VCAR compositions and their uses

Abstract

To provide VCAR compositions and methods for using the same.SOLUTION: Disclosed are VHH chimeric antigen receptors (VCARs), VCAR transposons encoding VCARs of the disclosure, cells modified to express VCARs of the disclosure, and methods of making and methods of using the same for adoptive cell therapy.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] This application claims the benefit of provisional application USSN 62 / 608,571, filed December 20, 2017, and provisional application USSN 62 / 608,894, filed December 21, 2017, the contents of each of which are incorporated herein by reference in their entirety.

[0002] The present disclosure is directed to molecular biology, and more particularly to chimeric antigen receptors and transposons containing one or more VCARs, as well as methods for making and using the same.

[0003] Including an array list by reference The file named "POTH-034_001WO_SeqListing_ST25_R.txt" was created on December 19, 2018, is 54.4 MB in size, and is incorporated herein by reference in its entirety. [Background technology]

[0004] There is a long-felt and unmet need in the art for methods to direct immune cell specificity without using traditional antibody sequences or fragments thereof. The present disclosure provides superior chimeric antigen receptors. Summary of the Invention

[0005] The present disclosure provides chimeric antigen receptors (CARs) 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 sequence or a humanized sequence. In some embodiments, the single domain antibody comprises a non-naturally occurring sequence. The single domain antibody comprises a recombinant sequence or a chimeric sequence. In some embodiments, the single domain antibody comprises a VHH or a sequence encoding a VHH. In some embodiments, the single domain antibody comprises a VH of the present disclosure or a sequence encoding a VH of the present disclosure.

[0006] The present disclosure provides chimeric antigen receptors (CARs) comprising: (a) an ectodomain comprising an antigen-recognition region, where the antigen-recognition region comprises at least one VHH; (b) a transmembrane domain; and (c) an endodomain comprising at least one costimulatory domain. Throughout this disclosure, CARs comprising VHHs are referred to as VCARs. In some embodiments, the antigen-recognition region can comprise two VHHs to generate bispecific or tandem VCARs. In some embodiments, the antigen-recognition region can comprise three VHHs to generate trispecific VCARs.

[0007] In some embodiments of the VCARs of the present disclosure, the ectodomain can further comprise a signal peptide. Alternatively, or in addition, in some embodiments, the ectodomain can further comprise a hinge between the antigen recognition region and the transmembrane domain. In some embodiments, the ectodomain can further comprise a signal peptide. Alternatively, or in addition, in some embodiments, the ectodomain can further comprise a hinge between the antigen recognition region and the transmembrane domain.

[0008] In some embodiments of the VCAR of the present disclosure, the VHH has the amino acid sequence: malpvtallplallllhaarpevqllesggglvqpggslrlscaasgftfssyamnwvrqapgkglewvagiigsggstyyadsvkgrfsisrdnsknt ldlqmnslraedtavyycvkdwnttmitergqgtlvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvl llslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr (VH-A; SEQ ID NO: 18000). Alternatively, the VHH comprises or consists of the amino acid sequence:

[0009] In some embodiments of the VCAR of the present disclosure, the VHH has the amino acid sequence: malpvtallplallllhaarpevqllesggglvqpggsltlscaasgftfsnyamnwvrqapgkglewvsgiigsgattyyadsvkgrftisrdnsknt lnlqmnslraedtaiyycvkdwnttmitergqgtlvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvl llslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr (VH-B; SEQ ID NO: 18002). Alternatively, the VHH comprises or consists of the amino acid sequence:

[0010] In some embodiments of the VCAR of the present disclosure, the VHH has the amino acid sequence: malpvtallplallllhaarpevqllesggglvqpgeslrlscaasgftfsnyamnwvrqapgkglewvsgivggggtsyyadsvrgrftisrdnsknt lylqmnslraedtavyycvkdwnttmitergqgtlvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvl llslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr (VH-C; SEQ ID NO: 18004). Alternatively, the VHH comprises or consists of the amino acid sequence:

[0011] In some embodiments of the VCAR of the present disclosure, the VHH has the amino acid sequence: malpvtallplallllhaarpevqllesggglvqpggslrlscaasgftfsnyamtwirqapgkglewvsgitgdggstfyadsvkgrftisrdnsknt lylqmnslraedtavyycvkdwnttmitergqgtlvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvl llslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr (VH-D; SEQ ID NO: 18006). Alternatively, the VHH comprises or consists of the amino acid sequence:

[0012] In some embodiments of the VCAR of the present disclosure, the VHH has the amino acid sequence: malpvtalllplallllhaarpevqllesggglaqpggslrlscaasgftfssyamnwirqapgkglewvsgisgsggstyyadsvkgrftisrdnsknt vylqmnslraedtavyycvkdwnttmitergqgtlvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvl llslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr (VH-E; SEQ ID NO: 18008). Alternatively, the VHH comprises or consists of the amino acid sequence:

[0013] In some embodiments of the VCAR of the present disclosure, the VHH has the amino acid sequence: malpvtallplallllhaarpevqllesggglvqpgrslrlscaasgftftnyamnwvrqapgkglewvsgisggggstyyadsvkgrftisrdnsknt lylqmnslraedtavyycvkdwnttmitergqgtlvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvl llslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr (VH-F; SEQ ID NO: 18010). Alternatively, the VHH comprises or consists of the amino acid sequence:

[0014] The present disclosure provides chimeric antigen receptors (CARs) 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. Throughout this disclosure, CARs comprising a VH are referred to as VCARs. In some embodiments, the antigen recognition region can comprise two VHHs to generate bispecific or tandem VCARs. In some embodiments, the antigen recognition region can comprise three VHHs to generate trispecific VCARs. In some embodiments, the ectodomain can further comprise a signal peptide. Alternatively, or in addition, in some embodiments, the ectodomain can further comprise a hinge between the antigen recognition region and the transmembrane domain. In some embodiments, the ectodomain can further comprise a signal peptide. Alternatively, or in addition, in some embodiments, the ectodomain can further comprise a hinge between the antigen recognition region and the transmembrane domain.

[0015] Some embodiments of the VCARs of the present disclosure include VCARs that comprise an ectodomain that comprises an antigen recognition region, where the antigen recognition region comprises at least one VH, and the VH comprises a human sequence or a humanized sequence.

[0016] Some embodiments of VCARs of the present disclosure include VCARs that comprise an ectodomain that comprises an antigen recognition region, where the antigen recognition region comprises at least one VH, wherein the VH comprises a non-naturally occurring sequence.

[0017] In some embodiments of VCARs of the present disclosure, including VCARs that comprise an ectodomain that includes an antigen recognition region, where the antigen recognition region includes at least one VH, the VH is not naturally occurring.

[0018] Some embodiments of the VCARs of the present disclosure include VCARs that comprise an ectodomain that comprises an antigen recognition region, where the antigen recognition region comprises at least one VH, and the VH comprises a recombinant or chimeric sequence.

[0019] Some embodiments of the VCARs of the present disclosure include VCARs that comprise an ectodomain that includes an antigen recognition region, where the antigen recognition region includes at least one VH, and the VH is generated by an in vitro affinity selection and recombination procedure.

[0020] In some embodiments of the VCAR of the disclosure, the VH has the amino acid sequence: malpvtallplallllhaarpevqllesggglvqpggslrlscaasgftfssyamnwvrqapgkglewvagiigsggstyyadsvkgrfsisrdnsknt ldlqmnslraedtavyycvkdwnttmitergqgtlvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvl llslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr (VH-A; SEQ ID NO: 18000). Alternatively, the VH may have the nucleic acid sequence:

[0021] In some embodiments of the VCAR of the disclosure, the VH has the amino acid sequence: malpvtallplallllhaarpevqllesggglvqpggsltlscaasgftfsnyamnwvrqapgkglewvsgiigsgattyyadsvkgrftisrdnsknt lnlqmnslraedtaiyycvkdwnttmitergqgtlvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvl llslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr (VH-B; SEQ ID NO: 18002). Alternatively, the VH may have the nucleic acid sequence:

[0022] In some embodiments of the VCAR of the disclosure, the VH has the amino acid sequence: malpvtallplallllhaarpevqllesggglvqpgeslrlscaasgftfsnyamnwvrqapgkglewvsgivggggtsyyadsvrgrftisrdnsknt lylqmnslraedtavyycvkdwnttmitergqgtlvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvl llslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr (VH-C; SEQ ID NO: 18004). Alternatively, the VH may have the nucleic acid sequence:

[0023] In some embodiments of the VCAR of the disclosure, the VH has the amino acid sequence: malpvtallplallllhaarpevqllesggglvqpggslrlscaasgftfsnyamtwirqapgkglewvsgitgdggstfyadsvkgrftisrdnsknt lylqmnslraedtavyycvkdwnttmitergqgtlvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvl llslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr (VH-D; SEQ ID NO: 18006). Alternatively, the VH may have the nucleic acid sequence:

[0024] In some embodiments of the VCAR of the disclosure, the VH has the amino acid sequence: malpvtalllplallllhaarpevqllesggglaqpggslrlscaasgftfssyamnwirqapgkglewvsgisgsggstyyadsvkgrftisrdnsknt vylqmnslraedtavyycvkdwnttmitergqgtlvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvl llslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr (VH-E; SEQ ID NO: 18008). Alternatively, the VH may have the nucleic acid sequence:

[0025] In some embodiments of the VCAR of the disclosure, the VH has the amino acid sequence: malpvtallplallllhaarpevqllesggglvqpgrslrlscaasgftftnyamnwvrqapgkglewvsgisggggstyyadsvkgrftisrdnsknt lylqmnslraedtavyycvkdwnttmitergqgtlvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvl llslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr (VH-F; SEQ ID NO: 18010). Alternatively, the VH may have the nucleic acid sequence:

[0026] In some embodiments of the VCARs of the present disclosure, the VCAR comprises a single domain antibody, a VHH, a VH, or a combination thereof. In some embodiments, the single domain antibody, VHH, or VH comprises or consists of recombinant and / or chimeric sequences. In some embodiments, the single domain antibody, VHH, or VH comprises or consists of human and / or humanized sequences.

[0027] In some embodiments of a VCAR of the present disclosure, the VCAR comprises a single domain antibody. In some embodiments, the single domain antibody is a VHH antibody or a VH antibody. In some embodiments, the VH antibody is a UniDab antibody. In some embodiments, the VH antibody is not a fragment of a naturally occurring monoclonal antibody.

[0028] In some embodiments of a VCAR of the present disclosure, the signal peptide can comprise a sequence encoding a human CD2, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD8α, CD19, CD28, 4-1BB, or GM-CSFR signal peptide. In some embodiments of a VCAR of the present disclosure, the signal peptide can comprise a sequence encoding a human CD8α signal peptide. The human CD8α signal peptide can comprise an amino acid sequence comprising MALPVTALLLPLALLLHAARP (SEQ ID NO: 18012). The human CD8α signal peptide can comprise an amino acid sequence comprising MALPVTALLLPLALLLHAARP (SEQ ID NO: 18012), or a sequence at least 70%, 80%, 90%, 95%, or 99% identical to an amino acid sequence comprising MALPVTALLLPLALLLHAARP (SEQ ID NO: 18012). The human CD8α signal peptide can be encoded by a nucleic acid sequence comprising atggcactgccagtcaccgccctgctgctgcctctggctctgctgcacgcagctagacca (SEQ ID NO: 18013).

[0029] In some embodiments of the VCARs of the present disclosure, the transmembrane domain can comprise a sequence encoding a human CD2, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD8α, CD19, CD28, 4-1BB, or GM-CSFR transmembrane domain. In some embodiments of the VCARs of the present disclosure, the transmembrane domain can comprise a sequence encoding a human CD8α transmembrane domain. The CD8α transmembrane domain can comprise an amino acid sequence comprising IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 18014), or a sequence at least 70%, 80%, 90%, 95%, or 99% identical to an amino acid sequence comprising IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 18014). The CD8α transmembrane domain can comprise the amino acid sequence atctacatttgggcaccactggccgggacctgtggagtgctgctgctgagcctggtcatcacactgtactg c (SEQ ID NO: 18015).

[0030] In some embodiments of a VCAR of the present disclosure, the endodomain can comprise a human CD3ζ endodomain.

[0031] In some embodiments of the VCARs of the present disclosure, at least one costimulatory domain can comprise any of human 4-1BB, CD28, CD40, ICOS, MyD88, OX-40 intracellular segments, or any combination thereof. In some embodiments of the VCARs of the present disclosure, at least one costimulatory domain can comprise a CD28 and / or 4-1BB costimulatory domain. A CD3 zeta costimulatory domain can be an amino acid sequence comprising: RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 18016); or The CD3 zeta costimulatory domain may comprise a sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to an amino acid sequence comprising: RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 18016). The 4-1BB costimulatory domain can be encoded by a nucleic acid sequence comprising: cgcgtgaagtttagtcgatcagcagatgccccagcttacaaacagggacagaaccagctgtataacgagctgaatctgggccgccgagaggaatatgacgtgctggataagcggagaggacgcgaccccgaaatgggaggcaagcccaggcgcaaaaaccctcaggaaggcctgtataacgagctgcagaaggacaaaatggcagaagcctattctgagatcggcatgaagggggagcgacggagaggcaaagggcacgatgggctgtaccagggactgagcaccgccacaaaggacacctatgatgctctgcatatgcaggcactgcctccaagg (SEQ ID NO: 18017). The 4-1BB costimulatory domain may comprise an amino acid sequence comprising KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 18018), or a sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to an amino acid sequence comprising KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 18018). aagagaggcaggaagaaactgctgtatattttcaaacagcccttcatgcgccccgtgcagactacccaggaggaagacgggtgctcctgtcgattccctgaggaagaggaaggcgggtgtgagctg (SEQ ID NO: 18019). The 4-1BB costimulatory domain can be located between the transmembrane domain and the CD28 costimulatory domain.

[0032] In some embodiments of the VCARs of the present disclosure, the hinge can comprise sequences derived from human CD8α, IgG4, and / or CD4 sequences. In some embodiments of the VCARs of the present disclosure, the hinge can comprise sequences derived from human CD8α sequences. The hinge can comprise: Human CD8α amino acid sequence comprising TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 18020), or TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDF The human CD8α hinge amino acid sequence may be at least 70%, 80%, 90%, 95%, or 99% identical to an amino acid sequence comprising ACD (SEQ ID NO: 18020). It can be encoded by a nucleic acid sequence comprising actaccacaccagcacctagaccaccaactccagctccaaccatcgcgagtcagcccctgagtctgagacctgaggcctgcaggccagctgcaggaggagctgtgcacaccaggggcctggacttcgcctgcgac (SEQ ID NO: 18021).

[0033] The VHHs and / or VCARs of the present disclosure may be 10 -9 M or less, 10 -10 M or less, 10 -11 M or less, 10 -12 M or less, 10 -13 M or less, 10 -14 M or less, and 10 -15 K below M DIt can bind to an antigen with at least one affinity selected from K D can be determined by surface plasmon resonance.

[0034] The present disclosure provides an anti-BCMA VCAR. The present disclosure provides a composition comprising a VCAR of the present disclosure and at least one pharmaceutically acceptable carrier.

[0035] The present disclosure provides a transposon comprising a VCAR of the present disclosure.

[0036] The transposons of the present disclosure can include a selection gene for identifying, enriching, and / or isolating 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 to which the cell is sensitive (or which may 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 culture medium lacking one or more nutrients essential for cell viability and / or survival in the absence of the selection gene. Non-limiting examples of representative selection genes include neo (confers resistance to neomycin), DHFR (encodes dihydrofolate reductase and confers resistance to methotrexate), TYMS (encodes thymidylate synthetase), MGMT (encodes O(6)-methylguanine-DNA methyltransferase), multidrug resistance gene (MDR1), ALDH1 (encodes aldehyde dehydrogenase 1 family, member A1), FRANCF, RAD51C (encodes RAD51 paralog C), GCS (encodes glucosylceramide synthase), and NKX2.2 (encodes NK2 homeobox 2).

[0037] A transposon of the present disclosure can comprise an inducible pro-apoptotic polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a pro-apoptotic polypeptide, but not comprising a non-human sequence. In some embodiments, the non-human sequence comprises a restriction site. In some embodiments, the ligand binding region can comprise a multimeric ligand binding region. An inducible pro-apoptotic polypeptide of the present disclosure can also be referred to as an "iC9 safety switch." In some embodiments, a transposon of the present disclosure can comprise an inducible caspase polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a caspase polypeptide, but not comprising a non-human sequence. In some embodiments, a transposon of the present disclosure can comprise an inducible caspase polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a caspase polypeptide, but not comprising a non-human sequence. In some embodiments, a transposon of the present disclosure can comprise an inducible caspase polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a cleaved caspase 9 polypeptide, but not comprising a non-human sequence. In some embodiments of the inducible pro-apoptotic polypeptide, inducible caspase polypeptide, or cleaved caspase-9 polypeptide of the present disclosure, the ligand-binding region can comprise an FK506 binding protein 12 (FKBP12) polypeptide. In some embodiments, the amino acid sequence of the ligand-binding region comprising an FK506 binding protein 12 (FKBP12) polypeptide can comprise a modification at position 36 of the sequence. The modification can be a substitution of phenylalanine (F) at position 36 with valine (V) (F36V). In some embodiments, the FKBP12 polypeptide is In some embodiments, the FKBP12 polypeptide is encoded by an amino acid sequence comprising: GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE (SEQ ID NO: 18022). GGGGTCCAGGTCGAGACTATTTCACCAGGGGATGGGCGAACATTTCCAAAAAGGGGCCAGACTTGCGTCGTGCATTACACCGGGATGCTGGAGGACGGGAAGAAAGTGGACAGCTCCAGGGATCGCAACAAGCCCTTCAAGTTCATGCTGGGAAAGCAGGAAGTGATCCGAGGATGGGAGGAAGGCGTGGCACAGATGTCAGTCGGCCAGCGGGCCAAACTGACCATTAGCCCTGACTACGCTTATGGAGCAACAGGCCACCCAGGGATCATTCCCCCTCATGCCACCCTGGTCTTCGAT GTGGAACTGCTGAAGCTGGAG (SEQ ID NO: 18023). In some embodiments, an inducer specific for the ligand binding domain can comprise an FK506 binding protein 12 (FKBP12) polypeptide having a phenylalanine (F) to valine (V) substitution at position 36 (F36V). Inducing agents include AP20187 and / or AP1903 (both synthetic drugs).

[0038] In some embodiments of the inducible pro-apoptotic polypeptide, inducible caspase polypeptide, or cleaved caspase-9 polypeptide of the present disclosure, the linker region is encoded by an amino acid sequence comprising GGGGS (SEQ ID NO: 18024) or a nucleic acid sequence comprising GGAGGAGGAGGATCC (SEQ ID NO: 18025). In some embodiments, the nucleic acid sequence encoding the linker does not comprise a restriction site.

[0039] In some embodiments of the cleaved caspase-9 polypeptides of the present disclosure, the cleaved caspase-9 polypeptide is encoded by an amino acid sequence that does not include an arginine (R) at position 87. Alternatively, or in addition, in some embodiments of the inducible pro-apoptotic polypeptides, inducible caspase polypeptides, or cleaved caspase-9 polypeptides of the present disclosure, the cleaved caspase-9 polypeptide is encoded by an amino acid sequence that does not include an alanine (A) at position 282. In some embodiments of the inducible pro-apoptotic polypeptides, inducible caspase polypeptides, or cleaved caspase-9 polypeptides of the present disclosure, the cleaved caspase-9 polypeptide is an amino acid comprising GFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS (SEQ ID NO: 18026); or (SEQ ID NO: 18027).

[0040] In some embodiments of the inducible pro-apoptotic polypeptides of the present disclosure, including cleaved caspase-9 polypeptides, the inducible pro-apoptotic polypeptide comprises: an amino acid sequence comprising: GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGGSGFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS (SEQ ID NO: 18028); or

[0041] A transposon of the present disclosure can comprise at least one self-cleaving peptide, for example, located between one or more VHHs or VCARs of the present disclosure and a selection gene of the present disclosure. A transposon of the present disclosure can comprise at least one self-cleaving peptide, for example, located between one or more VHHs or VCARs of the present disclosure and an inducible apoptosis-promoting polypeptide of the present disclosure. A transposon of the present disclosure can comprise at least two self-cleaving peptides, for example, one self-cleaving peptide located upstream or immediately upstream of the inducible apoptosis-promoting polypeptide of the present disclosure and a second self-cleaving peptide located downstream or immediately downstream of the inducible apoptosis-promoting polypeptide of the present disclosure.

[0042] The at least one self-cleaving peptide can include, for example, a T2A peptide, a 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. The T2A peptide can include an amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18030), or a sequence at least 70%, 80%, 90%, 95%, or 99% identical to an amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18030). The GSG-T2A peptide can include an amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 18031), or a sequence at least 70%, 80%, 90%, 95%, or 99% identical to an amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 18031). The GSG-T2A peptide can comprise a nucleic acid sequence comprising ggatctggagagggaaggggaagcctgctgacctgtggagacgtggaggaaaacccaggacca (SEQ ID NO: 18032). The E2A peptide can comprise an amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO: 18033), or a sequence at least 70%, 80%, 90%, 95%, or 99% identical to an amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO: 18033). The GSG-E2A peptide can comprise an amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 18034), or a sequence at least 70%, 80%, 90%, 95%, or 99% identical to an amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 18034). The F2A peptide can comprise an amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 18035), or a sequence at least 70%, 80%, 90%, 95%, or 99% identical to an amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 18035).The GSG-F2A peptide can comprise an amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 18036), or a sequence at least 70%, 80%, 90%, 95%, or 99% identical to an amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 18036). The P2A peptide can comprise an amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 18037), or a sequence at least 70%, 80%, 90%, 95%, or 99% identical to an amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 18037). The GSG-P2A peptide can comprise an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 18038), or a sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 18038).

[0043] The transposon of the present disclosure can comprise a first and a second self-cleaving peptide, where the first self-cleaving peptide is located upstream of, for example, one or more VHHs or VCARs of the present disclosure, and the second self-cleaving peptide is located downstream of, for example, one or more VHHs or VCARs of the present disclosure. The first and / or second self-cleaving peptides can comprise, for example, a T2A peptide, a 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. The T2A peptide can comprise an amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18030), or a sequence at least 70%, 80%, 90%, 95%, or 99% identical to an amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18030). The GSG-T2A peptide can include an amino acid sequence comprising GSGEGRGSLTCGDVEENPGP (SEQ ID NO: 18031), or a sequence at least 70%, 80%, 90%, 95%, or 99% identical to an amino acid sequence comprising GSGEGRGSLTCGDVEENPGP (SEQ ID NO: 18031). The GSG-T2A peptide can include a nucleic acid sequence comprising ggatctggagagggaaggggaagcctgctgacctgtggagacgtggaggaaaacccaggacca (SEQ ID NO: 18032). The E2A peptide can include an amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO: 18033), or a sequence at least 70%, 80%, 90%, 95%, or 99% identical to an amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO: 18033). The GSG-E2A peptide can comprise an amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 18034), or a sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to an amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 18034).The F2A peptide can comprise an amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 18035), or a sequence at least 70%, 80%, 90%, 95%, or 99% identical to an amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 18035). The GSG-F2A peptide can comprise an amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 18036), or a sequence at least 70%, 80%, 90%, 95%, or 99% identical to an amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 18036). The P2A peptide can comprise an amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 18037), or a sequence at least 70%, 80%, 90%, 95%, or 99% identical to an amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 18037). The GSG-P2A peptide can comprise an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 18038), or a sequence at least 70%, 80%, 90%, 95%, or 99% identical to an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 18038).

[0044] The present disclosure provides a composition comprising a transposon of the present disclosure. In some embodiments, the composition can further comprise a plasmid comprising a sequence encoding a transposon enzyme. The sequence encoding the transposon enzyme can be an mRNA sequence.

[0045] The transposon of the present disclosure can comprise a piggyBac transposon. In some embodiments of this method, the transposon is a plasmid DNA transposon having a sequence encoding a chimeric antigen receptor flanked by two cis-regulatory insulator elements. In some embodiments, the transposon is a piggyBac transposon. The transposon enzyme of the present disclosure can comprise a piggyBac transposase, or an enzyme compatible therewith. In some embodiments, particularly embodiments in which the transposase is a piggyBac transposon, the transposase is a piggyBac transposase or a super-piggyBac (SPB) transposase. In some embodiments, particularly embodiments in which the transposase is a super-piggyBac (SPB) transposase, the sequence encoding the transposase is an mRNA sequence.

[0046] In some embodiments of the disclosed methods, the transposase enzyme is a piggyBac (PB) transposase enzyme. 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 IRCLRMDKS 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 It may comprise or consist of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or any percentage therebetween identical to 541 PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV ICREHNIDMC QSCF (SEQ ID NO: 14487).

[0047] In some embodiments of the disclosed methods, the transposase enzyme has 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 IRCLRMDKS 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 A piggyBac (PB) transposase enzyme comprising or consisting of an amino acid sequence having an amino acid substitution at one or more of positions 30, 165, 282, or 538 of QSCF (SEQ ID NO: 14487).

[0048] In some embodiments, the transposase enzyme is a piggyBac (PB) transposase enzyme comprising or consisting of an amino acid sequence having an amino acid substitution at one or more of positions 30, 165, 282, or 538 of the sequence of SEQ ID NO: 14487. In some embodiments, the transposase enzyme is a piggyBac (PB) transposase enzyme comprising or consisting 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 some embodiments, the transposase enzyme is a piggyBac (PB) transposase enzyme comprising or consisting of an amino acid sequence having an amino acid substitution at each of positions 30, 165, 282, and 538 of the sequence of SEQ ID NO: 14487. In some embodiments, the amino acid substitution at position 30 of the sequence of SEQ ID NO: 14487 is an isoleucine (I) to valine (V). In some embodiments, the amino acid substitution at position 165 of the sequence of SEQ ID NO: 14487 is a glycine (G) to serine (S). In some embodiments, the amino acid substitution at position 282 of the sequence of SEQ ID NO: 14487 is a methionine (M) to valine (V). In some embodiments, the amino acid substitution at position 538 of the sequence of SEQ ID NO: 14487 is an asparagine (N) to lysine (K).

[0049] In some embodiments of the disclosed methods, the transposase enzyme is a Super-piggyBac (SPB) transposase enzyme. In some embodiments, the Super-piggyBac (SPB) transposase enzyme of the disclosure can comprise or consist of the amino acid sequence of SEQ ID NO: 14487, in which the amino acid substitution at position 30 is an isoleucine (I) to valine (V), the amino acid substitution at position 165 is a glycine (G) to serine (S), the substitution at position 282 is a methionine (M) to valine (V), and the amino acid substitution at position 538 is an asparagine (N) to lysine (K). In some embodiments, the Super-piggyBac (SPB) transposase enzyme is 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 IRCLRMDKS 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 It may comprise or consist of an amino acid sequence that is at least 70%, 80%, 90%, 95%, 99%, or any percentage therebetween identical to 541 PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV ICREHNIDMC QSCF (SEQ ID NO: 14484).

[0050] The present disclosure provides a vector comprising a VCAR of the present disclosure. In some embodiments, the vector is a viral vector. The vector can be a recombinant vector.

[0051] Viral vectors of the present disclosure can include sequences isolated from or derived from retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, or any combination thereof. Viral vectors can include sequences isolated from or derived from adeno-associated viruses (AAV). Viral vectors can include recombinant AAVs (rAAVs). Representative adeno-associated viruses and recombinant adeno-associated viruses of the present disclosure include two or more inverted terminal repeats (ITRs) located in cis adjacent to the VHH or VCAR encoding sequence of the present disclosure. Non-limiting examples of representative adeno-associated viruses and recombinant adeno-associated viruses of the present disclosure include any serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9). Non-limiting examples of representative adeno-associated viruses and recombinant adeno-associated viruses of the present disclosure include self-complementary AAV (scAAV) and AAV hybrids containing a genome of one serotype and a capsid of another serotype (e.g., AAV2 / 5, AAV-DJ, and AAV-DJ8). Non-limiting examples of representative adeno-associated viruses and recombinant adeno-associated viruses of the present disclosure include rAAV-LK03.

[0052] The viral vectors of the present disclosure can include a selection gene. The selection gene can encode a gene product essential for cell viability and survival. The selection gene can encode a gene product essential for cell viability and survival when challenged by selective cell culture conditions. The selective cell culture conditions may contain a compound that is deleterious to cell viability or survival, and the gene product confers resistance to the compound. Non-limiting examples of representative selection genes of the present disclosure may include neo (confers resistance to neomycin), DHFR (encodes dihydrofolate reductase and confers resistance to methotrexate), TYMS (encodes thymidylate synthetase), MGMT (encodes O(6)-methylguanine-DNA methyltransferase), multidrug resistance gene (MDR1), ALDH1 (encodes aldehyde dehydrogenase 1 family, member A1), FRANCF, RAD51C (encodes RAD51 paralog C), GCS (encodes glucosylceramide synthase), NKX2.2 (encodes NK2 homeobox 2), or any combination thereof.

[0053] The viral vectors of the present disclosure can comprise an inducible apoptosis-promoting polypeptide comprising (a) a ligand-binding region, (b) a linker, and (c) a pro-apoptotic polypeptide, but not comprising a non-human sequence. In some embodiments, the non-human sequence comprises a restriction site. In some embodiments, the ligand-binding region can be a multimeric ligand-binding region. The inducible apoptosis-promoting polypeptides of the present disclosure can also be referred to as "iC9 safety switches." In some embodiments, the viral vectors of the present disclosure can comprise an inducible apoptosis-promoting polypeptide comprising (a) a ligand-binding region, (b) a linker, and (c) a caspase polypeptide, but not comprising a non-human sequence. In some embodiments, the viral vectors of the present disclosure can comprise an inducible apoptosis-promoting polypeptide comprising (a) a ligand-binding region, (b) a linker, and (c) a caspase polypeptide, but not comprising a non-human sequence. In some embodiments, the viral vectors of the present disclosure can comprise an inducible apoptosis-promoting polypeptide comprising (a) a ligand-binding region, (b) a linker, and (c) a cleaved caspase 9 polypeptide, but not comprising a non-human sequence. In some embodiments of the inducible pro-apoptotic polypeptide, inducible caspase polypeptide, or cleaved caspase-9 polypeptide of the present disclosure, the ligand-binding region can comprise an FK506 binding protein 12 (FKBP12) polypeptide. In some embodiments, the amino acid sequence of the ligand-binding region comprising an FK506 binding protein 12 (FKBP12) polypeptide can comprise a modification at position 36 of the sequence. The modification can be a substitution of phenylalanine (F) at position 36 with valine (V) (F36V). In some embodiments, the FKBP12 polypeptide is In some embodiments, the FKBP12 polypeptide is encoded by an amino acid sequence comprising: GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE (SEQ ID NO: 18022). GGGGTCCAGGTCGAGACTATTTCACCAGGGATGGGCGAACATTTCCAAAAAG GGGCCAGACTTGCGTCGTGCATTACACCGGGATGCTGGAGGACGGGAAGAAAGTGGACAGCTCCAGGGATCGCAACAAGCCCTTCAAGTTCATGCTGGGAAAGCAGGAAGTGATCCGAGGATGGGAGGAAGGCGTGGCACAGATGTCAGTCGGCCAGCGGGCCAAACTGACCATTAGCCCTGACTACGCTTATGGAGCAACAGGCCACCCAGGGATCATTCCCCCTCATGCCACCCTGGTCTTCGAT GTGGAACTGCTGAAGCTGGAG (SEQ ID NO: 18023). In some embodiments, an inducer specific for the ligand binding domain can comprise an FK506 binding protein 12 (FKBP12) polypeptide having a valine (V) instead of a phenylalanine (F) at position 36 (F36V). Inducers include AP20187 and / or AP1903 (both synthetic drugs).

