VCAR compositions and methods for use

Abstract

Disclosed are VHH chimeric antigen receptors (VCARs), VCAR transposons encoding VCARs of the disclosure, cells modified to express VCARs of the disclosure, as well as methods of making and methods of using the same for adoptive cell therapy. 1004815119 ABSTRACT2023 2023208176 27 Jul

Description

RELATED APPLICATIONS

[01] This application is a divisional of Australian patent application 2018393110 and claims the benefit of provisional applications USSN 62 / 608,571, filed December 20, 2017 and USSN 62 / 608,894, filed December 21, 2017, the contents of all of which are herein incorporated by reference in their entirety. FIELD OF THE DISCLOSURE

[02] The disclosure is directed to molecular biology, and more, specifically, to chimeric antigen receptors, and to transposons containing one or more VCARs, as well as methods of making and using the same. INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

[03] The contents of the file named “POTH-034_001WO_SeqListing_ST25_R.txt”, which was created on December 19, 2018, and is 54.4 MB in size are hereby incorporated by reference in their entirety. BACKGROUND

[04] There has been a long-felt but unmet need in the art for a method of directing the specificity of an immune cell without using traditional antibody sequences or fragments thereof. The disclosure provides a superior chimeric antigen receptor. SUMMARY

[05] 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 domain antibody; (b) a transmembrane domain, and (c) an endodomain comprising at least one costimulatory domain. In some embodiments, the single domain antibody comprises a human or humanized sequence. In some embodiments, the single domain antibody comprises a non-naturally occurring sequence. In some embodiments, the single domain antibody comprises a recombinant or chimeric sequence. In some embodiments, the single domain antibody comprises a VHH or a sequence encoding a VHH. In some 2023208176 27 Jul 2023 embodiments, the single domain antibody comprises a VH or a sequence encoding a VH. In some embodiments, the single domain antibody comprises a VH of the disclosure or a sequence encoding a VH of the disclosure.

[06] 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 VHH; (b) a transmembrane domain, and (c) an endodomain comprising at least one costimulatory domain. As used throughout the disclosure, a CAR comprising a VHH is referred to as a VCAR. In certain embodiments, the antigen recognition region may comprise two VHHs to produce a bi-specific or tandem VCAR. In certain embodiments, the antigen recognition region may comprise three VHHs to produce a tri-specific VCAR.

[07] In certain embodiments of the VCARs of the disclosure, 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. 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.

[08] In certain embodiments of the VCARs of the disclosure, the VHH comprises or consists of the amino acid sequence malpvtalllplalllhaarpevqllesggglvqpggslrlscaasgftfssyamnwvrqapgkglewvagiigsggstyyadsvkgrfsi srdnskntldlqmnslraedtavyycvkdwnttmitergqgtlvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfac diyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynel nlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalp pr (VH-A; SEQ ID NO: 18000) or the VHH comprises or consists of the nucleic acid sequence atggctctgcctgtgacagctctgctgctgcctctggctctgcttcttcatgcggcgcgccctgaagttcagctgcttgaatctggcggaggc ctggttcaacctggcggatctctgagactgagctgtgccgccagcggcttcacctttagcagctacgccatgaactgggtccgacaggccc ctggcaaaggactggaatgggtggccggaatcatcggcagcggcggcagcacatattacgccgattctgtgaagggccgcttcagcatc agccgggacaacagcaagaacaccctggacctgcagatgaacagcctgagagccgaggataccgccgtgtactactgcgtgaaggatt ggaacaccaccatgatcaccgagagaggccagggcacactggtcaccgtgtcctctacaacaacaccggcgcctcggcctccaacacc agctcctacaatcgcgagtcagcccctgtctctcagacccgaagcctgtagacctgctgctggcggagctgtgcataccagaggactggat ttcgcctgcgacatctacatctgggctcctctggctggcacatgcggagttttgctgctgagcctggtcatcaccctgtactgtaagagaggc 2023208176 27 Jul 2023 aggaagaagctgctgtatatcttcaagcagcccttcatgagacccgtgcagaccacacaggaggaggacggctgctcttgtaggttcccag aggaggaggagggaggatgcgagctgcgcgtgaagtttagccggtccgccgatgcacctgcatacaagcagggacagaaccagctgta taacgagctgaatctgggccggagagaggagtacgacgtgctggataagaggcggggccgggaccccgagatgggaggcaagcctc ggagaaagaacccacaggagggcctgtacaatgagctgcaaaaggacaagatggccgaggcctattctgagatcggcatgaagggaga gaggcgccggggcaagggacacgatggcctgtaccagggcctgagcaccgccacaaaggacacctatgatgccctgcacatgcaggc cctgccccctagatga (VH-A; SEQ ID NO: 18001).

[09] In certain embodiments of the VCARs of the disclosure, the VHH comprises or consists of the amino acid sequence malpvtalllplalllhaarpevqllesggglvqpggsltlscaasgftfsnyamnwvrqapgkglewvsgiigsgattyyadsvkgrfti srdnskntlnlqmnslraedtaiyycvkdwnttmitergqgtlvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfac diyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynel nlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalp pr (VH-B; SEQ ID NO: 18002) or the VHH comprises or consists of the nucleic acid sequence atggctctgcctgtgacagctctgctgctgcctctggctctgcttcttcatgcggcgcgccctgaagttcagctgcttgaatctggcggaggc ctggttcaacctggcggatctctgacactgagctgtgccgccagcggcttcaccttcagcaactacgccatgaactgggtccgacaggccc ctggcaaaggccttgaatgggtgtccggcatcattggctctggcgccaccacctactacgccgattctgtgaagggcagattcaccatcagc cgggacaacagcaagaacaccctgaacctgcagatgaacagcctgagagccgaggacaccgccatctactactgcgtgaaggactgga acaccaccatgatcaccgagagaggccagggcacactggtcaccgtgtcctctacaacaacaccggcgcctcggcctccaacaccagct cctacaatcgcgagtcagcccctgtctctcagacccgaagcctgtagacctgctgctggcggagctgtgcataccagaggactggatttcg cctgcgacatctacatctgggctcctctggctggcacatgcggagttttgctgctgagcctggtcatcaccctgtactgtaagagaggcagg aagaagctgctgtatatcttcaagcagcccttcatgagacccgtgcagaccacacaggaggaggacggctgctcttgtaggttcccagagg aggaggagggaggatgcgagctgcgcgtgaagtttagccggtccgccgatgcacctgcatacaagcagggacagaaccagctgtataa cgagctgaatctgggccggagagaggagtacgacgtgctggataagaggcggggccgggaccccgagatgggaggcaagcctcgga gaaagaacccacaggagggcctgtacaatgagctgcaaaaggacaagatggccgaggcctattctgagatcggcatgaagggagagag gcgccggggcaagggacacgatggcctgtaccagggcctgagcaccgccacaaaggacacctatgatgccctgcacatgcaggccct gccccctagatga (VH-B; SEQ ID NO: 18003).

[010] In certain embodiments of the VCARs of the disclosure, the VHH comprises or consists of the amino acid sequence malpvtalllplalllhaarpevqllesggglvqpgeslrlscaasgftfsnyamnwvrqapgkglewvsgivggggtsyyadsvrgrft isrdnskntlylqmnslraedtavyycvkdwnttmitergqgtlvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfa 2023208176 27 Jul 2023 cdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlyne Inlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqal ppr (VH-C; SEQ ID NO: 18004) or the VHH comprises or consists of the nucleic acid sequence atggctctgcctgtgacagctctgctgctgcctctggctctgcttcttcatgcggcgcgccctgaagttcagctgcttgaatctggcggaggc ctggttcagcctggcgaatctctgagactgagctgtgccgccagcggcttcaccttcagcaactacgccatgaactgggtccgacaggccc ctggcaaaggccttgaatgggtgtccggaatcgttggcggcggaggcacaagctactacgccgattctgtgcggggcagattcaccatca gccgggacaacagcaagaacaccctgtacctgcagatgaacagcctgagagccgaggacaccgccgtgtactactgcgtgaaggactg gaacaccaccatgatcaccgagagaggccagggcacactggtcaccgtgtcctctacaacaacaccggcgcctcggcctccaacacca gctcctacaatcgcgagtcagcccctgtctctcagacccgaagcctgtagacctgctgctggcggagctgtgcataccagaggactggattt cgcctgcgacatctacatctgggctcctctggctggcacatgcggagttttgctgctgagcctggtcatcaccctgtactgtaagagaggca ggaagaagctgctgtatatcttcaagcagcccttcatgagacccgtgcagaccacacaggaggaggacggctgctcttgtaggttcccaga ggaggaggagggaggatgcgagctgcgcgtgaagtttagccggtccgccgatgcacctgcatacaagcagggacagaaccagctgtat aacgagctgaatctgggccggagagaggagtacgacgtgctggataagaggcggggccgggaccccgagatgggaggcaagcctcg gagaaagaacccacaggagggcctgtacaatgagctgcaaaaggacaagatggccgaggcctattctgagatcggcatgaagggagag aggcgccggggcaagggacacgatggcctgtaccagggcctgagcaccgccacaaaggacacctatgatgccctgcacatgcaggcc ctgccccctagatga (VH-C; SEQ ID NO: 18005). [OH] In certain embodiments of the VCARs of the disclosure, the VHH comprises or consists of the amino acid sequence malpvtalllplalllhaarpevqllesggglvqpggslrlscaasgftfsnyamtwirqapgkglewvsgitgdggstfyadsvkgrftis rdnskntlylqmnslraedtavyycvkdwnttmitergqgtlvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfac diyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynel nlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalp pr (VH-D; SEQ ID NO: 18006) or the VHH comprises or consists of the nucleic acid sequence atggctctgcctgtgacagctctgctgctgcctctggctctgcttcttcatgcggcgcgccctgaagttcagctgcttgaatctggcggaggc ctggttcaacctggcggatctctgagactgagctgtgccgccagcggcttcaccttcagcaattacgccatgacctggatcagacaggccc ctggcaaaggcctggaatgggtgtccggaattacaggcgacggcggcagcaccttttacgccgattctgtgaagggcagattcaccatca gccgggacaacagcaagaacaccctgtacctgcagatgaacagcctgagagccgaggacaccgccgtgtactactgcgtgaaggactg gaacaccaccatgatcaccgagagaggccagggcacactggtcaccgtgtcctctacaacaacaccggcgcctcggcctccaacacca gctcctacaatcgcgagtcagcccctgtctctcagacccgaagcctgtagacctgctgctggcggagctgtgcataccagaggactggattt 2023208176 27 Jul 2023 cgcctgcgacatctacatctgggctcctctggctggcacatgcggagttttgctgctgagcctggtcatcaccctgtactgtaagagaggca ggaagaagctgctgtatatcttcaagcagcccttcatgagacccgtgcagaccacacaggaggaggacggctgctcttgtaggttcccaga ggaggaggagggaggatgcgagctgcgcgtgaagtttagccggtccgccgatgcacctgcatacaagcagggacagaaccagctgtat aacgagctgaatctgggccggagagaggagtacgacgtgctggataagaggcggggccgggaccccgagatgggaggcaagcctcg gagaaagaacccacaggagggcctgtacaatgagctgcaaaaggacaagatggccgaggcctattctgagatcggcatgaagggagag aggcgccggggcaagggacacgatggcctgtaccagggcctgagcaccgccacaaaggacacctatgatgccctgcacatgcaggcc ctgccccctagatga (VH-D; SEQ ID NO: 18007).

[012] In certain embodiments of the VCARs of the disclosure, the VHH comprises or consists of the amino acid sequence malpvtalllplalllhaarpevqllesggglaqpggslrlscaasgftfssyamnwirqapgkglewvsgisgsggstyyadsvkgrfti srdnskntvylqmnslraedtavyycvkdwnttmitergqgtlvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfa cdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlyne Inlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqal ppr (VH-E; SEQ ID NO: 18008) or the VHH comprises or consists of the nucleic acid sequence atggcactgcctgtgacagccctgctgctgcctctggccctgctgctgcacgcagcacggcccgaggtgcagctgctggagtccggagg aggcctggcccagcctggcggcagcctgaggctgtcctgcgccgcctctggcttcacctttagctcctacgccatgaactggatcagacag gcccctggcaagggcctggagtgggtgtccggcatctccggctctggaggctctacatactatgccgacagcgtgaagggccggttcacc atcagcagagataactccaagaataccgtgtacctccagatgaactctctgcgggccgaggacaccgccgtgtactattgcgtgaaggatt ggaataccacaatgatcacagagaggggccagggcaccctggtgacagtgtctagcaccacaacccctgcccccagacctcccacacc cgcccctaccatcgcgagtcagccactgtccctgcggcctgaggcctgccggcccgccgccggcggagcagtgcacacacggggcct ggactttgcctgtgacatctacatatgggcaccactggcaggaacctgcggcgtgctgctgctgagcctggtcatcaccctgtactgtaaga gaggcaggaagaagctgctgtatatcttcaagcagcccttcatgagacccgtgcagaccacacaggaggaggacggctgctcttgtaggt tcccagaggaggaggagggaggatgcgagctgcgcgtgaagtttagccggtccgccgatgcacctgcatacaagcagggacagaacc agctgtataacgagctgaatctgggccggagagaggagtacgacgtgctggataagaggcggggccgggaccccgagatgggaggca agcctcggagaaagaacccacaggagggcctgtacaatgagctgcaaaaggacaagatggccgaggcctattctgagatcggcatgaa gggagagaggcgccggggcaagggacacgatggcctgtaccagggcctgagcaccgccacaaaggacacctatgatgccctgcacat gcaggccctgccccctagatga (VH-E; SEQ ID NO: 18009).

[013] In certain embodiments of the VCARs of the disclosure, the VHH comprises or consists of the amino acid sequence malpvtalllplalllhaarpevqllesggglvqpgrslrlscaasgftftnyamnwvrqapgkglewvsgisggggstyyadsvkgrfti 2023208176 27 Jul 2023 srdnskntlylqmnslraedtavyycvkdwnttmitergqgtlvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfac diyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynel nlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalp pr (VH-F; SEQ ID NO: 18010) or the VHH comprises or consists of the nucleic acid sequence atggcactgcctgtgacagccctgctgctgcctctggccctgctgctgcacgcagcacggcccgaggtgcagctgctggagtctggagga ggcctggtgcagcccggccggtccctgagactgtcttgcgccgccagcggcttcacctttacaaactacgccatgaattgggtgcggcag gcccctggcaagggcctggagtgggtgtctggcatcagcggaggaggaggcagcacctactatgcagactccgtgaagggcaggttca ccatctcccgcgataactctaagaatacactgtacctccagatgaacagcctgagggcagaggacaccgccgtgtactattgcgtgaagga ttggaataccacaatgatcacagagaggggacagggcaccctggtgaccgtgagcagcaccacaacccctgcccccagacctcccaca cccgcccctaccatcgcgagtcagccactgtccctgcggcctgaggcctgccggcccgccgccggcggagcagtgcacacacggggc ctggactttgcctgtgacatctacatatgggcaccactggcaggaacctgcggcgtgctgctgctgagcctggtcatcaccctgtactgtaag agaggcaggaagaagctgctgtatatcttcaagcagcccttcatgagacccgtgcagaccacacaggaggaggacggctgctcttgtagg ttcccagaggaggaggagggaggatgcgagctgcgcgtgaagtttagccggtccgccgatgcacctgcatacaagcagggacagaacc agctgtataacgagctgaatctgggccggagagaggagtacgacgtgctggataagaggcggggccgggaccccgagatgggaggca agcctcggagaaagaacccacaggagggcctgtacaatgagctgcaaaaggacaagatggccgaggcctattctgagatcggcatgaa gggagagaggcgccggggcaagggacacgatggcctgtaccagggcctgagcaccgccacaaaggacacctatgatgccctgcacat gcaggccctgccccctagatga (VH-F; SEQ ID NO: 18011).

[014] 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 VH; (b) a transmembrane domain, and (c) an endodomain comprising at least one costimulatory domain. As used throughout the disclosure, a CAR comprising a VH is referred to as a VCAR. In certain embodiments, the antigen recognition region may comprise two VHs to produce a bi-specific or tandem VCAR. In certain embodiments, the antigen recognition region may comprise three VHs to produce a tri-specific VCAR. 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. 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. 2023208176 27 Jul 2023

[015] In certain embodiments of the VCARs of the disclosure, including those comprising an ectodomain comprising an antigen recognition region, wherein the antigen recognition region comprises at least one VH, the VH comprises a human or a humanized sequence.

[016] In certain embodiments of the VCARs of the disclosure, including those comprising an ectodomain comprising an antigen recognition region, wherein the antigen recognition region comprises at least one VH, the VH comprises a non-naturally occurring sequence.

[017] In certain embodiments of the VCARs of the disclosure, including those comprising an ectodomain comprising an antigen recognition region, wherein the antigen recognition region comprises at least one VH, the VH is not naturally occurring.

[018] In certain embodiments of the VCARs of the disclosure, including those comprising an ectodomain comprising an antigen recognition region, wherein the antigen recognition region comprises at least one VH, the VH comprises a recombinant or chimeric sequence.

[019] In certain embodiments of the VCARs of the disclosure, including those comprising an ectodomain comprising an antigen recognition region, wherein the antigen recognition region comprises at least one VH, the VH is produced by an in vitro procedure of affinity selection and recombination.

[020] In certain embodiments of the VCARs of the disclosure, the VH comprises or consists of the amino acid sequence malpvtalllplalllhaarpevqllesggglvqpggslrlscaasgftfssyamnwvrqapgkglewvagiigsggstyyadsvkgrfsi srdnskntldlqmnslraedtavyycvkdwnttmitergqgtlvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfac diyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynel nlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalp pr (VH-A; SEQ ID NO: 18000) or the VH comprises or consists of the nucleic acid sequence atggctctgcctgtgacagctctgctgctgcctctggctctgcttcttcatgcggcgcgccctgaagttcagctgcttgaatctggcggaggc ctggttcaacctggcggatctctgagactgagctgtgccgccagcggcttcacctttagcagctacgccatgaactgggtccgacaggccc ctggcaaaggactggaatgggtggccggaatcatcggcagcggcggcagcacatattacgccgattctgtgaagggccgcttcagcatc agccgggacaacagcaagaacaccctggacctgcagatgaacagcctgagagccgaggataccgccgtgtactactgcgtgaaggatt ggaacaccaccatgatcaccgagagaggccagggcacactggtcaccgtgtcctctacaacaacaccggcgcctcggcctccaacacc agctcctacaatcgcgagtcagcccctgtctctcagacccgaagcctgtagacctgctgctggcggagctgtgcataccagaggactggat ttcgcctgcgacatctacatctgggctcctctggctggcacatgcggagttttgctgctgagcctggtcatcaccctgtactgtaagagaggc 2023208176 27 Jul 2023 aggaagaagctgctgtatatcttcaagcagcccttcatgagacccgtgcagaccacacaggaggaggacggctgctcttgtaggttcccag aggaggaggagggaggatgcgagctgcgcgtgaagtttagccggtccgccgatgcacctgcatacaagcagggacagaaccagctgta taacgagctgaatctgggccggagagaggagtacgacgtgctggataagaggcggggccgggaccccgagatgggaggcaagcctc ggagaaagaacccacaggagggcctgtacaatgagctgcaaaaggacaagatggccgaggcctattctgagatcggcatgaagggaga gaggcgccggggcaagggacacgatggcctgtaccagggcctgagcaccgccacaaaggacacctatgatgccctgcacatgcaggc cctgccccctagatga (VH-A; SEQ ID NO: 18001).

[021] In certain embodiments of the VCARs of the disclosure, the VH comprises or consists of the amino acid sequence malpvtalllplalllhaarpevqllesggglvqpggsltlscaasgftfsnyamnwvrqapgkglewvsgiigsgattyyadsvkgrfti srdnskntlnlqmnslraedtaiyycvkdwnttmitergqgtlvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfac diyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynel nlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalp pr (VH-B; SEQ ID NO: 18002) or the VH comprises or consists of the nucleic acid sequence atggctctgcctgtgacagctctgctgctgcctctggctctgcttcttcatgcggcgcgccctgaagttcagctgcttgaatctggcggaggc ctggttcaacctggcggatctctgacactgagctgtgccgccagcggcttcaccttcagcaactacgccatgaactgggtccgacaggccc ctggcaaaggccttgaatgggtgtccggcatcattggctctggcgccaccacctactacgccgattctgtgaagggcagattcaccatcagc cgggacaacagcaagaacaccctgaacctgcagatgaacagcctgagagccgaggacaccgccatctactactgcgtgaaggactgga acaccaccatgatcaccgagagaggccagggcacactggtcaccgtgtcctctacaacaacaccggcgcctcggcctccaacaccagct cctacaatcgcgagtcagcccctgtctctcagacccgaagcctgtagacctgctgctggcggagctgtgcataccagaggactggatttcg cctgcgacatctacatctgggctcctctggctggcacatgcggagttttgctgctgagcctggtcatcaccctgtactgtaagagaggcagg aagaagctgctgtatatcttcaagcagcccttcatgagacccgtgcagaccacacaggaggaggacggctgctcttgtaggttcccagagg aggaggagggaggatgcgagctgcgcgtgaagtttagccggtccgccgatgcacctgcatacaagcagggacagaaccagctgtataa cgagctgaatctgggccggagagaggagtacgacgtgctggataagaggcggggccgggaccccgagatgggaggcaagcctcgga gaaagaacccacaggagggcctgtacaatgagctgcaaaaggacaagatggccgaggcctattctgagatcggcatgaagggagagag gcgccggggcaagggacacgatggcctgtaccagggcctgagcaccgccacaaaggacacctatgatgccctgcacatgcaggccct gccccctagatga (VH-B; SEQ ID NO: 18003).

[022] In certain embodiments of the VCARs of the disclosure, the VH comprises or consists of the amino acid sequence malpvtalllplalllhaarpevqllesggglvqpgeslrlscaasgftfsnyamnwvrqapgkglewvsgivggggtsyyadsvrgrft isrdnskntlylqmnslraedtavyycvkdwnttmitergqgtlvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfa 2023208176 27 Jul 2023 cdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlyne Inlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqal ppr (VH-C; SEQ ID NO: 18004) or the VH comprises or consists of the nucleic acid sequence atggctctgcctgtgacagctctgctgctgcctctggctctgcttcttcatgcggcgcgccctgaagttcagctgcttgaatctggcggaggc ctggttcagcctggcgaatctctgagactgagctgtgccgccagcggcttcaccttcagcaactacgccatgaactgggtccgacaggccc ctggcaaaggccttgaatgggtgtccggaatcgttggcggcggaggcacaagctactacgccgattctgtgcggggcagattcaccatca gccgggacaacagcaagaacaccctgtacctgcagatgaacagcctgagagccgaggacaccgccgtgtactactgcgtgaaggactg gaacaccaccatgatcaccgagagaggccagggcacactggtcaccgtgtcctctacaacaacaccggcgcctcggcctccaacacca gctcctacaatcgcgagtcagcccctgtctctcagacccgaagcctgtagacctgctgctggcggagctgtgcataccagaggactggattt cgcctgcgacatctacatctgggctcctctggctggcacatgcggagttttgctgctgagcctggtcatcaccctgtactgtaagagaggca ggaagaagctgctgtatatcttcaagcagcccttcatgagacccgtgcagaccacacaggaggaggacggctgctcttgtaggttcccaga ggaggaggagggaggatgcgagctgcgcgtgaagtttagccggtccgccgatgcacctgcatacaagcagggacagaaccagctgtat aacgagctgaatctgggccggagagaggagtacgacgtgctggataagaggcggggccgggaccccgagatgggaggcaagcctcg gagaaagaacccacaggagggcctgtacaatgagctgcaaaaggacaagatggccgaggcctattctgagatcggcatgaagggagag aggcgccggggcaagggacacgatggcctgtaccagggcctgagcaccgccacaaaggacacctatgatgccctgcacatgcaggcc ctgccccctagatga (VH-C; SEQ ID NO: 18005).

[023] In certain embodiments of the VCARs of the disclosure, the VH comprises or consists of the amino acid sequence malpvtalllplalllhaarpevqllesggglvqpggslrlscaasgftfsnyamtwirqapgkglewvsgitgdggstfyadsvkgrftis rdnskntlylqmnslraedtavyycvkdwnttmitergqgtlvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfac diyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynel nlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalp pr (VH-D; SEQ ID NO: 18006) or the VH comprises or consists of the nucleic acid sequence atggctctgcctgtgacagctctgctgctgcctctggctctgcttcttcatgcggcgcgccctgaagttcagctgcttgaatctggcggaggc ctggttcaacctggcggatctctgagactgagctgtgccgccagcggcttcaccttcagcaattacgccatgacctggatcagacaggccc ctggcaaaggcctggaatgggtgtccggaattacaggcgacggcggcagcaccttttacgccgattctgtgaagggcagattcaccatca gccgggacaacagcaagaacaccctgtacctgcagatgaacagcctgagagccgaggacaccgccgtgtactactgcgtgaaggactg gaacaccaccatgatcaccgagagaggccagggcacactggtcaccgtgtcctctacaacaacaccggcgcctcggcctccaacacca gctcctacaatcgcgagtcagcccctgtctctcagacccgaagcctgtagacctgctgctggcggagctgtgcataccagaggactggattt 2023208176 27 Jul 2023 cgcctgcgacatctacatctgggctcctctggctggcacatgcggagttttgctgctgagcctggtcatcaccctgtactgtaagagaggca ggaagaagctgctgtatatcttcaagcagcccttcatgagacccgtgcagaccacacaggaggaggacggctgctcttgtaggttcccaga ggaggaggagggaggatgcgagctgcgcgtgaagtttagccggtccgccgatgcacctgcatacaagcagggacagaaccagctgtat aacgagctgaatctgggccggagagaggagtacgacgtgctggataagaggcggggccgggaccccgagatgggaggcaagcctcg gagaaagaacccacaggagggcctgtacaatgagctgcaaaaggacaagatggccgaggcctattctgagatcggcatgaagggagag aggcgccggggcaagggacacgatggcctgtaccagggcctgagcaccgccacaaaggacacctatgatgccctgcacatgcaggcc ctgccccctagatga (VH-D; SEQ ID NO: 18007).

[024] In certain embodiments of the VCARs of the disclosure, the VH comprises or consists of the amino acid sequence malpvtalllplalllhaarpevqllesggglaqpggslrlscaasgftfssyamnwirqapgkglewvsgisgsggstyyadsvkgrfti srdnskntvylqmnslraedtavyycvkdwnttmitergqgtlvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfa cdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlyne Inlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqal ppr (VH-E; SEQ ID NO: 18008) or the VH comprises or consists of the nucleic acid sequence atggcactgcctgtgacagccctgctgctgcctctggccctgctgctgcacgcagcacggcccgaggtgcagctgctggagtccggagg aggcctggcccagcctggcggcagcctgaggctgtcctgcgccgcctctggcttcacctttagctcctacgccatgaactggatcagacag gcccctggcaagggcctggagtgggtgtccggcatctccggctctggaggctctacatactatgccgacagcgtgaagggccggttcacc atcagcagagataactccaagaataccgtgtacctccagatgaactctctgcgggccgaggacaccgccgtgtactattgcgtgaaggatt ggaataccacaatgatcacagagaggggccagggcaccctggtgacagtgtctagcaccacaacccctgcccccagacctcccacacc cgcccctaccatcgcgagtcagccactgtccctgcggcctgaggcctgccggcccgccgccggcggagcagtgcacacacggggcct ggactttgcctgtgacatctacatatgggcaccactggcaggaacctgcggcgtgctgctgctgagcctggtcatcaccctgtactgtaaga gaggcaggaagaagctgctgtatatcttcaagcagcccttcatgagacccgtgcagaccacacaggaggaggacggctgctcttgtaggt tcccagaggaggaggagggaggatgcgagctgcgcgtgaagtttagccggtccgccgatgcacctgcatacaagcagggacagaacc agctgtataacgagctgaatctgggccggagagaggagtacgacgtgctggataagaggcggggccgggaccccgagatgggaggca agcctcggagaaagaacccacaggagggcctgtacaatgagctgcaaaaggacaagatggccgaggcctattctgagatcggcatgaa gggagagaggcgccggggcaagggacacgatggcctgtaccagggcctgagcaccgccacaaaggacacctatgatgccctgcacat gcaggccctgccccctagatga (VH-E; SEQ ID NO: 18009).

[025] In certain embodiments of the VCARs of the disclosure, the VH comprises or consists of the amino acid sequence malpvtalllplalllhaarpevqllesggglvqpgrslrlscaasgftftnyamnwvrqapgkglewvsgisggggstyyadsvkgrfti 2023208176 27 Jul 2023 srdnskntlylqmnslraedtavyycvkdwnttmitergqgtlvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfac diyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynel nlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalp pr (VH-F; SEQ ID NO: 18010) or the VH comprises or consists of the nucleic acid sequence atggcactgcctgtgacagccctgctgctgcctctggccctgctgctgcacgcagcacggcccgaggtgcagctgctggagtctggagga ggcctggtgcagcccggccggtccctgagactgtcttgcgccgccagcggcttcacctttacaaactacgccatgaattgggtgcggcag gcccctggcaagggcctggagtgggtgtctggcatcagcggaggaggaggcagcacctactatgcagactccgtgaagggcaggttca ccatctcccgcgataactctaagaatacactgtacctccagatgaacagcctgagggcagaggacaccgccgtgtactattgcgtgaagga ttggaataccacaatgatcacagagaggggacagggcaccctggtgaccgtgagcagcaccacaacccctgcccccagacctcccaca cccgcccctaccatcgcgagtcagccactgtccctgcggcctgaggcctgccggcccgccgccggcggagcagtgcacacacggggc ctggactttgcctgtgacatctacatatgggcaccactggcaggaacctgcggcgtgctgctgctgagcctggtcatcaccctgtactgtaag agaggcaggaagaagctgctgtatatcttcaagcagcccttcatgagacccgtgcagaccacacaggaggaggacggctgctcttgtagg ttcccagaggaggaggagggaggatgcgagctgcgcgtgaagtttagccggtccgccgatgcacctgcatacaagcagggacagaacc agctgtataacgagctgaatctgggccggagagaggagtacgacgtgctggataagaggcggggccgggaccccgagatgggaggca agcctcggagaaagaacccacaggagggcctgtacaatgagctgcaaaaggacaagatggccgaggcctattctgagatcggcatgaa gggagagaggcgccggggcaagggacacgatggcctgtaccagggcctgagcaccgccacaaaggacacctatgatgccctgcacat gcaggccctgccccctagatga (VH-F; SEQ ID NO: 18011).

[026] In certain embodiments of the VCARs of the disclosure, the VCAR comprises a single domain antibody, VHH, VH or a combination thereof. In some embodiments, the single domain antibody, VHH or VH comprises or consists of a recombinant sequence and / or a chimeric sequence. In some embodiments, the single domain antibody, VHH or VH comprises or consists of a human sequence and / or a humanized sequence.

[027] In certain embodiments of the VCARs of the disclosure, the VCAR comprises a single domain antibody. In some embodiments, the single domain antibody is a VHH or a VH antibody. In some embodiments, the VH antibody is a UniDab antibody. In some embodiments, VH antibody is not a fragment of a naturally occurring monoclonal antibody.

[028] In certain embodiments of the VCARs of the disclosure, the signal peptide may comprise a sequence encoding a human CD2, CD38, CD3s, CD3y, CD3^, CD4, CD8a, CD 19, CD28, 4-1BB or GM-CSFR signal peptide. In certain embodiments of the VCARs of the disclosure, the signal peptide may comprise a sequence encoding a human CD8a signal peptide. The human 2023208176 27 Jul 2023 CD8a signal peptide may comprise an amino acid sequence comprising MALPVTALLLPLALLLHAARP (SEQ ID NO: 18012). The human CD8a signal peptide may comprise an amino acid sequence comprising MALPVTALLLPLALLLHAARP (SEQ ID NO: 18012) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the an amino acid sequence comprising MALPVTALLLPLALLLHAARP (SEQ ID NO: 18012). The human CD8a signal peptide may be encoded by a nucleic acid sequence comprising atggcactgccagtcaccgccctgctgctgcctctggctctgctgctgcacgcagctagacca (SEQ ID NO: 18013).

[029] In certain embodiments of the VCARs of the disclosure, the transmembrane domain may comprise a sequence encoding a human CD2, CD36, CD3s, CD3y, CD3^, CD4, CD8a, CD 19, CD28, 4-IBB or GM-CSFR transmembrane domain. In certain embodiments of the VCARs of the disclosure, the transmembrane domain may comprise a sequence encoding a human CD8a transmembrane domain. The CD8a transmembrane domain may comprise an amino acid sequence comprising IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 18014) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 18014). The CD8a transmembrane domain may be encoded by the nucleic acid sequence comprising atctacatttgggcaccactggccgggacctgtggagtgctgctgctgagcctggtcatcacactgtactgc (SEQ ID NO: 18015).

[030] In certain embodiments of the VCARs of the disclosure, the endodomain may comprise a human CD3^ endodomain.

[031] In certain embodiments of the VCARs of the disclosure, the at least one costimulatory domain may comprise a human 4-IBB, CD28, CD40, ICOS, MyD88, OX-40 intracellular segment, or any combination thereof. In certain embodiments of the VCARs of the disclosure, the at least one costimulatory domain may comprise a CD28 and / or a 4-IBB costimulatory domain. The CD3zeta costimulatory domain may comprise an amino acid sequence comprising RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGL YNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 18016) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGL YNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 2023208176 27 Jul 2023 (SEQ ID NO: 18016). The CD3zeta costimulatory domain may be encoded by the nucleic acid sequence comprising cgcgtgaagtttagtcgatcagcagatgccccagcttacaaacagggacagaaccagctgtataacgagctgaatctgggccgccgagag gaatatgacgtgctggataagcggagaggacgcgaccccgaaatgggaggcaagcccaggcgcaaaaaccctcaggaaggcctgtat aacgagctgcagaaggacaaaatggcagaagcctattctgagatcggcatgaagggggagcgacggagaggcaaagggcacgatgg gctgtaccagggactgagcaccgccacaaaggacacctatgatgctctgcatatgcaggcactgcctccaagg (SEQ ID NO: 18017). The 4-IBB costimulatory domain may comprise an amino acid sequence comprising KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 18018) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 18018). The 4-IBB costimulatory domain may be encoded by the nucleic acid sequence comprising aagagaggcaggaagaaactgctgtatattttcaaacagcccttcatgcgccccgtgcagactacccaggaggaagacgggtgctcctgt cgattccctgaggaagaggaaggcgggtgtgagctg (SEQ ID NO: 18019). The 4-IBB costimulatory domain may be located between the transmembrane domain and the CD28 costimulatory domain.

[032] In certain embodiments of the VCARs of the disclosure, the hinge may comprise a sequence derived from a human CD8a, IgG4, and / or CD4 sequence. In certain embodiments of the VCARs 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 TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 18020) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 18020). The human CD8a hinge amino acid sequence may be encoded by the nucleic acid sequence comprising actaccacaccagcacctagaccaccaactccagctccaaccatcgcgagtcagcccctgagtctgagacctgaggcctgcaggccagc tgcaggaggagctgtgcacaccaggggcctggacttcgcctgcgac (SEQ ID NO: 18021).

[033] VHHs and / or VCARs of the disclosure may bind an antigen with at least one affinity selected from a Kd of less than or equal to 10-9M, less than or equal to 10-10M, less than or equal to 10-1 XM, less than or equal to 10-12M, less than or equal to 10-13M, less than or equal to 10-14M, and less than or equal to 10-15M. The Kd may be determined by surface plasmon resonance. 2023208176 27 Jul 2023

[034] The disclosure provides an anti-BCMA VCAR. The disclosure provides a composition comprising the VCAR of the disclosure and at least one pharmaceutically acceptable carrier.

[035] The disclosure provides a transposon comprising the VCAR of the disclosure.

[036] Transposons of the disclosure may comprise a selection gene for identification, enrichment and / or isolation of 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 ( encoding O(6)-methylguanine-DNA methyltransferase), multidrug resistance gene (MDR1), ALDH1 (encoding Aldehyde dehydrogenase 1 family, member Al), FRANCE, RAD51C (encoding RAD51 Paralog C), GCS (encoding glucosylceramide synthase), and NKX2.2 (encoding NK2 Homeobox 2).

[037] Transposons of the disclosure may comprise an inducible proapoptotic polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a proapoptotic polypeptide, wherein the inducible proapoptotic polypeptide does not comprise a non-human sequence. In certain embodiments, the non-human sequence comprises a restriction site. In certain embodiments, the ligand binding region may be a multimeric ligand binding region. Inducible proapoptotic polypeptides of the disclosure may also be referred to as an “iC9 safety switch”. In certain embodiments, transposons of the disclosure may comprise an inducible caspase polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a caspase polypeptide, wherein the inducible proapoptotic polypeptide does not comprise a non-human sequence. In certain embodiments, transposons of the disclosure may comprise an inducible caspase polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a caspase polypeptide, wherein the inducible proapoptotic polypeptide does not comprise a non-human sequence. In certain embodiments, transposons of the disclosure may comprise an inducible caspase polypeptide 2023208176 27 Jul 2023 comprising (a) a ligand-binding region, (b) a linker, and (c) a truncated caspase 9 polypeptide, wherein the inducible proapoptotic polypeptide does not comprise a non-human sequence. In certain embodiments of the inducible proapoptotic polypeptides, inducible caspase polypeptides or truncated caspase 9 polypeptides of the disclosure, the ligand binding region may comprise a FK506 binding protein 12 (FKBP12) polypeptide. In certain embodiments, the amino acid sequence of the ligand binding region that comprise a FK506 binding protein 12 (FKBP12) polypeptide may comprise a modification at position 36 of the sequence. The modification may be a substitution of valine (V) for phenylalanine (F) at position 36 (F36V). In certain embodiments, the FKBP12 polypeptide is encoded by an amino acid sequence comprising GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRG WEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE (SEQ ID NO: 18022). In certain embodiments, the FKBP12 polypeptide is encoded by a nucleic acid sequence comprising GGGGTCCAGGTCGAGACTATTTCACCAGGGGATGGGCGAACATTTCCAAAAAGGGG CCAGACTTGCGTCGTGCATTACACCGGGATGCTGGAGGACGGGAAGAAAGTGGACA GCTCCAGGGATCGCAACAAGCCCTTCAAGTTCATGCTGGGAAAGCAGGAAGTGATC CGAGGATGGGAGGAAGGCGTGGCACAGATGTCAGTCGGCCAGCGGGCCAAACTGA CCATTAGCCCTGACTACGCTTATGGAGCAACAGGCCACCCAGGGATCATTCCCCCTC ATGCCACCCTGGTCTTCGAT GTGGAACTGCTGAAGCTGGAG (SEQ ID NO: 18023). In certain embodiments, the induction agent specific for the ligand binding region may comprise a FK506 binding protein 12 (FKBP12) polypeptide having a substitution of valine (V) for phenylalanine (F) at position 36 (F36V) comprises AP20187 and / or AP1903, both synthetic drugs.

[038] In certain embodiments of the inducible proapoptotic polypeptides, inducible caspase polypeptides or truncated caspase 9 polypeptides of the disclosure, the linker region is encoded by an amino acid comprising GGGGS (SEQ ID NO: 18024) or a nucleic acid sequence comprising GGAGGAGGAGGATCC (SEQ ID NO: 18025). In certain embodiments, the nucleic acid sequence encoding the linker does not comprise a restriction site.

[039] In certain embodiments of the truncated caspase 9 polypeptides of the disclosure, the truncated caspase 9 polypeptide is encoded by an amino acid sequence that does not comprise an arginine (R) at position 87 of the sequence. Alternatively, or in addition, in certain embodiments 2023208176 27 Jul 2023 of the inducible proapoptotic polypeptides, inducible caspase polypeptides or truncated caspase 9 polypeptides of the disclosure, the truncated caspase 9 polypeptide is encoded by an amino acid sequence that does not comprise an alanine (A) at position 282 the sequence. In certain embodiments of the inducible proapoptotic polypeptides, inducible caspase polypeptides or truncated caspase 9 polypeptides of the disclosure, the truncated caspase 9 polypeptide is encoded by an amino acid comprising GFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRF SSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPGAVY GTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNP EPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQ WAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS (SEQ ID NO: 18026) or a nucleic acid sequence comprising TTTGGGGACGTGGGGGCCCTGGAGTCTCTGCGAGGAAATGCCGATCTGGCTTACATC CTGAGCATGGAACCCTGCGGCCACTGTCTGATCATTAACAATGTGAACTTCTGCAGA GAAAGCGGACTGCGAACACGGACTGGCTCCAATATTGACTGTGAGAAGCTGCGGAG AAGGTTCTCTAGTCTGCACTTTATGGTCGAAGTGAAAGGGGATCTGACCGCCAAGA AAATGGTGCTGGCCCTGCTGGAGCTGGCTCAGCAGGACCATGGAGCTCTGGATTGCT GCGTGGTCGTGATCCTGTCCCACGGGTGCCAGGCTTCTCATCTGCAGTTCCCCGGAG CAGTGTACGGAACAGACGGCTGTCCTGTCAGCGTGGAGAAGATCGTCAACATCTTC AACGGCACTTCTTGCCCTAGTCTGGGGGGAAAGCCAAAACTGTTCTTTATCCAGGCC TGTGGCGGGGAACAGAAAGATCACGGCTTCGAGGTGGCCAGCACCAGCCCTGAGGA CGAATCACCAGGGAGCAACCCTGAACCAGATGCAACTCCATTCCAGGAGGGACTGA GGACCTTTGACCAGCTGGATGCTATCTCAAGCCTGCCCACTCCTAGTGACATTTTCG TGTCTTACAGTACCTTCCCAGGCTTTGTCTCATGGCGCGATCCCAAGTCAGGGAGCT GGTACGTGGAGACACTGGACGACATCTTTGAACAGTGGGCCCATTCAGAGGACCTG CAGAGCCTGCTGCTGCGAGTGGCAAACGCTGTCTCTGTGAAGGGCATCTACAAACA GATGCCCGGGTGCTTCAATTTTCTGAGAAAGAAACTGTTCTTTAAGACTTCC (SEQ ID NO: 18027).

