AU2017224111A1 - Transposon system and methods of use - Google Patents

Transposon system and methods of use Download PDF

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AU2017224111A1
AU2017224111A1 AU2017224111A AU2017224111A AU2017224111A1 AU 2017224111 A1 AU2017224111 A1 AU 2017224111A1 AU 2017224111 A AU2017224111 A AU 2017224111A AU 2017224111 A AU2017224111 A AU 2017224111A AU 2017224111 A1 AU2017224111 A1 AU 2017224111A1
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David Hermanson
Eric Ostertag
Devon SHEDLOCK
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Poseida Therapeutics Inc
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Abstract

Disclosed are methods for the

Description

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 antibodytargeted 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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PCT/US2017/019531 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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PCT/US2017/019531 transposase enzyme may be produced in vitro. A protein sPBo may be delivered following pre-incubation with transposon DNA.
[Oil] 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 Tlymphocyte. 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 Τ (NKT) cell.
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PCT/US2017/019531 [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 piggyBac™ (sPBo) transposase enzyme. The Super piggyBac (PB) transposase enzyme may comprise or consist of an amino acid sequence at least 75% identical to: MGSSLDDEHILSALLQSDDELVGEDSDSEVSDHVSEDDVQSDTEEAFIDEVHEVQPTS SGSEILDEQNVIEQPGSSLASNRILTLPQRTIRGKNKHCWSTSKSTRRSRVSALNIVRS QRGPTRMCRNIYDPLLCFKLFFTDEIISEIVKWTNAEISLKRRESMTSATFRDTNEDEI YAFFGILVMTAVRKDNHMSTDDLFDRSLSMVYVSVMSRDRFDFLIRCLRMDDKSIR PTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRVYIPNKPSKY GIKILMMCDSGTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDN WFTSIPLAKNLLQEPYKLTIVGTVRSNKREIPEVLKNSRSRPVGTSMFCFDGPLTLVSY KPKPAKMVYLLSSCDEDASINESTGKPQMVMYYNQTKGGVDTLDQMCSVMTCSRK TNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMSLTSSFMRKR LEAPTLKRYLRDNISNILPKEVPGTSDDSTEEPVMKKRTYCTYCPSKIRRKANASCKK 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: MGKSKEISQDLRKKIVDLHKSGSSLGAISKRLKVPRSSVQTIVRKYKHHGTTQPSYRS GRRRYLSPRDERTLVRKVQINPRTTAKDLVKMLEETGTKVSISTVKRVLYRHNLKGR SARKKPLLQNRHKKARLRFATAHGDKDRTFWRNVLWSDETKIELFGHNDHRYVWR KKGEACKPKNTIPTVKHGGGSIMLWGCFAAGGTGALHKIDGIMRKENYVDILKQHL KTSVRKLKLGRKWVFQMDNDPKHTSKVVAKWLKDNKVKVLEWPSQSPDLNPIENL WAELKKRVRARRPTNLTQLHQLCQEEWAKIHPTYCGKLVEGYPKRLTQVKQFKGN ATKY (SEQ ID NO: 2). In certain embodiments, including those wherein the Sleeping
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PCT/US2017/019531
Beauty transposase is a hyperactive Sleeping Beauty SBIOOXtransposase, 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 nonnaturally 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 nonnaturally 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 mini circle 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 doggy bone™ DNA sequence. Doggybone™ 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 nonnaturally 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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PCT/US2017/019531 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, CD35, CD3c, CD3y, Οϋβζ, 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, CD35, CD3c, CD3y, Οϋ3ζ, 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 CD3ζ 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-IBB 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 nonnaturally 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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PCT/US2017/019531 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 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 100 pg/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 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 75 pg/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 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 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.
[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 pg per 100 pL of an electroporation or nucleofection reaction. In certain embodiments, a concentration of the amount of the DNA sequence
-7WO 2017/147538
PCT/US2017/019531 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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PCT/US2017/019531 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 -9WO 2017/147538
PCT/US2017/019531 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 Cytokineinduced Killer (CIK) cell or a Natural Killer Τ (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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PCT/US2017/019531 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 noncovalently. In certain embodiments, the Cas9 is an inactivated Cas9 (dCas9). In certain embodiments, the inactivated Cas9 comprises D10A 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 mkrnyilglA igitsvgygi idyetrdvid agvrlfkean vermegrrsk rgarrlkrrr 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 lklvpkkvdl sqqkeipttl vddfilspvv krsfiqsikv inaiikkygl pndiiielar
481 eknskdaqkm inemqkrnrq tnerieeiir ttgkenakyl iekiklhdmq egkclyslea
541 ipledllnnp fnyevdhiip rsvsfdnsfn 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 (SEQ ID NO: 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 Cytokineinduced 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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PCT/US2017/019531 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 postnucleofection 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 piggyBac™. T cells were nucleofected with the indicated amounts of transposon and 5 pg sPBo mRNA. Cells were then stimulated on day 2 post-nucleofection through CD3
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PCT/US2017/019531 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 pg 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 Beauty™ 100X (SB100X) transposase. Primary human pan T cells were nucleofected with GFP plasmids encoding either the piggyBac™ (PB) or Sleeping Beauty™ (SB) ITRs. (A) Cells were nucleofected with the indicated amounts of SB transposon and 1 pg SB transposase mRNA. (B) Cells were nucleofected with the indicated amounts of SB transposase and 0.75 pg 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 atgctgcctgcaccaaagaacctggtggtgtctcatgtgacagaggatagtgccagactgtcatggactgctcccgacgcagccttcg atagttttatcatcgtgtaccgggagaacatcgaaaccggcgaggccattgtcctgacagtgccagggtccgaacgctcttatgacctg acagatctgaagcccggaactgagtactatgtgcagatcgccggcgtcaaaggaggcaatatcagcttccctctgtccgcaatcttcac 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; (f) 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 l0M. less than or equal to 10 M. less than or equal to 10 l2M. less than or equal to 10 l3M. less than or equal to 10 l4M. and less than or equal to 10 l5M. 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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PCT/US2017/019531 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 alphahelix 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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PCT/US2017/019531 [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 bispecific 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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PCT/US2017/019531 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 lipoproteinassociated coagulation inhibitor (LACI)).
[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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PCT/US2017/019531 [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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PCT/US2017/019531 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(ab')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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PCT/US2017/019531 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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PCT/US2017/019531 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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PCT/US2017/019531 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 deoxy ribo- 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 nonnaturally 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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PCT/US2017/019531 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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PCT/US2017/019531 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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PCT/US2017/019531 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 GAPIL VF
Polar - uncharged C S TMNQ
Polar - charged DEKR
Aromatic HF WY
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 WY
Sulfur-containing: M
Borderline: GY
Uncharged-polar Hydroxyl: STY
Amides: NQ
Sulfhydryl: C
Borderline: GY
Positively Charged (Basic): KRH
Negatively Charged (Acidic): DE
[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
He (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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PCT/US2017/019531 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 naturallyoccur, 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 (bl2seq), 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 2.0.