[0054] In some embodiments of the inducible apoptosis-promoting polypeptide, inducible caspase polypeptide, or cleaved caspase-9 polypeptide of the present disclosure, the linker region is encoded by an amino acid sequence comprising GGGGS (SEQ ID NO: 18024) or a nucleic acid sequence comprising GGAGGAGGAGGATCC (SEQ ID NO: 18025). In some embodiments, the nucleic acid sequence encoding the linker does not comprise a restriction site.

[0055] In some embodiments of the cleaved caspase-9 polypeptides of the disclosure, the cleaved caspase-9 polypeptide is encoded by an amino acid sequence that does not include an arginine (R) at position 87. Alternatively, or in addition, in some embodiments of the inducible apoptosis-promoting polypeptides, inducible caspase polypeptides, or cleaved caspase-9 polypeptides of the disclosure, the cleaved caspase-9 polypeptide is encoded by an amino acid sequence that does not include an alanine (A) at position 282. In some embodiments of the inducible apoptosis-promoting polypeptides, inducible caspase polypeptides, or cleaved caspase-9 polypeptides of the disclosure, the cleaved caspase-9 polypeptide is an amino acid sequence comprising: GFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS (SEQ ID NO: 18026); or (SEQ ID NO: 18027).

[0056] In some embodiments of an inducible apoptosis-promoting polypeptide comprising a cleaved caspase-9 polypeptide, the inducible apoptosis-promoting polypeptide comprises: an amino acid sequence comprising: GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGGSGFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS (SEQ ID NO: 18028); or

[0057] The viral vector of the present disclosure can comprise at least one self-cleaving peptide. In some embodiments, the vector can comprise at least one self-cleaving peptide, where one self-cleaving peptide is located between the CAR and the selection gene. In some embodiments, the vector can comprise at least one self-cleaving peptide, where one self-cleaving peptide is located upstream of the CAR and a second self-cleaving peptide is located downstream of the CAR. The viral vector of the present disclosure can comprise at least one self-cleaving peptide, for example, located between one or more of the VCAR, VCAR, or VCAR of the present disclosure and an inducible apoptosis-promoting polypeptide of the present disclosure. The viral vector of the present disclosure can comprise at least two self-cleaving peptides, where, for example, one self-cleaving peptide is located upstream or immediately upstream of the inducible apoptosis-promoting polypeptide of the present disclosure and a second self-cleaving peptide is located downstream or immediately downstream of the inducible apoptosis-promoting polypeptide of the present disclosure. The self-cleaving peptide can include, for example, a T2A peptide, a 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. The T2A peptide can include an amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18030), or a sequence at least 70%, 80%, 90%, 95%, or 99% identical to an amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18030). The GSG-T2A peptide can include an amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 18031), or a sequence at least 70%, 80%, 90%, 95%, or 99% identical to an amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 18031). The GSG-T2A peptide can comprise a nucleic acid sequence comprising ggatctggagagggaaggggaagcctgctgacctgtggagacgtggaggaaaacccaggacca (SEQ ID NO: 18032).The E2A peptide can include an amino acid sequence that includes QCTNYALLKLAGDVESNPGP (SEQ ID NO: 18033), or a sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to an amino acid sequence that includes QCTNYALLKLAGDVESNPGP (SEQ ID NO: 18033). The GSG-E2A peptide can include an amino acid sequence that includes GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 18034), or a sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to an amino acid sequence that includes GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 18034). The F2A peptide can comprise an amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 18035), or a sequence at least 70%, 80%, 90%, 95%, or 99% identical to an amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 18035). The GSG-F2A peptide can comprise an amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 18036), or a sequence at least 70%, 80%, 90%, 95%, or 99% identical to an amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 18036). The P2A peptide can comprise an amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 18037), or a sequence at least 70%, 80%, 90%, 95%, or 99% identical to an amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 18037). The GSG-P2A peptide can comprise an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 18038), or a sequence at least 70%, 80%, 90%, 95%, or 99% identical to an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 18038).

[0058] The present disclosure provides a vector comprising a VCAR of the present disclosure. In some embodiments, the vector is a nanoparticle. Non-limiting examples of representative nanoparticle vectors of the present disclosure include 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, carbon sheets, carbon fibers, carbon tubes), inorganic molecules (e.g., calcium phosphate or gold), or any combination thereof. Nanoparticle vectors can be passively or actively transported through cell membranes.

[0059] The nanoparticle vector of the present disclosure can include a selection gene. The selection gene can encode a gene product essential for cell viability and survival. The selection gene can encode a gene product essential for cell viability and survival when challenged by selective cell culture conditions. The selective cell culture conditions may contain a compound that is detrimental to cell viability or survival, and the gene product confers resistance to the compound. Non-limiting examples of representative selection genes of the present disclosure may include neo (confers resistance to neomycin), DHFR (encodes dihydrofolate reductase and confers resistance to methotrexate), TYMS (encodes thymidylate synthetase), MGMT (encodes O(6)-methylguanine-DNA methyltransferase), multidrug resistance gene (MDR1), ALDH1 (encodes aldehyde dehydrogenase 1 family, member A1), FRANCF, RAD51C (encodes RAD51 paralog C), GCS (encodes glucosylceramide synthase), NKX2.2 (encodes NK2 homeobox 2), or any combination thereof.

[0060] The nanoparticle vector of the present disclosure can comprise an inducible pro-apoptotic polypeptide comprising (a) a ligand-binding region, (b) a linker, and (c) a pro-apoptotic polypeptide, but not comprising a non-human sequence. In some embodiments, the non-human sequence comprises a restriction site. In some embodiments, the ligand-binding region can be a multimeric ligand-binding region. The inducible pro-apoptotic polypeptide of the present disclosure can also be referred to as an "iC9 safety switch." In some embodiments, the nanoparticle vector of the present disclosure can comprise an inducible pro-apoptotic polypeptide comprising (a) a ligand-binding region, (b) a linker, and (c) a caspase polypeptide, but not comprising a non-human sequence. In some embodiments, the nanoparticle vector of the present disclosure can comprise an inducible pro-apoptotic polypeptide comprising (a) a ligand-binding region, (b) a linker, and (c) a caspase polypeptide, but not comprising a non-human sequence. In some embodiments, the nanoparticle vector of the present disclosure can comprise an inducible pro-apoptotic polypeptide comprising (a) a ligand-binding region, (b) a linker, and (c) a cleaved caspase 9 polypeptide, but not comprising a non-human sequence. In some embodiments of the inducible pro-apoptotic polypeptide, inducible caspase polypeptide, or cleaved caspase-9 polypeptide of the present disclosure, the ligand-binding region can comprise an FK506 binding protein 12 (FKBP12) polypeptide. In some embodiments, the amino acid sequence of the ligand-binding region comprising an FK506 binding protein 12 (FKBP12) polypeptide can comprise a modification at position 36 of the sequence. The modification can be a substitution of phenylalanine (F) at position 36 with valine (V) (F36V). In some embodiments, the FKBP12 polypeptide is In some embodiments, the FKBP12 polypeptide is encoded by an amino acid sequence comprising: GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE (SEQ ID NO: 18022). GGGGTCCAGGTCGAGACTATTTCACCAGGGGATGGGCGAACATTTCCAAAAAGGGGCCAGACTTGCGTCGTGCATTACACCGGGATGCTGGAGGACGGGAAGAAAGTGGACAGCTCCAGGGATCGCAACAAGCCCTTCAAGTTCATGCTGGGAAAGCAGGAAGTGATCCGAGGATGGGAGGAAGGCGTGGCACAGATGTCAGTCGGCCAGCGGGCCAAACTGACCATTAGCCCTGACTACGCTTATGGAGCAACAGGCCACCCAGGGATCATTCCCCCTCATGCCACCCTGGTCTTCGAT GTGGAACTGCTGAAGCTGGAG (SEQ ID NO: 18023). In some embodiments, an inducer specific for the ligand binding domain can comprise an FK506 binding protein 12 (FKBP12) polypeptide having a valine (V) instead of a phenylalanine (F) at position 36 (F36V). Inducing agents include AP20187 and / or AP1903 (both synthetic drugs).

[0061] In some embodiments of the inducible apoptosis-promoting polypeptide, inducible caspase polypeptide, or cleaved caspase-9 polypeptide of the present disclosure, the linker region is encoded by an amino acid sequence comprising GGGGS (SEQ ID NO: 18024) or a nucleic acid sequence comprising GGAGGAGGAGGATCC (SEQ ID NO: 18025). In some embodiments, the nucleic acid sequence encoding the linker does not comprise a restriction site.

[0062] In some embodiments of the cleaved caspase-9 polypeptides of the disclosure, the cleaved caspase-9 polypeptide is encoded by an amino acid sequence that does not include an arginine (R) at position 87. Alternatively, or in addition, in some embodiments of the inducible apoptosis-promoting polypeptides, inducible caspase polypeptides, or cleaved caspase-9 polypeptides of the disclosure, the cleaved caspase-9 polypeptide is encoded by an amino acid sequence that does not include an alanine (A) at position 282. In some embodiments of the inducible apoptosis-promoting polypeptides, inducible caspase polypeptides, or cleaved caspase-9 polypeptides of the disclosure, the cleaved caspase-9 polypeptide is an amino acid sequence comprising: GFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS (SEQ ID NO: 18026); or (SEQ ID NO: 18027).

[0063] In some embodiments of an inducible apoptosis-promoting polypeptide comprising a cleaved caspase-9 polypeptide, the inducible apoptosis-promoting polypeptide comprises: an amino acid sequence comprising: GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGGSGFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS (SEQ ID NO: 18028); or

[0064] The nanoparticle vector of the present disclosure can comprise at least one self-cleaving peptide. In some embodiments, the nanoparticle vector can comprise at least one self-cleaving peptide, where one self-cleaving peptide is located between VCAR and the nanoparticle. In some embodiments, the nanoparticle vector can comprise at least one self-cleaving peptide, where one self-cleaving peptide is located upstream of VCAR and a second self-cleaving peptide is located downstream of VCAR. In some embodiments, the nanoparticle vector can comprise at least one self-cleaving peptide, where one self-cleaving peptide is located between VCAR and the nanoparticle and a second self-cleaving peptide is located downstream of VCAR. In some embodiments, the nanoparticle vector can comprise at least one self-cleaving peptide, where one self-cleaving peptide is located between VCAR and the nanoparticle and a second self-cleaving peptide is located downstream of VCAR, for example, between VCAR and a selection gene.

[0065] The nanoparticle vector of the present disclosure can include, for example, at least one self-cleaving peptide located between one or more VHHs or VCARs of the present disclosure and an inducible apoptosis-promoting polypeptide of the present disclosure. The nanoparticle vector of the present disclosure can include at least two self-cleaving peptides, for example, one self-cleaving peptide located upstream or immediately upstream of the inducible apoptosis-promoting polypeptide of the present disclosure, and a second self-cleaving peptide located downstream or immediately downstream of the inducible apoptosis-promoting polypeptide of the present disclosure. The self-cleaving peptide can include, for example, a T2A peptide, a 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. The T2A peptide can include an amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18030) or a sequence at least 70%, 80%, 90%, 95%, or 99% identical to an amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18030). The GSG-T2A peptide can include an amino acid sequence comprising GSGEGRGSLTCGDVEENPGP (SEQ ID NO: 18031), or a sequence at least 70%, 80%, 90%, 95%, or 99% identical to an amino acid sequence comprising GSGEGRGSLTCGDVEENPGP (SEQ ID NO: 18031). The GSG-T2A peptide can include a nucleic acid sequence comprising ggatctggagagggaaggggaagcctgctgacctgtggagacgtggaggaaaacccaggacca (SEQ ID NO: 18032). The E2A peptide can include an amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO: 18033), or a sequence at least 70%, 80%, 90%, 95%, or 99% identical to an amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO: 18033). The GSG-E2A peptide can comprise an amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 18034), or a sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to an amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 18034).The F2A peptide can comprise an amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 18035), or a sequence at least 70%, 80%, 90%, 95%, or 99% identical to an amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 18035). The GSG-F2A peptide can comprise an amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 18036), or a sequence at least 70%, 80%, 90%, 95%, or 99% identical to an amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 18036). The P2A peptide can comprise an amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 18037), or a sequence at least 70%, 80%, 90%, 95%, or 99% identical to an amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 18037). The GSG-P2A peptide can comprise an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 18038), or a sequence at least 70%, 80%, 90%, 95%, or 99% identical to an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 18038).

[0066] The present disclosure provides compositions comprising the vectors of the present disclosure.

[0067] The present disclosure provides a cell comprising a VCAR of the present disclosure. The present disclosure provides a cell comprising a transposon of the present disclosure. In some embodiments, a cell comprising a VCAR, transposon, or vector of the present disclosure can express VCAR on the cell surface. The cell can be any type of cell. Preferably, the cell is an immune cell. The immune cell can be a T cell, a natural killer (NK) cell, a natural killer (NK)-like cell, a cytokine-induced killer (CIK) cell, a hematopoietic 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 can be an early memory cell, a stem-like T cell, a ... SCM -like cells, TSCM , or T CM As T cells, T SCM The cells can be artificial antigen-presenting cells, which can optionally be used to stimulate and expand the modified immune cells or T cells of the present disclosure. The cells can be tumor cells, which can optionally be used as artificial or modified antigen-presenting cells.

[0068] The modified cells of the present disclosure that can be used in adoptive therapy can be autologous or allogeneic.

[0069] The present disclosure provides a method for expressing a VCAR on a cell surface, comprising: (a) obtaining a cell population; (b) contacting the cell population with a VCAR of the present disclosure, or a composition comprising a sequence encoding a VCAR, under conditions sufficient to introduce the CAR through the cell membrane of at least one cell within 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) growing and / or selecting at least one cell from the modified cell population that expresses the CAR on its cell surface.

[0070] In some embodiments of this method of expressing VCAR, the cell population is a leukocyte, and / or a CD4 + White blood cells + CD8 + The cell population may include white blood cells, CD4 + White blood cells and CD8 + Contains an optimal ratio of white blood cells. CD4 + White blood cells and CD8 + The optimal ratio of leukocytes does not occur naturally in vivo. The cell population can include tumor cells.

[0071] In some embodiments of this method of expressing VCAR, the transposon or vector comprises VCAR or a sequence encoding VCAR.

[0072] In some embodiments of this method of expressing VCAR, the conditions sufficient to introduce the sequence encoding VCAR across the cell membrane of at least one cell in the cell population comprise nucleofection.

[0073] In some embodiments of this method of expressing VCAR, the conditions sufficient to introduce the VCAR-encoding sequence through the cell membrane of at least one cell in the cell population include at least one of application of one or more electrical pulses at a specific voltage, a buffer, and one or more cofactors. In some embodiments, the buffer can include PBS, HBSS, OptiMEM, BTXpress, Amaxa Nucleofector, human T cell nucleofection buffer, or any combination thereof. In some embodiments, the one or more cofactors can include (a) recombinant human cytokines, chemokines, interleukins, or any combination thereof; (b) salts, minerals, metabolites, or any combination thereof; (c) cell culture medium; (d) inhibitors of cellular DNA sensing, metabolic, differentiation, signal transduction, one or more apoptotic pathways, or combinations thereof; or (e) one or more nucleic acid modifying or stabilizing reagents. Recombinant human cytokines, chemokines, interleukins, or any combination thereof, include 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-1F1, IL-1 beta / IL-1F2, IL-12 p70, IL-12 / IL-35 The proteins may include p35, IL-13, IL-17 / IL-17A, IL-17A / F heterodimer, IL-17F, IL-18 / IL-1F4, IL-23, IL-24, IL-32, IL-32beta, IL-32gamma, IL-33, LAP (TGF-beta 1), lymphotoxin-alpha / TNF-beta, TGF-beta, TNF-alpha, TRANCE / TNFSF11 / RANK L, or any combination thereof.The salts, minerals, metabolites, or any combination thereof can include HEPES, nicotinamide, heparin, sodium pyruvate, L-glutamine, MEM non-essential amino acid solution, ascorbic acid, nucleosides, FBS / FCS, human serum, serum replacers, antibiotics, pH adjusters, Earle's salts, 2-mercaptoethanol, human transferrin, recombinant human insulin, human serum albumin, Nucleofector PLUS supplement, KCL, MgCl2, Na2HPO4, NAH2PO4, sodium lactobionate, mannitol, sodium succinate, sodium chloride, CINa, glucose, Ca(NO3)2, Tris / HCl, K2HPO4, KH2PO4, polyethyleneimine, polyethylene glycol, poloxamer 188, poloxamer 181, poloxamer 407, polyvinylpyrrolidone, Pop313, Crown 5, or any combination thereof. The cell culture medium can include 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. Inhibitors of cellular DNA sensing, metabolism, differentiation, signal transduction, one or more apoptosis pathways, or combinations thereof, can include inhibitors of TLR9, MyD88, IRAK, TRAF6, TRAF3, IRF-7, NF-KB, type 1 interferons, proinflammatory cytokines, cGAS, STING, Sec5, TBK1, IRF-3, RNA polymerase III, RIG-1, IPS-1, FADD, RIP1, TRAF3, AIM2, ASC, caspase 1, Pro-IL1B, PI3K, Akt, Wnt3A, inhibitors of glycogen synthase kinase-3β (GSK-3β) (e.g., TWS119), bafilomycin, chloroquine, quinacrine, AC-YVAD-CMK, Z-VAD-FMK, Z-IETD-FMK, or any combination thereof.Reagents that modify or stabilize one or more nucleic acids include pH adjusters, DNA binding proteins, lipids, phospholipids, CaPO4, pure neutrally charged DNA binding peptides with or without NLS sequences, TREX1 enzyme, or any combination thereof.

[0074] In some embodiments of this method of expressing VCAR, the conditions suitable for incorporation of a VCAR of the present disclosure, or a sequence encoding a VCAR, include at least one of a buffer and one or more cofactors. In some embodiments, a transposon or vector of the present disclosure includes a VCAR of the present disclosure, or a sequence encoding a VCAR. In some embodiments, the buffer can include PBS, HBSS, OptiMEM, BTXpress, Amaxa Nucleofector, human T cell nucleofection buffer, or any combination thereof. In some embodiments, the one or more cofactors can include (a) recombinant human cytokines, chemokines, interleukins, or any combination thereof; (b) salts, minerals, metabolites, or any combination thereof; (c) cell culture medium; (d) inhibitors of cellular DNA sensing, metabolic, differentiation, signal transduction, or one or more apoptotic pathways, or combinations thereof; or (e) one or more nucleic acid modifying or stabilizing reagents. Recombinant human cytokines, chemokines, interleukins, or any combination thereof, include 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-1F1, IL-1 beta / IL-1F2, IL-12 p70, IL-12 / IL-35 These proteins may include p35, IL-13, IL-17 / IL-17A, IL-17A / F heterodimer, IL-17F, IL-18 / IL-1F4, IL-23, IL-24, IL-32, IL-32beta, IL-32gamma, IL-33, LAP (TGF-beta 1), lymphotoxin-alpha / TNF-beta, TGF-beta, TNF-alpha, TRANCE / TNFSF11 / RANKL, or any combination thereof.The salts, minerals, metabolites, or any combination thereof can include HEPES, nicotinamide, heparin, sodium pyruvate, L-glutamine, MEM non-essential amino acid solution, ascorbic acid, nucleosides, FBS / FCS, human serum, serum replacers, antibiotics, pH adjusters, Earle's salts, 2-mercaptoethanol, human transferrin, recombinant human insulin, human serum albumin, Nucleofector PLUS supplement, KCL, MgCl2, Na2HPO4, NAH2PO4, sodium lactobionate, mannitol, sodium succinate, sodium chloride, CINa, glucose, Ca(NO3)2, Tris / HCl, K2HPO4, KH2PO4, polyethyleneimine, polyethylene glycol, poloxamer 188, poloxamer 181, poloxamer 407, polyvinylpyrrolidone, Pop313, Crown 5, or any combination thereof. The cell culture medium can include 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. Inhibitors of cellular DNA sensing, metabolism, differentiation, signal transduction, one or more apoptosis pathways, or combinations thereof, can include inhibitors of TLR9, MyD88, IRAK, TRAF6, TRAF3, IRF-7, NF-KB, type 1 interferons, proinflammatory cytokines, cGAS, STING, Sec5, TBK1, IRF-3, RNA polymerase III, RIG-1, IPS-1, FADD, RIP1, TRAF3, AIM2, ASC, caspase 1, Pro-IL1B, PI3K, Akt, Wnt3A, inhibitors of glycogen synthase kinase-3β (GSK-3β) (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 includes a pH adjuster, a DNA binding protein, a lipid, a phospholipid, CaPO4, a pure neutrally charged DNA binding peptide with or without an NLS sequence, a TREX1 enzyme, or any combination thereof.

[0075] In some embodiments of this method of expressing VCAR, the expansion and selection steps are performed sequentially. Expansion can occur before selection. Expansion can occur after selection, and in some cases, a further (i.e., second) selection can occur after expansion.

[0076] In some embodiments of this method for expressing VCAR, the expansion and selection steps can be performed simultaneously.

[0077] In some embodiments of this method of expressing VCAR, expanding can include contacting at least one cell of the modified cell population with an antigen and stimulating the at least one cell via VCAR to generate an expanded cell population. The antigen can be presented on the surface of a substrate. The substrate can have any shape, non-limiting examples of which include a surface, a well, one or more beads, and a matrix. The substrate can further include a paramagnetic or magnetic component. In some embodiments of this method of expressing VCAR, the antigen can be presented on the surface of a substrate, the substrate being a magnetic bead, and the magnetic bead can be removed or separated from the modified and expanded cell population using a magnet. The antigen can be presented on the surface of a cell or an artificial antigen-presenting cell. Non-limiting examples of artificial antigen-presenting cells of the present disclosure include tumor cells and stem cells.

[0078] In some embodiments of this method of expressing a VCAR, the transposon or vector comprises a selection gene and the selection step is performed by contacting at least one cell of the modified cell population with a compound to which the selection gene confers resistance, thereby identifying cells that express the selection gene as surviving the selection and cells that fail to express the selection gene as not surviving the selection step.

[0079] In some embodiments of this method of expressing VCAR, the expression and / or selection steps are allowed to proceed over a period of 10 to 14 days, inclusive.

[0080] The present disclosure provides compositions comprising the modified, expanded, and selected cell populations of the disclosed methods.

[0081] The present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a composition of the present disclosure, wherein VCAR specifically binds to an antigen on a tumor cell. In some embodiments, the tumor cell can be a malignant tumor cell. In some embodiments involving administering to a subject a composition comprising a modified cell or cell population of the present disclosure, the cell or cell population can be autologous. In some embodiments involving administering to a subject a composition comprising a modified cell or cell population of the present disclosure, the cell or cell population can be allogeneic.

[0082] The present disclosure provides a method of treating an autoimmune condition in a subject in need thereof, comprising administering to the subject a composition of the present disclosure, wherein VCAR specifically binds to an antigen on the subject's autoimmune cells. In some embodiments, the autoimmune cells can be lymphocytes that specifically bind to an autoantigen on a target cell in the subject. In some embodiments, the autoimmune cells can be B lymphocytes (i.e., B cells). In some embodiments, the autoimmune cells can be T lymphocytes (i.e., T cells). In some embodiments involving administering to a subject a composition comprising an engineered cell or cell population of the present disclosure, the cells or cell population can be autologous. In some embodiments involving administering to a subject a composition comprising an engineered cell or cell population of the present disclosure, the cells or cell population can be allogeneic.

[0083] The present disclosure provides a method for treating an infectious disease in a subject in need thereof, comprising administering to the subject a composition of the present disclosure, wherein VCAR specifically binds to an antigen on a cell containing the infectious agent, or on a cell transmitted by the infectious agent, or on a cell exposed to the infectious agent. In some embodiments, the cell transmitted by the infectious agent may be airborne (e.g., the infectious agent is airborne or inhaled) or liquidborne (e.g., the infectious agent is carried in an aqueous or bodily fluid). The infectious agent causing infection in the host cell may be a bacterium, virus, yeast, or microorganism. The infectious agent may cause conditions in the cell or in the cell's host organism (subject), including, but not limited to, viral infection, an immunodeficiency state, an inflammatory state, or a proliferative disorder. In some embodiments, the infection causes tuberculosis, microencephalopathy, neurodegeneration, or malaria. In some embodiments, the infection causes microencephalopathy in a fetus of the subject. In some embodiments, including those in which the infection causes microencephalopathy in the subject's fetus, the infectious agent is a virus, and the virus is Zika virus. In some embodiments, the immunodeficiency state is acquired immune deficiency syndrome (AIDS). In some embodiments, the proliferative disorder is cancer. In some embodiments, the cancer is cervical cancer and the infectious agent is human papillomavirus (HPV). In some embodiments, involving administering to a subject a composition comprising a modified cell or cell population of the present disclosure, the cell or cell population can be autologous. In some embodiments, involving administering to a subject a composition comprising a modified cell or cell population of the present disclosure, the cell or cell population can be allogeneic.

[0084] The present disclosure provides a method of treating a mast cell disorder in a subject in need thereof, comprising administering to the subject a composition of the present disclosure, wherein VCAR specifically binds to an antigen on a mast cell. In some embodiments, VCAR specifically binds to an antigen on a mast cell in the subject. In some embodiments, non-limiting examples of mast cell disorders can include disorders associated with hyperproliferation of mast cells, disorders associated with mast cells with abnormal activity, and disorders associated with both an abnormal number of mast cells and an abnormal activity of mast cells. Non-limiting examples of representative disorders associated with hyperproliferation of mast cells include mastocytosis, cutaneous mastocytosis (e.g., urticaria pigmentosa or maculopapular cutaneous mastocytosis), systemic mastocytosis (including mast cell leukemia), and localized mast cell proliferation. Non-limiting examples of disorders associated with abnormally active mast cells include mast cell activation syndrome (MCAS) or mast cell activation disorder (MCAD), allergic diseases (including anaphylaxis), asthma, inflammatory diseases (including, for example, autoimmune-related inflammation of joint tissues, rheumatoid arthritis, etc.), or any combination thereof. In some embodiments involving administering to a subject a composition comprising the modified cells or cell populations of the present disclosure, the cells or cell populations can be autologous. In some embodiments involving administering to a subject a composition comprising the modified cells or cell populations of the present disclosure, the cells or cell populations can be allogeneic. The present disclosure provides a method for treating a degenerative disease in a subject in need thereof, comprising administering to the subject a composition of the present disclosure, wherein VCAR specifically binds to an antigen on a harmful cell or an antigen on a senescent cell. In some embodiments, non-limiting examples of degenerative diseases can include neurodegenerative disorders, metabolic disorders, vascular disorders, and aging. Non-limiting examples of representative neurodegenerative disorders include disorders associated with loss of function or efficacy of one or more neurons, glial cells, or microglia.Non-limiting examples of representative neurodegenerative disorders include disorders related to the accumulation of one or more signaling molecules, proteins, or prions that interfere with one or more functions or reduce the effectiveness of neurons, glial cells, or microglia. Non-limiting examples of representative metabolic disorders include mitochondrial disorders, interruptions in the electron transport chain, interruptions in cellular ATP production, and disorders related to the loss or reduction of one or more functions or effectiveness of neurons, glial cells, or microglia. Non-limiting examples of representative metabolic disorders include disorders related to the loss of circulating blood flow or reduced blood flow to neurons, glial cells, or microglia (e.g., stroke); disorders related to transient or chronic hypoxia in neurons, glial cells, or microglia (e.g., sufficient to release free radicals intracellularly); and disorders related to the loss of circulating CNS blood flow or reduced CNS flow to neurons, glial cells, or microglia during a sleep state sufficient to reduce the effectiveness of neurons, glial cells, or microglia in removing waste products while the subject is asleep. Non-limiting examples of representative aging disorders include disorders associated with increased or shortened telomeres on one or more chromosomes of neurons, glial cells, or microglia; disorders associated with loss of function or reduced efficacy of telomeres in neurons, glial cells, or microglia; and disorders associated with loss of function or reduced efficacy of DNA repair mechanisms in neurons, glial cells, or microglia. In some embodiments, harmful or senescent cells impair the function or reduce the efficacy of other cells in a network that includes the harmful or senescent cells, thereby improving or restoring the function or improving the efficacy of the network. In some embodiments, harmful or senescent cells may alter the function or efficacy of a second cell, thereby preventing the transformation of the second cell. In some embodiments, the degenerative disease is a neurodegenerative disorder, and the harmful or senescent cells are stem cells, immune cells, neurons, glia, or microglia.In some embodiments, the degenerative disease is a metabolic disorder and the harmful or senescent cells are stem cells, somatic cells, neurons, glia, or microglia. In some embodiments, the degenerative disease is a vascular disorder and the harmful or senescent cells are stem cells, somatic cells, immune cells, endothelial cells, neurons, glia, or microglia. In some embodiments, the degenerative disease is aging and the harmful or senescent cells are oocytes, sperm, stem cells, somatic cells, immune cells, endothelial cells, neurons, glia, or microglia. In some embodiments involving administering to a subject a composition comprising an engineered cell or cell population of the present disclosure, the cells or cell population can be autologous. In some embodiments involving administering to a subject a composition comprising an engineered cell or cell population of the present disclosure, the cells or cell population can be allogeneic.

[0085] The present disclosure provides a method for modifying cell therapy in a subject in need thereof, comprising administering to the subject a composition comprising cells comprising a transposon or vector of a composition comprising an inducible apoptosis-promoting polypeptide, wherein apoptosis can be selectively induced in the cells by contacting the cells with an inducer. In some embodiments, the cells are autologous. In some embodiments, the cells are allogeneic. In some embodiments of this method, the cell therapy is adoptive cell therapy. In some embodiments of this method, modifying the cell therapy comprises terminating the cell therapy. In some embodiments of this method, modifying the cell therapy comprises depleting a portion of the cells provided by the cell therapy. In some embodiments, the method further comprises administering an inhibitor of the inducer to prevent modification of the cell therapy, thereby restoring function and / or efficacy of the cell therapy.

[0086] The disclosed methods of modifying cell therapy can be used to terminate or attenuate treatment in response to, for example, signs of recovery, or signs of decreased disease severity / progression, signs of disease remission / termination, and / or the occurrence of adverse events. If signs or symptoms of disease return or an increase in severity and / or adverse events becomes evident, the disclosed cell therapy can be resumed by inhibiting the inducer. [Brief explanation of the drawings]

[0087] [Figure 1] FIG. 1 is a graph showing the relationship between weight change and days post-treatment for VCARs of the present disclosure.

[0088] [Figure 2] FIG. 2 is a graph showing tumor burden versus days post-treatment for VCARs of the present disclosure.

[0089] [Figure 3] FIG. 3 is a graph showing survival versus days post-treatment for VCARs of the present disclosure.