[040] In certain embodiments of the inducible proapoptotic polypeptides, wherein the polypeptide comprises a truncated caspase 9 polypeptide, the inducible proapoptotic polypeptide is encoded by an amino acid sequence comprising 2023208176 27 Jul 2023 GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRG WEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGGSGFGDV GALESLRGNADL AYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRF S SLHF MVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGC PVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDAT PFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSE DLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS (SEQ ID NO: 18028) or the nucleic acid sequence comprising ggggtccaggtcgagactatttcaccaggggatgggcgaacatttccaaaaaggggccagacttgcgtcgtgcattacaccgggatgctg gaggacgggaagaaagtggacagctccagggatcgcaacaagcccttcaagttcatgctgggaaagcaggaagtgatccgaggatggg aggaaggcgtggcacagatgtcagtcggccagcgggccaaactgaccattagccctgactacgcttatggagcaacaggccacccagg gatcattccccctcatgccaccctggtcttcgatgtggaactgctgaagctggagggaggaggaggatccggatttggggacgtgggggc cctggagtctctgcgaggaaatgccgatctggcttacatcctgagcatggaaccctgcggccactgtctgatcattaacaatgtgaacttctg cagagaaagcggactgcgaacacggactggctccaatattgactgtgagaagctgcggagaaggttctctagtctgcactttatggtcgaa gtgaaaggggatctgaccgccaagaaaatggtgctggccctgctggagctggctcagcaggaccatggagctctggattgctgcgtggtc gtgatcctgtcccacgggtgccaggcttctcatctgcagttccccggagcagtgtacggaacagacggctgtcctgtcagcgtggagaaga tcgtcaacatcttcaacggcacttcttgccctagtctggggggaaagccaaaactgttctttatccaggcctgtggcggggaacagaaagat cacggcttcgaggtggccagcaccagccctgaggacgaatcaccagggagcaaccctgaaccagatgcaactccattccaggagggac tgaggacctttgaccagctggatgctatctcaagcctgcccactcctagtgacattttcgtgtcttacagtaccttcccaggctttgtctcatggc gcgatcccaagtcagggagctggtacgtggagacactggacgacatctttgaacagtgggcccattcagaggacctgcagagcctgctgc tgcgagtggcaaacgctgtctctgtgaagggcatctacaaacagatgcccgggtgcttcaattttctgagaaagaaactgttctttaagacttc c (SEQ ID NO: 18029).

[041] Transposons of the disclosure may comprise at least one self-cleaving peptide(s) located, for example, between one or more VHH(s) or VCAR(s) of the disclosure and a selection gene of the disclosure. Transposons of the disclosure may comprise at least one self-cleaving peptide(s) located, for example, between one or more VHH(s) or VCAR(s) of the disclosure and an inducible proapoptotic polypeptide of the disclosure. Transposons of the disclosure may comprise at least two self-cleaving peptide(s), a first self-cleaving peptide located, for example, upstream or immediately upstream of an inducible proapoptotic polypeptide of the disclosure and a second first self-cleaving peptide located, for example, downstream or immediately upstream of an inducible proapoptotic polypeptide of the disclosure. 2023208176 27 Jul 2023

[042] The at least one 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 amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18030) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18030). A GSG-T2A peptide may comprise an amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 18031) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 18031). A GSG-T2A peptide may comprise a nucleic acid sequence comprising ggatctggagagggaaggggaagcctgctgacctgtggagacgtggaggaaaacccaggacca (SEQ ID NO: 18032). An E2A peptide may comprise an amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO: 18033) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO: 18033). A GSG-E2A peptide may comprise an amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 18034) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 18034). An F2A peptide may comprise an amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 18035) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 18035). A GSG-F2A peptide may comprise an amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 18036) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 18036). A P2A peptide may comprise an amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 18037) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 18037). A GSG-P2A peptide may comprise an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 18038) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 18038). 2023208176 27 Jul 2023

[043] 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 VHH(s) or VCAR(s) of the disclosure the second self-cleaving peptide located, for example, downstream of the one or more VHH(s) or VCAR(s) 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 amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18030) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18030). A GSG-T2A peptide may comprise an amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 18031) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 18031). A GSG-T2A peptide may comprise a nucleic acid sequence comprising ggatctggagagggaaggggaagcctgctgacctgtggagacgtggaggaaaacccaggacca (SEQ ID NO: 18032). An E2A peptide may comprise an amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO: 18033) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO: 18033). A GSG-E2A peptide may comprise an amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 18034) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 18034). An F2A peptide may comprise an amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 18035) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 18035). A GSG-F2A peptide may comprise an amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 18036) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 18036). A P2A peptide may comprise an amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 18037) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence 2023208176 27 Jul 2023 comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 18037). A GSG-P2A peptide may comprise an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 18038) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 18038).

[044] The disclosure provides a composition comprising the transposon the disclosure. In certain embodiments, the composition may further comprise a plasmid comprising a sequence encoding a transposase enzyme. The sequence encoding a transposase enzyme may be an mRNA sequence.

[045] Transposons of the disclosure may comprise piggyBac transposons. In certain embodiments of this method, the transposon is a plasmid DNA transposon with a sequence encoding the chimeric antigen receptor flanked by two cis-regulatory insulator elements. In certain embodiments, the transposon is a piggyBac transposon. Transposase enzymes of the disclosure may include piggyBac transposases or compatible enzymes. In certain embodiments, and, in particular, those embodiments wherein the transposon is a piggyBac transposon, the transposase is a piggyBac or a Super piggyBac (SPB) transposase. In certain embodiments, and, in particular, those embodiments wherein the transposase is a Super piggyBac (SPB) transposase, the sequence encoding the transposase is an mRNA sequence.

[046] In certain embodiments of the methods of the disclosure, the transposase enzyme is a piggyBac (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 LSALLQSDDE LVGEDSDSEI SDHVSEDDVQ SDTEEAFIDE VHEVQPTSSG 61 SEILDEQNVI EQPGSSLASN RILTLPQRTI RGKNKHCWST SKSTRRSRVS ALNIVRSQRG 121 PTRMCRNIYD PLLCFKLFFT DEIISEIVKW TNAEISLKRR ESMTGATFRD TNEDEIYAFF 181 GILVMTAVRK DNHMSTDDLF DRSLSMVYVS VMSRDRFDFL IRCLRMDDKS IRPTLRENDV 241 FTPVRKIWDL FIHQCIQNYT PGAHLTIDEQ LLGFRGRCPF RMYIPNKPSK YGIKILMMCD 301 SGTKYMINGM PYLGRGTQTN GVPLGEYYVK ELSKPVHGSC RNITCDNWFT SIPLAKNLLQ 361 EPYKLTIVGT VRSNKREIPE VLKNSRSRPV GTSMFCFDGP LTLVSYKPKP AKMVYLLSSC 421 DEDASINEST GKPQMVMYYN QTKGGVDTLD QMCSVMTCSR KTNRWPMALL YGMINIACIN 481 SFIIYSHNVS SKGEKVQSRK KFMRNLYMSL TSSFMRKRLE APTLKRYLRD NISNILPNEV 541 PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV ICREHNIDMC QSCF (SEQ ID NO: 14487) . 2023208176 27 Jul 2023

[047] In certain embodiments of the methods of the disclosure, the transposase enzyme is a piggyBac (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 SDHVSEDDVQ SDTEEAFIDE VHEVQPTSSG 61 SEILDEQNVI EQPGSSLASN RILTLPQRTI RGKNKHCWST SKSTRRSRVS ALNIVRSQRG 121 PTRMCRNIYD PLLCFKLFFT DEIISEIVKW TNAEISLKRR ESMTGATFRD TNEDEIYAFF 181 GILVMTAVRK DNHMSTDDLF DRSLSMVYVS VMSRDRFDFL IRCLRMDDKS IRPTLRENDV 241 FTPVRKIWDL FIHQCIQNYT PGAHLTIDEQ LLGFRGRCPF RMYIPNKPSK YGIKILMMCD 301 SGTKYMINGM PYLGRGTQTN GVPLGEYYVK ELSKPVHGSC RNITCDNWFT SIPLAKNLLQ 361 EPYKLTIVGT VRSNKREIPE VLKNSRSRPV GTSMFCFDGP LTLVSYKPKP AKMVYLLSSC 421 DEDASINEST GKPQMVMYYN QTKGGVDTLD QMCSVMTCSR KTNRWPMALL YGMINIACIN 481 SFIIYSHNVS SKGEKVQSRK KFMRNLYMSL TSSFMRKRLE APTLKRYLRD NISNILPNEV 541 PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV ICREHNIDMC QSCF (SEQ ID NO: 14487) .

[048] In certain embodiments, the transposase enzyme is a piggyBac (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: 14487. In certain embodiments, the transposase enzyme is a piggyBac (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: 14487. In certain embodiments, the transposase enzyme is a piggyBac (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: 14487. In certain embodiments, the amino acid substitution at position 30 of the sequence of SEQ ID NO: 14487 is a substitution of a valine (V) for an isoleucine (I). In certain embodiments, the amino acid substitution at position 165 of the sequence of SEQ ID NO: 14487 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: 14487 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: 14487 is a substitution of a lysine (K) for an asparagine (N).

[049] In certain embodiments of the methods of the disclosure, the transposase enzyme is a Super piggyBac (SPB) transposase enzyme. In certain embodiments, the Super piggyBac (SPB) transposase enzymes of the disclosure may comprise or consist of the amino acid sequence of the 2023208176 27 Jul 2023 sequence of SEQ ID NO: 14487 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 piggyBac (SPB) 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 LSALLQSDDE LVGEDSDSEV SDHVSEDDVQ SDTEEAFIDE VHEVQPTSSG 61 SEILDEQNVI EQPGSSLASN RILTLPQRTI RGKNKHCWST SKSTRRSRVS ALNIVRSQRG 121 PTRMCRNIYD PLLCFKLFFT DEIISEIVKW TNAEISLKRR ESMTSATFRD TNEDEIYAFF 181 GILVMTAVRK DNHMSTDDLF DRSLSMVYVS VMSRDRFDFL IRCLRMDDKS IRPTLRENDV 241 FTPVRKIWDL FIHQCIQNYT PGAHLTIDEQ LLGFRGRCPF RVYIPNKPSK YGIKILMMCD 301 SGTKYMINGM PYLGRGTQTN GVPLGEYYVK ELSKPVHGSC RNITCDNWFT SIPLAKNLLQ 361 EPYKLTIVGT VRSNKREIPE VLKNSRSRPV GTSMFCFDGP LTLVSYKPKP AKMVYLLSSC 421 DEDASINEST GKPQMVMYYN QTKGGVDTLD QMCSVMTCSR KTNRWPMALL YGMINIACIN 481 SFIIYSHNVS SKGEKVQSRK KFMRNLYMSL TSSFMRKRLE APTLKRYLRD NISNILPKEV 541 PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV ICREHNIDMC QSCF (SEQ ID NO: 14484) .

[050] The disclosure provides a vector comprising the VCAR of the disclosure. In certain embodiments, the vector is a viral vector. The vector may be a recombinant vector.

[051] 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 VHH or VCAR 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., AAV1, 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- 2023208176 27 Jul 2023 associated viruses and recombinant adeno-associated viruses of the disclosure include, but are not limited to, rAAV-LK03.

[052] 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 Reductase and conferring resistance to Methotrexate), TYMS (encoding Thymidylate Synthetase), MGMT (encoding 0(6)-methylguanine-DNA methyltransferase), multidrug resistance gene (MDR1), ALDH1 (encoding Aldehyde dehydrogenase 1 family, member Al), FRANCF, RAD51C (encoding RAD51 Paralog C), GCS (encoding glucosylceramide synthase), NKX2.2 (encoding NK2 Homeobox 2) or any combination thereof.

[053] Viral vectors of the disclosure may comprise an inducible proapoptotic polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a proapoptotic polypeptide, wherein the inducible proapoptotic polypeptide does not comprise a non-human sequence. In certain embodiments, the non-human sequence comprises a restriction site. In certain embodiments, the ligand binding region may be a multimeric ligand binding region. Inducible proapoptotic polypeptides of the disclosure may also be referred to as an “iC9 safety switch”. In certain embodiments, viral vectors of the disclosure may comprise an inducible caspase polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a caspase polypeptide, wherein the inducible proapoptotic polypeptide does not comprise a non-human sequence. In certain embodiments, viral vectors of the disclosure may comprise an inducible caspase polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a caspase polypeptide, wherein the inducible proapoptotic polypeptide does not comprise a non-human sequence. In certain embodiments, viral vectors of the disclosure may comprise an inducible caspase polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a truncated caspase 9 polypeptide, wherein the inducible proapoptotic polypeptide does not comprise a non-human sequence. In certain embodiments of the inducible proapoptotic polypeptides, inducible caspase polypeptides or truncated caspase 9 polypeptides of the disclosure, the ligand binding region may comprise a 2023208176 27 Jul 2023 FK506 binding protein 12 (FKBP12) polypeptide. In certain embodiments, the amino acid sequence of the ligand binding region that comprise a FK506 binding protein 12 (FKBP12) polypeptide may comprise a modification at position 36 of the sequence. The modification may be a substitution of valine (V) for phenylalanine (F) at position 36 (F36V). In certain embodiments, the FKBP12 polypeptide is encoded by an amino acid sequence comprising GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRG WEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE (SEQ ID NO: 18022). In certain embodiments, the FKBP12 polypeptide is encoded by a nucleic acid sequence comprising GGGGTCCAGGTCGAGACTATTTCACCAGGGGATGGGCGAACATTTCCAAAAAGGGG CCAGACTTGCGTCGTGCATTACACCGGGATGCTGGAGGACGGGAAGAAAGTGGACA GCTCCAGGGATCGCAACAAGCCCTTCAAGTTCATGCTGGGAAAGCAGGAAGTGATC CGAGGATGGGAGGAAGGCGTGGCACAGATGTCAGTCGGCCAGCGGGCCAAACTGA CCATTAGCCCTGACTACGCTTATGGAGCAACAGGCCACCCAGGGATCATTCCCCCTC ATGCCACCCTGGTCTTCGAT GTGGAACTGCTGAAGCTGGAG (SEQ ID NO: 18023). In certain embodiments, the induction agent specific for the ligand binding region may comprise a FK506 binding protein 12 (FKBP12) polypeptide having a substitution of valine (V) for phenylalanine (F) at position 36 (F36V) comprises AP20187 and / or AP1903, both synthetic drugs.

[054] In certain embodiments of the inducible proapoptotic polypeptides, inducible caspase polypeptides or truncated caspase 9 polypeptides of the disclosure, the linker region is encoded by an amino acid comprising GGGGS (SEQ ID NO: 18024) or a nucleic acid sequence comprising GGAGGAGGAGGATCC (SEQ ID NO: 18025). In certain embodiments, the nucleic acid sequence encoding the linker does not comprise a restriction site.

[055] In certain embodiments of the truncated caspase 9 polypeptides of the disclosure, the truncated caspase 9 polypeptide is encoded by an amino acid sequence that does not comprise an arginine (R) at position 87 of the sequence. Alternatively, or in addition, in certain embodiments of the inducible proapoptotic polypeptides, inducible caspase polypeptides or truncated caspase 9 polypeptides of the disclosure, the truncated caspase 9 polypeptide is encoded by an amino acid sequence that does not comprise an alanine (A) at position 282 the sequence. In certain embodiments of the inducible proapoptotic polypeptides, inducible caspase polypeptides or 2023208176 27 Jul 2023 truncated caspase 9 polypeptides of the disclosure, the truncated caspase 9 polypeptide is encoded by an amino acid comprising GFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRF SSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPGAVY GTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNP EPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQ WAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS (SEQ ID NO: 18026) or a nucleic acid sequence comprising TTTGGGGACGTGGGGGCCCTGGAGTCTCTGCGAGGAAATGCCGATCTGGCTTACATC CTGAGCATGGAACCCTGCGGCCACTGTCTGATCATTAACAATGTGAACTTCTGCAGA GAAAGCGGACTGCGAACACGGACTGGCTCCAATATTGACTGTGAGAAGCTGCGGAG AAGGTTCTCTAGTCTGCACTTTATGGTCGAAGTGAAAGGGGATCTGACCGCCAAGA AAATGGTGCTGGCCCTGCTGGAGCTGGCTCAGCAGGACCATGGAGCTCTGGATTGCT GCGTGGTCGTGATCCTGTCCCACGGGTGCCAGGCTTCTCATCTGCAGTTCCCCGGAG CAGTGTACGGAACAGACGGCTGTCCTGTCAGCGTGGAGAAGATCGTCAACATCTTC AACGGCACTTCTTGCCCTAGTCTGGGGGGAAAGCCAAAACTGTTCTTTATCCAGGCC TGTGGCGGGGAACAGAAAGATCACGGCTTCGAGGTGGCCAGCACCAGCCCTGAGGA CGAATCACCAGGGAGCAACCCTGAACCAGATGCAACTCCATTCCAGGAGGGACTGA GGACCTTTGACCAGCTGGATGCTATCTCAAGCCTGCCCACTCCTAGTGACATTTTCG TGTCTTACAGTACCTTCCCAGGCTTTGTCTCATGGCGCGATCCCAAGTCAGGGAGCT GGTACGTGGAGACACTGGACGACATCTTTGAACAGTGGGCCCATTCAGAGGACCTG CAGAGCCTGCTGCTGCGAGTGGCAAACGCTGTCTCTGTGAAGGGCATCTACAAACA GATGCCCGGGTGCTTCAATTTTCTGAGAAAGAAACTGTTCTTTAAGACTTCC (SEQ ID NO: 18027).

[056] In certain embodiments of the inducible proapoptotic polypeptides, wherein the polypeptide comprises a truncated caspase 9 polypeptide, the inducible proapoptotic polypeptide is encoded by an amino acid sequence comprising GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRG WEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGGSGFGDV GALESLRGNADL AYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRF S SLHF MVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGC 2023208176 27 Jul 2023 PVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDAT PFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSE DLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS (SEQ ID NO: 18028) or the nucleic acid sequence comprising ggggtccaggtcgagactatttcaccaggggatgggcgaacatttccaaaaaggggccagacttgcgtcgtgcattacaccgggatgctg gaggacgggaagaaagtggacagctccagggatcgcaacaagcccttcaagttcatgctgggaaagcaggaagtgatccgaggatggg aggaaggcgtggcacagatgtcagtcggccagcgggccaaactgaccattagccctgactacgcttatggagcaacaggccacccagg gatcattccccctcatgccaccctggtcttcgatgtggaactgctgaagctggagggaggaggaggatccggatttggggacgtgggggc cctggagtctctgcgaggaaatgccgatctggcttacatcctgagcatggaaccctgcggccactgtctgatcattaacaatgtgaacttctg cagagaaagcggactgcgaacacggactggctccaatattgactgtgagaagctgcggagaaggttctctagtctgcactttatggtcgaa gtgaaaggggatctgaccgccaagaaaatggtgctggccctgctggagctggctcagcaggaccatggagctctggattgctgcgtggtc gtgatcctgtcccacgggtgccaggcttctcatctgcagttccccggagcagtgtacggaacagacggctgtcctgtcagcgtggagaaga tcgtcaacatcttcaacggcacttcttgccctagtctggggggaaagccaaaactgttctttatccaggcctgtggcggggaacagaaagat cacggcttcgaggtggccagcaccagccctgaggacgaatcaccagggagcaaccctgaaccagatgcaactccattccaggagggac tgaggacctttgaccagctggatgctatctcaagcctgcccactcctagtgacattttcgtgtcttacagtaccttcccaggctttgtctcatggc gcgatcccaagtcagggagctggtacgtggagacactggacgacatctttgaacagtgggcccattcagaggacctgcagagcctgctgc tgcgagtggcaaacgctgtctctgtgaagggcatctacaaacagatgcccgggtgcttcaattttctgagaaagaaactgttctttaagacttc c (SEQ ID NO: 18029).

[057] 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 selfcleaving 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. Viral vectors of the disclosure may comprise at least one self-cleaving peptide(s) located, for example, between one or more of a VCAR, VCAR or VCAR of the disclosure and an inducible proapoptotic polypeptide of the disclosure. Viral vectors of the disclosure may comprise at least two self-cleaving peptide(s), a first self-cleaving peptide located, for example, upstream or immediately upstream of an inducible proapoptotic polypeptide of the disclosure and a second first self-cleaving peptide located, for example, downstream or immediately upstream of an inducible proapoptotic polypeptide of the disclosure. The self-cleaving peptide may comprise, for example, a T2A peptide, GSG-T2A peptide, an E2A peptide, a GSG-E2A 2023208176 27 Jul 2023 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: 18030) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18030). A GSG-T2A peptide may comprise an amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 18031) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 18031). A GSG-T2A peptide may comprise a nucleic acid sequence comprising ggatctggagagggaaggggaagcctgctgacctgtggagacgtggaggaaaacccaggacca (SEQ ID NO: 18032). An E2A peptide may comprise an amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO: 18033) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO: 18033). A GSG-E2A peptide may comprise an amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 18034) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 18034). An F2A peptide may comprise an amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 18035) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 18035). A GSG-F2A peptide may comprise an amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 18036) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 18036). A P2A peptide may comprise an amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 18037) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 18037). A GSG-P2A peptide may comprise an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 18038) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 18038).

[058] The disclosure provides a vector comprising the VCAR of the disclosure. In certain embodiments, the vector is a nanoparticle. Exemplary nanoparticle vectors of the disclosure 2023208176 27 Jul 2023 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 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.

[059] 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 0(6)-methylguanine-DNA methyltransferase), multidrug resistance gene (MDR1), ALDH1 (encoding Aldehyde dehydrogenase 1 family, member Al), FRANCE, RAD51C (encoding RAD51 Paralog C), GCS (encoding glucosylceramide synthase), NKX2.2 (encoding NK2 Homeobox 2) or any combination thereof.

[060] Nanoparticle vectors of the disclosure may comprise an inducible proapoptotic polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a proapoptotic polypeptide, wherein the inducible proapoptotic polypeptide does not comprise a non-human sequence. In certain embodiments, the non-human sequence comprises a restriction site. In certain embodiments, the ligand binding region may be a multimeric ligand binding region. Inducible proapoptotic polypeptides of the disclosure may also be referred to as an “iC9 safety switch”. In certain embodiments, nanoparticle vectors of the disclosure may comprise an inducible caspase polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a caspase polypeptide, wherein the inducible proapoptotic polypeptide does not comprise a nonhuman sequence. In certain embodiments, nanoparticle vectors of the disclosure may comprise an inducible caspase polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a caspase polypeptide, wherein the inducible proapoptotic polypeptide does not comprise a nonhuman sequence. In certain embodiments, nanoparticle vectors of the disclosure may comprise 2023208176 27 Jul 2023 an inducible caspase polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a truncated caspase 9 polypeptide, wherein the inducible proapoptotic polypeptide does not comprise a non-human sequence. In certain embodiments of the inducible proapoptotic polypeptides, inducible caspase polypeptides or truncated caspase 9 polypeptides of the disclosure, the ligand binding region may comprise a FK506 binding protein 12 (FKBP12) polypeptide. In certain embodiments, the amino acid sequence of the ligand binding region that comprise a FK506 binding protein 12 (FKBP12) polypeptide may comprise a modification at position 36 of the sequence. The modification may be a substitution of valine (V) for phenylalanine (F) at position 36 (F36V). In certain embodiments, the FKBP12 polypeptide is encoded by an amino acid sequence comprising GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRG WEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE (SEQ ID NO: 18022). In certain embodiments, the FKBP12 polypeptide is encoded by a nucleic acid sequence comprising GGGGTCCAGGTCGAGACTATTTCACCAGGGGATGGGCGAACATTTCCAAAAAGGGG CCAGACTTGCGTCGTGCATTACACCGGGATGCTGGAGGACGGGAAGAAAGTGGACA GCTCCAGGGATCGCAACAAGCCCTTCAAGTTCATGCTGGGAAAGCAGGAAGTGATC CGAGGATGGGAGGAAGGCGTGGCACAGATGTCAGTCGGCCAGCGGGCCAAACTGA CCATTAGCCCTGACTACGCTTATGGAGCAACAGGCCACCCAGGGATCATTCCCCCTC ATGCCACCCTGGTCTTCGAT GTGGAACTGCTGAAGCTGGAG (SEQ ID NO: 18023). In certain embodiments, the induction agent specific for the ligand binding region may comprise a FK506 binding protein 12 (FKBP12) polypeptide having a substitution of valine (V) for phenylalanine (F) at position 36 (F36V) comprises AP20187 and / or AP1903, both synthetic drugs.

[061] In certain embodiments of the inducible proapoptotic polypeptides, inducible caspase polypeptides or truncated caspase 9 polypeptides of the disclosure, the linker region is encoded by an amino acid comprising GGGGS (SEQ ID NO: 18024) or a nucleic acid sequence comprising GGAGGAGGAGGATCC (SEQ ID NO: 18025). In certain embodiments, the nucleic acid sequence encoding the linker does not comprise a restriction site.

[062] In certain embodiments of the truncated caspase 9 polypeptides of the disclosure, the truncated caspase 9 polypeptide is encoded by an amino acid sequence that does not comprise an 2023208176 27 Jul 2023 arginine (R) at position 87 of the sequence. Alternatively, or in addition, in certain embodiments of the inducible proapoptotic polypeptides, inducible caspase polypeptides or truncated caspase 9 polypeptides of the disclosure, the truncated caspase 9 polypeptide is encoded by an amino acid sequence that does not comprise an alanine (A) at position 282 the sequence. In certain embodiments of the inducible proapoptotic polypeptides, inducible caspase polypeptides or truncated caspase 9 polypeptides of the disclosure, the truncated caspase 9 polypeptide is encoded by an amino acid comprising GFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRF SSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPGAVY GTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNP EPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQ WAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS (SEQ ID NO: 18026) or a nucleic acid sequence comprising TTTGGGGACGTGGGGGCCCTGGAGTCTCTGCGAGGAAATGCCGATCTGGCTTACATC CTGAGCATGGAACCCTGCGGCCACTGTCTGATCATTAACAATGTGAACTTCTGCAGA GAAAGCGGACTGCGAACACGGACTGGCTCCAATATTGACTGTGAGAAGCTGCGGAG AAGGTTCTCTAGTCTGCACTTTATGGTCGAAGTGAAAGGGGATCTGACCGCCAAGA AAATGGTGCTGGCCCTGCTGGAGCTGGCTCAGCAGGACCATGGAGCTCTGGATTGCT GCGTGGTCGTGATCCTGTCCCACGGGTGCCAGGCTTCTCATCTGCAGTTCCCCGGAG CAGTGTACGGAACAGACGGCTGTCCTGTCAGCGTGGAGAAGATCGTCAACATCTTC AACGGCACTTCTTGCCCTAGTCTGGGGGGAAAGCCAAAACTGTTCTTTATCCAGGCC TGTGGCGGGGAACAGAAAGATCACGGCTTCGAGGTGGCCAGCACCAGCCCTGAGGA CGAATCACCAGGGAGCAACCCTGAACCAGATGCAACTCCATTCCAGGAGGGACTGA GGACCTTTGACCAGCTGGATGCTATCTCAAGCCTGCCCACTCCTAGTGACATTTTCG TGTCTTACAGTACCTTCCCAGGCTTTGTCTCATGGCGCGATCCCAAGTCAGGGAGCT GGTACGTGGAGACACTGGACGACATCTTTGAACAGTGGGCCCATTCAGAGGACCTG CAGAGCCTGCTGCTGCGAGTGGCAAACGCTGTCTCTGTGAAGGGCATCTACAAACA GATGCCCGGGTGCTTCAATTTTCTGAGAAAGAAACTGTTCTTTAAGACTTCC (SEQ ID NO: 18027).

[063] In certain embodiments of the inducible proapoptotic polypeptides, wherein the polypeptide comprises a truncated caspase 9 polypeptide, the inducible proapoptotic polypeptide 2023208176 27 Jul 2023 is encoded by an amino acid sequence comprising GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRG WEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGGSGFGDV GALESLRGNADL AYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRF S SLHF MVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGC PVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDAT PFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSE DLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS (SEQ ID NO: 18028) or the nucleic acid sequence comprising ggggtccaggtcgagactatttcaccaggggatgggcgaacatttccaaaaaggggccagacttgcgtcgtgcattacaccgggatgctg gaggacgggaagaaagtggacagctccagggatcgcaacaagcccttcaagttcatgctgggaaagcaggaagtgatccgaggatggg aggaaggcgtggcacagatgtcagtcggccagcgggccaaactgaccattagccctgactacgcttatggagcaacaggccacccagg gatcattccccctcatgccaccctggtcttcgatgtggaactgctgaagctggagggaggaggaggatccggatttggggacgtgggggc cctggagtctctgcgaggaaatgccgatctggcttacatcctgagcatggaaccctgcggccactgtctgatcattaacaatgtgaacttctg cagagaaagcggactgcgaacacggactggctccaatattgactgtgagaagctgcggagaaggttctctagtctgcactttatggtcgaa gtgaaaggggatctgaccgccaagaaaatggtgctggccctgctggagctggctcagcaggaccatggagctctggattgctgcgtggtc gtgatcctgtcccacgggtgccaggcttctcatctgcagttccccggagcagtgtacggaacagacggctgtcctgtcagcgtggagaaga tcgtcaacatcttcaacggcacttcttgccctagtctggggggaaagccaaaactgttctttatccaggcctgtggcggggaacagaaagat cacggcttcgaggtggccagcaccagccctgaggacgaatcaccagggagcaaccctgaaccagatgcaactccattccaggagggac tgaggacctttgaccagctggatgctatctcaagcctgcccactcctagtgacattttcgtgtcttacagtaccttcccaggctttgtctcatggc gcgatcccaagtcagggagctggtacgtggagacactggacgacatctttgaacagtgggcccattcagaggacctgcagagcctgctgc tgcgagtggcaaacgctgtctctgtgaagggcatctacaaacagatgcccgggtgcttcaattttctgagaaagaaactgttctttaagacttc c (SEQ ID NO: 18029).

[064] 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 VCAR 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 VCAR and a second self-cleaving peptide is located downstream of a VCAR. 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 VCAR and the nanoparticle and a second self-cleaving peptide is located downstream 2023208176 27 Jul 2023 of the VCAR. In some embodiments, the nanoparticle vector may comprise at least one selfcleaving peptide and wherein a first self-cleaving peptide is located between a VCAR and the nanoparticle and a second self-cleaving peptide is located downstream of the VCAR, for example, between the VCAR and a selection gene.

[065] Nanoparticle vectors of the disclosure may comprise at least one self-cleaving peptide(s) located, for example, between one or more VHH(s) or VCAR(s) of the disclosure and an inducible proapoptotic polypeptide of the disclosure. Nanoparticle vectors of the disclosure may comprise at least two self-cleaving peptide(s), a first self-cleaving peptide located, for example, upstream or immediately upstream of an inducible proapoptotic polypeptide of the disclosure and a second first self-cleaving peptide located, for example, downstream or immediately upstream of an inducible proapoptotic polypeptide of the disclosure. 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. A T2A peptide may comprise an amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18030) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18030). A GSG-T2A peptide may comprise an amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 18031) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 18031). A GSG-T2A peptide may comprise a nucleic acid sequence comprising ggatctggagagggaaggggaagcctgctgacctgtggagacgtggaggaaaacccaggacca (SEQ ID NO: 18032). An E2A peptide may comprise an amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO: 18033) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO: 18033). A GSG-E2A peptide may comprise an amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 18034) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 18034). An F2A peptide may comprise an amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 18035) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 18035). A GSG-F2A 2023208176 27 Jul 2023 peptide may comprise an amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 18036) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 18036). A P2A peptide may comprise an amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 18037) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 18037). A GSG-P2A peptide may comprise an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 18038) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 18038).

[066] The disclosure provides a composition comprising a vector of the disclosure.

[067] The disclosure provides a cell comprising a VCAR of the disclosure. The disclosure provides a cell comprising a transposon of the disclosure. In certain embodiments, the cell comprising a VCAR, a transposon, or a vector of the disclosure may express a VCAR on the cell surface. The cell may be any type of cell. Preferably, the cell is an immune cell. The immune cell may be a T-cell, a Natural Killer (NK) cell, a Natural Killer (NK)-like cell, a Cytokine Induced Killer (CIK) cell, a hematopoeitic progenitor cell, a peripheral blood (PB) derived T cell or an umbilical cord blood (UCB) derived T-cell. Preferably, the immune cell is a T-cell. The T-cell may be an early memory cell, a stem-like T-cell, a TscM-like cell, a Tscm or a Tcm. The T-cell may be a Tscm. The cell may be an artificial antigen presenting cell, which, optionally, may be used to stimulate and expand a modified immune cell or T cell of the disclosure. The cell may be a tumor cell, which, optionally, may be used as an artificial or modified antigen presenting cell.

[068] Modified cells of the disclosure that may be used for adoptive therapy may be autologous or allogeneic.

[069] The disclosure provides a method for expressing a VCAR on the surface of a cell, comprising: (a) obtaining a cell population; (b) contacting the cell population to a composition comprising a VCAR of the disclosure or a sequence encoding the VCAR, under conditions sufficient to transfer the VCAR across a cell membrane of at least one cell in the cell population, thereby generating a modified cell population; (c) culturing the modified cell population under conditions suitable for integration of the transposon; and (d) expanding and / or selecting at least one cell from the modified cell population that express the VCAR on the cell surface. 2023208176 27 Jul 2023

[070] In certain embodiments of this method of expressing a VCAR, the cell population may comprise leukocytes and / or CD4+ and CD8+ leukocytes. The cell population may comprise CD4+ and CD8+ leukocytes in an optimized ratio. The optimized ratio of CD4+ to CD8+ leukocytes does not naturally occur in vivo. The cell population may comprise a tumor cell.

[071] In certain embodiments of this method of expressing a VCAR, a transposon or vector comprises the VCAR or the sequence encoding the VCAR.

[072] In certain embodiments of this method of expressing a VCAR, the conditions sufficient to transfer the sequence encoding the VCAR across a cell membrane of at least one cell in the cell population comprise nucleofection.

[073] In certain embodiments of this method of expressing a VCAR, wherein the conditions sufficient to transfer the sequence encoding the VCAR across a cell membrane of at least one cell in the cell population comprise at least one of an application of one or more pulses of electricity at a specified voltage, a buffer, and one or more supplemental factor(s). In certain embodiments, the buffer may comprise PBS, HBSS, OptiMEM, BTXpress, Amaxa Nucleofector, Human T cell nucleofection buffer or any combination thereof. In certain embodiments, the one or more supplemental factor(s) may comprise (a) a recombinant human cytokine, a chemokine, an interleukin or any combination thereof; (b) a salt, a mineral, a metabolite or any combination thereof; (c) a cell medium; (d) an inhibitor of cellular DNA sensing, metabolism, differentiation, signal transduction, one or more apoptotic pathway(s) or combinations thereof; and (e) a reagent that modifies or stabilizes one or more nucleic acids. The recombinant human cytokine, the chemokine, the interleukin or any combination thereof may comprise IL2, IL7, IL12, IL15, IL21, IL1, IL3, IL4, IL5, IL6, IL8, CXCL8, IL9, IL10, IL11, IL13, IL14, IL16, IL17, IL18, IL19, IL20, IL22, IL23, IL25, IL26, IL27, IL28, IL29, IL30, IL31, IL32, IL33, IL35, IL36, GM-CSF, IFN-gamma, IL-1 alpha / IL-lFl, IL-1 beta / IL-lF2, IL-12 p70, IL-12 / IL-35 p35, IL-13, IL-17 / IL-17A, IL-17A / F Heterodimer, IL-17F, IL-18 / IL-1F4, IL-23, IL-24, IL-32, IL-32 beta, IL-32 gamma, IL-33, LAP (TGF-beta 1), Lymphotoxin-alpha / TNF-beta, TGF-beta, TNF-alpha, TRANCE / TNFSF11 / RANK L or any combination thereof. The salt, the mineral, the metabolite or any combination thereof may comprise HEPES, Nicotinamide, Heparin, Sodium Pyruvate, L-Glutamine, MEM Non-Essential Amino Acid Solution, Ascorbic Acid, Nucleosides, FBS / FCS, Human serum, serum-substitute, anti-biotics, pH adjusters, Earle’s Salts, 2-Mercaptoethanol, Human transferrin, Recombinant human insulin, Human serum 2023208176 27 Jul 2023 albumin, Nucleofector PLUS Supplement, KCL, MgC12, Na2HPO4, NAH2PO4, Sodium lactobionate, Manitol, Sodium succinate, Sodium Chloride, CINa, Glucose, Ca(NO3)2, Tris / HCl, K2HPO4, KH2PO4, Polyethylenimine, Poly-ethylene-glycol, Pol oxamer 188, Poloxamer 181, Poloxamer 407, Poly-vinylpyrrolidone, Pop313, Crown-5, or any combination thereof. The cell medium may comprise PBS, HBSS, OptiMEM, DMEM, RPMI 1640, AIM-V, X-VIVO 15, CellGro DC Medium, CTS OpTimizer T Cell Expansion SFM, TexMACS Medium, PRIME-XV T Cell Expansion Medium, ImmunoCult-XF T Cell Expansion Medium or any combination thereof. The inhibitor of cellular DNA sensing, metabolism, differentiation, signal transduction, one or more apoptotic pathway(s) or combinations thereof comprise inhibitors of TLR9, MyD88, IRAK, TRAF6, TRAF3, IRF-7, NF-KB, Type 1 Interferons, pro-inflammatory cytokines, cGAS, STING, Sec5, TBK1, IRF-3, RNA pol III, RIG-1, IPS-1, FADD, RIP1, TRAF3, AIM2, ASC, Caspasel, Pro-ILIB, PI3K, Akt, Wnt3A, inhibitors of glycogen synthase kinase-30 (GSK-3 0) (e.g., TWS119), Bafilomycin, Chloroquine, Quinacrine, AC-YVAD-CMK, Z-VAD-FMK, Z-IETD-FMK or any combination thereof. The reagent that modifies or stabilizes one or more nucleic acids comprises a pH modifier, a DNA-binding protein, a lipid, a phospholipid, CaPO4, a net neutral charge DNA binding peptide with or without a NLS sequence, a TREX1 enzyme or any combination thereof.

[074] In certain embodiments of this method of expressing a VCAR, the conditions suitable for integration of the VCAR or a sequence encoding the VCAR of the disclosure comprise at least one of a buffer and one or more supplemental factor(s). In certain embodiments, a transposon or vector of the disclosure comprise the VCAR or a sequence encoding the VCAR of the disclosure. In certain embodiments, the buffer may comprise PBS, HBSS, OptiMEM, BTXpress, Amaxa Nucleofector, Human T cell nucleofection buffer or any combination thereof. In certain embodiments, the one or more supplemental factor(s) may comprise (a) a recombinant human cytokine, a chemokine, an interleukin or any combination thereof; (b) a salt, a mineral, a metabolite or any combination thereof; (c) a cell medium; (d) an inhibitor of cellular DNA sensing, metabolism, differentiation, signal transduction, one or more apoptotic pathway(s) or combinations thereof; and (e) a reagent that modifies or stabilizes one or more nucleic acids. The recombinant human cytokine, the chemokine, the interleukin or any combination thereof may comprise IL2, IL7, IL12, IL15, IL21, IL1, IL3, IL4, IL5, IL6, IL8, CXCL8, IL9, IL10, IL11, IL13, IL14, IL16, IL17, IL18, IL19, IL20, IL22, IL23, IL25, IL26, IL27, IL28, IL29, IL30, IL31, 2023208176 27 Jul 2023 IL32, IL33, IL35, IL36, GM-CSF, IFN-gamma, IL-1 alpha / IL-lFl, IL-1 beta / IL-lF2, IL-12 p70, IL-12 / IL-35 p35, IL-13, IL-17 / IL-17A, IL-17A / F Heterodimer, IL-17F, IL-18 / IL-1F4, IL-23, IL-24, IL-32, IL-32 beta, IL-32 gamma, IL-33, LAP (TGF-beta 1), Lymphotoxin-alpha / TNF-beta, TGF-beta, TNF-alpha, TRANCE / TNFSF11 / RANK L or any combination thereof. The salt, the mineral, the metabolite or any combination thereof may comprise HEPES, Nicotinamide, Heparin, Sodium Pyruvate, L-Glutamine, MEM Non-Essential Amino Acid Solution, Ascorbic Acid, Nucleosides, FBS / FCS, Human serum, serum-substitute, anti-biotics, pH adjusters, Earle’s Salts, 2-Mercaptoethanol, Human transferrin, Recombinant human insulin, Human serum albumin, Nucleofector PLUS Supplement, KCL, MgC12, Na2HPO4, NAH2PO4, Sodium lactobionate, Manitol, Sodium succinate, Sodium Chloride, CINa, Glucose, Ca(NO3)2, Tris / HCl, K2HPO4, KH2PO4, Polyethylenimine, Poly-ethylene-glycol, Pol oxamer 188, Poloxamer 181, Poloxamer 407, Poly-vinylpyrrolidone, Pop313, Crown-5, or any combination thereof. The cell medium may comprise PBS, HBSS, OptiMEM, DMEM, RPMI 1640, AIM-V, X-VIVO 15, CellGro DC Medium, CTS OpTimizer T Cell Expansion SFM, TexMACS Medium, PRIME-XV T Cell Expansion Medium, ImmunoCult-XF T Cell Expansion Medium or any combination thereof. The inhibitor of cellular DNA sensing, metabolism, differentiation, signal transduction, one or more apoptotic pathway(s) or combinations thereof comprise inhibitors of TLR9, MyD88, IRAK, TRAF6, TRAF3, IRF-7, NF-KB, Type 1 Interferons, pro-inflammatory cytokines, cGAS, STING, Sec5, TBK1, IRF-3, RNA pol III, RIG-1, IPS-1, FADD, RIP1, TRAF3, AIM2, ASC, Caspasel, Pro-ILIB, PI3K, Akt, Wnt3A, inhibitors of glycogen synthase kinase-30 (GSK-3 0) (e.g., TWS119), Bafilomycin, Chloroquine, Quinacrine, AC-YVAD-CMK, Z-VAD-FMK, Z-IETD-FMK or any combination thereof. The reagent that modifies or stabilizes one or more nucleic acids comprises a pH modifier, a DNA-binding protein, a lipid, a phospholipid, CaPO4, a net neutral charge DNA binding peptide with or without a NLS sequence, a TREX1 enzyme or any combination thereof.

[075] In certain embodiments of this method of expressing a VCAR, the expansion and selection steps occur sequentially. The expansion may occur prior to selection. The expansion may occur following selection, and, optionally, a further (i.e. second) selection may occur following expansion.

