout the disclosure,
the term
"fragment" refers to a portion of the DNA sequence or a portion of the amino
acid sequence
and hence protein encoded thereby. Fragments of a DNA sequence comprising
coding
sequences may encode protein fragments that retain biological activity of the
native protein
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and hence DNA recognition or binding activity to a target DNA sequence as
herein
described. Alternatively, fragments of a DNA sequence that are useful as
hybridization
probes generally do not encode proteins that retain biological activity or do
not retain
promoter activity. Thus, fragments of a DNA sequence may range from at least
about 20
nucleotides, about 50 nucleotides, about 100 nucleotides, and up to the full-
length
polynucleotide of the invention.
[081] Nucleic acids or proteins of the disclosure can be constructed by a
modular approach
including preassembling monomer units and/or repeat units in target vectors
that can
subsequently be assembled into a final destination vector. Polypeptides of the
disclosure may
comprise repeat monomers of the disclosure and can be constructed by a modular
approach
by preassembling repeat units in target vectors that can subsequently be
assembled into a
final destination vector. The disclosure provides polypeptide produced by this
method as well
nucleic acid sequences encoding these polypeptides. The disclosure provides
host organisms
and cells comprising nucleic acid sequences encoding polypeptides produced
this modular
approach.
[082] The term "antibody" is used in the broadest sense and specifically
covers single
monoclonal antibodies (including agonist and antagonist antibodies) and
antibody
compositions with polyepitopic specificity. It is also within the scope hereof
to use natural or
synthetic analogs, mutants, variants, alleles, homologs and orthologs (herein
collectively
referred to as "analogs") of the antibodies hereof as defined herein. Thus,
according to one
embodiment hereof, the term "antibody hereof' in its broadest sense also
covers such
analogs. Generally, in such analogs, one or more amino acid residues may have
been
replaced, deleted and/or added, compared to the antibodies hereof as defined
herein.
[083] "Antibody fragment", and all grammatical variants thereof, as used
herein are defined
as a portion of an intact antibody comprising the antigen binding site or
variable region of the
intact antibody, wherein the portion is free of the constant heavy chain
domains (i.e. CH2,
CH3, and CH4, depending on antibody isotype) of the Fc region of the intact
antibody.
Examples of antibody fragments include Fab, Fab', Fab'- SH, F(ab1)2, and Fv
fragments;
diabodies; any antibody fragment that is a polypeptide having a primary
structure consisting
of one uninterrupted sequence of contiguous amino acid residues (referred to
herein as a
"single-chain antibody fragment" or "single chain polypeptide"), including
without limitation
(1) single-chain Fv (scFv) molecules (2) single chain polypeptides containing
only one light
chain variable domain, or a fragment thereof that contains the three CDRs of
the light chain
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variable domain, without an associated heavy chain moiety and (3) single chain
polypeptides
containing only one heavy chain variable region, or a fragment thereof
containing the three
CDRs of the heavy chain variable region, without an associated light chain
moiety; and
multispecific or multivalent structures formed from antibody fragments. In an
antibody
fragment comprising one or more heavy chains, the heavy chain(s) can contain
any constant
domain sequence (e.g. CHI in the IgG isotype) found in a non-Fc region of an
intact
antibody, and/or can contain any hinge region sequence found in an intact
antibody, and/or
can contain a leucine zipper sequence fused to or situated in the hinge region
sequence or the
constant domain sequence of the heavy chain(s). The term further includes
single domain
antibodies ("sdAB") which generally refers to an antibody fragment having a
single
monomeric variable antibody domain, (for example, from camelids). Such
antibody fragment
types will be readily understood by a person having ordinary skill in the art.
[084] "Binding" refers to a sequence-specific, non-covalent interaction
between
macromolecules (e.g., between a protein and a nucleic acid). Not all
components of a binding
interaction need be sequence-specific (e.g., contacts with phosphate residues
in a DNA
backbone), as long as the interaction as a whole is sequence-specific.
[085] The term "comprising" is intended to mean that the compositions and
methods include
the recited elements, but do not exclude others. "Consisting essentially of"
when used to
define compositions and methods, shall mean excluding other elements of any
essential
significance to the combination when used for the intended purpose. Thus, a
composition
consisting essentially of the elements as defined herein would not exclude
trace contaminants
or inert carriers. "Consisting of shall mean excluding more than trace
elements of other
ingredients and substantial method steps. Embodiments defined by each of these
transition
terms are within the scope of this invention.
[086] The term "epitope" refers to an antigenic determinant of a polypeptide.
An epitope
could comprise three amino acids in a spatial conformation, which is unique to
the epitope.