[0090] [Figure 4] Figure 4 is a plot of tumor burden showing the relationship between days and bioluminescence signal after treatment with VH-A (triangles), irrelevant CAR (squares), or no tumor / no CAR-T (circles).

[0091] [Figure 5] Figure 5 is a plot of tumor burden showing the relationship between days and bioluminescence signal after treatment with VH-B (triangles), irrelevant CAR (squares), or no tumor / no CAR-T (circles).

[0092] [Figure 6] Figure 6 is a plot of tumor burden showing the relationship between days and bioluminescence signal after treatment with VH-C (triangles), irrelevant CAR (squares), or no tumor / no CAR-T (circles).

[0093] [Figure 7] Figure 7 is a plot of tumor burden showing the relationship between days and bioluminescence signal after treatment with VH-D (triangles), irrelevant CAR (squares), or no tumor / no CAR-T (circles).

[0094] [Figure 8] Figure 8 is a plot of tumor burden showing the relationship between days and bioluminescence signal after treatment with VH-E (triangles), irrelevant CAR (squares), or no tumor / no CAR-T (circles).

[0095] [Figure 9] Figure 9 is a plot of tumor burden showing the relationship between days and bioluminescence signal after treatment with VH-F (triangles), irrelevant CAR (squares), or no tumor / no CAR-T (circles).

[0096] [Figure 10A] FIG. 10A is a table showing the frame consensus and CDR sequences for representative VHs of the disclosure.

[0097] [Figure 10B] Figure 10B is an alignment of representative VHs of the present disclosure. From top to bottom, the sequences correspond to the consensus sequences of 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).

[0098] [Figure 11]Figure 11 is a bar graph showing the knockout efficiency of checkpoint signaling proteins on armed T cells. Primary human T cells are typically in a resting state when isolated from normal healthy donors. Cas-CLOVER was used to knockout the checkpoint receptors PD-1, TGFβR2, LAG-3, TIM-3, and CTLA-4. The knockout rate is shown on the y-axis. Gene editing resulted in a 30-70% loss of protein expression on the cell surface as measured by flow cytometry.

[0099] [Figure 12]Figure 12 shows a schematic diagram of wild-type, null, and switch receptors and their effects on inhibitory or stimulatory intracellular signaling in primary T cells. When endogenously expressed wild-type inhibitory receptors on T cells bind to their endogenous ligands, they transmit inhibitory signals. These inhibitory signals, in part, reduce T cell effector function. However, mutations (mutation null) or deletions (truncated null) of the intracellular domains (ICDs) of checkpoint receptor proteins (PD1 (top) or TGFβRII (bottom)) reduce or eliminate their signaling ability when their cognate ligands bind. Therefore, when engineered mutant or truncated null receptors are expressed on the surface of engineered T cells, they compete with the endogenously expressed wild-type receptor for binding of free endogenous ligands, effectively reducing or eliminating the inhibitory signal delivery by the endogenously expressed wild-type receptor. Specifically, any binding by a mutant or truncated null receptor prevents the endogenous ligand from binding to the wild-type receptor, reducing the overall level of checkpoint signaling effectively delivered to the engineered T cell, thereby reducing or preventing checkpoint inhibition and functional exhaustion of the engineered T cell. Switch receptors are generated by replacing the wild-type ICD with an ICD from a costimulatory molecule (e.g., D3z, CD28, 4-1BB) or a different inhibitory molecule (e.g., CTLA4, PD1, Lag3). In the former case, binding of the engineered switch receptor to the endogenous ligand may deliver a positive signal to the T cell, helping to enhance stimulation of the engineered T cell and potentially enhancing target tumor cell killing. In the latter case, binding of the engineered switch receptor to the endogenous ligand may deliver a negative signal to the T cell, potentially eliminating stimulation of the engineered T cell and reducing target tumor cell killing. The signal peptide (purple arrow), extracellular domain (ECD) (light green), transmembrane domain (yellow), intracellular signaling domain (ICD) (orange), and substituted ICD (green) are shown in the receptor diagram. "*" indicates a mutated ICD. "+" indicates the presence of a checkpoint signal. "-" indicates the absence of a checkpoint signal.

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

[0101] [Figure 14]Figure 14 is a series of histograms showing the expression of PD1 and TGFβRII null receptors on the surface of primary human T cells as determined by flow cytometry. Each of the six truncated null constructs from Figure 13 was expressed on the surface of primary human T cells. T cells were stained with antibody PD1 (top; blue histograms), anti-TGFβRII (bottom; blue histograms), isotype control, or secondary alone (gray histograms). Cells staining positive for PD-1 or TGFβRII expression were gated (frequency indicated above the gate), and mean fluorescence intensity (MFI) values ​​are shown above each positive histogram. The name of the null receptor construct is indicated above each plot. Both null receptor gene strategies were successful when wild-type SP was replaced with the surrogate CD8a. 02.8aSP-PD-1 and 02.8aSP-TGFβRII resulted in the highest levels of expression on the surface of T cells. The 02.8aSP-PD-1 null receptor exhibited an MFI of 43,680, which was 177-fold higher than the expression of endogenous T cell PD-1 and 2.8-fold higher than that of wild-type PD-1 null receptors. The 02.8aSP-TGFβRII null receptor exhibited an MFI of 13,809, which was 102-fold higher than the expression of endogenous T cell TGFβRII and 1.8-fold higher than that of wild-type TGFβRII null receptors. Replacing the wild-type SPs for both PD1 and TGFβRII with the alternative CD8a SP increased the surface expression of the null or switched receptors, which helps maximize checkpoint inhibition or costimulation, respectively, upon binding of endogenous ligands.

[0102] [Figure 15A]Figures 15A-B are a pair of schematic diagrams showing NF-κB-inducible vectors for expression in T cells. Two T cell-activating NF-κB-inducible vectors were developed. One has a gene expression system (GES) in the forward orientation (A), and the other in the complementary orientation (B), both of which are preceded by a constitutive EF1a promoter. These vectors also direct the expression of a CAR molecule and a DHFR selection gene, separated by a T2A sequence. Both the conditional NF-κB-inducible system and the EF1a-directed gene are part of a PiggyBac transposon, which can be permanently inserted into T cells using electroporation (EP). Once integrated into the genome, the T cells constitutively express the CAR on the membrane surface and DHFR intracellularly, whereas expression of the NF-κB-inducible gene, GFP, is expressed at its highest level only upon T cell activation. [Figure 15B] Figures 15A-B are a pair of schematic diagrams showing NF-κB-inducible vectors for expression in T cells. Two T cell-activating NF-κB-inducible vectors were developed. One has a gene expression system (GES) in the forward orientation (A), and the other in the complementary orientation (B), both of which are preceded by a constitutive EF1a promoter. These vectors also direct the expression of a CAR molecule and a DHFR selection gene, separated by a T2A sequence. Both the conditional NF-κB-inducible system and the EF1a-directed gene are part of a PiggyBac transposon, which can be permanently inserted into T cells using electroporation (EP). Once integrated into the genome, the T cells constitutively express the CAR on the membrane surface and DHFR intracellularly, whereas expression of the NF-κB-inducible gene, GFP, is expressed at its highest level only upon T cell activation.

[0103] [Figure 16]Figure 16 is a pair of graphs showing NF-κB-inducible expression of GFP in activated T cells. T cells were nucleofected with a piggyBac vector expressing an anti-BCMA CAR and a DHFR mutant protein gene under the control of the EF1a promoter in the absence (no GES control) or presence of an NF-κB-inducible expression system driving GFP expression in the forward (pNFκB-GFP forward) or reverse (pNFκB-GFP reverse) orientation. Cells were cultured in the presence of methotrexate selection until the cells were nearly completely quiescent (day 19), and GFP expression was assessed on days 5 and 19. On day 5, all T cells were proliferating and were intensely stimulated with cells carrying an NF-κB-inducible expression cassette, which resulted in high levels of GFP due to strong NF-κB activity. No GES control cells did not express detectable levels of GFP. By day 19, GES T cells were nearly completely quiescent, and GFP expression was significantly less than on day 5 (approximately 1 / 8 of the MFI). GFP expression is still observed on day 19, which may be due to the long half-life of the GFP protein (approximately 30 hours) or to basal levels of NF-κB activity, for example, through TCR, CAR, cytokine receptor, or growth factor receptor signaling.

[0104] [Figure 17]Figure 17 is a series of graphs showing that NF-κB-inducible expression of GFP is activated by anti-BCMA CAR in the 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 mutant protein gene under the control of the EF1 promoter in the absence (no GES control) or presence of an NF-κB-inducible expression system driving GFP expression in the forward (pNFκB-GFP forward) or reverse (pNFκB-GFP reverse) orientation. All cells were cultured for 22 days with or without methotrexate selection (Mock T cells) until cells were nearly completely quiescent. Cells were then stimulated for 3 days in the absence (no stimulation) or presence of BCMA- (K562), BMCA+ (RPMI 8226), or a positive control anti-CD3 / anti-CD28 activating reagent (CD3 / 28 stimulation). GFP expression was undetectable under any condition using the no-GES control or mock T cells. However, pNFκB-GFP forward and retrotransfer cells showed little GFP expression above the unstimulated control when cultured with BCMA-K562 cells, although both showed dramatic upregulation of gene expression in the presence of BCMA+ tumor cells or under positive control conditions. Little difference in GFP expression was observed between pNFκB-GFP forward and retrotransfer cells cultured with BCMA+ tumor cells.

[0105] [Figure 18-1]Figure 18 is a series of graphs demonstrating that inducible gene expression levels can be regulated by the number of response elements before a piggyBac vector encoding anti-BCMA CARTyrin followed by a selection gene (both under the control of the human EF1 promoter) is nucleofected into promoter T cells. Furthermore, the vector additionally encodes a conditional NF-κB-inducible gene expression system driving expression of a truncated CD19 protein (dCD19), containing a number of NFκB response elements (REs) varying from 0 to 5, and either lacking a GES (No GES) or receiving an electroporation pulse but not piggyBac nucleic acid (Mock). Data are shown for only the reversed (opposite) direction / orientation of the GES. All cells were cultured for 18 days, including selection for piggyBac-modified T cells using methotrexate addition. Cells were then stimulated for 3 days with anti-CD3 / anti-CD28 bead activation reagent, and dCD19 surface expression was assessed by FACS on days 0, 3, and 18. Data are presented as FACS histograms and MFI of target protein staining. Surface dCD19 expression was detected at low levels on day 0 in all T cells transfected with a GES-encoding vector. On day 3 post-stimulation, a dramatic upregulation of dCD19 expression was observed in all GES-expressing T cells, with T cells containing a higher number of REs showing a greater fold increase in surface expression. Thus, surface dCD19 expression was directly proportional to the number of REs encoded by the GES. No dCD19 was detected on the surface of T cells that did not contain the GES (no GES, mock control). [Figure 18-2]Figure 18 is a series of graphs demonstrating that inducible gene expression levels can be regulated by the number of response elements before a piggyBac vector encoding anti-BCMA CARTyrin followed by a selection gene (both under the control of the human EF1 promoter) is nucleofected into promoter T cells. Furthermore, the vector additionally encodes a conditional NF-κB-inducible gene expression system driving expression of a truncated CD19 protein (dCD19), containing a number of NFκB response elements (REs) varying from 0 to 5, and either lacking a GES (No GES) or receiving an electroporation pulse but not piggyBac nucleic acid (Mock). Data are shown for only the reversed (opposite) direction / orientation of the GES. All cells were cultured for 18 days, including selection for piggyBac-modified T cells using methotrexate addition. Cells were then stimulated for 3 days with anti-CD3 / anti-CD28 bead activation reagent, and dCD19 surface expression was assessed by FACS on days 0, 3, and 18. Data are presented as FACS histograms and MFI of target protein staining. Surface dCD19 expression was detected at low levels on day 0 in all T cells transfected with a GES-encoding vector. On day 3 post-stimulation, a dramatic upregulation of dCD19 expression was observed in all GES-expressing T cells, with T cells containing a higher number of REs showing a greater fold increase in surface expression. Thus, surface dCD19 expression was directly proportional to the number of REs encoded by the GES. No dCD19 was detected on the surface of T cells that did not contain the GES (no GES, mock control). [Figure 18-3]Figure 18 is a series of graphs demonstrating that inducible gene expression levels can be regulated by the number of response elements before a piggyBac vector encoding anti-BCMA CARTyrin followed by a selection gene (both under the control of the human EF1 promoter) is nucleofected into promoter T cells. Furthermore, the vector additionally encodes a conditional NF-κB-inducible gene expression system driving expression of a truncated CD19 protein (dCD19), containing a number of NFκB response elements (REs) varying from 0 to 5, and either lacking a GES (No GES) or receiving an electroporation pulse but not piggyBac nucleic acid (Mock). Data are shown for only the reversed (opposite) direction / orientation of the GES. All cells were cultured for 18 days, including selection for piggyBac-modified T cells using methotrexate addition. Cells were then stimulated for 3 days with anti-CD3 / anti-CD28 bead activation reagent, and dCD19 surface expression was assessed by FACS on days 0, 3, and 18. Data are presented as FACS histograms and MFI of target protein staining. Surface dCD19 expression was detected at low levels on day 0 in all T cells transfected with a GES-encoding vector. On day 3 post-stimulation, a dramatic upregulation of dCD19 expression was observed in all GES-expressing T cells, with T cells containing a higher number of REs showing a greater fold increase in surface expression. Thus, surface dCD19 expression was directly proportional to the number of REs encoded by the GES. No dCD19 was detected on the surface of T cells that did not contain the GES (no GES, mock control).

[0106] [Figure 19] FIG. 19 is a schematic diagram of the Csy4-T2A-Clo051-G4S linker-dCas9 construct map (Embodiment 2).

[0107] [Figure 20] FIG. 20 is a schematic representation of the pRT1-Clo051-dCas9 dual NLS construct map (embodiment 1).

[0108] [Figure 21] Figure 21 is a pair of graphs comparing the efficiency of embodiment 1 (pRT1-Clo051-dCas9 dual NLS shown in Figure 20) or embodiment 2 (Csy4-T2A-Clo051-G4S linker-dCas9 shown in Figure 19) of the Cas-Clover fusion protein of the present disclosure to knock out expression of B2M in pan-T cells (left) or the alpha chain of the T cell receptor in Jurkat cells (right). In the graph on the right, the fusion protein is provided at 10 μg or 20 μg, as indicated.

[0109] [Figure 22] Figure 22 is a photograph of a gel electrophoresis analysis of mRNA in the presence of Embodiment 1 (pRT1-Clo051-dCas9 dual NLS shown in Figure 20) and Embodiment 2 (Csy4-T2A-Clo051-G4S linker-dCas9 shown in Figure 19). As shown, both are effective in knocking down mRNA expression.

[0110] [Figure 23] FIG. 23 is a diagram of the UniRat™ human heavy chain antibody production platform.

[0111] [Figure 24] Figure 24 is a diagram of the immunization, B cell isolation, mRNA purification, next-generation sequencing (NGS), bioinformatics analysis, high-throughput vector assembly, and high-throughput expression and screening pipeline used to identify the complete antigen-specific repertoire of heavy chain antibodies produced by UniRat™ following immunization. A proprietary gene assembly method converts antibody repertoire sequence information into a large collection of fully human heavy chain antibodies that can be screened for diverse functions.

[0112] [Figure 25]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 heat map indicates VH sequence families expanded by immunization. Some highly ranked VH sequences are unique to a single animal. Other highly ranked VH sequences appear in more than one animal, suggesting convergent selection of these sequence families.

[0113] [Figure 26]Figure 26 shows a series of flow cytometry plots demonstrating that CAR-T populations expressing P-PSMA-101 and containing a mixture of TSCM / TCM give rise to CAR+ TCM, TEMs, and Teffs that attack solid tumors. Following removal of the solid tumor, the CAR-T+ TSCM population persists. While demonstrated with CARTyrin, this principle demonstrated here applies to the VCAR+ TSCM and VCAR+ TCM populations of the present disclosure. Specifically, a mouse xenograft model using the luciferase-expressing LNCaP cell line (LNCaP.luc) injected subcutaneously (SC) into NSG mice was used to evaluate the in vivo antitumor efficacy of CARs (P-PSMA5-101 and P-PSMA8-101) at a "stress" dose (4 x 106) of total CAR-T cells. For these in vivo studies, all CAR-T cells were generated using PB delivery of P-PSMA5-101 or P-PSMA8-101 plasmids using the Poseida manufacturing method. Mice were injected with LNCaP tumors in the axilla and treated when tumors were established (100–300 mm3 by caliper measurement). To address any potential differences in efficacy between PSMA5 and PSMA8 CARs, mice were treated by intravenous injection of a "stress" dose (4 × 106) of P-PSMA-101. Antitumor activity was assessed by survival, proliferation and detection of CD8+ T cells in the blood, tumor volume assessment by caliper measurement, and bioluminescence of LNCaP tumors. Stress doses of P-PSMA5-101 and P-PSMA8-101 demonstrated significantly enhanced antitumor efficacy and survival in established SC LNCaP.luc solid tumors in NSG mice compared with T cell (no CAR) control mice. Specifically, T cell (no CAR) control mice had zero survival, P-BCMA-101-treated mice had 25% survival, P-PSMA5-101-treated mice had 75% survival, and mice treated with a "stress" dose of P-PSMA8-101 had 100% survival. In peripheral blood, P-PSMA5-101 and P-PSMA8-101 expanded and gave rise to differentiated effector CARTyrin+ T cells, while tumor burden decreased below the detection limit of calipers and bioluminescence imaging.These cells were then shrunk and still persisted in the peripheral blood. DETAILED DESCRIPTION OF THE INVENTION

[0114] The present disclosure provides a chimeric antigen receptor (CAR) comprising at least one VHH (VCAR). The chimeric antigen receptor of the present disclosure can comprise two or more VHHs. For example, a bispecific VCAR can comprise two VHHs that specifically bind to two different antigens.

[0115] The VHH proteins of the present disclosure specifically bind to antigens. Chimeric antigen receptors of the present disclosure, comprising one or more VHHs that specifically bind to an antigen, can be used to target the specificity of cells (e.g., cytotoxic immune cells) to a particular antigen.

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

[0117] The chimeric antigen receptor of the present disclosure can comprise human CD2, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD8α, CD19, CD28, 4-1BB, or GM-CSFR. The hinge / spacer domain of the present disclosure can comprise the hinge / spacer / stalk of human CD8α, IgG4, and / or CD4. The intracellular domain or endodomain of the present disclosure can comprise the intracellular signaling domain of human CD3ζ and can further comprise human 4-1BB, CD28, CD40, ICOS, MyD88, OX-40 intracellular segment, or any combination thereof. Non-limiting examples of representative transmembrane domains include human CD2, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD8α, CD19, CD28, 4-1BB, or GM-CSFR transmembrane domains.

[0118] The present disclosure provides genetically modified cells (e.g., T cells, NK cells, hematopoietic progenitor cells, peripheral blood (PB)-derived T cells (including T cells from peripheral blood mobilized by G-CSF), umbilical cord blood (UCB)-derived T cells) that have been made specific for one or more antigens by introducing a VCAR of the present disclosure. The cells of the present disclosure can be modified by electrotransfer of a plasmid containing a transposon encoding a VCAR of the present disclosure and a sequence encoding a transposase of the present disclosure (the sequence encoding the transposase of the present disclosure is preferably an mRNA sequence). VH of the present disclosure

[0119] The present disclosure provides chimeric antigen receptors (CARs) comprising single domain antibodies (VCARs). In some embodiments, the single domain antibodies comprise a VH. In some embodiments, the VH is isolated from or derived from human sequences. In some embodiments, the VH comprises human CDR sequences and / or human framework sequences and non-human or humanized sequences (e.g., a rat Fc domain). In some embodiments, the VH is a fully humanized VH. In some embodiments, the VH is not a naturally occurring antibody or 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 fully engineered using UniRat™ (TeneoBio).

[0120] In some embodiments, VHs are fully engineered using the UniRat™ (TeneoBio) system and "NGS-based discovery" to generate VHs. Using this method, specific VHs do not occur naturally but are generated using a fully engineered system. The VHs are not derived from naturally occurring monoclonal antibodies (mAbs) isolated directly from a host (e.g., mouse, rat, or human) or from a cell or cell line (hybridoma). These VHs were not subsequently cloned from the cell line. Instead, the UniRat™ system was used as a transgene containing a human variable region (VH domain) with a rat Fc domain, and the VH sequence was fully engineered; therefore, this VH sequence is a human / rat chimera lacking a light chain, which differs from the standard mAb format. Because the native rat gene was knocked out, the only antibodies expressed in rats are derived from a transgene containing a VH domain linked to a rat Fc (UniAb). These are the limited Abs expressed in UniRat. Next-generation sequencing (NGS) and bioinformatics are used to identify the complete antigen-specific repertoire of heavy chain antibodies generated by UniRat™ after immunization. A proprietary gene assembly method is then used to convert the antibody repertoire sequence information into a large collection of fully human heavy chain antibodies that can be screened in vitro for various functions. In some embodiments, fully humanized VHs are generated by fusing a human VH domain with a human Fcs in vitro (to generate a non-naturally occurring recombinant VH antibody). In some embodiments, the VHs are fully humanized but expressed in vivo as a human / rat chimera (human VH, rat Fc) without light chains. The fully humanized VH expressed in vivo as a human / rat chimera (human VH, rat Fc) without light chains is approximately 80 kDa (instead of 150 kDa).

[0121] A VCAR of the present disclosure can comprise at least one VH of the present disclosure. In some embodiments, a VH of the present disclosure can be modified to remove the Fc domain or a portion thereof. In some embodiments, the framework sequence of a VH of the present disclosure can be modified to, for example, improve expression, reduce immunogenicity, or improve function. Representative VCARs of the present disclosure

[0122] In some embodiments of the VCAR of the present disclosure, the VCAR comprises: VH-A: malpvtallplallllhaarpevqllesggglvqpggslrlscaasgftfssyamnwvrqapgkglewvagiigsggstyyadsvkgrfsisrdnskntldlqmnslraedta vyycvkdwnttmitergqgtlvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrp vqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr (SEQ ID NO: 18000) that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of

[0123] In some embodiments of a VCAR of the disclosure, the amino acid sequence of the VCAR is VH-A:

[0124] In some embodiments of the VCAR of the present disclosure, the VCAR comprises: VH-B: malpvtallplallllhaarpevqllesggglvqpggsltlscaasgftfsnyamnwvrqapgkglewvsgiigsgattyyadsvkgrftisrdnskntlnlqmnslraedta iyycvkdwnttmitergqgtlvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrp vqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr (SEQ ID NO: 18002) that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of

[0125] In some embodiments of a VCAR of the present disclosure, the amino acid sequence of the VCAR is VH-B:

[0126] In some embodiments of the VCAR of the present disclosure, the VCAR comprises: VH-C: malpvtallplallllhaarpevqllesggglvqpgeslrlscaasgftfsnyamnwvrqapgkglewvsgivggggtsyyadsvrgrftisrdnskntlylqmnslraedta vyycvkdwnttmitergqgtlvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrp vqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr (SEQ ID NO: 18004) that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of

[0127] In some embodiments of a VCAR of the present disclosure, the amino acid sequence of the VCAR is VH-C:

[0128] In some embodiments of the VCAR of the present disclosure, the VCAR comprises: VH-D: malpvtallplallllhaarpevqllesggglvqpggslrlscaasgftfsnyamtwirqapgkglewvsgitgdggstfyadsvkgrftisrdnskntlylqmnslraedta vyycvkdwnttmitergqgtlvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrp vqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr (SEQ ID NO: 18006) that contains at least one amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of

[0129] In some embodiments of a VCAR of the present disclosure, the amino acid sequence of the VCAR is VH-D:

[0130] In some embodiments of the VCAR of the present disclosure, the VCAR comprises: VH-E: malpvtalllplallllhaarpevqllesggglaqpggslrlscaasgftfssyamnwirqapgkglewvsgisgsggstyyadsvkgrftisrdnskntvylqmnslraedta vyycvkdwnttmitergqgtlvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrp vqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr (SEQ ID NO: 18008) that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of

[0131] In some embodiments of a VCAR of the present disclosure, the amino acid sequence of the VCAR is VH-E:

[0132] In some embodiments of the VCAR of the present disclosure, the VCAR comprises: VH-F: malpvtallplallllhaarpevqllesggglvqpgrslrlscaasgftftnyamnwvrqapgkglewvsgisggggstyyadsvkgrftisrdnskntlylqmnslraedta vyycvkdwnttmitergqgtlvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrp vqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr (SEQ ID NO: 18010) that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of

[0133] In some embodiments of a VCAR of the present disclosure, the amino acid sequence of the VCAR is VH-F:

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

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

[0136] In some embodiments, immune progenitor cells include any cell that can differentiate into one or more types of cells. In some embodiments, immune progenitor cells include pluripotent stem cells that can self-renew and become immune cells. In some embodiments, immune progenitor cells include hematopoietic stem cells (HSCs) or their progeny. In some embodiments, immune progenitor cells include progenitor cells that can become immune cells. In some embodiments, immune progenitor cells include hematopoietic progenitor cells (HPCs). Hematopoietic stem cells (HSC)

[0137] Hematopoietic stem cells (HSCs) are multipotent, self-renewing cells. All blood cells, differentiated from 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] The HSCs of the present disclosure can be isolated or derived from primary or cultured stem cells. The HSCs of the present disclosure can be isolated or derived from embryonic stem cells, pluripotent stem cells, multipotent stem cells, adult stem cells, or induced pluripotent stem cells (iPSCs).

[0139] Immune progenitor cells of the present disclosure can include HSCs or HSC progeny. Non-limiting examples of representative HSC progeny of the present disclosure include pluripotent stem cells, lymphoid progenitor cells, natural killer (NK) cells, T lymphocytes (T cells), B lymphocytes (B cells), myeloid progenitor cells, neutrophils, basophils, eosinophils, monocytes, and macrophages.

[0140] HSCs generated by the disclosed methods can retain the characteristics of "primitive" stem cells, which share the characteristics of embryonic stem cells, while isolated or derived from adult stem cells and committed to a single cell lineage. For example, "primitive" HSCs generated by the disclosed methods retain their "stemness" and do not differentiate after differentiation. As a result, as adoptive cell therapy, "primitive" HSCs generated by the disclosed methods not only replenish their numbers but also expand in vivo. "Primitive" HSCs generated by the disclosed methods can be therapeutically effective when administered as a single dose. In some embodiments, primitive HSCs of the disclosed methods express CD34 + In some embodiments, the primitive HSCs of the present disclosure are CD34 + and CD38 - In some embodiments, the primitive HSCs of the present disclosure are CD34 + , CD38 - , and CD90 + In some embodiments, the primitive HSCs of the present disclosure are CD34 + , CD38 - , CD90 + , and CD45RA - In some embodiments, the primitive HSCs of the present disclosure are CD34 + , CD38 -, CD90 + , CD45RA - , and CD49f + In some embodiments, the primitive HSCs of the present disclosure are CD34 + , CD38 - , CD90 + , CD45RA - , and CD49f + is.

[0141] In some embodiments of the present disclosure, primitive HSCs, HSCs, and / or HSC progeny cells can be modified according to the methods of the present disclosure to express a foreign sequence (e.g., a chimeric antigen receptor or a therapeutic protein). In some embodiments of the present disclosure, the modified primitive HSCs, modified HSCs, and / or modified HSC progeny cells can be further differentiated to generate modified immune cells. Non-limiting examples of modified immune cells include the modified T cells, modified natural killer cells, and / or modified B cells of the present disclosure. T cells

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

[0143] Unlike traditional biologics and chemotherapeutic agents, the engineered T cells of the present disclosure have the ability to rapidly replicate upon antigen recognition, potentially avoiding the need for repeated treatment. To accomplish this, in some embodiments, the engineered T cells of the present disclosure not only drive an initial response but also persist in the patient as a stable population of viable memory T cells that prevent potential relapse. Alternatively, in some embodiments, the engineered T cells of the present disclosure do not survive in the patient, if desired.

[0144] Antigen receptor molecules that do not cause T cell exhaustion through antigen-independent (sustained) signaling and early memory T cells (especially stem cell memory T cells (T SCMThe stem cell-like engineered T cells of the present disclosure are characterized as central memory (T CM ) T cells or T CM -like cells, effector memory T cells (T EM ), and effector T cells (T E ) to induce naive T cells (T) and demonstrate the greatest potential for self-renewal and pluripotency to enhance tumor eradication and long-term engraftment of engineered T cells. N )>T SCM >T CM >T EM >T E >T TE A linear differentiation pathway, such as T N is T SCM These are the parent progenitor cells that directly generate T CM The T cell compositions of the present disclosure may be used to directly generate T SCM Cells or T CM The parental T cell subsets may include one or more of each of the parental T cell subsets in which the cells are most abundant (e.g., T SCM >T CM >T EM >T E >T TE ).

[0145] In some embodiments of the disclosed methods, immune cell precursors differentiate to form early memory T cells, stem cell-like T cells, naive T cells (T N ), T SCM , T CM , T EM , T E , or T TE In some embodiments, the immune cell precursor is a primitive HSC, HSC, or HSC progeny cell of the present disclosure.

[0146] In some embodiments of the disclosed methods, the immune cells include early memory T cells, stem cell-like T cells, naive T cells (T N ), T SCM , T CM , TEM , T E , or T TE is.

[0147] In some embodiments of the methods of the present disclosure, the immune cells are early memory T cells.

[0148] In some embodiments of the methods of the present disclosure, the immune cells are stem cell-like T cells.

[0149] In some embodiments of the disclosed methods, the immune cells are T SCM is.

[0150] In some embodiments of the disclosed methods, the immune cells are T CM is.

[0151] In some embodiments of the methods of the present disclosure, the method modifies T cells and / or the method generates 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 therebetween of the plurality of modified T cells express one or more cell surface markers of early memory T cells. In some embodiments, the plurality of modified early memory T cells comprises at least one modified stem cell-like T cell. In some embodiments, the plurality of modified early memory T cells expresses at least one modified T cell surface marker. SCM In some embodiments, the plurality of modified early memory T cells comprises at least one modified T CM Contains:

[0152] In some embodiments of the methods of the disclosure, the method modifies T cells, and / or the method generates 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 therebetween, of the plurality of modified T cells express one or more cell surface markers of stem cell-like T cells. In some embodiments, the plurality of modified stem cell-like T cells expresses at least one modified T SCM In some embodiments, the plurality of engineered stem cell-like T cells comprises at least one engineered T CM Contains:

[0153] In some embodiments of the methods of the disclosure, the method modifies T cells, and / or the method generates 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 therebetween of the plurality of modified T cells are stem and memory T cells (T SCM ) In some embodiments, the cell surface markers comprise one or more of CD62L and CD45RA. In some embodiments, the cell surface markers comprise one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95, and IL-2Rβ. In some embodiments, the cell surface markers comprise one or more of CD45RA, CD95, IL-2Rβ, CCR7, and CD62L.

[0154] In some embodiments of the methods of the disclosure, the method modifies T cells, and / or the method generates 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 therebetween of the plurality of modified T cells are central memory T cells (T CM ) In some embodiments, the cell surface markers include one or more of CD45RO, CD95, IL-2Rβ, CCR7, and CD62L.