[076] In certain embodiments of this method of expressing a VCAR, the expansion and selection steps may occur simultaneously. 2023208176 27 Jul 2023

[077] In certain embodiments of this method of expressing a VCAR, the expansion may comprise contacting at least one cell of the modified cell population with an antigen to stimulate the at least one cell through the VCAR, thereby generating an expanded cell population. The antigen may be presented on the surface of a substrate. The substrate may have any form, including, but not limited to a surface, a well, a bead or a plurality thereof, and a matrix. The substrate may further comprise a paramagnetic or magnetic component. In certain embodiments of this method of expressing a VCAR, the antigen may be presented on the surface of a substrate, wherein the substrate is a magnetic bead, and wherein a magnet may be used to remove or separate the magnetic beads from the modified and expanded cell population. The antigen may be presented on the surface of a cell or an artificial antigen presenting cell. Artificial antigen presenting cells of the disclosure may include, but are not limited to, tumor cells and stem cells.

[078] In certain embodiments of this method of expressing a VCAR, wherein the transposon or vector comprises a selection gene and wherein the selection step comprises contacting at least one cell of the modified cell population with a compound to which the selection gene confers resistance, thereby identifying a cell expressing the selection gene as surviving the selection and identifying a cell failing to express the selection gene as failing to survive the selection step.

[079] In certain embodiments of this method of expressing a VCAR, the expansion and / or selection steps may proceed for a period of 10 to 14 days, inclusive of the endpoints.

[080] The disclosure provides a composition comprising the modified, expanded and selected cell population of the methods of the disclosure.

[081] The disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a composition of the disclosure, wherein the VCAR specifically binds to an antigen on a tumor cell. In certain embodiments, the tumor cell may be a malignant tumor cell. In certain embodiments, comprising administering to the subject the composition comprising a modified cell or cell population of the disclosure, the cell or cell population may be autologous. In certain embodiments, comprising administering to the subject the composition comprising a modified cell or cell population of the disclosure, the cell or cell population may be allogeneic.

[082] The disclosure provides a method of treating an autoimmune condition in a subject in need thereof, comprising administering to the subject a composition of the disclosure, wherein 2023208176 27 Jul 2023 the VCAR specifically binds to an antigen on an autoimmune cell of the subject. In certain embodiments, the autoimmune cell may be a lymphocyte that specifically binds to a self-antigen on a target cell of the subject. In certain embodiments, the autoimmune cell may be a B lymphocyte (i.e. a B cell). In certain embodiments, the autoimmune cell may be a T lymphocyte (i.e. a T cell). In certain embodiments, comprising administering to the subject the composition comprising a modified cell or cell population of the disclosure, the cell or cell population may be autologous. In certain embodiments, comprising administering to the subject the composition comprising a modified cell or cell population of the disclosure, the cell or cell population may be allogeneic.

[083] The disclosure provides a method of treating an infection in a subject in need thereof, comprising administering to the subject a composition of the disclosure, wherein the VCAR specifically binds to an antigen on a cell comprising an infectious agent, a cell in communication with an infectious agent or a cell exposed to an infection agent. In some embodiments, a cell in communication with an infectious agent may be in air communication (e.g., the infectious agent is airborne or inhaled) or fluid communication (e.g., the infectious agent is carried in an aqueous or a biological fluid) with the infectious agent. The infectious agent causing the infection of the host cell may be a bacterium, a virus, a yeast, or a microbe. The infectious agent may induce in the cell or the cell’s host organism (the subject), exemplary conditions including, but not limited to, a viral infection, an immunodeficiency condition, an inflammatory condition and a proliferative disorder. In certain embodiments, the infection causes tuberculosis, microencephaly, neurodegeneration or malaria. In certain embodiments, the infection causes microencephaly in a fetus of the subject. In certain embodiments, including those wherein the infection causes microencephaly in a fetus of the subject, the infectious agent is a virus and wherein the virus is a Zika virus. In certain embodiments, the immunodeficiency condition is acquired immune deficiency syndrome (AIDS). In certain embodiments, the proliferative disorder is a cancer. In certain embodiments, the cancer is cervical cancer and wherein the infectious agent is a human papilloma virus (HPV). In certain embodiments, comprising administering to the subject the composition comprising a modified cell or cell population of the disclosure, the cell or cell population may be autologous. In certain embodiments, comprising administering to the subject the composition comprising a modified cell or cell population of the disclosure, the cell or cell population may be allogeneic. 2023208176 27 Jul 2023

[084] The disclosure provides a method of treating a mast cell disease in a subject in need thereof, comprising administering to the subject a composition of the disclosure, wherein the VCAR specifically binds to an antigen on a mast cell. In certain embodiments, the VCAR specifically binds to an antigen on a mast cell of the subject. In certain embodiments, the mast cell disease may include, but is not limited to, disorders associated with an excessive proliferation of mast cells, disorders associated with mast cells having abnormal activity, and disorders associated with both abnormal numbers of mast cells and abnormal mast cell activity. Exemplary disorders associated with an excessive proliferation of mast cells include, but are not limited to, mastocytosis, cutaneous mastocytosis (e.g., urticaria pigmentosa or maculopapular cutaneous mastocytosis), systemic mastocytosis (including mast cell leukaemia), and localized mast cell proliferations. Exemplary disorders associated with mast cells having abnormal activity, include, but are not limited to, mast cell activation syndrome (MCAS) or mast cell activation disorder (MCAD), allergic disease (including anaphylaxis), asthma, inflammatory disease (including autoimmune related inflammation of, for examplejoint tissues, arthritis, etc.), or any combination thereof. In certain embodiments, comprising administering to the subject the composition comprising a modified cell or cell population of the disclosure, the cell or cell population may be autologous. In certain embodiments, comprising administering to the subject the composition comprising a modified cell or cell population of the disclosure, the cell or cell population may be allogeneic.The disclosure provides a method of treating a degenerative disease in a subject in need thereof, comprising administering to the subject a composition of the disclosure, wherein the VCAR specifically binds to an antigen on a deleterious cell or an aged cell. In certain embodiments, the VCAR specifically binds to an antigen on a deleterious cell or an aged cell of the subject. In certain embodiments, the degenerative disease may include, but is not limited to, a neurodegenerative disorder, a metabolic disorder, a vascular disorder and aging. Exemplary neurodegenerative disorders include, but are not limited to, disorders associated with a loss of a function or efficacy of one or more of a neuron, a glial cell or a microglia. Exemplary neurodegenerative disorders include, but are not limited to, disorders associated with an accumulation of one or more of a signaling molecule, a protein, or a prion that interferes with a function or decreases an efficacy of one or more of a neuron, a glial cell or a microglia. Exemplary metabolic disorders include, but are not limited to, disorders associated with mitochondrial disorders, interruptions of the electron transport chain, interruptions of cellular 2023208176 27 Jul 2023 production of ATP, a loss of a function or a decreased efficacy of one or more mitochondria of one or more of a neuron, a glial cell or a microglia. Exemplary metabolic disorders include, but are not limited to, disorders associated with a loss of circulating blood flow or a decreased blood flow to a neuron, a glial cell or a microglia (e.g., a stroke); a transient or permanent state of hypoxia in a neuron, a glial cell or a microglia (for example, sufficient to release free radicals in a cell); a loss of circulating CNS or a decreased CNS flow to a neuron, a glial cell or a microglia during a sleep state of the subject sufficient to decrease efficacy of removal of a waste product of a neuron, a glial cell or a microglia during that sleep state. Exemplary aging disorders include, but are not limited to, disorders associated with an increased shortened or shortened telomeres on one or more chromosomes of a neuron, a glial cell or a microglia; a loss of a function or a decreased efficacy of telomerase in a neuron, a glial cell or a microglia; or a loss of a function or a decreased efficacy of a DNA repair mechanism in a neuron, a glial cell or a microglia. In certain embodiments, the deleterious cell or the aged cell interferes with a function or decreases an efficacy of another cell in a network comprising the deleterious cell or the aged cell and the targeted removal of the deleterious cell or the aged cell improves or restores a function or increases an efficacy of the network. In certain embodiments, the deleterious cell or the aged cell may transform the function or efficacy of a second cell and the targeted removal of the deleterious cell or the aged cell prevents the transformation of the second cell. In certain embodiments, the degenerative disease is a neurodegenerative disorder and the deleterious cell or the aged cell is a stem cell, an immune cell, a neuron, a glia or a microglia. In certain embodiments, the degenerative disease is a metabolic disorder and the deleterious cell or the aged cell is a stem cell, a somatic cell, a neuron, a glia or a microglia. In certain embodiments, the degenerative disease is a vascular disorder and the deleterious cell or the aged cell is a stem cell, a somatic cell, an immune cell, an endothelial cell, a neuron, a glia or a microglia. In certain embodiments, the degenerative disease is aging and the deleterious cell or the aged cell is an oocyte, a sperm, a stem cell, a somatic cell, an immune cell, an endothelial cell, a neuron, a glia or a microglia. In certain embodiments, comprising administering to the subject the composition comprising a modified cell or cell population of the disclosure, the cell or cell population may be autologous. In certain embodiments, comprising administering to the subject the composition comprising a modified cell or cell population of the disclosure, the cell or cell population may be allogeneic. 2023208176 27 Jul 2023

[085] The disclosure provides a method of modifying a cell therapy in a subject in need thereof, comprising administering to the subject a composition comprising a cell comprising a transposon or vector of the composition comprising an inducible proapoptotic polypeptide, wherein apoptosis may be selectively induced in the cell by contacting the cell with an induction agent. In certain embodiments, the cell is autologous. In certain embodiments, the cell is allogeneic. In certain embodiments of this method, the cell therapy is an adoptive cell therapy. In certain embodiments of this method, modifying the cell therapy comprises a termination of the cell therapy. In certain embodiments of this method, modifying the cell therapy comprises a depletion of a portion of the cells provided in the cell therapy. In certain embodiments, the method further comprises the step of administering an inhibitor of the induction agent to inhibit modification of the cell therapy, thereby restoring the function and / or efficacy of the cell therapy.

[086] Methods of modifying a cell therapy of the disclosure may be used to terminate or dampen a therapy in response to, for example, a sign of recovery or a sign of decreasing disease severity / progression, a sign of disease remission / cessation, and / or the occurrence of an adverse event. Cell therapies of the disclosure may be resumed by inhibiting the induction agent should a sign or symptom of the disease reappear or increase in severity and / or an adverse event is resolved. BRIEF DESCRIPTION OF THE DRAWINGS

[087] Figure lisa plot showing weight change versus days post treatment for VCARs of the disclosure.

[088] Figure 2 is a plot showing tumor burden versus days post treatment in for VCARs of the disclosure.

[089] Figure 3 is a plot showing percent survival versus days post treatment for VCARs of the disclosure.

[090] Figure 4 is a plot of tumor burden as shown by bioluminescence signal versus days post treatment of VH-A (triangles), an irrelevant CAR (squares) or no tumor / no CAR-T (circles).

[091] Figure 5 is a plot of tumor burden as shown by bioluminescence signal versus days post treatment of VH-B (triangles), an irrelevant CAR (squares) or no tumor / no CAR-T (circles). 2023208176 27 Jul 2023

[092] Figure 6 is a plot of tumor burden as shown by bioluminescence signal versus days post treatment of VH-C (triangles), an irrelevant CAR (squares) or no tumor / no CAR-T (circles).

[093] Figure 7 is a plot of tumor burden as shown by bioluminescence signal versus days post treatment of VH-D (triangles), an irrelevant CAR (squares) or no tumor / no CAR-T (circles).

[094] Figure 8 a plot of tumor burden as shown by bioluminescence signal versus days post treatment of VH-E (triangles), an irrelevant CAR (squares) or no tumor / no CAR-T (circles).

[095] Figure 9 a plot of tumor burden as shown by bioluminescence signal versus days post treatment of VH-F (triangles), an irrelevant CAR (squares) or no tumor / no CAR-T (circles).

[096] Figure 10A is a table providing consensus sequences of framework and CDR sequences for exemplary VHs of the disclosure.

[097] Figure 10B is an alignment of exemplary VHs of the disclosure. From top to bottom, the sequences correspond with the consensus sequences of the frameworks VH-B (SEQ ID NO: 18050), VH-D (SEQ ID NO: 18051), VH-A (SEQ ID NO: 18052), VH-E (SEQ ID NO: 18053), VH-F (SEQ ID NO: 18054) and VH-C (SEQ ID NO: 18055).

[098] Figure 11 is a bar graph depicting the knock out efficiency of checkpoint signaling proteins on armored T-cells. Primary human T-cells are typically in resting state when isolated from normal healthy donors. Cas-CLOVER was used to knockout the checkpoint receptors, PD-1, TGFBR2, LAG-3, TIM-3 and CTLA-4. Percent knock-out is shown on the y-axis. Gene editing resulted in 30-70% loss of protein expression at the cell surface as measured by flow cytometry.

[099] Figure 12 are schematic diagrams of wildtype, null and switch receptors and their effects on intracellular signaling, either inhibitory or stimulatory, in primary T-cells. Binding of the wildtype inhibitory receptor expressed endogenously on a T-cell with its endogenous ligand results in transmission of an inhibitory signal which, in part, reduces T-cell effector function. However, mutation (Mutated null) or deletion (Truncated null) of the intracellular domain (ICD) of a checkpoint receptor protein, such as PD1 (top panel) or TGFBRII (bottom panel), reduces or eliminates its signaling capability when cognate ligand(s) is bound. Thus, expression of engineered mutated or truncated null receptors on the surface of modified T cells results in a competition with endogenously-expressed wildtype receptors for binding of the free endogenous ligand(s), effectively reducing or eliminating delivery of inhibitory signals by endogenously-expressed wildtype receptors. Specifically, any binding by a mutated or null receptor sequesters 2023208176 27 Jul 2023 the endogenous ligand(s) from binding the wildtype receptor and results in dilution of the overall level of checkpoint signaling effectively delivered to the modified T-cell, thereby reducing or blocking checkpoint inhibition and functional exhaustion of the modified T cells. A switch receptor is created by replacement of the wildtype ICD with an ICD from either a co-stimulatory molecule (such as CD3z, CD28, 4-1BB) or a different inhibitory molecule (such as CTLA4, PD1, Lag3). In the former case, binding of the endogenous ligand(s) by the modified switch receptor results in the delivery of a positive signal to the T-cells, thereby helping to enhance stimulation of the modified T cell and potentially enhance target tumor cell killing. In the latter case, binding of the endogenous ligand(s) by the modified switch receptor results in the delivery of a negative signal to the T-cells, thereby eliminating stimulation of the modified T cell and potentially reducing target tumor cell killing. The signal peptide (purple arrow), extracellular domain (ECD) (bright green), transmembrane domain (yellow), intracellular signaling domain (ICD)(orange), and replacement ICD (green) are displayed in the receptor diagrams. indicates a mutated ICD. “+” indicates the presence of a checkpoint signal. indicates the absence of a checkpoint signal.

[0100] Figure 13 is a schematic diagram showing the design of PD1 and TGFBRII null receptors. The signal peptide domain (SP), transmembrane domain (TM) and extracellular domain (ECD) of truncated null receptors for PD1 (top panel) and TGFBRII (bottom panel) are shown. The first of the top four molecules is the wildtype PD-1 receptor, which encodes the wildtype PD-1 SP and TM. For the PD1 null receptor, replacement of PD1 wildtype SP or TM domain (green; light green) with the SP or TM domain of a human T cell CD8a receptor (red) is depicted. The second molecule encodes the CD8a SP along with the native PD-1 TM, the third encodes the wildtype PD-1 SP and the alternative CD8a TM, and the fourth encodes both the alternative CD8a SP and TM. Similarly, for the null receptor of TGFPRII, replacement of the wildtype TGFBRII SP (pink) with a SP domain of a human T cell CD8a receptor (red). The names of the constructs and the amino acid lengths (aa) of each construct protein is listed on the left of the diagram.

[0101] Figure 14 is a series of histograms depicting the expression of the PD1 and TGFBRII null Receptors on the surface of primary human T cells determined by flow cytometry. Each of the six truncated null constructs from Figure 13 were expressed on the surface of primary human T cells. T cells were stained with either anti-PDl (top; blue histograms) or anti-TGFpRII 2023208176 27 Jul 2023 (bottom; blue histograms), or isotype control or secondary only (gray histograms). Cells staining positive for PD-1 or TGFPRII expression were gated (frequency shown above gate) and mean fluorescence intensity (MFI) value is displayed above each positive histogram. The names of the null receptor constructs are depicted above each plot. Both null receptor gene strategies, replacement of the wildtype SP with the alternative CD8a were successfully expressed. 02.8aSP-PD-1 and 02.8aSP-TGFpRII resulted in the highest level of expression at the T-cell surface. 02.8aSP-PD-l null receptor exhibited an MFI of 43,680, which is 177-fold higher than endogenous T cell PD-1 expression and 2.8-fold higher than the wildtype PD-1 null receptor. 02.8aSP-TGFPRII null receptor exhibited an MFI of 13,809, which is 102-fold higher than endogenous T cell TGFPRII expression and 1.8-fold higher than the wildtype TGFPRII null receptor. Replacement of wildtype SP with the alternative CD8a SP for both PD1 and TGRBRII results in enhanced surface expression of the null or Switch receptor, which helps to maximize checkpoint inhibition or co-stimulation, respectively, upon binding of the endogenous ligand(s).

[0102] Figure 15A-B is a pair of schematic diagrams depicting NF-KB inducible vectors for expression in T-cells. Two T cell activation NF-KB inducible vectors were developed; one with the gene expression system (GES) in the forward orientation (A) and the other in the complementary direction (B), both preceding the constitutive EFla promoter. These vectors also direct expression of a CAR molecule and a DHFR selection gene, separated by a T2A sequence. Both the conditional NF-KB inducible system and the EFla directed genes are a part of a piggyBac transposon that can be permanently integrated into T cells using electroporation (EP). Once integrated into the genome, the T cells will constitutively express the CAR on the membrane surface and the DHFR within the cell, while expression of the NF-KB inducible gene, GFP, will be expressed to the highest level only upon T cell activation.

[0103] Figure 16 is a pair of graphs depicting NF-KB inducible expression of GFP in activated T cells. T cells were nucleofected with a piggyBac vector expressing an anti-BCMA CAR and a DHFR mutein gene under control of an EFla promoter along with the absence (No GES control) or presence of an NF-KB inducible expression system driving GFP expression in either the forward (pNFKB-GFP forward) or reverse orientation (pNFKB-GFP reverse). Cells were cultured in the presence of methotrexate selection until the cells were almost completely resting (Day 19) and GFP expression was assessed at Day 5 and Day 19. At Day 5, all T cells are proliferating and highly stimulated, with cells harboring the NF-KB inducible expression 2023208176 27 Jul 2023 cassette producing high levels of GFP due to strong NFkB activity. The No GES control cells did not express detectable levels of GFP. By Day 19, the GES T cells were almost fully resting and GFP expression was significantly lower than Day 5 (—1 / 8 MFI), since NFkB activity is lower. GFP expression is still observed at Day 19, which may due to the long half-life of GFP protein (~30hr), or, basal level of NFkB activity through, for example, a TCR, a CAR, a cytokine receptor, or a growth factor receptor signal.

[0104] Figure 17 is a series of graphs depicting anti-BCMA CAR-mediated activation of NFKB inducible expression of GFP in presence of BCMA+ tumor cells. T cells were either unmodified (Mock T cells) or nucleofected with a piggyBac vector expressing an anti-BCMA CAR and a DHFR mutein gene under control of an EFla promoter along with the absence (No GES control) or presence of an NF-KB inducible expression system driving GFP expression in either the forward (pNFKB-GFP forward) or reverse orientation (pNFKB-GFP reverse). All cells were cultured for 22 days, either with or without methotrexate selection (Mock T cells), until the cells were almost completely resting. Cells were then stimulated for 3 days in the absence (No stimulation) or presence of BCMA- (K562), BMCA+ (RPMI 8226), or positive control anti-CD3 anti-CD28 activation reagent (CD3 / 28 stimulation). GFP expression was undetectable under all conditions with the No GES control or Mock T cells. However, while pNFKB-GFP forward- and reverse-transposed cells exhibited little GFP expression over the No stimulation control when cultured with BCMA- K562 cells, they both demonstrated dramatic upregulation of gene expression either in the presence of BCMA+ tumor cells or under positive control conditions. Little difference in GFP expression was observed between the pNFKB-GFP forward- and reverse-transposed cells that were cocultured with BCMA+ tumor cells.

[0105] Figure 18 is a series of graphs demonstrating that the Expression level of inducible gene can be regulated by number of response elements preceding the promoter T cells were nucleofected with a piggyBac vector encoding an anti-BCMA CARTyrin followed by a selection gene, both under control of a human EFla promoter. Further, vectors either additionally encoded the conditional NF-KB inducible gene expression system driving expression of a truncated CD 19 protein (dCD19) and included a number of NFKB response elements (RE) varying from 0 - 5, no GES (No GES), or received an electroporation pulse but no piggyBac nucleic acid (Mock). Data are shown for only the GES in the reverse (opposite) direction / orientation. All cells were cultured for 18 days and included selection for piggyBac- 2023208176 27 Jul 2023 modified T cells using methotrexate addition. Cells were then stimulated for 3 days using anti-CD3 anti-CD28 bead activation reagent and dCD19 surface expression was assessed by FACS at Days 0, 3 and 18, and data are shown as FACS histograms and MFI of target protein staining. Surface dCD19 expression was detected at low levels at Day 0 in all T cells transposed with vectors encoding the GES. At 3 days post-stimulation, dramatic upregulation of dCD19 expression was observed for all T cells expressing the GES, with a greater fold increase in surface expression in those with higher numbers of REs. Thus, surface dCD19 expression was directly proportional with the number of REs encoded in the GES. No dCD19 was detected on the surface of T cells that did not harbor the GES: No GES and Mock controls.

[0106] Figure 19 is a schematic depiction of the Csy4-T2A-Clo051-G4Slinker-dCas9 construct map (Embodiment 2).

[0107] Figure 20 is a schematic depiction of the pRTl-Clo051-dCas9 Double NLS construct map (Embodiment 1).

[0108] Figure 21 is a pair of graphs comparing the efficacy of knocking out expression of either B2M in Pan T-cells (left) or the a-chain of the T-cell Receptor in Jurkat cells (right) for either Embodiment 1 (pRTl-Clo051-dCas9 Double NLS, as shown in Figure 20) or Embodiment 2 (Csy4-T2A-Clo051-G4Slinker-dCas9, as shown in Figure 19) of a Cas-Clover fusion protein of the disclosure. For the right-hand graph, the fusion protein is provided at either 10 pg or 20 pg, as indicated.

[0109] Figure 22 is a photograph of a gel electrophoresis analysis of mRNA in the presence of each of Embodiment 1 (pRTl-Clo051-dCas9 Double NLS, as shown in Figure 20) and Embodiment 2 (Csy4-T2A-Clo051-G4Slinker-dCas9, as shown in Figure 19). As shown, both are effective at knocking down mRNA expression.

[0110] Figure 23 is a diagram of the UniRat™ Human Heavy Chain Antibody production platform.

[0111] Figure 24 is a diagram of the pipeline of immunization, B-cell isolation, mRNA purification, Next Generation Sequencing (NGS), bioinformatics analysis, high-throughput vector assembly and high-throughput expression and screening used to identify the full antigenspecific repertoire of heavy-chain antibodies generated by the UniRat™ after immunization. Unique gene assembly methods convert the antibody repertoire sequence information into large collections of fully human heavy chain antibodies that can be screened for a variety of functions. 2023208176 27 Jul 2023

[0112] Figure 25 is a pair of heat maps showing how next generation sequencing (NGS) analysis reveals expanded VH sequence lineages. The degree of red (high) or blue (low) in the heatmap indicates expanded VH sequence families due to immunization. Some highly ranked VH sequences are unique to a single animal. Other highly ranked VH sequences appear in more than one anitmal suggesting convergent selection of those sequence families.

[0113] Figure 26 is a series of flow cytometry plots showing that a CAR-T population expressing P-PSMA-101 and comprising a mixture of Tscm / Tcm, give rise to CAR+ Tcm, Tem, and Teff to attack solid tumor. After solid tumor elimination, a population of CAR-T+ Tscm persists. Although demonstrated with a CARTyrin, this principle shown here applies to VCAR+ Tscm and VCAR+ Tcm populations of the disclosure. Specifically, a murine xenograft model using a luciferase-expressing LNCaP cell line (LNCaP.luc) injected subcutaneously (SC) into NSG mice was utilized to assess in vivo anti-tumor efficacy of a CAR (P-PSMA5-101 and P-PSMA8-101) at a ‘stress’ dose (4xlOA6) total CAR-T cells. For these in vivo studies, all CAR-T cells were produced using PB delivery of either the P-PSMA5-101 or P-PSMA8-101 plasmid using the Poseida manufacturing process. Mice were injected in the axilla with LNCaP and treated when tumors were established (100-300 mm3 by caliper measurement). Mice were treated with a ‘stress’ dose (4x10A6) of P-PSMA-101 by IV injection in order to tease out any possible differences in efficacy between the PSMA5 and the PSMA8 CARs. Anti-tumor activity was evaluated by survival, CD8+ T cell expansion and detection in the blood, tumor volume assessment by caliper measurement, and bioluminescence of LNCaP tumor. P-PSMA5-101 and P-PSMA8-101 at a ‘stress’ dose demonstrated significantly enhanced anti-tumor efficacy and survival in comparison to the T cells (no CAR) control mice against established SC LNCaP.luc solid tumors in NSG mice. Specifically, there was no survival in T cells (no CAR) control animals, 25% survival in the P-BCMA-101 treated group, 75% survival in the P-PSMA5-101 treated group, and 100% survival in animals treated with a ‘stress’ dose of P-PSMA8-101. In the peripheral blood, P-PSMA5-101 and P-PSMA8-101 expanded and gave rise to differentiated effector CARTyrin+ T-cells that were concomitant with a decrease in tumor burden below detectable caliper and bioluminescent imaging limits. These cells then contracted, yet persisted in the peripheral blood. 2023208176 27 Jul 2023 DETAILED DESCRIPTION

[0114] The disclosure provides chimeric antigen receptors (CARs) comprising at least one VHH (VCAR). Chimeric antigen receptors of the disclosure may comprise more than one VHH. For example, a bi-specific VCAR may comprise two VHHs that specifically bind two distinct antigens.

[0115] VHH proteins of the disclosure specifically bind to an antigen. Chimeric antigen receptors of the disclosure comprising one or more VHHs that specifically bind an antigen may be used to direct the specificity of a cell, (e.g., a cytotoxic immune cell) towards the specific antigen.

[0116] The disclosure provides chimeric antigen receptors (CARs) an antigen recognition region comprising a single domain antibody (VCARs). In some embodiments, the single domain antibody is a VHH antibody. In some embodiments, the single domain antibody is a VH antibody.

[0117] Chimeric antigen receptors of the disclosure may comprise a signal peptide of human CD2, CD36, CD3s, CD3y, CD3^, CD4, CD8a, CD19, CD28, 4-lBBor GM-CSFR. A hinge / spacer domain of the disclosure may comprise a hinge / spacer / stalk of human CD8a, IgG4, and / or CD4. An intracellular domain or endodomain of the disclosure may comprise an intracellular signaling domain of human CD3^ and may further comprise human 4-1BB, CD28, CD40, ICOS, MyD88, OX-40 intracellular segment, or any combination thereof. Exemplary transmembrane domains include, but are not limited to a human CD2, CD36, CD3s, CD3y, CD3^, CD4, CD8a, CD19, CD28, 4-lBBor GM-CSFR transmembrane domain.

[0118] The disclosure provides genetically modified cells, such as T cells, NK cells, hematopoietic progenitor cells, peripheral blood (PB) derived T cells (including T cells from G-CSF-mobilized peripheral blood), umbilical cord blood (UCB) derived T cells rendered specific for one or more antigens by introducing to these cells a VCAR of the disclosure. Cells of the disclosure may be modified by electrotransfer of a transposon encoding a VCAR of the disclosure and a plasmid comprising a sequence encoding a transposase of the disclosure (preferably, the sequence encoding a transposase of the disclosure is an mRNA sequence). VHs of the disclosure

[0119] The disclosure provides chimeric antigen receptors (CARs) comprising a single domain antibody (VCARs). In some embodiments, the single domain antibody comprises a VH. In some 2023208176 27 Jul 2023 embodiments, the VH is isolated or derived from a human sequence. In some embodiments, VH comprises a human CDR sequence and / or a human framework sequence and a non-human or humanized sequence (e.g., a rat Fc domain). In some embodiments, the VH is a fully humanized VH. In some embodiments, the VH s neither a naturally occurring antibody nor a fragment of a naturally occurring antibody. In some embodiments, the VH is not a fragment of a monoclonal antibody. In some embodiments, the VH is a UniDab™ antibody (TeneoBio).

[0120] In some embodiments, the VH is fully engineered using the UniRat™ (TeneoBio) system and “NGS-based Discovery” to produce the VH. Using this method, the specific VH are not naturally-occurring and are generated using fully engineered systems. The VH are not derived from naturally-occurring monoclonal antibodies (mAbs) that were either isolated directly from the host (for example, a mouse, rat or human) or directly from a single clone of cells or cell line (hybridoma). These VHs were not subsequently cloned from said cell lines. Instead, VH sequences are fully-engineered using the UniRat™ system as transgenes that comprise human variable regions (VH domains) with a rat Fc domain, and are thus human / rat chimeras without a light chain and are unlike the standard mAb format. The native rat genes are knocked out and the only antibodies expressed in the rat are from transgenes with VH domains linked to a Rat Fc (UniAbs). These are the exclusive Abs expressed in the UniRat. Next generation sequencing (NGS) and bioinformatics are used to identify the full antigen-specific repertoire of the heavychain antibodies generated by UniRat™ after immunization. Then, a unique gene assembly method is used to convert the antibody repertoire sequence information into large collections of fully-human heavy-chain antibodies that can be screened in vitro for a variety of functions. In some embodiments, fully humanized VH are generated by fusing the human VH domains with human Fes in vitro (to generate a non-naturally occurring recombinant VH antibody). In some embodiments, the VH are fully humanized, but they are expressed in vivo as human / rat chimera (human VH, rat Fc) without a light chain. Fully humanized VHs areexpressed in vivo as human / rat chimera (human VH, rat Fc) without a light chain are about 80kDa (vs 150 kDa).

[0121] VCARs of the disclosure may comprise at least one VH of the disclosure. In some embodiments, the VH of the disclosure may be modified to remove an Fc domain or a portion thereof. In some embodiments, a framework sequence of the VH of the disclosure may be modified to, for example, improve expression, decrease immunogenicity or to improve function. Exemplary VCARs of the disclosure 2023208176 27 Jul 2023

[0122] In some embodiments of the VCARs of the disclosure, the VCAR comprises at least one of an amino acid sequence of at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of: VH-A: malpvtalllplalllhaarpevqllesggglvqpggslrlscaasgftfssyamnwvrqapgkglewvagiigsggstyyadsvkgrfsi srdnskntldlqmnslraedtavyycvkdwnttmitergqgtlvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfac diyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynel nlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalp pr(SEQIDNO: 18000).

[0123] In some embodiments of the VCARs of the disclosure, the amino acid sequence of the VCAR is encoded by a nucleotide sequence comprising a sequence of at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the nucleotide sequence of: VH-A: atggctctgcctgtgacagctctgctgctgcctctggctctgcttcttcatgcggcgcgccctgaagttcagctgcttgaatctggcggaggc ctggttcaacctggcggatctctgagactgagctgtgccgccagcggcttcacctttagcagctacgccatgaactgggtccgacaggccc ctggcaaaggactggaatgggtggccggaatcatcggcagcggcggcagcacatattacgccgattctgtgaagggccgcttcagcatc agccgggacaacagcaagaacaccctggacctgcagatgaacagcctgagagccgaggataccgccgtgtactactgcgtgaaggatt ggaacaccaccatgatcaccgagagaggccagggcacactggtcaccgtgtcctctacaacaacaccggcgcctcggcctccaacacc agctcctacaatcgcgagtcagcccctgtctctcagacccgaagcctgtagacctgctgctggcggagctgtgcataccagaggactggat ttcgcctgcgacatctacatctgggctcctctggctggcacatgcggagttttgctgctgagcctggtcatcaccctgtactgtaagagaggc aggaagaagctgctgtatatcttcaagcagcccttcatgagacccgtgcagaccacacaggaggaggacggctgctcttgtaggttcccag aggaggaggagggaggatgcgagctgcgcgtgaagtttagccggtccgccgatgcacctgcatacaagcagggacagaaccagctgta taacgagctgaatctgggccggagagaggagtacgacgtgctggataagaggcggggccgggaccccgagatgggaggcaagcctc ggagaaagaacccacaggagggcctgtacaatgagctgcaaaaggacaagatggccgaggcctattctgagatcggcatgaagggaga gaggcgccggggcaagggacacgatggcctgtaccagggcctgagcaccgccacaaaggacacctatgatgccctgcacatgcaggc cctgccccctagatga (SEQ ID NO: 18001).

[0124] In some embodiments of the VCARs of the disclosure, the VCAR comprises at least one of an amino acid sequence of at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of: VH-B: malpvtalllplalllhaarpevqllesggglvqpggsltlscaasgftfsnyamnwvrqapgkglewvsgiigsgattyyadsvkgrfti 2023208176 27 Jul 2023 srdnskntlnlqmnslraedtaiyycvkdwnttmitergqgtlvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfac diyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynel nlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalp pr(SEQIDNO: 18002).

[0125] In some embodiments of the VCARs of the disclosure, the amino acid sequence of the VCAR is encoded by a nucleotide sequence comprising a sequence of at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the nucleotide sequence of: VH-B atggctctgcctgtgacagctctgctgctgcctctggctctgcttcttcatgcggcgcgccctgaagttcagctgcttgaatctggcggaggc ctggttcaacctggcggatctctgacactgagctgtgccgccagcggcttcaccttcagcaactacgccatgaactgggtccgacaggccc ctggcaaaggccttgaatgggtgtccggcatcattggctctggcgccaccacctactacgccgattctgtgaagggcagattcaccatcagc cgggacaacagcaagaacaccctgaacctgcagatgaacagcctgagagccgaggacaccgccatctactactgcgtgaaggactgga acaccaccatgatcaccgagagaggccagggcacactggtcaccgtgtcctctacaacaacaccggcgcctcggcctccaacaccagct cctacaatcgcgagtcagcccctgtctctcagacccgaagcctgtagacctgctgctggcggagctgtgcataccagaggactggatttcg cctgcgacatctacatctgggctcctctggctggcacatgcggagttttgctgctgagcctggtcatcaccctgtactgtaagagaggcagg aagaagctgctgtatatcttcaagcagcccttcatgagacccgtgcagaccacacaggaggaggacggctgctcttgtaggttcccagagg aggaggagggaggatgcgagctgcgcgtgaagtttagccggtccgccgatgcacctgcatacaagcagggacagaaccagctgtataa cgagctgaatctgggccggagagaggagtacgacgtgctggataagaggcggggccgggaccccgagatgggaggcaagcctcgga gaaagaacccacaggagggcctgtacaatgagctgcaaaaggacaagatggccgaggcctattctgagatcggcatgaagggagagag gcgccggggcaagggacacgatggcctgtaccagggcctgagcaccgccacaaaggacacctatgatgccctgcacatgcaggccct gccccctagatgac(SEQ ID NO: 18003).

[0126] In some embodiments of the VCARs of the disclosure, the VCAR comprises at least one of an amino acid sequence of at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of: VH-C: malpvtalllplalllhaarpevqllesggglvqpgeslrlscaasgftfsnyamnwvrqapgkglewvsgivggggtsyyadsvrgrft isrdnskntlylqmnslraedtavyycvkdwnttmitergqgtlvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfa cdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlyne Inlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqal ppr(SEQIDNO: 18004). 2023208176 27 Jul 2023

[0127] In some embodiments of the VCARs of the disclosure, the amino acid sequence of the VCAR is encoded by a nucleotide sequence comprising a sequence of at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the nucleotide sequence of VH-C: atggctctgcctgtgacagctctgctgctgcctctggctctgcttcttcatgcggcgcgccctgaagttcagctgcttgaatctggcggaggc ctggttcagcctggcgaatctctgagactgagctgtgccgccagcggcttcaccttcagcaactacgccatgaactgggtccgacaggccc ctggcaaaggccttgaatgggtgtccggaatcgttggcggcggaggcacaagctactacgccgattctgtgcggggcagattcaccatca gccgggacaacagcaagaacaccctgtacctgcagatgaacagcctgagagccgaggacaccgccgtgtactactgcgtgaaggactg gaacaccaccatgatcaccgagagaggccagggcacactggtcaccgtgtcctctacaacaacaccggcgcctcggcctccaacacca gctcctacaatcgcgagtcagcccctgtctctcagacccgaagcctgtagacctgctgctggcggagctgtgcataccagaggactggattt cgcctgcgacatctacatctgggctcctctggctggcacatgcggagttttgctgctgagcctggtcatcaccctgtactgtaagagaggca ggaagaagctgctgtatatcttcaagcagcccttcatgagacccgtgcagaccacacaggaggaggacggctgctcttgtaggttcccaga ggaggaggagggaggatgcgagctgcgcgtgaagtttagccggtccgccgatgcacctgcatacaagcagggacagaaccagctgtat aacgagctgaatctgggccggagagaggagtacgacgtgctggataagaggcggggccgggaccccgagatgggaggcaagcctcg gagaaagaacccacaggagggcctgtacaatgagctgcaaaaggacaagatggccgaggcctattctgagatcggcatgaagggagag aggcgccggggcaagggacacgatggcctgtaccagggcctgagcaccgccacaaaggacacctatgatgccctgcacatgcaggcc ctgccccctagatga(SEQ ID NO: 18005).

[0128] In some embodiments of the VCARs of the disclosure, the VCAR comprises at least one of an amino acid sequence of at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of: VH-D: malpvtalllplalllhaarpevqllesggglvqpggslrlscaasgftfsnyamtwirqapgkglewvsgitgdggstfyadsvkgrftis rdnskntlylqmnslraedtavyycvkdwnttmitergqgtlvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfac diyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynel nlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalp pr(SEQIDNO: 18006).

[0129] In some embodiments of the VCARs of the disclosure, the amino acid sequence of the VCAR is encoded by a nucleotide sequence comprising a sequence of at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the nucleotide sequence of: VH-D atggctctgcctgtgacagctctgctgctgcctctggctctgcttcttcatgcggcgcgccctgaagttcagctgcttgaatctggcggaggc ctggttcaacctggcggatctctgagactgagctgtgccgccagcggcttcaccttcagcaattacgccatgacctggatcagacaggccc 2023208176 27 Jul 2023 ctggcaaaggcctggaatgggtgtccggaattacaggcgacggcggcagcaccttttacgccgattctgtgaagggcagattcaccatca gccgggacaacagcaagaacaccctgtacctgcagatgaacagcctgagagccgaggacaccgccgtgtactactgcgtgaaggactg gaacaccaccatgatcaccgagagaggccagggcacactggtcaccgtgtcctctacaacaacaccggcgcctcggcctccaacacca gctcctacaatcgcgagtcagcccctgtctctcagacccgaagcctgtagacctgctgctggcggagctgtgcataccagaggactggattt cgcctgcgacatctacatctgggctcctctggctggcacatgcggagttttgctgctgagcctggtcatcaccctgtactgtaagagaggca ggaagaagctgctgtatatcttcaagcagcccttcatgagacccgtgcagaccacacaggaggaggacggctgctcttgtaggttcccaga ggaggaggagggaggatgcgagctgcgcgtgaagtttagccggtccgccgatgcacctgcatacaagcagggacagaaccagctgtat aacgagctgaatctgggccggagagaggagtacgacgtgctggataagaggcggggccgggaccccgagatgggaggcaagcctcg gagaaagaacccacaggagggcctgtacaatgagctgcaaaaggacaagatggccgaggcctattctgagatcggcatgaagggagag aggcgccggggcaagggacacgatggcctgtaccagggcctgagcaccgccacaaaggacacctatgatgccctgcacatgcaggcc ctgccccctagatga(SEQ ID NO: 18007).

[0130] In some embodiments of the VCARs of the disclosure, the VCAR comprises at least one of an amino acid sequence of at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of: VH-E: malpvtalllplalllhaarpevqllesggglaqpggslrlscaasgftfssyamnwirqapgkglewvsgisgsggstyyadsvkgrfti srdnskntvylqmnslraedtavyycvkdwnttmitergqgtlvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfa cdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlyne Inlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqal ppr(SEQIDNO: 18008).

[0131] In some embodiments of the VCARs of the disclosure, the amino acid sequence of the VCAR is encoded by a nucleotide sequence comprising a sequence of at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the nucleotide sequence of: VH-E atggcactgcctgtgacagccctgctgctgcctctggccctgctgctgcacgcagcacggcccgaggtgcagctgctggagtccggagg aggcctggcccagcctggcggcagcctgaggctgtcctgcgccgcctctggcttcacctttagctcctacgccatgaactggatcagacag gcccctggcaagggcctggagtgggtgtccggcatctccggctctggaggctctacatactatgccgacagcgtgaagggccggttcacc atcagcagagataactccaagaataccgtgtacctccagatgaactctctgcgggccgaggacaccgccgtgtactattgcgtgaaggatt ggaataccacaatgatcacagagaggggccagggcaccctggtgacagtgtctagcaccacaacccctgcccccagacctcccacacc cgcccctaccatcgcgagtcagccactgtccctgcggcctgaggcctgccggcccgccgccggcggagcagtgcacacacggggcct ggactttgcctgtgacatctacatatgggcaccactggcaggaacctgcggcgtgctgctgctgagcctggtcatcaccctgtactgtaaga 2023208176 27 Jul 2023 gaggcaggaagaagctgctgtatatcttcaagcagcccttcatgagacccgtgcagaccacacaggaggaggacggctgctcttgtaggt tcccagaggaggaggagggaggatgcgagctgcgcgtgaagtttagccggtccgccgatgcacctgcatacaagcagggacagaacc agctgtataacgagctgaatctgggccggagagaggagtacgacgtgctggataagaggcggggccgggaccccgagatgggaggca agcctcggagaaagaacccacaggagggcctgtacaatgagctgcaaaaggacaagatggccgaggcctattctgagatcggcatgaa gggagagaggcgccggggcaagggacacgatggcctgtaccagggcctgagcaccgccacaaaggacacctatgatgccctgcacat gcaggccctgccccctagatga(SEQ ID NO: 18009).

