AU2020227020B2 - Modified stem cell memory T cells, methods of making and methods of using same - Google Patents

Modified stem cell memory T cells, methods of making and methods of using same Download PDF

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AU2020227020B2
AU2020227020B2 AU2020227020A AU2020227020A AU2020227020B2 AU 2020227020 B2 AU2020227020 B2 AU 2020227020B2 AU 2020227020 A AU2020227020 A AU 2020227020A AU 2020227020 A AU2020227020 A AU 2020227020A AU 2020227020 B2 AU2020227020 B2 AU 2020227020B2
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Eric Ostertag
Devon SHEDLOCK
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Poseida Therapeutics Inc
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Abstract

The disclosure provides a method of producing modified stem memory T cells (e.g. CAR-T cells) for administration to a subject as, for example an adoptive cell therapy.

Description

MODIFIED STEM CELL MEMORY T CELLS, METHODS OF MAKING AND METHODS OF USING SAME RELATED APPLICATIONS
[01] This application is a divisional application of Australian Application No. 2017337147, filed on October 2, 2017. The subject matter of this application is related to the applicant's International Patent Application No. PCT/US2017/054799, and claims priority to and the benefit of provisional applications USSN 62/402,707 filed September 30, 2016, USSN 62/502,508 filed May 5, 2017, USSN 62/553,058 filed August 31, 2017 and USSN 62/556,309 filed September 8, 2017, the contents of each of which are herein incorporated by reference in their entirety.
INCORPORATION OF SEQUENCE LISTING
[02] The contents of the text filed named "POTH-012_001WO_SeqList.txt", which was created on 2 October 2017 and is 110 KB in size, are hereby incorporated by reference in their entirety.
FIELD OF THE DISCLOSURE
[03] The disclosure is directed to molecular biology, and more, specifically, to methods of making and using modified stem-cell memory T cells.
BACKGROUND
[04] There has been a long-felt but unmet need in the art for a method of producing modified stem-cell memory T cells for administration to a subject as, for example, an adoptive cell therapy. The disclosure provides a solution to this long-felt but unmet need.
SUMMARY
[05] Unlike traditional biologics and chemotherapeutics, modified-T cells of the disclosure possess the capacity to rapidly reproduce upon antigen recognition, thereby potentially obviating the need for repeat treatments. To achieve this, modified-T cells of the disclosure must not only drive tumor destruction initially, but must also persist in the patient as a stable population of viable memory T cells to prevent potential cancer relapses. Thus, intensive efforts have been focused on the development of antigen receptor molecules that do not cause T cell exhaustion through antigen-independent (tonic) signaling, as well as of a modified-T cell product containing early memory cells, especially stem cell memory (TscM). Stem cell like modified-T cells of the disclosure exhibit the greatest capacity for self-renewal
- la- and multipotent capacity to derive central memory (Tci), effector memory (TEM) and effector T cells (TE), thereby producing better tumor eradication and long-term modified-T cell engraftment. Modified-T cells of the disclosure include, but are not limited to, those cells that express an antigen receptor comprising a protein scaffold of the disclosure. Modified-T cells of the disclosure include, but are not limited to, those cells that express a chimeric antigen receptor (CAR) (i.e. CAR-T cells of the disclosure). Chimeric antigen receptors (CARs) of the disclosure may comprise one or more sequences that each specifically bind an antigen, including, but not limited to, a single chain antibody (e.g. a scFv), a sequence comprising one or more fragments of an antibody (e.g. a VH-H, referred to in the context of a CAR as a VCAR), an antibody mimic, and a Centyrin (referred to in the context of a CAR as a CARTyrin).
[061 Modified cells of the disclosure may be further subjected to genomic editing. For example, a genomic editing construct may be introduced into the modified cells of the disclosure in a transposon or other means of delivery through electroporation or nucleofection and allowed to integrate into the genome of the cell during the following incubation phase. The resultant cell is a modified Tcell with an editedgenome that retains a stem-like phenotype. This modified T cell with an edited genome that retains a stem-like phenotype may be used as a cellular therapy. Alternatively, or in addition, modified cells of the disclosure may be subject to a first electroporation or nucleofection and a subsequent electroporation or nucleofection to introduce a genomic editing construct. 1071 Specifically, the disclosure provides a method of producing a modified stem memory T cell (TscM), comprising introducing into a primary humanTcell (a) a transposon composition comprising atransposon comprsingan antigen receptor or atherapeutic protein and (b) a transposase composition comprising a transposase or a sequence encoding the transposase; to produce a modified T cell, wherein the modified T cell expresses one or more cell-surface marker(s) of a stein memory T cell (TcM), thereby producing a modified stem memory T cell (TscM). The disclosure provides a method of producing a plurality ofmodified stem memory T cells (TscM), comprising introducing into a plurality of primary human T cell (a) a transposon composition comprising a transposon comprising an antigen receptor or a therapeutic protein and (b) a transposase composition comprising a transposase or a sequence encoding the transposase; to produce a plurality of modified T cells, wherein at least 2%, 5%, %, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, %, 99% orany percentage in between of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memoryTcell (TsCM), thereby producing a plurality of modified stem memory T cells (TscM). In certain embodiments, the method produces a plurality of modified T cells, wherein at least 25% of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (TscM), thereby producing a plurality of modified stem memory'Tcells (TscM). In certain embodiments, the method produces a plurality of modified T cells, wherein at least 50% ofthe plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tsc), thereby producing a plurality of modifiedstem memory'T cells (Tsci). In certain embodiments, the method produces a plurality of modified T cells, wherein at least 60% of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memory Tcell (TSCM), thereby producing a plurality of modified stem memory T cells (TSCM). In certain embodiments, the method produces a plurality of modified Tcells, wherein at least 75% of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (TsCM), thereby producing a plurality of modified stem memory T cells (TSCI). In certain embodiments, the method produces a plurality of modified T cells, wherein at least 80%of the plurality of modified Tcells expresses one or more cell-surface marker(s) of a stem memory T cell (Tsci),thereby producing a plurality of modified stem memory Tcells (TscM). In certain embodiments, the method produces a plurality of modified Tcells, wherein at least 85% of the plurality of modified'ITcells expresses one or more cell surface marker(s) of a stem memory T cell (TscM), thereby producing a plurality ofmodified stem memory T cells (Tsci). In certain embodiments, the method produces a plurality of modified T cells, wherein at least 90% of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memoryTcell (TsCM), thereby producing a plurality of modified steminmemory T cells (TscM). In certain embodiments, the method produces a plurality of modified T cells, wherein at least 95% of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality ofmodified stem memory T cells (TscM). In certain embodiments, the cell-surface markers comprise CD62L and CD45RA. In certain embodiments, the cell surface markers of the CAR-TSCM comprise one or more of CD62L. CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2R§. In certain embodiments, the cell-surface markers of the CAR-Tsc comprise one or more of CD45RA, CD95, IL-2Ry, CR7, and CD62L. In certain embodiments of this method, the transposon is a plasmid DNA transposon with a sequence encoding the antigen receptor or the therapeutic protein is flanked by two cis-regulatory insulator elements. In certain embodiments, the transposon is a piggyBac transposon. In certain embodiments, and, in particular, those embodiments wherein the TM transposon is a piggyBac transposon, the transposase is a piggyBac or a Super piggyBacT M (SPB) transposase.
[081 In certain embodiments of the methods of the disclosure, the transposon is a plasmid DNA transposon with a sequence encoding the antigen receptor or the therapeutic protein is flanked by two cis-regulatory insulator elements. In certain embodiments, the transposon is a piggyBac transposon. In certain embodiments, and, in particular, those embodiments wherein the transposon is a piggyBac transposon, the transposase is a piggyBacTM or a Super piggyBacTM (SPB) transposase. In certain embodiments, and, in particular, those embodiments wherein the transposase is a Super piggyBacTM (SPB) transposase. the sequence encoding the transposase is an mRNA sequence.
[09] In certain embodiments of the methods of the disclosure, the transposase enzyme is a piggvBac TM (PB) transposase enzyme. The piggyBac (PB) transposase enzyme may comprise or consist of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to: 1 MGSSLDDEHI LSALLOSDDE LVGEDSDSEI SDHVSEDDVQ SDTEEAFIDE VHEVQPTSSG
61 SEILDEQNVI EQPGSSLAESN RILTLPQRTI RGKNKHCWST SKSTRRSRVS ALNIVRSQRG 1 PRMCRNTYD PLLCFKLFFT DEIISETVKW TNAEISLKRR ESMTGATFRD TNEDEIYAFF 181 GTLVMTAVRK DNHMSTDDLF DRSLSMVVVS VMSRDRFDFL IRCLR4DDKS IRPTLRENDV 241 FPV KIWDL FHQCIQNYT PGAHLTIDEQ LLGFRGRCPF RMYIPNKPSK YGTKILJMMC D 301 SGYKYMINGM PYLGRGTQTN GVFLGEYYVK ELSKPVHGSC RNTCDNVFT SIPLAKNLLQ
361 EPYKLTIVGT VRSNKRETPE VLKNSRSRPV GTSMFCFDGP LTLVSYKPKP AKMVYLLSSC 421 DEDASINEST GKPQMVMYYN QTKGGVDTLD QMCSVMTCSR KTNRWPMALL YGMINTACIN 481 S' IIYS H N VS SKGEKVQSRK KFMRNLYMSL TSSFMRKRLE APTLKRYLRD NISNILPNEV 541 PGTSDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV ICREHNIDMC QSCF (SEQ ID NO: 4.
[010] In certain embodiments of the methods of the disclosure, the transposase enzyme is a piggyBacTM (PB) transposase enzyme that comprises or consists of an amino acid sequence having an amino acid substitution at one or more of positions 30, 165, 282, or 538 of the sequence: 1 MGSSLDDEHI LSALLQSDDE LVGEDSDSEI SDH:VSEDDVO SDTEEAFIDE VH:-EVQPTSSG
61 ,EILDEQNVI EQPGSSLASN RTLTLPQRTT RGKNKHCWST SKSTRRSRVS ALNIVRSORG 121 PTR4CRNTYD PLLCFKLFFT DEISEIVKW TNAEISLKRR ESMTGATFRD TNEDETYAFF 18'1 GILVMTAVR K DNHMSTDDLF DRSLSMVYVS VMSRDRFDFL TRCLRMDDKS TRPTLRENDV
241 FTPVRKIWDL FIHQCIQNYT PGAHLTIDEQ LLGFRGRCPF RMYIPNKPSK YGT.KILMMCD 301 SGYKYMINGM PYLGRGTQTN GVPLG-EYYVK ELSKPVHGSC RNITCDNWFT SIP1AKNLLQ 361 EPYKLTIVGT VRSNKREP E VLKNSRSRPV GTSMFCFD GP LTLVSYKPKP AKMVYLLSSC 421 DEDASINEST GKPOMV~vMYYN QTKGGVDTL'D QMCSVMTCSR KTNRWPMALL YGMINIACIN 481 SFIIYSHNVS SKGEKVQSRK K FMRNLYMSL TSSFMRKRLE APTLKRYLRD NISNILPNEV 541 PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV ICREHNTDMC QSCF (SEQ ID NO: 4>.
[011] In certain embodiments, the transposase enzyme is a piggyBacT M (PB) transposase enzyme that comprises or consists of an amino acid sequence having an amino acid substitution at two or more of positions 30, 165, 282, or 538 of the sequence of SEQ ID NO: 4. In certain embodiments, the transposase enzyme is a piggyBacTM (PB) transposise enzyme that comprises or consists of an amino acid sequence having an amino acid substitution at three or more of positions 30, 165, 282, or 538 of the sequence of SEQ ID NO: 4. In certain embodiments, thetransposase enzyme is a piggyBacT M (PB) transposase enzyme that comprises or consists of an amino acid sequence having an amino acid substitution at each of the following positions 30, 165, 282, and 538 of the sequence of SEQ ID NO: 4. In certain embodiments, the amino acid substitution at position 30 of the sequence of SEQ ID NO: 4 is a substitution of a valine (V) foran isoleucine (I). In certain embodiments, the amino acid substitution at position 165 of the sequence of SEQ ID NO: 4 is a substitution of a serine (S) for a glycine (G). In certain embodiments, the amino acid substitution at position 282 of the sequence of SEQ ID NO: 4 is a substitution of a valine (V) for a methionine (M). In certain embodiments, the amino acid substitution at position 538 of the sequence of SEQ ID NO: 4 is a substitution of a lysine (K) for an asparagine (N).
[012] In certain embodiments of the methods of the disclosure, the transposase enzyme is a Super piggyBacTM (SPB) transposase enzyme. In certain embodiments, the Super TM piggyBac (SPB) transposase enzymes of the disclosure may comprise or consist of the amino acid sequence of the sequence of SEQ ID NO: 4 wherein the amino acid substitution at position 30 is a substitution of a valine (V) for an isoleucine (I),the amino acid substitution at position 165 is a substitution of a serine (S) for a glycine (G),the amino acid substitution at position 282 is a substitution of a valine (V) for a methionine (M), and the amino acid substitution at position 538 is a substitution of a lysine (K) for an asparagine (N). In certain embodiments, the Super piggyBacTM (SPB) transposase enzyme may comprise or consist of an aminoacid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to: l MGSSLDDEHI LSALLQS DDE LVGEDSDSEV SD-HVSEDD-VQ SDTEEAFIDE VHEVQPTSSG
61 SEILDEQNVI EQPGSSLASN RILTLPQRTI RGKNKHCWST SKSTRRSRV'S ALNIVRSQRG 121 PTRMCRNIYD P LLCFKLFFT DEIISEIVKW TNAEISLIKRR ESMTSATFRD TNEDE1YAFF
181 GILVM4TAVRK DNHMSTDDLF DRSLSMVYVS VMSRDRF'DFL IRCLPIVDDKS IRPTLRENDV 241 FTPVRKIWDL FHQCIQNYT PGAHLTIDEQ LLGFRGRCPF RVYIPNKPSK YGIKILMM CD
301 SGTKYMINGM PYLGRGTQTN GVPLGEYYVK ELSKPVHGSC RNITCDNWFT SIPLAKNLLQ
361 EPYKLTIVGT VRSNKREPTE VLKNSRSRPV GTSMFCFDGP LTLVSYKPKP A.KMYFVYLLSSC
421 DEDASINEST GKfPQMVMYYN QTrKGGVDTLD QMCSVMTCSR KTNRWPMALL YGITNIACIN
481 SFIIYSHNVS SKGEKVQSRK KFMRNLYMSL TSSFMRKRLE APTLKRYLRD NISNILPKEV
541 PGTSDDSTEE PVMKKRTYCT YCPSKIRRFA NASCKKCKKV ICREHNIDMC QSCF (SEQ ID N:
10131 In certain embodiments of the methods of the disclosure, including those embodiments wherein the transposase comprises the above-described mutations at positions , 165, 282 and/or 538, the piggyBacTM or Super piggyBacTM transposase enzyme may further comprise an amino acid substitution at one or more of positions 3, 46, 82, 103, 119, 125, 177, 180, 185, 187, 200, 207, 209, 226, 235, 240, 241, 243, 258, 296, 298, 311, 315, 319, 327, 328, 340, 421, 436, 456, 470, 486, 503, 552, 570 and 591 of the sequence of SEQ ID NO: 4 or SEQ ID NO: 5. In certain embodiments, including those embodiments wherein the transposase comprises the above-described mutations at positions 30, 165, 282 and/or 538, the piggyBacTM or SuperpiggyBacT' transposase enzyme may further comprise an amino acid substitution at one or more of positions 46, 119, 125, 177 180, 185, 187, 200, 207, 209, 226, 235, 240,241, 243,296, 298, 311, 315, 319, 327, 328, 340, 421, 436, 456, 470, 485, 503, 552 and 570. In certain embodiments, the amino acid substitution at position 3 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an asparagine (N) for a seine (S). In certain embodiments, the amino acid substitution at position 46 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a serine (S) foran alanine (A). In certain embodiments, the amino acid substitution at position 46 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a threonine (T) for an alanine (A). In certain embodiments, the amino acid substitution at position 82 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a tryptophan (W) for an isoleucine (1). In certain embodiments, the amino acid substitution at position 103 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a praline (P) for a seine (S). In certain embodiments, the amino acid substitution at position 119 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a proline (P) for an arginine (R). In certain embodiments, the amino acid substitution at position 125 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an alanine (A) a cysteine (C). In certain embodiments, the amino acid substitution at position 125 of
SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a leucine (L) for a cysteine (C). In certain embodiments, the amino acid substitution at position 177of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a lysine (K) for a tyrosine (Y). In certain embodiments, the amino acid substitution at position 177 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a histidine (H) for atyrosine (Y). In certain embodiments, theamino acid substitution at position 180 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a leucine (L) for a phenylalanine (F). In certain embodiments, the amino acid substitution at position 180 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an isoleucine (I) for a phenylalanine (F). In certain embodiments, the amino acid substitution at position 180 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a valine (V) for a phenylalanine (F). In certain embodiments, the amino acid substitution at position 185 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a leucine (L) for amethionine (M). In certain embodiments, the amino acid substitution at position 187 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a glycine (G) for an alanine (A). In certain embodiments, the amino acid substitution at position 200 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a tryptophan (W) for a phenvialanine (F).In certain embodiments, the amino acid substitution at position 207of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a proline (P) for a valine (V). In certain embodiments, the amino acid substitution at position 209 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a phenylalanine (F) for a valine (V). In certain embodiments, the amino acid substitution at position 226 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a phenylalanine (F) for a methionine (M). In certain embodiments, the amino acid substitution at position 235 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an arginine (R) for a leucine (L). In certain embodiments, theamino acid substitution at position 240of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a lysine (K) for a valine (V). In certain embodiments, the aminoacid substitution at position 241 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a leucine (L) for a phenylalanine (F). In certain embodiments, the amino acid substitution at position 243 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a lysine (K) for a proline (P). In certain embodiments, the amino acid substitution at position 258 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a serine (S) for an asparagine (N). In certain embodiments, the amino acid substitution at position 296of SEQ ID NO:4 or SEQ ID NO: 5 is a substitution of a tryptophan (W) for a leucine (L). In certain embodiments, the amino acid substitution at position 296 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a tyrosine (Y) for a leucine (L). In certain embodiments, the amino acid substitution at position 296 of SEQ ID
NO: 4 or SEQ ID NO: 5 is a substitution of a phenylalanine (F) for a leucine (L). In certain embodiments, the amino acid substitution at position 298 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a leucine (L) for a methionine (M). In certain embodiments, the amino acid substitution at position 298 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an alanine (A) for a methionine (M). In certain embodiments, the amino acid substitution at position 298 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a valine (V) for a methionine (M). In certain embodiments, the amino acid substitution at position 311 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an isoleucine (I) for a proline (P). Incertain embodiments, the amino acid substitution at position 311 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a valine for a proline (P). In certain embodiments, the amino acid substitution at position 315 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a lysine (K) for an arginine (R).In certain embodiments, the amino acid substitution at position 319 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a glycine (G) for athreonine (T). In certain embodiments, the amino acid substitution at position 327 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an arginine (R) for a tyrosine (Y). In certain embodiments, the ainoacid substitution at position 328 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a valine (V) for a tyrosine (Y). In certain embodiments. the amino acid substitution at position 340 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a glycine (G) for a cysteine (C). In certain embodiments, the amino acid substitution at position 340 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a leucine (L) for a cysteine (C). In certain embodiments, the amino acid substitution at position 421 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a histidine (H) for the aspartic acid (D). In certain embodiments, the amino acid substitution at position 436 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an isoleucine (I) for a valine (V). In certain embodiments, the amino acid substitution at position 456 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a tyrosine (Y) for a methionine (M). In certain embodiments, the amino acid substitution at position 470 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a phenylalanine (F) for a leucine (L). In certain embodiments, the amino acid substitution at position 485 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a lysine (K) for a serine (S). In certain embodiments, the amino acid substitution at position 503 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a leucine (L) for a methionine (M). In certain embodiments, the amino acid substitution at position 503 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of anisoleucine (1) for a methionine (M). In certain embodiments, the amino acid substitution at position 552 of SEQ
ID NO: 4 or SEQ ID NO: 5 is a substitution of a lysine (K) for a valine (V). In certain embodiments, the amino acid substitution at position 570 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a threonine (T) for an alanine (A). In certain embodiments, the amino acid substitution at position 591 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a proline (P) for a glutamine (Q). In certain embodiments, the amino acid substitution at position 591 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an arginine (R) for a glutamine (Q). 10141 In certain embodiments of the methods of the disclosure, including those embodiments wherein the transposase comprises the above-described mutations at positions , 165, 282 and/or 538, the piggyBacTM transposase enzyme may comprise or the Super piggyBacTM transposase enzyme may further comprise an amino acid substitution at one or more of positions 103, 194, 372, 375, 450, 509 and 570 of the sequence of SEQ ID NO: 4 or SEQ ID NO: 5. In certain embodiments of the methods of the disclosure, including those embodiments wherein the transposase comprises the above-described mutations at positions , 165, 282 and/or 538, the piggyBac TM transposase enzyme may comprise or the Super piggyBacTM transposase enzyme may further comprise an amino acid substitution at two, three, four, five, six or more of positions 103, 194, 372, 375, 450, 509 and 570 of the sequence of SEQ ID NO: 4 or SEQ ID NO: 5. In certain embodiments, including those embodiments wherein the transposase comprises the above-described mutations at positions , 165, 282 and/or 538, the piggyBacTM transposase enzyme may comprise or the Super piggyBac T Mtransposase enzyme may further comprise an amino acid substitution at positions 103, 194, 372, 375, 450, 509 and 570 of the sequence of SEQ ID NO: 4 or SEQ ID NO: 5. In certain embodiments, the amino acid substitution at position 103 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a proline (P) for a seine (S). In certain embodiments, the amino acid substitution at position 194 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a valine (V) for a methionine (M). In certain embodiments, the amino acid substitution at position 372 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an alanine (A) for an arginine (R). II certain embodiments, the amino acid substitution at position 375 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an alanine (A) for a lysine (K). In certain embodiments, the amino acid substitution at position 450 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an asparagine (N) for an aspartic acid (D). In certain embodiments, the amino acid substitution at position 509 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a glycine (G) for a serine (S). In certain embodiments, the amino acid substitution at position 570 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a seine (S) for an asparagine (N). In certain embodiments, the piggyBacTM transposase enzyme may comprise a substitution of a valine (V) for a methionine (M) at position 194 of SEQ ID NO: 4. In certain embodiments, including those embodiments wherein the piggyBacrm transposase enzyme may comprise a substitution of a valine (V) for a methionine (M) at position 194 of SEQ ID NO: 4, the piggyBacTM transposase enzyme may further comprise an amino acidsubstitution at positions 372, 375 and 450 oftLhe sequence of SEQ ID NO: 4 or SEQ ID NO: 5. In certain embodiments, the piggyBac T M transposase enzyme may comprise a substitution of a valine (V) for a methionine (M) at position 194of SEQ ID NO: 4, a substitutionof an alanine (A) foran arginine (R) at position 372 of SEQ ID NO: 4, and a substitution of an alanine (A) for a lysine (K) at position 375 of SEQ ID NO: 4. In certain embodiments, the piggyBacTM transposase enzyme may comprise a substitution of a valine (V) for a methionine (M) at position 194 of SEQ ID NO: 4, a substitution of an alanine (A) for an arginine (R) at position 372 of SEQ ID NO: 4, a substitution of an alaine (A) for a lysine (K) at position 375 of SEQ ID NO: 4 arid a substitution of an asparagine (N) for an aspartic acid (D) at position 450 of SEQ ID NO: 4.
[015] The disclosure provides armethod ofproducing amodifiedstem memory T cell (TscM), comprising introducing into a primary human T cell (a) a transposon composition comprising a transposon comprising an antigen receptor or a therapeutic protein and (b) a transposase composition comprising a transposase or a sequence encoding the transposase; to produce a modified T cell, wherein the modified T cell expresses one or more cell-surface marker(s) of a stem memory T cell (TsCr), thereby producing a modified stein memory T cell (Tsc). The disclosure provides a method of producing a plurality of modified stem memory T cells (TSCM), comprising introducing into a pIurality of primary humanT cell (a) a transposon composition comprising a transposon comprising an antigen receptor or a therapeutic protein and (b) a transposase composition comprising a transposase or a sequence encoding the transposase; to produce a plurality of modified T cells, wherein at least 2%., 5%, %, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% 60%, 65%, 70%, 75%, 80%, 85%, 90%, %, 99% or any percentage in between of the pluralityofmodifiedTcellsexpressesoneor more cell-surface marker(s) of a stem memory Tcell (TsCM). thereby producing a plurality of modified stem memory T cells (TCM). In certain embodiments, the method produces a plurality of modified T cells, wherein at least 25% of the plurality ofmodified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tsv), thereby producingaplurality of modified stem memory ITcells (TCM). In certain embodiments, the method produces a plurality of modified Tcells, wherein at least 50% of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (TsCM), thereby producing a plurality of modified stem memory T cells (TscM). In certain embodiments, the method produces a plurality of modified T cells, wherein at least 60% of the plurality ofmodified"T cells expressesone or more cell-surface marker(s) of a stem memory T cell (TscM), thereby producing a plurality of modified stem memory T cells (TSCM). In certain embodiments, the method produces a plurality of modified T cells, wherein at least 75% of the plurality of modified Tcells expresses one or more cell-surface marker(s) of a stem memory T cell (TCM), thereby producing a plurality of modified stem memory T cells (TscM). In certain embodiments, the method produces a plurality of modified T cells, wherein at least 80% of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memoryT cell (TCM), thereby producing a plurality of modified stem memory T cells (TscM). In certain embodiments, the method produces a plurality of modified T cells, wherein at least 85% of the plurality of modified T cells expresses one or more cell surface marker(s) of a stem memory T cell (TscM), thereby producing a plurality of modified stem memory'ITcells (TscM). In certain embodiments, the method produces a plurality of modified T cells, wherein at least 90% of the plurality ofmodified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (TScM), thereby producing a plurality of modified stem memory'T cells (TCM). In certain embodiments, the method produces a plurality of modified T cells, wherein at least 95% of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tsm), thereby producing a plurality of modified stem memory T cells (Tscr). In certain embodiments, the cell-surface markers comprise CD62L and CD45RA. In certain embodiments, the cell surface markers of the CAR-TscM comprise one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2R§. In certain embodiments, the cell-surface markers of the CAR-TCM comprise one or more of CD45RA, CD95, IL-2Rf, CR7, and CD62L. In certain embodiments of this method, the transposon is a Sleeping Beauty transposon. In certain embodiments, and, in particular, those embodiments wherein the transposon is a Sleeping Beauty transposon, the transposase is a Sleeping Beauty transposase or a hyperactive Sleeping Beauty transposase (SB100X).
[016] In certain embodiments of the methods of the disclosure, the Sleeping Beauty transposase enzyme comprises an amino acid sequence at least 75%,80%, 85%, 90%,95%, 99% or any percentage in between identical to:
1 MGKSKEI SQD LRKKTVDLHK SGSSLGATSK RLKVPRSSVQ TIVRKYKHG TTQPSYRSCR 61 RRYLSPRDER TLVRKVQINP RTTAKDLVKM LEETGTKVSI STRVKRLYRH NLKGRSARKK 121 PLLQNRHKKA RLRFATAHGD KDRTFWRPNVL WSDETKIELF GHNDHPYVWR KKGEACKPKN 181 TIPTVKHGGGCC SIMLWGCFAA GGTGALHKID GIMRKENYVD ILKQHLKTSV RKLKLGRKWV 241 FQMDNDPKHT SKliVAKWLKD NKVKVLEWPS QSPDLNPIEN LWAELKKFRVR ARRTNLTOL 301 HQLCQEEWAK IHPTYCGKLV EGYPKRLTQV KQFKGNATKY (SEQ ID NO: 6).
[017] In certain embodiments of the methods of the disclosure, the hyperactive Sleeping Beauty (SB1OOX) transposase enzyme comprises an amino acid sequence at least 75%. 80%, %, 90%, 95%, 99% or any percentage in between identical to: 1 MGKSKE1SQD LRFPIVDLHK SGSSLGAISK RLAVPRSSVQ TIVRKYKHHG TTQPSYRSGR 61 RRYLSPRDER TLVRKVQINP RTTAKDLVKM LEETGTKVSI STVKRVLYRH NLKGHSAlRKK 121 PLLQNR:HKKA RLRFATAHGD KDRTF.'WRNVL WSDETKIELF GHNDHRYVWSR KKGEACKPKN 181 TTPTVKHGG SIMLWGCFAA GGTCALH-IKID GIMDAVQYVD ILKQHLKTSV RKLKLGRKWV 241. FQHDNDPK-fT SKVVAKVJLKD NKVKVLEWPS QSPDLNPTEN LVJAELKKRVR ARRPTNLTQL 301 HQLCQEEWAK IHPNYCGKLV EGYPKRLTQV KQFKGNATKY (SEQ ID NO: 7
[018] The disclosure provides a method of producing a modified stem memory T cell (TsCM), comprising introducing into a primary human T cell (a) a transposon composition comprising a transposon comprising an antigen receptoror a therapeutic protein and (b) a transposase composition comprising a transposase or a sequence encoding the transposase; to produce a modified T cell, wherein the modified T cell expresses one or more cell-surface marker(s) of a stem memory T cell (TscM), thereby producing a modified stein memory T cell (TsCM).Thedisclosure provides a method of producing a plurality of modified stem memory T cells (TsCM), comprising introducing into a plurality of primary human T cell (a) a transposon composition comprising a transposon comprising an antigen receptor and (b) a transposase composition comprising a transposase or a sequence encoding the transposase; to produce a plurality of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, %, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of modified T cells expresses one or more cell-surfacemarker(s) of a stem memory'Tcell (TCM), thereby producing a plurality ofmodified stem memoryI cells (TSCi).In certain embodiments, the method produces a plurality of modified T cells, wherein at least 25% of the plurality of modified T cells expresses one ormomre cell-surface marker(s) of a stem memoryTcell (TCM), thereby producing a plurality of modified stem memory T cells (Tscm). In certain embodiments, the method produces a plurality of modified T cells, wherein at least 50% of the plurality of modified T cells expresses one or more cell surface marker(s) of a stem memory T cell (TscM), thereby producing a plurality of modified stem memory Tcells (TscN). In certain embodiments, the method produces a plurality of modified T cells, wherein at least 60% of the plurality ofmodified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (TscM), thereby producing a plurality of modified stem memory T cells (TSCM). In certain embodiments, the method produces a pluralityof modified Tcells, wherein at least 75% of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (TscM), thereby producing a plurality of modified stem memory T cells (Tscr). In certain embodiments, the method produces a plurality of modified Tcells, wherein at least 80% of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (TscM), thereby producing a plurality of modified stem memory T cells (TscM). In certain embodiments, the method produces a plurality of modified T cells, wherein at least 85% of the plurality ofmodifiedTcells expresses one or more cell-surface marker(s) of a stem memory T cell (TscM), thereby producing a plurality of modified stem memoryT cells (TscM). In certain embodiments, the method produces a plurality of modified T cells, wherein at least 90% of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memory'Tcell (TCM), therebyproducing a plurality ofmodified stem memoryI cells (TscM). In certain embodiments, the method produces a plurality of modified T cells, wherein at least 95% of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memoryTcell (TscM), thereby producing a plurality of modified stem memory T cells (TscM). In certain embodiments, the cell-surface markers comprise CD62L and CD45RA. In certain embodiments, the cell-surface markers of the CAR-TscM comprise one or more of CD62L, CD45RA, CD28, CCR7. CD127, CD45RO, CD95, CD95 and IL 2Rp. In certain embodiments, the cell-surface markers of the CAR-TscM comprise one or more of CD45RA, CD95, IL-2RP, CR7, and CD62L. In certain embodiments of this method, the transposon is a Helraiser transposon. In certain embodiments, and, in particular, those embodiments wherein the transposon is a HeIraiser transposon, the transposase is a Helitron transposase. 10191 In certain embodiments of the methods of the disclosure, the transposase is a-lelitron transposase. Helitron transposases mobilize the Heiraiser transposon, an ancient element from the bat genome that was active about 30 to 36 million years ago. An exemplary Helraiser transposon of the disclosure includes Helibati, which comprises a nucleic acid sequence comprising: TCCTATATAA TAAAGAGAA ACATGCAJmT TGACCATCCC TCCGCTACGC TCAAGCCACG
61 CCCACCAGCC A'ATCAGAAGT GACTATGCAA ATTAACCCAA CAAAGATGGC AGTTAAATTT 121 GCTl-ACGCAG GTGCAAG-CG CCCCAGGAGG CAACGGCGGC CGCGGGCTC C CAGGACCTTC 181 GcrTGGCCCG GGAGCGCGAGG ccCCcGC CTAGCCACAC CCGCGGGCTC CCGGGACCTT 241 CGCCAGCAA GAGCAGAGCG GGAGAGCGGG CGGAGAGCGG GAGGTTTGGA GGACTTGGCA 301 GAGCAGGAGG CCGCTGGACA TAGAGCAGAG CGAGAGAGAG GGTGGCTTGG GGGCGTGGOC 361 TCCCTCTGTC ACCCCAGCTT CCTCATCACA GCTGTGGAA CTGACAGCAG GGAGGAGGAA 421 GTCCCACCCC CACAGAATCA GCCAGATCA GCCGTTGGTC AGACAG-CTCT CAGCGGCCTG 481 ACAGCAGGA CTCTCATTCA CCTGCATCTC ACACCGTGAC AGTACAGAGG TGGGACTATG c41 TCTAAAGAAC AACTGTTGAT ACPACGTAGC TCTGCAsGCCG AAAGATGCCG GCGTTATCGA 601 CAGAAATGT CTGCAGA GCA ACGTGCGTCT GATCTTGAAA GAAGGCGGCG CCTGCAACAG 661 AATGTATCTG AAGAGCAGCT ACTGGAAAAA CGTCGCTCTG PAGCCGAAA ACAGCGGCGT 721 CATCGACAGA JJTGTCTAA AGACCAACGT GCCTTTGAAG TTGAAAGAAG GCGGTGGCGA 781 CGACAGAATA TGTCTAGAGA ACAGTCATCA ACAAGTACTA CCAATACCGG TAGGAACTGC 841 CTTCTCAGCAAAAATCGAGT ACATGAGGAT GCAATTCTCG AACATAGTTG TGGTGGAATG 901 ACTGTTCGAT GTGAATTTTG CCTATCACTA AATTTCTCTG ATGA AAACC ATCCGATGGG 961 AAATTTACTC GATGTTGTAG CAAAGGGAAA GTCTGTCC A AGATATACA TTTTCCAGAT 10211 TACCCGGCAT ATTTAAAAAG ATTAATGACA AACGAAGATT CTGACAGT'AA AAATTTCATG 1081 GAAAATATTC GTTCCATAAA TAGTTCTTTT GCTTTTGCTT CCATrGGGTGC AAATATTGCA 1141 TCGCcATCA.G GATATGGGCCA.TACTGTTTT AGAATTACACG GACAAGTTTA TCACCGTACT 1201 GGAACTTTAC ATCCTTCGGA TGGTGTTTCT CGGAAGTTTG CTCAACTCTA TATTTTGGAT 1261 ACAGCCGAAG CTACAAGTAA AAGATTAGCA ATGCCAGAAAA ACCAGGGCTG CTCAGAAAGA 1321 CTCATGATCA ACATCAACAA CCCATGCIAT GAAATAAATG AATTAACAAA ATCGTACAAG 1381 ATGCTACATG AGGTAGAAAA GGAAGCCCAA TCTCGAAG-CAG CAGCAAAAGG TATTGCTCCC 1441 AC.AGAAGTAA CAATGGCGAT TAAATACGAT CGTAACAGTG ACCCAGGTAG ATATAATTCT 1:01 CCCCGTGTAA CCGAGGTTGC TGTCATATTC AGAAACGAAG ATGGAGAA CC TCCTTTTGAA 1561 AGGGACTTGC TCATTCATTG TAAACCAGAT CCCAATAATC CAAATGCCAC TAuAAATGAAA 1621 CAAATCAGTA TCCTGTTTCC TACATTAGAT GCAATGACAT ATCCTATTCT TTTTCCACAT 16811 GGTGAAAAAG GCTGGGGAAC AGATATIGCA TTAAGACTCA GAGACAACAG TGTAATCOAC 1741 AATAATACTA GACAAATGT AAGGACACGA GTCACACAAA TGCAGTATTA TGGATTTCAT 1801 CTCTCTGTGC GGACACGTT CAATCCTATT TTAAATGCAG GAPAAATTAAC TCAACAGTTT 1861 ATTGTGGATT CATATTCAAA AATGGAGGCC AATCGGATAAAP.TTTCATCAA AGCAA CCAA 1921 TCTAAGTTG A GAGTTGAAA ATATAGTGGT TTGATGGATT'ATCTCAAATC TAGATCTGAA 1981 AA'TGACAATG TGCCGATTGG TAAAATGAA ATACTTCCA' CATCTTTTA GGGTAGOTCCC 2041 AGAAATATGC AGCAGCGATA TCAGGATGCT ATGGCAATTG TAACGAAGTA TGGCAAGCCc. 2101 GATTATTCA TAACCATGAC ATGCAACCC AAATGGGCArG ATATTAcAAA CAATTTACAA 2161 CGCTGGCAAA AAGTTGAAAA cAGACCTGAC TTGGTAGCCA GAGTTTTTAA TATTAAGCTG 2221 AATGCTCTTT TAAATGATAT ATGTAAATTC CATTTATTTG GCAAAGTAAT AGCTAAAATT 22811 CATGTCATTG AATTTCAGAA ACGCGOACTG CCTCACGCTC ACATATTATT GATATTAGAT 2341 AGTGAGTCCA AATTACGTTC AGAAGATCAC ATTGACCGTA TAGTTAAGGC AGAAATTCCA 2401 GATGAAGACC AGTGTCCTc.G ACTTTTTCAA ATTGTAAAAT CAAATATGGT ACATGGACCA
461 TTGGAATAC AJ AATCCJACA TAGTCCATGT ATGGAAAT'G GAAJJTGTC AAAGGGATAT 2521 CCAANAAAAT TTCAAPATGC GACCATTGGA AATATTGATG GATATOCCAA ATACAAACGA 2581 AGATCTGGTA GCACATGTC TATTGGAAAT AAAGTTGTCG ATAAOACTTG CATTCTCCCT 2641 TATAACCCGT ATTTGTGCCT TAAATATAAC TGTCATATPAA TTTGAAGT CTGTGCATCA 2701 ATTAAAAGTG TCAATATTT AT T ATCTATAAA GGCACGATTG TGCAAATAT T 2761 CAAATTTCTCG AAJ<AATAT TATCAATCAT GACGAAGTAC AGGACTTCAT TGACTCCAGG
2821 TATGTGAGCG CTCCTGAGGC TGTTTGGAGA CTTTTTGCA TGCGPATGCA TGACCAATCT
2881 CATGCAATCA CAAGATTAGC TATTCATTTC CCAAATGATC AGAATTTCTA TTTTCATACC 2941 GATGATTTTC CTGA.AGTTTT AGATAGGGCT AAAAGGCATA ACTCGACTTT GATGGCTTGG 3001 TTCTTATTGA ATAGAGAAGA TTCCTGATG CTAATTATT ATTAT`GGGA GATTCACACG 3061 CATTATGTGT TTAATlATTC TTTCTGGACA AAACGOCCGAAA GGGTGGGA TAAAGTATTA 3121 GGTAGACTGT TCACTGTGAG CTTTAGAGAA CCAGAACGAT ATTACCTTAG ACTTTTGCTT 3181 CTGCATGTAA AAGGTGCGAT AAGTTTTGAG GATC.TGCGAA CTGTAGGAGG TGTAACTTAT 3241 GATACATTTC ATGAAGCTGC TAAACACCCA GGATTATTAC TTGATGACAC TATCTGGAAA 3301 GATACGATTG ACGATGCAAT CATCCTTAA'TATTGCCCAAAC AACTACGGCA ACTTTTTGCA 3361 TATATATGTG TGTTTGCATG TCCTTCTGCT GCAGACAAAT TATGGGATCA GAATAAATCT
3421 CATTTTATTG AAGATTTCTG TTPGGATTA CACCGAAGAG AAGGTGCCTG TGTGAACTG 3481 GAAATGCATG C CTTAACA AATTOAGGAG GTATTCAAT TGCATGGAAT GAAATGTTCA 3541 CATTTAAAC TTCCGGACTA TCCTTTATTA ATGAATCAA ATACATCTGA TCAATTGTAC
3601 GAGCAACAAC AGGCAGCGT TTTGATAAAT TCTCTGAATG ATGAACAGTT GGCAGCCTTT 3661 CAGACTATAA CTTCAGC(CAT CGAGATCA ACTGTACCOO OOAAATGCTT TTTCTTGGAT 3721 GGTCCAGGTG GTAGTGGAA AAATAYTCTGT AACTTT T'AACACATTA TATTAGAGGT
3781 CGTTGGTGGA CTGTTTTACC CACAGCATCT ACAGGAATTG CTGCAAATTT ACTTCTTGGT 3841 GGCAAGAACT TTCATTCCCA ATATAAATTA CCAATTCCAT TAAATAAC TTAATTTT 3901 AGACTCGATA TAAAGAGTGA AGTTGCTAA ACCATTAAAA AGGCCCAACT TCTCATTATT
3961 GATCAATGCA CCATGGCATC CAGTCATGCT ATAAACGCCA TAGATAGATT AOTAACAGJAA 4021 ATTATGAATT TGATGTTGC ATTTGGTGGG AAGTTCTCC TTCTCGGAGG GGATTTTCGA 40811 CAAGTCTCA GTATTGTACC ACATGCTATG CCATCGGCCA TAGTACAAAC GAGTTTAAG 4141 TACTAATC TTTGGGGATG TTTCAGAAG TTGTCTCTTA AAACAAATAT GAGATCAGG 4201 GATTCTGCTT ATAGTGPATG GTTAGTAAAA CTTGGGATG GCAAACTTGA TAGCACTTTT
4261 CATTTAGGAA TGGATATTAT TGAAATCCC CATGCAATGA TTTGTAACGG ATCTATTATT 4321 GAAGCTACCT TTGGAATAG TATATCTATA GATAATTATA PAAATATATC TAAACGTGCA 4381 ATTCTTTGTC CAAAAATGA GCATGT CAA AAATTAAATG AAGAAATTTT CCATATACTT
4441 GATGGAGATT TTCACACATA TTTCACTGAT GATTCCATTG ATTCAACACA TGATGCTGAA 4501 AAGGAAAATT TTCCCATCGA ATTTCTTAAT AGTATTACTC CTTCCGGAAT GCCGTTCAT m 4S61 AA TPAAAT TGPAAGTGGG TGCAATCATC ATGCTATTGA GAAATCTTAA TAGTAAATCG 4621 GGCTTTTGTA ATGGTACTAG ATTTATTATC AAAAGATTAC CAACTCAT TATCGAAGCT 4681 GAAGTPTTAA CAGGATCTGC AGAGGGAGAG GTTGTTCTGA TTCCPAGAAT TGATTTGTCC 4741 CCATCTGACA CTGGCCCTCCC ATTTAATTA ATTCGAAGAC AGTTTCCCGT GATGCCAGCA 4801 TTTCGATA CTATTAATAA ATCACAAGGA CAAACTCTAG ACAACTAGG AATATTCCTA
4861 CCTGAACCCG TTTTCCGCACA TGGTCAGTTA TATGTTGCT TCTCTCGAGT TCGAAGAGCA 4921 TGTGCGTTA AAGTTAAAGT GcC TGTATJJACT TCATCACAAG GGJTAGT CAAGCACTCT 4981 GAAAGTGTTT TTACTCTTAA TGTGGTATAC AGGGAATAT TAGAATAAGT TTAATCACTT 4 TCAGTCAT TCTTTGCATC AATCTTGTTT TTATATCATC TTTTTGTTCT TTTTATATCA 10 TCTTTCTT GTTGTTATAT CATGTTGTTA TTGTTTA2TTT ATTTJAANTAAAT TTATGTATTA 16 TTT CATATA CATTTTACTC ATTTCCTTTC ATCTCTCACA CTTCTATTAT (GAGAAAGGG 5221 CAAATACCAA TATTAAAATA TTTCCTCTAA TTAATTCC TTCAATGTGC ACGAATTTCG
5281 "GCACGGGCCACTAG (SEQ ID NO: 27)
[020] Unlike other transposases, the Helitron transposase does not contain an RNase-H like catalytic domain, but instead comprises a RepHel motif made up of a replication initiator domain (Rep) and a DNA helicase domain. The Rep domain is a nuclease domainof the HUH superfamily of nucleases.
10211 An exemplary Helitron transposase of the disclosure comprises an amino acid sequence comprising: 1 MSKEQLLIQR SSAAERCRRY RQKMSAEQRA SDLERRRRLQ QNVSEEQLLE KRRSEAEKQR 61 RHRQKMSKIDQ RAFEVERRRW RRQNMSREQS STSTTNTGRN CLLSKNGVHE DAILESCCGG 121 MTVRCEFCLS LNFSDEKPSD GKFTRCCSKG KVCPNDIHFP DYPAYLKRLM TNEDSDSKNE 181 MENIRSINSS FAFASMCANI ASPSGYCPYC FRIHGQVYHR TGTLHPSDGV SRKFAQLYIL
241 DTAEATSKRL AMPENQGCSE RLMININNLM HEINELTKSY KMLHEVEKEA OSEAAAKGIA
301 PTEVTMAIKY DRNSDPGRYN SPRVTEVAVI FRNEDGEPPF ERDLLIHCKP DPNNPNATKM 361 QISILFPTL DAMrTYPIFP HGEKGWGTDI ALRLRDNSVI DNNTRQNVRT RVTQMQYYGF 421 HLSVRDTFNP ILNAGKLTQQ FIVDSYSKME ANRINFKAN QSKLRVEKYS GLMDYLKSRS 481 ENDNVPICKM I ILPSSFEGS PRNMQQRYQD AMAIVTKYGK PDLEITMTCN PKWADITNNL 241 QRWQKVENRP DLVARVENIK LNALLNDICK FHLFGKVIAK IHVIEFQKRG LEPHAHILLIL
601 DSESKLRSED DIDRIVKAEI PDEDQCPRLF Q'IVKSNMVG PCCGIQNPNSP CMENGKCSKG 661 YPKEQNATI GNIDGYPKYK RESGSTMSIG NKVVDNTWIV PYNPYLCLKY NCHINVEVCA 721 SIKSVKYLFK YIYGHDCAN IQISEKNIIN HDEVQDFIDS RYVSAPEAVW RLFAMEMHDQ 781 SHAITRLAIH LPNDQNLYFH TDDFAEVLDR AKRHNSTLMA WFLLNREDSD A.NYYYWEIP 841 QHYVFNNSLW TKRCKGGNKV LGRLFTVSFR EPERYYLRLL LLHVKGAISE EDLRTVGGVT 901 YDTFHEAAKH RGLLLDDTIW KDTIDDAIIL NMPKQLRQLF AYICVFGCPS AADKLWDENK 961 SHF-IEDFCWK LHRREGACVN CEMHALNEIQ EVFTLHGMKC SHFKLPDYPL LMNANTCDQL
1021 YEQQQAEVLI NSLNDEQLAA FQTITSAIED QTVHPKCFFL DGPGGSGKTY LYKVLTHYIR 1081 GRGGTVLPTA. STGIAANLLL GGRTFHSQYK LfPIPLNETSI SRLDIKSEVA KTIKKAQLLI 1141 IDECTMASSH AINAIDRLLR EIMNLNVAFG GKVLLLGDF RQCLS T VPHA MRSAIVQTSL 1201 KYCNVWGCFR KLSLKTNMRS EDSAYSEWLV KLGDGKLDSS FHLGMDIIEI PHEMICNGSI 1261 TEATCFGNSIS IDNIKNISKR AILCPKNEHV QKLNEEILDI LDGDFHTYLS DDSIDSTDDA 1321 EKENFPIEFL NSTTPSGMPC HKLKLEVGAI IMILLRNLNSE WGLCNGTRFI IKRLRPNITE 1381 AEVLTGSAEG EVVLIPRIDL SPSDTGLPFK LIRRQFPVIMP AFAMTINKSQ GQTLDVGIE
1441 LPEPVFAHGQ LYVA'FSRVRR ACDVKVKV\/N TSSQGKLVKH SESVFTLNVV YREILE (SEQ ID
NO: 28).
10221 In Helitron transpositions, a hairpin close to the 3' end of the transposon functions as a terminator. However, this hairpin can be bypassed by the transposase, resulting in the transduction of flanking sequences. In addition, Heraiser transposition generates covalently closed circular intermediates. Furthermore, Helitron transpositions can lack target site duplications. In the Helraiser sequence, the transposase is flanked by left and right terminal sequences termed LTS and RTS. These sequences terminate with a conserved 5'-TC/CTAG 3' motif. A 19 bp palindromic sequence with the potential to form the hairpin termination structure is located I1 nucleotides upstream of the RTS and consists of the sequence GTGCACG\AAT TT GTGCACGGGCC(CTAG (SEQ ID NO: 29). 10231 The disclosure provides a method of producing a modified stein memory T cell (TSCM), comprising introducing into a primary human Tcell (a) a transposon composition comprising a transposon comprising an antigen receptor or a therapeutic protein and (b) a transposase composition comprising a transposase or a sequence encoding the transposase; to produce a modified T cell, wherein the modified T cell expresses one or more cell-surface marker(s) of a stem memory T cell (TscN), thereby producing a modified stem memory T cell (TscM). The disclosure provides amethod of producing a plurality ofmodified stem memory T cells (Tsc), comprising introducing into a plurality of primary human T cell (a) a transposon composition comprising a transposon comprising an antigen receptor and (b) a transposase composition comprising a transposase or a sequence encoding the transposase; to produce a plurality of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%,30%, %, 40%, 45%, 501, 60%, 65%70%, 75%, 80%, 85%, 90%, 95%. 99% or any percentage in between of the pluralityof modified Tcells expresses one or more cell-surface marker(s) of a stem memory T cell (TSCM), thereby producing a plurality of modified stem memory T cells (TscM). In certain embodiments, the method produces a plurality of modified T cells, wherein at least 25% of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stein memory Tcell (TscM). thereby producing a plurality of modified stem memory T cells (TscM), In certain embodiments, the method produces a plurality of modified T cells, wherein at least 50% of the plurality of modified T cells expresses one or more cell surface marker(s) of a stem memory T cell (TscM), thereby producing a plurality of modified stem memory"T cells (TscM). In certain embodiments, the method produces a plurality of modified T cells, wherein at least 60% of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memoryTcell (TsCM), thereby producing a plurality of modified stem memory T cells (TscM). In certain embodiments. the method produces a plurality of modified T cells, wherein at least 75% of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (TscM), thereby producing a plurality of modified stem memory'Tcells (TscM). In certain embodiments, the method produces a plurality of modified T cells, wherein at least 80% ofthe plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tsc), thereby producing a plurality of modifiedstem memoryT cells (Tsci). In certain embodiments, the method produces a plurality of modified T cells, wherein at least 85% of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memory Tcell (TSCM), thereby producing a plurality of modified stem memoryT cells (TSCM). In certain embodiments, the method produces a plurality of modified Tcells, wherein at least 90% of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (TsCM), thereby producing a plurality of modified stem memory T cells (TSCM). In certain embodiments, the method produces a plurality of modified T cells, wherein at least 95%of the plurality of modified Tcells expresses one or more cell-surface marker(s) of a stem memory T cell (Tsci),thereby producing a plurality of modified stem memory T cells (TscM). In certain embodiments, the cell-surface markers comprise CD62L and CD45RA. In certain embodiments, the cell-surface markers of the CAR-TsM comprise one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL 2R§. In certain embodiments, the cell-surface markers of the CAR-TscM comprise one or more of CD45RA, CD95, IL-2R , CRT and CD62L. In certain embodiments of this method, the transposon is a.Tol2 transposon. In certain embodiments, including those embodiments wherein the transposon is a To12 transposon, the transposase is a Tol2 transposase.
[024] In certain embodiments of the methods of the disclosure, the transposase is a To12 transposase. Tol2 transposons may be isolated or derived from the genome of the medaka fish, and may be similar to transposons of the hAT family. Exemplary Tol2 transposons of the disclosure are encoded by a sequence comprising about 4.7 kilobases and contain a gene encoding the Tol2 transposase, which contains four exons. An exemplary Tol2 transposase of the disclosure comprises an amino acid sequence comprising the following: 1 MEEVCDSSAA ASSTVQNQPQ DQEHPWPYLR EFFSLSGVNK DSFKMKIKCVLC LPLNKEISAF'
61KSSPSNLRKH TEP11HNTYLK NYSKLTAQKR KIGTSTHASS SKQLKVDSVF PVKHVSPVTV 121 NKAILRYTTQ GLHPFSTVDL PSFKELTSTL QPGTSVITRP TLRSKTAEAA LTMKQKVTAA
181 MSEVEWIATT TIDCWTARRKS FIGVTAHWIN PGSLERHSAA LACKRLMGSH TFEVLASAIMN 241 DIHSEYEIRD KVVCTTTIDSG SNFMKAFRVF GVENNDIETE ARRCESDDTD SEGCGEGSDG 301 VEFQDASRVL DQDDGFEFQL PKHQKCACHL LNLVSSVDAQ KALSNEHYKK LYPSVIFGKCQ 361 ALWNKSSRSA LAAEAVESES RLQLLRPNQT RWNSTFMAVD RILQICKEAG EGALRNICTS 421 LEVEWENPAE MLELTEWANT MIRPVAKVLDI LQAETNTQLG WLLPSVHQLS LKLQRLHHSL
481 RYCDPLVDAL QQGIQTRFKH MFEDPEIIAA AILLPKFRTS WTNDETIIKR GMDYIRVHLE 541 PLDHKKELAN SSSDDEDFFA SLKPTTHEAS KELDGYLACV SDTRESLLTF PAICSLSIKT
601 NTPLPASAAC ERLFSTAGLL FSPKRAPLDT NNFENQLLLK LNLRFYNFE (SEQ ID NO: 30)
[025] An exemplary To12 transposon of the disclosure, including inverted repeats, subterminal sequences and the Toi2 transposase, is encoded by a nucleic acid sequence comprising the following: I CAGAGGTGTA AAGTACTTGA GTAATTTTAC TTGATTACTG TACTTAAGTA TTATTTTTGG
61 GGATTTTTAC TTTACTTGAG TACAATTAAA AATCAATACT TTTACTTTTA% CTTAATTACA
121 TTTTTTTAGA AAAIJJ AGTA CTTTTTACTC CTTACAATTT TATTTACAGT CAAAAAGTAC
181 TTATTTTTTCG GAGATCACTT CATTCTATTT TCCCTTGCTA TTACCAJA.CC IATTGAATTG 241 CGCTGATOGCC CAGTTAATT TAAATGTTAT TTATTCTGCC TATGPAATC GTTTTCACAT 301 TATATGAAAT TGGTCAGACA TGTTCATTGG TCCTTTGGAA GTGACGTCAT GTCACATCTA 361 TTJACCACAAT GCACAGCACC TTGACCTGGA AATTAGGGAA ATTATAACAG TCAATCAGTG 421 GAAGAAATG GAGGAAGTAT GTGATTCATC AGCAGCTGCG AGCAGCACAG TCCAAATCA 481 GCCACAGGAT CAAGAGCACC CGTGGCCGTA TCTTCGCGAA TCTTTCTT TAAGTGGTGT S41 AAATAAAGAT TCATTCAAGA TGAAATGTGT CCTCTGTCTC CCGCTTAATA AAGAAATATC 601 GGCCTTCAAA AGTTCGCCAT CAAACCTAAG GAAGCATATT GAGGTAAGTA CATTAAGTAT 661 TTTGTTTTAC TGATAGTTTT TTTTTTTTTT TTTTTTTTTT TTTTTGGGTG TGCATGTTTT
721 GACGTTGATG GCGCGCCTTT TATATGTGTA GTAGGCCTAT TTTCACTAAT GCATGCGATT 781 GACAATATAA GGCTCACGTA ATAAAATGCT AAITATC/ST TGTAATTGGT PACGTTAGG-T 841 CCACGGGAAA TTTGGCGCCT A.TTGC.AGCTT TCAA.TAATCA TTATCATTCC GTGCTCTCAT 901 TGTGTTTGAA TTCATGCAAA ACACAAGAAA ACCAAGCGAG AAATTTTTTT CCAAACATGT 961 TGTATTGTCA AAACGGTAAC ACTTTACAAT GAGGTTGATT AGTTCATGTA TTAACTAACA 1021 TTIAATAACC ATGAGCAATA CATTTGTTAC TGTATCTGTT 1ATCTTTGTT AACGTTAGTT 1081 AA'TAGAAATA CAGATGTTCA. TTGTTTGTTC ATGTTAGTTC ACAGTGCATT AACTAATGTT 1141 AACAAGATAT AAAGTATTAG TAAATGTTCA AATTAACATG TATACGTGCA GTTCATTATT 1201 AGTTCATGTT AA.CTAATGTA GTTAA.CTAAC GAACCTTATT GTAAAAGTGT TACCATCAAA 1261 ACTAATGTAA TGPAATCAAT TCACCCTGTC ATGTCAGCCT TACAGTCCTG TGTTTTTGTC 1321 AATATAATCA GAAATAAAT TAATGTTTGA TTGTCACTA ATGCTACTGT A TTTCT A A 1381 TCAACAAGTA TTTAACATTA TAAAGTGTGC AASTTGGCTGC AAATGTCAGT TTTATTAAG 1441 GGTTAGTTCA, CCCAAAATGOAAATARTGT CATTAATGAC TCGCCCTCAT GTCGTTCCAA 1501 GCCCGTAAGA CCTCCGTTCA TCTTCAGAAC ACAGTTAAG ATATTTTAGA TTTAGTCCGA 1561 GAGCTTTCTG TGCCTCCATT GAGAATGTAT GTACGGTATA CTGTCCATGT CCAGAAAGGT 1621 AATAAAAACA TCAAAGTAGT CCATGTGACA TCAGTGGGTT AGTTAGAATT TTTTGAAGCA
1681 TCGAATACAT TTTGGTCCAA AATAACAAA ACCTACGACT TTATTCCGGCA TTGTATTCTC
1741 TTCCGGGTCT GTTGTCAATC CGCGTTCACG ACTTCGCAGT GACGCTACAA TGCTGAATAA 1801 AGTCGTAGGT TTTCTTATTT TTGGACCAAA ATGTATTTTC GATGCTTCAA ATAATTCTAC 1861 CTAACCCACT CATCTCACAT GGACTACTTT GATGTTTTTA TTACCTTTCTCGGACATGGAC 1921 AGTATACCGT ACATACATTT TCAGTGGAGG GACAGAAAC TCTCGGACTA AATCTAAAAT 1981 ATCTTAA-ACT GTGTTCCCAA GATGAACGGA GGTGTTACGG GCTTGGAACG ACATGAGGGT 2041 GAGTCPTTAA TGACATCT TCATTTTTGG GTGAACTAAC CCTTTAATGC TGTAATCAGA 2101 GAGTGTATGT GTAATTGTTA CATTTATTGC ATACAATATA AATArTTATT TGTTGTTTTT 2161 ACAGAGAATG CACCCAAATT ACCTCAAAAA CTACTCTPAt TTGACAGCAC AGAAGAGAAA 2221 GATCGGACC TCCACCCATG CTTCCACCACG TAACACTC AAAGTTCACT CACTTTTCCC 2281 ACTCAAACAT GTCTCTCCAG TCACTGTGA CAAAGCTATA TTAAGGTACA TCATTCAAGG 231 7CT'CATCCT TTCAGCACTG TTGATCTCC ATCAT1'TTAAA GAGCTGA1TTA GTACACTGCA 2401 G CCTGGCATT TCTCTCATTA CAAGCCTAC TTTACGCCTCC ACAGI'AGCTG AAGCTCTCT 2461 GATCATGAAA CAGAAAGTGA CTCCTGCCAT GAGTGAACTT CAAJGGATTG CAACCACAAC 2521 GGATCTTGG ACTCACGTA GAAAGTCATT CATTGGTTA ACTGCTCACT GGATCAACCC 2581 TGGAGTCTOTT GAAGACAATT CCGCTGCACT TGCCTGCA AACATTAATGG GCTCTCATAC 2 64 1CTGAGGTA CTGGCCAGTG CCATGPATGA TATCCACTCA GAGTATGAAA TACGTGACAA 2701 GGT TGTTGC ACAACCACAG ACAGTGGTTC CAACTTTATC AAGCTTTCA GAGTTTTGG 2761 TGTGGAAAAC AATGATATCG AGACTCAGGC AAGAAGGTGT GAAAGTGATG ACACTGATTC 2821 TGAAGCTCT GGTGAGGGAA CTCATGTGT GGAATTCCAA GATGCCTCAC GAGTCCTGGA 881 CCIAGACGAT GGCTTCGAAT TCCAGCTACC AAACATCAA AGCTGTGCCT GTCACTTACT 2941 TAACCTAGTC TCAAGCGTTG ATGCCCAAAA AGCTCTCTCA AATGAACACT ACAAGAJJ.CT 3001 CTACAGATCT GTCTTTGGCA A-ATGCCAAGC TTTATGAAT AAAAGCAGCC GATCGGCTCT AGCAGCTGAA GCTGTTCAAT CACAACCC CCTTCACCTT TTAACGCCAAACCA.AACCG
3 12 GTGAATTCA ACTTTTATG CTGTTGACAG AATTCTTCAA ATTTGCAAAG AGCAGGAGA 3181 AGGCGCACTT CGAATATAT GCACCTCTCT TCAGGTTCCA ATGTAGTGT TTTTCCCCTC 3241 TGACATGTA AACAAATCTG GTTGTTTTT GTTTAATACT CTTTTATTAT GCTGATTTCT 33011 CTGTAGGTT TAATCCAGCA GAAATGCTGT TCTTGACAGA GTGGGCCAAC ACAA.TGCGTC 3361 CAGTTGCAAA AGTACTCGAC ATCTTGCAAG CGGAAACGAA TACACAGCTG GGGTGGCTC
3421 TGCCTAGTGT CCATCAGTTA AGCTTGAAAC TTCAGCGACT CCACCATTCT CTCAGTACT 3481 GTCACCCACT TGTGGATGCC CTACAACAAG GAATCCIAAC ACGATTCAG CATATGTTTG 3541 ACATCCTGA GATCATAGCA GCTGCCATCC TTCTCCCTAA ATTTCGGACC TCTTGGCAA 360 ATGArTGAAAC CATCATAAJJ CGAGGTAAAT GAATGCAAGC AACATACACT TGACGAATTC 31 TAATCTGGGC AACCTTTCGAG C.ATA.CCAAA A.TTATTTTT TATTTATTTA TTTTTGCACT 3721 TTTAGGAAT GTTATATCCC AT CTTTCTCGTGATCTCAA TATGAATATT GATGTAAAGT 3781 ATTCTTGCAG CAGCTTGTAG TTATCCCTCA GTCTTTCTTC AAACCAAACT ACPTATCTATC 3841 ATATGTGGTT TGGJATGCA GTTAGATTTT ATGCTAAAAT AAGGGATTTG CATGATTTTA 3901 GATGTAGATG ACTGCACGTAAATGTAGTTA ATGACAAAAT CCATAIACTT 'IGTTCCCAGT 39611 CAGAAGCCCC TCAACCAAAC TTTTCTTTCTGTCTCTCAO TGTGCTTCTA GGCATGACT 4021 ACATCAGAGT GCATCTGGAG CCTTTGGACC ACAGAAGGA ATTGGCCAAC AGTTCATCTG
4081 ATGATGOAAGA TTT'I"TTCG-CT TCTTTGAAAC CGACAACACA TGAAGCCAGC AAAGAG-TTGG 4141 ATGGATATCT GGCCTGTGTT TCAGACACCA GGGAGTCTCT GCT CACGTTT CCTGCTAT"T 4201 GCAGCCTCTC TATCAAGACT AATACACCC TTCCCGCATC GGCTGCCTGT GAGAGGCTTT
4261 TCAGCACTGC AGGATTGCTT TTCAGCCCCA AAAGAGCTAG GCTTGACACT PACAATTTTG 4321 AGAATCAGCT TCTACTGAAG TTAAATCTGA GGTTTTACAAk CTTTGAGTAG CGTGTACTGG 4381 CATTAGATTG TCTGTCTTAT AGTTTGATAA TTATACAkA CAGTTCTAA AGCAGGATAA 4441 AACCTTGTAT GCATTCATT TAATGTTTTT TGAGATTA AA GCTTAAACA AGAACTCTA 4501 GTTTTCTTTC TTGCTTTTAC TTTTACTTCC T'A1ACTCA AGTACAATTT TAATGGAGTA 4561 CTTTTTTACT TTTACTCAAG TAAGATTCTA GCCAGATACT TTTACTTTTA ATTG AGTAAA 4621 ATTTTCCCTA AGTACTTGTA CTTTCACTTG AGTAALATTT TTGAGTACTT TTTACACCTC
4681 TG (SEQ ID NO: 31).
[026] The disclosure provides a method of producing a modified central memory'T-cell (TcM), comprising introducing into a primary human T cell (a) a transposon composition comprising a transposon comprising an antigen receptor or a therapeutic protein and (b) a transposase composition comprising a transposase or a sequence encoding the transposase; to produce a modified Tcell, wherein the modified T cell expresses one or more cell-suface marker(s) of a central memory T-cell (Tci), thereby producing a modified central memory T cell (Tci). The disclosure provides a method of producing a plurality of modified central memory T-cells (TCM), comparing introducing intoa plurality of primary human T cell (a) a transposon composition comprising a transposon comprising an antigen receptor and (b) a transposase composition comprising a transposase or a sequence encoding the transposase; to produce a plurality of modified T cells, wherein at least 2% 5%, 10%, 15%, 20%, 25%, 30%, %,40%,45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or anypercentage in between of the plurality of modified T cells expresses one or more cell-surface marker(s) of a central memory T-cell (Tci), thereby producing a plurality of modified central memory T-cells (Tc). In certain embodiments, the method produces a plurality of modified T cells, wherein at least 25%of the plurality of modified Tcells expresses one or more cell-surface marker(s) of central memory T-cell (TcM), thereby producing a plurality of modified central memory T-cells (Tcx). In certain embodiments, the method produces a plurality of modified Tcells, wherein at least 50% of the plurality of modified T cells expresses one or more cell surface marker(s) of central memory T-cell (TcM), thereby producing a plurality ofmodified central memory T-cells (TcM). In certain embodiments, the method produces a plurality of modified T cells, wherein at least 60% of the plurality of modified T cells expresses one or more cell-surface marker(s) of central memory T-cell (TcM), thereby producing a plurality of modified central memory T-cells (TCM). In certain embodiments, the method produces a plurality of modified T cells, wherein at least 75% of the plurality ofmodified T cells expresses one or more cell-surface marker(s) of central memory T-cell (TM), thereby producing a plurality of modified central memory T-cells (CM).In certain embodiments, the method produces a plurality of modified'T cells, wherein at least 80% of the plurality of modified T cells expresses one or more cell-surface marker(s) of central memory T-cell (TC), thereby producing a plurality of modified central memory T-cells (TCM). In certain embodiments, the method produces a plurality of modified T cells, wherein at least 85%of the plurality of modified T cells expresses one or more cell-surface marker(s) of central memoryT-cel(TCM)therebyproducing a plurality of modified central memory T-cells (TC). In certain embodiments, the method produces a plurality of modified T cells, wherein at least 90% of the plurality of modified Tcells expresses one or more cell-surface marker(s) of central memory T-cell (TM), thereby producing a plurality of modified central memory T cells (TCM). In certain embodiments, the method produces a plurality of modified T cells, wherein at least 95% of the plurality of modified T cells expresses one or more cell-surface marker(s) of central memoryT-cell (TCm), thereby producing a plurality of modified central memory T-cells (TCM). In certain embodiments, the cell-surface markers comprise one or more of CD45RO, CD95, IL-2R3, CCR7, and CD62L. In certain embodiments of this method, the transposon is a plasmid DNA transposon with a sequence encoding the antigen receptor or the therapeutic protein is flanked by two cis-regulatory insulator elements. In certain embodiments, the transposon is a piggyBac transposon. In certain embodiments, and, in particular, those embodiments wherein the transposon is a piggyBac transposon, the transposase is a pggyBac oraSuperpiggyBacT (SPB) transposase. I certain embodiments ofthis method, the transposon is a Sleeping Beauty transposon. In certain embodiments, and, in particular, those embodiments wherein the transposon is a Sleeping Beauty transposon, the transposase is a Sleeping Beauty transposase or a hyperactive Sleeping Beauty transposase (SBIOOX). In certain embodiments ofthis method, the transposon is a Helraiser transposon. In certain embodiments, and, in particular, those embodiments wherein the transposon is a HeIraiser transposon, the transposase is a Helitron transposase. In certain embodiments of this method, the transposon is a'Tol2 transposon. In certain embodiments, including those embodiments wherein the transposon is a To2 transposon, the transposase is a To2 transposase.
[027] The disclosure provides a method of producing a composition comprising aplurality ofmodified stem memory T-cells (TsM) and a plurality of modified central memory T-cells (TCm), comprising introducing into a plurality of primary human T cell (a) a transposon composition comprising a transposon comprising an antigen receptor or a therapeutic protein and (b) a transposase composition comprising a transposase or a sequence encoding the transposase; to produce a composition comprising a plurality of modified TSCM and a plurality of modified TCM, wherein the plurality of modified TSCM expresses one or more CD62L, CD45RA, CD28, CCR7, CD127. CD45RO, CD95, CD95 and IL-2R and the plurality of modified TCM expresses one or more CD45RO, CD95, IL-RCCR7. and CD62L. thereby producing a composition comprising a plurality of modified TscM and a plurality of modified TCM. In certain embodiments of this method, the modified stem memory T-cells (TscM) comprise at least 1%, 2% 5%, 7%, 10%, 15%, 20%, 25%,30%, 35%, 40%, %, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or any percentage of cells in between of the total number of cells of the composition. In certain embodiments of this method, the modified central memory T-cells (TCM) comprise at least 1%, 2%, 5%, 7% %, 15%,20%,25%, 30%,35%, 40%, 45%,50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, %, 95%, 97%, 99%or any percentage of cells in between of the total number of cells of the composition. In certain embodiments of this method, the modified stem memory T-cells (TscM) comprise at least 10% ofthe total number of cellsof the composition andthe modified central memory T-cells (Tc) comprise at least 90% of the total number of cells of the composition. In certain embodiments of this method, the modified stem memory T-cells (TscM) comprise at least 90% of the total number of cells of the composition and the modified central memory T-cells (TcM) comprise at least 10%of the total number of cells of the composition. In certain embodiments of this method, the modified stem memory T-cells (Tsc) comprise at least 20% of the total number of cells of the composition and the modified central memory T-cells (Tc) comprise at least 80% of the total number of cells of the composition. In certain embodiments of this method, the modified stein memory T-cells (TscM) comprise at least 80%of the total number of cells of the composition and the modified central memory T-cells (Tc) comprise at least 20% of the total number of cells of the composition. In certain embodiments of this method, the modified stem memoryT-cells (TscM) comprise at least 30% of the total number of cells of the composition and the modifiedcentralmemoryfT-cells (TcM) comprise at least 70% of the total number of cells of the composition. In certain embodiments of this method, the modified stem memory T-cells
(Tsc) comprise at least 70% of the total number of cells of the composition and the modified central memory T-cells (TCM) comprise at least 30% ofthe total number of cells of the composition. In certain embodiments of this method, the modified stem memory T-cells (TsCM) comprise at least 40%of the total number of cells of the composition and the modified central memory-T-cells (Tc) comprise at least 60% of the total number of cells of the composition. In certain embodiments of this method, the modified stem memory T-cells (TSCM) comprise at least 60%of the total number of cells of the composition and the modifiedcentral memoryT-ceIls (Tc) comprise at least 40%of the total number of cells of the composition. In certain embodiments of this method, the modified stem memory T-cells (TscM) comprise at least 50% of the total number of cells of the composition and the modified central memory T-cells (Tc) comprise at least 50% of the total number of cells of the composition. In certain embodiments of this method, the transposon is a plasmid DNA transposon with a sequence encoding the antigen receptor or the therapeutic protein is flanked by two cis-regulatory insulator elements. In certain embodiments, the transposon is a piggyBac transposon. In certain embodiments, and, in particular, those embodiments wherein the transposon is a piggyBac transposon, the transposase is a piggyBacTM or a Super piggyBacTM (SPB) transposase. In certain embodiments of this method, the transposon is a Sleeping Beauty transposon. In certain embodiments, and, in particular, those embodiments wherein the transposon is a Sleeping Beauty transposon, the transposase is a Sleeping Beauty transposase or a hyperactive Sleeping Beauty transposase (SBIOOX). In certain embodiments of this method, the transposon is a Heraiser transposon. In certain embodiments, and, in particular, those embodiments wherein the transposon is a Heraiser transposon, the transposase is a Helitron transposase. In certain embodiments of this method, the transposon is a ToI2 transposon. In certain embodiments, including those embodiments wherein the transposon is a Tol2 transposon, the transposase is a To2 transposase.
[028] In certain embodiments of the methods of the disclosure, the transposon may be derived or reconbined from any species. Alternatively, or in addition, thetransposon may be synthetic.
10291 In certain embodiments of the methods of the disclosure, the antigen receptor is a T cell receptor. In certain embodiments, the T-cell receptor is naturally-occurring. In certain embodiments, the T-cell receptor is not naturally-occurrming. In certain embodiments, and, in particular, those embodiments wherein the T-cell receptor is not naturally-occurring, the T cell receptor comprises one or more mutation(s) compared to a wild-typeT-cell receptor. In certain embodiments, and, in particular,those embodiments wherein the T-cell receptor is not naturally-occurring, the T-cell receptor is a recombinant T-cell receptor. In certain embodiments of this method, the antigen receptor is a Chimeric Antigen Receptor (CAR). In certain embodiments, the CAR is a CARTyrin. In certain embodiments, the CAR comprises oneor more VHH sequence(s). In certain embodiments, the CAR is a VCAR.
[030] In certain embodiments of the methods of the disclosure, including those wherein the method comprises introducing into a primary human T cell (a) a transposon composition comprising a transposon comprising an antigen receptor and (b) a transposase composition comprising a transposase or a sequence encoding the transposase, the methods further comprise introducing into a primary human T cell (c) a second transposon composition comprising a transposon comprising a therapeutic protein, to produce a modified T cell, wherein the modified T cell is capable of expressing the therapeutic protein. In certain embodiments, the therapeutic protein is a secretable protein and the method produces a modified T cell capable of secreting the therapeutic protein. In certain embodiments, the transposase composition of (b) transposes the transposon of (a) and the transposon of (c). In certain embodiments, this methods further comprises introducing into the primary humanT cell (d) a second transposase composition comprising a transposase or a sequence encoding the transposase. In certain embodiments, the second transposase composition transposes the transposon of (c). In certain embodiments, the transposase composition of (b) transposes the transposon of (a) and the transposase composition of (d) transposes the transposon of (c). In certain embodiments of this method, the transposon is a plasmid DNA transposon with a sequence encoding the antigen receptor or the therapeutic protein flanked by two cis regulatory insulator elements. In certain embodiments, the transposon is a piggyBac transposon. In certain embodiments, and, in particular, those embodiments wherein the transposon is a piggyBac transposon, the transposase is a piggyBac TM or a Super piggyBacTM (SPB) transposase. In certain embodiments of this method. the transposon is a Sleeping Beauty transposon. In certain embodiments, and, in particular, those embodiments wherein the transposon is a Sleeping Beauty transposon, the transposase is a Sleeping Beauty transposase or a hyperactive Sleeping Beauty transposase (SB100X). In certain embodiments of this method, the transposon is a Heiraiser transposon. In certain embodiments, and, in particular, those embodiments wherein the transposon is a Helraiser transposon, the transposase is a Helitron transposase. In certain embodiments of this method, the transposon is a'To2 transposon. In certain embodiments, including those embodiments wherein the transposon is a Tol2 transposon, the transposase is a Tol2 transposase.
[031] The disclosure provides a method of producing a modified stem memory T cell (Tsc), comprising: (a) introducing into a primary human Tcell a composition comprising an antigen receptor to produce a modified Tcell, wherein the antigen receptor or the therapeutic protein is not contained in a transposon, and (b) contacting the modified T-cell and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetraineric antibody complex, an anti-human CD28 monospecific tetrameric antibody complexand an activation supplement to produce an activated modified T-cell, wherein the activated modified T-cell expresses one or more cell-surface marker(s) of a stem memory T cell (TscM), thereby producing a modified stem memory T cell (TscM). The disclosure provides a method of producing a plurality of modified stem nenoryTcells (TCM), comprising: (a) introducing into a plurality of primary human T cells a composition comprising an antigen receptor to produce a plurality of modified T cells, wherein the antigen receptor or the therapeutic protein is not contained in a transposon, and (b) contacting the plurality of modified T-cells and aT-cell activator composition comprising one or more of an anti-human CD3 monospecific tetraneric antibody complex, an anti-human CD28 monospecific tetramric antibody complex and an activation supplement to produce a
plurality of activated modified T-cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, %,40%,45%,50%,60%,65%,70%,75%, 80%,85%,90%,95%,99%orany7percentage in between of the plurality of activated modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (TSCM), thereby producing a plurality of activated modified stem memoryTcells (TCNi). In certain embodiments, the method produces a plurality of activated modified T cells, wherein at least 25% of the plurality of activated modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (TscM), thereby producing a plurality of activated modified stem memory T cells(Tsc4). In certain embodiments, the method produces a plurality of activated modified T cells, wherein at least 50% of the plurality of activated modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (TscM), thereby producing a plurality of activated modified stem memoryTcells (TsCNi). In certain embodiments, the method produces a plurality of activated modified T cells, wherein at least 60% of the plurality of activated modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tsc), thereby producing a plurality of activated modified stem memory T cells (Tsc). In certain embodiments, the method produces a plurality of activated modified T cells, wherein at least 75% of the plurality of activated modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tsci), thereby producing a plurality of activated modified steinm emory T cells (TSCM). In certain embodiments, the method produces a pluralityof activated modified Tcells, wherein at least 80% of the plurality of activated modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (TSCM), thereby producing a plurality of activated modified stein memory T cells (TSCM). In certain embodiments, the method produces a plurality of activated modified T cells, wherein at least 85% of the plurality of activated modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tsci), thereby producing a plurality of activated modified stein memory T cells (TscM). In certain embodiments, the method produces a pluralityof activated modified T cells, wherein at least 90% of the plurality ofactivated modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (TscM), thereby producing a plurality of activated modified stein memory T cells (TscM). In certain embodiments. the method produces a plurality of activated modified T cells, wherein at least 95% of the plurality of activated modifiedT cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tsci), thereby producing a plurality of activated modified stein memory T cells (TscM). In certain embodiments, the cell-surface markers comprise CD62L and CD45RA. In certain embodiments, the cell-surface markers of the activated modified TscMcomprise one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2Rp. In certain embodiments, the cell-surface markers of the activated modified TsCM comprise one or more of CD45RA, CD95, IL-2R3. CR7, and CD62L.
[032] In certain embodiments of the methods of the disclosure of producing a modified stein memory T cell (TscM), comprising: (a) introducing into a primary human T cell a composition comprising an antigen receptor to produce a modified T cell, wherein the antigen receptor or the therapeutic protein is not contained in a transposon, and (b) contacting the modified T-cell and a T-cell activator composition comprising one or more of an anti human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement to produce an activated modified T-cell, the T-cell activator composition of (b) further comprises an anti-human CD2 monospecifictetramericantibody complex. In certain embodiments, this method further comprises the step of (c) contacting the activated modifiedT-cell and a T-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin., human transferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement to produce a plurality of expanded modified T-cells. wherein at least 2% of the plurality of expanded modifiedT-cells expresses one or more cell-surface marker(s) of a steinm emory T cell (Tscx). In certain embodiments of this method, at least 2%. 5%, 10%, 15%, 20%, 25%,
%, 350, 40%, 45%, 500, 60%, 65%, 70, 75% 80%, 85 0 , 90%, 95%, 99% orany percentage in between of the plurality of expanded modified T-cells expresses cell-surface marker(s) of a stein memory T cell (TscM). In certain embodiments of this method, at least % of the plurality of expanded modified T-cells expresses cell-surface marker(s) of a stem memory T cell (Tscu). In certain embodiments, this method further comprises the step of (d) enriching the plurality of expanded modified T-cells to produce a composition comprising at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 60%, 65%, 70%, 75%, %,85%,90%,95%,99% orany percentage in between of modified T-cells that express cell-surface marker(s) of a stem memory T cell (TscM). In certain embodiments, this method further comprises the step of (d) enriching the plurality ofexpanded modified T-cells to produce a composition comprising at least 60% ofinodified T-cells that express cell-surface marker(s) of a stein memory T cell (Tsci). In certain embodiments of this method, the enriching step comprises isolating modified T-cells that express one or more cell-surface marker(s) of a stem memoryTcell (TCM) from the plurality of enriched modified T-cells. In certain embodiments of this method, the enriching step further comprises contacting the isolated modified Tscm and a T-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement to produce a plurality of expanded enriched modified Tscm.In certain embodiments of this method, the T-cell expansion composition further comprises one or more of octanoic acid, nicotinamide 2,4,7.9-tetramethyl-5-decyn 4,7-diol (TMDD). diisopropyl adipate (DIPA), n-butyl-benzenesulfonamide, 1,2 benzenedicarboxylicacid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearic acid hvdrazide, oleamide, a sterol and an alkane. In certain embodiments of this method, the T-cell expansion composition further comprises one or more of octanoic acid, palmitic acid, linoleicacid, oleicacid and a sterol. In certain embodiments of this method, the T-cell expansion composition further comprises one or more of octanoic acid at a concentration of between 0.9 mg/kg to 90 mg/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints;oleic acid at a concentration of 0.2 mg/kg to 20 mg/kg, inclusiveof the endpoints; and a sterol at a concentration of about 0.1 mg/kg to 10 mg/k'g, inclusive of the endpoints. In certain embodiments of this method, the T-cell expansion composition further comprises one or more of octanoic acid at a concentration of about 9 mg/kg, palmitic acid at a concentration of about 2 mg/kg, linoleic acid at a concentration of about 2 mg/kg, oleic acid at a concentration of about 2 mg/kg and a sterol at a concentration of about 1 mg/kg. In certain embodiments of this method, theT-cell expansion composition further comprises one or more of octanoic acid at a concentration of between 6.4 pmo/kg and 640 mol/kg, inclusive of tie endpoints; palmitic acid at a concentration of between 0.7 tmo/kg and 70 pLmol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 pmol/kg and 75 mol/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 pmol/kg and 75 pimol/kg, inclusive of the endpoints; and a sterol at a concentration of between 0.25 pmol/kg and 25 pmo/kg, inclusive of the endpoints. In certain embodiments of this method, the T-cell expansion composition further comprises one or more of octanoic acid at a concentration of about 64 mo/kg, palmitic acid at a concentration of about 7 imokg, linoleic acid at a concentration of about 7.5 pmol/kg, oleic acid at a concentration of about 7.5 pmol/kg and a sterol at a concentration of about 2.5 pmol/kg.
[033] The disclosure provides a methodofproducing a modified central memory T-cell (TcM), comprising: (a) introducing into a primary human T cell a composition comprising an antigen receptor to produce a modified T cell, wherein the antigen receptor or the therapeutic protein is not contained in a transposon, and (b) contacting the modified T-cell and aT-cell activator composition comprisingone or moreof an anti-human CD3monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complexand an activation supplement to produce an activated modified T-cell, wherein the activated modifiedT-cell expresses one or more cell-surface marker(s) of a central memory T-cell (TCM), thereby producing a central memory T-cell (Tci). The disclosure provides a method of producing a plurality ofmodified centralmemory T-cell (TCM), comprising: (a) introducing into a plurality of primary human T cells a composition comprising an antigen receptor to produce a plurality of modified Tcells, wherein the antigen receptor or the therapeutic protein is not contained in a tranSposon, and (b) contacting the plurality of modified T-cells and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement to produce a plurality of activated modified T-cells, wherein at least 2%, 5% 10%, 15% 20%, 25%, 30%, 35%, 40%, 45%, %, 60%, 65%-,70%, 75%, 80%, 85%, 90%, 95%. 99% or any percentage in between of the plurality of activated modified T cells expresses one or more cell-surface marker(s) of a central memoryT-cell (Tci), thereby producing a plurality of activated modified central memory T-cell (Tci). In certain embodiments, the method produces a plurality of activated modified T cells, wherein at least 25% of the plurality of activated modified T cells expresses one or more cell-surface marker(s) of a central memory'ITcell (Te). thereby producing a plurality of activated modified central memory T cell (TM). In certain embodiments, the method produces a plurality of activated modified T cells, wherein at least % of the plurality of activated modified T cells expresses one or more cell-surface marker(s) of a central memory T'cell(Tes),thereby producing a plurality of activated modified central memory T cell (TM). In certain embodiments, the method produces a plurality of activated modified T cells, wherein at least 60% of the plurality of activated modified T cells expresses one or more cell-surface marker(s) of a central memory T cell (TcM), thereby producing a plurality of activated modified central memory Tcell (TcM). In certain embodiments, the method produces a plurality of activated modified T cells, wherein at least 75% of the plurality of activated modified T cells expresses one or more cell-surface marker(s) of a central memory T'cell(Tes),thereby producing a plurality of activated modified central memory T cell (TcM). In certain embodiments, the method produces a plurality of activated modified T cells, wherein at least 80% of the plurality of activated modified T cells expresses one or more cell-surface marker(s) of a central memory T cell (TcM), thereby producing a plurality of activated modified central memory Tcell (TcM). In certain embodiments, the method produces a plurality of activated modified T cells, wherein at least 85% of the plurality of activated modified T cells expresses one or more cell-surface marker(s) of a central memory Icell (Tct), thereby producing a plurality of activated modified central memory T cell (TM). In certain embodiments, the method produces a plurality of activated modified T cells, wherein at least 90% of the plurality of activated modified T cells expresses one or more cell-surface marker(s) of a central memory T cell (TcM), thereby producing a plurality of activated modified central memory Tcell (TcM). In certain embodiments, the method produces a plurality of activated modified T cells, wherein at least 95% of the plurality of activated modified T cells expresses one or more cell-surface marker(s) of a central memory Icell(ce). thereby producing a plurality of activated modified central memory Tcell (TcM). In certain embodiments, the cell-surface markers of the activated modified TCM comprise one or more of CD45RO, CD95, IL-2RP, CCR7, and CD62L.
[034] In certain embodiments of the methods of the disclosure of producing a modified central memoryTcell (TcM), comprising: (a) introducing into a primary human'Tcell a composition comprising an antigen receptor to produce a modified T cell, wherein the antigen receptor or the therapeutic protein is not contained in a transposon, and (b) contacting the modified T-cell and aT-cell activator composition comprising one or more of an anti human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement to produce an activated modified T-cell, the T-cell activator composition of (b) further comprises an anti-human CD2 monospecifictetramericantibody complex. In certain embodiments, this method further comprises the step of (c) contacting the activated modified T-cell and a T-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin. human transferring, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement to produce a plurality of expanded modifiedT-cells, wherein at least 2%of the plurality of expanded modified T-cells expresses one or more cell-surface marker(s) of a central memory T cell (TCM). In certain embodiments of this method, at least 2%,5%, 10%, 15%, 20%, 25%, %, 35%,40%, 45%,50%,60%, 65%, 70%, 75%, 80%, 85%,90%, 95%,99% or any percentage in between of the plurality of expanded modified T-cells expresses cell-surface marker(s) of a central memory T cell (TcM). In certain embodiments of this method, at least % of the plurality of expanded modifiedT-cells expresses cell-surface marker(s) of a central memory Tcell (Tci). In certain embodiments, this method further comprises the step of (d) enriching the plurality of expanded modified T-cells to produce a composition comprising at least 2%. 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, %, 75%, 80%, 85%, 90%, 95% 99% or any percentage between of modified T-cells that express cell-surface marker(s) of a central memory T cell (Tci). In certain embodiments, this method further comprises the step of (d) enriching the plurality of expanded modified T-cells to produce a composition comprising at least 60% of modified T-cells that express cell surface marker(s) of a central memory'T cell (TCM). In certain embodimentsof this method, the enriching step comprises isolating modified T-cells that express one or more cell-surface marker(s) of a central memory T cell (TcM)from the plurality of enriched modified T-cells. In certain embodiments of this method, the enriching step further comprises contacting the isolated modifiedTcMand aTceliexpansion composition comprising one or more of human serum albumin, recombinant human insulin, humantransferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement to produce a plurality of expanded enriched modified TcN. In certain embodiments of this method, the T-cell expansion composition further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetrainethyl-5-decyn-4,7-dio (TMDD), diisopropyl adipate (DIPA), n-butvl-benzenesulfonamide, 1,2-benzenedicarboxvlic acid, bis(2-inethylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, a sterol and an alkane. In certain embodiments of this method, thef-cell expansion composition further comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid and a sterol. In certain embodiments of this method, the T-cell expansion composition further comprises one or more of octanoic acid at a concentration of between 0.9 mg/kg to 90 mg/kg, inclusive of the endpoints; palmiticacid at a concentration of between 0.2 mg/kg to mg/kg, inclusive of the endpoints;linoleic acid at a concentration of between 0.2 mg/kg to mg/kg, inclusive of the endpoints; oleic acid at a concentration of 0.2 mg/kg to 20 mg/kg inclusive of the endpoints; and a sterol at a concentration of about 0.1 mg/kg to 10mg/kg, inclusive of the endpoints. In certain embodiments of this method, the T-cell expansion composition further comprises one or more of octanoic acid at a concentration of about 9 mg/kg, palmitic acid at a concentration of about 2 mg/kg, linoleic acid at a concentration of about 2 mg/kg oleic acid at a concentration of about 2 mg/kg and a sterol at a concentration of about I mng/kg. In certain embodiments of this method, the T-cell expansion composition further comprises one or more of octanoic acid at a concentration of between 6.4 pmol/kg and 640 pmol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 pmol/kg and 70 pumol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 pmol/kg and 75 pmol/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 pmol/kg and 75 pmol/kg, inclusive of the endpoints; and a sterol at a concentration of between 0.25 pmol/kg and 25 pmol/kg, inclusive of the endpoints. In certain embodiments of this method, the T-cell expansion composition further comprises one or more of octanoic acid at a concentration of about 64 pLmol/kg, palmitic acid at a concentration of about 7 pmol/kg, linoleic acid at a concentration of about 7.5 Ltmol/kg, oleic acid at a concentration of about 7.5 pmol/kg and a sterol at a concentration of about 2.5 pumol/kg.
[035] The disclosure provides amethodofproducing acomposition comprising aplurality of modified stem memory T-cells (TScM) and a plurality of modified central memoryT-cells
(TcM), comprising: (a) introducing into a plurality of primary humanT cells acomposition comprising an antigen receptor to produce a plurality of modified T cells, wherein the antigen receptor or the therapeutic protein is not contained in a transposon, and (b) contacting the plurality of modified T-cells and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetraneric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement to produce a composition comprising a plurality of activated modified stem memory T-cells (TSCM) and a plurality of activated modified central memory T-cells (TCM), wherein the plurality of activated modified Tsci expresses one or more CD62L. CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2R3 and the plurality of activated modified TCM expresses one or more CD45RO, CD95., IL-2R, CCRT and CD62L, thereby producing a composition comprising a plurality of modified TsCM and a plurality of modified TM. In certain embodiments of this method, the modified stem memory T-cells (Tscm) comprise at least 1%, 2%. 5%, 7%, 10%, 15%, 20%. 25%,30%, 35%, 40%, 45%, 50%. 55%, 60%, 65%,70%, %, 80%, 85%, 90%, 95%, 97%, 99% or any percentage of cells in between of the total number of cells of the composition. In certain embodimentsof this method, the modified central memory T-cells (TCM) comprise at least 1%2%, 5%,.7%, 10%, 15%, 20%, 25%, %, 35 5%40%45, 50%, 55%60%, 65%, 70% 75%, 80 5%,85 90 95%, 97, 99% or any percentage of cells in between of the total number of cells of the composition. In certain embodiments of this method, the modified stem memory T-cells (TscM) comprise at least 10% of the total number of cells of the composition and the modified central memory T cells (TCM) comprise at least 90% of the total number of cells of the composition. In certain embodimentsof this method, the modified stem memory T-cells (TSCM) comprise at least % of the total number of cells of the composition and the modified central memory T-cells (TCm) comprise at least 10% of the total number of cells of the composition. In certain embodiments of this method, the modified stem memory T-cells (TsCM) comprise at least % of the total number of cells of the composition and the modified central memory T-cells (TcM) comprise at least 80% of the total number of cells of the composition. In certain embodiments of this method, the modified stem memory T-cells (TCM) comprise at least % of the total number of cells of the composition and the modified central memoryT-cells (TcM) comprise at least 20% of the total number of cells of the composition. In certain embodiments of this method, the modified stem memory T-cells (Tsm) comprise at least % of the total number of cells of the composition and the modified central memory T-cells
(TcM) comprise at least 70% of the total number of cells of the composition. In certain embodiments of this method, the modified stem memory T-cells (TscM) comprise at least % of the total number of cells of the composition and the modified central memory T-cells (TCM) comprise at least 30% of the total number of cells of the composition. In certain embodiments of this method, the modified stem memoryT-cells (TscM) comprise at least % of the total number of cells of the composition and the modified central memory T-cells (TcM) comprise at least 60% of the total number of cells of the composition. In certain embodimentsof this method, the modified stem memory T-cells (TscM) comprise at least % of the total number of cells of the composition and the modified central memory T-cells (TCMi) comprise at least 40% of the total number of cells of the composition. In certain embodiments of this method, the modified stem memoryT-cells (TscM) comprise at least % of the total number of cells of the composition and the modified central memory T-cells (TcM) comprise at least 50% of the total number of cells of the composition.
