Inducible Caspases and Methods of Use

Description

1 / 145 Inducible Caspases and Methods of Use Divided from BR 11 2019 006865, deposited on 06 / 10 / 2017. RELATED ORDERS

[001] This application claims the benefit of provisional application USSN 62 / 405,184 filed on October 6, 2016, the contents of which are incorporated herein by reference in their entirety. INCORPORATION OF SEQUENCE LISTING

[002] The contents of the archived text named POTH-011_001WO_SeqList.txt, which was created on October 6, 2017 and is 61 KB in size, are incorporated herein by reference in their entirety. FIELD OF DISSEMINATION

[003] The disclosure is directed to molecular biology, and more specifically, to compositions containing at least one sequence encoding an inducible caspase protein, as well as methods for producing and using them. BACKGROUND

[004] There has long been a felt but technically unmet need for a method of selectively inducing apoptosis in genetically modified cells, and in particular, in those modified cells intended for administration to an individual as, for example, adoptive cell therapy. Disclosure provides a solution. Petition 870260041395, dated 04 / 05 / 2026, page 9 / 185 2 / 145 for this long-felt but unmet need. SUMMARY

[005] Disclosure provides an inducible proapoptotic polypeptide operatively linked to a ligand-binding region that can be optimized to bind to a chemical dimerization inducer. When the ligand-binding region specifically binds to the inducing agent, pro-apoptotic target molecules are cross-linked and consequently activated to selectively induce apoptosis in a cell containing a Disclosure inducible proapoptotic polypeptide. Preferred Disclosure inducible proapoptotic polypeptides include, but are not limited to, caspase-induced polypeptides. Preferred Disclosure inducible caspase polypeptides include, but are not limited to, inducible caspase 9 polypeptides.Possible disclosure inducible caspase 9 polypeptides may comprise a truncated caspase 9 polypeptide encoded by a truncated or modified amino acid and / or nucleic acid sequence encoding the truncated caspase 9 polypeptide.

[006] The induced pro-apoptotic polypeptides described are superior to existing inducible polypeptides because the inducible pro-apoptotic polypeptides described are much less immunogenic. Although the inducible pro-apoptotic polypeptides of Petition 870260041395, dated 04 / 05 / 2026, page 10 / 185 3 / 145 disclosure are recombinant polypeptides and therefore do not occur naturally, the sequences that are recombined to produce the inducible pro-apoptotic disclosure polypeptides do not comprise non-human sequences that the host human immune system can recognize as Non-self and consequently induce an immune response in the individual receiving an inducible pro-apoptotic disclosure polypeptide, a cell comprising the inducible pro-apoptotic disclosure polypeptide or a composition comprising the inducible pro-apoptotic disclosure polypeptide or the cell comprising the inducible pro-apoptotic disclosure polypeptide.

[007] The disclosure provides an inducible proapoptotic polypeptide comprising (a) a ligand-binding region, (b) a ligand, and (c) a proapoptotic polypeptide, wherein the inducible proapoptotic polypeptide does not comprise a non-human sequence. In certain embodiments, the non-human sequence comprises a restriction site. In certain embodiments, the ligand-binding region may be a multimeric ligand-binding region.

[008] The disclosure provides an inducible caspase polypeptide comprising (a) a ligand-binding region, (b) a ligand, and (c) a caspase polypeptide, wherein the inducible pro-apoptotic polypeptide does not Petition 870260041395, dated 04 / 05 / 2026, p. 11 / 185 4 / 145 comprise a non-human sequence.

[009] The disclosure provides an inducible caspase polypeptide comprising (a) a ligand-binding region, (b) a ligand, and (c) a truncated caspase polypeptide 9, wherein the inducible pro-apoptotic polypeptide does not comprise a non-human sequence. In certain embodiments, the non-human sequence comprises a restriction site. In certain embodiments, the ligand-binding region may be a multimeric ligand-binding region.

[0010] In certain embodiments of inducible caspase polypeptide, the ligand-binding region can specifically bind to an induction agent and activate the transcription of the pro-apoptotic (e.g., caspase polypeptide) disclosure polypeptide. For example, the ligand-binding region does not bind to a therapeutic agent. Induction agents specifically bound by the ligand-binding region of inducible disclosure polypeptides do not directly induce the transcription of endogenous genes.

[0011] Inducible pro-apoptotic (e.g., caspase) disclosure polypeptides may be under the control of one or more transcription regulatory elements, including but not limited to a promoter capable of initiating caspase polypeptide transcription at a Petition 870260041395, dated 04 / 05 / 2026, p. 12 / 185 5 / 145 A modified cell containing an inducible caspase disclosure polypeptide. For example, inducible caspase disclosure polypeptides may be under the control of one or more transcriptional regulatory elements, including but not limited to a mammalian promoter capable of initiating caspase polypeptide transcription in a mammalian cell modified to contain an inducible caspase disclosure polypeptide. For example, inducible caspase disclosure polypeptides may be under the control of one or more transcriptional regulatory elements, including but not limited to a heterologous or exogenous promoter capable of initiating caspase polypeptide transcription in a mammalian cell modified to contain an inducible caspase disclosure polypeptide. Preferred mammalian cells include, but are not limited to, human cells.

[0012] The description provides an inducible caspase polypeptide comprising (a) a ligand-binding region, (b) a ligand, and (c) a truncated caspase 9 polypeptide, wherein the inducible pro-apoptotic polypeptide does not comprise a non-human sequence. In certain embodiments, the ligand-binding region may be a multimeric ligand-binding region.

[0013] In certain forms of inducible proapoptotic polypeptides, inducible caspase polypeptides Petition 870260041395, dated 04 / 05 / 2026, p. 13 / 185 6 / 145 or truncated caspase 9 disclosure polypeptides, the ligand-binding region may comprise an FK506 12-binding protein polypeptide (FKBP12). In certain embodiments, the amino acid sequence of the ligand-binding region comprising an FK506 12-binding protein polypeptide (FKBP-12) may comprise a modification at position 36 of the sequence. The modification may be a substitution of valine (V) for phenylalanine (F) at position 36 (F36V). In certain embodiments, the FKBP12 polypeptide is encoded by an amino acid sequence comprising GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGW EEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE (SEQ ID NO: 3).In certain embodiments, the FKBP12 polypeptide is encoded by a nucleic acid sequence comprising GGGGTCCAGGTCGAGACTATTTCACCAGGGGATGGGCGAACATTTCCAAAAAGGGGCCA GACTTGCGTCGTGCATTACACCGGGATGCTGGAGGACGGGAAGAAAGTGGACAGCTCCA GGGATCGCAACAAGCCCTTCAAGTTCATGCTGGGAAAGCAGGAAGTGATCCGAGGATGG GAGGAAGGCGTGGCACAGATGTCAGTCGGCCAGCGGGCCAAACTGACCATTAGCCCTGA CTACGCTTATGGAGCAACAGGCCACCCAGGGATCATTCCCCCTCATGCCACCCTGGTCT TCGAT GTGGAACTGCTGAAGCTGGAG (SEQ ID NO: 4). In certain embodiments, the specific induction agent for the ligand-binding region may comprise an FK506 12 binding protein polypeptide (FKBP12) having a valine (V) to phenylalanine (F) substitution at position 36 (F36V) comprising AP20187 and / or AP1903 (Rimiducid), both. Petition 870260041395, dated 04 / 05 / 2026, p. 14 / 185 7 / 145 synthetic drugs.

[0014] In certain embodiments of inducible proapoptotic polypeptides, inducible caspase polypeptides, or truncated caspase 9 disclosure polypeptides, the binding region is encoded by an amino acid comprising GGGGS (SEQ ID NO: 5) or a nucleic acid sequence comprising GGAGGAGGAGGATCC (SEQ ID NO: 6). In certain embodiments, the nucleic acid sequence encoding the ligand does not comprise a restriction site.

[0015] In certain embodiments of truncated caspase 9 disclosure polypeptides, the truncated caspase 9 polypeptide is encoded by an amino acid sequence that does not comprise an arginine (R) at position 87 of the sequence. Alternatively, or additionally, in certain embodiments of inducible pro-apoptotic polypeptides, inducible caspase polypeptides, or truncated caspase 9 disclosure polypeptides, the truncated caspase 9 polypeptide is encoded by an amino acid sequence that does not comprise an alanine (A) at position 282 of the sequence. In certain embodiments of induced pro-apoptotic polypeptides, inducible caspase polypeptides, or truncated caspase 9 disclosure polypeptides, the truncated caspase 9 polypeptide is encoded by an amino acid comprising GFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRR Petition 870260041395, dated 04 / 05 / 2026, p. 15 / 185 8 / 145 FSSLHFMVEVKGDLTAKKMVLALLELAQDHGALDCCVVVILSHGCQASHLQFPGAVYG TDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSN PEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYRDATVSWSTWSTWSGYWPK FEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS (SEQ ID NO: 7) or a nucleic acid sequence comprising TTTGGGGACGTGGGGCCCTGGAGTCTCTGCGAGGAAATGCCGATCTGGCTTACATCCT GAGCATGGAACCCTGCGGCCACTGTCTGATCATTAACAATGTGAACTTCTGCAGAGAAA GCGGACTGCGAACACGGACTGGCTCCAATATTGACTGTGAGAAGCTGCGGAGAAGGTTC TCTAGTCTGCACTTTATGGTCGAAGTGAAAGGGGATCTGACCGCCAAGAAAATGGTGCT GGCCCTGCTGGAGCTGGCTCAGCAGGACCATGGAGCTCTGGATTGCTGCGTGGTCGTGA TCCTGTCCCACGGGTGCCAGGCTTCTCATCTGCAGTTCCCCGGAGCAGTGTACGGAACA GACGGCTGTCCTGTCAGCGTGGAGAAGATCGTCAACATCTTCAACGGCACTTCTTGCCC TAGTCTGGGGGGAAAGCCAAAACTGTTCTTTATCCAGGCCTGTGGCGGGGAACAGAAAG ATCACGGCTTCGAGGTGGCCAGCACCAGCCCTGAGGACGAATCACCAGGGAGCAACCCT GAACCAGATGCAACTCCATCCAGGAGGGACTGAGGACCTTTGACCAGCTGGATGCTAT CTCAAGCCTGCCCACTCCTAGTGACATTTTCGTGTCTTACAGTACCTTCCCAGGCTTTG TCTCATGGCGCGATCCCAAGTCAGGGAGCTGGTACGTGGAGACACTGGACGACATCTTT GAACAGTGGGCCCATTCAGAGGACCTGCAGAGCCTGCTGCTGCGAGTGGCAAACGCTGT CTCTGTGAAGGGCATCTACAAACAGATGCCCGGGTGCTTCAATTTTCTGAGAAAGAAAC TGTTCTTTAAGACTTCC (SEQ ID NO: 8).

[0016] In certain embodiments of inducible proapoptotic polypeptides, wherein the polypeptide comprises a truncated caspase 9 polypeptide, the inducible proapoptotic polypeptide is encoded by an amino acid sequence comprising GVQVETISPGDGRTFPKR Petition 870260041395, dated 04 / 05 / 2026, p. 16 / 185 9 / 145 GQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTIS PDYAYGATGHPGIIPPHATLVFDVELLKLEGGGGSGFGDVGALESLRGNADLAYILSME PCGHCLIINNVNFCRESGLRTRTGSNIDCEKKRRFSDRNKLPKFMLGKLKLL ELAQQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLG GKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSL PTPSDIFVSYSTFPGFVSWRDPKGGSWYVETLDDIFEQSWKVLVLL GIYKQMPGCFNFLRKKLFFKTS (SEQ ID NO: 9) or the nucleic acid sequence comprising GGGGTCCAGGTCGAGACTATTTCACCAGGGGATGGGCGAACATTTCCAAAAAGGGCCA GACTTGCGTCGTGCATTACACCAGGGGGCTGGAGAGAGACCAGCAGGAGGAGGGGGGGGGGGGGGGGGCGAACATTTCCAAAAAGGGCCA GGGATCGCAACAAGCCCTTCAAGTTCATGCTGGGAAAGCAGGAAGTGATCCGAGGATGG GAGGAAGGCGTGGCACAGATGTCAGTCGGCCAGCGGGCCAAACTGACCATTAGCCCTGA CTACGCTTATGGAGCAACAGGCCACCCAGGGATCATTCCCCCTCATGCCACCCTGGTGCACCCT TCGATGTGGAACTGCTGAAGCTGGAGGGAGGAGGAGGAGGATCCGGATTTGGGGAAATGCCGATCTGGCTTACATCCTGAGCATGGAACCCTG CGGCCACTGTCTGATCATTAACAATGTGAACTTCTGAAGGAAGCGAAGGCGAACGAACG GGACTGGCTCCAATATTGACTGTGAGAAGCTGCGGAGAAGGTTCTCTAGTCTGCACTTTATGGTCGAAGTGAAAGGGGATCTGACCGCCAAGAAAATGGTGCTGGCCCTGCTGGAGCT GGCTCAGCAGGACCATGGAGCTCTGGATTGCTGCGTGGTCGTGATCCTGTCCCACGGGT GCCAGGCTTCTCATCTGCAGTTCCCCGGAGCAGTGTACGGAACAGACGGCTGTCCTGTC AGCGTGGAGAAGATCGTCAACATCTTCAACGGCACTTCTTGCCCTAGTCTGGGGGGAAA GCCAAAACTGTTCTTTATCCAGGCCTGTGGCGGGGAACAGAAAGATCACGGCTTCGAGG TGGCCAGCACCAGCCCTGAGGACGAATCACCAGGGAGCAACCCTGAACCAGATGCAACT CCATTCCAGGAGGGACTGAGGACCTTTGACCAGCTGGATGCTATCTCAAGCCTGCCCAC TCCTAGTGACATTTTCGTGTCTTACAGTACCTTCCCAGGCTTTGTCTCATGGCGCGATC Petição 870260041395, de 04 / 05 / 2026, pág. 17 / 185 10 / 145 CCAAGTCAGGGAGCTGGTACGTGGAGACACTGGACGACATCTTTGAACAGTGGGCCCAT TCAGAGGACCTGCAGAGCCTGCTGCTGCGAGTGGCAAACGCTGTCTCTGTGAAGGGCAT CTACAAACAGATGCCCGGGTGCTTCAATTTTCTGAGAAAGAAACTGTTCTTTAAGACTT CC (SEQ ID NO: 10).