Generally, an epitope consists of at least 4, 5, 6, or 7 such amino acids, and
more usually,
consists of at least 8, 9, or 10 such amino acids. Methods of determining the
spatial
conformation of amino acids are known in the art, and include, for example, x-
ray
crystallography and two-dimensional nuclear magnetic resonance.
[087] As used herein, "expression" refers to the process by which
polynucleotides are
transcribed into mRNA and/or the process by which the transcribed mRNA is
subsequently
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being translated into peptides, polypeptides, or proteins. If the
polynucleotide is derived from
genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.
[088] "Gene expression" refers to the conversion of the information, contained
in a gene,
into a gene product. A gene product can be the direct transcriptional product
of a gene (e.g.,
mRNA, tRNA, rRNA, antisense RNA, ribozyme, shRNA, micro RNA, structural RNA or
any other type of RNA) or a protein produced by translation of an mRNA. Gene
products
also include RNAs which are modified, by processes such as capping,
polyadenylation,
methylation, and editing, and proteins modified by, for example, methylation,
acetylation,
phosphorylation, ubiquitination, ADP-ribosylation, myristilation, and
glycosylation.
[089] "Modulation" or "regulation" of gene expression refers to a change in
the activity of a
gene. Modulation of expression can include, but is not limited to, gene
activation and gene
repression.
[090] The term "operatively linked" or its equivalents (e.g., "linked
operatively") means
two or more molecules are positioned with respect to each other such that they
are capable of
interacting to affect a function attributable to one or both molecules or a
combination thereof
[091] Non-covalently linked components and methods of making and using non-
covalently
linked components, are disclosed. The various components may take a variety of
different
forms as described herein. For example, non-covalently linked (i.e.,
operatively linked)
proteins may be used to allow temporary interactions that avoid one or more
problems in the
art. The ability of non-covalently linked components, such as proteins, to
associate and
dissociate enables a functional association only or primarily under
circumstances where such
association is needed for the desired activity. The linkage may be of duration
sufficient to
allow the desired effect.
[092] A method for directing proteins to a specific locus in a genome of an
organism is
disclosed. The method may comprise the steps of providing a DNA localization
component
and providing an effector molecule, wherein the DNA localization component and
the
effector molecule are capable of operatively linking via a non-covalent
linkage.
[093] The term "scFv" refers to a single-chain variable fragment. scFv is a
fusion protein of
the variable regions of the heavy (VH) and light chains (VL) of
immunoglobulins, connected
with a linker peptide. The linker peptide may be from about 5 to 40 amino
acids or from
about 10 to 30 amino acids or about 5, 10, 15, 20, 25, 30, 35, or 40 amino
acids in length.
Single-chain variable fragments lack the constant Fc region found in complete
antibody
molecules, and, thus, the common binding sites (e.g., Protein G) used to
purify antibodies.
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The term further includes a scFv that is an intrabody, an antibody that is
stable in the
cytoplasm of the cell, and which may bind to an intracellular protein.
[094] The term "single domain antibody" means an antibody fragment having a
single
monomeric variable antibody domain which is able to bind selectively to a
specific antigen.
A single-domain antibody generally is a peptide chain of about 110 amino acids
long,
comprising one variable domain (VH) of a heavy-chain antibody, or of a common
IgG, which
generally have similar affinity to antigens as whole antibodies, but are more
heat-resistant
and stable towards detergents and high concentrations of urea. Examples are
those derived
from camelid or fish antibodies. Alternatively, single-domain antibodies can
be made from
common murine or human IgG with four chains.
[095] The terms "specifically bind" and "specific binding" as used herein
refer to the ability
of an antibody, an antibody fragment or a nanobody to preferentially bind to a
particular
antigen that is present in a homogeneous mixture of different antigens. In
certain
embodiments, a specific binding interaction will discriminate between
desirable and
undesirable antigens in a sample, in some embodiments more than about ten- to
100-fold or
more (e.g., more than about 1000- or 10,000-fold). "Specificity" refers to the
ability of an
immunoglobulin or an immunoglobulin fragment, such as a nanobody, to bind
preferentially
to one antigenic target versus a different antigenic target and does not
necessarily imply high
affinity.
[096] A "target site" or "target sequence" is a nucleic acid sequence that
defines a portion of
a nucleic acid to which a binding molecule will bind, provided sufficient
conditions for
binding exist.
[097] The terms "nucleic acid" or "oligonucleotide" or "polynucleotide" refer
to at least two
nucleotides covalently linked together. The depiction of a single strand also
defines the
sequence of the complementary strand. Thus, a nucleic acid may also encompass
the
complementary strand of a depicted single strand. A nucleic acid of the
disclosure also
encompasses substantially identical nucleic acids and complements thereof that
retain the
same structure or encode for the same protein.