10361 In certain embodiments of methods of the disclosure of producing a composition comprisingaplurality of modified stem memoryT-cells (Tsc) and aplurality ofmodified central memory T-cells (TCM), comprising: (a) introducing into a plurality of primary human T cells a composition comprising an antigen receptor to produce a plurality of modified T cells, wherein the antigen receptor or the therapeutic protein is not contained in a transposon, and (b) contacting the plurality of modifiedT-cells and aT-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement to produce a composition comprising a plurality of activated modified sten memory T-cells (TscM) and a plurality of activated modified central memory T-cells (Tci), the T-cell activator composition of (b) further comprises an anti-human CD2 monospecific tetrameric antibody complex. In certain embodiments, this method further comprises the step of (c) contacting the composition the plurality of activated modified stem memory T-cells (TsCM) and the plurality of activated modified central memory T-cellS (TcM) with a T-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement to produce a plurality of expanded modifiedT-cells, wherein the plurality of expanded modified TSCMexpresses one or more CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2R3 and the plurality of expanded modified TM expresses one or more CD45RO, CD95., IL-2R, CCRT and CD62L, thereby producing a composition comprising a plurality of expanded modified TscM and a pluralityof expanded modified TC4. In certain embodiments of this method, the enriching step comprises isolating modified T cells that express one or more cell-surface marker(s) of a stem memory T cell (TscM) from the plurality of enriched modified T-cells or isolating modified'T-cells that express one or more cell-surface marker(s) of a central memory'T cell (TCNi) from the plurality of enriched modified T-cells. In certain embodiments of this method, the enriching step comprises isolating modified T-cells that express one or more cell-surface marker(s) of a stemmemory T cell (TSCM) from the plurality of enriched modified T-cells and isolating modified T-cells that express one or more cell-surface marker(s) of a central memory T cell (TCM) from the plurality of enriched modified T-cells. In certain embodiments of this method, the enriching step further comprises contacting the composition comprising the isolated modifiedTSCM and the isolated modified TCM with aT-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscoves MDI, and an expansion supplement to produce a composition comprising a plurality of expanded enriched modified TsCM and a plurality of expanded enriched modified TCNi. In certain embodiments of this method, theT-cell expansion composition further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethiyl-5-decyn-4,7-dio (TMDD), diisopropyl adipate (DIPA), n-butyl-benzenesulfonamide, 12-benzenedicarboxvlic acid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid. stearic acid hydrazide, oleamide, a sterol and an alkane. In certain embodiments of this method, the T-cell expansion composition further comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid and a sterol. In certain embodiments of this method, thei T-cell expansion composition further comprises one or more of octanoic acid at a concentration of between 0.9 mg/kg to 90 mg/kg, inclusive of the endpoints; palmitic acid at a concentration ofbetween0.2mg/kg to mg/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.2 mg/kg to mg/kg, inclusive of the endpoints; oleic acid at a concentration of 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; and a sterol at a concentration of about 0.1 mg/kg to 10mg/kg, inclusive of the endpoints. In certain embodiments of this method, the T-cell expansion composition further comprises one or more of octanoic acid at a concentration of about 9 mg/kg, palmitic acid at a concentration of about 2 mg/g, linoleic acid at a concentration of about 2 mg/kg, oleic acid at a concentration of about 2 mg/kg and a sterol at a concentration of about 1 mg/kg. In certain embodiments of this method, the T-cell expansion composition further comprises one or more of octanoic acid at a concentration of between 6.4 pmol/kg and 640 ptmol/kg, inclusive of the endpoints; palinitic acid at a concentration of between 0.7 umol/kg and 70 pmol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 pmol/kg and 75 pmol/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 pmol/g and 75 pmol/kg, inclusive of the endpoints; and a sterol at a concentration of between 0.25 punol/kg and 25 pmol/kg, inclusive of the endpoints. In certain embodiments of this method, the T-cell expansion composition further comprises one or more of octanoic acid at a concentration of about 64 pmol/kg, palmitic acid at a concentration of about 7 pmol/kg, linoleic acid at a concentration of about 7.5 pmol/kg. oleic acid at a concentration of about 7.5 umol/kg and a sterol at a concentration of about 2.5 pumol/kg. In certain embodiments of this method, the modified stem memory T-cells (Tsci) comprise at least 1%, 2%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, %,65%,70%,75%,80%,85%, 90%,95%,97%, 99%oranypercentage of cells in between of the total number of cells of the composition. In certain embodiments of this method, the modified central memory T-cells (Tc) comprise at least 1%, 2%, 5%, 7%, 10%, %, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%,90%, %, 97%, 99% or any percentage of cells in between of the total number of cells of the composition. In certain embodiments of this method, the modified stem memory T-cells (Tsc) comprise at least 10%of the total number of cells of the composition and the modified central memory-T-cells (Tc) comprise at least 90%of the total number of cells of the composition. In certain embodiments of this method, the modified stein memory T-cells (TscM) comprise at least 90%of the total number of cells of the composition and the modified central memory T-cells (TCM)comprise at least 10%of the total number of cells of the composition. In certain embodiments of this method, theinodified stem memoryT-cells (TscM) comprise at least 20%of the total number of cells of the composition and the modifiedcentralmemoryT-cells (TCM) comprise at least 80% of the total number of cells of the composition. In certain embodiments of this method, the modified stem memory T-cells (TscM) comprise at least 80% of the total number of cells of the composition and the modified central memory T-cells (TCM) comprise at least 20% of the total number of cells of the composition. In certain embodiments of this method, the modified stem memoryIT-cells (TscM) comprise at least 30% of the total number of cells of the composition and the modified central memory T-cells (TCM) comprise at least 70% ofthe total number of cells of the composition. In certain embodiments of this method, the modified stem memory T-cells (Tsc) comprise at least 70% of the total number of cells of the composition and the modified central memory T-cells (TCM) comprise at least 30%of the total number of cells of the composition. In certain embodiments of this method, the modified stem memory T-cells (TscM) comprise at least 40% of the total number of cells of the composition and the modified central memory T-cells (TcM) comprise at least 60% of the total number of cells of the composition. In certain embodiments of this method, the modified stem memory T-cells (TscM) comprise at least 60% of the total number of cells of the composition and the modified central memory T-cells (TC) comprise at least 40% of the total number of cells of the composition. In certain embodiments of this method, the modified stem memoryT-cells (Tsci) comprise at least 50%of the total number of cells of the composition and the modified central memory T-cells (TcM) comprise at least 50% of the total number of cells of the composition.
[037] In certain embodiments of the methods of producing an activatedmodified TSCM or TCM of the disclosure, including those methods compTising (a) introducing into a plurality of primary human T cells a composition comprising an antigen receptor to produce a plurality of modified T cells, wherein the antigen receptor or the therapeutic protein is not contained in a transposon, and (b) contacting the plurality of modified T-cells and aT-cell activator composition comprising one or more ofananti-human CD3 monospecific tetrameric antibody complex, the introducing step comprises a homologous recombination. In certain embodiments of the introduction step comprising a homologous recombination, agenomic editing composition contacts a genomic sequence of at least one primary T cell of the plurality of T cells. In certain embodiments ofthe introduction step comprising a homologous recombination, a genomic editing composition contacts a genomic sequence of a portion of primary Tcells of the plurality ofTcells. In certain embodiments, the portion of primary'T cells5is atleast1%2%5%7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, %, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%. 99% or any percentage in between of the total number of primary T cells in the plurality of Tcells. In certain embodiments of the introduction step comprising a homologous recombination, a genomic editing composition contacts a genomic sequence of each primary T cell of the plurality of T cells. In certain embodiments of the introduction step comprising a homologous recombination, a genomic editing composition induces a single strand break. In certain embodimentsof the introduction step comprising a homologous recombination, a genomic editing composition induces a double strand break. In certain embodiments of the introduction step comprising a homologous recombination, the introduction step further comprises a donor sequence composition. In certain embodiments, the donor sequence composition comprises a sequence encoding the antigen receptor. In certain embodiments, the donor sequence composition comprises a sequence encoding the antigen receptor, a 5' genomic sequence and a 3' genomic sequence, wherein the 5' genomic sequence is homologous or identical to a genomic sequence of the primaryTcell that is 5'to the break point induced bythe genomic editing composition aid the 3' genomic sequence is homologous or identical to a genomic sequence of the primary T cell that is 3' to the break point induced by the genomic editing composition. In certain embodiments of the introduction step comprising a homologous recombination, the genomic editing composition and donor sequence composition are contacted with the genomic sequence simultaneously or sequentially. In certain embodiments of the introduction step comprising a homologous recombination, the genomic editing composition and donor sequence composition are contacted with thegenomic sequence sequentially, and the genomic editing composition is provided first, In certain embodiments of the introduction step comprising a homologous recombination, the genomic editing composition comprises a sequence encoding a DNA binding domain and a sequence encoding a nuclease domain. In certain embodiments of the introduction step comprising a homologous recombination, the genomic editing composition comprises a DNA binding domain and a nuclease domain. In certain embodiments of the genomic editing composition, the DNA binding domain comprises a guide RNA (gRNA). In certain embodiments of the genomic editing composition, the DNA binding domain comprises a DNA-binding domain of a TALEN. In certain embodiments of the genomic editing composition, the DNA binding domain comprises a DNA-binding domain of a ZFN. In certain embodiments of the genomic editing composition, the nuclease domain comprises a Cas9 nuclease or a sequence thereof. In certain embodiments of the genomic editing composition, the nuclease domain comprises an inactive Cas9 (SEQ ID NO: 33, comprising a substitution of a Alanine (A) for Aspartic Acid (D) at position 10 (DI0A) and a substitution of Alanine (A) for Histidine (H)at position 840 (H840A)). In certain embodiments of the genomic editing composition, the nuclease domain comprises a short and inactive Cas9 (SEQ ID NO: 32, comprising a substitution of an Alanine (A) for an Aspartic Acid (D) at position 10 (DIOA) and a substitution of an Alanine (A) for an Asparagine (N) at position 540 (N540A)). In certain embodiments of the genomic editing composition, the nuclease domain comprises or further comprises a type IIS endonuclease. In certain embodiments of the genomic editing composition, the type IIS endonuclease comprises Acil Mnl, AlwI, Bbl, Bccl, BeceAl, BsmAI. BsmFI, BspCNI,
BsrI, BtsC, HgaI, Hphl, HpyAV, Mboll, Myll, Plel, SfaNI, Acul, BeiVI, BfuAI, BmgBI, BmrI, BpmI, BpuEl, BsaI, BseRI, BsgI, BsmI, BspMI, BsrBI, BsrBI, BsrDI, BLtgZI, Btsl, Earl, Ecil, MmeI, NmeAIIl, BbvCI, Bpul0I, BspQI, SapI, Bael, BsaXI, CspCI Bfi, MboII, Acc361, FokI or Clo051. In certain embodiments, the type IIS endonuclease comprises CoO51 In certain embodiments of the genomic editing composition, the nuclease domain comprises or further comprises a TALEN or a nuclease domain thereof. In certain embodiments of the genomic editing composition, the nuclease domain comprises or further comprises a ZFN or a nuclease domain thereof. In certain embodiments of the introduction step comprising a homologous recombination, the genomic editing composition induces a break in a genomic sequence and the donor sequence composition is inserted using the endogenous DNA repair mechanisms of the primary T cell. In certain embodiments of the introduction step comprising a homologous recombination, the insertion of the donor sequence composition eliminates a DNA binding site of the genomic editing composition, thereby preventing further activity of the genomic editing composition.
10381 In certain embodiments of the methods of producing an activated modifiedTScM or TcN of the disclosure, including those methods comprising (a) introducing into a plurality of primary human T cells a composition comprising an antigen receptor to produce a plurality of modified T cells, wherein the antigen receptor or the therapeutic protein is not contained in a transposon, and (b) contacting the plurality of modified T-cells and a T-cell activator composition comprising one or more of an anti-human CD3monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement, a viral vector comprises the antigen receptor. In certain embodiments., the viral vector comprises one or more sequences isolated, derived, or recombined from an RNA virus. In certain embodiments, the RNAs vis is a single-stranded or a double-stranded virus. In certain embodiments, the viral vector comprises one or more sequences isolated, derived, or recombined from a DNA virus. In certain embodiments, the DNA virus is a single-stranded or a double-stranded virus. In certain embodiments, the virus is replication defective.
10391 In certain embodiments of the methods of producing an activated modified TScv or TcNI of the disclosure, including those methods comprising (a) introducing into a plurality of primary human T cells a composition comprising an antigen receptor to produce a plurality of modified T cells, wherein the antigen receptor or the therapeutic protein is not contained in a transposon, and (b) contacting the plurality of modified T-cells and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement, a viral vector comprises the antigen receptor. In certain embodiments, the viral vector comprises a sequence isolated or derived from a retrovirus. In certain embodiments, the viral vector comprises a sequence isolated or derived from a lentivirus.
[040] In certain embodiments of the methods of producing an activatedmodified TCM or TCM of the disclosure, including those methods comprising (a) introducing into a plurality of primary human Tcells a composition comprisingan antigen receptor to produce a plurality of modified T cells, wherein the antigen receptor or the therapeutic protein is not contained in a transposon, and (b) contacting the plurality of modified T-cells and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement, a viral vector comprises the antigen receptor.In certain embodiments, the viral vector comprises a sequence isolated or derived from a retrovirus. In certain embodiments, the viral vector comprises a sequence isolated or derived from a gamma retrovirus.
[041] In certain embodiments of the methods of producing an activated modified Tscm or Tcmof the disclosure, including those methods comprising (a) introducing into a plurality of primary human Tcells a composition comprising an antigen receptor to produce a plurality of modified T cells, wherein the antigen receptor or the therapeutic protein is not contained in a transposon, and (b) contacting the plurality of modified T-cells and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement, a viral vector comprises the antigen receptor. In certain embodiments., the viral vector comprises a sequence isolated or derived from an adeno-associated virus (AAV). In certain embodiments, the AAV is a serotype A-V1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 or AAV11. In certain embodiments, the AAV comprises a sequence from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAVS, AAV9, AAV10 or AAV11. In certain embodiments, the AAV comprises a sequence isolated, derived, or recombined from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7. AAV8, AAV9, AAV10 or AAV11 In certain embodiments, the AAV comprises a sequence isolated, derived, or recombined from AAV2. In certain embodiments, including those in which the vector crosses the blood brain barrier
(BBB), the AAV comprises a sequence isolated, derived, or recombined from AAV9. Exemplary adeno-associated viruses aid recombinant adeno-associated viruses of the disclosure include, but are not limited to, self-complementary AAV (scAAV) and AAV hybrids containing the genome of one serotype and the capsid of another serotype (e.g. AAV2/5, AAV-DJand AAV-DJ8). Exemplary adeno-associated virusesand recombinant adeno-associated viruses of the disclosure include, butare not limited to, rAAV-LK03, rAAV-NP59and rAAV-NP84.
[042] In certain embodiments of the methods of producing an activated modified TSCM or TcM of the disclosure, a nucleic acid vector comprises the antigen receptor. In certain embodiments, a DNA vector comprises the antigen receptor. In certain embodiments, an mRNA vector comprises the antigen receptor. In certain embodiments, the nucleic acid vector is a plasmid or aminicircle vector.
10431 In certain embodiments of the methods of producing an activated modified TSCM or TCMof the disclosure, a nanoparticle vector comprises the antigen receptor. Nanoparticles may be comprised of polymers disclosed in, for example, International Patent Publication No. WO 2012/094679, International Patent Publication No. WO 2016/022805, International Patent Publication No. WO/2011/133635, International Patent Publication No. WO/2016/090111, International Patent Publication No. WO/2017/004498, WO/2017/004509 International Patent Application No. PCT/US2017/030271, US PatentNo. 6,835,394, US Patent No. 7,217,427, and US Patent No. 7,867,512.
10441 In certain embodiments of the methods of producing an activated modified TScM or TCM of the disclosure, the antigen receptor is a T-cell receptor. In certain embodiments, the T-cell receptor is naturally-occurring. In certain embodiments, the T-cell receptor is not naturally-occurring. In certain embodiments, and, in particular, those embodiments wherein theT-cell receptor is not naturally-occurring, the T-cell receptor comprises one or more mutation(s) compared to a wild-type T-cell receptor. In certain embodiments, and, in particular, those embodiments wherein the T-cell receptor is not naturally-occurring, the T cell receptor is a recombinant T-cell receptor. In certain embodiments of this method, the antigen receptor is a Chimeric Antigen Receptor (CAR). In certain embodiments, the CAR is a CARTyrin. In certain embodiments, the CAR comprises one or more VH-I sequence(s). In certain embodiments, the CAR is a VCAR.
[045] In certain embodiments of the methods of producing an activated modified TscM or TcM ofthe disclosure, including those methods comprising (a) introducinginto plurality of primary human T cells a composition comprising an antigen receptor to produce a plurality of modified T cells, wherein the antigen receptor or the therapeutic protein is not contained in a transposon, and (b) contacting the plurality of modified T-cells and a T-cell activator composition comprising one or more of an anti-human CD3monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement, the method further comprises introducing into the primary human T cell, a composition comprising a therapeutic protein to produce a modified T cell capable of expressing the therapeutic protein. In certain embodiments, the therapeutic protein is a secretable protein and the method produces a modified T cell capable of secreting the therapeutic protein. In certain embodiments, the introducing step comprises a homologous recombination and a donor sequence comprises a sequence encoding the therapeutic protein. In certain embodiments, the donor sequence that comprises the antigen receptor further comprises the therapeutic protein. In certain embodiments, a first donor sequence comprises the antigen receptor and a second donor sequence comprises the therapeutic protein. In certain embodiments, a vector comprises a sequence encoding the therapeutic protein. In certain embodiments, the vector is a viral vector. In certain embodiments, the vector is a nanoparticle. In certain embodiments, the vector that comprises the antigen receptor further comprises the therapeutic protein. In certain embodiments, a first vector comprises the antigen receptor and a second vector template comprises the therapeutic protein. 10461 The disclosure provides a method of producing a modified stein memory T cell (Tsc~), comprising: (a) introducing into a primary human T cell a composition comprising an antigen receptor to produce a modified T cell, wherein a transposon comprises theantigen receptor, and (b) contacting the modified T cell and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement to produce an activated modified T-cell, wherein the activated modified-T cell expresses one or more cell-surface marker(s) of a stem memory T cell (TscM), thereby producing a modified stem memory T cell (TscM). The disclosure provides a method of producing a plurality of modified stem memory T cells (Tsc), comprising: (a) introducing into a plurality of primary human T cells a composition comprising an antigen receptor to produce a plurality of modified T cells, wherein a transposon comprises the antigen receptor., and (b) contacting the pluralityof modified Tcells and aT-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement to produce a plurality of activated modified T-cells, wherein at least 25%, 50%, %, 75%, 80%, 85%, 90%, 95% or 99%of the plurality of activated modified -Tcells expresses one or more cell-surface marker(s) of a stein memory T cell (TscM), thereby producing a modified stem memory T cell (TscM). In certain embodiments of this method, at least 60% of the plurality of activated modified -T cells expresses one or more cell-surface marker(s) of a stein memory T cell (TscM). In certain embodiments of this method, the T-cell activator composition of (b) further comprises an anti-human CD2 monospecific tetrameric antibody complex. The disclosure provides a method of producing a modified stem memory Tcell (TscM). comprising: (a) introducing into a primary human T cell a composition comprising a chimeric antigen receptor (CAR) to produce a CAR-T cell and (b) contacting the CAR-T cell and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetramneric antibody complex, an anti-human CD28 monospecific tetramneric antibody complex, an anti-human CD2 inonospecific tetrameric antibody complex and an activation supplement to produce an activated CAR-T cell, wherein the activated CAR-T cell expresses one or more cell-surface marker(s) of a stem memoryTcell (TScM). thereby producing a CAR-expressing stemmemoryTcell (TCM) (CAR-TscM). The disclosure provides a method of producing a plurality of modified stem memory T cells (TscM), comprising: (a) introducing into a plurality of primary human T cells a composition comprising a chimeric antigen receptor (CAR) to produce a plurality of CAR-T cells and(b) contacting the plurality of CAR-T cells and a.T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetramneiic antibody complex, an anti-human CD28 monospecific tetrameric antibody complex, an anti-human CD2 monospecific tetrameric antibody complex and an activation supplement to produce a plurality of activated CAR-T cells, wherein at least 2%,.5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%45%,50%,60% %, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of activated CAR-Tcells expresses one or more cell-surface marker(s) of a stem memory T cell (Tsc~), thereby producing a pluralityof activated CAR stem memoryT cells (TsCM). In certain embodimnents, the methods further comprises the step of: (c) contacting the activated modified T cell and a T-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement to produce a plurality of expanded modified T-cells, whereinat least 2% of the plurality of expanded modified T-cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tsci). In certain embodiments, the T-cell expansion composition comprises or further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decyn-4,7-diol (TMDD), diisopropyl adipate (DIPA), n-butyl benzenesulfonamide, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, a sterol and an alkane. In certain embodiments, theT-cell expansion composition comprises one or more of octanoicacid, palmitic acid, linoleic acid, oleic acid and a sterol (e.g. cholesterol). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of between 0.9 mg/kg to 90 mg/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; oleic acid at a concentration of 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; and a sterol at a concentration of about 0.1 mg/kg to 10 mg/kg, inclusive of the endpoints (wherein mg/kg= parts per million). Incertain embodiments, theT-cell expansion composition comprises one or more of octanoic acid at a concentration of about 9 mg/kg, palmitic acid at a concentration of about 2 mg/kg, linoleic acid at a concentration of about 2 mg/kg, oleic acid at a concentration of about 2 mg/kg, and a sterol at a concentration ofabout 1 mg/kg (wherein mg/kg = parts per million). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of 9.19 mg/kg, palmitic acid at a concentration of 1.86 mg/kg, linoleic acid at a concentration of about 2.12 mg/kg, oleic acid at a concentration of about 2.13 mg/kg, and a sterol at a concentration of about 1.01 mg/kg (wherein mg/kg = parts per million). In certain embodiments, the T-cell expansion composition comprises octanoic acid at a concentration of 9.19 mg/kg, palmitic acid at a concentration of 1.86 mg/kg, linoleic acid at a concentration of 2.12 mg/kg, oleic acid at a concentration of about 2.13 mg/kg, and a sterol at a concentration of 1.01 mg/kg (wherein mg/kg = parts per million). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of between 6.4 Pmol/kg and 640 pimol/kg inclusive of the endpoints; palmitic acid at a concentration of between 0.7 mnol/kg and 70 pnol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 pLmol/kg and 75 mol/kg, inclusive of tie endpoints; oleic acid at a concentration of between 0.75 Pmol/kg and 75 pmol/kg, inclusive of the endpoints; and a sterol at a concentration of between 0.25 pmol/kg and 25 pmol/kg, inclusive of theendpoints. In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 64 pmoi/kg, palmitic acid at a concentration of about 7 mol/kg. linoleic acid at a concentration of about 7.5 pmol/kg oleic acid at a concentration of about 7.5 pmol/kg and a sterol at a concentration of about 2.5 pmol/kg. In certain embodiments, the'T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 63.75 pmol/kg, palmitic acid at a concentration of about 7.27 pmol/kg, linoleic acid at a concentration of about 7.57 pmoi/kg, oleic acid at a concentration of about 7.56 pmol/kg and a sterol at a concentration of about 2.61 mol/kg In certain embodiments, the T-cell expansion composition comprises octanoic acid at a concentration of about 63.75 pmol/kg, palmitic acid at a concentration of about 7.27 imol/kg, linoleic acid at a concentration of about 7.57 pmol/kg, oleic acid at a concentration of 7.56 pmol/kg and a sterol at a concentration of 2.61 jmol/kg. In certain embodiments, at least 2%, 5%, 10%15%, 0 20% %, 30%, 35%,40%, 45%, 50%, 60%, 65%, 70%, 75%,/80%, 85%, 90%,/95%, 99% or anv percentage in between of the plurality of expanded modified T-cells expresses cell-surface marker(s) of a stem memory Tcell (Tscm). In certain embodiments, at least 60% of the plurality of expanded modified T-cells expresses cell-surface marker(s) of a stem memory T cell (Tsc). In certain embodiments, the method further comprises the step of: (d) enriching the plurality of expanded modified T-cells to produce a composition comprising at least 2%, %, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%,70%, 75%, 80%, 85%, %, 95%, 99% or any percentage in between ofmodified T-cells that express cell-surface marker(s) of a stem memory T cell (TscM). In certain embodiments, the method further comprises the step of (d) enriching the plurality of expanded modified T-cells to produce a composition comprising at least 60% of modified T-cells that express cell-surface marker(s) of a stem memory T cell (TscM). In certain embodiments, the enriching step further comprises isolating modified T-cells that express one or more cell-surface marker(s) of a stem memory T cell (TscM) from the plurality of enriched modified T-cells. In certain embodiments, the enriching step further comprises contacting the isolated modified Tscm and aIT-celi expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transferring, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement to produce a plurality of expanded enriched modified TscM. In certain embodiments, the T-cell expansion composition further comprises one or more of octanoic acid, nicotinamide, 2,4.7,9-tetrametli-5-decyn-4,7-dioI (TMDD), diisopropyl adipate
(DIPA), n-butyl-benzenesulfonamide, 1,2-benzenedicarboxylic acid. bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, a sterol and an alkane. In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid and a sterol (e.g. cholesterol). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of between 0.9 mg/kg to 90 mg/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints;oleic acid at a concentration of 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; and a sterol at a concentration of about 0.1 mg/kg to 10 mg/kg, inclusive of the endpoints (wherein mg/kg = pails per million). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 9 mg/kg, palmitic acid at a concentration of about 2 mg/kg, linoleic acid at a concentration of about 2 mg/kg, oleic acid at a concentration of about 2 mg/kg, and a sterol at a concentration of about1 mg/kg (wherein mg/kg= parts per million). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of 9.19 mg/kg, palmitic acidata concentration of 1.86 mg/kg, linoleic acid at a concentration of about 2.12 mg/kg, oleic acid at a concentration of about 2.13 mg/kg, and a sterol at a concentration of about 1.01 mg/kg (wherein mg/kg = parts per million). In certain embodiments, theT-cel expansion composition comprises octanoic acid at a concentration of 9.19 mg/kg, palmitic acid at a concentration of 1.86 mg/kg, linoleic acid at a concentration of 2.12 mg/kg, oleic acid at a concentration of about 2.13 mg/kg, and a sterol at a concentration of 1.01 mg/kg (wherein mg/kg = parts per million). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of between 6.4 mol/kg and 640 pLmol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 [mol/kg and 70 pmol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 pmol/kg and 75 pmo/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 pmol/kg and 75 pmol/kg, inclusive of the endpoints; and a sterol at a concentration of between 0.25 pmol/kg and 25 pmol/kg, inclusive of the endpoints. In certain embodiments. the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 64 pmoi/kg, palmitic acid at a concentration of about 7 mol/kg,inoleic acid ata concentration of about 7.5 pmol/kg, oleic acid at a concentration of about 7.5 imol/kg and a sterol at a concentration of about 2.5 tmol/kg. In certain embodiments, the T-cell expansion composition comprisesone or more of octanoic acid at a concentration of about 63.75 pumol/kg, palmitic acid at a concentration of about 7.27 pno/kg. linoleic acid at a concentration of about 7.57 moi/kg, oleic acid at a concentration of about 7.56 pmol/kg and a sterol at a concentration of about 2.61 moil/kg. In certain embodiments, the T-cell expansion composition comprises octanoic acid at a concentration ofabout 63.75 pmo/kg, palmitic acid at a concentration of about 7.27 pmol/kg, linoleic acid at a concentration of about 7.57 pumol/kg, oleic acid at a concentration of 7.56 Vmol/kg and a sterol at a concentration of 2.61 pmol/kg.
[047] The disclosure provides a method of producing a modified central memory T cell (Tcm), comprising:(a) introducing into a primary human T cell a composition comprising an antigen receptor to produce a modified Tcell, wherein a transposon comprises the antigen receptor, and (b) contacting the modified Tcell and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement to produce an activated modified T-cell, wherein the activated modified-T cell expresses one or more cell-surface marker(s) of a central memoryTcell (Tei). thereby producing a modified central memory T cell (Tcd). The disclosure provides a method of producing a plurality of modified central memory T cells (Tc).,comprising: (a) introducing into a plurality of primary human'T cells a composition comprising an antigen receptor to produce a plurality of modified T cells, wherein a transposon comprises the antigen receptor, and (b) contacting the plurality of modified T cells and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement to produce a plurality of activated modified T-cells, wherein at least 25%,50%, %, 75%, 80%, 85%, 90%, 95% or 99% of the plurality of activated modified -T cells expresses one or more cell-surface marker(s) of a central memory T cell (TcM), thereby producing a modified central memoryT cell (TcM). In certain embodiments of this method, at least 60% of the plurality of activated modified -T cells expresses one or more cell-surface marker(s) of a central memory T cell (TcM). In certain embodiments of this method, the T cell activator composition of (b) further comprises an anti-human CD2monospecific tetramneric antibody complex. In certain embodiments, the methods further comprises the step of: (c) contacting the activated modified T cell and a T-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement to produce a plurality of expanded modified T-cells, wherein at least 2% of the plurality of expanded modified T-cells expresses one or more cell-surface marker(s) of a central memory T cell (TC). In certain embodiments, the T-cell expansion composition comprises or further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetraincthyl-5-decyn-4,7-dio (TMDD), diisopropyl adipate (DIPA), n-butvl-benzenesulfonamide, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, a sterol and an alkane. In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid and a sterol (e.g. cholesterol). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of between 0.9 mg/kg to 90 mg/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; oleic acid at a concentration of 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; and a sterol at a concentration of about 0.1 mg/kg to 10 mg/kg., inclusive of the endpoints (wherein mg/kg = parts per million). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 9 mg/kg, palmitic acid at a concentration of about 2 mg/kg, linoleic acid at a concentration of about 2 mg/kg, oleic acid at a concentration of about 2 mg/kg, and a sterol at a concentration of about 1 mg/kg (wherein mg/kg = parts per million . In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of 9.19 mg/kg palmitic acid at a concentration of 1.86 mg/kg, linoleic acid at a concentration of about 2.12 ig/kg, oleic acid at a concentration of about 2.13 mg/kg, and a sterol at a concentration of about 1.01 mg/kg (wherein mg/kg =partsper million). In certain embodiments, the T-cell expansion composition comprises octanoic acid at a concentration of 9.19 mg/kg, palmitic acid at a concentration of 1.86 mg/kg, linoleic acid at a concentration of 2.12 mg/kg, oleic acid at a concentration of about 2.13 mg/kg, and a sterol at a concentration of 1.01 mg/kg (wherein mg/kg = parts per million). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of between 6.4 pmol/kg and 640 pmol/kg, inclusive of the endpoints; palmiticacid at a concentration of between 0.71umol/kg and 70 imol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 pmol/kg and 75 pmiol/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 imol/kg and 75 pmol/kg, inclusive of the endpoints; and a sterol at a concentration of between 0.25 pmolg and 25 pmol/kg, inclusive of the endpoints. In certain embodiments, the T-cell expansion composition comprises one ormore of octanoic acid at a concentration of about 64 pmol/kg, palmitic acid at a concentration of about 7 mol/kg,linoleic acid at a concentration of about 7.5 pmol,g, oleic acid at a concentration of about 7.5 pmol/kg and a sterol at a concentrationof about 2.5 nmo1/kg. In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 63.75 [mol/kg, palmitic acid at a concentration of about 7.27 pmol/kg. linolcic acid at a concentration of about 7.57 pmol/kg, olcic acid at a concentration of about 7.56 pmol/kg and a sterol ata concentration of about 2.61 pno/kg. In certain embodiments, the T-cell expansion composition comprises octanoic acid at a concentration of about 63.75 Pmol/kg, palmitic acid at a concentration of about 7.27 pmol/kg, linoleic acid at a concentration of about 7.57 pmol/kg, oleic acid at a concentration of 7.56 imoi/kgand a sterol at a concentration of 2.61 pimol/kg. In certain embodiments. at least 2%, 5%, 10%, %, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or anypercentage in between of the plurality of expanded modified T-cells expresses cell-surface marker(s) of a central memory Tcell (TcM). In certain embodiments, at least % of the plurality of expanded modified T-cells expresses cell-surface marker(s) of a central memory T cell (Tcu). In certain embodiments, the method further comprises the step of (d) enriching the plurality of expanded modified'T-cells to produce a composition comprising at least 2%, 5%, 10%, 15% 20%, 25%,30%, 35% 40% 45% 50% 60%, 65%, %, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of modified T-cells that express cell-surface marker(s) of a central memoryTcell (Tcu). In certain embodiments, the method further comprises the stepof (d) enriching the pluralityof expanded modified T cells to produce a composition comprising at least 60% ofmodified T-cells that express cell surface marker(s) of a central memory T cell (TM). In certain embodiments, the enriching step further comprises isolating modifiedT-cells that express one or more cell-surface marker(s) of a central memory T cell (Tci) from the plurality of enriched modified T-cells. In certain embodiments, the enriching step further comprises contacting the isolated modified TCMand a T-cell expansion composition comprisingone or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement to produce a plurality of expanded enriched modified TCM. In certain embodiments, the T-cell expansion composition further comprises one or more of octanoic acid, nicotinamide, 2.4,7,9-tetranethli-5-decyn-4,7-dioI (IMDD). diisopropyl adipate
(DIPA), n-butyl-benzenesulfonamide, 1,2-benzenedicarboxylic acid. bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, a sterol and an alkane. In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid and a sterol (e.g. cholesterol). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of between 0.9 mg/kg to 90 mg/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; oleic acid at a concentration of 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; and a sterol at a concentration of about 0.1 mg/kg to 10 mg/kg, inclusive of the endpoints (wherein mg/kg = pails per million). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 9 mg/kg, palmitic acid at a concentration of about 2 mg/kg, linoleic acid at a concentration of about 2 mg/kg, oleic acid at a concentration of about 2 mg/kg, and a sterol at a concentration of about1 mg/kg (wherein mg/kg= parts per million). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of 9.19 mg/kg, palmitic acidata concentration of 1.86 mg/kg, linoleic acid at a concentration of about 2.12 mg/kg, oleic acid at a concentration of about 2.13 mg/kg, and a sterol at a concentration of about 1.01 mg/kg (wherein mg/kg = parts per million). In certain embodiments, theT-cel expansion composition comprises octanoic acid at a concentration of 9.19 mg/kg, palmitic acid at a concentration of 1.86 mg/kg, linoleic acid at a concentration of 2.12 mg/kg, oleic acid at a concentration of about 2.13 mg/kg, and a sterol at a concentration of 1.01 mg/kg (wherein mg/kg = parts per million). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of between 6.4 mol/kg and 640 pLmol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 [mol/kg and 70 pmol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 pmol/kg and 75 pmo/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 pmol/kg and 75 pmol/kg, inclusive of the endpoints; and a sterol at a concentration of between 0.25 pmol/kg and 25 pmol/kg, inclusive of the endpoints. In certain embodiments. the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 64 pmoi/kg, palmitic acid at a concentration of about 7 pmol/kg,inoleic acid ata concentration of about 7.5 pmol/kg, oleic acid at a concentration of about 7.5pmol/kg and a sterol at a concentration of about 2.5 pmol/kg. In certain embodiments, the T-cell expansion composition comprisesone or more of octanoic acid at a concentration of about 63.75 pumol/kg, palmitic acid at a concentration of about 7.27 pno/kg. linoleic acid at a concentration of about 7.57 moi/kg, oleic acid at a concentration of about 7.56 pmol/kg and a sterol at a concentration of about 2.61 moil/kg. In certain embodiments, the T-cell expansion composition comprises octanoic acid at a concentration ofabout 63.75 pmo/kg, palmitic acid at a concentration of about 7.27 pmol/kg, linoleic acid at a concentration of about 7.57 pumol/kg, oleic acid at a concentration of 7.56 Vmol/kg and a sterol at a concentration of 2.61 pmol/kg.
[048] The disclosureprovidesa methodofproducing acompositioncomprising aplurality of modified stem memory T-cells (TscM) and a plurality ofmodified central memory T-cells (TCM).comprising: (a) introducing into a plurality of primary human T cells a composition comprising an antigen receptor to produce a composition comprising a pluralityof modified stem memory T-cells (TsCM) and a plurality of modified central memory T-cellS (TM), wherein a transposon comprises the antigen receptor, and (b) contacting the composition and a'T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complexand an activation supplement to produce a composition comprising a plurality of activated modified stem memory T-cells (TscM) and a plurality of activated modified central memory T-cells (Tci). wherein the plurality of activated modifiedISCM expresses one or more CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2RP and the plurality of activated modified Tc[ expresses one or more CD45RO, CD95, IL-2R[, CCR7, and CD62L, thereby producing a composition comprising a plurality of modified'TSCM and a plurality of modified"TcM. In certain embodiments of this method, theT-cell activator composition of (b) further comprises an anti-human CD2 monospecific tetrameric antibody complex. In certain embodiments, the methods further comprises the step of: (c) contacting the composition and a T-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptothanol, Iscove's MDM, and an expansion supplement to produce a plurality of expanded modified T-cells, wherein at least 2% of the composition comprising a plurality ofexpanded modifiedT-cells expresses one or more cell-surface marker(s) of a stein memoryTcell (TscM). In certain embodiments, the methods further comprises the step of: (c) contacting the composition and a T-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement to produce a plurality of expanded modifiedT-cells, wherein at least 2% of the composition comprising a plurality of expanded modified T-cells expresses one or more cell surface marker(s) of a central memory T cell (TM). In certain embodiments, the T-cell expansion composition comprises or further comprises oneor more of octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decyn-4,7-dioI (TMDD), diisopropyl adipate (DIPA), n butyl-benzenesulfonamide, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, a sterol and an alkane. In certain embodiments, theT-cell expansion composition comprises one or more ofoctanoic acid,palmitic acid, linoleic acid, oleic acid and asterol (e.g. cholesterol). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of between 0.9 mg/kg to 90 mg/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; oleic acid at a concentration of 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; and a sterol at a concentration of about 0.1 mg/kg to 10 mg/kg, inclusive of the endpoints (wherein mg/kg= parts per million). In certain embodimentstheT-cell expansion composition comprises one or more of octanoic acid at a concentration of about 9 mg/kg, palmitic acid at a concentration of about 2 mg/kg, linoleic acid at a concentration of about 2 mg/kg, oleic acid at a concentration of about 2 mg/kg, and a sterol at a concentration of about1 mg/kg (wherein mg/kg = parts per million). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of 9.19 mg/kg, palmitic acid at a concentration of 1.86 mg/kg, linoleic acid at a concentration of about 2.12 mg/kg, oleic acid at a concentration of about 2.13 mg/kg, and a sterol at a concentration of about 1.01 mg/kg (wherein mg/kg = parts per million). In certain embodiments, the T-cell expansion composition comprises octanoic acid at a concentration of 9.19 mg/kg, palmitic acid at a concentration of 1.86 mg/kg, linoleic acid at a concentration of 2.12 mg/kg, oleic acid at a concentration of about 2.13 mg/kg, and a sterol at a concentration of 1.01 mg/kg (wherein mg/kg parts per million). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of between 6.4 Pmol/kg and 640 imol/kg. inclusive of the endpoints; palmitic acid at a concentration of between 0.7 pmol/kg and 70 nnol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 pLmol/kg and 75 mol/kg, inclusive of tie endpoints; oleic acid at a concentration of between 0.75 mol/kg and 75 pmol/kg, inclusive of the endpoints; and a sterol at a concentration of between 0.25 pmol/kg and 25 pmol/kg, inclusive of theendpoints. In certain embodiments., the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 64 mioi/kg, palmitic acid at a concentration of about 7 mol/kg. linoleic acid at a concentration of about 7.5 pmol/kg, oleic acid at a concentration of about 7.5 pmol/kg and a sterol at a concentration of about 2.5 pmol/kg. In certain embodiments, the'T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 63.75 pmol/kg, palmitic acid at a concentration of about 7.27 pmol/kg, linoleic acid at a concentration of about 7.57 pmo/kg, oleic acid at a concentration of about 7.56 pmolkg and a sterol at a concentration of about 2.61 mol/kg. In certain embodiments, the T-cell expansion composition comprises octanoic acid at a concentration of about 63.75 pmol/kg, palmitic acid at a concentration of about 7.27 pmol/kg, linoleic acid at a concentration of about 7.57 pmol/kg, oleic acid at a concentration of 7.56 pmol/kg and a sterol at a concentration of 2.61 tmol/kg. In certain embodiments, at least 2%, 5%, 10%15% ,020, %, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of cells the composition comprising a plurality of expanded modified TscM and a plurality of expanded modified TCM expresses cell-surface marker(s) of a stem memory T cell (Tsci). In certain embodiments, at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, %, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of cells the composition comprising a plurality of expanded modified TCM and a plurality of expanded modified TcM expresses cell-surface marker(s) of a central memory T cell (Tci). In certain embodiments, the method further comprises the step of (d) enriching the composition to produce a composition comprising at least 2%, 5%,10%, 15%, 20%, %, 30%, 35%,40%, 45%,50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,99% or any percentage in between of modified T-cellS that express cell-surface marker(s) of a stem memory T cell (Tscu). In certain embodiments, the method further comprises the step of (d) enriching the composition to produce a composition comprising at least 2%, 5%, 10%, 15%, %,25%, 30%, 35%,40%, 45%, 50%,60%, 65%, 70%,75%, 80%, 85%,90%,95%,99% or any percentage in between of modified T-cells that express cell-surface marker(s) of a central memory T cell (TC). In certain embodiments, the enriching step further comprises isolating modified'T-cells that express on or more cell-surface marker(s) of a stem e ory T cell (TscM) from the composition or isolating modified T-cellS that express one or more cell-surface marker(s) of a central memory T cell (Tc) from the composition. In certain embodiments, the enriching step further comprises isolating modified T-cells that express one or more cell-surface marker(s) of a stem memoryT cell (TCM) from the composition and isolating modified T-cells that express one or more cell-surface markers) of a central memory T cell (TcM) from the composition. In certain embodiments, the enriching step further comprises contacting the isolated modified TSCM and/orTCMand a T-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement to produce a composition comprising a plurality of expanded enriched modified Tsx and/or Tc i.In certain embodiments, the T-cell expansion composition further comprises one or more of octanoic acid,nicotinamide,2,4,7,9-tetramethyl-5-decyn-4,7-dioI (TMDD), diisopropyl adipate (DIPA), n-butil-benzenesulfonamide, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, a sterol andan alkane. In certain embodiments, theT-cell expansion composition comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid and asterol (e.g. cholesterol). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of between 0.9 mg/kg to 90 mg/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.2mng/kg to 20 mg/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; oleic acid at a concentration of 0.2 mg/kg to 20mg/kg, inclusive of the endpoints; and a sterol at a concentration of about 0.1 mg/kg to 10mg/kg, inclusive of the endpoints (wherein mg/kg = parts per million). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 9 mg/kg, palmitic acid at a concentration of about 2 mg/kg, linoleic acid at a concentration of about 2 mng/kg olic acid at a concentration of about 2 mg/kg, and a sterol at a concentration of about 1mg/kg (wherein mg/kg = parts per million). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of 9.19 mg/kg, palmitic acid at a concentration of 1.86 mg/kg, linoleic acid at a concentration of about 2.12 mg/kg, oleic acid at a concentration of about 2.13 mg/kg, and a sterol at a concentration of about 1.01 mg/kg (wherein mg/kg = parts per million). In certain embodiments, the T-cell expansion composition comprises octanoic acid at a concentration of 9.19 mg/kg, palnitic acid at a concentration of 1.86 mg/kg, linolcic acid at a concentration of 2.12 mg/kg, oleic acid at a concentration ofabout 2.13 mg/kg, and a sterol at a concentration of 1.01 mg/kg (wherein mg/kg = parts per million). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of between 6.4 pmol/kg and 640 piol/kg, inclusive of the endpoints; paliiticacid at a concentration of between 0.7 umol/kg and 70 pmoi/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 pmol/kg and 75 pmol/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 pmol/kg and 75 pmol/kg, inclusive of the endpoints; and asterol at a concentration of between 0.25 pmol/kg and 25 pmol/kg, inclusive of the endpoints. In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 64 pmol/kg, palmitic acid at a concentration of about 7 pmol/1g, linoleic acid at a concentration of about 7.5 pmo/kg, oleic acid at a concentration of about 7.5 imol/kg and a sterol at a concentration of about 2.5 mol/kg. In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 63.75 pmol/kg, palmitic acid at a concentration of about 7.27 pLmolkg, linoleic acid at a concentration of about 7.57 pmol/kg, oleicacid at a concentration of about 7.56 pmol/kg and a sterol at a concentration of about 2.61 pmol/kg. In certain embodiments, the T-cell expansion composition comprises octanoic acid at a concentration of about 63.75 pmol/lg, palmitic acid at a concentration of about 7.27 pnol/kg, linoleic acid at a concentration of about 7.57 pmolg, oleic acid at a concentration of 7.56 pmol/kg and a sterol at a concentration of 2.61 pmol/kg. In certain embodiments of this method, the modified stein memory T-cells (TsCM) comprise at least 1%. 2%, 5%, 7%, 10%, 15%, 20%, %,30%,35%, 40%,45%, 50% 55%,60%, 65%, 70%,75%, 80%, 85%,90%,95%, 97%, 99% or any percentage of cells in between of the total number of cells of the composition. In certain embodiments of this method, the modified central memory T-cells (TcM) comprise at least 1%, 2%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, %, 75%, 80%, 85%, 90%, 95%, 97%, 99%or any percentage of cells in between of the total number of cells of the composition. In certain embodiments of this method, the modified stem memory T-cells (TscM) comprise at least 10% of the total number of cells of the composition and the modified central memory T-cells (TcM) comprise at least 90% Of the total number of cells of the composition. In certain embodiments of this method, the modified stem memory T-cells (TscM) comprise at least 90%oof the total number of cells of the composition and the modified central memory T-cells(Tc) comprise at least 10% of the total number of cells of the composition. In certain embodiments of this method, the modified stem memory T-cells (TsCM) compTise at least 20%of the total number of cells of the composition and the modified central memory T-cells (Tcv) comprise at least 80% of the total number of cells of the composition. In certain embodiments of this method, the modified stem memoryT-cells (Tsc) comprise at least 80%of the total number of cells of the composition and the modified central memory T-cells (TCM) comprise at least 20% of the total number of cells of the composition. In certain embodiments of this method, the modified stein memory T-cells (Tsc) comprise at least 30% of the total number of cells of the composition and the modified central memoryT-cells (TcM) compriseat least 70% of the total number of cells of the composition. In certain embodiments of this method, the modified stem memory T-cells (TSCM) comprise at least 70%of the total number of cells of the composition and the modified central memory T-cells (TC) comprise at least 30% of the total number of cells of the composition. In certain embodiments of this method, the modified stein memory T-cells (TsCM) comprise at least 40% of the total number of cells of the composition and the modified central memory T-cells (TCM) comprise at least 60% of the total number of cells of the composition. In certain embodiments of this method, the modified stem memory T-cells (TscM) comprise at least 60% of the total number of cells of the composition and the modified central memory T-cells (T)comprise at least 40% of the total number of cells of the composition. In certain embodiments of this method, the modified stem memoryT-cells (TscM) compriseat least 50% of the total number of cells of the composition and the modified central memory T-cells (TCh) comprise at least 50% of the total number of cells of the composition.
[049] In certain embodiments of the methods of the disclosure, including those wherein the method comprises introducing into a primary human T cell (a) introducing into a primary human T cell a composition comprising an antigen receptor to produce a modified T cell, wherein atransposon comprises the antigen receptor, and (b) contacting the modified T cell and a T-cell activator composition comprising one or more of an anti-human CD3 monospecifictetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement to produce an activated modified T-cell, the method further comprises introducing into the primary humanTcell (c) a second transposon composition comprising a transposon comprising a therapeutic protein, to produce a modified T cell, wherein the modified T cell is capable of expressing the therapeutic protein. In certain embodiments, the therapeutic protein is a secretable protein and the method produces a modified'cell capable of secreting the therapeutic protein. In certain embodiments, the method further comprises introducing a transposase composition. In certain embodiments, the transposase composition transposes the transposon of (a) and the second transposon. In certain embodiments, the method comprises introducing a first transposase composition and a second transposase composition. In certain embodiments, including those wherein the method comprises introducing a first transposase composition and a second transposase composition, the first transposase composition transposes the transposon of (a) and the second transposase composition transposes the second transposon. In certain embodiments of this method, the transposon is a plasmid DNA transposon with a sequence encoding the antigen receptor or the therapeutic protein flanked by two cis-regulatory insulator elements. In certain embodiments, the transposon is a piggyBac transposon. In certain embodiments, and, in particular, those embodiments wherein the transposon is a piggyBac transposon, the TM transposase is a piggyBac or a Super piggyBacTM(SP embodiments of this method, the transposon is a Sleeping Beauty transposon. In certain embodiments, and, in particular, those embodiments wherein the transposon is a Sleeping Beauty transposon, the transposase is a Sleeping Beauty transposase or ahyperactive Sleeping Beauty transposase (SBIOOX). In certain embodiments ofthis method, the transposon is a Helraiser transposon. In certain embodiments, and, in particular, those embodiments wherein the transposon is a Helraiser transposon, the transposase is a Helitron transposase. In certain embodiments of this method, the transposon is aTol2 transposon. In certain embodiments, including those embodiments wherein the transposon is a To12 transposon, the transposase is a Tol2 transposase.
[050] In certain embodiments of the methods ofthe disclosure, including those wherein the method comprises introducing into a primary human T cell (a) introducing into a primary human T cell a composition comprising an antigen receptor to produce a modified T cell, wherein a transposon comprises the antigen receptor, and (b) contacting the modified Tcell and a T-cell activator composition comprising one or more of an anti-human CD3 monospecifictetrameric antibody complex, an anti-human CD28 monospecific tetrameric
antibody complex and an activation supplement to produce an activated modified T-cell, the method further comprises introducing into the primary humanTcell a sequence encoding a therapeutic protein, to produce a modified T cell, wherein the modified T cell is capable of expressing the therapeutic protein. In certain embodiments of introducing a sequence encoding a therapeutic protein, the introducing step comprises a homologous recombination. In certain embodiments of introducing a sequence encoding a therapeutic protein, a vector comprises the sequence encoding the therapeutic protein. In certain embodiments, the vector is a viral vector. In certain embodiments, the vector is a nanoparticle.
[051] In certain embodiments ofthe methods of the disclosure, the introducing step further comprises a composition comprising a genomic editing construct. In certain embodiments, the genomic editing construct comprises a guide RNA and a clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9) DNA endonuclease. In certain embodiments, the genomic editing construct comprises a DNA binding domain and a type IS endonuclease. In certain embodiments, the genomic editing construct encodes a fusion protein. In certain embodiments, the genomic editing construct encodes the DNA binding domain and the type IIS endonuclease and wherein the expressed DNA binding domain and the expressed type IS endonuclease are non-covalently linked. In certain embodiments, including those embodiments wherein the genomic editing construct comprises a DNA binding domain and a type IIS endonuclease, the genomic editing construct comprises a sequence derived from a Cas9 endonuclease. In certain embodiments, including those embodiments wherein the genomic editing construct comprises a DNA binding domain and a type IIS endonuclease, the sequence derived from a Cas9 endonuclease is the DNA binding domain. In certain embodiments, including those embodiments wherein the sequence derived from a Cas9 endonuclease is the DNA binding domain, the sequence derived from a Cas9 endonuclease encodes an inactive Cas9. In certain embodiments, including those embodiments wherein the sequence derived from a Cas9 endonuclease is the DNA binding domain, the sequence derived from a Cas9 endonuclease encodes a truncated Cas9. In certain embodiments, the sequence derived from a Cas9 endonuclease comprises an amino acid substitution of an Alanine (A) for an Aspartic Acid (D) at position 10 (DI0A). In certain embodiments, the sequence derived from a Cas9 endonuclease comprises an amino acid substitution of an Alanine (A) for a Histidine (H) at position 840 (H840A). In certain embodiments, the sequence derived from a Cas9 endonuclease comprises dCas9 (SEQ ID NO: 33). In certain embodiments, the sequence derived from a Cas9 endonuclease comprises an amino acid substitution of an Alanine (A) for an Asparagine (N) at position 580 (N580A). In certain embodiments, the sequence derived from a Cas9 endonuclease comprises dSaCas9 (SEQ ID NO: 32). In certain embodiments, includingthose embodiments wherein the genomic editing construct comprises a DNA binding domain and a type IS endonuclease, the
genomic editing construct comprises a sequence derived from a transcription activator-like effector nuclease (TALEN). In certain embodiments, including those embodiments wherein the genomic editing construct comprises a DNA binding domain and a type IIS endonuclease, the sequence derived from a TALEN is the DNA binding domain. In certain embodiments, the genomic editing construct comprises a.TALEN. In certain embodiments, including those embodiments wherein the genomic editing construct comprises a DNA binding domain and a type IIS endonuclease, the genomic editing construct comprises a sequence derived from a zinc-finger nuclease (ZFN). In certain embodiments., including those embodiments wherein the genomic editing construct comprises a DNA binding domain and a type IIS endonuclease, the sequence derived from a ZFN is the DNA binding domain. In certain embodiments, the genomic editing construct comprises a zinc-finger nuclease (ZFN).
[052] In certain embodiments ofthe methods of the disclosure, the transposon is aplasmid DNA transposon with a sequence encoding the antigen receptor or the therapeutic protein flanked by two cis-regulatory insulator elements. In certain embodiments of this method, the introducing step further comprises a composition comprising an mRNA sequence encoding a transposase. In certain embodiments, the transposon is a piggyBac transposon. In certain embodiments, and, in particular, those embodiments wherein the transposon is a piggyBac transposon, the transposase is a Super piggyBacTM (SPB) transposase. In certain embodiments, and, in particular, those embodiments wherein the transposase is a Super piggyBacTM (SPB) transposase, the sequence encoding the transposase is an mRNA sequence. In certain embodiments, the piggyBac transposase comprises an amino acid sequence comprising SEQ ID NO: 4. In certain embodiments, the piggyBac transposase is a hyperactive variant and the hyperactive variant comprises an amino acid substitution atone or more of positions 30, 165, 282 and 538 of SEQ ID NO: 4. In certain embodiments, the amino acid substitution at position 30 of SEQ ID NO: 4 is a substitution of a valine (V) for an isoleucine (I) (130V). In certain embodiments, the amino acid substitution at position 165 of SEQ ID NO: 4 is a substitution of a shrine (S) for agRlycine (G) (G165S). In certain embodiments, the amino acid substitution at position 282 of SEQ ID NO: 4 is a substitution of a valine (V) for amethionine (M) (M282V). In certain embodiments, the amino acid substitution at position 538 of SEQ ID NO: 4 is a substitution of a lysine (K) for an asparagine (N) (N538K). In certain embodiments, the Super piggyBac (SPB) transposase comprises an amino acid sequence comprising SEQ ID NO: 5. In certain embodiments, the transposon is a Sleeping Beauty transposon. In certain embodiments, and, in particular, those embodiments wherein the transposon is a Sleeping Beauty transposon, the transposase is a Sleeping Beauty transposase or a hyperactive Sleeping Beauty transposase (SBIOOX). In certain embodiments, the transposon is a Helraiser transposon. In certain embodiments, in particular those embodiments wherein the transposon is a Helraiser transposon, the transposase is a Helitron transposase. In certain embodiments, the transposon is a Tol2 transposon. In certain embodiments. in particular those embodiments wherein the transposon is a Tol2 transposon, the transposase is a Tol2 transposase. In certain embodiments, the sequence encoding the transposase is an mRNA sequence. In certain embodiments, the transposon may be derived or recombined from any species. Alternatively, oraddition, the transposon may be synthetic. 10531 In certain embodiments of the methods of the disclosure, the transposon further comprises a selection gene. In certain embodiments, the'T-cell expansion composition further comprises a selection agent.
[054] In certain embodiments of the methods of the disclosure, the antigen receptor is a T cell receptor. In certain embodiments, the T-cell receptor is naturally-occurring. In certain embodiments, the T-cell receptoris not naturally-occurring. In certain embodiments, and, in particular, those embodiments wherein the T-cell receptor is not naturally-occurring, the T cell receptor comprises one or more mutation(s) compared to a wild-type T-cell receptor. In certain embodiments, and, in particular, those embodiments wherein the T-cell receptor is not naturally-occurring, the T-cell receptor is a recombinantT-cell receptor. In certain embodiments of this method, the antigen receptor is a Chimeric Antigen Receptor (CAR). In certain embodiments, the CAR is a CARTyrin. In certain embodiments, the CAR comprises one or more VHH sequence(s). In certain embodiments, the CAR is a VCAR.
[055] In certain embodiments of the methods of the disclosure, the cell-surface markers of the modified TscM comprise CD62L and CD45RA. In certain embodiments, the cell-surface markers of the modified TsCM comprise one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2R§. In certain embodiments, the cell-surface markers of the modified TscM comprise one or more of CD45RA, CD95,IL-2RP, CR7,and CD62L.
[056] In certain embodiments of the methods of the disclosure, the plurality of expanded modified T-cells comprises a naive T-cell (modified TN) and the cell-surface markers of the CAR-TN comprise one or more of CD45RA. CCR7 and CD62L. In certain embodiments, the plurality of expanded modified T-cells comprises a central memory T-cll (modified TcM) and the cell-surface markers of the CAR-TCe comprise one or more of CD45RO, CD95, IL RD. CCR7 and CD62L. In certain embodiments, the plurality of expanded modified T-cells comprises an effector memory T-cell (modified TEM) and the cell-surface markers of the CAR-TEMcomprise one or more of CD45RO, CD95, and IL-2Rf. In certain embodiments, plurality of expanded modified T-cells comprises an effector T-cell (modifiedTfiF) and the cell-surface markers of the CAR-TEFF comprise one or more of CD45RA, CD95, and IL-2RP.
[057] In certain embodiments of the methods of the disclosure, the plurality of expanded modified T-cells comprises a central memory T-cell (modifiedCM) and the cell-surface markers of the CAR-TcM comprise one or more of CD45RO, CD95, IL-2R, CCR7, and CD62L. In certain embodiments, the most abundant cell in the plurality of expanded modified T-cells is a central memory T-cell (modified TCM) and the cell-surface markers of the CAR-TcM comprise one or more of CD45RO, CD95, IL-2RP, CCR7, and CD62L. In certain embodiments, wherein the most abundant cell in the plurality of expanded modified T-cells is a central memory T-cell (modified TCM), the plurality of expanded modified T-cells comprises a TsCM cell and the cell-surface markers of the TscE cell comprise one or more of CD62L, CD45RA, CD28, CCR7. CD127, CD45RO, CD95, CD95and IL-2RQ.
[058] The disclosure provides amethod of producinga modified stem memory T cell (TSCM),comprising: (a) introducing into a primary human T cell a composition comprising chimeric antigen receptor (CAR) to produce a CAR-T cell and (b) contacting the CAR-Tcell and a T-cell activator composition comprising one or more of an anti-human CD3 monospecifictetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex, an anti-human CD2 monospecific tetrameric antibody complex and an activation supplement to produce an activated CAR-Tcell, wherein the activated CAR-Tcell expresses one or more cell-surface marker(s) of a stein memory T cell (TsCM), thereby producing a CAR-expressing stem memory T cell (TscM) (CAR-TsCm).Thedisclosure provides a method of producing a plurality of modified stem memory T cells (TscM), comprising: (a) introducing into a plurality of primary human T cells a composition comprising a chineric antigen receptor (CAR) to produce a plurality of CAR-T cells and (b) contacting the plurality of CAR-T cells and a T-cell activator composition comprising one or more ofan anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex, ananti-human CD2 monospecific tetrameric antibody complex and an activation supplement to produce a plurality of activated CAR-T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, %, 70%, 75%, 80%, 85%, 90%, 95%, 99%or any percentage in between of the plurality of activated CAR-T cells expresses one or more cell-surface marker(s) of a stein memory T cell (Tsc), thereby producing a plurality of activated CAR stem memory T cells (Tsm). In certain embodiments, the method produces a plurality of activated CAR-Tcells, wherein at least 25% of the plurality of activated CAR-T cells expresses one ormore cell-surface marker(s) of a stem memory T cell (TscM), thereby producing a plurality of activated CAR stem memory T cells (Tsc4). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 50% of the plurality of activated CAR-T cells expresses one or more cell-surface marker(s) of a stem memory Tcell (TscM), thereby producing a plurality of activated CAR stem memory T cells (TscM). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 60% of the plurality of activated CAR-Tcells expresses one or more cell-surface marker(s) of a stem memory T cell (TscM), thereby producing a plurality of activated CAR stem memory T cells (TscM). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 75% of the plurality of activated CAR-Tcells expresses one or more cell surface marker(s) of a stem memoryTcell (TSCM), thereby producing a plurality of activated CAR stem memory T cells (TscM). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 80% of the plurality of activated CAR-T cells expresses one or more cell-surface marker(s) of a stem memory T cell (TCM), thereby producing a plurality of activated CAR stem memory Tcells (Tsm). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 85% of the plurality of activated CAR-T cells expresses one or more cell-surface marker(s) of a stem memoryI'cell (TsCM), thereby producing a plurality of activated CAR stem memoryTcells (TscM). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 90% of the plurality of activated CAR-T cells expresses one or more cell surface marker(s) of a stem memory T cell (TSCM), thereby producing a plurality of activated CAR stem memory"T cells (TscM). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 95% of the plurality of activated CAR-T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tsi), thereby producing a plurality of activated CAR stein memoIy T cells (TscM). In certain embodiments, the cell-surface markers comprise CD62L and CD45RA. In certain embodiments, the cell surface markers of the activated CAR TscM comprise one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2R3. In certain embodiments, the cell surface markers of the activated CAR TsCMcomprise one or more of CD45RA, CD95, IL 2R ,CR7, and CD62L. The disclosure provides a method of producing a modified stem memory T cell (TscM), comprising: (a) introducing into a primary human T cell a composition comprising a chimeric antigen receptor (CAR) to produce a CAR-T cell and (b) contacting the CAR-T cell and aT-cell activator composition comprising one or more ofan anti-human CD3 monospecific tetramneric antibody complex, an anti-human CD28 monospecific tetraeric antibody complex and an activation supplement to producean activated CAR-T cell, wherein the activated CAR-T cell expresses one or more cell-surface marker(s) of a stemmemoryT cell (TSCN), thereby producing a CAR-expressing stem memory T cell (TscM) (CAR-Tsci).
10591 The disclosure provides a method of producing a plurality of modified stem memory T cells (TSCM),omprising: (a) introducing intoa plurality of primary humanTcells a composition comprising a chimeric antigen receptor (CAR) to produce a plurality of CAR-T cells and (b) contacting the plurality of CAR-T cells and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement to produce a plurality of activated CAR-T cells, wherein at least 2%, 5%, 10%, %, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or anypercentage in between of the plurality of activated CAR-T cells expresses one or more cell-surface marker(s) of a stem memory T cell (TscM), thereby producing a plurality of activated CAR stem memory T cells (Tsc4). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 25% of the pluraity of activated CAR-T cells expresses one or more cell-surface marker(s) of a stein memory Tcell (TSCM), thereby producing a plurality of activated CAR stem memory T cells (TscM). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 50% of the plurality of activated CAR-Tcells expresses one or more cell-surface marker(s) of a stem memory Tcell (TscM), thereby producing a plurality of activated CAR stein memory T cells (Tsci). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 60% of the plurality of activated CAR-T cells expresses one or more cell surface marker(s) of a stem memory T cell (TSCi), thereby producing a plurality of activated CAR stem memory T cells (Tsci). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 75% of the plurality of activated CAR-T cells expresses one or more cell-surface marker(s) of a stem memory T cell (TscM), thereby producing a plurality of activated CAR stein memory Tcells (Tsc). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 80% of the plurality of activated CAR-T cells expresses one or more cell-surface marker(s) of a stem memory Tcell (Tsc), thereby producing a plurality of activated CAR stem memory Tcells
(Tsc). In certain embodiments, the method produces a plurality of activated CAR-Tcells, wherein at least 85% of the plurality of activated CAR-T cells expresses one or more cell surface marker(s) of a stem memory T cell (Tsci), thereby producing a plurality of activated CAR stem memory T cells (Tscr). In certain embodiments, the method produces a plurality of activated CART cells, wherein at least 90% of the plurality of activated CAR-T cells expresses one or more cell-surface marker(s) of a stem memory T cell (TscM), thereby producing a plurality of activated CAR stem memory T cells (TsM). In certain embodiments, the method produces a plurality of activated CAR-Tcells, wherein at least 95%of the plurality of activated CAR-T cells expresses one ormore cell-surface marker(s) of astem memory T cell (Tsci), thereby producing a plurality of activated CAR stem memory T cells (Tsc). In certain embodiments, the cell-surface markers comprise CD62L and CD45RA. In certain embodiments, the cell-surface markers of the activated CAR TCM comprise one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2R3. In certain embodiments, the cell-surface markers of the activated CAR TSCM comprise one or more of CD45RA, CD95, IL-2R , CR7, and CD62L.
[060] In certain embodiments, this method may further comprise thestep of: (c) contacting the activated CAR-T cell and a T-cell expansion composition comprising one or more of human serumalbumin, recombinant human insulin, human transferring, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement to produce a plurality of expanded CAR-T cells, wherein at least 2% of the pluralityof expanded CAR-T cells expresses one or more cell-surface marker(s) of a stem memory T cell (TCM) (CARTscM). In certain embodiments, the T-cell expansion composition further comprises one or more of octanoic acid, nicotnamide, 2,4,7,9-tetranethyl-5-decyn-4.7-diol (TMDD), diisopropyl adipate (DIPA), n
butyl-benzenesulfonamide, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, steaic acid hydrazide, oleamide, a sterol and an alkane. In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid and a sterol (e.g. cholesterol). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of between 0.9 mg/kg to 90 mg/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.2 mg/kg to 20mg/kg, inclusive of the endpoints;,linoleic acid at a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; oleic acid at a concentration of 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; and a sterol at a concentration of about 0.1 mg/kg to 10 mg/kg, inclusive of the endpoints (wherein mg/kg = parts per million). In certain embodiments, theT-cell expansion composition comprises one or more of octanoic acid at a concentration of about 9 mg/kg, palmitic acid at a concentration of about 2 mg/kg, linoleic acid at a concentration of about 2 ig/kg, oleic acid at a concentration of about 2 mg/kg, and a sterol at a concentration of about1 mg/kg (wherein mg/kg = parts per million). In certain embodiments, theT-cell expansion composition comprises one or more of octanoic acid at a concentration of 9.19 mg/kg, palmitic acid at a concentration of 1.86 mg/kg, linoleic acid at a concentration of about 2.12 mg/kg, oleic acid at a concentration of about 2.13 mg/kg, and a sterol at a concentration of about 1.01 mg/kg (wherein mg/kg = parts per million). In certain embodiments, the T-cell expansion composition comprises octanoic acid at a concentration of 9.19 mg/kg, palmitic acid at a concentration of 1.86 mg/kg, linoleic acid at a concentration of 2.12 mg/kg, oleic acid at a concentration of about 2.13 mg/kg, and a sterol at a concentration of 1.01ng/kg (wherein mg/kg = parts per million). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of between 6.4 pmo/kg and 640 p.1mol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 pmol/kg and 70 pnol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 .umol/kg and 75 pmol/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 Pmol/kg and 75 pmol/kg, inclusive of the endpoints; and a sterol at a concentration of between 0.25 pmol/kg and 25 pmolg, inclusive ofthe endpoints. In certainembodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 64 pmoi/kg, palmitic acid at a concentration of about 7 pmol/kg, linoleic acid at a concentration of about 7.5 pmol/kg, oleic acid at a concentration of about 7.5 pmol/kg and a sterol at a concentration of about 2.5 pmol/kg. In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 63.75 pLmol/kg, palmitic acid at a concentration of about 7.27 pmo/kg. linoleic acid at a concentration of about 7.57 imol/kg, oleic acid at a concentration of about 7.56 pmolkg and a sterol at a concentration of about 2.61 pmol/kg. In certain embodiments, the T-cell expansion composition comprises octanoic acid at a concentration of about 63.75 piolkg, palmitic acid at a concentration of about 7.27 tnmol/kg, linoleic acid at a concentration of about 7.57 mol/kg, oleic acid at a concentration of 7.56 mol/kg and a sterol at a concentration of 2.61 pmol/kg. In certain embodiments, at least 2%, 5%, 10%, 15%, 20%, %, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of expanded CAR-T cells expresses cell-surface marker(s) of a stem memory Tcell (TscM) (CAR-TScM). In certain embodiments, the plurality of expanded CAR-T cells may be enriched for CAR-T cells that express cell-surface marker(s) of a stem memory T cell (TscM) (CAR-Tscu), and, therefore, following an enrichment step, the method may produce an enriched composition comprising at least 2%, %, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, %,95% 99% orany percentage in between of CAR-T cells that express cell-surface marker(s) of a stem memory T cell (TSCM) (CAR-TscM). In certain embodiments, the cell surface markers comprise CD62L and CD45RA. In certain embodiments, the cell-surface markers of the CAR-Tsci comprise one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2R§. In certain embodiments, the cell-surface markers of the CAR-TSCM comprise one or more of CD45RA, CD95, IL-2Rf, CR7, and CD62L. In certain embodiments, the plurality of expanded CAR-Tcells comprises a naiveT cell (CAR-TN) and the cell-surface markers of the CAR-TN comprise one or more of CD45RA, CCR7 and CD62L. In certain embodiments, the plurality of expanded CAR-T cells comprises a central memory T-cell (CAR-TCM) and the cell-surface markers of the CAR-TCM comprise one or more of CD45RO, CD95, IL-2RD, CCR7. and CD62L. In certain embodiments, the plurality of expanded CAR-T cells comprises an effector memory T-cell (CAR-TEM) and the cell-surface markers of the CAR-Ti comprise one or more of CD45RO, CD95, and IL-2RQ. In certain embodiments, the plurality of expanded CAR-Tcells comprises an effector T-cell (CAR-TEFF) and the cell-surface markers of the CAR-TEi comprise one or more of CD45RA, CD95, and IL-R[3. Additional cell-surface markers are described in Gattinoniet al. (Nat Med. 2011 Sep 1S; 17(10): 1290-7; the contents of which are incorporated herein by reference in their entirety).
[061] The disclosure provides a method of producing a modified stem memory T cell (Tsc), comprising: (a) introducing into a primary human T cell a composition comprising a chimeric antigen receptor (CAR) to produce a CAR-Tcell and (b) contacting the CAR-T cell and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetramericantibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement to produce an activated CAR-T cell, wherein the activated CAR-T cell expresses one or more cell-surface marker(s) of a stem memoryT cell (TscM), thereby producing a CAR-expressing stem memory T cell (TSi) (CAR-TCM). The disclosure provides a method of producing a plurality ofmodified stem memory T cells (Tsc), comprising: (a) introducing into a plurality of primary humanTcells a composition comprising a chiineric antigen receptor (CAR) to produce a plurality of CAR-T cells and (b) contacting the plurality of CAR-T cells and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement to produce a plurality of activated CAR-T cells, wherein at least 2%. 5%, 10%,I 15% 20%, 25%, 30%, %,40%,45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of activated CAR-T cells expresses one or more cell-surface marker(s) of a stein memory Tcell (TscM). thereby producing a pluralityof activated CAR steminmemory T cells (TCM). In certain embodimnents, the method produces a plurality of activated CAR-T cells. wherein at least 25% of the plurality of activated CAR-T cells expresses one or more cell-surface marker(s) of a stem memory T cell (TscM), thereby producing a plurality ofactivated CAR stem memory- Tcells (Tscm). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 50% ofthe plurality of activated CAR-T cells expresses one or more cell-surface marker(s) of a stem memory T cell (TscM), thereby producing a plurality of activated CAR stem memory T cells (Tsc). In certain embodiments, the method produces a plurality ofactivated CAR-Tcells, wherein at least 60% ofthe plurality of activated CAR-T cells expresses one or more cell surface marker(s) of a stem memory T cell (TscM), thereby producing a plurality of activated CAR stem memory T cells (Tscu). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 75% of the plurality ofactivated CAR-T cells expresses one or more cell-surface marker(s) of a stem memory T cell (TscM), thereby producing a plurality of activated CAR stem memory T cells (TsCM). Incertain embodiments., the method produces a plurality ofactivated CAR-Tcells, wherein at least 80%of the plurality of activated CAR-T cells expresses one or more cell-surface marker(s) of asten memory T cell (TscM), thereby producing a plurality of activated CAR stem memory T cells (TsCM). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 85% ofthe plurality of activated CAR-T cells expresses one ormore cell surface marker(s) of a stem memory T cell (TsCM), thereby producing a plurality of activated CAR stem memory Icells(TSCM). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 90% of the plurality of activated CAR-Tcells expresses one or more cell-surface marker(s) of a stem memory T cell (TscM), thereby producing a plurality of activated CAR steinm emory T cells (TscM). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 95% of the plurality of activated CAR-Tcells expresses one or more cell-surface marker(s) of a stem memory T cell (TscM), thereby producing a plurality of activated CAR ste memory T cells (TsCM). In certain embodiments, the cell-surface markers comprise CD62L and CD45RA. In certain embodiments, the cell-surface markers of the activated CAR TsCm comprise one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2RP. In certain embodiments, the cell-surface markers of the activated CAR TSCM comprise one or more of CD45RA, CD95, IL-2R[3, CR7, and CD62L.
[062] In certain embodiments of the methods of the disclosure, the plurality of expanded CAR-T cells comprises a naive T-cell (CAR-TN) and the cell-surface markers of the CAR-TN comprise one or more of CD45RA, CCR7 and CD62L. In certain embodiments, the plurality of expanded CAR-T cells comprises a central memory T-cell (CAR-TC-t) and the cell-surface markers of the CAR-TCNi comprise one or more of CD45RO, CD95, IL-2R, CCR7, and CD62L. In certain embodiments, the plurality of expanded CAR-T cells comprises an effector memory T-cell (CAR-TE) and the cell-surface markers of the CAR-TEM comprise one or more of CD45RO, CD95, and IL-2R . In certain embodiments, the plurality of expanded CAR-Tcells comprises an effector T-cell (CAR-TFiF) and the cell-surface markers of the CAR-TEFF comprise one or more of CD45RA, CD95, and IL-2RP.
[063] In certain embodiments ofthe methods ofthe disclosure, a transposon comprises a chimeric antigen receptor (CAR) ofthe disclosure. The transposon may be a plasmid DNA transposon with a sequence encoding the CAR flanked by to cis-regulatory insulator elements. In certain preferred embodiments, the transposon is a piggyBac transposon. In certain embodiments, a step introducing a composition comprising a chimeric antigen receptor (CAR) ofthe disclosure may further a composition comprising anmRNA sequence encoding a transposase. In certain preferred embodiments, the transposase is a Super piggyBacTM (SPB) transposase.
[064] In certain embodiments, a transposon of the disclosure may further comprise a selection gene. When a transposon of the disclosure comprises a selection gene, the T-cell expansion composition ofthe methods ofthe disclosure may further comprise a selection agent to simultaneously select and expand an activated or modified T cell ofthe disclosure.
[065] In certain embodiments a CAR ofthe disclosure may be a CARTvrin. In certain embodiments, the CAR comprises one or more VHH sequence(s). In certain embodiments, the CAR is a CAR.
[066] In certain embodiments of the methods of producing a modified'TscN of the disclosure, the introducing step may comprise an electroporation or a nucleofection. When the introducing step comprises a nucleofection, the nucleofection may comprise the steps of (a) contacting a transposon composition, a transposase composition, and a composition comprising a plurality of primary human'T cells in a cuvette; (b) applying one or more electrical pulses to the cuvette, and (c) incubatingthe composition comprising the purality of primary human T cells in a composition comprising a T-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement at 37°C. In certain embodiments, the T-cell expansion composition further comprises one or more of octanoicacid, nicotinamide, 2,4,7,9-tetramethyl-5-decyn-4,7-diol (TMDD), diisopropyl adipate (DIPA), n-butv-benzenesulfonamide, I.,2-benzenedicarboxyiic acid, bis(2 methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, a sterol and an alkane. In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid and a sterol (e.g. cholesterol). In certain embodiments, the T-cell expansion composition comprises one or more of octanoicacidat a concentration of between 0.9 mg/kg to 90 mg/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; oleic acid at a concentration of 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; and a sterol at a concentration of about 0.1 mg/kg to 10 mg/kg, inclusive of the endpoints (wherein mg/kg = parts per million). In certain embodiments, the T-cell expansion composition comprisesone or more of octanoic acid at a concentration of about 9 mg/kg palmitic acid at a concentration of about 2 mg/kg, linoleic acid at a concentration of about 2 mg/kg, oleic acid at a concentration of about 2 mg/kg, and a sterol at a concentration of about 1mg/kg (wherein mg/kg = parts per million). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of 9.19 mg/kg, palmitic acid at a concentration of 1.86mng/kg, linoleic acid at a concentration of about 2.12 mg/kg oleic acid at a concentration of about 2.13 mg/kg, and a sterol at a concentration of about 1.01 mg/kg (wherein mg/kg = parts per million). In certain embodiments, the T-cell expansion composition comprises octanoic acid at a concentration of 9.19 mg/kg, palmitic acid at a concentration of 1.86 mg/k'g, linoleic acid at a concentration of 2.12 ing/kg, oleic acid at a concentration of about 2.13 mg/kg, and a sterol at a concentration of 1.01 mg/kg
(wherein mg/kg = parts per million). In certain embodiments, the'T-cell expansion composition comprises one or more of octanoic acid at a concentration of between 6.4 pLmol/kg and 640 pmol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 pmol/kg and 70 pmol,g, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 pmol/kg and 75 pmol/kg, inclusiveof the endpoints; oleic acid at a concentration of between 0.75 imol/kg and 75 pmol/kg, inclusive of the endpoints; and a sterol at a concentration of between 0.25pmoikg and 25 pmol/kg, inclusive of the endpoints. In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 64 tmol/kg, palmitic acid at a concentration of about 7 pmoi/kg, linoleic acid at a concentration of about 7.5 mo/g. oleic acid at a concentration of about 7.5 pmol/kg and a sterol at a concentration of about 2.5 pmol/kg. In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about63.75 pmol/kg, palmitic acid at a concentration of about 7.27 pmol/kg, linoleic acid at a concentration of about 7.57 moil/kg, oleic acid at a concentration of about 7.56 pmol/kg and a sterol at a concentration of about 2.61 pmol/kg. In certain embodiments, theT-cell expansion composition comprises octanoic acid at a concentration of about 63.75 pimol/kg, palmitic acid at a concentration of about 7.27 pmol/kg, linoleic acid at a concentration of about 7.57 pmol/kg, oleic acid at a concentration of 7.56 pmolkg and a sterol at a concentration of 2.61 pimol/kg. In certain embodiments of the nucleofection, the transposon composition is a 0.5 pg/l solution comprising nuclease free waterand the cuvette comprises'2 p. of the transposon composition to yield I g of transposon. The transposon composition may comprise a piggyBac transposon. The transposon composition may comprise a Sleeping Beauty transposon. In certain embodiments of the nucleofection, the transposase composition comprises 5 pg of transposase. The transposase composition may comprise a hyperactive piggyBach or Super piggyBacT h (SPB) transposase. The transposase composition may comprise a hyperactive Sleeping Beauty (SB100X) transposase. In certain enbodiments, the transposon may comprise a Helraiser transposon and the transposase composition may comprise a Helitron transposase. In certain embodiments, the transposon may comprise a Tol2 transposon and the transposase composition comprises a Tol2 transposase.
[067] In certain embodiments of the methods of the disclosure, including those embodiments wherein the introducing step comprises a nucleofection or an electroporation, the nucleofection comprises contacting a first transposon composition and a first transposase composition and a composition comprising a plurality of primary human Tcells in a cuvette. In certain embodiments of the methods of the disclosure, including those embodiments wherein the introducing step comprises a nucleofection or an electroporation, the nucleofection comprises contacting a first transposon composition, a second transposon composition, a first transposase composition and a composition comprising a plurality of primary human T cells in a cuvette. In certain embodiments of themethods of the disclosure, including those embodiments wherein the introducing step comprises a nucleofection or an electroporation, the nucleofection comprises contacting a first transposon composition, a second transposon composition, a first transposase composition, a secondtransposase composition and a composition comprising a plurality of primary human T cells in a cuvette. In certain embodiments, the first transposon comprises a sequence encoding an antigen receptor. In certain embodiments, the second transposon comprises a sequence encoding a therapeutic protein. In certain embodiments, the first transposon composition and the second transposon composition are identical. In certain embodiments, the first transposon composition and the second transposon composition are not identical. In certain embodiments, the first transposase mobilizes the first transposon composition and the second transposon composition. In certain embodiments, the first transposase mobilizes the first transposon composition but not the second transposon composition. In certain embodiments, the second transposase mobilizes the second transposon composition but not the first transposon composition. In certain embodiments, the first transposase mobilizes the first transposon composition and the second transposase mobilizes the second transposon composition. In certain embodiments, the first transposon composition or the second transposon composition comprises a sequence encoding an antigen receptor. In certain embodiments, the first transposon composition or the second transposon composition comprises a sequence encoding a therapeutic protein. In certain embodiments, the first transposon composition comprises a sequence encoding an antigen receptorand the second transposon composition comprises a sequence encoding a therapeutic protein. In certain embodiments, the therapeutic protein is a secreted or secretable protein. In certain embodiments of the methods of the disclosure, including those embodiments wherein the introducing step comprises a nucleofection or anelectroporation, thenucleofection comprises contacting a transposon composition. a first transposase composition, a second transposase composition and a composition comprising a plurality of primary human T cells in a cuvette. In certain embodiments, the transposon composition comprises a sequence encoding the antigen receptor. In certain embodiments, the transposon composition comprises a sequence encoding the therapeutic protein. In certain embodiments of the methods of the disclosure, including those embodiments wherein the introducing step comprises a nucleofection or an electroporation, the nucleofection further comprises contacting a composition capable of inducing homologous recombination at a specific site in the genome with a composition comprising a plurality of primary hIman T cells in a cuvete. In certain embodiments, the composition capable of inducing homologous recombination comprises an exogenous donor molecule. In certain embodiments, the exogenous donor molecule comprises a sequence encoding the antigen receptorand the transposon comprises a sequence encoding the therapeutic protein. In certain embodiments, the exogenous donor molecule comprises a sequence encoding the therapeutic protein and the transposon comprises a sequence encoding the antigen receptor. In certain embodiments, the composition comprising the transposon, the composition comprising the transposase and the composition capable of inducing homologous recombination at a specific site in the genome are contacted with the composition comprising a plurality of primary human T cells simultaneously. In certain embodiments, the composition comprising the transposon and the composition comprising the transposase are contacted with the composition comprising a plurality of primary human T cells first, and the composition capable of inducing homologous combination at a specific site in the genome is contacted with the composition comprising a plurality of primary human T cells second. In certain embodiments, the composition capable of inducing homologous recombination at a specific site in the genome is contacted with the composition comprising a plurality of primary human T cells first and the composition comprising the transposon and the composition comprising the transposase are contacted with the composition comprising a plurality of primary human T cells second. In certain embodiments of the methods of producing a modified TscM of the disclosure, the composition comprising primary human T cells comprises a buffer that maintains or enhances a level of cell viability and/or a stem-like phenotype of the primary human T cells. In certain embodiments, the buffer maintains or enhances a level of cell viability and/or a stem-like phenotype of the primary human T cells prior to the nucleofection. In certain embodiments, the buffer maintains or enhances a level of cell viabilityand/or a stem-like phenotype of the primary human T cells during the nucleofection. In certain embodiments, the buffer maintains or enhances a level of cell viability and/or a stem-like phenotype of the primary human T cells following the nucleofection. In certain embodiments, the buffer comprises a P3 primary cell solution
(Lonza). In certain embodiments, the buffer comprises one or more of KCMgCI 2, CNa, Glucose and Ca(NO3)2 in any absolute or relative abundance or concentration, and, optionally, the buffer further comprises a supplement selected from the group consisting of HEPES,'Tris/HCI, and a phosphate buffer. In certain embodiments, the buffer comprises 5 mM KCi, 15 mM MgCl2, 90 mM CNa, 10 mM Glucose and 0.4 mM Ca(NO3)2. In certain embodiments, the buffer comprises 5 mM KC, 15 mM MgCl2, 90 mM CiNa. 10 mM Glucose and 0.4 mM Ca(N03)2 and a supplement comprising20 mM HEPES and 75 mM Tris/HCl. In certain embodiments, the buffer comprises 5 mM KCl, 15 mMM 12, 90 mM CINa, 10 mM Glucose and 0.4 mM Ca(NO3)2 and a supplement comprising 40 mM Na2IPO4NaH2PO4 at pH7.2. In certain embodiments, the composition comprising primary human T cells comprises 100 l of the buffer and between 5x10 6 and 25x10 6 cells.
[068] In certain embodiments of the methods of producing a modified TSCM ofthe disclosure, the composition comprising primary human T cells is depleted of cells expressing CD14, CD56, and/or CD19. In certain embodiments, the composition comprising primary humanTcells comprises 100 g of the buffer and between 5x10 6 and 25x10 6 cells.
[069] As used herein, the terms"supplemented T-cell expansion composition" or "T-cell expansion composition" may be used interchangeably with a media comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM,an an expansion supplement at37°C. Alternatively, or in addition, the terms "supplemented T-cell expansion composition" or "T-cell expansion composition" may be used interchangeably with a media comprising one or more of phosphorus, an octanoic fatty acid, a palmitic fatty acid, a linoleic fatty acid and an oleic acid. In certain embodiments, the media comprises an amount of phosphorus that is 10-fold higher than may be found in. for example, Iscove's Modified Dulbecco's Medium ((IMDM); available at ThennoFisher Scientific as Catalog number 12440053).
[070] As used herein, the terms "supplemented T-cell expansion composition" or "T-cell expansion composition" may be used interchangeably with a media comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement at 370 C. Alternatively, or in addition, the terms "supplemented'I-cellexpansion composition" or "T-cell expansion composition" may be used interchangeably with a media comprising one or more of the following elements: boron, sodium, magnesium, phosphorus, potassium, and calcium. In certain embodiments, the terms "supplemented T-cell expansion composition" or "T-cell expansion composition" may be used interchangeably with a media comprising one ormore of the following elements present in the corresponding average concentrations: boron at 3.7 mg/L, sodium at 3000 mg/L, magnesium at 18 mg/L, phosphorus at 29 mg/L, potassium at 15 mg/L and calcium at 4 mg/L.
[071] As used herein, the terms "supplemented'T-cell expansion composition" or "T-cell expansion composition" may be used interchangeably with a media comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement at 37°C. Alternatively, or in addition, the terms "supplemented T-cell expansion composition" or "T-cell expansion composition" may be used interchangeably with a media comprising one or more of the following components: octanoic acid (CAS No. 124-07-2), nicotinamide (CAS No. 98-92-0), 2,4,7,9-ttramethyl-5 decyn-4,7-diol (TMDD) (CAS No. 126-86-3). diisopropyl adipate (DIPA) (CAS No. 6938 94-9), n-butyl-benzenesulfonamide (CAS No. 3622-84-2) 1,2-benzenedicarboxylicacid, bis(2-methylpropyl) ester (CAS No. 84-69-5), palmitic acid (CAS No. 57-10-3), linoleic acid (CAS No. 60-33-3), oleic acid (CAS No. 112-80-1), stearic acid hydrazide (CAS No. 4130 54-5),oleamide (CAS No. 3322-62-1), sterol (e.g., cholesterol) (CAS No. 57-88-5), and alkanes (e.g., nonadecane) (CAS No. 629-92-5). In certain embodiments, the terms "supplemented T-cell expansion composition" or "T-cell expansion composition" may be used interchangeably with a media comprising one or more of the following components: octanoic acid (CAS No. 124-07-2), nicotinamide (CAS No. 98-92-0) 2,4,7,9-tetramethy-5 decyn-4,7-diol (TMDD) (CAS No. 126-86-3), diisopropyl adipate (DIPA) (CAS No. 6938 94-9), n-butyi-benzenesulfonamide (CAS No. 3622-84-2), 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester (CAS No. 84-69-5), palmitic acid (CAS No. 57-10-3), linoleic acid (CAS No. 60-33-3), oleic acid (CAS No. 112-80-1), stearic acid hydrazide (CAS No. 4130 54-5), oleamide (CAS No. 3322-62-1), sterol (e.g., cholesterol) (CAS No. 57-88-5), alkanes (e.g., nonadecane) (CAS No. 629-92-5), and phenol red (CAS No. 143-74-8). In certain embodiments, the terms "supplemented T-cell expansion composition" or "T-cell expansion composition" may be used interchangeably with a media comprising oneormoreof the following components: octanoic acid (CAS No. 124-07-2), nicotinamide (CAS No. 98-92-0), 2,4,7,9-tetramethyl-5-decyn-4.7-diol (TMDD) (CAS No. 126-86-3). diisopropyl adipate (DIPA) (CAS No. 6938-94-9). n-butyi-benzenesulfonamide (CAS No. 3622-84-2), 1,2 benzenedicarboxylic acid, bis(2-methylpropyl) ester (CAS No. 84-69-5), palmitic acid (CAS No. 57-10-3), linoleic acid (CAS No. 60-33-3), oleic acid (CAS No. 112-80-1). stearic acid hydrazide (CAS No. 4130-54-5), oleamide (CAS No. 3322-62-1), phenol red (CAS No. 143 74-8) and lanolin alcohol.
[072] As used herein, the terms "supplemented T-cell expansion composition" or "T-cell expansion composition" may be used interchangeably with a media comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM,adan expansion supplement at 37C. Alternatively, or in addition, theterms "supplemented T-cell expansion composition" or "T-cell expansion composition" may be used interchangeably with a media comprising one or more of the following ions: sodium, ammonum, potassium, magnesium, calcium, chloride, sulfate and phosphate.
[073] As used herein, the terms "supplemented T-cell expansion composition" or "T-cell expansion composition" may be used interchangeably with a media comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM,adan expansion supplement at 37C. Alternatively, or in addition, theterms "supplemented T-cell expansion composition" or "T-cell expansion composition" may be used interchangeably with a media comprising one or more of the following free amino acids: histidine, asparagine, seine, glutamate, arginine. glycine, aspartic acid, glutamicacid, threonine, alanine, proline, cysteine, lysine, tyrosine, methionine, valine, isoleucine, leucine, phenylalanine and tryptophan. In certain embodiments, the terms"supplemented T-cell expansion composition" or"T-cell expansion composition" may be used interchangeably with a media comprising one or more of the following free amino acids in the corresponding average mole percentages: histidine (about 1%), asparagine (about 0.5%), serine (about 1.5%), glutamine (about 67%), arginine (about 1.5%), glycine (about 1.5%), aspartic acid (about 1%). glutamic acid (about 2%), threonine (about 2%), alanine (about 1%), proline (about 1.5%), cysteine (about 1.5%), lysine (about 3%)tyrosine (about 1.5%), methionine (about 1%), valine (about 3.5%). isoleucine (about 3%). leucine (about 3.5%), phenylalanine (about 1.5%) and tryptophan (about 0.5%). In certain embodiments, the terms 'supplemented T-cell expansion composition" or "T-cell expansion composition" may be used interchangeably with a media comprising one or more of the following free amino acids in the corresponding average mole percentages: histidine (about 78%)asparagine (about 0.4%), seine (about 1.6%), glutamine (about 67.01%), arginine (about 1.67%), glcine (about 1.72%), aspartic acid (about 1.00%), glutamic acid (about 1.93%), threonine (about 2.38%), alanine (about 1.11%), proline (about 1.49%), cysteine (about 1.65%), lysine (about 2.84%), tyrosine (about 1.62%)., methionine (about 0.85%), valine (about 3.45%), isoleucine
(about 3.14%). leucine (about 3.3%), phenylalanine (about 1.64%) and tryptophan (about 0.37%).
[074] As used herein, the terms "supplemented T-cell expansion composition" or "T-cell expansion composition" may be used interchangeably with a media comprising one or more of octanoic acid, paliniticacid, linoleicacid, oleic acid and a sterol (e.g. cholesterol). In certain embodiments, the terms "supplemented T-cell expansion composition" or "T-cell expansion composition" may be used interchangeably with a media comprising one or more of octanoic acid at a concentration of between 0.9 mg/g to 90 mg/kg, inclusive ofthe endpoints; palmitic acid at a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.2 mg/kg to 20 mg/kg. inclusive of the endpoints; oleic acid at a concentration of 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; and a sterol at a concentration of about 0.1 mg/kg to 10 mg/kg, inclusive of the endpoints (wherein mg/kg = parts per million). In certain embodiments, the tenns "supplemented T-cell expansion composition" or "T-cell expansion composition" may be used interchangeably with a media comprising one or more of octanoic acid at a concentration of about 9 mg/kg, palmitic acid at a concentration of about 2 mg/kg,linoleic acid at a concentration of about 2 mg/kg, oleic acid at a concentration of about 2mg/kg, and asterol at a concentration of about I mgkg (wherein mg/kg = parts per million). ). In certain embodiments, the terms "supplemented T-cell expansion composition"or'"T-cell expansion composition" may be used interchangeably with a media comprising one or more of octanoic acid at a concentration of 9.19 mg/kg, palmitic acid at a concentration of 1.86 mg/kg, linoleic acid at a concentration of about 2.12 mg/kg, oleic acid at a concentration of about 2.13 ing/kg. and a sterol at a concentration of about 1.01 mg/kg (wherein mg/kg = parts per million). In certain embodiments, the terms "supplemented T-cell expansion composition" or "T-cell expansion composition" may be used interchangeably with a media comprising one or more of octanoic acid at a concentration of 9.19 mg/kg, palmitic acid at a concentration of 1.86mg/kg, linoleic acid at aconcentration of 2.12 mg/kg, oleic acid at concentration of about 2.13 mg/kg, and a sterol at a concentration of 1.01 mg/kg (whereinmg/kg = parts per million). In certain embodiments, the terms"supplemented T-cell expansion composition" or "IT-cell expansion composition" may be used interchangeably with amedia comprising one or more of octanoic acid at a concentration of between 6.4 pmol/kg and 640 pmol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 pmol/kg and 70 pmol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 pmol/kg and 75 pmol/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 .umol/kg and 75 pmoi/kg, inclusive of the endpoints; and a sterol at a concentration of between 0.25 pmolkg and 25 pmol/kg, inclusive of the endpoints. In certain embodiments, the terms "supplemented T-cell expansion composition" or"T-cell expansion composition" may be used interchangeably with a media comprising one or more of octanoic acid at a concentration of about 64 pmol/kg, palmitic acid at a concentration of about 7 tmol/kg, linoleic acid at a concentration of about7.5 pmol/kg, oleic acid at a concentration of about 7.5 pmol/kg and a sterol at a concentration of about 2.5 mol/g. In certain embodiments, the terns "supplemented T-cell expansion composition" or "T-cell expansion composition" may be used interchangeably with a media comprising one or more of octanoic acid at a concentration of about 63.75 imol/kg, palmitic acid at a concentration of about 7.27 pimoilkg, inoeic acid at a concentration of about 7.57 pmol/kg, oleicacid at a concentration of about 7.56 pmol/kg anda sterol at a concentration of about 2.61 ino/kg. In certain embodiments, the terms "supplemented T-cell expansion composition" or "T-cell expansion composition"may be used interchangeably with a media comprising one or more of octanoic acid at a concentration of about 63.75 pnol/kg, paliniticacid at a concentration of about 727 umol/kg, linoleic acid at a concentration of about 7.57 pnol/kg, oleic acid at a concentration of 7.56 pmol/kg and a sterol at a concentration of 2.61 pmol/kg.