[0017] The disclosure provides a composition comprising an inducible pro-apoptotic polypeptide, an inducible caspase polypeptide, an inducible caspase 9 polypeptide and / or a truncated inducible caspase 9 polypeptide from the disclosure.

[0018] The description provides a transposon comprising an inducible pro-apoptotic polypeptide, inducible caspase polypeptide, inducible caspase 9 polypeptide and / or inducible truncated caspase 9 disclosure polypeptide.

[0019] Disclosure provides a transposon comprising an inducible pro-apoptotic polypeptide, inducible caspase polypeptide, inducible caspase 9 polypeptide, and / or inducible truncated caspase 9 polypeptide of disclosure. In certain embodiments of disclosure transposons, the transposon further comprises a sequence encoding a therapeutic protein. In certain embodiments, the therapeutic protein is naturally occurring. In certain embodiments, the therapeutic protein is an endogenous protein. In certain embodiments, the therapeutic protein is an exogenous protein. Petition 870260041395, dated 04 / 05 / 2026, page 18 / 185 11 / 145

[0020] The disclosure provides a transposon comprising an inducible pro-apoptotic polypeptide, inducible caspase polypeptide, inducible caspase 9 polypeptide, and / or inducible truncated caspase 9 polypeptide of the disclosure. In certain embodiments of the disclosure transposons, the transposon further comprises a sequence encoding a therapeutic protein. In certain embodiments, the therapeutic protein is not natural. In certain embodiments, the therapeutic protein is an endogenous protein. In certain embodiments, the therapeutic protein is an exogenous protein. In certain embodiments, the therapeutic protein is a synthetic protein. In certain embodiments, the therapeutic protein is a chimeric or recombinant protein. In certain embodiments, the therapeutic protein is a fusion protein. In certain embodiments, the therapeutic protein is a human protein, a wild-type protein, or a sequence variant thereof.

[0021] Disclosure provides a transposon comprising an inducible pro-apoptotic polypeptide, inducible caspase polypeptide, inducible caspase 9 polypeptide, and / or inducible truncated caspase 9 polypeptide of disclosure. In certain embodiments of disclosure transposons, the transposon further comprises a sequence encoding a therapeutic protein. In certain embodiments, the therapeutic protein comprises a surface protein. Petition 870260041395, dated 04 / 05 / 2026, p. 19 / 185 12 / 145 cellular, a membrane-bound protein, an extracellular membrane-bound protein, an intracellular membrane-bound protein, an intracellular protein, a protein located in the nucleus, a nuclear protein, a cytoplasmic protein, a cytosolic protein, a secreted protein, a lysosomal protein, an endosomal protein, a vesicle-associated protein, a mitochondrial protein, an endoplasmic reticulum protein, a cytoskeletal protein, a protein involved in intracellular signaling and / or a protein involved in extracellular signaling.

[0022] The description provides a transposon comprising an inducible pro-apoptotic polypeptide, inducible caspase polypeptide, inducible caspase 9 polypeptide, and / or inducible truncated caspase 9 polypeptide of the disclosure. In certain embodiments of the disclosure transposons, the transposon further comprises a sequence encoding a therapeutic protein. In certain embodiments, the therapeutic protein comprises an antigen receptor. In certain embodiments, the antigen receptor comprises a T cell receptor. In certain embodiments, the antigen receptor comprises an isolated or derived T cell receptor. In certain embodiments, the antigen receptor comprises one or more sequence variations and / or mutation(s) compared to a T cell receptor of the Petition 870260041395, dated 04 / 05 / 2026, p. 20 / 185 13 / 145 wild type. In certain embodiments, the T cell receptor is a recombinant T cell receptor.

[0023] The description provides a transposon comprising an inducible pro-apoptotic polypeptide, inducible caspase polypeptide, inducible caspase 9 polypeptide, and / or inducible truncated caspase 9 polypeptide of the disclosure. In certain embodiments of the disclosure transposons, the transposon further comprises a sequence encoding a therapeutic protein. In certain embodiments, where the antigen receptor is a Chimeric Antigen Receptor (CAR). In certain embodiments, the CAR comprises one or more Centirin sequences. In certain embodiments, the CAR is a CARTirin. In certain embodiments, the CAR comprises one or more VHH sequences. In certain embodiments, the CAR is a VCAR.

[0024] In certain embodiments, a disclosure transposon may further comprise at least one self-cleaving peptide. In certain embodiments, a disclosure transposon may comprise at least one self-cleaving peptide, wherein a self-cleaved peptide is located between the inducible pro-apoptotic polypeptide, inducible caspase polypeptide, inducible caspase 9 polypeptide, and / or truncated inducible caspase 9 polypeptide of the disclosure and another sequence in the transposon. In certain embodiments, a disclosure transposon may comprise Petition 870260041395, dated 04 / 05 / 2026, page 21 / 185 14 / 145 at least one self-cleaving peptide wherein one self-cleaving peptide is located upstream of the inducible pro-apoptotic polypeptide, inducible caspase polypeptide, inducible caspase 9 polypeptide and / or the inducible truncated caspase 9 polypeptide of disclosure and a second self-cleaving peptide is located downstream of the inducible pro-apoptotic polypeptide, inducible caspase polypeptide, inducible caspase 9 polypeptide and / or the inducible truncated caspase 9 polypeptide of disclosure.In certain embodiments, a disclosure transposon may comprise at least one self-cleaved peptide, wherein one self-cleaved peptide is located immediately upstream of the inducible pro-apoptotic polypeptide, inducible caspase polypeptide, inducible caspase 9 polypeptide, and / or inducible truncated caspase 9 polypeptide of disclosure, and a second self-cleaved peptide is located immediately downstream of the inducible pro-apoptotic polypeptide, inducible caspase polypeptide, inducible caspase 9 polypeptide, and / or inducible truncated caspase 9 polypeptide of disclosure. The at least one self-cleaving peptide may comprise 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. In certain forms, the T2A peptide comprises a sequence of amino acids. Petition 870260041395, dated 04 / 05 / 2026, page 22 / 185 15 / 145 comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 11). In certain embodiments, the GSG-T2A peptide comprises an amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 12). In certain embodiments, the E2A peptide comprises an amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO: 13). In certain embodiments, the GSG-E2A peptide comprises an amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 14). In certain embodiments, the F2A peptide comprises an amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 15). In certain embodiments, the GSG-F2A peptide comprises an amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 16). In certain embodiments, the P2A peptide comprises an amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 17). In certain embodiments, the GSG-P2A peptide comprises an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 18).

[0025] The disclosure provides a composition comprising a transposon of the disclosure. In certain embodiments of the compositions comprising a transposon, the composition may further comprise a plasmid comprising a sequence encoding a transposase enzyme. The sequence encoding a transposase enzyme may be an mRNA sequence. In certain embodiments, the transposon is a Petition 870260041395, dated 04 / 05 / 2026, p. 23 / 185 16 / 145 transposon piggyBac. In certain embodiments, the transposon is a transposon piggyBac and the transposase is a transposase Super piggyBac.

[0026] Disclosure transposons may comprise piggyBac transposons. In certain embodiments of the present method, the transposon is a plasmid DNA transposon with a sequence encoding the inducible caspase polypeptide of disclosure flanked by two cis regulatory insulator elements. In certain embodiments, the transposon is a piggyBac transposon. Disclosure transposase enzymes may include piggyBac transposases or compatible enzymes. In certain embodiments, and in particular those embodiments in which the transposon is a piggyBac transposon, the transposase is a piggyBac™ or Super piggyBac™ (SPB) transposase. In certain embodiments, and in particular those embodiments in which the transposase is a Super piggyBac™ (SPB) transposase, the sequence encoding the transposase enzyme is an mRNA sequence.

[0027] In certain embodiments of the disclosure methods, the transposase enzyme is a piggyBac™ (PB) transposase enzyme. The piggyBac (PB) transposase enzyme may comprise or consist of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or any percentage thereof identical to: Petition 870260041395, dated 04 / 05 / 2026, p. 24 / 185 17 / 145 MGSSLDDEHI LSALLQSDDE LVGEDSDSEI SDHVSEDDVQ SDTEEAFIDE VHEVQPTSSG 61 SEILDEQNVI EQPGSSLASN RILTLPQRTI RGKNKHCWST SKSTRRSRVS ALNIVRSQRG 121 PTRMCRNIYD PLLCFKLFFT DEIISEIVKW TNAEISLKRR ESMTGATFRD TNEDEIYAFF 181 GILVMTAVRK DNHMSTDDLF DRSLSMVYVS VMSRDRFDFL IRCLRMDDKS IRPTLRENDV 241 FTPVRKIWDL FIHQCIQNYT PGAHLTIDEQ LLGFRGRCPF RMYIPNKPSK YGIKILMMCD 301 SGTKYMINGM PYLGRGTQTN GVPLGEYYVK ELSKPVHGSC RNITCDNWFT SIPLAKNLLQ 361 EPYKLTIVGT VRSNKREIPE VLKNSRSRPV GTSMFCFDGP LTLVSYKPKP AKMVYLLSSC 421 DEDASINEST GKPQMVMYYN QTKGGVDTLD QMCSVMTCSR KTNRWPMALL YGMINIACIN 481 SFIIYSHNVS SKGEKVQSRK KFMRNLYMSL TSSFMRKRLE APTLKRYLRD NISNILPNEV 541 PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV ICREHNIDMC QSCF (SEQ ID NO: 1).

[0028] In certain embodiments of the disclosure methods, 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 the 30, 165, 282, or 538 positions of the sequence: 1 MGSSLDDEHI LSALLQSDDE LVGEDSDSEI SDHVSEDDVQ SDTEEAFIDE VHEVQPTSSG 61 SEILDEQNVI EQPGSSLASN RILTLPQRTI RGKNKHCWST SKSTRRSRVS ALNIVRSQRG 121 PTRMCRNIYD PLLCFKLFFT DEIISEIVKW TNAEISLKRR ESMTGATFRD TNEDEIYAFF 181 GILVMTAVRK DNHMSTDDLF DRSLSMVYVS VMSRDRFDFL IRCLRMDDKS IRPTLRENDV 241 FTPVRKIWDL FIHQCIQNYT PGAHLTIDEQ LLGFRGRCPF RMYIPNKPSK YGIKILMMCD 301 SGTKYMINGM PYLGRGTQTN GVPLGEYYVK ELSKPVHGSC RNITCDNWFT SIPLAKNLLQ 361 EPYKLTIVGT VRSNKREIPE VLKNSRSRPV GTSMFCFDGP LTLVSYKPKP AKMVYLLSSC 421 DEDASINEST GKPQMVMYYN QTKGGVDTLD QMCSVMTCSR KTNRWPMALL YGMINIACIN 481 SFIIYSHNVS SKGEKVQSRK KFMRNLYMSL TSSFMRKRLE APTLKRYLRD NISNILPNEV 541 PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV ICREHNIDMC QSCF (SEQ ID NO: 1).