[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 nonnaturally occurring genome location.
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PCT/US2017/019531 [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 extrachromosomal) 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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PCT/US2017/019531 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 pm” is intended to mean “about 20 pm.” [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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PCT/US2017/019531 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 mManiatis etal., 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 piggyBac™ (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 pmaxGFP™ 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 EFl alpha promoter driving GFP. Transfection efficiency, as measured by GFP
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PCT/US2017/019531 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 postelectroporation, 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 pmaxGFP™ 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 pg of plasmid DNA), cell viability decreased as measured at both day 1 and 5 postnucleofection. Even 1.3 pg 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 pg 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 pg 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 piggyBac™ (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 pg 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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PCT/US2017/019531 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 pg of sPBo mRNA. Seven (7) days following the nucleofection reaction and the addition of IL7 and IL 15, 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 doggybone™ 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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PCT/US2017/019531 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 IL 15, as well as the addition of anti-CD3 anti-CD28 Dynabeads® 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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PCT/US2017/019531 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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Claims (109)

  1. What is claimed is:
    1. A 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.
  2. 2. The method of claim 1, wherein the sequence encoding a transposase enzyme is an mRNA sequence.
  3. 3. The method of claim 1, wherein the sequence encoding a transposase enzyme is a DNA sequence.
  4. 4. The method of claim 1, wherein the sequence encoding a transposase enzyme is an amino acid sequence.
  5. 5. The method of any one of claims 1-4, wherein the delivering step comprises electroporation or nucleofection of the immune cell.
  6. 6. The method of any one of claims 1-5, wherein the method further comprises the step of stimulating the immune cell with one or more cytokine(s).
  7. 7. The method of claim 6, wherein the step of stimulating the immune cell with one or more cytokine(s) occurs following the delivering step.
  8. 8. The method of claim 6, wherein the step of stimulating the immune cell with one or more cytokine(s) occurs prior to the delivering step.
  9. 9. The method of any one of claims 6-8, wherein the one or more cytokine(s) comprise(s) IL-2, IL-21, IL-7 and/or IL-15.
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  10. 10. The method of any one of the preceding claims, wherein the immune cell is an autologous immune cell.
  11. 11. The method of any one of claims 1-10, wherein the immune cell is a T-lymphocyte.
  12. 12. The method of any one of claims 1-10, wherein the immune cell is a Natural Killer (NK) cell.
  13. 13. The method of any one of claims 1-10, wherein the immune cell is a Cytokineinduced Killer (CIK) cell.
  14. 14. The method of any one of claims 1-10, wherein the immune cell is a Natural Killer T (NKT) cell.
  15. 15. The method of any one of claims 2 and 5-14, wherein the mRNA sequence encoding a transposase enzyme is produced in vitro.
  16. 16. The method of any one of the preceding claims, wherein the transposase enzyme is a Super piggyBac™ (sPBo) transposase enzyme.
  17. 17. The method of claim 16, wherein the Super piggyBac (PB) transposase enzyme comprises an amino acid sequence at least 75% identical to:
    MGSSLDDEHILSALLQSDDELVGEDSDSEVSDHVSEDDVQSDTEEAFIDEVHEVQPTS SGSEILDEQNVIEQPGSSLASNRILTLPQRTIRGKNKHCWSTSKSTRRSRVSALNIVRS QRGPTRMCRNIYDPLLCFKLFFTDEIISEIVKWTNAEISLKRRESMTSATFRDTNEDEI YAFFGILVMTAVRKDNHMSTDDLFDRSLSMVYVSVMSRDRFDFLIRCLRMDDKSIR
    PTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRVYIPNKPSKY
    GIKILMMCDSGTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDN
    WFTSIPLAKNLLQEPYKLTIVGTVRSNKREIPEVLKNSRSRPVGTSMFCFDGPLTLVSY
    KPKPAKMVYLLSSCDEDASINESTGKPQMVMYYNQTKGGVDTLDQMCSVMTCSRK
    TNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMSLTSSFMRKR
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    LEAPTLKRYLRDNISNILPKEVPGTSDDSTEEPVMKKRTYCTYCPSKIRRKANASCKK CKKVICREHNIDMCQSCF (SEQ ID NO: 1).
  18. 18. The method of any one of claims 1-15, wherein the transposase enzyme is a Sleeping Beauty transposase enzyme.
  19. 19. The method of claim 18, wherein the Sleeping Beauty transposase is a hyperactive Sleeping Beauty SB 100X transposase.
  20. 20. The method of claim 18 or 19, wherein the Sleeping Beauty transposase enzyme comprises an amino acid sequence at least 75% identical to:
    MGKSKEISQDLRKKIVDLHKSGSSLGAISKRLKVPRSSVQTIVRKYKHHGTTQ PSYRSGRRRYLSPRDERTLVRKVQINPRTTAKDLVKMLEETGTKVSISTVKRVLYRH NLKGRSARKKPLLQNRHKKARLRFATAHGDKDRTFWRNVLWSDETKIELFGHNDH RYVWRKKGEACKPKNTIPTVKHGGGSIMLWGCFAAGGTGALHKIDGIMRKENYVDI LKQHLKTSVRKLKLGRKWVFQMDNDPKHTSKVVAKWLKDNKVKVLEWPSQSPDL NPIENLWAELKKRVRARRPTNLTQLHQLCQEEWAKIHPTYCGKLVEGYPKRLTQVK QFKGNATKY (SEQ ID NO: 2).
  21. 21. The method of claim 18 or 19, wherein the Sleeping Beauty transposase enzyme comprises an amino acid sequence at least 75% identical to:
    MGKSKEISQDLRKRIVDLHKSGSSLGAISKRLAVPRSSVQTIVRKYKHHGTTQ PSYRSGRRRYLSPRDERTLVRKVQINPRTTAKDLVKMLEETGTKVSISTVKRVLYRH NLKGHSARKKPLLQNRHKKARLRFATAHGDKDRTFWRNVLWSDETKIELFGHNDH RYVWRKKGEACKPKNTIPTVKHGGGSIMLWGCFAAGGTGALHKIDGIMDAVQYVD ILKQHLKTSVRKLKLGRKWVFQHDNDPKHTSKVVAKWLKDNKVKVLEWPSQSPDL NPIENLWAELKKRVRARRPTNLTQLHQLCQEEWAKIHPNYCGKLVEGYPKRLTQVK QFKGNATKY (SEQ ID NO: 3).
  22. 22. The method of any one of the preceding claims, wherein the recombinant and nonnaturally occurring DNA sequence comprising a DNA sequence encoding a transposon is circular.
    -38WO 2017/147538
    PCT/US2017/019531
  23. 23. The method of claim 22, wherein the recombinant and non-naturally occurring DNA sequence encoding a transposon is contained in a plasmid vector.