[098] Probes of the disclosure may comprise a single stranded nucleic acid
that can
hybridize to a target sequence under stringent hybridization conditions. Thus,
nucleic acids of
the disclosure may refer to a probe that hybridizes under stringent
hybridization conditions.
[099] Nucleic acids of the disclosure may be single- or double-stranded.
Nucleic acids of
the disclosure may contain double-stranded sequences even when the majority of
the
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molecule is single-stranded. Nucleic acids of the disclosure may contain
single-stranded
sequences even when the majority of the molecule is double-stranded. Nucleic
acids of the
disclosure may include genomic DNA, cDNA, RNA, or a hybrid thereof Nucleic
acids of the
disclosure may contain combinations of deoxyribo- and ribo-nucleotides.
Nucleic acids of the
disclosure may contain combinations of bases including uracil, adenine,
thymine, cytosine,
guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine. Nucleic
acids of the
disclosure may be synthesized to comprise non-natural amino acid
modifications. Nucleic
acids of the disclosure may be obtained by chemical synthesis methods or by
recombinant
methods.
[0100] Nucleic acids of the disclosure, either their entire sequence, or any
portion thereof,
may be non-naturally occurring. Nucleic acids of the disclosure may contain
one or more
mutations, substitutions, deletions, or insertions that do not naturally-
occur, rendering the
entire nucleic acid sequence non-naturally occurring. Nucleic acids of the
disclosure may
contain one or more duplicated, inverted or repeated sequences, the resultant
sequence of
which does not naturally-occur, rendering the entire nucleic acid sequence non-
naturally
occurring. Nucleic acids of the disclosure may contain modified, artificial,
or synthetic
nucleotides that do not naturally-occur, rendering the entire nucleic acid
sequence non-
naturally occurring.
[0101] Given the redundancy in the genetic code, a plurality of nucleotide
sequences may
encode any particular protein. All such nucleotides sequences are contemplated
herein.
[0102] As used throughout the disclosure, the term "operably linked" refers to
the expression
of a gene that is under the control of a promoter with which it is spatially
connected. A
promoter can be positioned 5' (upstream) or 3' (downstream) of a gene under
its control. The
distance between a promoter and a gene can be approximately the same as the
distance
between that promoter and the gene it controls in the gene from which the
promoter is
derived. Variation in the distance between a promoter and a gene can be
accommodated
without loss of promoter function.
[0103] As used throughout the disclosure, the term "promoter" refers to a
synthetic or
naturally-derived molecule which is capable of conferring, activating or
enhancing
expression of a nucleic acid in a cell. A promoter can comprise one or more
specific
transcriptional regulatory sequences to further enhance expression and/or to
alter the spatial
expression and/or temporal expression of same. A promoter can also comprise
distal
enhancer or repressor elements, which can be located as much as several
thousand base pairs
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from the start site of transcription. A promoter can be derived from sources
including viral,
bacterial, fungal, plants, insects, and animals. A promoter can regulate the
expression of a
gene component constitutively or differentially with respect to cell, the
tissue or organ in
which expression occurs or, with respect to the developmental stage at which
expression
occurs, or in response to external stimuli such as physiological stresses,
pathogens, metal
ions, or inducing agents. Representative examples of promoters include the
bacteriophage T7
promoter, bacteriophage T3 promoter, SP6 promoter, lac operator-promoter, tac
promoter,
SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, EF-
1
Alpha promoter, CAG promoter, SV40 early promoter or SV40 late promoter and
the CMV
IE promoter.
[0104] As used throughout the disclosure, the term "substantially
complementary" refers to a
first sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%,
98% or 99%
identical to the complement of a second sequence over a region of 8,9, 10, 11,
12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,
75, 80, 85, 90, 95,
100, 180, 270, 360, 450, 540, or more nucleotides or amino acids, or that the
two sequences
hybridize under stringent hybridization conditions.
[0105] As used throughout the disclosure, the term "substantially identical"
refers to a first
and second sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%,
98% or
99% identical over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24,
25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270,
360, 450, 540 or more
nucleotides or amino acids, or with respect to nucleic acids, if the first
sequence is
substantially complementary to the complement of the second sequence.
[0106] As used throughout the disclosure, the term "variant" when used to
describe a nucleic
acid, refers to (i) a portion or fragment of a referenced nucleotide sequence;
(ii) the
complement of a referenced nucleotide sequence or portion thereof; (iii) a
nucleic acid that is
substantially identical to a referenced nucleic acid or the complement
thereof; or (iv) a
nucleic acid that hybridizes under stringent conditions to the referenced
nucleic acid,
complement thereof, or a sequences substantially identical thereto.