[0155] In some embodiments of the methods of the disclosure, the method modifies T cells, and / or the method generates 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 therebetween of the plurality of modified T cells are naive T cells (T N ) In some embodiments, the cell surface markers include one or more of CD45RA, CCR7, and CD62L.

[0156] In some embodiments of the methods of the disclosure, the method modifies T cells, and / or the method generates 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 therebetween of the plurality of modified T cells are effector T cells (modified T EFF ) In some embodiments, the cell surface markers include one or more of CD45RA, CD95, and IL-2Rβ.

[0157] In some embodiments of the methods of the disclosure, the method modifies T cells, and / or the method generates 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 therebetween of the plurality of modified T cells are stem cell-like T cells, stem memory T cells (T SCM ), or central memory T cells (T CM ) express one or more cell surface markers.

[0158] In some embodiments of the disclosed methods, the buffer comprises immune cells or their precursors. The buffer maintains or enhances the level of cell viability and / or stem-like phenotype of the immune cells or their precursors (including T cells). In some embodiments, the buffer maintains or enhances the level of cell viability and / or stem-like phenotype of primary human T cells before nucleofection. In some embodiments, the buffer maintains or enhances the level of cell viability and / or stem-like phenotype of primary human T cells during nucleofection. In some embodiments, the buffer maintains or enhances the level of cell viability and / or stem-like phenotype of primary human T cells after nucleofection. In some embodiments, the buffer comprises one or more of KCl, MgCl, ClNa, glucose, and Ca(NO) in any absolute or relative amount or concentration, and optionally further comprises a supplement selected from the group consisting of HEPES, Tris / HCl, and phosphate buffer. In some embodiments, the buffer comprises 5 mM KCl, 15 mM MgCl, 90 mM ClNa, 10 mM glucose, and 0.4 mM Ca(NO). In some embodiments, the buffer comprises 5 mM KCl, 15 mM MgCl, 90 mM ClNa, 10 mM glucose, and 0.4 mM Ca(NO), with supplements comprising 20 mM HEPES and 75 mM Tris / HCl. In some embodiments, the buffer comprises 5 mM KCl, 15 mM MgCl, 90 mM ClNa, 10 mM glucose, and 0.4 mM Ca(NO), with supplements comprising 40 mM NaHPO / NaHPO at pH 7.2. In some embodiments, the composition comprising primary T cells comprises 100 μl of buffer and 5×10 6 ~25×10 6 In some embodiments, the composition contains an expanded ratio of 250 x 10 cells per milliliter of buffer or other medium during the transfer step. 6 Contains primary T cells.

[0159] In some embodiments of the methods of the present disclosure, the methods comprise contacting immune cells of the present disclosure (including T cells of the present disclosure) with a T cell expansion composition. In some embodiments of the methods of the present disclosure, the step of introducing a transposon and / or transposase of the present disclosure into an immune cell can further comprise contacting the immune cell with a T cell expansion composition. In some embodiments, including embodiments in which the introducing step of the method comprises an electroporation step or a nucleofection step, the electroporation step or the nucleofection step can be performed with the immune cell in contact with the T cell expansion composition of the present disclosure.

[0160] In some embodiments of the disclosed methods, the T cell expansion composition comprises, consists essentially of, or consists of phosphorus; one or more of octanoic acid, palmitic acid, linolenic acid, and oleic acid; sterols; and alkanes.

[0161] In some embodiments of the methods of generating modified T cells of the present disclosure, the growth supplement comprises one or more cytokines. The one or more cytokines can include any cytokine, non-limiting examples of which include lymphokines. Non-limiting examples of representative lymphokines include 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-γ (INFγ). The one or more cytokines can include IL-2.

[0162] In some embodiments of the disclosed methods, the T cell expansion composition comprises human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, and a proliferation supplement. In some embodiments of the methods, the T cell expansion composition further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD), diisopropyl adipate (DIPA), n-butyl-benzenesulfonamide, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl)ester, palmitic acid, linolenic acid, oleic acid, stearic acid hydrazide, oleamide, a sterol, and an alkane. In some embodiments of the methods, the T cell expansion composition further comprises one or more of octanoic acid, palmitic acid, linolenic acid, oleic acid, and a sterol. In some embodiments of this method, the T cell expansion composition further comprises octanoic acid at a concentration of 0.9 mg / kg to 90 mg / kg (inclusive); palmitic acid at a concentration of 0.2 mg / kg to 20 mg / kg (inclusive); linoleic acid at a concentration of 0.2 mg / kg to 20 mg / kg (inclusive); oleic acid at a concentration of 0.2 mg / kg to 20 mg / kg (inclusive); and a sterol at a concentration of 0.1 mg / kg to 10 mg / kg (inclusive). In some 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 some embodiments of this method, the T cell expansion composition further comprises octanoic acid at a concentration of 6.4 micromoles / kg to 640 micromoles / kg, inclusive; palmitic acid at a concentration of 0.7 micromoles / kg to 70 micromoles / kg, inclusive; linoleic acid at a concentration of 0.75 micromoles / kg to 75 micromoles / kg, inclusive; oleic acid at a concentration of 0.75 micromoles / kg to 75 micromoles / kg, inclusive; and sterol at a concentration of 0.25 micromoles / kg to 25 micromoles / kg, inclusive.In some embodiments of this method, the T cell expansion composition further comprises octanoic acid at a concentration of about 64 micromoles / kg (inclusive), palmitic acid at a concentration of about 7 micromoles / kg, linolenic acid at a concentration of about 7.5 micromoles / kg (inclusive), oleic acid at a concentration of about 7.5 micromoles / kg, and a sterol at a concentration of about 2.5 micromoles / kg.

[0163] In some embodiments, the T cell expansion composition comprises one or more of human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, and a proliferation supplement to generate a plurality of expanded engineered T cells, wherein at least 2% of the plurality of engineered T cells are early memory T cells, stem cell-like T cells, stem memory T cells (T SCM ), and / or central memory T cells (T CM) express one or more cell surface markers of T cell proliferation. In some embodiments, the T cell proliferation composition comprises or further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD), diisopropyl adipate (DIPA), n-butyl-benzenesulfonamide, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester, palmitic acid, linolenic acid, oleic acid, stearic acid hydrazide, oleamide, a sterol, and an alkane. In some embodiments, the T cell proliferation composition comprises one or more of octanoic acid, palmitic acid, linolenic acid, oleic acid, and a sterol (e.g., cholesterol). In some embodiments, the T cell expansion composition comprises one or more of octanoic acid at a concentration of 0.9 mg / kg to 90 mg / kg (inclusive), palmitic acid at a concentration of 0.2 mg / kg to 20 mg / kg (inclusive), linolenic acid at a concentration of 0.2 mg / kg to 20 mg / kg (inclusive), oleic acid at a concentration of 0.2 mg / kg to 20 mg / kg (inclusive), or a sterol at a concentration of 0.1 mg / kg to 10 mg / kg (inclusive) (mg / kg = parts per million). In some 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, linolenic 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 (mg / kg = parts per million). In some 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, linolenic acid at a concentration of 2.12 mg / kg, oleic acid at a concentration of about 2.13 mg / kg, and sterols at a concentration of 1.01 mg / kg (mg / kg = parts per million). In some 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, linolenic acid at a concentration of 2.12 mg / kg, oleic acid at a concentration of about 2.13 mg / kg, and sterols at a concentration of 1.01 mg / kg (mg / kg = parts per million).In some embodiments, the T cell expansion composition comprises one or more of octanoic acid at a concentration of 6.4 micromoles / kg to 640 micromoles / kg, inclusive; palmitic acid at a concentration of 0.7 micromoles / kg to 70 micromoles / kg, inclusive; linolenic acid at a concentration of 0.75 micromoles / kg to 75 micromoles / kg, inclusive; oleic acid at a concentration of 0.75 micromoles / kg to 75 micromoles / kg, inclusive; and a sterol at a concentration of 0.25 micromoles / kg to 25 micromoles / kg, inclusive. In some embodiments, the T cell expansion composition comprises one or more of octanoic acid at a concentration of about 64 micromoles / kg, palmitic acid at a concentration of about 7 micromoles / kg, linolenic acid at a concentration of about 7.5 micromoles / kg, oleic acid at a concentration of about 7.5 micromoles / kg, and a sterol at a concentration of about 2.5 micromoles / kg. In some embodiments, the T cell expansion composition comprises one or more of octanoic acid at a concentration of about 63.75 micromoles / kg, palmitic acid at a concentration of about 7.27 micromoles / kg, linolenic acid at a concentration of about 7.57 micromoles / kg, oleic acid at a concentration of about 7.56 micromoles / kg, and a sterol at a concentration of about 2.61 micromoles / kg. In some embodiments, the T cell expansion composition comprises octanoic acid at a concentration of about 63.75 micromoles / kg, palmitic acid at a concentration of about 7.27 micromoles / kg, linolenic acid at a concentration of about 7.57 micromoles / kg, oleic acid at a concentration of about 7.56 micromoles / kg, and a sterol at a concentration of about 2.61 micromoles / kg.

[0164] As used herein, the terms "supplemented T cell proliferation composition" or "T cell proliferation composition" can be used interchangeably with a medium comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, and a proliferation supplement at 37°C. Alternatively, or in addition, the terms "supplemented T cell proliferation composition" or "T cell proliferation composition" can be used interchangeably with a medium comprising one or more of phosphorus, octanoic fatty acid, palmitic fatty acid, linolenic fatty acid, and oleic acid. In some embodiments, the medium contains 10-fold more phosphorus than can be found in, for example, Iscove's Modified Dulbecco's Medium (IMDM; available from ThermoFisher Scientific as catalog number 12440053).

[0165] As used herein, the terms "supplemented T cell proliferation composition" or "T cell proliferation composition" can be used interchangeably with a medium containing one or more of human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iscove's MDM, and proliferation supplements at 37°C. Alternatively, or in addition, the terms "supplemented T cell proliferation composition" or "T cell proliferation composition" can be used interchangeably with a medium containing one or more of the following elements: boron, sodium, magnetite, phosphorus, potassium, and calcium. In some embodiments, the terms "supplemented T cell proliferation composition" or "T cell proliferation composition" can be used interchangeably with a medium containing one or more of the following elements present at corresponding average concentrations: 3.7 mg / L boron, 3000 mg / L sodium, 18 mg / L magnetite, 29 mg / L phosphorus, 15 mg / L potassium, and 4 mg / L calcium.

[0166] As used herein, the terms "supplemented T cell proliferation composition" or "T cell proliferation composition" can be used interchangeably with media containing one or more of human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, and proliferation supplements at 37° C. Alternatively, or in addition, the terms "supplemented T cell proliferation composition" or "T cell proliferation composition" can be used interchangeably with media containing 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-decyne-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), linolenic 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), sterols (e.g., cholesterol) (CAS No. 57-88-5), and alkanes (e.g., nonadecane) (CAS No. 629-92-5).In some embodiments, the terms "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably with a medium 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-decyne-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), linolenic 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), sterols (e.g., cholesterol) (CAS No. 57-88-5), alkanes (e.g., nonadecane) (CAS No. 629-92-5), and phenol red (CAS No. 143-74-8). In some embodiments, the terms "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably with a medium 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-decyne-4,7-diol (TMDD) (CAS No. 126-86-3), diisopropyl adipate (DIPA) (CAS No. 6938-94-9), n-butyl-benzenesulfonyl ester (NBS) (CAS No. 126-86-3), methylparaben (MRSA ... Sulfonamide (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), linolenic acid (CAS No. 60-33-3), oleic acid (CAS No. 112-80-1), stearic acid hydrazide (CAS No. 4130-54-5), oleamide (CAS No. 3322-62-1), phenol red (CAS No. 143-74-8), and lanolin alcohol.

[0167] In some embodiments, the terms "supplemented T cell proliferation composition" or "T cell proliferation composition" can be used interchangeably with media containing one or more of human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, and proliferation supplements at 37° C. Alternatively, or in addition, the terms "supplemented T cell proliferation composition" or "T cell proliferation composition" can be used interchangeably with media containing 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 proliferation composition" or "T cell proliferation composition" can be used interchangeably with a medium containing one or more of human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, and a proliferation supplement at 37° C. Alternatively, or in addition, the terms "supplemented T cell proliferation composition" or "T cell proliferation composition" can be used interchangeably with a medium containing one or more of the following free amino acids: histidine, asparagine, serine, glutamine, arginine, glycine, aspartic acid, glutamic acid, threonine, alanine, proline, cysteine, lysine, tyrosine, methionine, valine, isoleucine, leucine, phenylalanine, and tryptophan. In some embodiments, the terms "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably with a medium containing one or more of the following free amino acids at the corresponding average molar 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%), tryptophan (about 0.5%).In some embodiments, the terms "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably with a medium containing one or more of the following free amino acids at the corresponding average molar percentages: histidine (about 0.78%), asparagine (about 0.4%), serine (about 1.6%), glutamine (about 67.01%), arginine (about 1.67%), glycine (about 1.72%), aspartic acid (about 1.01%), arginine (about 1.01%), glycine ... 0.00%), glutamic acid (approx. 1.93%), threonine (approx. 2.38%), alanine (approx. 1.11%), proline (approx. 1.49%), cysteine ​​(approx. 1.65%), lysine (approx. 2.84%), tyrosine (approx. 1.62%), methionine (approx. 0.85%), valine (approx. 3.45%), isoleucine (approx. 3.14%), leucine (approx. 3.3%), phenylalanine (approx. 1.64%), tryptophan (approx. 0.37%).

[0169] As used herein, the terms "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably with a medium comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iscove's MDM, and a proliferation supplement at 37°C. Alternatively, or in addition, the terms "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably with a medium comprising one or more of phosphorus, octanoic fatty acid, palmitic fatty acid, linolenic fatty acid, and oleic acid. In some embodiments, the medium contains 10-fold more phosphorus than found in, for example, Iscove's Modified Dulbecco's Medium (IMDM) (available from ThermoFisher Scientific as catalog number 12440053).

[0170] In some embodiments, the terms "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably to refer to a medium comprising one or more of octanoic acid, palmitic acid, linolenic acid, oleic acid, and a sterol (e.g., cholesterol). In some embodiments, the terms "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably to refer to a medium comprising one or more of octanoic acid at a concentration of 0.9 mg / kg to 90 mg / kg (inclusive), palmitic acid at a concentration of 0.2 mg / kg to 20 mg / kg (inclusive), linolenic acid at a concentration of 0.2 mg / kg to 20 mg / kg (inclusive), oleic acid at a concentration of 0.2 mg / kg to 20 mg / kg (inclusive), and a sterol at a concentration of 0.1 mg / kg to 10 mg / kg (inclusive) (mg / kg = parts per million). In some embodiments, the terms "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably with a medium 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, linolenic 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 (mg / kg = parts per million). In some embodiments, the terms "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably with a medium 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, linolenic 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 (mg / kg = parts per million). In some embodiments, the terms "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably with a medium containing octanoic acid at a concentration of 9.19 mg / kg, palmitic acid at a concentration of 1.86 mg / kg, linolenic acid at a concentration of 2.12 mg / kg, oleic acid at a concentration of about 2.13 mg / kg, and sterols at a concentration of 1.01 mg / kg (mg / kg = parts per million).In some embodiments, the terms "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably with media comprising one or more of: octanoic acid at a concentration of 6.4 micromoles / kg to 640 micromoles / kg, inclusive; palmitic acid at a concentration of 0.7 micromoles / kg to 70 micromoles / kg, inclusive; linolenic acid at a concentration of 0.75 micromoles / kg to 75 micromoles / kg, inclusive; oleic acid at a concentration of 0.75 micromoles / kg to 75 micromoles / kg, inclusive; and sterols at a concentration of 0.25 micromoles / kg to 25 micromoles / kg, inclusive. In some embodiments, the terms "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably with a medium comprising one or more of octanoic acid at a concentration of about 64 micromoles / kg, palmitic acid at a concentration of about 7 micromoles / kg, linolenic acid at a concentration of about 7.5 micromoles / kg, oleic acid at a concentration of about 7.5 micromoles / kg, and sterols at a concentration of about 2.5 micromoles / kg.

[0171] In some embodiments, the terms "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably with a medium comprising one or more of octanoic acid at a concentration of about 63.75 micromoles / kg, palmitic acid at a concentration of about 7.27 micromoles / kg, linolenic acid at a concentration of about 7.57 micromoles / kg, oleic acid at a concentration of about 7.56 micromoles / kg, and a sterol at a concentration of about 2.61 micromoles / kg. In some embodiments, the terms "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably with a medium comprising one or more of octanoic acid at a concentration of about 63.75 micromoles / kg, palmitic acid at a concentration of about 7.27 micromoles / kg, linolenic acid at a concentration of about 7.56 micromoles / kg, oleic acid at a concentration of about 2.61 micromoles / kg.

[0172] The modified T cells of the present disclosure (e.g., stem cell-like T cells, T SCM and / or TCM In some embodiments of the method for generating the modified T cells of the present disclosure (the modified stem cell-like T cells of the present disclosure, T SCM and / or T CM (including, but not limited to, TWS119 (also known as GSK 3B inhibitor XII; chemical formula C)) can be cultured, grown, stored, or combined with growth medium containing one or more inhibitors of PI3K pathway components at any step of the procedural method. Non-limiting examples of representative inhibitors of PI3K pathway components include inhibitors of GSK3β (e.g., TWS119 (also known as GSK 3B inhibitor XII; chemical formula C) 18 H 14N4O2), CAS number 601514-19-6. Non-limiting examples of representative inhibitors of PI3K pathway components include bb007 (BLUEBIRDBIO™). Non-limiting examples of additional representative inhibitors of PI3K pathway components include allosteric Akt inhibitor VIII (also known as Akti-1 / 2, having compound number 10196499), ATP-competitive inhibitors (protein kinase C). Orthosteric inhibitors targeting the ATP-binding pocket of Akt (B), isoquinoline-5-sulfonamides (H-8, H-89, and NL-71-101), azepane derivatives (a series of structures derived from (-)-balanol), aminofurazan (GSK690693), heterocycles (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), phenylalanine derivatives, and phenylalanine derivatives. Lupyrazole derivatives (AT7867 and AT13148), thiophenecarboxamide derivatives (afuresertib (GSK2110183), 2-pyrimidyl-5-amidothiophene derivative (DC120), uprosertib (GSK2141795)), allosteric inhibitors (which offer greater specificity, fewer side effects, and less toxicity than orthosteric inhibitors), 2,3-diphenylquinoxaline analogs (2,3-diphenylquinoxaline derivatives, triazolo[3,4-f][1,6]naphthyridin-3(2H)-one derivative (MK-2206)), alkylphospholipids (edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine, ET-18-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-[(1-methyl-1H-pyrazol-4-yl)carbonyl]-N'-(3-bromophenylbromophenyl)-thiourea), purine derivatives (triciribine (TCN, NSC 154020), triciribine monophosphate active analogue (TCN-P), 4-amino-pyrido[2,3-d]pyrimidine derivative API-1, 3-phenyl-3H-imidazo[4,5-b]pyridine derivative, ARQ 092), BAY 1125976, 3-methyl-xanthine, quinoline-4-carboxamide and 2-[4-(cyclohexa-1,3-dien-1-yl)-1H-pyrazol-3-yl]phenol, 3-oxo-tirucallic acid, 3α- and 3β-acetoxy-tirucalic acid, acetoxy-tirucalic acid, irreversible inhibitors (antibiotics, lactoquinomycin, frenolicin B, kalafungin, medelmicin, Boc-Phe-vinyl ketone, 4-hydroxynonenal (4-HNE), 1,6-naphthyridinone derivatives, and imidazo-1,2-pyridine derivatives).

[0173] The modified T cells of the present disclosure (e.g., stem cell-like T cells, T SCM and / or T CM In some embodiments of the methods for generating T cells, the methods include contacting the modified T cells with an inhibitor of T cell effector differentiation. Non-limiting examples of representative inhibitors of T cell effector differentiation include BET inhibitors (e.g., the hyenotriazolodiazepine JQ1) and / or inhibitors of the BET family of proteins (e.g., BRD2, BRD3, BRD4, BRDT).

[0174] The modified T cells of the present disclosure (e.g., stem cell-like T cells, T SCM and / or T CM In some embodiments of the method for producing Acss1, the method includes contacting the modified T cell with an agent that reduces nuclear-cytoplasmic acetyl-CoA. Non-limiting examples of agents that reduce nuclear-cytoplasmic acetyl-CoA include 2-hydroxy-citrate (2-HC) and agents that increase expression of Acss1.

[0175] The modified T cells of the present disclosure (e.g., stem cell-like T cells, T SCM and / or T CM In some embodiments of the method for generating a histone deacetylase (HDAC), the method comprises contacting the modified T cells with a composition comprising a histone deacetylase (HDAC). In some embodiments, the composition comprising an HDAC inhibitor comprises or consists of valproic acid, sodium phenylbutyrate (NaPB), or a combination thereof. In some embodiments, the composition comprising an HDAC inhibitor comprises or consists of valproic acid. In some embodiments, the composition comprising an HDAC inhibitor comprises or consists of sodium phenylbutyrate (NaPB).

[0176] The modified T cells of the present disclosure (e.g., stem cell-like T cells, T SCM and / or T CMIn some embodiments of the method for producing IL-1, the activation supplement can include one or more cytokines. The one or more cytokines can include any cytokine, non-limiting examples of which include lymphokines. Non-limiting examples of representative lymphokines include 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-γ (INFγ). The one or more cytokines can include IL-2.

[0177] The modified T cells of the present disclosure (e.g., stem cell-like T cells, T SCM and / or T CM In some embodiments of the method for generating CD3, the activation supplement can comprise one or more activator complexes. Representative and non-limiting examples of activator complexes can include monomeric, dimeric, trimeric, or tetrameric antibody complexes that bind to one or more of CD3, CD28, and CD2. In some embodiments, the activation supplement comprises or consists of an activator complex comprising a human antibody, a humanized antibody, a recombinant antibody, or a chimeric antibody. In some embodiments, the activation supplement comprises or consists of an activator complex that binds to CD3 and CD28. In some embodiments, the activation supplement comprises or consists of an activator complex that binds to CD3, CD28, and CD2. Natural killer (NK) cells

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

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

[0180] In some embodiments, the non-activated NK cells are (CD14 / CD19 / CD56 + derived from CD3-depleted leukoreduction (including cells)

[0181] In some embodiments, NK cells are electroporated using a Lonza 4D Nucleofector or BTX ECM 830 (500 V, 700 microsecond pulse length, 0.2 mm electrode gap, 1 pulse). All Lonza 4D Nucleofector programs are considered within the scope of the disclosed methods.

[0182] In some embodiments, 5 x 10 ATP in 100 μl of P3 buffer in a cuvette per electroporation. 6 cells were electroporated, but this ratio of cells per volume is scalable to the scale of commercial manufacturing processes.

[0183] In some embodiments, the NK cells are stimulated by culturing with an additional cell line. In some embodiments, the additional cell line comprises an artificial antigen-presenting cell (aAPC). In some embodiments, the stimulation occurs on day 1, day 2, day 3, day 4, day 5, day 6, or day 7 after electroporation. In some embodiments, the stimulation occurs on day 2 after electroporation.

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

[0185] In some embodiments, the modified immune cells or immune precursor cells of the present disclosure are B cells. B cells are a type of lymphocyte that express a B cell receptor on the cell surface. The B cell receptor binds to a specific antigen.

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

[0187] In some embodiments, HSPCs are modified using the methods of the disclosure and then initiate differentiation into B cells in the 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 some embodiments, HSPCs are modified using the methods of the disclosure and then initiate differentiation into B cells in the presence of human IL-3, Flt3L, TPO, SCF, and G-CSF for 5 days.

[0188] In some embodiments, after priming, the modified HPSC cells are transferred to a layer of feeder cells and fed every two weeks, and transferred to a fresh feeder layer once a week. In some embodiments, the feeder cells are MS-5 feeder cells.

[0189] In some embodiments, the modified HPSC cells are cultured with MS-5 feeder cells for at least 7 days, 14 days, 21 days, 28 days, 30 days, 33 days, 35 days, 42 days, or 48 days. In some embodiments, the modified HPSC cells are cultured with MS-5 feeder cells for 33 days. Metastasis

[0190] Non-limiting examples of representative transposon / transposase systems of the present disclosure include piggyBac transposons and piggyBac transposases, piggyBac-like transposons and piggyBac-like transposases, Sleeping Beauty transposons and Sleeping Beauty transposases, Helraiser transposons and Helraiser transposases, and Tol2 transposons and Tol2 transposases.

[0191] The piggyBac transposase recognizes transposon-specific inverted terminal repeats (ITRs) at either end of the transposon and moves the contents between the ITRs into the TTAA chromosomal site. The piggyBac transposon system has no payload limitations regarding the gene of interest that can be included between the ITRs. In some embodiments, particularly in embodiments where the transposon is a piggyBac transposon, the transposase is a piggyBac transposase or a super-piggyBac (SPB) transposase. In some embodiments, particularly in embodiments where the transposon is a super-piggyBac (SPB) transposon, the sequence encoding the transposase is an mRNA sequence.

[0192] In some embodiments of the disclosed methods, the transposase enzyme is a PiggyBac (PB) transposase enzyme. 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 IRCLRMDKS 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 It may comprise or consist of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or any percentage therebetween identical to 541 PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV ICREHNIDMC QSCF (SEQ ID NO: 14487).

[0193] In some embodiments of the disclosed methods, the transposase enzyme has 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 IRCLRMDKS 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 A piggyBac (PB) transposase enzyme comprising or consisting of an amino acid sequence having an amino acid substitution at one or more of positions 30, 165, 282, or 538 of QSCF (SEQ ID NO: 14487).

[0194] In some embodiments, the transposase enzyme is a piggyBac (PB) transposase enzyme comprising or consisting of an amino acid sequence having an amino acid substitution at one or more of positions 30, 165, 282, or 538 of the sequence of SEQ ID NO: 14487. In some embodiments, the transposase enzyme is a piggyBac (PB) transposase enzyme comprising or consisting 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 some embodiments, the transposase enzyme is a piggyBac (PB) transposase enzyme comprising or consisting of an amino acid sequence having an amino acid substitution at each of positions 30, 165, 282, and 538 of the sequence of SEQ ID NO: 14487. In some embodiments, the amino acid substitution at position 30 of the sequence of SEQ ID NO: 14487 is an isoleucine (I) to valine (V). In some embodiments, the amino acid substitution at position 165 of the sequence of SEQ ID NO: 14487 is a glycine (G) to serine (S). In some embodiments, the amino acid substitution at position 282 of the sequence of SEQ ID NO: 14487 is a methionine (M) to valine (V). In some embodiments, the amino acid substitution at position 538 of the sequence of SEQ ID NO: 14487 is an asparagine (N) to lysine (K).

[0195] In some embodiments of the disclosed methods, the transposase enzyme is a Super-piggyBac (SPB) transposase enzyme. In some embodiments, the Super-piggyBac (SPB) transposase enzyme of the disclosure can comprise or consist of the amino acid sequence of SEQ ID NO: 14487, in which the amino acid substitution at position 30 is an isoleucine (I) to valine (V), the amino acid substitution at position 165 is a glycine (G) to serine (S), the substitution at position 282 is a methionine (M) to valine (V), and the amino acid substitution at position 538 is an asparagine (N) to lysine (K). In some embodiments, the Super-piggyBac (SPB) transposase enzyme is 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 IRCLRMDKS 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 It may comprise or consist of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or any percentage therebetween identical to 541 PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV ICREHNIDMC QSCF (SEQ ID NO: 14484).