  24. 24. The method of claim 22, wherein the recombinant and non-naturally occurring DNA sequence encoding a transposon is contained in a minicircle DNA vector.
  25. 25. The method of any one of claims 1-21, wherein the recombinant and non-naturally occurring DNA sequence encoding a transposon is linear.
  26. 26. The method of any one of the preceding claims, wherein the recombinant and nonnaturally occurring DNA sequence encoding a transposon is produced in vitro.
  27. 27. The method of claim 25 or 26, wherein the recombinant and non-naturally occurring DNA sequence encoding a transposon is a product of a restriction digest of a circular DNA.
  28. 28. The method of claim 27, wherein the circular DNA is a plasmid vector or a minicircle DNA vector
  29. 29. The method of claim 25 or 26, wherein the recombinant and non-naturally occurring DNA sequence encoding a transposon is a product of a polymerase chain reaction (PCR).
  30. 30. The method of claim 25 or 26, wherein the recombinant and non-naturally occurring DNA sequence encoding a transposon is a double-stranded doggybone™ DNA sequence
  31. 31. The method of claim 30, wherein the doggybone™ DNA sequence is produced by an enzymatic process that solely encodes an antigen expression cassette, comprising antigen, promoter, poly-A tail and telomeric ends.
  32. 32. The method of any one of the preceding claims, wherein the immune cell is isolated or derived from a human.
  33. 33. The method of any one of claims 1-31, wherein the immune cell is isolated or derived from a non-human mammal.
    -39WO 2017/147538
    PCT/US2017/019531
  34. 34. The method of claim 33, wherein the non-human mammal is a rodent, a rabbit, a cat, a dog, a pig, a horse, a cow, a camel or a primate.
  35. 35. The method of any one of the preceding claims, wherein the recombinant and nonnaturally occurring DNA sequence encoding a transposon further comprises a sequence encoding a chimeric antigen receptor or a portion thereof.
  36. 36. The method of claim 35, wherein the portion of the sequence encoding a chimeric antigen receptor encodes an antigen recognition region.
  37. 37. The method of claim 35 or 36, wherein the antigen recognition region comprises one or more complementarity determining region(s).
  38. 38. The method of claim 35 or 36, wherein the antigen recognition region comprises an antibody, an antibody mimetic, a protein scaffold or a fragment thereof.
  39. 39. The method of claim 38, wherein the antibody is a chimeric antibody, a recombinant antibody, a humanized antibody or a human antibody.
  40. 40. The method of claim 39, wherein the antibody is affinity-tuned.
  41. 41. The method of claim 38, wherein the antibody comprises or consists of a single-chain variable fragment (scFv), a VHH, a single domain antibody (sdAB), a small modular immunopharmaceutical (SMIP) molecule or a nanobody.
  42. 42. The method of claim 41, wherein the VHH is camelid.
  43. 43. The method of claim 41 or 42, wherein the VHH is humanized.
  44. 44. The method of claim 38, wherein the antibody fragment comprises or consists of a complementary determining region, a variable region, a heavy chain, a light chain, or any combination thereof.
    -40WO 2017/147538
    PCT/US2017/019531
  45. 45. The method of claim 38, wherein the antibody mimetic comprises or consists of an affibody, an afflilin, an affimer, an affitin, an alphabody, an anticalin, an avimer, a DARPin, a Fynomer, a Kunitz domain peptide, or a monobody.
  46. 46. The method of claim 38, wherein the protein scaffold comprises or consists of Centyrin.
  47. 47. The method of any one of claims 5-46, (a) wherein the nucleic acid sequence encoding the transposase enzyme is a DNA sequence, and (b) wherein 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 pg per 100 pL of an electroporation or nucleofection reaction.
  48. 48. The method of claim 48, wherein a concentration of the amount of the DNA sequence encoding the transposase enzyme and the amount of the DNA sequence encoding the transposon in the electroporation or nucleofection reaction is equal to or less than 100 pg/mL.
  49. 49. The method of any one of claims 5-46, (a) wherein the nucleic acid sequence encoding the transposase enzyme is a DNA sequence, and (b) wherein 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 pg per 100 pL of an electroporation or nucleofection reaction.
  50. 50. The method of claim 49, wherein a concentration of the amount of the DNA sequence encoding the transposase enzyme and the amount of the DNA sequence encoding the transposon in the electroporation or nucleofection reaction is equal to or less than 75 pg/mL.
  51. 51. The method of any one of claims 5-46,
    -41WO 2017/147538
    PCT/US2017/019531 (a) wherein the nucleic acid sequence encoding the transposase enzyme is a DNA sequence, and (b) wherein 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 pg per 100 pL of an electroporation or nucleofection reaction.
  52. 52. The method of claim 51, wherein a concentration of the amount of the DNA sequence encoding the transposase enzyme and the amount of the DNA sequence encoding the transposon in the electroporation or nucleofection reaction is equal to or less than 60 pg/mL.
  53. 53. The method of claim 50 or 51, wherein the transposase is a Sleeping Beauty transposase.
  54. 54. The method of claim 53, wherein the Sleeping Beauty transposase is a Sleeping Beauty 100X (SB 100X) transposase.
  55. 55. The method of any one of claims 5-46, (a) wherein the nucleic acid sequence encoding the transposase enzyme is a DNA sequence, and (b) wherein 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 pg per 100 pL of an electroporation or nucleofection reaction.
  56. 56. The method of claim 55, wherein a concentration of the amount of the DNA sequence encoding the transposase enzyme and the amount of the DNA sequence encoding the transposon in the electroporation or nucleofection reaction is equal to or less than 50 pg/mL.
  57. 57. The method of any one of claims 5-46, (a) wherein the nucleic acid sequence encoding the transposase enzyme is a DNA sequence, and (b) wherein 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.
    -42WO 2017/147538
    PCT/US2017/019531
  58. 58. The method of claim 57, wherein a concentration of the amount of the DNA sequence encoding the transposase enzyme and the amount of the DNA sequence encoding the transposon in the electroporation or nucleofection reaction is equal to or less than 25 pg/mL.
  59. 59. The method of any one of claims 5-46, (a) wherein the nucleic acid sequence encoding the transposase enzyme is a DNA sequence, and (b) wherein 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.
  60. 60. The method of claim 59, wherein a concentration of the amount of the DNA sequence encoding the transposase enzyme and 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.
  61. 61. The method of claim 59 or 60, wherein the transposase is a Super piggyBac (PB) transposase.
  62. 62. The method of any one of claims 5-46, (a) wherein the nucleic acid sequence encoding the transposase enzyme is a DNA sequence, and (b) wherein 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.
  63. 63. The method of claim 62, wherein a concentration of the amount of the DNA sequence encoding the transposase enzyme and 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.
  64. 64. The method of any one of claims 5-46,
    -43WO 2017/147538
    PCT/US2017/019531 (a) wherein the nucleic acid sequence encoding the transposase enzyme is a DNA sequence, and (b) wherein 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.