[0107] As used throughout the disclosure, the term "vector" refers to a
nucleic acid sequence
containing an origin of replication. A vector can be a viral vector,
bacteriophage, bacterial
artificial chromosome or yeast artificial chromosome. A vector can be a DNA or
RNA vector.
A vector can be a self-replicating extrachromosomal vector, and preferably, is
a DNA
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plasmid. A vector may comprise a combination of an amino acid with a DNA
sequence, an
RNA sequence, or both a DNA and an RNA sequence.
[0108] As used throughout the disclosure, the term "variant" when used to
describe a peptide
or polypeptide, refers to a peptide or polypeptide that differs in amino acid
sequence by the
insertion, deletion, or conservative substitution of amino acids, but retain
at least one
biological activity. Variant can also mean a protein with an amino acid
sequence that is
substantially identical to a referenced protein with an amino acid sequence
that retains at least
one biological activity.
[0109] A conservative substitution of an amino acid, i.e., replacing an amino
acid with a
different amino acid of similar properties (e.g., hydrophilicity, degree and
distribution of
charged regions) is recognized in the art as typically involving a minor
change. These minor
changes can be identified, in part, by considering the hydropathic index of
amino acids, as
understood in the art. Kyte et al., J. Mol. Biol. 157: 105-132 (1982). The
hydropathic index of
an amino acid is based on a consideration of its hydrophobicity and charge.
Amino acids of
similar hydropathic indexes can be substituted and still retain protein
function. In one aspect,
amino acids having hydropathic indexes of 2 are substituted. The
hydrophilicity of amino
acids can also be used to reveal substitutions that would result in proteins
retaining biological
function. A consideration of the hydrophilicity of amino acids in the context
of a peptide
permits calculation of the greatest local average hydrophilicity of that
peptide, a useful
measure that has been reported to correlate well with antigenicity and
immunogenicity. U.S.
Patent No. 4,554,101, incorporated fully herein by reference.
[0110] Substitution of amino acids having similar hydrophilicity values can
result in peptides
retaining biological activity, for example immunogenicity. Substitutions can
be performed
with amino acids having hydrophilicity values within 2 of each other. Both
the
hyrophobicity index and the hydrophilicity value of amino acids are influenced
by the
particular side chain of that amino acid. Consistent with that observation,
amino acid
substitutions that are compatible with biological function are understood to
depend on the
relative similarity of the amino acids, and particularly the side chains of
those amino acids, as
revealed by the hydrophobicity, hydrophilicity, charge, size, and other
properties.
[0111] As used herein, "conservative" amino acid substitutions may be defined
as set out in
Tables A, B, or C below. In some embodiments, fusion polypeptides and/or
nucleic acids
encoding such fusion polypeptides include conservative substitutions have been
introduced
by modification of polynucleotides encoding polypeptides of the invention.
Amino acids can
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be classified according to physical properties and contribution to secondary
and tertiary
protein structure. A conservative substitution is a substitution of one amino
acid for another
amino acid that has similar properties. Exemplary conservative substitutions
are set out in
Table A.
[0112] Table A -- Conservative Substitutions I
Side chain characteristics Amino Acid
Aliphatic Non-polar GAPILVF
Polar-uncharged CSTMNQ
Polar- charged DEKR
Aromatic HFWY
Other NQDE
[0113] Alternately, conservative amino acids can be grouped as described in
Lehninger,
(Biochemistry, Second Edition; Worth Publishers, Inc. NY, N.Y. (1975), pp. 71-
77) as set
forth in Table B.
[0114] Table B -- Conservative Substitutions II
Side Chain Characteristic Amino Acid
Non-polar (hydrophobic) Aliphatic: ALIVP
Aromatic: F W Y
Sulfur-containing:
Borderline: G Y
Uncharged-polar Hydroxyl: S T Y
Amides: NQ
Sulfhydryl:
Borderline: G Y
Positively Charged (Basic): K R H
Negatively Charged (Acidic): D E
[0115] Alternately, exemplary conservative substitutions are set out in Table
C.