[075] As used herein, the term "P3 buffer" may be used interchangeably with a buffer comprising one or more of KC, MgCl2, ClNa, Glucose and Ca(NO3)2 in any absolute or relative abundance or concentration, and, optionally, the further comprising a supplement selected from the group consisting of HEPES, Tris/HC, and a phosphate buffer. The term "P3 buffer" may be used interchangeably with a buffer comprising 5 mM KC, 15 mM
MgCl2, 90 mM CiNa, 10 mM Glucose and 0.4 mM Ca(N03)2, and, optionally, the further comprising a supplement selected from the group consisting of HEPES, Tris/ICi, and a phosphate buffer.The term "P3 buffer" may be used interchangeably with a buffer comprising 5 mM KC, 15 mM MgCl2, 90 mM CINa. 10 mM Glucose and 0.4 mM Ca(N03)2 and a supplement comprising 20 mM HEPES and 75 mM Tris/HCI. The term "P3 buffer" may be used interchangeably with a buffer comprising 5 mM KCI, 15 mM igCl2, 90 mM CiNa, 10 mM Glucose and 0.4 mM Ca(N03)2and a supplement comprising 40 mM Na2HPO4/NaH2PO4 at pH 7.2.
[076] As used herein, the terms "supplemented RPMI-1640 media" or "T-cell conditioned media (TCCM)" may be used interchangeably with a media comprising one or more of water, fetal bovine serum, HEPES, sodium pyruvate, one or more non-essential amino acids, a phenol red indicator, calcium nitrate, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, sodium phosphate dibasic (anhydrous), L-Alanyl-L-Glutamine, L-Arginine, L-Asparagine (anhydrous), L-Aspartic acid, L-Cysteine 2HCI, L-Glutamic acid, Glveine, L-Histidine, Hydroxy-L-Proline, L-Isoleucine, L-Leucine, L-Lysine HCi, L Methionine, L-Phenylalanine, L-Proline, L-Serine, L-Threonine, L-Tryptophan, L-Tyrosine 2Na 2120, L-Valine, D-Biotin. choline chloride, folic acid, Myo-Inositol, niacinamide, p Aminobenzoicacid, D-Panthothenic acid (hemicalcium), pyridoxine HC], riboflavin, thiamine HCi, vitamin B12, D-Glucose, Glutathione (reduced), L-Glutamine and 2 Mercaptoethanol in any absolute or relative abundance or concentration. The terms "supplemented RPMI-1640 media" or "T-cell conditioned media (TCCM)" may be used interchangeably with a media comprising water, fetal bovine serum, HEPES, sodium pyruvate, one or more non-essential amino acids, a phenol red indicator, calcium nitrate, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, sodium phosphate dibasic (anhydrous), L-Alanvl-L-Glutamine, L-Arginine, L-Asparagine (anhydrous), L-Aspartic acid, L-Cysteine 2HCl, L-Glutamic acid, Glycine, .- Histidine, Hydroxy-L-Proline, L-Isoleucine, L-Leucine, L-Lysine1-C, L-Methionine, L-Phenylalanine, L-Proline, L-Serine, L-Threonine, L-Tryptophan, L-Tyrosine 2Na 2H20, L-Vaine., D-Biotin, choline chloride, folic acid, Myo-Inositol, niacinamide, p-Aminobenzoic acid, D Panthothenic acid (hemicalcium), pyridoxine HC, riboflavin, thiamine HCI, vitamin B12, D Glucose, Glutathione (reduced), L-Glutamine and 2-Mercaptoethanol in any absolute or relative abundance or concentration.
[077] As used herein, the terms "supplemented AIM-V" or "supplemented AIMV" media may be used interchangeably with a media comprising one or more of water, human serum albumin, streptomycin sulfate, gentamicin, fetal bovine seru-, HEPES, sodium pyruvate, one or more non-essential amino acids, a phenol red indicator, calcium nitrate, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, sodium phosphate dibasic (anhydrous), L-Alanyl-L-Glutamine, L-Arginine, L-Asparagine (anhydrous), L-Aspartic acid L-Cysteine 2HCl, L-Glutamic acid, Glycine, L-Histidine, Hydroxy-L-Proline, L Isoleucine, L-Leucine, L-Lysine HCi, L-Methionine., L-Phenylalanine, L-Proline, L-Serine, L-Threonine, L-Tryptophan, L-Tyrosine 2Na 2H2O, L-Valine, D-Biotin, choline chloride, folic acid, Myo-Inositol, niacinamide, p-Aminobenzoic acid, D-Panthothenic acid (hemicalcium), pyridoxine HCl, riboflavin, thiamine HCI. vitamin B12, D-Glucose glutathione (reduced), L-Glutamine and 2-Mercaptoethanolinanyabsoluteorrelative abundance or concentration. The terms "supplemented AIM-V" or "supplemented AIMV" media may be used interchangeably with a media comprising water, human serum albumin, streptomycin sulfate, gentamicin, fetal bovine serum, HEPES, sodium pyruvate, one or more non-essential amino acids, a phenol red indicator, calcium nitrate, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, sodium phosphate dibasic (anhydrous), L-Alanyl-L-Glutamine, L-Arginine, L-Asparagine (anhydrous), L-Aspartic acid. L-Cysteine 2HCl. L-Glutamic acid, Glycine, L-Histidine, Hydroxy-L-Proline,L Isoleucine, L-Leucine, L-Lysine HCi, L-Methionine, L-Phenylalanine, L-Proine, L-Serine, L-Threonine, L-Tryptophan, L-Tvrosine 2Na 21-H0, L-Valine, D-Biotin, choline chloride, folie acid, Myo-Inositol, niacinamide, p-Aminobenzoic acid, D-Panthothenic acid (hericalcium), pyridoxine HCl, riboflavin, thiarme HCl, vitamin B12, D-Glucose, glutathione (reduced), L-Glutamine and 2-Mercaptoethanol in any absolute or relative abundance or concentration.
10781 As used herein, the term"ImmunoCultTm medium" may be usedinterchangeably with a medium comprising one or more of water, human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, L-Glutamine, phenol red, glycine, L-Alanine, L-Arginine hydrochloride, L-Asparagine, L-Aspartic acid, L-Cystene 2HCl, L-Glutamic acid, L-Glutamine, L-Histidine hydrochloride H20, L-Isoleucine, L-Leucine, L-Lysine hydrochloride, L-Methionine, L-Phenylalanine, L-Proline, L-Serine, L-Threonine, L Tryptophan, L-Tyrosine disodium silt, L-Valine, biotin, choline chloride, D-Calcium pantothenate, folic acid niacinamide, pyridoxal hydrochloride, riboflavin, thiamine hydrochloride, vitamin B12, i-Inositol, calcium chloride (anhydrous), magnesium sulfate (Anhydrous), potassium chloride, potassium nitrate, sodium bicarbonate, sodium chloride, sodium phosphate monobasic, sodium selenite, D-Glucose, HEPES and Sodium pyruvate in any absolute or relative abundance or concentration. The term"ImmunoCultTM medium" may be used interchangeably with a medium comprising water, human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, L-Glutamine, phenol red, glycine, L-Alanine, L-Arginine hydrochloride, L-Asparagine, L-Aspartic acid, L-Cysteine 2HCI, L-Glutamic acid, L-Glutamine, L-Histidine hydrochloride H20, L-Isoleucine, L Leucine, L-Lysine hydrochloride, L-Methionine, L-Phenylalanine, L-Proline, L-Serine, L Threonine, L-Tryptophan, L-Tyrosine disodium salt, L-Valine, biotin, choline chloride, D Calcium pantothenate, folic acid, niacinamide. pyridoxal hydrochloride, riboflavin, thiamine hydrochloride, vitamin B12, i-Inositol, calcium chloride (anhydrous), magnesium sulfate (Anhydrous), potassium chloride, potassium nitrate, sodium bicarbonate, sodium chloride, sodium phosphate monobasic, sodium selenite, D-Glucose, HEPES and Sodium pyruvate in any absolute or relative abundance or concentration.
[079] Modified T-cells of the disclosure, including modified TSCM and/Or TCM ofthe disclosure, may be incubated, cultured, grown, stored, or otherwise, combined at any step in the methods of the procedure with a growth medium comprising one or more inhibitors a component of a P13K pathway. Exemplary inhibitors a component of a Pi13K pathway include, but are not limited to, an inhibitor of GSK3P such as TWS119 (also known as GSK 3B inhibitor XII; CAS Number 601514-19-6 having a chemical formula Ci1iN402). Exemplary inhibitors a component of a PI3K pathway include, but are not limited to, bb007 (BLUEBIRDBIOTM)
[080] As used herein, the terms "electroporation" and "nucleofection" are meant to describe alternative means to deliver a nucleic acid, transposon, vector or composition of the disclosure to a cell by providing an electric pulse that induces a cell membrane (the cell membrane, nuclear membrane, or both) to become permeable or to become more permeable to the nucleic acid, transposon, vector or composition of the disclosure.
[081] In certain embodiments of the nucleofection, the method is performed one or more cuvette(s) simultaneously. In certain embodiments of the nucleofection, the method is performed in two cuvettes simultaneously. For a process performed on a larger scale for clinical or commercial applications, for example, the nucleofections may be performed in a large-volume cassette with many procedures ongoing simultaneously. In certain embodiments of the nucleofection, the incubating step comprises incubating the composition comprising the plurality of primary human T cells in a pre-warmed T-cell expansion composition. The incubation step may have a period of at least 1, 2, 3.4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, or any number/portion of hours in between, The incubation step may have a period of at least 1, 2, 3, 4, 5, 6 or 7 days or any number/portion of days in between. The incubation step may have a period of at least I week. In certain embodiments of the nucleofection, the incubation step has a period of two days. In certain embodiments of thenucleofection, the applying step may comprise applying one or more of the following programs) ET-]15, EI-151, EI-156, EI-158, EG-i15, EG-142, EG-151, ES-115, ES-151, EO-151. EO-148 EO-156, EO-210, EO-213, and FI-156. In certain embodiments, the applying step may comprise applying one or more of the following prograr(s) EI-15, EI-151, EI-156, EI-158, EG-l15, EG-142, EG-151, ES-I15, ES-151, EO 151 EO-148, EO-156, EO-210, EO-213, and FI-156, or a programthat provides the same number of electrical pulses, each pulse having the same duration and intensity, and a substantially similar interpulse duration of time. In certain embodiments, the applying step may be performed using a knownelectroporation/nucleofection device, including, but not limited to, Lonza Amaxa, MaxCyte technology, BTX PulseAgile, and BioRad GenePulser. In certain embodiments of the nucleofection, the applying step may comprise applying at least one electrical pulse. In certain embodiments of the nucleofection, the applying step may comprise applying at least one electrical pulse sufficient to induce the cell membrane and/or nuclear membrane of a cell to become permeable to a composition of the disclosure.
[082] While the amounts provided herein are exemplary and non-limiting, the relationship between these amounts (e.g. ratios or relative abundances) may be used to modify the methods exemplified herein for larger-scale processes and manufacturing.
10831 In certain embodiments of the methods of producing a modified T cell (e.g. a TSCM and/or TcM) of the disclosure, the activation supplement comprises one or more cytokine(s). The one ormore cytokine(s) may compriseany cytokine. including but not limited to, lymphokines. Exemplary lympokines include, but are not limited to, interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-15 (IL-15), intereukin-21 (IL-21), granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon-gamma (INFy). The one or more cytokine(s) may comprise IL-2.
10841 In certain embodiments of the methods of producing a modified T cell (e.g. a TscM and/orTcM) of the disclosure, the expansion supplement comprises one or more cytokine(s). The one or more cytokine(s) may comprise any cytokine, including but not limited to, lymphokines. Exemplary lympokines include, but are not limited to, interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-15 (IL-15). interleukin-21 (IL-21), granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon-gamma (INF). The one or more cvtokine(s) may comprise IL-2.
[085] In certain embodiments of the methods of producing a modified'Tcell (e.g. aTscM and/or TC) of the disclosure.the primary human I cell is a naive T cell. The naive T cell may express CD45RA, CCR7 and CD62L. In certain embodiments, the method is applied to a cell population comprising at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
%,60%,65%, 70%,75%,80%,85%.90%,95%, 99%. orany percentage inbetween of naive T cells. In certain embodiments, the efficiency of production ofmodified TsM and/or TcM of the disclosure may be increased by increasing a proportion or percentage of naive T cells in a cell population to which the methods of the disclosure are applied.
[086] In certain embodiments of the methods of producing a modified TscM and/orTcMof the disclosure, the primary human T cell is a memory T cell.
[0871 In certain embodiments of the methods of producing a modified TscM and/or TCM of the disclosure, the primary human T cell expresses one ormore of CD62L, CD45RA, CD28 CCR7, CD127, CD45RO, CD95, CD95 and IL-2Rp.
[088] In certain embodiments of the methods of producing a modified TscM and/or TcM of the disclosure, the primary humanTcell is a naive T-cell (modified TN) and the modified TN expresses one or more of CD45RA, CCR7 and CD62L. In certain embodiments of the methods of producing a modified TscMiand/or TcM of the disclosure, the pimary human T cell is a modified Tsc- a T memory stem cell (modified TscM) and the modified TsCM expresses one or more of CD45RA, CD95, IL-2R, CR7 .and CD62L. In certain embodiments of the methods of producing a modified Tsci and/or TCM of the disclosure, the primary human T cell is a central memory T-cell (modified Tm) and the modified Tm expresses one or more of CD45RO, CD95, IL-2R , CCR7,and CD62L. In certain embodiments of the methods of producing a modifiedTscM and/orTcM of the disclosure, the primary human T cell is an effector memory T-cell (modified TE) and the modified TEM expresses one or more of CD45RO, CD95, and IL-2R[. In certain embodiments of the methods of producing a modified TscM andor TcI of the disclosure, the primary human T cellis an effectorT-cell (modified TEFF) and the modified TEFFexpresses one or more of CD45RA, CD95, and IL-2R .
[089] In certain embodiments of the methods of producing a modified TscM and/orTCM of the disclosure, the primary humanTcell may express CD4 and/or CD8. In certain embodiments, the primary human T cell may express CD4 and/or CD8 at various ratios. In certain embodiments, the primary human T cell may express CD4 and/or CD8 at various ratios that are not naturally-occurring. In certain embodiments, the primary humanTcells that express CD4 and/or CD8 at various ratios, that may be not naturally occurring, are a heterologous cell population.
[090] In certain embodiments of the methods of producing a modified TscM and/or ICM of the disclosure, the primary humanTcell may be isolated, prepared or derived from for example, whole blood, peripheral blood, umbilical cord blood, lymph fluid, lymph node tissue, bone marrow, and cerebral spinal fluid (CSF). The term "peripheral blood" as used herein, refers to cellular components of blood (e.g., red blood cells, white blood cells and platelets), which are obtained or prepared from the circulating pool of blood and not sequestered within the lymphatic system, spleen, liver or bone marrow. Umbilical cord blood is distinct from peripheral blood and blood sequestered within the lymphatic system, spleen, liver or bone marrow. The terms "umbilical cord blood", "umbilical blood" or "cord blood", which can be used interchangeably, refers to blood that remains in the placenta and in the attached umbilical cord after child birth. Cord blood often contains stem cells including hematopoietic cells.
[091] Primary human T cells of the disclosure may comprise pan ' cells. As used herein, pan T-cells include all T lymphocytes isolated from a biological sample, without sorting by subtype, activation status, maturation state, or cell-surface marker expression.
10921 In certain embodiments of the methods of the disclosure, the method further comprises introducing into a modified TscM or''c cell a composition comprising a genomic editing construct or composition. In certain embodiments, the genomic editing construct comprises a guide RNA and a clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9) DNA endonuclease. In certain embodiments, the genomic editing construct comprises a DNA binding domain and a type ITS endonuclease. In certain embodiments, the genomic editing construct encodes a fusion protein. In certain embodiments, the genomic editing construct encodes the DNA binding domain and the type IIS endonuclease and wherein the expressed DNA binding domain and the expressed type ITS endonuclease are non-covalently linked. In certain embodiments, including those embodiments wherein the genomic editing construct comprises a DNA binding domain and a type IIS endonuclease, the genomic editing construct comprises a sequence derived from a Cas9 endonuclease. In certain embodiments, including those embodiments wherein the genomic editing construct comprises a DNA binding domain and a type IIS endonuclease, the sequence derived from a Cas9 endonuclease is the DNA binding domain. In certain embodiments, including those embodiments wherein the sequence derived from a Cas9 endonuclease is the DNA binding domain, the sequence derived from a Cas9 endonuclease encodes an inactive Cas9. In certain embodiments, including those embodiments wherein the sequence derived from a Cas9 endonuclease is the DNA binding domain, the sequence derived from a Cas9 endonuclease encodes a truncated Cas9. In certain embodiments, the sequence derived from a Cas9 endonuclease comprises an amino acid substitution of an Alanine (A) for an Aspartic Acid (D) at position 10 (DOA). In certain embodiments. the sequence derived from a Cas9 endonuclease comprises or further comprises an amino acid substitution of an Alanine (A) for a Histidine (H) at position 840 (H840A). In certain embodiments, the sequence derived from a Cas9 endonuclease comprises an inactivated Cas9 (dCas9) (SEQ ID NO: 33). In certain embodiments, the sequence derived from a Cas9 endonuclease comprises an amino acid substitution of an alanine(A) for an Asparagine (N) at position 580 (N580A). In certain embodiments, the sequence derived from a Cas9 endonuclease comprises a truncated and inactivated Cas9 (dSaCas9) (SEQ ID NO: 32). In certain embodiments, including those embodiments wherein the genomic editing construct comprises a DNA binding domain and a type IIS endonuclease, the genomic editing construct comprises a sequence derived from a transcription activator-like effector nuclease (TALEN). In certain embodiments, including those embodiments wherein the genomic editing construct comprises a DNA binding domain and a type IIS endonuclease, the sequence derived from a TALEN is the DNA binding domain. In certain embodiments, the genomic editing construct comprises a TALEN. In certain embodiments, including those embodiments wherein the genonic editing construct comprises a DNA binding domain and a type ITSendonuclease, the genomic editing construct comprises a sequence derived from a zinc-finger nuclease(ZFN) In certain embodiments, including those embodiments wherein the genomic editing construct comprises a DNA binding domain and a type IS endonuclease, the sequence derived from a ZFN is the DNA binding domain. In certain embodiments, the genomic editing construct comprises a zinc-finger nuclease (ZFN).
[093] The methods of making modified'TsNM and/or'Tc cells of the disclosure may be optimized to produce a greater number or greater proportion of modified TSCM and/or TM1 cells. For example, the population of cells subjected to the methods of the disclosure may be enriched to contain an increased number or greater proportion of naive T cells. As the number and/or proportion of naive T cells increases in the population of T cells subjected to the methods of the disclosure, the number and/or proportion of modified TscM and/or TcM cells of the disclosure produced also increases. Alternatively, or in addition, as the length of time or duration required for a method of disclosure to precede decreases, the number and/or proportion of modified TscM and/or TCM cells of the disclosure produced by the method increases. The length of time or duration required for a method of disclosure to precede, or the "manufacturing period" may also be referred to as the "out-of-life period" of the Tcells subjected to the methods of the disclosure.
[094] In certain embodiments of the methods of making modified T-cells ofthe disclosure, the primary human T cell expresses one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2R. In certain embodiments, the primary humanTcell is a naive T-cell (TN) and the TN expresses one or more of CD45RA, CCR7 and CD62L. In certain embodiments, the primary huran T cell is a T memory stem cell (TCM) and the TCM expresses one or more of CD45RA, CD95. IL-2RP, CR7. and CD62L. In certain embodiments, the primary human T cell is a central memory T-cell (TCM) and wherein the TCMexpresses one or more of CD45RO, CD95, IL-2RP, CCR7, and CD62L. In certain
embodiments, the primary human Tcell is an effector memory T-cell(TEM)and theFM expresses one or more of CD45RO, CD95, and IL-2RQ. In certain embodiments, the primary human T cell is an effector T-cell (TEi) and the TLFF expresses one or more of CD45RA, CD95, and IL-2R[. In certain embodiments, the primary human T cell expresses CD4 and/or CD8.
[095] The disclosure provides a composition comprising amodifiedTsM produced a method of the disclosure. The disclosure provides a composition comprising a modified Tci produced a method of the disclosure. The disclosure provides a composition comprising a modified TSCMand a modified'TcM produced a method of the disclosure. In certain embodiments of the composition comprising a modified TscM and a modified TCM produced a method of the disclosure, a plurality of TscM may comprise at least 1%, 2O, 5%, 10%, 15%. %, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%or 99% or the composition. . In certain embodiments of the composition comprising a modified TscM and a modified TCM produced a method of the disclosure, a plurality of TcM may comprise at least 1%, 2%, 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, %, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% or the composition. 10961 The disclosure provides a use of a composition comprising a modified TscM andor TCMproduced a method of the disclosure for the manufacture of a medicament to treat a subject in need thereof. In certain embodiments of this use, themodified TscM and/OrTcM is autologous. In certain embodiments of this use, themodified TscM and/or Tci is allogeneic. In certain embodiments, the antigen receptor is a T-cell receptor. In certain embodiments, the T-cell receptor is naturally-occurring. In certain embodiments, the T-cell receptor is not naturally-occurring. In certain embodiments, and, in particular, in those embodiments wherein the T-cell receptor is not naturally-occurring, the T-cell receptor comprises one or more mutation(s) compared to a wild-type T-cell receptor. In certain embodiments, and, in particular, in those embodiments wherein the T-cell receptor is not naturally-occurring, the T cell receptor is a recombinant T-cell receptor. In certain embodiments, the antigen receptor is a Chimeric Antigen Receptor (CAR). In certain embodiments, the CAR is a CARTyrin. In certain embodiments, the CAR comprises one or more VHI- sequence(s). In certain embodiments, the CAR is a VCAR.
[097] The disclosure provides a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition comprising a modified TscN and/or TcMproduced a method of the disclosure. In certain embodiments of this method, the modified Tsct and/orTcM is autologous. In certain embodiments of this method, the modified Tsci and/or TcM is allogeneic. In certain embodiments, the antigen receptor is a T-cell receptor. In certain embodiments, the T-cell receptor is naturally-occuring. In certain embodiments, the T-cell receptor is not naturally occurring. In certain embodiments, and, in particular, in those embodiments wherein theT cell receptor is not naturally-occurring, the T-cell receptor comprises one or more mutation(s) compared to a wild-type T-cell receptor. In certain embodiments, and, in particular, in those embodiments wherein the'T-cell receptor is not naturally-occurring, the T-cell receptor is a recombinant T-cell receptor. In certain embodiments, the antigen receptor is a Chimeric Antigen Receptor (CAR). In certain embodiments, the CAR is a CARTyrin. In certain embodiments, the CAR comprises one or more VHH sequence(s). In certain embodiments, the CAR is a VCAR. In certain embodiments of this method, the disease or disorder is cancer and the antigen receptor specifically targets a cancer antigen. In certain embodiments of this method, the disease or disorder is an infectious disease or disorder and the antigen receptor specifically targets a viral, bacterial, yeast or microbial antinen. In certain embodiments, the disease or disorder is a disease or disorder caused by a lack of an activity oran insufficient amount of a secretory protein. In certain embodiments, the disease or disorder is a disease or disorder treated by a replacement of an activity of a therapeutic protein or by an increase in an amount of the therapeutic protein. In certain embodiments, the therapeutic protein is a secreted protein. In certain embodiments, the secretory protein is lacking an activity or a sufficient amount within a local area of a body. In certain embodiments, the local area of a body is accessible by a native T-cellor a modified T-cell. In certain embodiments, the modified T-cell is produced in vivo, ex vivo, in vitro or in situ.
BRIEF DESCRIPTION OF THE DRAWINGS
[098] Figure 1 is a series of plots depicting the emergence of the CARTSCM phenotype at Day 11 of the method of Example 1. Cells were nucleofected with a surrogate CARTyrin plasniid. CAR-TscMcells express CD62Land CD45RA as shown in the bottom two plots.
[099] Figure 2 is a series of plots depicting the purity of the CAR-TscMproduced by the method of Example I at day 19. The population of CAR-TcM cells produced by the method described in Example 1 at day 19 contained no B cells or lymphocytes. The majority of the cells are CD3+ T-cells. Only 1.1% are Natural Killercellsand 1.7%are Natural Killer T cells.
[01001 Figure 3 is a plot showing that at Day I Iof the method described in Example 1, the majority of the T-cells produced express the CARTyrin. 101011 Figure 4 is a series of plots depicting an enrichment of the CAR-TscM phenotype at Day 19 of the method described in Example 1. Cells were nucleofected with a surrogate CARTyrin plasmid. CAR-Tsc[ cells express CD62L and CD45RA as shown in the bottom two plots.
[01021 Figure 5 is a series of plots depicting the absence ofT-cell exhaustion at Day 19 of the method described in Example 1. At Day 19, the cell population produced by this method does not express PD1, which is a marker for T cell activation and exhaustion. These cells expressing the CARTvrin have almost successfully reached a resting state post-manufacture. They do not exhibit signs of antigen -independent (tonic) signaling which would otherwise drive higher levels of PDi expression. Tonic signaling ishypothesized to be caused by some CAR molecules that lead to early exhaustion and reduced efficacy of a CAR T-cell therapy.
[01031 Figure 6A is a series of plots depicting T cells transposed with aplasmid containing a sequence encoding a transposon comprising a sequence encoding an inducible caspase polypeptide (a safety switch, "iC9"), a CARTyrin (anti-BCMA), and a selectable marker. Left-hand plots depict live T cells exposed to transposase in the absence of the plasmid. Right-hand plots depict liveT cells exposed to transposase in the presence of the plasmid. Cells were exposed to either a hyperactive transposase (the "Super piggyBac") or a wild type piggyBac transposase.
[01041 Figure 6B is a series of plots depictingT cells transposed with a plasmid containing a sequence encoding a green fluorescent protein (GFP). Left-hand plots depict live T cells exposed to transposase in the absence of the plasmid. Right-hand plots depict live T cells exposed to transposase in the presence of the plasmid. Cells were exposed to either a hyperactive transposase (the "Super piggyBac") or a wild type piggyBac transposase.
[01051 Figure 6C is a table depicting the percent of transformed T cells resulting from transposition with WT versus hyperactive piggyBac transposase. T cells contacted with the hyperactive piggvBac transposase (the Super piggyBac transposase) were transformed at a rate 4-fold greater than WT transposase.
[01061 Figure 6D is a table depicting the percent of transformed T cells resulting from transposition with WT versus hyperactive piggyBac transposase 5 days after nucleofection. T cells contacted with the hyperactive piggyBac transposase (the Super piggyBac transposase) were transformed at a rate far greater than WT transposase. 101071 Figure 7 is a graph showing a phenotypic difference between piggyBacT M - and lentivirus-produced CAR+ T cells. CAR-i- T cells were produced using either piggyBac transposition or lentivirus transduction. Human pan"T cells were transposed with piggyBac encoding CAR, stimulated with anti-CD3/CD28 beads at day 2 post-transposition, expanded, and examined on day 19 post-transposition. For production using lentivirus, pan T cells were stimulated with aCD3/CD28 beads, transduced with lentivirus encoding CAR (MOI 5), expanded, and examined on day 18 post-stimulation. Then, each population of CAR- T cells was characterized based on their expression of the standard memory markers CD62L, CD45RA and CD95. The percentage of each CAR- T cell subsetwas defined as naive (CD62L+CD45RA+),'Tcn (CD62L+CD45RA-), Tern (CD62L-CD45RA-) and Teff (CD62L-CD45RA+). All CAR+ T cells were CD95+.
[01081 Figure 8A-B is a pair of graphs showing that piggyBacTM preferentially transposes naive T cells. Human pan T cells were soiled (using a BD FACSAria I1 flow cytometer) into naive (CD62L+CD45RA+), Tern (CD62L+CD45RA-), Tem (CD62L-CD45RA-), and Teff (CD62L-CD45RA+) subsets. The soiled subsets were each either transposed with piggyBac GFP or transduced with lentivirus-GFP. For the former, each soiled subset was transposed with PiggyBac-GFP, stimulated with anti-CD3/CD28 beads at day 2 post-transposition, expanded, and examined on day 19 post-transposition. For the latter, the sorted subsets were stimulated with aCD3/CD28 beads, transduced with lentivirus encoding GFP (MOI 5), expanded, and examined on day 19 post-stimulation. n:=3 donors.
[01091 Figure 9 is a pair of graphs showing that thepiggyBacM manufacturing process yields high levels of TcM in samples from multiple myeloma (MM) patients even when naive T cells are rare. T cells from MM patients (triangles) and healthy donors (circles) were characterized for memory marker expression by flow cytometry before (left) and after (right) the Poseida manufacturing process. Expression of CD45RA and CD62L was assessed by FACS and plots are shown for the MM patients and a healthy donor. It is known that T cells from MM patients generally have lower frequencies of naive and TscM cells, but higher frequencies ofTeff, unlike those from healthy normal donors which are the opposite. Regardless of the input frequency of naive and Tsem from different MM patients, production of P-BCMA-101 using the Poseida manufacturing process resulted in a product that exhibited a high level of CD8+ Tscm (E). This was also true for a MM patient who was actively receiving treatment (red triangle).
[01101 Figure 10 is a series of Fluorescence Activated Cell Sorting (FACs) plots characterizing T and TscM cell markers in human pan T cells transformed with the Sleeping Beauty (SB100x) transposition system and the methods of the disclosure. Sleeping Beauty (SB100x) Transposition yields predominatelyTscm phenotype using Poseida manufacture process. Human pan T cells were transposed using Ipg of either aSleeping Beauty or piggyBac transposon plasmid and SBO0x or SPB mRNA, respectively as shown. Following transposition, cells were expanded ex vivo and all non-transposed cells were depleted using the Poseida manufacture drig selection system. Following 18 days in culture, cells were stained with the phenotypic markers CD4, CD8, CD45RA, and CD62L. Stem cellmemory phenotype (Tscm) is defined by CD45RA and CD62L double positive cells and make up >65% of the cells in all of samples. All panels in a column share common x-axis and y-axis parameters. In each row, from top to bottom, are shown data from T cells transposed with (top), 2.5 microgram (pg) of the Sleeping Beauty transposonSBOOx., (second from top) 5 pg of SBI 0x, (3 rd from top) 10 pg ofSB1OOx, (second from bottom) 5 pg of thepiggyBac transposon P-BCMA-101 and at bottom, an unstained control. The x-axis, in order from left to right, in the first and second columns shows Forward Scatter (FSC), units from 0 to 250 thousand (abbreviated "k"), in increments of 50k. The x axis of the third column from the left shows CD8 expression, with markings reading from 0 to 10' incrementing by powers of 10. The final right hand column shows CD62L expression, with markings reading from 0 to 10' incrementing by powers of 10. The y-axis, in the first column, shows Side Scatter (SSC), in units from 0 to 250k in increments of 50k. The y-axis in the second column from the left shows expression of the cell viability marker 7 aminoactinomycinD (7AAD), from 0 to 105 incrementing by powers of 10. The y-axis of the third column from the left shows the expression of the marker CD4, from 0 to 105 incrementing by powers of 10. The y-axis in the right hand column show expression of the marker CD45RA, from 0 to 0I incrementing by powers of 10.
[0111] Figure i I is a schematic diagram showing the human coagulation pathway leading to blood clotting. Contact activation, for example by damaging an endothelium, activates an intrinsic clotting pathway. Tissue factors activate an extrinsic clotting pathway, for example following trauma. Both pathways converge onto the conversion of Prothrombin into Thrombin, which catalyzes the conversion of fibrinogen into fibrin. Polymerized fibrin together with platelets forms a clot. In the absence of Factor IX (circled), clotting is defective. Factor VIII (FVIII) deficiency leads to development of Hemophilia A. Factor IX (FIX) deficiency leads to development of Hemophilia B. Hemophilia B is a rare disease, occurring with a frequency of about one in between 25,000 and 30,000. Sixty percent of hemophilia B cases are severe. Fewer than one percent of individuals with Hemophilia B have normal FIX levels. Prior to the compositions and methods of the disclosure, the standard treatment for hemophilia B involved an infusion of recombinant FIX every 2 to 3 days, at an expense of approximately $250,000 per year. In sharp contrast to this standard treatment option,TSCM cells of the disclosure are maintained in humans for several decades.
[0112] Figure 12 is a series of Fluorescence-Activated Cell Sorting (FACS plots) depicting FIX-secreting T cells. T cells encoding a human Factor IX transgene showed a TSCM phenotype in approximately 80% of cells. The 6 panels are described in order from left to right. (1) Forward scatter (FSC)on the x-axis versus side scatter (SSC) on the y-axis.The x axis is from 0 to 250 thousand (abbreviated k) in increments of 50k, the v-axis is for 0 to 250k, in increments of 50k. (2) FSC on the x-axis versus the cell viability marker 7 aminoactinoininD (7AAD).The x-axis is labeled from 0 to 250k in increments of 50k.The y-axis reads, from top to bottom, -103, 0, 10 104, 105. (3) On the x-axis is shown anti-CD56 APC conjugated to a Cy7 dye (CDC56-APC-Cy7), units from 0 to 10" incrementing in powers of 10. On the v-axis is shown anti-CD3 conjugated to phycoerythrin (PE), units from to 105 incrementing in powers of 10. (4) On the x-axisis shown anti-CD8 conjugated to fluoresci isicyaT(FITC), units from0to10incrementing in powers of 10.Ithe y-axis is shown anti-CD4 conjugated to Brilliant Violet 650 dye (BV650), units from 0 to 105 incrementing in powers of 10. (5) On the x-axis is shown an anti CD62L antibody conjugated to a Brilliant Violet 421 dye (BV421), units from 0 to 105 incrementing in powers of 10. On the y-axis is shown ananti-CD45RA antibody conjugated to PE and Cy7, units from 0 to 105 incrementing in powers of 10. This panel is boxed. (6) On the x-axis is shown an anti-CCR7 antibody conjugated to Brilliant Violet 786 (BV786), units from 0 to 105 incrementing in powers of 10. On the y-axis is shown anti-CD45RA conjugated to PE and Cy7, units from 0 to 105 incrementing in powers of 10.
101131 Figure 13A is a graph showing human FactorX secretionduring production of modified T cells of the disclosure. On the y-axis, Factor IX concentration in nanograms (ng) per milliliter (mL) from 0 to 80 in increments of 20. On the x-axis are shown 9 day and 12 day T cells.
[01141 Figure 13B is a graph showing the clotting activity of the secreted Factor IX produced by the'Tcells. On the y-axis is shown percent Factor IX activity relative to human plasma, from 0 to 8 in increments of 2. On the x-axis are 9 and 12 day T cells.
101151 Figures 14A-E are a series of plasmid maps for site-specific integration into the AAVS Isite using either HR or MMEJ and corresponding sequences. Donor plasmids for testing stable integration into the genome of human pan Tcells via A) site-specific (AAVSl) homologous recombination (HR), B) site-specific (AAVS1) nicrohomoogy-mediated end
joining (MMEJ) recombination and C) TTAA-specific piggyBaci transposition. For HR and MMEJ donor plasmids, GFP-2A-DHFR gene expression cassettes were flanked by CRISPR/Cas9 targeting sites and homology arms for AAVS Isite integration; for piggyBac Ti donor plasmid, GFP-2A-DHFR gene expression cassette is flanked by piggyBac T i transposon elements. The homology arms for the HR and MMEJ plasmids are 500 bpand 25 bp, respectively. Panels D and E, and F depict SEQ ID NOs 41 and 42 respectively.
[01161 Figure 15 is a graph showing transgene (GFP) expression in primary human pan T cells 3 days post-nucleofection. HR or MMEJ donor plasmids were co-delivered with or without CRISPR ribonucleoprotein (RNP) targeting reagents into pan T cells via nucleofection. T cells receiving donor plasmids alone were included as controls. Pan T cells were also modified using the piggyBacTM transposon delivery system. T cells were activated via TCR stimulation on Day 0 and GFP+T'cell percentage was accessed at day3 post nucleofection by flow cytometryand data are summarized in bar graph.
[01171 Figure 16 is a graph showing transgene (GFP) expression in primary human pan T cells 11 days post-nucleofection and selection. Activated T cells with stable integrated transgenes were selected by methotrexate addition using the DHFR selection gene encoded in the bi-cistronic GFP-2A-DHFR integration cassettes. GFP+ cell percentage was assessed at Day 11 post-nucleofection by flow cytometry and data are summarized in bar graph. GFP+ cellsxwerc highly enriched via selection in panTcells receiving transposition reagents, RNP pius HR or MMEJ donor plasmids, but notin Tcells receiving donor plasmids alone.
[01181 Figure 17A-C is a series of graphs showing the phenotype of primary human pan T cells modified by HR and MMEJ at the AAVS Isite. The phenotype of GFP+ CD8+ pan T cells was analyzed at Day 11 post-nucleofection by flow cytometry. A) Cells were stained with 7AAD (cell viability). CD4, CD8, CD45RA and CD62L, and FACS plots show gating strategy. CD8+ T cell subsets were defined by expression of CD45RA+CD62L+ (stem cell memory T cells ('sem)), CD45RA-CD62L+ (central memoryTcells (Tem)), CD45RA CD62L- (effector memoryTcells (Tem)), and CD45RA+CD62L- (T effectors (Teft)). B) Percentage of total GFP+ CD8+ T cells in each T cell subset is summarized in bar graph. An enriched population of GFP+ Tsem was achieved in all cases using either the piggyBaTM transposon system, or HR and MMEJ in combination with Cas9 RNP. C) The total number of pan Tcells was analyzed at day 13 post-nucleofection and data are summarized in bar graph.
[01191 Figure 18A-B is a pair of photographs ofgel electrophoresis results showing site specific integrationintothe AAVSi site. Selected cells from each group were harvested and genomic DNA was extractedand used as template for PCR to confirm site-specific integration into the AAVS1 site for A) HR and B) MMEJ. Two pairs of primers individually amplif the 5'-end junction (with one primer priming the promoter region of the insertion EFla-2r CACCGGAGCCAATTCCCACT (SEQ ID NO: 36) and the other priming the AAVS Iregion beyond the 500 bp homologue ann at the 5'-end AAVS-3r CTGCACCACGTGATGTCCTC (SEQ ID NO: 37), yielding a 0.73 kb DNA fragm tfor both HRorMMEJ) and 3'-endjunction (with one primerpriming the polyA signaling region SV40pA-lr GTAACCATTATAAGCTGCAATAAACAAG (SEQ ID NO: 38) and the other priming the AAVS Iregion beyond the 500 bp 5'-honologue arm AAVS-2f CTGGGGACTCTTTAAGGAAAGAAG (SEQ ID NO: 39), yielding a 0.76 kb DNA fragment for HR or MMEJ) of the AAVS Itarget site. PCR products were displayed on Agarose gel. Non-specific bands in HR samples are the result of onlya single round of PCR and would likely have been resolved given additional rounds.
DETAILED DESCRIPTION
[01201 The disclosure provides a method for producing human chimeric antigen receptor (CAR) expressing-T cells using the piggyBacTM Transposon System under conditions that preserve or induce stem-cell memory T cells (TSCM) with potent CAR activity (referred to herein as a CAR-Tscr. Compositions comprising CAR-TSCM produced using the methods of the disclosure comprise ;> 60% CAR-Tscx and exhibit a distinct functional profile that is consistent with this T cell subset. Other T cell subsets found in the compositions of the disclosure include, but are not limited to, central memory CAR-T cells (CAR-TM), effector memory CAR-T cells (CAR-TEM), effector CART cells (CAR-TE), and terminally differentiated effector CAR-T cells (CAR-TTE), A linear pathway of differentiation may be responsible for generating these cells: Naive T cells (TN)> TscM > TM > TEM> TE> TTE,
whereby TN is the parent precursor cell that directly gives rise to TSCA.which then, in turn, directly gives rise to TCM, etc. Compositions comprising CAR-TscM, CARTyrin-TscM and/or VCAR-TSc of the disclosure may comprise one or more of each parental CAR-T cell subset with CAR-Tscm being the most abundant (e.g. TSCM > TcM > TEM > TE> TTE). While, the absolute quantities/abundances and relative proportions of each parental T cell subset may vary among samples of patient blood and naturally-occurring cell populations, and naturally occurring cell populations may have a high abundance and/or proportion of TscM, compositions of the disclosure comprising non-naturally occurring CAR-TscM are more potent and efficacious in treating patients against diseases and cancers.
[01211 Immunotherapy using chimeric-antigen receptor (CAR)-T cells is emerging as an exciting therapeutic approach for cancer therapies. Autologous CAR-modified T cells targeting a tumor-associated antigen (Ag) can result in robust tumor killing, in some cases resulting in complete remission of CD19'hematological malignancies. Unlike traditional biologics and chemotherapeutics, CAR-T cells possess the capacity to rapidly reproduce upon Ag recognition, thereby potentially obviating the need for repeat treatments. To achieve this. CAR-T cells must not only drive tumor destruction initially, but must also persist in the patient as a stable population of viable memory T cells to prevent potential cancer relapses. Thus, intensive efforts have been focused on the development of CAR molecules that do not cause T cell exhaustion through Ag-independent (tonic) signaling, as well as of a CAR-T product containing early memory cells, especially stem cell memory (TCM). Astem cell-like CAR-T would exhibit the greatest capacity for self-renewal andmultipotent capacity to derive central memory (TcM), effector memory (TEM) and effector T cells (TE), thereby producing better tumor eradication and long-term CAR-Tengraftment.
[01221 CAR-TscM of the disclosure may comprise a Centyrin-based CAR, referred to as a CARTyrin (and hence, the cell may be referred to as a CARTyrin-TscM). Centyrins are alternative scaffold molecules based on human consensus tenascin FN3 domain, are smaller than scFv molecules, and can be selected for monomeric properties that favor stability and decrease the likelihood of tonic signaling in CAR molecules. CARTyrins of the disclosure may be introduced to T cells using a plasmid DNA transposon encoding the CARTyrin that is flanked by two cis-regulatory insulator elements to help stabilize CARTyrin expression by blocking improper gene activation or silencing.
[01231 CAR-TscM of the disclosure may comprise aVHH-based CAR, referred to as a VCAR (and hence, the cell may be referred to as a VCAR-TscM). VCARs of the disclosure may be introduced to T cells using a plasmid DNA transposon encoding the VHH that is flanked by two cis-regulatory insulator elements to help stabilize VHH expression by blocking improper gene activation or silencing.
101241 In certain embodiments ofthe methods of the disclosure, the piggyBacT M (PB) Transposon System may be used for stable integration of antigen-specific (including cancer antigen-specific) CARTyrinor VCAR into resting panT cells, whereby the transposon was co-delivered along with an mRNA transposase enzyme (although the transposon and transposase would be comprised in separate compositions until they were introduced into a cell), called SuperpiggyBacM (SPB),in single electroporation reaction Delivery of piggyBacTI transposon into untouched, resting primary human pan T cells resulted in 20 % of cells with stable integration and expression of PB-deliveredgenes. Unexpectedly, a majority ofthese modified CARTyrin-expressing T cells were positive for expression of CD62L and CD45RA, markers commonly associated with stem memory T-cells (TSCM cells). To confirm that this phenotype was retained upon CAR-T cell stimulation and expansion, the modified CARTyrin-expressing T cells positive for expression of CD62L and CD45RA were activated via stimulation of CD3 and CD28. As a result of stimulation of CD3 and CD28, > % of CARTyrin- T cells exhibited a stem-cell memory phenotype. Furthermore, these cells, which expressed a CARTyrin specific for a cancer antigen, were fully capable of expressing potent anti-tumor effector function.
[01251 To determine whetheror not the PB system directly contributed to enhancing the expression of stem-like markers, the phenotype of CAR-T cells generated either by PB transposition or lentiviral (LV) transduction was compared. To do this, a new vector was constructed by subcioning the CARTvrin transgene into a common LV construct for production of virus. Following introduction of the CARTyrin to untouched restingTcells either by PB-transposition or LV-transduction, the CAR-Tyrin cells were expanded and then allowed toreturn toaresting state. A variety of phenotypic and functional characteristics were measured including kinetic analysis of memory and exhaustion-associated markers, secondary proliferation in response to homeostatic cytokine or tumor-associated AR, cytokine production, and lytic capability in response to target tuorcells.Unlike the PB-transposed CARTyrin+ T cells, the LV-transduced CARTyrin' T cells did not exhibit an augmented memory phenotype. In addition, PB-transposed cells exhibited a comparable or greater capability for secondary proliferation and killing of target tumor cells. Together, these data demonstrate that CAR-T cells produced by PB transposition are predominantly TSCM cells, a highly desirable product phenotype in the CAR-Tfield. Furthermore, these CARTyrin+ T cells exhibit strong anti-tumor activity and may give rise to cells that persist longer in vivo due to the use of a Centyrin-based CAR, which may be less prone to tonic signaling and functional exhaustion. ChimericAntigen Receptors
[01261 The disclosure provides a chimeric antigen receptor (CAR) comprising: (a) an ectodomain comprising an antigen recognition region, wherein the antigen recognition region comprises one or more sequences that each specifically bind an antigen; (b) a transmembrane domain, and (c) an endodomain comprising at least one costimulatory domain. In certain emnbodiments, the antigen recognition region may comprise two sequences that each specifically bind an antigen to produce a bi-specific or tandem CAR. In certain embodiments, the antigen recognition region may comprise three sequences that each specifically bind an antigen to produce a tri-specific CAR. In certain embodiments, the ectodomain may further comprise a signal peptide. Alternatively, or in addition, in certain embodiments, the ectodomain may further comprise a hinge between the antigen recognition region and the transmembrane domain. Sequences that each specifically bind an antigen may include, but not limited to, a single chain antibody (e.g. a seFv), a sequence comprising one or more fragments of an antibody (e.g. a VHIH, referred to in the context of a CAR as a VCAR), an antibody mimic, and a Centyrin (referred to in the context of a CAR as a CARTyrin).
[01271 In certain embodiments of the CARS of the disclosure, the signal peptide may comprise a sequence encoding a human CD2, CD36, CD3F, CD3y, CD3(, 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. The human CD8a signal peptide maycomprise an amino acid sequence comprising MALPVTALLLPLALLLI-IAARP (SEQ ID NO: 8). The human CD8a signal peptide may comprise an amino acid sequence comprising MALPVTALLLPLALLLHAARP (SEQ ID NO: 8) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the an amino acid sequence comprising MALPVTALLLPLALLLHAARP (SEQ ID NO: 8). The human CD8a signal peptide may be encoded by a nucleic acid sequence comprising atggcactgccagtcaccgccctgctgcgcctctggctctgctgctgcacgagtagacca (SEQ ID NO: 9).
[01281 In certain embodiments of the CARs of the disclosure, the transmembrane domain may comprise a sequence encoding a human CD2, CD36, CD3R, CD37,CD3(, 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 transmenibrane domain. The CD8a transineinbrane domain may comprise an amino acid sequence comprising YIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 10) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 10). The CD8 transineinbrane domain may be encoded by the nucleic acid sequence comprising atctacatttggcaccactggccgggacctg.tggagtgtgtgctgagcctggcatcacactgtactgc (SEQ ID NO: 11).
[01291 In certain embodiments of the CARs of the disclosure, the endodoinain may comprise a human CD3( endodoman.
101301 In certain embodiments of the CARs ofthe disclosure, theat least one costimulatory domain may comprise a human 4-IBB, CD28, CD40, ICOS, MyD88, OX-40 intracellular segment, orany combination thereof. In certain embodimentsof the CARsof the disclosure, the at least one costimulatory domain may comprise a CD28 and/or a 4-BB costimulatory domain. The CD28 costimulatory domain may comprise an amino acid sequence comprising RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKCHDGLYQGLSTATKDTYDALlIMQALP PR (SEQ ID NO: 12) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ ECLYNELQKDKMA EAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP PR (SEQ ID NO: 12). The CD28 costimulatory domain may be encoded by the nucleic acid sequence comprising cgcgtgaagtttagtcgatcagcagatgccccagcttacaaacagggacagaac.cagctgtataacgagctgaattgggccgccga gaggaatatgacgtgctggataagcggagaggacgcgaccccgaaatgggaggcaagcccaggcgcaaaaaccctcaggaagg cctgtataacgagctgcagaaggacaaaatggcagaagcctattctgagatcggcatgaagggggagcgacggagaggcaaagg gcacgatgggctgtaccagggactgagcaccgccacaaaggacacctatgatgctctgcatatgcaggcactgcctccaagg (SEQ ID NO: 13). The 4-1BB costimulatory domain may comprise an amino acid sequence comprising KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 14) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 14). 'Te 4 1BB costimulatory domain may be encoded by the nucleic acid sequence comprising aagagaggcaggaagaaactgctgtatattttcaaacagccettcatgcgccccgtgcagactacccaggaggaagagggtgctce tgtegattccctgaggaagaggaagggggtgtgagtg (SEQ ID NO: 15). The 4-1BB costimulatory domain may be located between the transimembrane domain and the CD28 costimulatory domain.
101311 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. The hinge may comprise a human CD8a amino acid sequence comprising TTTPAPRPPTPAPTIASQPLSLRPEACRIAAGGAVHTRGLDFACD (SEQ ID NO: 16) or a sequence having at least 70%, 80%, 90% 95%, or 99% identity to the amino acid sequence comprising TTTPAPRPPTPAPTIASQPLSLRPEACRPAAG(VHI-ITRGLDFACD (SEQ ID NO: 16). The human CD8a hinge amino acid sequence may be encoded by the nucleic acid sequence comprising actaccacaccagcacctagaccaccaactcagctccaaccatcgegagtcagcccetgagtctgagacctgaggcctgcaggcc agctgcaggaggagctgtgeacaccaggggcctggacttgcctggac (SEQ ID NO: 17).
[01321 The disclosure provides a composition comprising the CAR of the disclosure and at least one pharnaceutically acceptable carrier
101331 The disclosure provides a transposon comprising the CAR of the disclosure. Transposons of the disclosure be episomally maintained or integrated into the genome of the recombinant/modified cell. The transposon may be part of a two component piggyBac system that utilizes a transposon and transposase for enhanced non-viral gene transfer.
[01341 Transposons of the disclosure may comprise a selection gene foridentification, enrichment and/or isolation of cells that express the transposon. Exemplary selection genes encode anygene product (e.g. transcript, protein, enzyme) essential for cell viability and survival. Exemplary selection genes encode any gene product (e.g. transcript, protein, enzyme) essential for conferring resistance to a drug challenge against which the cell is sensitive (or which could be lethal to the cell) in the absence of the gene product encoded by the selection gene. Exemplary selection genes encode any gene product(e.g. transcript, protein, enzyme) essential for viability and/or survival in a cell media lacking one or more nutrients essential for cell viability and/or survival in the absence of the selection gene. Exemplary selection genes include, but are not limited to, neo (conferring resistance to neomycin), DHFR (encoding Dihydrofolate Reductase and conferring resistance to Methotrexate), TYMS (encoding Thymidylate Synthetase), MGMT ( encoding 0(6) methlviguanine-DNA methyltransferase), multidrug resistance gene (MDRI), ALDHi (encoding Aldehyde dehydrogenase I family, member Al), FRANCE RAD5IC (encoding RAD51 Paralog C), GCS (encoding glucosyceramide synthase), and NKX2.2 (encoding NK2 Homeobox 2). 101351 Transposons of the disclosure may comprise at least one self-cleaving peptide(s) located, for example, between oneor moreof a sequence that specifically binds an antigen and a selection gene of the disclosure. The at least one self-cleaving peptide may comprise, for example, aT2A peptide, GSG-T2A peptide, an E2A peptide, a GSG-E2A peptide, an F2A peptide, a GSG-F2A peptide, a P2A peptide, or a GSG-P2A peptide. AT2A peptide may comprise an amino acid sequence comprising EGRCSLLTCGDVEENPGP (SEQ ID NO: 18) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18). A GSG-T2A peptide may comprise an amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19) or a sequence having at least 70%, 80%, 90%, 95%, or 99%identity to the amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19). A GSG-T2A peptide may comprise a nucleic acid sequence comprising ggatctggagagggaggggaagcctgctgacctgtggagacgtggaggaaaacccaggacca (SEQ ID NO: 20). An E2A peptide may comprise an amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO: 21) or a sequence having at least 70%, 80%, %, 95%, or 99% identity to the amino acid sequence comprising QCTNYALLKLACDVESNPGP (SEQ ID NO: 21). ACSG-E2A peptide may comprise an amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22) or a sequence having at least 70%, 80%, 90%, 95%, or 99%identity to the amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22). An F2A peptide may comprise anamino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP(SEQ ID NO: 23) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identitv to the amino acid sequence cmprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23). A GSG F2A peptide may comprise an amino acid sequence comprising CSGVKQTLNFDLLKLADVESNPGP (SEQ ID NO: 24) or a sequence having at least %, 80%,90%,95%, or 99% identity to the amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24). A P2A peptide may comprise an ano acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO 25) or a sequencehiaving at least 70%,80%, 90%, 95%, or99% identity to the amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25). A GSG-P2A peptide may compriseanaminoacid sequence comprising GSGATNFSLLIKQAGDVEENPGP (SEQ ID NO: 26) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acidsequence comprising (ISGATNFSLLKQAGDVEENPGP (SEQ ID NO:26). 101361 Transposons of the disclosure may comprise a first and a second self-cleaving peptide,the first self-cleaving peptide located, forexample, upstreamofone orimoreof a sequence that specifically binds an antigen of the disclosure the second self-cleaving peptide located, for example, downstream of the one or more of a sequence that specifically binds an antigen of the disclosure. The first and/or the second self-cleaving peptide may comprise, for example, a T2A peptide, GSG-T2A peptide, an E2A peptide, a GSG-E2A peptide, an F2A peptide, a GSG-F2A peptide, a P2A peptide, or a GSG-P2A peptide. A T2A peptide may comprise an aminoacid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18)or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18). A GSG-T2A peptide may comprise an amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19) or a sequence having at least 70%, 80%, 90%, 95%. or 99% identity to the amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19). A GSG-T2A peptide may comprise a nucleic acid sequence comprising ggatctggagagggaaggggaagcctgctgacetgtggagacgtggaggaaaacccaggacca (SEQ ID NO: 20). An E2A peptide may comprise an amino acid sequence comprising QCTNYALLKLA GDVESNPGP (SEQ ID NO: 21) or a sequence having at least 70%, 80%, %, 95%, or 99% identity to the amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO: 21). A GSG-E2A peptide may comprise an aminoacid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22)or a sequence having at least 70%, 80%, 90%,95%, or 99% identity to the amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO:22). An F2A peptide may comprisean amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to theamino acid sequence comprising VKQTLNFDLLKLACDVESNPGP (SEQ ID NO: 023). A GSG F2A peptide may comprise an amino acid sequence comprising GSGVKQTNFDLLKLAGDVESNPGP (SEQ ID NO: 24) or a sequence having at least %, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24). A P2A peptide may comprise an amino acid sequence comprising A'TNFSLLKQAGDVEENPGP (SEQ ID NO: 25) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25). A GSG-P2A peptide may comprise an amino acid sequence comprising (IS(ATNFSLLKQAGDVEENPGP (SEQ ID NO: 26) or a sequence havingat least 70%, 80%,90%, 95%, or 99% identity to the amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 26).
[01371 The disclosure provides composition comprising the transposon the disclosure. In certain embodiments, a method introducing the composition may further comprise a composition comprising a plasmid comprising a sequence encoding a transposase enzyme. The sequence encoding a transposase enzyme may be an mRNA sequence. 101381 Transposons of the disclosure may comprise piggyBac transposons. Transposase enzvmes of the disclosure may include piggyBac transposases or compatible enzymes.
[01391 The disclosure provides a vector comprising the CAR of the disclosure. In certain embodiments, the vector is a viral vector. The vector may be a recombinant vector.
[01401 Viral vectors of the disclosure may comprise a sequence isolated or derived from a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus or any combination thereof The viral vector may comprise a sequence isolated or derived from an adeno-associated virus (AAV). The viral vector may comprise a recombinant AAV (rAAV). Exemplary adeno associated viruses and recombinant adeno-associated viruses of the disclosure comprise two or more inverted terminal repeat (ITR) sequences located in cis next to one or more of a sequence that specifically binds an antigen. Exemplary adeno-associated viruses and recombinant adeno-associated viruses of the disclosure include, but are not limited to all serotypes (e.g. AAV, AAV2. AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9).
Exemplary adeno-associated viruses and recombinant adeno-associated viruses of the disclosure include, but are not limited to, self-complementary AAV (scAAV) and AAV hybrids containing the genome of one serotype and the capsid of another serotype (e.g. AAV2/5, AAV-DJ and AAV-DJS). Exemplary adeno-associated viruses and recombinant adeno-associated viruses of the disclosure include, but are not limited to, rAAV-LK03.
[0141] Viral vectors of the disclosure may comprise a selection gene. The selection gene may encode a gene product essential for cell viability and survival. The selection gene may encode a gene product essential for cell viability and survival when challenged by selective cell culture conditions. Selective cell culture conditions may comprise a compound harmful to cell viability or survival and wherein the gene product confers resistance to the compound. Exemplary selection genes of the disclosure may include, but are not limited to, neo (conferring resistance to neomycin), DHFR (encoding Dihydrofolate Reductaseand conferring resistance to Methotrexate), TYMS (encoding Thymidylate Synthetase), MGMT( encoding O(6)-methylguanine-DNA methyltransferase), multidrug resistance gene (MDRi), ALDH1 (encoding Aldehyde dehydrogenase I family, member A1), FRANCF, RAD51C (encoding RAD51 Paralog C), GCS (encoding glucosylceramide synthase), NKX2.2 (encoding NK2 Homeobox 2) orany combination thereof.
[0142] Viral vectors of the disclosure may comprise at least one self-cleaving peptide. In some embodiments, the vector may comprise at least one self-cleaving peptide and wherein a self-cleaving peptide is located between a CAR and a selection gene. In some embodiments, the vector may comprise at least one self-cleaving peptide and wherein a first self-cleaving peptide is located upstream of a CAR and a second self-cleaving peptide is located downstream of a CAR.The self-cleaving peptide may comprise, for example, aT2A peptide, GSC-T2A peptide, an E2A peptide, a GSG-E2A peptide, an F2A peptide, a GSG-F2A peptide, a P2A peptide. or a GSG-P2A peptide. A T2A peptide may comprise an amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18). A GSG-T2A peptide may comprise an amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identityto the amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19). A GSG-T2A peptide may comprise a nucleic acid sequence comprising ggatctggagagggaaggggaagcctgctgacctgtggagacgtggaggaaaacccaggacca (SEQ ID NO: 20). An
E2A peptide may comprise an amino acid sequence comprising QCTNYALLKLA GDVESNPGP (SEQ ID NO: 21) or a sequence having at least 70%, 80%, %, 95%, or 99% identity to the amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO: 21). A GSG-E2A peptide may comprise an amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22). An F2A peptide may comprise an amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to tie amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23). A GSG F2A peptide may comprise an amino acid sequence comprising GSGNVKQTLNFDLLKLAGDVESNPGP(SEQ ID NO: 24) or a sequence having at least %, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24). A P2A peptide may comprise an amino acidsequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to theamino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25). A GSG-P2A peptide may comprisean amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 26) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 26).
101431 The disclosure provides a vector comprising the CAR of the disclosure. In certain embodiments, the vector is an mRNA vector. The vector may be a recombinant mRNA vector.'T cells ofthe disclosure may be expanded priorto contacting the T-cell andthe mRNA vector comprising the CAR of the disclosure. The T cell comprising the mRNA vector, the modified T cell, may then be administered to a subject.
[01441 The disclosure provides a vector comprising the CAR of the disclosure. In certain embodiments, the vector is a nanoparticle. Exemplary nanoparticle vectors of the disclosure include, but are not limited to,nucleic acids (e.g. RNA, DNA, synthetic nucleotides, modified nucleotides or any combination thereof ), amino acids (L-amino acids, D-amino acids, synthetic amino acids, modified amino acids, or anycombination thereof), polymers (e.g. polymersomes), micelles, lipids (e.g. liposomes), organic molecules (e.g. carbon atoms, sheets, fibers, tubes), inorganic molecules (e.g. calcium phosphate or gold) or any combination thereof A nanoparticle vector may be passively or actively transported across a cell membrane.
[01451 Nanoparticle vectors of the disclosure may comprise a selection gene. The selection gene may encode a gene product essential for cell viability and survival. The selection gene may encode a gene product essential for cell viability and survival when challenged by selective cell culture conditions. Selective cell culture conditions may comprise a compound harmful to cell viability or survival and wherein the gene product confers resistance to the compound. Exemplary selection genes of the disclosure may include, but are not limited to, neo conferringg resistance to neomycin), DHFR (encoding Dihydrofolate Reductase and conferring resistance to Methotrexate), TYMS (encoding Thymidylate Synthetase), MGMT( encoding O(6)-methvguanine-DNA methyltransferase), multidrug resistance gene (MDRi), ALDH I(encoding Aldehyde dehydrogenase I family, member A1). FRANCE RAD5IC (encoding RAD51 Paralog C), GCS (encoding glucosylceramide synthase), NKX2.2 (encoding NK2 Homeobox 2) or any combination thereof.
101461 Nanoparticle vectors of the disclosure may comprise at least one self-cleaving peptide. In some embodiments, the nanoparticle vector may comprise at least one self cleaving peptide and wherein a self-cleaving peptide is located between a CAR and the nanoparticle. In some embodiments, the nanoparticle vector may comprise at least one self cleaving peptide and wherein a first self-cleaving peptide is located upstream of a CAR and a second self-cleaving peptide is located downstream of a CAR. In son embodiments, the nanoparticle vector may comprise at least one self-cleaving peptide and wherein a first self cleaving peptide is located between a CARand the nanoparticle and a second self-cleaving peptide is located downstream ofthe CAR. In some embodimentsthe nanoparticle vector may comprise at least one self-cleaving peptide and wherein a first self-cleaving peptide is located between a CAR and the nanoparticle and a second self-cleaving peptide is located downstream of the CAR, for example, between the CAR and a selection gene. The self cleaving peptide may comprise, for example, a T2A peptide, GSG-T2A peptide, an E2A peptide, a GSG-E2A peptide, an F2A peptide, a GSG-F2A peptide, a P2A peptide, or a GSG P2A peptide. AT2A peptide may comprise an amino acid sequence comprising EGRGSLIITCGDVEENPGP (SEQ ID NO: 18) or a sequence having at least 70%, 80%, %, 95%, or 99%identity to the amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18). A GSG-T2A peptide may comprise an amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19) or a sequence having at least 70%, 80%, 90%, 95%,or 99% identity to the amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19). A GSG-T2A peptide may comprise a nucleic acid sequence comprising ggatctggagagggaaggggaagcctgctgacctgtggagacgtggaggaaaacccaggacca (SEQ ID NO: 20). An E2A peptide may comprise an amino acid sequence comprising QCTNYA LLKLAGDVESNPGP (SEQ ID NO: 21) or a sequence having at least 70%,80%, %, 95%, or 99% identity to the amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO: 21). A GSG-E2A peptide may comprise an aino acid sequence comprising GSCQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22). An F2A peptide may comprise an amino acid sequence comprising VKQTUNFDLLKLAGDVESNPGP (SEQ ID NO: 23) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising VKQTLNFDLLK.LAGDVESNPGP (SEQ ID NO: 23). A GSG F2A peptide may comprise an amino acid sequence comprising GSGVKQTNFDLLKLAGDVESNPGP (SEQ ID NO: 24) or a sequence having at least %, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24). A P2A peptide may comprise an amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25). A GSG-P2A peptide may comprise an aminoacid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 26) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 26).
[01471 The disclosure provides a composition comprising a vector of the disclosure. CARTyrins
[01481 The disclosure provides a chimericantigen receptor (CAR) comprising: (a) an ectodomain comprising an antigen recognition region, wherein the antigen recognition region comprises at least one Centyrin; (b) a transmembrane domain, and (c)an endodomain comprisingat least one costinulatory domain. As used throughout the disclosure, a CAR comprising a Centyrin is referred to as a CARTyrin. In certain embodiments, the antigen recognition region may comprise two Centyrins to produce a bi-specific or tandem CAR. In certain embodiments, the antigen recognition region may comprise three Centyrins to produce a tri-specific CAR. In certain embodiments, the ectodomain may further comprise a signal peptide. Alternatively, or in addition, in certain embodiments, the ectodomain may further comprise a hinge between the antigen recognition region and the transmembrane domain.
[01491 The disclosure provides a chimeric antigen receptor (CAR) comprising: (a) an ectodomain comprising an antigen recognition region, wherein the antigen recognition region comprises at least one protein scaffold or antibody mimetic; (b) a transmembrane domain, and (c) an endodomain comprising at least one costimulatory domain. In certain embodiments, the antigen recognition region may comprise two scaffold proteins or antibody mimetics to produce a bi-specific or tandem CAR. In certain embodiments, the antigen recognition region may comprise three protein scaffolds or antibody mimetics to produce a tr-specific CAR. In certain embodiments, the ectodomain may further comprise a signal peptide. Alternatively, or in addition, in certain embodiments, the ectodomain may further comprise a hinge between the antigen recognition region andthe transmembrane domain.
101501 In certain embodiments of the CARs of the disclosure, the signal peptide may comprise a sequence encoding a human CD2, CD36, CD3F, CD3., CD3(, CD4, CD8a, CD19, CD28, 4-IBB 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. The human CD8a signal peptide may comprise an amino acid sequence comprising MALPVTALLLPLALLLHAARP (SEQ ID NO: 8). The human CD8a signal peptide may comprise an amino acid sequence comprising MALPVTALLLPLALLLHAARP (SEQ ID NO: 8) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the an amino acid sequence comprising MALPVTALLLPLALLLHAARP (SEQ ID NO: 8).The human CD8a signal peptide may be encoded by a nucleic acid sequence comprising atggcactgccagtcaccgccctgctgctgcctctggctctgtgtgeacgcagctagacca (SEQ ID NO: 9).
[01511 In certain embodiments of the CARs of the disclosure, the transmembrane domain may comprise a sequence encoding a human CD2 CD36, CD3, CD3, CD3(, 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 CD8. transmembrane domain.The CD8a transmembrane domain may comprise an amino acid sequence comprising IYIWAPLAGTCGVLISLVITLYC (SEQ ID NO: 10) or a sequencehaving at least 70%,80%, 90%, 95%, or99% identityto the amino acid sequence comprising IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 10). The CD8a.
transmembrane domain may be encoded by the nucleic acid sequence comprising atctacatttgggaccactggccgggacctgtggagtgetgctgctgagcctggtcatcacactgtactgc (SEQ ID NO: 11).
[01521 In certain embodiments of the CARs of the disclosure, the endodomain may comprise a human CD3( endodomain.
[01531 In certain embodiments of the CARs ofthe disclosure, the at least one costimulatory domain may comprise a human 4-IBB, CD28, CD40, ICOS, MyD88, OX-40 intracellular segment, orany combination thereof. In certain embodimentsof the CARsof the disclosure, the at least one costimulatory domain may comprise a CD28 and/or a 4-BB costimulatory domain. The CD28 costimulatory domain may comprise an amino acid sequence comprising RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP PR (SEQ ID NO: 12) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising R/KFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP PR (SEQ ID NO: 12). The CD28 costimulatory domain may be encoded by the nucleic acid sequence comprising cgcgtgaagtttagtcgatcagcagatgccccagcttacaaacagggacagaaccagctgtataacgagctgaatctgggccgccga gaggaatatgacgtgctggatagcggagggacggaccgaaatgggaggcaagcccagggaaaaacctcaggaagg cctgtataacgagctgcagaaggacaaaatggcagaagcetattctgagatcggcatgaaggggagcgacggagaggcaaagg gcacgatgggetgtaccagggactgageaccgecacaaaggacacctatgatgctctgcatatgcaggcactgcctecaagg (SEQ ID NO: 13). The 4-IBB costimulatory domain may comprise an amino acid sequence comprising KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 14) or a sequence having at least 70%, 80%. 90%, 95%, or 99% identity to the amino acid sequence comparing KRCRKKLLYIFKQPFMRPVQTTQEEDCCSCRFPEEEEGGCEL (SEQ ID NO: 14). The 4 IBB costimulatory domain may be encoded by the nucleic acid sequence comprising aagagaggcaggaagaaactgctgtatattttcaaacagcccttcatgcgccccgtgcagactaccaggaggaagacgggtgctcc tgtcgattccctgaggaagaggaaggcgggtgtgagctg (SEQ ID NO: 15). The 4-1BB costimulatory domain may be located between the transmembrane domain andthe CD28 costimulatory domain.
[01541 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 CDSa sequence. The hinge may comprise a human CD8a amino acid sequence comprising TTTPAPRPPTPAPTIASQPLSLRPEACRPAA GGAVHTRGLDFACD (SEQ ID NO: 16) or a sequence having at least 70%, 80%, 90% 95%, or 99% identity to the amino acid sequence comprising TTTPAPRPPTPAPTIASQPLSLRPEACRPAAG(GVHTRGLDFACD (SEQ ID NO: 16).'The human CD8a hinge aminoacid sequence may be encoded by the nucleic acid sequence comprising actaccacaccagcacetagaccaccaaetecagetccaaccategegagtcagccctgagtetggacetgaggectgeaggcc agctgcaggaggagetgtgcacaccaggggctggacttcgcctgcgac (SEQ ID NO: 17).
[01551 Centyrins ofthe disclosure may comprise aprotein scaffold, wherein the scaffold is capable of specifically bindinganantigen. 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 TGLIKPGTEYTVSIYGVKGGHRSNPLSAEFTT (SEQ ID NO: 1) or MLPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYD LTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT (SEQ ID NO: 2). The consensus sequence may encoded by a nucleic acid sequence comprising atgctgctgcaccaaagaacctggtggtgtctcatgtgacagaggatagtgccagactgtcatggatgctcccgacgcagcettcg atagtttatcatcgtgtacegggagaacatcgaaaccggegaggccattgtcctgacagtgccagggtccgaacgctcttatgactg acagatctgaagcccggaactgagtactatgtgcagatcgccggcgtcaaaggaggcaatatcagcttccctctgccgcaatttcac caca (SEQ ID NO: 3). 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 at positions 13-16 of the consensus sequence; (b) a B-C loop comprising or consisting of the amino acid residues TAPDAAF at positions 22-28 of the consensus sequence; (c) a C-D loop comprising or consisting of the amino acid residues SEKVGE at positions 38-43 of the consensus sequence; (d)aD-E loop comprising or consisting of the amino acid residues GSER at positions 51-54 of the consensus sequence; (e) a E-F loop comprising or consisting of the amino acid residues GLKPG at positions 60-64 of the consensus sequence; (f) a F-G loop comprising or consisting of the amino acid residues KGGHRSN 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 111 (FN3) domains or at least 15 fibronectin type II (FN3) domains.The scaffold may bind an antigen with at least one affinity selected from a K of less than or equal to 10-'M, less than or equal to I 10 -M, less than or equal to 10- 1 M. less than or equal to 10-IM. less than or equal to 10-"IM, less than or equal to 10-IM, and less than or equal to --M. The KD may be determined by surface plasmon resonance.
[01561 The disclosure provides acomposition comprising the CAR of the disclosure and at least one pharmaceutically acceptable carrier.
[01571 The disclosure provides a transposon comprising the CAR of the disclosure. Transposons ofthe disclosure be episomally maintained or integrated into the genome ofthe recombinant/modified cell. The transposon may be part of atwo component piggyBac system that utilizes a transposon and transposase for enhanced non-viral gene transfer.
101581 Transposons of the disclosure may comprise a selection gene for identification, enrichmentand/or isolationof cells that express the transposon. Exemplary selection genes encode any gene product (e.g. transcript, protein, enzyme) essential for cell viability and survival. Exemplary selection genes encode any gene product (e.g. transcript, protein, enzyme) essential for conferring resistance to a drug challenge against which the cell is sensitive (or which could be lethal to the cell) in the absence of the gene product encoded by the selection gene. Exemplary selection genes encode any gene product (e.g. transcript, protein, enzyme) essential for viability and/or survival in a cell media lacking one or more nutrients essential for cell viability and/or survival in the absence of the selection gene. Exemplary selection genes include, but are not limited to, neo (conferring resistance to neomycin), DHFR (encoding Dihydrofolate Reductase and conferring resistance to Methotrexate),TYMS (encoding Thymidylate Synthetase), MGMT(encoding0 (6) methylguanie-DNA methyltransferase), multidrug resistance gene (MDRI), ALDHI (encoding Aldehyde dehydrogenase I family, member A1), FRANCF, RAD5IC (encoding RAD51 Paralog C), GCS (encoding glucosvlceramide synthase), and NKX2.2 (encoding NK2 Homeobox 2).
[01591 Transposons ofthe disclosure may comprise at least one self-cleaving peptides) located, for example, between on or more of a protein scaffold, Centyrin or CARTyrin of the disclosure and a selection gene ofthe disclosure. The at least one self-cleaving peptide may comprise, for example, aT2A peptide, GSG-T2A peptide, an E2A peptide, a GSG-E2A peptide, an F2A peptide, a GSG-F2A peptide, a P2A peptide, or a GSG-P2A peptide. A T2A peptide may comprise an amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18) or a sequence having at least 70%, 80%, 90%, 95%, or 99%)identity to the amino acid sequence comprising EGRGSLI'CGDVEENPGP (SEQ ID NO: 18). A GSG T2A peptide may comprise anamino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19) or a sequence having at least 70%, 80%, %, 95%, or 99% identity to the amino acid sequence comprising GSGEGRGSLTCGDVEE'NPGP (SEQ ID NO: 19). A GSG-T2A peptide may comprise a nucleic acid sequence comprising ggatctggagagggaaggggaagcctgctgacctgtggagacgtggaggaaaacccaggacca (SEQ ID NO: 20). An E2A peptide may comprise an amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO: 21) or a sequence having at least 70%,80%, %, 95%, or 99% identity to the amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO: 21). A GSG-E2A peptide may comprise an amino acid sequence comprising GSGQC'TNYALLKLAGDVESNPGP (SEQ ID NO: 22)or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22). An F2A peptide may compriseanaminoacid sequence comprising VKQTUNFDLLKLAGDVESNPGP (SEQ NO: 23) or a sequence havingat least70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23). A GSG-F2A peptide may comprise an amino acid sequence comprising GSGVKQTNFDLLKLAGDVESNPGP (SEQ ID NO: 24) or a sequence having at least %, 80% 90%, 95%, or 99% identity to the amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24). A P2A peptide may comprise an amino acid sequence comprising A'TNFSLLKQAGDVEENPGP (SEQ ID NO: 25) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25). A GSG-P2A peptide may comprise an aminoacid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 26) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 26).
[01601 Transposons of the disclosure may comprise a first and a second self-cleaving peptide, the first self-cleaving peptide located, for example, upstream of one or more of a protein scaffold, Centyrinor CARTyrin of the disclosure the second self-cleaving peptide located, for example, downstreamof the one or more of a protein scaffold, Centyrin or CARTyrin of the disclosure. The first and/or the second self-cleaving peptide may comprise, for example, aT2A peptide, GSG-T2A peptide, an E2A peptide, a GSG-E2A peptide, an F2A peptide, a GSG-F2A peptide, a P2A peptide, or a GSG-P2A peptide. AT2A peptide may comprise anamino acid sequence comprising EGRSLLTCGDVEENPGP (SEQ ID NO: 18) or a sequence having at least 70%, 80%, 90%, 95%, or 99%identity to the amino acid sequence comprising EGRGSLITCGDVEENPGP (SEQ ID NO: 18). A GSG-T2A peptide may comprise an amino acid sequence comprising GSGEGRGSLLTCGDVEENPCP (SEQ ID NO: 19) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19). A GSG-T2A peptide may comprise a nucleic acid sequence comprising ggtctggagagggaaggggaagcctgctgacctgtggagacgtggaggaaaacccaggacca (SEQ ID NO: 20). An E2A peptide may comprise an amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO: 21) or a sequence having at least 70%, 80%, %, 95%, or 99% identity to the amino acid sequence comprising QCTNYALLKLACDVESNPGP (SEQ ID NO:21). A GSG-E2A peptide may comprise an amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22) or a sequence having at least 70%, 80%, 90%, 95%, or 99%identity to the amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22).An F2A peptide may comprise an amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23) or a sequence having at least 70%, 80%, 90%, 95%, or 99%identity to the amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23). A GSG F2A peptide may comprise an amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24) or a sequence having at least %, 80%, 90%, 95%, or 99%)identity to the amino acid sequence comprising CSGVKQTLNFDLLKLADVESNPGP (SEQ ID NO: 24). A P2A peptide may comprise an amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25). A GSG-P2A peptide may compriseanaminoacid sequence comprisingGSGATNFSLLKQAGDVEENPGP (SEQ ID
NO: 26) or a sequence having at least 70%, 80%. 90%, 95%, or 99% identity to the amino acid sequence cmprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 26).
[01611 The disclosure provides a composition comprising the transposon the disclosure. In certain embodiments, a method introducing the composition may further comprise a composition comprising a plasmid comprising a sequence encoding a transposase enzyme. The sequence encoding a transposase enzyme may be an mRNA sequence.
[01621 Transposons of the disclosure may comprise piggyBac transposons. Transposase enzymes of the disclosure may include piggyBac transposases or compatible enzymes. 101631 The disclosure provides a vector comprising the CAR of the disclosure. In certain embodiments, the vector is a viral vector. The vector may be a recombinant vector.
[01641 Viral vectors of the disclosure may comprise a sequence isolated or derived from a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus or any combination thereof. The viral vector may comprise a sequence isolated or derived from an adeno-associated virus (AAV). The viral vector may comprise a recombinant AAV (rAAV). Exemplary adeno associated viruses and recombinant adeno-associated viruses of the disclosure comprise two or more inverted terminal repeat (ITR) sequences located in cis next to a sequence encoding a protein scaffold, Centyrin or CARTyrin of the disclosure. Exemplary adeno-associated viruses and recombinant adeno-associated viruses of the disclosure include, but are not limited to all serotypes (e.g. AAVI, AAV2 AAV3 AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9). Exemplary adeno-associated viruses and recombinant adeno-associated viruses of the disclosure include, but are not limited to, self-complementary AAV (scAAV) and AAV hybrids containing the genome of one serotype and the capsid of another serotype (e.g. AAV2/5, AAV-DJ and AAV-DJ8). Exemplary adeno-associated viruses and recombinant adeno-associated viruses of the disclosure include, but are not limited to, rAAV-LK03.
[01651 Viral vectors of the disclosure may comprise a selection gene. The selection gene may encode a gene product essential for cell viability and survival. The selection gene may encode a gene product essential for cell viability and survival when challenged by selective cell culture conditions. Selective cell culture conditions may comprise a compound harmful to cell viability or survival and wherein the gene product confers resistance to the compound. Exemplary selection genes of the disclosure may include, but are not limited to, neo (conferring resistance to neomycin), DHFR (encoding Dihvdrofolate Reductase and conferring resistance to Methotrexate),"TYMS (encodingThyidylate Synthetase), MGMT( encoding O(6)-nethylguanine-DNA methyltransferase), multidrug resistance gene (MDR-F), ALDHI1 (encoding Aldehyde dehydrogenase I family, member A1), FRANCF, RAD5IC
- 111l-
(encoding RAD51 Paralog C), GCS (encoding glucosylceramide synthase), NKX2.2 (encoding NK2 Homeobox 2) orany combination thereof.
[01661 Viral vectors of the disclosure may comprise at least one self-cleaving peptide. In some embodiments, the vector may comprise at least one self-cleaving peptide and wherein a self-cleaving peptide is located between a CAR and a selection gene. In some embodiments, the vector may comprise at least one self-cleaving peptide and wherein a first self-cleaving peptide is located upstream of a CAR and a second self-cleaving peptide is located downstream of a CAR. The self-cleaving peptide may comprise, for example, aT2A peptide, GSC-T2A peptide, an E2A peptide, a GSG-E2A peptide, an F2A peptide, a GSG-F2A peptide, a P2A peptide. or a GSG-P2A peptide. A T2A peptide may comprise an amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18). A GSG-T2A peptide may comprise an amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP(SEQ ID NO: 19) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP(SEQ ID NO: 19). A GSG-T2A peptide may comprise a nucleic acid sequence comprising ggatctggagagggaaggggaagcctgctgacctgtggagacgtggaggaaaacccaggacca (SEQ ID NO: 20). An E2A peptide may comprise an amino acid sequence comprising QCTNYA LLKLAGDVESNPGP (SEQ ID NO: 21) or a sequence having at least 70%,80%, %, 95%, or 99% identity to the amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO: 21). A GSG-E2A peptide may comprise an amino acid sequence comprising GSGQC'TNYALLKLAGDVESNPGP (SEQ ID NO: 22)or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO:22). An F2A peptide may comprisean amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising VKQTLNFDLLIKLAGDVESNPGP (SEQ ID NO: 23). A GSG F2A peptide may comprise an amino acid sequence comprising GSGVKQTNFDLLKLAGDVESNPGP (SEQ ID NO: 24) or a sequence having at least %, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24). A P2A peptide may comprise an amino acid sequence comprising A'TNFSLLKQAGDVEENPGP (SEQ ID NO: 25) or a sequence having at least 70%, 80%, 90%, 95%,or 99% identity to the amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25). A GSG-P2A peptide may comprise an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 26) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGATNFSLLIKQAGDVEENPGP (SEQ ID NO: 26).
[01671 The disclosure provides avector comprising the CAR of the disclosure. In certain embodiments, the vector is an mRNA vector. The vector may be a recombinant mRNA vector.'T cells of the disclosure may be expanded priorto contacting theT-cell and the mRNA vector comprising the CAR of the disclosure. The T cell comprising the mRNA vector, the modified T cell, may then be administered to a subject.
[01681 The disclosure provides a vector comprising the CAR of the disclosure. In certain embodiments, the vector is a nanoparticle. Exemplary nanoparticle vectors of the disclosure include, butare not limited to, nucleic acids (e.g. RNA, DNA, synthetic nucleotides, modified nucleotides or any combination thereof ), amino acids (L-amino acids, D-amino acids, synthetic amino acids, modified amino acids. or any combination thereof), polymers (e.g. polymersomes), micelles, lipids (e.g. liposomes), organic molecules (e.g. carbon atoms, sheets, fibers, tubes), inorganic molecules (e.g. calcium phosphate or gold) or any combination thereof A nanoparticle vector may be passively or actively transported across a cell membrane.
[01691 Nanoparticle vectors of the disclosure may comprise a selection gene. The selection gene may encode a gene product essential for cell viability and survival. The selection gene may encode a gene product essential for cell viability and survival when challenged by selective cell culture conditions. Selective cell culture conditions may comprise a compound harmful to cell viability or survival and wherein the gene product confers resistance to the compound. Exemplary selection genes of the disclosure may include, but are not limited to, neo (conferring resistance to neomycin), DHFR (encoding Dihydrofolate Reductase and conferring resistance to Methotrexate), TYMS (encoding Thymidylate Synthetase), MGMT (encoding O(6)-methylguanine-DNA methyltransferase), multidrug resistance gene (MDRi), ALDH1 (encoding Aldehyde dehydrogenase I family, member A1), FRANCF, RAD51C (encoding RAD51 Paralog C), GCS (encoding glucosylceramide synthase), NKX2.2 (encoding NK2 Hlomeobox 2) orany combination thereof.
[01701 Nanoparticle vectors of the disclosure may comprise at least oneself-cleaving peptide. In some embodiments, the nanoparticle vector may comprise at least one self cleaving peptide and wherein a self-cleaving peptide is located between a CAR and the nanoparticle. In some embodiments, the nanoparticle vector may comprise at least one self cleaving peptide and wherein a first self-cleaving peptide is located upstream of a CAR and a second self-cleaving peptide is located downstream of a CAR. In some embodiments, the nanoparticle vector may comprise at least one self-cleaving peptide and wherein a first self cleaving peptide is located between a CAR and the nanoparticle and a second self-cleaving peptide is located downstream of the CAR. In some embodiments, the nanoparticle vector may comprise at least one self-cleaving peptide and wherein a first self-cleaving peptide is located between a CAR and the nanoparticle and a second self-cleaving peptide is located downstream of the CAR, for example, between the CAR and a selection gene. The self cleaving peptide may comprise, for example, aT2A peptide, GSG-12A peptide., an E2A peptide, a GSG-E2A peptide, an F2A peptide, a GSG-F2A peptide, a P2A peptide, or a GSG P2A peptide. A T2A peptide may comprise an amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18) or a sequence having at least 70%, 80%, %, 95%, or 99% identity to the amino acid sequence comprising EGRGSLIITCGDVEENPGP (SEQ ID NO: 18). A GSG-T2A peptide may comprise an aminoacid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19) or a sequence having at least 70%, 80%, 90%, 95%, or 99%identity to the amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19). A GSG-T2A peptide may comprise a nucleic acid sequence comprising ggatctggagagggaaggggaagcetgtgactgtggagacgtggaggaaaacecaggacca (SEQ ID NO: 20). An E2A peptide may comprise an amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO: 21) or a sequence havingat least 70%, 80%, %, 95%, or 99% identity to the amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO: 21). A GSG-E2A peptide may comprise an amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22). An F2A peptide may comprise an amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23). A GSG F2A peptide may comprise an amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24) or a sequence having at least
%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24). A P2A peptide may comprisean amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25) or a sequence havingat least 70%, 80%, 90%, 95%, or 99%identity to the amino acid sequence comprising AINFSLLKQAGDVEENPGP (SEQ ID NO: 25). A GSG-P2A peptide may comprise an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 26) or a sequence having at least 70%, 80%, 90%, 95%, or 99%identity to the amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 26).
[01711 The disclosure provides a composition comprisinga vector of the disclosure.
ScaffoldProteins
[01721 A Centyrin is one example of a protein scaffold of the disclosure. An antigen recognition region of a CAR of the disclosure may comprise at least one protein scaffold.
[01731 Protein scaffolds ofthe disclosure maybe derivedfrom afibronectin type III (FN3) repeat protein, encoding or complementary nucleic acids, vectors, host cells, compositions, combinations, formulations, devices, and methods of making and using them. In apreferred embodiment, the protein scaffold is comprised of a consensus sequence of multiple FN3 domains from human Tenascin-C (hereinafter "Tenascin"). In a further preferred embodiment, the protein scaffold of the present invention is a consensus sequence of 15 FN3 domains. The protein scaffolds of the disclosure can be designed to bind various molecules, for example, a cellular target protein. In a preferred embodiment, the protein scaffolds of the disclosure can be designed to bind an epitope of a wild type and/or variant form of an antigen.
[01741 Protein scaffoldsof the disclosure may include additional molecules or moieties., for example, the Fe region of an antibody, albumin binding domain, or othermoiety influencing half-life. In further embodiments, the protein scaffolds of the disclosure may be bound to a nucleic acid molecule that may encode the protein scaffold.
[01751 The disclosure provides at least one method for expressing at least one protein scaffold based on a consensus sequence of multiple FN3 domains, in a host cell, comprising culturing a host cell as described herein under conditions wherein at least one protein scaffold is expressed in detectable and/or recoverable amounts.
[01761 The disclosure provides at least one composition comprising (a) aprotein scaffold based on a consensus sequence of multiple FN3 domains and/or encoding nucleic acid as described herein; and (b) a suitable and/or pharmaceutically acceptable carrier or diluent.
[01771 The disclosure provides method of generating libraries of aprotein scaffold based on a fibronectin type III (FN3) repeat protein, preferably, a consensus sequence of multiple FN3 domains and, more preferably, a consensus sequence of multiple FN3 domains from human Tenascin. The library is formed by making successive generations of scaffolds by altering (by mutation) the amino acids or the number ofamino acids in themolecules in particular positions in portions of the scaffold, eg., loop regions. Libraries can be generated by altering the amino acid composition of a single loop or the simultaneous alteration of multiple loops or additional positions of the scaffold molecule. The loops that are altered can be lengthened or shortened accordingly. Such libraries can be generated to include all possible amino acids at each position, or a designed subset of amino acids. The library members can be used for screening by display, such as in vitro or CIS display (DNA, RNA, ribosome display, etc.). yeast, bacterial, and phage display.
[01781 Protein scaffolds ofthe disclosure provide enhanced biophysical properties, such as stability under reducing conditions and solubility at high concentrations; they may be expressed and folded in prokarvotic systems, such as K col, ineukaryotic systems. such as yeast, and in in vitro transcription/translation systems, such as the rabbit reticulocyte lysate system.
[01791 The disclosure provides an isolated, recombinantand/orsynthetic protein scaffold based on a consensus sequence of fibronectin type III (FN3) repeat protein, including, without limitation, mammalian-derived scaffold, as well as compositions and encoding nucleic acid molecules comprising at least one polynucleotide encoding protein scaffold based on the consensus FN3 sequence. The disclosure further includes, but is not limited to, methods of making and using such nucleic acids and protein scaffolds, including diagnostic and therapeutic compositions, methods and devices.
[01801 The protein scaffolds of the disclosure offer advantages over conventional therapeutics, such as ability to administer locally, orally, or cross the blood-brain barrier, ability to express in E Coli allowing for increased expression of protein as a fiction of resources versus mammalian cell expression ability to be engineered into bispecific or tandem molecules that bind to multiple targets or multiple epitopes of the same target, ability to be conjugated to drugs, polymers, and probes, ability to be formulated to high concentrations, and the ability of such molecules to effectively penetrate diseased tissues and tumors.
[01811 Moreover, the protein scaffolds possess many ofthe properties of antibodies in relation to their fold that mimics the variable region ofan antibody. This orientation enables the FN3 loops to be exposed similar to antibody complementarity determining regions (CDRs). They should be able to bind to cellular targets and the loops can be altered,e.g., affinity matured, to improve certain binding or related properties.
[01821 Three of the six loops of the protein scaffold of the disclosure correspond topologically to the complementarity determining regions (CDRs 1-3) i.e., antigen-binding regions, of an antibody, while the remaining three loops are surface exposed in amanner similarto antibody CDRs. These loops span at orabout residues 13-16, 22-28, 38-43, 51-54, -64, and 75-81 of SEQ ID NO: 1. Preferably, the loop regions at or about residues 22-28, 51-54, and 75-81 are altered for binding specificity and affinity. One or more of these loop regions are randomized with other loop regions and/orother strands maintaining their sequence as backbone portions to populate a library and potent binders can be selected from the library having high affinity for a particular protein target. One or more of the loop regions can interact with a target protein similar to an antibody CDR interaction with the protein.
[01831 Scaffolds of the disclosure may comprise a single chain antibody (e.g. a scFv). Single chain antibodies of the disclosure may comprise three light chain and three heavy chain CDRs of an antibody. In certain embodiments, the single chain antibodies of the disclosure comprise three light chain and three heavy chain CDRs of an antibody, wherein the complementarit-determining regions (CDRs) of the single chainantibody are human sequences. The disclosure provides a chimeric antigen receptor (CAR) comprising: (a) an ectodomain comprising an antigen recognition region, wherein the antigen recognition region comprises at least one single chain antibody (e.g. a scFv); (b) atransmembrane domain, and (c) an endodomain comprising at least one costimulatory domain. In certain embodiments, the antigen recognition region may comprise two single chain antibodies (e.g. two scFvs) to produce a bi-specific or tandem CAR. In certain embodiments, the antigen recognition region may comprise three single chain antibodies (e.g. three scFvs) to produce a tn-specific CAR. In certain embodiments, the ectodomain may further comprise a signal peptide. Altematively, or in addition, in certain embodiments, the ectodomain may further comprise a hinge between the antigen recognition region and the transmembrane domain.
[01841 Scaffolds of the disclosure may comprise a sequence comprising one or more fragments of an antibody (e.g. a VHI-). Sequence comprising one or more fragments of an antibody of the disclosure may comprise two heavy chain variable regions of an antibody. In certain embodiments, the sequence comprises two heavy chain variable regions of an antibody, wherein the complementarity-determining regions (CDRs) of the VHH are human sequences. Scaffolds of the disclosure may comprise a sequence comprising one or more fragments of an antibody (e.g. a VHH). The disclosure provides a chimeric antigen receptor (CAR) comprising: (a) an ectodonain comprising an antigen recognition region, wherein the antigen recognition region comprises at least one a sequence comprising one or more fragments of an antibody (e.g. a V-H); (b) a transmembrane domain, and (c) an endodomain comprising at least one costimulatory domain. Incertain embodiments, theantigen recognition region may comprise two sequences comprising one or more fragments of an antibody (e.g. two VHHs) to produce a bi-specific or tandem CAR. In certain embodiments, the antigen recognition region may comprise three sequences comprising one or more fragments of an antibody (e.g. three VHHs) to produce a tri-specific CAR. In certain embodiments, the ectodomain may further comprise a signal peptide. Alternatively, or in addition, in certain embodiments, the ectodomain may further comprise a hinge between the antigen recognition region and the transmembrane domain.
[01851 Scaffolds of the disclosure may comprise an antibody mimetic.
[01861 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. resistance to enzymatic degradation), and lower production costs. Exemplary antibody miinetics 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.
[01871 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 affibodv molecule of the disclosure may comprise an immunoglobulin binding domain. An affibody molecule of the disclosure may comprise the Z domain of protein A.
[01881 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 aproperly-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.
[01891 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 specific target sequence. Exemplary affimer molecules of the disclosure comprise a protein scaffold based upon a cystatin protein or tertiary structure thereof. Exemplary affimer molecules of the disclosure may share a common tertiary structure of comprising an alpha helix lying on top of an anti-parallel beta-sheet.
[01901 Affitinmoleculesofthedisclosurecompriseanartificialprotein scaffoldthe 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. Exemplay 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.
101911 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 (includingantigens). 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 ofthe 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 ofthe molecule, alphabody molecules of the disclosuremaintain correct folding and thermostability,
[01921 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 eightantiparallel beta-strands pairwise connected by loops and an attached alpha helix.
[01931 Avimer molecules of the disclosure comprise an artificial protein that specifically binds to a target sequence (which may also be an antigen). Avimers of the disclosure may recognize multiple binding sites within the same target or within distinct targets. When an avimer of the disclosure recognize more than one target, the avimer mimics function of a bi specific antibody. The artificial protein avimer may comprise two or more peptide sequences of approximately 30-35 amino acids each. These peptides may be connected via one or more linker peptides. Amino acid sequences of one or more of the peptides of the avimer may be derived from an A domain of a membrane receptor. Avimers have a rigid structure that may optionally comprise disulfide bonds and/or calcium. Avimers of the disclosure may demonstrate greater heat stability compared to an antibody. 101941 DARPins (Designed Ankyrin Repeat Proteins) of the disclosure comprise genetically-engineered, recombinant, or chimeric proteins having high specificity and high affinityfor a target sequence. In certain embodiments, DARPins of the disclosure are derived from ankyrin proteins and, optionally, comprise at leastthree 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. 101951 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 specificityas an antibody.