  65. 65. The method of claim 64, wherein a concentration of the amount of the DNA sequence encoding the transposase enzyme and 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.
  66. 66. The method of any one of claims 5-46, (a) wherein the nucleic acid sequence encoding the transposase enzyme is a DNA sequence, and (b) wherein 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 0.1 pg per 100 pL of an electroporation or nucleofection reaction.
  67. 67. The method of claim 66, wherein a concentration of the amount of the DNA sequence encoding the transposase enzyme and 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.
  68. 68. The method of any one of claims 5-46, (a) wherein the nucleic acid sequence encoding the transposase enzyme is a RNA sequence, and (b) wherein an DNA sequence encoding the transposon is equal to or less than 10 pg per 100 pL of an electroporation or nucleofection reaction.
  69. 69. The method of claim 68, wherein 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.
  70. 70. The method of any one of claims 5-46,
    -44WO 2017/147538
    PCT/US2017/019531 (a) wherein the nucleic acid sequence encoding the transposase enzyme is a RNA sequence, and (b) wherein an DNA sequence encoding the transposon is equal to or less than 7.5 pg per 100 pL of an electroporation or nucleofection reaction.
  71. 71. The method of claim 70, wherein 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.
  72. 72. The method of any one of claims 5-46, (a) wherein the nucleic acid sequence encoding the transposase enzyme is a RNA sequence, and (b) wherein an DNA sequence encoding the transposon is equal to or less than 6.0 pg per 100 pL of an electroporation or nucleofection reaction.
  73. 73. The method of claim 72, wherein 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.
  74. 74. The method of claim 72 or 73, wherein the transposase is a Sleeping Beauty transposase.
  75. 75. The method of claim 74, wherein the Sleeping Beauty transposase is a Sleeping Beauty 100X (SB 100X) transposase.
  76. 76. The method of any one of claims 5-46, (a) wherein the nucleic acid sequence encoding the transposase enzyme is a RNA sequence, and (b) wherein an DNA sequence encoding the transposon is equal to or less than 5.0 pg per 100 pL of an electroporation or nucleofection reaction.
    -45WO 2017/147538
    PCT/US2017/019531
  77. 77. The method of claim 76, wherein 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.
  78. 78. The method of any one of claims 5-46, (a) wherein the nucleic acid sequence encoding the transposase enzyme is a RNA sequence, and (b) wherein an DNA sequence encoding the transposon is equal to or less than 2.5 pg per 100 pL of an electroporation or nucleofection reaction.
  79. 79. The method of claim 78, wherein 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.
  80. 80. The method of any one of claims 5-46, (a) wherein the nucleic acid sequence encoding the transposase enzyme is a RNA sequence, and (b) wherein an DNA sequence encoding the transposon is equal to or less than 1.67 pg per 100 pL of an electroporation or nucleofection reaction.
  81. 81. The method of claim 80, wherein 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.
  82. 82. The method of claim 80 or 81, wherein the transposase is a Super piggyBac (PB) transposase.
  83. 83. The method of any one of claims 5-46, (a) wherein the nucleic acid sequence encoding the transposase enzyme is a RNA sequence, and (b) wherein an DNA sequence encoding the transposon is equal to or less than 0.55 pg per 100 pL of an electroporation or nucleofection reaction.
    -46WO 2017/147538
    PCT/US2017/019531
  84. 84. The method of claim 83, wherein 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.
  85. 85. The method of any one of claims 5-46, (a) wherein the nucleic acid sequence encoding the transposase enzyme is a RNA sequence, and (b) wherein an DNA sequence encoding the transposon is equal to or less than 0.19 pg per 100 pL of an electroporation or nucleofection reaction.
  86. 86. The method of claim 85, wherein 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.
  87. 87. The method of any one of claims 5-46, (a) wherein the nucleic acid sequence encoding the transposase enzyme is a RNA sequence, and (b) wherein an DNA sequence encoding the transposon is equal to or less than 0.1 pg per 100 pL of an electroporation or nucleofection reaction.
  88. 88. The method of claim 87, wherein 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.
  89. 89. An immune cell modified according to the method of any one of claims 1-88.
  90. 90. The immune cell of claim 89, wherein the immune cell is a T-lymphocyte, a Natural Killer (NK) cell, a Cytokine-induced Killer (CIK) cell or a Natural Killer T (NKT) cell.
  91. 91. The immune cell of claim 89 or 90, further modified by a second gene editing tool.
  92. 92. The immune cell of claim 91, wherein the second gene editing tool comprises an endonuclease operably-linked to either a Cas9 or a TALE sequence.
    -47WO 2017/147538
    PCT/US2017/019531
  93. 93. The immune cell of claim 92, wherein the endonuclease is operably-linked to either a Cas9 or a TALE sequence covalently.
  94. 94. The immune cell of claim 92, wherein the endonuclease is operably-linked to either a Cas9 or a TALE sequence non-covalently.
  95. 95. The immune cell of any one of claims 89-94, wherein the Cas9 is an inactivated Cas9 (dCas9).
  96. 96. The immune cell of claim 95, wherein the inactivated Cas9 comprises D10A and N580A within the catalytic site.
  97. 97. The immune cell of claim 95 or 96, wherein the Cas9 is a small and inactivated Cas9 (dSaCas9).
  98. 98. The immune cell of claim 97, wherein the dSaCas9 comprises the amino acid sequence of mkmyilglaigitsvgygiidyetrdvidagvrlfkeanvennegrrskrgarrlkrrrrhriqrvkkllfdynlltdhselsginpyea rvkglsqklseeefsaallhlakrrgvhnvneveedtgnelstkeqismskaleekyvaelqlerlkkdgevrgsinrfktsdyvke akqllkvqkayhqldqsfidtyidlletrrtyyegpgegspfgwkdikewyemlmghctyfpeelrsvkyaynadlynalndln nlvitrdenekleyyekfqiienvfkqkkkptlkqiakeilvneedikgyrvtstgkpeftnlkvyhdikditarkeiienaelldqia kiltiyqssediqeeltnlnseltqeeieqisnlkgytgthnlslkainlildelwhtndnqiaifnrlklvpkkvdlsqqkeipttlvddfi lspvvkrsfiqsikvinaiikkyglpndiiielareknskdaqkminemqkmrqtnerieeiirttgkenakyliekiklhdmqegk clysleaipledllnnpfiiyevdhiiprsvsfdnsfiinkvlvkqeeaskkgnrtpfqylsssdskisyetfkkhilnlakgkgrisktk keylleerdinrfsvqkdfinmlvdtryatrglmnllrsyfrvnnldvkvksinggftsflrrkwkfkkemkgykhhaedaliiana dfifkewkkldkakkvmenqmfeekqaesmpeieteqeykeifitphqikhikdfkdykyshrvdkkpnrelindtlystrkdd kgntlivnnlnglydkdndklkklinkspekllmyhhdpqtyqklklimeqygdeknplykyyeetgnyltkyskkdngpvik kikyygnklnahlditddypnsmkvvklslkpyrfdvyldngvykfvtvknldvikkenyyevnskcyeeakklkkisnqaefi asfynndlikingelyrvigvnndllnrievnmidityreylenmndkrppriiktiasktqsikkystdilgnlyevkskkhpqiik kg (SEQ ID NO: 4).