[0029] In certain embodiments, the transposase enzyme is a piggyBac™ (PB) transposase enzyme comprising or Petition 870260041395, dated 04 / 05 / 2026, p. 25 / 185 18 / 145 consists of an amino acid sequence having an amino acid substitution at two or more of the 30, 165, 282, or 538 positions of the SEQ ID sequence. NO: 1. In certain 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 the 30, 165, 282, or 538 positions of the SEQ ID sequence. NO: 1. In certain 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 the following positions: 30, 165, 282, and 538 of the SEQ ID sequence. NO: 1. In certain embodiments, the amino acid substitution at position 30 of the SEQ ID NO: 1 sequence is a substitution of a valine (V) for an isoleucine (I).In certain embodiments, the amino acid substitution at position 165 of the SEQ ID NO: 1 sequence is a substitution of a serine (S) for a glycine (G). In certain embodiments, the amino acid substitution at position 282 of the SEQ ID NO: 1 sequence is a substitution of a valine (V) for a methionine (M). In certain embodiments, the amino acid substitution at position 538 of the SEQ ID NO: 1 sequence is a substitution of a lysine (K) for an asparagine (N).

[0030] In certain embodiments of the dissemination methods, the transposase enzyme is a transposase enzyme. Petition 870260041395, dated 04 / 05 / 2026, p. 26 / 185 19 / 145 Super piggyBac™ (SPB). In certain embodiments, the Super piggyBac™ (SPB) transposase enzymes of disclosure may comprise or consist of the amino acid sequence of sequence SEQ ID NO: 1, wherein the amino acid substitution at position 30 is a substitution of a valine (V) for an isoleucine (I), the amino acid substitution at position 165 is a substitution of a serine (S) for a glycine (G), the amino acid substitution at position 282 is a substitution of a valine (V) for a methionine (M), and the amino acid substitution at position 538 is a substitution of a lysine (K) for an asparagine (N).In certain embodiments, the Super piggyBac™ transposase enzyme (SPB) may comprise or consist of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or any percentage thereof identical to: 1 MGSSLDDEHI LSALLQSDDE LVGEDSDSEV SDHVSEDDVQ SDTEEAFIDE VHEVQPTSSG 61 SEILDEQNVI EQPGSSLASN RILTLPQRTI RGKNKHCWST SKSTRRSRVS ALNIVRSQRG 121 PTRMCRNIYD PLLCFKLFFT DEIISEIVKW TNAEISLKRR ESMTSATFRD TNEDEIYAFF 181 GILVMTAVRK DNHMSTDDLF DRSLSMVYVS VMSRDRFDFL IRCLRMDDKS IRPTLRENDV 241 FTPVRKIWDL FIHQCIQNYT PGAHLTIDEQ LLGFRGRCPF RVYIPNKPSK YGIKILMMCD 301 SGTKYMINGM PYLGRGTQTN GVPLGEYYVK ELSKPVHGSC RNITCDNWFT SIPLAKNLLQ 361 EPYKLTIVGT VRSNKREIPE VLKNSRSRPV GTSMFCFDGP LTLVSYKPKP AKMVYLLSSC 421 DEDASINEST GKPQMVMYYN QTKGGVDTLD QMCSVMTCSR KTNRWPMALL YGMINIACIN 481 SFIIYSHNVS SKGEKVQSRK KFMRNLYMSL TSSFMRKRLE APTLKRYLRD NISNILPKEV 541 PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV ICREHNIDMC QSCF (SEQ ID NO: 2).

[0031] In certain modalities of the methods of Petition 870260041395, dated 04 / 05 / 2026, p. 27 / 185 20 / 145 disclosure, including embodiments in which the transposase comprises the mutations described above at positions 30, 165, 282 and / or 538, the piggyBac™ or Super piggyBac™ transposase enzyme may further comprise an amino acid substitution at one or more of the positions 3, 46, 82, 103, 119, 125, 177, 180, 185, 187, 200, 207, 209, 226, 235, 240, 241, 243, 258, 296, 298, 311, 315, 319, 327, 328, 340, 421, 436, 456, 470, 486, 503, 552, 570 and 591 of the SEQ ID NO: 1 or SEQ ID NO: 2 sequence. In certain embodiments, including those embodiments in which the transposase comprises the mutations described above at positions 30, 165, 282 and / or 538, the piggyBac™ or Super piggyBac™ transposase enzyme may also comprise an amino acid substitution at one or more positions 46, 119, 125, 177, 180, 185, 187, 200, 207, 209, 226, 235, 240, 241, 243, 296, 298, 311, 315, 319, 327, 328, 340, 421, 436, 456, 470, 485, 503, 552 and 570.In certain embodiments, the amino acid substitution at position 3 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of an asparagine (N) for a serine (S). In certain embodiments, the amino acid substitution at position 46 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of a serine (S) for an alanine (A). In certain embodiments, the amino acid substitution at position 46 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of a threonine (T) for an alanine (A). In certain embodiments, the amino acid substitution at position... Petition 870260041395, dated 04 / 05 / 2026, p. 28 / 185 21 / 145 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of tryptophan (W) for isoleucine (I). In certain embodiments, the amino acid substitution at position 103 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of proline (P) for serine (S). In certain embodiments, the amino acid substitution at position 119 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of proline (P) for arginine (R). In certain embodiments, the amino acid substitution at position 125 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of alanine (A) for cysteine ​​(C). In certain embodiments, the amino acid substitution at position 125 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of leucine (L) for cysteine ​​(C). In certain embodiments, the amino acid substitution at position 177 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of a lysine (K) for a tyrosine (Y).In certain embodiments, the amino acid substitution at position 177 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of a histidine (H) for a tyrosine (Y). In certain embodiments, the amino acid substitution at position 180 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of a leucine (L) for a phenylalanine (F). In certain embodiments, the amino acid substitution at position 180 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of an isoleucine (I) for a phenylalanine (F). In certain embodiments, the amino acid substitution at position 180 of SEQ ID NO: 1. Petition 870260041395, dated 04 / 05 / 2026, p. 29 / 185 22 / 145 or SEQ ID NO: 2 is a substitution of a valine (V) for a phenylalanine (F). In certain embodiments, the amino acid substitution at position 185 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of a leucine (L) for a methionine (M). In certain embodiments, the amino acid substitution at position 187 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of a glycine (G) for an alanine (A). In certain embodiments, the amino acid substitution at position 200 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of a tryptophan (W) for a phenylalanine (F). In certain embodiments, the amino acid substitution at position 207 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of a proline (P) for a valine (V). In certain embodiments, the amino acid substitution at position 209 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of a phenylalanine (F) for a valine (V).In certain embodiments, the amino acid substitution at position 226 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of phenylalanine (F) for methionine (M). In certain embodiments, the amino acid substitution at position 235 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of arginine (R) for leucine (L). In certain embodiments, the amino acid substitution at position 240 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of lysine (K) for valine (V). In certain embodiments, the amino acid substitution at position 241 of SEQ ID NO: 1 or SEQ ID NO: Petition 870260041395, dated 04 / 05 / 2026, p. 30 / 185 23 / 145 is a substitution of leucine (L) for phenylalanine (F). In certain embodiments, the amino acid substitution at position 243 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of lysine (K) for proline (P). In certain embodiments, the amino acid substitution at position 258 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of serine (S) for asparagine (N). In certain embodiments, the amino acid substitution at position 296 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of tryptophan (W) for leucine (L). In certain embodiments, the amino acid substitution at position 296 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of tyrosine (Y) for leucine (L). In certain embodiments, the amino acid substitution at position 296 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of a phenylalanine (F) for a leucine (L).In certain embodiments, the amino acid substitution at position 298 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of leucine (L) for methionine (M). In certain embodiments, the amino acid substitution at position 298 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of alanine (A) for methionine (M). In certain embodiments, the amino acid substitution at position 298 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of valine (V) for methionine (M). In certain embodiments, the amino acid substitution at position 311 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of a... Petition 870260041395, dated 04 / 05 / 2026, p. 31 / 185 24 / 145 isoleucine (I) for proline (P). In certain embodiments, the amino acid substitution at position 311 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of valine for proline (P). In certain embodiments, the amino acid substitution at position 315 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of lysine (K) for arginine (R). In certain embodiments, the amino acid substitution at position 319 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of glycine (G) for threonine (T). In certain embodiments, the amino acid substitution at position 327 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of arginine (R) for tyrosine (Y). In certain embodiments, the amino acid substitution at position 328 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of a valine (V) for a tyrosine (Y). In certain embodiments, the amino acid substitution at position 340 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of a glycine (G) for a cysteine ​​(C).In certain embodiments, the amino acid substitution at position 340 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of a leucine (L) for a cysteine ​​(C). In certain embodiments, the amino acid substitution at position 421 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of a histidine (H) for aspartic acid (D). In certain embodiments, the amino acid substitution at position 436 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of a... Petition 870260041395, dated 04 / 05 / 2026, p. 32 / 185 25 / 145 isoleucine (I) for valine (V). In certain embodiments, the amino acid substitution at position 456 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of tyrosine (Y) for methionine (M). In certain embodiments, the amino acid substitution at position 470 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of phenylalanine (F) for leucine (L). In certain embodiments, the amino acid substitution at position 485 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of lysine (K) for serine (S). In certain embodiments, the amino acid substitution at position 503 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of leucine (L) for methionine (M). In certain embodiments, the amino acid substitution at position 503 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of an isoleucine (I) for a methionine (M).In certain embodiments, the amino acid substitution at position 552 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of lysine (K) for valine (V). In certain embodiments, the amino acid substitution at position 570 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of threonine (T) for alanine (A). In certain embodiments, the amino acid substitution at position 591 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of proline (P) for glutamine (Q). In certain embodiments, the amino acid substitution at position 591 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of arginine (R) for glutamine (Q). Petition 870260041395, dated 04 / 05 / 2026, p. 33 / 185 26 / 145

[0032] In certain embodiments of the disclosure methods, including embodiments in which the transposase comprises the mutations described above at positions 30, 165, 282 and / or 538, the piggyBac™ transposase enzyme may comprise, or the Super piggyBac™ transposase enzyme may further comprise, an amino acid substitution at one or more of positions 103, 194, 372, 375, 450, 509 and 570 of the SEQ ID NO: 1 or SEQ ID NO: 2 sequence. In certain embodiments of the disclosure methods, including embodiments in which the transposase comprises the mutations described above at positions 30, 165, 282 and / or 538, the piggyBac™ transposase enzyme may comprise, or the Super piggyBac™ transposase enzyme may further comprise, an amino acid substitution at two, three, four, five, six or more of the positions 103, 194, 372, 375, 450, 509 and 570 of the sequence SEQ ID NO: 1 or SEQ ID NO: 2.In certain embodiments, including embodiments in which the transposase comprises the mutations described above at positions 30, 165, 282 and / or 538, the piggyBac™ transposase enzyme may comprise, or the piggyBac™ Super transposase enzyme may further comprise, an amino acid substitution at positions 103, 194, 372, 375, 450, 509 and 570 of the SEQ ID NO: 1 or SEQ ID NO: 2 sequence. In certain embodiments, the amino acid substitution at position 103 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitute for... Petition 870260041395, dated 04 / 05 / 2026, p. 34 / 185 27 / 145 a proline (P) for a serine (S). In certain embodiments, the amino acid substitution at position 194 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of a valine (V) for a methionine (M). In certain embodiments, the amino acid substitution at position 372 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of an alanine (A) for an arginine (R). In certain embodiments, the amino acid substitution at position 375 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of an alanine (A) for a lysine (K). In certain embodiments, the amino acid substitution at position 450 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of an asparagine (N) for an aspartic acid (D). In certain embodiments, the amino acid substitution at position 509 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of a glycine (G) for a serine (S).In certain embodiments, the amino acid substitution at position 570 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of a serine (S) for an asparagine (N). In certain embodiments, the piggyBac™ transposase enzyme may comprise a substitution of a valine (V) for a methionine (M) at position 194 of SEQ ID NO: 1. In certain embodiments, including those embodiments in which the piggyBac™ transposase enzyme may comprise a substitution of a valine (V) for a methionine (M) at position 194 of SEQ ID NO: 1, the piggyBac™ transposase enzyme may still comprise an amino acid substitution at the positions. Petition 870260041395, dated 04 / 05 / 2026, p. 35 / 185 28 / 145 372, 375 and 450 of the sequence SEQ ID NO. : 1 or SEQ ID NO: 2. In certain embodiments, the piggyBac™ transposase enzyme may comprise a valine (V) substitution for a methionine (M) at position 194 of SEQ ID NO: 1, an alanine (A) substitution for an arginine (R) at position 372 of SEQ ID NO: 1, and an alanine (A) substitution for a lysine (K) at position 375 of SEQ ID NO: 1. In certain embodiments, the piggyBac™ transposase enzyme may comprise a valine (V) substitution for a methionine (M) at position 194 of SEQ ID NO: 1, an alanine (A) substitution for an arginine (R) at position 372 of SEQ ID NO: 1, an alanine (A) substitution for a lysine (K) at position 375 of SEQ ID NO: 1, and an asparagine substitution. (N) by an aspartic acid (D) at position 450 of SEQ ID NO: 1.