[0132] In some embodiments of the VCARs of the disclosure, the VCAR at least one of comprises an amino acid sequence of at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of: VH-F: malpvtalllplalllhaarpevqllesggglvqpgrslrlscaasgftftnyamnwvrqapgkglewvsgisggggstyyadsvkgrfti srdnskntlylqmnslraedtavyycvkdwnttmitergqgtlvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfac diyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynel nlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalp pr(SEQIDNO: 18010).

[0133] In some embodiments of the VCARs of the disclosure, the amino acid sequence of the VCAR is encoded by a nucleotide sequence comprising a sequence of at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the nucleotide sequence of: VH-F atggcactgcctgtgacagccctgctgctgcctctggccctgctgctgcacgcagcacggcccgaggtgcagctgctggagtctggagga ggcctggtgcagcccggccggtccctgagactgtcttgcgccgccagcggcttcacctttacaaactacgccatgaattgggtgcggcag gcccctggcaagggcctggagtgggtgtctggcatcagcggaggaggaggcagcacctactatgcagactccgtgaagggcaggttca ccatctcccgcgataactctaagaatacactgtacctccagatgaacagcctgagggcagaggacaccgccgtgtactattgcgtgaagga ttggaataccacaatgatcacagagaggggacagggcaccctggtgaccgtgagcagcaccacaacccctgcccccagacctcccaca cccgcccctaccatcgcgagtcagccactgtccctgcggcctgaggcctgccggcccgccgccggcggagcagtgcacacacggggc ctggactttgcctgtgacatctacatatgggcaccactggcaggaacctgcggcgtgctgctgctgagcctggtcatcaccctgtactgtaag agaggcaggaagaagctgctgtatatcttcaagcagcccttcatgagacccgtgcagaccacacaggaggaggacggctgctcttgtagg ttcccagaggaggaggagggaggatgcgagctgcgcgtgaagtttagccggtccgccgatgcacctgcatacaagcagggacagaacc agctgtataacgagctgaatctgggccggagagaggagtacgacgtgctggataagaggcggggccgggaccccgagatgggaggca agcctcggagaaagaacccacaggagggcctgtacaatgagctgcaaaaggacaagatggccgaggcctattctgagatcggcatgaa 2023208176 27 Jul 2023 gggagagaggcgccggggcaagggacacgatggcctgtaccagggcctgagcaccgccacaaaggacacctatgatgccctgcacat gcaggccctgccccctagatga(SEQ ID NO: 18011).

[0134] In some embodiments of the VCARs of the disclosure, the VCAR comprises a sequence encoding VH-A, VH-B, VH-C, VH-D, VH-E, or VH-F. In some embodiments of the VCARs of the disclosure, the VCAR comprises two sequences encoding en VH-A, VH-B, VH-C, VH-D, VH-E, or VH-F. Immune and Immune Precursor Cells

[0135] In certain embodiments, immune cells of the disclosure comprise lymphoid progenitor cells, natural killer (NK) cells, T lymphocytes (T-cell), stem memory T cells (Tscm cells), central memory T cells (Tcm), stem cell-like T cells, B lymphocytes (B-cells), myeloid progenitor cells, neutrophils, basophils, eosinophils, monocytes, macrophages, platelets, erythrocytes, red blood cells (RBCs), megakaryocytes or osteoclasts.

[0136] In certain embodiments, immune precursor cells comprise any cells which can differentiate into one or more types of immune cells. In certain embodiments, immune precursor cells comprise multipotent stem cells that can self renew and develop into immune cells. In certain embodiments, immune precursor cells comprise hematopoietic stem cells (HSCs) or descendants thereof. In certain embodiments, immune precursor cells comprise precursor cells that can develop into immune cells. In certain embodiments, the immune precursor cells comprise hematopoietic progenitor cells (HPCs). Hematopoietic Stem Cells (HSCs)

[0137] Hematopoietic stem cells (HSCs) are multipotent, self-renewing cells. All differentiated blood cells from the lymphoid and myeloid lineages arise from HSCs. HSCs can be found in adult bone marrow, peripheral blood, mobilized peripheral blood, peritoneal dialysis effluent and umbilical cord blood.

[0138] HSCs of the disclosure may be isolated or derived from a primary or cultured stem cell. HSCs of the disclosure may be isolated or derived from an embryonic stem cell, a multipotent stem cell, a pluripotent stem cell, an adult stem cell, or an induced pluripotent stem cell (iPSC).

[0139] Immune precursor cells of the disclosure may comprise an HSC or an HSC descendent cell. Exemplary HSC descendent cells of the disclosure include, but are not limited to, multipotent stem cells, lymphoid progenitor cells, natural killer (NK) cells, T lymphocyte cells 2023208176 27 Jul 2023 (T-cells), B lymphocyte cells (B-cells), myeloid progenitor cells, neutrophils, basophils, eosinophils, monocytes, and macrophages.

[0140] HSCs produced by the methods of the disclosure may retain features of “primitive” stem cells that, while isolated or derived from an adult stem cell and while committed to a single lineage, share characteristics of embryonic stem cells. For example, the “primitive” HSCs produced by the methods of the disclosure retain their “sternness” following division and do not differentiate. Consequently, as an adoptive cell therapy, the “primitive” HSCs produced by the methods of the disclosure not only replenish their numbers, but expand in vivo. “Primitive” HSCs produced by the methods of the disclosure may be therapeutically-effective when administered as a single dose. In some embodiments, primitive HSCs of the disclosure are CD34+. In some embodiments, primitive HSCs of the disclosure are CD34+ and CD38-. In some embodiments, primitive HSCs of the disclosure are CD34+, CD38- and CD90+. In some embodiments, primitive HSCs of the disclosure are CD34+, CD38-, CD90+ and CD45RA-. In some embodiments, primitive HSCs of the disclosure are CD34+, CD38-, CD90+, CD45RA-, and CD49f+. In some embodiments, the most primitive HSCs of the disclosure are CD34+, CD38-, CD90+, CD45RA-, and CD49f+.

[0141] In some embodiments of the disclosure, primitive HSCs, HSCs, and / or HSC descendent cells may be modified according to the methods of the disclosure to express an exogenous sequence (e.g., a chimeric antigen receptor or therapeutic protein). In some embodiments of the disclosure, modified primitive HSCs, modified HSCs, and / or modified HSC descendent cells may be forward differentiated to produce a modified immune cell including, but not limited to, a modified T cell, a modified natural killer cell and / or a modified B-cell of the disclosure. T Cells

[0142] Modified T cells of the disclosure may be derived from modified hematopoietic stem and progenitor cells (HSPCs) or modified HSCs.

[0143] Unlike traditional biologies 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, in some embodiments, modified-T cells of the disclosure not only drive an initial response, but also persist in the patient as a stable population of viable memory T cells to prevent potential relapses. Alternatively, in some embodiments, when it is not desired, modified-T cells of the disclosure do not persist in the patient. 2023208176 27 Jul 2023

[0144] 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 T cells, especially stem cell memory (Tscm) or stem cell-like T cells. Stem cell-like modified-T cells of the disclosure exhibit the greatest capacity for self-renewal and multipotent capacity to derive central memory (Tcm) T cells or Tcm like cells, effector memory (Tem) and effector T cells (Te), thereby producing better tumor eradication and long-term modified-T cell engraftment. A linear pathway of differentiation may be responsible for generating these cells: Naive T cells (Tn) > Tscm > Tcm > Tem > Te > Tte, whereby Tn is the parent precursor cell that directly gives rise to Tscm, which then, in turn, directly gives rise to Tcm, etc. Compositions of T cells of the disclosure may comprise one or more of each parental T cell subset with Tscm or Tcm cells being the most abundant (e.g., Tscm > Tcm > Tem > Te > Tte).

[0145] In some embodiments of the methods of the disclosure, the immune cell precursor is differentiated into or is capable of differentiating into an early memory T cell, a stem cell like T-cell, a Naive T cells (Tn), a Tscm, a Tcm, a Tem, a Te, or a Tte. In some embodiments, the immune cell precursor is a primitive HSC, an HSC, or a HSC descendent cell of the disclosure.

[0146] In some embodiments of the methods of the disclosure, the immune cell is an early memory T cell, a stem cell like T-cell, a Naive T cells (Tn), a Tscm, a Tcm, a Tem, a Te, or a Tte.

[0147] In some embodiments of the methods of the disclosure, the immune cell is an early memory T cell.

[0148] In some embodiments of the methods of the disclosure, the immune cell is a stem cell like T-cell.

[0149] In some embodiments of the methods of the disclosure, the immune cell is a Tscm.

[0150] In some embodiments of the methods of the disclosure, the immune cell is a Tcm.

[0151] In some embodiments of the methods of the disclosure, the methods modify and / or the methods produce a plurality of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 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 expresses one or more cell-surface marker(s) of an early memory T cell. In certain embodiments, the plurality of modified early memory T cells comprises at least one modified stem cell-like T cell. In certain embodiments, the plurality of modified early memory T cells comprises at least one modified Tscm. In certain 2023208176 27 Jul 2023 embodiments, the plurality of modified early memory T cells comprises at least one modified Tcm.

[0152] In some embodiments of the methods of the disclosure, the methods modify and / or the methods produce a plurality of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 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 expresses one or more cell-surface marker(s) of a stem cell-like T cell. In certain embodiments, the plurality of modified stem celllike T cells comprises at least one modified Tscm. In certain embodiments, the plurality of modified stem cell-like T cells comprises at least one modified Tcm.

[0153] In some embodiments of the methods of the disclosure, the methods modify and / or the methods produce a plurality of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 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 expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm). In certain embodiments, the cell-surface markers comprise CD62L and CD45RA. In certain embodiments, the cell-surface markers comprise one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2Rp. In certain embodiments, the cell-surface markers comprise one or more of CD45RA, CD95, IL-2Rp, CCR7, and CD62L.

[0154] In some embodiments of the methods of the disclosure, the methods modify and / or the methods produce a plurality of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 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 expresses one or more cell-surface marker(s) of a central memory T cell (Tcm). In certain embodiments, the cell-surface markers comprise one or more of CD45RO, CD95, IL-2RP, CCR7, and CD62L.

[0155] In some embodiments of the methods of the disclosure, the methods modify and / or the methods produce a plurality of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 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 expresses one or more cell-surface marker(s) of a naive T cell (Tn). In certain embodiments, the cell-surface markers comprise one or more of CD45RA, CCR7 and CD62L. 2023208176 27 Jul 2023

[0156] In some embodiments of the methods of the disclosure, the methods modify and / or the methods produce a plurality of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 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 expresses one or more cell-surface marker(s) of an effector T-cell (modified Teff). In certain embodiments, the cell-surface markers comprise one or more of CD45RA, CD95, and IL-2R0.

[0157] In some embodiments of the methods of the disclosure, the methods modify and / or the methods produce a plurality of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 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 expresses one or more cell-surface marker(s) of a stem cell-like T cell, a stem memory T cell (Tscm) or a central memory T cell (Tcm).

[0158] In some embodiments of the methods of the disclosure, a buffer comprises the immune cell or precursor thereof. The buffer maintains or enhances a level of cell viability and / or a stemlike phenotype of the immune cell or precursor thereof, including 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 viability and / 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 one or more of KC1, MgCh, CINa, 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 / HCl, and a phosphate buffer. In certain embodiments, the buffer comprises 5 mM KC1, 15 mM MgCh, 90 mM CINa, 10 mM Glucose and 0.4 mM Ca(NO3)2. In certain embodiments, the buffer comprises 5 mM KC1, 15 mM MgCh, 90 mM CINa, 10 mM Glucose and 0.4 mM Ca(NO3)2 and a supplement comprising 20 mM HEPES and 75 mM Tris / HCl. In certain embodiments, the buffer comprises 5 mM KC1, 15 mM MgCh, 90 mM CINa, 10 mM Glucose and 0.4 mM Ca(NO3)2 and a supplement comprising 40 mM Na2HPO4 / NaH2PO4 at pH 7.2. In certain embodiments, the composition comprising primary human T cells comprises 100 pl of the buffer and between 5xl06 and 25xl06 cells. In certain embodiments, the composition 2023208176 27 Jul 2023 comprises a scalable ratio of 250xl06 primary human T cells per milliliter of buffer or other media during the introduction step.

[0159] In some embodiments of the methods of the disclosure, the methods comprise contacting an immune cell of the disclosure, including a T cell of the disclosure, and a T-cell expansion composition. In some embodiments of the methods of the disclosure, the step of introducing a transposon and / or transposase of the disclosure into an immune cell of the disclosure may further comprise contacting the immune cell and a T-cell expansion composition. In some embodiments, including those in which the introducing step of the methods comprises an electroporation or a nucleofection step, the electroporation or a nucleofection step may be performed with the immune cell contacting T-cell expansion composition of the disclosure.

[0160] In some embodiments of the methods of the disclosure, the T-cell expansion composition comprises, consists essentially of or consists of phosphorus; one or more of an octanoic acid, a palmitic acid, a linoleic acid, and an oleic acid; a sterol; and an alkane.

[0161] In certain embodiments of the methods of producing a modified T cell 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 (INFy). The one or more cytokine(s) may comprise IL-2.

[0162] In some embodiments of the methods of the disclosure, the T-cell expansion composition comprises human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, and an expansion supplement. 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-benzenedicarboxylic 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, 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 2023208176 27 Jul 2023 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. 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, 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 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. 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 pmol / kg and a sterol at a concentration of about 2.5 pmol / kg.

[0163] In certain embodiments, the T-cell expansion composition comprises one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, and an expansion supplement to produce a plurality of expanded modified T-cells, wherein at least 2% of the plurality of modified T-cells expresses one or more cell-surface marker(s) of an early memory T cell, a stem cell-like T cell, a stem memory T cell (Tscm) and / or a central memory T cell (Tcm). 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, 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; 2023208176 27 Jul 2023 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 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 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 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 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. 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 pmol / 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, the T-cell expansion composition comprises octanoic acid at a concentration of about 63.75 pmol / kg, palmitic acid at a 2023208176 27 Jul 2023 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 pmol / kg.

[0164] 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, 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 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 ThermoFisher Scientific as Catalog number 12440053).

[0165] 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 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 or more 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.

[0166] 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, 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-tetramethyl-5-decyn-4,7-diol (TMDD) (CAS No. 12686-3), diisopropyl adipate (DIPA) (CAS No. 6938-94-9), n-butyl-benzenesulfonamide (CAS No. 2023208176 27 Jul 2023 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. 112-801), 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-tetramethyl-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-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. 143-748). 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-tetramethyl-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-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. 143-748) and lanolin alcohol.

[0167] 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 human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, 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 ions: sodium, ammonium, potassium, magnesium, calcium, chloride, sulfate and phosphate.

[0168] 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 2023208176 27 Jul 2023 human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, 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 free amino acids: histidine, asparagine, serine, glutamate, arginine, glycine, aspartic acid, glutamic acid, 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%), serine (about 1.6%), glutamine (about 67.01%), arginine (about 1.67%), glycine (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%).

[0169] 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 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, 2023208176 27 Jul 2023 Iscove's Modified Dulbecco's Medium ((IMDM); available at ThermoFisher Scientific as Catalog number 12440053).

[0170] 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, palmitic acid, linoleic acid, 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 / 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 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 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 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 mg / 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 1.86 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 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 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 2023208176 27 Jul 2023 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, 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 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.

[0171] 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 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 about 7.56 pmol / kg and a sterol at a concentration of about 2.61 pmol / 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 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 pmol / kg.

[0172] In certain embodiments of the methods of producing a modified T cell (e.g., a stem celllike T cell, a Tscm and / or a Tcm) of the disclosure, the method comprises contacting a modified T cell and an inhibitor of the P13K-Akt-mTOR pathway. Modified T-cells of the disclosure, including modified stem cell-like T cells, Tscm and / or Tcm of the 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 PI3K pathway. Exemplary inhibitors a component of a PI3K 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 C18H14N4O2). Exemplary inhibitors of a component of a PI3K pathway include, but are not limited to, bb007 (BLUEBIRDBIO™). Additional Exemplary inhibitors of a component of a PI3K pathway include, but are not limited to, an allosteric Akt inhibitor VIII (also referred to as Akti-1 / 2 having Compound number 10196499), ATP competitive inhibitors (Orthosteric inhibitors targeting the ATP-binding pocket of the protein 2023208176 27 Jul 2023 kinase B (Akt)), Isoquinoline-5-sulfonamides (H-8, H-89, and NL-71-101), Azepane derivatives (A series of structures derived from (-)-balanol), Aminofurazans (GSK690693), Heterocyclic rings (7-azaindole, 6-phenylpurine derivatives, pyrrolo[2,3-d]pyrimidine derivatives, CCT128930, 3-aminopyrrolidine, anilinotriazole derivatives, spiroindoline derivatives, AZD5363, ipatasertib (GDC-0068, RG7440), A-674563, and A-443654), Phenylpyrazole derivatives (AT7867 and AT13148), Thiophenecarboxamide derivatives (Afuresertib (GSK2110183), 2-pyrimidyl-5-amidothiophene derivative (DC120), uprosertib (GSK2141795)), Allosteric inhibitors (Superior to orthosteric inhibitors providing greater specificity, reduced side-effects and less toxicity), 2,3-diphenylquinoxaline analogues (2,3-diphenylquinoxaline derivatives, triazolo[3,4-f][l,6]naphthyridin-3(2H)-one derivative (MK-2206)), Alkylphospholipids (Edelfosine (l-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine, ET-I8-OCH3) ilmofosine (BM 41.440), miltefosine (hexadecylphosphocholine, HePC), perifosine (D-21266), erucylphosphocholine (ErPC), erufosine (ErPC3, erucylphosphohomocholine), Indole-3-carbinol analogues (Indole-3-carbinol, 3-chloroacetylindole, diindolylmethane, diethyl 6-methoxy-5,7-dihydroindolo [2,3-b]carbazole-2,10-dicarboxylate (SR13668), OSU-A9), Sulfonamide derivatives (PH-316 and PHT-427), Thiourea derivatives (PIT-1, PIT-2, DM-PIT-1, N-[(l-methyl-lH-pyrazol-4-yl)carbonyl]-N'-(3-bromophenyl)-thiourea), Purine derivatives (Triciribine (TCN, NSC 154020), triciribine mono-phosphate active analogue (TCN-P), 4-amino-pyrido[2,3-d]pyrimidine derivative API-1, 3-phenyl-3H-imidazo[4,5-b]pyridine derivatives, ARQ 092), BAY 1125976, 3-methyl-xanthine, quinoline-4-carboxamide and 2-[4-(cyclohexa-l,3-dien-l-yl)-lH-pyrazol-3-yl]phenol, 3-oxo-tirucallic acid, 3a- and 3P-acetoxy-tirucallic acids, acetoxy-tirucallic acid, and irreversible inhibitors (antibiotics, Lactoquinomycin, FrenolicinB, kalafungin, medermycin, Boc-Phe-vinyl ketone, 4-hydroxynonenal (4-HNE), 1,6-naphthyridinone derivatives, and imidazo-l,2-pyridine derivatives).

[0173] In certain embodiments of the methods of producing a modified T cell (e.g., a stem celllike T cell, a Tscm and / or a Tcm) of the disclosure, the method comprises contacting a modified T cell and an inhibitor of T cell effector differentiation. Exemplary inhibitors of T cell effector differentiation include, but are not limited to, a BET inhibitor (e.g., JQ1, a hienotriazolodiazepine) and / or an inhibitor of the BET family of proteins (e.g., BRD2, BRD3, BRD4, and BRDT). 2023208176 27 Jul 2023

[0174] In certain embodiments of the methods of producing a modified T cell (e.g., a stem celllike T cell, a Tscm and / or a Tcm) of the disclosure, the method comprises contacting a modified T cell and an agent that reduces nucleo-cytoplasmic Acetyl-CoA. Exemplary agents that reduce nucleo-cytoplasmic Acetyl-CoA include, but are not limited to, 2-hydroxy-citrate (2-HC) as well as agents that increase expression of Acssl.

[0175] In certain embodiments of the methods of producing a modified T cell (e.g., a stem celllike T cell, a Tscm and / or a Tcm) of the disclosure, the method comprises contacting a modified T cell and a composition comprising a histone deacetylase (HDAC) inhibitor. In some embodiments, the composition comprising an HD AC inhibitor comprises or consists of valproic acid, Sodium Phenylbutyrate (NaPB) or a combination thereof. In some embodiments, the composition comprising an HD AC inhibitor comprises or consists of valproic acid. In some embodiments, the composition comprising an HD AC inhibitor comprises or consists of Sodium Phenylbutyrate (NaPB).

[0176] In certain embodiments of the methods of producing a modified T cell (e.g., a stem celllike T cell, a Tscm and / or a Tcm) of the disclosure, the activation supplement may comprise 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 (INFy). The one or more cytokine(s) may comprise IL-2.

[0177] In certain embodiments of the methods of producing a modified T cell (e.g., a stem celllike T cell, a Tscm and / or a Tcm) of the disclosure, the activation supplement may comprise one or more activator complexes. Exemplary and nonlimiting activator complexes may comprise a monomeric, dimeric, trimeric or tetrameric antibody complex that binds one or more of CD3, CD28, and CD2. In some embodiments, the activation supplement comprises or consists of an activator complex that comprises a human, a humanized or a recombinant or a chimeric antibody. In some embodiments, the activation supplement comprises or consists of an activator complex that binds CD3 and CD28. In some embodiments, the activation supplement comprises or consists of an activator complex that binds CD3, CD28 and CD2. Natural Killer INK) cells 2023208176 27 Jul 2023

[0178] In certain embodiments, the modified immune or immune precursor cells of the disclosure are natural killer (NK) cells. In certain embodiments, NK cells are cytotoxic lymphocytes that differentiate from lymphoid progenitor cells.

[0179] Modified NK cells of the disclosure may be derived from modified hematopoietic stem and progenitor cells (HSPCs) or modified HSCs.

[0180] In certain embodiments, non-activated NK cells are derived from CD3-depleted leukopheresis (containing CD14 / CD19 / CD56+ cells).

[0181] In certain embodiments, NK cells are electroporated using a Lonza 4D nucleofector or BTX ECM 830 (500V, 700 usee pulse length, 0.2 mm electrode gap, one pulse). All Lonza 4D nucleofector programs are contemplated as within the scope of the methods of the disclosure.

[0182] In certain embodiments, 5xlOE6 cells were electroporated per electroporation in 100 pL P3 buffer in cuvettes. However, this ratio of cells per volume is scalable for commercial manufacturing methods.

[0183] In certain embodiments, NK cells were stimulated by co-culture with an additional cell line. In certain embodiments, the additional cell line comprises artificial antigen presenting cells (aAPCs). In certain embodiments, stimulation occurs at day 1, 2, 3, 4, 5, 6, or 7 following electroporation. In certain embodiments, stimulation occurs at day 2 following electroporation.

[0184] In certain embodiments, NK cells express CD56. B cells

[0185] In certain embodiments, the modified immune or immune precursor cells of the disclosure are B cells. B cells are a type of lymphocyte that express B cell receptors on the cell surface. B cell receptors bind to specific antigens.

[0186] Modified B cells of the disclosure may be derived from modified hematopoietic stem and progenitor cells (HSPCs) or modified HSCs.

[0187] In certain embodiments, HSPCs are modified using the methods of the disclosure, and then primed for B cell differentiation in presence of human IL-3, Flt3L, TPO, SCF, and G-CSF for at least 3 days, at least 4 days, at least 5 days, at least 6 days or at least 7 days. In certain embodiments, HSPCs are modified using the methods of the disclosure, and then primed for B cell differentiation in presence of human IL-3, Flt3L, TPO, SCF, and G-CSF for 5 days. 2023208176 27 Jul 2023

[0188] In certain embodiments, following priming, modified HSPC cells are transferred to a layer of feeder cells and fed bi-weekly, along with transfer to a fresh layer of feeders once per week. In certain embodiments, the feeder cells are MS-5 feeder cells.

[0189] In certain embodiments, modified HSPC cells are cultured with MS-5 feeder cells for at least 7, 14, 21, 28, 30, 33, 35, 42 or 48 days. In certain embodiments, modified HSPC cells were cultured with MS-5 feeder cells for 33 days. Transposition Systems

[0190] Exemplary transposon / transposase systems of the disclosure include, but are not limited to, piggyBac transposons and transposases, piggyBac-like transposons and transposases, Sleeping Beauty transposons and transposases, Helraiser transposons and transposases and Tol2 transposons and transposases.

[0191] The piggyBac transposase recognizes transposon-specific inverted terminal repeat sequences (ITRs) on the ends of the transposon, and moves the contents between the ITRs into TTAA chromosomal sites. The piggyBac transposon system has no payload limit for the genes of interest that can be included between the ITRs. In certain embodiments, and, in particular, those embodiments wherein the transposon is a piggyBac transposon, the transposase is a piggyBac or a Super piggyBac (SPB) transposase. In certain embodiments, and, in particular, those embodiments wherein the transposase is a Super piggyBac (SPB) transposase, the sequence encoding the transposase is an mRNA sequence.

[0192] In certain embodiments of the methods of the disclosure, the transposase enzyme is a piggyBac (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 LSALLQSDDE LVGEDSDSEI SDHVSEDDVQ SDTEEAFIDE VHEVQPTSSG 61 SEILDEQNVI EQPGSSLASN RILTLPQRTI RGKNKHCWST SKSTRRSRVS ALNIVRSQRG 121 PTRMCRNIYD PLLCFKLFFT DEIISEIVKW TNAEISLKRR ESMTGATFRD TNEDEIYAFF 181 GILVMTAVRK DNHMSTDDLF DRSLSMVYVS VMSRDRFDFL IRCLRMDDKS IRPTLRENDV 241 FTPVRKIWDL FIHQCIQNYT PGAHLTIDEQ LLGFRGRCPF RMYIPNKPSK YGIKILMMCD 301 SGTKYMINGM PYLGRGTQTN GVPLGEYYVK ELSKPVHGSC RNITCDNWFT SIPLAKNLLQ 361 EPYKLTIVGT VRSNKREIPE VLKNSRSRPV GTSMFCFDGP LTLVSYKPKP AKMVYLLSSC 421 DEDASINEST GKPQMVMYYN QTKGGVDTLD QMCSVMTCSR KTNRWPMALL YGMINIACIN 481 SFIIYSHNVS SKGEKVQSRK KFMRNLYMSL TSSFMRKRLE APTLKRYLRD NISNILPNEV 2023208176 27 Jul 2023 541 PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV ICREHNIDMC QSCF (SEQ ID NO: 14487).

[0193] In certain embodiments of the methods of the disclosure, the transposase enzyme is a piggyBac (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 SDHVSEDDVQ SDTEEAFIDE VHEVQPTSSG 61 SEILDEQNVI EQPGSSLASN RILTLPQRTI RGKNKHCWST SKSTRRSRVS ALNIVRSQRG 121 PTRMCRNIYD PLLCFKLFFT DEIISEIVKW TNAEISLKRR ESMTGATFRD TNEDEIYAFF 181 GILVMTAVRK DNHMSTDDLF DRSLSMVYVS VMSRDRFDFL IRCLRMDDKS IRPTLRENDV 241 FTPVRKIWDL FIHQCIQNYT PGAHLTIDEQ LLGFRGRCPF RMYIPNKPSK YGIKILMMCD 301 SGTKYMINGM PYLGRGTQTN GVPLGEYYVK ELSKPVHGSC RNITCDNWFT SIPLAKNLLQ 361 EPYKLTIVGT VRSNKREIPE VLKNSRSRPV GTSMFCFDGP LTLVSYKPKP AKMVYLLSSC 421 DEDASINEST GKPQMVMYYN QTKGGVDTLD QMCSVMTCSR KTNRWPMALL YGMINIACIN 481 SFIIYSHNVS SKGEKVQSRK KFMRNLYMSL TSSFMRKRLE APTLKRYLRD NISNILPNEV 541 PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV ICREHNIDMC QSCF (SEQ ID NO: 14487) .

[0194] In certain embodiments, the transposase enzyme is a piggyBac (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: 14487. In certain embodiments, the transposase enzyme is a piggyBac (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: 14487. In certain embodiments, the transposase enzyme is a piggyBac (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: 14487. In certain embodiments, the amino acid substitution at position 30 of the sequence of SEQ ID NO: 14487 is a substitution of a valine (V) for an isoleucine (I). In certain embodiments, the amino acid substitution at position 165 of the sequence of SEQ ID NO: 14487 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: 14487 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: 14487 is a substitution of a lysine (K) for an asparagine (N).

[0195] In certain embodiments of the methods of the disclosure, the transposase enzyme is a Super piggyBac (SPB) transposase enzyme. In certain embodiments, the Super piggyBac (SPB) 72 2023208176 27 Jul 2023 transposase enzymes of the disclosure may comprise or consist of the amino acid sequence of the sequence of SEQ ID NO: 14487 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 piggyBac (SPB) 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 LSALLQSDDE LVGEDSDSEV SDHVSEDDVQ SDTEEAFIDE VHEVQPTSSG 61 SEILDEQNVI EQPGSSLASN RILTLPQRTI RGKNKHCWST SKSTRRSRVS ALNIVRSQRG 121 PTRMCRNIYD PLLCFKLFFT DEIISEIVKW TNAEISLKRR ESMTSATFRD TNEDEIYAFF 181 GILVMTAVRK DNHMSTDDLF DRSLSMVYVS VMSRDRFDFL IRCLRMDDKS IRPTLRENDV 241 FTPVRKIWDL FIHQCIQNYT PGAHLTIDEQ LLGFRGRCPF RVYIPNKPSK YGIKILMMCD 301 SGTKYMINGM PYLGRGTQTN GVPLGEYYVK ELSKPVHGSC RNITCDNWFT SIPLAKNLLQ 361 EPYKLTIVGT VRSNKREIPE VLKNSRSRPV GTSMFCFDGP LTLVSYKPKP AKMVYLLSSC 421 DEDASINEST GKPQMVMYYN QTKGGVDTLD QMCSVMTCSR KTNRWPMALL YGMINIACIN 481 SFIIYSHNVS SKGEKVQSRK KFMRNLYMSL TSSFMRKRLE APTLKRYLRD NISNILPKEV 541 PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV ICREHNIDMC QSCF (SEQ ID NO: 14484) .

[0196] 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 piggyBac 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: 14487 or SEQ ID NO: 14484. In certain embodiments, including those embodiments wherein the transposase comprises the above-described mutations at positions 30, 165, 282 and / or 538, the piggyBac or Super piggyBac 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 is a substitution of a 2023208176 27 Jul 2023 serine (S) for an alanine (A). In certain embodiments, the amino acid substitution at position 46 of SEQ ID NO: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 is a substitution of a proline (P) for a serine (S). In certain embodiments, the amino acid substitution at position 119 of SEQ ID NO: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 is a substitution of an alanine (A) a cysteine (C). In certain embodiments, the amino acid substitution at position 125 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a leucine (L) for a cysteine (C). In certain embodiments, the amino acid substitution at position 177 of SEQ ID NO: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 is a substitution of a histidine (H) for a tyrosine (Y). In certain embodiments, the amino acid substitution at position 180 of SEQ ID NO: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 is a substitution of a phenylalanine (F) for a methionine (M). In certain embodiments, the amino acid 2023208176 27 Jul 2023 substitution at position 235 of SEQ ID NO: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 is a substitution of a serine (S) for an asparagine (N). In certain embodiments, the amino acid substitution at position 296 of SEQ ID NO: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 is a substitution of a valine for a proline (P). In certain embodiments, the amino acid substitution at position 315 of SEQ ID NO: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 is a 2023208176 27 Jul 2023 substitution of a glycine (G) for a cysteine (C). In certain embodiments, the amino acid substitution at position 340 of SEQ ID NO: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 is a substitution of an arginine (R) for a glutamine (Q).

[0197] 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 piggyBac transposase enzyme may comprise or the Super piggyBac 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: 14487 or SEQ ID NO: 14484. 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 piggyBac transposase enzyme may comprise or the Super piggyBac transposase enzyme may further comprise an amino acid substitution at two, three, four, five, six or more of positions 103, 2023208176 27 Jul 2023 194, 372, 375, 450, 509 and 570 of the sequence of SEQ ID NO: 14487 or SEQ ID NO: 14484. In certain embodiments, including those embodiments wherein the transposase comprises the above-described mutations at positions 30, 165, 282 and / or 538, the piggyBac transposase enzyme may comprise or the Super piggyBac transposase 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: 14487 or SEQ ID NO: 14484. In certain embodiments, the amino acid substitution at position 103 of SEQ ID NO: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 is a substitution of a serine (S) for an asparagine (N). In certain embodiments, the piggyBac transposase enzyme may comprise a substitution of a valine (V) for a methionine (M) at position 194 of SEQ ID NO: 14487. In certain embodiments, including those embodiments wherein the piggyBac transposase enzyme may comprise a substitution of a valine (V) for a methionine (M) at position 194 of SEQ ID NO: 14487, the piggyBac transposase enzyme may further comprise an amino acid substitution at positions 372, 375 and 450 of the sequence of SEQ ID NO: 14487 or SEQ ID NO: 14484. In certain embodiments, the piggyBac transposase enzyme may comprise a substitution of a valine (V) for a methionine (M) at position 194 of SEQ ID NO: 14487, a substitution of an alanine (A) for an arginine (R) at position 372 of SEQ ID NO: 14487, and a substitution of an alanine (A) for a lysine (K) at position 375 of SEQ ID NO: 14487. In certain embodiments, the piggyBac transposase enzyme may comprise a substitution of a valine (V) for a methionine (M) at position 194 of SEQ ID NO: 14487, a substitution of an alanine (A) for an arginine (R) at position 372 of SEQ ID NO: 14487, a substitution of an 2023208176 27 Jul 2023 alanine (A) for a lysine (K) at position 375 of SEQ ID NO: 14487 and a substitution of an asparagine (N) for an aspartic acid (D) at position 450 of SEQ ID NO: 14487.

[0198] The sleeping beauty transposon is transposed into the target genome by the Sleeping Beauty transposase that recognizes ITRs, and moves the contents between the ITRs into TA chromosomal sites. In various embodiments, SB transposon-mediated gene transfer, or gene transfer using any of a number of similar transposons, may be used in the compositions and methods of the disclosure.

[0199] 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).

[0200] 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 LRKKIVDLHK SGSSLGAISK RLKVPRSSVQ TIVRKYKHHG TTQPSYRSGR 61 RRVLSPRDER TLVRKVQINP RTTAKDLVKM LEETGTKVSI STVKRVLYRH NLKGRSARKK 121 PLLQNRHKKA RLRFATAHGD KDRTFWRNVL WSDETKIELF GHNDHRYVWR KKGEACKPKN 181 TIPTVKHGGG SIMLWGCFAA GGTGALHKID GIMRKENYVD ILKQHLKTSV RKLKLGRKWV 241 FQMDNDPKHT SKWAKWLKD NKVKVLEWPS QSPDLNPIEN LWAELKKRVR ARRPTNLTQL 301 HQLCQEEWAK IHPTYCGKLV EGYPKRLTQV KQFKGNATKY (SEQ ID NO: 14485).

[0201] In certain embodiments of the methods of the disclosure, the hyperactive Sleeping Beauty (SB100X) transposase enzyme comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to: 1 MGKSKEISQD LRKRIVDLHK SGSSLGAISK RLAVPRSSVQ TIVRKYKHHG TTQPSYRSGR 61 RRVLSPRDER TLVRKVQINP RTTAKDLVKM LEETGTKVSI STVKRVLYRH NLKGHSARKK 121 PLLQNRHKKA RLRFATAHGD KDRTFWRNVL WSDETKIELF GHNDHRYVWR KKGEACKPKN 181 TIPTVKHGGG SIMLWGCFAA GGTGALHKID GIMDAVQYVD ILKQHLKTSV RKLKLGRKWV 241 FQHDNDPKHT SKWAKWLKD NKVKVLEWPS QSPDLNPIEN LWAELKKRVR ARRPTNLTQL 301 HQLCQEEWAK IHPNYCGKLV EGYPKRLTQV KQFKGNATKY (SEQ ID NO: 14486).