  99. 99. A composition comprising the immune cell according to any one of claims 89-98.
    -48WO 2017/147538
    PCT/US2017/019531
  100. 100. The use of the composition according to claim 99 for the treatment of a disease or disorder in a subject in need thereof.
  101. 101. The use of claim 100, wherein the disease or disorder is a cancer.
  102. 102. The use of claim 100, wherein the disease or disorder is an infectious disease.
  103. 103. The use of any one of claims 99-102, wherein the immune cell is autologous.
  104. 104. The use of any one of claims 99-102, wherein the immune cell is allogeneic.
  105. 105. A culture media for enhancing viability of a modified immune cell comprising IL-2, IL-21, IL-7, IL-15 or any combination thereof.
  106. 106. The culture media of claim 105, wherein the modified immune cell is a Tlymphocyte, a Natural Killer (NK) cell, a Cytokine-induced Killer (CIK) cell or a Natural Killer T (NKT) cell.
  107. 107. The culture media of claim 105 or 106, wherein the modified immune cell contains one or more exogenous DNA sequences.
  108. 108. The culture media of claim 105 or 106, wherein the modified immune cell contains one or more exogenous RNA sequences.
  109. 109. The culture media of any one of claims 105-108, wherein the modified immune cell has been electroporated or nucleofected.
    -49WO 2017/147538
    PCT/US2017/019531
    1/34 >
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    Neon electroporation system 4E6 100μΙ Solution T 2400V/20ms/1 pulse 03 LO Gold-plated tip Lonza 4D nucleofector 1E6 20μΙ (16 well strip) P3 kit Several recommended CT -Λί. Polymer electrodes Cell number Transfection volume Transfection solution Transfection program DNA amount Electroporation technology
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    FIGURE 2
    DAY 2
    7AAD 7AAD 7AAD
    DNA is cytotoxic to T ceiis
    10ug pMAX Plamsid
    10ug GFP Transposon
    0 50K 100K 150K 200K 250K 0 103 104 105
    FSC
    GFP
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    FIGURE 2 (cont.)
    DNA is cytotoxic to T cells
    DAYS
    7AAD 7AAD 7AAD
    10gg pMAX Plasmid
    0 SOK 100K 150K 200K 250K
    10gg GFP Transposon
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    7AAD 7AAD 7AAD
    DAY 6
    FIGURE 2 (cont.)
    DNA is cytotoxic to T celis
    10(ug pMAX Plasmid
    10pg GFP Transposon
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    FIGURE 3
    DNA-mediated cytotoxicity is dose dependent
    DAY 1 20gg Transposon
    10(ug Transposon
    0 SOK 100K 150K 200K 250K 0 103 104 105
    FSC GFP*
    5pg Transposon
    0 SOK 100K 150K 200K 250K
    FSC
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    FIGURE 3 (cont.)
    DNA-mediated cytotoxicity is dose dependent
    2.5gg Transposon
    6/34
    DAY1
    1,3gg Transposon
    GFP*
    FSC
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    FIGURE 3 (cont.)
    DNA-mediated cytotoxicity is dose dependent
    7/34
    DAYS
    20iig Transposon
    FSC GFP*
    10(ug Transposon
    0 SOK 100K 150K 200K 250K 0 103 104 105
    FSC GFP+
    5tug Transposon
    0 SOK 100K 150K 200K 250K
    FSC
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    FIGURE 3 (cont.)
    DNA-mediated cytotoxicity is dose dependent
    8/34
    DAYS
    2.5gg Transposon
    FSC
    50K 100K 150K 200K 250K
    1,3gg Transposon
    Mock
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    FIGURE 3 (cont.)
    DNA-mediated cytotoxicity is dose dependent
    DNA Toxicity in T cells
    Transfection Efficacy (%)
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    FIGURE 4 block Nucleofection and 5μο DNA to media added 45 later
    DAY 1 Live/Dead
    GFP*
    0 102 103 104 105
    No Nucleofection and 5μο DNA added to media
    Nucleofection with 5μο DNA
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    FIGURE 4 (cont.) 11/34 block Nucleofection and 5μο DNA to media added 45 later
    DAY 4 Live/Dead GFP*
    No Nucleofection and 5μο DNA added to media
    Nucleofection with 5μο DNA
    Live/Dead GFP+
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    FIGURE 6
    T cell Transposition with sPBo plasmid DNA or mRNA c: o < O CM O o CD Q_ σι to
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    FIGURE 7
    PB002G 5ug PB002G 5 ug + sPBo (3:1) ”” >
    nucleofection addition of beads)
    GFP + 72.9 -·¥ S S J S J J
    ί Ύ f Ύ i Γ Tf Ύ i Sj£ Γ Tf T Ϊ Ύ’Τ,Ύ θ o CD CD θ LO g> LO CD LO CM CM τ—
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    OSS
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    FIGURE 8
    Testing of additional post-nucleofection conditions (Day 4)
    15/34
    PB-EF1a-GFP 10ug (Untouched)
    Quinacrine 50uM
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    FIGURE 8
    Testing of additional post-nucleofection conditions (Day 4)
    16/34
    Bafilomycin A1 50nM
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    Testing of additional post-nucleofection conditions (Day 4)
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    Bafilomycin A1 5nM
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    Testing of additional post-nucleofection conditions (Day 4)
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    Testing of additional post-nucleofection conditions (Day 4)
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    Chloroquine 10uM
    0 SOK 100K 150K 200K 250K
    FSC
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    FIGURE 8
    Testing of additional post-nucleofection conditions (Day 4)
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    FIGURE 8
    Testing of additional post-nucleofection conditions (Day 4)
    21/34
    SUBSTITUTE SHEET (RULE 26)
    WO 2017/147538
    PCT/US2017/019531
    22/34
    FIGURE 8
    Testing of additional post-nucleofection conditions (Day 4)
    Z-ETD-FmK2um
    0 50K WOK 150K 200K 250K
    FSC
    SUBSTITUTE SHEET (RULE 26)
    WO 2017/147538
    PCT/US2017/019531
    FIGURE 8
    Testing of additional post-nucleofection conditions (Day 4)
    23/34
    20ng/mL IL15/L7 1 hr post
    FSC
    GFP*
    SUBSTITUTE SHEET (RULE 26)
    WO 2017/147538
    PCT/US2017/019531
    24/34
    FIGURE 8
    Testing of additional post-nucleofection conditions (Day 4)
    AH samples are donor #59 nucloefected on the 2B with program U-014 and
    1 Dug PB-EF1-GFP (except for mock). Five reactions were performed with the PB-EF1-GFP and were pooled and then split into 18 samples.