[0196] In some embodiments of the disclosed methods, the piggyBac transposase enzyme or super-piggyBac transposase enzyme comprises a mutation at positions 3, 46, 82, 103, 119, 125, 177, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1190, 1250, 1770, The amino acid sequence may further comprise an amino acid substitution at one or more of the following positions: 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. In some embodiments, including embodiments in which the transposase comprises the above-described mutations at positions 30, 165, 282, and / or 538, the piggyBac transposase enzyme or super-piggyBac transposase enzyme can 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 of SEQ ID NO:14487 or SEQ ID NO:14484. In some embodiments, the amino acid substitution at position 3 of SEQ ID NO:14487 or SEQ ID NO:14484 is a serine (S) to asparagine (N) substitution. In some embodiments, the amino acid substitution at position 46 of SEQ ID NO:14487 or SEQ ID NO:14484 is an alanine (A) to serine (S) substitution. In some embodiments, the amino acid substitution at position 46 of SEQ ID NO:14487 or SEQ ID NO:14484 is an alanine (A) to threonine (T) substitution. In some embodiments, the amino acid substitution at position 82 of SEQ ID NO:14487 or SEQ ID NO:14484 is an isoleucine (I) to tryptophan (W) substitution. In some embodiments, the amino acid substitution at position 103 of SEQ ID NO:14487 or SEQ ID NO:14484 is a serine (S) to proline (P) substitution.In some embodiments, the amino acid substitution at position 119 of SEQ ID NO:14487 or SEQ ID NO:14484 is an arginine (R) to proline (P) substitution. In some embodiments, the amino acid substitution at position 125 of SEQ ID NO:14487 or SEQ ID NO:14484 is a cysteine ​​(C) to alanine (A) substitution. In some embodiments, the amino acid substitution at position 125 of SEQ ID NO:14487 or SEQ ID NO:14484 is a cysteine ​​(C) to leucine (L) substitution. In some embodiments, the amino acid substitution at position 177 of SEQ ID NO:14487 or SEQ ID NO:14484 is a tyrosine (Y) to lysine (K) substitution. In some embodiments, the amino acid substitution at position 177 of SEQ ID NO:14487 or SEQ ID NO:14484 is a tyrosine (Y) to histidine (H) substitution. In some embodiments, the amino acid substitution at position 180 of SEQ ID NO:14487 or SEQ ID NO:14484 is a phenylalanine (F) to leucine (L). In some embodiments, the amino acid substitution at position 180 of SEQ ID NO:14487 or SEQ ID NO:14484 is a phenylalanine (F) to isoleucine (I). In some embodiments, the amino acid substitution at position 180 of SEQ ID NO:14487 or SEQ ID NO:14484 is a phenylalanine (F) to valine (V). In some embodiments, the amino acid substitution at position 185 of SEQ ID NO:14487 or SEQ ID NO:14484 is a methionine (M) to leucine (L). In some embodiments, the amino acid substitution at position 187 of SEQ ID NO:14487 or SEQ ID NO:14484 is an alanine (A) to glycine (G). In some embodiments, the amino acid substitution at position 200 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a phenylalanine (F) to tryptophan (W). In some embodiments, the amino acid substitution at position 207 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a valine (V) to proline (P) substitution. In some embodiments, the amino acid substitution at position 209 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a valine (V) to phenylalanine (F).In some embodiments, the amino acid substitution at position 226 of SEQ ID NO:14487 or SEQ ID NO:14484 is a methionine (M) to phenylalanine (F). In some embodiments, the amino acid substitution at position 235 of SEQ ID NO:14487 or SEQ ID NO:14484 is a leucine (L) to arginine (R). In some embodiments, the amino acid substitution at position 240 of SEQ ID NO:14487 or SEQ ID NO:14484 is a valine (V) to lysine (K). In some embodiments, the amino acid substitution at position 241 of SEQ ID NO:14487 or SEQ ID NO:14484 is a phenylalanine (F) to leucine (L). In some embodiments, the amino acid substitution at position 243 of SEQ ID NO:14487 or SEQ ID NO:14484 is a proline (P) to lysine (K). In some embodiments, the amino acid substitution at position 258 of SEQ ID NO:14487 or SEQ ID NO:14484 is an asparagine (N) to serine (S) substitution. In some embodiments, the amino acid substitution at position 296 of SEQ ID NO:14487 or SEQ ID NO:14484 is a leucine (L) to tryptophan (W) substitution. In some embodiments, the amino acid substitution at position 296 of SEQ ID NO:14487 or SEQ ID NO:14484 is a leucine (L) to tyrosine (Y) substitution. In some embodiments, the amino acid substitution at position 296 of SEQ ID NO:14487 or SEQ ID NO:14484 is a leucine (L) to phenylalanine (F) substitution. In some embodiments, the amino acid substitution at position 298 of SEQ ID NO:14487 or SEQ ID NO:14484 is a methionine (M) to leucine (L) substitution. In some embodiments, the amino acid substitution at position 298 of SEQ ID NO:14487 or SEQ ID NO:14484 is a methionine (M) to alanine (A). In some embodiments, the amino acid substitution at position 298 of SEQ ID NO:14487 or SEQ ID NO:14484 is a methionine (M) to valine (V). In some embodiments, the amino acid substitution at position 311 of SEQ ID NO:14487 or SEQ ID NO:14484 is a proline (P) to isoleucine (I). In some embodiments, the amino acid substitution at position 311 of SEQ ID NO:14487 or SEQ ID NO:14484 is a proline (P) to valine.In some embodiments, the amino acid substitution at position 315 of SEQ ID NO:14487 or SEQ ID NO:14484 is an arginine (R) to lysine (K). In some embodiments, the amino acid substitution at position 319 of SEQ ID NO:14487 or SEQ ID NO:14484 is a threonine (T) to glycine (G). In some embodiments, the amino acid substitution at position 327 of SEQ ID NO:14487 or SEQ ID NO:14484 is a tyrosine (Y) to arginine (R). In some embodiments, the amino acid substitution at position 328 of SEQ ID NO:14487 or SEQ ID NO:14484 is a tyrosine (Y) to valine (V). In some embodiments, the amino acid substitution at position 340 of SEQ ID NO:14487 or SEQ ID NO:14484 is a cysteine ​​(C) to glycine (G). In some embodiments, the amino acid substitution at position 340 of SEQ ID NO:14487 or SEQ ID NO:14484 is a cysteine ​​(C) to leucine (L). In some embodiments, the amino acid substitution at position 421 of SEQ ID NO:14487 or SEQ ID NO:14484 is an aspartic acid (D) to histidine (H). In some embodiments, the amino acid substitution at position 436 of SEQ ID NO:14487 or SEQ ID NO:14484 is a valine (V) to isoleucine (I). In some embodiments, the amino acid substitution at position 456 of SEQ ID NO:14487 or SEQ ID NO:14484 is a methionine (M) to tyrosine (Y). In some embodiments, the amino acid substitution at position 470 of SEQ ID NO:14487 or SEQ ID NO:14484 is a leucine (L) to phenylalanine (F). In some embodiments, the amino acid substitution at position 485 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a serine (S) to lysine (K). In some embodiments, the amino acid substitution at position 503 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a methionine (M) to leucine (L). In some embodiments, the amino acid substitution at position 503 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a methionine (M) to isoleucine (I). In some embodiments, the amino acid substitution at position 552 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a valine (V) to lysine (K).In some embodiments, the amino acid substitution at position 570 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is an alanine (A) to threonine (T). In some embodiments, the amino acid substitution at position 591 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a glutamine (Q) to proline (P). In some embodiments, the amino acid substitution at position 591 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a glutamine (Q) to arginine (R).

[0197] In some embodiments of the disclosed methods, the piggyBac transposase enzyme can comprise an amino acid substitution, or the super-piggyBac transposase enzyme can further comprise an amino acid substitution, at one or more of positions 103, 194, 372, 375, 450, 509, and 570 of SEQ ID NO:14487 or SEQ ID NO:14484, including embodiments in which the transposase comprises the above-described mutations at positions 30, 165, 282, and / or 538. In some embodiments of the disclosed methods, the piggyBac transposase enzyme can comprise an amino acid substitution, or the super-piggyBac transposase enzyme can further comprise an amino acid substitution, at two, three, four, five, six, or more of positions 103, 194, 372, 375, 450, 509, and 570 of SEQ ID NO:14487 or SEQ ID NO:14484, including embodiments in which the transposase comprises the above-described mutations at positions 30, 165, 282, and / or 538. In some embodiments of the disclosed methods, the piggyBac transposase enzyme can comprise an amino acid substitution, or the super-piggyBac transposase enzyme can further comprise an amino acid substitution, at positions 103, 194, 372, 375, 450, 509, and 570 of SEQ ID NO: 14487 or SEQ ID NO: 14484, including embodiments in which the transposase comprises the above mutations at positions 30, 165, 282, and / or 538. In some embodiments, the amino acid substitution at position 103 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a serine (S) to proline (P) substitution. In some embodiments, the amino acid substitution at position 194 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a methionine (M) to valine (V) substitution. In some embodiments, the amino acid substitution at position 372 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is an arginine (R) to alanine (A). In some embodiments, the amino acid substitution at position 375 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a lysine (K) to alanine (A).In some embodiments, the amino acid substitution at position 450 of SEQ ID NO:14487 or SEQ ID NO:14484 is an aspartic acid (D) to asparagine (N). In some embodiments, the amino acid substitution at position 509 of SEQ ID NO:14487 or SEQ ID NO:14484 is a serine (S) to glycine (G). In some embodiments, the amino acid substitution at position 570 of SEQ ID NO:14487 or SEQ ID NO:14484 is an asparagine (N) to serine (S). In some embodiments, the piggyBac transposase enzyme can comprise a methionine (M) to valine (V) substitution at position 194 of SEQ ID NO:14487. In some embodiments, the piggyBac transposase enzyme can further comprise amino acid substitutions at positions 372, 375, and 450 of SEQ ID NO: 14487 or SEQ ID NO: 14484, including embodiments in which the piggyBac transposase enzyme can comprise a methionine (M) to valine (V) substitution at position 194 of SEQ ID NO: 14487, an arginine (R) to alanine (A) substitution at position 372 of SEQ ID NO: 14487, or a lysine (K) to alanine (A) substitution at position 375 of SEQ ID NO: 14487. In some embodiments, the piggyBac transposase enzyme can comprise a methionine (M) to valine (V) substitution at position 194 of SEQ ID NO: 14487, an arginine (R) to alanine (A) substitution at position 372 of SEQ ID NO: 14487, a lysine (K) to alanine (A) substitution at position 375 of SEQ ID NO: 14487, and an aspartic acid (D) to asparagine (N) substitution at position 450 of SEQ ID NO: 14487.

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

[0199] In some embodiments, particularly in embodiments where the transposon is a Sleeping Beauty transposon, the transposase is a Sleeping Beauty transposase or a hyperactive Sleeping Beauty transposase (SB100X).

[0200] In some embodiments of the disclosed methods, the Sleeping Beauty transposase enzyme is 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 SKVVAKWLKD NKVKVLEWPS QSPDLNPIEN LWAELKKRVR ARRPTNLTQL It contains an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or any percentage between thereof, identical to 301 HQLCQEEWAK IHPTYCGKLV EGYPKRLTQV KQFKGNATKY (SEQ ID NO: 14485).

[0201] In some embodiments of the disclosed methods, the hyperactive Sleeping Beauty (SB100X) transposase enzyme comprises: 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 SKVVAKWLKD NKVKVLEWPS QSPDLNPIEN LWAELKKRVR ARRPTNLTQL It contains an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or any percentage between thereof, identical to 301 HQLCQEEWAK IHPNYCGKLV EGYPKRLTQV KQFKGNATKY (SEQ ID NO: 14486).

[0202] The Helraiser transposon is transposed by the Helitron transposase. The Helitron transposase mobilizes the Helraiser transposon, an ancient element derived from the genome of bats and active approximately 30-36 million years ago. An example of a representative Helraiser transposon of the present disclosure is Helibat1, which is 1 TCCTATATAA TAAAAGAGAA ACATGCAAAT TGACCATCCC TCCGCTACGC TCAAGCCACG 61 CCCACCAGCC AATCAGAAGT GACTATGCAA ATTAACCCAA CAAAGATGGC AGTTAAATTT 121 GCATACGCAG GTGTCAAGCG CCCCAGGAGG CAACGGCGGC CGCGGGCTCC CAGGACCTTC 181 GCTGGCCCCG GGAGGCGAGG CCGGCCGCGC CTAGCCACAC CCGCGGGCTC CCGGGACCTT 241 CGCCAGCAGA GAGCAGAGCG GGAGAGCGGG CGGAGAGCGG GAGGTTTGGA GGACTTGGCA 301 GAGCAGGAGG CCGCTGGACA TAGAGCAGAG CGAGAGAGAG GGTGGCTTGG AGGGCGTGGC 361 TCCCTCTGTC ACCCCAGCTT CCTCATCACA GCTGTGGAAA CTGACAGCAG GGAGGAGGAA 421 GTCCCACCCC CACAGAATCA GCCAGAATCA GCCGTTGGTC AGACAGCTCT CAGCGGCCTG 481 ACAGCCAGGA CTCTCATTCA CCTGCATCTC AGACCGTGAC AGTAGAGGG TGGGACTATG 541 TCTAAAGAAC AACTGTTGAT ACAACGTAGC TCTGCAGCCG AAAGATGCCG GCGTTATCGA 601 CAGAAAATGT CTGCAGAGCA ACGTGCGTCT GATCTTGAAA GAAGGCGGCG CCTGCCAACAG 661 AATGTATCTG AAGAGCAGCT ACTGGAAAAA CGTCGCTCTG AAGCCGAAAA ACAGCGGCGT 721 CATCGACAGA AAATGTCTAA AGACCAACGT GCCTTTGAAG TTGAAAGAAG GCGGTGGCGA 781 CGACAGAATA TGTCTAGAGA ACAGTCATCA ACAAGTACTA CCAATACCGG TAGGAACTGC 841 CTTCTCAGCA AAAATGGAGT ACATGAGGAT GCAATTCTCG AACATAGTTG TGGTGGAATG 901 ACTGTTCGAT GTGAATTTTG CCTATCACTA AATTTCTCTG ATGAAAAACC ATCCGATGGG 961 AAATTTACTC GATGTTGTAG CAAAGGGAAA GTCTGTCCAA ATGATATACA TTTTCCAGAT 1021 TACCCGGCAT ATTTAAAAAG ATTAATGACA AACGAAGATT CTGACAGTAA AAATTTCATG 1081 GAAAATATTC GTTCCATAAA TAGTTCTTTT GCTTTTGCTT CCATGGGTGC AAATATTGCA 1141 TCGCCATCAG GATATGGGCC ATACTGTTTT AGAATACACG GACAAGTTTA TCACCGTACT 1201 GGAACTTTAC ATCCTTCGGA TGGTGTTTCT CGGAAGTTTG CTCAACTCTA TATTTTGGAT 1261 ACAGCCGAAG CTACAAGTAA AAGATTAGCA ATGCCAGAAA ACCAGGGCTG CTCAGAAAGA 1321 CTCATGATCA ACATCAACAA CCTCATGCAT GAAATAAATG AATTAACAAA ATCGTACAAG 1381 ATGCTACATG AGGTAGAAAA GGAAGCCCAA TCTGAAGCAG CAGCAAAAGG TATTGCTCCC 1441 ACAGAAGTAA CAATGGCGAT TAAATACGAT CGTAACAGTG ACCCAGGTAG ATATAATTCT 1501 CCCCGTGTAA CCGAGGTTGC TGTCATATTC AGAAACGAAG ATGGAGAACC TCCTTTTGAA 1561 AGGGACTTGC TCATTCATTG TAAACCAGAT CCCAATAATC CAAATGCCAC TAAAATGAAA 1621 CHAPTER TCCTGTTTCC CHAPTER GCATCACAT ATCCTATTCT TTTTCCACAT 1681 GGTGAAAAG GCTGGGGAAC AGATATTGCA TTAAGACTCA GAGACAACAG TGTAATCGAC 1741 AATAATTACT REPLACEMENT ATGTGT ARRANGEMENT GTCACACAAA TGCATTA TGGATTTCAT 1801 CTCTCTGTGC GGGACACGTT CAATCCTATT TTAAATGCAG GAAAATTAAC TCAACAGTTT 1861 ATTGTGGATT CATATTCAAA AATGGAGGCC AATCGGATAA ATTTCATCAA ACAAACCAA 1921 TCTAAGTTGA GAGTTGAAAA ATATAGTGGT TTGATGGATT ATCTCAAATC TAGATCTGAA 1981 AATGACAATG TGCCGATTGG TAAAATGATA ATACTTCCAT CATCTTTTGA GGGTAGTCCC 2041 AGAAATATGC AGCAGCGATA TCAGGATGCT ATGGCAATTG TAACGAAGTA TGGCAAGCCC 2101 COMMUNICATION ASSISTANT ATGCAACCCC AAATGGGCAG ATTATTCAAA ASSISTANT 2161 CGCTGGCAAA AAGTTGAAAA CAGACCTGAC TTGGTAGCCA GAGTTTTTAA TATTAAGCTG 2221 AATGCTCTTT TAAATGATAT ATGTAAATTC ASSISTANCE GCAAAGTAAT AGCTAAAAT 2281 CATGTCATTG AATTTCAGAA ACGCGGACTG CCTCACGCTC ACATATTATT GATATTAGAT 2341 AGTGAGTCCA AATTACGTTC AGAAGATGAC ATTGACCGTA TAGTTAAGGC AGAAATTCCA 2401 CONNECT AGTGTCCTCG ACTTTTTCAA ATTGTAAAAT CAATATGGT ACATGCCCA 2461 TGTGGAATAC AAAATCCAAAA TAGTCCATGT ATGGAAAATG GAAAATGTTC AAAGGGATAT 2521 CCAAAAGAAT TTCAAAATGC ANSWER AATATTGATG GATATCCCAA ATCAAAACGA 2581 AGATCTGGTA GCACCATGTC TATTGGAAT AAAGTTCG ATAACACTTG GATTGTCCCT 2641 TATAACCCGT ATTTGTGCCT TAAATATAAC TGTCATATAA ATGTTGAAGT CTGTGCATCA 2701 ATTAAAGTG TCAAATTATT ATTAAAATAC ATTAAAAG GGCACGATTG TGCAAATTATT 2761 CAAATTTCTG AAAAAAATAT TATCAATCAT AGGACTTCAT TGACTCCAGG 2821 TATGTGAGCG CTCCTGAGGC TGTTTGGAGA CTTTTTGCAA TGCGAATGCA TGACCAATCT 2881 CATGCAATCA CAAGATTAGC TATTCATTTG CCAAATGATC AGAATTTGTA TTTTCATACC 2941 GATGATTTTG CTGAAGTTTT AGATAGGGCT AAAAGGCATA ACTCGACTTT GATGGCTTGG 3001 TTCTTATTGA ATAGGAAGA TTCTGATGCA CGTAATTATT ATTATTGGGA GATTCCACAG 3061 CATTATGTGT TTAATAATTC TTTGTGGACA AAACGCCGAA AGGGTGGGAA TAAAGTATTA 3121 GGTAGACTGT TCACTGTGAG CTTTAGAGAA CCAGAACGAT ATTACCTTAG ACTTTTGCTT 3181 CTGCATGTAA AAGGTGCGAT AAGTTTTGAG GATCTGCGAA CTGTAGGAGG TGTAACTTAT 3241 GATACATTTC ATGAAGCTGC TAAACACCGA GGATTATTAC TTGATGACAC TATCTGGAAA 3301 GATACGATTG ACGATGCAAT CATCCTTAAT ATGCCCAAAC AACTACGGCA ACTTTTTGCA 3361 TATATATGTG TGTTTGGATG TCCTTCTGCT GCAGACAAAT TATGGGATGA GAATAAATCT 3421 HELP AAGATTTCTG TTGGAAATTA CACCGAAGAG AAGGTGCCTG TGTGAACTGT 3481 GAAATGCATG CCCTTAACGA AATTCAGGAG GTATTCACAT TGCATGGAAT GAAATGTCA 3541 CATTTCAAAC TTCCGGACTA TCCTTTATTA ATGAATGCAA ATACATGTGA TCAATTGTAC 3601 GAGCAACAAC AGGCAGAGGT TTTGATAAAT TCTCTGAATG ATGAACAGTT GGCAGCCTTT 3661 CAGACTATAA CTTCAGCCAT CGAACTCAA ACTGTACACC CCAAATGCTT TTTCTTGGAT 3721 GGTCCAGGTG GTAGTGGAAA AACATATCTG TATAAAGTTT TAACACATTA TATTAGAGGT 3781 CGTGGTGGTA CTGTTTTACC CACAGCATCT ACAGGAATTG CTGCAAATTT ACTTCTTGGT 3841 GGAAGAACCT TTCATTCCCA ATATAAATTA CCAATTCCAT TAAATGAAAC TTCAATTTCT 3901 AGACTCGATA TAAAGAGTGA AGTTGCTAAA ACCATTAAAA AGGCCCAACT TCTCATTATT 3961 GATGAATGCA CCATGGCATC CAGTCATGCT ATAAACGCCA TAGATAGATT ACTAAGAGAA 4021 ATTATGAATT TGAATGTTGC ATTTGGTGGG AAAGTTCTCC TTCTCGGAGG GGATTTTCGA 4081 CAATGTCTCA GTATTGTACC ACATGCTATG CGATCGGCCA TAGTACAAAC GAGTTTAAAG 4141 TACTGTAATG TTTGGGGATG TTTCAGAAAG TTGTCTCTTA AAACAAATAT GAGATCAGAG 4201 GATTCTGCTT ATAGTGAATG GTTAGTAAAA CTTGGAGATG GCAAACTTGA TAGCAGTTTT 4261 CATTTAGGAA TGGATATTAT TGAAATCCCC CATGAAATGA TTTGTAACGG ATCCATTATT 4321 GAAGCTACCT TTGGAAATAG TATATCTATA GATAATATTA AAAATATATC TAAACGTGCA 4381 ATTCTTTGTC CAAAAAATGA GCATGTTCAA AAATTAAATG AAGAAATTTT GGATATACTT 4441 GATGGAGATT TTCACACATA TTTGAGTGAT GATTCCATTG ATTCAACAGA TGATGCTGAA 4501 AAGGAAAATT TTCCCATCGA ATTTCTTAAT AGTATTACTC CTTCGGGAAT GCCGTGTCAT 4561 AAATTAAAT TGAAAGTGGG TGCAATCATC ATGCTATTGA GAAATCTTAA TAGTAAATGG 4621 GGTCTTTGTA ATGGTACTAG ATTTATTATC AAAAGATTAC GACCTAACAT TATCGAAGCT 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 TTGTTTTATT 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, Helitron transposase does not contain an RNase-H-like catalytic domain, but instead contains a RepHel motif consisting of a replication initiation domain (Rep) and a DNA helicase domain. The Rep domain is a nuclease domain of the HUH superfamily of nucleases.

[0204] An example of a representative Helitron transposase of the present disclosure is: 1 MSKEQLLIQR SSAAERCRRY RQKMSAEQRA SDLERRRRLQ QNVSEEQLLE KRRSEAEKQR 61 RHRQKMSKDQ RAFEVERRRW RRQNMSREQS STSTTNTGRN CLLSKNGVHE DAILEHSCGG 121 MTVRCEFCLS LNFSDEKPSD GKFTRCSKG 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 NKVVDNTWIV 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 1201 KYCNVWGCFR KLSLKTNMRS EDSAYSEWLV KLGDGKLDSS FHLGMDIIEI PHEMICNGSI 1261 IEATFGNSIS IDNIKNISKR AILCPKNEHV QKLNEEILDI LDGDFHTYLS DDSIDSTDDA 1321 EKENFPIEFL NSITPSGMPC HKLKLKVGAI IMLLRNLNSK WGLCNGTRFI IKRLRPNIIE 1381 AEVLTGSAEG EVVLIPRIDL SPSDTGLPFK LIRRQFPVMP AFAMTINKSQ GQTLDRVGIF It contains an amino acid sequence containing 1441 LPEPVFAHGQ LYVAFSRVRR ACDVKVKVVN TSSQGKLVKH SESVFTLNVV YREILE (SEQ ID NO: 14501).

[0205] In Helitron transposition, a hairpin near the 3' end of the transposon functions as a terminator. However, this hairpin can be bypassed by the transposase, resulting in transduction of adjacent sequences. In addition, Helraiser transposition generates a covalently closed circular intermediate. Furthermore, Helitron transposition may lack target site duplication. In the Helraiser sequence, the transposase is flanked by left and right terminal sequences, designated LTS (5' terminal sequence) and RTS (3' terminal sequence). These sequences end with a conserved 5'-TC / CTAG-3' motif. A 19-bp palindrome capable of forming a hairpin termination structure is located 11 nucleotides upstream of the RTS and consists of the sequence GTGCACGAATTTCGTGCACCGGGCCACTAG (SEQ ID NO: 14500).

[0206] The Tol2 transposon can be isolated from or derived from the medaka genome and may resemble transposons of the hAT family. A representative Tol2 transposon of the present disclosure is encoded by a sequence comprising approximately 4.7 kilobases and contains a gene encoding the Tol2 transposase. This gene contains four exons. An example of a representative Tol2 transposase of the present disclosure is 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 KVVCTTTDSG 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 It contains the amino acid sequence 601 NTPLPASAAC ERLFSTAGLL FSPKRARLDT NNFENQLLLK LNLRFYNFE (SEQ ID NO: 14502).

[0207] An example of a representative Tol2 transposon of the present disclosure includes an inverted repeat subterminal sequence and a Tol2 transposase, and is shown below: 1 CAGAGGTGTA AAGTACTTGA GTAATTTTAC TTGATTACTG TACTTAAGTA TTATTTTTGG 61 GGATTTTTAC TTTACTTGAG TACAATTAAA AATCAATACT TTTACTTTTA CTTAATTACA 121 TTTTTTTAGA AAAAAAAGTA CTTTTTACTC CTTACAATTT TATTTACAGT CAAAAAGTAC 181 TTATTTTTTG GAGATCACTT CATTCTATTT TCCCTTGCTA TTACCAAACC AATTGAATTG 241 CGCTGATGCC CAGTTTAATT TAAATGTTAT TTATTCTGCC TATGAAAATC GTTTTCACAT 301 TATATGAAAT TGGTCAGACA TGTTCATTGG TCCTTTGGAA GTGACGTCAT GTCACATCTA 361 TTACCACAAT GCACAGCACC TTGACCTGGA AATTAGGGAA ATTATAACAG TCAATCAGTG 421 GAAGAAAATG REPETITION GTGATTCATC AGCAGCTGCG AGCAGCACAG TCCAAAATCA 481 GCCACAGGAT CAAGAGCACC CGTGGCCGTA TCTTCGCGAA TTCTTTTCTT TAAGTGGTGT 541 AAATAAAGAT TCATTCAAGA TGAAATGTGT CCTCTGTCTC CCGCTTAATA AAGAAATATC 601 GGCCTTCAAA AGTTCGCCAT CAAACCTAAG REQUIREMENTS REQUIREMENTS 661 TTTGTTTTAC TGATAGTTTT TTTTTTTTT TTTTTTTTT TTTGGGTG TGCATGTTTT 721 GACGTTGATG GCGCGCCTTT TATATGTGTA GTAGGCCTAT TTTCACTAAT GCATGCGATT 781 GACAATAA GGCTCACGTA ATAAAATGCT AAAATGCATT TGTAATTGGT AACGTTAGGT 841 CCACGGGAAA TTTGGCGCCT ATTGCAGCTT TGAATAATCA TTATCATTCC GTGCTCTCAT 901 TGTGTTTGAA TTCATGCAAAA ACCAAGCGAG AAATTTTTTT CCAAACATGT 961 TGTATTGTCA AAACGGTAAC ACTTTACAAT GAGGTTGATT AGTTCATGTA TTAACTAACA 1021 TTAAATAACC ATGAGCAATA CATTTGTTAC TGTATCTGTT AATCTTTGTT AACGTTAGTT 1081 AATAGAAATA CAGATGTTCA TTGTTTGTTC ATGTTAGTTC ACAGTGCATT AACTAATGTT 1141 AACAAGATAT AAAGTATTAG TAAATGTTGA AATTAACATG TATACGTGCA GTTCATTATT 1201 AGTTCATGTT AACTAATGTA GTTAACTAAC GAACCTTATT GTAAAAGTGT TACCATCAAA 1261 ACTAATGTAA TGAAATCAAT TCACCCTGTC ATGTCAGCCT TACAGTCCTG TGTTTTTGTC 1321 AATATAATCA GAAATAAAAT TAATGTTTGA TTGTCACTAA ATGCTACTGT ATTTCTAAAA 1381 TCAACAAGTA TTTAACATTA TAAAGTGTGC AATTGGCTGC AAATGTCAGT TTTATTAAAG 1441 GGTTAGTCA CCCAAAAATG AAAATAATGT CATTAATGAC TCGCCCTCAT GTCGTTCCAA 1501 GCCCGTAAGA CCTCCGTTCA TCTTCAGAAC ACAGTTTAAG ATATTTTAGA TTTAGTCCGA 1561 GAGCTTTCTG TGCCTCCATT GAGAATGTAT GTACGGTATA CTGTCCATGT CCAGAAAGGT 1621 AATAAAAACA TCAAAGTAGT CCATGTGACA TCAGTGGGTT AGTTAGAATT TTTTGAAGCA 1681 TCGAATACAT TTTGGTCCAA AAATAACAAA ACCTACGACT TTATTCGGCA TTGTATTCTC 1741 TTCCGGGTCT GTTGTCAATC CGCGTTCACG ACTTCGCAGT GACGCTACAA TGCTGAATAA 1801 AGTCGTAGGT TTTGTTATTT TTGGACCAAA ATGTATTTTC GATGCTTCAA ATAATTCTAC 1861 CTAACCCACT GATGTTCACAT GGACTACTTT GATGTTTTTA TTACCTTTCT GGACATGGAC 1921 AGTATACCGT ACATACATTT TCAGTGGAGG GACAGAAAGC TCTCGGACTA AATCTAAAAAT 1981 ATCTTAAACT GTGTTCCGAA GATGAACGGA GGTGTTACGG GCTTGGAACG ACATGAGGGT 2041 GAGTCATTAA TGACATCTTT TCATTTTTGG GTGAACTAAC CCTTTAATGC TGTAATCAGA 2101 GAGTGTATGT GTAATTGTTA CATTTATTGC ATACAATATA AATATTTATT TGTTGTTTTT 2161 INTERRUPTG REPEAT ACCTCAAAAA CTACTCTAAA TTINTRODUCTION REVIEW 2221 GATCGGGACC TCCACCCATG CTTCCAGCAG TAAGCAACTG AAAGTTGACT CAGTTTTCCC 2281 AGTCAAACAT GTGTCTCCAG TCACTGTGAA CAAAGCTATA TTAAGGTACA TCATTCAAGG 2341 ACTTCATCCT TTCAGCACTG TTGATCTGCC ATCATTTAAA GAGCTGATTA GTACACTGCA 2401 GCCTGGCATT TCTGTCATTA CAAGGCCTAC TTTACGCTCC AAGATAGCTG AAGCTGCTCT 2461 CONNECTIVITY INTERSECTION CTGCTGCCAT INTEGRATED INTEGRATED INTEGRATED STORM 2521 GGATTGTTGG ACTGCACGTA GAAAGTCATT CATTGGTGTA ACTGCTCACT GGATCAACCC 2581 TGGAAGTCTT GAAAGACATT CCGCTGCACT TGCCTGCAAAA AGATTAATGG GCTCTCATAC 2641 TTTTGAGTA CTGGCCAGTG CCATGAATGA TATCCACTCA GAGTATGAAA TACGTGACAA 2701 GGTTGTTTGC ACAACCACAG ACAGTGGTTC CAACTTTATG AAGGCTTTCA GAGTTTTTGG 2761 TGTGGAAAAC AATGATATCG AGACTGAGGC AAGAAGGTGT GAAAGTGATG ACACTGATTC 2821 TGAAGGCTGT GGTGAGGGAA GTGATGGTGT GGAATTCCAA GATGCCTCAC GAGTCCTGGA 2881 CCAAGACGAT GGCTTCGAAT TCCAGCTACC AAAACATCAA AAGTGTGCCT GTCACTTACT 2941 TAACCTAGTC TCAAGCGTTG ATGCCCAAAA AGCTCTCTCA AATGAACACT ACAAGAAACT 3001 CTACAGATCT GTCTTTGGCA AATGCCAAGC TTTATGGAAT AAAAGCAGCC GATCGGCTCT 3061 AGCAGCTGAA GCTGTTGAAT CAGAAAGCCG GCTTCAGCTT TTAAGGCCAA ACCAAACGCG 3121 GTGGAATTCA ACTTTTATGG CTGTTGACAG AATTCTTCAA ATTTGCAAAG AAGCAGGAGA 3181 AGGCGCACTT CGGAATATAT GCACCTCTCT TGAGGTTCCA ATGTAAGTGT TTTTCCCCTC 3241 TATCGATGTA AACAAATGTG GGTTGTTTTT GTTTAATACT CTTTGATTAT GCTGATTTCT 3301 CCTGTAGGTT TAATCCAGCA GAAATGCTGT TCTTGACAGA GTGGGCCAAC ACAATGCGTC 3361 CAGTTGCAAA AGTACTCGAC ATCTTGCAAG CGGAAACGAA TACACAGCTG GGGTGGCTGC 3421 TGCCTAGTGT CCATCAGTTA AGCTTGAAAC TTCAGCGACT CCACCATTCT CTCAGGTACT 3481 GTGACCCACT TGTGGATGCC CTACAACAAG GAATCCAAAC ACGATTCAAG CATATGTTTG 3541 AAGATCCTGA GATCATAGCA GCTGCCATCC TTCTCCCTAA ATTTCGGACC TCTTGGACAA 3601 ATGATGAAAC CATCATAAAA CGAGGTAAAT GAATGCAAGC AACATACACT TGACGAATTC 3661 TAATCTGGGC AACCTTTGAG CCATACCAAA ATTATTCTTT TATTTATTTA TTTTTGCACT 3721 TTTTAGGAAT GTTATATCCC ATCTTTGGCT GTGATCTCAA TATGAATATT GATGTAAAGT 3781 ATTCTTGCAG CAGGTTGTAG TTATCCCTCA GTGTTTCTTG AAACCAAACT CATATGTATC 3841 ATATGTGGTT TGGAAATGCA GTTAGATTTT ATGCTAAAAT AAGGGATTTG CATGATTTTA 3901 GATGTAGATG ACTGCACGTA AATGTAGTTA ATGACAAAAT CCATAAAATT TGTTCCCAGT 3961 CAGAAGCCCC TCAACCAAAC TTTTCTTTGT GTCTGCTCAC TGTGCTTGTA GGCATGGACT 4021 ACATCAGAGT GCATCTGGAG CCTTTGGACC ACAAGAAGGA ATTGGCCAAC AGTTCATCTG 4081 ATGATGAAGA TTTTTTCGCT TCTTTGAAAC CGACAACACA TGAAGCCAGC AAAGAGTTGG 4141 ATGGATATCT GGCCTGTGTT TCAGACACCA GGGAGTCTCT GCTCACGTTT CCTGCTATTT 4201 GCAGCCTCTC TATCAAGACT AATACACCTC TTCCCGCATC GGCTGCCTGT GAGAGGCTTT 4261 TCAGCACTGC AGGATTGCTT TTCAGCCCCA AAAGAGCTAG GCTTGACACT AACAATTTTG 4321 AGAATCAGCT TCTACTGAAG TTAAATCTGA GGTTTTACAA CTTTGAGTAG CGTGTACTGG 4381 CATTAGATTG TCTGTCTTAT AGTTTGATAA TTAAATACAA ACAGTTCTAA AGCAGGATAA 4441 AACCTTGTAT GCATTTCATT TAATGTTTTT TGAGATTAAA AGCTTAAACA AGAATCTCTA 4501 GTTTTCTTTC TTGCTTTTAC TTTTACTTCC TTAATACTCA AGTACAATTT TAATGGAGTA 4561 CTTTTTTACT TTTACTCAAG TAAGATTCTA GCCAGATACT TTTACTTTTA ATTGAGTAAA 4621 ATTTTCCCTA AGTACTTGTA CTTTCACTTG AGTAAAATTT TTGAGTACTT TTTACACCTC 4681 TG (SEQ ID NO: 17007).