[0116] Table C -- Conservative Substitutions III
Original Residue Exemplary Substitution
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Ala (A) Val Leu Ile Met
Arg (R) Lys His
Asn (N) Gln
Asp (D) Glu
Cys (C) Ser Thr
Gln (Q) Asn
Glu (E) Asp
Gly (G) Ala Val Leu Pro
His (H) Lys Arg
Ile (I) Leu Val Met Ala Phe
Leu (L) Ile Val Met Ala Phe
Lys (K) Arg His
Met (M) Leu Ile Val Ala
Phe (F) Trp Tyr Ile
Pro (P) Gly Ala Val Leu Ile
Ser (S) Thr
Thr (T) Ser
Trp (W) Tyr Phe Ile
Tyr (Y) Trp Phe Thr Ser
Val (V) Ile Leu Met Ala
[0117] It should be understood that the polypeptides of the disclosure are
intended to include
polypeptides bearing one or more insertions, deletions, or substitutions, or
any combination
thereof, of amino acid residues as well as modifications other than
insertions, deletions, or
substitutions of amino acid residues. Polypeptides or nucleic acids of the
disclosure may
contain one or more conservative substitution.
[0118] As used throughout the disclosure, the term "more than one" of the
aforementioned
amino acid substitutions refers to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, or
20 or more of the recited amino acid substitutions. The term "more than one"
may refer to 2,
3, 4, or 5 of the recited amino acid substitutions.
[0119] Polypeptides and proteins of the disclosure, either their entire
sequence, or any
portion thereof, may be non-naturally occurring. Polypeptides and proteins of
the disclosure
may contain one or more mutations, substitutions, deletions, or insertions
that do not
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naturally-occur, rendering the entire amino acid sequence non-naturally
occurring.
Polypeptides and proteins of the disclosure may contain one or more
duplicated, inverted or
repeated sequences, the resultant sequence of which does not naturally-occur,
rendering the
entire amino acid sequence non-naturally occurring. Polypeptides and proteins
of the
disclosure may contain modified, artificial, or synthetic amino acids that do
not naturally-
occur, rendering the entire amino acid sequence non-naturally occurring.
[0120] As used throughout the disclosure, "sequence identity" may be
determined by using
the stand-alone executable BLAST engine program for blasting two sequences
(b12seq),
which can be retrieved from the National Center for Biotechnology Information
(NCBI) ftp
site, using the default parameters (Tatusova and Madden, FEMS Microbiol Lett.,
1999, 174,
247-250; which is incorporated herein by reference in its entirety). The terms
"identical" or
"identity" when used in the context of two or more nucleic acids or
polypeptide sequences,
refer to a specified percentage of residues that are the same over a specified
region of each of
the sequences. The percentage can be calculated by optimally aligning the two
sequences,
comparing the two sequences over the specified region, determining the number
of positions
at which the identical residue occurs in both sequences to yield the number of
matched
positions, dividing the number of matched positions by the total number of
positions in the
specified region, and multiplying the result by 100 to yield the percentage of
sequence
identity. In cases where the two sequences are of different lengths or the
alignment produces
one or more staggered ends and the specified region of comparison includes
only a single
sequence, the residues of single sequence are included in the denominator but
not the
numerator of the calculation. When comparing DNA and RNA, thymine (T) and
uracil (U)
can be considered equivalent. Identity can be performed manually or by using a
computer
sequence algorithm such as BLAST or BLAST 2Ø
[0121] As used throughout the disclosure, the term "endogenous" refers to
nucleic acid or
protein sequence naturally associated with a target gene or a host cell into
which it is
introduced.
[0122] As used throughout the disclosure, the term "exogenous" refers to
nucleic acid or
protein sequence not naturally associated with a target gene or a host cell
into which it is
introduced, including non-naturally occurring multiple copies of a naturally
occurring nucleic
acid, e.g., DNA sequence, or naturally occurring nucleic acid sequence located
in a non-
naturally occurring genome location.
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[0123] The disclosure provides methods of introducing a polynucleotide
construct
comprising a DNA sequence into a host cell. By "introducing" is intended
presenting to the
plant the polynucleotide construct in such a manner that the construct gains
access to the
interior of the host cell. The methods of the invention do not depend on a
particular method
for introducing a polynucleotide construct into a host cell, only that the
polynucleotide
construct gains access to the interior of one cell of the host. Methods for
introducing
polynucleotide constructs into bacteria, plants, fungi and animals are known
in the art
including, but not limited to, stable transformation methods, transient
transformation
methods, and virus-mediated methods.
[0124] By "stable transformation" is intended that the polynucleotide
construct introduced
into a plant integrates into the genome of the host and is capable of being
inherited by
progeny thereof By "transient transformation" is intended that a
polynucleotide construct
introduced into the host does not integrate into the genome of the host.
[0125] As used throughout the disclosure, the term "genetically modified plant
(or transgenic
plant)" refers to a plant which comprises within its genome an exogenous
polynucleotide.