    SUBSTITUTE SHEET (RULE 26)
    WO 2017/147538
    PCT/US2017/019531
    25/34
    OS w fe
    CO $
    O
    o χ S E o 5= Ό o''·’ CO co O OJ
    • θ * KO : c-i •
    i/3 o oo c: o o CT. 00 o_ cz CD E ro •Hr™’ CL. CD LL. JO O 05 o _______i CT E Σ3 CT ch ·*“ CZ .CT CO CM
    awz
    V-O o'CO ’ssj”
    }»oc{cco{gC.»{>.aE co co i
    awz
    SUBSTITUTE SHEET (RULE 26)
    WO 2017/147538
    PCT/US2017/019531
    26/34
    FSC GFP+ FSC GFP +
    IL7/IL15 were each dosed at 20ng/mL at the time points shown Cells were transfected with 10ug of GFP transposon
    FIGURE 9 (cont.)
    SUBSTITUTE SHEET (RULE 26)
    WO 2017/147538
    PCT/US2017/019531
    27/34
    FIGURE 10
    T Cell Transposition of pre-stimulated cells Mock PB002G 5ug (T-023) tn θ
    FSC GFP +
    (Λ Ό c- ro G> s d o O 71777 d eg co Σ5 Q. E cs X. ω OO <D Ό ro c£ ω M_ o ro sn ro d >Q Q oo CM CM CO Q O
    awz awz
    SUBSTITUTE SHEET (RULE 26)
    WO 2017/147538
    PCT/US2017/019531
    28/34
    1 + iLO % LL-θ'3 CU 1 1
    FIGURE 10 (cont)
    T Cell Transposition of pre-stimulated cells Mock PB002G 5ug (T-023) r sc:
    Day 5 Post-Nucleofection
    ‘ i J ‘ O> o U I j 3 i O θ . ί-γCM O “ΤΤγ“ΤΊΤΊΓ“ O O> IO o lo o θ 04 QQ ’
    awz . θ „ Ο-* . U3 awz
    SUBSTITUTE SHEET (RULE 26)
    WO 2017/147538
    29/34
    PCT/US2017/019531
    FIGURE 10 (cont)
    T Cell Transposition of pre-stimulated cells Mock PB002G 5ug (T-023) j
    τ—ρτηΊΠΓΊ!—γπτγγίγτ---ρτηΓΓτητρτητΓίη
    Λ CL·4^ ll
    4- Ό n . LL.O e00
    awz awz
    SUBSTITUTE SHEET (RULE 26)
    WO 2017/147538
    PCT/US2017/019531
    30/34 w
    fe
    SUBSTITUTE SHEET (RULE 26)
    WO 2017/147538
    PCT/US2017/019531
    31/34 z-°°°X
    CO CO CQ ¢/0 θ co co cm
    SUBSTITUTE SHEET (RULE 26)
    WO 2017/147538
    PCT/US2017/019531
    CM
    IB
    32/34
    XZZZZZZZZZZZZZZZZZZZ7
    FIGURE 11 (cont.)
    Ο
    C □ ο
    Ε
    GJ
    Ο)
    C
    S SSSSS
    2?
    >
    r θ co ^ΖΖΖεΖΫ22ΖΪ7Ζ2ΖΖΖΖΖδ
    4Ζ ο
    Ο /ρ· ο
    ϊϋΖ7ΖΖΖΖΖΣ2ΖΖΜ/ΕΕΖΕ7Λ ~
    ---...................................................................... 41
    ΕΖΖΖΖΞΖΖ^ .
    θ
    WZZZZZZZZlZZZZZZZmZ £Ζ
    Ο <
    £Ζ
    Ο σ> ο ο. σ> c:
    X 900001
    BS
    C ο
    X 900001 ω
    ο Q.
    W £ <β
    Η ~Ί----------1----------1Γ θ θ θθ
    CO CO ’<ίCN (%) S||901 θνβιλ (%) S||90 1 9AR!S0d ο
    W3 Ο
    Ο., co ε:: to
    CQ
    SUBSTITUTE SHEET (RULE 26)
    WO 2017/147538
    PCT/US2017/019531
    33/34
    FIGURE 12
    A T cell Transposition with low DNA amounts using another transposase
    Transposon 0 pg PB'. 0.84 pg SS:Syg LO
    cd o o cd CD CD ID o UD CD UD CM CM τ— τ™
    1 1 i 1 1 isC ' “ 1 1 ' ‘Ύ*¥“|Ύ*Τ k *| I CD CD CD CD CD UD CD UD CD UD CM CM X™ •τ™
    :: tn C~~ CC o O Z5 O Q_ o E M CD < o CD CO O Q ZD
    sc: cd CD CD CD CD o Wj CD UD CD UD CM CM v— —
    CT «-3 isi CD g>
    LO
    CD CD CD CD LO CD UD CD CM CM ?— V
    SUBSTITUTE SHEET (RULE 26)
    WO 2017/147538
    PCT/US2017/019531
    34/34
    FIGURE 12 (cont.)
    θ θ θ tn o LO o CM CM X—
    + ~~e * o'· £ Q_ uo * i ! CM * Ο <o “i—γτί—rn—Γτη—r—j—rj—
    isi isi o θ <o o LO o uo θ CM CM •»T“ V““
    < CZ 4_J Οί O GO O CZ Z5 O Ξ CL GO E EQ CZ < co 03 5,..
    ’ O ra o Q Σ3
    SUBSTITUTE SHEET (RULE 26)
    POTH-007001WO_SeqList.txt SEQUENCE LISTING <110> Poseida Therapeutics, Inc.