[0202] The Helraiser transposon is transposed by the Helitron transposase. Helitron transposases mobilize the Helraiser 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 Helibatl, which comprises a nucleic acid sequence comprising: 1 TCCTATATAA TAAAAGAGAA ACATGCAAAT TGACCATCCC TCCGCTACGC TCAAGCCACG 61 CCCACCAGCC AATCAGAAGT GACTATGCAA ATTAACCCAA CAAAGATGGC AGTTAAATTT 2023208176 27 Jul 2023 121 GCATACGCAG GTGTCAAGCG CCCCAGGAGG 181 GCTGGCCCCG GGAGGCGAGG CCGGCCGCGC 241 CGCCAGCAGA GAGCAGAGCG GGAGAGCGGG 301 GAGCAGGAGG CCGCTGGACA TAGAGCAGAG 361 TCCCTCTGTC ACCCCAGCTT CCTCATCACA 421 GTCCCACCCC CACAGAATCA GCCAGAATCA 481 ACAGCCAGGA CTCTCATTCA CCTGCATCTC 541 TCTAAAGAAC AACTGTTGAT ACAACGTAGC 601 CAGAAAATGT CTGCAGAGCA ACGTGCGTCT 661 AATGTATCTG AAGAGCAGCT ACTGGAAAAA 721 CATCGACAGA AAATGTCTAA AGACCAACGT 781 CGACAGAATA TGTCTAGAGA ACAGTCATCA 841 CTTCTCAGCA AAAATGGAGT ACATGAGGAT 901 ACTGTTCGAT GTGAATTTTG CCTATCACTA 961 AAATTTACTC GATGTTGTAG CAAAGGGAAA 1021 TACCCGGCAT ATTTAAAAAG ATTAATGACA 1081 GAAAATATTC GTTCCATAAA TAGTTCTTTT 1141 TCGCCATCAG GATATGGGCC ATACTGTTTT 1201 GGAACTTTAC ATCCTTCGGA TGGTGTTTCT 1261 ACAGCCGAAG CTACAAGTAA AAGATTAGCA 1321 CTCATGATCA ACATCAACAA CCTCATGCAT 1381 ATGCTACATG AGGTAGAAAA GGAAGCCCAA 1441 ACAGAAGTAA CAATGGCGAT TAAATACGAT 1501 CCCCGTGTAA CCGAGGTTGC TGTCATATTC 1561 AGGGACTTGC TCATTCATTG TAAACCAGAT 1621 CAAATCAGTA TCCTGTTTCC TACATTAGAT 1681 GGTGAAAAAG GCTGGGGAAC AGATATTGCA 1741 AATAATACTA GACAAAATGT AAGGACACGA 1801 CTCTCTGTGC GGGACACGTT CAATCCTATT 1861 ATTGTGGATT CATATTCAAA AATGGAGGCC 1921 TCTAAGTTGA GAGTTGAAAA ATATAGTGGT 1981 AATGACAATG TGCCGATTGG TAAAATGATA 2041 AGAAATATGC AGCAGCGATA TCAGGATGCT 2101 GATTTATTCA TAACCATGAC ATGCAACCCC 2161 CGCTGGCAAA AAGTTGAAAA CAGACCTGAC 2221 AATGCTCTTT TAAATGATAT ATGTAAATTC 2281 CATGTCATTG AATTTCAGAA ACGCGGACTG 2341 AGTGAGTCCA AATTACGTTC AGAAGATGAC CAACGGCGGC CGCGGGCTCC CAGGACCTTC CTAGCCACAC CCGCGGGCTC CCGGGACCTT CGGAGAGCGG GAGGTTTGGA GGACTTGGCA CGAGAGAGAG GGTGGCTTGG AGGGCGTGGC GCTGTGGAAA CTGACAGCAG GGAGGAGGAA GCCGTTGGTC AGACAGCTCT CAGCGGCCTG AGACCGTGAC AGTAGAGAGG TGGGACTATG TCTGCAGCCG AAAGATGCCG GCGTTATCGA GATCTTGAAA GAAGGCGGCG CCTGCAACAG CGTCGCTCTG AAGCCGAAAA ACAGCGGCGT GCCTTTGAAG TTGAAAGAAG GCGGTGGCGA ACAAGTACTA CCAATACCGG TAGGAACTGC GCAATTCTCG AACATAGTTG TGGTGGAATG AATTTCTCTG ATGAAAAACC ATCCGATGGG GTCTGTCCAA ATGATATACA TTTTCCAGAT AACGAAGATT CTGACAGTAA AAATTTCATG GCTTTTGCTT CCATGGGTGC AAATATTGCA AGAATACACG GACAAGTTTA TCACCGTACT CGGAAGTTTG CTCAACTCTA TATTTTGGAT ATGCCAGAAA ACCAGGGCTG CTCAGAAAGA GAAATAAATG AATTAACAAA ATCGTACAAG TCTGAAGCAG CAGCAAAAGG TATTGCTCCC CGTAACAGTG ACCCAGGTAG ATATAATTCT AGAAACGAAG ATGGAGAACC TCCTTTTGAA CCCAATAATC CAAATGCCAC TAAAATGAAA GCAATGACAT ATCCTATTCT TTTTCCAGAT TTAAGACTCA GAGACAACAG TGTAATCGAC GTCACACAAA TGCAGTATTA TGGATTTCAT TTAAATGCAG GAAAATTAAC TCAACAGTTT AATCGGATAA ATTTCATCAA AGCAAACCAA TTGATGGATT ATCTCAAATC TAGATCTGAA ATACTTCCAT CATCTTTTGA GGGTAGTCCC ATGGCAATTG TAACGAAGTA TGGCAAGCCC AAATGGGCAG ATATTACAAA CAATTTACAA TTGGTAGCCA GAGTTTTTAA TATTAAGCTG CATTTATTTG GCAAAGTAAT AGCTAAAATT CCTCACGCTC ACATATTATT GATATTAGAT ATTGACCGTA TAGTTAAGGC AGAAATTCCA 2023208176 27 Jul 2023 2401 GATGAAGACC AGTGTCCTCG ACTTTTTCAA 2461 TGTGGAATAC AAAATCCAAA TAGTCCATGT 2521 CCAAAAGAAT TTCAAAATGC GACCATTGGA 2581 AGATCTGGTA GCACCATGTC TATTGGAAAT 2641 TATAACCCGT ATTTGTGCCT TAAATATAAC 2701 ATTAAAAGTG TCAAATATTT ATTTAAATAC 2761 CAAATTTCTG AAAAAAATAT TATCAATCAT 2821 TATGTGAGCG CTCCTGAGGC TGTTTGGAGA 2881 CATGCAATCA CAAGATTAGC TATTCATTTG 2941 GATGATTTTG CTGAAGTTTT AGATAGGGCT 3001 TTCTTATTGA ATAGAGAAGA TTCTGATGCA 3061 CATTATGTGT TTAATAATTC TTTGTGGACA 3121 GGTAGACTGT TCACTGTGAG CTTTAGAGAA 3181 CTGCATGTAA AAGGTGCGAT AAGTTTTGAG 3241 GATACATTTC ATGAAGCTGC TAAACACCGA 3301 GATACGATTG ACGATGCAAT CATCCTTAAT 3361 TATATATGTG TGTTTGGATG TCCTTCTGCT 3421 CATTTTATTG AAGATTTCTG TTGGAAATTA 3481 GAAATGCATG CCCTTAACGA AATTCAGGAG 3541 CATTTCAAAC TTCCGGACTA TCCTTTATTA 3601 GAGCAACAAC AGGCAGAGGT TTTGATAAAT 3661 CAGACTATAA CTTCAGCCAT CGAAGATCAA 3721 GGTCCAGGTG GTAGTGGAAA AACATATCTG 3781 CGTGGTGGTA CTGTTTTACC CACAGCATCT 3841 GGAAGAACCT TTCATTCCCA ATATAAATTA 3901 AGACTCGATA TAAAGAGTGA AGTTGCTAAA 3961 GATGAATGCA CCATGGCATC CAGTCATGCT 4021 ATTATGAATT TGAATGTTGC ATTTGGTGGG 4081 CAATGTCTCA GTATTGTACC ACATGCTATG 4141 TACTGTAATG TTTGGGGATG TTTCAGAAAG 4201 GATTCTGCTT ATAGTGAATG GTTAGTAAAA 4261 CATTTAGGAA TGGATATTAT TGAAATCCCC 4321 GAAGCTACCT TTGGAAATAG TATATCTATA 4381 ATTCTTTGTC CAAAAAATGA GCATGTTCAA 4441 GATGGAGATT TTCACACATA TTTGAGTGAT 4501 AAGGAAAATT TTCCCATCGA ATTTCTTAAT 4561 AAATTAAAAT TGAAAGTGGG TGCAATCATC 4621 GGTCTTTGTA ATGGTACTAG ATTTATTATC ATTGTAAAAT CAAATATGGT ACATGGACCA ATGGAAAATG GAAAATGTTC AAAGGGATAT AATATTGATG GATATCCCAA ATACAAACGA AAAGTTGTCG ATAACACTTG GATTGTCCCT TGTCATATAA ATGTTGAAGT CTGTGCATCA ATCTATAAAG GGCACGATTG TGCAAATATT GACGAAGTAC AGGACTTCAT TGACTCCAGG CTTTTTGCAA TGCGAATGCA TGACCAATCT CCAAATGATC AGAATTTGTA TTTTCATACC AAAAGGCATA ACTCGACTTT GATGGCTTGG CGTAATTATT ATTATTGGGA GATTCCACAG AAACGCCGAA AGGGTGGGAA TAAAGTATTA CCAGAACGAT ATTACCTTAG ACTTTTGCTT GATCTGCGAA CTGTAGGAGG TGTAACTTAT GGATTATTAC TTGATGACAC TATCTGGAAA ATGCCCAAAC AACTACGGCA ACTTTTTGCA GCAGACAAAT TATGGGATGA GAATAAATCT CACCGAAGAG AAGGTGCCTG TGTGAACTGT GTATTCACAT TGCATGGAAT GAAATGTTCA ATGAATGCAA ATACATGTGA TCAATTGTAC TCTCTGAATG ATGAACAGTT GGCAGCCTTT ACTGTACACC CCAAATGCTT TTTCTTGGAT TATAAAGTTT TAACACATTA TATTAGAGGT ACAGGAATTG CTGCAAATTT ACTTCTTGGT CCAATTCCAT TAAATGAAAC TTCAATTTCT ACCATTAAAA AGGCCCAACT TCTCATTATT ATAAACGCCA TAGATAGATT ACTAAGAGAA AAAGTTGTCG TTCTCGGAGG GGATTTTCGA CGATCGGCCA TAGTACAAAC GAGTTTAAAG TTGTCTCTTA AAACAAATAT GAGATCAGAG CTTGGAGATG GCAAACTTGA TAGCAGTTTT CATGAAATGA TTTGTAACGG ATCTATTATT GATAATATTA AAAATATATC TAAACGTGCA AAATTAAATG AAGAAATTTT GGATATACTT GATTCCATTG ATTCAACAGA TGATGCTGAA AGTATTACTC CTTCGGGAAT GCCGTGTCAT ATGCTATTGA GAAATCTTAA TAGTAAATGG AAAAGATTAC GACCTAACAT TATCGAAGCT 2023208176 27 Jul 2023 4681 GAAGTATTAA CAGGATCTGC AGAGGGAGAG GTTGTTCTGA TTCCAAGAAT TGATTTGTCC 4741 CCATCTGACA CTGGCCTCCC ATTTAAATTA ATTCGAAGAC AGTTTCCCGT GATGCCAGCA 4801 TTTGCGATGA CTATTAATAA ATCACAAGGA CAAACTCTAG ACAGAGTAGG AATATTCCTA 4861 CCTGAACCCG TTTTCGCACA TGGTCAGTTA TATGTTGCTT TCTCTCGAGT TCGAAGAGCA 4921 TGTGACGTTA AAGTTAAAGT TGTAAATACT TCATCACAAG GGAAATTAGT CAAGCACTCT 4981 GAAAGTGTTT TTACTCTTAA TGTGGTATAC AGGGAGATAT TAGAATAAGT TTAATCACTT 5041 TATCAGTCAT TGTTTGCATC AATGTTGTTT TTATATCATG TTTTTGTTGT TTTTATATCA 5101 TGTCTTTGTT GTTGTTATAT CATGTTGTTA TTGTTTATTT ATTAATAAAT TTATGTATTA 5161 TTTTCATATA CATTTTACTC ATTTCCTTTC ATCTCTCACA CTTCTATTAT AGAGAAAGGG 5221 CAAATAGCAA TATTAAAATA TTTCCTCTAA TTAATTCCCT TTCAATGTGC ACGAATTTCG 5281 TGCACCGGGC CACTAG (SEQ ID NO: 17006).

[0203] 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 domain of the HUH superfamily of nucleases.

[0204] An exemplary Helitron transposase of the disclosure comprises an amino acid sequence comprising: 1 MSKEQLLIQR SSAAERCRRY RQKMSAEQRA SDLERRRRLQ QNVSEEQLLE KRRSEAEKQR 61 RHRQKMSKDQ RAFEVERRRW RRQNMSREQS STSTTNTGRN CLLSKNGVHE DAILEHSCGG 121 MTVRCEFCLS LNFSDEKPSD GKFTRCCSKG KVCPNDIHFP DYPAYLKRLM TNEDSDSKNF 181 MENIRSINSS FAFASMGANI ASPSGYGPYC FRIHGQVYHR TGTLHPSDGV SRKFAQLYIL 241 DTAEATSKRL AMPENQGCSE RLMININNLM HEINELTKSY KMLHEVEKEA QSEAAAKGIA 301 PTEVTMAIKY DRNSDPGRYN SPRVTEVAVI FRNEDGEPPF ERDLLIHCKP DPNNPNATKM 361 KQISILFPTL DAMTYPILFP HGEKGWGTDI ALRLRDNSVI DNNTRQNVRT RVTQMQYYGF 421 HLSVRDTFNP ILNAGKLTQQ FIVDSYSKME ANRINFIKAN QSKLRVEKYS GLMDYLKSRS 481 ENDNVPIGKM IILPSSFEGS PRNMQQRYQD AMAIVTKYGK PDLFITMTCN PKWADITNNL 541 QRWQKVENRP DLVARVFNIK LNALLNDICK FHLFGKVIAK IHVIEFQKRG LPHAHILLIL 601 DSESKLRSED DIDRIVKAEI PDEDQCPRLF QIVKSNMVHG PCGIQNPNSP CMENGKCSKG 661 YPKEFQNATI GNIDGYPKYK RRSGSTMSIG NKWDNTWIV PYNPYLCLKY NCHINVEVCA 721 SIKSVKYLFK YIYKGHDCAN IQISEKNIIN HDEVQDFIDS RYVSAPEAVW RLFAMRMHDQ 781 SHAITRLAIH LPNDQNLYFH TDDFAEVLDR AKRHNSTLMA WFLLNREDSD ARNYYYWEIP 841 QHYVFNNSLW TKRRKGGNKV LGRLFTVSFR EPERYYLRLL LLHVKGAISF EDLRTVGGVT 901 YDTFHEAAKH RGLLLDDTIW KDTIDDAIIL NMPKQLRQLF AYICVFGCPS AADKLWDENK 961 SHFIEDFCWK LHRREGACVN CEMHALNEIQ EVFTLHGMKC SHFKLPDYPL LMNANTCDQL 1021 YEQQQAEVLI NSLNDEQLAA FQTITSAIED QTVHPKCFFL DGPGGSGKTY LYKVLTHYIR 1081 GRGGTVLPTA STGIAANLLL GGRTFHSQYK LPIPLNETSI SRLDIKSEVA KTIKKAQLLI 1141 IDECTMASSH AINAIDRLLR EIMNLNVAFG GKVLLLGGDF RQCLSIVPHA MRSAIVQTSL 81 2023208176 27 Jul 2023 1201 KYCNVWGCFR KLSLKTNMRS EDSAYSEWLV KLGDGKLDSS FHLGMDIIEI PHEMICNGSI 1261 IEATFGNSIS IDNIKNISKR AILCPKNEHV QKLNEEILDI LDGDFHTYLS DDSIDSTDDA 1321 EKENFPIEFL NSITPSGMPC HKLKLKVGAI IMLLRNLNSK WGLCNGTRFI IKRLRPNIIE 1381 AEVLTGSAEG EWLIPRIDL SPSDTGLPFK LIRRQFPVMP AFAMTINKSQ GQTLDRVGIF 1441 LPEPVFAHGQ LYVAFSRVRR ACDVKVKWN TSSQGKLVKH SESVFTLNW YREILE (SEQ ID NO: 14501) .

[0205] 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 (5’ terminal sequence) and RTS (3’ terminal sequence). 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 11 nucleotides upstream of the RTS and consists of the sequence gtgcacgaatttcgtgcaccgggccactag (SEQ ID NO: 14500).

[0206] 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 DSFKMKCVLC LPLNKEISAF 61 KSSPSNLRKH IERMHPNYLK NYSKLTAQKR KIGTSTHASS SKQLKVDSVF PVKHVSPVTV 121 NKAILRYIIQ GLHPFSTVDL PSFKELISTL QPGISVITRP TLRSKIAEAA LIMKQKVTAA 181 MSEVEWIATT TDCWTARRKS FIGVTAHWIN PGSLERHSAA LACKRLMGSH TFEVLASAMN 241 DIHSEYEIRD KWCTTTDSG SNFMKAFRVF GVENNDIETE ARRCESDDTD SEGCGEGSDG 301 VEFQDASRVL DQDDGFEFQL PKHQKCACHL LNLVSSVDAQ KALSNEHYKK LYRSVFGKCQ 361 ALWNKSSRSA LAAEAVESES RLQLLRPNQT RWNSTFMAVD RILQICKEAG EGALRNICTS 421 LEVPMFNPAE MLFLTEWANT MRPVAKVLDI LQAETNTQLG WLLPSVHQLS LKLQRLHHSL 481 RYCDPLVDAL QQGIQTRFKH MFEDPEIIAA AILLPKFRTS WTNDETIIKR GMDYIRVHLE 541 PLDHKKELAN SSSDDEDFFA SLKPTTHEAS KELDGYLACV SDTRESLLTF PAICSLSIKT 601 NTPLPASAAC ERLFSTAGLL FSPKRARLDT NNFENQLLLK LNLRFYNFE (SEQ ID NO: 14502).

[0207] An exemplary Tol2 transposon of the disclosure, including inverted repeats, subterminal sequences and the Tol2 transposase, is encoded by a nucleic acid sequence comprising the following: 2023208176 27 Jul 2023 1 61 121 181 241 301 361 421 481 541 601 661 721 781 841 901 961 1021 1081 1141 1201 1261 1321 1381 1441 1501 1561 1621 1681 1741 1801 1861 1921 1981 2041 2101 2161 2221 CAGAGGTGTA AAGTACTTGA GTAATTTTAC TTGATTACTG TACTTAAGTA TTATTTTTGG GGATTTTTAC TTTACTTGAG TACAATTAAA AATCAATACT TTTACTTTTA CTTAATTACA TTTTTTTAGA AAAAAAAGTA CTTTTTACTC CTTACAATTT TATTTACAGT CAAAAAGTAC TTATTTTTTG GAGATCACTT CATTCTATTT TCCCTTGCTA TTACCAAACC AATTGAATTG CGCTGATGCC CAGTTTAATT TAAATGTTAT TTATTCTGCC TATGAAAATC GTTTTCACAT TATATGAAAT TGGTCAGACA TGTTCATTGG TCCTTTGGAA GTGACGTCAT GTCACATCTA TTACCACAAT GCACAGCACC TTGACCTGGA AATTAGGGAA ATTATAACAG TCAATCAGTG GAAGAAAATG GAGGAAGTAT GTGATTCATC AGCAGCTGCG AGCAGCACAG TCCAAAATCA GCCACAGGAT CAAGAGCACC CGTGGCCGTA TCTTCGCGAA TTCTTTTCTT TAAGTGGTGT AAATAAAGAT TCATTCAAGA TGAAATGTGT CCTCTGTCTC CCGCTTAATA AAGAAATATC GGCCTTCAAA AGTTCGCCAT CAAACCTAAG GAAGCATATT GAGGTAAGTA CATTAAGTAT TTTGTTTTAC TGATAGTTTT TTTTTTTTTT TTTTTTTTTT TTTTTGGGTG TGCATGTTTT GACGTTGATG GCGCGCCTTT TATATGTGTA GTAGGCCTAT TTTCACTAAT GCATGCGATT GACAATATAA GGCTCACGTA ATAAAATGCT AAAATGCATT TGTAATTGGT AACGTTAGGT CCACGGGAAA TTTGGCGCCT ATTGCAGCTT TGAATAATCA TTATCATTCC GTGCTCTCAT TGTGTTTGAA TTCATGCAAA ACACAAGAAA ACCAAGCGAG AAATTTTTTT CCAAACATGT TGTATTGTCA AAACGGTAAC ACTTTACAAT GAGGTTGATT AGTTCATGTA TTAACTAACA TTAAATAACC ATGAGCAATA CATTTGTTAC TGTATCTGTT AATCTTTGTT AACGTTAGTT AATAGAAATA CAGATGTTCA TTGTTTGTTC ATGTTAGTTC ACAGTGCATT AACTAATGTT AACAAGATAT AAAGTATTAG TAAATGTTGA AATTAACATG TATACGTGCA GTTCATTATT AGTTCATGTT AACTAATGTA GTTAACTAAC GAACCTTATT GTAAAAGTGT TACCATCAAA ACTAATGTAA TGAAATCAAT TCACCCTGTC ATGTCAGCCT TACAGTCCTG TGTTTTTGTC AATATAATCA GAAATAAAAT TAATGTTTGA TTGTCACTAA ATGCTACTGT ATTTCTAAAA TCAACAAGTA TTTAACATTA TAAAGTGTGC AATTGGCTGC AAATGTCAGT TTTATTAAAG GGTTAGTTCA CCCAAAAATG AAAATAATGT CATTAATGAC TCGCCCTCAT GTCGTTCCAA GCCCGTAAGA CCTCCGTTCA TCTTCAGAAC ACAGTTTAAG ATATTTTAGA TTTAGTCCGA GAGCTTTCTG TGCCTCCATT GAGAATGTAT GTACGGTATA CTGTCCATGT CCAGAAAGGT AATAAAAACA TCAAAGTAGT CCATGTGACA TCAGTGGGTT AGTTAGAATT TTTTGAAGCA TCGAATACAT TTTGGTCCAA AAATAACAAA ACCTACGACT TTATTCGGCA TTGTATTCTC TTCCGGGTCT GTTGTCAATC CGCGTTCACG ACTTCGCAGT GACGCTACAA TGCTGAATAA AGTCGTAGGT TTTGTTATTT TTGGACCAAA ATGTATTTTC GATGCTTCAA ATAATTCTAC CTAACCCACT GATGTCACAT GGACTACTTT GATGTTTTTA TTACCTTTCT GGACATGGAC AGTATACCGT ACATACATTT TCAGTGGAGG GACAGAAAGC TCTCGGACTA AATCTAAAAT ATCTTAAACT GTGTTCCGAA GATGAACGGA GGTGTTACGG GCTTGGAACG ACATGAGGGT GAGTCATTAA TGACATCTTT TCATTTTTGG GTGAACTAAC CCTTTAATGC TGTAATCAGA GAGTGTATGT GTAATTGTTA CATTTATTGC ATACAATATA AATATTTATT TGTTGTTTTT ACAGAGAATG CACCCAAATT ACCTCAAAAA CTACTCTAAA TTGACAGCAC AGAAGAGAAA GATCGGGACC TCCACCCATG CTTCCAGCAG TAAGCAACTG AAAGTTGACT CAGTTTTCCC 2023208176 27 Jul 2023 2281 2341 2401 2461 2521 2581 2641 2701 2761 2821 2881 2941 3001 3061 3121 3181 3241 3301 3361 3421 3481 3541 3601 3661 3721 3781 3841 3901 3961 4021 4081 4141 4201 4261 4321 4381 4441 4501 AGTCAAACAT GTGTCTCCAG TCACTGTGAA CAAAGCTATA TTAAGGTACA ACTTCATCCT TTCAGCACTG TTGATCTGCC ATCATTTAAA GAGCTGATTA GCCTGGCATT TCTGTCATTA CAAGGCCTAC TTTACGCTCC AAGATAGCTG GATCATGAAA CAGAAAGTGA CTGCTGCCAT GAGTGAAGTT GAATGGATTG GGATTGTTGG ACTGCACGTA GAAAGTCATT CATTGGTGTA ACTGCTCACT TGGAAGTCTT GAAAGACATT CCGCTGCACT TGCCTGCAAA AGATTAATGG TTTTGAGGTA CTGGCCAGTG CCATGAATGA TATCCACTCA GAGTATGAAA GGTTGTTTGC ACAACCACAG ACAGTGGTTC CAACTTTATG AAGGCTTTCA TGTGGAAAAC AATGATATCG AGACTGAGGC AAGAAGGTGT GAAAGTGATG TGAAGGCTGT GGTGAGGGAA GTGATGGTGT GGAATTCCAA GATGCCTCAC CCAAGACGAT GGCTTCGAAT TCCAGCTACC AAAACATCAA AAGTGTGCCT TAACCTAGTC TCAAGCGTTG ATGCCCAAAA AGCTCTCTCA AATGAACACT CTACAGATCT GTCTTTGGCA AATGCCAAGC TTTATGGAAT AAAAGCAGCC AGCAGCTGAA GCTGTTGAAT CAGAAAGCCG GCTTCAGCTT TTAAGGCCAA GTGGAATTCA ACTTTTATGG CTGTTGACAG AATTCTTCAA ATTTGCAAAG AGGCGCACTT CGGAATATAT GCACCTCTCT TGAGGTTCCA ATGTAAGTGT TATCGATGTA AACAAATGTG GGTTGTTTTT GTTTAATACT CTTTGATTAT CCTGTAGGTT TAATCCAGCA GAAATGCTGT TCTTGACAGA GTGGGCCAAC CAGTTGCAAA AGTACTCGAC ATCTTGCAAG CGGAAACGAA TACACAGCTG TGCCTAGTGT CCATCAGTTA AGCTTGAAAC TTCAGCGACT CCACCATTCT GTGACCCACT TGTGGATGCC CTACAACAAG GAATCCAAAC ACGATTCAAG AAGATCCTGA GATCATAGCA GCTGCCATCC TTCTCCCTAA ATTTCGGACC ATGATGAAAC CATCATAAAA CGAGGTAAAT GAATGCAAGC AACATACACT TAATCTGGGC AACCTTTGAG CCATACCAAA ATTATTCTTT TATTTATTTA TTTTAGGAAT GTTATATCCC ATCTTTGGCT GTGATCTCAA TATGAATATT ATTCTTGCAG CAGGTTGTAG TTATCCCTCA GTGTTTCTTG AAACCAAACT ATATGTGGTT TGGAAATGCA GTTAGATTTT ATGCTAAAAT AAGGGATTTG GATGTAGATG ACTGCACGTA AATGTAGTTA ATGACAAAAT CCATAAAATT CAGAAGCCCC TCAACCAAAC TTTTCTTTGT GTCTGCTCAC TGTGCTTGTA ACATCAGAGT GCATCTGGAG CCTTTGGACC ACAAGAAGGA ATTGGCCAAC ATGATGAAGA TTTTTTCGCT TCTTTGAAAC CGACAACACA TGAAGCCAGC ATGGATATCT GGCCTGTGTT TCAGACACCA GGGAGTCTCT GCTCACGTTT GCAGCCTCTC TATCAAGACT AATACACCTC TTCCCGCATC GGCTGCCTGT TCAGCACTGC AGGATTGCTT TTCAGCCCCA AAAGAGCTAG GCTTGACACT AGAATCAGCT TCTACTGAAG TTAAATCTGA GGTTTTACAA CTTTGAGTAG CATTAGATTG TCTGTCTTAT AGTTTGATAA TTAAATACAA ACAGTTCTAA AACCTTGTAT GCATTTCATT TAATGTTTTT TGAGATTAAA AGCTTAAACA GTTTTCTTTC TTGCTTTTAC TTTTACTTCC TTAATACTCA AGTACAATTT TCATTCAAGG GTACACTGCA AAGCTGCTCT CAACCACAAC GGATCAACCC GCTCTCATAC TACGTGACAA GAGTTTTTGG ACACTGATTC GAGTCCTGGA GTCACTTACT ACAAGAAACT GATCGGCTCT ACCAAACGCG AAGCAGGAGA TTTTCCCCTC GCTGATTTCT ACAATGCGTC GGGTGGCTGC CTCAGGTACT CATATGTTTG TCTTGGACAA TGACGAATTC TTTTTGCACT GATGTAAAGT CATATGTATC CATGATTTTA TGTTCCCAGT GGCATGGACT AGTTCATCTG AAAGAGTTGG CCTGCTATTT GAGAGGCTTT AACAATTTTG CGTGTACTGG AGCAGGATAA AGAATCTCTA TAATGGAGTA 2023208176 27 Jul 2023 4561 CTTTTTTACT TTTACTCAAG TAAGATTCTA GCCAGATACT TTTACTTTTA ATTGAGTAAA 4621 ATTTTCCCTA AGTACTTGTA CTTTCACTTG AGTAAAATTT TTGAGTACTT TTTACACCTC 4681 tg (SEQIDNO: 17007).

[0208] Exemplary transposon / transposase systems of the disclosure include, but are not limited to, piggyBac and piggyBac-like transposons and transposases.

[0209] PiggyBac and piggyBac-like transposases recognizes transposon-specific inverted terminal repeat sequences (ITRs) on the ends of the transposon, and moves the contents between the ITRs into TTAA or TTAT chromosomal sites. The piggyBac or piggyBac-like transposon system has no payload limit for the genes of interest that can be included between the ITRs.

[0210] In certain embodiments, and, in particular, those embodiments wherein the transposon is a piggyBac transposon, the transposase is a piggyBac, Super piggyBac (SPB) transposase. In certain embodiments, and, in particular, those embodiments wherein the transposase is a piggyBac, Super piggyBac (SPB), the sequence encoding the transposase is an mRNA sequence.

[0211] In certain embodiments of the methods of the disclosure, the transposase enzyme is a piggyBac or piggyBac-like transposase enzyme.

[0212] In certain embodiments of the methods of the disclosure, the transposase enzyme is a piggyBac or a piggyBac-like transposase enzyme. The piggyBac (PB) or piggyBac-like transposase enzyme may comprise or consist of an amino acid sequence at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to: 1 MGSSLDDEHI LSALLQSDDE LVGEDSDSEI SDHVSEDDVQ SDTEEAFIDE VHEVQPTSSG 61 SEILDEQNVI EQPGSSLASN RILTLPQRTI RGKNKHCWST SKSTRRSRVS ALNIVRSQRG 121 PTRMCRNIYD PLLCFKLFFT DEIISEIVKW TNAEISLKRR ESMTGATFRD TNEDEIYAFF 181 GILVMTAVRK DNHMSTDDLF DRSLSMVYVS VMSRDRFDFL IRCLRMDDKS IRPTLRENDV 241 FTPVRKIWDL FIHQCIQNYT PGAHLTIDEQ LLGFRGRCPF RMYIPNKPSK YGIKILMMCD 301 SGTKYMINGM PYLGRGTQTN GVPLGEYYVK ELSKPVHGSC RNITCDNWFT SIPLAKNLLQ 361 EPYKLTIVGT VRSNKREIPE VLKNSRSRPV GTSMFCFDGP LTLVSYKPKP AKMVYLLSSC 421 DEDASINEST GKPQMVMYYN QTKGGVDTLD QMCSVMTCSR KTNRWPMALL YGMINIACIN 481 SFIIYSHNVS SKGEKVQSRK KFMRNLYMSL TSSFMRKRLE APTLKRYLRD NISNILPNEV 541 PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV ICREHNIDMC QSCF (SEQ ID NO: 14487) .

[0213] In certain embodiments of the methods of the disclosure, the transposase enzyme is a piggyBac or piggyBac-like transposase enzyme that comprises or consists of an amino acid 2023208176 27 Jul 2023 sequence having an amino acid substitution at 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 ALNIVRSQRG 121 PTRMCRNIYD PLLCFKLFFT DEIISEIVKW TNAEISLKRR ESMTGATFRD TNEDEIYAFF 181 GILVMTAVRK DNHMSTDDLF DRSLSMVYVS VMSRDRFDFL IRCLRMDDKS IRPTLRENDV 241 FTPVRKIWDL FIHQCIQNYT PGAHLTIDEQ LLGFRGRCPF RMYIPNKPSK YGIKILMMCD 301 SGTKYMINGM PYLGRGTQTN GVPLGEYYVK ELSKPVHGSC RNITCDNWFT SIPLAKNLLQ 361 EPYKLTIVGT VRSNKREIPE VLKNSRSRPV GTSMFCFDGP LTLVSYKPKP AKMVYLLSSC 421 DEDASINEST GKPQMVMYYN QTKGGVDTLD QMCSVMTCSR KTNRWPMALL YGMINIACIN 481 SFIIYSHNVS SKGEKVQSRK KFMRNLYMSL TSSFMRKRLE APTLKRYLRD NISNILPNEV 541 PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV ICREHNIDMC QSCF (SEQ ID NO: 14487) .

[0214] In certain embodiments, the transposase enzyme is a piggyBac or piggyBac-like 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: 14487. In certain embodiments, the transposase enzyme is a piggyBac or piggyBac-like 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: 14487. In certain embodiments, the transposase enzyme is a piggyBac or piggyBac-like 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: 14487. In certain embodiments, the amino acid substitution at position 30 of the sequence of SEQ ID NO: 14487 is a substitution of a valine (V) for an isoleucine (I). In certain embodiments, the amino acid substitution at position 165 of the sequence of SEQ ID NO: 14487 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: 14487 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: 14487 is a substitution of a lysine (K) for an asparagine (N).

[0215] In certain embodiments of the methods of the disclosure, the transposase enzyme is a Super piggyBac (SPB) or piggyBac-like transposase enzyme. In certain embodiments, the Super piggyBac (SPB) or piggyBac-like transposase enzyme of the disclosure may comprise or consist of the amino acid sequence of the sequence of SEQ ID NO: 14487 wherein the amino acid 2023208176 27 Jul 2023 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 piggyBac (SPB) or piggyBac-like transposase enzyme may comprise or consist of an amino acid sequence at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to: 1 MGSSLDDEHI LSALLQSDDE LVGEDSDSEV SDHVSEDDVQ SDTEEAFIDE VHEVQPTSSG 61 SEILDEQNVI EQPGSSLASN RILTLPQRTI RGKNKHCWST SKSTRRSRVS ALNIVRSQRG 121 PTRMCRNIYD PLLCFKLFFT DEIISEIVKW TNAEISLKRR ESMTSATFRD TNEDEIYAFF 181 GILVMTAVRK DNHMSTDDLF DRSLSMVYVS VMSRDRFDFL IRCLRMDDKS IRPTLRENDV 241 FTPVRKIWDL FIHQCIQNYT PGAHLTIDEQ LLGFRGRCPF RVYIPNKPSK YGIKILMMCD 301 SGTKYMINGM PYLGRGTQTN GVPLGEYYVK ELSKPVHGSC RNITCDNWFT SIPLAKNLLQ 361 EPYKLTIVGT VRSNKREIPE VLKNSRSRPV GTSMFCFDGP LTLVSYKPKP AKMVYLLSSC 421 DEDASINEST GKPQMVMYYN QTKGGVDTLD QMCSVMTCSR KTNRWPMALL YGMINIACIN 481 SFIIYSHNVS SKGEKVQSRK KFMRNLYMSL TSSFMRKRLE APTLKRYLRD NISNILPKEV 541 PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV ICREHNIDMC QSCF (SEQ ID NO: 14484) .

[0216] 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 piggyBac, Super piggyBac or piggyBac-like 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: 14487 or SEQ ID NO: 14484. In certain embodiments, including those embodiments wherein the transposase comprises the above-described mutations at positions 30, 165, 282 and / or 538, the piggyBac, Super piggyBac or piggyBac-like 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 2023208176 27 Jul 2023 14484 is a substitution of a serine (S) for an alanine (A). In certain embodiments, the amino acid substitution at position 46 of SEQ ID NO: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 is a substitution of a proline (P) for a serine (S). In certain embodiments, the amino acid substitution at position 119 of SEQ ID NO: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 is a substitution of an alanine (A) a cysteine (C). In certain embodiments, the amino acid substitution at position 125 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a leucine (L) for a cysteine (C). In certain embodiments, the amino acid substitution at position 177 of SEQ ID NO: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 is a substitution of a histidine (H) for a tyrosine (Y). In certain embodiments, the amino acid substitution at position 180 of SEQ ID NO: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 is a substitution of a phenylalanine (F) for a methionine (M). In certain embodiments, the 2023208176 27 Jul 2023 amino acid substitution at position 235 of SEQ ID NO: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 is a substitution of a serine (S) for an asparagine (N). In certain embodiments, the amino acid substitution at position 296 of SEQ ID NO: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 is a substitution of a valine for a proline (P). In certain embodiments, the amino acid substitution at position 315 of SEQ ID NO: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 2023208176 27 Jul 2023 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 is a substitution of an arginine (R) for a glutamine (Q).

[0217] 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 piggyBac or piggyBac-like transposase enzyme or may comprise or the Super piggyBac 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: 14487 or SEQ ID NO: 14484. 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 piggyBac or piggyBac-like transposase enzyme may comprise or the Super piggyBac transposase enzyme may further comprise an amino acid substitution at two, 2023208176 27 Jul 2023 three, four, five, six or more of positions 103, 194, 372, 375, 450, 509 and 570 of the sequence of SEQ ID NO: 14487 or SEQ ID NO: 14484. In certain embodiments, including those embodiments wherein the transposase comprises the above-described mutations at positions 30, 165, 282 and / or 538, the piggyBac or piggyBac-like transposase enzyme may comprise or the Super piggyBac transposase 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: 14487 or SEQ ID NO: 14484. In certain embodiments, the amino acid substitution at position 103 of SEQ ID NO: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 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: 14487 or SEQ ID NO: 14484 is a substitution of a serine (S) for an asparagine (N). In certain embodiments, the piggyBac or piggyBac-like transposase enzyme may comprise a substitution of a valine (V) for a methionine (M) at position 194 of SEQ ID NO: 14487. In certain embodiments, including those embodiments wherein the piggyBac or piggyBac-like transposase enzyme may comprise a substitution of a valine (V) for a methionine (M) at position 194 of SEQ ID NO: 14487, the piggyBac or piggyBac-like transposase enzyme may further comprise an amino acid substitution at positions 372, 375 and 450 of the sequence of SEQ ID NO: 14487 or SEQ ID NO: 14484. In certain embodiments, the piggyBac or piggyBac-like transposase enzyme may comprise a substitution of a valine (V) for a methionine (M) at position 194 of SEQ ID NO: 14487, a substitution of an alanine (A) for an arginine (R) at position 372 of SEQ ID NO: 14487, and a substitution of an alanine (A) for a lysine (K) at position 375 of SEQ ID NO: 14487. In certain embodiments, the piggyBac or piggyBac-like transposase enzyme may comprise a substitution of a valine (V) for a methionine (M) at position 194 of SEQ ID NO: 14487, a substitution of an 2023208176 27 Jul 2023 alanine (A) for an arginine (R) at position 372 of SEQ ID NO: 14487, a substitution of an alanine (A) for a lysine (K) at position 375 of SEQ ID NO: 14487 and a substitution of an asparagine (N) for an aspartic acid (D) at position 450 of SEQ ID NO: 14487.

[0218] In certain embodiments, the piggyBac or piggyBac-like transposase enzyme is isolated or derived from an insect. In certain embodiments, the insect is Trichoplusia ni (GenBank Accession No. AAA87375; SEQ ID NO: 16796), Argyrogramma agnata (GenBank Accession No. GU477713; SEQ ID NO: 14534, SEQ ID NO: 16797), Anopheles gambiae (GenBank Accession No. XP_312615 (SEQ ID NO: 16798); GenBank Accession No. XP 320414 (SEQ ID NO: 16799); GenBank Accession No. XP_310729 (SEQ ID NO: 16800)), Aphis gossypii (GenBank Accession No. GU329918; SEQ ID NO: 16801, SEQ ID NO: 16802), Acyrthosiphon pisum (GenBank Accession No. XP_001948139; SEQ ID NO: 16803), Agrotis ipsilon (GenBank Accession No. GU477714; SEQ ID NO: 14537, SEQ ID NO: 16804), Bombyx mori (GenBank Accession No. BAD11135; SEQ ID NO: 14505), Chilo suppressalis (GenBank Accession No. JX294476; SEQ ID NO: 16805, SEQ ID NO: 16806), Drosophila melanogaster (GenBank Accession No. AAL39784; SEQ ID NO: 16807), Helicoverpa armigera (GenBank Accession No. ABS18391; SEQ ID NO: 14525), Heliothis virescens (GenBank Accession No. ABD76335; SEQ ID NO: 16808), Macdunnoughia crassisigna (GenBank Accession No. EU287451; SEQ ID NO: 16809, SEQ ID NO: 16810), Pectinophora gossypiella (GenBank Accession No. GU270322; SEQ ID NO: 14530, SEQ ID NO: 16811), Tribolium castaneum (GenBank Accession No. XP 001814566; SEQ ID NO: 16812), Ctenoplusia agnata (also called Argyrogramma agnata). Messour bouvieri, Megachile rotundata, Bombus impatiens, Mamestra brassicae. Mayetiola destructor or Apis mellifera.

[0219] In certain embodiments, the piggyBac or piggyBac-like transposase enzyme is isolated or derived from an insect. In certain embodiments, the insect is Trichoplusia ni (AAA87375).

[0220] In certain embodiments, the piggyBac or piggyBac-like transposase enzyme is isolated or derived from an insect. In certain embodiments, the insect is Bombyx mori (BAD11135).

[0221] In certain embodiments, the piggyBac or piggyBac-like transposase enzyme is isolated or derived from a crustacean. In certain embodiments, the crustacean is Daphniapulicaria (AAM76342, SEQ ID NO: 16813).

[0222] In certain embodiments, the piggyBac or piggyBac-like transposase enzyme is isolated or derived from a vertebrate. In certain embodiments, the vertebrate is Xenopus tropicalis 2023208176 27 Jul 2023 (GenBank Accession No. BAF82026; SEQ ID NO: 14518), Homo sapiens (GenBank Accession No. NP_689808; SEQ ID NO: 16814), Mus musculus (GenBank Accession No. NP_741958; SEQ ID NO: 16815), Macacafascicularis (GenBank Accession No. AB179012; SEQ ID NO: 16816, SEQ ID NO: 16817), Rattus norvegicus (GenBank Accession No. XP 220453; SEQ ID NO: 16818) ox Myotis lucifugus.

[0223] In certain embodiments, the piggyBac or piggyBac-like transposase enzyme is isolated or derived from a urochordate. In certain embodiments, the urochordate is Ciona intestinalis (GenBank Accession No. XP_002123602; SEQ ID NO: 16819).

[0224] In certain embodiments, the piggyBac or piggyBac-like transposase inserts a transposon at the sequence 5’-TTAT-3’ within a chromosomal site (a TTAT target sequence).

[0225] In certain embodiments, the piggyBac or piggyBac-like transposase inserts a transposon at the sequence 5’-TTAA-3’ within a chromosomal site (a TTAA target sequence).

[0226] In certain embodiments, the target sequence of the piggyBac or piggyBac-like transposon comprises or consists of 5’-CTAA-3’, 5’-TTAG-3’, 5’-ATAA-3’, 5’-TCAA-3’, 5’AGTT-3’, 5’-ATTA-3’, 5’-GTTA-3’, 5’-TTGA-3’, 5’-TTTA-3’, 5’-TTAC-3’, 5’-ACTA-3’, 5’-AGGG-3’, 5’-CTAG-3’, 5’-TGAA-3’, 5’-AGGT-3’, 5’-ATCA-3’, 5’-CTCC-3’, 5’-TAAA-3’, 5’-TCTC-3’, 5’TGAA-3’, 5’-AAAT-3’, 5’-AATC-3’, 5’-ACAA-3’, 5’-ACAT-3’, 5’-ACTC-3’, 5’-AGTG-3’, 5’-ATAG-3’, 5’-CAAA-3’, 5’-CACA-3’, 5’-CATA-3’, 5’-CCAG-3’, 5’-CCCA-3’, 5’-CGTA-3’, 5’-GTCC-3’, 5’-TAAG-3’, 5’-TCTA-3’, 5’-TGAG-3’, 5’-TGTT-3’, 5’-TTCA-3’5’-TTCT-3’ and 5’-TTTT-3’.

[0227] In certain embodiments of the methods of the disclosure, the transposase enzyme is a piggyBac or piggyBac-like transposase enzyme. In certain embodiments, the piggyBac or piggyBac-like transposase enzyme is isolated or derived from Bombyx mori. The piggyBac or piggyBac-like transposase enzyme may comprise or consist of an amino acid sequence at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to: 1 MDIERQEERI RAMLEEELSD YSDESSSEDE TDHCSEHEVN YDTEEERIDS VDVPSNSRQE 61 EANAIIANES DSDPDDDLPL SLVRQRASAS RQVSGPFYTS KDGTKWYKNC QRPNVRLRSE 121 NIVTEQAQVK NIARDASTEY ECWNIFVTSD MLQEILTHTN SSIRHRQTKT AAENSSAETS 181 FYMQETTLCE LKALIALLYL AGLIKSNRQS LKDLWRTDGT GVDIFRTTMS LQRFQFLQNN 241 IRFDDKSTRD ERKQTDNMAA FRSIFDQFVQ CCQNAYSPSE FLTIDEMLLS FRGRCLFRVY 301 IPNKPAKYGI KILALVDAKN FDWNLEVYA GKQPSGPYAV SNRPFEWER LIQPVARSHR 361 NVTFDNWFTG YELMLHLLNE YRLTSVGTVR KNKRQIPESF IRTDRQPNSS VFGFQKDITL 421 VSYAPKKNKV VWMSTMHHD NSIDESTGEK QKPEMITFYN STKAGVDWD ELSANYNVSR 481 NSKRWPMTLF YGVLNMAAIN ACIIYRANKN VTIKRTEFIR SLGLSMIYEH LHSRNKKKNI 2023208176 27 Jul 2023 541 PTYLRQRIEK QLGEPSPRHV NVPGRYVRCQ DCPYKKDRKT KHSCNACAKP ICMEHAKFLC 601 ENCAELDSSL (SEQ ID NO: 14504) .

[0228] The piggyBac (PB) or piggyBac-like transposase enzyme may comprise or consist of an amino acid sequence at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to: 1 MDIERQEERI RAMLEEELSD YSDESSSEDE TDHCSEHEVN YDTEEERIDS VDVPSNSRQE 61 EANAIIANES DSDPDDDLPL SLVRQRASAS RQVSGPFYTS KDGTKWYKNC QRPNVRLRSE 121 NIVTEQAQVK NIARDASTEY ECWNIFVTSD MLQEILTHTN SSIRHRQTKT AAENSSAETS 181 FYMQETTLCE LKALIALLYL AGLIKSNRQS LKDLWRTDGT GVDIFRTTMS LQRFQFLQNN 241 IRFDDKSTRD ERKQTDNMAA FRSIFDQFVQ CCQNAYSPSE FLTIDEMLLS FRGRCLFRVY 301 IPNKPAKYGI KILALVDAKN FYWNLEVYA GKQPSGPYAV SNRPFEWER LIQPVARSHR 361 NVTFDNWFTG YELMLHLLNE YRLTSVGTVR KNKRQIPESF IRTDRQPNSS VFGFQKDITL 421 VSYAPKKNKV VWMSTMHHD NSIDESTGEK QKPEMITFYN STKAGVDWD ELCANYNVSR 481 NSKRWPMTLF YGVLNMAAIN ACIIYRTNKN VTIKRTEFIR SLGLSMIYEH LHSRNKKKNI 541 PTYLRQRIEK QLGEPSPRHV NVPGRYVRCQ DCPYKKDRKT KRSCNACAKP ICMEHAKFLC 601 ENCAELDSSL (SEQ ID NO: 14505) .