[0033] The disclosure provides a composition comprising a disclosure transposon. In certain embodiments of the present method, the transposon is a plasmid DNA transposon with a sequence encoding the disclosure-inducible caspase polypeptide flanked by two cis-regulatory insulator elements. In certain embodiments of the compositions comprising a transposon, the composition may further comprise a plasmid comprising a sequence encoding a transposase enzyme. The sequence encoding a transposase enzyme may be a Petition 870260041395, dated 04 / 05 / 2026, p. 36 / 185 29 / 145 mRNA sequence. In certain embodiments, the transposon is a Sleeping Beauty transposon. In certain embodiments, the transposon is a Sleeping Beauty transposon and the transposase is either a Sleeping Beauty transposase or a hyperactive Sleeping Beauty transposase (SB100X).

[0034] Disclosure transposons may comprise Sleeping Beauty transposons. In certain embodiments, and in particular those embodiments in which the transposon is a Sleeping Beauty transposon, the composition further comprises a plasmid comprising a sequence encoding a transposase enzyme. In certain embodiments, the sequence encoding the transposase enzyme is a sequence encoding a Sleeping Beauty transposase or a hyperactive Sleeping Beauty transposase (SB100X). In certain embodiments, the sequence encoding the transposase enzyme is an mRNA sequence.

[0035] In certain embodiments of the disclosure methods, the Sleeping Beauty transposase enzyme comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or any percentage thereof identical to: 1 MGKSKEISQD LRKKIVDLHK SGSSLGAISK RLKVPRSSVQ TIVRKYKHHG TTQPSYRSGR 61 RRVLSPRDER TLVRKVQINP RTTAKDLVKM LEETGTKVSI STVKRVLYRH NLKGRSARKK 121 PLLQNRHKKA RLRFATAHGD KDRTFWRNVL WSDETKIELF GHNDHRYVWR KKGEACKPKN 181 TIPTVKHGGG SIMLWGCFAA GGTGALHKID GIMRKENYVD ILKQHLKTSV RKLKLGRKWV 241 FQMDNDPKHT SKVVAKWLKD NKVKVLEWPS QSPDLNPIEN LWAELKKRVR ARRPTNLTQL 301 HQLCQEEWAK IHPTYCGKLV EGYPKRLTQV KQFKGNATKY (SEQ ID NO: 19). Petition 870260041395, dated 04 / 05 / 2026, p. 37 / 185 30 / 145

[0036] In certain embodiments of the disclosure methods, the Sleeping Beauty hyperactive transposase enzyme (SB100X) comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or any percentage thereof identical to: 1 MGKSKEISQD LRKRIVDLHK SGSSLGAISK RLAVPRSSVQ TIVRKYKHHG TTQPSYRSGR 61 RRVLSPRDER TLVRKVQINP RTTAKDLVKM LEETGTKVSI STVKRVLYRH NLKGHSARKK 121 PLLQNRHKKA RLRFATAHGD KDRTFWRNVL WSDETKIELF GHNDHRYVWR KKGEACKPKN 181 TIPTVKHGGG SIMLWGCFAA GGTGALHKID GIMDAVQYVD ILKQHLKTSV RKLKLGRKWV 241 FQHDNDPKHT SKVVAKWLKD NKVKVLEWPS QSPDLNPIEN LWAELKKRVR ARRPTNLTQL 301 HQLCQEEWAK IHPNYCGKLV EGYPKRLTQV KQFKGNATKY (SEQ ID NO: 20).

[0037] The disclosure provides a composition comprising a disclosure transposon. In certain embodiments of the present method, the transposon is a plasmid DNA transposon with a sequence encoding the disclosure-inducible caspase polypeptide flanked by two cis-regulatory isolator elements. In certain embodiments of the compositions comprising a transposon, the composition may further comprise a plasmid comprising a sequence encoding a transposase enzyme. The sequence encoding a transposase enzyme may be an mRNA sequence. In certain embodiments, the transposon is a Helraiser transposon. In certain embodiments, the transposon is a Helraiser transposon and the transposase is a Helitron transposase.

[0038] Transposons of disclosure may include Petition 870260041395, dated 04 / 05 / 2026, p. 38 / 185 31 / 145 Helraiser transposons. In certain embodiments of this method, the transposon is a plasmid DNA transposon with a sequence encoding the inducible disclosure caspase polypeptide flanked by two cis-regulatory isolator elements. In certain embodiments of this method, the transposon is a Helraiser transposon. In certain embodiments, and in particular in those embodiments where the transposon is a Helraiser transposon, the composition further comprises a plasmid comprising a sequence encoding a transposase enzyme. In certain embodiments, the sequence encoding the transposase enzyme comprises a sequence encoding a Helitron transposase. In certain embodiments, the sequence encoding the transposase enzyme is an mRNA sequence.

[0039] In certain embodiments, the transposase is a Helitron transposase. Helitron transposases mobilize the Helraiser transposon, an ancient element of the bat genome that was active about 30 to 36 million years ago. An example of a Helraiser transposon from disclosure includes Helibat1, which comprises a nucleic acid sequence comprising: 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 Petition 870260041395, dated 04 / 05 / 2026, p. 39 / 185 32 / 145 CGGAGAGCGG GAGGTTTGGA GGACTTGGCA CGAGAGAGAG GGTGGCTTGG AGGGCGTGGC GCTGTGGAAA CTGACAGCAG GGAGGAGGAA GCCGTTGGTC AGACAGCTCT CAGCGGCCTG AGACCGTGAC AGTAGAGAGG TGGGACTATG TCTGCAGCCG AAAGATGCCG GCGTTATCGA GATCTTGAAA GAAGGCGGCG CCTGCAACAG CGTCGCTCTG AAGCCGAAAA ACACGGCGCGT GCCTTTGAAG TTGAAAGAAG GCGGTGGCGA ACAAGTACTA CCAATACCGG TAGGAACTGC GCAATTCTCG AACATAGTTG TGGTGGAATG AATTTCTCTG ATGAAAAACC ATCCGATGGG GTCTGTCCAA ATGATATACA TTTTCCAGAT AACGAAGATT CTGACAGTAA AAATTTCATG GCTTTTGCTT CCATGGGTGC AAATATTGCA AGAATACACG GACAAGTTTA TCACCGTACT CGGAAGTTTG CTCAACTCTA TATTTTGGAT ATGCCAGAAA ACCAGGGCTG CTCAGAAAGA GAAATAAATG AATTAACAAA ATCGTACAAG TCTGAAGCAG CAGCAAAAGG TATTGCTCCC CGTAACGTG ACCCAGGTAG ATATAATTCT AGAAACGAAG ATGGAGAACC TCCTTTTGAA CCCATAATC CAAATGCCAC TAAAATGAAA GCAATGACAT ATCCTATTCT TTTTCCACAT TAAGACTCA GAGACAACAG TGTAATCGAC GTCACACAAA TGCAGTATTA TGGATTTCAT TTAAATGCAG GAAAATTAAC TCAACAGTTT AATCGGATAA ATTTCATCAA AGCAAACCAA TTGATGGATT ATCTCAAATC TAGATCTGAA ATACTTCCAT CATCTTTTGA GGGTAGTCCC 241 CGCCAGCAGA GAGCAGAGCG GGAGAGCGGG 301 GAGCAGGAGG CCGCTGGACA TAGAGCAGAG 361 TCCCTCTGTC ACCCCAGCTT CCTCATCACA 421 GTCCCACCCC CACAGAATCA GCCAGAATCA 481 ACAGCCAGGA CTCTCATTCA CCTGCATCTC 541 TCTAAAGAAC AACTGTTGAT ACAACGTAGC 601 CAGAAAATGT CTGCAGAGCA ACGTGCGTCT 661 AATGTATCTG AAGAGCAGCT ACTGGAAAAA 721 CATCGACAGA AAATGTCTAA AGACCAACGT 781 CGACAGAATA TGTCTAGAGA ACAGTCATCA 841 CTTCTCAGCA AAAATGGAGT ACATGAGGAT 901 ACTGTTCGAT GTGAATTTTG CCTATCACTA 961 AAATTTACTC GATGTTGTAG CAAAGGGAAA 1021 TACCCGGCAT ATTTAAAAAG ATTAATGACA 1081 GAAAATATTC GTTCCATAAA TAGTTCTTTT 1141 TCGCCATCAG GATATGGGCC ATACTGTTTT 1201 GGAACTTTAC ATCCTTCGGA TGGTGTTTCT 1261 ACAGCCGAAG CTACAAGTAA AAGATTAGCA 1321 CTCATGATCA ACATCAACAA CCTCATGCAT 1381 ATGCTACATG AGGTAGAAAA GGAAGCCCAA 1441 ACAGAAGTAA CAATGGCGAT TAAATACGAT 1501 CCCCGTGTAA CCGAGGTTGC TGTCATATTC 1561 AGGGACTTGC TCATTCATG TAAACCAGAT 1621 CAAATCAGTA TCCTGTTTCC CAPTIONS 1681 GGTGAAAAAG GCTGGGGAAC AGATATTGCA 1741 AATAATACTA GACAAAATGT AAGGACACGA 1801 CTCTCTGTGC GGGACACGTT CAATCCTATT 1861 ATTGTGGATT CATATTCAAA AATGGAGGCC 1921 TCTAAGTTGA GAGTTGAAAA ATATAGTGGT 1981 AATGACAATG TGCCGATTGG TAAAATGATA Petition 870260041395, of 04 / 05 / 2026, p. 40 / 185 33 / 145 2041 AGAAATATGC AGCAGCGATA TCAGGATGCT 2101 GATTTATTCA TAACCATGAC ATGCAACCCC 2161 CGCTGGCAAA AAGTTGAAAA CAGACCTGAC 2221 AATGCTCTTT TAAATGATAT ATGTAAATTC 2281 CATGTCATTG AATTTCAGAA ACGCGGACTG 2341 AGTGAGTCCA AATTACGTTC AGAAGATGAC 2401 CONNECT CC AGTGTTCCTCG ACTTTTTCAA 2461 TGTGGAATAC AAAATCCAAAA TGTCCATGT 2521 CCAAAAGAAT TTCAAAATGC GACCATTGGA 2581 AGATCTGGTA GCACCATGTC TATTGGAAAT 2641 TATAACCCGT ATTTGTGCCT TAAATATAAC 2701 ATTACKGG SECURITY ATTACK 2761 CAUTIONTTCTG AAAAAAATTACT CAUTION CATCH 2821 TATGTGAGCG CTCCTGAGGC TGTTTGGAGA 2881 CATGCAATCA CAAGATTAGC TATTCATTTG 2941 GATGATTTTG CTGAAGTTTT AGATAGGGCT 3001 TTCTTATTGA ATAGGAAGA TTCTGATGCA 3061 CATTATGTGT TTAATAATTC TTTGTGGACA 3121 GGTAGACTGT TCACTGTGAG CTTTAGAGAA 3181 CTGCATGTAA AAGGTGCGAT AAGTTTTGAG 3241 GATACATTTC ATGAAGCTGC TAAACACCGA 3301 GATACGATTG ACGATGCAAT CATCCTTAAT 3361 TATATATGTG TGTTTGGATG TCCTTCTGCT 3421 HELP AAGATTTCTG TTGGAAATTA 3481 GAAATGCATG CCCTTAACGA AATTCAGGAG 3541 CATTTCAAAC TTCCGGACTA TCCTTTATTA 3601 GAGCAACAAC AGGCAGAGGT TTTGATAAAT 3661 CAGACTATAA CTTCAGCCAT CGAAGATCAA 3721 GGTCCAGGTG GTAGTGGAAA AACATATCTG 3781 CGTGGTGGTA CTGTTTTACC CACAGCATCT ATGGCAATTG TAACGAAGTA TGGCAAGCCC AAATGGGCAG ATATTACAAA CAATTTACAA TTGGTAGCCA GAGTTTTTAA TATTAAGCTG CATTTATTTG GCAAAGTAAT AGCTAAAATT CCTCACGCTC ACATATTATT GATATTAGAT ATTGACCAGTAGTA GTAGTATTCATTAGTA CAAATATGGT ACATGGACCA ATGGAAAATG GAAAATGTTC AAAGGGATAT AATATTGATG GATATCCCAA ATACAAACGA AAAGTTGTCG ATAACACTTG GATTGTCCCT TGTCATATAA ATGTTGAAGT CTGTGCATCA ATCTATAAAG GGCACATAGATTGATTGATCGATTGATCGATT TGACTCCAGG CTTTTTGCAA TGCGAATGCA TGACCAATCT CCAAATGATC AGAATTTGTA TTTTCATACC AAAAGGCATA ACTCGACTTT GATGGCTTGG CGTAATTATT ATTATTGGGA GATTCCACAG AAACGCCGAA AGGGTGGGAA TAAAGTAAGTCGTACCTATTTAGCCTATTAG GATCTGCGAA CTGTAGGAGG TGTAACTTAT GGATTATTTAC TTGATGACAC TATCTGGAAA ATGCCCAAAC AACTACGGCA ACTTTTTGCA GCAGACAAAT TATGGGATGA GAATAAATCT CACCGAAGAG AAGGTGCCTG TGTGAACTGT GTATTCACAT GATGATCGAAT GATGATCAA ATACATGTGA TCAATTGTAC TCTCTGAATG ATGAACAGTT GGCAGCCTTT ACTGTACACC CCAAATGCTT TTTCTTGGAT TATAAAGTTT TAACACATTA TATTAGAGGT ACAGGAATTG CTGCAAATTT ACTTCTTGGT Petition 870260041395, of 04 / 05 / 2026, p. 41 / 185 34 / 145 3841 GGAAGAACCT TTCATTCCCA ATATAAATTA CCAATTCCAT TAAATGAAAC TTCAATTTCT 3901 AGACTCGATA TAAAGAGTGA AGTTGCTAAA ACCATTAAA 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: 21).