[01961 Kunitz domain peptides of the disclosure comprise aprotein 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 ofthe disclosure may comprise Ecallantide (derived from a human lipoprotein associated coagulation inhibitor (LACI)).
[01971 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. Productionand Generationof Scafold Proteins
[01981 At least one scaffold protein of the disclosure can be optionally produced by a cell line, a mixed cell line, an immortalized cell or clonal population of immortalized cells, as well known in the art. See, e.g., Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y. (1987-2001); Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor, N.Y. (1989); Harlow and Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor, N.Y. (1989); Colligan, etal., eds., Current Protocols in Immunology, John Wiley & Sons, Inc., NY (1994-2001); Colligan et al., Current Protocols in Protein Science, John Wiley & Sons, NY N.Y., (1997-2001).
[01991 Amino acids from a scaffold protein can be altered, added and/or deleted to reduce immunogenicity or reduce, enhance or modify binding, affinity, on-rate, off-rate, avidity, specificity, half-life, stability, solubility or any other suitable characteristic, as known in the art.
[02001 Optionally, scaffold proteins can be engineered with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, the scaffold proteins can be optionally prepared by a process of analysis of the parental sequences and various conceptual engineered products using three-dimensional models of the parental and engineered sequences. Three-dimensional models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate sequences and can measure possible immunogenicity (e.g.Immunofilter program of Xencor, Inc. of Monrovia, Calif.). Inspection of these displays permits analysis ofthe likely role ofthe residues in the functioning of the candidate sequence, i.e., the analysis of residues that influence the ability ofthe candidate scaffold protein to bind its antigen. In this way, residues can be selected and combined from the parent and reference sequences so that the desired characteristic, such as affinity for the target antigen(s), is achieved. Alternatively, or in addition to, the above procedures, other suitable methods of engineering can be used. piggyBac TransposonSystem
[0201] The methods of the disclosure produce a modified TSCM of the disclosure regardless of the method used for introducing an antigen receptor into a primary human T cell of the disclosure. The methods ofthe disclosure produce a modified TCM of the disclosure with greater efficacy and/oragreater abundance, proportion, yield of modified -SCM ofthe
disclosure when the antigen receptor or the therapeutic protein of the disclosure is introduced to the primary human T cell using the piggyBac transposon system. A piggyBac transposon system of the disclosure maycomprise a piggyBac transposon comprising an antigen receptor of the disclosure. Preferably, the primary human T cell contacts a piggyBac transposon comprising an antigen receptor of the disclosure and a transposase of the disclosure simultaneously (or in very close temporal proximity, e.g. the primary human T cell, the transposon and the transposase are contained in the same container (such as a cuvette) prior to introduction of the transposon and transposase into the cell - however they would not be permitted to interact in the absence of the cell. Preferably, the primary human T cell contacts a piggyBac transposon comprising an antigen receptor of the disclosure and a Super TM piggyBac (SPB) transposase of the disclosure simultaneously prior to introduction of the transposon and transposase into the cell. In certain preferred embodiments, the Super piggyBac" (SPB) transposase is an m-NA sequence encoding the Super piggyBacT M (SPB) transposase.
[02021 Additional disclosure regarding piggyBac transposons and Super piggyBacTM (SPB) transposases may be found in International Patent Publication WO 2010/099296, US Patent No. 8,399,643, US Patent No. 9,546,382, US Patent No. 6,218,185, US Patent No. 6,551,825,
US Patent No. 6,962,810, and US PatentNo. 7,105,343, the contents of which are each herein incorporated by reference in their entireties.
[02031 The disclosure provides methods of introducing a polynucleotide construct comprising a DNA sequence into a host cell. Preferably, the introducing steps are mediated by the piggyBac transposon system.
[02041 In certain embodiments of the methods ofthe disclosure, the transposon is aplasmid DNA transposon with a sequence encoding the antigen receptor or the therapeutic protein flanked by two cis-regulatory insulator elements. In certain embodiments, the transposon is a piggyBac transposon. In certain embodiments, and, in particular, those embodiments wherein the transposon is a piggyBac transposon, the transposase is a piggyBacTM or a Super piggyBacTM(SPB) transposase. In certain embodiments, and, in particular, those embodiments wherein the transposase is a Super piggyBacTM (SPB) transposase, the sequence encoding the transposase is an mRNA sequence.
102051 In certain embodiments of the methods of the disclosure, the transposase enzyme is a piggyBac'M (PB)transposase enzyme. The piggyBac (PB) transposase enzyme may comprise or consist of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or any percentage inbetween identical to. MGSSLDDEHI LSALLQSDDE LVGEDSDSEI SDHVSEDDVQ SDTEEAFIDE VHEVQPTSScG 61 SEILDEQNVI EQPGSSLASN RILTLPQRTI RGKNKHCWST SKSTRRSRVS ALNI VRSQRG 121 PTPM CRNTYD PLLC'KLFFT DEIISEIVKW TNAEISLKRR E SMTGATFRD TNEDEIYA'F 181 GILVI'TAVRK DNHMSTDDLF DRS LSMV VS VMSPDRFDFL IRCLRMfDDKS IRPTLRENDV 41 FTPVRKIWDL FIHQCIQNYT PGAHLTIDEQ LLGFRGRCPF RM YIPNKPSK YGTKILMMCD 301 SGYKYMINGM PYLGRGTQTN GVPLGEYYVK ELSKPVHGSC RNITCDNFT SIPLAKNLLQ 361 EPYKLTIVGT VRSNKREIPE VLKNSRSRPV GTSMFCFDGP LTLVSYKPKP AKMVYLLSSC 421 DEDASINEST GKPQMVvMYYN QTKGGVDTLD QMCSVMTCSR KTNRWPMALL YGMINTACIN 481 SFIIYSHNVS SKGEKVQSRK KFMRNLYMSL TSSFMRKRLE AETLKRYLRD NISNILPNEV 541 PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV ICREHNIDMC QSCF (SEQ ID NO: 4>.
[02061 In certain embodiments of the methods of the disclosure, the transposase enzyme is a piggyBacTM (PB) transposase enzyme that comprises or consists of an amino acid sequence having an amino acid substitutionat one or more of positions 30, 165, 282, or 538 of the sequence: 1 MGSSLDDEHI LSALLQSDDE LVGEDSDSEI SDHVSEDDVQ SDTEEAFIDE VHEVQPTSSG 61 SEILDEQNVI EQPGSSLASN RILTLPQRTI RGKNKHCWST SKSTRRSRVS ALNIVRSQPG 121 PTRICRNTYD PLLCFKLFFT DEIISEIVKW TNAEISLKRR ESMTGATFRD TNEDEIYAFF
181 GITLVMTAVRK DNHMSTDDLF DRSLSMVVS VMSRDRFDFL IRCLRJ4DDKS IPETLRENDV FTPVRKIWDL FIHQCIQNYT PGAHLTIDEQ LLGFRGRCPF RMYIPNKPSK YGTKILMMCD 301 SGYKYMINGM PYLGRGTQT2N GVfPLGEYYVK ELSKPVHGSC RNTCDNWFT SIPLAKNLLr 361 EPYKLTIVGT VRSNKREPE VLKNSRSRPV GTSMFCFDGP LTLVSYKPKP AKMVYLLSSC 421 DEDASINEST GKPQMVMYYN QTKGGVDTLD QMCSVMTCSR KTNRWPMALL YGMINIACIN 481 SFIIYSHNVS SKGEKVQSRK KFMRNLYMSL TSSFMRKRLE APTLKRYLRD NSNILPNEV 541 PGTSDDSTEE PVMKKRTYCT YCPSEIRRKANASCKKCKKV ICREHNIDMC QSCF (SEQ ID NO: 4).
[02071 Incertainembodiments, the transposase enzyme is apiggyBacT M (PB) transposase enzyme that comprises or consists of an amino acid sequence having an amino acid substitution at two or more of positions 30, 165, 282, or 538 of the sequence of SEQ ID NO: 4. In certain embodiments, the transposase enzyme is a piggyBacTM (PB) transposase enzyme that comprises or consists of an amino acid sequence having an amino acid substitution at three or more of positions 30, 165, 282. or 538 of the sequence of SEQ ID NO: 4. In certain embodiments, the transposase enzyme is a piggyBacM (PB)transposase enzyme that comprises or consists of an amino acid sequence having an amino acid substitution at each of the following positions 30, 165, 282, and 538 of the sequence of SEQ ID NO: 4. In certain embodiments, the amino acid substitution at position 30 of the sequence of SEQ ID NO: 4 is a substitution of a valine (V) foran isoleucine (1). In certain embodiments, the amino acid substitution at position 165 of the sequence of SEQ ID NO: 4 is a substitution of a serine (S) for a glycine (G). In certain embodiments, the amino acid substitution at position 282 of the sequence of SEQ ID NO: 4 is a substitution of a valine (V) for a methionine (M). In certain embodiments, the amino acid substitution at position 538 of the sequence of SEQ ID NO: 4 is a substitution of a lysine (K)for an asparagine (N).
[02081 In certain embodiments of the methods of the disclosure, the transposase enzyme is a Super piggyBacTM (SPB) transposase enzyme. In certain embodiments, the Super TM piggyBac (SPB) transposase enzymes of the disclosure may comprise or consist of the amino acid sequence of the sequence of SEQ ID NO: 4 wherein the amino acid substitution at position 30 is a substitution of a valine (V) for an isoleucine (I). the amino acid substitution at position 165 is a substitution of a serine (S) for a glycine (G), the amino acid substitution at position 282 is a substitution of a valine (V) for a methionine (M), and the amino acid substitution at position 538 is a substitution of a lysine (K) for an asparagine (N). In certain embodiments, the Super piggyBacrM (SPB) transposase enzyme may comprise or consist of an amino acid sequence at least 75%. 80%, 85%, 90%, 95%, 99% orany percentage in between identical to: I MGSSLDDEHI LSALLQSDDE LVGTEDSDV SDHVSEDDVQ SDTEEAFIDE VHEVQPTSSG 61 SEILDEQNVI EQPGSSLASN RILTLPQRTI RGKNKHCWST SKSTRRSRVS ALNIVRSQRG 121 PTRMCRNIYD PLLCFKLFFT DEIISEIVKW TNAEISLKRR ESMTSATFRD TNEDETYAFF 181 GTLVMTAVRK DNHMSTDDLF DRSLSMVVS VMSRDRFDFL IRCLRj4DDKS IPETLRENDV FTPVRKIWDL FIHQCIQNYT PGAHLTIDEQ LLGFRGRCPF RVYIPNKPSK Y G1KILMMCD 301 SGTKYMINGM PYLGRGTQTN GVfPLGEYYVK ELSKPVHGSC RNITCDNWFT SIPLAKNLLQ 361 EPYKLTIVGT VRSNKRETPE VLKNSRSRPV GTSMFCFDGP LTLVSYKPKP AKMVYLLSSC 421 DEDASINEST GKPQMVMYYN QTKGGVDTLD QMCSVMTCSR KTNRWPMALL YGMIN1ACIN 48 1 SFIIYSHNVS SKGEKVQSRK KFMRNLYMSL TSSFMRKRLE APTLKRYLRD NISNILPKEV 541 PGTSDDSTEE PVMKKRTYCT YCPSKIRRKANASCKKCKKV ICREHNIDMC QSCF (SEQ ID NO:
[0209 In certain embodiments of the methods of the disclosure, including those embodiments wherein the transposase comprises the above-described mutations at positions , 165, 282 and/or 538, the piggyBacl or Super piggyBac'' transposase enzyme may further comprise an amino acid substitution at one or more of positions 3, 46, 82, 103, 119, 125, 177, 180, 185, 187,200, 207, 209, 226, 235, 240, 241, 243, 258, 296, 298, 311, 315, 319, 327, 328, 340, 421, 436, 456, 470, 486, 503, 552, 570 and 591 of the sequence of SEQ ID NO: 4 or SEQ ID NO: 5. In certain embodiments, including those embodiments wherein the transposase comprises the above-described mutations at positions 30, 165, 282 and/or 538, the piggyBacTM or Super piggy Bac T M transposase enzyme may further comprise an amino acid substitution at one or more of positions 46, 119, 125, 177, 180, 185, 18T 200, 207, 209, 226, 235, 240, 241, 243, 296, 298, 311, 315, 319, 327, 328, 340, 421, 436, 456, 470, 485, 503, 552 and 570. In certain embodiments, the amino acid substitution at position 3 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an asparagine (N) for a serine (S). In certain embodiments, the amino acid substitution at position 46 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a seine (S) for an alanine (A). In certain embodiments, the amino acid substitution at position 46of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a threonine (T) for an alanine (A). In certain embodiments, the amino acid substitution at position 82 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a tryptophan (W) for an isoleucine (I). In certain embodiments. the amino acid substitution at position 103 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a proline (P) for a seine (S). In certain embodiments, the amino acid substitution at position 119of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a proline (P) for an arginine (R). In certain embodiments, the amino acid substitution at position 125 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an alanine (A) a cysteine (C). In certain embodiments, the amino acid substitution at position 125 of SEQ ID NO: 4 or SEQ ID NO: 5is a substitution of a leucine (L) for a cysteine (C). In certain embodiments, the amino acid substitution at position 177 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a lysine (K) for a tyrosine (Y). In certain embodiments, the amino acid substitution at position 177 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a histidine (-) for a tyrosine (Y). In certain embodiments, the amino acid substitution at position 180 of SEQ ID NO: 4or SEQ ID NO: 5 is a substitution of a leucine (L) for a phenylalanine (F). In certain embodiments, the amino acid substitution at position 180 of SEQ ID NO: 4 or SEQ ID NO: 5is a substitution of an isoleucine (1)for a phenylalanine (F). In certain embodiments, the amino acid substitution at position 180 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a valine (V) for a phenylalanine (F). In certain embodiments, the amino acid substitution at position 185 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a leucine (L) for a methionine (M). In certain embodiments, the amino acid substitution at position 187 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a glycine (G) foran alanine (A). In certain embodiments, the amino acid substitution at position 200 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a tryptophan (W) for a phenylalanine (F).In certain embodiments, the amino acid substitution at position 207 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a proline (P) for a valne (V). In certain embodiments, the amino acid substitutionatpoition 209 of SEQ ID NO: 4 or SEQ ID NO: 5 isasubstitution of a phenylalanine (F) for a valine (V). In certain embodiments, the amino acid substitution at position 226 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a phenylalanine (F) for a inethionine (M). In certain embodiments, theamino acid substitution at position 235 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an arginine (R) for a leucine (L). In certain embodiments, the amino acid substitution at position 240 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a lysine (K) for a valine (V). In certain embodiments, the amino acid substitution at position 241 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a leucine (L) for a phenylalanine (F). In certain embodiments, the amino acid substitution at position 243 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a lysine (K) for a proline (P). In certain embodiments, the amino acid substitution at position 258of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a seine (S) foran asparagine (N). In certain embodiments, the amino acid substitution at position 296 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a tryptophan (W) for a leucine (L). In certain embodiments, the amino acid substitution at position 296 of SEQID NO: 4 or SEQ IDNO: 5 is a substitution of a tyrosine (Y)for a leucine (L). In certain embodiments, the amino acid substitution at position 296 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a phenylalanine (F) for a leucine (L). In certain embodiments, the amino acid substitution at position 298 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a leucine (L) for amethionine (M). In certain embodiments, the amino acid substitution atposition298 of SEQ ID NO: 4 or SEQ ID NO: 5isasubstitution of an alanine (A) for a methionine (M). In certain embodiments, the amino acid substitution at position 298 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a valine (V) for a methionine (M). In certain embodiments, the amino acid substitution at position 311 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an isoleucine (I) for a proline (P). In certain embodiments, the amino acid substitution at position 311 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a valine for a proine (P). In certain embodiments, the amino acid substitution at position 315 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a lysine (K) for an arginine (R).In certain embodiments, the amino acid substitution at position 319 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a glycine (G) for a threonine (T). In certain embodiments, the amino acid substitution at position 327 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an arginine (R) for a tyrosine (Y). In certain embodiments, the amino acid substitution at position 328 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a valine (V) for a tyrosine (Y). In certain embodiments, the aminoacid substitution at position 340 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a glycine (G) for a cysteine (C). In certain embodiments, the amino acid substitution at position 340 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a leucine (L) for a cysteine (C). In certain embodiments, the amino acid substitution at position 421 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a histidine (H) for the aspartic acid (D). In certain embodiments, the amino acid substitution at position 436 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an isoleucine (1) for a valine (V). In certain embodiments, the amino acid substitution at position 456 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a tyrosine (Y) fora methionine (M). In certain embodiments, the amino acid substitution at position 470 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a phenylalanine (F) for a leucine (L). In certain embodiments, the amino acid substitution at position 485 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a lysine (K) for a seine (S). In certain embodiments, theamino acid substitution at position 503 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a leucine (L) for a methionine (M). In certain embodiments, the amino acid substitution at position 503 of SEQ ID NO: 4or SEQ ID NO: 5 is a substitution of an isoleucine(I) for a methionine (M). In certain embodiments, the amino acid substitution at position 552 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a lysine (K) for a valine (V). In certain embodiments, the amino acid substitution at position 570 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a threonine (T) for an alanine (A). In certain embodiments, the amino acid substitution at position 591 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a proline (P) for a glutamine (Q). In certain embodiments, the amino acid substitution at position 591 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an arginine (R) for a gutamine (Q).
[0210 In certain embodiments of the methods of the disclosure, including those embodiments wherein the transposase comprises the above-described mutations at positions , 165, 282 and/or 538, the piggyBacrM transposase enzyme may comprise or the Super piggyBacTMi transposase enzyme may further comprisean amino acid substitution at one or more of positions 103, 194, 372, 375, 450, 509 and 570 of the sequence of SEQ ID NO: 4 or SEQ ID NO: 5. In certain embodiments of the methods of the disclosure, including those embodiments wherein the transposase comprises the above-described mutations at positions 30. 165, 282 and/or 538, the piggyBacTu transposase enzyme may comprise or the Super piggyBacTM transposase enzyme may further comprise anaminor acid substitution at to three, four, five, six or more of positions 103, 194, 372, 375, 450, 509 and 570 of the sequence of SEQ ID NO: 4 or SEQ ID NO: 5. In certain embodiments, including those embodiments wherein the transposase comprises the above-described mutations at positions , 165, 282 and/or 538, the piggyBac TM transposase enzyme may comprise or the Super piggyBac" transposase enzyme may further comprise an amino acid substitution atpositions 103, 194, 372375,450, 509and 570 of the sequence of SEQ ID NO: 4 or SEQ ID NO: 5. In certain embodiments, the amino acid substitution at position 103 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a proline (P) for a serine (S). In certain embodiments, the amino acid substitution at position 194 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a valine (V) for a mthionine (M). In certain embodiments, the amino acid substitution at position 372 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an alanine (A) for an arginine (R). In certain embodiments, the amino acid substitution at position 375 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an alanine (A) for a lysine (K). In certain embodiments, the amino acid substitution at position 450 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an asparagine (N) for an aspartic acid (D). In certain embodiments, the amino acid substitution at position 509 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a glycine (G) for a serine (S). In certain embodiments, the amino acid substitution at position 570 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a serine (S) for an asparagine (N). In certain embodiments, the piggyBacTM transposase enzyme may comprise a substitution of a valine (V) for a methionine (M) at position 194 of SEQ ID NO: 4. In certain embodiments, including those embodiments wherein the piggyBacTM transposase enzyme may comprise a substitution of a valine (V) for a methionine (M) at position 194 of SEQ ID NO: 4, the piggyBacTM transposase enzyme may further comprise animino acid substitution atpositions 372,375and450ofthesequence ofSEQIDNO:4 orSEQIDNO: 5.Incertain embodiments, the piggyBacTM transposase enzyme may comprise a substitution of a valine (V') for a methionine (M) at position 194 of SEQ ID NO: 4, a substitution of an alanine (A) foran arginine (R)at position 372 ofSEQIDNO:4, andasubstitutionof analanine(A) for a lysine (K) at position 375 of SEQ ID NO: 4. In certain embodiments, the piggyBaclI transposase enzyme may comprise a substitution of a valine (V) for a methioine (M) at position 194 of SEQ ID NO: 4, a substitution of an alanine (A) for an arginine (R) at position 372 of SEQ ID NO: 4, a substitution of an alanine (A) for a lysine (K) at position 375 of SEQ ID NO: 4 and a substitution of an asparagine (N) foran aspartic acid (D) at position 450 of SEQ ID NO: 4.
[02111 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 particularmethod 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.
[02121 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.
[02131 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
[02141 By "transient transformation" isintended thatapolynucleotideconstructintroduced into the host does not integrate into the genome of the host.
[02151 In preferred embodiments, the piggyBac transposon system is used tointroduce exogenous sequences into a primary human T cell by stable transformationtogeneratea modified TscM or ICM. Additional Transposon Systems
[0216 In certain embodiments of the methods of the disclosure, the transposon is a Sleeping Beauty transposon. In certain embodiments, and, in particular,those embodiments wherein the transposon is a Sleeping Beauty transposon, the transposase is a Sleeping Beauty transposase ora hyperactive Sleeping Beauty transposase (SB1OX).
[02171 The disclosure provides a method of producing a modified stem memory T-cell (TscM) or a modified central memory T-cell (TcM), comprising introducing into a primary human T cell (a) a transposon composition comprising a transposon comprising an antigen receptor or a therapeutic protein and (b) a transposase composition comprising a transposase or a sequence encoding the transposase; to produce a modified T cell, wherein the modified T cell expresses one or more cell-surface marker(s) of a modified stem memory T-cell (TscM) or a modified central memory T-cell (TCM), thereby producing a modified stem memory T cell (TscM) or a modified central memoryT-cell (IcM).'The disclosure provides a method of producing a plurality of modified stem memory T-cells (TscM) or a plurality of modified central memoryT-cells (TC), comprising introducing into a plurality of primary human T cells (a) a transposon composition comprising a transposon comprising an antigen receptor and (b) a transposase composition comprising a transposase or a sequence encoding the transposase; to produce a plurality of modified T cells, wherein at least 2%, 5%, 10%, 15%, %, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of modified T cells expressesone or more cell surface marker(s) of a stem memory T-cell (TscM) or a central memory T-cell (Tcm), thereby producing a plurality of modified stem memory T-cells (TscM) or a plurality of modified central memoryT-cells (TCMI).
[02181 In certain embodiments of the methods of the disclosure, the transposon is a Sleeping Beauty transposon. In certain embodiments, and, in particular, those embodiments wherein the transposon is a Sleeping Beauty transposon, the transposase is a Sleeping Beauty transposase or a hyperactive Sleeping Beauty transposase (SB1OX).
[02191 In certain embodiments of the methods of the disclosure, the Sleeping Beauty transposase enzyme comprises an amino acid sequence at least 75%, 80%, 85%, 90%,95%, 99% or any percentage in between identical to:
1 MGKSKEISQD LRKKIVDILHK SGSSLGATSK RLKVPRSSVQ TIVRKYKHHG TTQPSYRSCR 61 PRYLSPRDER TLVRKVQINP RTTAKDLVKM LEETGTKVSI STVKRVLYRH NLKGRSARKK 121 PLLQNRHKKA RLPFATAHGD KDRTF'WPNVL WSDETKIELF GHNDHPYVWR KKGEACKPKN 181 TIPTVKHCCC SIMLWGCAA GTGALHKID GIMRKENYVD ILKQHLKTSV RKLKLGRKIV 241 FQMDNDPKHT SK/VAKWLKD NKVKVLEWPS QSPDLNPlEN LWAELKKRVR ARRPTNLTOL 301 HQLCQEEWAK IHPTYCGKLV EGYPKRLTQV KQFKGNATKY (SEQ ID NO: 6).
[02201 In certain embodiments of the methods of the disclosure, the hyperactive Sleeping Beauty (SB1OOX) transposase enzyme comprises an amino acid sequence at least 75%. 80%, %, 90%, 95%, 99% or any percentage in between identical to: 1 MGKSKEISQD LRPIVDLHK SGSSLGAISK RLAVPRSSVQ TIVRKYKHHG TTQPSYRSGR 61 RRYLSPRDER TLVRKVQINP RTTAKDLVKM LEETGTKVSI STVKRVLYRH NLKGHSARKK 121 PLLQNRHKKA RLRFATAHGD KDRTFWPNVL WSDETKIELF GHNDHRYVWR KKCEACKPKN 181 TTPTVKHGG SIMLWGCFAA GTCALHKID GIMDAVQYVD ILKQHLKTSV RKLKLGRKW4V 241. FQHDNDPKHT SNVAKWLKD NKVKVLEWPS QSPDLNPIEN LWAELKKRVR ARRPTNLTQL 301 HQLCQEEWAK IHPNYCGKLV EGYPKRLTQV KQFKGNATKY (SEQ ID NO: 7)
[02211 In certain embodiments of the methods of the disclosure, the transposase is a Helitron transposase. Helitron transposases mobilize the HeIraiser transposon, an ancient element from the bat genome that was active about 30 to 36 million years ago. An exemplary Heraiser transposon of the disclosure includes Hlelibat, which comprises a nucleic acid sequence comprising: 1 TCCTATATAA TAAAGAGAAA ACATGCAAAT TGACCATCCC TCCGCTACGC TCAAGCCACG 61 CCCACCACC AT.TCAGAAGT GACTATCAA ATTAACCCAA CAAA.GATGGC AGTTAAT.TT 121 GC.ATACGCAG GTGTAAGCG CCCCAGGAGG CAA2.GGCGGC CGCGGGCTCC CAGCGACCTTC 181 CCTGGCCCCG GAGGCGCAG CCCCCCGC CTAGCCACAC CCGCGGCTC CCGGCACCTT 241 CGCCAGCAGA GAGCAGAGCG GGAGGCGGG CGGGAGCG GAGGTTTGCA GGACTTGGCA
301 GAGCAGGAGG CCGCTCGACA TAGAGCAGAG CGAGAGAGAC GGTGGCCTTGG AGGCGTGGC
361 TCCCTCTGTC ACCCCAGCTT CCTCATCACA GCTGTGGAAA CTGACAGCAG GGAGGAGGAA
421 GTCCCACCCC CACAGAATCA CCAGAATCA GCCCTTGGTC AGACAGCTCT CAGCGCCTG
481 ACAGCCAGGA CTCCAT TCA CCTCATCTC AGACCGTGAC AGTAGAGACG TGGGACTATC
841 TCTAAACAAC ACTGTT GAT ACAACGTAGC TCTCCAGCCCG PAAATGCCG GCGTTATCCA
601 CAGAAAATGT CTCGCAGAGCA ACTGCCGTCT GATCTTGAAA GAAGGCGGCG CCTGCAACAG
661 AATGTATCTG AAGAGCAGCT ACTGGAAAAA CGTCGCTTCTG AG-CCCGAAAA ACAGCGGCGT T 721 CATCGACAGA AATGTC AA ACACCAA.CGT GCCTTTGAAG TTGAAAGAAG GCGTGGCGA
'71 CGACAGAATA TTCTAGAGA ACAGTCATCA ACAAGTACTA CCAATACCGG TAGGAACTGC
841i CT T CT CAGCA AAAAT GCAGT ACAT GGA AT CAT TCT CG AACATAGTTG T GGT GGAAT G
901 ACTGTTCGAT GTGAATTTTG CCTATCACTA AATTTCTCTG ATCAAAAACC ATCCGATGGG
961 AAATTTACTC GATGTTGTAG CAAAGGGAAA GCTGTCCAIN ATGATATACA ' TTCCAGAT
1021 TACCCGGCAT ATTTAAAAAG ATTAATGACA AACGAAGATT CTGACAGTAA AAA.TTT.ATG
1081 GAAAATATTC GTTCCATAATACTTCTTTT GCTTTTGCCTT CCATCGGGTGC AAATATTGCA 1141 TCGCCATCAG GATATGGGCC ATACTGTTTT AGAATACACG GACAAGTTTA TCACCGTACT 1201 GGAACTTTAC ATCCTTCGGATTGGTGTTTCT CGGAAGTTTG CTCAACTCTA TATTTTGGAT 1261 ACAGCCGAAG CTACAAGCTAA AAGATTAGCA ATCCAGAAACA CCAGGCTG CTCACAAAGA 1321 CTCATGATCA ACAA CATCAACA CCCATCAT GAJ'ATAAATG AATTAACAAA TCTACACG 1381 ATGCTACATG AGGTAAGAAAA GGAAGCCCA TCTGAAGCACG CACCAAAGG TATTGCTOCC 1441 ACAGAAGTAA CAATGGCGAT TAAATACGAT CCTAA.CAGTCG ACCCAGGTAG ATATAATTCT 1501 CCCCGTGTAA CCCGAGGTTGC TCTCATATTC ACAAACGAAG ATGAGAACC TCCTTTTGAA 1561 AGGGACTTGC TCATTCATTG TAAACCAGAT CCCAATAATC CPAATGCCAC TAAAATGAAA 1621 CAAATCAGTA TCCTGTTTCC TACATTAGAT GCAATGACAT ATCCTATTCT TTTTCCACAT 1681 GGTCAAAAG GCTGGGGAAC ACATATTGCA TTAACACTCA CAGACAACAG TGTPATCGAC 1741 AATAATACTA GACAAAATGT AAGGACACGA GTCACACAAA TGCAGTATTA TGGATTTCAT 1801 CTCTCTCTGC GGG.ACCGTT CAATCCTATT TTAAATGCAG GAAATTAAC TCAACAGTTT 1861 ATTGTGGATT CATATTCAAA AATGGAGGCC AATCGGATAA ATTTCATCAA AGCAAACCAA 1921 TCTAAGTTCA GAGTTGAAAA ATATAGTGGT TTGATCCATT ATCTCAAKTC T AGATCTGAA 1981 AATGACAATG TGCCGATTGG TAAAATGATA ATACTTCCAT CATCTTTTGA GGGTAGTCC' 2041 AGAAATATGC AGCAGCGATA TCAGGATGCT ATGGCAATTG TAACGAAGTA TGGCAAGCCC 2101 GATTTATTCA TAACCATGAC ATGCAACCCC AAATC.AG ATATTACAAA CAATTTACAA 2161 CGCTGGCAAA AAGTTGAAAA CAGACCTGAC TTGGTAGCCA GAGTTTTTAA TATTAAGCTG 2221 AATGCTCTTT TAPATGATAT ATCTAPATTC CATTT ATTTG GCAAACTAAT AGCTAAAATT 2281 CATCTCATT AATTTCAGAAACCGGACTG CCTC ACGCTC ACATATTATT GATATTAGAT
31 A G'TGAGTCCAAATTACGTTC AGAAGATGAC ATTGACCGTA TAGTTAAGGC AGAAATTCCA 41 GAG-AAGACC AGGTCCCTCG ACTTTTTCAA ATTGTAAAT CAATATGGT ACATGGACCA 2461 TGTGGAATAC AAAATCCAAA TAGTCCATGT ATGGAAATTG GAAATGTTC AAAGGG AT 2521 CCAAAAGAAT TTCAAAATGC GACCATTGGA ATATTGATC GATATCCCAA ATACAAACGA 2581 AGATCTGGTA GCACCATGTC TATTGGAAT AAAGTTGTCG ATAACACTTG GATTGT'CCCT 2641 TATAACCCGT ATTTGTGCCT TAATATAAC TGTCATATPA ATGTTGAAGT CTGTGCATCA 2701 ATTAAAAGTG TCAIATATTT ATTTAAATAC ATCTATAAAG GGCACGATTG TGCAAATATT 2761 CAAATTTCTG AAAAAAATAT TATCAATCAT GACGAAGTAC AGGACTTCAT TGACTCCACGG 2821 TATGTGAGCG CTCCTGAGGC TGTTTGGAGA CTTTTTCCAA TGCGAATGCA TGACCAATCT 2881 CATGCAATCAp CAAGATTAGC TATTCAT'TTG CCAAATGATC AGAATTTGTA TTTTCATACC 241 GATGATTTTG CTGAAGTTTT AGATAGGGCT AAAACATA CTCGACTTT GATGGCTTGG 01 TT CTATTAATAAGAAGA TTCTGATGCA CGTAATTATT ATTATTGGAGATTCCACAG 36CAATCTC'G TTAATAATTC TTTTGGACA AAAC.GCCCGAA AGGGTGGGAA TAAAGTATTA 3 121 'GTAGACTGT TCATCTGTGAG CTTTAGAGAA CCAGAACGAT ATTACCTTAG ACTTTTGCTT 3181 CTGCATGTAA AAGTGCGAT AAGTTTTCAG GATCTGCGAA CTGTACCAGG TGTAACTTAT 3241 GATACATTTC ATGJAAGCTGC TAAACA(CCGA GATTATTAC TT'GATGACAC TATCTCGGiA 3301 GATACGATTG ACGATGCAAT CATCCTTAAT ATGCCCAAAC AACTACGGCA ACTTTGCA 3361 TATATATCTG TGTTCGGATG TCCTTCTGCT GCAGACAAAT TATGGGATGA CAATAATCT 3421 CATTTTATTG AAGATTTCTG TTCCAAATTA ACCGAAGAG AAGGTGCCG TG'TGAACTGT
3481 GAAATGCATG CCCTTACGA AATTCA GGA TATTCACAT TOOATAGGAT cAAATGTTCA 3541 CATTTCAAAC TTCCGGACTA TCCTTTATTA ATGAATGCAA ATACATGTGA TCAATTGTAC 36U1 GAGCAACAAC AGGCAGAGGT TTTGATAAAT TOTCTGAATG ATGAACAGTT GGCAGCCTTT 3661 CAGACTATAA CTTCAGCCAT CGAAGATCAA ACTGTACACC CCAAATGCTT TTTCTTGGAT 3721 GGTCCAGGTG GTAGTGGAAA AACATATCTG TATAAAGTTT TAACAOATTA TATTAGAGGT 3781 CGTGGTGGTA CTGTTTTACC CACAGCATCT ACAGGAATTG CTGCAATTT ACTTCTTGGT 3841 GGAAGACCT TTCATTCCCA ATATAPATTA CCAATTCCAT TAAATAAC TTCAATTTCT 3901 AGACTCGATA TAAAGGTGA AGTTGCTAAA ACCATTAAAA AGGCCCAACT TCTOATTATT 3961 GATGAATGCA CCATGGCATC CAGTCATGCT ATAAACGCCA TAGATAGATT ACTAAGAGAA 4021 ATTATGAATT TCAATGTTGC ATTTGGTGGG AAAGTTCTCC TTCTCGGAGG GGATTTTCGA 4081 CAATGTCTC GTATTGTACC ACATGCTATG CGATCGGCCA TAGTACAAC GAGTTTAAAG 4141 TACTGTAATG TTTGGGGATG TTTCAGAAAG TTGTCTCTTA AAACAAATAT GAGATCAGAG 4201 GATTCTGCTT ATAGTGAATG GTTAGTAAAA CTTGGAGATG GCAAACTTGA TAGCAGTTTT 4261 CATTTAGGAA TGGATATTAT TGAAATCCCC CATGAAATGA TTTGTAACGG ATCTATTATT 4321 GAAGCTACCT TTGGAAATAG TATATCTATA GATAATATTA AAAATATATC TAAACGTGCA 438' ATTCTTTGTC CIAAAATGA GCATGTTCAA AATTAAATG AAGAAATTTT GGATATACTT 4441 GATGAGATT TTCACACATA TTTGAGTGAT GAd/TTCCATTG ATTCAIACAGA TGATGCTGIA 4501 A.AGGAAAATT TTCCCATCGA ATTTCTTAAT AGTATTACTC CTTCGGGAAT GCCGTGTCAT 4561 A ATTAA.T TGAAAGTGGG TGCAATCATC ATGCTATTGAGAAATCTTAA TAGTAAATGG 4621 GGTCTTTGTA ATGGTACTAG ATTTATTATC AAAAGATTAC GACCTAACAT TATCGAAGCT 4681 GAAOTATTAA CAGGATCTGC AGAGGGAGAG GTTGTTCTGA TTCCAACAT TGATTTGTCC 4741 CCATCTGACA CTGGCCTCCC ATTTAAATTA ATTCGAAGAC AGTTTCCCGT GATGCCAGCA 4801 TTTGCGATGA CTATTAAITAAT ACACAAGGA CAAACTCTAG ACAGAGTAGG AATATTCCTA 4861 CCTGAACCCG TTTTCGCACA TGGTCAGTTA TATGTTGCTT TOCTCICAGT TCG/AAGAGCA 4921 TGTGACGTTA AAGTTAAAGT TGTAAATACT TCATCACAAG GGAAATTAGT CAAGCACTCT 498i GAAAGTGTTT TTCTCTTAA TGTGGTATAC AGGGACATAT TAGAATAGT TTAAT CACTT 5041 TATCAGTCAT TGTTTGCATC AATGTTGTTT TTATATCATG TTTTTGTTGT TTTTATATCA 5101 TGTCTTTGTT GTTGTTATAT CATGTTGTTA TTGTTTATTT ATTAAITAAAT TTATCTATTA 5161 TTTTCATATA CATTTTACTC ATTTCCr'r'TTC ATCCTCACA CTT CTATTAT AGAGAAAGGG 5221 CAAATAGCAA TATTAAAATA TTTCCTCTAA TTAATTCCCT TTCAATGTGC ACGAATTTCG 5281 TGCACCGGGC CACTAG (SEQ ID NO: 27).
[02221 Unlike other transposases, the Helitron transposase does not contain an RNase-H like catalytic domain, but instead comprises a R-eplel motif made up of a replication initiator domain (Rep) and a DNA helicase domain. The Rep domain is a nuclease domain of the HUH superfamily of nucleases.
[02231 An exemplary Helitron transposase of the disclosure compares an amino acid sequence comprising: 1 MSKEQLLIQR SSAAERCRRY RQKMSAEQRA SDLERRRRLQ QNVSEEQLLE KRRSEAEKQR 61 RHRQKMSKDQ RAFEVERRR' RRQNMSREQS STSTTNTGPN CLLSKNGVHE DAILEHSCGG
121 MTVRCEF'CLS LNFSDEKPSD GKFTRCCSKG KVCPNDIHFP DYPAYLKRLM TNEDSDSKNF 181 MENIRSTNSS FAFASMGANI ASPSGYGPYC FR'THGQVYHR TGTLHPSDGV SRKFAQLYTL 241 DTAEATSKRL AMPENQGCSE RLMININNLM HEINELTKSY KMLHEVEKEA QSEAAAKGIA 301 PTEVTMAIKY DRNSDPGRYN SPRVTEVAVI FP:TEDGEPPF ERDLLIHCKP DPNNPNATKM 361 KQISILFPTL DAMTYPILFT HGEKGWGTDT ALRLRDNSVI DNNTRQNVRT RVTQMQYYGF 421 HLSVRDTFNP ILNAGKLTQQ FIVDSYSKME ANRINIKAN QSKLRVEKYS GLMDYLKSRS 481. ENDNVPIGKM TILPSSTfEGS PRNMQQRYQD AMAAIVTKYGK PDLFITMTCN PKWADITNNL 541 QPWQKVENP.P DLVARVFNIK LNALLNDICK FHLFGKVIAK IHVIEFQKPG LPHAHILLIL 601 DSESKLRSED DIDPIVKAEI PDEDQCPRLF QIVKSNMVHG PCCIQNPNSP CMENGCSKG 661 YPKEFQNATI GNIDGYPKYK RRSCSTMSIG NKVVDNTWIV PYNPYLCLKY NCHINVEVCA 721 SIKSVKYLEK YIYKGHDCAN IOTSEKNIIN HDEVQDFIDS RYVSAPEAVW RL FAMPAHDQ 781 SIHAITRLAIH LPNDQNLYFH TDDFAEVLDR AKRHNSTLMA WFLLNREDSD ARNYYYWET.P 841 QHYVFNNSLW TKPRKGGNKV LGRLFI'VSFR EPERYYLRLL LLHVKGAISF EDLRTVGGVT 901 YDTFHEAAKH RGLLLDDTIW KDTIDDAIIL NMPKQLRQLF AYICVFGCPS AADKLWDENK 961 SHFIED'CWK LHRREGACVN CEMHALNEIQ EVFTLHCMKC SHF'KLPDYPL LMNANTCDQL 1021 YEQQQAEVLI NSLNDEQLAA FQTITSAIED QTVHPKCFFL DGPGGSGKTY LYKVLTHYIR 1081 GRGGTVLPTA. STG I ILLL GGIRTFSQYK LPIPLNETSI SRLDIKSEVA KTIKKAQLLI 1141 IDECTMASSH AINAIDLLR EIMNLNVAFG GKVLLLGGDF RQCLSIVPHA MRSAIVQTSL 1201 KYCNVCFIR KSLKTNMRS EDSAYSEWLV KLGDGKLDSS FHL GMDIIEI PHEMI-CNGSI
1261 IEATF'GNSIS IDNIKNSKR AILCPKNEHV QKLNEEILDI LDG-DFHTYLS DDSIDSTDDA
1321 EKENFPIEFL NSITPSGMPC HKLKLEAIA IMLLRNNSK WGC'NGTRFTI IKPLRPNIIE
1381 AEVLTGSAEG EVVLIPRIDL SPSDTCLPFK LIRPQPVMP AFAMTINKSQ GQTLDRVCI.F
1441 LPEPVFAHGQ LYVAFSPVRP ACDVKVKVVN TSSQGLVKH SESVFTLNVV YREILE (SEQ ID
NO: 28).
[02241 In Helitron transpositions, a hairpin close to the 3' end of the transposon functions as a terminator. However, this hairpin can be bypassed by the transposase, resulting in the transduction of flanking sequences. In addition, Helraiser transposition generates covalently closed circular intermediates. Furthermore, Helitron transpositions can lack target site duplications. In the Helraiser sequence, the transposase is flanked by left and right terminal sequences termed LTS and RTS. These sequences tenninate with a conserved 5'-TC/CTAG 3' motif. A 19 bp palindromic sequence with the potential to forn the hairpin termination structure is located i I nucleotides upstream of the RTS and consists of the sequence TGCTACGAAT TTCGTGCACCGCGCCACTAG (SEQ ID NO: 29).
[02251 In certain embodiments of the methods of the disclosure, the transposase is a To2 transposase. Tolt2 ransposons may be isolated or derived from the genome of the medaka fish, and may be similar to transposons of the hAT family. Exemplary To2 transposons of the disclosure are encoded by a sequence comprising about 47 kilobases and contain a gene encoding the To2 transposase, which contains four exons. An exemplaryTol2 transposaseof the disclosure comprises an amino acid sequence comprising the following: 1 MEEVCDSSAA ASSTVQNQPQ DQEHPWPYLR EFFSLSGVNK DSEKMKCVLC LPLNKETSAF 61 KSSPSNLRKH IERIHPNYLK NYSKLTAQKR KIGTSTHASS SKQLKVDSVF PVKHVSPVTV
121 NKAILRYIIQ GLHPEFSTVDL PSFKELISTL QPGISVITRP TLRSKIAE1A LIMKQKVTAA 181 MSEVEWIATT TIDCWTARRKS FIGVTAHWIN PGSLERHSAA LACKRLMGSH TF'EVLASAMN
241 DIHSEYEIRD KVVCTTTDSG SNEMKAFRVE GVENNDIETE ARRCESDDTD SEGCGEGSDG
301 VEFQDASRVL DQDDGFEFQL PKHQKCACHL LNLVSSVDAQ KALSNEHYKK LYRSVF'GcKCQ
361 ALWNKSSRSA LAAEAVESES RLQLLRPNQT RWNSTFMAVD RILQICKEAG EGALRNICTS 421 LEVEYRNPAE MLFLTEWANT MREVAKVLDI LQAETNTQLG WLLPSVHQLS LKLQRLHHSL 481 RYCDPLVDAL QQGIQTRFKH MFEDPEIIAA AILLPKFRTS WTNDETIIKR GMDYIVHLE 541 PLDHKELAN SSSDDEDFFA SLKPTTHEAS KELDGYLACV SDTRESLLTF PAICSLSIKT
601 NTPLPASAAC ERLFSTAGLL ESPKRARLDT NNFENQLLLK LNLRF'YNFE (SEQ ID NO: 30).
[02261 An exemplary To12 transposon of the disclosure, including inverted repeats, subterminal sequences and the Toi2 transposase, is encoded by a nucleic acid sequence comprising the following: I CAGAGGTGTA AAGTACTTGA GTAATTTTAC TTGATTACTG TACTTAAGTA TTATTTTTGG 61 GGATTTTTAC TTTACTTGAG TACAATTAAA AATCAATACT TTTACTTTTA% CTTAATTACA 121 TTTTTTTAGA AAAVJJAAGTA CTTTTACTC CTTACAATTT TATTTACAGT CAJ AAGTAC 181 TTATTTTTTCG GAGATCACTT CATTCTATTT TCCCTTGCTA TTACCAJ.CC AATTGAATTG 241 CGCTGATcGCC CAGr'rTTAATT TAAATGTTAT TTATTCTGCC TATGAAAATC GTTTTCACAT 301 TATATGAAT TGGTCAGACA TGTTCATTGG TCCTTTGGAA GTGACGTCAT GTCACATCTA 361 TTACCACAAT GCACAGCACC TTGACCTGGA AATTAGGGAA ATTATAACAG TCAATCAGTG 421 GAAGAAAATG GAGGAAGTAT GTGATTCATC AGCAGCTGCG AGCAGCACAG TCCAAAATCA 481 GCCACAGGAT CAAGAGCACC CGTGGCCGTA TCTTCGCGAA TTCTTTTCTT TAAGTG-GTGT 541 AAATAAAGAT TCATTCAAGA TCAAA.TGTGT CCTCTGTCTC CCGCTTAATA AAGAAATATC 601 GGCCTTCAAA AGTTCGCCAT CAAACCTAAG GPAGCATATT GAGGTAAGTA CATTAAGTAT 661 TTTGTTTTAC TGATAGTTTT TTTTTTTTTT TTTTTTTTTT TTTTTGGGTG TGCATGTTTT 721 GACGTTGATG GCGCGCCTTT TATATGTGTA GTAGGCCTAT TTTCACTAAT GCATGCGATT 781 GACAATATAA GGCTCA.CGTA ATAAAATGCT AAATGCATT TAPATTrGGT AACGTTAGGT 841 CCACGGGAAA TTTGGCGCCT ATTGCAGCTT TGAATAATCA TTATCATTCC GTGCTCTCAT 901 TGTGTTTGAA TTCATGCAAA ACACAAGAAA ACCAA.GCGAG AAATTTTTT CCAAACATGT 961 TGTATTGTCA AAACGGTAAC ACTTTACAAT GAGGTTGATT AGTTCATGTA TTAACTAACA 1021 TTIAATAACC ATGAGCAATA CATTTGTTAC TGTATCTGTT PATCTTTGTT AACGTTAGTT 1081 AATAGAAATA CAGATGTTCA. TTGTTTGTTC ATGTTAGTTC ACAGTGCATT AACTAATGTT 1141 AACAAGATAT AAAGTATTAG TAAATGTTGA AATTAACATG TATACGTGCA GTTCATTATT 1201 AGTTCATGTT AACTAATGTA GTTAACTAAC GAACCTTATT GTAAAAGTGT TACCATCAAA 1261 ACTAATGTAA TGPAATCAAT TCACCCTGTC ATGTC!AGCCT TACAGTCCTG TGTTTTTGTC
1321 AATATAATCA GAAATAVJT TAPATGTTTGA TTGTCACTAA ATGCTACTGT ATTTCTAAAA 1381 "CAACAAGTA TTTAACATTA TAAAGTGTGC AA.TGGCCTGC AATGTCAGT TTTATTAAAG 14 GGTTAGTTCA CCCAAAAATG AAAATAATGT CATTAATGAC TCGCCCTCT CTCGTTCCAA 1C0 CCCTAGA CCTCCTTCA. TCTTCAAAC ACAGTTTAA -ATATTTTAGA TTTAGTCCGA 1561 GAGCTTTCTG TGCCTCCATT GAGAATGTAT GTACGGTATA CTGTCCATGT CCAGAAAGT 1621 AATAAAACA TCAAGTAGT CCATGTGACA TCAGTGGGTT AGTTAGAATT TTTTGAACCA 1681 TCGAATACAT TTTC-GTCCAA AAATAACAAA ACCTACGACT TTATTCGGCA TTGTATTCTC 1741 TTCCGGGTCT GTTGTCAATC GCGT'CACG ACTTCGCAGT GACGCCTACAA TGCTGAATAA 1801 AGTCCTAGGT TTTGTTATTT TTGGACCAAA ATCTATTTTC GATGCTTCAA ATAATTCTAC 1861 CTAACCCACT GATGTCACAT GGACTACTTT GATGTTTTTA TTACCTTTCT GGAC ATGGAC
1921 AGTATACCGT ACATACATTT TCAGTGGAGG GACAGAAAGC TCTCGGACTA AATCTAAAAT
81 ATC TTAAACT GTGTTCCGAA GATGAACGGA GGTGTTACGG GClGGAACG ACATGAGGGT 2041 GAG-CATTAA TGACATCTTT TCATTTTTGG GTGAACTAAC CCTTTAATGC TGTAATCAGA 2101 GAGTGTATGT GTAATTGTTA CATTTATTGC ATACAATATIAAATATTTATT TGTTGTTTTT
2161 AAGAGPATG CACCCAAATT ACCTCAAAA CTACTCTAAA TTGACAGCAC AGAAGAGAAA
2221 GATCGGGACC TCCACCCTG C TTCCCAG TAGCAACTG AAAGTTGACT CAGTTTTCCC 2281 AGTCAAACAT GTGTCTCCAG TCACTGTGAA CAAAGCTATA TTAAGGTACA TCATTCAAG-G 2341 ACTTCATCCT TTCAGCACTG TTGATCTGCC ATCATTTAAA GAGCTGATA GrTACACTGCA 2401 GCCTGGCATT TCTCTCATTA CAAGGCCTAC TTTACGCTCC AAGATAGCTG AAGCTGCTCT 2461 CATCATCAAA CAGAAAGTCA CTGCTGCCAT CACTGPAGTT GAATGGATTC CAACCACAAC
2521 GGATTGTTGG ACTGCACGTA GAAGTCATT CATTGGTGTAA CTGCTCACT GGATCAACCC 281 T' GGAAGTCTT GAAAGACATT CCGCTGCACT TGCCTGCAAA AATTAATCGG CTCTCATAC 2661 1T"TGAG GTA CTGGCCAGTG CCATGAATGA TATCCACTCA GAGTATGAA TACCTGACAA
2701 G GTl TTTGC ACAACCACAG ACAGTGGTTC CAACTTTAT.G AAGGCTTTCA GAGTTTTTG
2761 T GTGGAAAC AATGATATCG AGACTGAGGC AAGAAGGTGT GAAAGTCATG ACACTCATTC 2821 TGAAGGCTGT GGTGAGGGAA GTCATGGTGT GGAATTCCA GATGCOCTCAC CAGTCCTGGA 2881 CCAAGACGAT GGCTTCGAAT TCCAGCTACC AAAACATC7A AAGTGTGCCT GTCACTT ACT 2941 TAA.CCTAGTC TCAAGCGTTG ATGCCCAAAA AGCTCTCTCA AATGAACACT ACAA.GAAACT 3001 CTACACATCT GTCTTGGCA AATGCCAAGC TTTATGGAAT AAAAGCAGCC GATCGGCTCT
3061 AGCACCTGAA CCTCTTGAAT CAGAAAGCCG GCTTCACGCTT TTAAGGCCCA ACCAAACCG 3121 GTGGAATTCA ACTTTTATGG CTGTTGAACG AATTCTTCAAATTTCCAACAACAGACA '318 AGGCGCACTT CGGAATATAT GCACCTCTCT TGAGGTTCCA TGTAACTGT TTTTCCCCTC 1 324 A1 TA TCCTGTA AACAATCGG GGTTGT TT GTTTAATACT CTTTGATTAT GCTGATTTCT
2301 CCTGTAGGTT TAATC.CAGCA GAAATGCTGT TCTTGACAGA GTGGGCC.AAC ACAATGCGTC
3361 CAGTTGCAAA AGTACTCGAC ATCTTGCAAG CGGAMACGAA TACACAGCTG GGTGGCTC 3/421 TGCCTAGTGT CCATCAGTTA AGCTTGAAAC TTCAGCACT CCACCATTCT CTCAGGTACT 3481 GTGACCCACT TGTGGATGCC CTACAACAAG GATCCAAAC ACCATTCAAG CATATGTTTG 3541 AAGATCCTGA GATCATAGCA GCTGCCATCC TTCTCCCTAA ATTTCCGGACC TCTTGGACAA 3601 ATGATGAAA.C CATCATAAAA CGAGGTAAAT GAATGCAAGC AACATACACT TGACGAATTC 3661 TAATCTGGGC AACCTTTGAG CCATACCAAA ATTATTCTTT TATTTATTTA TTTTTGCACT
3721 TTTTAGGAAT GTTATATCCC A'TCTTTGGCT GTGA'TCTCAA TATGA1ATATT GATGTAAAGT 3781 ATTCTTGCAG CAGGTTGTAG TTATCCCTCA GTGTTTCTTG AAACCAAACT CATATGTATC 3841 ATATGTGGTT TGGAAATGCA GTTAGATTTT ATGCTAAAT AAG G GATTTG CATGATTTTA 3901 GATGTAGATG ACTGCACGTA AATGTAGTTA ATGACAAAAT CCATPkAAATT TGTTCCCAGT 3961 CAGAAGCCCC TC1ACCAAAC TTTTCTTTGT GTCTGCTCAC TGTGCTTGTA GGCATGGACT 4021 ACATCAGAGT GCATCTGGAG CCTTTGGACC ACAAGAAGGA ATTGGCCIAAC AGTTCATCTG 4081 ATGATGAAGA TTTTTTCGCT TCTTTCIAAAC CGACA ACACA TGAAGCCAGC AAAGAGTTGG 4141 ATGGATATCT GGCCTGTGTT TCAGACACCA GGGAGTCTCT GCTCACGTTT CCTGCTArTTT 4201 GCAGCCTCTC TATCAAGACT AATACACCTC TTCCCGCATC GGCTGCCTCT GAGAGGCTTT 4261 TCAGCACTGC AGGATTGCTT TTCAGCCCCA AAAGAGCTAG GCTTGACACT AACPATTTTG 4321 AGAATCAGCT TCTACTGAAG TTAAATCTGA GGTTTTACA CTTTGAGTAG CGTGTACTGG 4381 CATTAGATTG TCTGTCTTAT AGTTT'GATAA JTTAAATACAA ACAGTTCTAA AGCAGGATIAA 4441 AACCTTGTAT GCATTTCATT TAATG1TTTTT TGAGATTAAA AGCTTAAACA AGAATCTCTA 4501 GTTTTCTTTC TTGCTTTTAC TTTTACTTCC TTAATACTCA AGTACAATTT TAATGGAGTA 4S61 CTTTTTTACT TTTACTCA-AG TAAGATTCTA GCCAGATACT TTTACTTTTA ATTGAGTPAA 4621 ATTTCCCTA AGTACTTGTA CTTTCACTTG AGTAAATTT TTGAGTACTT TTTACACCTC
4681 TG (SEQ ID NO: 31). HomologousRecombination
[02271 In certain embodiments of the methods of the disclosure, a modified CAR-Tsci or CAR-TcM of the disclosure is produced by introducing an antigen receptor into a primary human T cell of the disclosure by homologous recombination. In certain embodiments of the disclosure, the homologous recombination is induced by a single or double strand break induced by a genomic editing composition or construct of the disclosure. Homologous recombination methods of the disclosure comprise contacting a genomic editing composition or construct of the disclosure to a genomic sequence to induce at least one break in the sequence and to provide an entrypoint in the genomic sequence for an exogenous donor sequence composition. Donor sequence compositions of the disclosure are integrated into the genomic sequence at the induced entry point by the cell's native DNA repairmachinery.
102281 In certain embodiments of the methods of the disclosure, homologous recombination introduces a sequence encoding an antigen receptor and/or a donor sequence composition of the disclosure into a "genomic safe harbor" site. In certain embodiments. a mammalian genomic sequence comprises the genomic safe harbor site. In certain embodiments, a primate genomic sequence comprises the genomic safe harbor site. In certain embodiments, a human genomic sequence comprises the genomic safe harbor site.
102291 Genomic safe harbor sites are able to accommodate the integration of new genetic material in a manner that ensures that the newly inserted genetic elements function reliably
(for example, are expressed at a therapeutically effective level of expression) and do not cause deleterious alterations to the host genome that cause a risk to the host organism. Potential genomic safe harbors include, but are not limited to, intronic sequences of the human albumin gene, the adeno-associated virus site 1 (AAVS1), a naturally occurring site of integration of AAV virus on chromosome 19, the site of the chemokine (C-C motif) receptor (CCR5) gene and the site of the human ortholog of the mouse Rosa26 locus.
102301 In certain embodiments ofthe methods of the disclosure, homologous recombination introduces a sequence encoding an antigen receptor and/or a donor sequence composition of the disclosure into a sequence encoding one or more components of an endogenous T-cell receptor or a major histocompatibility complex (MH C). In certain embodiments, inducing homologous recombination within a genomic sequence encoding the endogenous T-cell receptor or the MIHC disrupts the endogenous gene, and optionally, replaces partof the coding sequence ofthe endogenous gene with a donor sequence composition ofthe disclosure. In certain embodiments, inducing homologous recombination within a genomic sequence encoding the endogenousT-cell receptor or the MHC disrupts the endogenous gene, andoptionally, replaces the entire coding sequence of the endogenous gene with a donor sequence composition of the disclosure. In certain embodiments of the methods of the disclosure, introduction of a sequence encoding an antigen receptor or a donor sequence composition of the disclosure by homologous recombination operably links the antigen receptor to an endogenous T cell promoter. In certain embodiments of the methods of the disclosure, introduction of a sequence encoding an antigen receptor or a donor sequence composition of the disclosure by homologous recombination operably links the antigen receptor or the therapeutic protein to a transcriptional or translational regulatory element. In certain embodiments of the methods of the disclosure, introduction of a sequence encoding an antigen receptor or a donor sequence composition of the disclosure by homologous recombination operably links the antigen receptor or the therapeutic protein to a transcriptional regulatory element. In certain embodiments, the transcriptional regulatory element comprises an endogenous T cell 5' UTR.
102311 In certain embodiments of the introduction step comprising a homologous recombination, a genomic editing composition contacts a genomic sequence of at least one primary T cell of the plurality of T cells. In certain embodiments of the introduction step comprising a homologous recombination, a genomic editing composition contacts a genomic sequence of a portion of primary Tcells of the plurality of T cells. In certain embodiments, the portion of primaryTcells isat least 1%, 2%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, %, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 97%, 99% or any percentage in between of the total number of primary T cells in the plurality of T cells. In certain embodiments of the introduction step comprising a homologous recombination, a genomic editing composition contacts a genomic sequenceof each primary T cell of the plurality of T cells. In certain embodiments of the introduction step comprising a homologous recombination, a genomic editing composition induces a single strand break. In certain embodiments of the introduction step comprising a homologous recombination, a genomic editing composition induces a double strand break. In certain embodiments of the introduction step comprising a homologous recombination, the introduction step further comprises a donor sequence composition. In certain embodiments, the donor sequence composition comprises a sequence encoding the antigen receptor. In certain embodiments., the donor sequence composition comprises a sequence encoding the antigen receptor, a5' genomic sequence and a 3' genomic sequence, wherein the 5' genomic sequence is homologous or identical to a genomic sequence of the primary T cell that is 5' to the break point induced by the genomic editing composition and the 3' genomic sequence is homologous or identical to a genomic sequence of the primary T cell that is 3' to the break point induced by the genomic editing composition. In certain embodiments, the 5' genomic sequence and/or the 3' genomic sequence comprises at least 50 bp, 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 600 bp, at least 700 bp, at least 800 bp, at least 900 bp, at least 1000 bp, at least I100 bp, at least 1200 bp, at least 1300 bp, at least 1400, or at least 1500 bp, at least 1600 bp, at least 1700 bp, at least 1800 bp, at least 1900 bp, at least 2000 bp in length or any length of base pairs (bp)in between, inclusive of the end points. In certain embodiments of the introduction step comprising a homologous recombination, the genomic editing composition and donor sequence composition are contacted with the genomic sequence simultaneously or sequentially. In certain embodiments of the introduction step comprising a homologous recombination, the genomic editing composition and donor sequence composition are contacted with the genomic sequence sequentially, and the genomic editing composition is provided first. In certain embodiments of the introduction step comprising a homologous recombination, the genomic editing composition comprises a sequence encoding a DNA binding domain and a sequence encoding a nuclease domain. In certain embodiments of the introduction step comprising a homologous recombination, the genomic editing composition comprises a DNA binding domain and a nuclease domain. In certain embodiments of the genomic editing composition, the DNA binding domain comprises a guide RNA (gRNA). In certain embodiments of the genomic editing composition, the DNA binding domain comprises a DNA-binding domain of aTALEN. In certain embodiments of the genomic editing composition, the DNA binding domain comprises a DNA-binding domain of a ZFN. In certain embodiments of the genomic editing composition, the nuclease domain comprises a Cas9 nuclease or a sequence thereof. In certain embodiments of the genomic editing composition, the nuclease domain comprises an inactive Cas9 (SEQ ID NO: 33, comprising a substitution of a Alanine (A) for Aspartic Acid (D) at position 10 (DIA) and a substitution of Alanine (A) for Histidine (H) at position 840 (-1840A)). In certain embodiments of the genomic editing composition, the nuclease domain comprises a short and inactive Cas9 (SEQ ID NO: 32, comprising a substitution of an Alanine (A) for an Aspartic Acid (D) at position 10 (D10A) and a substitution of an Alanine (A) for an Asparagine (N) at position 540 (N540A)). In certain embodiments of the genomic editing composition. the nuclease domain comprises or further comprises a type S endonuclease. In certain embodiments of the genomic editing composition, the type IIS endonuclease comprises Acil, Mu1, Alwl, BbvI, Bec, BceA, BsmA, BsmFlI BspCNI, Bsrl, BtsCI, 1gal, Hphl, HpyAV, MbolI, MylI, Plel, SfaNI, Acul, BciVI, BfuAI, BmgBI, Bmrl, Bpml BpuEI, Bsal, BseRl, BsgI, BsmI, BspMI, BsrBI, BsrBI, BsrDI, BtgZI, Bts, Earl, Ecil, Mme, NmeAIII, BbvCI, Bpul0I, BspQI, Sapi, Bael, BsaXI, CspCI, Bfil, MboII, Acc361, FokI or C1o051 In certain embodiments, the type IS endonuclease comprises C1o051. In certain embodiments of the genomic editing composition, the nuclease domain comprises or further comprises a TALEN or a nuclease domain thereof In certain embodimentsof the genomic editing composition. the nuclease domain comprises or further comprises a ZFN or anuclease domain thereof. In certain embodiments of the introduction step comprising a homologous recombination, the genomic editing composition induces a break in a genomic sequence and the donor sequence composition is inserted using the endogenous DNA repair mechanisms of the primary T cell. In certain embodiments of the introduction step comprising a homologous recombination, the insertion of the donor sequence composition eliminates a DNA binding site of the genomic editing composition, thereby preventing further activity of the genomic editing composition.
[02321 In certain embodinentsofthemethodsofhomologousrecombination ofthe disclosure, the nuclease domain of a genomic editing composition or construct is capable of introducing a break at a defined location in a genomic sequence of the primary human'Icell, and, furthermore, may comprise, consist essentially of or consist of, a homodimer or a heterodimer. In certain embodiments, the nuclease is an endonuclease. Effector molecules, including those effector molecules comprising a hoinodimer or aheterodimer, may comprise, consist essentially of or consist of, a Cas9, a Cas9 nuclease domain or a fragment thereof In certain embodiments, the Cas9 is a catalytically inactive or "inactivated" Cas9 (dCas9). In certain embodiments, the Cas9 is a catalytically inactive or "inactivated" nuclease domain of Cas9. In certain embodiments, the dCas9 is encoded by a shorter sequence that is derived from a full length, catalytically inactivated, Cas9, referred to herein as a "small" dCas9 or dSaCas9.
[02331 Incertainembodiments, the inactivated, small, Cas9 (dSaCas9) operatively-linkedto an active nuclease. In certain embodiments, the disclosure provides a fusion protein comprising, consisting essentially of or consisting of a DNA binding domain and molecule nuclease, wherein the nuclease comprises a small, inactivated Cas9 (dSaCas9)) In certain embodiments, the dSaCas9 of the disclosure comprises the mutations DI0A and N580A (underlined and bolded) which inactivate the catalytic site. In certain embodiments, the dSaCas9 of the disclosure comprises the amino acid sequence of 1 MKRNYILcGLA IGITSVGYGI IDYETRDVID AGVRLKEAN VENNEGRRSK RGARRLKRRR 61 RHRIQRVKKL LFDYNLLTDH SELSGINPYE ARVKGLSQKL SEEEFSAALL HLAKRRGVHN 121 VNEVEEDTGN ELSTKE0TSR NSKALEEKYV AELQLERLKK DGEVRGSINR FKTSDYVKEA 181 fIQLKVQKAY HQLDQSFIDT YIIDLLETRRT YYEGPGEGSP FGWKDIKEWY EMIIL MGHCTYF 241 PEELRSVKYA YNADLYNALN DLNNLVITRD ENEKLEYYEK FQIIENVFKQ KKKTLKQIA KEILVNEEDI KGYRVTSTGK PEE-TNLKVYH DI KDITARKE IIENAELLDQ IAKILTIYQS
361 SEDIQEELTN LNSELTQEEI EQISNLKGYT GTHNLSLKAI NLILDELWHT NDNQIAIFNR 421 LKLVPKKVDL SQQKEIPTTL VDDFILSPVV KRSFIQSIKV INAIIKKYGL PNDIIIELAR 481 EKNSKDAQKM INEMQKRNRQ TNERIEE II TTG-KENAKYL IEKRIKLHDMQ EGKCLYSLEA 541 IPLEDLLNNP FNYEVDHIIP RSVSFDNSFN NKVLVKQEEA SKKGNRTPFQ YLSSSDSKIS 601 YETFKKHILN LAKGKGRISK TKEYLLEER DINRFSVQKD FINRNLVDTR YATRGLMNLL 661 RSY FRVNNLD VKVKSINrGF TSFLRRKWKF KKERNKGYKH HAEDALIIAN ADFIFKEWKK 721 LDKAKKVMEN QMFEEKQAES MPETETEQEY KEIFITPHQI KHITKDF'KDYK YSHRVDKKPN 781 RELINDTLYS TRKDDKGNTL IVNNLNGLYD KDNDKLKKLI NKSPELLMY HHDPQTYQKL 841 KLIMEQYGDE KNPLYKYYEE TGNYLTKYSK KDNGPVIKKT KYYGNKLNAH LDITDDYPNS 901 RNKVVKLSLK PYREDVYLDN GVYKFVTVKN LDVIKKENYY EVNSKCYEEA KKLKKISNQA 961 EFIASFYNND LIKINGELYR VIGVNNDLLN RIEVNMIDIT YREYLENMND KRPPRIIKTI
1021 ASKTQSIKKY STDILGNLYE VKSKKHEQIT KKG (SEQ ID NO: 32).
[02341 In certain embodiments, the dCas9 of the disclosure comprises a dCas9 isolated or derived from Staphyloccocus pyogenes. In certain embodiments, the dCas9 comprises a dCas9 with substitutions at positions 10 and 840 of the amino acid sequence of the dCas9 which inactivate the catalytic site. In certain embodiments, these substitutions are DOA and H1840A. In certain embodiments, the amino acid sequence of the dCas9 comprises the sequence of XDKKYSIGLA IGTNSVGWAV ITDEYKVPSK KF'KVLGNTDR HSIKKNLIGA LLFDSGETAE
61 ATRLKPTARR RYTRRKNRIC YLQEIFSNEM AKVDDSFFHR LEESFLVEED KKHERHPTFG 121 INIVDEVAYHE KYPTIYHLPK KLVDSTDKAD LRLITYLAAH MIKFRGHFL1 EGDLNPDNSD 181 VDKLFIQLVQTYNQLFEENP INASGVDAKA ILSARLSKSR RLELAQLP GEKKNGLFG.N 241 LIALSLGLTP NFKSNFDLAE DAKLQLSKDT YDDDLDNLLA Q:GDQYADLF LAAKNLSDAI 301 LLSDILRVNT EITKAPLSAS MIKRYDEHHQ DLTLLKALVR QQLPEKYKEI FFDQSKNGYA 361 GYIDGGASQE EFYKF'IKPIL EKMDGTEELL VKLNREDLLR KQRTFDNGSI PHQIHLGELH 421 AILRRQEDFY PFLKDNREKI EKILTFRIPY YVGPLARGNS RFAWMTRKSE ETITPWNEEE 481 VVDKGASAQS FIERMTNEDK NLPNEKVLPK HSLLYEYFTV YNELTKVKYV TEGIMRKPAFL 541 SGEQKKA7VD LLEFKTNRKVT VKQLTKEDYFK KIECFDSVEI SGVEDRFNAS LGTYHDLLKI
601 TKDKDFLDNE ENEDILEDIV LTLTLFEDRE M'IEERLKTYA MLFDDKVMKQ LKRRRYTGWG
661 RLSRKLINGI RDKQSGKTIL DFLKSDGFAN RNFMQLIHDD SLTF'KEDIQK AQVSGQGDSL
721 HEHIANLAGS PATfKKGILQT VKVVDELVKV MGRHKPENIV IEMARENQTT QKGQKNSRER
781 MKREEGIKE LGSQILKEP VENTQLQNEK LYLYYLQNGR DMYVDQELDI NRLSDYDVDA
841 IVPQSFLKDD SIDNKVLTRS DKNRGKSDNV PSEEVVKKMK NYWRQLLNAK LITQRKFDNL
901 TAEARGGLSE LDKAGF'KTRO LVETROITKH VAQILDSRMEN TKYDENDKLI REVEVaITLKS
961 KLVSDFRKDF QFYKVREINN YHHAHDAYLN AVVGTALIKK YPKLESEFVY GDYEVlYDVRK
1021 MIAKSEQEIG KATAKYFFYS NIMNFFKTEI TLANGEIRKR PLIETNGETG EIVWDKGRDF 1081 ATVRKVLSMP QVNIVKKTEV Q' TGGFSKESI LPKRNSDKL1 ARKKDWDPKK YGGFDSPTVA
1141 YSVLVVAKVE KGKSKKKSV KELLGITIME RSSFEKNPID FLEAKGYKEV KKDLIIKLPK
1201 YSLFELENGR KRMVLASAGEL QKGNELALPS KYVNFLYLAS MYEKLKGSPE DNEQKQLFVE 1261 QHKHYLDEII EQISEFSKRV ILADANLDKV LSANYNKHRfDK PIREQAENII HLFTLTNLGA
1321 PAAFKYFDTT IDRKRYTSTK EVLDATLIHQ SITGLYETRI DLSQLGGD (SEQ ID NO: 33).
[02351 In certain embodiments of the disclosure, the nuclease domain may comprise, consist essentially of or consist of a dCas9 or a dSaCas9 and a type IIS endonuclease. In certain embodiments of the disclosure, the nuclease domain may comprise, consist essentially of or consist of a dSaCas9 and a type IIS endonuclease, including, but not limited to, AciI, MnlI. AlI, BbvI, Bccl, BceAI, BsmAl. BsmF, BspCNI, Bsrl, BtsCI, Hgal, Hph, HpyAV, MboM, MyI1, Plel, SfaNI, Acul, BeiVI, BfuAl, BmgBI, BmrI, BpmI, BpuEI. BsaI, BseRI, BsgI, BsmI, BspMI, BsrBI, BsrBI, BsrDI, BtgZI, Btsl, Earl, Ecil, Mmel, NmeAIII. BbvCI, Bpul01, BspQI. SapI, Bae, BsaX, CspCI, BfiI. Mbol, Acc36I, FokI or Clo051. In certain embodiments of the disclosure, the nuclease domain may comprise, consist essentially of or consistof a dSaCas9 and Clo05I.Anexemplary C10051 nuclease domain may comprise, consist essentially of or consist of, the amino acid sequence of: EGIKSNISLLKDELRGQISHISI-EYLSLIDLAFDSKQNRLFEMKVLELLVNEYGFKGRI LGGSRKPDGIVYSTTLEDNFGIIVDTKAYSEGYSLPISQADEMERYVRENSNRDEEVN PNKWWENFSEEVKKYYFVFISGSFKGKFEEQLRRLSMTTGVrNGSAVNVVNLLLGAE KIRSGEMTIEELERAMFNNSEFILKY (SEQ ID NO: 34).
102361 An exemplarydCas9-Clo051 nuclease domain may comprise. consistessentiallyof or consist of, the amino acid sequence of (Co05 sequence underlined, linker bold italics, dCas9 sequence in italics): MAPKKKRKVEGIKSNISLLKDELRGQISHISHEYLSLIDLAFDSKQNRLFEMKVLELL VNEYGFKGIMLGGSRKPDGIVYSTI'LEDNFGIIVDTKAYSEGYSLPISQADEMERY\VR ENSNRDEEVNPNKXWENFSEEVKKYYFVFISGSFKGKFEEQRRSMTj NGSAV NVVNLLLGAEKIRSGEMTIEELERAMFNNSEFILKYGGGGSDKKYSGLAIGTSVGWA VITDEYKVPSKKF KVLGNTDRH SIKKNLIG ALLPDSGE7AEATRLKRARRRYTRRKNRICY LQl§IFSNEMAKVDDSFFHRLELESFLVEEDKKHR RHPIFGNIV'DEV'AYHEKP1YHLRKKL VD)STDKAD)LRLYL ALAIIK1RGF JLEGDLNPDNSDVKLF[QLVQTYNQLF1EENPIA SGDAAILSARLSKSRRLENLAQLPGFKKNTGLFGNIALSLGLTPNFKSNA/F)IAEDAKLQ LSKDTYDDDLDNLLAQIGDQY ADLFLA AKNLSDAILSDILRNTETKAPLJSASMIKRYDE HHfQDLTLLKALVRQQLPEKYKEJFDQKGYGYDGGASQEEYKFIKPILEKMvDG1E ELLVKLNREDLLRKQRTF)NGSPHQILGELH-IAJILRRQEDFYPFLKDNREKIEKILTFRIP YYV'GPL ARGNSRFAWMITIRKE EIJ77PWFEEVVDKGASAQSFiERM~flTDKNLPNE§KVLP KH1-SLLYEYTVNELTKVKYVEGMRKPAF'LSGEQKKAIVDLLFKTNRKV"TVKQLKEDYF
IEERLKTYA LFDDKMKQLKRRRYTCWGRLSRKJJvGIRDKQSGKTIDFLKSDGFANTR NF'MQLIH-DDSLTFKEDIQOKQVSGQGD)SLHEH-IANLAGiSPAIKKGILQTVKVDELV'KVM GRHKPELNIVJI ARENQTTQKGQKSRERMJKRIEEGK ELGSQILKHPVENT'QLQN'EKL YLYYLQNGRDVEDQELDINRLSDEDVD)AIVPQSF LKDDSDNKVT RSKNRGKSDNVP SEEVVKKMJKNYRQLNAKLTQRKFDLTKAERGGLSELDKAFKRQLVETRQITKH-V AQI LDSRMV7KYDE NDKIREKVI7LKSKLVSDRKDQFKVR AE-YHLHAHDAYLNAV VG A LIKKYPKLESEFVYGDYEKVYDRKMLFAKSEQEIGK4AKYFbFYSNLANFFKIEIT LAN GEIRKRPLJKTNGETGEIVWDKGFRDFATVRKVLSMVPQV'NIVKKT EVQTGGSKSIL.PKR N SDKLIAIRKKDWDPKKYGGF'DSPTVAEWSLVVAKiTE KGKSKKLKSVKELLGI77MERSSJEK NPIDFLEAKGYKEVKK)LHJKLPKYSL'ELLNGRKRML4SAGELQKGNLL4LPSKYVNFLY
LASHYEKLKGSPEDNEQKQLFVEQHKHYLL)ElEQISFSKRVILADA'LDKVLSAYNKHR DKPIRkQAEH'IHLFTTNLGAPAAFKYFJ)TTJZDRKRYTSTKEVLD ATIJIJQSITGLYETRiJIDL SQLGGDGSPKKKRKVSS (SEQ ID NO: 40).
[0237] In certain embodiments, the nuclease capable of introducing a break at a defined location in the genomic DNA of the primary human Tcell may comprise, consist essentially of or consist of, a homodimer or a heterodimer. Nuclease domains of the genomic editing compositions or constructs of the disclosure may comprise, consist essentially of or consist of a nuclease domain isolated, derived or recombined from a transcription-activator-like effector nuclease (TALEN). TALENs are transcription factors with programmable DNA binding domains that provide a means to create designer proteinsthat bind to pre-determined DNA sequences or individual nucleic acids. Modular DNA binding domains have been identified in transcriptional activator-like (TAL) proteins, or, more specifically, transcriptional activator-like effector nucleases (TALENs), thereby allowing for the de novo creation of synthetic transcription factors that bind to DNA sequences of interest and, if desirable, also allowing a second domain present on the protein or polypeptide to perform an activity related to DNA. TAL proteins have been derived from the organisms Xanthomonas and Ralsonia.
[0238] In certain embodiments ofthe disclosure, the nuclease domain of the genomic editing composition or construct may comprise, consist essentially of or consist of a nuclease domain isolated, derived or recombined from a TALEN and a type IIS endonuclease. In certain embodiments of the disclosure, the type IIS endonuclease may comprise, consist essentially of or consist of Acil, MnllI, AlwI, BbvI, BccI, BceAI, BsmAI, BsmFI, BspCNI, Bsrl, BtsCl, Hgal, Hphl, HpyAV, Mboll, Myll, PleI, SfaNI, Acul, BciVI, BfuA, BmgBI, Bmr, BpmI, BpuEI, Bsal, BseRI, BsgI, Bsml, BspMI, BsrBI, BsrBI, BsrDI, BtgZI, Bts, Earl, EciI, MmeI, NmeAIll, BbvCI, Bpul01, BspQI, SapI, Bael, BsaXI, CspCI, Bfil, MboII, Acc36I, FokI or C1o051. In certain embodiments ofthe disclosure, the type IIS endonuclease may comprise, consist essentially of or consist of Clo051 (SEQ ID NO: 34).
[0239] In certain embodiments of the disclosure, the nuclease domain of the genomic editing composition or construct may comprise, consist essentially of or consist of a nuclease domain isolated, derived or recombined from a zinc finger nuclease (ZFN) and a type IIS endonuclease. In certain embodiments of the disclosure, the type IIS endonuclease may comprise, consist essentially of or consist of Acil, Mnll, AlwI, BbvI, Bccl, BceAl, BsmAl, BsmFI, BspCNI, Bsr, BtsCI, Hgal, iphI, HpyAV, MbolI, MylI, Plel, SfaN, Acul, BciVI, BfuAl, BmgBI, BmrI, BpmL BpuEl, BsaL, BseRI, BsgL, BsmI, BspML BsrB, BsrBl, BsrDI,
BtgZI, Bts, Earl, Ecil, MmeI, NmeAIll. BbvCI, Bpu101, BspQl, Sapi, Bael, BsaXI, CspCI, BfiI, MboHI, Acc361, FokI or Clo051. In certain embodiments of the disclosure, the type IIS endonuclease may comprise, consist essentially of or consist of Co051 (SEQ ID NO: 34).
[02401 In certain embodiments of the genomic editing compositions or constructs of the disclosure, the DNA binding domain and the nuclease domain may be covalently linked. For example, a fusion protein may comprise the DNA binding domain and the nuclease domain. In certain embodiments of the genomic editing compositions or constructs of the disclosure, the DNA binding domain and the nuclease domain may be operably linked through anon covalent linkage. Secreted PrteinsfromModfied I'Cells
[02411 In certain embodiments of the composition and methods of the disclosure, modified T-cells express therapeutic proteins. Therapeutic proteins of the disclosure include secreted proteins. Preferably, in a therapeutic context, the therapeutic protein is a human protein, including a secreted human protein. When expressed or secreted by CAR-T cells of the disclosure, the combination comprising the CAR-T cell and the therapeutic protein secreted therefrom may be considered a monotherapy. However, the CAR-T cells of the disclosure may be administered as a combination therapy with a second agent. A database of human secreted proteins that may be expressed or secreted by modified T-cell of the disclosure can be found at proteinatlas.org/search/proteinclass:Predicted%20secreted%20proteins, the contents of which are incorporated herein by reference. Exemplary human secreted proteins are provided, but are not limited to the human secreted proteins, in Table 1.