[0208] Non-limiting examples of representative transposon / transposase systems of the present disclosure include piggyBac transposons and piggyBac transposases, and piggyBac-like transposons and piggyBac-like transposases.

[0209] The piggyBac transposase and piggyBac-like transposases recognize transposon-specific inverted terminal repeats (ITRs) at both ends of the transposon and move the contents between the ITRs into the TTAA or TTAT chromosomal locus. The piggyBac or piggyBac-like transposon systems are not limited in payload for the gene of interest that may be contained between the ITRs.

[0210] In some embodiments, particularly in embodiments where the transposon is a piggyBac transposon, the transposase is a piggyBac transposase, a super-piggyBac (SPB) transposase, etc. In some embodiments, particularly in embodiments where the transposase is a piggyBac transposase, a super-piggyBac (SPB) transposase, etc., the sequence encoding the transposase is an mRNA sequence.

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

[0212] In some embodiments of the disclosed methods, the transposase enzyme is a piggyBac transposase enzyme or a piggyBac-like transposase enzyme. A piggyBac (PB) transposase enzyme or a piggyBac-like transposase enzyme is 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 IRCLRMDKS 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 It may comprise or consist of an amino acid sequence that is 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 therebetween, identical to 541 PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV ICREHNIDMC QSCF (SEQ ID NO: 14487).

[0213] In some embodiments of the disclosed methods, the transposase enzyme has 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 IRCLRMDKS 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 A piggyBac transposase enzyme or a piggyBac-like transposase enzyme comprising or consisting of an amino acid sequence having an amino acid substitution at one or more of positions 30, 165, 282, or 538 of QSCF (SEQ ID NO: 14487).

[0214] In some embodiments, the transposase enzyme is a piggyBac or piggyBac-like transposase enzyme comprising or consisting 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 some embodiments, the transposase enzyme is a piggyBac or piggyBac-like transposase enzyme comprising or consisting 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 some embodiments, the transposase enzyme is a piggyBac or piggyBac-like transposase enzyme comprising or consisting of an amino acid sequence having an amino acid substitution at each of positions 30, 165, 282, and 538 of the sequence of SEQ ID NO: 14487. In some embodiments, the amino acid substitution at position 30 of the sequence of SEQ ID NO: 14487 is a substitution of isoleucine (I) to valine (V). In some embodiments, the amino acid substitution at position 165 of the sequence of SEQ ID NO: 14487 is a substitution of glycine (G) to serine (S). In some embodiments, the amino acid substitution at position 282 of the sequence of SEQ ID NO: 14487 is a substitution of methionine (M) to valine (V). In some embodiments, the amino acid substitution at position 538 of the sequence of SEQ ID NO: 14487 is a substitution of asparagine (N) to lysine (K).

[0215] In some embodiments of the disclosed methods, the transposase enzyme is a Super-piggyBac (SPB) transposase enzyme or a piggyBac-like transposase enzyme. In some embodiments, the Super-piggyBac (SPB) transposase enzyme or piggyBac-like transposase enzyme of the disclosure can comprise or consist of the amino acid sequence of SEQ ID NO: 14487, in which the amino acid substitution at position 30 is an isoleucine (I) to valine (V), the amino acid substitution at position 165 is a glycine (G) to serine (S), the amino acid substitution at position 282 is a methionine (M) to valine (V), and the amino acid substitution at position 538 is an asparagine (N) to lysine (K). In some embodiments, the Super-piggyBac (SPB) transposase enzyme or piggyBac-like transposase enzyme is 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 IRCLRMDKS 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 It may comprise or consist of an amino acid sequence that is 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 therebetween, identical to 541 PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV ICREHNIDMC QSCF (SEQ ID NO: 14484).

[0216] In some embodiments of the disclosed methods, the piggyBac transposase enzyme, super piggyBac transposase enzyme, or piggyBac-like transposase enzyme comprises a mutation at positions 3, 46, 82, 103, 119, 125 of SEQ ID NO: 14487 or SEQ ID NO: 14484, including embodiments in which the transposase comprises a mutation at positions 30, 165, 282, and / or 538 as described above. , 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. In some embodiments, the piggyBac transposase enzyme, super piggyBac transposase enzyme, or piggyBac-like transposase enzyme comprises a mutation at positions 46, 119, 125, 177, 180, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 2 , 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 some embodiments, the amino acid substitution at position 3 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a serine (S) to asparagine (N) substitution. In some embodiments, the amino acid substitution at position 46 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is an alanine (A) to serine (S) substitution. In some embodiments, the amino acid substitution at position 46 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is an alanine (A) to threonine (T). In some embodiments, the amino acid substitution at position 82 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is an isoleucine (I) to tryptophan (W).In some embodiments, the amino acid substitution at position 103 of SEQ ID NO:14487 or SEQ ID NO:14484 is a serine (S) to proline (P) substitution. In some embodiments, the amino acid substitution at position 119 of SEQ ID NO:14487 or SEQ ID NO:14484 is an arginine (R) to proline (P) substitution. In some embodiments, the amino acid substitution at position 125 of SEQ ID NO:14487 or SEQ ID NO:14484 is a cysteine ​​(C) to alanine (A) substitution. In some embodiments, the amino acid substitution at position 125 of SEQ ID NO:14487 or SEQ ID NO:14484 is a cysteine ​​(C) to leucine (L) substitution. In some embodiments, the amino acid substitution at position 177 of SEQ ID NO:14487 or SEQ ID NO:14484 is a tyrosine (Y) to lysine (K) substitution. In some embodiments, the amino acid substitution at position 177 of SEQ ID NO:14487 or SEQ ID NO:14484 is a tyrosine (Y) to histidine (H) substitution. In some embodiments, the amino acid substitution at position 180 of SEQ ID NO:14487 or SEQ ID NO:14484 is a phenylalanine (F) to leucine (L) substitution. In some embodiments, the amino acid substitution at position 180 of SEQ ID NO:14487 or SEQ ID NO:14484 is a phenylalanine (F) to isoleucine (I) substitution. In some embodiments, the amino acid substitution at position 180 of SEQ ID NO:14487 or SEQ ID NO:14484 is a phenylalanine (F) to valine (V) substitution. In some embodiments, the amino acid substitution at position 185 of SEQ ID NO:14487 or SEQ ID NO:14484 is a methionine (M) to leucine (L) substitution. In some embodiments, the amino acid substitution at position 187 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is an alanine (A) to glycine (G). In some embodiments, the amino acid substitution at position 200 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a phenylalanine (F) to tryptophan (W). In some embodiments, the amino acid substitution at position 207 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a valine (V) to proline (P) substitution.In some embodiments, the amino acid substitution at position 209 of SEQ ID NO:14487 or SEQ ID NO:14484 is a valine (V) to phenylalanine (F). In some embodiments, the amino acid substitution at position 226 of SEQ ID NO:14487 or SEQ ID NO:14484 is a methionine (M) to phenylalanine (F). In some embodiments, the amino acid substitution at position 235 of SEQ ID NO:14487 or SEQ ID NO:14484 is a leucine (L) to arginine (R). In some embodiments, the amino acid substitution at position 240 of SEQ ID NO:14487 or SEQ ID NO:14484 is a valine (V) to lysine (K). In some embodiments, the amino acid substitution at position 241 of SEQ ID NO:14487 or SEQ ID NO:14484 is a phenylalanine (F) to leucine (L). In some embodiments, the amino acid substitution at position 243 of SEQ ID NO:14487 or SEQ ID NO:14484 is a proline (P) to lysine (K) substitution. In some embodiments, the amino acid substitution at position 258 of SEQ ID NO:14487 or SEQ ID NO:14484 is an asparagine (N) to serine (S) substitution. In some embodiments, the amino acid substitution at position 296 of SEQ ID NO:14487 or SEQ ID NO:14484 is a leucine (L) to tryptophan (W) substitution. In some embodiments, the amino acid substitution at position 296 of SEQ ID NO:14487 or SEQ ID NO:14484 is a leucine (L) to tyrosine (Y) substitution. In some embodiments, the amino acid substitution at position 296 of SEQ ID NO:14487 or SEQ ID NO:14484 is a leucine (L) to phenylalanine (F) substitution. In some embodiments, the amino acid substitution at position 298 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a methionine (M) to leucine (L). In some embodiments, the amino acid substitution at position 298 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a methionine (M) to alanine (A). In some embodiments, the amino acid substitution at position 298 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a methionine (M) to valine (V).In some embodiments, the amino acid substitution at position 311 of SEQ ID NO:14487 or SEQ ID NO:14484 is a proline (P) to isoleucine (I) substitution. In some embodiments, the amino acid substitution at position 311 of SEQ ID NO:14487 or SEQ ID NO:14484 is a proline (P) to valine substitution. In some embodiments, the amino acid substitution at position 315 of SEQ ID NO:14487 or SEQ ID NO:14484 is an arginine (R) to lysine (K) substitution. In some embodiments, the amino acid substitution at position 319 of SEQ ID NO:14487 or SEQ ID NO:14484 is a threonine (T) to glycine (G) substitution. In some embodiments, the amino acid substitution at position 327 of SEQ ID NO:14487 or SEQ ID NO:14484 is a tyrosine (Y) to arginine (R) substitution. In some embodiments, the amino acid substitution at position 328 of SEQ ID NO:14487 or SEQ ID NO:14484 is a tyrosine (Y) to valine (V). In some embodiments, the amino acid substitution at position 340 of SEQ ID NO:14487 or SEQ ID NO:14484 is a cysteine ​​(C) to glycine (G). In some embodiments, the amino acid substitution at position 340 of SEQ ID NO:14487 or SEQ ID NO:14484 is a cysteine ​​(C) to leucine (L). In some embodiments, the amino acid substitution at position 421 of SEQ ID NO:14487 or SEQ ID NO:14484 is an aspartic acid (D) to histidine (H). In some embodiments, the amino acid substitution at position 436 of SEQ ID NO:14487 or SEQ ID NO:14484 is a valine (V) to isoleucine (I). In some embodiments, the amino acid substitution at position 456 of SEQ ID NO:14487 or SEQ ID NO:14484 is a methionine (M) to tyrosine (Y). In some embodiments, the amino acid substitution at position 470 of SEQ ID NO:14487 or SEQ ID NO:14484 is a leucine (L) to phenylalanine (F). In some embodiments, the amino acid substitution at position 485 of SEQ ID NO:14487 or SEQ ID NO:14484 is a serine (S) to lysine (K). In some embodiments, the amino acid substitution at position 503 of SEQ ID NO:14487 or SEQ ID NO:14484 is a methionine (M) to leucine (L).In some embodiments, the amino acid substitution at position 503 of SEQ ID NO:14487 or SEQ ID NO:14484 is a methionine (M) to isoleucine (I). In some embodiments, the amino acid substitution at position 552 of SEQ ID NO:14487 or SEQ ID NO:14484 is a valine (V) to lysine (K). In some embodiments, the amino acid substitution at position 570 of SEQ ID NO:14487 or SEQ ID NO:14484 is an alanine (A) to threonine (T). In some embodiments, the amino acid substitution at position 591 of SEQ ID NO:14487 or SEQ ID NO:14484 is a glutamine (Q) to proline (P). In some embodiments, the amino acid substitution at position 591 of SEQ ID NO:14487 or SEQ ID NO:14484 is a glutamine (Q) to arginine (R).

[0217] In some embodiments of the disclosed methods, the piggyBac transposase enzyme or piggyBac-like transposase enzyme can comprise an amino acid substitution, or the super-piggyBac transposase enzyme can further comprise an amino acid substitution, at one or more of positions 103, 194, 372, 375, 450, 509, and 570 of SEQ ID NO:14487 or SEQ ID NO:14484, including embodiments in which the transposase comprises the above-described mutations at positions 30, 165, 282, and / or 538. In some embodiments of the disclosed methods, the piggyBac or piggyBac-like transposase enzyme can comprise an amino acid substitution, or the super-piggyBac transposase enzyme can further comprise an amino acid substitution, at two, three, four, five, six, or more of positions 103, 194, 372, 375, 450, 509, and 570 of SEQ ID NO:14487 or SEQ ID NO:14484, including embodiments in which the transposase comprises the above-described mutations at positions 30, 165, 282, and / or 538. In some embodiments of the disclosed methods, the piggyBac or piggyBac-like transposase enzyme can comprise an amino acid substitution, or the super-piggyBac transposase enzyme can further comprise an amino acid substitution, at positions 103, 194, 372, 375, 450, 509, and 570 of SEQ ID NO: 14487 or SEQ ID NO: 14484, including embodiments in which the transposase comprises the above-described mutations at positions 30, 165, 282, and / or 538. In some embodiments, the amino acid substitution at position 103 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a serine (S) to proline (P) substitution. In some embodiments, the amino acid substitution at position 194 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a methionine (M) to valine (V) substitution. In some embodiments, the amino acid substitution at position 372 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is an arginine (R) to alanine (A). In some embodiments, the amino acid substitution at position 375 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a lysine (K) to alanine (A).In some embodiments, the amino acid substitution at position 450 of SEQ ID NO:14487 or SEQ ID NO:14484 is an aspartic acid (D) to asparagine (N). In some embodiments, the amino acid substitution at position 509 of SEQ ID NO:14487 or SEQ ID NO:14484 is a serine (S) to glycine (G). In some embodiments, the amino acid substitution at position 570 of SEQ ID NO:14487 or SEQ ID NO:14484 is an asparagine (N) to serine (S). In some embodiments, the piggyBac transposase enzyme can comprise a methionine (M) to valine (V) substitution at position 194 of SEQ ID NO:14487. In some embodiments, the piggyBac transposase enzyme can further comprise amino acid substitutions at positions 372, 375, and 450 of SEQ ID NO: 14487 or SEQ ID NO: 14484, including embodiments in which the piggyBac transposase enzyme can comprise a methionine (M) to valine (V) substitution at position 194 of SEQ ID NO: 14487, an arginine (R) to alanine (A) substitution at position 372 of SEQ ID NO: 14487, or a lysine (K) to alanine (A) substitution at position 375 of SEQ ID NO: 14487. In some embodiments, the piggyBac transposase enzyme can comprise a methionine (M) to valine (V) substitution at position 194 of SEQ ID NO: 14487, an arginine (R) to alanine (A) substitution at position 372 of SEQ ID NO: 14487, a lysine (K) to alanine (A) substitution at position 375 of SEQ ID NO: 14487, and an aspartic acid (D) to asparagine (N) substitution at position 450 of SEQ ID NO: 14487.

[0218] In some embodiments, the piggyBac transposase enzyme or piggyBac-like transposase enzyme is isolated or derived from an insect. In some embodiments, the insect is selected from the group consisting of Trichoplusia ni (nettle looper) (GenBank Accession No. AAA87375; SEQ ID NO: 16796), Argyrogramma agnata (honeycomb looper) (GenBank Accession No. GU477713; SEQ ID NOs: 14534, 16797), Anopheles gambiae (anopheles mosquito) (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 (cotton aphid) (GenBank Accession No. GU329918; SEQ ID NOs: 16801, 16802), Acyrthosiphon pisum (pea aphid) (GenBank accession number XP_001948139; SEQ ID NO: 16803), Agrotis ipsilon (cutworm moth) (GenBank accession number GU477714; SEQ ID NO: 14537, SEQ ID NO: 16804), Bombyx mori (silkworm) (GenBank accession number BAD11135; SEQ ID NO: 14505), Chilo suppressalis (rice suppressor moth) (GenBank accession number JX294476; SEQ ID NO: 16805, SEQ ID NO: 16806), Drosophila melanogaster (fruit fly) (GenBank accession number AAL39784; SEQ ID NO: 16807), Helicoverpa armigera (cotton bollworm) (GenBank accession number ABS18391; SEQ ID NO: 14525), Heliothis virescens (false tobacco budworm) (GenBank accession no. ABD76335; SEQ ID NO: 16808), Macdunnoughia crassisigna (large silvergrass moth) (GenBank accession no. EU287451; SEQ ID NO: 16809, SEQ ID NO: 16810), Pectinophora gossypiella (pink bollworm) (GenBank accession no. GU270322;SEQ ID NO: 14530, SEQ ID NO: 16811), Tribolium castaneum (red flour beetle) (GenBank accession number XP_001814566; SEQ ID NO: 16812), Ctenoplusia agnata (honey looper) (also known as Argyrogramma agnata), Messour bouvieri (ant), Megachile rotundata (monk's leafcutter), Bombus impatiens (bumblebee), Mamestra brassicae (army moth), Mayetiola destructor (Hessian fly), or Apis mellifera (western honeybee);

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

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

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

[0222] In some embodiments, the piggyBac transposase enzyme or piggyBac-like transposase enzyme is isolated from or derived from a vertebrate, such as Xenopus tropicalis (GenBank Accession No. BAF82026; SEQ ID NO: 14518), Homo sapiens (GenBank Accession No. NP_689808; SEQ ID NO: 16814), Mus musculus (mouse) (GenBank Accession No. NP_741958; SEQ ID NO: 16815), Macaca fascicularis (cynomolgus monkey) (GenBank Accession No. AB179012; SEQ ID NOs: 16816, 16817), Rattus norvegicus (rat) (GenBank Accession No. XP_220453; SEQ ID NO: 16818), or Myotis lucifugus (little brown bat).

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

[0224] In some embodiments, the piggyBac transposase enzyme or piggyBac-like transposase enzyme is the sequence 5'-TTAT-3' within the chromosomal site (TTAT target sequence).

[0225] In some embodiments, the piggyBac transposase enzyme or piggyBac-like transposase enzyme is the sequence 5'-TTAA-3' within the chromosomal site (TTAA target sequence).

[0226] In some embodiments, the target sequence of a piggyBac transposon or piggyBac-like transposon is 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'-AAA and 5'-TTTT-3'.

[0227] In some embodiments of the disclosed methods, the transposase enzyme is a piggyBac transposase enzyme or a piggyBac-like transposase enzyme. In some embodiments, the piggyBac transposase enzyme or piggyBac-like transposase enzyme is isolated from or derived from Bombyx mori. The piggyBac transposase enzyme or piggyBac-like transposase enzyme is 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 FDVVNLEVYA GKQPSGPYAV SNRPFEVVER LIQPVARSHR 361 NVTFDNWFTG YELMLHLLNE YRLTSVGTVR KNKRQIPESF IRTDRQPNSS VFGFQKDITL 421 VSYAPKKNKV VVVMSTMHHD NSIDESTGEK QKPEMITFYN STKAGVDVVD ELSANYNVSR 481 NSKRWPMTLF YGVLNMAAIN ACIIYRANKN VTIKRTEFIR SLGLSMIYEH LHSRNKKKNI 541 PTYLRQRIEK QLGEPSPRHV NVPGRYVRCQ DCPYKKDRKT KHSCNACAKP ICMEHAKFLC It can comprise or consist of an amino acid sequence that is 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 therebetween, identical to 601 ENCAELDSSL (SEQ ID NO: 14504).

[0228] piggyBac (PB) transposase enzyme or PiggyBac-like transposase enzyme 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 FYVVNLEVYA GKQPSGPYAV SNRPFEVVER LIQPVARSHR 361 NVTFDNWFTG YELMLHLLNE YRLTSVGTVR KNKRQIPESF IRTDRQPNSS VFGFQKDITL 421 VSYAPKKNKV VVVMSTMHHD NSIDESTGEK QKPEMITFYN STKAGVDVVD ELCANYNVSR 481 NSKRWPMTLF YGVLNMAAIN ACIIYRTNKN VTIKRTEFIR SLGLSMIYEH LHSRNKKKNI 541 PTYLRQRIEK QLGEPSPRHV NVPGRYVRCQ DCPYKKDRKT KRSCNACAKP ICMEHAKFLC It can comprise or consist of an amino acid sequence that is 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 therebetween, identical to 601 ENCAELDSSL (SEQ ID NO: 14505).

[0229] In some embodiments, the piggyBac transposase or piggyBac-like transposase is fused to a nuclear localization signal. In some embodiments, the piggyBac transposase or piggyBac-like transposase fused to a nuclear localization signal is 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 gtccggaac 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 tgccccgtaca aaaaggaccg caaaaccaaa agatcgtgta acgcgtgcgc caaacctatc Encoded by a polynucleotide sequence comprising 1801 tgcatggagc atgccaaatt tctgtgtgaa aattgtgctg aactcgattc ctccctg (SEQ ID NO: 14629).

[0230] In some embodiments, the piggyBac transposase or piggyBac-like transposase is hyperactive. A hyperactive piggyBac transposase or piggyBac-like transposase is a transposase that is more active than the naturally occurring variant from which it is derived. In some embodiments, the hyperactive piggyBac transposase or piggyBac-like transposase is isolated from or derived from Bombyx mori. In some embodiments, the piggyBac transposase or piggyBac-like transposase is a hyperactive variant of sequence 14505. In some embodiments, the hyperactive piggyBac transposase or piggyBac-like transposase is 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 SNRPFEVVER LIQPVARSHR 361 NVTFDNWFTG YELMLHLLNE YRLTSVGTVR KNKRQIPESF IRTDRQPNSS VFGFQKDITL 421 VSYAPKKNKV VVVMSTMHHD NSIDESTGEK QKPEMITFYN STKAGVDVVD ELCANYNVSR 481 NSKRWPMTLF YGVLNMAAIN ACIIYRTNKN VTIKRTEFIR SLGLSMIYEH LHSRNKKKNI 541 PTYLRQRIEK QLGEPSPRHV NVPGRYVRCQ DCPYKKDRKT KRSCNACAKP ICMEHAKFLC 601 ENCAELDSHL (SEQ ID NO: 14576) contains a sequence that is at least 90% identical to ENCAELDSHL.

[0231] In some embodiments, the hyperactive piggyBac transposase or piggyBac-like transposase comprises SEQ ID NO: 14576. In some embodiments, the hyperactive piggyBac transposase or piggyBac-like transposase comprises 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 SNRPFEVVER LIQPVARSHR 361 NVTFDNWFTG YEVMLHLLNE YRLTSVGTVR KNKRQIPESF IRTDRQPNSS VFGFQKDITL 421 VSYAPKKNKV VVVMSTMHHD NSIDESTGEK QKPEMITFYN STKAGVDVVD ELCANYNVSR 481 NSKRWPMTLF YGVLNMAAIN ACIIYRTNKN VTIKRTEFIR SLGLSMIYEH LHSRNKKKNI 541 PTYLRQRIEK QLGEPSPRHV NVPGRYVRCQ DCPYKKDRKT KRSCNACAKP ICMEHAKFLC 601 ENCAHLDS (SEQ ID NO: 14630).

[0232] In some embodiments, the hyperactive piggyBac transposase or piggyBac-like transposase is 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 SNRPFEVVER LIQPVARSHR 361 NVTFDNWFTG YELMLHLLNE YRLTSVGTVR KNKRQIPESF IRTDRQPNSS VFGFQKDITL 421 VSYAPKKNKV VVVMSTMHHD NSIDESTGEK QKPEMITFYN STKAGVDVVD ELCANYNVSR 481 NSKRWPMTLF YGVLNMAAIN ACIIYRTNKN VTIKRTEFIR SLGLSMIYEH LHSRNKKKNI 541 PTYLRQRIAM QLGEPSPRHV NVPGRYVRCQ DCPYKKDRKT KRSCNACAKP ICMEHAKFLC 601 ENCAELDSSL (SEQ ID NO: 14631).

[0233] In some embodiments, the hyperactive piggyBac transposase or piggyBac-like transposase is 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 SNRPFEVVER LIQPVARSHR 361 NVTFDNWFTG YELMLHLLNE YRLTSVGTVR KNKTQIPENF IRTDRQPNSS VFGFQKDITL 421 VSYAPKKNKV VVVMSTMHHD NSIDESTGEK QKPEMITFYN STKAGVDVVD ELQANYNVSR 481 NSKRWPMTLF YGVLNMAAIN ACIIYRTNKN VTIKRTEFIR SLGLSMIYEH LHSRNKKKNI 541 PTYLRQRIEK QLGEPSPRHV NVPGRYVRCQ DCPYKKDRKT KRSCNACAKP ICMEHAKFLC 601 ENCAELDSSL (SEQ ID NO: 14632).

[0234] In some embodiments, the hyperactive piggyBac transposase or piggyBac-like transposase is 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 SNRPFEVVER LIQPVARSHR 361 NVTFDNWFTG YELMLHLLNE YRLTSVGTVR KNKRQIPESF IRTDRQPNSS VFGFQKDITL 421 VSYAPKKNKV VVVMSTMHHD NSIDESTGEK QKPEMITFYN STKAGVDVVD ELCANYNVSR 481 NSKRWPMTLF YGVLNMAAIN ACIIYRTNKN VTIKRTEFIR SLGLSMIYEH LHSRNKKKNI 541 PTYLRQRIEK QLGEPSPRHV NVPGRYVRCQ DCPYKKDRKT KRSCNACAKP ICMEHAKFLC 601 ENCAELDSSL (SEQ ID NO: 14633) sequence.

[0235] In some embodiments, the hyperactive piggyBac transposase or piggyBac-like transposase is 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 DYVVNLEVYA GKQPSGPYAV SNRPFEVVER LIQPVARSHR 361 NVTFDNWFTG YELMLHLLNE YRLTSVGTVR KNKRQIPESF IRTDRQPNSS VFGFQKDITL 421 VSYAPKKNKV VVVMSTMHHD NSIDESTGEK QKPEMITFYN STKAGVDVVD ELCANYNVSR 481 NSKRWPMTLF YGVLNMAAIN ACIIYRTNKN VTIKRTEFIR SLGLSMIYEH LHSRNKKKNI 541 PTYLRQRIEK QLGEPSSRHV NVKGRYVRCQ DCPYKKDRKT KRSCNACAKP ICMEHAKFLC 601 ENCAELDSSL (SEQ ID NO: 14634) sequence.

[0236] In some embodiments, the hyperactive piggyBac transposase or piggyBac-like transposase is more active than the transposase of sequence 14505. In some embodiments, the hyperactive piggyBac transposase or piggyBac-like transposase is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or any percentage in between, identical to sequence 14505.

[0237] In some embodiments, the hyperactive piggyBac transposase or piggyBac-like transposase comprises an amino acid substitution at positions 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, 523, 527, 528, 543, 549, 550, 557, 601, 605, 607, 609, 610, or a combination thereof (relative to SEQ ID NO: 14505). In some embodiments, the hyperactive piggyBac transposase or piggyBac-like transposase is selected from the group consisting of Q92A, V93L, V93M, P96G, F97H, F97C, H165E, H165W, E178S, E178H, C189P, A196G, L200I, A201Q, L211A, W215Y, G219S, Q235Y, Q235G, Q23 8L, K246I, K253V, M258V, F261L, S263K, C271S, N303R, F321W, F321D, V324K, V324H, A330V, L373C, L 373V, V389L, S399N, R402K, T403L, D404Q, D404S, D404M, N441R, G448W, E449A, V469T, C473Q, R484K The amino acid substitutions include T507C, G523A, I527M, Y528K Y543I, E549A, K550M, P557S, E601V, E605H, E605W, D607H, S609H, L610I, or any combination thereof.In some embodiments, the hyperactive piggyBac transposase or piggyBac-like transposase is selected from the group consisting of Q92A, V93L, V93M, P96G, F97H, F97C, H165E, H165W, E178S, E178H, C189P, A196G, L200I, A201Q, L211A, W215Y, G219S, Q235Y, Q235G, Q23 8L, K246I, K253V, M258V, F261L, S263K, C271S, N303R, F321W, F321D, V324K, V324H, A330V, L373C, L 373V, V389L, S399N, R402K, T403L, D404Q, D404S, D404M, N441R, G448W, E449A, V469T, C473Q, R484K It contains the following amino acid substitutions: T507C, G523A, I527M, Y528K Y543I, E549A, K550M, P557S, E601V, E605H, E605W, D607H, S609H, and L610I.

[0238] In some embodiments, the hyperactive piggyBac transposase or piggyBac-like transposase comprises one or more substitutions of a non-wild-type amino acid, wherein the one or more amino acid substitutions with a wild-type amino acid are selected from the group consisting of (relative to SEQ ID NO: 14505): 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, Q8X, Q9X, Q10X, Q11X, Q12X, Q13X, Q14X, Q15X, Q16X, Q17X, Q18X, Q19X, Q20X, Q21X, Q22X, Q23X, Q24X, Q25X, Q26X, Q27X, Q28X, Q29X, Q30X, Q31X, Q32X, Q33X, Q34X, Q35X, Q36X, Q37X, Q38X, Q39X, Q41X, Q42X, Q43X, Q44X, Q45X, Q46X, Q46X, Q48X, Q49X, Q58X, Q59X, Q51X, Q52X, Q53X, Q54X, Q55X, Q56X, Q57X, Q58X, Q59X, Q60X, Q61X, Q62X, Q63X 5X, A87X, S88X, Q92X, V93X, S94X, G95X, P96X, F97X, Y98X, T99X, I145X, S 149X, D150X, L152X, E154X, T157X, N160X, S161X, S162X, H165X, R166X, T 168X, K169X, T170X, A171X, E173X, S175X, S176X, E178X, T179X, M183X, Q 184X, T186X, T187X, L188X, C189X, L194X, I195X, A196X, L198X, L200X, A 201X, 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, 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, P4 07X, 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, E 471X, L472X, C473X, A474X, K483X, W485X, T488X, L489X, Y491X, G492X, V493X, M496X, I499X, C502X, I503X, T507X, K50 9X, 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, H and / or L610X substitutions. A list of highly active amino acid substitutions can be found in U.S. Patent No. 10,041,077, the contents of which are incorporated herein by reference in their entirety.