[0040] Unlike other transposases, the Helitron transposase does not contain an RNase-H as a catalytic domain, but instead comprises a RepHel motif composed of a replication initiator domain (Rep) and a Petition 870260041395, dated 04 / 05 / 2026, p. 42 / 185 35 / 145 DNA helicase domain. The Rep domain is a nuclease domain of the HUH superfamily of nucleases.

[0041] An exemplary Helitron transposase from disclosure comprises an amino acid sequence comprising: 1 MSKEQLLIQR SSAAERCRRY RQKMSAEQRA SDLERRRRLQ QNVSEEQLLE KRRSEAEKQR 61 RHRQKMSKDQ RAFEVERRRW RRQNMSREQS STSTTNTGRN CLLSKNGVHE DAILEHSCGG 121 MTVRCEFCLS LNFSDEKPSD GKFTRCCSKG KVCPNDIHFP DYPAYLKRLM TNEDSDSKNF 181 MENIRSINSS FAFASMGANI ASPSGYGPYC FRIHGQVYHR TGTLHPSDGV SRKFAQLYIL 241 DTAEATSKRL AMPENQGCSE RLMININNLM HEINELTKSY KMLHEVEKEA QSEAAAKGIA 301 PTEVTMAIKY DRNSDPGRYN SPRVTEVAVI FRNEDGEPPF ERDLLIHCKP DPNNPNATKM 361 KQISILFPTL DAMTYPILFP HGEKGWGTDI ALRLRDNSVI DNNTRQNVRT RVTQMQYYGF 421 HLSVRDTFNP ILNAGKLTQQ FIVDSYSKME ANRINFIKAN QSKLRVEKYS GLMDYLKSRS 481 ENDNVPIGKM IILPSSFEGS PRNMQQRYQD AMAIVTKYGK PDLFITMTCN PKWADITNNL 541 QRWQKVENRP DLVARVFNIK LNALLNDICK FHLFGKVIAK IHVIEFQKRG LPHAHILLIL 601 DSESKLRSED DIDRIVKAEI PDEDQCPRLF QIVKSNMVHG PCGIQNPNSP CMENGKCSKG 661 YPKEFQNATI GNIDGYPKYK RRSGSTMSIG NKVVDNTWIV PYNPYLCLKY NCHINVEVCA 721 SIKSVKYLFK YIYKGHDCAN IQISEKNIIN HDEVQDFIDSRYVSAPEAVW 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 Petition 870260041395, on 04 / 05 / 2026, page. 43 / 185 36 / 145 1441 LPEPVFAHGQ LYVAFSRVRR ACDVKVKVVN TSSQGKLVKH SESVFTLNVV YREILE (SEQ ID NO: 22).

[0042] In Helitron transpositions, a hairpin near the 3' end of the transposon functions as a terminator. However, this hairpin can be bypassed by the transposase, resulting in the transduction of flanking sequences. Furthermore, Helraiser transposition generates covalently closed circular intermediates. Additionally, Helitron transpositions may lack target-site duplications. In the Helraiser sequence, the transposase is flanked by left- and right-terminal sequences called LTS and RTS. These sequences terminate with a conserved 5'-TC / CTAG-3' motif. A 19 bp palindromic sequence with the potential to form the hairpin termination structure is located 11 nucleotides upstream of the RTS and consists of the sequence GTGCACGAATTTCGTGCACCGGGCCACTAG (SEQ ID NO: 23).

[0043] The disclosure provides a composition comprising a disclosure transposon. In certain embodiments of this method, the transposon is a plasmid DNA transposon with a sequence encoding the disclosure-inducible caspase polypeptide flanked by two cis-regulatory isolator elements. In certain embodiments of the compositions comprising a transposon, the composition may further comprise a plasmid comprising Petition 870260041395, dated 04 / 05 / 2026, p. 44 / 185 37 / 145 a sequence that encodes a transposase enzyme. The sequence that encodes a transposase enzyme can be an mRNA sequence. In certain embodiments, the transposon is a Tol2 transposon. In certain embodiments, the transposon is a Tol2 transposon and the transposase is a Tol2 transposase.

[0044] Disclosure transposons may comprise Tol2 transposons. In certain embodiments of this method, the transposon is a plasmid DNA transposon with a sequence encoding the inducible caspase polypeptide of disclosure flanked by two cis-regulatory isolator elements. In certain embodiments of this method, the transposon is a Tol2 transposon. In certain embodiments, and in particular those embodiments in which the transposon is a Tol2 transposon, the composition further comprises a plasmid comprising a sequence encoding a transposase enzyme. In certain embodiments, the sequence encoding the transposase enzyme comprises a sequence encoding a Tol2 transposase. In certain embodiments, the sequence encoding the transposase enzyme is an mRNA sequence.

[0045] Tol2 transposons can be isolated from or derived from the medaka fish genome, and may be similar to hAT family transposons. The Tol2 transposons exemplifying disclosure are encoded by a sequence comprising approximately 4.7 kilobases and contain a Petition 870260041395, dated 04 / 05 / 2026, p. 45 / 185 38 / 145 gene encoding the Tol2 transposase, which contains four exons. An exemplary Tol2 transposase from disclosure comprises an amino acid sequence comprising the following: 1 MEEVCDSSAA ASSTVQNQPQ DQEHPWPYLR EFFSLSGVNK DSFKMKCVLC LPLNKEISAF 61 KSSPSNLRKH IERMHPNYLK NYSKLTAQKR KIGTSTHASS SKQLKVDSVF PVKHVSPVTV 121 NKAILRYIIQ GLHPFSTVDL PSFKELISTL QPGISVITRP TLRSKIAEAA LIMKQKVTAA 181 MSEVEWIATT TDCWTARRKS FIGVTAHWIN PGSLERHSAA LACKRLMGSH TFEVLASAMN 241 DIHSEYEIRD 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 601 NTPLPASAAC ERLFSTAGLL FSPKRARLDT NNFENQLLLK LNLRFYNFE (SEQ ID NO: 24).

[0046] An exemplary Tol2 transposon of the disclosure, including inverted repeats, subterminal sequences and the Tol2 transposase, is encoded by a nucleic acid sequence comprising the following: 1 CAGAGGTGTA AAGTACTTTGA GTATTATTACTTAG TTATTTTTGG 61 GGATTTTTAC TTTACTTGAG TACAATTAAA AATCAATACT TTTACTTTTA CTTAATTACA 121 TTTTTTTAGA AAAAAAAGTA CTTTTTACTC CTTACAATTT TATTTACAGT CAAAAAGTAC 181 TTATTTTG GAGATTATTTG CATTCATTATTCTTTACCTTTA AATTGAATTG 241 CGCTGATGCC CAGTTTAATT TAAATGTTAT TTATTCTGCC TATGAAAATC GTTTTCACAT 301 TATATGAAAT TGGTCAGACA TGTTCATTGG TCCTTTGGAA GTGACGTCAT GTCACATCTA 361 TACCACAGCCAT GCCGATTGATTGATT AATTAGGGAA ATTATAACAG TCAATCAGTG 421 GAAGAAAATG GAGGAAGTAT GTGATTCATC AGCAGCTGCG AGCAGCACAG TCCAAAATCA Petition 870260041395, of 04 / 05 / 2026, p. 46 / 185 39 / 145 481 GCCACAGGAT CAAGAGCACC CGTGGCCGTA TCTTCGCGAA TTCTTTTCTT TAAGTGGTGT 541 AAATAAAGAT TCATTCAAGA TGAAATGTGT CCTCTGTCTC CCGCTTAATA AAGAAATATC 601 GGCCTTCAAA AGTTCGCCAT CAAACCTAAG GAAGCATATT GAGGTAAGTA CATTAAGTAT 661 TTTGTTTTAC TGATAGTTTT TTTTTTTTTT TTTTTTTTTT TTTTTGGGTG TGCATGTTTT 721 GACGTTGATG GCGCGCCTTT TATATGTGTA GTAGGCCTAT TTTCACTAAT GCATGCGATT 781 GACAATATAA GGCTCACGTA ATAAAATGCT AAAATGCATT TGTAATTGGT AACGTTAGGT 841 CCACGGGAAA TTTGGCGCCT ATTGCAGCTT TGAATAATCA TTATCATTCC GTGCTCTCAT 901 TGTGTTTGAA TTCATGCAAA ACACAAGAAA 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 AATATACTCA GAAATAAAAT TAATGTTTGA TTGTCACTAA ATGCTACTGT ATTTCTAAAA 1381 TCWINDSTAKE TTTACK TAAAGTGTGC AATTGGCTGC AAATGTCAGT TTTTAG 1441 GGTTAGTTCA CCCAAAAATG AAAATAATGT ASSISTANTGAC TCGCCCTCAT GTCGTTCCAA 1501 GCCCGTAAGA CCTCCGTTCA TCTTCAGAAC ACAGTTTAAG ATATTTTAGA TTTAGTCCGA 1561 GAGCTTTCTG TGCCTCCATT GAGAATGTAT GTACGGTATA CTGTCCATGT CCAGAAAGGT 1621 AAAAAAACA TCAAAGTAGT CCATGTGACA TCAGTGGGTT AGTTAGT TTTTGACA 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 ACAGAGAATG CACCCAAATT ACCTCAAAAA CTACTCTAAA TTGACAGCAC AGAAGAGAAA 2221 GATCGGGACC TCCACCCATG CTTCCAGCAG TAAGCAACTG AAAGTTGACT CAGTTTTCCC Petition 870260041395, of 04 / 05 / 2026, p. 47 / 185 40 / 145 2281 2341 2401 2461 2521 2581 2641 2701 2761 2821 2881 2941 3001 3061 3121 3181 3241 3301 3361 3421 3481 3541 3601 3661 3721 3781 3841 3901 3961 4021AGTCAAACAT GTGTCTCCAG TCACTGTGAA CAAAGCTATA TTAAGGTACA TCATTCAAGG ACTTCATCCT TTCAGCACTG TTGATCTGCC ATCATTTAAA GAGCTGATTA GTACACTGCA GCCTGGCATT TCTGTCATTA CAAGGCCTAC AAGTCATGACTGAA AAGTCATGACTGACC CAGAAAGTGA CTGCTGCCAT GAGTGAAGTT GAATGGATTG CAACCAAC GGATTGTTGG ACTGCACGTA GAAAGTCATT CATTGGTGTA ACTGCTCACT GGATCAACCC TGGAAGTCTT GAAAGACATT CCGCTGCTGACAG GCCTGATCAAT GCATTCGAATAG CCATGAATGA TATCCACTCA GAGTATGAAA TACGTGACAA GGTTGTTTGC ACAACCACAG ACAGTGGTTC CAACTTTATG AAGGCTTTCA GAGTTTTTGG TGTGGAAAAC AATGATATCG AGACTGAGGC AAGAGGTGT GAAAGTGGATG T GGAGATGATTC GGAATTCCAA GATGCCTCAC GAGTCCTGGA CCAAGACGAT GGCTTCGAAT TCCAGCTACC AAAACATCAA AAGTGTGCCT GTCACTTACT TAACCTAGTC TCAAGCGTTG ATGCCCAAAA AGCTCTCTCA AATGAACACT ACAAGAACT ACTACGAGATCACT AAAAGCAGCC GATCGGCTCT AGCAGCTGAA GCTGTTGAAT CAGAAAGCCG GCTTCAGCTT TTAAGGCCAA ACCAAACGCG GTGGAATTCCA ACTTTTATGG CTGTTGACAG AATTCTTCAA ATTTGCAAAG AAGCAGGAGA AGGCGCACTTCGGAATATAT GCACCTCTCT TGAGGTTCCA ATGTAAGTGT TTTTCCCCTC TATCGATGTA AACAAATGTG GGTTGTTTTT GTTTAATACT CTTTGATTAT GCTGATTTCT CCTGTAGGTT TAATCCAGCA GAAATGCTGT TCTTGACAGA GTGGGCCAAC ACAATGCGTC CAGTTGCAAA AGTACTCGAC ATCTTGCAAG CGGAAACGAA TACACAGCTG GGGTGGCTGC TGCCTAGTGT CCATCAGTTA AGCTTGAAAC TTCAGCGACT CCACCATTCT CTCAGGTACT GTGACCCACT TGTGGATGCC CTACAACAAG GAATCCAAAC ACGATTCAAG CATATGTTTG AAGATCCTGA GATCATAGCA GCTGCCATCC TTCTCCCTAA ATTTCGGACC TCTTGGACAA ATGATGAAAC CATCATAAAA CGAGGTAAAT GAATGCAAGC AACATACACT TGACGAATTC TAATCTGGGC AACCTTTGAG CCATACCAAA ATTATTCTTT TATTTATTTA TTTTTGCACT TTTTAGGAAT GTTATATCCC ATCTTTGGCT GTGATCTCAA TATGAATATT GATGTAAAGT ATTCTTGCAG CAGGTTGTAG TTATCCCTCA GTGTTTCTTG AAACCAAACT CATATGTATC ATATGTGGTT TGGAAATGCA GTTAGATTTT ATGCTAAAAT AAGGGATTTG CATGATTTTA GATGTAGATG ACTGCACGTA AATGTAGTTA ATGACAAAAT CCATAAAATT TGTTCCCAGT CAGAAGCCCC TCAACCAAAC TTTTCTTTGT GTCTGCTCAC TGTGCTTGTA GGCATGGACT ACATCAGAGT GCATCTGGAG CCTTTGGACC ACAAGAAGGA ATTGGCCAAC AGTTCATCTG Petition 870260041395, de 04 / 05 / 2026, pág. 48 / 185 41 / 145 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 TTTCACCTC 4681 TG (SEQ ID NO: 25).