102421 TABLE 1. Exemplary Human Secreted Proteins Gene Ensemb] ID Gene description AIBG ENSG00000121410 Alpha-I-B glycoprotein A2M ENSG00000175899 Alpha-2-macroglobulin A2MLI ENSG00000166535 Alpha-2-macroglobulin-like 1 A4GNT ENSG00000118017 Alpha-1,4-N-acetylgiucosaminyltransfemse AADACL2 ENSG00000197953 Atylacetamide deacetylase-like 2 AANAT ENSG00000 129673 Aralkylamine N-acetyitransferase ABCGI ENSG00000160179 ATP-binding cassette, sub-famly G(WIifTE), members ABHDI ENSG00000143994 Abhydrolase domain containing I ABIHD1)10 ENSG00000144827 Abhydrolase domain containing 10 ABID14A ENSG00000248487 Abhydrolase domain containing 14A ABHD15 ENSG00000168792 Abhvdrolase domain containing 15 AB13BP ENSG00000154175 ABI family, member 3 (NESH) binding protein AC008641.1 ENSG00000279109
AC009133.22 ENSG00000277669 AC009491.2 ENSG00000279664 AC011513.3 ENSG00000267881 AC136352.5 ENSG00000277666 AC145212.4 ENSG00000277400 MaFF-interacting protein AC233755.1 ENSG00000275063 ACACB ENSG00000076555 Acetyl-CoA carboxylase beta ACAN ENSGO000015766 Aggrecar ACE ENSGO0000159640 Angiotensin . converting enzyme ACHE ENSG00000087085 Acetylcholinesterase (Yt blood group) ACP2 ENSGO0000 134575 Acid phosphalase 2, lysosotal ACP5 ENSG00000102575 Acid phosphatase 5, tartrate resistant ACP6 ENSG00000162836 Acid phosphatase 6, iysophosphatidic ACPP ENSG00000014257 Acid phosphatase, prostate ACR ENSG0000100312 Acrosin ACRBP ENSGOO00111644 Acrosiri binding protein ACRV1 ENSG00000134940 Acrosomal vesicle protein I ACSF2 ENSG00000167107 Acyl-CoA synthetase family member 2 ACTLio ENSG00000182584 Actin-like 10 ACVRI ENSG00000115170 Activin A receptor, type I ACVTRC ENSGOO00123612 Activin A oceptor, type IC ACVRLI ENSGO0000139567 Activin A receptor type II-like I ACYPi ENSGO0000119640 Acylphosphntase 1, erYthrocyte (connon) ty'pe ACYP2 ENSGO0000170634 Acylphosphatase 2, muscle type ADAM10 ENSG00000137845 ADAM metallopeptidase domain 10 ADAM12 ENSG0000148848 ADAM netallopeptidase domain 12 ADAM15 ENSG00000143537 ADAM muetallopeptidase domain 15 ADAM17 ENSGO0000151694 ADAM metallopepldase domain 17 ADAM18 ENSG00000168619 ADAM metallopeptidase domain 18 ADAM22 ENSG00000008277 ADAM metallopeptidase domain 22 ADAM28 ENSG00000042980 ADAM rmetallopeptidase domain 28 ADAM29 ENSG00000168594 ADAM muetallopeptidase domain 29 ADAM32 ENSGO0000197140 ADAM mnetallopeptidase domain32 ADAM33 ENSG00000149451 ADAM metallopeptidase domain 33 ADAM7 ENSG00000069206 ADAM metallopeptidase domain 7 ADAM8 ENSG0000151651 ADAM metallopeptidase domain 8 ADAM9 ENSG00000168615 ADAM metallopeptidase domain 9 ADAMDECI ENSGO0000134028 ADAM-like, decysin I ADAMTS1 ENSG00000154734 ADAM mnetallopeptidase with thrombospondin type 1 rmotif, 1 ADAMTS10 ENSG00000142303 ADAM metallopeptidase with thrombospondin type I motif. 10 ADAMITS12 ENSGOOOOO151388 ADAM rnetallopeptidase with thrombospondintype I motif, 12 ADAMTS13 ENSGO0000160323 ADAM netallopeptidase with thrornbospondin type I motif, 13 ADAMTS14 ENSG0000138316 ADAM metallopeptidase with thrombospondin type I motif, 14 ADAMTS15 ENSG00000166106 ADAM mnetallopeptidase with thrombospondin type 1 motif. 15
ADAMTS16 ENSG000145536 ADAM mnetallopeptidase with thronbospondin type I motif, 16 ADAMTS17 ENSG00000140470 ADAM metallopeptidase with thrombospondin type I motif, 17 ADAMTS18 ENSiO0000140873 ADAM metallopeptidase with throimbospondin type 1 motif, 18 ADAMTS19 ENSG00000145808 ADAM metallopeptidase with thrombospondin type I motif. 19 ADAMTS2 ENSG00000087116 ADAM metallopeptidase with thrombospondintype I motifE 2 ADAMTS20 EN SG00000173157 ADAM metallopeptidase with thronbospondin type I motif, 20 ADAMTS3 ENSG00000156140 ADAM metallopeptidase with thrombospondin type I motif 3 ADAMTS5 ENSG00000154736 ADAM metallopeptidase with thrombospondin type 1 motif, 5 ADAMTS6 ENSG00000049192 ADAM metallopeptidase with thrombospondin type 1 motif. 6 ADAiMTS7 ENSGO0000136378 ADAM metallopeptidase with thrombospondintype I motif 7 ADAMTS8 ENSG00000134917 ADAM metallopeptidase with thrombospondin type I motif, 8 ADAMTS9 ENSG00000163638 ADAM metallopeptidase with thrombospondin type I motif, 9 ADAMTSL1 ENSG00000178031 ADAMTS-like I ADAMTSL2 ENSG00000197859 ADAMTS-like 2 ADAMTSL3 ENSGO0000156218 ADAMTS-iike 3 ADAMTSL4 ENSG0000143382 ADAMTS-iike 4 ADAMTSL5 ENSG00000185761 ADAMTS-like 5 ADCKI ENSG00000063761 AarF domain containing kinase 1 ADCYAPI ENSG00000141433 Adenylate cyclase activating polypeptide 1 (pituitary) ADCYAPIRI ENSG00000078549 Adenvylate cyclase activating polypeptide I (pituitary) receptor type I ------------ ADGRA3 ENSG00000152990 Adhesion G protein-coupled receptor A3 ADGRB2 ENSG00000121753 Adhesion G protein-coupled receptor B2 ADGIRDI ENSG00000111452 Adhesion G3 protein-coupled receptor 1 ADGRE3 ENSG00000131355 Adhesion G protein-coupled receptor E3 ADGRE5 ENSG00000 123146 Adhesion G protein-coupled receptors 5 ADGRFI ENSG00000153292 Adhesion G protein-coupled receptor F1 ADGRGI ENSG00000205336 Adhesion G protein-coupled receptor G1 ADGR(G5 ENSG00000159618 Adhesion G protein-coupled receptor G35 ADGRG6 ENSG00000112414 Adhesion G protein-coupled receptor G6 ADGRVI ENSG0000164199 Adhesion G protein-coupled receptor VI ADII ENSG00000182551 Acieductone dioxygenase 1 ADIG ENSG00000182035 Adipogenin ADIPOQ ENSG00000181092 Adiponectin, C IQ and collagen domain containing ADM ENSG00000148926 Adrenomedullin ADM2 ENSG0000 128165 Adrenomedullin2 ADM5 ENSG00000224420 Adrenomedullin 5 (putative) ADPGK ENSG00000159322 ADP-dependent glucokinase ADPRIL2 ENSGOOOOO116863 ADP-ribosvlhvdrolase like 2 AEBPI ENSGOO000 106624 AE binding protein I AFM ENSG00000079557 Afamin AFP ENSG00000081051 Alpha-fetoprotein AGA ENSG00000038002 Aspartylglucosaminidase ACER ENSG00000204305 Advanced glycosylation end product-specific receptor AGK ENSG00000006530 Acylglycerol kinase
AGPS EN SG00000018510 Alkylglycerone phosphate synthase AGR2 ENSG00000 106541 Anterior gradient 2, protein disulphide isomerase family member AGR3 ENSG00000173467 Anterior gradient 3. protein disulphideisomlem-se family member AGRN ENSG00000188157 Agrin AGRP ENSGO0000159723 Agouti related neuropeptide AGT ENSG00000135744 Angiotensinogen (serpinpeptidase inhibitor, clade A, member
AGTPBPI ENSG00000135049 ATPi/GTP binding protein AGTRAP ENSG00000 177674 Angiotensin II receptor-associated protein AfCYL2 ENSG00000 158467 Adenlosy [homocy steinase-like 2 AHSG ENSG00000145192 Alpha-2-H{S-glycoprotein AIGI ENSGOOOOO146416 Androgen-induced I AK4 ENSG00000162433 AdenylatemkiMase 4 AKAPIO ENSG00000108599 A kinase (PRKA) anchorprotein 10 AKR 1C1 ENSG00000187134 Aldo-keto reductase family 1, member C1 AL356289.1 ENSG00000279096 AL589743.1 ENSG00000279508 ALAS2 ENSG00000158578 5-aminolevlinate synthase 2 ALB ENSGO0000163631 Albumin ALDH9A1 ENSG00000)143149 Aldehyde dehydrogenase 9 family, member Al ALDOA ENSG00000 149925 Aldolase A, fructose-bisphosphate ALGI ENSGO0000033011 ALGI, ciitobiosyldiphosphodolichol beta-mannosyltmnsferase ALG5 ENSG00000120697 ALG5, dolichyl-phosphate beta-glucosiransfemse ALG9 ENSG00000086848 ALG9, alpha-1,2-mannosltransferase ALKBHI1 ENSGOOOOO100601 AlkB homolog 1, histone H2A dioxygenase ALKBH5 ENSG00000091542 AlkB homolog 5. RNA demethylase ALPI ENSG00000163295 Alkaline phosphatase, intestinal ALPL ENSG0000162551 Alkaline phosphatase, liver/bone/kidney ALPP ENSG00000163283 Alkaline phosphatase, placental ALPPL2 ENSGOOOOO163286 Alkaline phosphatase, placental-like 2 AMBN ENSG00001178522 Ameloblastin (enamel matrix protein) AMBP ENSG00000106927 Alpha-1-microglobuiin/bikunin precursor AMELX ENSG0000125363 Amelogenin, X-linked AMELY ENSGO0000099721 Amelogenin Y-inked AMH-1 ENSGOOOOO104899 Anti-Mullerianhonnone AMICAI ENSG00000)160593 Adhesion molecule, interacts with CXADR antigen I AMPDI ENS G00000116748 Adenosine monophosphate deaminase I AMTN ENSGO0000187689 Amelotin AMYIA ENSG00000237763 Amylase, alpha IA (salivary) AMYlB ENSGOOOOO174876 Amylase, alpha 1B (salivaiy) AMY IC ENSG00000 187733 Amylase, alpha 1C (sai vaC. )
AMY2A ENSG00000243480 Amylase alpha 2A (pancreatic) AMY2B ENSG00000240038 Amylase, alpha 2B (pancreatic) ANG ENS G00000214274 Augiogenin, ribonouclease, RNase A family, 5
ANGELI ENSG00000013523 Angel homolog I (Drosophila) ANGPTI ENSG00000154188 Angiopoietin I ANCPT2 ENSG00000091879 Angiopoietin 2 ANGPT4 ENSG00000101280 Angiopoietin 4 ANGPTL1 ENSG0000116194 Angiopoictin-like I ANGPTL2 ENSGO00000136859 Angiopoietin-like 2 ANGPTL3 ENSGO0000132855 Angiopoietin-like 3 ANGPTL4 ENSG000016772 Angiopoietin-like 4 ANGPTL5 ENSG00000187151 Angiopoietin-like 5 ANGPTL6 ENSG0000 130812 Angiopoictin-like 6 ANGPTL7 ENSG00000 171819 Angiopoletin-like 7 ANKI ENSG00000029534 Ankyrin 1, erythrocytic ANKDD1A ENSG00000166839 Ankynn rireeat and death domain containing IA ANKRD54 ENSG00000100124 Ankyrin repeat domain 54 ANKRD60 ENSG0000 124227 Ankyrin repeat domain 60 ANO7 ENSG00000 146205 Anoctarnir ANOSI ENSG00000011201 Anosmin 1 ANTXR I ENSGO0000169604 Anthrax toxin receptor I AOAH ENSG00000136250 Acyloxyacyi hydrolase neutrophili) AOCI ENSG00000002726 Amine oxidase, copper containing I AOC2 ENSG00000131480 Amine oxidase, copper containing 2 (retina-specific) AOC3 ENSG00000131471 Amine oxidase, copper containing 3 AP000721.4 ENSG00000256100 AP000866.1 ENSG00000279342 APBBI ENSG00000166313 Amyloid beta (A4) precursor protein-binding, family B, member I (Fe65) APCDD1 ENSGO00000154856 Adenomnatosis polyposis coli down-regulated 1 APCS ENSG00000132703 Amyloid P component, serum APELA ENSG00000248329 Apelin receptor early endogenous ligand APLN ENSG00000171388 Apelin APLP2 ENSG00000084234 Amyloid beta (A4) precursor-like protein 2 APOA I ENSG00000118137 Apolipoproteini A-i APOAIBP ENSG00000163382 Apolipoprotein A-I binding protein APOA2 ENSG00000158874 Apolipoprotein A-II APOA4 ENSG00000110244 Apolipoprotein A-IV APOA5 ENSGO0000110243 Apolipoprotein A-V APOB ENSG00000084674 Apolipoproteir B APOCI ENSG00000130208 Apolipoprotein C-i APOC2 ENSG00000234906 Apolipoprotein C-lI APOC3 ENSG00000110245 Apolipoprotein C-IlI APOC4 ENSG00000267467 Apolipoprotein C-IV APOC4-APOC2 ENSG00000224916 APOC4-APOC2 readthrough (NM]D candidate) APOD ENSG00000189058 Apolipoprotein D APOE ENSG00000 130203 Apolipoprotein E APOF ENSG00000175336 Apolipoprotein F
APOH ENSG00000091583 Apolipoprotein H (beta-2-glycoprotein I) APOL1 ENSG00000100342 Apolipoprotein L, I APOL3 ENSG0000128284 Apolipoprotein L, 3 APOI ENSG00000204444 Apolipoprotein M APOOL ENSGO0000155008 Apolipoprotein O-like ARCNI ENSG000000095139 Archain I ARFIP2 ENSG00000132254 ADP-ribosylation factor interactingprotein 2 ARHGAP36 ENSG00000147256 Rho GTPase activating protein 36 ARIHGAP6 ENSG00000047648 Rho GTPase activating protein 6 ARHGEF4 ENSG00000136002 Rho guanine nucleotide exchange factor (GEF) 4 ARL.16 ENSG00000214087 ADP-rbosylation fIctor-like 16 ARMC5 ENSG00000140691 Annadillo repeat containing 5 ARNTL ENSG0000133794 A[y Ihydrocarbon receptor nuclear translocator-like ARSA ENSG00000100299 Atylsuilfatase A ARSB ENSGOOOOO113273 Arylsulfatase B ARSE ENSG00000 157399 Arvlsulfatase E (chondrodysplasia punctata 1) ARSG ENSG00000141337 Arylsulfatase G ARSI ENSG00000183876 AryIsulfatase family. member I ARSK ENSG00000164291 Aryisulfatase family, member K ART3 ENSG00000156219 ADP-ribosyltransferase 3 ART4 ENSGOOOO 111339 ADP-bosyltransfemase 4 (Dombrock blood group) ART5 ENSG00000167311 ADP-ribosyltransferase 5 ARTN ENSG00000117407 Artemin ASA-I1 ENSGOOOOO104763 N-acylsphingosine amidohydrolase (acid cemmrnidase) I ASAH2 ENSG00000188611 N-acylsphingosine amidohydrolase (non-lysosomal ceramidase) 2 ASCL1 ENSG00000139352 Achaete-scute family blE-H transcription factor 1 ASIP ENSG00000101440 Agouti signaling protein ASPN ENSiO0000106819 Asporin ASTL ENSG00000188886 Astacin-like metallo-endopeptidase (M12 family) ATAD5 ENSGOOOOO176208 ATPase family, AAA domain containing 5 ATATI ENSG00000137343 Alpha tnbulin acetyltransferase I ATG2A ENSG00000110046 Autophagy related 2A ATG5 ENSG00000057663 Atnophagy related 5 A TMIN ENSG00000166454 ATM interactor ATP13AI ENSG00000105726 ATPase type 13A1 ATP5F1 ENSG00000116459 ATP synthase, H+ transporting, mitochondrial Fo complex subunit B I ATP6AP I ENSG00000071553 ATPase, H transportinig,l ysosonrial accessory protein I A TP6AP2 ENSG0000182220 ATPase, H+ transporting, lysosonal accessory protein 2 ATPAFI ENSG00000123472 ATP synthase mitochondrial Fl complex assembly factor I AUH ENSGOOOOO 148090 AU RNA binding protein/enoyi-CoA hydratase AVP EN SG00000101200 Arginine vasopressin AXIN2 ENSG00000168646 Axin 2 AZGPI ENSii00000160862 Alha-2-glycoprotein 1, zinc-binding
AZUl ENSG0000(0172232 Azurocidin I B2M ENSG00000166710 Beta-2-microglobulin B3GALNT1 ENSG00000169255 Beta-1,3-N-acetylgalactosaninyltmisferase 1 (globoside blood group) B3GALNT2 ENSG00000162885 Beta-1,3-N-acetylgalactosaminvltransferase 2 B30ALT1 ENSG00000172318 UDP-Gal:betaGlcNAc beta 1,3-galactosyltransferase, polypeptide 1 B3(GALT4 ENSG00000235863 UDP-Gal:betaGicNAc beta 13-galactosvlmnsferase _______________ ___________________polypeptide 4 B3GALT5 ENSG00000183778 UDP-Gal:betaGlcNAc beta 1,3-galactosyltransferase, polypeptide 5 B30sALT6 ENSG00000176022 UDP-Gia:betaGal beta 1,3-galactosyltransferase polypeptide 6 B3GAT3 ENSGOt000149541 Beta-1,3-glucuronytmnsfease 3 B3GLCT ENSG00000187676 Beta 3-glucosyltransferase B!GNT3 BNS(100000179913 U P-cNcba(ibea1,3-N acelylglucosamninyltransferase 3 B3GNT4 ENSGO0000 176383 UDP-GicNAc:betaGal beta-1,3-N acetylgiucosaminyltransferase 4 B3GNT6 ENSGO0t00198488 UDP-GIcNAc:betaGal beta-1,3-N acetylglucosaminltransferase 6 B3GNT7 ENSG00000 156966 UDP-GlcNAc:betaGalbeta-1,3-N acetylglucosamninyltransferase 7 B3GNT8 ENSG00000 177191 UDP-GIcNAc:betaGal beta-1,3-N acetvlglucosaminyiltransferase 8 B3GNT9 ENSG00000237172 UDP-GIcNAc:betaGal beta-1,3-N acetylglucosaminltransferase 9 B4GALNT1 ENSGO0000135454 Beta-l,4-N-acety'l-galactosaminyl transferase I B4GALNT3 ENSG00000139044 Beta-i.4-N-acetyl-gaiactosamninyl transferase 3 B4GALNT4 ENSGO0000182272 Beta-1,4-N-acet yl-galactosaminyl transferase 4 B4GALT4 ENSGO0000121578 UDP-Gal:betaGlcNAc beta 1,4- galactosyitransferase polypeptide 4 B4GALT5 ENSG00000158470 UDP-Gal:betaGicNAc beta 1,4- galactosyltransferase, polypeptide 5 B4GALT6 ENSGO0000118276 UDP-Gal:betaGlcNAc beta 1,4- galactosytransfease, ------- _-------------_ polypeptide 6 B4GATI ENSGO0000 174684 Beta-1,4-glucuronyitransferase 1 B9D1) ENSG0000108641 B9 protein domain 1 BACE2 ENSG00000182240 Beta-site APP-cleaving enzyme 2 BAGE5 ENSG00000279973 B melanoma antigen family, member 5 BCAM ENSG00000187244 Basal cell adhesion molecule (Lutheran blood group) BCAN ENSGO000132692 Brevican BCAP29 ENSG0000075790 B-cell receptor-associated protein 29 BCAR1 ENSG0000050820 Breast cancer anti-estrogen resistance I BCIE ENSG00000114200 Butyrylcholiresterase BCKDHB ENSGO0000083123 Branched chain keto acid dehvdrogenase El, beta polypeptide BDNF ENSGOOOOO 176697 Brain-derived neurotrophic factor BGILAP ENSG00000242252 Bone ganma-carboxyglutanate (gla) protein BGN ENSG00000182492 Biglycan BL.VRB ENSG00000090013 Biliverdin reductase B BMPI ENSGO0000168487 Bone morphogenetic protein 1
BMP10 ENSG00000163217 Bone morphogenetic protein 10 BMP15 ENSG00000130385 Bone morphogenetic protein 15 BMP2 ENSG00000125845 Bone morphogenetic protein 2 BMP3 ENSG00000152785 Bone morphogenetic protein 3 BMP4 ENSG00000125378 Bone mnorphogenetic protein 4 BMP6 ENSG00000153162 Bone morphogenetic protein 6 BMP7 ENSG00000101144 Bone morphogenetic protein 7 BMP8A ENSG00000183682 Bone rmorphogenetic protein 8a BMP8B ENSG00000 116985 Bone norphogenetic protein 8b BMIIPER ENSG0000 164619 BMP binding endothelial regulator BNC1 ENSG00000169594 Basonuclin I BOC ENSGO0000144857 BOC cell adhesion associated, oncogene regulated BODI ENSG00000145919 Biorientation of chromosomes in celi division 1 BOLAI ENSG 0000178096 BoIA familymember I BPI ENSG0000010 1425 Bactericidai/penneability-incrasing protein BPIFA1 ENSG00000198183 BPI fold containing fImily A, member I BPIFA2 ENSG00000131050 BPIfold containing-family A, member BPIFA3 ENSG00000131059 BPI fold containing family A, member 3 BPIF13 ENSG00000125999 BPI fold containing family B, member 1 BPIFB2 ENSG00000078898 BPI fold containing family B, member 2 BPIFB3 ENSG00000186190 BPI fold containing family B, member 3 BPIFB4 ENSG00000186191 BPI fold containing family B, member 4 BPIFB6 ENSG00000167104 BPI fold containing family B, member 6 BPIFC ENSG00000184459 BPI fold containing family C BRFI ENSG00000 185024 BRF1. RNA polymerase III transcription initiation factor 90 kDa subunit BRINP1 ENSG00000078725 Bone morphogenetic protein/retinoic acid inducible neural specific 1 BRINP2 ENSGO0000198797 Bone rorphogenetic protei/retinoic acid inducible neural specific 2 BRINP3 ENSG00000162670 Bone morphogenetic protein/retinoic acid inducible neural specific 3 BSG ENSGOO0 172270 Basigin (Ok blood group) BSPHI ENSG00000188334 Binder of sperm protein homolog 1 BST] ENSG00000109743 Bone marrow stromal cell antigen I BT-lBD17 ENSG00000204347 BIB (POZ) domain containing 17 BTD ENSGOOOOO169814 Biotiidase BTN2A2 ENSG00000124508 Butyrophiln, subfamily 2, member A2 BrN3AI ENSGO0000026950 Butyrophiin, subfamily 3, member Al BTN3A2 ENSG00000186470 Butyropilin, subfamily 3, memberA2 B1TN3A3 ENSGOOOOOI11801 Butyrophilin, subfarily 3, member A3 C1OorfI ENSG00000165507 Chromosome 10 open reading frame 10 CI01f99 ENSGO0000188373 Chromosome 10 open reading frame 99 Cl lorfi ENSG00000137720 Chromosome 11 open reading frame 1 C lIorf24 ENSG00000171067 Chromosome II open reading frame 24 CIorf45 ENSG1i00000174370 Chromosome II open reading frame 45
C11o1f94 ENSG00000234776 Chromiosome II open reading frame 94 C1201f10 ENSG00000139637 Chromosome 12 open reading frame 10 C12orf49 ENSG00000111412 Chromosome 12 open reading frame 49 C12orf73 ENSG00000204954 Chromosome 12 open reading frame 73 CI2orf76 ENSG00000 174456 Chrornosome 12 open reading frame 76 C14orf80 ENSG00000185347 Chromosome 14 open reading frame 80 C14oif93 ENSG00000100802 Chromosome 14 open reading frame 93 C16orf89 ENSGO0000153446 Chromosome 16 open reading frame 89 C16orf90 ENSG00000215131 Chromosome 16 open reading frame 90 C17orf67 ENSG00000214226 Chrornosome 17 open reading frame 67 C17oIf75 ENSG00000108666 Chromosome 17 open reading frame 75 C17of99 ENSG00000187997 Chromosome 17 open reading frame 99 C18orf54 ENSG00000166845 Chromosome 18 open reading frame 54 C19or47 ENSG00000160392 Chromosome 19 open reading frame 47 CI9orf70 ENSG00000174917 Chromosome 19 open reading frame70 C19If80 ENSG00000130173 Chromosome 19 open reading frame 80 C1GALTI ENSG00000106392 Core I synthase, glycoprotein-N-acetylgalactosamine 3-beta galactosyltransferase I
Clorf127 ENSG00000175262 Chromosome 1 open reading frame 127 Clorf159 ENSG00000131591 Chromosome 1 open reading frame 159 Clolfl98 ENSG00000119280 Chromosome 1 open reading frame 198 Clorf234 ENSG00000227868 Chromosome I open reading frame 234 Clorf54 ENSG00000118292 Chromosome 1 open reading frme 54 Clorf56 ENSG00000143443 Chromosome 1 open reading frame 56 CIQA ENSGO0000173372 Complement compooenm1,qsnconponent, A chain ClQB ENSG00000173369 Comnplement component 1, q subcomponent, B chain CIQC ENSG00000159189 Complement component 1, q subcomponent, C chain CIQLI ENSG00000131094 Complement component 1, q subcornponent-like I CIQL2 ENSG00000144119 Complement component 1, q subcomponent-like 2 CIQL3 ENSG00000165985 Complement component 1,o subcomponent-like 3 C1QL4 ENSG00000186897 Complement component 1, q subcomponent-like 4 C1QTNF1 ENSG00000173918 Clq and tumor necrosis factorrelated protein 1 CIQTNF2 ENSG00000 145861 Ciq and tumor necrosis factor related protein 2 CIQTNF3 ENSG00000082196 Ciq and tumor necrosis factor related protein 3 CIQTNF4 ENSGO0000172247 Clq and tumornecrosis factor related protein 4 C1QTNF5 ENSG00000223953 C lg and tumor necrosis factor related protein 4 CIQTNF7 ENSG00000163145 C I qand tumor necrosis factor related protein 7 C IQTNF8 ENSG00000 184471 Ciq and tumor necrosis factor related protein 8 CIQ'NF9 ENSG00000240654 C1 and humor necrosis factor related protein 9 CIQTNF9B ENSG00000205863 CIq and tumor necrosis factor related protein 9B C1R ENSG00000159403 Complement corponent 1, r subcompornent CIRL ENSGO0000139178 Complement component 1, r subcomponent-like CIS ENSG00000182326 Complement component 1, s subcomponent C2 ENSGO0000166278 Complemet component2
C2lorf33 ENSGO0000160221 Chromosome 21 open reading frame 33 C2lorf62 ENSG00000205929 Chromosome 21 open reading frame 62 C22orf15 ENSG00000169314 Chromosome 22 open reading frame 15 C22orf46 ENSGO0000184208 Chromosome 22 open reading frame 46 C2CD2 ENSGOOOOO157617 C2 calciumn-dependent domain containing 2 C2orf4() ENSG00000119147 Chromosome 2 open reading frame 40 C2orf66 ENSG00000187944 Chromosome 2 open reading frame 66 Co2f69 ENSG00000178074 Chromosome 2 open reading frame 69 C2orf78 ENSG00000187833 Chromosome 2 open reading fmme 78 C3 ENSG00000125730 Complement component 3 C3orf33 ENSGO0000174928 Chromosome 3 open reading frame 33 C3orf58 ENSG00000181744 Chromosome 3 open reading frame 58 C4A ENSG00000244731 Complement coniponernt 4A (Rodgers blood group) C4B ENSG00000224389 Complement component 4B (Chido blood group) C4BPA ENSG00000123838 Complement component 4 binding protein, alpha C4BPB ENSGO0000123843 Complemenrtcomponent4bindingprotein, beta C4orf26 ENSG00000174792 Chromosome 4 open reading frame 26 C4orf48 ENSG00000243449 Chromosome 4 open reading frame 48 C5 ENSG00000106804 Complement component 5 C5orf46 ENSG00000178776 Chromosome 5 open reading frame 46 C6 ENSG00000039537 Complenent component6 C6orfl2O ENSG00000185127 Chromosome 6 open reading frame 120 C6orf15 ENSG00000204542 Chromosome 6 open reading frame 15 C6orf25 ENSG00000204420 Chromosome 6 open reading [mne 25 C6orf58 ENSG00000184530 Chromosome 6 open reading frame 58 C7 ENSG000 112936 Comlementcomponent2 C-orf57 ENSGO0000164746 Chromosome 7 open reading frame 57 C7orf73 ENSG00000243312 Chromosome 2 open reading frame 73 C8A ENSG0000157131 Complement componern 8, alpha polypeptide C8B ENSG00000021852 Complement component 8, beta polypeptide C80 ENSGOOOO 176919 Complerment component 8, gamma polypeptide C9 EN SGO0000113600 Complement component 9 C9orf47 ENSGO0000186354 Chromosome 9 open reading frame 47 CA 10 ENSGO0000154975 Carbonic anhydrase X CAl1 ENSG00000063180 Carbonic anhydrase X1 CA6 ENSGOOOOO 131686 Carbonic anhydrase Vi CA9 ENSGO0000107159 Carbonic arhydrase IX CABLESI ENSG00000134508 Cdk5 and Abl enzyme substrate I CABP1 ENSGOOOOO157782 Calcium binding protein I CACNA2DI ENSG00000153956 Calcium channel, voltage-dependent, alpha 2/delta subunit I CACNA2D4 ENSGOOOOOI151062 Calcium channel, voltage-dependent, alpha 2/delta subunit 4 CAIDI3 ENSGO0000162706 Cell adhesion molecule 3 CALCA ENSG00000110680 Calcitonin-related polypeptide alpha CALCB ENSGO0000175868 Calcitomin-related polypeptide beta
CALCR EN SG00000004948 Calcitonin receptor CALCRL ENSG00000064989 Calcitonin receptor-like CALR ENSGOOOOO179218 Caireticulin CALR3 ENSG00000269058 Calreticulin 3 CALU ENSGOOOOO128595 Calumenin CAMK2) ENSGOOOO145349 Calcium/calmodulin-dependent protein kinase II delta CAMP ENSGO0000164047 Catheicidin antimicrobial peptide CANX ENSG00000127022 Calnexin CARKI) ENSGO0000213995 Carbohydrate kinase domain containing CARMI ENSG00000 142453 Coactivator-associated arginine methyltransferase I CARNS1 ENSG00000172508 Carnosine syrthase 1 CARTPT ENSGO0000164326 CART prepropeptide CASQ] ENSG0000 143318 Calsequestrin 1 (fIasi-twitch, skeletal muscle) CASQ2 ENSG00000118729 Calsequestrin 2 (cardiac muscle) CATSPERG ENSG00000099338 Catsper channel auxiliary subunit garnma CBLN1 ENSG00000102924 Cerebellin 1 precursor CBLN2 ENSG00000141668 Cerebellin 2 precursor CBLN3 ENSGO0000139899 Cerebeilin 3 precursor CBLN4 ENSGO00000054803 Cerebelhn 4 precursor CCBEI ENSG00000183287 Collagen and calcium binding EGF domains I CCDC108 ENSGO000 181378 Codled-coil domain containing 108 CCDC112 ENSG00000164221 Coiled-coil domain containing 112 CCDC129 ENSGOOOOO 180347 Coiled-coil domain containing 129 CCDC134 ENSG00000100147 Coiled-coil domain containing 134 CCDC149 ENSG0000 181982 Coiled-coil domain containing 149 CCDC3 ENSGO0000 151468 Coiled-coil domain containing 3 CCDC80 ENSGOO00091986 Coiled-coil domain containing 80 CCDC85A ENSGO0000055813 Coiled-coil domain containing 85A CCDC88B ENSG00000 168071 Coiled-coil domain containing 88B CCER2 ENSG00000262484 Coiled-coil glutamate-rich protein 2 CCK ENSGO00000187094 C1holecystokinin CCL1 ENSGO0000108702 Chenmokine (C-C imotil) ligand 1 CCLII ENSG00000172156 Ch.mokine (C-C motif) ligand 11 CCL13 ENSG00000181374 Che-mokine (C-C motif, ligand 13 CCL14 ENSG00000276409 Chemokine (C-C motif) ligand 14 CCL15 ENSG00000275718 Chemokine (C-C motif) ligand 15 CCL16 ENSG000275152 Chemookine (C-C motif) ligaid 16 CCL17 ENSG00000102970 Chemokine (C-C motif) ligand 17 CCL18 ENSG00000275385 Chemokine (motif) ligand 18 (pulmonary and activation regulated) CCL19 ENSGO0000172724 Chemokine (C-C motif) ligand 19 CCL2 ENSGO0000108691 C1hemokine (C-C motif) ligand 2 CCL20 EN SGOOOOO115009 Chemookine (C-C imoti) ligand 20 CCL21 ENSGO0000137077 Chemokine (C-C moti) ligand 21 CCL22 ENSGO0000102962 Chemokine (C-C motif) ligand 22
CCL23 ENSGO0000274736 Chemokine (C-C motif) ligand 23 CCL24 ENSG00000106178 Chemokine (C-C motif) ligand 24 CCL25 ENSG00000131142 Chemokine (C-( motif) ligand 25 CCL26 ENSG00000006606 Chemokine (C-C motif) ligand 26 CCL27 ENSGO0000213927 Chernokine (C-C motif) ligand 27 CCL28 ENSGO0000151882 Chemokine (C-C mnoti) ligand 28 CCL3 ENSG00000277632 Chemokine (C-C motif) ligand 3 CCL3L3 ENSG00000276085 Chemokine (C-( noi) ligand 3-like 3 CCL4 ENSG00000275302 Chemokine (C-C moti') ligand 4 CCL4L2 ENSG00000276070 Chernokine (C-C motif) ligand 4-like 2 CCL5 ENSGO0000271503 Chemokine (C-C motif) ligand5 CCL7 ENSG00000108688 Chemokine (C-C motif) ligand 7 CCL8 ENSG00000108700 Chemokie (C-C motif) ligand 8 CCNBlIP1 ENSGO0000 100814 Cyclin B1 interacting protein 1, E3 ubiquitin protein ligase CCNL I ENSGOOOOO 163660 Cycling LI CCNL2 ENSGO0000221978 Cyciin L2 CD14 ENSG00000170458 CD14 molecule CD160 ENSGO0000117281 CD160 molecule CID164 ENSGOOO00135535 CD164 molecule. sialomucin CD177 ENSG00000204936 CD177 molecule CD1E ENSGO0000158488 CDle molecule CD2 ENSG00000116824 CD2 molecule CD200 ENSGO0000091972 CD200 molecule CID200R1 ENSGO0000163606 CD200 receptor 1 CD22 ENSGO00000 12124 CD22 molecule CD226 ENSG00000150637 CD226 molecule CD24 ENSG00000272398 CD24 molecule CD276 ENSG00000103855 CD276 molecule CD300A ENSG00000167851 CD300a molecule CD300LB ENSGO0000178789 CD300 molecule-like family memberb CD300LF ENSGO0000186074 CD300 molecule-like fam-ily member f CD300LG ENSG000161649 CD300 molecule-like family member g CD3D ENSGOOOOO167286 CD3d molecule, delta (CD3-TCR complex) CiD4 ENSGOOOOO010610 CD4 molecule CD40 ENSGO0000101017 CD40 molecule, TNF receptor superfamily member 5 CD44 ENSG00000026508 CD44 molecule (Indian blood group) CID48 EN SGOOOOO117091 CD48 molecule CD5 ENSG00000110448 CD5 molecule C1D55 ENSG0000196352 (D55 molecule, decay accelemting factor for complement (Crorer blood group)
C:D59 ENSG00000085063 ()59 molecule, complementregulato protein C)5L ENSG00000073754 CD5 molecule-like CD6 ENSGOOOOOO13725 CD6 molecule ()68 ENSGOOOO129226 CD68 molecule
CD7 ENSG00000173762 CD7 molecule CD79A ENSG00000105369 CD79a molecule, immunoglobulin-associated alpha C-D80 ENSG00000121594 CD80 molecule CD86 ENSGO0000114013 CD86 molecule CD8A ENSGO0000153563 CD8a molecule CD8B ENSGO0000172116 CD8b molecule CD99 ENSG00000002586 CD99 molecule CDC23 ENSG00000094880 Cel! division cycle 23 CDC40 ENSG00000 168438 Cell division cycle 40 CDC45 ENSG00000093009 Cell division cycle 45 CDCP1 ENSG00000 163814 CUB dornai containing protein I CDCP2 ENSGO0000157211 CUB domain containing protein 2 CDH1 ENSG00000039068 Cadhmerir 1, type I CDI-1iI ENSG00000140937 Cadherin 11, type 2. OB-cadherin osteoblastt) CDHI13 ENSG00000140945 Cadhern 13 CDH 17 ENSG00000079112 Cadherin 17, LI cadherin (liver-intestine) CDH18 ENSG00000145526 Cadherin 18, type 2 CDH19 ENSG00000071991 Cadherin 19, type 2 CID123 ENSG00000107736 Cadherin-related 23 CDH5 ENSG00000179776 Cadherin 5, type 2 (vascular endothelium) CDHRI ENSGO0000148600 Cadherin-related fanlv member I CDHR4 ENSG00000187492 Cadherin-related family member 4 CDHR5 ENSG00000099834 Cadherin-related family member CDKN2A ENSGO00147889 Cyclin-dependent kinase inhibitor 2A CDNF ENSG00000185267 Cerebral dopamine neurotrophic factor CDON ENSG00000064309 Cell adhesionassociated, orcogene regulated CDSN ENSG00000204539 Corneodesmosin CEACAM16 ENSGO0000213892 Carcinoembryonic antigen-related cell adhesion molecule 16 fA CAM18 ENSG00000213822 Carcinoermbryonic antigen-related cell adhesion molecule 18 CEACAM19 ENSG00000 186567 Carcinoembryonic antigen-related cell adhesion molecule 19 CEACAM5 ENSGOOOO 105388 Carcinoembryonic anigen-related cell adhesion molecule 5 CEACAM7 ENSG00000007306 Carcinoembryonic antigen-related cell adhesion molecule 7 CEACAM8 ENSGO0000124469 Carcinoembryonic antigen-related cell adhesion molecule 8 CIECR I ENSG00000093072 Cat eye syndrome chroosonme region, candidate 1 CECR5 ENSG00000069998 Cat eye syndrome chromosome region, candidate 5 CEL ENSGOOOOO170835 Carboxyl ester lipase CELA2A ENSG00000142615 Chymotrypsin-like elastase family, member 2A CELA2B ENSG00000215704 Chymotrypsin-like elastase family, member 2B CE3LA3A ENSG00000142789 Chymotrypsin-like elastase family, member 3A CELA3B ENSGO0000219073 Chymotrypsin-like elastase family, member 3B CEMIP ENSGOOOOO103888 Cell migration inducing protein, hyaluronan binding CEP89 ENSGOOOOO 121289 Centrosomal protein 89kDa CERI ENSG00000147869 Cerberus 1, DAN family BIP antagonist \4RCAM ENSGOOOOO167123 Cerebralendothelial cell adhesion molecule
CERS1 ENSG00000223802 Ceraimde synthase 1 CES1 ENSG00000198848 Carboxylesterase 1 CES3 ENSG00000172828 Carboxylesterase 3 CES4A ENSGO0000172824 Carboxylesterase 4A CESSA ENSGOOOOO159398 Carboxy'lesterase 5A CETP ENSG00000087237 Ciolesteryi ester transfer protein, plasma CFB ENSG00000243649 Complement factor B CFC1 ENSG00000136698 Cripto, FRL-1, cryptic family 1 CFCIB ENSGO0000152093 Cripto, FRL-1, cryptic family 1B CFD ENSGOOO0197766 Complement factor D (adipsin) CFDP1 ENSG00000)153774 Craniofacial development protein 1 CFH ENSG00000000971 Complement factor H CFHR I ENSG00000244414 Comnlement factor H-related 1 CFHR2 ENSG00000080910 Complement factor1-1-related 2 CFH1R3 ENSG00000 116785 Complement factor H-related 3 CFHR4 ENSG00000 134365 Complement factor H-related4 CFHR5 ENSG0000134389 Complement factor H-related 5 COI ENSG00000205403 Complement factor I CFP ENSGO00000126759 Complement factor properditn CGA ENSG00000135346 Glycoprotein hormones, alpha polypeptide CGB ENSG00000104827 Chorionic gonadotropin, beta polypeptide CGBI ENSG00000267631 Chorionic gonadotropin, beta polypeptide I CGB2 ENSG00000104818 Chorionic gonadotropin, beta polypeptide 2 CGB5 ENSGO00000189052 Chorionic gonadotropin, beta polIpeptide 5 CGB7 ENSGO0000196337 Chorionic gonadotropin, beta polypeptide 7 CGB8 ENSG00000213030 Cho tionic gonadotropin, beta polypeptide 8 CGREF ENSG00000138028 Cell growth regulator with EF-hand domain 1 CH507-9B2.3 ENSG00000280071 CHAD ENSG00000136457 Chondroadherin CHADL ENSG00000100399 Chondroadherin-like CiHEK2 ENSG00000183765 Checkpoint kinase 2 CHGA ENSG00000100604 Chromogranin A CHGB ENSG00000089199 Chromogranin B CHI3L1 ENSG0000133048 Chitinase 3-like I (cartilage glycoprotein-39) CHI3L2 ENSG00000064886 Chitinase 3-like 2 CHIA ENSG0000 134216 Chitinase, acidic C-11)I ENSG00000177830 Chitinase domain containing 1 CHITI ENSG00000133063 Chitinase I (chitotriosidase) CHI.I ENSG00000134121 Celladhesion molecule Li -like CHNI ENSG00000 128656 Chimerin I CHPF ENSGO0000123989 Chondroitin pov merizing factor C-IPF2 ENSG00000033100 Chondroitin polyrmerizigh factor 2 CHRD ENSG00000090539 Chordin CHRDL ENSi00000101938 Chordi-like I
C-1RDL2 ENSG00000054938 Chordin-like 2 CIRNA2 ENSG00000120903 Cholinergic receptor, nicotinic, aloha 2 (neuronal) CHRNA5 ENSG00000169684 Cholinergic receptor, icotinic, alpha 5 (neuronal) CHRNBI ENSG00000 170175 Cholinergic receptor, nicotinic, beta I (muscle) CI-IRND ENSGO0000135902 Cholinergic receptor, nicotinic, delta (muscle) C1]ST ENSG00000175264 Carbohydrate (keratan sulfate Gial-6) sulfbtransferase 1 CHST10 ENSG00000115526 Carbohydrate sulfotransfemse 10 CHST11I ENSG00000171310 Carbohydrate (chondroitin 4) suli'otransferase II CHST13 ENSG00000180767 Carbohydrate (chondroitin 4) sulbtransferase 13 CI-ST4 ENSGO0000 140835 Carbohydrate (N-acetylglucosarnine 6-0) suilfotransferase 4 C-IST5 ENSG00000 135702 Carbohydrate (N-acetylglucosamirne 6-0) sulf'otransferase 5 CHST6 ENSG0000183196 Carbohydrate (N-acetylgiucosamine 6-0) sulfotransferase 6 CHST7 ENSG00000 147119 Carbohydrate (N-acetylglucosamine 6-0) sulfotransferase 7 CHST8 ENSG00000124302 Carbohydrate (N-acetylgalactosamine 4-0) sulfotransferase 8 CI-ISYI ENSG00000131873 Chondroitin sulfate synthase 1 C-ISY3 ENSG00000198108 Chondroitin sulfate synthase 3 CITF8 ENSG00000168802 Chromosome transmission fidelity factor 8 CILP ENSG0000138615 Cartilage intermediate layer proein, nucleotide py rophosphohydrolase CILP2 ENSG00000 160161 Cartilage intermediate layer protein 2 CIRHIA ENSGOOOOO 141076 Cirthosis, atosonal recessive 1A (cih-in) CKLF ENSG00000217555 Chemokine-like factor CKMTIA ENSG00000223572 Creatine kinase, mitochondrial lA CKMT1B ENSG00000237289 Creatine kinase, mitochondrial lB CL CA IENSG00000016490 Chloride channel accessory 1 CLCFI ENSGO0000175505 Cardiotrophin-like cytokine factor 1 CLDN15 ENSG00000106404 Claudin 15 CLDN7 ENSG00000181885 Claudin7 CLDNDI ENSG00000080822 Claudin domain containing 1 CLEC11IA ENSGO0000105472 C-type lectin domain family 11, member A CLEC16A ENSGO0000038532 C-type lectin domain family 16, member A CLEC18A ENSG00000157322 C-type lectin domain family 18, memberA CLEC18B ENSG00000140839 C-type lectin domain family 18. member B CLEC18C ENSG00000157335 C-typelectin domain family 18, member C CLEC19A ENSG0000026121) C-type lectin domain family 19, member A CLEC2B ENSGOOOOO 110852 C-type lectin domain family 2, member B CLEC3 A ENS00000166509 C-type lectin domain family 3, member A CLEC3B ENSG00000163815 C-type lectin domain family 3, member B CLGN ENSG00000153132 Caimegin CILN5 ENSGOOOOO 102805 Ceroid-lipofuscinosis, neuronal 5 CLPS ENSGO0000137392 Colipase, pancreatic CLPSLI ENSG00000204140 Colipase-like 1 CLPSL2 ENSG00000196748 Colipase-like 2 CLPX ENSG00000166855 Caseinolytic mitochondrial matrix peptidase chaperone subunit CLSTN3 ENSG00000139182 Calsyntenin 3
CLU ENSGO0000120885 Clustenn CLUL1 ENSG00000079101 Clusterin-like I (retinal) CMA1 ENSG00000092009 Chymase 1, mast cell CMPK1 ENSG00000162368 Cytidine monophosphate (UMP-CMP) kinase 1, cytosolic CNBDI ENSGOOOOO176571 Cy ciic nucleotide binding domain containing I CNDPI ENSG00000 150656 Canosine dipeptidase I (metallopeptidase M20 family) CNPY2 ENSG00000257727 Canopy FGF signaling rgulator2 CNPY3 ENSG00000137161 Canopy FGF signaling mgulator 3 CNPY4 ENSG00000166997 Canopy FGF signaling regulator 4 CNTFR ENSG00000 122756 Ciiiarv neurotrophic factor receptor CNTN1 ENSG00000018236 Contactin I CNTN2 ENSG00000184144 Contactin 2 (axonal) CNTN3 ENSG00000113805 Contactir3 (plasmacvoma associated) CNTN4 ENSG00000144619 Contactin 4 CNTN5 ENSG0000149972 Contactin 5 CNTNAP2 ENSG00000174469 Contactin associated protein-like 2 CNTNAP3 ENSG00000106714 Contactin associated protein-like 3 CNTNAP3B ENSG00000154529 Contactin associated protein-like 3B COASY ENSG00000068120 CoA synthase COCH ENSG00000100473 Cochlin COG3 ENSGO0000136152 Cormponent of oligomeric golgi complex 3 COL10A1 ENSG00000123500 Collagen, tpe X, alpha I COLIIAI ENSG00000060718 Collagci type XT, alpha I COL11A2 ENSG00000204248 Collagen, type Xi, alpha 2 COL12A1 ENSG00000111799 Collagen, type XII, alpha I COL.14AI ENSG00000 187955 Collagen, type XIV, alpha I COL15A1 ENSG00000204291 Collagen, type XV, alpha 1 COI16A1 ENSG00000084636 Collagcn, type XVI, alpha I COL18A1 ENSG00000182871 Collagen, type XVII, apha 1 COL19AI ENSG00000082293 Collagen, type XIX, alpha I COL.1AI ENSG00000 108821 Collagen, type 1, alpha I COL A2 ENSG00000164692 Collagen, type ", alpha 2 COL20A1 ENSG00000101203 Collagen, type XX, alpha 1 COL21A1 ENSG00000124749 Collagen, type XXI, alpha 1 COL22AI ENSG00000169436 Collagen, type XXII, alpha I COL24AI ENSGOOOOO171502 Collagen, type XXIV, alpha I COL,26A1 ENSG00000160963 Collagen, type XXVI, alpha 1 COL27A1 ENSG00000196739 Collagen, type XXVII, alpha I COL28A1 ENSG00000215018 Collagen, type XXV[II, alpha I COL2AI ENSG00000139219 Collagen, type II, alpha I COL3AI ENSG00000168542 Collagen, type III, alpha COL4A1 ENSG00000 182498 Collagen, type IV, alha I COL4A2 ENSG00000134871 Collagen, type IV. alpha 2 COL4A3 ENSG00000169031 Collagen, type IV, alpha 3 (Goodpasture antiger)
COL.4A4 ENSGO0000081052 Collagen, type iV, alpha 4 COL4A5 ENSGO0000188153 Collagen, type IV. alpha 5 COL4A6 ENSG3000197565 Collagen, type IV, alpha 6 COL5AI ENSG00000130635 Collagen, type V. alpha 1 COL5A2 ENSG00000204262 Collagen, type V, alpha 2 COL.5A3 ENSG00000080573 Collagen, type V, alpha 3 COL6A1 ENSG00000142156 Collagen, type VI, alpha 1 COL6A2 ENSG00000142173 Collagen, type Vi, alpha 2 COL6A3 ENSG00000163359 Collagen, type Vi, alpha 3 COL6A5 ENSG00000 172752 Collagen, type Vi, alpha 5 COL.6A6 ENSG00000206384 Collagen, type V, alpha 6 COL7A1 ENSG00000114270 Collagen, t-oe VII, alpha I COL8A1 ENSG00000144810 Collagen, type III, apha COL8A2 ENSG00000171812 Collagen, type VIII, alha 2 COL9AI ENSG00000 112280 Collagen, type IX, alha I COL.9A2 ENSG00000049089 Collagen., type IX, alpha 2 COL9A3 ENSG00000092758 Collagen, type IX. alpha 3 COLEC10 ENSG00000184374 Collectin sub-family member 10 (C-type lectin) COLEC11 ENSG00000118004 Collectin sub-family member II COLGALTI ENSG00000130309 Collagen beta(1-O)galactosyltransferase I COLGAL T2 ENSG0000198756 Collagen beta(1-O)galactosyl transferase 2 COLQ ENSG00000206561 Collagen-like tail subunit (single strand of homotimer) of asynmmetric acetylcholinesterase COMP ENSG00000105664 Cartilage oligomueric matrix protein COPS6 ENSG00000 168090 COP9 signalosome subunit 6 COQ6 ENSG00000119723 Coenzytme Q6 monooxygernase CORT ENSG00000241563 Cortistatin CP ENSG00000047457 Ceruloplasrnin (ferroxidase) CPA] ENSG00000091704 Carboxypeptidase Al (pancreatic) CPA2 ENSG00000158516 Carboxypeptidase A2 (pancreatic) CPA3 ENSG00000163751 Carboxypeptidase A3 (mast cell) CPA4 ENSG00000128510 Caiboxypeptidase A4 CPA6 ENSGO000165078 Carboxypeptidase A6 CPAMID8 EN SG00000160111 C3 and PZP-like, alpha-2-macroglobulin domain containing 8 CPBI ENSG00000153002 Carboxypeptidase BI (tissue) CPB2 ENSG00000080618 Carboxypeptidase B2 (plasma) CPE ENSGO0000109472 Carboxypeptidase E CPM ENSG0000135678 Carboxypeptidase M CPNI ENSG00000120054 Carboxypeptidase N, polypeptide 1 CPN2 ENSG00000178772 Carboxypeptidase N, polypeptide 2 CPO ENSG0000144410 Carboxypeptidase O CPQ ENSG00000104324 Carboxypeptidase Q CPVL ENSG0000 106066 Carboxypeptidase, vitellogenic-like CPXMI ENSG00000088882 Carboxypepiase X (M14 fartiily), member 1
CPXM2 ENSG0000121898 Carboxypeptidase X (M14 family), member 2 CPZ ENSG00000109625 Carboxypeptidase Z CR [L ENSG00000197721 Complerment component (3b/4b) receptor 1-like CRB2 ENSGO0000148204 Crumbs family member 2 CREGI ENSG0000 143162 Ceililarrepressor of EIA-stimuiated genes I CREG2 ENSGOO000175874 Cellular repressor ofElA-stimulated genes 2 CRELDI ENSGO0000163703 Cy steine-rich with EGF-like domains I CRELD2 ENSG00000184164 Cysteirne-rich with EGF-like domains 2 CRH ENSG00000147571 Corticotropin releasing hormone CRHBP ENSGO0000145708 Corticotropin releasing hormone binding protein CRH-R I ENSG00000 120088 Corticotropin releasing hormone receptor 1 CRHR2 ENSG00000106113 Corticoropin releasing hormone receptor 2 CRISPI ENSG00000124812 Cysteine-rich secretary protein 1 CRISP2 ENSG0000 124490) Cysteine-rich secretory protein 2 CRISP3 ENSG00000096006 Cysteine-ich secretory protein I CRISPLI)2 ENSGO0000103196 C steine-rich secretory protein LCCL domain containing 2 CRLF1 ENSG0000006016 Cytokine receptor-like factor I CRP ENSGO0000132693 C-reactive protein, pentraxin-related CRTAC1 ENSG00000095713 Cartilage acidic protein I CRTAP ENSGO0000170275 Cartilage associated protein CRY2 ENSG0000121671 Cryptochrorne circadian clock 2 CSAD ENSG00000139631 Cysteine sulfinic acid decarboxylase CSF1 ENSGO0000184371 Colony stimulating factor I (macrophage) CSFIR ENSGO0000182578 Colony stimulating factor 1 receptor CSF2 ENSG00000164400 Colony stimulating factor 2 (girnlocyte-macrophage) CSF2RA ENSGO0000198223 Colony stimulating factor 2 receptor, alpha. low-affinity (granulocyte-macrophage) CSF3 ENSGOOOOO 108342 Colony stimulating factor 3 (granulocyte) CSGALNACT1 ENSG00000147408 Chondroitin sulfate N-acetigalactosaminyltransferase 1 CSHI ENSGOOOOO 136488 Choionic somatomammotropin hormone I (placental lactogen) CSHf 2 ENSG00000213218 Choriontic somatornammotropin hormone 2 CSHL I ENSGO0000204414 Chorionic somatomammotropin hormone-like I CSNISI ENSGOOOOO126545 Casein alpha s i CSN2 ENSG000135222 Casein beta CSN3 ENSG00000171209 Casein kappa CST1 ENSGO0000170373 Cystatln SN CSTII ENSG00000125831 Cystatin 11 CST2 ENSGOOOOO170369 Cystatin SA CST3 ENSGO0000 101439 Cystatin C CST4 ENSG00000101441 Cystatin S CST5 ENSG00000170367 Cystatin D CST6 ENSGO0000175315 Cystatin E/M CST7 ENSG00000077984 Cystatin F (leukocystatin) CST8 ENSG00000125815 Cystatin 8 (cystatin-related epididy-mal specific)
CST9 EN SGOOOOO173335 Cystatin 9(testatin) CST9L ENSG00000101435 Cystatin 9-like CSTII ENSG00000125823 (ystatin-like 1 CT55 ENSGO0000169551 Cancer/testis antigen 55 CTB-60B18.6 ENSG00000267335 CTBS ENSGO0000117151 Chitobiase, di-N-acetvi CTD-23 13N18.7 ENSG00000225805 CTD-2370N5.3 ENSG00000265118 CTGF ENSG00000118523 Connective tissue growth factor CTHRCI ENSG00000164932 Collagen triple helix repeat containing I CTLA4 ENSG00000163599 Cytotoxic T-lymphocyte-associated protein 4 CTNS ENSG00000040531 Cystinosin, lysosomal cystine transporter CTRB1 ENSG00000168925 Chymotly psinoger B1 CTRB2 ENSG00000168928 Chymotrypsinogen B2 CTRC ENSG00000162438 Chymotrypsin C (caldecrin) CTRL ENSG00000141086 Ch Inotrypsin-like CTSA ENSG00000064601 Cathepsin A CTSB ENSG00000164733 CathepsinB CTSC ENSG00000 109861 Cathepsin C CTSD ENSG00000117984 Cathepsin D CTSE ENSG00000 196188 Cathepsir E CTSF ENSG00000174080 Cathepsin F CTSG ENSG00000100448 Cathepsin G CTSH ENSG00000103811 Cathepsin -1 CTSK ENSG00000143387 Cathepsin K CTSL ENSGO000 135047 Cathepsir L CTSO ENSG00000256043 Cathepsin )
CTSS ENSGO0000163131 Cathepsin S CTSV ENSG00000136943 Cathepsin V CTSW ENSG00000 172543 Cathepsin W CTSZ ENSGO000 101160 Cathepsin Z CUBN ENSGO0000 107611 Cubilin (intrinsic factor-cobalamin receptor) CTUTA ENSGOOOOO112514 CutA divalent cation tolerance homolog (E. coli) CX3CL1 ENSGO0000006210 Chemokie (C-X3-C motif) ligand I CXADR ENSG00000154639 Coxsackie virus and adenovirus receptor CXCL1 ENSGO0000163739 Chernokine(C-X-Cmotif)ligandI(melanomagrowth stimulating activity, alpha)
CXCLI ENSGOOOOO169245 Chernokine (C-X-C motif) ligand 10 CXCL 11 ENSGO0000169248 Chenmokine (C-X-C motif) ligand 11 CXCL 12 ENSGOOOO0107562 Chemokine (C-X-C motif) ligand 12 CXCLI3 ENSGO0000156234 Chemoke (C-X-C motif) ligand 13 CXCL14 ENSGO0000145824 Chemokine (C-X-C motif) ligand 14 CXCL 17 ENSGOOOOO 189377 Chernokine (C-X-C motif) ligand 17 CXCL2 ENSGOOOOOO81041 CIenmokine (C-X-C motif) ligand 2
CXCL3 EN SGO00163734 Chemokine (C-X-C motif) ligand 3 CXCL5 ENSG00000163735 Chemokine (C-X-C motif) ligand 5 CXCL6 ENSGO0000124875 Chemokie (C-X-C motif) ligand 6 CXCL8 ENSG00000169429 Chemokine (C-X-C moti) ligand 8 CXCL9 ENSGO0000138755 Chemokine (C-X-C motif) ligand 9 CXorD36 ENSGO0000147113 Chromosome X open reading frame 36 CYB5D2 ENSG00000167740 Cytochrome b5 domain containing 2 CY-R1 ENSG00000187954 Cysteire/histidine-rich 1 CYP17AI ENSGO0000148795 Cytochrome P450. family 17, subfamily A. polypeptide I CYP20AI ENSG00000119004 Cvtochrome P450, family 20, subfamily A, polypeptide 1 CYP21A2 ENSG00000231852 Cytochrorme P450, f[anily 21, sublamily A, poly)eptide 2 CYP26BI ENSG00000003137 Cytochrome P450, family 26, subfamily B, polypeptide I CYP2A6 ENSG00000255974 Cytochrome P450, family 2, subfamily A, polypeptide 6 CYP2A7 ENSGO0000198077 Cytochrome P450, family 2, subfarily A, polypeptide 7 CYP2B6 ENSG00000192408 Cvtochrome P450, famil 2, subfamily B, polypeptide 6 CYP2C18 ENSGO0000108242 Cytochrome P450, fiarlym 2, subfamily C, polypeptide 18 CYP2CI9 ENSGO0000165841 Cytochrome P450, family 2, subfamily C, polypeptide 19 CYP2C8 ENSGOOOOO138115 (ytochrome P450, family 2, subfamily C, polypeptide 8 CYP2C9 ENSGO0000138109 Cytochrome P450, family 2, subfamily C, polypeptide 9 CYP2E1 ENSGO0000130649 Cytochrome P450, family 2, subfamily E, polypeptide 1 CYP2F1 ENSGO0000192446 Cytochrone P450, family 2, subflrtily F, polypeptide I CYP2J2 ENSG00000134716 Cytochrome P450, family 2, subfamily J, polypeptide 2 CYP2R1 ENSGO0000186104 (ytochrome P450, family 2, subfamily R, polypeptide 1 CYP2S1 ENSG00000167600 Cytochrome P450, family 2, subfamily S, polypeptide 1 CYP2W1 ENSG00000073067 Cytochrome P450, family 2, subfamily W, polypeptide I CYP46A1 ENSG00000036530 Cytochrorme P450, family 46, subfamily A, polypeptide I CYP4FII ENSG0000171903 Cytochrome P450, family 4, subfamily F, polypeptide II CYP4F2 ENSGO0000186115 Cytochrome P450, family 4, subfamily F, polypeptide 2 CYR61 ENS(i00000142871 Cysteirie-rich arigiogeric inducer, 61 CYTLI ENSG00000170891 Cytokine-like 1 D2HGDHi- ENSGOOOOO180902 D-2-hydroxyglutarate dehydrogenase DAG1 ENSGOO000173402 Dystroglycan 1 (dystrophin-associated glycoprotein 1) DAND5 ENSGO0000179284 DAN domain family member 5, BMP antagonist DAO ENSG00000110887 D-amito-acid oxidase DAZAP2 ENSG00000183283 DAZ associated orotein2 DBH ENSGOOO0123454 Dopamine beta-hydroxy'lase (dopamine beta-monooxygenase) DBNL ENSGOOOO136279 Direbrin-like DCD ENSG00000161634 Dermcidin DCN ENSGOOOOOO11465 Decorin DDIAS ENSG00000165490 DNA damage-induced apoptosis suppressor DDOST ENSG00000244038 Dolichyl-dipho sphooligosaccharide--protein glycosyltransferase subunit (non-cataiytic)
DDR I ENSG00000204580 Discoidin domain rceptor tyrosine kinase I DDR2 ENSGOOOO 162233 Discoidin domain receptor tyrosine kinase 2
DDT EN SG00000099977 D-d opachrome tautomerase DDX17 ENSG00000100201 DEAD (Asp-GIu-Ala-Asp) box helicase 17 DDX20 ENSG00000064703 DEAD (Asp)-Giu-Ala-Asp) box polypeptide 20 DDX25 ENSG00000109832 DEAD (Asp-Glu-Ala-Asp) box helicase 25 DDX28 ENSGO0000182810 DEAD (Asp-Gl-Ala-Asp) box polvpeptide 28 DEAF1 ENSGOCOO177030 DEAFl transcritionfactor DEF8 ENSG00000140995 Differentially expressed in FDCP 8 homolog (mouse) DEFAI ENSG00000206047 Deferisin, apia I DEFA1B ENSG00000240247 Defensin, alphia lB DEFA3 ENSG00000239839 Defensin, alpha 3, neutrophil-specific DEFA4 ENSG00000 164821 Defeinsin, lpha 4, corticostatin DEFA5 ENSG00000164816 Defensin, alpha 5, Paneth cell-specific DEFA6 ENSG00000164822 Defensin, alpha 6, Paneth cell-specific DEFBI ENSG00000 164825 Defensin, beta 1 DEFBI03A ENSGOOOOO 176797 Defensin, beta 103A DEFB103B ENSGO0000177243 Defensin, beta 103B DEFB104A ENSG00000176782 Defensin, beta 104A DEFB104B ENSG0000177023 Defensin, beta 104B DEFB105A ENSGOOOOO186562 Defensin, beta 105A DEFB105B ENSG00000186599 Defensin, beta 105B DEFB106A ENSG00000 186579 Deifnsin, beta 106A DEFB106B ENSGOOO0187082 Defensin, beta 106B DEFB1O7A ENSGOOOOO186572 Defensin, beta 107A DEFFB107B ENSGOOOO198129 Defensin, beta 107B DEFB108B ENSGO0000184276 Defensin, beta 108B DEFB110 ENSG00000203970 Defensir, beta 110 DEFB113 ENSGOOOO214642 Defensin, beta 113 DEFB114 ENSGO0000177684 Defensin, beta 114 DEFBI15 ENSGO0000215547 Deferisin, beta 115 DEFB116 ENSGO0000215545 Defensin, beta 116 DEFBI18 ENSGOOOOO131068 Defensir, beta 118 DEFB119 ENSGO0000180483 Defensin, beta 119 DEFB121 ENSG00000204548 Defensin, beta 121 DEFB123 ENSGO0000180424 Deferisin, beta 123 DEFB124 ENSGO0000180383 Defensin, beta 124 DEFB125 ENSGOOOOO178591 Defensin, beta 125 DEFB126 EN SG)00 125788 Defensin, beta 126 DEFB127 ENSG00000088782 Defensin, beta 127 DEFB128 ENSGO0000185982 Deferisin, beta 128 DEFB129 ENSGO0000125903 Defensin, beta 129 DEFB130 ENSG00000232948 Defensin, beta 130 DEFB131 ENSGOOOO186146 Defensir, beta 131 DEFB132 ENSG00000186458 Defensin, beta 132 DEFB133 ENSGO0000214643 Deferisin, beta 133
DEFB134 EN SG00000205882 Defensin, beta 134 DEFB135 ENSG00000205883 Defensin, beta 135 DEFB136 ENSG00000205884 Defensin, beta 136 DEFB4A ENSG000001711711 Defensin, beta 4A DEFB4B ENSG00000177257 Defensin, beta4B DFNA5 ENSG00000105928 Deafness, autosomal dominant 5 DFNB31 ENSG00000095397 Deafness, autosomal recessive 31 DGCR2 ENSG00000070413 DiGeorge syndrome crtical region gene 2 DK1 ENSG00000139549 Desert hedgehog DHRS4 ENSG00000157326 Dehydrogenase/reductase (SDR family) member 4 DHRS4L2 ENSGO0000187630 Dehydrogenase/reductase (SDR family) member 4 like 2 DHRS7 ENSG00000100612 Dehydrogenase/reductase (SDR family) member 7 DIRS7C ENSG00000184544 Dehy drogenase/rductase (SDR family) mer'ber7 D.RS9 ENSG00000073 Deh3ydrogenase/reductase (SDR family) member 9 DHRSX ENSGOOOOO169084 Dehydrogenase/reductase (SDR family) X-linked DHX29 ENSG00000067248 DEA- (Asp-Glu-A]a-His) box polypeptide 29 DHX30 ENSG00000132153 DEAH (Asp-Gi-Ala-His) box helicase 30 DHX8 ENSG00000067596 DEAH (Asp-Gin-Ala-His) box polypeptide 8 )12 ENSG00000211448 Deiodinase. iodothyronine. type II DIXDCI ENSG00000150764 DIX domain containing 1 DKKI ENSG000001107984 Dickkopf WNT signaling pathway inhibitor I DKK2 ENSG00000155011 Dickkopf WNT signaling pathway inhibitor 2 DKK3 ENSG00000050165 Dickkopf WNT signaling pathway inhibitor 3 DKK4 ENSGO0000104371 Diclkopf WNT signaling pathway inhibitor 4 DKKL1 ENSG00000104901 Dickkopf-like I DLG4 ENSG00000 132535 Discs, large homolog 4 (Drosophila) DLKI ENSG000000185559 Delta-like 1 homolog (Drosophila) DLLI ENSG00000198719 Delta-like I (Drosophia) DLL3 ENSG00000090932 Delta-like 3 (Drosophila) DMBTI ENSG00000 187908 Deleted in malignant brain tumors I DMKN ENSG00000161249 Dermokine DMTP1 ENSG00000152592 Dentin matrix acidic phosphoprotein 1 DMRTA2 ENSG00000142700 DMRT-like arily A2 DNAAF5 ENSGOOOOO164818 Dynein, axoncnai, assenbl factor DNAH14 ENSG00000185842 Dynein, axonemal, heavy chain 14 DNAJBII ENSG0000090520 DnaJ (Hsp40) homolog, subfamily B, mernber 11 DNAJB9 ENSG00000128590 DinaJ (-Isp40) homolog, subfamily B, member 9 DNAJC25-GNG10 ENSG00000244115 DNAJC25-GNGO10 readthrough DNAJC3 ENSG00000102580 DnaJ (Hsp40) homolog, subfamily C, member 3 DNASE1 ENSGO0000213918 Deoxyribonuclease I DNASEILI ENSG00000013563 Deoxvribonuclease i-like I DNASE1L2 ENSG0000167968 Deoxyribonuclease i-like 2 DNASEIL3 ENSG00000163687 Deoxyribonuclease I-like 3 DNASE2 ENSG00000105612 Deoxyribonuclease Ii, lvsosomal
DNASE2B ENSG00000137976 Deoxyribonuclease II beta DPEPI ENSG00000015413 Dipeptidase 1 (renal) DPEP2 ENSG00000167261 Dipeptidase 2 DPEP3 ENSGO0000141096 Dipeptidase 3 DPF3 ENSG00000205683 D4, zinc and double PHD fingers family 3 DPP4 ENSG00000197635 Dipeptidyl-peptidase 4 DPP'7 ENSG00000176978 Dipeptidvl-peptidase 7 DPT ENSG0000143196 Dermnatoponinm DRAXIN ENSG00000 162490) Dorsal inhibitory axon guidance protein DSE ENSG00000111817 Dernatan sulfate epimerase DSG2 ENSG00000046604 Desmogleir 2 DSPP ENSG00000 152591 Dentin sialophosphoprotein DST ENSG00000151914 Dystonlin DUOXI ENSG00000137857 Dual oxidase 1 DYNLT3 ENSG00000 165169 Dynein, light chain, Tctex-type 3 E2F5 ENSG00000 133740 E2F transcription factor 5, p130-binding EBAG9 ENSG00000147654 Estrogen receptor binding site associated, antigen, 9 EBI3 ENSG00000105246 Epstein-Barr virus induced 3 ECHDC1 ENSG00000093144 Ethylialonyl-CoA decarboxylase 1 ECMI ENSG00000143369 Extracellular matrix protein I ECM42 ENSG00000106823 Extracellular matrix protein 2, female organ and adipocyte specific ECSIT ENSG00000 130159 ECSIT signalling integrator EDDM3A ENSG00000181562 Epididymal protein 3A EDDM3B ENSG00000181552 Epididymal protein 3B EDEM2 ENSG00000088298 ER degradation enhancer, manmosidase alha-like 2 EDEM3 ENSG00000116406 ER degradation enhancer mannosidase alpha-like 3 EDIL3 ENSG00000164176 EGF-iike repeats and discoidin I-like domains 3 EDNI ENSG00000078401 Endothelin1 EDN2 ENSG00000127129 Endothelin 2 EDN3 ENSG0000124205 Endothelin 3 EDNRB ENSG00000)136160 Endothelin receptor type B EFEMPI ENSG00000115380 EGF containing fibulin-like extracellular matrix protein I EFEMIP2 ENSGO0000172638 EGF containing fibulin-like extracellular matrix protein 2 EFNAI ENSG00000169242 Ephrin-Al EFNA2 ENSGO0000099617 Ephrin-A2 EFNA4 ENSG00000243364 Ephrin-A4 EGFL6 ENSG00000198759 EGF-like-domain, multiple 6 EGFL7 ENSG00000172889 EGF-like-domain, multiple 7 EGFL8 ENSGO0000241404 EGF-like-domain. multiple 8 EGFLAM ENSG00000164318 EGF-like, fibronectin type III and laminin G domains EGFR ENSG0000146648 Epidermal growth factor receptor EHBP1 ENSG0000115504 EH domain binding protein 1 EHF ENSG00000135373 Ets homologous factor EHMTi1 ENSG00000181090 Euchromatic histone-lVsine N-methyltransferase I
EHMT2 ENSG00000204371 Euchromatic histone-lysine N-methyltransferase 2 EIF2AK1 ENSG00000086232 Eukaryotic translation initiation factor 2-alpha kinase 1 EL ANE ENSG00000 197561 Elastase, neutrophil expressed ELN ENSG00000049540 Elastin ELP2 ENSG0000134759 Elongator acetyLtransferase complex subunit 2 EL SPBPI EN SGC00169393 Epididyinal sperm binding protein I EMC1 ENSG00000127463 ER membrane protein complex subunit I EMC10 ENSG00000161671 ER membrane protein complex sbumt 10 EMC9 ENSG00000100908 ER membrane protein complex subunit 9 EMCN ENSGO0000164035 Endomucin EMIlD ENSG00000186998 EMI dorain contaning I EMILINI ENSG00000138080 Elastin microfibil interfacer I EMILIN2 ENSG00000132205 Elastin mcrofibril interfacer 2 EMILIN3 ENSG00000183798 Elastin microfibrilinterfacer 3 ENAM ENSG00000132464 Enamelin EN[DOi ENSG00000167136 Endonuclease G ENDOU ENSG00000111405 Endonuclease, polvU-specific ENHO ENSG00000168913 Energy homneostasis associated EN04 ENSGO0000188316 Enolasefamily menber4 ENPP6 ENSGO0000164303 Ectonuclcotide pvrophosphatase/phosphodiesterase 6 ENPP7 ENSG00000 182156 Ectonucleotideprophospatasephosphodiestemse7 ENTPD5 ENSG00000187097 Ectonucleoside triphosphate diphosphohydrolase 5 ENTPD8 ENSG00000188833 Ectonucleoside triphosphate diphosphohydrolase 8 EOGT ENSGO0000163378 EGF domain-specific 0-linked N-acetylglucosamine (GlcNAc) transferase
EPCAM ENSGOOOOO119888 Epithelia! cell adhesion molecule EPDRI ENSG00000086289 Ependymin related I EPGN ENSGOOOOO182585 Epithelial mitogen EPHAIO ENSG00000183317 EPH receptor A10 EPHA3 ENSG00000044524 EPH receptor A3 EPIA4 ENSGOOOOO116106 EPH receptor A4 EPHA7 ENSGO0000135333 EPH receptor A7 EPHA8 ENSG00000070886 EPH receptor A8 EPHB2 ENSG00000133216 EPH receptor B2 EPHB4 ENSG00000196411 EPH receptor B4 EPIX3 ENSGO0000105131 Epoxide hydrolase 3 EPO ENSG00000130427 Ervthropoietin EPPIN ENSGO0000101448 Epididymal peptidase inhibitor EPPIN-WFDC6 ENSG00000249139 EPPIN-WF[)C6 readthrough EPS15 ENSG00000085832 Epidermal growth factor receptor pathway substrate 15 EPS8L1 ENSG00000131037 EPS8-like 1 EPX ENSG00000121053 Eosinophil peroxidase EPYC ENSG00000083782 Epiphycan EQTN ENSG00000-120160 Equatorin, sperm acrosome associated
ER AP I ENSG00000164307 Endoplasmic reticulum aminopeptidase I ERAP2 ENSG00000164308 Endoplasmic reticulum aminopeptidase 2 ERBB3 ENSG00000065361 Erb-b2 receptor tyrosinie kinase 3 ERLINI ENSGO0000 107566 ER lipid raft associated I ERLIN2 ENSGO0000 147475 ER lipid raft associated 2 ERNI ENSG00000178607 Endoplasmic reticulum to nucleus signaling 1 ERN2 ENSG0000134398 Endoplasmic reticulum to nucleus signaling 2 EROIA ENSG00000197930 Endoplasmic reticulum oxidoreductase alpha ERO1B ENSG00000086619 Endoplasmic reticulum oxidoreductase beta ERP27 ENSGO000 139055 Endoplasmic reticulurn protein 27 ERP29 ENSG00000089248 Endoplasmic reticulum protein '29 ERP44 ENSG00000023318 Endoplasmic reticulum protein 44 ERV3-1 ENSG00000213462 Endogenous retrovirus group 3, member I ESMI ENSG00000164283 Endothelial cell-specific molecule 1 ESRPI ENSGO0000104413 Epithelial splicing regulatory protein 1 EXOG ENSGO0000157036 Endo/exonuclease (5'-3), endonuclease G-like EXTL I ENSG00000158008 Exostosin-like glycosyltransferase I EXTL2 ENSG000162694 Exostosin-like glycosyitransferase 2 F10) ENSGOOOOO126218 Coagulation factor X F11 ENSG00000088926 Coagulation factor XI F12 ENSGO0000 131187 Coagulation factor XII (Hageman factor) F13B ENSG00000143278 Coagulation factor XIII. B polypeptide F2 ENSGO0000180210 Coagulation factor II (thrombin) F2R ENSGOOOOO181104 Coagulation factor II (thrombin) receptor F2RL3 ENSG00000127533 Coagulation factor II (thrombin) receptor-like 3 F5 ENSGOOOOO 198734 Coagulation factor V (proaccelerin, labile factor) F7 ENSGOOOOO57593 Coagulation factor VII (serum prothrombin conversion accelerator) F8 ENSG0000O1850 10 Coagulation factor VIII, procoagulant component F9 ENSGOOOOO101981 Coagulation factor IX FABP6 ENSGO0000170231 Fatty acid binding protein 6, ileal FAMI107B ENSG00000065809 Family with sequence similarity 107, member B FAM1\4131A ENSG00000175182 Family with sequence similarity 131, member A FAM132A ENSGOOOOOI84163 Family with sequence similarity 132, member A FAN132B ENSGOOOOO178752 Family with sequence similarity 132, member B FAM150A ENSGOOOOO196711 Family with sequence similarity 150, member A FAMI150B ENSGO00000189292 Family with sequence similarity 150, member B FAM1\4171A1 ENSG00000148468 Family with sequence similarity 17 1, member Al FAM171B ENSGO0000144369 Family with sequence sirnilaity 17 1, memberB FANI72A ENSGOOOOO113391 Family with sequence simuilarity 172, member A FAM175A ENSGOOOOO163322 Family with sequence similarity 175, member A FAMI177A ENSGO00000151327 Family with sequence similarity 177, member Al FAM179B ENSGOi00198718 Family with sequence similarity 179, memberB FAM180A ENSGOOOOO189320 Family with sequence sirnilaity 180, member A FAN189AI ENSG00000104059 Family with sequence simuilarity 189, member Al
FAM198A ENSG00000144649 Family with sequence similarity 198, member A FA\I19A1 ENSG00000183662 Family with sequence similarity 19 (chemokine (C-C motif) like), member Al FA\419A2 ENSG00000198673 Family with sequence similarity 19 (chemokine (C-C motif) like), member A2 FA\l19A3 ENSG00000184599 Family with sequence similarity 19 (chemokine (C-C motif) like), member A3 FAMI9A4 ENSG00000163377 Family with sequence similarity 19 (chemokine (C-C motif like), member A4 FAMI9A5 ENSG00000219438 Family with sequence similarity 19 (chemokine (C-C motif) like), member A5
FAM20A ENSG0000108950 Family with sequence similarity 20, member A FA\420C ENSG00000177706 Family with sequence similant 20, member C FAN213A ENSG00000122378 Family with sequence similarity 213, member A FAN26D ENSG0000164451 Family with sequence similarity 26, member
) FAM46B ENSG00000158246 Family with sequence similarity 46, member B FAM57A ENSG00000)167695 Family wih sequence similarity 57, member A FA\478A ENSG00000126882 Family with sequence similarity 78, member A FAN96A ENSG00000166797 Family with sequence similarity 96, member A FAN9B ENSG00000177138 Family with sequence similarity 9, member B FAP ENSG00000078098 Fibroblast activation protein, alpha FAS ENSG00000026103 Fas cell surface death receptor FATI ENSG00000083857 FAT atypical cadherin I FBLNI ENSG00000077942 Fibulin 1 FBLN2 ENSG00000 163520 Fibulin 2 FBLN5 ENSG00000 140092 Fibulin 5 FBLN7 ENSG00000 144152 Fibulin 7 FBNI ENSG00000166147 Fibrillin I FBN2 ENSG00000138829 Fibrillin 2 FBN3 ENSG00000142449 Fibrillin 3 FBXW7 ENSG0000109670 F-box and WD repeat domain containing 7, E3 ubiquitin protein ligase FCAR ENSG00000186431 Fc fragment ofIgA receptor FCGBP ENSG0000275395 Fe fragment of Ig binding protein FCGR I B ENSG00000198019 Fc fragment of igG, high affinity lb, receptor (CD64) FCGR3A ENSG00000203747 Fc fragment of IgG, low affinity II1a, receptor (CD16a) FCGRT ENSGO0000 104870 Fc fragment of IgG, receptor, transporter, alpha FCMR ENSG00000162894 Fc fragment of TgM receptor FCNI ENSG00000085265 Ficolin (collagen/fibrinogen domain containing) I FCN2 ENSG00000160339 Ficolirn (collagen/fibrinogern domain containing lectin) 2 FCN3 ENSG00000142748 Ficolin (collagen/fibrinogen domain containing) 3 FCRLI ENSGOOOOO 163534 Fc receptor-like I FCRL3 ENSG00000 160856 Fc receptor-Iike 3 FCRL5 ENSG00000143297 Fe receptor-like 5
- 17()-
FCRLA ENSG00000132185 Fc receptor-like A FCRLB ENSG00000 162746 Fc receptor-like B FDCSP ENSG00000181617 Follicular dendritic cell secreted protein FETUB ENSG00000090512 Fetuin B FGA ENSG00000171560 Fibrinogen alpha chain FGB ENSG00000171564 Fibrinogenbeta chain FGF1i0 ENSG0000070 193 Fibroblast growth factor 10 FIF]7 ENSG00000158815 Fibroblast growth factor 17 FGF18 ENSG00000156427 Fibroblast growth factor 18 FGF19 ENSG0000162344 Fibroblast growth factor 19 FGF2I ENSGOO00105550 Fibroblast growth factor 21 FGF22 ENSG00000070388 Fibrobiast growth factor 22 FGF23 ENSGO0000118972 Fibroblast growth factor 23 FGF3 ENSG00000186895 Fibroblast growth factor 3 FGF4 ENSG00000075388 Fibroblast growth factor 4 FGF5 ENSG0000138675 Fibroblast growth factor 5 FGF7 ENSG00000140285 Fibrobiast growth factor 7 FGF8 ENSG00000107831 Fibroblast growth factor 8 (androgen-induced) FGFBPl ENSGOOOOO 137440) Fibroblast growth factor binding protein I FGFBP2 ENSG00000 137441 Fibroblast growth factor bindingprotein 2 FGFBP3 ENSGOOOO 174721 Fibroblast growth factor binding protein 3 FGFR1 ENSG00000077782 Fibrobiast growth factor receptor 1 FGFR2 ENSG00000066468 Fibroblast growth factor receptor 2 FGFR3 ENSGO00000068078 Fibroblast growth factor receptor 3 FGFR4 ENSG00000160867 Fibroblast growth factor receptor 4 FGFRLI ENSGOOOO 122418 Fibroblast growth factor receptor-like I FGG ENSGO0000171557 Fibrinogen garnma chain FGL1 ENSG00000104760 Fibrinogen-like 1 FGL2 ENSG00000127951 Fibrinogen-like 2 FHL I ENSG00000022267 Four and a halfLIM domains 1 FHIOD3 ENSGOOOO134775 Fonnin homology 2 domain containing 3 FIBIN ENSGO00000176971 Fin bud initiation factorhonolog (zebrafish) FICD ENSG00000198855 FIC domain containing FIGF ENSG00000165197 C-fos induced growth factor (vascular endothelial growth factor
FJXI ENSGOOOOO179431 Fourjointed box I FKBP1O ENSGOOOOO141756 FK506 binding protein 10, 65 kDa FKBP11 ENSG00000134285 FK506 binding protein 11, 19 kDa FKBP14 ENSGOOOOOI06080 FK506 binding protein 14, 22 kDa FKBP2 ENSGO0000173486 FK506 binding protein 2, 13kDa FKBP7 ENSGOOOO0079150 FK506 binding protein 7 FKBP9 ENSGOOOOO122642 FK506 binding protein 9, 63 kDa FL T I ENSGO00000102755 Fus-related tyrosine kinase 1 FLT4 ENSGO0000037280 Fns-related tyrosine kinase 4 FMO1 ENSGO0000010932 Flavin containing monooxygenase 1
FMO2 ENSG00000094963 Flavin containing monooxygenase 2 (non-functional) FMO3 ENSG00000007933 Flavin containing monooxygenase 3 FMO5 ENSGOO00 131781 Flavin containing monooxygeniase 5 FMOD ENSG00000122176 Fibromodulin FNI ENSG00000115414 Fibronectin I FNDC1 ENSG00000164694 Fibronectin type III domain containing 1 FNDC7 ENSG00000143107 Fibronectin type III domain containing 7 FOCAD ENSG00000188352 Focadlesin FOLR2 ENSGO0000165457 Folate receptor (fetal) FOLR3 ENSGO000 110203 Folate receptor 3 (gamma) FOXRED2 ENSGOO00 100350 FAD-dependent oxidoreductase domain containing 2 FP325331.1 ENSG00000278881 Uncharacterized protein 7UNQ6126/PRO20091 FPGS ENSG0000136877 Folylpolyglutamate synthase FRASI ENSG00000138759 Fraser extracelilar miatrix complex subunit 1 FREMI ENSG00000 164946 FRAS Irelated extracelilar matrix 1 FREM3 ENSGOO00183090 FRAS1 related extracellular matrix 3 FRMPD2 ENSG00000170324 FERM and PDZ domain containing 2 FRZB ENSGO0000162998 Frizzled-related protein FSHB ENSGO0000131808 Follicle stimuating hormone, beta polypeptide FSHR ENSG00000170820 Follicle stimulating hormone receptor FSTI ENSG00000134363 Follistatin FSTLI ENSG00000163430 Follistatin-like I FSTL ENSG00000070404 Follistatin-like 3 (secreted glycoprotein) FSTL.4 ENSG00000053108 Follistatin-like 4 FSTL5 ENSG00000 168843 Follistatin-like 5 FTCDNL1 ENSG00000226124 Fo'riminotransferase cyclodeaminase N-ternntial like FUCAI ENSG00000179163 Fucosidase. alpha-L- 1, tissue FUCA2 ENSG00000001036 Fucosidase, alpha-L- 2, plasma FUJRITN ENSG0000140564 Furin (paired basic amino acid cleaving enzyine) FUT10 ENSG00000172728 Fucosyltransferase 10 (alpha (1,3) fucosyitransferase) FITII ENSGOOOO196968 Fucosyltransferase II (alpha (1,3) fucosy transferase) FXN ENSG00000165060 Frataxin FXRI ENSGOOOOO114416 Fragile X mental retardation, autosomal homolog I FXYD3 ENSG00000089356 FXYD domain containing ion transport regulator 3 GABBRI ENSG00000204681 Gannna-aminobutyric acid (GABA) B receptor, I GABRA1 ENSG00000022355 Gamma-aminobnuytic acid (GABA) A receptor, alpha I GABRA2 ENSG00000151834 Gamma-aninobutyric acid (GABA) A receptor, alpha 2 GABRA5 ENSG00000186297 Gamma-aminobutyric acid (GABA) A receptor, alpha 5 GABRG3 ENSG0000182256 Gamma-aminobutyric acid (GABA) A receptor, gamma 3 GABRP ENSG00000094755 Gamma-aminobutyric acid (GABA) A receptor, pi GAL ENSG00000069482 Galanin/GMAP prepropeptide GAL3ST1 ENSG00000128242 Galactose-3-0-sulfotransferase 1 GAL3ST2 ENSG00000154252 Gaiactose-3-O-suifotransferase 2 GAL3ST3 ENSGOOOOO175229 Gaiactose-3-O-sulfotransferase 3
GALC ENSG00000054983 Galactosylceramidase GALNS ENSG00000141012 Galactosamine (N-acetyl)-6-sulfatase GALNT10 ENSG00000164574 Polypeptide N-acetylgalactosamiivltmsferase 10 GALNT12 ENSG00000119514 Polypeptide N-acetylgalactosaminyltransferase 12 GALNT15 ENSGOOOOO131386 Polypeptide N-acelylgalactosaminyltransferase 15 GALNT2 ENSGO0000143641 Polypeptide N-acetvlgalactosamninyltransferase 2 GALNT6 ENSG00000139629 Polypeptide N-acetylgalactosaminyltransferase 6 GALNT8 ENSG00000130035 Poly peptide N-aceylgalactosaminivltransferase 8 GALNTL6 ENSGOOOOO174473 Polypeptide N-acetygalactosaminyltrnsferase-like 6 GALP ENSGOOOOO197487 Galanin-like peptide GANAB ENSG00000089597 Glucosidase, alpha; neutral AB GARS ENSG00000106105 Glvcyl-tRNA synthetase GAS1 ENSG0000180447 Growtharrest-specific 1 GAS6 ENSGO0000183087 Growtharrest-specific 6 GAST ENSGOOOOO184502 Gastrin GBA ENSGOOOO177628 Glucosidase, beta, acid GBGTI ENSG00000148288 Globoside aloha-1,3-N-acetylgalactosaminvtransferase I GC ENSGO0000145321 Group-specific component (vitamin D binding protein) GCG ENSG00000115263 Glucagon GCGR ENSG00000215644 Glucagon receptor GCNT7 ENSGOOOO124091 Glucosaminyl (N-acetyl) trarsferase family member 7 GCSH ENSG00000140905 Glycine cleavage system protein H (aminomethyl carrier) GDFI ENSGO0000130283 Growth differentiation factor I GDFiO ENSG00000266524 Growth differentiation factor 10 GDFI ENSGO0000135414 Growth differentiation factor 11 GDF15 ENSGOOOO130513 Growth differentiation factor 15 GDF2 ENSG00000263761 Growth differentiation factor 2 GDF3 ENSGO0000184344 Growth differentiation factor 3 GDF5 ENSG0000125965 Growth dilferentiation factor 5 GDF6 ENSG00000156466 Growth differentiation factor 6 GDF7 ENSGO0000143869 Growth differentiation factor GDF9 ENSGO0000164404 Growth differentiation "actor 9 GDNF ENSGO0000168621 Glial cell derived neurotrophic factor GFOD2 ENSGOOOOO 141098 Glucose-fructose oxidoreductase domain containing 2 GFPT2 ENSGO0000131459 Glutamine-fructose-6-phosphate transaminase 2 GFRA2 ENSGOOOOO168546 GDNTF family receptor alpha 2 GFRA4 ENSGO0000125861 GDNF family receptor alpha 4 GGA2 ENSG00000103365 Golgi-associated, gamna adaptin ear containing ARF binding protein 2 GGH ENSGO0000137563 Gamna-glutamhyl vdrolase (conjugase, folyipolygamnaglutanl hydrolase) 1 GGTI ENSGOOOOOi10003 Gamna-glutarmyItransferase 1 GGT5 ENSG00000099998 Gamma-glutamyltransferase 5 GH1I ENSG00000259384 Growtl hormone 1 GH2 ENSGO00000136487 Growth hormone 2
GHDC ENSGO00000167925 GH3 domain containing GHRH ENSG00000118702 Growth hormone releasing hormone G-IRHR ENSG00000106128 Growth hormone releasing hormone receptor GHRL ENSG00000157017 Ghrelin/obestatinprepropeptide GIF ENSG0000134812 Gastric intrinsic factor (vitamin B synthesis) GIP ENSGOO00159224 Gastric inhibitory polypeptide GKNI ENSG00000169605 Gastrokine I GKN2 ENSG00000183607 Gastrokine 2 GLA ENSGO0000102393 Galactosidase, alpha GLBI ENSGO0000170266 Galactosidase, beta I GLB1L ENSG00000163521 Galactosidase, beta 1-like GLBIL2 ENSGO0000149328 Galactosidase, beta 1-like 2 GLCE ENSGO0000138604 Glucuronic acid epimerase GLG1 ENSG00000090863 Golgi gly coprotein 1 GLIPRI ENSGOOOOO139278 GLI pathogenesis-melated I GLIPRiL1 ENSGOO00173401 GLI patlhogenesis-related 1 like 1 GLIS3 ENSG00000107249 GLIS family zinc finger 3 GLMP ENSGOOOOO198715 Glvcosylated lysosomal membrane protein GLRB ENSGO0000109738 Glycine receptor, beta GLS ENSG00000115419 Glutaminase GLT6D I ENSGO0000204007 Glycosyl ransferase 6 domain containing 1 GLTPD2 ENSG00000 182327 Glvcoliiod transfer protein domain containing 2 GLUDI ENSGO0000148672 Glutamate dehydrogenase 1 GM2A ENSGO0000196743 GM2 ganglioside activator GVIL ENSGOOOOO 104499 Glycosylphosphatidylinositol anchored molecule like GNAS ENSG00000087460 GNAS corniex locus GNLY ENSG00001115523 Granulysin GNPTG ENSGO000009058I N-acetylglucosamine-1-phosphate transferase, gamma subunit GNRH1 ENSGO0000147437 Gonadotropir-releasing hormone 1(luteinizing-releasing hormone) GNRH2 ENSGO0000125787 Gonadotropin-releasing honnone 2 GNS ENSGOOOOO135677 Glucosarnire (N-acetyl)-6-sulfatase GOLMI ENSG00000135052 Golgi membrane protein 1 GORAB ENSGO00001203710 Golgin, RAB6-intemcting GOT2 ENSGO0000125166 Glutamic-oxaloacetic transaiinase 2. mitochondrial GP2 ENSGOOOOO169347 Glycoprotein 2 (zymogen granule mnembrane) GP6 ENSG0000088053 Glycoprotein VI (platelet) GPC2 ENSG00000213420 Glypican 2 GPC5 ENSG00000179399 Glvpican 5 GPC6 ENSGOOOOO 183098 Glypican 6 GPD2 ENSGO0000115159 Glycerol-3-phosphate dehydrogenase 2 (mitochondrial) GPER1 ENSGO0000164850 G protein-coupled estrogen receptor 1 GPHA2 ENSGOOOO 149735 GIcoprotein hormone alpha 2 GPHB5 ENSGO0000 179600 Glycoprotein hormone beta 5
GPIHBPl ENSG00000277494 Glicosyphosphatidylinositol anchored high density lipoprotein binding protein I GPLD1 ENSGOOOO112293 Glycosylphosphatidylinositol specific phospholipase D1 GPNMB ENSG00000136235 Glycoprotein (transmembrane) nmb GPR162 ENSG00000250510 G protein-coupled receptor 162 GPX3 ENSGO0000211445 Glutathione peroxidase 3 GPX4 ENSGO0000 167468 Giutathione peroxidase 4 GPX5 ENSG00000224586 Glutathione peroxidase 5 GPX6 ENSG00000198704 Glutathioneperoxidase 6 GPX7 ENSGOOOOO116157 Glutathione peroxidase G(REMI ENSG00000166923 Gremlin 1. DAN family BMP antagonist GREM2 ENSG00000180825 Gremlin 2. DAN family BMP antagonist GRHL3 ENSGOOOOO158055 Grainhead-like transcription factor 3 GRIA2 ENSG00000120251 Glutamate receptor, ionotropic, AMPA 2 GRIA3 ENSGOOOOO125625 Glutamate receptor, ionotropic, AMPA 3 GRIA4 ENSG00000152578 Glutamate receptor, ionotropic, AMPA 4 GRIK2 ENSG00000 164418 Glutamate receptor, ionotropic, kainate 2 GRIN2B ENSG00000273079 Glutamate receptor, ionotropic, N-methyl D-aspartate 2B GRM2 ENSG00000164082 Glutamate receptor, metabotropic 2 GRM3 ENSGO0000198822 Glutamate receptor, metabotropic 3 GRM5 ENSG0000168959 Glutamate receptor, metabotropic 5 GRN ENSG00000030582 Granulin GRP ENSGO00000134443 Gastrin-releasing peptide GSG I ENSGOO000111305 Gen cell associated 1 GSN ENSGO0000148180 Gelsolin GTDCI ENSG00000 121964 Glycosyltransferase-like domain containing I GTPBPIO ENSG00000105793 GTP-binding protein 10 (putative) GUCA2A ENSGOOOOO197273 Guarylate cvclase activator 2A (guanylin) GUCA2B ENSG00000044012 Guarylate cvclase activator 2B (uroguanyin) GUSB ENSG00000169919 Glucuronidase, beta GVQWI ENSGO0000241043 GVQW motifcontaining 1 GXYLT1 ENSG00000151233 Glucoside xylosyltransferase I GXYLT2 ENSGOOOOO172986 Glucoside xylosyitransferase 2 GYLTLlB ENSGOO000165905 Glycosyitnsferase-like 1B GYPB ENSG00000250361 Glycophorin B (MNS blood group) GZMA ENSGOOOOO145649 Gtrriyme A (granzyme 1, cytotoxicT-Ivymphoc3te-associated seic esterase 3) GZMB ENSGOOOOOI00453 Gravyme B (granzvymc 2, ctotoxic T-ymphocyte-associated shrine esterase 1)
GZMHrI ENSGOOOOO100450 Granzymc H (cathepsin G-like 2, protein h-CCPX) GZMK ENSG0000113088 Grnzriyme K (granzVme3tptaseII) GZIV ENSGO0000197540 Granzyme M (lymphocyte met-ase 1) 1-16PD ENSG00000049239 Hexose-6-phosphate dehydrogenase (glucose 1-dehydrogenase) IABP2 ENSGOOOO 148702 Hyaluronan binding protein 2
HIADHB ENSG00000138029 Hydroxvacyl-CoA dehvdrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA bydratase (trifunctional protein), beta subunit HAMP ENSGO000105697 Hepcidin antimicrobial peptide IAPLNi ENSG00000145681 Hyaluronan and proteoglycan link protein 1 HAPLN2 ENSG00000132702 Hyaluronan andproteoglycan link protein 2 H APLN3 ENS00000 140511 -Ivaluronarand proteogly can link protein 3 H APLN4 ENSG00000 187664 Hyaluronan and proteoglycan link protein 4 HARS2 ENSG00000112855 Histidyl-tRNA synthetase 2, mitochondrial IAVCRi ENSGO0000 113249 Hepatitis A vius cellular receptor 1 HCCS ENSG00000004961 Holocytochrome c synthase H CRT ENSG00000161610 Hypocretir (orexin) neuropeptide precursor HEATR5A ENSG00000129493 HEAT repeat containing 5A HEPH ENSG00000089472 Hephaestin IEXA ENSG00000213614 Hexosaminidase A (alpha polypeptide) HEXB ENSG00000049860 Hexosaminidase B (beta polypeptide) HFE2 ENSGO0000168509 Hemochrornatosis type 2 (juvenile) HGF ENSG00000019991 Hepatocyte growth factor (hepapoietin A; scatter factor) HGFAC ENSG00000109758 HGF activator 1-HIP ENSG00000164161 Hedgehog interacting protein HHIPL1 ENSG00000182218 HHIP-like 1 HHIPL2 ENSG00000143512 HHIP-like 2 -HLAI ENSG00000132297 HIERV-H{ LTR-associating I HHLA2 ENSG000000114455 HERV-H LTR-associating 2 IBADH ENSG0000106049 3-iy droxy isobut rate dehydrogenase I-INT2 ENSG0000137133 Histidine triad nucleotide binding protein 2 HLA-A ENSG00000206503 Major histocomnpatibility complex, class I, A HLA-C ENSi00000204525 Major lnstocotmpatibility complex, class I, C HLA-DOA ENSG00000204252 Major histocompatibility complex, class II, DO alpha ILA-DPA I ENSG00000231389 Major histocomipatibility complex, class II, DIP alpha 1 -ILA-DQA1 ENSG0000)0196735 Major histocompattoitity complex, class II, DQ alpha 1 HLA-DQBI ENSG00000 179344 Major histocompatibility oplex, class IiDQbeta HLA-DQB2 ENSG00000232629 Major histocompatibility complex, class II, DQ beta 2 H1MCNI ENSG00000143341 Hemicentin 1 HMCN2 ENSGOOOOO148357 Hemicentin 2 HMGCL EN SG00000117305 3-hydroxytethyl-3-metiylglutaryl-CoA ivase HMHAI ENSG00000 180448 Histocompatibility (minor) HA-I H{MSD ENSG00000221887 Histocompatibity (minor) sepindoman containing HP ENSG00000257017 Haptoglobin HPR ENSGO000026170 1 Haptoglobin-related protein IPSE ENSG00000173083 Heparanase HPSE2 ENSG00000172987 Heparanase 2 (inactive) IHPX ENSG00000110169 Hemopexin HRC ENSG00000130528 Histidine rich calcium binding protein HRG ENSGO0000 113905 Histidine--ich glycoprotein
IRSP12 ENSG0000132541 Hfeat-responsive protein 12 HS2STI ENSGO0000153936 Heparan sulfate 2-0-sulfotransferase I H S3 STI ENSG00000002587 Heparan sulfate (glucosamine) 3-O-sulfottarsferase I HS6STI ENSG00000136720 Heparan sulfate 6-0-sulfotransferase I HS6ST3 ENSGOOOOO185352 Heparan sulfate 6-0-sulfotransferase 3 ISD 1113 IL EN SGO000167733 Hydroxysteroid (t1-beta) dehydrogenase 1-like HSD17B11 ENSG00000198189 Hydroxysteroid (17-beta) dehydrogenase I1 f SD17B7 ENSG00000132196 Hydroxysteroid (17-beta) dehydrogenase 7 HSP90B1 ENSG00000166598 H-eat shock protein 90k1)a beta (Grp94), member 1 HSPA13 ENSG00000155304 Heat shock protein 70kDa family, member 13 H-SPA5 ENSG00000044574 Heat shock 7OkDa protein 5 (glucose-regulated protein, 78kDa) HSPG2 ENSG00000142798 Heparan sulfate proteoglycan 2 HTATIP2 ENSG00000109854 I-IV-1 Tat interactive protein 2, 30kDa HTN ENSG00000126-550) Histatin 1 HTN3 ENSG00000205649 Histatin 3 H-TRA1 ENSG00000166033 HtrA seine peptidase I HTRA3 ENSG00000170801 HtrA seine peptidase 3 HTRA4 ENSG00000169495 HtrA serine peptidase 4 HYALI ENSG00000114378 H-tyaluronoglucosaminidase 1 HYAL2 ENSG00000068001 Hvaluronoglucosaminidase 2 HYAL3 ENSG00000)186792 Hyalurornoglucosatmntidase 3 HYOUI ENSG00000149428 Hypoxia up-regulated 1 IAPP ENSGO0000121351 Islet amyloid polypeptide BSP ENSG00000029559 Integri -binding sialoprotein ICAMI ENSG00000090339 Intercelular adhesion molecule I iCAM2 ENSG00000108622 ntercellular adhesion rtolecule2 ICAM4 ENSG00000105371 ntercelularadhesionmolecule 4 (Landsteiner-Wienerblood ________________ ___________________group)
ID1 ENSG00000125968 ithibitor of DNA binding 1, dominant riegative helix-oop-helix protein IDE ENSGOOO0 119912 Insulin-degrading enzyme IDNK ENSGOOOO148057 idnK, glucortokinase hornolog (E. coli) IDS EN SG00000010404 iduronate 2-sulfatase IDUA ENSG000001274I5 iduronidase, alpha-L IFI27L2 ENSGOOOOO119632 Interfero., alpha-inducible protein 27-like 2 II30 ENSG00000216490 Interferon, gamma-inducible protein 30 IFNA I ENSGOOOOO197919 Interferon, alpha 1 IFNA10 ENSGOO000186803 interferon, alpha 10 IFNA13 ENSGI0000233816 interferon, alpha 13 IFNA14 ENSGM000228083 ITnterfero, alpha 14 IFNA16 ENSG00000147885 Interferon, alpha 16 INA17 ENSG0000234829 Interferon, alpha 17 IFNA2 ENSG00000188379 interfenonalph 2 IFNA21 ENSG00000137080 Interferon, alpha 21 IFNA4 ENSG00000236637 Interferon, alpha 4
IFNA5 ENSGO0000147873 interferonalpha 5 FNA6 ENSG00000120235 Interferon, alpha 6 IFNA7 ENSG00000214042 interferon, alpha 7 FNA8 ENSG00000120242 Interferon, alpha 8 IFNARI ENSGO0000142166 interferon (alpha, beta and omega) receptor 1 IFNBI ENSGO0000171855 interferon, beta 1, fibroblast FNE ENSG00000184995 Interferon, epsilon IFNG ENSC0000011537 interferon, gamma FNG R1 ENSG00000027697 Interferon gamma receptor 1 IFNL ENSGOOOOO182393 interferon, lambda I IFNL2 ENS.00000183709 Interferon, lambda 2 TFNL 3 ENSG00000197110 Interferon, lambda 3 FNLRI ENSG00000185436 interferon, lanbda receptor I IFNW1 ENSG00000177047 Interferon, omega I 1GF11 ENSG00000017427 Insulin-like growth factor 1 (somatomedin C) 1GF2 ENSG0000167244 Insulin-like growth factor 2 IGFALS ENSG0000099769 Insulin-like growth factor bindingprotein, acid labile subunit IGFBP I ENSG00000146678 Insulin-like growthfactorbindingprotein i iGFBP2 ENSG00000115457 Insulin-hike growth factorbinding protein 2, 36kDa iGFBP3 ENSGO0000146674 insulin-hke growth factorbinding protein 3 iGFBP4 ENSG0000141753 Insulin-like growth factor binding protein 4 TIGFBP5 ENSG00000115461 insulin-like growth factor binding protein 5 iGFBP6 ENSG0000167779 insulin-like growth factor binding protein6 iGFBP7 ENSG00000163453 Insulin-hike growth factorbinding protein 7 IGFBPL1 ENSG00000137142 insulin-hke growth factor binding protein-like I iGFL.1 3NSG00000 188293 IGF-like family member 1 IGF3L2 ENSG00000204866 iGF-likefamily member TGFL3 ENSG00000188624 iGF-likefamilymember3 iGFLRI ENSG00000126246 iGF-like family receptor 1 iGiP ENSG00000182700 IgA-inducing protein iGLON5 ENSG0000142549 igLON Iairily member 5 IGSF1 ENSG0000147255 immunoglobulin superfamily. member 1 1GSF10 ENSG00000152580 Immunoglobulin superfamily, member 10 GSF11 ENSG00000144847 imrunoglobulin superfaImily, member 11 IGSF21 ENSG00000117154 immunoglobin superfamily, member 21 iGSF8 ENSGO0000162729 immuinoglobulin superfamily, member 8 IGSF9 ENSG00000085552 immunoglobulin superfamily, member 9 IHH ENSG00000163501 indian hedgehog IL10 ENSG00000136634 Interleukmn 10 ILl ENSG00000095752 Interleukin I1 IL11RA ENSGO0000137070 Interleukin 11 receptor, alpha IL 12B ENSG60000113302 Interleukin 12B IL 12RBI ENSG00000096996 Interleukin 12 receptor, beta I iL 12RB2 ENSG00000081985 interleukin 12 receptor, beta 2
IL13 ENSG00000169194 Interleukin 13 13RA1 ENSG00000131724 Interleukin 13 receptor, alpha 1 L15RA ENSG00000134470 Interleukin 15 receptor, alpha IL 17A ENSG00000112115 Interleukin 17A IL 17B ENSG0000122743 Interleukin 17B IL17C ENSG00000124391 Interleukin 17C ILI17D ENSG00000172458 Interleukin17D L 7F ENSG00000112116 Interleukn 17F L 1RA ENSG00000177663 Interleukin 17 receptor A IL17RC ENSG00000163202 Interleukin 17 receptor C IL 17RE ENSG00000)163701 Interleukin 17 eceptor E IL18BP ENSG00000137496 Interleukin 18 bindingprotein 1L18RI ENSG00000115604 Interleukin 18 receptor 1 LI8RAP ENSG00000115607 Interleukin 18 receptor accessory protein IL19 ENSGO0000142224 Interleukin 19 ILIRI ENSG00000115594 Interleukin 1 receptor, type I IL1R2 ENSGOOOO115590 Interleukin I receptor, type II ILIRAP ENSGO0000 196083 interleukin I receptor accessory protein iLIRL1 ENSG00000115602 Interleukin 1 receptor-like 1 IL IRL2 ENSG00000115598 Interleukin I receptor-like 2 IL IRN ENSGOOOOO136689 Interleukin 1 receptor antagonist L2 ENSG00000109471 Interleukin2 L20 ENSGO0000162891 Interleukin 20 L20RA ENSGO0000016402 Interleukin 20 receptor, alpha 121 ENSG00000138684 Interleukin 21 IL22 ENSGOOOO127318 Interleukin 22 IL22RA2 ENSGOO0000164485 Interleukin 22 receptor, alpha 2 1L23A ENSGO0000110944 interleukin 23, alpha subunit p19 1L24 ENSG0000162892 Interleuki 24 125 ENSG00000166090 Interleukin 25 IL26 ENSGOOOO111536 Interleukin 26 1L22 ENSGO0000197272 Interleukin 27 IL2RB ENSG00000100385 interIeukin 2 rceptor, beta 1L3 ENSGO0000164399 Interleukni3 131 ENSG00000204671 Interleukin 31 IL3IRA ENSGO0000164509 InterleukinI I receptor A 1L32 ENSGO0000008517 Interleukin 32 134 ENSG00000157368 Interleukin 34 IL3RA ENSG00000185291 Interleukin 3 receptor, alpha (low affinity) 14 ENSGO0000113520 Interleukin 4 1L4Il ENSGOOOOO104951 Interleukin4induced1 IL4R ENSG00000077238 Interleukin 4 receptor L5 ENSG00000113525 Interleukin5 IL5RA ENSG00000091181 Interleukin 5 receptor, alpha
IL6 ENSGO0000136244 interleukin6 IL6R ENSG00000160712 Interleukin 6 receptor 1L6ST ENSG00000134352 interleukin 6 signal transducer 17 ENSG00000104432 Interleukin7 IL7R ENSG0000168685 Interleukin 7 receptor IL9 ENSG00000145839 interleukin 9 ILDRI ENSG00000145103 Immunoglobuin-ike domain containing receptor I ILDR2 ENSG00000143195 inuunoglobulin-like domain containing receptor 2 MP4 ENSGO0000136718 INP4, U3 small nucleolar ribonucleoprotein IMPG1 ENSG0000112706 Interphotoreceptor matrix proteoglycan I INIA ENSG00000123999 inhibin, alpha INHBA ENSG00000122641 Inhibin, beta A INIIBB ENSGM000163083 inhibin, beta B IHBC ENSG00000175189 Inhibin, beta C INHBE ENSGOOOOO 139269 1hibin, beta E INPP5A ENSG00000068383 inositol polyphosphate-5-phosphatase A EN ENSG00000254647 Insuin INS-IGF2 ENSG00000129965 INS-IGF2 readthrough INSL3 ENSG00000248099 Insulin-like 3 (Leydig cell) INSL4 ENSG00000120211 Insulin-like 4 (placenta) INSL5 ENSG00000172410 insulin-like5 INSL6 ENSG00000120210 Insulin-like 6 INTS3 ENSGO0000143624 Integrator complex subunit 3 IPO11 ENSGO0000086200 Importin I1 P09 ENSGO0000198700 Importin 9 iQCF6 ENSG00000214686 IQ motifcontaining F6 IRAK3 ENSG00000090376 interleukin-1 receptor-associated kinase 3 IRS4 ENSG00000133124 Insulin receptor substrate 4 ISLR ENSG00000 129009 imrunoglobuli superfanily containing leucine-rich repeat ISLR2 ENSGOOOOO 167178 Immunoglobulin superfamily containing leucine-rich repeat 2 iSMI ENSG00000 101230 Isthmin 1, angiogenesis inhibitor ISM2 ENSG00000100593 Isthmnin2 ITGA4 ENSGOOOOO115232 Integrin, alpha 4 (antigen CD49D, alpha 4 subunit of VLA-4 receptor) TGA9 ENSG00000144668 Integrin, alpha 9 ITGAL ENSG00000005844 Integrin, alpha L (antigen CDIIA (p80), lymphocyte fimctiot associated antigen 1; alpha polypeptide)