Generally, and preferably, the exogenous polynucleotide is stably integrated
into the genome
such that the polynucleotide is passed on to successive generations. The
exogenous
polynucleotide may be integrated into the genome alone or as part of a
recombinant
expression cassette. "Transgenic" is used herein to include any cell, cell
line, callus, tissue,
plant part or plant, the genotype of which has been altered by the presence of
exogenous
nucleic acid including those trans genies initially so altered as well as
those created by sexual
crosses or asexual propagation from the initial transgenic. The term
"transgenic" as used
herein does not encompass the alteration of the genome (chromosomal or extra-
chromosomal) by conventional plant breeding methods or by naturally occurring
events such
as random cross-fertilization, non-recombinant viral infection, non-
recombinant bacterial
transformation, non-recombinant transposition, or spontaneous mutation.
[0126] As used throughout the disclosure, the term "modifying" is intended to
mean that the
sequence is considered modified simply by the binding of the polypeptide. It
is not intended
to suggest that the sequence of nucleotides is changed, although such changes
(and others)
could ensue following binding of the polypeptide to the nucleic acid of
interest. In some
embodiments, the nucleic acid sequence is DNA. Modification of the nucleic
acid of interest
(in the sense of binding thereto by a polypeptide modified to contain modular
repeat units)
could be detected in any of a number of methods (e.g. gel mobility shift
assays, use of
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labelled polypeptides--labels could include radioactive, fluorescent, enzyme
or
biotin/streptavidin labels). Modification of the nucleic acid sequence of
interest (and
detection thereof) may be all that is required (e.g. in diagnosis of disease).
Desirably,
however, further processing of the sample is performed. Conveniently the
polypeptide (and
nucleic acid sequences specifically bound thereto) is separated from the rest
of the sample.
Advantageously the polypeptide-DNA complex is bound to a solid phase support,
to facilitate
such separation. For example, the polypeptide may be present in an acrylamide
or agarose gel
matrix or, more preferably, is immobilized on the surface of a membrane or in
the wells of a
microtitre plate.
[0127] All percentages and ratios are calculated by weight unless otherwise
indicated.
[0128] All percentages and ratios are calculated based on the total
composition unless
otherwise indicated.
[0129] Every maximum numerical limitation given throughout this disclosure
includes every
lower numerical limitation, as if such lower numerical limitations were
expressly written
herein. Every minimum numerical limitation given throughout this disclosure
will include
every higher numerical limitation, as if such higher numerical limitations
were expressly
written herein. Every numerical range given throughout this disclosure will
include every
narrower numerical range that falls within such broader numerical range, as if
such narrower
numerical ranges were all expressly written herein.
[0130] The values disclosed herein are not to be understood as being strictly
limited to the
exact numerical values recited. Instead, unless otherwise specified, each such
value is
intended to mean both the recited value and a functionally equivalent range
surrounding that
value. For example, a value disclosed as "20 m" is intended to mean "about 20
m."
[0131] Every document cited herein, including any cross referenced or related
patent or
application, is hereby incorporated herein by reference in its entirety unless
expressly
excluded or otherwise limited. The citation of any document is not an
admission that it is
prior art with respect to any invention disclosed or claimed herein or that it
alone, or in any
combination with any other reference or references, teaches, suggests or
discloses any such
invention. Further, to the extent that any meaning or definition of a term in
this document
conflicts with any meaning or definition of the same term in a document
incorporated by
reference, the meaning or definition assigned to that term in this document
shall govern.
[0132] While particular embodiments of the disclosure have been illustrated
and described,
various other changes and modifications can be made without departing from the
spirit and
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scope of the disclosure. The scope of the appended claims includes all such
changes and
modifications that are within the scope of this disclosure.
EXAMPLES
[0133] In order that the invention disclosed herein may be more efficiently
understood,
examples are provided below. It should be understood that these examples are
for illustrative
purposes only and are not to be construed as limiting the invention in any
manner.
Throughout these examples, molecular cloning reactions, and other standard
recombinant
DNA techniques, were carried out according to methods described in Maniatis et
al.,
Molecular Cloning - A Laboratory Manual, 2nd ed., Cold Spring Harbor Press
(1989), using
commercially available reagents, except where otherwise noted.
EXAMPLE 1: Ex vivo genetic modification of T cells
[0134] The piggyBacTM (PB) transposon system was used for genetically
modifying human
lymphocytes for production of autologous CAR-T immunotherapies and other
applications.
T Lymphocytes purified from patient blood or apheresis product was
electroporated with a
plasmid DNA transposon and a transposase. Several different electroporation
systems have
been used for T cell delivery of the transposon system, including the Neon
(Thermo Fisher),
BTX ECM 830 (Harvard Apparatus), Gene Pulser (BioRad), MaxCyte PulseAgile
(MaxCyte), and the Amaxa 2B and Amaxa 4D (Lonza). Some were tested using
manufacturer provided or recommended electroporation buffer, as well as
several in-house
developed buffers. Results were consistent with the prevailing dogma that
resting T
lymphocytes are particularly refractory to DNA transfection and that there
appeared to be an
inverse relationship between electroporation efficiency, as measured by GFP
expression from
the electroporated plasmid, and cell viability. Figure 1 shows an example of
an experiment
testing multiple electroporation systems and nucleofection programs.