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

[0240] In some embodiments, the excision-competent integration-deficient piggyBac transposase or piggyBac-like transposase comprises one or more substitutions of a non-wild-type amino acid, wherein the one or more amino acid substitutions with a wild-type amino acid are (relative to SEQ ID NO: 14505) 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, 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 substitutions. A list of under-incorporated amino acid substitutions is provided in U.S. Pat. No. 10,041,No. 077, the entire contents of which are incorporated herein by reference.

[0241] In some embodiments, the integration-deficient piggyBac transposase or piggyBac-like transposase is 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 FYVVNLEVYA GKQPSGPYAV SNRPFEVVER LIQPVARSHR 361 NVTFDNWFTG YELMLHLLNE YRLTSVGTVR KNKRQIPESF IRTDRQPNSS VFGFQKDITL 421 VSYAPKKNKV VVVMSTMHHD NSIDESTGEK QKPEMITFYN STKAGVDVVD ELCANYNVSR 481 NSKKWPMTLF YGVLNMAAIN ACIIYRTNKN VTIKRTEFIR SLGLSMMYEH LHSRNKKKNI 541 PTYLRQRIEK QLGEPVPRHV NVPGRYVRCQ DCPYKKDRKT KRSCNACAKP ICMEHAKFLC 601 ENCAELDSSL (SEQ ID NO: 14606). In some embodiments, the integration-deficient piggyBac transposase or piggyBac-like transposase is 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 FYVVNLEVYA GKQPSGPYAV SNRPFEVVER LIQPVARSHR 361 NVTFDNWFTG YELMLHLLNE YRLTSVGTVR KNKRQIPESF IRTDRQPNSS VFGFQKDITL 421 VSYAPKKNKV VVVMSTMHHD NSIDESTGEK QKPEMITFYN STKAGVDVVD ELCANYNVSR 481 NSKRWPMTLF YGVLNMAAIN ACIIYRTNKN VTIKRTEFIR SLGLSMIYEH LHSRNKKKNI 541 PTYLRQRIEK QLGEPSPRHV NYPGRYVRCQ DCPYKKDRKT KRSCNACAKP ICMEHAKFLC 601 VNCAELDSSL (SEQ ID NO: 14607). In some embodiments, the integration-deficient piggyBac transposase or piggyBac-like transposase is 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 DYVVNLEVYA GKQPSGPYAV SNRPFEVVER LIQPVARSHR 361 NVTFDNWFTG YECMLHLLNE YRLTSVGTVR KNKRQIPESF IRTDRQPNSS VFGFQKDITL 421 VSYAPKKNKV VVVMSTMHHD NSIDESTGEK QKPEMITFYN STKAGVDVVD ELCANYNVSR 481 NSKKWPMTLF YGVLNMAAIN ACIIYRTNKN VTIKRTEFIR SLGLSMIKEH LHSRNKKKNI 541 PTYLRQRIEK QLGEPSPRHV NVPGRYVRCQ DCPYKKDRKT KRSCNACAKP ICMEHAKFLC 601 ENCAELDSSL (SEQ ID NO: 14608). In some embodiments, the integration-deficient transposase comprises a sequence at least 90% identical to SEQ ID NO:14608.

[0242] In some embodiments, the piggyBac transposon or piggyBac-like transposon is isolated from or derived from Bombyx mori. In some embodiments, the piggyBac transposon or piggyBac-like transposon is 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 It contains the sequence 361 cagtttttga tcaaa (SEQ ID NO: 14506). In some embodiments, the piggyBac transposon or piggyBac-like transposon is 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 cgggttat (SEQ ID NO: 14507). In some embodiments, the piggyBac transposon or piggyBac-like transposon is 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 tcat (SEQ ID NO: 14508) sequence. In some embodiments, the piggyBac (PB) transposon or piggyBac-like transposon is 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) sequence.

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

[0244] In some 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 some 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:14507 or SEQ ID NO:14509. In some embodiments, the piggyBac or piggyBac-like transposon comprises one end that is at least 90% identical to SEQ ID NO:14506 or SEQ ID NO:14508. In some embodiments, the piggyBac or piggyBac-like transposon comprises one end that is at least 90% identical to SEQ ID NO:14507 or SEQ ID NO:14509.

[0245] In some embodiments, the piggyBac transposon or piggyBac-like transposon is 1 ttaacccggc 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 It contains the sequence 361 cagtttttga tcaaa (SEQ ID NO: 14515).

[0246] In some embodiments, the piggyBac transposon or piggyBac-like transposon is 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 cttacaccg acgtccgatc ccctccgagg tcagaacccg gatgaggtag 541 gggggctacc gcggtcaaca ctacaaccag acggcgcggc tcaccccaag gacgcccagc 601 cgacggagcc ttcgaggcga atcgaaggct ctgaaacgtc ggccgtctcg gtacggcagc 661 ccgtcgggcc gcccagacgg tgccgctggt gtccggaat accccgctgg accagaacca 721 gcctgccggg tcgggacgcg atacaccgtc gaccggtcgc tctaatcact ccacggcagc 781 contains the sequence gcgctagagt gctggta (sequence number 14516).

[0247] In some embodiments, the piggyBac transposon or piggyBac-like transposon is CCCGGCGAGCATGAGG (SEQ ID NO: 14510). In some embodiments, the piggyBac transposon or piggyBac-like transposon comprises the ITR of SEQ ID NO: 14510. In some embodiments, the piggyBac transposon or piggyBac-like transposon comprises the sequence of TTATCCCGGCGAGCATGAGG (SEQ ID NO: 14511). In some embodiments, the piggyBac transposon or piggyBac-like transposon comprises at least 16 contiguous nucleotides from SEQ ID NO: 14511. In some embodiments, the piggyBac transposon or piggyBac-like transposon comprises the sequence of CCTCATGCTCGCCGGGTTAT (SEQ ID NO: 14512). In some embodiments, the piggyBac transposon or piggyBac-like transposon comprises at least 16 contiguous nucleotides from SEQ ID NO: 14512. In some embodiments, the piggyBac or piggyBac-like transposon comprises 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 some embodiments, the piggyBac or piggyBac-like transposon comprises SEQ ID NO: 14511 and SEQ ID NO: 14512. In some embodiments, the piggyBac or piggyBac-like transposon comprises the sequence TTAACCCGGCGAGCATGAGG (SEQ ID NO: 14513). In some embodiments, the piggyBac or piggyBac-like transposon comprises the sequence CCTCATGCTCGCCGGGTTAA (SEQ ID NO: 14514).

[0248] In some embodiments, the piggyBac or piggyBac-like transposon can have ends comprising SEQ ID NO:14506 and SEQ ID NO:14507, or a variant of one or both thereof 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 identity of 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 therebetween, to SEQ ID NO:14504 or SEQ ID NO:14505. In some embodiments, the piggyBac or piggyBac-like transposon comprises a heterologous polynucleotide inserted between a pair of inverted repeats, and the transposon is capable of being transposed by a piggyBac or piggyBac-like transposase that is 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 therebetween, identical to SEQ ID NO:14504 or SEQ ID NO:14505. In some embodiments, the transposon comprises two transposon ends, each of which contains SEQ ID NO:14510 in opposite orientations within the two transposon ends. In some embodiments, each inverted terminal repeat (ITR) is at least 90% identical to SEQ ID NO:14510.

[0249] In some embodiments, a piggyBac or piggyBac-like transposon can be inserted into a target nucleic acid at the sequence 5'-TTAT-3' by a piggyBac or piggyBac-like transposase. In some embodiments, one end of the piggyBac or piggyBac-like transposon comprises at least 16 contiguous nucleotides from SEQ ID NO:14506, and the other end of the transposon comprises at least 16 contiguous nucleotides from SEQ ID NO:14507. In some embodiments, one end of the piggyBac transposon or piggyBac-like transposon comprises at least 17, at least 18, at least 19, at least 20, at least 22, at least 25, or 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, or at least 30 contiguous nucleotides from SEQ ID NO:14507.

[0250] In some embodiments, the piggyBac or piggyBac-like transposon contains transposon ends corresponding to SEQ ID NO:14506 and SEQ ID NO:14507 (each end containing an ITR) and has a target sequence corresponding to 5'-TTAT3'. In some embodiments, the piggyBac or piggyBac-like transposon also contains a sequence encoding a transposase (e.g., SEQ ID NO:14505). In some embodiments, the piggyBac or piggyBac-like transposon contains 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 larger insertion shortly before the ITR. The ITR sequences for the two transposon ends are the same (both are the same as 14510), but have different target sequences. Thus, the second transposon has a target sequence corresponding to 5'-TTAA-3', providing evidence that an unchanged ITR sequence is required to alter target sequence specificity. The piggyBac or piggyBac-like transposase (SEQ ID NO: 14504) associates with a 5'-TTAA-3' target site and differs from the 5'-TTAT-3' related transposase (SEQ ID NO: 14505) by only four amino acids (D322Y, S473C, A507T, H582R). In some embodiments, the piggyBac or piggyBac-like transposase (SEQ ID NO: 14504) associates with a 5'-TTAA-3' target site and has less activity against transposons with 5'-TTAT-3' ends than the piggyBac or piggyBac-like transposase (SEQ ID NO: 14505) associated with a 5'-TTAA-3' target site. In some embodiments, a piggyBac transposon or piggyBac-like transposon having a 5'-TTAA-3' target site can be converted to a piggyBac transposase or piggyBac-like transposase having a 5'-TTAT-3' target site by replacing the 5'-TTAA-3' target site with a 5'-TTAT-3' target site.Such transposons can be used in conjunction with piggyBac or piggyBac-like transposases (such as SEQ ID NO: 14504) that recognize the 5'-TTAT-3' target sequence, or with variants of the transposase originally related to the 5'-TTAA-3' transposon. In some embodiments, the strong similarity between the 5'-TTAA-3' piggyBac or piggyBac-like transposases and the 5'-TTAT-3' piggyBac or piggyBac-like transposases demonstrates that only small changes in the amino acid sequence of the piggyBac or piggyBac-like transposase can alter the target sequence specificity. In some embodiments, the modification of any piggyBac transposon-transposase gene delivery system or piggyBac-like transposon-transposase gene delivery system is the result of low-level mutagenesis to introduce mutations into the transposase, in which the 5'-TTAA-3' target sequence in the gene delivery system is replaced with a 5'-TTAT-3' target sequence, the ITRs remain the same, and the transposase is the original piggyBac transposase or a piggyBac-like transposase, or a variant thereof. In some embodiments, a piggyBac transposon-transposase delivery system or a piggyBac-like transposon-transposase delivery system can be formed by modifying a 5'-TTAT-3' active piggyBac transposon-transposase gene delivery system or a piggyBac-like transposon-transposase gene delivery system in which the 5'-TTAT-3' target sequence is replaced with a 5'-TTAA-3' target sequence, the ITRs remain the same, and the piggyBac transposase or piggyBac-like transposase is the original transposase or a variant thereof.

[0251] In some embodiments, the piggyBac transposon or piggyBac-like transposon is isolated from or derived from Bombyx mori. In some embodiments, the piggyBac transposon or piggyBac-like transposon is 1 cccggcgagc atgaggcagg gtatctcata ccctggtaaa attttaaagt tgtgtatttt 61 ataaaatttt cgtctgacaa cactagcgcg ctcagtagct ggaggcagga gcgtgcggga 121 ggggatagtg gcgtgatcgc agtgtggcac gggacaccgg cgagatattc gtgtgcaaac It contains the sequence 181 ctgtttcggg tatgttatac cctgcctcat tgttgacgta t (sequence number 14577). In some embodiments, the piggyBac transposon or piggyBac-like transposon is 1 tttaagaaaa agattaataa ataataataa tttcataatt aaaaacttct ttcattgaat 61 gccattaaat aaaccattat tttacaaaat aagatcaaca taattgagta aataataata 121 agaacaatat tatagtacaa caaaatatgg gtatgtcata ccctgccaca ttcttgatgt It contains the sequence 181 aacttttttt cacctcatgc tcgccggg (SEQ ID NO: 14578). In some embodiments, the transposon comprises at least 16 contiguous bases from SEQ ID NO: 14577, at least 16 contiguous bases from SEQ ID NO: 14578, and an inverted terminal repeat that is at least 87% identical to CCCGGCGAGCATGAGG (SEQ ID NO: 14510). In some embodiments, the piggyBac transposon or piggyBac-like transposon is 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 It contains the sequence 361 ttttgatcaa a (SEQ ID NO: 14595). In some embodiments, the piggyBac transposon or piggyBac-like transposon is 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 It contains the sequence 361 cggg (sequence number 14596).

[0252] In some 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 some embodiments, the ITRs of SEQ ID NO: 14595 and SEQ ID NO: 14596 are not flanked by a 5'-TTAA-3' sequence. In some embodiments, the ITRs of SEQ ID NO: 14595 and SEQ ID NO: 14596 are flanked by a 5'-TTAT-3' sequence.

[0253] In some embodiments, the piggyBac transposon or piggyBac-like transposon is 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 It contains the sequence of 301 g (sequence number 14597). In some embodiments, the piggyBac transposon or piggyBac-like transposon is 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) sequence. In some embodiments, the piggyBac transposon or piggyBac-like transposon is 1 cagggtatct cataccctgg taaaatttta aagttgtgta ttttataaaa ttttcgtctg 61 acaacactag cgcgctcagt agctggaggc aggagcgtgc gggaggggat agtggcgtga 121 tcgcagtgtg gcacgggaca ccggcgagat attcgtgtgc aaacctgttt cgggtatgtt 181 ataccctgcc tcattgttga cgtat (SEQ ID NO: 14599) sequence. In some embodiments, the 5' end of the piggyBac transposon or piggyBac-like transposon comprises the sequence of SEQ ID NO: 14577, SEQ ID NO: 14595, or SEQ ID NOs: 14597-14599. In some embodiments, the 5' end of the piggyBac transposon or piggyBac-like transposon is preceded by a 5' targeting sequence. In some embodiments, the piggyBac transposon or piggyBac-like transposon is 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 some embodiments, the piggyBac transposon or piggyBac-like transposon is 1 tttaagaaaa agattaataa ataataataa tttcataatt aaaaacttct ttcattgaat 61 gccattaaat aaaccattat tttacaaaat aagatcaaca taattgagta aataataata 121 agaacaatat tatagtacaa caaaatatgg gtatgtcata ccctgccaca ttcttgatgt It contains the sequence 181 aacttttttt ca (SEQ ID NO: 14601). In some embodiments, the piggyBac transposon or piggyBac-like transposon is 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: 14602).

[0254] In some embodiments, the 3' end of the piggyBac or piggyBac-like transposon comprises the sequence of SEQ ID NO: 14578, SEQ ID NO: 14596, or SEQ ID NOs: 14600-14601. In some embodiments, the 3' end of the piggyBac or piggyBac-like transposon is followed by a 3' target sequence. In some embodiments, the transposon is transposable by the transposase of SEQ ID NO: 14505. In some 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 opposite orientations that flanks the target sequence. In some embodiments, the 5' end of the transposon begins with SEQ ID NO: 14510 and the 3' end of the transposon ends with the reverse complement of SEQ ID NO: 14510, 5'-CCTCATGCTCGCCGGG-3' (SEQ ID NO: 14603). In some embodiments, the piggyBac or piggyBac-like transposon comprises ITRs that are at least 93%, at least 87%, at least 81%, or any percentage therebetween, identical to SEQ ID NO:14510 or SEQ ID NO:14603. In some embodiments, the piggyBac or piggyBac-like transposon comprises a target sequence followed by a 5' end of the transposon comprising a sequence selected from SEQ ID NOs:88, 105, or 107, and a 3' end of the transposon comprising SEQ ID NO:14578 or 106, followed by the target sequence. In some embodiments, the piggyBac or piggyBac-like transposon comprises one end comprising a sequence that is at least 90%, at least 95%, at least 99%, or any percentage therebetween, identical to SEQ ID NO:14577, and another end comprising a sequence that is at least 90%, at least 95%, at least 99%, or any percentage therebetween, identical to SEQ ID NO:14578.In some 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 some embodiments, the piggyBac or piggyBac-like transposon comprises two transposon ends, each of which contains a sequence in reverse orientation that is at least 81%, at least 87%, at least 93%, or any percentage in between, identical to SEQ ID NO: 14510. One end can 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 can 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 can be transposed by the transposase of SEQ ID NO: 14505, which can optionally be fused to a nuclear localization signal.

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

[0257] In some embodiments, the piggyBac or piggyBac-like transposon comprises a 5' end comprising one, two, three, four, five, six, or seven 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), ATTTATAAAAT (SEQ ID NO: 14619), TCATACCCTG (SEQ ID NO: 14620), and TAAATAATAATAA (SEQ ID NO: 14621). In some embodiments, the piggyBac or piggyBac-like transposon comprises a 3' end comprising one, two, or three sequences selected from SEQ ID NO: 14617, SEQ ID NO: 14620, and SEQ ID NO: 14621.

[0258] In some embodiments of the disclosed methods, the transposase enzyme is a piggyBac transposase enzyme or a piggyBac-like transposase enzyme. In some embodiments, the piggyBac transposase enzyme or piggyBac-like transposase enzyme is isolated from or derived from Xenopus tropicalis. The piggyBac transposase enzyme or piggyBac-like transposase enzyme is 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 It may comprise or consist of an amino acid sequence that is 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 therebetween, identical to 541 GKQRPQKGCK VCRKRGIRRD TRYYCPKCPR NPGLCFKPCF EIYHTQLHY (SEQ ID NO: 14517).

[0259] In some embodiments, the piggyBac transposase or piggyBac-like transposase is a hyperactive variant of SEQ ID NO: 14517. In some embodiments, the piggyBac transposase or piggyBac-like transposase is an integration-deficient variant of SEQ ID NO: 14517. The piggyBac transposase enzyme or piggyBac-like transposase enzyme is 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 It may comprise or consist of an amino acid sequence that is 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 therebetween, identical to 541 GKQRPQKGCK VCRKRGIRRD TRYYCPKCPR NPGLCFKPCF EIYHTQLHY (SEQ ID NO: 14518).

[0260] In some embodiments, the piggyBac transposase or piggyBac-like transposase is isolated from or derived from Xenopus tropicalis. In some embodiments, the piggyBac transposase or piggyBac-like transposase is a hyperactive piggyBac transposase or piggyBac-like transposase. In some embodiments, the hyperactive piggyBac transposase or piggyBac-like transposase is 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 Contains a sequence that is at least 90% identical to 541 GKQRPQKGCK VCRKRGIRRD TRYYCPKCPR NPGLCRKPCF EIYHTQLHY (SEQ ID NO: 14572).

[0261] In some embodiments, the piggyBac transposase or piggyBac-like transposase is a hyperactive piggyBac transposase or piggyBac-like transposase. A hyperactive piggyBac transposase or piggyBac-like transposase is a transposase that is more active than the naturally occurring variant from which it is derived. In some embodiments, the hyperactive piggyBac transposase or piggyBac-like transposase is more active than the transposase of SEQ ID NO: 14517. In some embodiments, the hyperactive piggyBac transposase or piggyBac-like transposase is 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 It contains the sequence 541 GKQRPQKGCK VCRKRGIRRD TRYYCPKCPR NPGLCRKPCF EIYHTQLHY (SEQ ID NO: 14572).

[0262] In some embodiments, the hyperactive piggyBac transposase or piggyBac-like transposase is 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 It contains the sequence 541 GKQRPQKGCK VCRKRGIRRD TRYYCPKCPR NPGLCRKPCF EIYHTQLHY (SEQ ID NO: 14624).

[0263] In some embodiments, the hyperactive piggyBac transposase or piggyBac-like transposase is 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 It contains the sequence 541 GKQRPQKGCK VCRKRGIRRD TRYYCPKCPR NPGLCFKPCF EIYHTQLHY (SEQ ID NO: 14625).

[0264] In some embodiments, the hyperactive piggyBac transposase or piggyBac-like transposase is 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 It contains the sequence 541 GKQRPQKGCK VCRKRGIRRD TRYYCPKCPR NPGLCRKPCF EIYHTQLHY (SEQ ID NO: 14627).

[0265] In some embodiments, the hyperactive piggyBac transposase or piggyBac-like transposase is 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 It contains the sequence 541 GKQRPQKGCK VCRKRGIRRD TRYYCPKCPR NPGLCRKPCF EIYHTQLHY (SEQ ID NO: 14628).

[0266] In some embodiments, the hyperactive piggyBac transposase or piggyBac-like transposase is 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 It contains the sequence 541 GKQRPQKGCK VCRKRGIRRD TRYYCPKCPR NPGLCRKPCF EIYHTQLHY (SEQ ID NO: 16820).

[0267] In some embodiments, a hyperactive piggyBac transposase or piggyBac-like transposase has the following amino acids (relative to SEQ ID NO: 14517): 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, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 226, 228, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 2 and containing amino acid substitutions at positions selected from 98, 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, and 587.In some embodiments, a hyperactive piggyBac transposase or piggyBac-like transposase is selected from the group consisting of (relative to SEQ ID NO: 14517): 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, T20 0W, L210H, F212N, N218E, A248N, L263M, Q270L, S294T, T297M, S308R, L310R, L333M, Q336M, A354H, C357V, L358F, D359N, L377I, V and the amino acid substitutions include 423H, P426K, K428R, S438A, T447G, T447A, L450V, A462H, A462Q, I469V, I472L, Q498M, L502V, E5171, P520D, P520G, N523S, I533E, D534A, 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.

[0268] In some embodiments, the hyperactive piggyBac transposase or piggyBac-like transposase comprises one or more substitutions of a non-wild-type amino acid, wherein the one or more substitutions with a wild-type amino acid are (relative to SEQ ID NO: 14517) 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, L 54X, E55X, E56X, P57X, M58X, E59X, E62X, D63X, V64X, D65X, D66X, L67X, E68X, D69X, Q70X, E71X, A72X, G73X, D74X, R7 5X, A76X, D77X, A78X, A79X, A80X, G81X, G82X, E83X, P84X, A85X, W86X, G87X, P88X, P89X, C90X, N91X, F92X, P93X, E9 5X, I96X, P97X, P98X, F99X, T100X, T101X, P103X, G104X, V105X, K106X, V107X, D108X, T109X, N111X, P114X, I115X, N116X, F117X, F118X, Q119X, 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, L18 2X, D187X, T188X, T189X, T190X, L192X, S193X, I194X, P195X, V196X, S1 98X, A199X, T200X, S202X, L208X, L209X, L210X, R211X, F212X, F215X, N 217X, 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、S293X、S294X、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、V482X、Y483X、K484X、A485X、A486X、V487X、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 substitutions. A list of highly active amino acid substitutions can be found in U.S. Patent No. 10,041,077, the contents of which are incorporated herein by reference in their entirety.

[0269] In some embodiments, the piggyBac transposase or piggyBac-like transposase is integration-deficient. In some embodiments, an integration-deficient piggyBac transposase or piggyBac-like transposase is a transposase that can excise a corresponding transposon but integrates the excised transposon less frequently than a corresponding naturally occurring transposase. In some embodiments, the piggyBac transposase or piggyBac-like transposase is an integration-deficient variant of SEQ ID NO: 14517. In some embodiments, an integration-deficient piggyBac transposase or piggyBac-like transposase is deficient compared to SEQ ID NO: 14517.

[0270] In some embodiments, the piggyBac transposase or piggyBac-like transposase is excision active but integration deficient. In some embodiments, the integration deficient piggyBac transposase or piggyBac-like transposase is 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 Contains a sequence that is at least 90% identical to the sequence of 541 GKQRPQKGCK VCRKRGIRRD TRYYCPKCPR NPGLCFKPCF EIYHTQLHYG RR (SEQ ID NO: 14605).

[0271] In some embodiments, the integration-deficient piggyBac transposase or piggyBac-like transposase is 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 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 Contains a sequence that is at least 90% identical to the sequence of 541 GKQRPQKGCK VCRKRGIRRD TRYYCPKCPR NPGLCFKPCF EIYHTQLHY (SEQ ID NO: 14604).

[0272] In some embodiments, the integration-deficient piggyBac transposase or piggyBac-like transposase is 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 Contains a sequence that is at least 90% identical to the sequence of 541 GKQRPQKGCK VCRKRGIRRD TRYYCPKCPR NPGLCFKPCF EIYHTQLHYG RR (SEQ ID NO: 14611).

[0273] In some embodiments, the integration-deficient piggyBac transposase or piggyBac-like transposase comprises SEQ ID NO: 14611. In some embodiments, the integration-deficient piggyBac transposase or piggyBac-like transposase comprises SEQ ID NO: 14611. 1 MAKRFYSAEE AAAHCMASSS EEFSGSDSEY VPPASESDSS TEESWCSSST VSALEEPMEV 61 DEDVDDLEDQ EAGDRADAAP GGEPAWGPPC NFPPEIPPFT TVPGVKVDTS NFEPINFFQL 121 FMTEAILQDM VLYTNVYAEQ YLTQVPLPRY ARAHAWHPTD IAEMKRFVGL TLAMGLIKAN 181 SLESYWDTTT VLSIPVFSAT MSRNRYQLLL RFLHFNNEAT 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 Contains a sequence that is at least 90% identical to the sequence of 541 GKQRPQKGCK VCRKRGIRRD TRYYCPKCPR NPGLCFKPCF EIYHTQLHYG RR (SEQ ID NO: 14612).

[0274] In some embodiments, the integration-deficient piggyBac transposase or piggyBac-like transposase comprises SEQ ID NO: 14612. In some embodiments, the integration-deficient piggyBac transposase or piggyBac-like transposase comprises SEQ ID NO: 14612. 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 RFLEFNNNAT 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 Contains a sequence that is at least 90% identical to the sequence of 541 GKQRPQKGCK VCRKRGIRRD TRYYCPKCPR NPGLCFKPCF EIYHTQLHYG RR (SEQ ID NO: 14613).

[0275] In some embodiments, the integration-deficient piggyBac or piggyBac-like transposase comprises SEQ ID NO: 14613. In some embodiments, the integration-deficient piggyBac or piggyBac-like transposase comprises an amino acid substitution in which Asn at position 218 (relative to SEQ ID NO: 14517) is substituted with Glu or Asp (N218D or N218E).

[0276] In some embodiments, the excision-competent integration-deficient piggyBac transposase or piggyBac-like transposase comprises one or more substitutions of a non-wild-type amino acid, wherein the substitution or substitutions with a wild-type amino acid are selected from the group consisting of (relative to SEQ ID NO: 14517): A2X, K3X, R4X, F5X, Y6X, S7X, A8X, E9X, E10X, A11X, A12X, A13X, H14X, C15X, M16X, A17X, S18X, S19X, S20X, E21X, E22X, F23X, S24X, G25X, 26X, D27X, D28X, D29X, D30X, D31X, D32X, D33X, D34X, D35X, D36X, D37X, D38X, D39X, D40X, D41X, D42X, D43X, D44X, D45X, D46X, D47X, D48X, D49X, D50X, D51X, D52X, D53X, D54X, D55X, D56X, D57X, D58X, D59X, D60X, D61X, D62X, D63X, D64X, D65X, D66X, D67X, D68X, D69X, D70X, D71X, D72X, D73X, D74X, D75X, D76X, D77X, D78X, D79X, D80X, D81X, D82X, D83X, D84X, D85X, D86X, D87X, D88X X, S28X, E29X, V31X, P32X, P33X, A34X, S35X, E36X, S37X, D38X, S39X, S40X, T41X, E42X, E43X, S44X, W45X, C46X, S47X, S48X, S49X, T50X, V51X, S52X, A53 X, L54X, E55X, E56X, P57X, M58X, E59X, V60X, M122X, T123X, E124X, A125X, L 127X, Q128X, D129X, L132X, Y133X, V126X, Y127X, E139X, Q140X, Y141X, L142 X, T143X, Q144X, N145X, P146X, L147X, P148X, R149X, Y150X, A151X, H154X, H157X, P158X, T159X, D160X, I161X, A162X, E163X, M164X, K165X, R166X, F16 7X, V168X, G169X, L170X, T171X, L172X, A173X, M174X, G175X, L176X, I177X , K178X, A179X, N180X, S181X, L182X, S184X, Y185X, D187X, T188X, T189X, T1 90X, V191X, L192X, S193X, I194X, P195X, V196X, F197X, S198X, A199X, T200 X, M201X, S202X, R203X, N204X, R205X, Y206X, Q207X, L208X, L209X, L210X, R 211X, F212X, L213X, H241X, F215X, N216X, N217X, N218X, A219X, T220X, A22 1X, V222X, P223X, P224X, D225X, Q226X, P227X, G228X, H229X, D230X, R231X,H233X、K234X、L235X、R236X、L238X、I239X、D240X、L242X、S243X、E244X、R244X、F246X、A247X、A248X、V249X、Y250X、T251X、P252X、C253X、Q254X、N255X、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、S293X、S294X、G295X、Y296X、T297X、S298X、Y299X、F300X、I302X、E304X、G305X,K306X、D307X、S308X、K309X、 L310X、D311X、P312X、P313X、G314X、C315X、P316X、P317X、D318X、L319X、T320X、V321X、S322X、G323X、K324X、I325X、V326X、W327X、E328X、L329X、I330X、S331X、P332X、L333X、L334X、G335X、Q336X、F338X、H339X、L340X、V342X、N344X、F345X、Y346X、S347X、S348X、I349X、L351X、T353X、A354X、Y356X、C357X、L358X、D359X、T360X、P361X、A362X、C363X、G364X、I366X、N367X、R368X、D369X、K371X、G372X、L373X、R375X、A376X、L377X、L378X、D379X、K380X、K381X、L382X、N383X、R384XG385X、T387X、Y388X、A389X、L390X、K392X、N393X、E394X、A397X、K399X、F400X、F401X、D402X、N405X、L406X、L409X、R422X、Y423X、G424X、E425X、P426X、K428X、N429X、K430X、P431X、L432X、S434X、K435X, E436X, S438X, K439X, Y440X, G442X, G443X, V444X, R446X, T447X, L450X, Q451X, H452X, N455X, T457X, R458X, T460X, R4 61X, A462X, Y464X, K465X, V467X, G468X, I469X, L471X, I472X, Q473X, M474X, L476X, R477X, N478X, S479X, Y480X, V482X, Y483 X, K484X, A485X, A486X, V487X, P488X, G489X, P490X, K491X, L492X, S493X, Y494X, Y495X, K496X, Q498X, L499X, Q500X, I501X, L502X, P503X, A504X, L505X, L506X, F507X, G508X, G509X, V510X, E511X, E512X, Q513X, T514X, V515X, E517X, M518X, P519X, P5 20X, S521X, D522X, N523X, V524X, A525X, L527X, I528X, G529X, K530X, F532X, I533X, D534X, T535X, L536X, P537X, P538X, T539 X, P540X, G541X, F542X, Q543X, R544X, P545X, Q546X, K547X, G548X, C549X, K550X, V551X, C552X, R553X, K554X, R555X, G556X, and Y589X. A list of excision-competent, incorporation-deficient amino acid substitutions can be found in U.S. Patent No. 10,041,077, the contents of which are incorporated herein by reference in their entirety. ,

[0277] In some embodiments, the piggyBac transposase or piggyBac-like transposase is fused to a nuclear localization signal. In some embodiments, SEQ ID NO: 14517 or SEQ ID NO: 14518 is fused to the nuclear localization signal. In some embodiments, the amino acid substitution in the piggyBac transposase or piggyBac-like transposase fused to the nuclear localization signal is 1 atggcaccca aaaagaaacg taaagtgatg gccaaaagat tttacagcgc cgaagaagca 61 gcagcacatt gcatggcatc gtcatccgaa gaattctcgg ggagcgattc cgaatatgtc 121 ccaccggcct cggaaagcga ttcgagcact gaggagtcgt ggtgttcctc ctcaactgtc 181 tcggctcttg aggagccgat ggaagtggat gaggatgtgg acgacttgga ggaccaggaa 241 gccggagaca gggccgacgc tgccgcggga ggggagccgg cgtggggacc tccatgcaat 301 tttcctcccg aaatcccacc gttcactact gtgccgggag tgaaggtcga cacgtccaac 361 ttcgaaccga tcaatttctt tcaactcttc atgactgaag cgatcctgca agatatggtg 421 ctctacacta atgtgtacgc cgagcagtac ctgactcaaa acccgctgcc tcgctacgcg 481 agagcgcatg cgtggcaccc gaccgatatc gcggagatga agcggttcgt gggactgacc 541 ctcgcaatgg gcctgatcaa ggccaacagc ctcgagtcat actgggatac cacgactgtg 601 cttagcattc cggtgttctc cgctaccatg tccgtaacc gctaccaact cctgctgcgg 661 ttcctccact tcaacaacaa tgcgaccgct gtgccacctg accagccagg acacgacaga 721 ctccacaagc tgcggccatt gatcgactcg ctgagcgagc gattcgccgc ggtgtacacc 781 ccttgccaaa acatttgcat cgacgagtcg cttctgctgt ttaaaggccg gcttcagttc 841 cgccagtaca tcccatcgaa gcgcgctcgc tatggtatca aattctacaa actctgcgag 901 tcgtccagcg gctacacgtc atacttcttg atctacgagg ggaaggactc taagctggac 961 ccaccggggt gtccaccgga tcttactgtc tccggaaaaa tcgtgtggga actcatctca 1021 cctctctcg gacaaggctt tcatctctac gtcgacaatt tctactcatc gatccctctg 1081 ttcaccgccc tctactgcct ggatactcca gcctgtggga ccattaacag aaaccggaag 1141 ggtctgccga gagcactgct ggataagaag ttgaacaggg gagactta cgcgctgaga 1201 aagaacgaac tcctcgccat caaattcttc gacaagaaaa atgtgttat gctcacctcc 1261 atccacgacg aatccgtcat ccgggagcag cgcgtgggca ggccgccgaa aaacaagccg 1321 ctgtgctcta aggaatactc caagtacatg gggggtgtcg accggaccga tcagctgcag 1381 cattactaca acgccactag aaagacccgg gcctggtaca agaaagtcgg catctacctg 1441 atccaaatgg cactgaggaa ttcgtatatt gtctacaagg ctgccgttcc gggcccgaaa 1501 ctgtcatact acaagtacca gcttcaaatc ctgccggcgc tgctgttcgg tggagtggaa 1561 gaacagactg tgcccgagat gccgccatcc gacaacgtgg cccggttgat cggaaagcac 1621 ttcattgata ccctgcctcc gacgcctgga aagcagcggc cacagaaggg atgcaaagtt 1681 tgccgcaagc gcggaatacg gcgcgatacc cgctactatt gcccgaagtg cccccgcaat Encoded by a polynucleotide sequence comprising 1741 cccggactgt gtttcaagcc ctgttttgaa atctaccaca cccagttgca ttac (SEQ ID NO: 14626).