ITGAX ENSG00000140678 Integrin, alpha X (complement component 3 receptor 4 subunit) iTGBI ENSG00000 150093 Integrinibeta I (fibronectin receptor, beta polypeptide, antgen CD29 includes MDF2, MSK12) iTGB 2 ENSG00000160255 Integrin, beta 2 complementt coiponett 3 receptor 3 and 4 subunit) ITGB3 ENSG0000259207 Integrin. beta 3 (platelet glycoproteinilla, antigenCD61) ITGB7 ENSG00000139626 Integrin. beta iTGBL NSG00000198542 Integriti, beta-like I (with EGF-like repeat domains)
111-1 ENSG00000055957 inter-alpha-trypsin inhibitor heavy chain I TH2 ENSG00000151655 Inter-alpha-trypsininhibitor heavy chain 2 1T1H3 ENSG00000162267 inter-alpha-try psin inhibitor heavy chain 3 IT14 ENSGO0000055955 Inter-aIpha-tryosin inhibitor heavy chain family, member 4 11115 ENSG00000123243 Inter-alpha-trypsin inhqibitor heavy chain family, member 5 1111-16 ENSG00000102313 inter-alpha-trypsin inhibitor heavy chain family, member 6 ITLN1 ENSG00000179914 Intelectin I (galactofuranosebinding) ITLN2 ENSG00000158764 Intelectin2 iZUMO1R ENSG00000183560 IZUMO1 receptor, JUNO IZUMO4 ENSG00000099840 IZUM4Ofamily member 4 JCHAIN ENSG00000 132465 Joining chain of mnulmneric IgA and IgM JMJD8 ENSG00000 161999 Jumonji domain containing 8 JSRP1 ENSG00000167476 JunctionalIsarcopasmicreticulum protei KANSL2 ENSG00000139620 KAT8 regulatory NSL complex subunit 2 KAZALDI ENSG0000 107821 Kazal-type serine peptidase inhibitor domain I KCNIP3 ENSGO0000115041 Kv channel interacting protein 3, caisenilin KCNK7 ENSG00000173338 Potassium channel, two pore domain subfamily K, member KCNN4 ENSG00000104783 Potassium channel, c naium activated internediate/small conductance subfamily N alpha member 4
KCNUI ENSG00000215262 Potassium channel, subfamily U, member 1 KCP ENSGM000135253 Kielin/chordin-like protein KDEL CI ENSG00000134901 KDEL (Lys-Asp-Glu-Leu) containing I KDELC2 ENSG00000178202 KDEL (Lys-Asp-Glu-Lci) continirig 2 KDMIA ENSG000000004487 Lysine (K)-specific deimethylase IA KDM3B ENSGO0000120733 Lysine (K)-specific demethylase 3B iKDN46A ENSIOO00O142050 Ly si ce(K)spcifiu deniethylaise6A KDM7A ENSG00000006459 Lysine (K)-specific demethylase 7A KDSR ENSGO000 119537 3-ketodihydrosphingosine reductase KERA ENSGO0000139330 Keratocan KIAA0100 ENSG00000007202 KIAAO100 KIAA0319 ENSG00000137261 KIAA0319 KIAA1324 ENSG00000116299 KIAA1324 KIFC2 ENSGO0000167702 Kinesin family member C2 KiR2)L4 ENSG00000189013 Killer cell irnunoglobulin-like receptor, two domains, long cytoplasmic tail, 4
KIR3'DXI ENSG00000104970 Killer cell immnoglobuin-likereceptor, three domains, XI KIRREL2 ENSG00000126259 Kin ofIRRE like 2 (Drosophila) KISSI ENSGO0000170498 KiSS-1 metastasis-suppressor KLiBILII ENSG00000178502 Kelch-like family member II KLHL22 ENSG00000099910 Kelch-like family member 22 KLK1 ENSG00000 167748 Kallikreini KLK1O ENSG000000129451 Kallikreiin-related peptidase 10 KLK]1 ENSGOOOOO167757 Kallikrein-related peptidase 11 KLK12 ENSG00000186474 Kallikrein-related peptidase 12
KLK13 ENSGO00167759 Kallikrein-related peptidase 13 KLK14 ENSG00000129437 Kallikrein-related peptidase 14 KLK15 ENSG00000174562 Kallikrein-reiated peptidase 15 KLK2 ENSG00000167751 Kallikrein-related peptidase 2 KLK3 ENSG00000 142515 Kallikrein-related peptidase 3 KLK4 ENSGOOO0 167749 Kallikrein-related peptidase 4 KLK5 ENSG00000 167754 Kallikrein-related peptidase 5 KLK6 ENSG00000 167755 Kalikrein-reiated peptidase 6 KLK7 ENSG00000 169035 Kallikrein-related peptidase 7 KLK8 ENSG00000 129455 Kallikrein-related peptidase 8 KLK9 ENSG00000213022 Kallikrein-related peptidase 9 KLKB1 ENSGO0000164344 Kallikrein B, plasma (Fletcher factor) 1 KNDCI ENSG00000171798 Kinase noni-catalytic C-lobe domain (KIN)) containnig 1 KNG IENSG0000113889 Kinmogen 1 KRBA2 ENSG00000184619 KRAB-A domain containing 2 KREMEN2 ENSG00000 131650 Kringie containing transmembrane protein 2 KRTDAP ENSG00000188508 Kemtinocvte differentiation-associated protein LICAM ENSGO0000198910 L Icell adhesionmolecule L,3MBTL2 ENSG00000100395 L(3)nbt-like 2 (Drosophila) LA16c-380H5.3 ENSG00000270168 LACEI ENSGO0000 135537 Lactationelevated 1 LACRT ENSG00000135413 Lacritin LACTB ENSG00000103642 Lactamase, beta LAG3 ENSG00000089692 Lymphocyte-activation gene 3 LAIR2 ENSG00000167618 Leukocyte-associated immunoglobulin-like receptor 2 LALBA ENSGOOOO 167531 Lactalbumnin, alpha LAMAI ENSGOOOOO101680 Laininin. alpha I LAMA2 ENSG00000196569 Laminin, alpha 2 LAIA3 ENSG00000053747 Laminin, alpha 3 LAMA4 ENSG00000112769 Laminin, alpha 4 LAMA5 ENSGO000130702 Laminiin, alpha 5 LAMB1 ENSGO0000091136 Laninin, beta 1 LAMB2 ENSGO0000172037 Laminin, beta 2 (laminin S) LAMB3 ENSG00000196878 Larinin, beta 3 LAMB4 ENSG00000091128 Lamimn, beta 4 LAMC1 ENSGO0000135862 Laminin, gamma I (formerly LAMB2) LAMC2 ENSG00000058085 Laininin. gamma 2 LAMC3 ENSG00000050555 Laminin, gamma 3 LAMP3 ENSG00000078081 Lysosomal-associated membrane protein 3 LAT ENSGO0000213658 Linker for activation of T cells LAT2 ENSG00000086730 Linker for activation of T cells family, member LBP ENSGOOOO129988 Lipopolysaccharide binding protein LCAT ENSGO0000213398 Lecithin-cholesterol acytransferase LCNI ENSGO000160349 Lipocalin I
LCNIO ENSG00000187922 Lipocalin 10 LCN12 ENSG00000184925 Liiocalin 12 LCN15 ENSG00000177984 Lipocalin 15 LCN2 ENSG00000148346 Lipocalin 2 LCN6 ENSG00000267206 Lipocalin 6 LCN8 ENSG00000204001 Lipocalin 8 LCN9 ENSG0000148386 Li-oocalin 9 LCORL ENSG00000178177 Ligand dependent nuclear receptor corpressor-like LDLR ENSG00000130164 Low density lipoprotein receptor LDLRAD2 ENSGOOOOO 187942 Low density lipoproteins receptor class A domain containing 2 LEAP2 ENSGOOOOO164406 Liver expressed antimicrobial peptide 2 LECT2 ENSG00000145826 Leukocyte cell-derived chemotaxin 2 LEFTYI ENSG00000243709 Left-rght determination factor 1 LEFTY2 ENSG00000143768 Left-right determination [actor 2 LEP ENSG0000174697 Leptin LFNG ENSGOOOOO106003 LFNG O-fucosylpeptide 3-beta-N acetylglucosaminyltransferase LGALS3BP ENSGOOOOO108679 Lectin, galactoside-binding, soluble,3 binding protein LGII ENSGO0000108231 Leucine-rich, glioma inactivated 1 LGI2 ENSG00000153012 Leucine-rich repeat LGI family, member 2 LGI3 ENSGOOOOO 168481 Leucine-rich repeat LGI family, member 3 LGI4 ENSG00000153902 Leucine-rich repeat LGI family, member 4 LGMN ENSGOOOOO100600 Legumain LGR4 ENSGOO000205213 Leucine-rich repeat containing G protein-coupled eceptor 4 LIH1B ENSGO0000104826 Luteinizing hormone beta polypeptide LHCGR ENSGO0000138039 Luteinizing hormone/choriogonadotropin receptor LIF ENSGOO000 128342 Leukemia inhibitory factor LIFR ENSGOOOOO 113594 Leukemia inhibitory factor receptor alpha LILRAI ENSGO0000104974 Leukocyte immunogobulin-like receptor, subfamily A (with TM domain), member I
LILRA2 ENSG00000239998 Leukocyte imnunoglobulin-like receptor, subfamily A (with TM domain), ember2
LILRB3 ENSG00000204577 Leukocyte immunoglobulin-like receptor, subfamily B (with TM and ITIM domains), member 3
LIMEI ENSG00000203896 Lck interacting transmembrane adaMor I LINGOI ENSG0000169783 Leucine rich repeat and ig domain containing 1 LIPA ENSGOOOOO 107798 Lipase A lysosomal acid, cholesterol esterase LIPC ENSGOOOOO166035 Lipase, hepatic LIPF ENSGO0000182333 Lipase, gastric LIPG ENSG00000101670 Lipoase, endothelial LIPH ENSC00000163898 Lipase, member H LIPK ENSGO0000204021 Lipase, family member K LIPM ENSGOOOOO 173239 Lipase, family ember M LIPN ENSG00000204020 Lipase, family member N LMAN2 ENSGO0000169223 Lectin, mannose-binding 2
LMNTDI ENSG00000152936 Lamin tail domain containing I LNXI ENSG00000072201 Ligand of numb-protein X 1. E3 ubiquitin-protein ligase LOX ENSG00000113083 Lysyl oxidase LOXL1 ENSG00000129038 Lysyl oxidase-like 1 LOXL2 ENSGOOOOO134013 Lysyl oxidase-like 2 LOXL3 ENSG0000115318 Lysyl oxidase-like 3 LOXL4 ENSG00000138131 Lysyl oxidase-like 4 LPA ENSGO0000198670 Lipoproteirn, Lp(a) LPL ENSG00000175445 Lipoprotein lipase LPO ENSGO0000167419 Lactoperoxidase LRAT ENSGOOOOO121207 Lecithin retinol acyltransflerase (phosphatidylcholine--retinol 0 acyitransferase)
LRCI-13 ENSGOO000 186001 Leucine-ich repeatsand calporin horology (CH) doman containing 3 LRCOL I ENSG00000204583 Leucine rich colipase-like I LRFN4 ENS(1i0000017 3621 Lencine nebrepeatarid fibroriectinltype III]domainicontlain-ing 4 LRFN5 ENSGOOOOO 165379 Leucine rich repeat and fibronectin type III domain containing 5 LRGI ENSGO(0000171236 Leucine-rich alpha-2-glycoprotein I LRP1 ENSG000000123384 Low density lipoprotein receptor-related protein I LRPII ENSG00000120256 Low density lipoprotein receptor-related protein 11 LRPlB ENSGO0000168702 Low densitylipoprotein receptor-related protein IB LRP2 ENSGO0000081479 Low density lipoprotein receptor-related protein 2 LRP4 ENSGO0000134569 Low density lipoprotein receptor-related protein 4 LRPAP1 ENSGO00000163956 Low density lipoprotein receptor-related protein associated protein 1 LRRCI7 ENSGOOOOO 128606 Leucine rich repeat containing 17 LRRC32 ENSG00000 137507 Leucine rich repeat containing 32 LRRC3B ENSGO0000179796 Leucine rich repeat containing 3B LRRC4B ENSG00000131409 Leucine rich repeat containing 4B LRRC70 EN SG00000186105 Leucine rich repeat containing 20 LRRN3 ENSG00000173114 Leucine rich repeat neuronal 3 LRRTMI ENSG00000162951 Leucine nich repeat transmembrane neuronal 1 LRRTM2 ENSGO0000146006 Leucine rich repeat transmembrane neuronal 2 LRRTM4 ENSGOOOOO 176204 Leucine rich repeat transmembrane neuronal 4 LR'TM2 ENSG0000(0166159 Leucine-rich repeatsand transmembnie domains 2 LSR ENSGOOOOO105699 Lipolysis stimulated lipoprotein receptor LSTI ENSG00000204482 Leukocyte specific transcrpti LTA ENSG00000226979 Lymphotoxin alpha LTBPI ENSG00000049323 Latent transforming growth factor beta binding protein 1 LTBP2 EN SG00000119681 Latent transforming growth factor beta binding protein 2 LTBP3 ENSG00000168056 Latent transforming growth factor beta binding protein 3 LTBP4 ENSG00000090006 Latent transfo niringgrowthfactor beta binding protein 4 LTBR ENSG00000111321 Lyrnphotoxin beta receptor (TNFR superfamily, member 3) LTF ENSGO000012223 Lactotransferrin LTK ENSG00000062524 Leukocyte receptor tyrosine kinase
LUM ENSG t00139329 Lumican LUZP2 ENSG00000187398 Leucine zipper protein 2 LVRN ENSG00000172901 Laeverin LY6E ENSG00000160932 Lymphocyte antigen 6 complex, locus E LY6G5B ENSGO0000240053 Lymphocyte antigen 6 complex, locus 05B LY6G6) ENSG00000244355 Lymphocyte antigen 6 complex, locus G61) LY6G6E ENSG00000255552 Lymphocyte antigen 6 complex, locus G6E (pseudogene) LY6-l ENSG00000176956 Lymphocyte antigen 6 complex, locus -I LY6K ENSG00000160886 Lymphocyte antigen 6 complex, locus K LY86 ENSG00000 112799 Lymphocyte antigen 86 LY96 ENSG00000 154589 Lymphocyte antigen 96 LYGI ENSG00000144214 Lvsozyme G-like I LYG2 ENSG00000185674 Lysozyme G-like 2 LYNXI ENSGO0000180155 Ly6/neurotoxin 1 LYPDI ENSG00000150551 LY6/PLAUR domain containing I LYPD2 ENSGOOOO197353 LY6/PLAUR domain containing 2 LYPD4 ENSG00000273111 LY6/PLAUR domain containing 4 LYPD6 ENSGOOOOO187123 LY6/PLAUR domaincontaining 6 LYPD6B ENSGO0000150556 LY6/PLAUR domain containing 6B LYPD8 ENSG00000259823 LY6/PL AUR domain containing 8 LYZ ENSG00000090382 Lysozyme LYZL4 ENSG00000157093 Lysozyme-like 4 LYZL6 ENSG00000275722 Lysozyrne-like 6 NM6PR ENSG00000003056 Mannose-6-phosphate receptor (cation dependent) MADIL1 ENSG00000002822 MADI mitotic arrest deficient-like I (yeast) MAG ENSGOOOO105695 My elin associated gly coprotein MAGTi ENSG00000102158 Magnesium transporter I MALSUI ENSG00000156928 Mitochondrial assembly of ribosomal large subunit I MAMDC2 ENSG0000165072 MAM domain containing 2 MAN2BI ENSGO0000104774 Mamosidase, alpha, class 2B, member 1 MAN2B2 ENSG00000013288 Mannosidase, alpha, class 2B, member 2 MANBA ENSGOOOO 109323 Manosidase. beta A, lysosomal MANEAL ENSG00000185090 Mannosidase, endo-alpha-like MANF ENSG0000145050 Mesencephaic astrocyte-derived neurotrophic factor MANSCI ENSGO0000111261 MANSC domain containing I MAP3K9 ENSG00000006432 Mitogen-activated proteinkinase 9 MASPI ENSG0000127241 Mannman-binding lectin series peptidase I (C4/C2 activating component ofRa-reactiveactor) MASP2 ENSG00000009724 Mannan-binding lectin serinie peptidase 2 MATNI ENSG00000162510 Matrilin 1, cartilage matrixprotein MATN2 ENSG00000132561 MatInlin 2 MATN3 ENSG00000132031 Matnlin 3 MATN4 ENSGOOOOO124159 Matrilin 4 MATR3 ENSG00000015479 Matrinm3
MATR3 EN SG00000280987 Matrin 3 MAU2 ENSGO0000129933 MAU2 sister chromatid cohesion factor MAZ ENSG00000103495 MYC-associated zinc finger protein (purine-binding transcription factor) MBD6 ENSG00000166987 Methyl-CpG binding domain protein 6 MIBL2 ENSG00000165471 Mannose-binding lectin (protein C) 2, soluble MBNLI ENSG00000152601 Muscleblind-like splicing regulator I MCCCI ENSG00000078070 Methylcrotonoyl-CoA carboxylase I (alpha) MCCDI ENSGO0000204511 Mitochondrial coiled-coil domain I MCEE ENSGO0000124370 Methylmalonyl CoA epimerase MCF2L ENSGO0000126217 MCF.2 cell line derived transfoming sequence-like MCFD2 ENSGO0000180398 Multiple coagulation factor deficiency 2 MDFIC ENSG00000135272 MyoD family inhibitor domain containing MDGA I ENSG1i00000112139 MAM domain containing glycosylphosphatidylnositoi anchor I MDK ENSG0000110492 Midkine (neurite growth-promoting factor) MED20 ENSG0000 124641 Mediator complex subunit20 MEGF10) ENSG00000145794 Multiple EGF-like-domains 10 MEGF6 ENSGO0000162591 Multiple EGF-like-domains 6 ME]I ENSG00000167077 eiotic double-standed break formationprotein 1 MEI4 ENSG00000269964 Meiotic double-stranded break formation protein 4 MEISI ENSGO0000143995 Meishomcobox I MEIS3 ENSGO0000105419 Meishoeraobox 3 IVEPE ENSG00000152595 Matrix extracellularphosphoglycoprotein MESDC2 ENSG00000117899 Mesoderm development candidate 2 MEST ENSGO0000106484 Mesoderm specific transcript MET ENSGOOOOO105976 MET proto-oncogene, receptor tyrosine kinase METRN ENSGOO000103260 Meteorin, glial cell diflerentiation regulator METRNL ENSG00000176845 Meteorin, glial cell differentiation regulator-like METT12 ENSG0000165792 MeItiyltransferase like 17 METTL24 ENSGO0000053328 Methyltransferase like 24 METTL7B ENSGOOOOO170439 Methy'ltmnsferase like 7B METTL9 ENSGOOOO192006 Methyltransferase like 9 MEX3C ENSG00000176624 Mex-3 RNA binding family member C MFAP2 ENSGOOOOO 117122 Microfibrillar-associated protein 2 MFAP3 ENSG00000037749 Microfibrillar-associated protein 3 MFAP3L ENSGOOOOO 198948 Microfibrillar-associated protein 3-like MFAP4 ENSG0000 166482 Microfib rilar-associated protein 4 1MFAP5 ENSG00000197614 Microfibrillar associated protein 5 MFGE8 ENSG00000140545 Milk fat globule-EGF factor 8 protein MFI2 ENSGO0000163975 Antigenp97 (melanomaassociated)identifiedby monoclonal antibodies 133.2 and 96.5
MFNG ENSGO0000100060 MFNG O-fucosylpeptide 3-beta-N acetyIglucosaminyiltransferase MGA ENSGOOOOO174197 MGA, MAX dimerization protein
MGAT2 ENSG00000168282 Mannosyl (alpha-1.6-)-glycoprotein bea-1,2-N acetylIglucosaminyltransferase M3\GAT3 ENSG00000 128268 Manosyl (beta-1,4-)-glycoprotein beta-1,4-N acetylglucosaminyltransfemse
VIGAT4A ENSG00000071073 Nannosyl (atpha-1,3)gycoprotein beta-1,4-N acetylglucosaminyltraisferase, isozvme A
]MI(iAT4B ENSG00000161013 Nannosyl(alpha-1,3 glycoproteinbeta-1,4-N acetylglucosainyltranisferase, isozvme B
MGAT4D ENSG00000205301 MGAT4 family, member D MGLL ENSG00000074416 Monoglyceride lipase MGP ENSG00000 111341 Matrix Gia Protein MGST2 ENS(100000085871 M icrosomal ghutahiorie S-tiansferase2 MIA ENSG00000261857 Melanoma inhibitory activity MIA2 ENSG00000150526 Melanoma inhibitory activity 2 MIA3 ENSG00000154305 Melanoma inhibitory activity family, inember 3 MICUI ENSG00000107745 Mitochondrial calcium uptake I NMIERI ENSG00000198160 Mesoderminduction early response 1,tmarscptional regulator MINOS I-NBL1 ENSG00000270136 MINOS I-NBL I readthrough MINPPI ENSG00000107789 Multiple inositol-polyphosphate phosphatase I MLEC ENSG00000110917 Malectin MLN ENSG00000096395 Motilin MLXIP ENSG0000175727 MLX interacting protein MLXIPL ENSG00000009950 MLX interacting protein-like MMvIPI ENSG00000 196611 Matrix metallopeptidase I MMP10 ENSG00000(166670 Matrix metallopeptidase 10 MiMPII ENSG00000099953 Matrix metallopeptidase 11 MMP12 ENSG00000262406 Matrix rnetallopeptidase 12 NMMP13 ENSG00000137745 Matrix uetallopeptidase 13 MMvIP14 ENSG00000 157227 Matrix metallopeptidase 14 (membmne-inserted) MMP17 ENSG00000 198598 Matrix metallopeptidase 17 (membmne-inserted) MiMP19 ENSG00000123342 Matrix metallopeptidase 19 MMP2 ENSG00000087245 Matrix mnetallopeptidase 2 NMMP20 ENSG00000137674 Matrix uetallopeptidase 20 MMP21 ENSG00000154485 Matrix metallopeptidase 21 MMP25 ENSG00000008516 Matrix metallopeptidase 25 MMP26 ENSG00000167346 Matrix metallopeptidase 26 MMP27 ENSG00000137675 Matrix mnetallopeptidase 27 NMMP28 ENSG00000271447 Matrix uetallopeptidase 28 MMLP3 ENSG00000 149968 Matrix metallopeptidase 3 MMP7 ENSG00000 137673 Matrix metallopeptidase 7 MMP8 EN SGO0000 118113 Matrix metallopeptidase 8 MMP9 ENSGO0000 100985 Matrix mnetallopeptidase 9 MMR N1 ENSG00000138722 Multimerin I MMRN2 ENSG00000173269 Multimerin 2
MOXDi ENSG00000079931 Monooxygease, DBI-like 1 MPO ENSG00000005381 Myeloperoxidase MPPED I ENSG00000186732 Metallophosphoesterase domain containing 1 MPZLI ENSGO0000197965 Myelin protein zero-like I MRI1 ENSG00000 153029 Major histocornpatibility complex, class I-rolated MRPL2 ENSGO0000112651 Mitochondrial ribosomal protein L2 MRPL21 ENSG00000197345 Mitochondrial ibosomal protein L2I NMRPL.22 ENSG0000082515 Mitochoridrial rbosomal protein L22 MRPL24 ENSG00000143314 Mitochondrial ribosomal protein L24 MRkPL27 ENSG00000 108826 Mitochondrial ribosomal protein L27 MRPL32 ENSG00000106591 Mitochondral ribosomal protem L32 MPL34 ENSG00000130312 Mitochondrial ibosomalprotein L34 NMRPL.35 ENSG00000132313 Mitochoridrial rbosomal protein L35 MRPL52 ENSGO00000172590 Mitochondrial nbosomal protein L52 MRPL5 ENSGO0000162910 Mitochondrial ribosomal protein L55 MRPS14 ENSGO00000120333 Mitochondrial ribosomal protein S14 MPS22 ENSG00000175110 Mitochondrial ribosomalprotein S22 MRPS28 ENSGO0000147586 Mitochondrial rPbosomal protein S28 MS4A14 ENSG00000166928 Membrane-spanning 4-domains, subfamily A, member 14 MS4A3 ENSG00000149516 Membrane-spanning 4-domains, subfamily A, member 3 (hematopoietic cell-specific) MSH3 ENSG00000113318 MutS homolog 3 MSH5 ENSGO0000204410 MutS homolog 5 MSLN ENSG t00102854 Mesothelin MISMBS ENSGO0000263639 Microsemninopro'einbeta NMSRA ENSGOOOOO175806 Methionine sulfoxde reductase A MSRB2 ENSG00000148450 Methionine sulfoxide reductase B2 MSRB3 ENSGOOOOO174099 Methionine sulfoxide reductase B3 MST1 ENSGOO000173531 Macrophage stimulating 1 MSTN ENSG00000138379 Myostatin MT1G ENSG00000125144 Metallothionein i1G MTHFD2 ENSGO0000065911 Methylenetetrahydrofolate dehydrogenase (NADP+ dependent 2, netheinytetrahy drofolate cyclohydrolase MTMR 14 ENSGOOOOO163719 Myotubularin related protein 14 MTRNR2L11 ENSG00000270188 MT-RNR2-like I (pseudogene) MTRR ENSG00000124275 5-methy[etrahy drofolate-homocysteine methyltransferase reductase MTTP ENSGO0000138823 Microsomal triglyceride transfer protein NMTX2 ENSG00000128654 Metaxin 2 MUCI ENSGO0000185499 Mucin 1, cell surface associated MU(JCi3 ENSGO0000173702 Mucin 13, cell suface associated MUC20 ENSGO0000176945 Mucin 20, cell surface associated MUC3A ENSGO0000169894 Mucin 3A, cell surface associated MUC5AC ENSG00000215182 Mucir 5AC, oligomeric mucus/gel-forming MUC5B ENSGOOOOO117983 Mucin 5B, oligomeric mucus/gel-foiming
MIIUC6 ENSGOO000184956 Mucin 6, oligo-meric mucus/gel-forming MUC7 ENSG00000171195 Mucin 7. secreted MUCLI ENSG00000172551 Mucin-like 1 MXRA5 ENSG00000101825 Matrix-remodelling associated 5 MXRA7 ENSGO0000182534 Matrix-remodelling associated 7 MY)GF ENSG00000074842 Myeloid-derived growth [actor MYL I ENSG00000168530 Myosin, light chain 1, alkali; skeletal, fast MYOC ENSG00000034971 MVocilii trabecular meshwork mducible glucocorticoid response MYRFL ENSGO0000166268 Mvelin regulatory factor-like MZBI ENSGO0000170476 Marginal zone B and B I cell-specific protein N4BP2L2 ENSG00000244754 NEDD4 binding protein 2-like 2 NAA38 ENSGO00001830 11 N(alpha)-acetyltransfemse 38, NatC auxiliary subunit NAAA ENSG0000138744 N-acylethanolamineacid amiase NAGA ENSGO0000198951 N-acetylgalactosaminidase, alpha NAGLU ENSGOOOOO 108784 N-acetylglucosaminidase, alpha NAGS ENSG000161653 N-acetyglutamate synthase NAPSA ENSGO0000131400 Napsin A aspartic peptidase NBLI ENSG00000158747 Neuroblastorna 1, DAN frnily BMP antagonist NCAM1I ENSG00000 149294 Neural cell adhesion molecule I NCAN ENSGOOOOO130287 Neurocan NCBP2-AS2 ENSG000270170 NCBP2 antisense RNA 2 (head to head) NCSTN ENSG00000162736 Nicastrin NDNF ENSGO00000173376 Neuron-derived neurotrophic factor NDP ENSGO0000124479 Norrie disease (pseudoglioma) NDUFAI0 ENSGOOOOO 130414 NADH dehydrogenase (ubiquinone) I alpha subcomplex, 10, 42kDa NDUFB5 ENSG00000136521 NADH dehydrogenase (ubiquinone) I beta subcomplex, 5. 16kDa ND[JFS8 ENSGOOOOO10717 NAD-1 dehydrogenase (ubiqimnone) Fe-S protein 8, 23kDa (NADIHI-coenzyme Q reductase)
NDIJFVI ENSGOOOOO167792 NADH dehydrogenase (ubiquinone) flavopromen 1, 51 kDa NECAB3 ENSGOOOO0125967 N-terminal EF-hand calcium binding protein 3 NELLI ENSGOOOOO165973 NeuralEGFL like I NELL2 ENSGOOOOO184613 Neural EGFL like 2 NENF ENSGOOOOO117691 Neudesin neurotrophic factor NETOL ENSG00000166342 Neuropilin (NRP) and tolloid (TL)-like I NFASC ENSGO0000163531 Neurofascin NFE2L1 ENSG00000082641 Nuclear factor, erythroid 2-like 1 NFE2L3 ENSG00000050344 Nuclear factor, erythroid 2-like 3 NGEF ENSG00000066248 Neuronal guanine nucleotide exchange factor NGF ENSGO0000134259 Nerve growth factor (beta polypeptide) NGLYI ENSG00000151092 N-glvcanase I NGRN ENSGOOOO182768 Neugrin, neurite outgrowth associated NILRC3 ENSGOOOOO 188811 NI-L repeat containing 3 NIDI EN SGOOOO 116962 Nidogen I
NiD2 ENSG0000087303 Nidogen 2 (osteonidoger) NKG7 ENSG00000105374 Natural killer cell granule protein 7 NLGN3 ENSG00000196338 Neuroligin 3 NLGN4Y ENSG00000165246 Neuroligin 4, Y-linked NLRP5 ENSG00000 171487 NLR family, pyrin domain containing 5 NMB ENSGO0000197696 Neuromedin B NMEI ENSG00000239672 N\E/NM23 nucleoside diphosphate kinase I NMEII-NME2 ENSG00000011052 NMEl-NME2 readthrough NIME3 ENSG00000103024 NME/NM23 nucleoside diphosphate kinase 3 NMS ENSG00000204640 Neuromedin S NMU ENSG00000109255 Neurotmedin U NOA1 ENSG00000084092 Nitric oxide associated 1 NODAL ENSG00000156574 Nodal growth differentiation factor NOG ENSG00000183691 Noggin NOMO ENSG00000103226 NODAL modulator 3 NOS1AP ENSG00000198929 Nitrc oxide synthase I (neuronal) adaptor protein NOTCH3 ENSG00000074181 Notch 3 NOTUlJM ENSGO0000185269 Notum pectinacetylesterase homolog (Drosophila) NOV ENSG00000136999 Nephroblastotma overexpressed NPB ENSG00000183979 Neuropeptide B NPC2 ENSG00000119655 Nienartn-Pick disease, type C2 NPFF ENSG00000139574 Neuropeptide FF-amide peptide precursor NPFFR2 ENSG00000056291 Neuropeptide FF receptor 2 NPHS1 ENSG00000161270 Nephrosis 1, congenital, Finnish type (nephrin) NPNT ENSG00000168743 Nephronectin NPPA ENSG00000175206 Natriuretic peptide A NPPB ENSGO0000120937 Nattiuretic peptide B NPPC ENSGO0000163273 Natriuretic peptide C NPS ENSG00000214285 Neuropeptide S NPTX1 ENSG00000171246 Neuronal pentraxin I NPTX2 ENSGO0000106236 Neuronal penttnxin II NPTXR ENSG0000221890 Neuronal pentraxin receptor NPVF ENSGO0000105954 Neuropeptide VF pmcursor NPW ENSG00000183971 Neuropeptide W NPY ENSG00000122585 Neuropeptide Y NQO2 ENSGOOOOO 124588 NAD(P)H dehydrogenase, quinone 2 NRCAM ENSGO0000091129 Neuronal cell adhesion molecule NRGI ENSG00000157168 Neuregulin I NRN1L ENSG00000188038 Neuritin 1-like NRP1 ENSG00000099250 Neuropilin I NRP2 ENSGOOOOO118257 Neuropilin 2 NRTN ENSG00000171119 Neu1turin NRXNI ENSG0000179915 Neurexin 1 NRXN2 ENSG00000 110076 Neurexin2
NT5C3A ENSG00000122643 5-nucleotidase, cytosolic IA NT5DC3 ENSG00000111696 5nucleotidase domain containing 3 NT5E ENSG00000 135318 5-nucleotidase, ecto(CD73) NTF3 ENSG00000185652 Neurotrophin 3 NTF4 ENSG00000225950 Neurotrophin 4 NTM ENSG00000182667 Neurotrimin NTNI ENSG00000065320 Netrin 1 NTN3 ENSG00000162068 Netrin 3 NTN4 ENSG00000074527 Netrin 4 NTN5 ENSG00000142233 Netrin 5 NTNG1 ENSG00000162631 Netrin G1 NTNG2 ENSG00000196358 Netrin G2 NTS ENSG00000133636 Neurotensin NUI-3PL ENSG00000151413 Nucleotide binding protein-like NUCB1 ENSGO0000 104805 Nucleobindin I NUCB2 ENSG00000070081 Nucleobindin 2 NUDTI9 ENSG00000213965 Nudix (nucleoside diphosphate linked moiety X)-type motif 19 NUDT9 ENSG00000170502 Nudix (nucleoside diphosphate linked moiety X)-type motif 9 NUP155 ENSG00000113569 Nucleoorin 155kDa NUP214 ENSG00000126883 Nucleooorin 214kDa NUP85 ENSG00000125450 Nucleoporin 85kDa NXPE3 ENSG00000144815 Neurexophlin and PC-esterase domain family, member 3 NXPE4 ENSG00000137634 Neurexophilin and PC-esterase domain family, member 4 NXPI-J- ENSG00000122584 Nenrexophilin 1 NXPH2 ENSG00000144227 Neurexophilin 2 NXPHf3 ENSG00000182575 Neurexophilin 3 NXPH4 ENSG00000182379 Neurexophilin 4 NYX ENSG00000188937 Nyctalopin OAF ENSG00000184232 Out at first homolog OBP2A ENSG00000122 136 Odorant binding protein 2A OBP2B ENSG00000171102 Odorant binding protein 2B OC90 ENSG00000253117 Otoconin 90 OCLN ENSG00000197822 Occildin ODAM ENSG00000109205 Odontogemc, amleloblast asssociated OGGI ENSG00000114026 8-oxoguanine DNA glycosylase OGN ENSGOOOOO106809 Osteoglycin T3 ENSG00000138315 Oncoprotein induced transcript 3 OLFM1 ENSG00000130558 Olfactomedin I OLFN2 ENSG00000105088 Olfactormedin 2 OLFM3 ENSG00000118733 Olfactomedin 3 OLFM4 ENSG000001(2837 Olfactomedin 4 OLFMLI ENSG00000183801 Olfactomedin-like 1 OLFML2A ENSG00000185585 Olfactomedin-like 2A OLFNL2B ENS00000162745 Olfactormedin-like 2B
OLFM,1L3 ENSGOOOOO116774 Olfactomedin-like 3 OID ENSG00000127083 Osteomodulin OMG ENSG0000126861 Oligoderdrocyte moyelin glycoproteirn OOSP2 ENSGO0000149507 Oocyte secreted protein 2 OPCML ENSG00000183715 Opioidbinding protein/cell adhesion molecule-like OPTC ENSG0000188770 Opticin ORAII ENSG00000276045 ORAI calcium release-activated calcium modulator I ORM41 ENSG00000229314 Orosomucoid 1 ORM/'2 ENSG00000228278 Orosomucoid 2 ORMDL2 ENSG00000123353 ORMDL sphingolipid biosynthesis regulator 2 OS9 ENSG0000135506 Osteosarcoma amplified 9, endoplasmic reticulum lectin OSCAR ENSG00000170909 Osteoclast associated, imunuogiobuin-like receptor OSM ENSG00000099985 Oncostatin M OSMR ENSGO0000145623 Oncostatin M receptor OSTN ENSGO000188729 Osteocrin OTOA ENSG00000155719 Otoancorin OTOG ENSG00000188162 Otogelin OTOGL ENSGO0000165899 Otogelin-like OTOLI ENSG00000182447 Otolin 1 OTOR ENSG00000125879 Otoranlin OTOS ENSG00000)178602 Otospiralin OVCH1 ENSG00000187950 Ovochymase 1 OVCH2 ENSGOOOOO183378 Ovochvmase 2 (gene/pseudogene) OVGPI ENSG00000085465 Oviductal glycoprotein 1, 120kDa OXCTI ENSG00000083720 3-oxoacid CoA transferase1 OXCT2 ENSGOOOOO198754 3-oxoacid CoA transferase 2 OXNAD1 ENSG00000154814 Oxidoreductase NAD-binding domain containing I OXT ENSG00000101405 Oxvtocin/neurophv sin I prepropeptide P3111 ENSG0000117385 Prolyl 3-hydroxylase 1 P3H2 ENSG00000090530 Prolyl 3-hydroxylase 2 P31-13 ENSGOOOO110811 Proly] 3-hydroxvase 3 P31-14 ENSGOO000141696 Prolyl 3-hydroxylase family member 4 (non-enzynatic) P4HA1 ENSGOOOOO122884 ProIv 4-hydroxylase, alpha polypeptide I P41HA2 ENSG00000072682 Prolyl 4-hydroxylase, alpha polypeptide II P4HA3 ENSG00000149380 Prolyl 4-hydroxylase, alpha polypeptide III P4HB ENSGOOOOO 185624 Prolyi 4-hydroxylase, beta polypeptide PAEP ENSG00000122133 Progestagen-associated endometrial protein PAM1\4 ENSG00000145730 Peptidylglycine alpha-amidating monooxvgenase PAMR 1 ENSGOOOOO 149090 Peptidase dornai conaining associatedwith muscle receneration 1 PAPL ENSGO0000183760 Iron/zinc purple acid phosphatase-like protein PAPLN ENSG00000100767 Papilin, proteoglycan-hke sulfated glycoproteirn PAPPA ENSGOOOO 182752 Pregnancy-associated plasma protein A, pappalysin I PAPPA2 ENSGOOOOO116183 Pappalysin 2 PARP15 ENSGO0000173200 Poly (ADP-rbose) polvinerase family, member 15
PAR VB ENSG00000188677 Parvin, beta PATEI ENSG00000171053 Prostate and testis expressed I PATE2 ENSG00000196844 Prostate and testis expressed 2 PATE3 ENSG00000236027 Prostate and testis expressed 3 PATE4 ENSG00000237353 Prostate and testis expressed 4 PATL2 ENSG00000229474 Protein associated with topoisomerase II homolog 2 (yeast) PAX2 ENSG00000075891 Paired box 2 PAX4 ENSG00000106331 Paired box 4 PCCB ENSG00000114054 Propionyl CoA carboxylase, beta polypeptide PCDHI ENSG0000156453 Protocadherin I PCD-112 ENSGOO00113555 Protocadherin 12 PCDH15 ENSG00000150275 Protocadherin-related 15 PCDHA1 ENSG00000204970 Protocadherin alpha I PCDHA10 ENSG00000250120 Protocadhein alpha 10 PCDHA 11 ENSG00000249158 Protocadherin alpha 11 PCDIA6 ENSG00000081842 Protocadherin alpha 6 PCDHB12 ENSG00000120328 Protocadherin beta 12 PCDHGAI11 ENSG00000253873 Protocadherin gamrna subfamnily A, 11 PCF11 ENSG00000165494 PCF11 cleavage and polyadenylation factor subunit PCOLCE ENSGO0000106333 Procollagen C-endopeptidase enhancer PCOLCE2 ENSG0000163710 Procollagen C-endopeptidase enhancer 2 PCSKI ENSG00000175426 Proprotein convertase subtilisin/kexin type I PCSKIN ENSG00000102109 Proprotein convertase subtilisinlkexin type 1 inhibitor PCSK2 ENSG00000125851 Proprotein convertase subtilisin/kexin type 2 PCSK4 ENSG00000115257 Proprotein convertase subtilisin/kexin type 4 PCSK5 ENSG00000099139 Proproteir convertase subtilisin/kexmn type 5 PCSK9 ENSGO0000169174 Proprotein convertase subtilisin/exin type 9 PCYOXI ENSG00000116005 PrenvIcysteine oxidase I PCYOX1L ENSG00000145882 Prenyicysteineoxidase 1 like PDDCI ENSG0000177225 Parkinson disease 7 domain containing I PDE1IA ENSG00000)128655 Phosphodiesterase HA PIDE2A ENSG00000186642 Phosphodiestease 2A. cGMP-stimulated PDE7A ENSG00000205268 Phosphodiesterase 7A PDF ENSG00000258429 Peptide deformylase (Initochondrial) PDGFA ENSG00000 197461 Platelet-derived growth factor alpa polypeptide PDGFB ENSG00000 100311 Platelet-derived growth factor beta polypeptide P)GFC ENSG00000145431 Platelet derived growth factor C PDGFD ENSG00000170962 Platelet derived growth factor D PDGFRA ENSG00000134853 Platelet-derived growth factor receptor, alpha polypeptide PDGFRB ENSG00000113721 Platelet-derived growth factor receptor, beta polypeptide PDGFRL ENSG00000104213 Platelet-derived growth factor receptor-like PDIA I ENSG00000)131828 Pyruvate dehydrogenase (lipoamide)alpha I PDIA2 ENSG00000185615 Protein disulfide isomerase family A, member 2 PDIA3 ENSG00000 167004 Protein disulfide isomnerase family A, member 3
PDIA4 ENSGOO000155660 Protein disulfide isomerase family A. member 4 PDIA5 ENSG00000065485 Protein disulfide isomerase family A, member 5 PDIA6 ENSG0000143870 Protein disulfide isornerase family A, member 6 PDILT ENSG00000 169340 Protein disulfide isomerase-like, testis expressed PDYN ENSG00000101327 Prodynouphin P)ZD8 ENSG0000165650 PDZ domain containing 8 PDZRN4 ENSG00000165966 PDZ domain containing ring finger 4 PEARl ENSG00000187800 Platelet endothelial aggregation receptor I PEBP4 ENSG00000134020 Phosphatidylethanolamine-binding protein PECAMI ENSG00000261371 Platelet/endothelial cell adhesion molecule I PENK ENSGOOOO0181195 Proerkephalin PET117 ENSG0000232838 PET117 homolog PF4 ENSGO0000163737 Platelet factor4 PF4V1 ENSG00000109272 Platelet factor 4 variant 1 PFKP ENSG00000067057 Phosphofructokinase, platelet PFN1 ENSGOOOO108518 Profilin I PGA3 ENSG00000229859 Pepsinogen 3, group I (pepsinogen A) PGA4 ENSG00000229183 Pepsinogen 4, group I (pepsinogen A) PGA5 ENSG00000256713 Pepsinogen 5. group I (pepsinogen A) PGAMI5 ENSG00000247077 PGAM family member 5, seine/threonine protein phosphatase, mitochondrial
PGAP3 ENSG00000161395 Post-GPI attachment to proteins 3 PGIC ENSG00000096088 Progastricsin (pepsiniogen C) PGF EN SG00000119630 Placental growth factor PGLYRP1 ENSG00000008438 Peptidoglycan recognition protein I PGLYRP2 ENSG00000161031 Peptidoglycan recognition protem 2 PGLYRP3 ENSG00000159527 Peptidoglvcan recognitionprotein 3 PG3LYRP4 ENSG(00000163218 Peptidoglycan recognition protein 4 PHACTIR EN SG00000112137 Phosphatase and acting regulator I PHB ENSG00000167085 Prohibitin P115 ENSG00000137558 Peptidase inhibitor 15 P13 ENSG00000124102 Pentidase inhibitor 3. skin-derived PIANP ENSGOOOOO 139200 PILR alpha associated neural protein P1GK EN SG00000)142892 Phosphatidylinositol glycan anchor biosynthesis, class K PIGL ENSG00000108474 Phosphatidylinositol glycan anchor biosynthesis class L PIGT ENSG0000124155 Phosphatidylinosito] glycan anchor biosynthesis, class T PIGZ ENSG00000119227 Phosphatidylinositol glycan anchorbiosynthesis, class Z PIK3API ENSGOOOOO155629 Phosphoinositide-3-kinase adaptor protein I PIKIPI EN SG00000)100100 Phosphoinositide-3-kinase interacting protein 1 PILRA ENSG00000085514 Paired immunoglobin-like type 2 receptoralpha PILRB ENSG00000121716 Paired immunoglobin-like type 2 receptor beta PINLYP ENSG00000234465 Phospholpase A2 inhibitor and LY6/PLAJR domain containing PIP ENSGOOOO0159763 Prolactin-induced protein PIWIL4 ENSG00000134627 Piwi-like RNA-mediated gene silencing 4
PKDCC ENSG00000162878 Proteinkinase domain containing, cytoplasmic PKHDI ENSG00000170927 Polycystic kidney and hepatic disease I (autosomal recessive) PLA1A ENSG00000144837 Phospholipase A I member A PLA2G0IO ENSG00000069764 Phospholipase A2. group X PLA2G12A ENSGO0000123739 Phospholipase A2, group XIIA PLA212B ENSG00000138308 Phospholipase A2, group XI13 PLA2GI5 ENSG00000103066 Phospholipase A2, group XV PLA2GIB ENSG00000170890 Phospholipase A2, group IB (pancreas) PLA2G2A ENSG00000188257 Phospholipase A2. group IIA (platelets, synovial fluid) PLA2G2C ENSG00000187980 Phospholipase A2, group IIC PLA2G2D ENSG00000117215 Phospholipase A2, group IID PLA2G2E ENSG00000188784 Phospholipase A2, group IIE PLA2G3 ENSGO0000100078 Phospholipase A2, group III PLA2G5 ENSG00000127472 Phospholipase A2 group V PLA2G7 ENSGOOOOO 146070 Phospholipase A2, group VII (platelet-activating factor acetvlhvdrolase, plasma) PLA2R1 ENSGO0000153246 Phospholipase A2 receptor 1, 18OkDa PLACI ENSG00000170965 Placenta-specific 1 PLAC9 ENSG000000189129 Placenta-specific 9 PLAT ENSG00000104368 Plasminogen activator, tissue PLAU ENSG0000122861 Piasminogen activator, urokinase PLAUR ENSGO0000011422 Plasminogen activator, urokinase receptor PLBDI ENSG00000121316 Phospholipase B domain contaiming I PLBD2 EN SG00000151176 Phospholipase B domain containing 2 PLG ENSG00000122194 Plasminogen PLGLB1 ENSG00000183281 Piasminogen-like B11 PLGLB2 ENSG00000125551 Piasminogen-like B2 PLODI ENSG00000083444 Procollagen-lysine, 2-oxoglutarate 5-dioxygenase I PLOD2 ENSG00000152952 Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 PLOD3 ENSG00000106397 Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3 PLTP ENSG00000100979 Phospholipid transfer protein PLXNA4 ENSG00000221866 Plexin A4 PLXNB2 ENSGOOOOO196576 Plexin1B2 PM201O ENSG00000162877 Peptidase M20 domain containing I PMCH ENSG00000183395 Pro-melanin-concentrating hormone PMEL ENSGOO00185664 Preielanosome protein PIMEPA I ENSGOOO0124225 Prostate transmembrane protein .androgen induced 1 PNLIP ENSG00000175535 Pancreatic lipase PNLIPRP1 ENSG00000187021 Pancreatic lipase-related protein I PNLIPRP3 ENSG00000203837 Pancreatic lipase-related protein 3 PNOC ENSGO0000168081 Prepronociceptirn PNP ENSGO0000198805 Purine nucleoside phosphorylase PNPLA4 ENSG00000006757 Patatin-like phosphohpase domain containing 4 PODNL1 ENSG00000132000 Podocan-like1
POFUT ENSG00000)101346 Protein O-facosyltransferase1 POFUT2 ENSG00000186866 Protein O-fucosyltransferase 2 POGLUTI ENSG00000163389 Protein 0-glucosyltarinsferase I POLL ENSGO0000166169 Polvmerase (DNA directed), lambda POMC ENSG00000115138 Proopiomelanocorin POMGNT2 ENSG00000144647 Protein 0-linked inannose N-acetlglucosamiinyltransferase 2 (beta 1,4-) PONI ENSG00000005421 Paraoxonase 1 PON2 ENSG00000105854 Paraoxonase 2 PON3 ENSG00000105852 Paraoxonase 3 POSTN ENSG00000133110 Periostir, osteoblast specific factor PPBP ENSG00000)163736 Pro-platelet basic protein (chemokine (C-X-C motif) ligand 7) PPIB ENSGOOOOO166794 Peptidylprolyl isomerase B (cyclophilin B) PPI C ENSG00000168938 Peptidylprolyl isomerase C (cyclophilin C) PPOX ENSG00000143224 Protoporphyrinogen oxidase PPPICA ENSG00000172531 Protein phosphatase 1, catalytic subunit, alpha isozyme PPT1 ENSG00000131238 PahnitoyI-protein tioesterase I PPT2 ENSG00000221988 Paimitoyl-protein thioesterase 2 PPY ENSG00000108849 Pancreatic polypeptide PRAC2 ENSG00000229637 Prostate cancer susceptibility candidate 2 PRADC1 ENSG00000135617 Protease-associated domain containing 1 PRAPI ENSG00000165828 Proline-rich acidic protein 1 PRBI ENSG00000251655 Proline-rich protein BstNI subfamily I PRB2 ENSGO0000121335 Proline-rich protein BstNI subfanily 2 PR133 ENSG00000197870 Proline-rich protein BstNI subfamily 3 PRB4 ENSG00000230657 Proline-rich protein BstNI subfamily 4 PRCD ENSG00000214140 Progressive rod-cone degeneration PRCP ENSG00000137509 Prolvlcarboxvpeptidase (angiotensinase C) PRDM12 ENSG00000130711 PR domain containing 12 PRDX4 ENSGOOOOO123131 Peroxiredoxin 4 PRELP ENSGO0000188783 Proline/arginine-rich end leucine-rich repeat protein PRF1 ENSG00000 180644 Perfori 1 (pore forming protein) PRG2 ENSG00000186652 Proteoglycan 2, bone marrow (natural killer cell activator, eosinophil granule majorbasic protein)
PRG3 ENSG00000 156575 Proteoglycan 3 PRG4 ENSG00000 116690 Proteoglycan 4 PR1- ENSG00000231887 Proline-rich protein HaeIII subfamnily 1 PRH2 ENSG00000134551 Proline-rich protein HaeIII subfamily 2 PRKAGI ENSG00000181929 Proteinkinase, AMP-activated, gamma 1 no-catalytic subunit PRKCSHI ENSG00000130175 Protein kinase C subsite 80K-l PRKDI ENSG00000184304 Protein kinase DI PRL ENSGOOOOO172179 Prolactin PRLH ENSG00000071677 Prolactin releasing hormone PRLR ENSG00000113494 Prolactin receptor PRNP EN SG00000171867 Prion protein
PRNT ENSG00000180259 Prion protein (testis specific) PROC ENSGOOOOO115718 Protein C (inactivator of coagulation factors Va and VllIa) PROK 1 ENSG00000143125 Prokineticin 1 PROK2 ENSG00000163421 Prokineticin 2 PROL I ENSG00000 171199 Proline rich, lacrimal I PROMI ENSG00000007062 Prormirin 1 PROSI ENSG00000184500 Protein S (alpha) PROZ ENSG0000126231 Protein Z, vitamnn K-dependent plasma glycoproteirn PRR27 ENSGO0000187533 Proline rich 27 PRR4 ENSGOOOOO111215 Proline rich4(lacrimal) PRRG2 ENSG00000 126460 Proline rich Ga (G-catrboxvgtutamic acid) 2 PRRT3 ENSG00000163704 Proline-rich transmembrane protein 3 PRRT4 ENSG00000224940 Prolirne-rich iransrmembrane protein 4 PRSS1 ENSG00000204983 Protease, seine, 1 (trypsin 1) PRSS12 ENSG00000164099 Protease, serine, 12 (neurotrypsin, motopsin) PRSS16 ENSG00000112812 Protease, serine, 16 (thym-ius) PRSS2 ENSG00000275896 Protease, seine, 2 (trypsin 2) PRSS21 ENSG00000007038 Protease, shrine, 21 (Jestisin) PRSS22 ENSG00000005001 Protease, seine, 22 PRSS23 ENSG00000150687 Protease,serine,23 PRSS27 ENSG00000172382 Protease, serine 27 PRSS3 ENSG00000010438 Protease, serine. 3 PRSS33 ENSG00000103355 Protease, shrine, 31 PRSS35 ENSG0000014625) Protease, serine, 35 PRSS36 ENSG00000178226 Protease, seine, 36 PRSS37 ENSG00000165076 Protease, serine, 37 PRSS38 ENSG00000185888 Protease. seine, 38 PRSS42 ENSG00000178055 Protease, shrine, 42 PRSS48 ENSG00000189099 Protease, serine, 48 PRSS50 ENSG00000206549 Protease, seine, 50 PRSS53 ENSG00000151006 Protease, serine, 53 PRSS54 ENSG0000103023 Protease. seine 54 PRSS55 ENSG00000184647 Protease shrine 55 PRSS56 ENSG00000237412 Protease serine, 56 PRSS57 ENSG00000185198 Protease, seine, 57 PRSS58 ENSG00000258223 Protease, serine, 58 PRSS8 ENSG0000052344 Protease. seine, 8 PRTG ENSG00000166450 Protogenin PRTN3 ENSG00000196415 Proteinase 3 PSAP ENSG00000197746 Prosaposin PSAPL1 ENSG00000178597 Prosaposin-like I (gene/pseudogene) PS1i ENSG00000231924 Pregnancy specific beta-I-giycoprotein I PSG11 ENSG00000243130 Pregnancy specific beta-l-glycoprotein 11 PSG2 ENSG00000242221 Pregnancy specific beta-i-glycoproten 2
PSG3 ENSG00000221826 Pregnancy specific beta-I-glycoprotein 3 PSG4 ENSG00000243137 Pregnancy specific beta-1-glycoprotein 4 PSG5 ENSG00000204941 Pregnancy specific beta-I-gvcoprotein 5 PSG6 ENSG00000170848 Pregnancy specific beta-1-glycoprotein 6 PSG7 ENSG00000221878 Pregnancy specific beta-1-glycoprotein 7 (gene/pseudogene) PSG8 ENSG00000124467 Pregnancy specific beta-I-glycoprotein 8 PSG9 ENSG00000183668 Pregnancy specific beta-1-glycoprotein 9 PSMD1 ENSG00000173692 Proteasone 26S subunit, non-ATPase 1 PSORS1C2 ENSG00000204538 Psoriasis susceptibility 1 candidate 2 PSPN ENSG00000125650 Persephin PTGDS ENSG00000 107317 Prostaglandin D2 syrthase 2IkDa (bmin) PTGIR ENSG00000160013 Prostaglandin 12 (prostacyclin) receptor (IP) PTGSI ENSG00000095303 Prostaglandin-endoperoxide sy1ase 1 (prostagiandiri Glf synthase and cyclooxygenase)
PTGS2 ENSG00000073756 Prostaglandin-endoperoxide svnthase 2 (prostaglandin G H synthase and cyclooxygenase)
PTH ENSG00000152266 Parathyroid hormone PTI-12 ENSG0000142538 Parathyroid hormone 2 PTHLH ENSG00000087494 Parathyroid hormone-like hormone PTK7 ENSG00000 112655 Protein tvrosine kinase 7 (inactive) PTN ENSG00000105894 Pleiotrophin PTPRA ENSG00000132670 Protein tyrosine phosphatase, receptor type A PTPRB ENSG00000127329 Protein tyrosine phosphatase, receptor type, B PTPRC ENSG00000081237 Protein tyrosine phosphatase, receptor type, C PTPRCAP ENSG00000213402 Protein tyrosine phosphatase, receptor type. C-associated protein PTPRD ENSG00000153707 Protein tyrosine phosphatase, receptor type. D PTPRF ENSG00000142949 Protein tyrosine phosphatase, receptor type F PTPRJ ENSGOO000149177 Protein tyrosine phosphatase, receptor type J PTPRO ENSG0000015149) Protein tyrosine phosphatase, receptor type, 0 PTPRS ENSG00000105426 Protein tyrosine phosphatase, receptor type, S PTTGlIP ENSG00000183255 Pituitary tumor-transforming I interacting protein PTX3 ENSG00000 163661 Pentraxin 3, long PTX4 ENSG00000251692 Pentnixin 4, long PVR ENSG00000073008 Poliovinis receptor PVRL 1 ENSGO0000 110400 Poliovinis receptor-related I (herpesvirus entry mediator C) PXDN ENSG00000)130508 Peroxidasin PXDNL ENSG00000147485 Peroxidasin-like PXYLPI ENSG00000155893 2-phosphoxylose phosphatase 1 PYY ENSG00000131096 Peptide YY PZP ENSG00000126838 Pregnancy-zone protein QPCT ENSG00000115828 Glutarmiryl-peptide cyclotranstemse QPRT ENSG00000103485 Quinolinate phosphoibosyltransferase QRPP ENSG00000 188710 Pyroglutamylated RFanide peptide QSOXI ENSG00000 116260 Quiescin Q6 sulfhydryl oxidase 1
R13HDNL EN SGO0000101074 R3-1 domain containing-like RAB26 ENSG0000 167964 RAB26, member RAS oncogene family RAB36 ENSG00000100228 RAB36, member RAS oncogene family RAB9B ENSG00000123570 RAB9B. member RAS oncogene family RAETIE ENSG0000164520 Retinoic acid early transcript lE RAETIG ENSG00000203722 Retinoic acid early transcript IG RAMP2 ENSG00000131477 Receptor (G protein-coupled) activity modifying protein 2 RAPGEF5 ENSG00000136237 Rap guanmne nucleotide exchange factor (GET) 5 RARRESI ENSG0000 118849 Retinoic acid receptor respoider tazarotene induced) 1 RARRES2 ENSG0000 106538 Retinoic acid receptor responder (tazarotene induced) 2 RASA2 ENSG00000155903 RAS p 2 1 protein activator 2 RBM3 ENSG00000102317 RNA binding motif(RNP1, RRM) protein RBP3 ENSG00000265203 Retinol binding protein 3, interstitial RBP4 ENSGO0000138207 Retinol binding protein 4, plasma RCNI ENSG00000049449 Reticulocalbin 1, EF-hand calcium binding domain RCN2 ENSG000 1,17906 Reliculocalbin 2, EF-hand calcium binding domain RCN3 ENSG00000142552 Reticulocalbin 3, EF-hand calcium binding domain RCORI ENSG00000089902 REST corepressor I RD-I11 ENSG00000072042 Retinol dehydrogenase II (all-trans/9-cis/1 I-cis) RDH12 ENSG00000139988 Retinol dehydrogenase 12 (all-trans/9-cis/l1-cis) RDH13 ENSGO00000160439 Retinol dehydrogenase 13 (all-tmans/9-cis) RDH5 ENSG00000135437 Retinol dehydrogenase 5 (11-cis/9-cis) RDH8 ENSGOOO00080511 Retinol dehydrogenase 8 (all-trans) REGIA ENSGOOOOO115386 Regeneratii islet-denved 1 alpha REGIB ENSG00000172023 Regenerating islet-derived I beta REG3A ENSGOOOO172016 Regenerating islet-derived 3 alpha REG3G ENSG00000143954 Regeneratingisle-derived 3 gamma REG4 ENSG0000134193 Regenerating islet-derived family, member 4 RELN ENSG00000189056 Reelin RELT ENSG00000054967 RELT tumor necrosis factor receptor REN ENSGOOOOO143839 Remind REPINI ENSG00000214022 Replication initiator I REPS2 ENSG0000169891 RALBPI associatedEpsdomain containing 2 RET ENSGO0000165731 Ret proto-oncogene RETN ENSGO0000104918 Resistn RETNLB ENSGOOOOO 163515 Resistin like beta RETSAT ENSG00000042445 Retinol saturase (aill-trans-retinol 13,14-reductase) RFNG ENSGOOOOO 169733 RFNG 0-fucosylpeptide 3-beta-N acetylglucosaminyltransferase RGCC ENSG0000102760 Regulatorof cell cycle RGL4 ENSGO0000159496 Ral uaninenucleotide dissociation stimulator-like 4 RGMA ENSG00O0 182175 Repulsive guidance molecule family member a RGMB ENSG00000174136 Repuisive guidance molecule family meniberb RH1OQ ENSGOOOOO 119729 Ras homolog family member Q RIC3 ENSGO0000166405 RIC3 acelvicholine receptor chaperone
RIMS1 ENSG00000079841 Regulating synaptic membrane exocytosis 1 RIPPLYl ENSG00000147223 Ripply transcriptional repressor I RLNI ENSC000107018 Relaxin I RLN2 ENSG00000107014 Relaxin 2 R LN3 ENSG00000171136 Relaxin 3 RM)N] ENSG00000176623 Regulator of microtubule dyniam ics I RNASEI ENSG00000129538 Ribonuclease, RNase A family, I (pancreatic) RNASEIO ENSG00000182545 Riborncase, RNase A family, 10 '(on-active) RNASElI ENSG00000173464 Ribonuclease. RNase A family, 11 (non-active) RNASE12 ENSG00000258436 Ribonuclease, RNase A family, 12 (non-active) RNASE13 ENSG00000206150 Ribonuciease, RNase A family, 13 (nor-active) RNASE2 ENSG00000169385 Ribonuclease, RNase A family, 2 (liver, eosinophil-derived neurotoxin) RNASE3 ENSG00000169397 Ribornuclease, RNase A family, 3 RNASE4 ENSG00000258818 Ribonuclease, RNase A family, 4 RNASE6 ENSG00000169413 Ribonuclease RNase A family, k6 RNASE7 ENSG00000165799 Ribonuclease, RNase A.family 7 RNASE8 ENSG00000173431 Ribonuclease, RNase Alfamily. 8 RNASE9 ENSG00000188655 Ribornucease, RNase A family, 9 (non-active) RNASEHI1 ENSG00000171865 Ribonuclease HI RNASET2 ENSG00000026297 Ribonuclease T2 RNF146 ENSG00000118518 Ring finger protein 146 RNF148 ENSG00000235631 Ring finger protein 148 RNF150 ENSG00000170153 Ring finger protein 150 RNF167 ENSG00000108523 Ring finger protein 167 RNF220 ENSG00000187147 Ring finger protein 220 RNF34 ENSG0000170633 Ring finger protein 34, E3 ubiquitin protein ligase RNL S ENSG00000184719 Renalase, FAD-depndent amine oxidase RNPEP ENSG00000176393 Arginyl arminiopepidase (aminopeptidase B) ROR I ENSG00000185483 Receptor tyrosine kinase-like orphan receptor 1 RP11-1236K1.1 ENSG00000233050 RPIl-14J7.7 ENSG00000259060 RPII-196G11.1 ENSG00000255439 RP11-350014.18 ENSG00000261793 RPil-520P18.5 ENSG00000261667 RPil-812E19.9 ENSG00000259680 RP11-903H12.5 ENSG00000259171 RP11-977G19.10 ENSG00000144785 RP4-576H24.4 ENSG00000260861 RP4-608015.3 ENSG00000276911 Completent factor -- related protein 2 RPL3 ENSG00000100316 Ribosomal protein L3 RPLP2 ENSG00000177600 Ribosomal protein, large, P2 RPN2 EN SG00000118705 Ribophorin IL RPS27L ENSGOOOOO185088 Ribosomal protein S27-like RQCD1 ENSG00000144580) R CDI required for cell differentiation homolog (S. pombe)
RS1 ENSG00000102104 Retinoschisin 1 RSF1 ENSG00000048649 Remodeling and spacing factor I RSPO1 ENSG00000169218 R-spondin 1 RSPO2 ENSG00000147655 R-spondin 2 RSPO3 ENSG00000146374 R-spondin 3 RSPO4 ENSG00000101282 R-spondin4 RSPRYI ENSG00000159579 Ring finger and SPRY domain containing 1 RTBDN ENSG00000132026 Retbiridin RTN4RL.1 ENSG0000185924 Reticulon 4 receptor-like 1 RTN4RL2 ENSG00000 186907 Reticulon 4 receptor-like 2 SAA1 ENSG00000173432 Sem amyloid Al SAA2 ENSGO0000134339 Serum amyloid A2 SAA4 ENSG00000148965 Seram amyloid A4, constituirve SAP30 ENSG00000164105 Sin3A-associated protein, 30k1 )a SARIA ENSG00000079332 Secretion associated, Ras related GTPase 1A SARAF ENSG00000)133872 Store-operated calcium entry-associated regulatory factor SARMI ENSG00000004139 Sterile alpha and TIR motif containing 1 SATBI ENSG00000182568 SATB horneobox I SAXO2 ENSG00000188659 Stabilizerofaxonemal microtubuiles 2 SBSN ENSG00000189001 Suprabasin SBSPON ENSG00000164764 Somatoredin B and throrbospondin, type 1 domain containing SCARF1 ENSG00000074660 Scavenger receptor class F. member I SCG2 ENSGO0000171951 Secretogranin II SC G3 ENSG00000104112 Secretogranin III SCG5 ENSG00000 166922 Secretogranin V SCGB1A1 ENSG00000149021 Secretoglobin, family IA, member 1 (uteroglobin) SCGBICI ENSG00000188076 Secretogiobin, family IC, member 1 SCGB IC2 ENSG00000268320 Secretoglobin, family IC. member 2 SCGB11 ENSG00000168515 Secretoglobin, family 1D, member 1 SCGB1D2 ENSG00000 124935 Secretoglobin, family ID. member2 SCGBD114 ENSG00000 197745 Secretogoobin, family 1), member 4 SCGB2A1 ENSG00000124939 Secretoglobin, family 2A, member I SCGB2A2 ENSG00000110484 Secretoglobin, family 2A, member 2 SCGB2B2 ENSG00000205209 Secretoglobirn, family 2B, member 2 SCGB3A1 ENSG00000 161055 Secretoglobin, family 3A. member 1 SCGB3A2 ENSG00000 16-4265 Secretoglobin, family 3A, member2 SCNIB ENSG00000105711 Sodium channel, voltage gated, type I beta subunit SCN3B ENSG00000166257 Sodium channel, voltage gated, type III beta subunit SCPEPI ENSG00000121064 Serine carboxypeptidase 1 SCRGI ENSG00000164106 Stimulatorof chondrogenesis I SCT ENSG0000007003I Secretin SCUBE] ENSG00000159307 Signal peptide, CIB domain, EGF-like I SCUBE2 ENSG00000175356 Signal peptide, CUB domain. EGF-like 2 SCIJBE3 ENSG00000146197 Signal peptide, CUB domain, EiF-like 3
SDC1 ENSG00000115884 Syndecan 1 SDF2 ENSG00000132581 Stromal cell-derived factor 2 SDF2L. ENSG00000128228 Stromal cell-derived factor 2-like 1 SDF4 ENSG00000078808 Stromal cell derived factor 4 SDHAF2 ENSG00000167985 Succinate dehydrogenase complex assembly factor 2 SIDHAF4 ENSG00000154079 Succinate dehydrogenase complex assembly factor 4 SDHB ENSG00000117118 Succinate dehydrogenase complex, subunit B, iron sulfur (Ip) SDHD ENSG00000204370 Succinate dehydrogenase complex, subunit ), integral mernbrane protein SEC14L3 ENSGU0000100012 SEC14-like lipid binding 3 SEC16A ENSGO0000148396 SEC16 homolog A, endoplasmuic reticulum export factor SEC16B ENSG00000120341 SEC16 homolog B, endoplasmic reticulum export factor SEC22C ENSG00000093183 SEC22 homolog C, vesicle trafficking protein SEC31A ENSG00000138674 SEC31 honolog A, COPIl coat complex component SECISBP2 ENSG00000187742 SECIS binding protein 2 SECTMI ENSGOOOOO 141574 Secreted and tmnsmernbranc I SELIL ENSG00000071537 Sel-I suppressor of lin-2-like (C. elegans) SELM ENSGO0000198832 Sclenoprotein M SELO ENSG00000073169 Selenioprotein (O) SEMA3A ENSG00000075213 Sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3A
SEMA3B ENSG00000012171 Sema domain, immunoglobulin domain (ig), short basic domain, secreted, (semaphorin) 3B
SEMA3C ENSG00000075223 Sema domain, immunoglobulin domain (ig), short basic domain, secreted, (semaphorin) 3C
SEMA3E ENSG00000170381 Sema domain, immunoglobulin dornain (ig), short basic domain. secreted, (semaphorin) 3E SEMA3F ENSGO000001617 Sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3F SEMA3G ENSG00000010319 Sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3i
SEMA4A ENSG00000196189 Sema domain, immunoglobulin domain (Ig), transmenibrane domain (TM) and short cytoplasmic domain, (semaphorin) 4A
SEMA4B ENSGO0000185033 Sema domain, immunoglobulin domain (Tg) t.an.embrane domain (TM) and short cytoplasmic domain, (semaphorin) 4B
SEMA4C ENSG00000168758 Sema domain, immunoglobulin domain(.g) trnembrane domain (TM) and short cytoplasmic domain, (semaphorin) 4C
SEMA4D ENSG00000187764 Sema domain, immunoglobulin domain (.g)trnsmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 4D SEMA4F ENSGO0000135622 Semadomainimmunoglobulin domain(Ig) tranmembrane domain (P) and short cytoplasmic domain, (semaphorin) 4F
SEMA4G ENSG00000095539 Sema domain, immunoglobulin domain (Ig), transmembrane domain (P)and short cytoplasmic domain, (semaphorin) 4G
SEMA5A ENSG00000112902 Sena domain, seven thrombospondin repeats (type I and type 1 like), transnembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5A SEMA6A ENSG0000009242I Sema domain, transmembrane domain (TM), and cytoplasmic domain, (semaphorin) 6A
SEMA6C ENSG00000143434 Sema domain, transmembrane domain (TM), and cytoplasmnic domain, (semaphorin) 6C
SEMA6D ENSG00000137872 Sema domain, transmembrane domain (TM), and cytoplasmic domain, (semaphorin) 6D
SEMG1 ENSG00000124233 Semenogelin I SEMG2 ENSG00000 124157 Sernenogelin II 15-Sep ENSG0000(0183291 15 kDa selenoprotein SEPN I ENSGO0000162430 SelenoproteinN. I SEPPI ENSG00000250722 Selernoproteir P, plasma, I 9-Sep ENSG00000184640 Septin 9 SERPINAI ENSG00000197249 Serpn peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member I
SERPINA10 ENSG00000140093 Serpin peptidase inhibitor, clade A (alpha-1 antiproteinase antitrypsin), member 10
SERPINAII ENSG00000186910 Serpin peptidase inhibitor, clade A (alpha-1 antiproteinase antitrypsin), member II SERPINA12 ENSG00000165953 Serpin peptidase inhibitor, clade A (alpha-1 antiproteinase antitrypsin), member 12
SERPINA3 ENSG00000196136 Serpin peptidase inhibitor, clade A(alpha- antiproteinase antitrypsin), member 3
SERPINA3 ENSG00000273259 Serpin peptidase inhibitor, clade A(alpha- antiproteinase antitrypsin), member 3
SERPINA4 ENSG0000100665 Serpin peptidase inhibitor, clade A(alpha- antiproteinase antitrypsin). member 4
SER PINA5 ENSG00000188488 Serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin.) mener 5
SERPINA6 ENSG00000170099 Serpinpeptidaseinhibitor, clade A(alpha-1 antiproteinase antitrypsin). member 6
SER PINA7 ENSG00000123561 Serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrvpsin), member 7
SERPINA9 ENSGO0000170054 Sepin peptidase irhibitor, cade A(alpha- antiproteinase, antitrvpsin), member 9 SERPINB2 ENSGO0000197632 Serpin peptidase inhibitor, cade B (ovaibuinin), meraber 2 SERPINC I ENSG00000 117601 Serpin peptidase inhibitor, clade C (antitLrombin), member I SERPINDI ENSG00000099937 Serpin peptidase inhibitor, clade D (heparin cofactor), member I SERPINE ENSGOOOOO106366 Serpminpeptidase inhibitor, clade E (nexin, plasminiogen activator inhibitor type I),member I
SERPINE2 ENSGOOOO 135919 Serpmnpeptiase inhibitor, clade E (nexin, plasninogen activator inhibitor type 1), member
SERPINE3 ENSG00000253309 Serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type I), member 3
SERPINF ENSGOOOO132386 Serpin peptidase inhibitor, clade F (alpha-2 antiplasmin, pigrment epithelium derived factor), member 1
SERPINF2 ENSG0000167711 Serpin peptidase inhibitor, clade F (alpha-2antiplasmin, pigment epithelium derived factor), member
SERPINGi ENSG0000149131 Sertn peptidase inhibitor, clade i ((1 inhibitor), member i SERPINHI EN SGOOOOO149257 Serpin peptidase inhibitor, clade 11 (heatshock protein 47), member 1, (collagen binding proteni 1)
SERPINIl ENSG000163536 Serpin peptidase inhibitor, clade I (neuroserpin), member I SERPINI2 ENSGO0000114204 Serpin peptidase inhibitor, clade I (pancpin), mernber 2 SETDs ENS(100000183955 SE-T dom-.ain conl1aing (lysine inethlybransferase) 8 SEZ6L2 ENSG00000174938 Seizure related 6 homolog (mouse)-like 2 SFRP1 ENSG00000104332 Secreted frizzled-related protein I SFRP2 ENSGOO000145423 Secreted frizzled-related protein 2 SFRP4 ENSG00000106483 Secreted frizzled-related protein 4 SFRP5 ENSG00000120057 Secreted frizzled-related protein 5 SFTA2 ENSG00000196260 Surfactantassociated 2 SFTPAI ENSG00000 122852 Surfactant protein A l SFTPA2 ENSG00000)185303 Sufactant protein A2 SFTPB ENSG00000168878 Surifactant proteinB SFTPD ENSG00000133661 Surfactant protein )
SFXN 5 ENSG00000144040) Sideroflexin 5 SGCA ENSG0000 108823 Sarcoglycan, alpha (50kDa dystrophin-associated glycoprotein) SGSH ENSGOOOOO 181523 N-sulfoglucosamine sulfohydrolase SH3RF3 ENSG00000172985 SH3 domain containing ring finger 3 SHBG ENSGO0000129214 Sex hormone-binding globulin SHE ENSG00000169291 Src homology 2 domain contaiFi E SHH ENSGOOOOO164690 Sonic hedgehog SHKBPI ENSGO0000160410 S-I3KBPI binding protein I SIAE ENSG00000110013 Sialic acid acetylesterase SIDT2 ENSG00000149577 SlID transmembrane family, member SIGLEC1 , ENSGO0000142512 Sialic acid binding Ig-like lectin 10 SIGLEC6 ENSG00000 105492 Sialic acid binding Ig-like lectin 6 SIGLEC7 ENSGOOOO 168995 Salic acid binding ig-like lectin 7 SIGLECLI ENSG00000179213 SIGLEC family like 1 SIGMARI ENSG00000147955 Sigma non-opioid intracellular receptor I SIL1 ENSGO0000120725 SIE l nucleotide exchange factor SIRPBI ENSGOOOOO101307 Signal-regulatory protein beta 1 SIRPD ENSG0000125900 Signal-regulatory protein delta StAMFI EN SGOOOOO117090 Signaling Iymphocytic activation molecule family member I SLAMF7 ENSG00000026751 SLAM family member 7 SLCIOA3 ENSG0000126903 Solute carrier family 10, member 3 SLC15A3 ENSG00000110446 Solute carrier family 15 (oligopeptide transporter), member 3
SLC25A14 ENSGO0000102078 Solute carrierfamily 25 (mitochondrial carrier, brain), member 14 SLC25A25 ENSGO0000148339 Solute carrier family 25 mitochondriall carrier; phosphate carrier), member 25
SL C2A5 ENSG00000142583 Solute carrier family 2 (facilitated glucose/fructose transporter) member 5
SLC35E3 ENSG0000175782 Solute carrier family 35, member E3 SLC39A10 ENSG00000196950 Solute carrier family 39 (zinc transporter), member 10 SLC39A14 ENSG00000104635 Solute carrierfamily 39 (zinc transporter), member 14 SLC39A4 ENSG00000 147804 Solute carrier family 39 (zinc transporter), member 4 StC39A5 ENSG00000139540 Solute carrier family 39 (zinc transporter), member 5 SLC3A 1 ENSG00000138079 Solute carrier family 3 (amino acid transporter heavy chain), member I SLC51A ENSGO0000163959 Solute carrier family 51, alpha subunit SLC52A2 ENSG00000 185803 Solute carrier family 52 transporter), member 2 SL.C5A6 ENSGO0000 138074 Solute carrier fiarnily 5 (soditum/multivitamin and iodide cotansporter), member 6
SL.C6A9 ENSGO0000 196517 Solute carrier firnily 6 (neurottonsittertransporter, glycmne), _member 9 SLZC8AI ENSGO0000183023 Solute carrier family 8 (sodium/calcium exchanger), member 1 SLC8Bl ENSG-0000089060 Solut carrier family 8 (sodium/lithium/calciumexchanger), memberBI SLC9A6 ENSG00000198689 Solute carrier family 9, subfamily A (NHE6, cationproton antiporter 6), member 6 SLCO1A2 ENSG00000084453 Solute carrier organic anion transporter family, menfer 1A2 SLI ENSGOOOOO187122 Slit guidance ligand 1 SLI.T2 ENSGOOOO145147 Slit guidance ligand 2 SLIT3 ENSGO0000184347 Slit guidance igand 3 SLiTRK3 ENSGOOOOO 121871 SLTand NTRK- ke family, member 3 SLPI ENSGOOOOO 124107 Secretory leukocyte peptidase inhibitor SLTM ENSGO0000137776 SAFB-like, transcription modulator SLURPI ENSGO0000126233 Secreted LY6/PLA1R domain containing 1 SMARCA2 ENSG0000080503 SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 2
SMG6 ENSG00000070366 SMG6 nonsense mediated mRNA decav factor SMIM7 ENSG00000214046 Small integral membrane protemn 7 SMOCI ENSGOOOOO198732 SPARC related modular calcium binding I SMOC2 ENSGOOOOO 112562 SPARC related modular calcium binding 2 SMPDL3A ENSGO0000172594 Sphingomyelin phosphodiesterase,;acid-like 3A SMPDL3B ENSG00000130768 Sphingomyelin phosphodiesterase, acid-like 3B SMR3A ENSGOOOOO 109208 Submaxillary gland androgen regulated protein 3A SMR3B ENSGOOOOO171201 Submaxillary gland androgen regulated protein 3B SNEDI ENSGOOOOO162804 Sushi, nidogen and EGF-like domains I SNTBI ENSGOOOOO172164 Syntrophin, beta I (dystrophin-associated protein A 1, 59kDa, basic component 1)
SNTB2 ENSG00000168807 Syntrophin. beta 2 (dystrophin-associated protein Al 59kDa basic component 2) SNX14 ENSG00000135317 Sorting nexin 14 SOD3 ENSG00000109610 Superoxide dismutase 3, extracellular SOST ENSG00000167941 Sclerostin SOSTDCi ENSG00000 171243 Sclerostin domain containing I SOWAHA ENSG0000 198944 Sosondowah ankyrin repeat domain family member A SPACA3 ENSG00000141316 Sperm acrosome associated 3 SPACA4 ENSG00000177202 Sperm acrosome associated 4 SPACA5 ENSGO0000171489 Sperm acrosome associated 5 SPACA5B ENSG00000171478 Sperm acrosome associated 5B SPACA7 ENSG00000153498 Sperm acrosome associated 7 SPAGIlA ENSG00000178287 Sperm associated antigen 11A SPAGIIB ENSG00000164871 Sperm associated antigen 11B SPARC ENSG00000113140 Secreted protein, acidic, cysteine-rich (osteonectin) SPARCL1 ENSG00000152583 SPARC-like I (hevin) SPATA20 ENSG00000006282 Spermatogenesis associated 20 SPESPI ENSG00000258484 Spermr equatorial segment protein I SPINKI ENSGO0000164266 Serine peptidase inhibitor, Kazal type 1 SPINK13 ENSGO0000214510 Serine peptidase inhibitor, Kazal type 13 (putative) SPINK 14 ENSG00000196800 Serine peptidase inhibitor, Kazal type 14 (putatnve) SPINK2 ENSG00000128040 Serine peptidase inhibitor, Kazal type 2 (acrosin-trysin inhibitor) SPINK4 ENSG00000122711 Serine peptidase inhibitor, Kazal type 4 SPINK5 ENSG00000133710 Serine peptidase inhibitor, Kazal type 5 SPINK6 ENSGO0000178172 Serine peptidase inhibitor, Kazal type 6 SPINK7 ENSGO0000145879 Serine peptidase inhibitor, Kazal type 7 (putative) SPINK8 ENSG00000229453 Serine peptidase inhibitor, Kazal type 8 (putative) SPINK9 ENSG00000204909 Serirre peptidase inhibitor, Kazai type 9 SPINTI ENSGOOOOO 166145 Serine peptidase inhibitor, Kunitz t-pe 1 SPINT2 ENSGOOOOO 167642 Serine peptidase inhibitor, Kurnitz type, 2 SPINT3 ENSG00000101446 Serine peptidase inhibitor, Kunitz type, 3 SPINT4 ENSGOOOOO 149651 Serine peptidase inhibitor, Kunitz type 4 SPOCK1 ENSGOOOO0 152377 Sparc/osteonectin, cwcv and kazal-ilke domains proteoglycan (testican) 1 SPOCK2 ENSGO0000107742 Sparc/osteonectin, cwcv and kazal-like domains proteoglycan (testican) 2 SPOCK3 ENSGOOOOO 196104 Sparc/osteonectin, cwcv and kazal-like domaiis proteoglycan (testican) 3 SPONI ENSG00000262655 Spondin 1, extracellular matrix protein SPON2 ENSGO0000159674 Spondin 2, extracellular matrix protein SPPI ENSGOOOOO118785 Secrted phosphoprotein I SPP2 ENSGO0000072080 Secreted phosphoprotein 2, 24kDa SPRN ENSG00000203772 Shadow of pion protein homolog (zebrafish) SPRYD3 ENSGOOOOO 167778 SPRY domain containing 3 SPRYD4 ENSGO0000176422 SPRY domain containing 4
SPTY2DI-ASI ENSG00000247595 SPTY2D1 antisense RNA 1 SPX ENSG00000134548 Spexin hormone SRGN ENSG0000122862 Serglycin SRL ENSGO0000185739 Sarcalumenin SRP14 ENSG00000140319 Signal recognition particle 14kDa (homologous Alu RNA binding protein) SRPX ENSG00000101955 Sushi-repeat containing protein X-liked SRPX2 ENSGOOOO0102359 Sushi-repeat containing protein, X-linked 2 SSC41) ENSG0000014670) Scavenger receptor cysteine rich family. 4 domains SSC5D ENSG00000179954 Scavenger receptor cysteine rich family, 5 domains SSPO ENSG00000)197558 SCO-spondin SSR2 ENSG00000163479 Signal sequence receptor, beta (translocon-associated protein beta) SST ENSG00000157005 Somatostatn ST3GAL1 ENSG00000008513 ST3 beta-galactosidealpha-23-sialyltmnsfemse1 ST3GAL4 ENSG00000110080 ST3 beta-galactoside alph a-2,3-sialyitmnsferase 4 ST6GAL1 ENSG00000073849 ST6 beta-galactosamide alpha-2,6-sialyltranfemase 1 ST6GALNAC2 ENSG00000070731 ST6 (alpha-N-acetyl-nieurminyl-2,3-beta-galactosyl-1,3)-N acetylgalactosarnirde alpha-2,6-sialyltransferase 2 ST6GALNAC5 ENSG00000117069 ST6(alpha-N-acetyl-neuramnyl-3beta-galactosyl-1,3)-N acetylgalactosaminde alpha-2,6-sialyltransferase 5 ST6GALNAC6 ENSGO0000160408 ST6(alpha-Nacetyl-n inl-,3-etagaactosyl-,3)-N acetyigalactosarinide alpha-2,6-sialytransferase 6
ST8SIA2 ENSG0000(0140557 ST8 alpha-N-acetyl-neumminide alpha-2,8-sialyltransferase 2 ST8SIA4 ENSGO0000113532 ST8 alpha-N-acetv-neuraminide alpha-2,8-sialyltransferase 4 ST8SIA6 ENSG00000148488 ST8 alpha-N-acetyl-neumminide alpha-2,8-sialytransferase 6 STARD7 ENSG00000084090 StAR-related hipid transfer (START) domain containing 7 STATH ENSG00000126549 Statherin STC I EN SG00000159167 Stanniocalcin 1 STC2 ENSG00000113739 Stanniocalcin 2 STMNDI ENSGO000230873 Stathnin dormai coitaining I STOMTL2 ENSG00000165283 Stomatin (EPB72)-like 2 STOXI ENSG00000 165730 Storkheadbox 1 STRC ENSG00000242866 Stereocilin SUCLGI ENSG00000163541 Succinate-CoA ligase, alpha subunit SIDS3 ENSG00000111707 SIDS3 homolog, SIN3A corepressor complex component SULF1 ENSGO0000137573 Sulfatase 1 SULF2 ENSG00000196562 Suifatase 2 SUMIF ENSGOOOOO144455 Sulfatasemodifying factor I SIUMiVF2 ENSG00000129103 Sulfatase modifying factor 2 SJSDI ENSGOOOOO 106868 Sushi domain containing I SUSD5 ENSGO0000173705 Sushi domain containing'5 SVEPI ENSGOOOOO165124 Sushi, von Willebrand factor type A, EGF and pentraxin domain contaning I
SWSAPI ENSG0000173928 SWIM-type zinc finger 7 associated protein I
SYAPI ENSG00000169895 Synapse associated protein I SYCN ENSG00000179751 Syncollin TAC1 ENSG00000006128 Tachykinin, precursor I TAC3 ENSGO0000166863 Tachykinin 3 TAC4 ENSG00000176358 Tachykinin 4 (hemokinin) TAGLN2 ENSGOO0158710 Transgelin 2 TAPBP ENSG00000231925 TAPbinding protein (tapasin) TAPBPL ENSG00000139192 TAP binding protein-like TIL2 ENSG00000106638 transducin (beta)-like 2 TBX10 ENSG0000 167800 T-box 10 TCF12 ENSGOO00140262 Transcription factor 12 TCN1 ENSG00000134827 Transcobalamin I (vitaminB12 binding protein, R binder _ ily) fam ------------------- TCN2 ENSG0000185339 Transcobalarnin II TCTN1 ENSG00000204852 Tectonic family member I TCTN3 ENSGOOOOO 119977 Tectonic family member 3 TDP~2 ENS00000111802 Tyrosyl-DNA phosphodiesterase 2 TEK ENSG00000120156 TEK tvrosine kinase, endothelial TEPP ENSGO0000159648 Testis,prostate and placenta expressed TEX101 ENSG00000131126 Testis expressed 101 TEX264 ENSGOOOOO164081 Testis expressed 264 TF ENSGO0000091513 Tmsfern TFAM ENSG00000 108064 Transcription factor A, mitochondrial TFF1 ENSG0000160182 Trefoil factor 1 TFF2 ENSGO00160181 Trefoil factor 2 TFF3 ENSGOOOOO 160180 Trefoil factor 3 (intestinal) TFFI ENSG00000003436 Tissue factor pathway inhibitor (lipoproten-associated coagulation inhibitor) TFPI2 ENSGOOOO105825 Tissue factor pathway inhibitor 2 TG ENSG00000042832 Thyroglobulin TGFBI ENSGOOOOO 105329 Transforming growth factor, beta 1 TGFB2 ENSG00000092969 Transfonning growth factor, beta 2 TGFB3 ENSGOOOOO 119699 Transforming growth factor, beta 3 TGFBI ENSG00000120708 Transforming growth factor, beta-induced, 68k)a TGFBR1 ENSG00000106799 Transforming growth factor, beta receptor 1 TGFBR3 ENSG00000069702 Transforming growth factor, beta receptor III TUBS1 ENSG00000137801 Thrombospondin I THBS2 ENSGO0000186340 Throibospondin 2 TI]BS3 ENSGOOOO169231 Thrombospondin 3 THBS4 EN SGOOOOO113296 Thrombospondin4 THOC3 ENSGO0000051596 THO complex 3 TIPO ENSG00000090534 Thrombonoletin THSD4 ENSGOOOOO187720 Thrombospondin, type 1, domain containing 4 TIY1 ENSGOOOOO154096 Thy-1 cell surface antigen
TIE EN.SG00000066056 Tyrosinekinasewithimmunoglobulin-likeandEGF-like domains 1 TIMMDCI ENSGOOOOO113845 Translocase of inner mitochondrial membrane domain containing I TIMI ENSGU0000102265 TIMP metallopeptidase inhibitor I TIMP2 ENSG00000035862 TIMIP metaliopeptidase inhibitor 2 TIMP3 EN SG00000100234 TIMP metallopeptidase inhibitor 3 TIMP4 ENSG00000157150 TIP metal"opeptidase inhibitor 4 TINAGL1 ENSG00000142910 Tubulointerstitial nephi antigen-like I TINF2 ENSG00000092330 TERFI (TRF1)-interacting nuclear factor 2 TLL2 ENSG00000095587 Tolloid-like 2 TLRi ENSG00000174125 Toll-like receptor I TLR3 ENSG00000164342 Toll-like receptor 3 TM2D2 ENSG00000169490 TM2 domain containing 2 TM2D3 ENSG00000184277 TM2 domain containing 3 TM7SF3 ENSG00000064115 Transmembrane 7 superfamily member 3 TM9SF1 ENSG00000100926 Tmnsmrembrane 9 superfrmily mrember 1 TMCO6 ENSG00000113119 Transmembrane and coiled-coil domains 6 IMEiDI ENSG00000099203 Transmeimbone p24 trafficking protein 1 TMED2 ENSG00000086598 Transnembrane p24 trafficking protein 2 TMED3 ENSGOOOOO 166557 Transmembrane p24 trafficking protein 3 TMEDI4 ENSGOOOO 158604 Transmnernbrane p24 trafficking protein 4 TME~D5 ENSG00000 117500 Transmembrane p24 trafficking protein 5 TMED7 ENSG00000134970 Transmentomne p24 trafficking protein7 1'ED7-TICAM2 ENSG00000251201 TMEID7-TICAN2 readthrough TMEM108 ENSG00000144868 Transmenbrane protein 108 TMEM116 ENSG00000)198270 Thnsmernbrane protein 116 TIEM119 ENSG00000183160 Transmembrane protein 119 TMEM155 ENSG00000164112 Transmenombane protein 155 TMEM168 ENSG0000146802 Transmembrane protein 168 TMEM178A ENSG00000152154 Transmenbrane protein 178A TMEMI179 ENSG00000258986 Transmermnbrane protein 179 TMEM196 ENSG00000)173452 Transmembrane protein 196 TMEM199 ENSGO0000244045 Transmentmane protein 199 TMEM205 ENSG0000 105518 Transtembrane protein 205 TMEM213 ENSG00000214128 Transmenbrane protein 213 TMEM25 ENSGOOOO 149582 Transmnemnbrane protein 25 TMEMI30C ENSG00000235156 Transmembrane protein 30C TMEM38B ENSG00000095209 Transmenombane protein 38B TMEM44 ENSG00000 145014 Transmembtane protein 44 TMEM52 ENSG0000178821 Transmenbrane protein 52 TMEM52B ENSG00000165685 Transmembrane protein 52B TIMEM59 EN SG00000 116209 Transmembrane protein 59 TIEM467 ENSG00000 164953 Transmembrane protein 67 TMEM70 ENSG00000175606 Transmembtane protein 70
TMEM87A ENSGC.000103978 Transmembrane protein 87A TMEM94 ENS00000177728 Transmenmbrane protein 94 TMEM95 ENSG00000 182896 Tratsmembmne protein 95 TMIGDI ENSG00000182271 Transmembrane and immunoglobulin domain containing I TMPRSS12 ENSGO0000186452 Transmembrane (C-terminal) protease, serine 12 TMPRSS5 ENSG00000166682 Transmembrane protease. serine 5 TMUBI ENSG00000164897 Transmembrane and ubiquitin-like domain containing I TMX2 ENSG00000213593 Thioredoxin-related tmnsmembnare protein 2 TMX3 ENSG00000166479 Thioredoxin-related transmembrane protein 3 TNC ENSGO0000041982 Tenascin C TNFAIP6 ENSGO0000 123610 Tumor necrosis factor, alpha-induced protein 6 TNFRSFIIA ENSG0000 141655 Tumor necrosis factor receptor supeifamily, member IIa., NFKB activator
TNFRSF1lB ENSG00000164761 Tumor necrosis factor receptor supeifamily. member lb TNFRSF12A ENSG00000006327 Tumor necrosis factor receptor superfamily, member 12A TNFRSF14 ENSG00000157873 Tumornecrosis factor receptor superamily, member 14 TNFRSF18 ENSGO0000186891 Tumor necrosis factor receptor superfamily, member 18 TNFRSFIA ENSG00000067182 Tumor necrosis factor receptor superfamiy, member IA TNFR SFlIB EN8SG00000028137 Tumor necrosis factor receptor superfamily. member lB TNFRSF25 ENSG00000215788 Tumor necrosis factor receptor superfamily, member 25 TNIFRSF6B ENSG00000243509 Tumor necrosis factor receptor superfimily, member 6b, decoy TNFSF11 ENSGO0000120659 Tumor necrosis factor (ligand) superfamily, member 11 TNFSF12 ENSG00000239697 Tumor necrosis factor ([igand) superfamily, member 12 TNFSF12-TNFSF13 ENSG00000248871 TNFSF12-TNFSF13 readthrough TNFSF15 ENSG00000181634 Tumornecrosis factor (ligand) superfamily, member 15 TNN ENSG00000120332 Tenascin N TNR ENSG00000 116147 Tenascin R TNXB ENSG0000 168477 Tenascin XB TOMM7 ENSG00000196683 Translocase of outer mitochondrial membrane 7 homolog ------------ (yeast) TOP1MT ENSG00000184428 Topoisomrerase (DNA) I, mitochondrial TOR IA ENSG00000136827 Torsin family 1. member A (torsin A) TORIB ENSGOOOOO 136816 Torsin family 1, member B (torsin B) TOR2A ENSG00000160404 Torsin family 2, member A TOR3A ENSG00000186283 Torsin family 3, member A TPD52 ENSG00000076554 Tumor protein D52 TPO ENSGOOOOO115705 Thyroid peroxidase TPPI ENSGO0000 166340 Tripeptidyl peptidase I TPSAB1 ENSG00000172236 Try ptase alpha/beta I TPSB2 ENSG00000197253 Tryptase beta 2 (gene/pseudogene) TPSDI ENSG00000095917 Tiyptase delta I TPST1 ENSGOOOOO169902 Tyrosylprotein sulfotransferase I TPST2 ENSGO0000128294 Tyrosylnrotein sulfotransferase 2 TRABD2A ENSG00000186854 TraB dornaini containitg 2A TRABD2B ENSG00000269113 ThB domain containing 2B
TREHI EN SG0000118094 Trehalase (brush-border membrane glycoprotein) TREMI ENSG00000124731 Triggering receptorexpressed on myeloid cells 1 TREN2 ENSG00000095970 Triggering receptor expressed onmyeloid cells 2 TRH ENSG00000170893 Thyrotropin-releasing hormone TRIM24 ENSG00000122779 Tripartite motifcontaining 24 TRIM28 ENSGOCOO130726 Tripartite motiffcontaining 28 TRIO ENSG00000038382 Trio Rho guamne nucleotide exchange factor TRNP1 ENSG00000253368 TMFi-regulated nuclear protein 1 TSC22D4 ENSG00000166925 TSC22 domain family, member 4 TSHB ENSG00000 134200 Thyroid stimulating honnone, beta TSIR ENSGO000165409 Thyrod stimulating hormone receptor TSKU ENSG00000182704 Tsukushi, small leucine rich proteogiycan TSLP ENSGO0000145777 Thymic strormal ltymphopoietin TSPAN3 ENSG0000140391 Tetraspanin 3 TSPAN31 ENSG0000135452 Tetraspanin 31 TSPEAR ENSG000175894 Thrombospondin-lype laminin G domain and EAR repeats TTC13 ENSG00000143643 Tetratricopeptide repeat domain 13 TTC19 ENSG00000011295 Tetratricopeptide repeat domain 19 TTC9B ENSGOO000174521 Tetratricopeptide repeat domain 9B TTLLII ENSG00000175764 Tubulin tyrosine ligase-like family member 11 TTR ENSGOOOOO118271 Transtiyretin TWSG1 ENSG00000128791 Twisted gastrulation BMP signaling modulator 1 TXNDC[2 ENSG00000117862 Thioredoxin domain containing 12 (endoplasmic reticulum) TXNIDC15 ENSGOO000113621 Thioredoxin domain containing 15 TXNDC5 ENSG00000239264 Thioredoxin domain containing 5 endoplasmicc reticulum) TXNRD2 ENSG00000 184470 Thioredoxin reductase 2 TYRPI ENSG00000107165 Tyrosinase-related protein 1 UBAC2 ENSG00000134882 UBA domain containing 2 UBALDI ENSGOOOOO153443 UBA-like domain containing 1 UBAP2 ENSG00000137073 Ubiquitin associated protein 2 UBXN8 ENSG00000 104691 UBX domain protein 8 UCMA ENSG00000165623 Upper zone of growth plate and cartilage matrix associated UCN ENSG00000163794 Urocortin UCN2 ENSGOOOOO145040 Urocortin 2 UCN3 ENSGOOOOO178473 Urocortin 3 UGGT2 ENSGOOOOO 102595 UDP-glucose glycoprotein glucosyltransfemase 2 UGTIA1O ENSG00000242515 UDP glucuronosyltmansfeise I family, polypeptide AIO UGT2AI ENSG00000173610 UDP glucuronosyltransfemse 2 family, polypeptide Al, complex locus U GT2Bi I ENSG00000213759 UDP giucuronosyltmnsferase 2 family, polypeptide B11 UGT2B28 ENSGOOOOO135226 UDP glucuronosyltransferase 2 family, polypeptide B28 UGMT2B4 ENSG00000 156096 UDP gucuronosyltransferase 2 family, poly peptide B4 UGT2B7 ENSG00000171234 UDP glucuronosyltansferase 2 family, polypeptide B7 UGT3AI ENSG00000145626 UDP glycosyitransferase 3 family, polypeptide Al UGT3A2 ENSGOO000168671 U)P glycosyltransferase 3 family, polypeptide A2
UGT8 ENSG000174607 UDP glycosyltransferase 8 ULBP3 ENSG00000131019 UL16 binding protein 3 UMOI) ENSG00000169344 Uromnoduin UNC5C ENSGO0000182168 Unc-5 netrin receptor C UPK3B ENSGO0000243566 Uroplaki 3B USPI ENSG000102226 Ubiquitn specific peptidase II USP14 ENSG00000101557 Ubiquii specific peptidase 14 (tRNA-guanine transg.ycosylase) U SP3 ENSGO0000140455 Ubiquitin specific peptidase 3 UTS2 ENSG00000049247 Urotensin 2 UTS2B ENSGOOOO 188958 Urotensin 2B UTY ENSGO000183878 Ubiquitously transcribed tetratricopeptide repeat containing, Y linked UXS1 ENSGOOOOO115652 UDP-glucuronate decarboxylase 1 VAS-11 ENSGO0000071246 Vasolbin 1 VCAN ENSGO0000038427 Versican VEGFA ENSGOOOO112715 Vascular endothelial growth factor A VEGFB ENSG00000173511 Vascular endothelial growth factor B VEGFC ENSG0000150630 Vascular endotheijal growth factor C VGF ENSGOOO00128564 VGF nerve growth factor inducible VIP ENSGOOOOO146469 Vasoactive intestinal peptide V1P2 ENSG00000106018 Vasoactive intestinal peptide receptor 2 VIT ENSG00000205221 Vitrin VKOR CI ENSG00000167397 Vitamin K epoxide reductase complex, subunit 1 VLDLR ENSGO0000147852 Very low density lipoprotein receptor VMIO ENSGO0000182853 Vitelline membrane outer layerI 1homolog (chicken) VNNI ENSGOOOOO112299 Vanin 1 VNN2 ENSG00000112303 Vanin 2 VNN3 ENSGO0000093134 Vanin 3 VOPP1 ENSGO0000154978 Vesicular, overexpressed in cancer, prostrvival protein I VPREBI ENSGO0000169575 Pre-B lymphocyte I VPREB3 ENSG00000128218 Pre-B lynnhocyte 3 VPS37B ENSG00000139722 Vacuolarprotein sorting 37 homologo B (S. cerevisiae) VPS51 ENSGO0000149823 Vacuolar protein sorting 51 homolog (S. cerevisiae) VSIG1 ENSGOOOOO101842 V-set1andimmunoglobulindomancontaining1 VSIG10 ENSGO0000176834 V-set and immunoglobulin domain containing 10 VSTM1 ENSGOOOOO189068 V-set and transnerbrane domain containing 1 VSTM2A ENSG00000170419 V-set and trnsmembrane domain containing 2A VSTM2B ENSG00000187135 V-set and transmenmmne domain containing 2B VSTN2L ENSGOOOOO 132821 V-set and transmembne domain containing like VSTM4 ENSGO0000165633 V-set and transmembrane domain containing 4 VTN ENSGOOOOO109072 Vitronectin VWAI ENSG00000179403 Von Willebrand factor A domain containin I VWA2 ENSG00000165816 Von Willebrand factor A domain containing 2 VWA5B2 ENSGO0000145198 Von Willebrand factor A domain containing 5B2
VWA7 ENSG00000204396 Von Willebrand factor A domain containing 7 VWC2 ENSGO0000188730 Von Willebrand factor C domain containing 2 VWC2L ENSG00000174453 Von Willebrand factor C domain containing protein 2-like VWCE ENSG00000167992 Von Wiliebrand factor Cand EGF domains VWDE ENSGO000 146530 Von Willebrand factor D and EGF domains VWF ENSG00000110799 Von Willebrand factor WDR25 ENSG00000176473 WD repeat domain 25 WDR8I ENSG00000167716 WD repeat domain 81 WDR90 ENSG00000161996 WD repeat domain 90 WFDCI ENSGO0000 103175 WAP four-disulfide core domain I WFDC1OA ENSG00000 180305 WAP four-disulfide core domain 10A WFDC1OB ENSG00000182931 WAP four-disulfide core domain 1OB WFDCI1 ENSG00000180083 WAP four-disulfide core domain I WFIDC12 ENSG00000168703 WAP four-disufide core domain 12 WFDC13 ENSGO0000 168634 WAP four-disulfide core domain 13 WFDC2 ENSG00000101443 WAP four-disulfide core domain 2 WFDC3 ENSG00000124116 WAP four-disulfide core domain 3 WFDC5 ENSGO0000175121 WAP four-disulfide core domnan 5 WFDC6 ENSG00000243543 WAP four-disulfide core domain 6 WFDC8 ENSGO0000158901 WAP four-disulfide core domain 8 WFIKKN1 ENSGO0000 127578 WAP, follistatin/kazal, immnunoglobulin, kunitzand netrn domain containing 1
WFIKKN2 ENSGO0000173714 WAP, follistatin/kazal, immnunoglobulin, kunitzand netrn domain containing 2
WIF1 ENSGO0000156076 WNT inhibitory factor I WISP1 ENSGOO000104415 WNTI inducible signaling pathway protein 1 WISP2 ENSG00000064205 WNTIinducible signaling pathway protein 2 WISP3 ENSG00000112761 WNTI inducible signaling pathway protein 3 WNKI ENSG00000060237 WNK lysine deficient protein kinase I WNITI ENSGOOOOO 125084 Wingless-type MMTV integration site family, member I WNTIOB ENSG000169884 Wingless-type MMTV integration site family, member 10B WNTI1 ENSG00000085741 Wingless-type MMTIV integration site family, member I1 WNTI6 ENSG00000002745 Wingless-type MMITV integration site fimrnily, member 16 WNT2 ENSGO00000105989 Wingless-type MMTV integration site famil member WNIT3 ENSGOOOOO 108379 Wingless-type MMTV integration site family, member 3 WNT3A ENSGOOOO154342 Wingless-typeMMITV integration site family, member 3A WNT5A ENSG00000114251 Wingliess-type 'MTV integration site family, member 5A WNT5B ENSGOOOOO111186 Wingless-type MMTV integration site family, member 5B WNT6 ENSG00000115596 Wingliess-type MMTV integration site family, member 6 WNIT7A ENSG00000154764 Wingless-type MTVintegration site family, member 7A WNTI7B ENSGOOOO188064 Wingless-type MMITV integmtion site family, member 7B WNT8A ENSG00000061492 Winlwess-type MMTV integration site family, member 8A WNT8B ENSGO0000075290 Wingless-type MMTV integration site family, member 8B WNT9A ENSGO0000143816 Wingless-type MMTV integration site family, member 9A
WNT9B EN SGOOOO158955 Wingless-type MMTV integration site family, member 9B WSB1 ENSG00000109046 WD repeat and SOCS box containing 1 WSCD1 ENSG00000179314 WSC domain containing I WSCD2 ENSG00000075035 WSC domain containing 2 XCLI ENSG00000143184 Chemokine (C motif) ligand I XCL2 ENSGO0000143185 Cheniokine (C motif ligand 2 XPNPEP2 ENSG00000122121 X-prolyl aminopeptidase (aminopeptidase P) 2, membrane bound XXbac- ENSG00000244255 BPG116M5.17 XXbac- ENSG00000248993 BPG181M17.5 XXbac-BPG32J3.20 ENSG00000204422 XXYLT1 ENSGO0000173950 Xyloside xylosyitransferase I XYLT1 ENSG00000103489 Xylosyltransfemase I XYLT2 ENSG00000015532 Xylosyltransferase II ZFYVE21 ENSG00000100711 Zinc finger, FYVE domain containing 21 ZG16 ENSG00000174992 Zymogen granule protein 16 ZG16B ENSG00000162078 Zymogen granule protein 16B ZIC4 ENSG00000174963 Zic fairly member 4 ZNF207 ENSG00000010244 Zinc finger protein 207 ZNF26 ENSG00000)198393 Zinc finger protein 26 ZNF34 ENSG00000196378 Zinc finger protein 34 ZNF419 ENSGO0000105136 Zinc finger protein 419 ZNF433 ENSG00000 197647 Zinc finger protein 433 ZNF449 ENSG00000173275 Zinc finger protein 449 ZNF488 ENSG00000265763 Zinc finger protein 488 ZNF511 ENSG00000 198546 Zinc finger protein 511 ZNF570 ENSG0000171827 Zinc finger protein 570 ZNF691 ENSG00000164011 Zinc finger protein 691 ZNF98 ENSG00000197360 Zinc finger protein 98 ZPBP ENSG00000042813 Zona pellucida binding protein ZPBP2 ENSG00000186075 Zona pellucida binding protein 2 ZSCAN29 ENSG00000140265 Zinc finger and SCAN domain containing 29
[02431 In some embodiments of the disclosure, T cells are modified to express therapeutic proteins, including secreted proteins and secreted human proteins. In some embodiments of the methods of the disorder, compositions comprising CAR-T cells modified to express or to secrete a human protein are used to treat a clotting disorder. Blood clotting occurs through a multistep process known as the coagulation cascade. In the extrinsicpathway, Tissue Factor (also known as factor III or thromboplastin) comes into contact with factor VII to form an activated Vila complex. This initiates a coagulation protease cascade, converting the inactive Factor X to an active protease Factor Xa, which, with activated Factor V, produces thrombin
-2 14-
(Ila) from Prothrombin (II). In the intrinsic pathway, collagen forms a complex with high molecular-weight-kininogen, prekallikrein and Factor XII, leading to the conversion of Factor XII into Factor XIla. Factor XIIa converts Factor XI into Factor XIa, and Factor XIa activates Factor IX to produce Factor IXa. which, together with FVIIIa form the tenase complex, which activates Factor X, which helps convert Prothrombin (II) into Thrombin (I1a). Thrombin in turn leads to the conversion of Fibrinogen (I) into Fibrin, which together with Factor XIIIa forms a cross-linked fibrin clot. Many clotting disorders are the result of low levels of secreted proteins in the blood that are involved in the coagulation cascade. Clotting disorders can drastically increase the amount of blood leaving the body upon injury, or cause bleeding to occur under the skin or in vital organs. These disorders are frequently genetic. Exemplary, but non-limiting diseases caused by deficiencies in clotting factors include Hemophilias, von Willebrand disease and deficiencies in Antithrombin III, protein C or protein S. Hemopila A and B are X-linked, and are caused by insufficient levels of clotting factor VIII and factor IX (FIX) respectively. Hemophila C is caused by insufficient factor XI. Factor II, VII, X or XII deficiencies can also cause bleeding disorders. Von Willebrand disease is due to a low level of the von Willebrand clotting factor inthe blood. In some cases, deficiencies in blood proteins that regulate clotting lead can lead to too much clotting. Factor V Leiden is a genetic disorder, where the factor V Leiden protein overreacts, causing the blood to clot too often or too much. Deficiencies in Antithrombin III, protein C or protein S, which help regulate bleeding, can also cause excessive clotting. Currently, clotting disorders such as Hemophilia are treated with blood transfusions or infusions of the missing clotting factor (replacement therapy). However, complications of replacement therapy include developing antibodies to the clotting factor, contracting viral infections from blood derived products and damage to joints. There thus exists a need for additional therapies.