[0135] To further test whether or not plasmid DNA was toxic to T cells during
nucleofection,
primary human T lymphocytes were electroporated with two different DNA
plasmids. The
first plasmid was a pmaxGFPTM plasmid that is provided as a control plasmid in
the Lonza
Amaxa nucleofection kit. It is highly purified by HPLC and does not contain
endotoxin at
detectable levels. The second plasmid was our in-house produced PB transposon
encoding a
human EF1 alpha promoter driving GFP. Transfection efficiency, as measured by
GFP
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expression from the electroporated plasmid, and cell viability was assessed by
FACS at days
2, 3, and 6 post-electroporation. Data are displayed in Figure 2. While mock
electroporated
cells (no plasmid DNA) exhibited relatively high levels of cell viability by
day 6 post-
electroporation, 54%, T cells electroporated with either plasmid were only 1.4-
2.6% viable.
These data show that plasmid DNA was cytotoxic to T lymphocytes. In addition,
these data
show that DNA-mediated toxicity was not due to transposon element such as the
ITR regions
or the core insulators since the pmaxGFPTM plasmid are devoid of these
elements and was
also cytotoxic at the same DNA concentration. Both plasmids are approximately
the same
size, meaning that similar amounts of DNA were electroporated into the T
cells.
[0136] To test whether or not DNA-mediated toxicity in T cells was dose
dependent, we
performed a titration of our PB-GFP plasmid. Figure 3 shows that as the dose
of plasmid
DNA added to the nucleofection reaction was increased incrementally (1.3, 2.5,
5.0, 10.0, and
20.0 [ig of plasmid DNA), cell viability decreased as measured at both day 1
and 5 post-
nucleofection. Even 1.3 [ig of plasmid DNA was responsible for a 2.4-fold
decrease in T cell
viability by day 4.
[0137] Since it was clear that plasmid DNA is toxic to T cells during
nucleofection, we
considered whether or not extracellular plasmid DNA was contributing to cell
death. Figure
4 shows that extracellular plasmid DNA was not cytotoxic to T cells. In that
experiment, 5
[ig of plasmid DNA was added to the cells 45 min post-electroporation and
little cell death
was observed at day 1 or day 4. Similarly, when 5 [ig of plasmid DNA was added
to the
nucleofection reaction in the absence of electroporation, little cell death
was observed.
However, when the plasmid DNA was added before the electroporation reaction,
the cells
exhibited a 2.0-fold reduction in cell viability at day 1 and a 13.2-fold
reduction at day 4.
[0138] Since DNA-mediated toxicity is dose dependent, we next focused our
attention on
ways to reduce the total amount of DNA delivered to the T cells that is
required for
transposition. One relatively straightforward way of achieving this would be
to deliver the
transposase as encoded in mRNA instead of encoded in DNA. mRNA delivery to
primary
human T cells is very efficient, resulting in high transfection efficiency and
high viability.
We subcloned the Super piggyBacTM (sPBo) transposase enzyme into our in-house
mRNA
production vector and produced high quality sPBo mRNA. Co-delivery of PB-GFP
transposon with various doses of sPBo mRNA (30, 10, 3.3, 3, 1, 0.33 [ig mRNA)
in Jurkat
cells demonstrated strong transposition at all doses tested (Figure 5). These
data show that
sPBo transposase can be delivered and are equally effective as either plasmid
DNA or
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mRNA. In addition, that the amount of sPBo mRNA makes little difference in
overall
transposition efficiency in Jurkats, in either overall percentage of GFP+
cells or in the MFI of
GFP expression. To see if this also holds true for T lymphocytes, we delivered
PB-GFP with
either sBPo plasmid DNA, at a 3:1 ratio, or 5 lig of sPBo mRNA. Seven (7) days
following
the nucleofection reaction and the addition of IL7 and IL15, GFP transposition
was assessed.
Figure 6 shows that sPBo mRNA efficiently mediated transposition of the GFP
transposon
into T lymphocytes. Importantly, T cell viability was improved when co-
delivering the sPBo
as an mRNA as opposed to a pDNA; 32.4% versus 25.4%, respectively. These data
suggest
that co-delivery of sPBo as mRNA would be dose-sparing in the total amount of
plasmid
DNA being delivered to T cells and is thus less cytotoxic.