[0229] In certain embodiments, the piggyBac or piggyBac-like transposase is fused to a nuclear localization signal. In certain embodiments, the amino acid sequence of the piggyBac or piggyBac-like transposase fused to a nuclear localization signal is encoded by a polynucleotide sequence comprising: 1 atggcaccca aaaagaaacg taaagtgatg gacattgaaa gacaggaaga aagaatcagg 61 gcgatgctcg aagaagaact gagcgactac tccgacgaat cgtcatcaga ggatgaaacc 121 gaccactgta gcgagcatga ggttaactac gacaccgagg aggagagaat cgactctgtg 181 gatgtgccct ccaactcacg ccaagaagag gccaatgcaa ttatcgcaaa cgaatcggac 241 agcgatccag acgatgatct gccactgtcc ctcgtgcgcc agcgggccag cgcttcgaga 301 caagtgtcag gtccattcta cacttcgaag gacggcacta agtggtacaa gaattgccag 361 cgacctaacg tcagactccg ctccgagaat atcgtgaccg aacaggctca ggtcaagaat 421 atcgcccgcg acgcctcgac tgagtacgag tgttggaata tcttcgtgac ttcggacatg 481 ctgcaagaaa ttctgacgca caccaacagc tcgattaggc atcgccagac caagactgca 541 gcggagaact catcggccga aacctccttc tatatgcaag agactactct gtgcgaactg 601 aaggcgctga ttgcactgct gtacttggcc ggcctcatca aatcaaatag gcagagcctc 661 aaagatctct ggagaacgga tggaactgga gtggatatct ttcggacgac tatgagcttg 721 cagcggttcc agtttctgca aaacaatatc agattcgacg acaagtccac ccgggacgaa 781 aggaaacaga ctgacaacat ggctgcgttc cggtcaatat tcgatcagtt tgtgcagtgc 841 tgccaaaacg cttatagccc atcggaattc ctgaccatcg acgaaatgct tctctccttc 901 cgggggcgct gcctgttccg agtgtacatc ccgaacaagc cggctaaata cggaatcaaa 961 atcctggccc tggtggacgc caagaatttc tacgtcgtga atctcgaagt gtacgcagga 1021 aagcaaccgt cgggaccgta cgctgtttcg aaccgcccgt ttgaagtcgt cgagcggctt 1081 attcagccgg tggccagatc ccaccgcaat gttaccttcg acaattggtt caccggctac 1141 gagctgatgc ttcaccttct gaacgagtac cggctcacta gcgtggggac tgtcaggaag 1201 aacaagcggc agatcccaga atccttcatc cgcaccgacc gccagcctaa ctcgtccgtg 1261 ttcggatttc aaaaggatat cacgcttgtc tcgtacgccc ccaagaaaaa caaggtcgtg 1321 gtcgtgatga gcaccatgca tcacgacaac agcatcgacg agtcaaccgg agaaaagcaa 1381 aagcccgaga tgatcacctt ctacaattca actaaggccg gcgtcgacgt cgtggatgaa 1441 ctgtgcgcga actataacgt gtcccggaac tctaagcggt ggcctatgac tctcttctac 1501 ggagtgctga atatggccgc aatcaacgcg tgcatcatct accgcaccaa caagaacgtg 1561 accatcaagc gcaccgagtt catcagatcg ctgggtttga gcatgatcta cgagcacctc 1621 cattcacgga acaagaagaa gaatatccct acttacctga ggcagcgtat cgagaagcag 1681 ttgggagaac caagcccgcg ccacgtgaac gtgccggggc gctacgtgcg gtgccaagat 1741 tgcccgtaca aaaaggaccg caaaaccaaa agatcgtgta acgcgtgcgc caaacctatc 2023208176 27 Jul 2023 1801 tgcatggagc atgccaaatt tctgtgtgaa aattgtgctg aactcgattc ctccctg (SEQ ID NO: 14629).

[0230] In certain embodiments, the piggyBac or piggyBac-like transposase is hyperactive. A hyperactive piggyBac or piggyBac-like transposase is a transposase that is more active than the naturally occurring variant from which it is derived. In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase enzyme is isolated or derived from Bombyx mori. In certain embodiments, the piggyBac or piggyBac-like transposase is a hyperactive variant of SEQ ID NO: 14505. In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises a sequence that is at least 90% identical to: 1 MDIERQEERI RAMLEEELSD YSDESSSEDE TDHCSEHEVN YDTEEERIDS VDVPSNSRQE 61 EANAIIANES DSDPDDDLPL SLVRQRASAS RQMSGPHYTS KDGTKWYKNC QRPNVRLRSE 121 NIVTEQAQVK NIARDASTEY ECWNIFVTSD MLQEILTHTN SSIRWRQTKT AAENSSASTS 181 FYMQETTLCE LKALIGLLYI AGLIKSNRQS LKDLWRTDGT GVDIFRTTMS LQRFQFLQNN 241 IRFDDKSTRD ERKQTDNMAA FRSIFDQFVQ SCQNAYSPSE FLTIDEMLLS FRGRCLFRVY 301 IPNKPAKYGI KILALVDAKN FYVKNLEVYA GKQPSGPYAV SNRPFEWER LIQPVARSHR 361 NVTFDNWFTG YELMLHLLNE YRLTSVGTVR KNKRQIPESF IRTDRQPNSS VFGFQKDITL 421 VSYAPKKNKV VWMSTMHHD NSIDESTGEK QKPEMITFYN STKAGVDWD ELCANYNVSR 481 NSKRWPMTLF YGVLNMAAIN ACIIYRTNKN VTIKRTEFIR SLGLSMIYEH LHSRNKKKNI 541 PTYLRQRIEK QLGEPSPRHV NVPGRYVRCQ DCPYKKDRKT KRSCNACAKP ICMEHAKFLC 601 ENCAELDSHL (SEQ ID NO: 14576).

[0231] In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises SEQ ID NO: 14576. In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises a sequence of: 1 MDIERQEERI RAMLEEELSD YSDESSSEDE TDHCSEHEVN YDTEEERIDS VDVPSNSRQE 61 EANAIIANES DSDPDDDLPL SLVRQRASAS RQVSGPFYTS KDGTKWYKNC QRPNVRLRSE 121 NIVTEQAQVK NIARDASTEY ECWNIFVTSD MLQEILTHTN SSIRWRQTKT AAENSSAETS 181 FYMQETTLCE LKALIGLLYI AGLIKSNRQS LKDLWRTDGT GVDIFRTTMS LQRFQFLLNN 241 IRFDDKSTRD ERKQTDNMAA FRSIFDQFVQ SCQNAYSPSE FLTIDEMLLS FRGRCLFRVY 301 IPNKPAKYGI KILALVDAKN FYVHNLEVYA GKQPSGPYAV SNRPFEWER LIQPVARSHR 361 NVTFDNWFTG YEVMLHLLNE YRLTSVGTVR KNKRQIPESF IRTDRQPNSS VFGFQKDITL 421 VSYAPKKNKV VWMSTMHHD NSIDESTGEK QKPEMITFYN STKAGVDWD ELCANYNVSR 481 NSKRWPMTLF YGVLNMAAIN ACIIYRTNKN VTIKRTEFIR SLGLSMIYEH LHSRNKKKNI 541 PTYLRQRIEK QLGEPSPRHV NVPGRYVRCQ DCPYKKDRKT KRSCNACAKP ICMEHAKFLC 601 ENCAHLDS (SEQ ID NO: 14630).

[0232] In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises a sequence of: 1 MDIERQEERI RAMLEEELSD YSDESSSEDE TDHCSEHEVN YDTEEERIDS VDVPSNSRQE 61 EANAIIANES DSDPDDDLPL SLVRQRASAS RQVSGPFYTS KDGTKWYKNC QRPNVRLRSE 121 NIVTEQAQVK NIARDASTEY ECWNIFVTSD MLQEILTHTN SSIRWRQTKT AAENSSASTS 181 FYMQETTLCE LKALIGLLYI AGLIKSNRQS LKDLWRTDGT GVDIFRTTMS LQRFQFLLNN 241 IRFDDKSTRD ERKQTDNMAA FRSIFDQFVQ SCQNAYSPSE FLTIDEMLLS FRGRCLFRVY 301 IPNKPAKYGI KILALVDAKN FYVKNLEVYA GKQPSGPYAV SNRPFEWER LIQPVARSHR 361 NVTFDNWFTG YELMLHLLNE YRLTSVGTVR KNKRQIPESF IRTDRQPNSS VFGFQKDITL 421 VSYAPKKNKV VWMSTMHHD NSIDESTGEK QKPEMITFYN STKAGVDWD ELCANYNVSR 481 NSKRWPMTLF YGVLNMAAIN ACIIYRTNKN VTIKRTEFIR SLGLSMIYEH LHSRNKKKNI 541 PTYLRQRIAM QLGEPSPRHV NVPGRYVRCQ DCPYKKDRKT KRSCNACAKP ICMEHAKFLC 2023208176 27 Jul 2023 601 ENCAELDSSL (SEQ ID NO: 14631) .

[0233] In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises a sequence of: 1 MDIERQEERI RAMLEEELSD YSDESSSEDE TDHCSEHEVN YDTEEERIDS VDVPSNSRQE 61 EANAIIANES DSDPDDDLPL SLVRQRASAS RQVSGPFYTS KDGTKWYKNC QRPNVRLRSE 121 NIVTEQAQVK NIARDASTEY ECWNIFVTSD MLQEILTHTN SSIRWRQTKT AAENSSAETS 181 FYMQETTLCE LKALIGLLYI AGLIKSNRQS LKDLWRTDGT GVDIFRTTMS LQRFQFLLNN 241 IRFDDKSTRD ERKQTDNMAA FRSIFDQFVQ SCQNAYSPSE FLTIDEMLLS FRGRCLFRVY 301 IPNKPAKYGI KILALVDAKN FYVKNLEVYA GKQPSGPYAV SNRPFEWER LIQPVARSHR 361 NVTFDNWFTG YELMLHLLNE YRLTSVGTVR KNKTQIPENF IRTDRQPNSS VFGFQKDITL 421 VSYAPKKNKV VWMSTMHHD NSIDESTGEK QKPEMITFYN STKAGVDWD ELQANYNVSR 481 NSKRWPMTLF YGVLNMAAIN ACIIYRTNKN VTIKRTEFIR SLGLSMIYEH LHSRNKKKNI 541 PTYLRQRIEK QLGEPSPRHV NVPGRYVRCQ DCPYKKDRKT KRSCNACAKP ICMEHAKFLC 601 ENCAELDSSL (SEQ ID NO: 14632).

[0234] In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises a sequence of: 1 MDIERQEERI RAMLEEELSD YSDESSSEDE TDHCSEHEVN YDTEEERIDS VDVPSNSRQE 61 EANAIIANES DSDPDDDLPL SLVRQRASAS RQVSGPFYTS KDGTKWYKNC QRPNVRLRSE 121 NIVTEQAQVK NIARDASTEY ECWNIFVTSD MLQEILTHTN SSIRWRQTKT AAENSSAETS 181 FYMQETTLCE LKALIGLLYI AGLIKSNRQS LKDLWRTDGT GVDIFRTTMS LQRFQFLQNN 241 IRFDDKSTRD ERKQTDNMAA FRSIFDQFVQ SCQNAYSPSE FLTIDEMLLS FRGRCLFRVY 301 IPNKPAKYGI KILALVDAKN FYVKNLEVYA GKQPSGPYAV SNRPFEWER LIQPVARSHR 361 NVTFDNWFTG YELMLHLLNE YRLTSVGTVR KNKRQIPESF IRTDRQPNSS VFGFQKDITL 421 VSYAPKKNKV VWMSTMHHD NSIDESTGEK QKPEMITFYN STKAGVDWD ELCANYNVSR 481 NSKRWPMTLF YGVLNMAAIN ACIIYRTNKN VTIKRTEFIR SLGLSMIYEH LHSRNKKKNI 541 PTYLRQRIEK QLGEPSPRHV NVPGRYVRCQ DCPYKKDRKT KRSCNACAKP ICMEHAKFLC 601 ENCAELDSSL (SEQ ID NO: 14633).

[0235] In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises a sequence of: 1 MDIERQEERI RAMLEEELSD YSDESSSEDE TDHCSEHEVN YDTEEERIDS VDVPSNSRQE 61 EANAIIANES DSDPDDDLPL SLVRQRASAS RQVSGPFYTS KDGTKWYKNC QRPNVRLRSE 121 NIVTEQAQVK NIARDASTEY ECWNIFVTSD MLQEILTHTN SSIRHRQTKT AAENSSAETS 181 FYMQETTLCE LKALIALLYL AGLIKSNRQS LKDLWRTDGT GVDIFRTTMS LQRFQFLQNN 241 IRFDDKSTRD ERKQTDNMAA FRSIFDQFVQ CCQNAYSPSE FLTIDEMLLS FRGRCLFRVY 301 IPNKPAKYGI KILALVDAKN DYWNLEVYA GKQPSGPYAV SNRPFEWER LIQPVARSHR 361 NVTFDNWFTG YELMLHLLNE YRLTSVGTVR KNKRQIPESF IRTDRQPNSS VFGFQKDITL 421 VSYAPKKNKV VWMSTMHHD NSIDESTGEK QKPEMITFYN STKAGVDWD ELCANYNVSR 481 NSKRWPMTLF YGVLNMAAIN ACIIYRTNKN VTIKRTEFIR SLGLSMIYEH LHSRNKKKNI 541 PTYLRQRIEK QLGEPSSRHV NVKGRYVRCQ DCPYKKDRKT KRSCNACAKP ICMEHAKFLC 601 ENCAELDSSL (SEQ ID NO: 14634).

[0236] In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase is more active than the transposase of SEQ ID NO: 14505. In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or any percentage in between identical to SEQ ID NO: 14505. 2023208176 27 Jul 2023

[0237] In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises an amino acid substitution at a position selected from 92, 93, 96, 97, 165, 178, 189, 196, 200, 201, 211, 215, 235, 238, 246, 253, 258, 261, 263, 271, 303, 321, 324, 330, 373, 389, 399, 402, 403, 404, 448, 473, 484, 507,5 23, 527, 528, 543, 549, 550, 557,6 01, 605, 607, 609, 610 or a combination thereof (relative to SEQ ID NO: 14505). In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises an amino acid substitution of Q92A, V93L, V93M, P96G, F97H, F97C, H165E, H165W, E178S, E178H, C189P, A196G, L200I, A201Q, L211A, W215Y, G219S, Q235Y, Q235G, Q238L, K246I, K253V, M258V, F261L, S263K, C271S, N303R, F321W, F321D, V324K, V324H, A330V, L373C, L373V, V389L, S399N, R402K, T403L, D404Q, D404S, D404M, N441R, G448W, E449A, V469T, C473Q, R484K T507C, G523A, I527M, Y528K Y543I, E549A, K550M, P557S, E601V, E605H, E605W, D607H, S609H, L610I or any combination thereof. In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises an amino acid substitution of Q92A, V93L, V93M, P96G, F97H, F97C, H165E, H165W, E178S, E178H, C189P, A196G, L200I, A201Q, L211A, W215Y, G219S, Q235Y, Q235G, Q238L, K246I, K253V, M258V, F261L, S263K, C271S, N303R, F321W, F321D, V324K, V324H, A330V, L373C, L373V, V389L, S399N, R402K, T403L, D404Q, D404S, D404M, N441R, G448W, E449A, V469T, C473Q, R484K T507C, G523A, I527M, Y528K Y543I, E549A, K550M, P557S, E601V, E605H, E605W, D607H, S609H and L610I.

[0238] In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises one or more substitutions of an amino acid that is not wild type, wherein the one or more substitutions a for wild type amino acid comprises a substitution of E4X, A12X, M13X,L14X, E15X, D20X, E24X, S25X, S26X, S27X, D32X, H33X, E36X, E44X, E45X, E46X, I48X, D49X, R58X, A62X, N63X, A64X, I65X, I66X, N68X, E69X, D71X, S72X, D76X, P79X, R84X, Q85X, A87X, S88X, Q92X, V93X, S94X, G95X, P96X, F97X, Y98X, T99X, I145X, S149X, D150X, L152X, E154X, T157X, N160X, S161X, S162X, H165X, R166X, T168X, K169X, T170X, A171X, E173X, S175X, S176X, E178X, T179X, M183X, Q184X, T186X, T187X, L188X, C189X, L194X, I195X, A196X, L198X, L200X, A201X, L203X, I204X, K205X, A206X, N207X, Q209X, S210X, L211X, K212X, D213X, L214X, W215X, R216X, T217X, G219X, V222X, D223X, I224X, T227X, M229X, Q235X, L237X, Q238X, N239X, N240X, P302X, N303X, P305X, A306X, K307X, Y308X, I310X, K311X, 2023208176 27 Jul 2023 I312X, L313X, A314X, L315X, V316X,D317X, A318X, K319X, N320X, F321X, Y322X, V323X, V324X, L326X, E327X, V328X, A330X, Q333X, P334X, S335X, G336X, P337X, A339X, V340X, S341X, N342X, R343X, P344X, F345X, E346X, V347X, E349X, I352X, Q353X, V355X, A356X, R357X, N361X, D365X, W367X, T369X, G370X, L373X, M374X, L375X, H376X, N379X, E380X, R382X, V386X, V389X, N392X, R394X, Q395X, S399X, F400X, I401X, R402XT403X, D404X, R405X, Q406X, P407X, N408X, S409X, S410X, V411X, F412X, F414X, Q415X, I418X, T419X, L420X, N428XV432X, M434X, D440X, N441X, S442X, I443X, D444X, E445X, G448X, E449X, Q451X, K452X, M455X, I456X, T457X, F458X, S461X, A464X, V466X, Q468X, V469X, E471X, L472X, C473X, A474X, K483X, W485X, T488X, L489X, Y491X, G492X, V493X, M496X, I499X, C502X, I503X, T507X, K509X, N510X, V511X, T512X, I513X, R515X, E517X, S521X, G523X, L524X, S525X, I527X, Y528X, E529X, H532X, S533X, N535X, K536X, K537X, N539X, I540X, T542X, Y543X, Q546X, E549X, K550X, Q551X, G553X, E554X, P555X, S556X, P557X, R558X, H559X, V560X, N561X, V562X, P563X, G564X, R565X, Y566X, V567X, Q570X, D571X, P573X, Y574X, K576X, K581X, S583X, A586X, A588X, E594X, F598X, L599X, E601X, N602X, C603X, A604X, E605X, L606X, D607X, S608X, S609X or L610X (relative to SEQ ID NO: 14505). A list of hyperactive amino acid substitutions can be found in US patent No. 10,041,077, the contents of which are incorporated herein by reference in their entirety.

[0239] In certain embodiments, the piggyBac or piggyBac-like transposase is integration deficient. In certain embodiments, an integration deficient piggyBac or piggyBac-like transposase is a transposase that can excise its corresponding transposon, but that integrates the excised transposon at a lower frequency than a corresponding wild type transposase. In certain embodiments, the piggyBac or piggyBac-like transposase is an integration deficient variant of SEQ ID NO: 14505.

[0240] In certain embodiments, the excision competent, integration deficient piggyBac or piggyBac-like transposase comprises one or more substitutions of an amino acid that is not wild type, wherein the one or more substitutions a for wild type amino acid comprises a substitution of R9X, A12X, M13X, D20X, Y21K, D23X, E24X, S25X, S26X, S27X, E28X, E30X, D32X, H33X, E36X, H37X, A39X, Y41X, D42X, T43X, E44X, E45X, E46X, R47X, D49X, S50X, S55X, A62X, N63X, A64X, I66X, A67X, N68X, E69X, D70X, D71X, S72X, D73X, P74X, D75X, D76X, D77X,I78X, S81X,V83X, R84X, Q85X, A87X, S88X, A89X,S90X,R91X, 2023208176 27 Jul 2023 Q92X, V93X, S94X, G95X, P96X, F97X, Y98X, T99X, W012X, G103X, Y107X, K108X, L117X, I122X, Q128X, I312X, D135X, S137X, E139X, Y140X, I145X, S149X, D150X, Q153X, E154X, T157X, S161X, S162X, R164X, H165X, R166X, Q167X, T168X, K169X, T170X, A171X, A172X, E173X, R174X, S175X, S176X, A177X, E178X, T179X, S180X,Y182X, Q184X, E185X, T187X, L188X, C189X, L194X, I195X, A196X, L198X, L200X, A201X, L203X, I204X, K205X, N207X, Q209X, L211X, D213X, L214X, W215X, R216X, T217X, G219X, T220X, V222X, D223X, I224X, T227X, T228X, F234X, Q235X, L237X, Q238X, N239X, N240X, N303X, K304X, I310X, I312X, L313X, A314X, L315X, V316X,D317X, A318X, K319X, N320X, F321X, Y322X, V323X, V324X, N325X, L326X, E327X, V328X, A330X, G331X, K332X, Q333X, S335X, P337X, P344X, F345X, E349X, H359X, N361X, V362X, D365X, F368X, Y371X, E372X, L373X, H376X, E380X, R382X, R382X, V386X, G387X, T388X, V389X, K391X, N392X, R394X, Q395X, E398X, S399X, F400X, I401X, R402XT403X, D404X, R405X, Q406X, P407X, N408X, S409X, S410X, Q415X,K416X, A424X, K426X, N428X, V430X, V432X, V433X, M434X, D436X, D440X, N441X, S442X, I443X, D444X, E445X, S446X, T447X, G448X, E449X, K450X, Q451X, E454X, M455X, I456X, T457X, F458X, S461X, A464X, V466X, Q468X, V469X, C473X, A474X, N475X, N477X, K483X, R484X, P486X, T488X, L489X, G492X, V493X, M496X, I499X, I503X, Y505X, T507X, N510X, V511X, T512X, I513X, K514X, T516X, E517X, S521X, G523X, L524X, S525X, I527X, Y528X, L531X, H532X, S533X, N535X, I540X, T542X, Y543X, R545X, Q546X, E549X, L552X, G553X, E554X, P555X, S556X, P557X, R558X, H559X, V560X, N561X, V562X, P563X, G564X, V567X, Q570X, D571X, P573X, Y574X, K575X, K576X, N585X, A586X, M593X, K596X, E601X, N602X, A604X, E605X, L606X, D607X, S608X, S609X or L610X (relative to SEQ ID NO: 14505). A list of integration deficient amino acid substitutions can be found in US patent No. 10,041,077, the contents of which are incorporated by reference in their entirety.

[0241] In certain embodiments, the integration deficient piggyBac or piggyBac-like transposase comprises a sequence of: 1 MDIERQEERI RAMLEEELSD YSDESSSEDE TDHCSEHEVN YDTEEERIDS VDVPSNSRQE 61 EANAIIANES DSDPDDDLPL SLVRQRASAS RQVSGPFYTS KDGTKWYKNC QRPNVRLRSE 121 NIVTEQAQVK NIARDASTEY ECWNIFVTSD MLQEILTHTN SSIRHRQTKT AAENSSAETS 181 FYMQETTLCE LKALIALLYL AGLIKSNRQS LKDLWRKDGT GVDIFRTTMS LQRFQFLLNN 241 IRFDDISTRD ERKQTDNMAA FRSIFDQFVQ CCQNAYSPSE FLTIDEMLLS FRGRCLFRVY 301 IPNKPAKYGI KILALVDAKN FYWNLEVYA GKQPSGPYAV SNRPFEWER LIQPVARSHR 361 NVTFDNWFTG YELMLHLLNE YRLTSVGTVR KNKRQIPESF IRTDRQPNSS VFGFQKDITL 421 VSYAPKKNKV VWMSTMHHD NSIDESTGEK QKPEMITFYN STKAGVDWD ELCANYNVSR 99 2023208176 27 Jul 2023 481 NSKKWPMTLF YGVLNMAAIN ACIIYRTNKN VTIKRTEFIR SLGLSMMYEH LHSRNKKKNI 541 PTYLRQRIEK QLGEPVPRHV NVPGRYVRCQ DCPYKKDRKT KRSCNACAKP ICMEHAKFLC 601 ENCAELDSSL (SEQ ID NO: 14606). In certain embodiments, the integration deficient piggyBac or piggyBac-like transposase comprises a sequence of: 1 MDIERQEERI RAMLEEELSD YSDESSSEDE TDHCSEHEVN YDTEEERIDS VDVPSNSRQE 61 EANAIIANES DSDPDDDLPL SLVRQRASAS RQVSGPFYTS KDGTKWYKNC QRPNVRLRSE 121 NIVTEQAQVK NIARDASTEY ECWNIFVTSD MLQEILTHTN SSIRHRQTKT AAENSSAETS 181 FYMQETTLCE LKALIGLLYL AGLIKSNRQS LKDLWRTDGT GVDIFRTTMS LQRFYFLQNN 241 IRFDDKSTLD ERKQTDNMAA FRSIFDQFVQ SCQNAYSPSE FLTIDEMLLS FRGRCLFRVY 301 IPNKPAKYGI KILALVDAKN FYWNLEVYA GKQPSGPYAV SNRPFEWER LIQPVARSHR 361 NVTFDNWFTG YELMLHLLNE YRLTSVGTVR KNKRQIPESF IRTDRQPNSS VFGFQKDITL 421 VSYAPKKNKV VWMSTMHHD NSIDESTGEK QKPEMITFYN STKAGVDWD ELCANYNVSR 481 NSKRWPMTLF YGVLNMAAIN ACIIYRTNKN VTIKRTEFIR SLGLSMIYEH LHSRNKKKNI 541 PTYLRQRIEK QLGEPSPRHV NYPGRYVRCQ DCPYKKDRKT KRSCNACAKP ICMEHAKFLC 601 VNCAELDSSL (SEQ ID NO: 14607). In certain embodiments, the piggyBac or piggyBac-like transposase that is is integration deficient comprises a sequence of 1 MDIERQEERI RAMLEEELSD YSDESSSEDE TDHCSEHEVN YDTEEERIDS VDVPSNSRQE 61 EANAIIANES DSDPDDDLPL SLVRQRASAS RQVSGPFYTS KDGTKWYKNC QRPNVRLRSE 121 NIVTEQAQVK NIARDASTEY ECWNIFVTSD MLQEILTHTN SSIRHRQTKT AAENSSAETS 181 FYMQETTLCE LKALIALLYL AGLIKSNRQS LKDLWRKDGT GVDIFRTTMS LQRFQFLLNN 241 IRFDDKSTRD ERKQTDNMAA FRSIFDQFVQ CCQNAYSPSE FLTIDEMLLS FRGRCLFRVY 301 IPNKPAKYGI KILALVDAKN DYWNLEVYA GKQPSGPYAV SNRPFEWER LIQPVARSHR 361 NVTFDNWFTG YECMLHLLNE YRLTSVGTVR KNKRQIPESF IRTDRQPNSS VFGFQKDITL 421 VSYAPKKNKV VWMSTMHHD NSIDESTGEK QKPEMITFYN STKAGVDWD ELCANYNVSR 481 NSKKWPMTLF YGVLNMAAIN ACIIYRTNKN VTIKRTEFIR SLGLSMIKEH LHSRNKKKNI 541 PTYLRQRIEK QLGEPSPRHV NVPGRYVRCQ DCPYKKDRKT KRSCNACAKP ICMEHAKFLC 601 ENCAELDSSL (SEQ ID NO: 14608) . In certain embodiments, the integration deficient transposase comprises a sequence that is at least 90% identical to SEQ ID NO: 14608.

[0242] In certain embodiments, the piggyBac or piggyBac-like transposon is isolated or derived from Bombyx mori. In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of: 1 ttatcccggc gagcatgagg cagggtatct cataccctgg taaaatttta aagttgtgta 61 ttttataaaa ttttcgtctg acaacactag cgcgctcagt agctggaggc aggagcgtgc 121 gggaggggat agtggcgtga tcgcagtgtg gcacgggaca ccggcgagat attcgtgtgc 181 aaacctgttt cgggtatgtt ataccctgcc tcattgttga cgtatttttt ttatgtaatt 241 tttccgatta ttaatttcaa ctgttttatt ggtattttta tgttatccat tgttcttttt 301 ttatgattta ctgtatcggt tgtctttcgt tcctttagtt gagttttttt ttattatttt 361 cagtttttga tcaaa (SEQ ID NO: 14506). In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of: 1 tcatattttt agtttaaaaa aataattata tgttttataa tgaaaagaat ctcattatct 61 ttcagtatta ggttgattta tattccaaag aataatattt ttgttaaatt gttgattttt 2023208176 27 Jul 2023 121 gtaaacctct aaatgtttgt tgctaaaatt 181 taataatttc ataattaaaa acttctttca 241 caaaataaga tcaacataat tgagtaaata 301 atatgggtat gtcataccct gccacattct 361 cgggttat (SEQ ID NO: 14507) . actgtgttta agaaaaagat taataaataa ttgaatgcca ttaaataaac cattatttta ataataagaa caatattata gtacaacaaa tgatgtaact ttttttcacc tcatgctcgc In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of: 1 ttatcccggc gagcatgagg cagggtatct 61 ttttataaaa ttttcgtctg acaacactag 121 gggaggggat agtggcgtga tcgcagtgtg 181 aaacctgttt cgggtatgtt ataccctgcc cataccctgg taaaatttta aagttgtgta cgcgctcagt agctggaggc aggagcgtgc gcacgggaca ccggcgagat attcgtgtgc teat (SEQ ID NO: 14508). In certain embodiments, the piggyBac (PB) or piggyBac-like transposon comprises a sequence of: 1 taaataataa taatttcata attaaaaact tctttcattg aatgccatta aataaaccat 61 tattttacaa aataagatca acataattga gtaaataata ataagaacaa tattatagta 121 caacaaaata tgggtatgtc ataccctgcc acattcttga tgtaactttt tttcacctca 181 tgctcgccgg gttat (SEQ ID NO: 14509) .

[0243] In certain embodiments, the piggyBac or piggyBac-like transposon comprises a 5’ sequence corresponding to SEQ ID NO: 14506 and a 3’ sequence corresponding to SEQ ID NO: 14507. In certain embodiments, one piggyBac or piggyBac-like transposon end is at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identical or any percentage in between identical to SEQ ID NO: 14506 and the other piggyBac or piggyBac-like transposon end is at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or any percentage in between identical to SEQ ID NO: 14507. In certain embodiments, the piggyBac or piggyBac-like transposon comprises SEQ ID NO: 14506 and SEQ ID NO: 14507 or SEQ ID NO: 14509. In certain embodiments, the piggyBac or piggyBac-like transposon comprises SEQ ID NO: 14508 and SEQ ID NO: 14507 or SEQ ID NO: 14509. In certain embodiments, the 5’ and 3’ transposon ends share a 16 bp repeat sequence at their ends of CCCGGCGAGCATGAGG (SEQ ID NO: 14510) immediately adjacent to the 5'-TTAT-3 target insertion site, which is inverted in the orientation in the two ends. In certain embodiments, 5’ transposon end begins with a sequence comprising 5'-TTATCCCGGCGAGCATGAGG-3 (SEQ ID NO: 14511), and the 3’ transposon ends with a sequence comprising the reverse complement of this sequence: 5'-CCTCATGCTCGCCGGGTTAT-3' (SEQ ID NO: 14512).

[0244] In certain embodiments, the piggyBac or piggyBac-like transposon comprises one end comprising at least 14, 16, 18, 20, 30 or 40 contiguous nucleotides of SEQ ID NO: 14506 or SEQ ID NO: 14508. In certain embodiments, the piggyBac or piggyBac-like transposon 2023208176 27 Jul 2023 comprises one end comprising at least 14, 16, 18, 20, 30 or 40 contiguous nucleotides of SEQ ID NO: 14507 or SEQ ID NO: 14509. In certain embodiments, the piggyBac or piggyBac-like transposon comprises one end with at least 90% identity to SEQ ID NO: 14506 or SEQ ID NO: 14508. In certain embodiments, the piggyBac or piggyBac-like transposon comprises one end with at least 90% identity to SEQ ID NO: 14507 or SEQ ID NO: 14509.

[0245] In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of: 1 ttaacccggc gagcatgagg cagggtatct 61 ttttataaaa ttttcgtctg acaacactag 121 gggaggggat agtggcgtga tcgcagtgtg 181 aaacctgttt cgggtatgtt ataccctgcc 241 tttccgatta ttaatttcaa ctgttttatt 301 ttatgattta ctgtatcggt tgtctttcgt 361 cagtttttga tcaaa (SEQ ID NO: 145 cataccctgg taaaatttta aagttgtgta cgcgctcagt agctggaggc aggagcgtgc gcacgggaca ccggcgagat attcgtgtgc tcattgttga cgtatttttt ttatgtaatt ggtattttta tgttatccat tgttcttttt tcctttagtt gagttttttt ttattatttt 15) .

[0246] In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of: 1 tcatattttt agtttaaaaa aataattata tgttttataa tgaaaagaat ctcattatct 61 ttcagtatta ggttgattta tattccaaag aataatattt ttgttaaatt gttgattttt 121 gtaaacctct aaatgtttgt tgctaaaatt actgtgttta agaaaaagat taataaataa 181 taataatttc ataattaaaa acttctttca ttgaatgcca ttaaataatt cattatttta 241 caaaataaga tcaacataat tgagtaaata ataataagaa caatattata gtacaacaaa 301 atatgggtat gtcataccct tttttttttt tttttttttt ttttttcggg tagagggccg 361 aacctcctac gaggtccccg cgcaaaaggg gcgcgcgggg tatgtgagac tcaacgatct 421 gcatggtgtt gtgagcagac cgcgggccca aggattttag agcccaccca ctaaacgact 481 cctctgcact cttacacccg acgtccgatc ccctccgagg tcagaacccg gatgaggtag 541 gggggctacc gcggtcaaca ctacaaccag acggcgcggc tcaccccaag gacgcccagc 601 cgacggagcc ttcgaggcga atcgaaggct ctgaaacgtc ggccgtctcg gtacggcagc 661 ccgtcgggcc gcccagacgg tgccgctggt gtcccggaat accccgctgg accagaacca 721 gcctgccggg tcgggacgcg atacaccgtc gaccggtcgc tctaatcact ccacggcagc 781 gcgctagagt gctggta (SEQ ID NO: 14516).

[0247] In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of CCCGGCGAGCATGAGG (SEQ ID NO: 14510). In certain embodiments, the piggyBac or piggyBac-like transposon comprises an ITR sequence of SEQ ID NO: 14510. In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of TTATCCCGGCGAGCATGAGG (SEQ ID NO: 14511). In certain embodiments, the piggyBac or piggyBac-like transposon comprises at least 16 contiguous nucleotides from SEQ ID NO: 14511. In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of CCTCATGCTCGCCGGGTTAT (SEQ ID NO: 14512). In certain embodiments, the piggyBac or piggyBac-like transposon comprises at least 16 contiguous nucleotides from SEQ ID NO: 14512. In certain embodiments, the piggyBac or piggyBac-like transposon comprises 102 2023208176 27 Jul 2023 one end comprising at least 16 contiguous nucleotides from SEQ ID NO: 14511 and one end comprising at least 16 contiguous nucleotides from SEQ ID NO: 14512. In certain embodiments, the piggyBac or piggyBac-like transposon comprises SEQ ID NO: 14511 and SEQ ID NO: 14512. In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of TTAACCCGGCGAGCATGAGG (SEQ ID NO: 14513). In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of CCTCATGCTCGCCGGGTTAA (SEQ ID NO: 14514).

[0248] In certain embodiments, the piggyBac or piggyBac-like transposon may have ends comprising SEQ ID NO: 14506 and SEQ ID NO: 14507, or a variant of either or both of these having at least 90% sequence identity to SEQ ID NO: 14506 or SEQ ID NO: 14507, and the piggyBac or piggyBac-like transposase has the sequence of SEQ ID NO: 14504 or SEQ ID NO: 14505, or a sequence at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identity to SEQ ID NO: 14504 or SEQ ID NO: 14505. In certain embodiments, the piggyBac or piggyBac-like transposon comprises a heterologous polynucleotide inserted between a pair of inverted repeats, where the transposon is capable of transposition by a piggyBac or piggyBac-like transposase having at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identity to SEQ ID NO: 14504 or SEQ ID NO: 14505. In certain embodiments, the transposon comprises two transposon ends, each of which comprises SEQ ID NO: 14510 in inverted orientations in the two transposon ends. In certain embodiments, each inverted terminal repeat (ITR) is at least 90% identical to SEQ ID NO: 14510.

[0249] In certain embodiments, the piggyBac or piggyBac-like transposon is capable of insertion by a piggyBac or piggyBac-like transposase at the sequence 5'-TTAT-3 within a target nucleic acid. In certain embodiments, one end of the piggyBac or piggyBac-like transposon comprises at least 16 contiguous nucleotides from SEQ ID NO: 14506 and the other transposon end comprises at least 16 contiguous nucleotides from SEQ ID NO: 14507. In certain embodiments, one end of the piggyBac or piggyBac-like transposon comprises at least 17, at least 18, at least 19, at least 20, at least 22, at least 25, at least 30 contiguous nucleotides from SEQ ID NO: 14506 and the other transposon end comprises at least 17, at least 18, at least 19, at least 20, at least 22, at least 25, at least 30 contiguous nucleotides from SEQ ID NO: 14507. 2023208176 27 Jul 2023

[0250] In certain embodiments, the piggyBac or piggyBac-like transposon comprises transposon ends (each end comprising an ITR) corresponding to SEQ ID NO: 14506 and SEQ ID NO: 14507, and has a target sequence corresponding to 5'-TTAT3'. In certain embodiments, the piggyBac or piggyBac-like transposon also comprises a sequence encoding a transposase (e.g. SEQ ID NO: 14505). In certain embodiments, the piggyBac or piggyBac-like transposon comprises one transposon end corresponding to SEQ ID NO: 14506 and a second transposon end corresponding to SEQ ID NO: 14516. SEQ ID NO: 14516 is very similar to SEQ ID NO: 14507, but has a large insertion shortly before the ITR. Although the ITR sequences for the two transposon ends are identical (they are both identical to SEQ ID NO: 14510), they have different target sequences: the second transposon has a target sequence corresponding to 5'-TTAA-3', providing evidence that no change in ITR sequence is necessary to modify the target sequence specificity. The piggyBac or piggyBac-like transposase (SEQ ID NO: 14504), which is associated with the 5'-TTAA-3’ target site differs from the 5'-TTAT-3'-associated transposase (SEQ ID NO: 14505) by only 4 amino acid changes (D322Y, S473C, A507T, H582R). In certain embodiments, the piggyBac or piggyBac-like transposase (SEQ ID NO: 14504), which is associated with the 5'-TTAA-3’ target site is less active than the 5'-TTAT-3'-associated piggyBac or piggyBac-like transposase (SEQ ID NO: 14505) on the transposon with 5'-TTAT-3' ends. In certain embodiments, piggyBac or piggyBac-like transposons with 5'-TTAA-3’ target sites can be converted to piggyBac or piggyBac-like transposases with 5'-TTAT-3 target sites by replacing 5'-TTAA-3’ target sites with 5'-TTAT-3'. Such transposons can be used either with a piggyBac or piggyBac-like transposase such as SEQ ID NO: 14504 which recognizes the 5'-TTAT-3’ target sequence, or with a variant of a transposase originally associated with the 5'-TTAA-3' transposon. In certain embodiments, the high similarity between the 5'-TTAA-3' and 5'-TTAT-3' piggyBac or piggyBac-like transposases demonstrates that very few changes to the amino acid sequence of a piggyBac or piggyBac-like transposase alter target sequence specificity. In certain embodiments, modification of any piggyBac or piggyBac-like transposon-transposase gene transfer system, in which 5'-TTAA-3’ target sequences are replaced with 5'-TTAT-3'-target sequences, the ITRs remain the same, and the transposase is the original piggyBac or piggyBac-like transposase or a variant thereof resulting from using a low-level mutagenesis to introduce mutations into the transposase. In certain embodiments, piggyBac or piggyBac-like transposon transposase transfer systems can be formed by the modification of a 5'- 2023208176 27 Jul 2023 TTAT-3'-active piggyBac or piggyBac-like transposon-transposase gene transfer systems in which 5'-TTAT-3’ target sequences are replaced with 5'-TTAA-3'-target sequences, the ITRs remain the same, and the piggyBac or piggyBac-like transposase is the original transposase or a variant thereof.

[0251] In certain embodiments, the piggyBac or piggyBac-like transposon is isolated or derived from Bombyx mori. In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of: 1 cccggcgagc atgaggcagg gtatctcata ccctggtaaa attttaaagt tgtgtatttt 61 ataaaatttt cgtctgacaa cactagcgcg ctcagtagct ggaggcagga gcgtgcggga 121 ggggatagtg gcgtgatcgc agtgtggcac gggacaccgg cgagatattc gtgtgcaaac 181 ctgtttcggg tatgttatac cctgcctcat tgttgacgta t (SEQ ID NO: 14577) . In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of: 1 tttaagaaaa agattaataa ataataataa tttcataatt aaaaacttct ttcattgaat 61 gccattaaat aaaccattat tttacaaaat aagatcaaca taattgagta aataataata 121 agaacaatat tatagtacaa caaaatatgg gtatgtcata ccctgccaca ttcttgatgt 181 aacttttttt cacctcatgc tcgccggg (SEQ ID NO: 14578) . In certain embodiments, the transposon comprises at least 16 contiguous bases from SEQ ID NO: 14577 and at least 16 contiguous bases from SEQ ID NO: 14578, and inverted terminal repeats that are at least 87% identical to CCCGGCGAGCATGAGG (SEQ ID NO: 14510). In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of: 1 cccggcgagc atgaggcagg gtatctcata ccctggtaaa attttaaagt tgtgtatttt 61 ataaaatttt cgtctgacaa cactagcgcg ctcagtagct ggaggcagga gcgtgcggga 121 ggggatagtg gcgtgatcgc agtgtggcac gggacaccgg cgagatattc gtgtgcaaac 181 ctgtttcggg tatgttatac cctgcctcat tgttgacgta ttttttttat gtaatttttc 241 cgattattaa tttcaactgt tttattggta tttttatgtt atccattgtt ctttttttat 301 gatttactgt atcggttgtc tttcgttcct ttagttgagt ttttttttat tattttcagt 361 ttttgatcaa a (SEQ ID NO: 14595) . In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of: 1 tcatattttt agtttaaaaa aataattata tgttttataa tgaaaagaat ctcattatct 61 ttcagtatta ggttgattta tattccaaag aataatattt ttgttaaatt gttgattttt 121 gtaaacctct aaatgtttgt tgctaaaatt actgtgttta agaaaaagat taataaataa 181 taataatttc ataattaaaa acttctttca ttgaatgcca ttaaataaac cattatttta 241 caaaataaga tcaacataat tgagtaaata ataataagaa caatattata gtacaacaaa 301 atatgggtat gtcataccct gccacattct tgatgtaact ttttttcacc tcatgctcgc 361 cggg (SEQ ID NO: 14596).