[0047] Disclosure provides a vector comprising the disclosure inducible pro-apoptotic polypeptide, inducible caspase polypeptide, inducible caspase 9 polypeptide and / or truncated inducible caspase 9 polypeptide. In certain embodiments, the vector is a viral vector.

[0048] Viral vectors for dissemination may comprise a single or derived sequence of a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus (AAV), or any combination thereof. In certain embodiments, the viral vector comprises a single or derived sequence of a retrovirus. In certain embodiments, the retrovirus is a gamma retrovirus. In certain embodiments, the retrovirus is a lentivirus. In certain embodiments, viral vectors for dissemination may be recombinant vectors.

[0049] Viral vectors for dissemination may comprise an isolated or derived sequence of a virus. Petition 870260041395, dated 04 / 05 / 2026, page 49 / 185 42 / 145 adeno-associated. In certain embodiments, the AAV comprises an AAV of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11. In certain embodiments, the AAV comprises a sequence of one or more AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11. In certain embodiments, the AAV comprises a single, derived, or recombined sequence of one or more AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11. In certain embodiments, the AAV comprises a single, derived, or recombined sequence of AAV2. In certain modalities, including those in which the vector crosses the blood-brain barrier (BBB), the AAV comprises an isolated, derived, or recombined AAV9 sequence.Exemplary adeno-associated viruses and recombinant adeno-associated viruses of dissemination include, but are not limited to, autocomplementary AAVs (scAAVs) and hybrid AAVs containing the genome of one serotype and the capsid of another serotype (e.g., AAV2 / 5, AAV-DJ, and AAV-DJ8). Examples of adeno-associated viruses and recombinant adeno-associated viruses of dissemination include, but are not limited to, rAAV-LK03, rAAV-NP59, and rAAV-NP84. In certain embodiments, the AAV comprises an isolated or derived sequence of rAAV-LK03, rAAV-NP59, or rAAV-NP84. In certain embodiments, the viral dissemination vectors may be recombinant vectors.

[0050] The description provides a vector comprising the Petition 870260041395, dated 04 / 05 / 2026, p. 50 / 185 43 / 145 inducible pro-apoptotic polypeptide, inducible caspase polypeptide, inducible caspase 9 polypeptide and / or inducible truncated caspase 9 polypeptide of disclosure. In certain embodiments, the vector is a nanoparticle vector.

[0051] The nanoparticle vectors of the disclosure may comprise a nucleic acid, an amino acid, a polymer, a micelle, a lipid, an organic molecule, an inorganic molecule, or any combination thereof. The nanoparticles may be composed of polymers described, for example, in International Patent Publication No. WO 2012 / 094679, International Patent Publication No. WO 2016 / 022805, International Patent Publication No. WO / 2011 / 133635, International Patent Publication No. WO / 2016 / 090111, International Patent Publication No. WO / 2017 / 004498, WO / 2017 / 004509, International Patent Application No. PCT / US2017 / 030271, US Patent No. 6,835,394. US Patent No. 7,217,427 and US Patent No. 7,867,512.

[0052] Dissemination nanoparticle vectors may further comprise at least one self-cleaving peptide. In certain embodiments, a dissemination nanoparticle vector may comprise at least one self-cleaving peptide, wherein a self-cleaving peptide is located between an inducible pro-apoptotic polypeptide, an inducible caspase polypeptide, and a caspase polypeptide. Petition 870260041395, dated 04 / 05 / 2026, page 51 / 185 44 / 145 inducible and / or truncated inducible caspase 9 polypeptide of the disclosure and another sequence linked to the nanoparticle. In certain embodiments, a disclosure nanoparticle vector may comprise at least one self-cleaving peptide, wherein one self-cleaving peptide is located upstream of the inducible pro-apoptotic polypeptide, inducible caspase polypeptide, inducible caspase 9 polypeptide, and / or truncated inducible caspase 9 polypeptide of the disclosure, and a second self-cleaving peptide is located downstream of the inducible pro-apoptotic polypeptide, inducible caspase polypeptide, inducible caspase 9 polypeptide, and / or truncated inducible caspase 9 polypeptide of the disclosure. At least one self-cleaving peptide may comprise 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.In certain embodiments, the T2A peptide comprises an amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 11). In certain embodiments, the GSG-T2A peptide comprises an amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 12). In certain embodiments, the E2A peptide comprises an amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO: 13). In certain embodiments, the GSG-E2A peptide comprises an amino acid sequence comprising... Petition 870260041395, dated 04 / 05 / 2026, page 52 / 185 45 / 145 GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 14). In certain embodiments, the F2A peptide comprises an amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 15). In certain embodiments, the GSG-F2A peptide comprises an amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 16). In certain embodiments, the P2A peptide comprises an amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 17). In certain embodiments, the GSG-P2A peptide comprises an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 18).

[0053] The disclosure provides a composition comprising a vector of disclosure.

[0054] The description provides a cell comprising an inducible pro-apoptotic polypeptide, an inducible caspase polypeptide, an inducible caspase 9 polypeptide and / or an inducible truncated caspase 9 polypeptide of disclosure. The disclosure provides a cell comprising a disclosure transposon. The disclosure provides a cell comprising a disclosure vector. In certain embodiments, the cell expresses the inducible caspase protein of the following description upon contact with an induction agent.

[0055] In certain embodiments, the dissemination cells comprising a dissemination polypeptide, transposon or vector may be human cells. Petition 870260041395, dated 04 / 05 / 2026, page 53 / 185 46 / 145

[0056] In certain embodiments, the disclosure cells comprising a disclosure polypeptide, transposon, or vector may be immune cells. Examples of immune cells include, but are not limited to, T cells, natural killer (NK) cells, natural killer (NK-like) cells, hematopoietic progenitor cells, peripheral blood (PB)-derived T cells, and umbilical cord blood (UCB)-derived T cells. In certain embodiments, the disclosure cells comprising a disclosure polypeptide, transposon, or vector may be T cells. In certain embodiments, the disclosure cells comprising a disclosure polypeptide, transposon, or vector may be activated T cells. In certain embodiments, the disclosure cells comprising a disclosure polypeptide, transposon, or vector may be activated T cells expressing an iC9 disclosure sequence.In certain embodiments, dissemination cells comprising a dissemination polypeptide, transposon, or vector may be artificial antigen-presenting cells (APCs). Dissemination immune cells may also include any commercially available cell line or modified cell line, including, but not limited to, dendritic cell lines, B cells, macrophages, and monocytes.

[0057] In certain forms, the cells of Petition 870260041395, dated 04 / 05 / 2026, page 54 / 185 47 / 145 Dissemination cells comprising a polypeptide, transposon, or dissemination vector may be immune cells. Examples of immune cells include, but are not limited to, T cells, natural killer (NK) cells, natural killer (NK-like) cells, hematopoietic progenitor cells, peripheral blood-derived (PB) T cells, and umbilical cord blood-derived (UCB) T cells. In certain embodiments, the dissemination cells comprising a polypeptide, transposon, or dissemination vector may be T cells. In certain embodiments, the cell is an artificial antigen-presenting cell.

[0058] In certain embodiments, the disclosure cells comprising a disclosure polypeptide, transposon, or vector may be stem cells. Disclosure stem cells may be human stem cells. Disclosure stem cells may be embryonic or adult stem cells. Disclosure stem cells may be totipotent, pluripotent, or multipotent. In certain embodiments, disclosure stem cells may be induced pluripotent stem cells (iPSCs).

[0059] In certain embodiments, the dissemination cells comprising a dissemination polypeptide, transposon, or vector may be somatic cells. Somatic dissemination cells may be isolated from or derived from Petition 870260041395, dated 04 / 05 / 2026, page 55 / 185 48 / 145 any part of a body and, preferably, a human body, including, but not limited to, a human heart; skeletal or smooth muscle; blood vessel, vein or capillary; spleen; thyroid; lymph node or lymphatic vessel; bone or bone marrow; skin or endothelium; adrenal gland; esophagus; larynx; brain or spinal cord; peripheral nervous system; eye; hypothalamus; liver; olfactory tissue; prostate; stomach; large or small intestine; lung or bronchi; kidney; pancreas; thymus gland; ureter or urethra; bladder; auditory tissue; bladder; parathyroid gland; salivary gland; or trachea.Somatic cell dissemination can be isolated from or derived from a precursor or stem cell that can differentiate into any part of a body, and preferably a human body including, but not limited to, a human heart; skeletal or smooth muscle; blood vessel, vein, or capillary; spleen; thyroid; lymph node or lymphatic vessel; bone or bone marrow; skin or endothelium; adrenal gland; esophagus; larynx; brain or spinal cord; peripheral nervous system; eye; hypothalamus; liver; olfactory tissue; prostate; stomach; large or small intestine; lung or bronchi; kidney; pancreas; thymus gland; ureter or urethra; bladder; auditory tissue; bladder; parathyroid gland; salivary gland; or trachea. Somatic cell dissemination can be isolated from or derived from transdifferentiated cells. Petition 870260041395, dated 04 / 05 / 2026, page 56 / 185 49 / 145

[0060] Disclosure provides a composition comprising a disclosure cell comprising a disclosure polypeptide, transposon or vector.

[0061] The disclosure provides a use of the disclosure compositions for an adoptive cell therapy. In certain modalities, the composition cells may be autologous. In certain modalities, the composition cells may be allogeneic.

[0062] Disclosure provides a use of disclosure compositions for ex vivo gene therapy. In certain embodiments, the composition cells may be autologous. In certain embodiments, the composition cells may be allogeneic.