[02441 In some embodiments of the disclosure, T cells are modified to express therapeutic proteins, including secreted proteins and secreted human proteins. In some embodiments of the methods of the disorder, compositions comprising CAR-T cells modified to express or to secrete a human protein are used for enzyme replacement therapy. Enzyme replacement therapy typically involves intravenous infusions of therapeutically effective amounts of compositions comprising enzymes that balance underlying enzyme deficiencies that cause the symptoms of the disease. The missing enzyme activity is thus supplied exogenously in this manner. Exemplary diseases that can be treated by modified T cells of the disclosure include, but are not limited to, lysosomal storage diseases Gaucher's disease (glucocerebrosidase enzyme), Fabry disease, mucopolysaccharidosis I (MPS I), mucopolysaccharidosis I (MPS II, or Hunter syndrome, caused by iduronate-2-sulfatase deficiency), mucopolysaccharidosis VI (MPS VI, caused by arylsulfatase B deficiency) and Pompe disease (or glycogen stoarage disease type II, caused by a deficiency in acid alpha-glucosidase). Additional diseases treatable with enzyme replacement therapy include but are not limited to Adenosine deaminase (ADA) deficiency, Hyperammonemia due to the deficiency of the hepatic enzyme N-acetylglutamate synthetase (NAiS), Hlypophosphatasia, Lysosomal acid lipase deficiency, Morquio Syndrome A. Woinan LAL Lysosomal Acid Lipase deficiency, AlAT(Alphal Antitrypsin) deficiency and Urea cycle disorder. Enzymes supplied to patients during enzyme replacement therapy include, but are not limited to Alphal-Antitrypsin, 3 Glucocerebrosidase, Adenosine Deaminase, Alpha-Galactosidase A. a-L-Iduronidase, Iduronate-2-Sulfatase, N-Acetylgalactosamine-6 Sulfatase, -Acetylgalactosamine-4 Sulfatase and Lysosomal Acid Lipase.
102451 In some embodiments of the disclosure, T cells are modified to express therapeutic proteins, including secreted proteins and secreted human proteins. In some embodiments of the methods of the disorder, compositions comprising CAR-T cells modified to express or to secrete a human protein are used to produce human antibodies. In sone embodiments, the disease to be treated by modified T cells expressing secreted proteins is a disease that can be treated through the intravenous infusion or injection of an antibody or an antibody fragment. Antibody based therapies are used in the treatment of many types of diseases in addition to cancer, including immune-based diseases such as arthritis and asthma, and infections, as well as other diseases. Exemplary, but non-limiting list of diseases that can be treated with the modified Tcells of the disclosure include platelet aggregation, Clostridium difficile infection, Rheumatoid arthritis, Crohn's Disease, Plaque Psoriasis, Psoriatic Arthritis, Ankylosing Spondylitis, Juvenile Idiopathic Arthritis, Alzheimer's disease, sepsis, Multiple Sclerosis,
hvpercholesterolemia, systemic lupus erythematosus, prevention of organ transplant rejections, viral infections, asthma, severe allergic disorders, retinopathy, osteoporosis, inflammatory bowel diseases, inflammatory diseases, influenza A, paroxysmal nocturnal hemoglobinuria, sepsis caused by Gram-negative bacteria, psoriasis, invasive Candida infection, ulcerative colitis, hypocholesteroleimia, respiratory syncytial virus infection, focal segmental glomenilosclerosis, graft versus host disease, ankylosing spondylitis, HIV infection, ulcerative colitis. autoimmune diseases, chronic asthma, reduction of scarring after glaucoma surgery, hypercholesterolemia, white blood cell diseases, systemic scleroderma, respiratory syncytial virus (prevention). lupus ervthematosus, diabetes mellitus type 1, inflammation, Pseudomonas aeruginosa infection, macular degeneration, anthrax, cytomegalovirus infection, inflammations of the airways, skin and gastrointestinal tract, systemiic lupus erythematosus. rheumatic diseases. uveitis cytomegalovirus infection, dermatomyositis, polymyositis, fibrosis, choroidal and retinal neovascularization., muscular dystrophy, Staphylococcus aureus infection, lupus nephritis, follicular lymphoma, chronic hepatitis B and ulcerative colitis. Infirsion of Modified Cells as Adoptive CellTherapy
[02461 In certain embodiments ofthe disclosure, modified cells of the disclosure are delivered to a patient via injection or intravenous infusion. In certain embodiments, a therapeutically effective dose of a composition of the disclosure or of compositions comprising modified cells of the disclosure comprises between 2x10 5and 5x10' cells per kg of body weight of the patient per adiniistration, or any range, value or fraction thereof.
102471 In certain embodiments of the disclosure, modified cells of the disclosure are delivered to a patient via injection or intravenous infusion. In certain embodiments, a therapeutically effective dose of a composition of the disclosure or of compositions comprising modified cells of the disclosure comprises between 0.2x106 to 20x106 cells per kg of body weight of the patient per administration. In certain embodiments, a therapeutically effective dose of a composition of the disclosure or of compositions comprising modified cells of the disclosure comprises 0.2x10 6 cells per kg of body weight of the patient per 6 administration, 2x106 cells per kg of body weight of the patient per administration,20x10 cells per kg of body weight of the patient per administration, or any cells per kg of body weight of the patient per administration in between.
[02481 In certain embodiments ofthe disclosure, modified cells of the disclosure are delivered to a patient via injection or intravenous infusion. In certain embodiments, a therapeutically effective dose of a composition of the disclosure or of compositions comprising modified cells of the disclosure comprises 1xi06 cells oraboutL x106 cells per kg of body weight of the patient per administration.
102491 In certain embodiments of the disclosure, modified cells of the disclosure are delivered to a patient via injection or intravenous infusion. In certain embodiments, a therapeutically effective dose of a composition of the disclosure or of compositions comprising modified cells of the disclosure comprises 3x10 6 cells or about 3x10 6 cellsperkg of body weight of the patient per administration.
[02501 In certain embodiments of the disclosure, modified cells of the disclosure are delivered to a patient via injection orintravenous infusion. In certain embodiments, a therapeutically effective dose of a composition of the disclosure or of compositions comprising modified cells of the disclosure comprises between 0.7x10 6 to 6.7x106 cells per kg of body weight of the patient per administration. In certain embodiments, a therapeutically effective dose of a composition of the disclosure or of compositions comprising modified cells of the disclosure comprises 0.7x10 6cells perkg of body weight of the patient per administration, 6.7x10 6cells per kg of body weight of the patient peradministration or any cells per kg of body weight of the patient peradministration in between.
[02511 In certain embodiments of the disclosure, modified cells of the disclosure are delivered to a patient via injection or intravenous infusion. In certain embodiments, a therapeutically effective dose of a composition of the disclosure or of compositions comprising modified cells of the disclosure comprises between 0.7x10 6 to 16x10 6 cells per kg of body weight of the patient per administration. In certain embodiments, a therapeutically effective dose of a composition of the disclosure or of compositions comprising modified cells of the disclosure comprises 0.7x0 6 cellsper kg of body weight of the patient per administration, 2x106 cells per kg of body weight of the patient per administration, 6x106 cells per kg of body weight of the patient per administration, 10.7x10 6 cells per kg of body weight ofthe patient per administration, 16x106cells per kg of body weight of the patient per administration or any cells per kg of body weight of the patient per administration in between. 102521 In certain embodiments of the disclosure, modified cells of the disclosure are delivered to a patient via injection or intravenous infusion. In certain embodiments, a therapeutically effective dose of a composition of the disclosure or of compositions comprising modified cells of the disclosure comprises .2x106 to 7.ix1 6 cells per kg of body weight of the patient per administration. In certain embodiments, a therapeutically effective dose of a composition of the disclosure or of compositions comprising modified cells of the disclosure comprises i.2x10 6 cells per kg of body weight of the patient per administration, 7.1x106 cells per kg of body weight of the patient per administration or any number of cells per kg of body weight of the patient per administration. In certain embodiments, a therapeutically effective dose of a composition of the disclosure or of compositions comprising modified cells of the disclosure comprises between 2x106 to 3x106cells per kg of body weight of the patient per administration.
[02531 In certain embodiments of the disclosure, modified cells of the disclosure are delivered to a patient via injection orintravenous infusion. In certain embodiments, a therapeutically effective dose of a composition of the disclosure or of compositions comprising modified cells of the disclosure comprises 1106x10 6 to 2106x10 6 cells per kg of body weight of the patient per administration. In certain embodiments, a therapeutically effective dose of a composition of the disclosure or of compositions comprising modified cells of the disclosure comprises 1106x106 cells per kg of body weight of the patient per administration, 2106x100 cells per kg of body weight of the patient per administration or any number of cells per kg of body weight of the patient peradministration in between. In certain embodiments of the disclosure, modified cells of the disclosure are delivered to a patient via injection or intravenous infusion. In certain embodiments. a therapeutically effective dose of a composition of the disclosure or of compositions comprising modified cells of the disclosure comprises 0.7x10 6 to 1.3x10 6cells per kg of body weight of the patient per administration. In certain embodiments, a therapeutically effective dose of a composition of the disclosure or of compositions comprising modified cells of the disclosure comprises 0.7x0 6 cellsper kg of body weight of the patient per administration, 13x10 6 cellsper kg of body weight of the patient per administration or any number of cells per kg of body weight of the patient per administration in between.
[02541 In certain embodiments of the disclosure, modified cells of the disclosure are delivered to a patient via injection or intravenous infusion. In certain embodiments, a therapeutically effective dose of a composition of the disclosure or of compositions comprising modified cells of the disclosure comprises a single or multiple doses. In certain embodiments, a therapeutically effective dose of a compositionof the disclosure or of compositions comprising modified cells of the disclosure comprises a split dose. In certain embodiments, a therapeutically effective dose of a composition of the disclosure or of compositions comprising modified cells of the disclosure comprises an initial dose and a maintenance dose. 102551 In certain embodiments of the disclosure, the modified cells are T cells and the T cells may be sorted according to T cell markers prior to either in vitro expansion or formulation with a pharmaceutically acceptable carrier. In some embodiments, modified T cells may be sorted on using CD8+ and/or CD4+ markers. NucleicAcid Molecules
-"19-
[02561 Nucleic acid molecules of the disclosure encoding protein scaffolds can be in the form of RNA, such as mRNA, hnRNA, tRNA or any other form, or in the form of DNA, including, but not limited to, cDNA and genomic DNA obtained by cloning or produced syntheticalIy, or any combinations thereof The DNA can be triple-stranded, double-stranded or single-stranded .or any combination thereof. Any portion of at leastone strand of the DNA or RNA can be the coding strand, also known as the sense strand, or it can be the non-coding strand, also referred to as the anti-sense strand.
[02571 Isolated nucleic acid molecules of the disclosure can include nucleic acid molecules comprising an open reading frame (ORF), optionally, with one or more introns, e.g., but not limited to, at least one specified portion of at least one protein scaffold; nucleic acid molecules comprising the coding sequence for a protein scaffold or loop region that binds to the target protein; and nucleic acid molecules which comprise a nucleotide sequence substantially different from those describedabove but which, due to the degeneracy of the genetic code, still encode the protein scaffold, Centyrin, CAR, CARTyrin, transposon, and/or transposase as described herein and/or as known in the art. Of course, the genetic code is well known in the art. Thus, it would be routine for one skilled in the art to generate such degenerate nucleic acid variants that code for specific protein scaffolds of the present invention. See, e.g., Ausubel, et al., supra. and such nucleic acid variants are included in the present invention.
[02581 As indicated herein, nucleic acid molecules ofthe disclosure which comprise a nucleic acid encoding a protein scaffold, Centyrin, CAR, CARTyrin, transposon, and/or transposase can include, but are not limited to, those encoding the amino acid sequence of a protein scaffold, Centyrin, CAR, CARTyrin, transposon, and/or transposase fragment, by itself; the coding sequence for the entire protein scaffold, Centyrin, CAR, CARTyrin, transposon, and/or transposase or a portion thereof; the coding sequence for a protein scaffold, Centyrin, CAR, CARTyrin, transposon, and/or transposase. fragment or portion, as well as additional sequences, such as the coding sequence of at least one signal leader or fusion peptide, with or without the aforementioned additional coding sequences, such as at least one intron, together with additional, non-coding sequences, including but not limited to, non-coding 5' and 3' sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA processing, including splicing and polvadenylation signals (for example, ribosome binding and stability of mRNA); an additional coding sequence that codes for additional amino acids, such as those that provide additional functionalities. Thus, the sequence encoding a protein scaffold, Centyrin, CAR, CARTvrin, transposon, and/or transposase can be fused to a marker sequence, such as a sequence encoding a peptide that facilitates purification of the fused protein scaffold, Centyrin, CAR, CARTyrin, transposon, and/or transposase comprising a protein scaffold fragment or portion. ConstructionofNucleic Acids
[0259] The isolated nucleic acids of the disclosure can be made using (a) recombinant methods, (b) synthetic techniques, (c) purification techniques, and/or (d) combinations thereof, as well-known in the art.
[02601 The nucleic acids can conveniently comprise sequences in addition to a polynucleotide of the present invention. For example, a multi-cloning site comprising one or more endonuclease restriction sites can be inserted into the nucleic acid to aid in isolation of the polynucleotide. Also, translatable sequences can be inserted to aid in the isolation of the translated polynucleotide of the disclosure. For example, a hexa-histidine marker sequence provides a convenient means to purify the proteins of the disclosure. The nucleic acid of the disclosure, excluding the coding sequence, is optionally a vector, adapter, or linkerfor cloning and/or expression of a polynucleotide of the disclosure.
[0261] Additional sequences can be added to such cloning and/or expression sequences to optimize their function in cloning and/or expression, to aid in isolation of the polvnucleotide, or to improve the introduction of the polynucleotide into a cell. Use of cloning vectors, expression vectors, adapters, and linkers is well known in the art. (See, e.g., Ausubel, supra; or Sambrook, supra). Recombinant Methodsfor ConstructingNucleicAcids
[02621 The isolated nucleic acid compositions of this disclosure, such as RNA, cDNA, genonic DNA, or any combination thereof, can be obtained from biological sources using any number of cloning methodologies known to those of skill in the art. In some embodiments, oligonucleotide probes that selectively hybridize, under stringent conditions, to the polynucleotides of the present invention are used to identify the desired sequence in a cDNA or genomic DNA library. The isolation of RNA, and construction of cDNA and genomic libraries are well known to those of ordinary skill in the art. (See, e.g., Ausubel, supra; or Sambrook, supra). Vectors and Host Cells
[02631 The disclosure also relates to vectors that include isolated nucleic acid molecules of the disclosure, host cells that are genetically engineered with the recombinant vectors, and the production of at leastone protein scaffold by recombinant techniques, as is well known in the art. See, e.g., Sambrook, et al., supra; Ausubel, et al., supra, each entirely incorporated herein by reference.
[0264] For example, the PB-EFla vector may be used. The vector comprises the following nucleotide sequence: tgtacatagattaaccctagaaagataatcatattgtgacgtacgttaaagataatcatgcgtaaaattgacgcatgtgttttatggtetgt atatef~ggtttttattatttgaatagatattaagtttattatatttatcactacatactaataataaatteaacaaacaatttatttatgtttatt tatttattaaaaaaaaaaaaactcaaaatttcttctataaagtaacaaaacttttatcgaatacctgcagccccgggggatccagaggga cagccccccccaaageccccagggatgtaattacgtccctcccccgetagggggcagcagcgagccgcccgggctccgctcc ggtccgcgctecccccgcatccccgagcggcaggtggggg acagccgggcacggggaaggtggcacgggatcgctttc ctctgaacgcttctcgctgetctttgagcetgcagacacctggggggatacggggaaaagttgactgtgcctttcgatgaaccatgga cagttagctttgcaaagatggataaagttttaaacagagaggaatctttgcagctaatggaccttctaggtcttgaaaggagtgggaattg gctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggagggggtcggcaatgaaccggtg cetaigagaaggtgggggggtaatg ggaaagtga tgtegtg tatctggeteegcetttttccgagggggaacgta
taagtgcagtagtcgccgtgaacgttetttttcgcaacgggtttgecgccagaacacaggtaagtgccgtgtgtggttcccgegggcct ggcctctttacgggttatggcccttgcgtgccttgaattacttecactggtgagtacg.tgattttgatccgagttcgggttggaag tgggtgggagagttcgaggcettgcgcttaaggagcccCttegcctcgtgcttgagttgaggctggcctgggcgctggggcgcg cgtgcgaatctggtggcaccttcgcgcctgtctcgctgctttcgataagtctctagccatttaaaatttttgatgacctgctggacgettttt ttctggcaagatagtcttgtaaatgcgggcaagattgcacactggtatttgg ttggggcecgggcggcgacggggeccgt, cgtcccagcgcacatgttcggcgaggcggggcctgcgagcgcggccaccgagaaitcggacgggggtagtctcaagtggccgge
ctgetetggtgectgygcetegegcegcegtg tategcccegccetgggelgggcaaggetggccggteggcaccag ttgelgtgageg gaaagatggccgcttcccggccetgetgcagggagetcaaaatggaggacgcggcgetcgggagagcgggegggtgagtcaccc acacaaaggaaaagggcctttcg.tcectcagccgtcgcttcatgtgactccacggagtaccgggcgccgtccaggcacctgattagt tctcgagcttttggagtacgtcgtcttlaggttggggggaggggtttagegatggagittecccacactgagtgggtggagacgaag ttaggccagcttggcacttgatgtaatlctccttggaatttgccctttttgagtttggatcttggtteattctcaagcctcagacagtggttcaa agtttttttettccatttcaggtgtcgtgagaattctaatacgactcactatagggtgtgctgtctcatcattttggcaaagattggccaccaa gcttgtectgcaggagggtcgacgectctagacgggcggccgctccggatccacgggtaccgatcacatatgccttaaitaaacact agtctatagtgtcacctaaattccctttag I gggtaatggccgtaggccgccgaatg gtcc gacatgataagatacatt gatg agtttggacaaaccacaactagaatgcagtgaaaaaaatgctttatttgtgaaatttgtgatgctattgctttatttgtaaccattataagtg caataaacaagttaacaacaacaattgcattcattttatgtttcaggttcagggggaggtgtgggaggttttcggactctaggacctgcg catgcgcttggcgtaatcatggtcatagetgttteetgtttccccgtatecccccaggtgictgcaggtcaaagagaggagaagg ttcagaggaaagcgitcccgtgccacttccgtgcceggctgtccgcacgtgcggtcggggatgegggggaggcc ggaccggageggagcccegggcggctcgctgctgccecetagcggggagggacgtaattacatccetgggggctttggggggg ggctgtccctctcaccgcggtggagctccagettttgttegaattggggcccccccteg.agggtatcgatgatatctataacaagaaaat atatatataatagttatcacgtaagtaaacatgaaataacaatataattategtatgagitaaatettaaaagtcacgtaaaagataatcat gcgtcattttgactcacgcggtcgttatagttcaaaatcagtgacacttaccgcattgacaagcacgctcacgggagctccaagcggc gactgagatgtcctaaatgcacagcgacggattcgcgctatttagaaagagagagcaatatttcaagaatgeatgcgtcaattttacgca gactatctttctagggttaatctagctagcettaagggcgcctattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgc cagetgcattaatgaategca~acgegecggggya.gaggeggctttgegiyattygggeeteecietegtccga ,ctgtge gctcggtcgttcggctgcggcgacggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaa agaacatgaccaaaatcccttaacgtgagtttcgttccactgagcgtcagaccccg.tagaaaagatcaaagatcttcttgagatccttt tt1ttgcgcgtaatctgctecttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactcttttc cgaaggtaaetggetteagcagagegcagataccaaatactgttettetagtgtagcegtagttaggecaccactaagaaetetgtag caccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagac gatagttaccggataaggcgcagcggtcgggctgaacggggggttgtgcacacagcccagcttggagcgaacgacctacaccga actgagatacctacagcgtgagctatgagaaagcgccacgctteccgaaggagaaaggcggacaggtatceggtaagcegcagg gteggaacaggagagegcacgagggagettcagggggaaacgectggtatetttatagtcetgtegggttgccacetetgacttg agcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttctggccttttgct ggccttttgctcacatgagattatcaaaaagatttcactagatcctttaaattaaaaatgaagttttaaatcaatctaaagtatatatga gtaaacttggtctgacagtcagaagaactegtcaagaaggcgatagaaggatgegctgeaatgggageggegatccgtaaa gcacgaggaagcggtcagcccattcgccgccaagctcttcagcaatatcacgggtagccaacgctatgtcctgatagcggtccgcca cacccagccggccacagtcgatgaatccagaaaagcgeccattttccaccatgatattcggcaagcagcatcgccatgggtcacga cgagatcctcgccgtcgggcatgctegcttgagcctggegaacagteggtggegcgagcccctgatgctcttgtcagatcat ctgategaca agaceggettecatecg ag tacgtgetege1teg atgegatgtttegecttggtggtegaatggicaggtagecgg atca agcgtatgcagccgccgcattgcatcagccatgatggatactttctcggcaggagcaaggtgagatgacaggagatcctgccccggc acttcgcccaatagcagccagtecetteccgcttcagtgacaacgtcgagcacagtgeaaggaacgcccgtcgtggccagcca egatagccgcgctgcctcgtgcttgeagttcattcagggcaccggacaggteggtcttgacaaagaaccgggcgcccctgcgctga cagccggaacacggeggcacagagcagccgattgttgttgtgccagtcatagcgaatagcctctccacccaagcggccggag aacctgcgtgcaatecatcttgttcaatcataatattattgaagcatttatcagggttcgtctcgtcccggttcctccaatgcatgtcaata itggccattagccatattatteattggttatatagcataaatcatacattggcattgcatacgttgtatetatatcataata (SEQ ID NO: 35).
102651 The polynucleotides can optionally be joined to a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it can be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.
[02661 The DNA insert should be operatively linked to an appropriate promoter. The expression constructs will further contain sites for transcription initiation,terminationnd,in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs will preferably include a translation initiating at the beginning and a termination codon (e.g., UAA, UGA or UAG) appropriately positioned at the end of the mRNA to be translated, with UAAand UAG preferred for mammalian or eukaryotic cell expression.
[02671 Expression vectors will preferably but optionally include at least one selectable marker. Such markers include, e.g. but are not limited to, ampicillin, zeocin (Sh bla gene), puromycin (pac gene), hygromycin B (hygB gene), G418/Geneticin (neo gene), mycophenolic acid, or glutamine synthetase (GS, U.S. Pat. Nos. 5,122,464 5,770,359; ,827,739), blasticidin (bsd gene), resistance genes for eukarvotic cell culture as well as ampicillin, zeocin (Sh bla gene), puromycin (pac gene), hygromvcin B (hygB gene), G418/Geneticin (neo gene), kanamycin, spectinomycin, streptomvcin, carbenicillin, bleomvcin, erythromycin, polvmvxin B, or tetracycline resistance genes for culturing in E. coli and other bacteria or prokarvotics (the above patents are entirely incorporated herebyby reference). Appropriate culture mediums and conditions forthe above-described host cells are known in the art. Suitable vectors will be readily apparent to the skilled artisan. Introduction of a vector construct into a host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other known methods. Such methods are described in the art, such as Sambrook, supra, Chapters 1-4 and 16-18; Ausubel, supra, Chapters 1, 9, 13, 15, 16.
[02681 Expression vectors will preferably but optionally include at least one selectable cell surface marker for isolation of cells modified by the compositions and methods of the disclosure. Selectable cell surface markers of the disclosure comprise surface proteins, glycoproteins, or group of proteins that distinguish a cell or subset of cells from another defined subset of cells. Preferably the selectable cell surface marker distinguishes those cells modified by a composition or method of the disclosure from those cells that are not modified by a composition or method of the disclosure. Such cell surface markers include, e.g., but are not limited to, "cluster of designation" or "classification determinant" proteins (often abbreviated as "CD") such as a truncated or full length form of CD19, CD271, CD34, CD22,
CD2.0 CD33, CD52, or any combination thereof Cell surface markers further include the suicide gene marker RQR8 (Philip B et al. Blood. 014 Aug 21; 124(8):1277-87).
[02691 Expression vectors will preferably but optionally include at least one selectable drug resistance marker for isolation of cells modified by the compositions and methods of the disclosure. Selectable drug resistance markers of the disclosure may comprise wild-type or mutant Neo, DHFR, TYMS, FRANCF, RAD5I C, GCS, MDR 1, ALDH1, NKX2.2, or any combination thereof.
[02701 At least one protein scaffold of the disclosure can be expressed in a modified form, such as a fusion protein, and can include not only secretion signals, butalso additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, can be added to the N-terminus of a protein scaffold to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties can be added to a protein scaffold of the disclosure to facilitate purification. Such regions can be removed prior to final preparation of a protein scaffold or at least one fragment thereof Such methods are described in many standard laboratory manuals, such as Sambrook, supra, Chapters 17.29-17.42 and 18.1-18.74; Ausubel, supra, Chapters 16, 17 and 18.
[02711 Those of ordinary skill in theart are knowledgeable inthe numerous expression systems available for expression of a nucleic acid encoding a protein of the disclosure. Alternatively, nucleic acids of the disclosure can be expressed in a host cell by tuning on (by manipulation) in a host cell that contains endogenous DNA encoding a protein scaffold of the disclosure. Such methods are well known in the arte.g. as described in U.S. Pat. Nos. ,580,734, 5,641,670, 5,733,746, and 5,733,761, entirely incorporatedhereinbreference.
[02721 Illustrative of cell cultures useful for the production of the protein scaffolds, specified portions or variants thereof, are bacterial, yeast, and mammalian cells as known in the art. Mammalian cell systems often will be in the form of monolayers of cells although mammalian cell suspensions or bioreactors can also be used. A number of suitable host cell lines capable of expressing intact glycosylated proteins have been developed in the art, and include the COS-1 (e.g., ATCC CRL 1650)., COS-7 (e.g., ATCC CRL-1651), HEK293, BHK21 (e.g., ATCC CRL-10), CHO (e.g., ATCC CRL 1610) and BSC-1 (e.g., ATCC CRL 6) cell lines, Cos-7 cells, CHO cells, hep G2 cells, P3X63Ag8.653, SP2/0-Ag14, 293 cells, HeLa cells and the like, which are readily available from, for example, American Type Culture Collection, Manassas, Va. (www.atc.org). Preferred host cells include cells of lymphoid origin, such as mveloma and lymphoma cells. Particularly preferred host cells are P3X63Ag8.653 cells (ATCC Accession Number CRL-1580) and SP2/0-Ag14 cells (ATCC Accession Number CRL851). In a particularly preferred embodiment, the recombinant cell is a P3X63Ab8653 or an SP2/0-Ag14 cell.
[02731 Expression vectors for these cells can include one or more of the following expression control sequences, such as, but not limited to, an origin of replication; a promoter (e.g., late or early SV40 promoters, the CMV promoter (U.S. Pat. Nos. 5,168,062; ,385,839), an HSV tk promoter, a pgk (phosphoglycerate kinase) promoter, an EF-1 alpha
promoter (U S. Pat. No. 5,266,491). at least one human promoter; an enhancer, and/or processing information sites, such as ribosome binding sites, RNA splice sites, polvadenvlation sites (e.g., an SV40 large T Ag poly A addition site), and transcriptional terminator sequences. See, e.g., Ausubel et al., supra; Sambrook, et al., supra. Other cells useful for production of nucleic acids or proteins of the present invention are known and/or available, for instance, from the American Type Culture Collection Catalogue of Cell Lines and Hybridomas (www.atcc.org) or other known or commercial sources.
[02741 When eukaryotichost cells are employed, polyadenlyation ortranscription terminator sequences are typically incorporated into the vector. An example of a tenninator sequence is the polyadenlyation sequence from the bovine growth hormone gene. Sequences for accurate splicing of the transcript can also be included. An example of a splicing sequence is the VPi intron from SV40 (Sprague, et al., J. Virol. 45:773-781 (1983)). Additionally, gene sequences to control replication in the host cell can be incorporated into the vector, as known in the art. Purificationof a ProteinScaffold
[02751 A protein scaffold can be recovered and purified from recombinant cell cultures by well-known methods including, but not limited to, protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. High performance liquid chromatography ("HPLC") can also be employed for purification. See,. e.g., Colligan, Current Protocols in Immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY, N.Y, (1997-2001), e.g., Chapters 1. 4, 6, 8, 9, 10, each entirely incorporated herein by reference.
[02761 Protein scaffolds of the disclosureinclude naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokarvotic or eukaryotic host, including, for example, E. coli,yeast, higher plant, insect and manmalian cells. Depending upon the host employed in a recombinant production procedure, the protein scaffold of the disclosure can be glycosylated or can be non glycosylated. Such methods are described in many standard laboratory manuals, such as Sambrook, supra, Sections 17.37-17.42; Ausubel, supra, Chapters 10, 12, 13, 16, 18 and 20, Colligan, Protein Science, supra, Chapters 12-14. all entirely incorporated herein by reference. Variants
[02771 The amino acids that make up protein scaffolds of the disclosure are often abbreviated. The amino acid designations can be indicated by designating the amino acid by its single letter code, its three letter code, name, or three nucleotide codon(s) as is well understood in the art (see Alberts, B., et al., Molecular Biology ofThe Cell, Third Ed., Garland Publishing, Inc., New York, 1994). A protein scaffold of the disclosure can include one or more amino acid substitutions, deletions or additions, either from natural mutations or human manipulation, as specified herein. Amino acids in a protein scaffold of the disclosure that are essential for function can be identified by methods known in the art, such as site directed mutagenesis or alanine-scanning mutagenesis (e.g., Ausubel, supra, Chapters 8. 15: Cunningham and Wells, Science 244:1081-1085 (1989)). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity, such as, but not limited to. at least one neutralizing activity. Sites that are critical for protein scaffold binding can also be identified by structural analysis, such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith, et al., J. Mol. Biol.224:899-904 (1992) and de Vos, et al., Science 255:306-312 (1992)).
[02781 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, , 100, 180, 270, 360, 450, 540, ormore nucleotides or amino acids, orthat the two sequences hybridize under stringent hybridization conditions.
[02791 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, , '0, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540 ormore nucleotides or amino acids, or with respect to nucleic acids, if the first sequence is substantially complementary to the complement ofthe second sequence.
[02801 As used throughoutthe 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 asequences substantially identical thereto.
[02811 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, a bacteriophage, a bacterial artificial chromosome or a 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 plasmid.
[02821 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.
102831 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,aminoacids 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 hvdrophilicity of that peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. US.Patent No. 4,554,101, incorporated fully herein by reference.
[02841 Substitution of amino acids having similarhydrophilicity values can result in peptides retaining biological activity, for example immunogenicitv. Substitutionscanbe performed with amino acids having hydrophilicity values within _2 of each other. Both the hydrophobicity 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.
[02851 As used herein, "conservative" amino acid substitutions may be defined as set out in Tables A, B, or C below. In some embodiments, fusion polvpeptides 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 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 acidthat has similar properties. Exemplary conservative substitutions are set outing Table A.
[02861 Table A -- Conservative Substitutions I Side chain characteristics Amino Acid Aliphatic Non-polar G A P ILVF
Polar - uncharged CSTMN Q Polar - charged DEKR Aromatic HFWY Other NQDE
[02871 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.
[02881 Table B -- Conservative Substitutions II Side Chain Characteristic Amino Acid
Non-polar (hydrophobic) Aliphatic: ALIVP Aromatic: FWY Sulfur-containing: M Borderline: GY Uncharged-polar Hydroxyl: STY Amides: NQ Sulfhydryl: C Borderline: GY Positively Charged (Basic): KRH Negatively Charged (Acidic): D E
102891 Alternately, exemplary conservative substitutions are set out inTable C.
[02901 Table C -- Conservative Substitutions III Original Residue Exemplary Substitution Ala (A) Val Leu Ile Met Arg (R) Lvs His Asn (N) Gin Asp (D) Glu Cys (C) Ser Thr Gin (Q) Asn Glu (E) Asp Gly (G) Ala Val Leu Pro His (H) Lvs Arg Ile (1) Leu Val Met Ala Phe Leu (L) Ile Val Met Ala Phe Lys (K) Arg His Met (M) Leu Ile Val Ala Phe (F) TrpTyr Ile Pro (P) Giy Ala Val Leu lIle Ser (S) Thr Thr (T) Ser 1Trp (W) Tyr Phe Ile
Val (V) Ile Leu Met Ala
[02911 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 asmodifications other than insertions, deletions, or substitutions of amino acid residues. Polypeptides or nucleic acids of the disclosure may contain one or more conservative substitution.
[02921 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 or more of the recited amino acid substitutions. The term "more than one" may refer to2, 3, 4, or 5 of the recited amino acid substitutions.
102931 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 naturally-occur, rendering the entire amino acid sequence non-naturally occurring. Polypeptides and proteins of the disclosure may contain one or more duplicated, inverted or repeated sequences, the resultant sequence of which does not naturally-occur, rendering the entire amino acid sequence non-naturally occurring. Polypeptides and proteins of the disclosure may contain modified, artificial, or synthetic amino acids that do not naturally occur, rendering the entire amino acid sequence non-naturally occurring.
[02941 As used throughout the disclosure, "sequence identity" may be determined by using the stand-alone executable BLAST engine program for blasting two sequences (b2seq), 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 ormore 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 toyield 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.
EXAMPLES Example 1: Production of stein-like modified T-cells
[02951 The following is an illustrative but nonlimiting example of one protocol for modifyingTcells to express a chimeric antigen receptor (CAR) under conditions thatinduce or preserve desirable sten-like properties of theT cells.
[02961 Day 0: Nucleofection of T cells
102971 Pre-warm ImmunoCultTM-XF T cell expansion medium (Stemcell Technologies, Cat R: 10981) in 37° C, 5% C02, high humidity incubator. For 5x1O6 T cells/reaction (100 pL cuvette size) warm 3 mL of media/reaction in a single well of a 6-well plate. For 25x10 6 T cells/reaction (100 iLcuvette size) warm 20 nL ofmedia/reaction in a G-Rex10 (Wilson Wolf, Cat #: 80040S).
[02981 Warm P3 primary cell solution (Lonza, Cat #: PBP3-02250) up to room temperature and add supplement if necessary.
102991 Tum on the core unit (Lonza, Cat-#: AAF-1002B) of the 4D-NucleofectorTM System, which controls the X-unit (Lonza, Cat #: AAF-1002X). Program the number of nucleofections required to use P3 buffer. Program EO-210.
[03001 Label cuvettes, pre-open transferpipettes (supplied with the LonzaP3 kit), and prepare proper dilutions of nucleic acids prior to working with the cells.
[03011 For a transposon plasmid, make a 0.5 g/pL solution in nuclease free H20.
[03021 CountCD14, CD56, and CD19depleted cells collectedusing the CliniMACs Prodigy and calculate the volume needed for the required cell number.
103031 Centrifuge T cells at 90 g for 10 minutes with brake at 7 on alHeraeus Multifuge X3R benchtop centrifuge (Thermofisher Scientific). If performing multiple reactions using the same number of cells/reaction, centrifuge all the necessary cells in a single centrifuge tube. Either a 15 mL (Fisher,Cat#: 14-959-49B) or 50 mL (Fisher, Cat #: 14-959-49A) conical tube can be used depending on volume. During centrifugation add nucleic acids directly to the bottom of cuvettes that come with the P3 primary cell solution box (Lonza, Cat #.PBP3-02250). Add 2 pL of the 0.5 ig/uL transposon plasmid solution made instep 4 for a total of I g transposon to one of the bottom corners of the cuvette. Add 5 g of Super piggyBac'' (SPB) transposase mRNA to the other corner of the cuvette.
[03041 Because mRINA can be rapidly degraded, itis optimalto minimize the time it is in contact with othernucleic acid solutions and with cells priorto electroporation due to the potential presence of RNases. This is why, for example, the transposon and transposase are delivered to opposite corners of the cuvette to preventmixing. In addition, it is optimal to keep the total volume of nucleic acids under 10pL (10%) of the total reaction volume.
[03051 The amount of both transposon (I pg) and transposase (5 tg) stays the same regardless of the number of cells/reaction. Transposition efficiencies remain unchanged between 5x10 6 cells/100 L reaction and 25x10 6 cells/100ul reaction.
[03061 Following centrifugation, completely aspirate off the media without disturbing the cell pellet. 103071 Suspend the cell pellet in 100 pL of room temperature P3 buffer containing the supplement/reaction.
[03081 Transfer 100uL of cells in P3 buffer to a cuvette containing the appropriate nucleic acids, optimally, taking care not to introduce any air bubbles into the solution. It is recommended thatonly up to 2 cuvettes should loaded with cells at a time. After the addition of cells to the cuvette, it is optimal to work quickly and efficiently to reduce contact time of mRNA with cells prior to nucleofection. While no decrease in transposition efficiency has observed for cells resting in P3 buffer for up to 10 minutes, it is recommended to minimize the amount of time cells remain in P3.
[03091 Mix the contents of the cuvette by flicking several times and load up to two cuvettes into the 4D-NucleofectorTM X-unit.
[03101 Pulse the cells with program EO-210 and ensure there was no error recorded by the machine. 103111 Immediately transfer the nucleofected cells into either the 6-well plate or G-Rex10 using the transfer pipettes provided with the Lonza P3 kit. To transfer the cells, first draw up a small amount of pre-warmed media into the transfer pipette from either the 6-well plate or the G-Rex flask. Then pipette the media into the cuvette and transfer the entire contents of the cuvette using the pipette into the final culture dish. It is recommended not to pipette the cells up and down in either the cuvette or the final culture dish.
[03121 Repeat protocol from the transfer of cells in P3 buffer to a cuvette containing the appropriate nucleic acids through the mixing, pulsing, and transfer of the nucleofected cells into either the 6-well plate or G-Rex10 for any remaining reactions.
[03131 Place cells in incubator at 37 C, 5% C02, high humidity.
[03141 Da_- T celActivation
[03151 Add 25 pL/mL of ImmunoCuItTM Human CD3/CD28/CD2 T cell Activator (Stemcell Technologies, Cat .: 10970) to the nucleofected cells.
[03161 Mix cells gently by pipetting.
[03171 Place cells back into the incubator at 37° C, 5% CO2, high humidity.
[03181 For cellsbeing grown in G-RexflaskC: It is essential not to disturb the cultures until visible cell clumping is observed. Thus, it is recommended to separate the media additions and changes from the disrption/mixing/pipetting of the cells.
[03191 Culture medianotes: For growing cells in the G-Rex flask, media addition and/or changes should be done based off of glucose and other metabolite levels. If the glucose level (or another indicating metabolite) falls to a critical level (~100 mg/dL of glucose, for example) media volume should be doubled and/or replenished by a half-media change using pre-warmed ImmunoCultTM-XF T cell expansion median. Media addition should be performed slowly and care taken to disrupt the cells as little as possible. Half media changes should be performed at least 12 hours post mechanical disruption of the cell culture to allow the cells to fully settle to the bottom of the culture flask.
103201 Cell Sampling and disruption: Cells should be left undisturbed during much ofthe culture period.
[03211 The firstdisruption ofthe cell culture following activation reagentaddition should occur once large visible aggregates of cells have formed (aggregates will measure 3-4 squares by 3-4 squares of the grid that can be seen on the G-Rex membrane).
[03221 Once cell aggregates have reached the required size, they can be mechanically disrupted using a 10 nL serological pipette. This time point may occur between 11-14 days depending on donor and transposition efficiency. In certain circumstances, this time point may occur closer to day 14 than day 11, for example, when using a manual cassette, a large volume and/or a large cell number for nucleofection. A sampling of cells should be collected at this point for cell counts, viability, and flow analysis. Ideally the volume of culture medium at this point will have no more than doubled from the initial volume used (200mL for a G-Rex100). It is recommended to collect all of the cells needed at once so that the cells do not need to be disturbed again.
[03231 Once the cells have been disrupted they should be left undisturbed for 12 hours in the same volume of media they started in. Cells should re-aggregate at this point; however, the aggregates will be smaller and more numerous. These aggregates should measure 1-2 squares by 1-2 squares on the G-Rexmembrane grid.
103241 Three days following the first disruption (day 14-17 depending on the culture) of the cells they can be pipetted a second time. Samples should be taken again for cell counts, viability, and flow cytometry. Once again the cells should be left undisturbed for at least 12 hours post sampling. It is recommended to collect all of the cells needed at once so that the cells do not need to be disturbed again.
[03251 Following this second disruption, the cells will likely not forn any clumps and the rate of cell growth will slow considerably.
103261 Cell harvest should be performed 3 days after the second disruption of cells between day 17 and day 20 of the culture.
[03271 Flow Cvtometry
[03281 Flow should be run on Day 5, D-Day, D-Day + 3, and D-Day + 6.
[03291 For Day 5, D-Dav, and D-Day+ 3 use the CD45, CD4, CD8, and CARTyrin flow panel
[03301 For D-Day + 6. there are 3 target panels: a. Panel 1: CD3, CD8. CD4, CARTyrin, CD45RA, CD45RO, CD62L b. Panel 2: CD3, CD8., CD4, CARJyrin, CD25, CXCR4, PD-I c. Panel.3: CD45, CD14, CD20, CD56, CD8, CD4. CD3
Example 2: Functional characterization of CARTvrin+ stem memory T cells
[03311 CARTyrins of the disclosure may be introduced toTcells using a plasmid DNA transposon encoding the CARTyrin that is flanked by two cis-regulatory insulator elements to help stabilize CARTyrin expression by blocking improper gene activation or silencing. 103321 In certain embodiments of the methods of the disclosure, the piggyBacT M (PB) Transposon System may be used for stable integration of antigen-specific (includingcancer antigen-specific) CAR-Tyrin into resting pan T cells, whereby the transposon was co delivered along with an mRNA transposase enzyme, called Super piggyBacT M (SPB), in a single electroporation reaction. Delivery of piggyBacTM transposon into untouched, resting primary human panT cells resulted in20-30% of cells with stable integration and expression of PB-delivered genes. Unexpectedly, majority of these modified CARTyrin-expressing T cells were positive for expression of CD62L and CD45RA, markers commonly associated with stem memory T-cells (TScM cells). To confirm that this phenotype was retained upon CAR-T cell stimulation and expansion, the modified CARTvrin-expressing T cells positive for expression of CD62L and CD45RA were activated via stimulation of CD3 and CD28. As a result of stimulation of CD3 and CD28,> 60% of CARTyrin+ T cells exhibited a stem-cell memory phenotype. Furthermore, these cells, which expressed a CARTyrin specific for a cancer antigen, were fully capable of expressing potent anti-tumor effector function.
[03331 To determine whether or not the PB system directly contributed to enhancing the expression of stem-like markers, the phenotype of CAR-Tcells generated either by PB transposition or lentiviral (LV) transduction was compared. To do this, a new vector was constructed by subcloning the CARTyrin transgene into a common LV construct for production of virus. Following introduction of the CARTyrin to untouched resting T cells either by PB-transposition or LV-transduction, the CARTrin- cells were expanded and then allowed to return to a resting state. A variety of phenotypic and functional characteristics were measured including kinetic analysis of memory and exhaustion-associated markers, secondary proliferation in response to homeostatie cytokine or tumor-associated Ag, cvtokine production, and lytic capability in response to target tumor cells. Unlike the PB-transposed CARTyrin' T cells, the LV-transduced CARTyrin+ T cells did not exhibit an augmented memory phenotype. In addition, PB-transposed cells exhibited a comparable or greater capability for secondary proliferation and killing of target tumor cells. Together, these data demonstrate that CAR-Tcells produced by PB transposition are predominantly Tscu cells, a highly desirable product phenotype inthe CAR-T field. Furthermore, these CARTyrin' T cells exhibit strong anti-tumor activity and may give rise to cells that persist longer in vivo due to the use of a Centyrin-based CAR, which may be less prone to tonic signaling and functional exhaustion.
Example 3: Sleeping Beauty Transposition Yields Predominantly TscM phenotype
[03341 Sleeping Beauty (SBI00x)'Transposition yielded a predominately TSCM phenotype using the methods ofthe disclosure. Human pan T cells were transposed using Ipg of either a Sleeping Beauty or piggyBac transposon plasmid and. SB0Oxor SPB mRNA, respectively as shown in Figure 10. Following transposition, cells were expanded ex vivo and all non transposed cells were depleted using a drug selection system. Following 18 days in culture, cells were stained with the phenotypic markers CD4, CD8, CD45RA, and CD62L Stem cell memory phenotype (TscM) is defined by CD45RA and CD62L double positive cells and make up >65% of the cells in all of samples.
Example 4: Expression of Factor IX in modified T-cells
103351 Genetic deficiencies in Factor IX (Figure 11) lead to a life threatening disease called Hemophila B. Henophila B is a rare disease thataffects I in 25,000 to I in 30,00 people. Current Hemophilia B treatments involve an infusion of recombinant Factor IX protein every 2-3 days, at a cost of around $250,000 per year.
[03361 Stem memory T cells (Tscrcells) are maintained in humans for several decades,and are therefore an ideal vehicle to secrete Factor IX, supplying the Factor IX missing in Henophilia B patients without the need for frequent transfusions. T cells were transformed with PiggyBac to secrete Factor IX. When transgenic T cells encoding a human Factor IX transgene were examined for T and Tsc Cell markers using FACS, approximately 80% of all cells showed a TscM phenotype (Figure 12). These modified T cells were able to secrete human Factor IX (Figure 13A), and this secreted Factor IX provided clotting activity (Figure 13B).
INCORPORATION BY REFERENCE
103371 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 orotherwise 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 withany 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.
OTHER EMBODIMENTS
[03381 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 scope of the disclosure. The scope of the appended claims includes all such changes and modifications that are within the scope of this disclosure.
[0339] Other embodiments of the invention as described herein are defined in the following paragraphs. 1. A method of producing a modified stem memory T cell (TscM), comprising introducing into a primary human T cell (a) a transposon composition comprising a transposon comprising an antigen receptor, a therapeutic protein or a sequence encoding the same and (b) a transposase composition comprising a transposase or a sequence encoding the transposase to produce a modified T cell, wherein the modified T cell expresses one or more cell-surface marker(s) of a stem memory T cell (TscM), thereby producing a modified stem memory T cell (TscM).
2. A method of producing a plurality of modified stem memory T cells (TscM), comprising introducing into a plurality of primary human T cells (a) a transposon composition comprising a transposon comprising an antigen receptor, a therapeutic protein or a sequence encoding the same and (b) a transposase composition comprising a transposase or a sequence encoding the transposase to produce a plurality of modified T cells, wherein at least 25%, 50%, 60%, 75%, 80%, 85%, 90%, 95% or 99% of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (TscM), thereby producing a plurality of modified stem memory T cell (TscM).
3. The method of paragraph 2, wherein at least 60% of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (TscM).
4. A method of producing a modified central memory T cell (TcM), comprising introducing into a primary human T cell (a) a transposon composition comprising a transposon comprising an antigen receptor, a therapeutic protein or a sequence encoding the same and (b) a transposase composition comprising a transposase or a sequence encoding the transposase to produce a modified T cell, wherein the modified T cell expresses one or more cell-surface marker(s) of a central memory T cell (TcM), thereby producing a modified central memory T cell (TcM).
5. A method of producing a plurality of modified central memory T cells (TcM), comprising introducing into a plurality of primary human T cells (a) a transposon composition comprising a transposon comprising an antigen receptor, a therapeutic protein or a sequence encoding the same and (b) a transposase composition comprising a transposase or a sequence encoding the transposase to produce a plurality of modified T cells, wherein at least 25%, 50%, 60%, 75%, 80%, 85%, 90%, 95% or 99% of the plurality of modified T cells expresses one or more cell-surface marker(s) of a central memory T cell (TcM), thereby producing a plurality of modified central memory T cell (TcM).
6. The method of paragraph 5, wherein at least 60% of the plurality of modified T cells expresses one or more cell-surface marker(s) of a central memory T cell (TcM).
7. The method of any one of paragraphs 1-6, wherein the transposon is a plasmid DNA transposon with a sequence encoding the antigen receptor or the therapeutic protein flanked by two cis-regulatory insulator elements.
8. The method of any one of paragraphs 1-7, wherein the transposon is a piggyBac transposon.
9. The method of any one of paragraphs 1-8, wherein the transposase is a piggyBac transposase.
10. The method of paragraph 9, wherein the piggyBac transposase comprises an amino acid sequence comprising SEQ ID NO: 4.
11. The method of paragraph 9 or 10, wherein the piggyBac transposase is a hyperactive variant and wherein the hyperactive variant comprises an amino acid substitution at one or more of positions 30, 165, 282 and 538 of SEQ ID NO: 4.
12. The method of paragraph 11, wherein the amino acid substitution at position 30 of SEQ ID NO: 4 is a substitution of a valine (V) for an isoleucine (I)(130V).
13. The method of paragraph 11, wherein the amino acid substitution at position 165 of SEQ ID NO: 4 is a substitution of a serine (S) for a glycine (G) (G165S).
14. The method of paragraph 11, wherein the amino acid substitution at position 282 of SEQ ID NO: 4 is a substitution of a valine (V) for a methionine (M) (M282V).
15. The method of paragraph 11, wherein the amino acid substitution at position 538 of SEQ ID NO: 4 is a substitution of a lysine (K) for an asparagine (N) (N538K).
16. The method of any one of paragraphs 1-15, wherein the transposase is a Super piggyBac (SPB) transposase.
17. The method of paragraph 16, wherein the Super piggyBac (SPB) transposase comprises an amino acid sequence comprising SEQ ID NO: 5.
18. The method of any one of paragraphs 1-17, wherein the sequence encoding the transposase is an mRNA sequence.
19. The method of any one of paragraphs 1-6, wherein the transposon is a Sleeping Beauty transposon.
20. The method of any one of paragraphs 1-6 or 19, wherein the transposase is a Sleeping Beauty transposase or a hyperactive Sleeping Beauty transposase (SB10OX).
21. The method of any one of paragraphs 1-6, wherein the transposon is a Helraiser transposon.
22. The method of any one of paragraphs 1-6 or 21, wherein the transposase is a Helitron transposase.
23. The method of any one of paragraphs 1-6, wherein the transposon is a Tol2 transposon.
24. The method of any one of paragraphs 1-6 or 23, wherein the transposase is a Tol2 transposase.
25. The method of any one of paragraphs 1-6, wherein the transposon is derived or recombined from any species.
26. The method of any one of paragraphs 1-6 or 25, wherein the transposon is synthetic.
27. The method of any one of paragraphs 1-26, wherein the antigen receptor is a T-cell receptor.
28. The method of paragraph 27, wherein the T-cell receptor is naturally-occurring.
29. The method of paragraph 27, wherein the T-cell receptor is not naturally-occurring.
30. The method of paragraph 29, wherein the T-cell receptor comprises one or more mutation(s) compared to a wild-type T-cell receptor.
31. The method of paragraph 29 or 30, wherein the T-cell receptor is a recombinant T-cell receptor.
32. The method of any one of paragraphs 1-31, wherein the antigen receptor is a Chimeric Antigen Receptor (CAR).
33. The method of paragraph 32, wherein the CAR is a CARTyrin.
34. The method of paragraph 32, wherein the CAR comprises one or more VHH sequence(s).
35. The method of paragraph 34, wherein the CAR is a VCAR.
33. The method of any one of paragraphs 1-32, further comprising introducing into the primary human T cell (c) a composition comprising a second transposon comprising a sequence encoding a therapeutic protein, to produce a modified T cell capable of expressing the therapeutic protein.
34. The method of paragraph 33, wherein the therapeutic protein is a secreted or secretable protein.
35. The method of paragraph 33 or 34, wherein the sequence encoding the therapeutic protein is a nucleic acid sequence.
36. The method of paragraph 35, wherein the sequence encoding the therapeutic protein is a DNA sequence.
37. The method of any one of paragraphs 33-36, wherein the transposase composition of (b) mobilizes the transposon of (a) and the second transposon of (c).
38. The method of any one of paragraphs 1-37, further comprising introducing into the primary human T cell (d) a second transposase composition comprising a transposase or a sequence encoding the transposase, wherein the second transposase of (d) is capable of transposing the transposon of (c), and wherein the second transposase composition of (d) and the transposase composition of (b) are not identical.
39. The method of paragraph 38, wherein the transposase composition of (b) mobilizes the transposon of (a) and the transposase composition of (d) mobilizes the transposon of (c).
40. A method of producing a modified stem memory T cell (TscM), comprising: (a) introducing into a primary human T cell a composition comprising an antigen receptor, a therapeutic protein or a sequence encoding the same to produce a modified T-cell, wherein the antigen receptor or the therapeutic protein is not contained in a transposon, and (b) contacting the modified T-cell and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement to produce an activated modified T-cell, wherein the activated modified T-cell expresses one or more cell-surface marker(s) of a stem memory T cell (TscM), thereby producing a modified stem memory T cell (TscM).
41. A method of producing a plurality of modified stem memory T cells (TscM), comprising: (a) introducing into a plurality of primary human T cells a composition comprising an antigen receptor, a therapeutic protein or a sequence encoding the same to produce a plurality of modified T-cells, wherein the antigen receptor or the therapeutic protein is not contained in a transposon, and (b) contacting the plurality of modified T-cells and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement to produce a plurality of activated modified T-cells, wherein at least 25%, 50%, 60%, 75%, 80%, 85%, 90%, 95% or 99% of the plurality of activated modified T-cells expresses one or more cell-surface marker(s) of a stem memory T cell (TscM), thereby producing a plurality of modified stem memory T cells (TscM).
42. The method of paragraph 41, wherein at least 60% of the plurality of activated modified T-cells expresses one or more cell-surface marker(s) of a stem memory T cell (TscM).
43. A method of producing a modified central memory T cell (TcM), comprising: (a) introducing into a primary human T cell a composition comprising an antigen receptor, a therapeutic protein or a sequence encoding the same to produce a modified T-cell, wherein the antigen receptor or the therapeutic protein is not contained in a transposon, and (b) contacting the modified T-cell and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement to produce an activated modified T-cell, wherein the activated modified T-cell expresses one or more cell-surface marker(s) of a central memory T cell (TcM), thereby producing a modified central memory T cell (TcM).
44. A method of producing a plurality of modified central memory T cells (TcM), comprising: (a) introducing into a plurality of primary human T cells a composition comprising an antigen receptor, a therapeutic protein or a sequence encoding the same to produce a plurality of modified T-cells, wherein the antigen receptor or the therapeutic protein is not contained in a transposon, and (b) contacting the plurality of modified T-cells and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement to produce a plurality of activated modified T-cells, wherein at least 25%, 50%, 60%, 75%, 80%, 85%, 90%, 95% or 99% of the plurality of activated modified T-cells expresses one or more cell-surface marker(s) of a central memory T cell (TcM), thereby producing a plurality of modified central memory T cells (TcM).
45. The method of paragraph 44, wherein at least 60% of the plurality of activated modified T-cells expresses one or more cell-surface marker(s) of a central memory T cell (TcM).
46. The method of any one of paragraphs 40-45, wherein a viral vector comprises the antigen receptor or the therapeutic protein.
47. The method of paragraph 46, wherein the viral vector comprises a sequence isolated or derived from a lentivirus.
48. The method of paragraph 46, wherein the viral vector comprises a sequence isolated or derived from a retrovirus.
49. The method of paragraph 48, wherein the retrovirus is a gammaretrovirus.
50. The method of any one of paragraphs 40-46, wherein the viral vector comprises a sequence isolated or derived from an adeno-associated virus (AAV).
51. The method of any one of paragraphs 40-45, wherein a nucleic acid vector comprises the antigen receptor or the therapeutic protein.
52. The method of paragraph 51, wherein an mRNA vector comprises the antigen receptor or the therapeutic protein.
53. The method of any one of paragraphs 40-45, wherein a nanoparticle vector comprises the antigen receptor or the therapeutic protein.
54. The method of any one of paragraphs 40-45, wherein the introducing step comprises a homologous recombination.
55. The method of paragraph 54, wherein the homologous recombination comprises contacting the composition comprising the antigen receptor or the therapeutic protein, a genomic editing construct, and a genomic sequence of at least one primary human T cell of the plurality of primary human T cells.
56. The method of paragraph 55, wherein a vector comprises the antigen receptor or the therapeutic protein.
57. The method of paragraph 56, wherein the vector is an adeno-associated vector (AAV).
58. The method of any one of paragraphs 54-57, wherein the genomic editing construct comprises a guide RNA and a clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9) DNA endonuclease.
59. The method of paragraph 58, wherein the genomic editing construct comprises a DNA binding domain and a type IIS endonuclease.
60. The method of paragraph 59, wherein the genomic editing construct encodes a fusion protein.
61. The method of paragraph 59, wherein the genomic editing construct encodes the DNA binding domain and the type IIS endonuclease and wherein the expressed DNA binding domain and the expressed type IIS endonuclease are non-covalently linked.
62. The method of any one of paragraphs 58-62, wherein the genomic editing construct comprises a sequence derived from a Cas9 endonuclease.
63. The method of paragraph 62, wherein the sequence derived from a Cas9 endonuclease is the DNA binding domain.
64. The method of paragraph 62 or 63, wherein the sequence derived from a Cas9 endonuclease encodes an inactive Cas9.
65. The method of paragraph 64, wherein the sequence derived from a Cas9 endonuclease comprises an amino acid substitution of an Alanine (A) for a Histidine (H) at position 840 (H840A).
66. The method of any one of paragraphs 62-65 wherein the sequence derived from a Cas9 endonuclease encodes a truncated Cas9.
67. The method of paragraph 66, wherein the sequence derived from a Cas9 endonuclease comprises an amino acid substitution of an Alanine (A) for an Asparagine (N) at position 580 (N580A).
68. The method of any one of paragraphs 62-67, wherein the sequence derived from a Cas9 endonuclease comprises an amino acid substitution of an Alanine (A) for an Aspartic Acid (D) at position 10 (D1OA).
69. The method of any one of paragraphs 58-61, wherein the genomic editing construct comprises a sequence derived from a transcription activator-like effector nuclease (TALEN).
70. The method of paragraph 69, wherein the sequence derived from a TALEN is the DNA binding domain.
71. The method of paragraph 58, wherein the genomic editing construct comprises a TALEN.
72. The method of any one of paragraphs 58-61, wherein the genomic editing construct comprises a sequence derived from a zinc-finger nuclease (ZFN).
73. The method of paragraph 72, wherein the sequence derived from a ZFN is the DNA binding domain.
74. The method of paragraph 58, wherein the genomic editing construct comprises a zinc finger nuclease (ZFN).
75. The method of any one of paragraphs 58-74, wherein genomic editing construct targets a safe harbor site on a mammalian chromosome.
76. The method of any one of paragraphs 58-74, wherein genomic editing construct targets a safe harbor site on a human chromosome.
77. The method of paragraph 75 or 76, wherein the chromosome is in vivo, in situ, ex vivo or in vitro.
78. The method of any one of paragraphs 58-77, wherein genomic editing construct targets a sequence encoding a component of an endogenous T-cell receptor or a sequence encoding a component of an endogenous major histocompatibility complex (MHC) on a mammalian chromosome.
79. The method of any one of paragraphs 58-77, wherein genomic editing construct targets a sequence encoding a component of an endogenous T-cell receptor or a sequence encoding a component of an endogenous major histocompatibility complex (MHC) on a human chromosome.
80. The method of any one of paragraphs 40-79, wherein the antigen receptor is a T-cell receptor.
81. The method of paragraph 80, wherein the T-cell receptor is naturally-occurring.
82. The method of paragraph 80, wherein the T-cell receptor is not naturally-occurring.
83. The method of paragraph 82, wherein the T-cell receptor comprises one or more mutation(s) compared to a wild-type T-cell receptor.
84. The method of paragraph 82 or 83, wherein the T-cell receptor is a recombinant T-cell receptor.
85. The method of any one of paragraphs 40-79, wherein the antigen receptor is a Chimeric Antigen Receptor (CAR).
86. The method of paragraph 85, wherein the CAR comprises one or more Centyrin sequence(s).
87. The method of paragraph 86, wherein the CAR is a CARTyrin.
88. The method of paragraph 85, wherein the CAR comprises one or more VHH sequence(s).
89. The method of paragraph 88, wherein the CAR is a VCAR.
90. The method of any one of paragraphs 40-89, further comprising introducing into the primary human T cell a composition comprising a sequence encoding a therapeutic protein, to produce a modified T cell capable of expressing the therapeutic protein.
91. The method of any one of paragraphs 40-90, wherein the therapeutic protein is a secreted or secretable protein.
92. The method of paragraph 90 or 91, wherein the sequence encoding the therapeutic protein is a nucleic acid sequence.
93. The method of paragraph 92, wherein the sequence encoding the therapeutic protein is a DNA sequence.
94. The method of any one of paragraphs 90-93, wherein the introducing comprises a homologous recombination.
95. The method of any one of paragraphs 40-94, wherein the T-cell activator composition of (b) further comprises an anti-human CD2 monospecific tetrameric antibody complex.
96. The method of any one of paragraphs 40-42 or 46-95, further comprising the step of: (c) contacting the activated modified T-cell and a T-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement to produce a plurality of expanded modified T-cells, wherein at least 2% of the plurality of expanded modified T-cells expresses one or more cell-surface marker(s) of a stem memory T cell (TscM).
97. The method of paragraph 96, wherein at least 2%, 5 0%, 10%,15%, 20%, 25%, 30%, 35%, %, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of expanded modified T-cells expresses cell-surface marker(s) of a stem memory T cell (TscM).
98. The method of paragraph 97, wherein at least 60% of the plurality of expanded modified T-cells expresses cell-surface marker(s) of a stem memory T cell (TscM).
99. The method of any one of paragraphs 43-95, further comprising the step of: (c) contacting the activated modified T-cell and a T-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement to produce a plurality of expanded modified T-cells, wherein at least 2% of the plurality of expanded modified T-cells expresses one or more cell-surface marker(s) of a central memory T cell (TcM).
100. The method of paragraph 99, wherein at least 2 %,5%,10%,15%,20%, 25%,30%,35%,
%,45%,50%,60%,65%,70%,75%,80%,85%,90%,95%,99% or any percentage in between of the plurality of expanded modified T-cells expresses cell-surface marker(s) of a central memory T cell (TcM).
101. The method of paragraph 99, wherein at least 60% of the plurality of expanded modified T-cells expresses cell-surface marker(s) of a central memory T cell (TcM).
102. The method of any one of paragraphs 40-42 or 46-101, wherein the method further comprises the step of: (d) enriching the plurality of expanded modified T-cells to produce a composition comprising at least 2%, 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 7 0% , 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of modified T-cells that express cell-surface marker(s) of a stem memory T cell (TscM).
103. The method of any one of paragraphs 40-42 or 46-101, wherein the method further comprises the step of: (d) enriching the plurality of expanded modified T-cells to produce a composition comprising at least 60% of modified T-cells that express cell-surface marker(s) of a stem memory T cell (TscM).
104. The method of any one of paragraphs 43-101, wherein the method further comprises the step of: (d) enriching the plurality of expanded modified T-cells to produce a composition comprising at least 2%, 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 7 0% , 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of modified T-cells that express cell-surface marker(s) of a central memory T cell (TcM).
105. The method of any one of paragraphs 43-101, wherein the method further comprises the step of:
(d) enriching the plurality of expanded modified T-cells to produce a composition comprising at least 60% of modified T-cells that express cell-surface marker(s) of a central memory T cell (TcM).
106. The method of paragraph 102 or 103, wherein the enriching step comprising isolating modified T-cells that express one or more cell-surface marker(s) of a stem memory T cell (TscM) from the plurality of enriched modified T-cells.
107. The method of paragraph 106, wherein the enriching step further comprises contacting the isolated modified TscM and a T-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement to produce a plurality of expanded enriched modified TscM.
108. The method of paragraph 104 or 105, wherein the enriching step comprising isolating modified T-cells that express one or more cell-surface marker(s) of a central memory T cell (scM) from the plurality of enriched modified T-cells.
109. The method of paragraph 108, wherein the enriching step further comprises contacting the isolated modified TcM and a T-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement to produce a plurality of expanded enriched modified TcM.
110. The method of any one of paragraphs 40-109, wherein the T-cell expansion composition further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decyn-4,7 diol (TMDD), diisopropyl adipate (DIPA), n-butyl-benzenesulfonamide, 1,2 benzenedicarboxylic acid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, a sterol and an alkane.
111. The method of any one of paragraphs 40-109, wherein the T-cell expansion composition further comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid and a sterol.
112. The method of paragraph 111, wherein the T-cell expansion composition further comprises one or more of octanoic acid at a concentration of between 0.9 mg/kg to 90 mg/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; oleic acid at a concentration of 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; and a sterol at a concentration of about 0.1 mg/kg to 10 mg/kg, inclusive of the endpoints.
113. The method of paragraph 111, wherein the T-cell expansion composition further comprises one or more of octanoic acid at a concentration of about 9 mg/kg, palmitic acid at a concentration of about 2 mg/kg, linoleic acid at a concentration of about 2 mg/kg, oleic acid at a concentration of about 2 mg/kg and a sterol at a concentration of about 1 mg/kg.
114. The method of paragraph 111, wherein the T-cell expansion composition further comprises one or more of octanoic acid at a concentration of between 6.4 tmol/kg and 640 tmol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 tmol/kg and tmol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 pmol/kg and 75 pLmol/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 pmol/kg and 75 pmol/kg, inclusive of the endpoints; and a sterol at a concentration of between 0.25 tmol/kg and 25 tmol/kg, inclusive of the endpoints.
115. The method of paragraph 111, wherein the T-cell expansion composition further comprises one or more of octanoic acid at a concentration of about 64 tmol/kg, palmitic acid at a concentration of about 7 pmol/kg, linoleic acid at a concentration of about 7.5 pmol/kg, oleic acid at a concentration of about 7.5 pmol/kg and a sterol at a concentration of about 2.5 gmol/kg.
116. A method of producing a modified stem memory T cell (TscM), comprising:
(a) introducing into a primary human T cell a composition comprising an antigen receptor or a therapeutic protein to produce a modified T cell, wherein a transposon comprises the antigen receptor, and (b) contacting the modified T cell and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement to produce an activated modified T-cell, wherein the activated modified -T cell expresses one or more cell-surface marker(s) of a stem memory T cell (TscM), thereby producing a modified stem memory T cell (TscM).
117. A method of producing a plurality of modified stem memory T cells (TscM), comprising: (a) introducing into a plurality of primary human T cells a composition comprising an antigen receptor or a therapeutic protein to produce a plurality of modified T cells, wherein a transposon comprises the antigen receptor, and (b) contacting the plurality of modified T cells and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement to produce a plurality of activated modified T-cells, wherein at least 25%, 50%, 60%, 75%, 80%, 85%, 90%, 95% or 99% of the plurality of activated modified -T cells expresses one or more cell-surface marker(s) of a stem memory T cell (TscM), thereby producing a modified stem memory T cell (TscM).
118. The method of paragraph 117, wherein at least 60% of the plurality of activated modified -T cells expresses one or more cell-surface marker(s) of a stem memory T cell (TscM).
119. A method of producing a modified central memory T cell (TcM), comprising:
(a) introducing into a primary human T cell a composition comprising an antigen receptor or a therapeutic protein to produce a modified T cell, wherein a transposon comprises the antigen receptor, and (b) contacting the modified T cell and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement to produce an activated modified T-cell, wherein the activated modified -T cell expresses one or more cell-surface marker(s) of a central memory T cell (TcM), thereby producing a modified central memory T cell (TcM).
120. A method of producing a plurality of modified central memory T cells (TcM), comprising: (a) introducing into a plurality of primary human T cells a composition comprising an antigen receptor or a therapeutic protein to produce a plurality of modified T cells, wherein a transposon comprises the antigen receptor, and (b) contacting the plurality of modified T cells and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement to produce a plurality of activated modified T-cells, wherein at least 25%, 50%, 60%, 75%, 80%, 85%, 90%, 95% or 99% of the plurality of activated modified -T cells expresses one or more cell-surface marker(s) of a central memory T cell (TcM), thereby producing a modified central memory T cells (TcM).
121. The method of paragraph 120, wherein at least 60% of the plurality of activated modified -T cells expresses one or more cell-surface marker(s) of a central memory T cells (TcM).
122. The method of any one of paragraphs 116-121, wherein the T-cell activator composition of (b) further comprises an anti-human CD2 monospecific tetrameric antibody complex.
123. The method of any one of paragraphs 116-118 or 122, further comprising the step of: (c) contacting the activated modified T-cell and a T-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement to produce a plurality of expanded modified T-cells, wherein at least 2% of the plurality of expanded modified T-cells expresses one or more cell-surface marker(s) of a stem memory T cell (TCM).
124. The method of paragraph 123, wherein at least 2%,5%,10%,15%,20%, 25%,30%, %,40%,45%,50%,60%,65%,70%,75%,80%,85%,90%,95%,99% or any percentage in between of the plurality of expanded modified T-cells expresses cell-surface marker(s) of a stem memory T cell (TSCM).
125. The method of paragraph 123, wherein at least 60% of the plurality of expanded modified T-cells expresses cell-surface marker(s) of a stem memory T cell (TCM).
126. The method of any one of paragraphs 119-122, further comprising the step of: (c) contacting the activated modified T-cell and a T-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement to produce a plurality of expanded modified T-cells, wherein at least 2% of the plurality of expanded modified T-cells expresses one or more cell-surface marker(s) of a stem memory T cell (TCM).
127. The method of paragraph 126, wherein at least 2%,5%,10%,15%,20%, 25%,30%, %,40%,45%,50%,60%,65%,70%,75%,80%,85%,90%,95%,99% or any percentage in between of the plurality of expanded modified T-cells expresses cell-surface marker(s) of a stem memory T cell (TCM).
128. The method of paragraph 126, wherein at least 60% of the plurality of expanded modified T-cells expresses cell-surface marker(s) of a stem memory T cell (TscM).
129. The method of any one of paragraphs 116-118 or 122-128, wherein the method further comprises the step of: (d) enriching the plurality of expanded modified T-cells to produce a composition comprising at least 2%, 5%, 10%,l15%,20%, 25%, 30%, 35%,40%, 45%, 50%, 60%, 65%, %, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of modified T-cells that express cell-surface marker(s) of a stem memory T cell (TscM).
130. The method of any one of paragraphs 116-118 or 122-128, wherein the method further comprises the step of: (d) enriching the plurality of expanded modified T-cells to produce a composition comprising at least 60% of modified T-cells that express cell-surface marker(s) of a stem memory T cell (TscM).
131. The method of any one of paragraphs 119-128, wherein the method further comprises the step of: (d) enriching the plurality of expanded modified T-cells to produce a composition comprising at least 2%, 5%,10%, 15 ,20%, 25%, 30%, 35%, 40%, 45%, 5 0%, 60%, 65%, 7 0% , 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of modified T-cells that express cell-surface marker(s) of a stem memory T cell (TscM).
132. The method of any one of paragraphs 119-128, wherein the method further comprises the step of: (d) enriching the plurality of expanded modified T-cells to produce a composition comprising at least 60% of modified T-cells that express cell-surface marker(s) of a stem memory T cell (TscM).
133. The method of paragraph 129 or 130, wherein the enriching step comprising isolating modified T-cells that express one or more cell-surface marker(s) of a stem memory T cell (TscM) from the plurality of enriched modified T-cells.
134. The method of paragraph 133, wherein the enriching step further comprises contacting the isolated modified TscM and a T-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement to produce a plurality of expanded enriched modified TscM.
135. The method of paragraph 131 or 132, wherein the enriching step comprising isolating modified T-cells that express one or more cell-surface marker(s) of a stem memory T cell (TscM) from the plurality of enriched modified T-cells.
136. The method of paragraph 135, wherein the enriching step further comprises contacting the isolated modified TscM and a T-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement to produce a plurality of expanded enriched modified TscM.
137. The method of any one of paragraphs 116-136, wherein the T-cell expansion composition further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decyn-4,7 diol (TMDD), diisopropyl adipate (DIPA), n-butyl-benzenesulfonamide, 1,2 benzenedicarboxylic acid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, a sterol and an alkane.
138. The method of any one of paragraphs 116-137, wherein the T-cell expansion composition further comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid and a sterol.
139. The method of paragraph 138, wherein the T-cell expansion composition further comprises one or more of octanoic acid at a concentration of between 0.9 mg/kg to 90 mg/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; oleic acid at a concentration of 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; and a sterol at a concentration of about 0.1 mg/kg to 10 mg/kg, inclusive of the endpoints.
140. The method of paragraph 138, wherein the T-cell expansion composition further comprises one or more of octanoic acid at a concentration of about 9 mg/kg, palmitic acid at a concentration of about 2 mg/kg, linoleic acid at a concentration of about 2 mg/kg, oleic acid at a concentration of about 2 mg/kg and a sterol at a concentration of about 1 mg/kg.
141. The method of paragraph 138, wherein the T-cell expansion composition further comprises one or more of octanoic acid at a concentration of between 6.4 pmol/kg and 640 pmol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 pmol/kg and pmol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 pmol/kg and 75 pmol/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 pmol/kg and 75 pmol/kg, inclusive of the endpoints; and a sterol at a concentration of between 0.25 pmol/kg and 25 pmol/kg, inclusive of the endpoints.
142. The method of paragraph 138, wherein the T-cell expansion composition further comprises one or more of octanoic acid at a concentration of about 64 pmol/kg, palmitic acid at a concentration of about 7 pmol/kg, linoleic acid at a concentration of about 7.5 pmol/kg, oleic acid at a concentration of about 7.5 pmol/kg and a sterol at a concentration of about 2.5 pmol/kg.
143. The method of any one of paragraphs 116-142, further comprising introducing into the primary human T cell (c) a composition comprising a second transposon comprising a sequence encoding a therapeutic protein, to produce a modified T cell capable of expressing the therapeutic protein.
144. The method of paragraph 143, wherein the therapeutic protein is a secreted or a secretable protein.
145. The method of paragraph 143 or 144, wherein the sequence encoding the therapeutic protein is a nucleic acid sequence.
146. The method of paragraph 143 or 144, wherein the sequence encoding the therapeutic protein is a DNA sequence.
147. The method of any one of paragraphs 143-146, wherein the transposase composition of (b) mobilizes the transposon of (a) and the second transposon of (c). 148. The method of any one of paragraphs 143-147, further comprising introducing into the primary human T cell (d) a second transposase composition comprising a transposase or a sequence encoding the transposase, and wherein the second transposase of (d) is capable of transposing the transposon of (c), and wherein the second transposase composition of (d) and the transposase composition of (b) are not identical.
149. The method of any one of paragraphs 1-148, wherein the introducing step further comprises a composition comprising a genomic editing construct.
150. The method of paragraph 149, wherein the genomic editing construct comprises a guide RNA and a clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9) DNA endonuclease.
151. The method of paragraph 150, wherein the genomic editing construct comprises a DNA binding domain and a type IIS endonuclease.
152. The method of paragraph 151, wherein the genomic editing construct encodes a fusion protein.
153. The method of paragraph 151, wherein the genomic editing construct encodes the DNA binding domain and the type IIS endonuclease and wherein the expressed DNA binding domain and the expressed type IIS endonuclease are non-covalently linked.
154. The method of any one of paragraphs 149-153, wherein the genomic editing construct comprises a sequence derived from a Cas9 endonuclease.
155. The method of paragraph 154, wherein the sequence derived from a Cas9 endonuclease is the DNA binding domain.
156. The method of paragraph 154 or 155, wherein the sequence derived from a Cas9 endonuclease encodes an inactive Cas9.
157. The method of paragraph 156, wherein the sequence derived from a Cas9 endonuclease comprises an amino acid substitution of an Alanine (A) for a Histidine (H) at position 840 (H840A).
158. The method of any one of paragraphs 154-157, wherein the sequence derived from a Cas9 endonuclease encodes a truncated Cas9.
159. The method of paragraph 158, wherein the sequence derived from a Cas9 endonuclease comprises an amino acid substitution of an Alanine (A) for an Asparagine (N) at position 580 (N580A).
160. The method of any one of paragraphs 154-159, wherein the sequence derived from a Cas9 endonuclease comprises an amino acid substitution of an Alanine (A) for an Aspartic Acid (D) at position 10 (D1OA).
161. The method of any one of paragraphs 149-153, wherein the genomic editing construct comprises a sequence derived from a transcription activator-like effector nuclease (TALEN).
162. The method of paragraph 161, wherein the sequence derived from a TALEN is the DNA binding domain.
163. The method of paragraph 149, wherein the genomic editing construct comprises a TALEN.
164. The method of any one of paragraphs 149-153, wherein the genomic editing construct comprises a sequence derived from a zinc-finger nuclease (ZFN).
165. The method of paragraph 164, wherein the sequence derived from a ZFN is the DNA binding domain.
166. The method of paragraph 149, wherein the genomic editing construct comprises a zinc finger nuclease (ZFN).
167. The method of any one of paragraphs 149-153, further comprising introducing into a primary human T cell a composition comprising a sequence encoding a therapeutic protein.
168. The method of paragraph 167, wherein the therapeutic protein is a secreted or a secretable protein.
169. The method of paragraph 168, wherein the therapeutic protein is an intracellular protein.
170. The method of paragraph 168, wherein the therapeutic protein is a cytosolic protein.
171. The method of paragraph 168, wherein the therapeutic protein is a membrane-bound protein.
172. The method of paragraph 168, wherein the therapeutic protein is a transmembrane protein.
173. The method of any one of paragraphs 1-172, wherein the cell-surface markers of the modified TscM comprise CD62L and CD45RA.