[0252] In certain embodiments, the piggyBac or piggyBac-like transposon comprises SEQ ID NO: 14595 and SEQ ID NO: 14596, and is transposed by the piggyBac or piggyBac-like transposase of SEQ ID NO: 14505. In certain embodiments, the ITRs of SEQ ID NO: 14595 and SEQ ID: 14596 are not flanked by a 5’-TTAA-3’ sequence. In certain embodiments, the ITRs of SEQ ID NO: 14595 and SEQ ID: 14596 are flanked by a 5’-TTAT-3’ sequence. 2023208176 27 Jul 2023

[0253] In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of: 1 cccggcgagc atgaggcagg gtatctcata 61 ataaaatttt cgtctgacaa cactagcgcg 121 ggggatagtg gcgtgatcgc agtgtggcac 181 ctgtttcggg tatgttatac cctgcctcat 241 cgattattaa tttcaactgt tttattggta 301 g (SEQ ID NO: 14597) . ccctggtaaa attttaaagt tgtgtatttt ctcagtagct ggaggcagga gcgtgcggga gggacaccgg cgagatattc gtgtgcaaac tgttgacgta ttttttttat gtaatttttc tttttatgtt atccattgtt ctttttttat In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of: 1 cagggtatct cataccctgg taaaatttta aagttgtgta ttttataaaa ttttcgtctg 61 acaacactag cgcgctcagt agctggaggc aggagcgtgc gggaggggat agtggcgtga 121 tcgcagtgtg gcacgggaca ccggcgagat attcgtgtgc aaacctgttt cgggtatgtt 181 ataccctgcc tcattgttga cgtatttttt ttatgtaatt tttccgatta ttaatttcaa 241 ctgttttatt ggtattttta tgttatccat tgttcttttt ttatg (SEQ ID NO: 14598) . In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of: 1 cagggtatct cataccctgg taaaatttta 61 acaacactag cgcgctcagt agctggaggc 121 tcgcagtgtg gcacgggaca ccggcgagat 181 ataccctgcc tcattgttga cgtat (SEQ aagttgtgta ttttataaaa ttttcgtctg aggagcgtgc gggaggggat agtggcgtga attcgtgtgc aaacctgttt cgggtatgtt ID NO: 14599) . In certain embodiments, the 5’ end of the piggyBac or piggyBac-like transposon comprises a sequence of SEQ ID NO: 14577, SEQ ID NO: 14595, or SEQ ID NOs: 14597-14599. In certain embodiments, the 5’ end of the piggyBac or piggyBac-like transposon is preceded by a 5’ target sequence. In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of: 1 tcatattttt agtttaaaaa aataattata tgttttataa tgaaaagaat ctcattatct 61 ttcagtatta ggttgattta tattccaaag aataatattt ttgttaaatt gttgattttt 121 gtaaacctct aaatgtttgt tgctaaaatt actgtgttta agaaaaagat taataaataa 181 taataatttc ataattaaaa acttctttca ttgaatgcca ttaaataaac cattatttta 241 caaaataaga tcaacataat tgagtaaata ataataagaa caatattata gtacaacaaa 301 atatgggtat gtcataccct gccacattct tgatgtaact ttttttcacc tcatgctcgc 361 cggg (SEQ ID NO: 14600). In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of: 1 tttaagaaaa agattaataa ataataataa 61 gccattaaat aaaccattat tttacaaaat 121 agaacaatat tatagtacaa caaaatatgg 181 aacttttttt ca (SEQ ID NO: 14601). tttcataatt aaaaacttct ttcattgaat aagatcaaca taattgagta aataataata gtatgtcata ccctgccaca ttcttgatgt In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of: 1 cccggcgagc atgaggcagg 61 ataaaatttt cgtctgacaa 121 ggggatagtg gcgtgatcgc 181 ctgtttcggg tatgttatac 241 cgattattaa tttcaactgt 301 gatttactgt atcggttgtc gtatctcata ccctggtaaa cactagcgcg ctcagtagct agtgtggcac gggacaccgg cctgcctcat tgttgacgta tttattggta tttttatgtt tttcgttcct ttagttgagt attttaaagt tgtgtatttt ggaggcagga gcgtgcggga cgagatattc gtgtgcaaac ttttttttat gtaatttttc atccattgtt ctttttttat ttttttttat tattttcagt 2023208176 27 Jul 2023 361 ttttgatcaa a (SEQ ID NO: 14602).

[0254] In certain embodiments, the 3’ end of the piggyBac or piggyBac-like transposon comprises a sequence of SEQ ID NO: 14578, SEQ ID NO: 14596, or SEQ ID NOs: 1460014601. In certain embodiments, the 3’ end of the piggyBac or piggyBac-like transposon is followed by a 3’ target sequence. In certain embodiments, the transposon is transposed by the transposase of SEQ ID NO: 14505. In certain embodiments, the 5’ and 3’ ends of the piggyBac or piggyBac-like transposon share a 16 bp repeat sequence of SEQ ID NO: 14510 in inverted orientation and immediately adjacent to the target sequence. In certain embodiments, the 5’ transposon end begins with SEQ ID NO: 14510, and the 3’ transposon end ends with the reverse complement of SEQ ID NO: 14510, 5’- CCTCATGCTCGCCGGG-3’ (SEQ ID NO: 14603). In certain embodiments, the piggyBac or piggyBac-like transposon comprises an ITR with at least 93%, at least 87%, or at least 81% or any percentage in between identity to SEQ ID NO: 14510 or SEQ ID NO: 14603. In certain embodiments, the piggyBac or piggyBac-like transposon comprises a target sequence followed by a 5’ transposon end comprising a sequence selected from SEQ ID NOs: 88, 105 or 107 and a 3’ transposon end comprising SEQ ID NO: 14578 or 106 followed by a target sequence, in certain embodiments, the piggyBac or piggyBac like transposon comprises one end that comprises a sequence that is at least 90%, at least 95% or at least 99% or any percentage in between identical to SEQ ID NO: 14577 and one end that comprises a sequence that is at least 90%, at least 95% or at least 99% or any percentage in between identical to SEQ ID NO: 14578. In certain embodiments, one transposon end comprises at least 14, at least 16, at least 18 or at least 20 contiguous bases from SEQ ID NO: 14577 and one transposon end comprises at least 14, at least 16, at least 18 or at least 20 contiguous bases from SEQ ID NO: 14578.

[0255] In certain embodiments, the piggyBac or piggyBac-like transposon comprises two transposon ends wherein each transposon ends comprises a sequence that is at least 81% identical, at least 87% identical or at least 93% identical or any percentage in between identical to SEQ ID NO: 14510 in inverted orientation in the two transposon ends. One end may further comprise at least 14, at least 16, at least 18 or at least 20 contiguous bases from SEQ ID NO: 14599, and the other end may further comprise at least 14, at least 16, at least 18 or at least 20 contiguous bases from SEQ ID NO: 14601. The piggyBac or piggyBac-like transposon may be 2023208176 27 Jul 2023 transposed by the transposase of SEQ ID NO: 14505, and the transposase may optionally be fused to a nuclear localization signal.

[0256] In certain embodiments, the piggyBac or piggyBac-like transposon comprises SEQ ID NO: 14595 and SEQ ID NO: 14596 and the piggyBac or piggyBac-like transposase comprises SEQ ID NO: 14504 or SEQ ID NO: 14505. In certain embodiments, the piggyBac or piggyBac-like transposon comprises SEQ ID NO: 14597 and SEQ ID NO: 14596 and the piggyBac or piggyBac-like transposase comprises SEQ ID NO: 14504 or SEQ ID NO: 14505. In certain embodiments, the piggyBac or piggyBac-like transposon comprises SEQ ID NO: 14595 and SEQ ID NO: 14578 and the piggyBac or piggyBac-like transposase comprises SEQ ID NO: 14504 or SEQ ID NO: 14505. In certain embodiments, the piggyBac or piggyBac-like transposon comprises SEQ ID NO: 14602 and SEQ ID NO: 14600 and the piggyBac or piggyBac-like transposase comprises SEQ ID NO: 14504 or SEQ ID NO: 14505.

[0257] In certain embodiments, the piggyBac or piggyBac-like transposon comprises a 5’ end comprising 1, 2, 3, 4, 5, 6, or 7 sequences selected from ATGAGGCAGGGTAT (SEQ ID NO: 14614), ATACCCTGCCTCAT (SEQ ID NO: 14615), GGCAGGGTAT (SEQ ID NO: 14616), ATACCCTGCC (SEQ ID NO: 14617), TAAAATTTTA (SEQ ID NO: 14618), ATTTTATAAAAT (SEQ ID NO: 14619), TCATACCCTG (SEQ ID NO: 14620) and TAAATAATAATAA (SEQ ID NO: 14621). In certain embodiments, the piggyBac or piggyBac-like transposon comprises a 3’ end comprising 1, 2 or 3 sequences selected from SEQ ID NO: 14617, SEQ ID NO: 14620 and SEQ ID NO: 14621.

[0258] In certain embodiments of the methods of the disclosure, the transposase enzyme is a piggyBac or piggyBac-like transposase enzyme. In certain embodiments, the piggyBac or piggyBac-like transposase enzyme is isolated or derived from Xenopus tropicalis. The piggyBac or piggyBac-like transposase enzyme may comprise or consist of an amino acid sequence at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to: 1 MAKRFYSAEE AAAHCMASSS EEFSGSDSEY VPPASESDSS TEESWCSSST VSALEEPMEV 61 DEDVDDLEDQ EAGDRADAAA GGEPAWGPPC NFPPEIPPFT TVPGVKVDTS NFEPINFFQL 121 FMTEAILQDM VLYTNVYAEQ YLTQNPLPRY ARAHAWHPTD IAEMKRFVGL TLAMGLIKAN 181 SLESYWDTTT VLSIPVFSAT MSRNRYQLLL RFLHFNNNAT AVPPDQPGHD RLHKLRPLID 241 SLSERFAAVY TPCQNICIDE SLLLFKGRLQ FRQYIPSKRA RYGIKFYKLC ESSSGYTSYF 301 LIYEGKDSKL DPPGCPPDLT VSGKIVWELI SPLLGQGFHL YVDNFYSSIP LFTALYCLDT 361 PACGTINRNR KGLPRALLDK KLNRGETYAL RKNELLAIKF FDKKNVFMLT SIHDESVIRE 421 QRVGRPPKNK PLCSKEYSKY MGGVDRTDQL QHYYNATRKT RAWYKKVGIY LIQMALRNSY 481 IVYKAAVPGP KLSYYKYQLQ ILPALLFGGV EEQTVPEMPP SDNVARLIGK HFIDTLPPTP 2023208176 27 Jul 2023 541 GKQRPQKGCK VCRKRGIRRD TRYYCPKCPR NPGLCFKPCF EIYHTQLHY (SEQ ID NO: 14517) .

[0259] In some embodiments, the piggyBac or piggyBac-like transposase is a hyperactive variant of SEQ ID NO: 14517. In certain embodiments, the piggyBac or piggyBac-like transposase is an integration defective variant of SEQ ID NO: 14517. The piggyBac or piggyBac-like transposase enzyme may comprise or consist of an amino acid sequence at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to: 1 MAKRFYSAEE AAAHCMAPSS EEFSGSDSEY VPPASESDSS TEESWCSSST VSALEEPMEV 61 DEDVDDLEDQ EAGDRADAAA GGEPAWGPPC NFPPEIPPFT TVPGVKVDTS NFEPINFFQL 121 FMTEAILQDM VLYTNVYAEQ YLTQNPLPRY ARAHAWHPTD IAEMKRFVGL TLAMGLIKAN 181 SLESYWNTTT VLSIPVFSAT MSRNRYQLLL RFLHFNNNAT AVPPDQPDHD RLHKLRPLID 241 SLSERFAAVY TPCQNICIDE SLLLFKGRLR FRQYIPSKRA RYGIKFYKLC ESSSGYTSYF 301 LIYEGKDSKL DPPGCPPDLT VSGKIVWELI SPLLGQGFHL YVDNFYSSIP LFTALYCLDT 361 PACGTINRTR KGLPRALLDK KLNRGETYAL RKNELLAIKF FDKKNVFMLT SIHDESVIRE 421 QRVGRPPKNK PLCSKEYSKY MGGVDRTDQL QHYYNATRKT SAWYKKVGIY LIQMALRNSY 481 IVYKAAVPGP KLSYYKYQLQ ILPALLFGGV EEQTVPEMLP SDNVARLIGK HFIDTLPPTP 541 GKQRPQKGCK VCRKRGIRRD TRYYCPKCPR NPGLCFKPCF EIYHTQLHY (SEQ ID NO: 14518) .

[0260] In certain embodiments, the piggyBac or piggyBac-like transposase is isolated or derived from Xenopus tropicalis. In certain embodiments, the piggyBac or piggyBac-like transposase is a hyperactive piggyBac or piggyBac-like transposase. In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises a sequence at least 90% identical to: 1 MAKRFYSAEE AAAHCSASSS EEFSGSDSEY VPPASESDSS TEESWCSSST VSALEEPMEV 61 DEDVDDLEDQ EAGDRADAAA GGEPAWGPPC NFPPEIPPFT TVPGVKVDTS NFEPINFFQL 121 FMTEAILQDM VLYTNVYAEQ YLTQNPLTRG ARAHAWHPTD IAEMKRFVGL TLAMGLIKAN 181 SIESYWDTTT VLSIPVFGAT MSRNRYQLLL RFLHFNNNAT AVPPDQPGHD RLHKLRPLID 241 SLSERFANVY TPCQNICIDE SLMLFKGRLQ FRQYIPSKRA RYGIKFYKLC ESSTGYTSYF 301 LIYEGKDSKL DPPGCPPDLT VSGKIVWELI SPLLGQGFHL YVDNFYSSIP LFTALYCLNT 361 PACGTINRNR KGLPRALLDK KLNRGETYAL RKNELLAIKF FDKKNVFMLT SIHDESVIRE 421 QRVGRPPKNK PLCSKEYSKY MGGVDRTDQL QHYYNATRKT RHWYKKVGIY LIQMALRNSY 481 IVYKAAVPGP KLSYYKYQLQ ILPALLFGGV EEQTVPEMPD SDNVARLIGK HFIDTLPPTP 541 GKQRPQKGCK VCRKRGIRRD TRYYCPKCPR NPGLCRKPCF EIYHTQLHY (SEQ ID NO: 14572) .

[0261] In certain embodiments, piggyBac or piggyBac-like transposase is a hyperactive piggyBac or piggyBac-like transposase. A hyperactive piggyBac or piggyBac-like transposase is a transposase that is more active than the naturally occurring variant from which it is derived. In certain embodiments, a hyperactive piggyBac or piggyBac-like transposase is more active than the transposase of SEQ ID NO: 14517. In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises a sequence of: 1 MAKRFYSAEE AAAHCSASSS EEFSGSDSEY VPPASESDSS TEESWCSSST VSALEEPMEV 2023208176 27 Jul 2023 61 DEDVDDLEDQ EAGDRADAAA GGEPAWGPPC NFPPEIPPFT TVPGVKVDTS NFEPINFFQL 121 FMTEAILQDM VLYTNVYAEQ YLTQNPLTRG ARAHAWHPTD IAEMKRFVGL TLAMGLIKAN 181 SIESYWDTTT VLSIPVFGAT MSRNRYQLLL RFLHFNNNAT AVPPDQPGHD RLHKLRPLID 241 SLSERFANVY TPCQNICIDE SLMLFKGRLQ FRQYIPSKRA RYGIKFYKLC ESSTGYTSYF 301 LIYEGKDSKL DPPGCPPDLT VSGKIVWELI SPLLGQGFHL YVDNFYSSIP LFTALYCLNT 361 PACGTINRNR KGLPRALLDK KLNRGETYAL RKNELLAIKF FDKKNVFMLT SIHDESVIRE 421 QRVGRPPKNK PLCSKEYSKY MGGVDRTDQL QHYYNATRKT RHWYKKVGIY LIQMALRNSY 481 IVYKAAVPGP KLSYYKYQLQ ILPALLFGGV EEQTVPEMPD SDNVARLIGK HFIDTLPPTP 541 GKQRPQKGCK VCRKRGIRRD TRYYCPKCPR NPGLCRKPCF EIYHTQLHY(SEQIDNO: 14572).

[0262] In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises a sequence of: 1 MAKRFYSAEE AAAHCMASSS EEFSGSDSEY VPPASESDSS TEESWCSSST VSALEEPMEV 61 DEDVDDLEDQ EAGDRADAAA GGEPAWGPPC NFPPEIPPFT TVPGVKVDTS NFEPINFFQL 121 FMTEAILQDM VLYTNVYAEQ YLTQNPLTRY ARAHAWHPTD IAEMKRFVGL TLAMGLIKAN 181 SLESYWDTTT VLSIPVFSAT MSRNRYQLLL RFLHFNNNAT AVPPDQPGHD RLHKLRPLID 241 SLSERFAAVY TPCQNICIDE SLLLFKGRLQ FRQYIPSKRA RYGIKFYKLC ESSSGYTSYF 301 LIYEGKDSKL DPPGCPPDLT VSGKIVWELI SPLLGQGFHL YVDNFYSSIP LFTALYCLNT 361 PACGTINRNR KGLPRALLDK KLNRGETYAL RKNELLAIKF FDKKNVFMLT SIHDESVIRE 421 QRVGRPPKNK PLCSKEYSKY MGGVDRTDQL QHYYNATRKT RHWYKKVGIY LIQMALRNSY 481 IVYKAAVPGP KLSYYKYQLQ ILPALLFGGV EEQTVPEMPP SDNVARLIGK HFIDTLPPTP 541 GKQRPQKGCK VCRKRGIRRD TRYYCPKCPR NPGLCRKPCF EIYHTQLHY (SEQ ID NO: 14624) .

[0263] In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises a sequence of: 1 MAKRFYSAEE AAAHCMASSS EEFSGSDSEY VPPASESDSS TEESWCSSST VSALEEPMEV 61 DEDVDDLEDQ EAGDRADAAA GGEPAWGPPC NFPPEIPPFT TVPGVKVDTS NFEPINFFQL 121 FMTEAILQDM VLYTNVYAEQ YLTQNPLPRY ARAHAWHPTD IAEMKRFVGL TLAMGLIKAN 181 SLESYWDTTT VLKIPVFSAT MSRNRYQLLL RFLHFNNNAT AVPPDQPGHD RLHKLRPLID 241 SLSERFAAVY TPCQNICIDE SLLLFKGRLQ FRQYIPSKRA RYGIKFYKLC ESSSGYTSYF 301 LIYEGKDSKL DPPGCPPDLT VSGKIVWELI SPLLGQGFHL YVDNFYSSIP LFTALYCLNT 361 PACGTINRNR KGLPRALLDK KLNRGETYAL RKNELLAIKF FDKKNVFMLT SIHDESVIRE 421 QRVGRPPKNK PLCSKEYSKY MGGVDRTDQL QHYYNATRKT RHWYKKVGIY LIQMALRNSY 481 IVYKAAVPGP KLSYYKYQLQ ILPALLFGGV EEQTVPEMPP SDNVARLIGK HFIDTLPPTP 541 GKQRPQKGCK VCRKRGIRRD TRYYCPKCPR NPGLCFKPCF EIYHTQLHY (SEQ ID NO: 14625) .

[0264] In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises a sequence of: 1 MAKRFYSAEE AAAHCMASSS EQTSGSDSEY VPPASESDSS TEESWCSSST VSALEEPMEV 61 DEDVDDLEDQ EAGDRADAAA GGEPAWGPPC NFPPEIPPFT TVPGVKVDTS NFEPINFFQL 121 FMTEAILQDM VLYTNVYAEQ YLTQNPLTRY ARAHAWHPTD IAEMKRFVGL TLAMGLIKAN 181 SIESYWDTTT VLSIPVFGAT MSRNRYQLLL RFLHFNNNAT AVPPDQPGHD RLHKLRPLID 241 SLSERFANVY TPCQNICIDE SLLLFKGRLQ FRQYIPSKRA RYGIKFYKLC ESSSGYTSYF 301 LIYEGKDSKL DPPGCPPDLT VSGKIVWELI SPLLGQGFHL YVDNFYSSIP LFTALYCLNT 361 PACGTINRNR KGLPRALLDK KLNRGETYAL RKNELLAIKF FDKKNVFMLT SIHDESVIRE 421 QRVGRKPKNK PLCSKEYSKY MGGVDRTDQL QHYYNATRKT RHWYKKVGIY LIQMALRNSY 481 IVYKAAVPGP KLSYYKYQLQ ILPALLFGGV EEQTVPEMPP SDNVARLIGK HFIDTLPPTP 541 GKQRPQKGCK VCRKRGIRRD TRYYCPKCPR NPGLCRKPCF EIYHTQLHY (SEQ ID NO: 14627) . 2023208176 27 Jul 2023

[0265] In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises a sequence of: 1 MAKRFYSAEE AAAHCSASSS EEFSGSDSEY VPPASESDSS TEESWCSSST VSALEEPMEV 61 DEDVDDLEDQ EAGDRADAAA GGEPAWGPPC NFPPEIPPFT TVPGVKVDTS NFEPINFFQL 121 FMTEAILQDM VLYTNVYAEQ YLTQNPLTRG ARAHAWHPTD IAEMKRFVGL TLAMGLIKAN 181 SLESYWDTTT VLSIPVFGAT MSRNRYQLLL RFLHFNNNAT AVPPDQPGHD RLHKLRPLID 241 SLSERFANVY TPCQNICIDE SLMLFKGRLQ FRQYIPSKRA RYGIKFYKLC ESSTGYTSYF 301 LIYEGKDSKL DPPGCPPDLT VSGKIVWELI SPLLGQGFHL YVDNFYSSIP LFTALYCLNT 361 PACGTINRNR KGLPRALLDK KLNRGETYAL RKNELLAIKF FDKKNVFMLT SIHDESVIRE 421 QRVGRPPKNK PLCSKEYSKY MGGVDRTDQL QHYYNATRKT RHWYKKVGIY LIQMALRNSY 481 IVYKAAVPGP KLSYYKYQLQ ILPALLFGGV EEQTVPEMPP SDNVARLIGK HFIDTLPPTP 541 GKQRPQKGCK VCRKRGIRRD TRYYCPKCPR NPGLCRKPCF EIYHTQLHY (SEQ ID NO: 14628) .

[0266] In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises a sequence of: 1 MAKRFYSAEE AAAHCMASSS EEFSGSDSEY VPPASESDSS TEESWCSSST VSALEEPMEV 61 DEDVDDLEDQ EAGDRADAAA GGEPAWGPPC NFPPEIPPFT TVPGVKVDTS NFEPINFFQL 121 FMTEAILQDM VLYTNVYAEQ YLTQNPLTRY ARAHAWHPTD IAEMKRFVGL TLAMGLIKAN 181 SLESYWDTTT VLSIPVFGAT MSRNRYQLLL RFLHFNNNAT AVPPDQPGHD RLHKLRPLID 241 SLSERFANVY TPCQNICIDE SLLLFKGRLQ FRQYIPSKRA RYGIKFYKLC ESSSGYTSYF 301 LIYEGKDSKL DPPGCPPDLT VSGKIVWELI SPLLGQGFHL YVDNFYSSIP LFTALYCLNT 361 PACGTINRNR KGLPRALLDK KLNRGETYAL RKNELLAIKF FDKKNVFMLT SIHDESVIRE 421 QRVGRPPKNK PLCSKEYSKY MGGVDRTDQL QHYYNATRKT RHWYKKVGIY LIQMALRNSY 481 IVYKAAVPGP KLSYYKYQLQ ILPALLFGGV EEQTVPEMPP SDNVARLIGK HFIDTLPPTP 541 GKQRPQKGCK VCRKRGIRRD TRYYCPKCPR NPGLCRKPCF EIYHTQLHY (SEQ ID NO: 16820) .

[0267] In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises an amino acid substitution at a position selected from amino acid 6, 7, 16, 19, 20, 21, 22, 23, 24, 26, 28, 31, 34, 67, 73, 76, 77, 88, 91, 141, 145, 146, 148, 150, 157, 162, 179, 182, 189, 192, 193, 196, 198, 200, 210, 212, 218, 248, 263, 270, 294, 297, 308, 310, 333, 336, 354, 357, 358, 359, 377, 423, 426, 428, 438, 447, 450, 462, 469, 472, 498, 502, 517, 520, 523, 533, 534, 576, 577, 582, 583 or 587 (relative to SEQ ID NO: 14517). In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises an amino acid substitution of Y6C, S7G, M16S, S19G, S20Q, S20G, S20D, E21D, E22Q, F23T, F23P, S24Y, S26V, S28Q, V31K, A34E, L67A, G73H, A76V, D77N, P88A, N91D, Y141Q, Y141A, N145E, N145V, P146T, P146V, P146K, P148T, P148H, Y150G, Y150S, Y150C, H157Y, A162C, A179K, L182I, L182V, T189G, L192H, S193N, S193K, V196I, S198G, T200W, L210H, F212N, N218E, A248N, L263M, Q270L, S294T, T297M, S308R, L310R, L333M, Q336M, A354H, C357V, L358F, D359N, L377I, V 423H, P426K, K428R, S438A, T447G, T447A, L450V, A462H, A462Q, I469V, I472L, Q498M, L502V, E5171, P520D, P520G, N523S, I533E, D534A, 2023208176 27 Jul 2023 F576R, F576E, K577I, I582R, Y583F, L587Y or L587W, or any combination thereof including at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or all of these mutations (relative to SEQ ID NO: 14517).

[0268] In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises one or more substitutions of an amino acid that is not wild type, wherein the one or more substitutions a for wild type amino acid comprises a substitution of A2X, K3X, R4X, F5X, Y6X, S7X, A11X, A13X, C15X, M16X, A17X, S18X, S19X, S20X, E21X, E22X, F23X, S24X, G25X, 26X, D27X, S28X, E29X, E42X, E43X, S44X, C46X, S47X, S48X, S49X, T50X, V51X, S52X, A53X, L54X, E55X, E56X, P57X, M58X, E59X, E62X, D63X, V64X, D65X, D66X, L67X, E68X, D69X, Q70X, E71X, A72X, G73X, D74X, R75X, A76X, D77X, A78X, A79X, A80X, G81X, G82X, E83X, P84X, A85X, W86X, G87X, P88X, P89X, C90X, N91X, F92X, P93X, E95X, I96X, P97X, P98X, F99X, T100X, T101X, P103X, G104X, V105X, K106X, V107X, D108X, T109X, N11IX, Pl 14X, Il 15X, N116X, Fl 17X, Fl 18X, QI 19X, M122X, T123X, E124X, A125X, I126X, L127X, Q128X, D129X, M130X, L132X, Y133X, V126X, Y127X, A138X, E139X, Q140X, Y141X, L142X, Q144X, N145X, P146X, L147X, P148X, Y150X, A151X, A155X, H157X, P158X , I161X, A162X, V168X, T171X, L172X, A173X, M174X, I177X, A179X, L182X, D187X, T188X, T189X, T190X, L192X, S193X, I194X, P195X, V196X, S198X, A199X, T200X, S202X, L208X, L209X, L210X, R211X, F212X, F215X, N217X, N218X, A219X, T220X, A221X, V222X, P224X, D225X, Q226X, P227X, H229X, R231X, H233X, L235X, P237X, I239X, D240X, L242X, S243X, E244X, R244X, F246X, A247X, A248X, V249X, Y250X, T251X, P252X, C253X, Q254X, I256X, C257X, I258X, D259X, E260X, S261X, L262X, L263X, L264X, F265X, K266X, G267X, R268X, L269X, Q270X, F271X, R272X, Q273X, Y274X, I275X, P276X, S277X, K278X, R279X, A280X, R281X, Y282X, G283X, I284X, K285X, F286X, Y287X, K288X, L289X, C290X, E291X, S292X, S293XS294X, G295X, Y296X, T297X, S298X, Y299X, F300X, E304X, L310X, P313X, G314X, P316X, P317X, D318X, L319X, T320X, V321X, K324X, E328X, I330X, S331X, P332X, L333X, L334X, G335X, Q336X, F338X, L340X, D343X, N344X, F345X, Y346X, S347X, L351X, F352X, A354X, L355X, Y356X, C357X, L358X, D359X, T360X, R422X, Y423X, G424X, P426X, K428X, N429X, K430X, P431X, L432X, S434X, K435X, E436X, S438X, K439X, Y440X, G443X, R446X, T447X, L450X, Q451X, N455X, T460X, R461X, A462X, K465X, V467X, G468X, I469X, Y470X, L471X, I472X, M474X, A475X, L476X, R477X, S479X, Y480X, V482XY483X, K484X, A485X, A486X, V487X, 2023208176 27 Jul 2023 P488X, P490X, K491X, S493X, Y494X, Y495X, K496X, Y497T, Q498X, L499X, Q500X, I501X, L502X, P503X, A504X, L505X, L506X, F507X, G508X, G509X, V510X, E511X, E512X, Q513X, T514X, V515X, E517X, M518X, P519X, P520X, S521X, D522X, N523X, V524X, A525X, L527X, I528X, K530X, H531X, F532X, I533X, D534X, T535X, L536X, T539X, P540X,Q546X, K550X, R553X, K554X, R555X, G556X, I557X, R558X, R559X, D560X, T561X, Y564X, P566X, K567X, P569X, R570X, N571X, L574X, C575X, F576X, K577X, P578X, F580X, E581X, I582X, Y583X, T585X, Q586X, L587X, H588X or Y589X (relative to SEQ ID NO: 14517). A list of hyperactive amino acid substitutions can be found in US patent No. 10,041,077, the contents of which are incorporated by reference in their entirety.

[0269] In certain embodiments, the piggyBac or piggyBac-like transposase is integration deficient. In certain embodiments, an integration deficient piggyBac or piggyBac-like transposase is a transposase that can excise its corresponding transposon, but that integrates the excised transposon at a lower frequency than a corresponding naturally occurring transposase. In certain embodiments, the piggyBac or piggyBac-like transposase is an integration deficient variant of SEQ ID NO: 14517. In certain embodiments, the integration deficient piggyBac or piggyBac-like transposase is deficient relative to SEQ ID NO: 14517.

[0270] In certain embodiments, the piggyBac or piggyBac-like transposase is active for excision but deficient in integration. In certain embodiments, the integration deficient piggyBac or piggyBac-like transposase comprises a sequence that is at least 90% identical to a sequence of 1 MAKRFYSAEE AAAHCMASSS EEFSGSDSEY VPPASESDSS TEESWCSSST VSALEEPMEV 61 DEDVDDLEDQ EAGDRVDAAA GGEPAWGPPC NFPPEIPPFT TVPGVKVDTS NFEPINFFQL 121 FMTEAILQDM VLYTNVYAEQ YLTQNPLPRY ARAHAWHPTD IAEMKRFVGL TLAMGLIKAN 181 SLESYWDTTT VLSIPVFSAT MSRNRYQLLL KFLHFNNEAT AVPPDQPGHD RLHKLRPLID 241 SLSERFAAVY TPCQNICIDE SLLLFKGRLQ FRQYIPSKRA RYGIKFYKLC ESSSGYTSYF 301 LIYEGKDSKL DPPGCPPDLT VSGKIVWELI SPLLGQGFHL YVDNFYSSIP LFTALYCLDT 361 PACGTINRNR KGLPRALLDK KLNRGETYAL RKNELLAIKF FDKKNVFMLT SIHDESVIRE 421 QRVGRPPKNK PLCSKEYSKY MGGVDRTDQL QHYYNATRKT RAWYKKVGIY LIQMALRNSY 481 IVYKAAVPGP KLSYYKYQLQ ILPALLFGGV EEQTVPEMPP SDNVARLIGK HFIDTLPPTP 541 GKQRPQKGCK VCRKRGIRRD TRYYCPKCPR NPGLCFKPCF EIYHTQLHYG RR (SEQ ID NO: 14605) .

[0271] In certain embodiments, the integration deficient piggyBac or piggyBac-like transposase comprises a sequence that is at least 90% identical to a sequence of: 1 MAKRFYSAEE AAAHCMASSS EEFSGSDSEY VPPASESDSS TEESWCSSST VSALEEPMEV 61 DEDVDDLEDQ EAGDRADAAA GGEPAWGPPC NFPPEIPPFT TVPGVKVDTS NFEPINFFQL 121 FMTEAILQDM VLYTNVYAEQ YLTQVPLPRY ARAHAWHPTD IAEMKRFVGL TLAMGLIKAN 181 SLESYWDTTT VLNIPVFSAT MSRNRYQLLL RFLEFNNEAT AVPPDQPGHD RLHKLRPLID 241 SLSERFAAVY TPCQNICIDE SLLLFKGRLQ FRQYIPSKRA RYGIKFYKLC ESSSGYTSYF 2023208176 27 Jul 2023 301 LIYEGKDSKL DPPGCPPDLT VSGKIVWELI SPLLGQGFHL YVDNFYSSIP LFTALYCLDT 361 PACGTINRNR KGLPRALLDK KLNRGETYAL RKNELLAIKF FDKKNVFMLT SIHDESVIRE 421 QRVGRPPKNK PLCSKEYSKY MGGVDRTDQL QHYYNATRKT RAWYKKVGIY LIQMALRNSY 481 IVYKAAVPGP KLSYYKYQLQ ILPALLFGGV EEQTVPEMPP SDNVARLIGK HFIDTLPPTP 541 GKQRPQKGCK VCRKRGIRRD TRYYCPKCPR NPGLCFKPCF EIYHTQLHY (SEQ ID NO: 14604) .

[0272] In certain embodiments, the integration deficient piggyBac or piggyBac-like transposase comprises a sequence that is at least 90% identical to a sequence of: 1 MAKRFYSAEE AAAHCMASSS EEFSGSDSEY VPPASESDSS TEESWCSSST VSALEEPMEV 61 DEDVDDLEDQ EAGDRADAAA GGEPAWGPPC NFPPEIPPFT TVPGVKVDTS NFEPINFFQL 121 FMTEAILQDM VLYTNVYAEQ YLTQNVLPRY ARAHAWHPTD IAEMKRFVGL TLAMGLIKAN 181 SLESYWDTTT VLSIPVFSAT MSRNRYQLLL RFLHFNNDAT AVPPDQPGHD RLHKLRPLID 241 SLTERFAAVY TPCQNICIDE SLLLFKGRLQ FRQYIPSKRA RYGIKFYKLC ESSSGYTSYF 301 LIYEGKDSKL DPPGCPPDLT VSGKIVWELI SPLLGQGFHL YVDNFYSSIP LFTALYCLDT 361 PACGTINRNR KGLPRALLDK KLNRGETYAL RKNELLAIKF FDKKNVFMLT SIHDESVIRE 421 QRVGRPPKNK PLCSKEYSKY MGGVDRTDQL QHYYNATRKT RAWYKKVGIY LIQMALRNSY 481 IVYKAAVPGP KLSYYKYQLQ ILPALLFGGV EEQTVPEMPP SDNVARLIGK HFIDTLPPTP 541 GKQRPQKGCK VCRKRGIRRD TRYYCPKCPR NPGLCFKPCF EIYHTQLHYG RR (SEQ ID NO: 14611) .

[0273] In certain embodiments, the integration deficient piggyBac or piggyBac-like transposase comprises SEQ ID NO: 14611. In certain embodiments, the integration deficient piggyBac or piggyBac-like transposase comprises a sequence that is at least 90% identical to a sequence of: 1 MAKRFYSAEE AAAHCMASSS EEFSGSDSEY VPPASESDSS TEESWCSSST VSALEEPME...

Claims

1. A chimeric antigen receptor (CAR) comprising:(a) an ectodomain comprising an antigen recognition region, wherein the antigen recognition region comprises at least one VH;(b) a transmembrane domain, and(c) an endodomain comprising at least one costimulatory domain.

2. The CAR of claim 1, wherein the VH comprises or consists of a recombinant or chimeric sequence.

3. The CAR of claim 1, wherein the VH comprises or consists of a human or humanized sequence.

4. The CAR of claim 3, wherein the VH is not naturally occurring.

5. The CAR of any one of claims 3-4, wherein the VH is not a naturally occurringantibody or a fragment of thereof.

6. The CAR of any one of claims claim 1-5, wherein the ectodomain of (a) further comprises a signal peptide.

7. The CAR of any one of claims claim 1 or 6, wherein the ectodomain of (a) further comprises a hinge between the antigen recognition region and the transmembrane domain.

8. The CAR of claim 6 or 7, wherein the signal peptide comprises a sequence encoding a human CD2, CD36, CD3e, CD3y, CD3^ CD4, CD8a, CD19, CD28, 4-1BB or GM-CSFR signal peptide.2023208176 27 Jul 20239. The CAR of claim 6 or 7, wherein the signal peptide comprises a sequence encoding a human CD8a signal peptide.

10. The CAR of claim 9, wherein the signal peptide comprises an amino acid sequence comprising MALPVTALLLPLALLLHAARP (SEQ ID NO: 18012).

11. The CAR of claim 9 or 10, wherein the signal peptide is encoded by a nucleic acidsequence comprising atggcactgccagtcaccgccctgctgctgcctctggctctgctgctgcacgcagctagacca (SEQ ID NO: 18013).

12. The CAR of any one of the preceding claims, wherein the transmembrane domain comprises a sequence encoding a human CD2, CD36, CD3e, CD3y, CD3^, CD4, CD8a, CD19, CD28, 4-1BB or GM-CSFR transmembrane domain.

13. The CAR of any one of the preceding claims, wherein the transmembrane domain comprises a sequence encoding a human CD8a transmembrane domain.

14. The CAR of claim 13, wherein the transmembrane domain comprises an amino acid sequence comprising IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 18014).

15. The CAR of claim 13 or 14, wherein the transmembrane domain is encoded by a nucleic acid sequence comprising atctacatttgggcaccactggccgggacctgtggagtgctgctgctgagcctggtcatcacactgtactgc (SEQ ID NO: 18015).

16. The CAR of any one of the preceding claims, wherein the endodomain comprises a human CD3^ endodomain.

17. The CAR of any one of the preceding claims, wherein the at least one costimulatory domain comprises a human 4-1BB, CD28, CD40, ICOS, MyD88, OX-40 intracellular segment, or any combination thereof.2023208176 27 Jul 202318. The CAR of any one of the preceding claims, wherein the at least one costimulatory domain comprises a human CD28 and / or a4-lBB costimulatory domain.

19. The CAR of claim 17 or 18, wherein the CD3zeta costimulatory domain comprises an amino acid sequence comprising RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP PR (SEQ ID NO: 18016).

20. The CAR of claim 19, wherein the CD3zeta costimulatory domain is encoded by a nucleic acid sequence comprising cgcgtgaagtttagtcgatcagcagatgccccagcttacaaacagggacagaaccagctgtataacgagctgaatctgggccgccga gaggaatatgacgtgctggataagcggagaggacgcgaccccgaaatgggaggcaagcccaggcgcaaaaaccctcaggaagg cctgtataacgagctgcagaaggacaaaatggcagaagcctattctgagatcggcatgaagggggagcgacggagaggcaaagg gcacgatgggctgtaccagggactgagcaccgccacaaaggacacctatgatgctctgcatatgcaggcactgcctccaagg (SEQ ID NO: 18017).

21. The CAR of claim 17 or 18, wherein the 4-1BB costimulatory domain comprises an amino acid sequence comprisingKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 18018).

22. The CAR of claim 21, wherein the 4-1BB costimulatory domain is encoded by a nucleic acid sequence comprisingaagagaggcaggaagaaactgctgtatattttcaaacagcccttcatgcgccccgtgcagactacccaggaggaagacgggtgctcc tgtcgattccctgaggaagaggaaggcgggtgtgagctg (SEQ ID NO: 18019).

23. The CAR of any one of claims 18-22, wherein the 4-1BB costimulatory domain is located between the transmembrane domain and the CD28 costimulatory domain.2023208176 27 Jul 202324. The CAR of any one of claims 6-23, wherein the hinge comprises a sequence derived from a human CD8a, IgG4, and / or CD4 sequence.

25. The CAR of any one of claims 6-23, wherein the hinge comprises a sequence derived from a human CD8a sequence.

26. The CAR of claim 24 or 25, wherein the hinge comprises an amino acid sequence comprising TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 18020).

27. The CAR of claim 26, wherein the hinge is encoded by a nucleic acid sequence comprisingactaccacaccagcacctagaccaccaactccagctccaaccatcgcgagtcagcccctgagtctgagacctgaggcctgcaggcc agctgcaggaggagctgtgcacaccaggggcctggacttcgcctgcgac (SEQ ID NO: 18021).

28. The CAR of any one of the preceding claims, wherein the at least one VC AR is capable of specifically binding an antigen.

29. The CAR of claim 28, wherein the VC AR binds an antigen with at least one affinity selected from a Kd of less than or equal to 10 9M. less than or equal to 10 l0M. less than or equal to 10 1 'M. less than or equal to 10 l2M. less than or equal to 10 l3M. less than or equal to 10 l4M. and less than or equal to 10 l5M.

30. The CAR of claim 29, wherein the Kd is determined by surface plasmon resonance.

31. A composition comprising the CAR of any one of the preceding claims and at least one pharmaceutically acceptable carrier.

32. A transposon comprising the CAR of any one of the preceding claims.2023208176 27 Jul 202333. The transposon of claim 32, wherein the transposon further comprises a selection gene.

34. The transposon of claim 33, wherein the selection gene encodes a gene product essential for cell viability and survival.

35. The transposon of claim 33, wherein the selection gene encodes a gene product essential for cell viability and survival when challenged by selective cell culture conditions.

36. The transposon of claim 35, wherein the selective cell culture conditions comprise a compound harmful to cell viability or survival and wherein the gene product confers resistance to the compound.

37. The transposon of claim 33, wherein the selection gene comprises neo, DHFR (Dihydrofolate Reductase),TYMS (Thymidylate Synthetase), MGMT (O(6)-methylguanine-DNA methyltransferase), multidrug resistance gene (MDR1), ALDH1 (Aldehyde dehydrogenase 1 family, member Al), FRANCF, RAD51C (RAD51 Paralog C), GCS (glucosylceramide synthase), NKX2.2 (NK2 Homeobox 2) or any combination thereof.

38. The transposon of any one of claims 32 to 37, wherein the transposon comprises an inducible caspase polypeptide comprising(a) a ligand binding region,(b) a linker, and(c) a truncated caspase 9 polypeptide, wherein the inducible caspase polypeptide does not comprise a non-human sequence.