[0063] The disclosure provides a use for the composition comprising a disclosure cell for ex vivo gene therapy. In certain embodiments of the use of the composition comprising disclosure cells, the cell is autologous. In certain embodiments, the cell is allogeneic.

[0064] The description provides a method of modifying a cell therapy in an individual in need thereof, comprising administering to the individual a composition comprising a cell containing a therapeutic agent and an inducible dissemination caspase polypeptide, wherein apoptosis can be selectively induced in the cell. Petition 870260041395, dated 04 / 05 / 2026, page 57 / 185 50 / 145 contacting the cell with an induction agent.

[0065] The description provides a method of modifying a cell therapy in an individual in need thereof, comprising administering to the individual a composition comprising a cell comprising a therapeutic agent and a dissemination composition, wherein apoptosis can be selectively induced in the cell by contact of the cell with an inducing agent.

[0066] Disclosure provides a method for modifying cell therapy in an individual in need thereof, comprising administering to the individual a composition comprising a cell comprising a therapeutic agent, a disclosure transposon and a composition comprising a disclosure transposase, wherein apoptosis can be selectively induced in the cell by contact of the cell with an inducing agent.

[0067] The description provides a method for modifying a cell therapy in an individual in need thereof, comprising administering to the individual a composition comprising a cell comprising a therapeutic agent and a dissemination vector, wherein apoptosis can be selectively induced in the cell by contact of the cell with an induction agent.

[0068] In certain modalities of the methods of modifying a cell dissemination therapy, the cells Petition 870260041395, dated 04 / 05 / 2026, page 58 / 185 51 / 145 of the composition may be autologous. In certain modalities, the cells of the composition may be allogeneic. In certain modalities of this method, cell therapy is adoptive cell therapy. In certain modalities, the therapeutic agent was introduced by ex vivo gene therapy. In certain modalities, the therapeutic agent is a sequence encoding a modified endogenous gene, an exogenous gene, or a portion thereof. In certain modalities of this method, the modification of cell therapy is a termination of cell therapy. In certain modalities of this method, the modification of cell therapy is a depletion of a portion of the cells supplied in cell therapy. This depletion may be transient or may be maintained for a period of time, for example, during a period of remission of a disease or disorder.In certain modalities, the method also includes the step of administering an inhibitor of the inducing agent to inhibit the modification of cell therapy, thereby restoring the function and / or efficacy of the cell therapy. For example, if a disease or disorder returns after remission or if an individual's adverse reaction diminishes, cell therapy can be resumed by administering an inhibitor of the inducing agent to the individual.

[0069] Methods for modifying a cell therapy's release can be used to interrupt or attenuate a therapy in response to, for example, a signal of Petition 870260041395, dated 04 / 05 / 2026, page 59 / 185 52 / 145 recovery or a sign of decreased severity / progression of the disease, a sign of remission / cessation of the disease, and / or the occurrence of an adverse event. Cell dissemination therapies may be resumed by inhibiting the inducing agent if a sign or symptom of the disease reappears or increases in severity and / or an adverse event is resolved.

[0070] In certain embodiments, a composition comprising a dissemination cell or a modified dissemination cell is administered to a patient by intravenous injection or infusion. In certain embodiments, a therapeutically effective dose of a dissemination composition or compositions comprising modified dissemination cells comprises between 2 x 10⁵ and 5 x 10⁸ cells per kg of the patient's body weight per administration, or any range, value or fraction thereof.

[0071] In certain embodiments, a composition comprising a dissemination cell or a modified dissemination cell is administered to a patient by intravenous injection or infusion. In certain embodiments, a therapeutically effective dose of a dissemination composition or compositions comprising modified dissemination cells comprises between 0.2 x 10⁶ and 20 x 10⁶ cells per kg of the patient's body weight. Petition 870260041395, dated 04 / 05 / 2026, page 60 / 185 53 / 145 administration. In certain embodiments, a therapeutically effective dose of a dissemination composition or compositions comprising modified dissemination cells comprises 0.2 x 10⁶ cells per kg of patient body weight per administration, 2 x 10⁶ cells per kg of patient body weight per administration, 20 x 10⁶ cells per kg of patient body weight per administration, or any number of cells per kg of patient body weight per administration in between.

[0072] In certain embodiments, a composition comprising a dissemination cell or a modified dissemination cell is administered to a patient by intravenous injection or infusion. In certain embodiments, a therapeutically effective dose of a dissemination composition or compositions comprising modified dissemination cells comprises 1 x 106 cells or approximately 1 x 106 cells per kg of the patient's body weight per administration.

[0073] In certain embodiments, a composition comprising a dissemination cell or a modified dissemination cell is administered to a patient by intravenous injection or infusion. In certain embodiments, a therapeutically effective dose of a dissemination composition or compositions comprising modified dissemination cells comprises 3 x 10⁶ cells. Petition 870260041395, dated 04 / 05 / 2026, page 61 / 185 54 / 145 or approximately 3 x 106 cells per kg of the patient's body weight per administration.

[0074] In certain embodiments, a composition comprising a dissemination cell or a modified dissemination cell is administered to a patient by intravenous injection or infusion. In certain embodiments, a therapeutically effective dose of a dissemination composition or compositions comprising modified dissemination cells comprises between 0.7 x 10⁶ and 6.7 x 10⁶ cells per kg of patient body weight per administration. In certain embodiments, a therapeutically effective dose of a dissemination composition or compositions comprising modified dissemination cells comprises 0.7 x 10⁶ cells per kg of patient body weight per administration, 6.7 x 10⁶ cells per kg of patient body weight per administration, or any number of cells per kg of patient body weight per administration in between.

[0075] In certain embodiments, a composition comprising a dissemination cell or a modified dissemination cell is administered to a patient by intravenous injection or infusion. In certain embodiments, a therapeutically effective dose of a dissemination composition or compositions comprising modified dissemination cells comprises between 0.7 x 106 Petition 870260041395, dated 04 / 05 / 2026, page 62 / 185 55 / 145 to 16 x 10⁶ cells per kg of patient body weight per administration. In certain embodiments, a therapeutically effective dose of a dissemination composition or compositions comprising modified dissemination cells comprises 0.7 x 10⁶ cells per kg of patient body weight per administration, 2 x 10⁶ cells per kg of patient body weight per administration, 6 x 10⁶ cells per kg of patient body weight per administration, 10.7 x 10⁶ cells per kg of patient body weight per administration, 16 x 10⁶ cells per kg of patient body weight per administration, or any cells per kg of patient body weight per administration in between.

[0076] In certain embodiments, a composition comprising a dissemination cell or a modified dissemination cell is administered to a patient by intravenous injection or infusion. In certain embodiments, a therapeutically effective dose of a dissemination composition or compositions comprising modified dissemination cells comprises 1.2 x 10⁶ to 7.1 x 10⁶ cells per kg of patient body weight per administration. In certain embodiments, a therapeutically effective dose of a dissemination composition or compositions comprising modified dissemination cells comprises 1.2 x 10⁶ cells per kg of body weight. Petition 870260041395, dated 04 / 05 / 2026, page 63 / 185 56 / 145 of the patient's body weight per administration, 7.1 x 10⁶ cells per kg of the patient's body weight per administration, or any number of cells per kg of the patient's body weight per administration. In certain embodiments, a therapeutically effective dose of a dissemination composition or compositions comprising modified dissemination cells comprises between 2 x 10⁶ and 3 x 10⁶ cells per kg of the patient's body weight per administration.

[0077] In certain embodiments, a composition comprising a dissemination cell or a modified dissemination cell is administered to a patient by intravenous injection or infusion. In certain embodiments, a therapeutically effective dose of a dissemination composition or compositions comprising modified dissemination cells comprises 1 x 10⁶ to 2 x 10⁶ cells per kg of patient body weight per administration. In certain embodiments, a therapeutically effective dose of a dissemination composition or compositions comprising modified dissemination cells comprises 1 x 10⁶ cells per kg of patient body weight per administration, 2 x 10⁶ cells per kg of patient body weight per administration, or any number of cells per kg of patient body weight per administration in between. In certain dissemination embodiments, the modified dissemination cells are delivered to a patient by Petition 870260041395, dated 04 / 05 / 2026, page 64 / 185 57 / 145 means of intravenous injection or infusion. In certain embodiments, a therapeutically effective dose of a dissemination composition or compositions comprising modified dissemination cells comprises from 0.7 x 10⁶ to 1.3 x 10⁶ cells per kg of patient body weight per administration. In certain embodiments, a therapeutically effective dose of a dissemination composition or compositions comprising modified dissemination cells comprises 0.7 x 10⁶ cells per kg of patient body weight per administration, 1.3 x 10⁶ cells per kg of patient body weight per administration, or any number of cells per kg of patient body weight per administration in between.

[0078] In certain embodiments, a composition comprising a dissemination cell or a modified dissemination cell is administered to a patient by intravenous injection or infusion. In certain embodiments, a therapeutically effective dose of a dissemination composition or compositions comprising modified dissemination cells comprises a single or multiple dose. In certain embodiments, a therapeutically effective dose of a dissemination composition or compositions comprising modified dissemination cells comprises a divided dose. In certain embodiments, a therapeutically effective dose of a Petition 870260041395, dated 04 / 05 / 2026, page 65 / 185 58 / 145 composition of the disclosure or of compositions comprising modified disclosure cells comprises an initial dose and a maintenance dose.

[0079] The description provides a method of treating a disease or disorder in an individual in need thereof comprising administering to the individual a composition comprising a cell comprising a kinetic agent and an inducible caspase-releasing polypeptide, wherein apoptosis can be selectively induced in the cell by contact of the cell with an inducing agent and wherein the cell comprising the kinetic agent induces local tissue toxicity in a target tissue of the individual, and selectively inducing apoptosis in the cell comprising the kinetic agent prior to inducing toxicity in a non-target tissue of the individual, thereby treating the elimination of the target tissue, preserving the non-target tissue and treating the disease or disorder in the individual.

[0080] As used herein, a kinetic agent is intended to describe a therapeutic agent with specificity for a target tissue and known / intentional or unknown / unintentional toxicity to non-target tissue, which, in either case, can be controlled or limited in such a way that the toxicity does not significantly affect non-target tissues. Kinetic agents specifically target a tissue, specifically and locally induce toxicity, but before Petition 870260041395, dated 04 / 05 / 2026, page 66 / 185 59 / 145 If a kinetic agent can induce significant toxicity or damage in a non-target tissue, a disclosure-inducing agent may be administered to reduce or prevent toxicity in the non-target tissue. The extent to which any toxicity affects target tissues (e.g., damage to non-target tissues) can be limited by administration of the inducing agent and may correspond to a duration of exposure of the non-target tissue to the kinetic agent. In certain disclosure modalities, cells comprising a therapeutic agent can react in both target tumor cells and non-target tumor cells simultaneously and in multiple different tissues. Non-target tumor cells are preserved through activation of disclosure-inducible caspase polypeptides in cells comprising a therapeutic agent to eliminate the cells comprising disclosure-inducible caspase polypeptides, for example, after the tumor has been eliminated.In certain modalities, non-tumor off-target effects may be due to cross-reactivity of the kinetic agent. In certain modalities, if the off-target effects are too large, the toxicity of the kinetic agent may be limited or eliminated by administering an induction agent before treatment of the individual's disease is complete.

[0081] Disclosure provides a method of treating a disease or disorder in an individual in need. Petition 870260041395, dated 04 / 05 / 2026, page 67 / 185 60 / 145 of the same comprising administering to the individual a composition comprising a cell comprising a kinetic agent and a dissemination composition, wherein apoptosis can be selectively induced in the cell by contact of the cell with an induction agent and wherein the cell comprising the kinetic agent induces local tissue toxicity in a target tissue of the individual and selectively inducing apoptosis in the cell comprising the kinetic agent prior to inducing significant toxicity in a cell of the individual's non-target tissue, thereby treating the elimination of the target tissue, preserving the non-target tissue and treating the disease or disorder in the individual.

[0082] Disclosure provides a method of treating a disease or disorder in an individual in need thereof comprising administering to the individual a composition comprising a cell comprising a kinetic agent, a disclosure transposon and a composition comprising a disclosure transposase, wherein apoptosis can be selectively induced in the cell by contacting the cell with an inducing agent and wherein the cell comprising the kinetic agent induces local tissue toxicity in a target tissue of the individual and selectively inducing apoptosis in the cell comprising the kinetic agent prior to inducing significant toxicity in a non-target tissue of the individual, thereby treating tissue shedding Petition 870260041395, dated 04 / 05 / 2026, page 68 / 185 61 / 145 target, preserving non-target tissue and treating the disease or disorder in the individual.