174. The method of any one of paragraphs 1-172, wherein the cell-surface markers of the modified TscMcomprise one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2R.
175. The method of any one of paragraphs 1-172, wherein the cell-surface markers of the modified TscMcomprise one or more of CD45RA, CD95, IL-2R, CR7, and CD62L.
176. The method of any one of paragraphs 1-172, wherein the cell-surface markers of the modified TcM comprise one or more of CD45RO, CD95, IL-2R, CCR7 and CD62L.
177. The method of any one of paragraphs 96-176, wherein the plurality of expanded modified T-cells comprises a naive T-cell (modified TN) and the cell-surface markers of the CAR-TN comprise one or more of CD45RA, CCR7 and CD62L.
178. The method of any one of paragraphs 96-176, wherein the plurality of expanded modified T-cells comprises a central memory T-cell (modified TcM) and the cell-surface markers of the CAR-TcM comprise one or more of CD45RO, CD95, IL-2R, CCR7, and CD62L.
179. The method of any one of paragraphs 96-176, wherein the plurality of expanded modified T-cells comprises an effector memory T-cell (modified TEM) and the cell-surface markers of the CAR-TEM comprise one or more of CD45RO, CD95, and IL-2R.
180. The method of any one of paragraphs 96-176, wherein the plurality of expanded modified T-cells comprises an effector T-cell (modified TEFF) and the cell-surface markers of the CAR TEFF comprise one or more of CD45RA, CD95, and IL-2R.
181. The method of any one of paragraphs 1-39 or 116-180, wherein the transposon is a plasmid DNA transposon with a sequence encoding the antigen receptor or the therapeutic protein flanked by two cis-regulatory insulator elements.
182. The method of paragraph 181, wherein the introducing further comprises a composition comprising an mRNA sequence encoding a transposase.
183. The method of paragraph 181 or 182, wherein the transposon is a piggyBac transposon.
184. The method of any one of paragraphs 181-183, wherein the transposase is a piggyBac transposase.
185. The method of paragraph 184, wherein the piggyBac transposase comprises an amino acid sequence comprising SEQ ID NO: 4.
186. The method of paragraph 184 or 185, wherein the piggyBac transposase is a hyperactive variant and wherein the hyperactive variant comprises an amino acid substitution at one or more of positions 30, 165, 282 and 538 of SEQ ID NO: 4.
187. The method of paragraph 186, wherein the amino acid substitution at position 30 of SEQ ID NO: 4 is a substitution of a valine (V) for an isoleucine (I)(130V).
188. The method of paragraph 186, wherein the amino acid substitution at position 165 of SEQ ID NO: 4 is a substitution of a serine (S) for a glycine (G) (G165S).
189. The method of paragraph 186, wherein the amino acid substitution at position 282 of SEQ ID NO: 4 is a substitution of a valine (V) for a methionine (M) (M282V).
190. The method of paragraph 186, wherein the amino acid substitution at position 538 of SEQ ID NO: 4 is a substitution of a lysine (K) for an asparagine (N) (N538K).
191. The method of any one of paragraphs 183-190, wherein the transposase is a Super piggyBac (SPB) transposase.
192. The method of paragraph 191, wherein the Super piggyBac (SPB) transposase comprises an amino acid sequence comprising SEQ ID NO: 5.
193. The method of any one of paragraphs 1-39 or 116-180, wherein the transposon is a Sleeping Beauty transposon.
194. The method of paragraph 193, wherein the transposase is a Sleeping Beauty transposase or a hyperactive Sleeping Beauty transposase (SB100X).
195. The method of any one of paragraphs 1-39 or 116-180, wherein the transposon is a Helraiser transposon.
196. The method of paragraph 195, wherein the transposase is a Helitron transposase.
197. The method of any one of paragraphs 1-39 or 116-180, wherein the transposon is a Tol2 transposon.
198. The method of paragraph 197, wherein the transposase is a Tol2 transposase.
199. The method of any one of paragraphs 1-39 or 116-198, wherein the sequence encoding the transposase is an mRNA sequence.
200. The method of any one of paragraphs 1-39 or 116-180, wherein the transposon is derived or recombined from any species.
201. The method of any one of paragraphs 1-39 or 116-180, wherein the transposon is synthetic.
202. The method of any one of paragraphs 1-39 or 116-180, wherein the transposon further comprises a selection gene.
203. The method of paragraph 202, wherein the T-cell expansion composition further comprises a selection agent.
204. The method of any one of paragraphs 1-203, wherein the antigen receptor is a T-cell receptor.
205. The method of paragraph 204, wherein the T-cell receptor is naturally-occurring.
206. The method of paragraph 204, wherein the T-cell receptor is not naturally-occurring.
207. The method of paragraph 206, wherein the T-cell receptor comprises one or more mutation(s) compared to a wild-type T-cell receptor.
208. The method of paragraph 206 or 207, wherein the T-cell receptor is a recombinant T-cell receptor.
209. The method of any one of paragraphs 1-203, wherein the antigen receptor is a Chimeric Antigen Receptor (CAR).
210. The method of paragraph 209, wherein the CAR comprises one or more Centyrin sequence(s).
211. The method of paragraph 210, wherein the CAR is a CARTyrin.
212. The method of paragraph 209, wherein the CAR comprises one or more VHH sequence(s).
213. The method of paragraph 212, wherein the CAR is a VCAR.
214. The method of any one of paragraphs 1-39 and 116-213, wherein the introducing step comprises an electroporation or a nucleofection.
215. The method of any one of paragraphs 1-39 and 116-213, wherein the introducing step comprises a nucleofection and wherein the nucleofection comprises the steps of: (a) contacting a transposon composition, a transposase composition, and a composition comprising a plurality of primary human T cells in a cuvette; (b) applying one or more electrical pulses to the cuvette, and (c) incubating the composition comprising the plurality of primary human T cells in a composition comprising a T-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement at 37C.
216. The method of paragraph 215, wherein the transposon is a first transposon or a second transposon.
217. The method of paragraph 215 or 216, wherein the transposase composition is a first transposase composition or a second transposase composition.
218. The method of any one of paragraphs 214-217, wherein the transposon composition is a 0.5 pg/tl solution comprising nuclease free water and wherein the cuvette comprises 2 pl of the transposon composition to yield 1 g of transposon.
219. The method of paragraph 218, wherein the transposon composition comprises a piggyBac transposon.
220. The method of paragraph 219, wherein the transposon composition comprises a Sleeping Beauty transposon.
221. The method of paragraph 219 or 220, wherein the transposase composition comprises 5 pg of transposase.
222. The method of paragraph 221, wherein the transposase composition comprises a Super piggyBac (SPB) transposase.
223. The method of paragraph 221, wherein the transposase composition comprises a hyperactive Sleeping Beauty (SB1OOX) transposase.
224. The method of paragraph 219, wherein the transposon comprises a Helraiser transposon.
225. The method of paragraph 224, wherein the transposase composition comprises a Helitron transposase.
226. The method of paragraph 219, wherein the transposon comprises a Tol2 transposon.
227. The method of paragraph 226, wherein the transposase composition comprises a Tol2 transposase.
228. The method of any one of paragraphs 215-227, wherein the composition comprising primary human T cells comprises a buffer that maintains or enhances a level of cell viability and/or a stem-like phenotype of the primary human T cells.
229. The method of paragraph 228, wherein the buffer maintains or enhances a level of cell viability and/or a stem-like phenotype of the primary human T cells prior to the nucleofection.
230. The method of paragraph 228, wherein the buffer maintains or enhances a level of cell viability and/or a stem-like phenotype of the primary human T cells during the nucleofection.
231. The method of paragraph 228, wherein the buffer maintains or enhances a level of cell viability and/or a stem-like phenotype of the primary human T cells following the nucleofection.
232. The method of any one of paragraphs 228-231, wherein the buffer comprises a P3 primary cell solution.
233. The method of any one of paragraphs 228-231, wherein the buffer comprises one or more of KCl, MgCl2, ClNa, Glucose and Ca(N03)2 in any absolute or relative abundance or concentration.
234. The method of paragraph 228, wherein the buffer further comprises a supplement selected from the group consisting of HEPES, Tris/HCl, and a phosphate buffer.
235. The method of paragraph 228 or 229, wherein the buffer comprises 5 mM KCl, 15 mM MgCl2, 90 mM ClNa, 10 mM Glucose and 0.4 mM Ca(N03)2.
236. The method of paragraph 235, wherein the buffer further comprises a supplement comprising 20 mM HEPES and 75 mM Tris/HCl.
237. The method of paragraph 236, wherein the buffer further comprises a supplement comprising 40 mM Na2HPO 4/NaH2PO 4 at pH 7.2.
238. The method of paragraph 215 or 228-237, wherein the composition comprising primary human T cells is depleted of cells expressing CD14, CD56, and/or CD19.
239. The method of any one of paragraphs 215-238, wherein the composition comprising primary human T cells comprises 100 1 of the buffer and between 5x10 6 and 25x106 cells.
240. The method of any one of paragraphs 215-239, wherein the method is performed in one or more cuvette(s) simultaneously.
241. The method of any one of paragraphs 215-240, wherein the incubating step comprises incubating the composition comprising the plurality of primary human T cells in a pre-warmed T-cell expansion composition.
242. The method of any one of paragraphs 215-241, wherein the incubation step has a period of 2 days.
243. The method of any one of paragraphs 40-242, wherein the activation supplement comprises one or more cytokine(s).
244. The method of paragraph 243, wherein the one or more cytokine(s) comprise IL-2.
245. The method of any one of paragraphs 96-244, wherein the expansion supplement comprises one or more cytokine(s).
246. The method of paragraph 245, wherein the one or more cytokine(s) comprise IL-2.
247. The method of any one of paragraphs 1-246, wherein the method further comprises introducing into a modified TscM cell or a modified TcM cell a composition comprising a genomic editing construct.
248. The method of paragraph 247, wherein the genomic editing construct comprises a guide RNA and a clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9) DNA endonuclease.
249. The method of paragraph 248, wherein the genomic editing construct comprises a DNA binding domain and a type IIS endonuclease.
250. The method of paragraph 249, wherein the genomic editing construct encodes a fusion protein.
251. The method of paragraph 249, wherein the genomic editing construct encodes the DNA binding domain and the type IIS endonuclease and wherein the expressed DNA binding domain and the expressed type IIS endonuclease are non-covalently linked.
252. The method of any one of paragraphs 247-251, wherein the genomic editing construct comprises a sequence derived from a Cas9 endonuclease.
253. The method of paragraph 252, wherein the sequence derived from a Cas9 endonuclease is the DNA binding domain.
254. The method of paragraph 253, wherein the sequence derived from a Cas9 endonuclease comprises an amino acid substitution of an Alanine (A) for a Histidine (H) at position 840 (H840A).
255. The method of any one of paragraphs 252-254, wherein the sequence derived from a Cas9 endonuclease encodes a truncated Cas9.
256. The method of paragraph 255, wherein the sequence derived from a Cas9 endonuclease comprises an amino acid substitution of an Alanine (A) for an Asparagine (N) at position 580 (N580A).
257. The method of any one of paragraphs 252-256, wherein the sequence derived from a Cas9 endonuclease comprises an amino acid substitution of an Alanine (A) for an Aspartic Acid (D) at position 10 (D1OA).
258. The method of any one of paragraphs 247-251, wherein the genomic editing construct comprises a sequence derived from a transcription activator-like effector nuclease (TALEN).
259. The method of paragraph 258, wherein the sequence derived from a TALEN is the DNA binding domain.
260. The method of paragraph 247, wherein the genomic editing construct comprises a TALEN.
261. The method of any one of paragraphs 247-251, wherein the genomic editing construct comprises a sequence derived from a zinc-finger nuclease (ZFN).
262. The method of paragraph 261, wherein the sequence derived from a ZFN is the DNA binding domain.
263. The method of paragraph 247, wherein the genomic editing construct comprises a zinc finger nuclease (ZFN).
264. The method of any one of paragraphs 1-263, wherein the primary human T cell expresses one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2R.
265. The method of any one of paragraphs 1-263, wherein the primary human T cell is a naive T-cell (TN) and wherein the TN expresses one or more of CD45RA, CCR7 and CD62L.
266. The method of any one of paragraphs 1-263, wherein the primary human T cell is a T memory stem cell (TscM) and wherein the TscMexpresses one or more of CD45RA, CD95, IL 2R, CR7, and CD62L.
267. The method of any one of paragraphs 1-263, wherein the primary human T cell is a central memory T-cell (TcM) and wherein the TcM expresses one or more of CD45RO, CD95, IL-2R, CCR7, and CD62L.
268. The method of any one of paragraphs 1-263, wherein the primary human T cell is an effector memory T-cell (TEM) and wherein the TEMexpresses one or more of CD45RO, CD95, and IL-2R.
269. The method of any one of paragraphs 1-263, wherein the primary human T cell is an effector T-cell (TEFF) and wherein the TEFF expresses one or more of CD45RA, CD95, and IL 2RP.
270. The method of any one of paragraphs 1-269, wherein the primary human T cell expresses CD4 and/or CD8.
271. A composition comprising a modified-Tscmproduced by the method of any one of paragraphs 1-270.
272. A composition comprising a modified-TcM produced by the method of any one of paragraphs 1-270.
273. A use of the composition of paragraph 271 or 272 for the manufacture of a medicament to treat a subject in need thereof.
274. The use of paragraph 273, wherein the modified TscM or modified TcM is autologous.
275. The use of paragraph 274, wherein the modified TscM or modified TcM is allogeneic.
276. The use of any one of paragraphs 273-275, wherein the antigen receptor is a T-cell receptor.
277. The use of paragraph 276, wherein the T-cell receptor is naturally-occurring.
278. The use of paragraph 276, wherein the T-cell receptor is not naturally-occurring.
279. The use of paragraph 278, wherein the T-cell receptor comprises one or more mutation(s) compared to a wild-type T-cell receptor.
280. The use of paragraph 278 or 279, wherein the T-cell receptor is a recombinant T-cell receptor.
281. The use of any one of paragraphs 273-275, wherein the antigen receptor is a Chimeric Antigen Receptor (CAR).
282. The use of paragraph 281, wherein the CAR comprises one or more Centyrin sequence(s).
283. The use of paragraph 282, wherein the CAR is a CARTyrin.
284. The method of paragraph 283, wherein the CAR comprises one or more VHH sequence(s).
285. The method of paragraph 284, wherein the CAR is a VCAR.
286. A method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the composition of paragraph 271 or 272.
287. The method of paragraph 286, wherein the modified TscM or modified TcM is autologous.
288. The method of paragraph 286, wherein the modified TscM or modified TcM is allogeneic.
289. The method of any one of paragraphs 286-288, wherein the antigen receptor is a T-cell receptor.
290. The method of paragraph 289, wherein the T-cell receptor is naturally-occurring.
291. The method of paragraph 289, wherein the T-cell receptor is not naturally-occurring.
292. The method of paragraph 291, wherein the T-cell receptor comprises one or more mutation(s) compared to a wild-type T-cell receptor.
293. The method of paragraph 291 or 292, wherein the T-cell receptor is a recombinant T-cell receptor.
294. The method of any one of paragraphs 286-288, wherein the antigen receptor is a Chimeric Antigen Receptor (CAR).
295. The method of paragraph 294, wherein the CAR comprises one or more Centyrin sequence(s).
296. The method of paragraph 295, wherein the CAR is a CARTyrin.
297. The method of paragraph 294, wherein the CAR comprises one or more VHH sequence(s).
298. The method of paragraph 297, wherein the CAR is a VCAR.
299. The method of paragraph any one of paragraphs 286-298, wherein the disease or disorder is cancer and the antigen receptor specifically targets a cancer antigen.
300. The method of paragraph any one of paragraphs 286-298, wherein the disease or disorder is an infectious disease or disorder and the antigen receptor specifically targets a viral, bacterial, yeast or microbial antigen.
301. The method of paragraph any one of paragraphs 286-298, wherein the disease or disorder is a disease or disorder characterized by a lack of an activity or low abundance of a secretory protein or wherein the disease or disorder is a disease or disorder is treated by increasing an activity or an abundance of a secretory protein.
302. The method of paragraph 301, wherein the secretory protein comprises a coagulation factor VIII or coagulation factor IX protein.
303. The method of paragraph 301 or 302, wherein the abundance of the secretory protein is determined at a local site.
304. The method of paragraph 303, wherein the local site is accessible by a modified TscM cell or a modified TcM cell.
[0339] Definitions of specific embodiments of the invention as claimed herein follow.
[0340] According to a first embodiment of the invention, there is provided a fusion protein comprising the amino acid sequence of SEQ ID NO: 40.
[0341] According to a second embodiment of the invention, there is provided a method of producing a modified cell comprising introducing into the cell the fusion protein of the first embodiment.
[0342] According to a third embodiment of the invention, there is provided a modified T cell comprising a fusion protein of the first embodiment.
[0343] According to a fourth embodiment of the invention, there is provided a modified T cell produced by the method of the second embodiment.
[0344] According to a fifth embodiment of the invention, there is provided a composition comprising a population of modified cells comprising the fusion protein of thefirst embodiment.
[0345] According to an sixth embodiment of the invention, there is provided a composition comprising a population of modified cells produced by the method of the second embodiment.
[0346] In the present specification and claims, the word 'comprising' and its derivatives including 'comprises' and 'comprise' include each of the stated integers but does not exclude the inclusion of one or more further integers.
[0347] The reference to any prior art in this specification is not, and should not be taken as an acknowledgement or any form of suggestion that the prior art forms part of the common general knowledge.
POTH‐012_001WO_SeqList.txt 01 Sep 2020
SEQUENCE LISTING
<110> Poseida Therapeutics OSTERTAG, Eric SHEDLOCK, Devon <120> MODIFIED STEM CELL MEMORY T CELLS, METHODS OF MAKING AND METHODS OF USING SAME 2020227020
<130> POTH‐012/01WO
<150> 62/402,707 <151> 2016‐09‐30
<150> 62/553,058 <151> 2017‐08‐31
<150> 62/556,309 <151> 2017‐09‐08
<150> 62/502,508 <151> 2017‐05‐05
<160> 42
<170> PatentIn version 3.5
<210> 1 <211> 89 <212> PRT <213> Artificial Sequence
<220> <223> FN3 domain consensus sequence
<400> 1
Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp Ser 1 5 10 15
Leu Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe Leu 20 25 30
Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu Ala Ile Asn Leu Thr 35 40 45
Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro Gly 50 55 60
Page 1
POTH‐012_001WO_SeqList.txt 01 Sep 2020
Thr Glu Tyr Thr Val Ser Ile Tyr Gly Val Lys Gly Gly His Arg Ser 65 70 75 80
Asn Pro Leu Ser Ala Glu Phe Thr Thr 85 2020227020
<210> 2 <211> 90 <212> PRT <213> Artificial Sequence
<220> <223> FN3 consensus sequence
<400> 2
Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp 1 5 10 15
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> 3 <211> 270 <212> DNA <213> Artificial Sequence
<220> <223> FN3 consensus sequence
<400> 3 Page 2
POTH‐012_001WO_SeqList.txt 01 Sep 2020
atgctgcctg caccaaagaa cctggtggtg tctcatgtga cagaggatag tgccagactg 60
tcatggactg ctcccgacgc agccttcgat agttttatca tcgtgtaccg ggagaacatc 120
gaaaccggcg aggccattgt cctgacagtg ccagggtccg aacgctctta tgacctgaca 180
gatctgaagc ccggaactga gtactatgtg cagatcgccg gcgtcaaagg aggcaatatc 240
agcttccctc tgtccgcaat cttcaccaca 270 2020227020
<210> 4 <211> 594 <212> PRT <213> Trichoplusia ni
<400> 4
Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln 1 5 10 15
Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Ile 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 Page 3
POTH‐012_001WO_SeqList.txt 01 Sep 2020
Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg 145 150 155 160
Glu Ser Met Thr Gly Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile 165 170 175 2020227020
Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn 180 185 190
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 Met Tyr Ile Pro Asn Lys Pro 275 280 285
Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Tyr 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 Page 4
POTH‐012_001WO_SeqList.txt 01 Sep 2020
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 2020227020
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
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 Asn 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 Page 5
POTH‐012_001WO_SeqList.txt 01 Sep 2020
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 2020227020
Cys Phe
<210> 5 <211> 594 <212> PRT <213> Artificial Sequence
<220> <223> Super Piggybac Transposase
<400> 5
Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln 1 5 10 15
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
Page 6
POTH‐012_001WO_SeqList.txt 01 Sep 2020
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 2020227020
145 150 155 160
Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile 165 170 175
Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn 180 185 190
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
Page 7
POTH‐012_001WO_SeqList.txt 01 Sep 2020
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 2020227020
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
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
Page 8
POTH‐012_001WO_SeqList.txt 01 Sep 2020
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 2020227020
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> 6 <211> 340 <212> PRT <213> Artificial Sequence
<220> <223> Sleeping Beauty Transposase
<400> 6
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 65 70 75 80
Arg Thr Thr Ala Lys Asp Leu Val Lys Met Leu Glu Glu Thr Gly Thr 85 90 95 Page 9
POTH‐012_001WO_SeqList.txt 01 Sep 2020
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 2020227020
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 Page 10
POTH‐012_001WO_SeqList.txt 01 Sep 2020
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 2020227020
Ala Thr Lys Tyr 340
<210> 7 <211> 340 <212> PRT <213> Artificial Sequence
<220> <223> hyperactive sleeping beauty transposase
<400> 7
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
Page 11
POTH‐012_001WO_SeqList.txt 01 Sep 2020
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 2020227020
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
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
Page 12
POTH‐012_001WO_SeqList.txt 01 Sep 2020
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> 8 2020227020
<211> 21 <212> PRT <213> Homo sapiens
<400> 8
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15
His Ala Ala Arg Pro 20
<210> 9 <211> 63 <212> DNA <213> Artificial Sequence
<220> <223> nucleotide sequence for human CD8alpha signal peptide
<400> 9 atggcactgc cagtcaccgc cctgctgctg cctctggctc tgctgctgca cgcagctaga 60
cca 63
<210> 10 <211> 24 <212> PRT <213> Homo sapiens
<400> 10
Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 1 5 10 15
Ser Leu Val Ile Thr Leu Tyr Cys 20
Page 13
POTH‐012_001WO_SeqList.txt 01 Sep 2020
<210> 11 <211> 72 <212> DNA <213> Artificial Sequence
<220> <223> nucleotide sequence coding for human CD8alpha transmembrane domain 2020227020
<400> 11 atctacattt gggcaccact ggccgggacc tgtggagtgc tgctgctgag cctggtcatc 60
acactgtact gc 72
<210> 12 <211> 112 <212> PRT <213> Homo sapiens
<400> 12
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly 1 5 10 15
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110
<210> 13 <211> 336 Page 14
POTH‐012_001WO_SeqList.txt 01 Sep 2020
<212> DNA <213> Artificial Sequenec
<220> <223> nucleotide sequence encoding the CD28 costimulatory domain
<400> 13 cgcgtgaagt ttagtcgatc agcagatgcc ccagcttaca aacagggaca gaaccagctg 60 2020227020
tataacgagc tgaatctggg ccgccgagag gaatatgacg tgctggataa gcggagagga 120
cgcgaccccg aaatgggagg caagcccagg cgcaaaaacc ctcaggaagg cctgtataac 180
gagctgcaga aggacaaaat ggcagaagcc tattctgaga tcggcatgaa gggggagcga 240
cggagaggca aagggcacga tgggctgtac cagggactga gcaccgccac aaaggacacc 300
tatgatgctc tgcatatgca ggcactgcct ccaagg 336
<210> 14 <211> 42 <212> PRT <213> Homo sapiens
<400> 14
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 1 5 10 15
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35 40
<210> 15 <211> 126 <212> DNA <213> Artificial Sequence
<220> <223> nucleotide sequence encoding the 4‐1BB costimulatory domain
<400> 15 aagagaggca ggaagaaact gctgtatatt ttcaaacagc ccttcatgcg ccccgtgcag 60
actacccagg aggaagacgg gtgctcctgt cgattccctg aggaagagga aggcgggtgt 120
Page 15
POTH‐012_001WO_SeqList.txt 01 Sep 2020
gagctg 126
<210> 16 <211> 45 <212> PRT <213> Homo sapiens
<400> 16 2020227020
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 1 5 10 15
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 20 25 30
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 35 40 45
<210> 17 <211> 135 <212> DNA <213> Artificial Sequence
<220> <223> nucleotide sequence encoding human CD8alpha hinge
<400> 17 actaccacac cagcacctag accaccaact ccagctccaa ccatcgcgag tcagcccctg 60
agtctgagac ctgaggcctg caggccagct gcaggaggag ctgtgcacac caggggcctg 120
gacttcgcct gcgac 135
<210> 18 <211> 18 <212> PRT <213> Thosea asigna
<400> 18
Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro 1 5 10 15
Gly Pro
Page 16
POTH‐012_001WO_SeqList.txt 01 Sep 2020
<210> 19 <211> 21 <212> PRT <213> Artificial Sequence
<220> <223> GSG‐T2A 2020227020
<400> 19
Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu 1 5 10 15
Glu Asn Pro Gly Pro 20
<210> 20 <211> 63 <212> DNA <213> Artificial Sequence
<220> <223> GSG‐T2A
<400> 20 ggatctggag agggaagggg aagcctgctg acctgtggag acgtggagga aaacccagga 60
cca 63
<210> 21 <211> 20 <212> PRT <213> Equine rhinitis A
<400> 21
Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp Val Glu Ser 1 5 10 15
Asn Pro Gly Pro 20
<210> 22 <211> 23 <212> PRT <213> Artificial Sequence Page 17
POTH‐012_001WO_SeqList.txt 01 Sep 2020
<220> <223> GSG‐E2A peptide
<400> 22
Gly Ser Gly Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp 1 5 10 15 2020227020
Val Glu Ser Asn Pro Gly Pro 20
<210> 23 <211> 22 <212> PRT <213> Foot and nout disease virus type O
<400> 23
Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val 1 5 10 15
Glu Ser Asn Pro Gly Pro 20
<210> 24 <211> 25 <212> PRT <213> Artificial Sequence
<220> <223> GSG‐F2A peptide
<400> 24
Gly Ser Gly Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala 1 5 10 15
Gly Asp Val Glu Ser Asn Pro Gly Pro 20 25
<210> 25 <211> 19 <212> PRT <213> Porcine teschovirus‐1
Page 18
POTH‐012_001WO_SeqList.txt 01 Sep 2020
<400> 25
Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn 1 5 10 15
Pro Gly Pro 2020227020
<210> 26 <211> 22 <212> PRT <213> Artificial Sequence
<220> <223> GSG‐P2A peptide
<400> 26
Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val 1 5 10 15
Glu Glu Asn Pro Gly Pro 20
<210> 27 <211> 5296 <212> DNA <213> Artificial Sequence
<220> <223> Helraiser transposon
<400> 27 tcctatataa taaaagagaa acatgcaaat tgaccatccc tccgctacgc tcaagccacg 60
cccaccagcc aatcagaagt gactatgcaa attaacccaa caaagatggc agttaaattt 120
gcatacgcag gtgtcaagcg ccccaggagg caacggcggc cgcgggctcc caggaccttc 180
gctggccccg ggaggcgagg ccggccgcgc ctagccacac ccgcgggctc ccgggacctt 240
cgccagcaga gagcagagcg ggagagcggg cggagagcgg gaggtttgga ggacttggca 300
gagcaggagg ccgctggaca tagagcagag cgagagagag ggtggcttgg agggcgtggc 360
tccctctgtc accccagctt cctcatcaca gctgtggaaa ctgacagcag ggaggaggaa 420
gtcccacccc cacagaatca gccagaatca gccgttggtc agacagctct cagcggcctg 480 Page 19
POTH‐012_001WO_SeqList.txt 01 Sep 2020
acagccagga ctctcattca cctgcatctc agaccgtgac agtagagagg tgggactatg 540
tctaaagaac aactgttgat acaacgtagc tctgcagccg aaagatgccg gcgttatcga 600
cagaaaatgt ctgcagagca acgtgcgtct gatcttgaaa gaaggcggcg cctgcaacag 660
aatgtatctg aagagcagct actggaaaaa cgtcgctctg aagccgaaaa acagcggcgt 720 2020227020
catcgacaga aaatgtctaa agaccaacgt gcctttgaag ttgaaagaag gcggtggcga 780
cgacagaata tgtctagaga acagtcatca acaagtacta ccaataccgg taggaactgc 840
cttctcagca aaaatggagt acatgaggat gcaattctcg aacatagttg tggtggaatg 900
actgttcgat gtgaattttg cctatcacta aatttctctg atgaaaaacc atccgatggg 960
aaatttactc gatgttgtag caaagggaaa gtctgtccaa atgatataca ttttccagat 1020
tacccggcat atttaaaaag attaatgaca aacgaagatt ctgacagtaa aaatttcatg 1080
gaaaatattc gttccataaa tagttctttt gcttttgctt ccatgggtgc aaatattgca 1140
tcgccatcag gatatgggcc atactgtttt agaatacacg gacaagttta tcaccgtact 1200
ggaactttac atccttcgga tggtgtttct cggaagtttg ctcaactcta tattttggat 1260
acagccgaag ctacaagtaa aagattagca atgccagaaa accagggctg ctcagaaaga 1320
ctcatgatca acatcaacaa cctcatgcat gaaataaatg aattaacaaa atcgtacaag 1380
atgctacatg aggtagaaaa ggaagcccaa tctgaagcag cagcaaaagg tattgctccc 1440
acagaagtaa caatggcgat taaatacgat cgtaacagtg acccaggtag atataattct 1500
ccccgtgtaa ccgaggttgc tgtcatattc agaaacgaag atggagaacc tccttttgaa 1560
agggacttgc tcattcattg taaaccagat cccaataatc caaatgccac taaaatgaaa 1620
caaatcagta tcctgtttcc tacattagat gcaatgacat atcctattct ttttccacat 1680
ggtgaaaaag gctggggaac agatattgca ttaagactca gagacaacag tgtaatcgac 1740
aataatacta gacaaaatgt aaggacacga gtcacacaaa tgcagtatta tggatttcat 1800
ctctctgtgc gggacacgtt caatcctatt ttaaatgcag gaaaattaac tcaacagttt 1860
attgtggatt catattcaaa aatggaggcc aatcggataa atttcatcaa agcaaaccaa 1920
tctaagttga gagttgaaaa atatagtggt ttgatggatt atctcaaatc tagatctgaa 1980
aatgacaatg tgccgattgg taaaatgata atacttccat catcttttga gggtagtccc 2040 Page 20
POTH‐012_001WO_SeqList.txt 01 Sep 2020
agaaatatgc agcagcgata tcaggatgct atggcaattg taacgaagta tggcaagccc 2100
gatttattca taaccatgac atgcaacccc aaatgggcag atattacaaa caatttacaa 2160
cgctggcaaa aagttgaaaa cagacctgac ttggtagcca gagtttttaa tattaagctg 2220
aatgctcttt taaatgatat atgtaaattc catttatttg gcaaagtaat agctaaaatt 2280 2020227020
catgtcattg aatttcagaa acgcggactg cctcacgctc acatattatt gatattagat 2340
agtgagtcca aattacgttc agaagatgac attgaccgta tagttaaggc agaaattcca 2400
gatgaagacc agtgtcctcg actttttcaa attgtaaaat caaatatggt acatggacca 2460
tgtggaatac aaaatccaaa tagtccatgt atggaaaatg gaaaatgttc aaagggatat 2520
ccaaaagaat ttcaaaatgc gaccattgga aatattgatg gatatcccaa atacaaacga 2580
agatctggta gcaccatgtc tattggaaat aaagttgtcg ataacacttg gattgtccct 2640
tataacccgt atttgtgcct taaatataac tgtcatataa atgttgaagt ctgtgcatca 2700
attaaaagtg tcaaatattt atttaaatac atctataaag ggcacgattg tgcaaatatt 2760
caaatttctg aaaaaaatat tatcaatcat gacgaagtac aggacttcat tgactccagg 2820
tatgtgagcg ctcctgaggc tgtttggaga ctttttgcaa tgcgaatgca tgaccaatct 2880
catgcaatca caagattagc tattcatttg ccaaatgatc agaatttgta ttttcatacc 2940
gatgattttg ctgaagtttt agatagggct aaaaggcata actcgacttt gatggcttgg 3000
ttcttattga atagagaaga ttctgatgca cgtaattatt attattggga gattccacag 3060
cattatgtgt ttaataattc tttgtggaca aaacgccgaa agggtgggaa taaagtatta 3120
ggtagactgt tcactgtgag ctttagagaa ccagaacgat attaccttag acttttgctt 3180
ctgcatgtaa aaggtgcgat aagttttgag gatctgcgaa ctgtaggagg tgtaacttat 3240
gatacatttc atgaagctgc taaacaccga ggattattac ttgatgacac tatctggaaa 3300
gatacgattg acgatgcaat catccttaat atgcccaaac aactacggca actttttgca 3360
tatatatgtg tgtttggatg tccttctgct gcagacaaat tatgggatga gaataaatct 3420
cattttattg aagatttctg ttggaaatta caccgaagag aaggtgcctg tgtgaactgt 3480
gaaatgcatg cccttaacga aattcaggag gtattcacat tgcatggaat gaaatgttca 3540
catttcaaac ttccggacta tcctttatta atgaatgcaa atacatgtga tcaattgtac 3600 Page 21
POTH‐012_001WO_SeqList.txt 01 Sep 2020
gagcaacaac aggcagaggt tttgataaat tctctgaatg atgaacagtt ggcagccttt 3660
cagactataa cttcagccat cgaagatcaa actgtacacc ccaaatgctt tttcttggat 3720
ggtccaggtg gtagtggaaa aacatatctg tataaagttt taacacatta tattagaggt 3780
cgtggtggta ctgttttacc cacagcatct acaggaattg ctgcaaattt acttcttggt 3840 2020227020
ggaagaacct ttcattccca atataaatta ccaattccat taaatgaaac ttcaatttct 3900
agactcgata taaagagtga agttgctaaa accattaaaa aggcccaact tctcattatt 3960
gatgaatgca ccatggcatc cagtcatgct ataaacgcca tagatagatt actaagagaa 4020
attatgaatt tgaatgttgc atttggtggg aaagttctcc ttctcggagg ggattttcga 4080
caatgtctca gtattgtacc acatgctatg cgatcggcca tagtacaaac gagtttaaag 4140
tactgtaatg tttggggatg tttcagaaag ttgtctctta aaacaaatat gagatcagag 4200
gattctgctt atagtgaatg gttagtaaaa cttggagatg gcaaacttga tagcagtttt 4260
catttaggaa tggatattat tgaaatcccc catgaaatga tttgtaacgg atctattatt 4320
gaagctacct ttggaaatag tatatctata gataatatta aaaatatatc taaacgtgca 4380
attctttgtc caaaaaatga gcatgttcaa aaattaaatg aagaaatttt ggatatactt 4440
gatggagatt ttcacacata tttgagtgat gattccattg attcaacaga tgatgctgaa 4500
aaggaaaatt ttcccatcga atttcttaat agtattactc cttcgggaat gccgtgtcat 4560
aaattaaaat tgaaagtggg tgcaatcatc atgctattga gaaatcttaa tagtaaatgg 4620
ggtctttgta atggtactag atttattatc aaaagattac gacctaacat tatcgaagct 4680
gaagtattaa caggatctgc agagggagag gttgttctga ttccaagaat tgatttgtcc 4740
ccatctgaca ctggcctccc atttaaatta attcgaagac agtttcccgt gatgccagca 4800
tttgcgatga ctattaataa atcacaagga caaactctag acagagtagg aatattccta 4860
cctgaacccg ttttcgcaca tggtcagtta tatgttgctt tctctcgagt tcgaagagca 4920
tgtgacgtta aagttaaagt tgtaaatact tcatcacaag ggaaattagt caagcactct 4980
gaaagtgttt ttactcttaa tgtggtatac agggagatat tagaataagt ttaatcactt 5040
tatcagtcat tgtttgcatc aatgttgttt ttatatcatg tttttgttgt ttttatatca 5100
tgtctttgtt gttgttatat catgttgtta ttgtttattt attaataaat ttatgtatta 5160 Page 22
POTH‐012_001WO_SeqList.txt 01 Sep 2020
ttttcatata cattttactc atttcctttc atctctcaca cttctattat agagaaaggg 5220
caaatagcaa tattaaaata tttcctctaa ttaattccct ttcaatgtgc acgaatttcg 5280
tgcaccgggc cactag 5296
<210> 28 2020227020
<211> 1496 <212> PRT <213> Artificial Sequence
<220> <223> Helitron transposase
<400> 28
Met Ser Lys Glu Gln Leu Leu Ile Gln Arg Ser Ser Ala Ala Glu Arg 1 5 10 15
Cys Arg Arg Tyr Arg Gln Lys Met Ser Ala Glu Gln Arg Ala Ser Asp 20 25 30
Leu Glu Arg Arg Arg Arg Leu Gln Gln Asn Val Ser Glu Glu Gln Leu 35 40 45
Leu Glu Lys Arg Arg Ser Glu Ala Glu Lys Gln Arg Arg His Arg Gln 50 55 60
Lys Met Ser Lys Asp Gln Arg Ala Phe Glu Val Glu Arg Arg Arg Trp 65 70 75 80
Arg Arg Gln Asn Met Ser Arg Glu Gln Ser Ser Thr Ser Thr Thr Asn 85 90 95
Thr Gly Arg Asn Cys Leu Leu Ser Lys Asn Gly Val His Glu Asp Ala 100 105 110
Ile Leu Glu His Ser Cys Gly Gly Met Thr Val Arg Cys Glu Phe Cys 115 120 125
Leu Ser Leu Asn Phe Ser Asp Glu Lys Pro Ser Asp Gly Lys Phe Thr 130 135 140 Page 23
POTH‐012_001WO_SeqList.txt 01 Sep 2020
Arg Cys Cys Ser Lys Gly Lys Val Cys Pro Asn Asp Ile His Phe Pro 145 150 155 160
Asp Tyr Pro Ala Tyr Leu Lys Arg Leu Met Thr Asn Glu Asp Ser Asp 165 170 175 2020227020
Ser Lys Asn Phe Met Glu Asn Ile Arg Ser Ile Asn Ser Ser Phe Ala 180 185 190
Phe Ala Ser Met Gly Ala Asn Ile Ala Ser Pro Ser Gly Tyr Gly Pro 195 200 205
Tyr Cys Phe Arg Ile His Gly Gln Val Tyr His Arg Thr Gly Thr Leu 210 215 220
His Pro Ser Asp Gly Val Ser Arg Lys Phe Ala Gln Leu Tyr Ile Leu 225 230 235 240
Asp Thr Ala Glu Ala Thr Ser Lys Arg Leu Ala Met Pro Glu Asn Gln 245 250 255
Gly Cys Ser Glu Arg Leu Met Ile Asn Ile Asn Asn Leu Met His Glu 260 265 270
Ile Asn Glu Leu Thr Lys Ser Tyr Lys Met Leu His Glu Val Glu Lys 275 280 285
Glu Ala Gln Ser Glu Ala Ala Ala Lys Gly Ile Ala Pro Thr Glu Val 290 295 300
Thr Met Ala Ile Lys Tyr Asp Arg Asn Ser Asp Pro Gly Arg Tyr Asn 305 310 315 320
Ser Pro Arg Val Thr Glu Val Ala Val Ile Phe Arg Asn Glu Asp Gly 325 330 335
Glu Pro Pro Phe Glu Arg Asp Leu Leu Ile His Cys Lys Pro Asp Pro 340 345 350 Page 24
POTH‐012_001WO_SeqList.txt 01 Sep 2020
Asn Asn Pro Asn Ala Thr Lys Met Lys Gln Ile Ser Ile Leu Phe Pro 355 360 365
Thr Leu Asp Ala Met Thr Tyr Pro Ile Leu Phe Pro His Gly Glu Lys 370 375 380 2020227020
Gly Trp Gly Thr Asp Ile Ala Leu Arg Leu Arg Asp Asn Ser Val Ile 385 390 395 400
Asp Asn Asn Thr Arg Gln Asn Val Arg Thr Arg Val Thr Gln Met Gln 405 410 415
Tyr Tyr Gly Phe His Leu Ser Val Arg Asp Thr Phe Asn Pro Ile Leu 420 425 430
Asn Ala Gly Lys Leu Thr Gln Gln Phe Ile Val Asp Ser Tyr Ser Lys 435 440 445
Met Glu Ala Asn Arg Ile Asn Phe Ile Lys Ala Asn Gln Ser Lys Leu 450 455 460
Arg Val Glu Lys Tyr Ser Gly Leu Met Asp Tyr Leu Lys Ser Arg Ser 465 470 475 480
Glu Asn Asp Asn Val Pro Ile Gly Lys Met Ile Ile Leu Pro Ser Ser 485 490 495
Phe Glu Gly Ser Pro Arg Asn Met Gln Gln Arg Tyr Gln Asp Ala Met 500 505 510
Ala Ile Val Thr Lys Tyr Gly Lys Pro Asp Leu Phe Ile Thr Met Thr 515 520 525
Cys Asn Pro Lys Trp Ala Asp Ile Thr Asn Asn Leu Gln Arg Trp Gln 530 535 540
Lys Val Glu Asn Arg Pro Asp Leu Val Ala Arg Val Phe Asn Ile Lys 545 550 555 560 Page 25
POTH‐012_001WO_SeqList.txt 01 Sep 2020
Leu Asn Ala Leu Leu Asn Asp Ile Cys Lys Phe His Leu Phe Gly Lys 565 570 575
Val Ile Ala Lys Ile His Val Ile Glu Phe Gln Lys Arg Gly Leu Pro 580 585 590 2020227020
His Ala His Ile Leu Leu Ile Leu Asp Ser Glu Ser Lys Leu Arg Ser 595 600 605
Glu Asp Asp Ile Asp Arg Ile Val Lys Ala Glu Ile Pro Asp Glu Asp 610 615 620
Gln Cys Pro Arg Leu Phe Gln Ile Val Lys Ser Asn Met Val His Gly 625 630 635 640
Pro Cys Gly Ile Gln Asn Pro Asn Ser Pro Cys Met Glu Asn Gly Lys 645 650 655
Cys Ser Lys Gly Tyr Pro Lys Glu Phe Gln Asn Ala Thr Ile Gly Asn 660 665 670
Ile Asp Gly Tyr Pro Lys Tyr Lys Arg Arg Ser Gly Ser Thr Met Ser 675 680 685
Ile Gly Asn Lys Val Val Asp Asn Thr Trp Ile Val Pro Tyr Asn Pro 690 695 700
Tyr Leu Cys Leu Lys Tyr Asn Cys His Ile Asn Val Glu Val Cys Ala 705 710 715 720
Ser Ile Lys Ser Val Lys Tyr Leu Phe Lys Tyr Ile Tyr Lys Gly His 725 730 735
Asp Cys Ala Asn Ile Gln Ile Ser Glu Lys Asn Ile Ile Asn His Asp 740 745 750
Glu Val Gln Asp Phe Ile Asp Ser Arg Tyr Val Ser Ala Pro Glu Ala 755 760 765 Page 26
POTH‐012_001WO_SeqList.txt 01 Sep 2020
Val Trp Arg Leu Phe Ala Met Arg Met His Asp Gln Ser His Ala Ile 770 775 780
Thr Arg Leu Ala Ile His Leu Pro Asn Asp Gln Asn Leu Tyr Phe His 785 790 795 800 2020227020
Thr Asp Asp Phe Ala Glu Val Leu Asp Arg Ala Lys Arg His Asn Ser 805 810 815
Thr Leu Met Ala Trp Phe Leu Leu Asn Arg Glu Asp Ser Asp Ala Arg 820 825 830
Asn Tyr Tyr Tyr Trp Glu Ile Pro Gln His Tyr Val Phe Asn Asn Ser 835 840 845
Leu Trp Thr Lys Arg Arg Lys Gly Gly Asn Lys Val Leu Gly Arg Leu 850 855 860
Phe Thr Val Ser Phe Arg Glu Pro Glu Arg Tyr Tyr Leu Arg Leu Leu 865 870 875 880
Leu Leu His Val Lys Gly Ala Ile Ser Phe Glu Asp Leu Arg Thr Val 885 890 895
Gly Gly Val Thr Tyr Asp Thr Phe His Glu Ala Ala Lys His Arg Gly 900 905 910
Leu Leu Leu Asp Asp Thr Ile Trp Lys Asp Thr Ile Asp Asp Ala Ile 915 920 925
Ile Leu Asn Met Pro Lys Gln Leu Arg Gln Leu Phe Ala Tyr Ile Cys 930 935 940
Val Phe Gly Cys Pro Ser Ala Ala Asp Lys Leu Trp Asp Glu Asn Lys 945 950 955 960
Ser His Phe Ile Glu Asp Phe Cys Trp Lys Leu His Arg Arg Glu Gly 965 970 975 Page 27
POTH‐012_001WO_SeqList.txt 01 Sep 2020
Ala Cys Val Asn Cys Glu Met His Ala Leu Asn Glu Ile Gln Glu Val 980 985 990
Phe Thr Leu His Gly Met Lys Cys Ser His Phe Lys Leu Pro Asp Tyr 995 1000 1005 2020227020
Pro Leu Leu Met Asn Ala Asn Thr Cys Asp Gln Leu Tyr Glu Gln 1010 1015 1020
Gln Gln Ala Glu Val Leu Ile Asn Ser Leu Asn Asp Glu Gln Leu 1025 1030 1035
Ala Ala Phe Gln Thr Ile Thr Ser Ala Ile Glu Asp Gln Thr Val 1040 1045 1050
His Pro Lys Cys Phe Phe Leu Asp Gly Pro Gly Gly Ser Gly Lys 1055 1060 1065
Thr Tyr Leu Tyr Lys Val Leu Thr His Tyr Ile Arg Gly Arg Gly 1070 1075 1080
Gly Thr Val Leu Pro Thr Ala Ser Thr Gly Ile Ala Ala Asn Leu 1085 1090 1095
Leu Leu Gly Gly Arg Thr Phe His Ser Gln Tyr Lys Leu Pro Ile 1100 1105 1110
Pro Leu Asn Glu Thr Ser Ile Ser Arg Leu Asp Ile Lys Ser Glu 1115 1120 1125
Val Ala Lys Thr Ile Lys Lys Ala Gln Leu Leu Ile Ile Asp Glu 1130 1135 1140
Cys Thr Met Ala Ser Ser His Ala Ile Asn Ala Ile Asp Arg Leu 1145 1150 1155
Leu Arg Glu Ile Met Asn Leu Asn Val Ala Phe Gly Gly Lys Val 1160 1165 1170 Page 28
POTH‐012_001WO_SeqList.txt 01 Sep 2020
Leu Leu Leu Gly Gly Asp Phe Arg Gln Cys Leu Ser Ile Val Pro 1175 1180 1185
His Ala Met Arg Ser Ala Ile Val Gln Thr Ser Leu Lys Tyr Cys 1190 1195 1200 2020227020
Asn Val Trp Gly Cys Phe Arg Lys Leu Ser Leu Lys Thr Asn Met 1205 1210 1215
Arg Ser Glu Asp Ser Ala Tyr Ser Glu Trp Leu Val Lys Leu Gly 1220 1225 1230
Asp Gly Lys Leu Asp Ser Ser Phe His Leu Gly Met Asp Ile Ile 1235 1240 1245
Glu Ile Pro His Glu Met Ile Cys Asn Gly Ser Ile Ile Glu Ala 1250 1255 1260
Thr Phe Gly Asn Ser Ile Ser Ile Asp Asn Ile Lys Asn Ile Ser 1265 1270 1275
Lys Arg Ala Ile Leu Cys Pro Lys Asn Glu His Val Gln Lys Leu 1280 1285 1290
Asn Glu Glu Ile Leu Asp Ile Leu Asp Gly Asp Phe His Thr Tyr 1295 1300 1305
Leu Ser Asp Asp Ser Ile Asp Ser Thr Asp Asp Ala Glu Lys Glu 1310 1315 1320
Asn Phe Pro Ile Glu Phe Leu Asn Ser Ile Thr Pro Ser Gly Met 1325 1330 1335
Pro Cys His Lys Leu Lys Leu Lys Val Gly Ala Ile Ile Met Leu 1340 1345 1350
Leu Arg Asn Leu Asn Ser Lys Trp Gly Leu Cys Asn Gly Thr Arg 1355 1360 1365 Page 29
POTH‐012_001WO_SeqList.txt 01 Sep 2020
Phe Ile Ile Lys Arg Leu Arg Pro Asn Ile Ile Glu Ala Glu Val 1370 1375 1380
Leu Thr Gly Ser Ala Glu Gly Glu Val Val Leu Ile Pro Arg Ile 1385 1390 1395 2020227020
Asp Leu Ser Pro Ser Asp Thr Gly Leu Pro Phe Lys Leu Ile Arg 1400 1405 1410
Arg Gln Phe Pro Val Met Pro Ala Phe Ala Met Thr Ile Asn Lys 1415 1420 1425
Ser Gln Gly Gln Thr Leu Asp Arg Val Gly Ile Phe Leu Pro Glu 1430 1435 1440
Pro Val Phe Ala His Gly Gln Leu Tyr Val Ala Phe Ser Arg Val 1445 1450 1455
Arg Arg Ala Cys Asp Val Lys Val Lys Val Val Asn Thr Ser Ser 1460 1465 1470
Gln Gly Lys Leu Val Lys His Ser Glu Ser Val Phe Thr Leu Asn 1475 1480 1485
Val Val Tyr Arg Glu Ile Leu Glu 1490 1495
<210> 29 <211> 30 <212> DNA <213> Artificial Sequence
<220> <223> Helraiser palindromic sequence
<400> 29 gtgcacgaat ttcgtgcacc gggccactag 30
<210> 30 <211> 649 Page 30
POTH‐012_001WO_SeqList.txt 01 Sep 2020
<212> PRT <213> Oryzias latipes
<400> 30
Met Glu Glu Val Cys Asp Ser Ser Ala Ala Ala Ser Ser Thr Val Gln 1 5 10 15 2020227020
Asn Gln Pro Gln Asp Gln Glu His Pro Trp Pro Tyr Leu Arg Glu Phe 20 25 30
Phe Ser Leu Ser Gly Val Asn Lys Asp Ser Phe Lys Met Lys Cys Val 35 40 45
Leu Cys Leu Pro Leu Asn Lys Glu Ile Ser Ala Phe Lys Ser Ser Pro 50 55 60
Ser Asn Leu Arg Lys His Ile Glu Arg Met His Pro Asn Tyr Leu Lys 65 70 75 80
Asn Tyr Ser Lys Leu Thr Ala Gln Lys Arg Lys Ile Gly Thr Ser Thr 85 90 95
His Ala Ser Ser Ser Lys Gln Leu Lys Val Asp Ser Val Phe Pro Val 100 105 110
Lys His Val Ser Pro Val Thr Val Asn Lys Ala Ile Leu Arg Tyr Ile 115 120 125
Ile Gln Gly Leu His Pro Phe Ser Thr Val Asp Leu Pro Ser Phe Lys 130 135 140
Glu Leu Ile Ser Thr Leu Gln Pro Gly Ile Ser Val Ile Thr Arg Pro 145 150 155 160
Thr Leu Arg Ser Lys Ile Ala Glu Ala Ala Leu Ile Met Lys Gln Lys 165 170 175
Val Thr Ala Ala Met Ser Glu Val Glu Trp Ile Ala Thr Thr Thr Asp 180 185 190
Page 31
POTH‐012_001WO_SeqList.txt 01 Sep 2020
Cys Trp Thr Ala Arg Arg Lys Ser Phe Ile Gly Val Thr Ala His Trp 195 200 205
Ile Asn Pro Gly Ser Leu Glu Arg His Ser Ala Ala Leu Ala Cys Lys 210 215 220 2020227020
Arg Leu Met Gly Ser His Thr Phe Glu Val Leu Ala Ser Ala Met Asn 225 230 235 240
Asp Ile His Ser Glu Tyr Glu Ile Arg Asp Lys Val Val Cys Thr Thr 245 250 255
Thr Asp Ser Gly Ser Asn Phe Met Lys Ala Phe Arg Val Phe Gly Val 260 265 270
Glu Asn Asn Asp Ile Glu Thr Glu Ala Arg Arg Cys Glu Ser Asp Asp 275 280 285
Thr Asp Ser Glu Gly Cys Gly Glu Gly Ser Asp Gly Val Glu Phe Gln 290 295 300
Asp Ala Ser Arg Val Leu Asp Gln Asp Asp Gly Phe Glu Phe Gln Leu 305 310 315 320
Pro Lys His Gln Lys Cys Ala Cys His Leu Leu Asn Leu Val Ser Ser 325 330 335
Val Asp Ala Gln Lys Ala Leu Ser Asn Glu His Tyr Lys Lys Leu Tyr 340 345 350
Arg Ser Val Phe Gly Lys Cys Gln Ala Leu Trp Asn Lys Ser Ser Arg 355 360 365
Ser Ala Leu Ala Ala Glu Ala Val Glu Ser Glu Ser Arg Leu Gln Leu 370 375 380
Leu Arg Pro Asn Gln Thr Arg Trp Asn Ser Thr Phe Met Ala Val Asp 385 390 395 400
Page 32
POTH‐012_001WO_SeqList.txt 01 Sep 2020
Arg Ile Leu Gln Ile Cys Lys Glu Ala Gly Glu Gly Ala Leu Arg Asn 405 410 415
Ile Cys Thr Ser Leu Glu Val Pro Met Phe Asn Pro Ala Glu Met Leu 420 425 430 2020227020
Phe Leu Thr Glu Trp Ala Asn Thr Met Arg Pro Val Ala Lys Val Leu 435 440 445
Asp Ile Leu Gln Ala Glu Thr Asn Thr Gln Leu Gly Trp Leu Leu Pro 450 455 460
Ser Val His Gln Leu Ser Leu Lys Leu Gln Arg Leu His His Ser Leu 465 470 475 480
Arg Tyr Cys Asp Pro Leu Val Asp Ala Leu Gln Gln Gly Ile Gln Thr 485 490 495
Arg Phe Lys His Met Phe Glu Asp Pro Glu Ile Ile Ala Ala Ala Ile 500 505 510
Leu Leu Pro Lys Phe Arg Thr Ser Trp Thr Asn Asp Glu Thr Ile Ile 515 520 525
Lys Arg Gly Met Asp Tyr Ile Arg Val His Leu Glu Pro Leu Asp His 530 535 540
Lys Lys Glu Leu Ala Asn Ser Ser Ser Asp Asp Glu Asp Phe Phe Ala 545 550 555 560
Ser Leu Lys Pro Thr Thr His Glu Ala Ser Lys Glu Leu Asp Gly Tyr 565 570 575
Leu Ala Cys Val Ser Asp Thr Arg Glu Ser Leu Leu Thr Phe Pro Ala 580 585 590
Ile Cys Ser Leu Ser Ile Lys Thr Asn Thr Pro Leu Pro Ala Ser Ala 595 600 605
Page 33
POTH‐012_001WO_SeqList.txt 01 Sep 2020
Ala Cys Glu Arg Leu Phe Ser Thr Ala Gly Leu Leu Phe Ser Pro Lys 610 615 620
Arg Ala Arg Leu Asp Thr Asn Asn Phe Glu Asn Gln Leu Leu Leu Lys 625 630 635 640 2020227020
Leu Asn Leu Arg Phe Tyr Asn Phe Glu 645
<210> 31 <211> 4682 <212> DNA <213> Oryzias latipes
<400> 31 cagaggtgta aagtacttga gtaattttac ttgattactg tacttaagta ttatttttgg 60
ggatttttac tttacttgag tacaattaaa aatcaatact tttactttta cttaattaca 120
tttttttaga aaaaaaagta ctttttactc cttacaattt tatttacagt caaaaagtac 180
ttattttttg gagatcactt cattctattt tcccttgcta ttaccaaacc aattgaattg 240
cgctgatgcc cagtttaatt taaatgttat ttattctgcc tatgaaaatc gttttcacat 300
tatatgaaat tggtcagaca tgttcattgg tcctttggaa gtgacgtcat gtcacatcta 360
ttaccacaat gcacagcacc ttgacctgga aattagggaa attataacag tcaatcagtg 420
gaagaaaatg gaggaagtat gtgattcatc agcagctgcg agcagcacag tccaaaatca 480
gccacaggat caagagcacc cgtggccgta tcttcgcgaa ttcttttctt taagtggtgt 540
aaataaagat tcattcaaga tgaaatgtgt cctctgtctc ccgcttaata aagaaatatc 600
ggccttcaaa agttcgccat caaacctaag gaagcatatt gaggtaagta cattaagtat 660
tttgttttac tgatagtttt tttttttttt tttttttttt tttttgggtg tgcatgtttt 720
gacgttgatg gcgcgccttt tatatgtgta gtaggcctat tttcactaat gcatgcgatt 780
gacaatataa ggctcacgta ataaaatgct aaaatgcatt tgtaattggt aacgttaggt 840
ccacgggaaa tttggcgcct attgcagctt tgaataatca ttatcattcc gtgctctcat 900
tgtgtttgaa ttcatgcaaa acacaagaaa accaagcgag aaattttttt ccaaacatgt 960
tgtattgtca aaacggtaac actttacaat gaggttgatt agttcatgta ttaactaaca 1020 Page 34
POTH‐012_001WO_SeqList.txt 01 Sep 2020
ttaaataacc atgagcaata catttgttac tgtatctgtt aatctttgtt aacgttagtt 1080
aatagaaata cagatgttca ttgtttgttc atgttagttc acagtgcatt aactaatgtt 1140
aacaagatat aaagtattag taaatgttga aattaacatg tatacgtgca gttcattatt 1200
agttcatgtt aactaatgta gttaactaac gaaccttatt gtaaaagtgt taccatcaaa 1260 2020227020
actaatgtaa tgaaatcaat tcaccctgtc atgtcagcct tacagtcctg tgtttttgtc 1320
aatataatca gaaataaaat taatgtttga ttgtcactaa atgctactgt atttctaaaa 1380
tcaacaagta tttaacatta taaagtgtgc aattggctgc aaatgtcagt tttattaaag 1440
ggttagttca cccaaaaatg aaaataatgt cattaatgac tcgccctcat gtcgttccaa 1500
gcccgtaaga cctccgttca tcttcagaac acagtttaag atattttaga tttagtccga 1560
gagctttctg tgcctccatt gagaatgtat gtacggtata ctgtccatgt ccagaaaggt 1620
aataaaaaca tcaaagtagt ccatgtgaca tcagtgggtt agttagaatt ttttgaagca 1680
tcgaatacat tttggtccaa aaataacaaa acctacgact ttattcggca ttgtattctc 1740
ttccgggtct gttgtcaatc cgcgttcacg acttcgcagt gacgctacaa tgctgaataa 1800
agtcgtaggt tttgttattt ttggaccaaa atgtattttc gatgcttcaa ataattctac 1860
ctaacccact gatgtcacat ggactacttt gatgttttta ttacctttct ggacatggac 1920
agtataccgt acatacattt tcagtggagg gacagaaagc tctcggacta aatctaaaat 1980
atcttaaact gtgttccgaa gatgaacgga ggtgttacgg gcttggaacg acatgagggt 2040
gagtcattaa tgacatcttt tcatttttgg gtgaactaac cctttaatgc tgtaatcaga 2100
gagtgtatgt gtaattgtta catttattgc atacaatata aatatttatt tgttgttttt 2160
acagagaatg cacccaaatt acctcaaaaa ctactctaaa ttgacagcac agaagagaaa 2220
gatcgggacc tccacccatg cttccagcag taagcaactg aaagttgact cagttttccc 2280
agtcaaacat gtgtctccag tcactgtgaa caaagctata ttaaggtaca tcattcaagg 2340
acttcatcct ttcagcactg ttgatctgcc atcatttaaa gagctgatta gtacactgca 2400
gcctggcatt tctgtcatta caaggcctac tttacgctcc aagatagctg aagctgctct 2460
gatcatgaaa cagaaagtga ctgctgccat gagtgaagtt gaatggattg caaccacaac 2520
ggattgttgg actgcacgta gaaagtcatt cattggtgta actgctcact ggatcaaccc 2580 Page 35
POTH‐012_001WO_SeqList.txt 01 Sep 2020
tggaagtctt gaaagacatt ccgctgcact tgcctgcaaa agattaatgg gctctcatac 2640
ttttgaggta ctggccagtg ccatgaatga tatccactca gagtatgaaa tacgtgacaa 2700
ggttgtttgc acaaccacag acagtggttc caactttatg aaggctttca gagtttttgg 2760
tgtggaaaac aatgatatcg agactgaggc aagaaggtgt gaaagtgatg acactgattc 2820 2020227020
tgaaggctgt ggtgagggaa gtgatggtgt ggaattccaa gatgcctcac gagtcctgga 2880
ccaagacgat ggcttcgaat tccagctacc aaaacatcaa aagtgtgcct gtcacttact 2940
taacctagtc tcaagcgttg atgcccaaaa agctctctca aatgaacact acaagaaact 3000
ctacagatct gtctttggca aatgccaagc tttatggaat aaaagcagcc gatcggctct 3060
agcagctgaa gctgttgaat cagaaagccg gcttcagctt ttaaggccaa accaaacgcg 3120
gtggaattca acttttatgg ctgttgacag aattcttcaa atttgcaaag aagcaggaga 3180
aggcgcactt cggaatatat gcacctctct tgaggttcca atgtaagtgt ttttcccctc 3240
tatcgatgta aacaaatgtg ggttgttttt gtttaatact ctttgattat gctgatttct 3300
cctgtaggtt taatccagca gaaatgctgt tcttgacaga gtgggccaac acaatgcgtc 3360
cagttgcaaa agtactcgac atcttgcaag cggaaacgaa tacacagctg gggtggctgc 3420
tgcctagtgt ccatcagtta agcttgaaac ttcagcgact ccaccattct ctcaggtact 3480
gtgacccact tgtggatgcc ctacaacaag gaatccaaac acgattcaag catatgtttg 3540
aagatcctga gatcatagca gctgccatcc ttctccctaa atttcggacc tcttggacaa 3600
atgatgaaac catcataaaa cgaggtaaat gaatgcaagc aacatacact tgacgaattc 3660
taatctgggc aacctttgag ccataccaaa attattcttt tatttattta tttttgcact 3720
ttttaggaat gttatatccc atctttggct gtgatctcaa tatgaatatt gatgtaaagt 3780
attcttgcag caggttgtag ttatccctca gtgtttcttg aaaccaaact catatgtatc 3840
atatgtggtt tggaaatgca gttagatttt atgctaaaat aagggatttg catgatttta 3900
gatgtagatg actgcacgta aatgtagtta atgacaaaat ccataaaatt tgttcccagt 3960
cagaagcccc tcaaccaaac ttttctttgt gtctgctcac tgtgcttgta ggcatggact 4020
acatcagagt gcatctggag cctttggacc acaagaagga attggccaac agttcatctg 4080
atgatgaaga ttttttcgct tctttgaaac cgacaacaca tgaagccagc aaagagttgg 4140 Page 36
POTH‐012_001WO_SeqList.txt 01 Sep 2020
atggatatct ggcctgtgtt tcagacacca gggagtctct gctcacgttt cctgctattt 4200
gcagcctctc tatcaagact aatacacctc ttcccgcatc ggctgcctgt gagaggcttt 4260
tcagcactgc aggattgctt ttcagcccca aaagagctag gcttgacact aacaattttg 4320
agaatcagct tctactgaag ttaaatctga ggttttacaa ctttgagtag cgtgtactgg 4380 2020227020
cattagattg tctgtcttat agtttgataa ttaaatacaa acagttctaa agcaggataa 4440
aaccttgtat gcatttcatt taatgttttt tgagattaaa agcttaaaca agaatctcta 4500
gttttctttc ttgcttttac ttttacttcc ttaatactca agtacaattt taatggagta 4560
cttttttact tttactcaag taagattcta gccagatact tttactttta attgagtaaa 4620
attttcccta agtacttgta ctttcacttg agtaaaattt ttgagtactt tttacacctc 4680
tg 4682
<210> 32 <211> 1053 <212> PRT <213> Artificial Sequence
<220> <223> dSaCas9
<400> 32
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
Page 37
POTH‐012_001WO_SeqList.txt 01 Sep 2020
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 100 105 110
Ala Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu Glu Asp Thr 2020227020
115 120 125
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
Page 38
POTH‐012_001WO_SeqList.txt 01 Sep 2020
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 2020227020
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 370 375 380
Asn Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser Leu Lys Ala Ile 385 390 395 400
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
Page 39
POTH‐012_001WO_SeqList.txt 01 Sep 2020
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 2020227020
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 645 650 655
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
Page 40
POTH‐012_001WO_SeqList.txt 01 Sep 2020
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 2020227020
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
Page 41
POTH‐012_001WO_SeqList.txt 01 Sep 2020
Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe Val Thr Val 915 920 925
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 2020227020
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> 33 <211> 1368 <212> PRT <213> Artificial Sequence
<220> <223> dCas9 with X at position 1, X can be any amino acid
<220> <221> misc_feature <222> (1)..(1) <223> Xaa can be any naturally occurring amino acid
<400> 33
Page 42
POTH‐012_001WO_SeqList.txt 01 Sep 2020
Xaa Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val 1 5 10 15
Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe 20 25 30
Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile 2020227020
35 40 45
Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 50 55 60
Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys 65 70 75 80
Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser 85 90 95
Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 100 105 110
His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 115 120 125
His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp 130 135 140
Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His 145 150 155 160
Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 165 170 175
Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 180 185 190
Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala 195 200 205
Page 43
POTH‐012_001WO_SeqList.txt 01 Sep 2020
Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 210 215 220
Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn 225 230 235 240
Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 2020227020
245 250 255
Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 260 265 270
Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 275 280 285
Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 290 295 300
Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser 305 310 315 320
Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 325 330 335
Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 340 345 350
Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 355 360 365
Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 370 375 380
Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg 385 390 395 400
Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 405 410 415
Page 44
POTH‐012_001WO_SeqList.txt 01 Sep 2020
Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 420 425 430
Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 435 440 445
Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 2020227020
450 455 460
Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu 465 470 475 480
Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 485 490 495
Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 500 505 510
Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 515 520 525
Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 530 535 540
Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr 545 550 555 560
Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp 565 570 575
Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 580 585 590
Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 595 600 605
Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 610 615 620
Page 45
POTH‐012_001WO_SeqList.txt 01 Sep 2020
Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala 625 630 635 640
His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 645 650 655
Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 2020227020
660 665 670
Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 675 680 685
Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 690 695 700
Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu 705 710 715 720
His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 725 730 735
Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 740 745 750
Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln 755 760 765
Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 770 775 780
Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro 785 790 795 800
Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 805 810 815
Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 820 825 830
Page 46
POTH‐012_001WO_SeqList.txt 01 Sep 2020
Leu Ser Asp Tyr Asp Val Asp Ala Ile Val Pro Gln Ser Phe Leu Lys 835 840 845
Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 850 855 860
Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys 2020227020
865 870 875 880
Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 885 890 895
Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 900 905 910
Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 915 920 925
Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp 930 935 940
Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser 945 950 955 960
Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 965 970 975
Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val 980 985 990
Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe 995 1000 1005
Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala 1010 1015 1020
Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe 1025 1030 1035
Page 47
POTH‐012_001WO_SeqList.txt 01 Sep 2020
Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala 1040 1045 1050
Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu 1055 1060 1065
Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val 2020227020
1070 1075 1080
Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr 1085 1090 1095
Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys 1100 1105 1110
Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro 1115 1120 1125
Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val 1130 1135 1140
Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys 1145 1150 1155
Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser 1160 1165 1170
Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys 1175 1180 1185
Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu 1190 1195 1200
Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly 1205 1210 1215
Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val 1220 1225 1230
Page 48
POTH‐012_001WO_SeqList.txt 01 Sep 2020
Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 1235 1240 1245
Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys 1250 1255 1260
His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys 2020227020
1265 1270 1275
Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala 1280 1285 1290
Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn 1295 1300 1305
Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala 1310 1315 1320
Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser 1325 1330 1335
Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr 1340 1345 1350
Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp 1355 1360 1365
<210> 34 <211> 199 <212> PRT <213> Clostridium sp. 7_2_43FAA
<400> 34
Glu Gly Ile Lys Ser Asn Ile Ser Leu Leu Lys Asp Glu Leu Arg Gly 1 5 10 15
Gln Ile Ser His Ile Ser His Glu Tyr Leu Ser Leu Ile Asp Leu Ala 20 25 30
Phe Asp Ser Lys Gln Asn Arg Leu Phe Glu Met Lys Val Leu Glu Leu Page 49
POTH‐012_001WO_SeqList.txt 01 Sep 2020
35 40 45
Leu Val Asn Glu Tyr Gly Phe Lys Gly Arg His Leu Gly Gly Ser Arg 50 55 60
Lys Pro Asp Gly Ile Val Tyr Ser Thr Thr Leu Glu Asp Asn Phe Gly 65 70 75 80 2020227020
Ile Ile Val Asp Thr Lys Ala Tyr Ser Glu Gly Tyr Ser Leu Pro Ile 85 90 95
Ser Gln Ala Asp Glu Met Glu Arg Tyr Val Arg Glu Asn Ser Asn Arg 100 105 110
Asp Glu Glu Val Asn Pro Asn Lys Trp Trp Glu Asn Phe Ser Glu Glu 115 120 125
Val Lys Lys Tyr Tyr Phe Val Phe Ile Ser Gly Ser Phe Lys Gly Lys 130 135 140
Phe Glu Glu Gln Leu Arg Arg Leu Ser Met Thr Thr Gly Val Asn Gly 145 150 155 160
Ser Ala Val Asn Val Val Asn Leu Leu Leu Gly Ala Glu Lys Ile Arg 165 170 175
Ser Gly Glu Met Thr Ile Glu Glu Leu Glu Arg Ala Met Phe Asn Asn 180 185 190
Ser Glu Phe Ile Leu Lys Tyr 195
<210> 35 <211> 4897 <212> DNA <213> Artificial Sequence
<220> <223> PB‐EF1a vector
<400> 35 Page 50
POTH‐012_001WO_SeqList.txt 01 Sep 2020
tgtacataga ttaaccctag aaagataatc atattgtgac gtacgttaaa gataatcatg 60
cgtaaaattg acgcatgtgt tttatcggtc tgtatatcga ggtttattta ttaatttgaa 120
tagatattaa gttttattat atttacactt acatactaat aataaattca acaaacaatt 180
tatttatgtt tatttattta ttaaaaaaaa acaaaaactc aaaatttctt ctataaagta 240
acaaaacttt tatcgaatac ctgcagcccg ggggatgcag agggacagcc cccccccaaa 300 2020227020
gcccccaggg atgtaattac gtccctcccc cgctaggggg cagcagcgag ccgcccgggg 360
ctccgctccg gtccggcgct ccccccgcat ccccgagccg gcagcgtgcg gggacagccc 420
gggcacgggg aaggtggcac gggatcgctt tcctctgaac gcttctcgct gctctttgag 480
cctgcagaca cctgggggga tacggggaaa agttgactgt gcctttcgat cgaaccatgg 540
acagttagct ttgcaaagat ggataaagtt ttaaacagag aggaatcttt gcagctaatg 600
gaccttctag gtcttgaaag gagtgggaat tggctccggt gcccgtcagt gggcagagcg 660
cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgaa ccggtgccta 720
gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc gcctttttcc 780
cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc tttttcgcaa 840
cgggtttgcc gccagaacac aggtaagtgc cgtgtgtggt tcccgcgggc ctggcctctt 900
tacgggttat ggcccttgcg tgccttgaat tacttccacc tggctgcagt acgtgattct 960
tgatcccgag cttcgggttg gaagtgggtg ggagagttcg aggccttgcg cttaaggagc 1020
cccttcgcct cgtgcttgag ttgaggcctg gcctgggcgc tggggccgcc gcgtgcgaat 1080
ctggtggcac cttcgcgcct gtctcgctgc tttcgataag tctctagcca tttaaaattt 1140
ttgatgacct gctgcgacgc tttttttctg gcaagatagt cttgtaaatg cgggccaaga 1200
tctgcacact ggtatttcgg tttttggggc cgcgggcggc gacggggccc gtgcgtccca 1260
gcgcacatgt tcggcgaggc ggggcctgcg agcgcggcca ccgagaatcg gacgggggta 1320
gtctcaagct ggccggcctg ctctggtgcc tggcctcgcg ccgccgtgta tcgccccgcc 1380
ctgggcggca aggctggccc ggtcggcacc agttgcgtga gcggaaagat ggccgcttcc 1440
cggccctgct gcagggagct caaaatggag gacgcggcgc tcgggagagc gggcgggtga 1500
gtcacccaca caaaggaaaa gggcctttcc gtcctcagcc gtcgcttcat gtgactccac 1560
Page 51
POTH‐012_001WO_SeqList.txt 01 Sep 2020
ggagtaccgg gcgccgtcca ggcacctcga ttagttctcg agcttttgga gtacgtcgtc 1620
tttaggttgg ggggaggggt tttatgcgat ggagtttccc cacactgagt gggtggagac 1680
tgaagttagg ccagcttggc acttgatgta attctccttg gaatttgccc tttttgagtt 1740
tggatcttgg ttcattctca agcctcagac agtggttcaa agtttttttc ttccatttca 1800
ggtgtcgtga gaattctaat acgactcact atagggtgtg ctgtctcatc attttggcaa 1860 2020227020
agattggcca ccaagcttgt cctgcaggag ggtcgacgcc tctagacggg cggccgctcc 1920
ggatccacgg gtaccgatca catatgcctt taattaaaca ctagttctat agtgtcacct 1980
aaattccctt tagtgagggt taatggccgt aggccgccag aattgggtcc agacatgata 2040
agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa atgctttatt 2100
tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt 2160
aacaacaaca attgcattca ttttatgttt caggttcagg gggaggtgtg ggaggttttt 2220
tcggactcta ggacctgcgc atgcgcttgg cgtaatcatg gtcatagctg tttcctgttt 2280
tccccgtatc cccccaggtg tctgcaggct caaagagcag cgagaagcgt tcagaggaaa 2340
gcgatcccgt gccaccttcc ccgtgcccgg gctgtccccg cacgctgccg gctcggggat 2400
gcggggggag cgccggaccg gagcggagcc ccgggcggct cgctgctgcc ccctagcggg 2460
ggagggacgt aattacatcc ctgggggctt tggggggggg ctgtccctct caccgcggtg 2520
gagctccagc ttttgttcga attggggccc cccctcgagg gtatcgatga tatctataac 2580
aagaaaatat atatataata agttatcacg taagtagaac atgaaataac aatataatta 2640
tcgtatgagt taaatcttaa aagtcacgta aaagataatc atgcgtcatt ttgactcacg 2700
cggtcgttat agttcaaaat cagtgacact taccgcattg acaagcacgc ctcacgggag 2760
ctccaagcgg cgactgagat gtcctaaatg cacagcgacg gattcgcgct atttagaaag 2820
agagagcaat atttcaagaa tgcatgcgtc aattttacgc agactatctt tctagggtta 2880
atctagctag ccttaagggc gcctattgcg ttgcgctcac tgcccgcttt ccagtcggga 2940
aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt 3000
attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg 3060
cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac 3120
Page 52
POTH‐012_001WO_SeqList.txt 01 Sep 2020
gcaggaaaga acatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac 3180
cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc 3240
ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca 3300
actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta 3360
gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct 3420 2020227020
ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg 3480
gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc 3540
acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta 3600
tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg 3660
gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt 3720
cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg 3780
cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg 3840
ccttttgctc acatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa 3900
tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag tcagaagaac 3960
tcgtcaagaa ggcgatagaa ggcgatgcgc tgcgaatcgg gagcggcgat accgtaaagc 4020
acgaggaagc ggtcagccca ttcgccgcca agctcttcag caatatcacg ggtagccaac 4080
gctatgtcct gatagcggtc cgccacaccc agccggccac agtcgatgaa tccagaaaag 4140
cggccatttt ccaccatgat attcggcaag caggcatcgc catgggtcac gacgagatcc 4200
tcgccgtcgg gcatgctcgc cttgagcctg gcgaacagtt cggctggcgc gagcccctga 4260
tgctcttcgt ccagatcatc ctgatcgaca agaccggctt ccatccgagt acgtgctcgc 4320
tcgatgcgat gtttcgcttg gtggtcgaat gggcaggtag ccggatcaag cgtatgcagc 4380
cgccgcattg catcagccat gatggatact ttctcggcag gagcaaggtg agatgacagg 4440
agatcctgcc ccggcacttc gcccaatagc agccagtccc ttcccgcttc agtgacaacg 4500
tcgagcacag ctgcgcaagg aacgcccgtc gtggccagcc acgatagccg cgctgcctcg 4560
tcttgcagtt cattcagggc accggacagg tcggtcttga caaaaagaac cgggcgcccc 4620
tgcgctgaca gccggaacac ggcggcatca gagcagccga ttgtctgttg tgcccagtca 4680
Page 53
POTH‐012_001WO_SeqList.txt 01 Sep 2020
tagccgaata gcctctccac ccaagcggcc ggagaacctg cgtgcaatcc atcttgttca 4740
atcataatat tattgaagca tttatcaggg ttcgtctcgt cccggtctcc tcccaatgca 4800
tgtcaatatt ggccattagc catattattc attggttata tagcataaat caatattggc 4860
tattggccat tgcatacgtt gtatctatat cataata 4897 2020227020
<210> 36 <211> 20 <212> DNA <213> Artificial Sequence
<220> <223> EF1a‐2r primer
<400> 36 caccggagcc aattcccact 20
<210> 37 <211> 20 <212> DNA <213> Artificial Sequence
<220> <223> AAVS‐3r primer
<400> 37 ctgcaccacg tgatgtcctc 20
<210> 38 <211> 28 <212> DNA <213> Artificial Sequence
<220> <223> SV40pA‐1r primer
<400> 38 gtaaccatta taagctgcaa taaacaag 28
<210> 39 <211> 24 <212> DNA <213> Artificial Sequence
<220> <223> AAVS‐2f primer Page 54
POTH‐012_001WO_SeqList.txt 01 Sep 2020
<400> 39 ctggggactc tttaaggaaa gaag 24
<210> 40 <211> 1591 <212> PRT <213> Artificial Sequence 2020227020
<220> <223> dCas9‐Clo051
<400> 40
Met Ala Pro Lys Lys Lys Arg Lys Val Glu Gly Ile Lys Ser Asn Ile 1 5 10 15
Ser Leu Leu Lys Asp Glu Leu Arg Gly Gln Ile Ser His Ile Ser His 20 25 30
Glu Tyr Leu Ser Leu Ile Asp Leu Ala Phe Asp Ser Lys Gln Asn Arg 35 40 45
Leu Phe Glu Met Lys Val Leu Glu Leu Leu Val Asn Glu Tyr Gly Phe 50 55 60
Lys Gly Arg His Leu Gly Gly Ser Arg Lys Pro Asp Gly Ile Val Tyr 65 70 75 80
Ser Thr Thr Leu Glu Asp Asn Phe Gly Ile Ile Val Asp Thr Lys Ala 85 90 95
Tyr Ser Glu Gly Tyr Ser Leu Pro Ile Ser Gln Ala Asp Glu Met Glu 100 105 110
Arg Tyr Val Arg Glu Asn Ser Asn Arg Asp Glu Glu Val Asn Pro Asn 115 120 125
Lys Trp Trp Glu Asn Phe Ser Glu Glu Val Lys Lys Tyr Tyr Phe Val 130 135 140
Phe Ile Ser Gly Ser Phe Lys Gly Lys Phe Glu Glu Gln Leu Arg Arg Page 55
POTH‐012_001WO_SeqList.txt 01 Sep 2020
145 150 155 160
Leu Ser Met Thr Thr Gly Val Asn Gly Ser Ala Val Asn Val Val Asn 165 170 175
Leu Leu Leu Gly Ala Glu Lys Ile Arg Ser Gly Glu Met Thr Ile Glu 180 185 190 2020227020
Glu Leu Glu Arg Ala Met Phe Asn Asn Ser Glu Phe Ile Leu Lys Tyr 195 200 205
Gly Gly Gly Gly Ser Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly 210 215 220
Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro 225 230 235 240
Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys 245 250 255
Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu 260 265 270
Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys 275 280 285
Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys 290 295 300
Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu 305 310 315 320
Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp 325 330 335
Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys 340 345 350
Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Page 56
POTH‐012_001WO_SeqList.txt 01 Sep 2020
355 360 365
Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly 370 375 380
Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu 385 390 395 400 2020227020
Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser 405 410 415
Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg 420 425 430
Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly 435 440 445
Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe 450 455 460
Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys 465 470 475 480
Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp 485 490 495
Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile 500 505 510
Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro 515 520 525
Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu 530 535 540
Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys 545 550 555 560
Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Page 57
POTH‐012_001WO_SeqList.txt 01 Sep 2020
565 570 575
Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu 580 585 590
Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu 595 600 605 2020227020
Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His 610 615 620
Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp 625 630 635 640
Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu 645 650 655
Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser 660 665 670
Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp 675 680 685
Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile 690 695 700
Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu 705 710 715 720
Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu 725 730 735
Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu 740 745 750
Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn 755 760 765
Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Page 58
POTH‐012_001WO_SeqList.txt 01 Sep 2020
770 775 780
Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn 785 790 795 800
Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys 805 810 815 2020227020
Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val 820 825 830
Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu 835 840 845
Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys 850 855 860
Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn 865 870 875 880
Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys 885 890 895
Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp 900 905 910
Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln 915 920 925
Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala 930 935 940
Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val 945 950 955 960
Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala 965 970 975
Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Page 59
POTH‐012_001WO_SeqList.txt 01 Sep 2020
980 985 990
Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu 995 1000 1005
Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu 1010 1015 1020 2020227020
Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln 1025 1030 1035
Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp Ala Ile 1040 1045 1050
Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val 1055 1060 1065
Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro 1070 1075 1080
Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu 1085 1090 1095
Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr 1100 1105 1110
Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe 1115 1120 1125
Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys His Val 1130 1135 1140
Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn 1145 1150 1155
Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys 1160 1165 1170
Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Page 60
POTH‐012_001WO_SeqList.txt 01 Sep 2020
1175 1180 1185
Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala 1190 1195 1200
Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser 1205 1210 1215 2020227020
Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met 1220 1225 1230
Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr 1235 1240 1245
Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr 1250 1255 1260
Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn 1265 1270 1275
Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala 1280 1285 1290
Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys 1295 1300 1305
Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu 1310 1315 1320
Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp 1325 1330 1335
Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr 1340 1345 1350
Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys 1355 1360 1365
Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Page 61
POTH‐012_001WO_SeqList.txt 01 Sep 2020
1370 1375 1380
Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly 1385 1390 1395
Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr 1400 1405 1410 2020227020
Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser 1415 1420 1425
Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys 1430 1435 1440
Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys 1445 1450 1455
Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln 1460 1465 1470
His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe 1475 1480 1485
Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu 1490 1495 1500
Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala 1505 1510 1515
Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro 1520 1525 1530
Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr 1535 1540 1545
Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser 1550 1555 1560
Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Page 62
POTH‐012_001WO_SeqList.txt 01 Sep 2020
1565 1570 1575
Gly Asp Gly Ser Pro Lys Lys Lys Arg Lys Val Ser Ser 1580 1585 1590
<210> 41 <211> 4073 2020227020
<212> DNA <213> Artificial Sequence
<220> <223> pHR‐GFP‐ selection gene plasmid
<400> 41 ggggccacta gggacaggat cggcgtcttc actcgctggg ttcccttttc cttctccttc 60
tggggcctgt gccatctctc gtttcttagg atggccttct ccgacggatg tctcccttgc 120
gtcccgcctc cccttcttgt aggcctgcat catcaccgtt tttctggaca accccaaagt 180
accccgtctc cctggcttta gccacctctc catcctcttg ctttctttgc ctggacaccc 240
cgttctcctg tggattcggg tcacctctca ctcctttcat ttgggcagct cccctacccc 300
ccttacctct ctagtctgtg ctagctcttc cagccccctg tcatggcatc ttccaggggt 360
ccgagagctc agctagtctt cttcctccaa cccgggcccc tatgtccact tcaggacagc 420
atgtttgctg cctccaggga tcctgtgtcc ccgagctggg accaccttat attcccaggg 480
ccggttaatg tggctctggt tctgggtact tttatctgtc ccgacgtccg atcgaaccat 540
ggacagttag ctttgcaaag atggataaag ttttaaacag agaggaatct ttgcagctaa 600
tggaccttct aggtcttgaa aggagtggga attggctccg gtgcccgtca gtgggcagag 660
cgcacatcgc ccacagtccc cgagaagttg gggggagggg tcggcaattg aaccggtgcc 720
tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt 780
cccgagggtg ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc 840
aacgggtttg ccgccagaac acaggtaagt gccgtgtgtg gttcccgcgg gcctggcctc 900
tttacgggtt atggcccttg cgtgccttga attacttcca cctggctgca gtacgtgatt 960
cttgatcccg agcttcgggt tggaagtggg tgggagagtt cgaggccttg cgcttaagga 1020
gccccttcgc ctcgtgcttg agttgaggcc tggcctgggc gctggggccg ccgcgtgcga 1080
Page 63
POTH‐012_001WO_SeqList.txt 01 Sep 2020
atctggtggc accttcgcgc ctgtctcgct gctttcgata agtctctagc catttaaaat 1140
ttttgatgac ctgctgcgac gctttttttc tggcaagata gtcttgtaaa tgcgggccaa 1200
gatctgcaca ctggtatttc ggtttttggg gccgcgggcg gcgacggggc ccgtgcgtcc 1260
cagcgcacat gttcggcgag gcggggcctg cgagcgcggc caccgagaat cggacggggg 1320
tagtctcaag ctggccggcc tgctctggtg cctggcctcg cgccgccgtg tatcgccccg 1380 2020227020
ccctgggcgg caaggctggc ccggtcggca ccagttgcgt gagcggaaag atggccgctt 1440
cccggccctg ctgcagggag ctcaaaatgg aggacgcggc gctcgggaga gcgggcgggt 1500
gagtcaccca cacaaaggaa aagggccttt ccgtcctcag ccgtcgcttc atgtgactcc 1560
acggagtacc gggcgccgtc caggcacctc gattagttct cgagcttttg gagtacgtcg 1620
tctttaggtt ggggggaggg gttttatgcg atggagtttc cccacactga gtgggtggag 1680
actgaagtta ggccagcttg gcacttgatg taattctcct tggaatttgc cctttttgag 1740
tttggatctt ggttcattct caagcctcag acagtggttc aaagtttttt tcttccattt 1800
caggtgtcgt gagaattcta atacgactca ctatagggtg tgctgtctca tcattttggc 1860
aaagattggc caccaagctt accgccatgg tgagcaaggg cgaggagctg ttcaccgggg 1920
tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg 1980
gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg 2040
gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct 2100
tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag 2160
gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg 2220
aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca 2280
aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct 2340
atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca 2400
tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg 2460
gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc 2520
ccaacgagaa gcgtgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc 2580
tcggcatgga cgagctgtac aaggaaggaa gaggcagcct gctgacatgt ggcgacgtgg 2640
Page 64
POTH‐012_001WO_SeqList.txt 01 Sep 2020
aggagaaccc tggcccaatg gtgggcagcc tgaattgtat cgtggccgtg tcccagaaca 2700
tgggcatcgg caagaatggc gattttcctt ggccccctct gagaaatgag tccagatact 2760
ttcagaggat gaccacaacc agctccgtgg agggcaagca gaacctggtc atcatgggca 2820
agaagacatg gttctctatc ccagagaaga accgccccct gaagggccgg atcaatctgg 2880
tgctgagcag ggagctgaag gagccacccc agggagcaca ctttctgtcc aggtctctgg 2940 2020227020
acgatgccct gaagctgacc gagcagcctg agctggccaa caaggtggac atggtgtgga 3000
tcgtgggcgg ctctagcgtg tataaggagg ccatgaatca ccctggccac ctgaagctgt 3060
tcgtgacacg gatcatgcag gactttgagt ccgatacctt ctttccagag atcgacctgg 3120
agaagtacaa gctgctgccc gagtatcctg gcgtgctgtc tgatgtgcag gaggagaagg 3180
gcatcaagta caagttcgag gtgtatgaga agaacgattg ataacatatg cctttaatta 3240
aacactagtt ctatagtgtc acctaaattc cctttagtga gggttaatgg ccgtaggccg 3300
ccagaattgg gtccagacat gataagatac attgatgagt ttggacaaac cacaactaga 3360
atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc 3420
attataagct gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt 3480
cagggggagg tgtgggaggt tttttcggac tctaggacct gcgcatgcgc ttggggtacc 3540
taggatatcg acagaaaagc cccatcctta ggcctcctcc ttcctagtct cctgatattg 3600
ggtctaaccc ccacctcctg ttaggcagat tccttatctg gtgacacacc cccatttcct 3660
ggagccatct ctctccttgc cagaacctct aaggtttgct tacgatggag ccagagagga 3720
tcctgggagg gagagcttgg cagggggtgg gagggaaggg ggggatgcgt gacctgcccg 3780
gttctcagtg gccaccctgc gctaccctct cccagaacct gagctgctct gacgcggccg 3840
tctggtgcgt ttcactgatc ctggtgctgc agcttcctta cacttcccaa gaggagaagc 3900
agtttggaaa aacaaaatca gaataagttg gtcctgagtt ctaactttgg ctcttcacct 3960
ttctagtccc caatttatat tgttcctccg tgcgtcagtt ttacctgtga gataaggcca 4020
gtagccagcc ccgtcctggc agggctgtgc ctctagggac aggattggtg acg 4073
<210> 42 <211> 3089 <212> DNA Page 65
POTH‐012_001WO_SeqList.txt 01 Sep 2020
<213> Artificial Sequence
<220> <223> pMMEJ‐GFP‐selection gene plasmid
<400> 42 ccaatcctgt ccctagtggc cccactgtgg ggacgtccga tcgaaccatg gacagttagc 60
tttgcaaaga tggataaagt tttaaacaga gaggaatctt tgcagctaat ggaccttcta 120 2020227020
ggtcttgaaa ggagtgggaa ttggctccgg tgcccgtcag tgggcagagc gcacatcgcc 180
cacagtcccc gagaagttgg ggggaggggt cggcaattga accggtgcct agagaaggtg 240
gcgcggggta aactgggaaa gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg 300
gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc 360
cgccagaaca caggtaagtg ccgtgtgtgg ttcccgcggg cctggcctct ttacgggtta 420
tggcccttgc gtgccttgaa ttacttccac ctggctgcag tacgtgattc ttgatcccga 480
gcttcgggtt ggaagtgggt gggagagttc gaggccttgc gcttaaggag ccccttcgcc 540
tcgtgcttga gttgaggcct ggcctgggcg ctggggccgc cgcgtgcgaa tctggtggca 600
ccttcgcgcc tgtctcgctg ctttcgataa gtctctagcc atttaaaatt tttgatgacc 660
tgctgcgacg ctttttttct ggcaagatag tcttgtaaat gcgggccaag atctgcacac 720
tggtatttcg gtttttgggg ccgcgggcgg cgacggggcc cgtgcgtccc agcgcacatg 780
ttcggcgagg cggggcctgc gagcgcggcc accgagaatc ggacgggggt agtctcaagc 840
tggccggcct gctctggtgc ctggcctcgc gccgccgtgt atcgccccgc cctgggcggc 900
aaggctggcc cggtcggcac cagttgcgtg agcggaaaga tggccgcttc ccggccctgc 960
tgcagggagc tcaaaatgga ggacgcggcg ctcgggagag cgggcgggtg agtcacccac 1020
acaaaggaaa agggcctttc cgtcctcagc cgtcgcttca tgtgactcca cggagtaccg 1080
ggcgccgtcc aggcacctcg attagttctc gagcttttgg agtacgtcgt ctttaggttg 1140
gggggagggg ttttatgcga tggagtttcc ccacactgag tgggtggaga ctgaagttag 1200
gccagcttgg cacttgatgt aattctcctt ggaatttgcc ctttttgagt ttggatcttg 1260
gttcattctc aagcctcaga cagtggttca aagttttttt cttccatttc aggtgtcgtg 1320
agaattctaa tacgactcac tatagggtgt gctgtctcat cattttggca aagattggcc 1380
Page 66
POTH‐012_001WO_SeqList.txt 01 Sep 2020
accaagctta ccgccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc 1440
ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag 1500
ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc 1560
gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac 1620
cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag 1680 2020227020
gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc 1740
gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc 1800
aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc 1860
gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc 1920
agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg 1980
ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag 2040
cgtgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac 2100
gagctgtaca aggaaggaag aggcagcctg ctgacatgtg gcgacgtgga ggagaaccct 2160
ggcccaatgg tgggcagcct gaattgtatc gtggccgtgt cccagaacat gggcatcggc 2220
aagaatggcg attttccttg gccccctctg agaaatgagt ccagatactt tcagaggatg 2280
accacaacca gctccgtgga gggcaagcag aacctggtca tcatgggcaa gaagacatgg 2340
ttctctatcc cagagaagaa ccgccccctg aagggccgga tcaatctggt gctgagcagg 2400
gagctgaagg agccacccca gggagcacac tttctgtcca ggtctctgga cgatgccctg 2460
aagctgaccg agcagcctga gctggccaac aaggtggaca tggtgtggat cgtgggcggc 2520
tctagcgtgt ataaggaggc catgaatcac cctggccacc tgaagctgtt cgtgacacgg 2580
atcatgcagg actttgagtc cgataccttc tttccagaga tcgacctgga gaagtacaag 2640
ctgctgcccg agtatcctgg cgtgctgtct gatgtgcagg aggagaaggg catcaagtac 2700
aagttcgagg tgtatgagaa gaacgattga taacatatgc ctttaattaa acactagttc 2760
tatagtgtca cctaaattcc ctttagtgag ggttaatggc cgtaggccgc cagaattggg 2820
tccagacatg ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa 2880
aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg 2940
Page 67
POTH‐012_001WO_SeqList.txt 01 Sep 2020
caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc agggggaggt 3000
gtgggaggtt ttttcggact ctaggacctg cgcatgcgct tggggtacct aggatatcgg 3060
atggggcttt tctgtcacca atcctgagg 3089 2020227020
Page 68

Claims (10)

1. A fusion protein comprising the amino acid sequence of SEQ ID NO: 40.
2. A method of producing a modified cell comprising introducing into the cell the fusion protein of claim 1.
3. The method of claim 2, wherein the modified cell is a T cell.
4. The method of claim 3, wherein the modified T cell is a modified stem memory T cell (TscM) or a modified central memory T cell (Tcm).
5. The method of claim 3, wherein the modified T cell is an allogeneic cell.
6. The method of claim 3, wherein the modified T cell is an autologous cell.
7. A modified T cell comprising a fusion protein of claim 1.
8. A modified T cell produced by the method of any one of claims claim 2-6.
9. A composition comprising a population of modified cells comprising the fusion protein of claim 1.
10. A composition comprising a population of modified cells produced by the method of claim 2.
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