39. The transposon of claim 38, wherein the non-human sequence is a restriction site.

40. The transposon of claim 37 or 39, wherein the ligand binding region induciblecaspase polypeptide comprises a FK506 binding protein 12 (FKBP12) polypeptide.2023208176 27 Jul 202341. The transposon of claim 40, wherein the amino acid sequence of the FK506 binding protein 12 (FKBP12) polypeptide comprises a modification at position 36 of the sequence.

42. The transposon of claim 41, wherein the modification is a substitution of valine (V) for phenylalanine (F) at position 36 (F36V).

43. The transposon of any one of claims 40-42, wherein the FKBP12 polypeptide is encoded by an amino acid sequence comprisingGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVI RGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE (SEQ ID NO: 18022).

44. The transposon of claim 43, wherein the FKBP12 polypeptide is encoded by a nucleicacid sequence comprisingGGGGTCCAGGTCGAGACTATTTCACCAGGGGATGGGCGAACATTTCCAAAAAGG GGCCAGACTTGCGTCGTGCATTACACCGGGATGCTGGAGGACGGGAAGAAAGTG GACAGCTCCAGGGATCGCAACAAGCCCTTCAAGTTCATGCTGGGAAAGCAGGAA GTGATCCGAGGATGGGAGGAAGGCGTGGCACAGATGTCAGTCGGCCAGCGGGCC AAACTGACCATTAGCCCTGACTACGCTTATGGAGCAACAGGCCACCCAGGGATC ATTCCCCCTCATGCCACCCTGGTCTTCGAT GTGGAACTGCTGAAGCTGGAG (SEQ ID NO: 18023).

45. The transposon of any one of claims 38-44, wherein the linker region of the inducible proapoptotic polypeptide is encoded by an amino acid comprising GGGGS (SEQ ID NO: 18024).

46. The transposon of claim 45, wherein the linker region of the inducible proapoptoticpolypeptide is encoded by a nucleic acid sequence comprising GGAGGAGGAGGATCC (SEQ ID NO: 18025).2023208176 27 Jul 202347. The transposon of any one of claims 38-46, wherein the truncated caspase 9 polypeptide of the inducible proapoptotic polypeptide is encoded by an amino acid sequence that does not comprise an arginine (R) at position 87 of the sequence.

48. The transposon of any one of claims 38-47, wherein the truncated caspase 9 polypeptide of the inducible proapoptotic polypeptide is encoded by an amino acid sequence that does not comprise an alanine (A) at position 282 the sequence.

49. The transposon of any one of claims 38-48, wherein the truncated caspase 9 polypeptide of the inducible proapoptotic polypeptide is encoded by an amino acid comprisingGFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRR RFSSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPG AVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDE SPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVE TLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS (SEQ ID NO: 18026).

50. The transposon of claim 49, wherein the truncated caspase 9 polypeptide of the inducible proapoptotic polypeptide is encoded by a nucleic acid sequence comprising TTTGGGGACGTGGGGGCCCTGGAGTCTCTGCGAGGAAATGCCGATCTGGCTTAC ATCCTGAGCATGGAACCCTGCGGCCACTGTCTGATCATTAACAATGTGAACTTCT GCAGAGAAAGCGGACTGCGAACACGGACTGGCTCCAATATTGACTGTGAGAAGC TGCGGAGAAGGTTCTCTAGTCTGCACTTTATGGTCGAAGTGAAAGGGGATCTGAC CGCCAAGAAAATGGTGCTGGCCCTGCTGGAGCTGGCTCAGCAGGACCATGGAGC TCTGGATTGCTGCGTGGTCGTGATCCTGTCCCACGGGTGCCAGGCTTCTCATCTG CAGTTCCCCGGAGCAGTGTACGGAACAGACGGCTGTCCTGTCAGCGTGGAGAAG ATCGTCAACATCTTCAACGGCACTTCTTGCCCTAGTCTGGGGGGAAAGCCAAAAC TGTTCTTTATCCAGGCCTGTGGCGGGGAACAGAAAGATCACGGCTTCGAGGTGG CCAGCACCAGCCCTGAGGACGAATCACCAGGGAGCAACCCTGAACCAGATGCAA CTCCATTCCAGGAGGGACTGAGGACCTTTGACCAGCTGGATGCTATCTCAAGCCT2023208176 27 Jul 2023GCCCACTCCTAGTGACATTTTCGTGTCTTACAGTACCTTCCCAGGCTTTGTCTCAT GGCGCGATCCCAAGTCAGGGAGCTGGTACGTGGAGACACTGGACGACATCTTTG AACAGTGGGCCCATTCAGAGGACCTGCAGAGCCTGCTGCTGCGAGTGGCAAACG CTGTCTCTGTGAAGGGCATCTACAAACAGATGCCCGGGTGCTTCAATTTTCTGAG AAAGAAACTGTTCTTTAAGACTTCC (SEQ ID NO: 18027).

51. The transposon of any one of claims 38-50, wherein of the inducible proapoptoticpolypeptide is encoded by an amino acid sequence comprisingGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVI RGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGGS GFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRR RFSSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPG AVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDE SPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVE TLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS (SEQ ID NO: 18028).

52. The transposon of claim 51, wherein of the inducible proapoptotic polypeptide isencoded by a nucleic acid sequence comprisingggggtccaggtcgagactatttcaccaggggatgggcgaacatttccaaaaaggggccagacttgcgtcgtgcattacaccgggatg ctggaggacgggaagaaagtggacagctccagggatcgcaacaagcccttcaagttcatgctgggaaagcaggaagtgatccgag gatgggaggaaggcgtggcacagatgtcagtcggccagcgggccaaactgaccattagccctgactacgcttatggagcaacagg ccacccagggatcattccccctcatgccaccctggtcttcgatgtggaactgctgaagctggagggaggaggaggatccggatttgg ggacgtgggggccctggagtctctgcgaggaaatgccgatctggcttacatcctgagcatggaaccctgcggccactgtctgatcatt aacaatgtgaacttctgcagagaaagcggactgcgaacacggactggctccaatattgactgtgagaagctgcggagaaggttctcta gtctgcactttatggtcgaagtgaaaggggatctgaccgccaagaaaatggtgctggccctgctggagctggctcagcaggaccatg gagctctggattgctgcgtggtcgtgatcctgtcccacgggtgccaggcttctcatctgcagttccccggagcagtgtacggaacaga cggctgtcctgtcagcgtggagaagatcgtcaacatcttcaacggcacttcttgccctagtctggggggaaagccaaaactgttctttat ccaggcctgtggcggggaacagaaagatcacggcttcgaggtggccagcaccagccctgaggacgaatcaccagggagcaaccc tgaaccagatgcaactccattccaggagggactgaggacctttgaccagctggatgctatctcaagcctgcccactcctagtgacatttt cgtgtcttacagtaccttcccaggctttgtctcatggcgcgatcccaagtcagggagctggtacgtggagacactggacgacatctttga2023208176 27 Jul 2023acagtgggcccattcagaggacctgcagagcctgctgctgcgagtggcaaacgctgtctctgtgaagggcatctacaaacagatgcc cgggtgcttcaattttctgagaaagaaactgttctttaagacttcc (SEQ ID NO: 18029).

53. The transposon of any one of claims 32 to 52, wherein the transposon comprises at least one self-cleaving peptide.

54. The transposon of any one of claims 33 to 52, wherein the transposon comprises at least one self-cleaving peptide and wherein a self-cleaving peptide is located between the CAR and the selection gene.

55. The transposon of any one of claims 38 to 54, wherein the transposon comprises at least one self-cleaving peptide and wherein a self-cleaving peptide is located between the CAR and the inducible proapoptotic polypeptide.

56. The transposon of any one of claims 38 to 55, wherein the transposon comprises at least two self-cleaving peptides and wherein a first self-cleaving peptide is located upstream of the inducible proapoptotic polypeptide and a second self-cleaving peptide is located downstream of the inducible proapoptotic polypeptide.

57. The transposon of any one of claims 32 to 56, wherein the transposon comprises at least one self-cleaving peptide and wherein a first self-cleaving peptide is located upstream of the CAR and a second self-cleaving peptide is located downstream of the CAR.

58. The transposon of any one of claims 53 to 57, wherein the at least one self-cleaving peptide comprises 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.

59. The transposon of claim 58, wherein the T2A peptide comprises an amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18030).2023208176 27 Jul 202360. The transposon of claim 58, wherein the GSG-T2A peptide comprises an amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 18031).

61. The transposon of claim 58, wherein the E2A peptide comprises an amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO: 18033).

62. The transposon of claim 58, wherein the GSG-E2A peptide comprises an amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 18034).

63. The transposon of claim 58 wherein the F2A peptide comprises an amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 18035).

64. The transposon of claim 58, wherein the GSG-F2A peptide comprises an amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 18036).

65. The transposon of claim 58, wherein the P2A peptide comprises an amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 18037).

66. The transposon of claim 58, wherein the GSG-P2A peptide comprises an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 18038).

67. The transposon of any one of claims 32-66, wherein the transposon is a piggyBac transposon.

68. A composition comprising the transposon of any one of claims 32 to 67.

69. The composition of claim 68, further comprising a plasmid comprising a sequence encoding a transposase enzyme.

70. The composition of claim 69, wherein the sequence encoding a transposase enzyme is an mRNA sequence.2023208176 27 Jul 202371. The composition of any one of claims 68 to 70, wherein the transposase is a piggyBac tiansposase.

72. The composition of claim 71, wherein the piggyBac transposase comprises an amino acid sequence comprising SEQ ID NO: 14487.

73. The composition of claim 71 or 72, 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: 14487.

74. The composition of claim 73, wherein the amino acid substitution at position 30 of SEQ ID NO: 14487 is a substitution of a valine (V) for an isoleucine (I) (BOV).

75. The composition of claim 73, wherein the amino acid substitution at position 165 of SEQ ID NO: 14487 is a substitution of a serine (S) for a glycine (G) (G165S).

76. The composition of claim 73, wherein the amino acid substitution at position 282 of SEQ ID NO: 14487 is a substitution of a valine (V) for a methionine (M) (M282V).

77. The composition of claim 73, wherein the amino acid substitution at position 538 of SEQ ID NO: 14487 is a substitution of a lysine (K) for an asparagine (N) (N538K).

78. The composition of any one of claims 71 to 77, wherein the transposase is a Super piggyBac (SPB) transposase.

79. The composition of claim 78, wherein the Super piggyBac (SPB) transposase comprises an amino acid sequence comprising SEQ ID NO: 14484.

80. A vector comprising the CAR of any one of claims 1-30.2023208176 27 Jul 202381.    The vector of claim 80, wherein the vector is a viral vector.

82. The vector of claim 81, wherein the viral vector comprises a sequence isolated orderived from a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus or any combination thereof.

83. The vector of claim 81 or 82, wherein the viral vector comprises a sequence isolated or derived from an adeno-associated virus.

84. The vector of any one of claims 81 to 83, wherein the viral vector is a recombinant vector.

85. The vector of claim 80, wherein the vector is a nanoparticle vector.

86. The vector of claim 85, wherein the nanoparticle vector comprises a nucleic acid, anamino acid, a polymers, a micelle, lipid, an organic molecule, an inorganic molecule or any combination thereof.

87. The vector of any one of claims 80 to 86, wherein the vector further comprises a selection gene.

88. The vector of claim 87, wherein the selection gene encodes a gene product essential for cell viability and survival.

89. The vector of claim 87, wherein the selection gene encodes a gene product essential for cell viability and survival when challenged by selective cell culture conditions.

90. The vector of claim 89, wherein the selective cell culture conditions comprise a compound harmful to cell viability or survival and wherein the gene product confers resistance to the compound.2023208176 27 Jul 202391. The vector of any one of claims 87 to 90, wherein the selection gene comprises neo, DHFR (Dihydrofolate Reductase),TYMS (Thymidylate Synthetase), MGMT (0(6)-methylguanine-DNA methyltransferase), multidrug resistance gene (MDR1), ALDH1 (Aldehyde dehydrogenase 1 family, member Al), FRANCF, RAD51C (RAD51 Paralog C), GCS (glucosylceramide synthase), NKX2.2 (NK2 Homeobox 2) or any combination thereof.

92. The vector of any one of claims 80 to 91, wherein the vector comprises an inducible caspase polypeptide comprising(a) a ligand binding region,(b) a linker, and(c) a truncated caspase 9 polypeptide, wherein the inducible caspase polypeptide does not comprise a non-human sequence.

93. The vector of claim 92, wherein the non-human sequence is a restriction site.

94. The vector of claim 92 or 93, wherein the ligand binding region inducible caspase polypeptide comprises a FK506 binding protein 12 (FKBP12) polypeptide.

95. The vector of claim 94, wherein the amino acid sequence of the FK506 binding protein 12 (FKBP12) polypeptide comprises a modification at position 36 of the sequence.

96. The vector of claim 95, wherein the modification is a substitution of valine (V) for phenylalanine (F) at position 36 (F36V).

97. The vector of any one of claims 94 to 96, wherein the FKBP12 polypeptide is encoded by an amino acid sequence comprisingGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVI RGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE (SEQ ID NO: 18022).2023208176 27 Jul 202398. The vector of claim 97, wherein the FKBP12 polypeptide is encoded by a nucleic acid sequence comprisingGGGGTCCAGGTCGAGACTATTTCACCAGGGGATGGGCGAACATTTCCAAAAAGG GGCCAGACTTGCGTCGTGCATTACACCGGGATGCTGGAGGACGGGAAGAAAGTG GACAGCTCCAGGGATCGCAACAAGCCCTTCAAGTTCATGCTGGGAAAGCAGGAA GTGATCCGAGGATGGGAGGAAGGCGTGGCACAGATGTCAGTCGGCCAGCGGGCC AAACTGACCATTAGCCCTGACTACGCTTATGGAGCAACAGGCCACCCAGGGATC ATTCCCCCTCATGCCACCCTGGTCTTCGAT GTGGAACTGCTGAAGCTGGAG (SEQ ID NO: 18023).

99. The vector of any one of claims 92 to 98, wherein the linker region of the inducible proapoptotic polypeptide is encoded by an amino acid comprising GGGGS (SEQ ID NO: 18024).

100. The vector of claim 99, wherein the linker region of the inducible proapoptotic polypeptide is encoded by a nucleic acid sequence comprising GGAGGAGGAGGATCC (SEQ ID NO: 18025).

101. The vector of any one of claims 92-100, wherein the truncated caspase 9 polypeptide of the inducible proapoptotic polypeptide is encoded by an amino acid sequence that does not comprise an arginine (R) at position 87 of the sequence.

102. The vector of any one of claims 92-101, wherein the truncated caspase 9 polypeptide of the inducible proapoptotic polypeptide is encoded by an amino acid sequence that does not comprise an alanine (A) at position 282 the sequence.

103. The vector of any one of claims 92-102, wherein the truncated caspase 9 polypeptide of the inducible proapoptotic polypeptide is encoded by an amino acid comprising GFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRR RFSSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPG AVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDE2023208176 27 Jul 2023SPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVE TLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS (SEQ ID NO: 18026).

104. The vector of claim 103, wherein the truncated caspase 9 polypeptide of the inducible proapoptotic polypeptide is encoded by a nucleic acid sequence comprising TTTGGGGACGTGGGGGCCCTGGAGTCTCTGCGAGGAAATGCCGATCTGGCTTAC ATCCTGAGCATGGAACCCTGCGGCCACTGTCTGATCATTAACAATGTGAACTTCT GCAGAGAAAGCGGACTGCGAACACGGACTGGCTCCAATATTGACTGTGAGAAGC TGCGGAGAAGGTTCTCTAGTCTGCACTTTATGGTCGAAGTGAAAGGGGATCTGAC CGCCAAGAAAATGGTGCTGGCCCTGCTGGAGCTGGCTCAGCAGGACCATGGAGC TCTGGATTGCTGCGTGGTCGTGATCCTGTCCCACGGGTGCCAGGCTTCTCATCTG CAGTTCCCCGGAGCAGTGTACGGAACAGACGGCTGTCCTGTCAGCGTGGAGAAG ATCGTCAACATCTTCAACGGCACTTCTTGCCCTAGTCTGGGGGGAAAGCCAAAAC TGTTCTTTATCCAGGCCTGTGGCGGGGAACAGAAAGATCACGGCTTCGAGGTGG CCAGCACCAGCCCTGAGGACGAATCACCAGGGAGCAACCCTGAACCAGATGCAA CTCCATTCCAGGAGGGACTGAGGACCTTTGACCAGCTGGATGCTATCTCAAGCCT GCCCACTCCTAGTGACATTTTCGTGTCTTACAGTACCTTCCCAGGCTTTGTCTCAT GGCGCGATCCCAAGTCAGGGAGCTGGTACGTGGAGACACTGGACGACATCTTTG AACAGTGGGCCCATTCAGAGGACCTGCAGAGCCTGCTGCTGCGAGTGGCAAACG CTGTCTCTGTGAAGGGCATCTACAAACAGATGCCCGGGTGCTTCAATTTTCTGAG AAAGAAACTGTTCTTTAAGACTTCC (SEQ ID NO: 18027).

105. The vector of any one of claims 92-104, wherein of the inducible proapoptotic polypeptide is encoded by an amino acid sequence comprisingGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVI RGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGGS GFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRR RFSSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPG AVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDE SPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVE2023208176 27 Jul 2023TLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS (SEQ ID NO: 18028).

106. The vector of claim 105, wherein of the inducible proapoptotic polypeptide is encoded by a nucleic acid sequence comprisingggggtccaggtcgagactatttcaccaggggatgggcgaacatttccaaaaaggggccagacttgcgtcgtgcattacaccgggatg ctggaggacgggaagaaagtggacagctccagggatcgcaacaagcccttcaagttcatgctgggaaagcaggaagtgatccgag gatgggaggaaggcgtggcacagatgtcagtcggccagcgggccaaactgaccattagccctgactacgcttatggagcaacagg ccacccagggatcattccccctcatgccaccctggtcttcgatgtggaactgctgaagctggagggaggaggaggatccggatttgg ggacgtgggggccctggagtctctgcgaggaaatgccgatctggcttacatcctgagcatggaaccctgcggccactgtctgatcatt aacaatgtgaacttctgcagagaaagcggactgcgaacacggactggctccaatattgactgtgagaagctgcggagaaggttctcta gtctgcactttatggtcgaagtgaaaggggatctgaccgccaagaaaatggtgctggccctgctggagctggctcagcaggaccatg gagctctggattgctgcgtggtcgtgatcctgtcccacgggtgccaggcttctcatctgcagttccccggagcagtgtacggaacaga cggctgtcctgtcagcgtggagaagatcgtcaacatcttcaacggcacttcttgccctagtctggggggaaagccaaaactgttctttat ccaggcctgtggcggggaacagaaagatcacggcttcgaggtggccagcaccagccctgaggacgaatcaccagggagcaaccc tgaaccagatgcaactccattccaggagggactgaggacctttgaccagctggatgctatctcaagcctgcccactcctagtgacatttt cgtgtcttacagtaccttcccaggctttgtctcatggcgcgatcccaagtcagggagctggtacgtggagacactggacgacatctttga acagtgggcccattcagaggacctgcagagcctgctgctgcgagtggcaaacgctgtctctgtgaagggcatctacaaacagatgcc cgggtgcttcaattttctgagaaagaaactgttctttaagacttcc (SEQ ID NO: 18029).

107. The vector of any one of claims 80 to 106, wherein the vector comprises at least one self-cleaving peptide.

108. The vector of any one of claims 80 to 106, wherein the vector comprises at least one self-cleaving peptide and wherein a self-cleaving peptide is located between the CAR and a selection gene.

109. The vector of any one of claims 92 to 108, wherein the transposon comprises at least one self-cleaving peptide and wherein a self-cleaving peptide is located between the CAR and the inducible proapoptotic polypeptide.2023208176 27 Jul 2023110. The vector of any one of claims 92 to 109, wherein the transposon comprises at least two self-cleaving peptides and wherein a first self-cleaving peptide is located upstream of the inducible proapoptotic polypeptide and a second self-cleaving peptide is located downstream of the inducible proapoptotic polypeptide.

111. The vector of any one of claims 80-110, wherein the vector comprises at least one self-cleaving peptide and wherein a first self-cleaving peptide is located upstream of the CAR and a second self-cleaving peptide is located downstream of the CAR.

112. The vector of any one of claims 107-111, wherein the at least one self-cleaving peptide comprises 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.

113. The vector of claim 112, wherein the T2A peptide comprises an amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18030).

114. The vector of claim 112, wherein the GSG-T2A peptide comprises an amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 18031).

115. The vector of claim 112, wherein the E2A peptide comprises an amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO: 18033).

116. The vector of claim 112, wherein the GSG-E2A peptide comprises an amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 18034).

117. The vector of claim 112, wherein the F2A peptide comprises an amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 18035).

118. The vector of claim 112, wherein the GSG-F2A peptide comprises an amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 18036).2023208176 27 Jul 2023119. The vector of claim 112, wherein the P2A peptide comprises an amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 18037).

120. The vector of claim 112, wherein the GSG-P2A peptide comprises an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 18038).

121. A composition comprising the vector of any one of claims 80 to 120.

122. A cell comprising the CAR of any one of claims 1-30.

123. A cell comprising the transposon or transposase of any one of claims 32 to 79.

124. A cell comprising the vector of any one of claims 80 to 120.

125. The cell of any one of claims 122 to 124, wherein the cell expresses the CAR on the cell surface.

126. The cell of any one of claims 122 to 125, wherein the cell is an immune cell.

127. The cell of claim 126, wherein the immune cell is a T-cell, a Natural Killer (NK) cell,a Natural Killer (NK)-like cell, a hematopoeitic progenitor cell, a peripheral blood (PB) derived T cell or an umbilical cord blood (UCB) derived T-cell.

128. The cell of claim 126, wherein the Natural Killer (NK)-like cell is a cytokine-induced killer (CIK) cell.

129. The cell of claim 126, wherein the immune cell is a T-cell.

130. The cell of claim 129, wherein the T-cell is a memory stem T-cell (Tscm).

131. The cell of claim 130, wherein the Tscm expresses one or more cell-surface marker(s).2023208176 27 Jul 2023132. The cell of claim 131, wherein the cell-surface markers comprise CD62L and CD45RA.

133. The cell of claim 131, wherein the cell-surface markers comprise CD45RA, CD95, IL-2RP, CR7, and CD62L.

134. The cell of claim 131, wherein the cell-surface markers comprise CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2Rp.

135. The cell of any one of claims 122 to 125, wherein the cell is an artificial antigen presenting cell.

136. The cell of any one of claims 122 to 125, wherein the cell is a tumor cell.

137. The cell of any one of claims 112 to 136, wherein the cell is autologous.

138. The cell of any one of claims 112 to 136, wherein the cell is allogeneic.

139. A composition comprising the cell of any one of claims 122-138.

140. A composition comprising a plurality of cells, wherein the plurality comprises at least one cell of any one of claims 122-138.

141. A composition comprising a plurality of cells, wherein the plurality comprises at least one cell of any one of claims 122-138.

142. The composition of claim 141, wherein the plurality comprises at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or any percentage in between of cells of any one of claims 122-138.2023208176 27 Jul 2023143. The composition of claim 141, wherein the each cell of the plurality of cells is a cell of any one of claims 122-138.

144. A method for expressing a chimeric antigen receptor (CAR) on the surface of a cell, comprising:(a) contacting a cell population to a composition comprising a CAR according to any one of claims 1-30 or a sequence encoding the CAR, under conditions sufficient to transfer the CAR across a cell membrane of at least one cell in the cell population, thereby generating a modified cell population;(b) culturing the modified cell population under conditions suitable for integration of the CAR;(c) expanding and / or selecting at least one cell from the modified cell population that express the CAR on the cell surface.

145. The method of claim 144, wherein the cell population comprises leukocytes.

146. The method of claim 145, wherein the cell population comprises CD4+ and CD8+leukocytes in an optimized ratio.

147. The method of claim 146, wherein the optimized ratio of CD4+ to CD8+ leukocytes does not naturally occur in vivo.

148. The method of claim 144, wherein a transposon or vector comprises the CAR or the sequence encoding the CAR.

149. The method of claim 144, wherein a transposon of any one of claims 38 to 73 comprises the CAR or the sequence encoding the CAR.

150. The method of claim 148 or 149, wherein the transposon comprises a piggyBac transposon.2023208176 27 Jul 2023151. The method of claim 150, further comprising a composition comprising a plasmid comprising a sequence encoding a transposase enzyme.

152. The method of claim 151, wherein the sequence encoding a transposase enzyme is an mRNA sequence.

153. The method of any one of claims 151 or 152, wherein the transposase is a piggyBac transposase.

154. The method of claim 153, wherein the piggyBac transposase comprises an amino acid sequence comprising SEQ ID NO: 14487.

155. The method of claim 153 or 154, 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: 14487.

156. The method of claim 155, wherein the amino acid substitution at position 30 of SEQ ID NO: 14487 is a substitution of a valine (V) for an isoleucine (I) (BOV).

157. The method of claim 155, wherein the amino acid substitution at position 165 of SEQ ID NO: 14487 is a substitution of a serine (S) for a glycine (G) (G165S).

158. The method of claim 155, wherein the amino acid substitution at position 282 of SEQ ID NO: 14487 is a substitution of a valine (V) for a methionine (M) (M282V).

159. The method of claim 155 wherein the amino acid substitution at position 538 of SEQ ID NO: 14487 is a substitution of a lysine (K) for an asparagine (N) (N538K).

160. The method of any one of claims 153-159, wherein the transposase is a Super piggyBac (SPB) transposase.2023208176 27 Jul 2023161. The method of claim 160, wherein the Super piggyBac (SPB) transposase comprises an amino acid sequence comprising SEQ ID NO: 14484.

162. The method of claim 144, wherein a vector of any one of claims 80 to 120 comprises the CAR or the sequence encoding the CAR.

163. The method of claim 144, 148, 149, or 162, wherein the conditions sufficient to transfer the sequence encoding the CAR across a cell membrane of at least one cell in the cell population comprise nucleofection.

164. The method of any one of claims 144 to 162, wherein the conditions sufficient to transfer the sequence encoding the CAR across a cell membrane of at least one cell in the cell population of (b) comprise at least one of an application of one or more pulses of electricity at a specified voltage, a buffer, and one or more supplemental factor(s).

165. The method of claim 164, wherein the buffer comprises PBS, HBSS, OptiMEM, BTXpress, Amaxa Nucleofector, Human T cell nucleofection buffer or any combination thereof.

166. The method of claim 164 or 165, wherein the one or more supplemental factor(s) comprise(a) a recombinant human cytokine, a chemokine, an interleukin or any combination thereof;(b) a salt, a mineral, a metabolite or any combination thereof;(c) a cell medium;(d) an inhibitor of cellular DNA sensing, metabolism, differentiation, signal transduction, one or more apoptotic pathway(s) or combinations thereof; and(e) a reagent that modifies or stabilizes one or more nucleic acids.2023208176 27 Jul 2023167. The method of claim 166, wherein the recombinant human cytokine, the chemokine, the interleukin or any combination thereof comprise IL2, IL7, IL12, IL15, IL21, IL1, IL3, IL4, IL5, IL6, IL8, CXCL8, IL9, IL10, IL11, IL13, IL14, IL16, IL17, IL18, IL19, IL20, IL22, IL23, IL25, IL26, IL27, IL28, IL29, IL30, IL31, IL32, IL33, IL35, IL36, GM-CSF, IFN-gamma, IL-1 alpha / IL-lFl, IL-1 beta / IL-lF2, IL-12 p70, IL-12 / IL-35 p35, IL-13, IL-17 / IL-17A, IL-17A / F Heterodimer, IL-17F, IL-18 / IL-1F4, IL-23, IL-24, IL-32, IL-32 beta, IL-32 gamma, IL-33, LAP (TGF-beta 1), Lymphotoxin-alpha / TNF-beta, TGF-beta, TNF-alpha, TRANCE / TNFSF11 / RANK L or any combination thereof.

168. The method of claim 166, wherein the salt, the mineral, the metabolite or any combination thereof comprise HEPES, Nicotinamide, Heparin, Sodium Pyruvate, L-Glutamine, MEM Non-Essential Amino Acid Solution, Ascorbic Acid, Nucleosides, FBS / FCS, Human serum, serum-substitute, anti-biotics, pH adjusters, Earle’s Salts, 2-Mercaptoethanol, Human transferrin, Recombinant human insulin, Human serum albumin, Nucleofector PLUS Supplement, KCL, MgC12, Na2HPO4, NAH2PO4, Sodium lactobionate, Manitol, Sodium succinate, Sodium Chloride, CINa, Glucose, Ca(NO3)2, Tris / HCl, K2HPO4, KH2PO4, Polyethylenimine, Poly-ethylene-glycol, Poloxamer 188, Poloxamer 181, Poloxamer 407, Poly-vinylpyrrolidone, Pop313, Crown-5, or any combination thereof.

169. The method of claim 166, wherein the cell medium comprises PBS, HBSS, OptiMEM, DMEM, RPMI 1640, AIM-V, X-VIVO 15, CellGro DC Medium, CTS OpTimizer T Cell Expansion SFM, TexMACS Medium, PRIME-XV T Cell Expansion Medium, ImmunoCult-XF T Cell Expansion Medium or any combination thereof.

170. The method of claim 166, wherein the inhibitor of cellular DNA sensing,metabolism, differentiation, signal transduction, one or more apoptotic pathway(s) or combinations thereof comprise inhibitors of TLR9, MyD88, IRAK, TRAF6, TRAF3, IRF-7, NF-KB, Type 1 Interferons, pro-inflammatory cytokines, cGAS, STING, Sec5, TBK1, IRF-3, RNA pol III, RIG-1, IPS-1, FADD, RIP1, TRAF3, AIM2, ASC, Caspasel, Pro-ILIB, PI3K, Akt, Wnt3A, glycogen synthase kinase-3p (GSK-3 P), TWS119, Bafilomycin,2023208176 27 Jul 2023Chloroquine, Quinacrine, AC-YVAD-CMK, Z-VAD-FMK, Z-IETD-FMK or any combination thereof.

171. The method of claim 166, wherein the reagent that modifies or stabilizes one or more nucleic acids comprises a pH modifier, a DNA-binding protein, a lipid, a phospholipid, CaPO4, a net neutral charge DNA binding peptide with or without aNLS sequence, a TREX1 enzyme or any combination thereof.

172. The method of any one of claims 144 to 162, wherein the conditions suitable for integration of the CAR or the sequence encoding the CAR comprise at least one of a buffer and one or more supplemental factor(s).

173. The method of claim 172, wherein the buffer comprises PBS, HBSS, OptiMEM, BTXpress, Amaxa Nucleofector, Human T cell nucleofection buffer or any combination thereof.

174. The method of claim 172 or 173, wherein the one or more supplemental factor(s) comprise(a) a recombinant human cytokine, a chemokine, an interleukin or any combination thereof;(b) a salt, a mineral, a metabolite or any combination thereof;(c) a cell medium;(d) an inhibitor of cellular DNA sensing, metabolism, differentiation, signal transduction, one or more apoptotic pathway(s) or combinations thereof; and(e) a reagent that modifies or stabilizes one or more nucleic acids.

175. The method of claim 174, wherein the recombinant human cytokine, the chemokine, the interleukin or any combination thereof comprise IL2, IL7, IL12, IL15, IL21, IL1, IL3, IL4, IL5, IL6, IL8, CXCL8, IL9, IL10, IL11, IL13, IL14, IL16, IL17, IL18, IL19, IL20, IL22, IL23, IL25, IL26, IL27, IL28, IL29, IL30, IL31, IL32, IL33, IL35, IL36, GM-CSF, IFN-gamma, IL-1 alpha / IL-lFl, IL-1 beta / IL-lF2, IL-12 p70, IL-12 / IL-35 p35, IL-13, IL-2023208176 27 Jul 202317 / IL-17A, IL-17A / F Heterodimer, IL-17F, IL-18 / IL-1F4, IL-23, IL-24, IL-32, IL-32 beta, IL-32 gamma, IL-33, LAP (TGF-beta 1), Lymphotoxin-alpha / TNF-beta, TGF-beta, TNF-alpha, TRANCE / TNFSF11 / RANK L or any combination thereof.

176. The method of claim 174, wherein the salt, the mineral, the metabolite or any combination thereof comprise HEPES, Nicotinamide, Heparin, Sodium Pyruvate, L-Glutamine, MEM Non-Essential Amino Acid Solution, Ascorbic Acid, Nucleosides, FBS / FCS, Human serum, serum-substitute, anti-biotics, pH adjusters, Earle’s Salts, 2-Mercaptoethanol, Human transferrin, Recombinant human insulin, Human serum albumin, Nucleofector PLUS Supplement, KCL, MgC12, Na2HPO4, NAH2PO4, Sodium lactobionate, Manitol, Sodium succinate, Sodium Chloride, CINa, Glucose, Ca(NO3)2, Tris / HCl, K2HPO4, KH2PO4, Polyethylenimine, Poly-ethylene-glycol, Poloxamer 188, Poloxamer 181, Poloxamer 407, Poly-vinylpyrrolidone, Pop313, Crown-5, or any combination thereof.

177. The method of claim 174, wherein the cell medium comprises PBS, HBSS, OptiMEM, DMEM, RPMI 1640, AIM-V, X-VIVO 15, CellGro DC Medium, CTS OpTimizer T Cell Expansion SFM, TexMACS Medium, PRIME-XV T Cell Expansion Medium, ImmunoCult-XF T Cell Expansion Medium or any combination thereof.

178. The method of claim 174, wherein the inhibitor of cellular DNA sensing, metabolism, differentiation, signal transduction, one or more apoptotic pathway(s) or combinations thereof comprise inhibitors of TLR9, MyD88, IRAK, TRAF6, TRAF3, IRF-7, NF-KB, Type 1 Interferons, pro-inflammatory cytokines, cGAS, STING, Sec5, TBK1, IRF-3, RNApol III, RIG-1, IPS-1, FADD, RIP1, TRAF3, AIM2, ASC, Caspasel, Pro-ILIB, PI3K, Akt, Wnt3A, glycogen synthase kinase-3p (GSK-3P), TWS119, Bafilomycin, Chloroquine, Quinacrine, AC-YVAD-CMK, Z-VAD-FMK, Z-IETD-FMK or any combination thereof.

179. The method of claim 164, wherein the reagent that modifies or stabilizes one or more nucleic acids comprises a pH modifier, a DNA-binding protein, a lipid, a phospholipid, CaPO4, a net neutral charge DNA binding peptide with or without aNLS sequence, a TREX1 enzyme or any combination thereof.2023208176 27 Jul 2023180. The method of any one of claims 144 to 179, wherein the expansion and selection of (d) occur sequentially.

181. The method of claim 180, wherein the expansion occurs prior to selection.

182. The method of claim 180, wherein the expansion occurs following selection.

183. The method of claim 182, wherein a further selection occurs following expansion.

184. The method of any one of claims 144 to 179, wherein the expansion and selection of (d) occur simultaneously.

185. The method of any one of claims 144 to 184, wherein the expansion comprises contacting at least one cell of the modified cell population with an antigen to stimulate the at least one cell through the CAR.

186. The method of claim 185, wherein the antigen is presented on the surface of a substrate.

187. The method of claim 186, wherein the substrate is a bead or a plurality of beads.

188. The method of claim 187, wherein the bead or plurality of beads is / are separated from the modified cell population following expansion.

189. The method of claim 185, wherein the antigen is presented on the surface of a cell.

190. The method of claim 189, wherein the antigen is presented on the surface of an artificial antigen presenting cell.

191. The method of any one of claims 144 to 190, wherein the transposon or vector comprises a selection gene and wherein the selection step comprises contacting at least one2023208176 27 Jul 2023cell of the modified cell population with a compound to which the selection gene confers resistance, thereby identifying a cell expressing the selection gene as surviving the selection and identifying a cell failing to express the selection gene as failing to survive the selection step.

192. The method of any one of claims 144 to 191, wherein the expansion and selection steps proceed for a period of 10 to 14 days, inclusive of the endpoints.

193. A composition comprising the expanded and selected cell population of any one of claims 144 to 192.

194. A method of treating cancer in a subject in need thereof, comprising administering to the subject the composition of any one of claims 31, 68-79, 121, 133 or 183, wherein the CAR specifically binds to an antigen on a tumor cell.

195. The method of claim 184, wherein the tumor cell is a malignant tumor cell.

196. The method of claim 184 or 185, comprising administering to the subject thecomposition of claim 133 or 179, wherein the cell or cell population is autologous.

197. The method of claim 184 or 185, comprising administering to the subject the composition of claim 133 or 179, wherein the cell or cell population is allogeneic.

198. A method of treating an autoimmune condition in a subject in need thereof, comprising administering to the subject the composition of any one of claims 31, 68-79, 121, 143 or 193, wherein the CAR specifically binds to an antigen on an autoimmune cell of the subject.

199. The method of claim 198, wherein the autoimmune cell is a lymphocyte that specifically binds to a self-antigen on a target cell of the subject.2023208176 27 Jul 2023200.   The method of claim 198 or 199, wherein the autoimmune cell is a B lymphocyte.

201. The method of claim 198 or 199, wherein the autoimmune cell is a T lymphocyte.

202. The method of any one of claims 198 to 201, comprising administering to the subjectthe composition of claim 133 or 179, wherein the cell or cell population is autologous.

203. The method of any one of claims 198 to 201, comprising administering to the subject the composition of claim 143 or 189, wherein the cell or cell population is allogeneic.

204. A method of treating or preventing an infection in a subject in need thereof, comprising administering to the subject the composition of any one of claims 31, 68-79, 121, 143 or 193, wherein the CAR specifically binds to an antigen on a cell comprising an infectious agent, a cell in communication with an infectious agent or a cell exposed to an infectious agent.

205. The method of claim 204, wherein the infectious agent is a bacterium, a virus, a yeast or a microbe.

206. The method of claim 204 or 205, wherein the infectious agent may induce one or more of an infection, an immunodeficiency condition, an inflammatory condition, and a proliferative disorder.

207. The method of claim 206, wherein the infection causes tuberculosis, microencephaly, neurodegeneration or malaria.

208. The method of claim 206 or 207, wherein the infection causes microencephaly in a fetus of the subject.

209. The method of claim 208, wherein the infectious agent is a virus and wherein the virus is a Zika virus.2023208176 27 Jul 2023210. The method of claim 206, wherein the immunodeficiency condition is acquired immune deficiency syndrome (AIDS).

211. The method of claim 206, wherein the proliferative disorder is a cancer.

212. The method of claim 211, wherein the cancer is cervical cancer and wherein the infectious agent is a human papilloma virus (HPV).

213. The method of any one of claims 204 to 212, comprising administering to the subject the composition of claim 143 or 189, wherein the cell or cell population is autologous.

214. The method of any one of claims 204 to 12, comprising administering to the subject the composition of claim 143 or 189, wherein the cell or cell population is allogeneic.

215. A method of treating a mast cell disease in a subject in need thereof, comprising administering to the subject the composition of any one of claims 31, 68-79, 121, 143 or 193, wherein the CAR specifically binds to an antigen on a mast cell.

216. The method of claim 215, wherein the mast cell disease is a disorder associated with an excessive proliferation of mast cells.

217. The method of claim 216, wherein the mast cell disease is mastocytosis.

218. The method of claim 217, wherein the mast cell disease is a disorder associated withan abnormal activity of a mast cell.

219. The method of claim 218, wherein the mast cell disease is mast cell activation syndrome (MCAS), an allergic disease, asthma or an inflammatory disease.2023208176 27 Jul 2023220. The method of any one of claims 215 to 19, comprising administering to the subject the composition of claim 143 or 189, wherein the cell or cell population is autologous.

221. The method of any one of claims 215 to 219, comprising administering to the subject the composition of claim 143 or 189, wherein the cell or cell population is allogeneic.

222. A method of treating a degenerative disease in a subject in need thereof, comprising administering to the subject the composition of any one of claims 31, 68-79, 121, 143 or 193, wherein the CAR specifically binds to an antigen on a deleterious cell or an aged cell.

223. The method of claim 222, wherein the degenerative disease is a neurodegenerative disorder, a metabolic disorder, a vascular disorder or aging.

224. The method of claim 222 or 223, wherein the degenerative disease is a neurodegenerative disorder and wherein the deleterious cell or the aged cell is a stem cell, an immune cell, a neuron, a glia or a microglia.

225. The method of claim 222 or 223, wherein the degenerative disease is a metabolic disorder and wherein the deleterious cell or the aged cell is a stem cell, a somatic cell, a neuron, a glia or a microglia.

226. The method of claim 222 or 223, wherein the degenerative disease is a vascular disorder and wherein the deleterious cell or the aged cell is a stem cell, a somatic cell, an immune cell, an endothelial cell, a neuron, a glia or a microglia.

227. The method of claim 222 or 223, wherein the degenerative disease is aging and wherein the deleterious cell or the aged cell is an oocyte, a sperm, a stem cell, a somatic cell, an immune cell, an endothelial cell, a neuron, a glia or a microglia.

228. The method of any one of claims 222 to 227, comprising administering to the subject the composition of claim 133 or 179, wherein the cell or cell population is autologous.2023208176 27 Jul 2023229. The method of any one of claims 222 to 227, comprising administering to the subject the composition of claim 143 or 189, wherein the cell or cell population is allogeneic.

230. A method of modifying a cell therapy in a subject in need thereof, comprising administering to the subject a composition comprising a cell comprising a transposon of any one of claims 32 to 67, wherein apoptosis may be selectively induced in the cell by contacting the cell with an induction agent.

231. A method of modifying a cell therapy in a subject in need thereof, comprising administering to the subject a composition comprising a cell comprising a vector of any one of claims 80 to 120, wherein apoptosis may be selectively induced in the cell by contacting the cell with an induction agent.

232. The method of claim 230 or 231, wherein the cell is autologous.

233. The method of claim 230 or 231, wherein the cell is allogeneic.

234. The method of claim any one of claims 230 to 233, wherein the cell therapy is anadoptive cell therapy.

235. The method of claim any one of claims 230 to 234, wherein the modifying is a termination of the cell therapy.

236. The method of claim any one of claims 230 to 234, wherein the modifying is a depletion of a portion of the cells provided in the cell therapy.

237. The method of claim any one of claims 230 to 236, further comprising the step of administering an inhibitor of the induction agent to inhibit modification of the cell therapy, thereby restoring the function and / or efficacy of the cell therapy.

Images