    <120> TRANSPOSON SYSTEM AND METHODS OF USE <130> POTH-007001WO <150> US 62/300,387 <151> 2016-02-26 <160> 13 <170> PatentIn version 3.5 <210> 1 <211> 594 <212> PRT <213> Artificial Sequence <220>
    <223> Synthetic Construct <400> 1
    Met Gly Ser 1 Ser Leu Asp Asp 5 Glu His Ile 10 Leu Ser Ala Leu Leu 15 Gln Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp 20 25 30 His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile 35 40 45 Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu 50 55 60 Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn 65 70 75 80 Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His 85 90 95 Cys Trp Ser Thr Ser Lys Ser Thr Arg Arg Ser Arg Val Ser Ala Leu 100 105 110 Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile 115 120 125 Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile 130 135 140 Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg 145 150 155 160 Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile
    165 170 175
    Page 1
    POTH-007001WO_SeqList.txt
    Tyr Ala Phe Phe Gly Ile 180 Leu Val Met 185 Thr Ala Val Arg Lys 190 Asp Asn His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr 195 200 205 Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu 210 215 220 Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val 225 230 235 240 Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile 245 250 255 Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu 260 265 270 Gly Phe Arg Gly Arg Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro 275 280 285 Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys 290 295 300 Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn 305 310 315 320 Gly Val Pro Leu Gly Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val 325 330 335 His Gly Ser Cys Arg Asn Ile Thr Cys Asp Asn Trp Phe Thr Ser Ile 340 345 350 Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val 355 360 365 Gly Thr Val Arg Ser Asn Lys Arg Glu Ile Pro Glu Val Leu Lys Asn 370 375 380 Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro 385 390 395 400 Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu 405 410 415 Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys 420 425 430 Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr 435 440 445
    Page 2
    POTH-007001WO_SeqList.txt
    Leu Asp Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg 450 455 460 Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn 465 470 475 480 Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val 485 490 495 Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Ser Leu Thr Ser 500 505 510 Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu 515 520 525 Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser 530 535 540 Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Thr 545 550 555 560 Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Asn Ala Ser Cys Lys Lys 565 570 575 Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys Gln Ser 580 585 590
    Cys Phe <210> 2 <211> 340 <212> PRT <213> Artificial Sequence <220>
    <223> Synthetic Construct <400> 2
    Met Gly Lys Ser Lys Glu Ile Ser Gln Asp Leu Arg Lys Lys Ile Val 1 5 10 15 Asp Leu His Lys Ser Gly Ser Ser Leu Gly Ala Ile Ser Lys Arg Leu 20 25 30 Lys Val Pro Arg Ser Ser Val Gln Thr Ile Val Arg Lys Tyr Lys His 35 40 45 His Gly Thr Thr Gln Pro Ser Tyr Arg Ser Gly Arg Arg Arg Tyr Leu 50 55 60 Ser Pro Arg Asp Glu Arg Thr Leu Val Arg Lys Val Gln Ile Asn Pro
    Page 3
    POTH-007001WO_SeqList.txt
    65 70 75 80
    Arg Thr Thr Ala Lys 85 Asp Leu Val Lys Met 90 Leu Glu Glu Thr Gly 95 Thr Lys Val Ser Ile Ser Thr Val Lys Arg Val Leu Tyr Arg His Asn Leu 100 105 110 Lys Gly Arg Ser Ala Arg Lys Lys Pro Leu Leu Gln Asn Arg His Lys 115 120 125 Lys Ala Arg Leu Arg Phe Ala Thr Ala His Gly Asp Lys Asp Arg Thr 130 135 140 Phe Trp Arg Asn Val Leu Trp Ser Asp Glu Thr Lys Ile Glu Leu Phe 145 150 155 160 Gly His Asn Asp His Arg Tyr Val Trp Arg Lys Lys Gly Glu Ala Cys 165 170 175 Lys Pro Lys Asn Thr Ile Pro Thr Val Lys His Gly Gly Gly Ser Ile 180 185 190 Met Leu Trp Gly Cys Phe Ala Ala Gly Gly Thr Gly Ala Leu His Lys 195 200 205 Ile Asp Gly Ile Met Arg Lys Glu Asn Tyr Val Asp Ile Leu Lys Gln 210 215 220 His Leu Lys Thr Ser Val Arg Lys Leu Lys Leu Gly Arg Lys Trp Val 225 230 235 240 Phe Gln Met Asp Asn Asp Pro Lys His Thr Ser Lys Val Val Ala Lys 245 250 255 Trp Leu Lys Asp Asn Lys Val Lys Val Leu Glu Trp Pro Ser Gln Ser 260 265 270 Pro Asp Leu Asn Pro Ile Glu Asn Leu Trp Ala Glu Leu Lys Lys Arg 275 280 285 Val Arg Ala Arg Arg Pro Thr Asn Leu Thr Gln Leu His Gln Leu Cys 290 295 300 Gln Glu Glu Trp Ala Lys Ile His Pro Thr Tyr Cys Gly Lys Leu Val 305 310 315 320 Glu Gly Tyr Pro Lys Arg Leu Thr Gln Val Lys Gln Phe Lys Gly Asn 325 330 335
    Ala Thr Lys Tyr
    Page 4
    POTH-007001WO_SeqList.txt
    340 <210> 3 <211> 340 <212> PRT <213> Artificial Sequence <220>
    <223> Synthetic Construct <400> 3
    Met Gly Lys Ser Lys Glu Ile Ser Gln Asp Leu Arg Lys Arg Ile Val 1 5 10 15 Asp Leu His Lys Ser Gly Ser Ser Leu Gly Ala Ile Ser Lys Arg Leu 20 25 30 Ala Val Pro Arg Ser Ser Val Gln Thr Ile Val Arg Lys Tyr Lys His 35 40 45 His Gly Thr Thr Gln Pro Ser Tyr Arg Ser Gly Arg Arg Arg Tyr Leu 50 55 60 Ser Pro Arg Asp Glu Arg Thr Leu Val Arg Lys Val Gln Ile Asn Pro 65 70 75 80 Arg Thr Thr Ala Lys Asp Leu Val Lys Met Leu Glu Glu Thr Gly Thr 85 90 95 Lys Val Ser Ile Ser Thr Val Lys Arg Val Leu Tyr Arg His Asn Leu 100 105 110 Lys Gly His Ser Ala Arg Lys Lys Pro Leu Leu Gln Asn Arg His Lys 115 120 125 Lys Ala Arg Leu Arg Phe Ala Thr Ala His Gly Asp Lys Asp Arg Thr 130 135 140 Phe Trp Arg Asn Val Leu Trp Ser Asp Glu Thr Lys Ile Glu Leu Phe 145 150 155 160 Gly His Asn Asp His Arg Tyr Val Trp Arg Lys Lys Gly Glu Ala Cys 165 170 175 Lys Pro Lys Asn Thr Ile Pro Thr Val Lys His Gly Gly Gly Ser Ile 180 185 190 Met Leu Trp Gly Cys Phe Ala Ala Gly Gly Thr Gly Ala Leu His Lys 195 200 205 Ile Asp Gly Ile Met Asp Ala Val Gln Tyr Val Asp Ile Leu Lys Gln
    210 215 220
    Page 5
    POTH-007001WO_SeqList.txt
    His Leu Lys Thr Ser Val Arg Lys Leu Lys Leu Gly Arg Lys Trp Val 225 230 235 240 Phe Gln His Asp Asn Asp Pro Lys His Thr Ser Lys Val Val Ala Lys 245 250 255 Trp Leu Lys Asp Asn Lys Val Lys Val Leu Glu Trp Pro Ser Gln Ser 260 265 270 Pro Asp Leu Asn Pro Ile Glu Asn Leu Trp Ala Glu Leu Lys Lys Arg 275 280 285 Val Arg Ala Arg Arg Pro Thr Asn Leu Thr Gln Leu His Gln Leu Cys 290 295 300 Gln Glu Glu Trp Ala Lys Ile His Pro Asn Tyr Cys Gly Lys Leu Val 305 310 315 320 Glu Gly Tyr Pro Lys Arg Leu Thr Gln Val Lys Gln Phe Lys Gly Asn 325 330 335
    Ala Thr Lys Tyr
    340 <210> 4 <211> 1053 <212> PRT <213> Artificial Sequence <220>
    <223> Synthetic Construct <400> 4
    Met Lys Arg Asn Tyr Ile Leu Gly Leu Ala Ile Gly Ile Thr Ser Val 1 5 10 15 Gly Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala Gly 20 25 30 Val Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg Arg 35 40 45 Ser Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg Ile 50 55 60 Gln Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp His 65 70 75 80 Ser Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val Lys Gly Leu 85 90 95 Ser Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His Leu