[0139] Since the current plasmid transposon also contains a backbone required
for plasmid
amplification in bacteria, it is possible to significantly reduce the total
amount of DNA by
excluding this sequence. This may be achieved by restriction digest of the
plasmid
transposon prior to the nucleofection reaction. In addition, this could be
achieved by
administering the transposon as a PCR product or as a doggyboneTM DNA, which
is a double
stranded DNA that is produced in vitro by a mechanism that excludes the
initial backbone
elements required for bacterial replication of the plasmid.
[0140] We performed a pilot experiment to see whether or not plasmid
transposon needed to
be circular, or if it could be delivered to the cell in a linear fashion. To
test this, transposon
was incubated overnight with a restriction enzyme (ApaLI) to linearize the
plasmid. Either
uncut or linearized plasmid was electroporated into primary T lymphocytes and
GFP
expression was assessed 2 days later. Figure 7 shows that linearized plasmid
was also
efficiently delivered to the cell nucleus. These data demonstrate that linear
transposon
products can also be efficiently electroporated into primary human T cells.
[0141] We show above that plasmid DNA is toxic in primary T lymphocytes, but
we have
observed that this toxic effect is not as dramatic in tumor cell lines and
other transformed
cells. Based upon this observation, we hypothesized that primary T lymphocytes
may be
refractory to plasmid DNA transfection due to heightened DNA sensing pathways,
which
would protect immune cells from infection by viruses and bacteria. If these
data are a result
of heightened DNA sensing mechanisms, then it may be possible to enhance
plasmid
transfection efficiency and/or cell viability by the addition of DNA sensing
pathway
inhibitors to the post-nucleofection reaction. Thus, we tested a number of
different reagents
that inhibited the TLR-9 pathway, caspase pathway, or those involved in
cytoplasmic double
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stranded DNA sensing. These reagents include Bafilomycin Al, which is an
autophagy
inhibitor that interferes with endosomal acidification and blocks NFkB
signaling by TLR9,
Chloroquine, which is a TLR9 antagonist, Quinacrine, which is a TLR9
antagonist and a
cGAS antagonist, AC-YVAD-CMK, which is a caspase 1 inhibitor targeting the
AIM2
pathway, Z-VAD-FMK, which is a pan caspase inhibitor, Z-IETD-FMK, which is a
caspase 8
inhibitor triggered by the TLR9 pathway. In addition, we also tested the
stimulation of
electroporated T cells by the addition of the cytokines IL7 and IL15, as well
as the addition of
anti-CD3 anti-CD28 Dynabeads0 Human T-Expander CD3/CD28 beads. Results are
displayed in Figure 8. We found that few of the compounds or caspase
inhibitors had any
positive effect on cell viability at day 4 post-nucleofection at the doses
tested. However, we
acknowledge that further dosing studies may be required to better test these
reagents. It may
also be more effective to inhibit these pathways genetically. Two post-
nucleofection
conditions did enhance viability of the T cells. The addition of IL7 and IL15,
whether they
were added either 1 hour or 1 day following electroporation, enhanced
viability over 3-fold
when compared with introduction of the plasmid transposon alone without
additional
treatment. Furthermore, stimulation of the T cells post-nucleofection using
either activator or
expander beads also dramatically enhanced T cell viability; stimulation was
better when the
beads were added 1 hour or 1 day post-nucleofection as compared to adding them
2 days
post. Lastly, we also tested ROCK inhibitor and the removal of dead cells from
the culture
using the Dead Cell Removal kit from Miltenyi, but saw no improvement in cell
viability.
[0142] To further expand upon these findings demonstrating that stimulation of
the T cells
post-nucleofection improves viability, we repeated the study using the
addition of the
cytokine IL7 and IL15. Figure 9 shows that the addition of these cytokines
each at a dose of
20 ng/mL either immediately following nucleofection or up to 1 hour post
enhanced cell
viability up to 2.9-fold when compared to no treatment. Addition of these
cytokines up to 1
day post-nucleofection also enhanced viability, but not as strong as the prior
time points.
[0143] Since we found that immediate stimulation of the T cells post-
nucleofection was able
to increase cell viability, we hypothesized that stimulating the cells prior
to nucleofection
may also enhance viability and transfection efficiency. To test this, we
stimulated primary T
lymphocytes either 2, 3, or 4 days prior to transposon nucleofection. Figure
10 shows that
some level of transposition occurs when the transposon and the transposase are
co-delivered
after the T cells have been stimulated prior to the nucleofection reaction.
The efficacy of pre-
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stimulation may be influenced by the kinetics of stimulation and may therefore
be dependent
upon the precise type of expander technology chosen.
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