[0278] In some embodiments, the piggyBac transposon or piggyBac-like transposon is isolated from or derived from Xenopus tropicalis. In some embodiments, the piggyBac transposon or piggyBac-like transposon is 1 ttaacctttt tactgccaat gacgcatggg atacgtcgtg gcagtaaaag ggcttaaatg 61 ccaacgacgc gtcccatacg ttgttggcat tttaagtctt ctctctgcag cggcagcatg 121 tgccgccgct gcagagagtt tctagcgatg acagcccctc tgggcaacga gccggggggg 181 ctgtc (SEQ ID NO: 14519) sequence.

[0279] In some embodiments, the piggyBac transposon or piggyBac-like transposon is 1 tttgcatttt tagacattta gaagcctata tcttgttaca gaattggaat tacacaaaaa 61 ttctaccata ttttgaaagc ttaggttgtt ctgaaaaaaa caatatattg ttttcctggg 121 taaactaaaa gtcccctcga ggaaaggccc ctaaagtgaa acagtgcaaa acgttcaaaa 181 actgtctggc aatacaagtt ccactttgac caaaacggct ggcagtaaaa gggttaa (SEQ ID NO: 14520) sequence.

[0280] In some embodiments, the piggyBac transposon or piggyBac-like transposon comprises SEQ ID NO: 14519 and SEQ ID NO: 14520. In some embodiments, the piggyBac transposon or piggyBac-like transposon comprises 1 ttaacccttt gcctgccaat cacgcatggg atacgtcgtg gcagtaaaag ggcttaaatg 61 ccaacgacgc gtcccatacg ttgttggcat tttaagtctt ctctctgcag cggcagcatg 121 tgccgccgct gcagagagtt tctagcgatg acagcccctc tgggcaacga gccggggggg 181 ctgtc (SEQ ID NO: 14521) sequence.

[0281] In some embodiments, the piggyBac transposon or piggyBac-like transposon is 1 tttgcatttt tagacattta gaagcctata tcttgttaca gaattggaat tacacaaaaa 61 ttctaccata ttttgaaagc ttaggttgtt ctgaaaaaaa caatatattg ttttcctggg 121 taaactaaaa gtcccctcga ggaaaggccc ctaaagtgaa acagtgcaaa acgttcaaaa 181 actgtctggc aatacaagtt ccactttggg acaaatcggc tggcagtgaa agggttaa (SEQ ID NO: 14522) sequence.

[0282] In some embodiments, the piggyBac transposon or piggyBac-like transposon is 1 ttaacctttt tactgccaat gacgcatggg atacgtcgtg gcagtaaaag ggcttaaatg 61 ccaacgacgc gtcccatacg ttgttggcat tttaattctt ctctctgcag cggcagcatg 121 tgccgccgct gcagagagtt tctagcgatg acagcccctc tgggcaacga gccggggggg 181 ctgtc (SEQ ID NO: 14523) sequence.

[0283] In some embodiments, the piggyBac transposon or piggyBac-like transposon comprises SEQ ID NO: 14520 and SEQ ID NO: 14519, SEQ ID NO: 14521, or SEQ ID NO: 14523. In some embodiments, the piggyBac transposon or piggyBac-like transposon comprises SEQ ID NO: 14522 and SEQ ID NO: 14519, SEQ ID NO: 14521, or SEQ ID NO: 14523. In some embodiments, the piggyBac transposon or piggyBac-like transposon comprises one end comprising at least 14, 16, 18, 20, 30, or 40 contiguous nucleotides from SEQ ID NO: 14519, SEQ ID NO: 14521, or SEQ ID NO: 14523. In some embodiments, the piggyBac or piggyBac-like transposon comprises one end comprising at least 14, 16, 18, 20, 30, or 40 contiguous nucleotides from SEQ ID NO:14520 or SEQ ID NO:14522. In some embodiments, the piggyBac or piggyBac-like transposon comprises one end that is at least 90% identical to SEQ ID NO:14519, SEQ ID NO:14521, or SEQ ID NO:14523. In some embodiments, the piggyBac or piggyBac-like transposon comprises one end that is at least 90% identical to SEQ ID NO:14520 or SEQ ID NO:14522. In one embodiment, one transposon end is at least 90% identical to SEQ ID NO:14519 and the other transposon end is at least 90% identical to SEQ ID NO:14520.

[0284] In some embodiments, the piggyBac transposon or piggyBac-like transposon comprises the sequence TTAACCTTTTTACTGCCA (SEQ ID NO: 14524). In some embodiments, the piggyBac transposon or piggyBac-like transposon comprises the sequence TTAACCCTTTGCCTGCCA (SEQ ID NO: 14526). In some embodiments, the piggyBac transposon or piggyBac-like transposon comprises the sequence TTAACCYTTTTACTGCCA (SEQ ID NO: 14527). In some embodiments, the piggyBac transposon or piggyBac-like transposon comprises the sequence TGGCAGTAAAAGGGTTAA (SEQ ID NO: 14529). In some embodiments, the piggyBac transposon or piggyBac-like transposon comprises the sequence TGGCAGTGAAAGGGTTAA (SEQ ID NO: 14531). In some embodiments, the piggyBac transposon or piggyBac-like transposon comprises the sequence TTAACCYTTTKMCTGCCA (SEQ ID NO: 14533). In some embodiments, one end of the piggyBac transposon or piggyBac-like transposon comprises a sequence selected from SEQ ID NO: 14524, SEQ ID NO: 14526, and SEQ ID NO: 14527. In some embodiments, one end of the piggyBac (PB) transposon or piggyBac-like transposon comprises a sequence selected from SEQ ID NO: 14529 and SEQ ID NO: 14531. In some embodiments, each inverted terminal repeat of the piggyBac transposon or piggyBac-like transposon comprises the ITR sequence CCYTTTKMCTGCCA (SEQ ID NO: 14563). In some embodiments, each end of the piggyBac (PB) transposon or piggyBac-like transposon comprises SEQ ID NO: 14563 in an inverted orientation. In some embodiments, one ITR of the piggyBac transposon or piggyBac-like transposon comprises a sequence selected from SEQ ID NO:14524, SEQ ID NO:14526, and SEQ ID NO:14527.In some embodiments, one ITR of the piggyBac transposon or piggyBac-like transposon comprises a sequence selected from SEQ ID NO: 14529 and SEQ ID NO: 14531. In some embodiments, the piggyBac transposon or piggyBac-like transposon comprises SEQ ID NO: 14533 in reverse orientation within two transposon ends.

[0285] In some embodiments, the piggyBac or piggyBac-like transposon can have ends comprising SEQ ID NO:14519 and SEQ ID NO:14520, or a variant of one or both thereof that is at least 90% identical to SEQ ID NO:14519 or SEQ ID NO:14520, and the piggyBac or piggyBac-like transposase has the sequence of SEQ ID NO:14517, or a variant that is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or any percentage in between, identical to SEQ ID NO:14517 or SEQ ID NO:14518. In some embodiments, one piggyBac or piggyBac-like transposon end comprises at least 14 contiguous nucleotides from SEQ ID NO:14519, SEQ ID NO:14521, or SEQ ID NO:14523, and the other transposon end comprises at least 14 contiguous nucleotides from SEQ ID NO:14520 or SEQ ID NO:14522. In some embodiments, one transposon end comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 22, at least 25, or at least 30 contiguous nucleotides from either SEQ ID NO:14519, SEQ ID NO:14521, or SEQ ID NO:14523, and the other transposon end comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 22, at least 25, or at least 30 contiguous nucleotides from SEQ ID NO:14520 or SEQ ID NO:14522.

[0286] In some embodiments, a piggyBac or piggyBac-like transposase recognizes one transposon end having a 5' sequence corresponding to SEQ ID NO: 14519 and a 3' sequence corresponding to SEQ ID NO: 14520. It excises the transposon from one DNA molecule by cleaving the DNA from the 5' TTAA-3' sequence at the 5' end of one transposon to the 5'-TTAA-3' sequence at the 3' end of a second transposon end (including any homologous DNA located in between), and inserts the excised sequence into a second DNA molecule. In some embodiments, the truncated modified versions of the 5' and 3' transposon ends will also function as part of a transposon that can be transposed by a piggyBac or piggyBac-like transposase. For example, the 5' transposon end can be replaced with a sequence corresponding to SEQ ID NO: 14521 or SEQ ID NO: 14523, and the 3' transposon end can be replaced with a shorter sequence corresponding to SEQ ID NO: 14522. In some embodiments, the 5' and 3' transposon ends share an 18 bp near-perfect repeat sequence (5'-TTAACCYTTTKMCTGCCA: SEQ ID NO: 14533) at their ends that contains a 5'-TTAA-3' insertion site (this sequence is inverted in the two ends). Thus, in SEQ ID NO:14519 and SEQ ID NO:14523, the 5' transposon end begins with the sequence 5'-TTAACCTTTTTACTGCCA-3' (SEQ ID NO:14524), and in SEQ ID NO:14521, the 5' transposon end begins with the sequence 5'-TTAACCCTTTGCCTGCCA-3' (SEQ ID NO:14526); the 3' transposon end, which has the approximate reverse complement of this sequence, ends with 5' TGGCAGTAAAAGGGTTAA-3' (SEQ ID NO:14529) in SEQ ID NO:14520 and 5'-TGGCAGTGAAAGGGTTAA-3' (SEQ ID NO:14531) in SEQ ID NO:14522.One embodiment of the invention is a transposon comprising a heterologous polynucleotide inserted between two transposon ends, each comprising SEQ ID NO: 14533 in opposite orientations within the two transposon ends. In some embodiments, one transposon end comprises a sequence selected from SEQ ID NO: 14524, SEQ ID NO: 14526, and SEQ ID NO: 14527. In some embodiments, one transposon end comprises a sequence selected from SEQ ID NO: 14529 and SEQ ID NO: 14531.

[0287] In some embodiments, the piggyBac transposon or piggyBac-like transposon is isolated from or derived from Xenopus tropicalis. In some embodiments, the piggyBac transposon or piggyBac-like transposon is 1 ccctttgcct gccaatcacg catgggatac gtcgtggcag taaaagggct taaatgccaa 61 cgacgcgtcc catacgtt (SEQ ID NO: 14573).

[0288] In some embodiments, the piggyBac transposon or piggyBac-like transposon is 1 cctgggtaaa ctaaaagtcc cctcgaggaa aggcccctaa agtgaaacag tgcaaaacgt 61 tcaaaaactg tctggcaata caagttccac tttgggacaa atcggctggc agtgaaaggg (SEQ ID NO: 14574) sequence.

[0289] In some embodiments, the piggyBac transposon or piggyBac-like transposon comprises at least 16 contiguous bases from SEQ ID NO: 14573 or SEQ ID NO: 14574 and the inverted end sequences CCYTTTBMCTGCCA (SEQ ID NO: 14575).

[0290] In some embodiments, the piggyBac transposon or piggyBac-like transposon is 1 ccctttgcct gccaatcacg catgggatac gtcgtggcag taaaagggct taaatgccaa 61 cgacgcgtcc catacgttgt tggcatttta agtcttctct ctgcagcggc agcatgtgcc 121 gccgctgcag agagtttcta gcgatgacag cccctctggg caacgagccg ggggggctgt It contains the sequence of 181 c (sequence number 14579).

[0291] The piggyBac transposon or piggyBac-like transposon, in some embodiments, 1 ccttttact gccaatgacg catgggatac gtcgtggcag taaaagggct taaatgccaa 61 cgacgcgtcc catacgttgt tggcatttta attcttctct ctgcagcggc agcatgtgcc 121 gccgctgcag agagtttcta gcgatgacag cccctctggg caacgagccg ggggggctgt It contains the sequence of 181 c (sequence number 14580).

[0292] In some embodiments, the piggyBac transposon or piggyBac-like transposon is 1 ccttttact gccaatgacg catgggatac gtcgtggcag taaaagggct taaatgccaa 61 cgacgcgtcc catacgttgt tggcatttta agtcttctct ctgcagcggc agcatgtgcc 121 gccgctgcag agagtttcta gcgatgacag cccctctggg caacgagccg ggggggctgt 181 c (SEQ ID NO: 14581).

[0293] In some embodiments, the piggyBac transposon or piggyBac-like transposon is 1 ccttttact gccaatgacg catgggatac gtcgtggcag taaaagggct taaatgccaa 61 cgacgcgtcc catacgttgt tggcatttta agtcttctct ctgcagcggc agcatgtgcc It contains the sequence 121 gccgctgcag agag (SEQ ID NO: 14582).

[0294] In some embodiments, the piggyBac transposon or piggyBac-like transposon is 1 ccttttact gccaatgacg catgggatac gtcgtggcag taaaagggct taaatgccaa 61 cgacgcgtcc catacgttgt tggcatttta agtctt (SEQ ID NO: 14583) sequence.

[0295] In some embodiments, the piggyBac transposon or piggyBac-like transposon is 1 ccctttgcct gccaatcacg catgggatac gtcgtggcag taaaagggct taaatgccaa 61 cgacgcgtcc catacgttgt tggcatttta agtctt (SEQ ID NO: 14584) sequence.

[0296] In some embodiments, the piggyBac transposon or piggyBac-like transposon is 1 ttatcctttt tactgccaat gacgcatggg atacgtcgtg gcagtaaaag ggcttaaatg 61 ccaacgacgc gtcccatacg ttgttggcat tttaagtctt ctctctgcag cggcagcatg 121 tgccgccgct gcagagagtt tctagcgatg acagcccctc tgggcaacga gccggggggg 181 ctgtc (SEQ ID NO: 14585) sequence.

[0297] In some embodiments, the piggyBac transposon or piggyBac-like transposon is 1 tttgcatttt tagacattta gaagcctata tcttgttaca gaattggaat tacacaaaaa 61 ttctaccata ttttgaaagc ttaggttgtt ctgaaaaaaa caatatattg ttttcctggg 121 taaactaaaa gtcccctcga ggaaaggccc ctaaagtgaa acagtgcaaa acgttcaaaa 181 actgtctggc aatacaagtt ccactttggg acaaatcggc tggcagtgaa aggg (SEQ ID NO: 14586) sequence.

[0298] In some embodiments, the piggyBac transposon or piggyBac-like transposon comprises a 5' transposon end sequence selected from SEQ ID NO: 14573 and SEQ ID NOs: 14579-14585. In some embodiments, the 5' transposon end sequence is preceded by a 5' targeting sequence. In some embodiments, the piggyBac transposon or piggyBac-like transposon comprises 1 tttgcatttt tagacattta gaagcctata tcttgttaca gaattggaat tacacaaaaa 61 ttctaccata ttttgaaagc ttaggttgtt ctgaaaaaaa caatatattg ttttcctggg 121 taaactaaaa gtcccctcga ggaaaggccc ctaaagtgaa acagtgcaaa acgttcaaaa 181 actgtctggc aatacaagtt ccactttgac caaaacggct ggcagtaaaa ggg (SEQ ID NO: 14587) sequence.

[0299] In some embodiments, the piggyBac transposon or piggyBac-like transposon is 1 ttgttctgaa aaaaacaata tattgttttc ctgggtaaac taaaagtccc ctcgaggaaa 61 ggcccctaaa gtgaaacagt gcaaaacgtt caaaaactgt ctggcaatac aagttccact It contains the sequence 121 ttgaccaaaa cggctggcag taaaaggg (SEQ ID NO: 14588).

[0300] In some embodiments, the piggyBac transposon or piggyBac-like transposon is 1 tttgcatttt tagacattta gaagcctata tcttgttaca gaattggaat tacacaaaaa 61 ttctaccata ttttgaaagc ttaggttgtt ctgaaaaaaa caatatattg ttttcctggg 121 taaactaaaa gtcccctcga ggaaaggccc ctaaagtgaa acagtgcaaa acgttcaaaa 181 actgtctggc aatacaagtt ccactttgac caaaacggct ggcagtaaaa gggttat (SEQ ID NO: 14589) sequence.

[0301] In some embodiments, the piggyBac transposon or piggyBac-like transposon is 1 ttgttctgaa aaaaacaata tattgttttc ctgggtaaac taaaagtccc ctcgaggaaa 61 ggcccctaaa gtgaaacagt gcaaaacgtt caaaaactgt ctggcaatac aagttccact It contains the sequence 121 ttgggacaaa tcggctggca gtgaaaggg (SEQ ID NO: 14590).

[0302] In some embodiments, the piggyBac transposon or piggyBac-like transposon comprises a 3' transposon end sequence selected from SEQ ID NOs: 14574 and 14587-14590. In some embodiments, the 3' transposon end sequence is followed by a 3' target sequence. In some embodiments, the 5' transposon end and the 3' transposon end share a 14 repeat sequence (SEQ ID NO: 14575) in reverse orientation within their two ends adjacent to the target sequence. In some embodiments, the piggyBac transposon or piggyBac-like transposon comprises a target sequence, a 5' target sequence comprising a sequence selected from SEQ ID NOs: 14582-14584 and 14573, and a 3' transposon end comprising a sequence selected from SEQ ID NOs: 14588-14590 and 14574, followed by a 3' target sequence.

[0303] In some embodiments, the 5' transposon end of the piggyBac transposon or piggyBac-like transposon is 1 atcacgcatg ggatacgtcg tggcagtaaa agggcttaaa tgccaacgac gcgtcccata 61 cgtt (SEQ ID NO: 14591) and ITRs. In some embodiments, the 5' transposon end comprises: 1 atgacgcatg ggatacgtcg tggcagtaaa agggcttaaa tgccaacgac gcgtcccata 61 cgttgttggc attttaagtc tt (SEQ ID NO: 14592) and ITRs. In some embodiments, the 3' transposon end of the piggyBac transposon or piggyBac-like transposon comprises 1 cctgggtaaa ctaaaagtcc cctcgaggaa aggcccctaa agtgaaacag tgcaaaacgt 61 tcaaaaactg tctggcaata caagttccac tttgggacaa atcggc (SEQ ID NO: 14593) and ITRs. In some embodiments, the 3' transposon end comprises: 1 ttgttctgaa aaaaacaata tattgttttc ctgggtaaac taaaagtccc ctcgaggaaa 61 ggcccctaaa gtgaaacagt gcaaaacgtt caaaaactgt ctggcaatac aagttccact 121 ttgaccaaaa cggc (SEQ ID NO: 14594) and contains the ITRs.

[0304] In some embodiments, one transposon end comprises a sequence that is at least 90%, at least 95%, at least 99%, or any percentage therebetween identical to SEQ ID NO: 14573, and the other transposon end comprises a sequence that is at least 90%, at least 95%, at least 99%, or any percentage therebetween identical to SEQ ID NO: 14574. In some embodiments, one transposon end comprises at least 14, at least 16, at least 18, at least 20, or at least 25 contiguous nucleotides from SEQ ID NO: 14573, and the other transposon end comprises at least 14, at least 16, at least 18, at least 20, or at least 25 contiguous nucleotides from SEQ ID NO: 14574. In some embodiments, one transposon end comprises at least 14, at least 16, at least 18, at least 20, or at least 25 contiguous nucleotides from SEQ ID NO: 14591, and the other transposon end comprises at least 14, at least 16, at least 18, at least 20, or at least 25 contiguous nucleotides from SEQ ID NO: 14593. In some embodiments, each transposon end comprises SEQ ID NO: 14575 in reverse orientation.

[0305] In some embodiments, the piggyBac transposon or piggyBac-like transposon comprises a sequence selected from SEQ ID NO:14573, SEQ ID NO:14579, SEQ ID NO:14581, SEQ ID NO:14582, SEQ ID NO:14583, and SEQ ID NO:14588, and a sequence selected from SEQ ID NO:14587, SEQ ID NO:14588, SEQ ID NO:14589, and SEQ ID NO:14586, and the piggyBac transposase or pi...

Claims

1. (a) an ectodomain comprising an antigen recognition region, wherein the antigen recognition region comprises at least one VHH domain that specifically binds to B cell maturation antigen (BCMA); (b) human CD8α transmembrane domain; and (c) an endodomain comprising a human 4-1BB costimulatory domain and a human CD3ζ endodomain; A cell comprising a chimeric antigen receptor (CAR) comprising: The VHH domain comprises: Complementarity determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 18056; A complementarity determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 18060; and A cell comprising a complementarity determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 18064.

2. The cell of claim 1 , wherein the VHH domain comprises or consists of a recombinant or chimeric sequence.

3. The cell of claim 1 , wherein the VHH domain comprises or consists of a human or humanized sequence.

4. The cell according to any one of claims 1 to 3, further comprising a human CD8α signal peptide.

5. The cell of claim 4, wherein the human CD8α signal peptide comprises the amino acid sequence of SEQ ID NO: 18012.

6. The cell of any one of claims 1 to 5, wherein the human CD8α transmembrane domain comprises the amino acid sequence of SEQ ID NO: 18014.

7. The cell of any one of claims 1 to 6, wherein the endodomain comprises at least one additional costimulatory domain comprising the amino acid sequence of human CD28, CD40, ICOS, MyD88, OX-40 intracellular segment, or any combination thereof.

8. A cell described in any one of claims 1 to 7, wherein the human CD3ζ endodomain comprises the amino acid sequence of SEQ ID NO: 18016.

9. The cell of any one of claims 1 to 8, wherein the human 4-1BB costimulatory domain comprises the amino acid sequence of SEQ ID NO: 18018.

10. The cell according to any one of claims 1 to 9, wherein the ectodomain of (a) further comprises a hinge between the antigen recognition region and the transmembrane domain.

11. The cell of claim 10 , wherein the hinge comprises the amino acid sequence of a human CD8α hinge, an IgG4 hinge, a CD4 hinge, or a combination thereof.

12. The cell of claim 11 , wherein the hinge comprises the amino acid sequence of a human CD8α hinge.

13. The cell described in claim 12, wherein the amino acid sequence of the human CD8α hinge comprises the amino acid sequence of SEQ ID NO: 18020.

14. The cell of any one of claims 1 to 13, wherein the VHH domain comprises the amino acid sequence of SEQ ID NO: 18056.

15. The cell of any one of claims 1 to 14, wherein the CAR comprises amino acids 22 to 362 of the amino acid sequence of SEQ ID NO: 18006.

16. The cell of any one of claims 1 to 15, wherein the CAR comprises the amino acid sequence of SEQ ID NO: 18006.

17. (a) an ectodomain comprising an antigen recognition region, wherein the antigen recognition region comprises at least one VHH domain that specifically binds to BCMA, and the VHH domain comprises the amino acid sequence of SEQ ID NO: 18051; (b) a hinge domain containing the human CD8α hinge domain; (c) a transmembrane domain comprising a human CD8α transmembrane domain; and (d) A cell comprising a chimeric antigen receptor (CAR) comprising an endodomain comprising a human 4-1BB costimulatory domain and a human CD3ζ endodomain.

18. The cell described in claim 17, wherein the human CD8α hinge domain comprises the amino acid sequence of SEQ ID NO: 18020, the human CD8α transmembrane domain comprises the amino acid sequence of SEQ ID NO: 18014, the human 4-1BB costimulatory domain comprises the amino acid sequence of SEQ ID NO: 18018, and the human CD3ζ endodomain comprises the amino acid sequence of SEQ ID NO: 18016.

19. The cell of claim 17 or 18, further comprising a human CD8α signal peptide comprising the amino acid sequence of SEQ ID NO: 18012.

20. The cell according to any one of claims 1 to 19, wherein the cell is an immune cell.

21. 21. The cell of claim 20, wherein the immune cell is a T cell, a natural killer (NK) cell, a natural killer (NK)-like cell, a cytokine-induced killer (CIK) cell, a hematopoietic progenitor cell, a peripheral blood (PB)-derived T cell, or an umbilical cord blood (UCB)-derived T cell.

22. The cell according to any one of claims 1 to 21, wherein the cell is a T cell.

23. The T cells are memory stem T cells (T SCM 23. The cell of claim 22, wherein

24. The cell of any one of claims 1 to 23, wherein the cell is autologous.

25. The cell according to any one of claims 1 to 23, wherein the cell is of allogeneic origin.

26. The cell of any one of claims 1 to 23, further comprising an exogenous gene that confers resistance to a harmful compound.

27. 27. The cell of claim 26, wherein the exogenous gene is a dihydrofolate reductase (DHFR) gene.

28. 28. The cell of claim 27, wherein the DHFR gene comprises the sequence of SEQ ID NO: 17012.

29. The cell according to any one of claims 1 to 28, further comprising an exogenously inducible apoptosis-promoting polypeptide.

30. The cell of claim 29 , wherein the exogenously induced apoptosis-promoting polypeptide comprises a cleaved caspase 9 polypeptide.

31. The cell of claim 30, wherein the cleaved caspase 9 polypeptide comprises the sequence of SEQ ID NO: 18028.

32. A composition comprising the cells of any one of claims 1 to 31 and at least one pharmaceutically acceptable carrier.

33. A composition comprising a population of cells, wherein a plurality of cells of said population comprises a cell according to any one of claims 1 to 31.

34. 34. The composition of claim 33, wherein the plurality of cells of the population 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 therebetween of the cells of claim 1.

35. 34. The composition of claim 33, wherein the cell or cell population is autologous.

36. 34. The composition of claim 33, wherein the cell or cell population is allogeneic.

37. (a) an ectodomain comprising an antigen recognition region, wherein the antigen recognition region comprises at least one VHH domain that specifically binds to B cell maturation antigen (BCMA); (b) a transmembrane domain; and (c) an endodomain comprising at least one costimulatory domain; 1. A polynucleotide encoding a chimeric antigen receptor (CAR) comprising: The VHH domain comprises: Complementarity determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 18056; A complementarity determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 18060; and A polynucleotide comprising a complementarity determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 18064.

38. 38. The polynucleotide of claim 37, wherein the ectodomain further comprises a signal peptide.

39. 39. The polynucleotide of claim 38, wherein the signal peptide is human CD8α signal peptide.

40. The polynucleotide of any one of claims 37 to 39, wherein the ectodomain of (a) further comprises a hinge between the antigen recognition region and the transmembrane domain.

41. The polynucleotide of claim 40, wherein the hinge comprises the amino acid sequence of a human CD8α hinge.

42. The polynucleotide of any one of claims 37 to 41, wherein the transmembrane domain comprises a human CD8α transmembrane domain.

43. A polynucleotide described in any one of claims 37 to 42, wherein the endodomain comprises a human CD3ζ endodomain.

44. The polynucleotide of any one of claims 37 to 43, wherein the at least one costimulatory domain comprises the amino acid sequence of a human 4-1BB costimulatory domain.

45. The polynucleotide of any one of claims 37 to 44, wherein the VHH domain comprises the amino acid sequence of SEQ ID NO: 18051.

46. The polynucleotide of any one of claims 37 to 45, wherein the CAR comprises the amino acid sequence of SEQ ID NO: 18006.

47. 47. The polynucleotide of any one of claims 37 to 46, comprising the sequence of SEQ ID NO: 18007.

48. A polynucleotide described in any one of claims 37 to 47, wherein the polynucleotide further comprises an exogenous gene that confers resistance to harmful compounds.

49. 49. The polynucleotide of claim 48, wherein the exogenous gene is a dihydrofolate reductase (DHFR) gene.

50. A polynucleotide described in any one of claims 37 to 47, wherein the polynucleotide further encodes an exogenously inducible apoptosis-promoting polypeptide, and the exogenously inducible apoptosis-promoting polypeptide comprises a cleaved caspase 9 polypeptide.

51. A transposon comprising the polynucleotide according to any one of claims 37 to 50.

52. 52. The transposon of claim 51, wherein the transposon is a piggyBac transposon.

53. A composition comprising the polynucleotide of any one of claims 37 to 50 and a pharmaceutically acceptable carrier.

54. 53. A composition comprising the transposon of claim 51 or 52 and a pharmaceutically acceptable carrier.

55. 55. The composition of any one of claims 32 to 36, 53 and 54 for use in the manufacture of a medicament for the treatment of cancer.

56. 56. The composition of claim 55, wherein the cancer is multiple myeloma.

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