    Page 6
    100 POTH-007001WO_SeqList.txt Thr 105 Glu Val Glu 125 110 Ala Lys Arg 115 Arg Gly Val His Asn 120 Val Asn Glu Asp Gly Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn Ser Lys Ala 130 135 140 Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg Leu Lys Lys 145 150 155 160 Asp Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr Ser Asp Tyr 165 170 175 Val Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala Tyr His Gln 180 185 190 Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu Glu Thr Arg 195 200 205 Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe Gly Trp Lys 210 215 220 Asp Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys Thr Tyr Phe 225 230 235 240 Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala Asp Leu Tyr 245 250 255 Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg Asp Glu Asn 260 265 270 Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu Asn Val Phe 275 280 285 Lys Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys Glu Ile Leu 290 295 300 Val Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser Thr Gly Lys 305 310 315 320 Pro Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys Asp Ile Thr 325 330 335 Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp Gln Ile Ala 340 345 350 Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln Glu Glu Leu 355 360 365 Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu Gln Ile Ser Page 7
    POTH-007001WO_SeqList.txt
    370 375 380
    Asn 385 Leu Lys Gly Tyr Thr 390 Gly Thr His Asn Leu 395 Ser Leu Lys Ala Ile 400 Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn Gln Ile Ala 405 410 415 Ile Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp Leu Ser Gln 420 425 430 Gln Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile Leu Ser Pro 435 440 445 Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile Asn Ala Ile 450 455 460 Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu Leu Ala Arg 465 470 475 480 Glu Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu Met Gln Lys 485 490 495 Arg Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile Arg Thr Thr 500 505 510 Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys Leu His Asp 515 520 525 Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile Pro Leu Glu 530 535 540 Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His Ile Ile Pro 545 550 555 560 Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val Leu Val Lys 565 570 575 Gln Glu Glu Ala Ser Lys Lys Gly Asn Arg Thr Pro Phe Gln Tyr Leu 580 585 590 Ser Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys Lys His Ile 595 600 605 Leu Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr Lys Lys Glu 610 615 620 Tyr Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val Gln Lys Asp 625 630 635 640 Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr Arg Gly Leu
    Page 8
    645 POTH-007001WO SeqList.txt 650 655 Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu Asp Val Lys 660 665 670 Val Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg Arg Lys Trp 675 680 685 Lys Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His His Ala Glu Asp 690 695 700 Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu Trp Lys Lys 705 710 715 720 Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe Glu Glu Lys 725 730 735 Gln Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln Glu Tyr Lys Glu 740 745 750 Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp Phe Lys Asp 755 760 765 Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg Glu Leu Ile 770 775 780 Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly Asn Thr Leu 785 790 795 800 Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn Asp Lys Leu 805 810 815 Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met Tyr His His 820 825 830 Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu Gln Tyr Gly 835 840 845 Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr Gly Asn Tyr 850 855 860 Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile Lys Lys Ile 865 870 875 880 Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile Thr Asp Asp 885 890 895 Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu Lys Pro Tyr 900 905 910 Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe Val Thr Val Page 9
    915 POTH-007001WO_SeqList.txt 920 925 Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu Val Asn Ser 930 935 940 Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser Asn Gln Ala 945 950 955 960 Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile Lys Ile Asn Gly 965 970 975 Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu Asn Arg Ile 980 985 990 Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu Glu Asn Met 995 1000 1005 Asn Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile Ala Ser Lys 1010 1015 1020 Thr Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn Leu 1025 1030 1035 Tyr Glu Val Lys Ser Lys ; Lys His Pro Gln Ile Ile Lys Lys Gly 1040 1045 1050 <210> 5 <211> 88 <212> PRT <213> 1 Homo sapiens <400> 5 Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp Ser Leu 1 5 10 15 Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe Leu Ile 20 25 30 Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu Ala Ile Asn Leu Thr Val 35 40 45 Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro Gly Thr 50 55 60 Glu Tyr Thr Val Ser Ile Tyr Gly Val Lys Gly Gly His Arg Ser Asn 65 70 75 80 Pro Leu Ser Ala Glu Phe Thr Thr 85
    <210> 6 <211> 90
    Page 10 <212> PRT <213> Homo sapiens <400> 6
    POTH-007001WO_SeqList.txt
    Met 1 Leu Pro Ala Pro 5 Lys Asn Leu Val Val 10 Ser Glu Val Thr Glu 15 Asp Ser Leu Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe 20 25 30 Leu Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu Ala Ile Asn Leu 35 40 45 Thr Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr Thr Val Ser Ile Tyr Gly Val Lys Gly Gly His Arg 65 70 75 80 Ser Asn Pro Leu Ser Ala Glu Phe Thr Thr 85 90
    <210> 7 <211> 270 <212> DNA <213> Homo sapiens
    <400> 7 atgctgcctg caccaaagaa cctggtggtg tctcatgtga cagaggatag tgccagactg tcatggactg ctcccgacgc agccttcgat agttttatca tcgtgtaccg ggagaacatc gaaaccggcg aggccattgt cctgacagtg ccagggtccg aacgctctta tgacctgaca gatctgaagc ccggaactga gtactatgtg cagatcgccg gcgtcaaagg aggcaatatc agcttccctc tgtccgcaat cttcaccaca
    120
    180
    240
    270
    <210> 8 <211> 4 <212> PRT <213> Homo sapiens <400> 8 Thr Glu 1 Asp Ser
    <210> 9 <211> 7 <212> PRT <213> Homo sapiens <400> 9
    Thr Ala Pro Asp
    Ala Ala Phe
    Page 11 <210> 10 <211> 6 <212> PRT <213> Homo sapiens <400> 10
    Ser Glu Lys Val Gly Glu
    1 5
    POTH-007001WO_SeqList.txt <210> 11 <211>4 <212> PRT <213> Homo sapiens <400>11
    Gly Ser Glu Arg <210> 12 <211>5 <212> PRT <213> Homo sapiens <400>12
    Gly Leu Lys Pro Gly <210>13 <211>7 <212> PRT <213> Homo sapiens <400>13
    Lys Gly Gly His Arg Ser Asn
    Page 12
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WO2017147538A1 (en) 2017-08-31
BR112018067536A2 (en) 2019-02-05
SG11201807134RA (en) 2018-09-27
CA3015642A1 (en) 2017-08-31
CN109312361A (en) 2019-02-05
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