[0083] Disclosure provides a method for treating a disease or disorder in an individual in need thereof, comprising administering to the individual a composition comprising a cell comprising a kinetic agent and a disclosure vector, wherein apoptosis can be selectively induced in the cell by contact of the cell with an inducing agent and wherein the cell comprising the kinetic agent induces local tissue toxicity in a target tissue of the individual and selectively inducing apoptosis in the cell comprising the kinetic agent prior to inducing significant toxicity in a non-target tissue of the individual, thereby treating the elimination of the target tissue, preserving the non-target tissue and treating the disease or disorder in the individual.

[0084] The term “significant toxicity in a non-target tissue” can describe a level of toxicity at which tissue cells are dead (e.g., died as a result of necrosis or apoptosis), have become non-viable, or have ceased to perform one or more essential physiological functions. In certain modalities, significant toxicity denotes permanent damage to a cell or tissue. In certain modalities, significant toxicity denotes a transient loss of a cell's function or Petition 870260041395, dated 04 / 05 / 2026, page 69 / 185 62 / 145 of a tissue. In certain modalities, significant toxicity induces symptoms in an individual that are recognizable as such by a specialist in the technique. In certain modalities, significant toxicity leads to death, reduced lifespan, or a reduction in the quality of life of an individual.

[0085] In certain modalities of the methods of treating a disease or disorder of dissemination, the disease or disorder is a proliferative disorder or cancer. In certain modalities, the target tissue comprises a tumor. In certain modalities, the tumor is benign. In certain modalities, the tumor is malignant. In certain modalities, the target tissue comprises an exposed tissue or margin of a resected tumor. In certain modalities, the target tissue comprises a likely site of metastasis. In certain modalities, the site of metastasis comprises one or more lymph nodes, lymphatic fluid, peripheral circulating blood, local circulating blood, bone, bone marrow, and cerebrospinal fluid (CSF).

[0086] In certain modalities of the methods of treating a disease or disorder of dissemination, the disease or disorder is an inflammatory disease or disorder. In certain modalities, the target tissue comprises a site of inflammation.

[0087] In certain modalities of the methods of Petition 870260041395, dated 04 / 05 / 2026, page 70 / 185 63 / 145 treatment of a disease or disorder of dissemination, the disease or disorder is an immune or autoimmune disease or disorder. In certain modalities, the target tissue comprises a site of exposed or infected tissue. In certain modalities, the target tissue comprises burned or injured tissue.

[0088] In certain modalities of the methods of treating a disease or disorder of dissemination, the disease or disorder is an infectious disease or disorder. In certain modalities, the target tissue comprises an infected tissue.

[0089] In certain modalities of methods for treating a disease or disorder of disclosure, the disease or disorder is a genetic or epigenetic disease or disorder. In certain modalities, the target tissue comprises one or more cells comprising the genetic or epigenetic modification when compared to a wild-type cell.

[0090] In certain modalities of the methods of treating a disease or disorder of dissemination, the disease or disorder is a metabolic disorder. In certain modalities, the target tissue comprises one or more cells with the metabolic disorder.

[0091] In certain modalities of the methods of treating a disease or disorder of dissemination, the disease or disorder is a vascular disorder. In certain modalities, the target tissue comprises one or more cells of a vein, blood vessel, capillary, or a component of blood. Petition 870260041395, dated 04 / 05 / 2026, p. 71 / 185 64 / 145 in circulation.

[0092] In certain modalities of the methods of treating a disseminating disease or disorder, the disease or disorder is a respiratory disorder. In certain modalities, the target tissue comprises one or more cells of a nasal passage, esophagus, or lung.

[0093] In certain modalities of treatment methods for a dissemination disease or disorder, the disease or disorder is a fibrotic disorder. In certain modalities, the target tissue comprises a fibroid mass or a cell in the vicinity of the uterine mass.

[0094] In certain modalities of methods for treating a disease or disorder of dissemination, an adoptive cell therapy comprises the cell comprising the kinetic agent. In certain modalities, the cell comprising the kinetic agent is autologous. In certain modalities, the cell comprising the kinetic agent is allogeneic. In certain modalities, the cell comprising the kinetic agent is a T cell. In certain modalities, the kinetic agent is a receptor that does not occur naturally. In certain modalities, the non-naturally occurring receptor is a synthetic, modified, recombinant, or chimeric receptor. In certain modalities, the chimeric receptor is a chimeric antigen receptor (CAR).

[0095] In certain modalities of the methods of Petition 870260041395, dated 04 / 05 / 2026, p. 72 / 185 65 / 145 treatment of a disease or disorder of dissemination, the disease or disorder is a proliferative disorder or a cancer. In certain embodiments, the target tissue comprises a tumor. In certain embodiments, the tumor treatment comprises a composition comprising a cell comprising an inducible caspase dissemination polypeptide. In certain embodiments, the composition comprising a cell comprising an inducible caspase dissemination polypeptide further comprises a chimeric antigen receptor (CAR). In certain embodiments, the chimeric antigen receptor (CAR) binds specifically to a sequence expressed on a tumor cell, thereby conferring CAR specificity to the tumor cell. In certain embodiments, the cell expressing the CAR that binds specifically to a tumor cell is a T cell, thus producing a CAR-T that binds specifically to a tumor cell.In certain modalities, compositions comprising CAR-T cells specifically directed at tumor cells will selectively kill only tumor cells expressing the antigen sequence. However, in certain modalities, the tumor antigen may be expressed in other normal tissues, leading to non-tumor activity in the CAR-T cell target in non-target tissues. For example, the antigen may be CD19, and CD19 is expressed almost exclusively on B cells. In the case of CD19, the activity... Petition 870260041395, dated 04 / 05 / 2026, page 73 / 185 66 / 145 off-target of anti-CD19 CAR-T cells is minimal. However, if, for example, the antigen is PSMA (folate hydrolase 1) and PSMA is expressed in several types of normal cells, anti-PSMA CAR-T cells can target normal cells in addition to pathological target cells in an activity called off-tumor target toxicity. In certain modalities, once the pathological target cells of dissemination (e.g., tumor cells) are eradicated by anti-PSMA CAR-T cells, treating the individual with the dissemination-inducing agent to induce programmed cell death in anti-PSMA CAR-T cells using inducible caspase dissemination polypeptides eliminates the non-tumor effects on the target.

[0096] In certain methods of dissemination, including those in which an adoptive cell therapy comprises the cell comprising the kinetic agent, the kinetic agent comprises an anticancer agent. In certain embodiments, the anticancer agent comprises an anti-CD19 agent. In certain embodiments, the anticancer agent comprises an anti-BCMA agent. In certain embodiments, the anticancer agent comprises an anti-PSMA agent. In certain embodiments, the anticancer agent comprises an anti-Muc1 agent. In certain embodiments, the kinetic agent comprises a non-naturally occurring receptor. In certain embodiments, the non-naturally occurring receptor comprises Petition 870260041395, dated 04 / 05 / 2026, page 74 / 185 67 / 145 a synthetic, modified, recombinant, or chimeric receptor. In certain embodiments, the chimeric receptor is a chimeric antigen receptor (CAR). In certain embodiments, the CAR comprises one or more VHH sequences. In certain embodiments, the CAR is a VCAR. In certain embodiments, the kinetic agent induces aplasia. In certain embodiments, the aplasia is not fatal. In certain embodiments, the inducing agent eliminates the cell comprising the kinetic agent. In certain embodiments, the cell is a T cell. In certain embodiments, the inducing agent eliminates or reduces a sign or symptom of aplasia. In certain embodiments, CAR-T therapy eliminates malignancy but continues to target healthy B cells, so the individual must be treated with IVIG infusions. In certain embodiments, anti-BCMA CAR-T therapy eliminates the individual's non-mutated plasma cells, so the individual must also be treated with IVIG.In certain modalities, the CAR-T cell that causes aplasia can be eliminated through the release and induction of caspase 9 polypeptides. In certain modalities, treatment of the individual with the release-inducing agent alleviates a sign or symptom of aplasia. BRIEF DESCRIPTION OF THE DRAWINGS

[0097] Figure 1 is a schematic diagram representing an exemplary inducible truncated caspase 9 polypeptide from disclosure. Petition 870260041395, dated 04 / 05 / 2026, p. 75 / 185 68 / 145

[0098] Figure 2 is a series of flow cytometry graphs showing the abundance of cells shifting from an area of ​​live cells (the closed lower right quadrant) to an area filled with apoptotic cells (the upper left quadrant) as a function of increasing dosage of the induction agent (AP1903, also called Rimiducid) in cells modified to express a therapeutic agent (a CARTirin) alone or in combination with an inducible caspase disclosure polypeptide (encoded by an iC9 construct (also known as a “safety switch”) introduced into cells by a piggyBac (PB) transposase) on day 12 after nucleofection.

[0099] Figure 3 is a series of flow cytometry graphs showing the abundance of cells shifting from an area of ​​live cells (the closed lower right quadrant) to an area filled with apoptotic cells (the upper left quadrant) as a function of increasing dosage of the induction agent (AP1903, also called Rimiducid) in cells modified to express a therapeutic agent (a CARTirin) alone or in combination with an inducible caspase disclosure polypeptide (encoded by an iC9 construct (also known as a safety switch) introduced into cells by a piggyBac (PB) transposase) on day 19 after nucleofection.

[00100] Figure 4 is a pair of graphs that describe Petition 870260041395, dated 04 / 05 / 2026, page 76 / 185 69 / 145 a quantification of the aggregated results shown in both Figure 2 (left graph) and Figure 3 (right graph). Specifically, these graphs show the impact of the iC9 safety switch on percent cell viability as a function of the concentration of the iC9 switch induction agent (AP1903, also called Rimiducid) for each modified cell type on day 12 (Figure 2 and left graph) or day 19 (Figure 3 and right graph).

[00101] Figure 5 is a diagram showing a timeline of the study and an outline for an in vivo study. NSG mice were injected IV with MM.1S / luciferase+ cells, placed on day 8, injected with T cells on day 9, and treated with AP1903 (Rimiducid) on day 12 at the indicated doses. 24 hours later, the mice were euthanized and blood, spleen, and bone marrow cells were collected and stained for the presence of huCD45+ cells.

[00102] Figure 6 is a graph demonstrating the highly efficient killing of cells comprising P-BCMA-101 using Rimiducid (AP1903) in vivo. Figure 6 shows the presence of CARTyrin+ cells in blood, spleen, and bone marrow after AP1903 treatment. Blood, spleen, and bone marrow cells were analyzed by flow cytometry for the presence of huCD45+ cells. Relative viability was determined by: (# of huCD45+ cells / # of cells Petition 870260041395, dated 04 / 05 / 2026, page 77 / 185 70 / 145 msCD45) / (Average huCD45 / msCD45 in no treatment group)) * 100% per 1,500 counting events for each sample. Each data point represents a different mouse. The x-axis plots the relative viability of CAR-T from 0 to 175 in increments of 25. The y-axis, from left to right for each panel, shows blood, spleen, and bone marrow after AP1903 (Rimiducid) treatment. AP1903 (Rimiducid) increases per panel from left to right in the following order: 0 mg / kg, 0.005 mg / kg, 0.05 mg / kg, 0.5 mg / kg, 5 mg / kg, and 5 mg / kg in the absence of the iC9 gene. DETAILED DESCRIPTION

[00103] Dissemination provides inducible pro-apoptotic polypeptides, as well as transposons, vectors, and cells comprising inducible pro-apoptotic dissemination polypeptides. Induced pro-apoptotic dissemination polypeptides can be introduced into a cell simultaneously or sequentially with a therapeutic agent. For example, induced pro-apoptotic dissemination polypeptides can be introduced into a cell simultaneously or sequentially with one or more sequences encoding a chimeric antigen receptor to produce a modified dissemination cell. Modified dissemination cells can be used in cell-based therapies. To control the activity of modified dissemination cells comprising a pro-apoptotic polypeptide Petition 870260041395, dated 04 / 05 / 2026, p. 78 / 185 71 / 145 Inducible apoptotic polypeptide and, optionally, a therapeutic agent, the cell and / or the inducible pro-apoptotic polypeptide can contact an induction agent that specifically binds to the ligand-binding region of the inducible pro-apoptotic polypeptide and ultimately triggers the initiation of apoptosis in the cell comprising the inducible pro-apoptotic polypeptide. When a modified disclosure cell is used as cell therapy, the induction agent can be administered locally or systemically to the individual receiving the cell therapy. An inhibitor of the induction agent can be administered locally or systemically to the individual receiving both the cell therapy and the induction agent.

[00104] As used herein, the term “therapeutic agent” may refer to any molecule, organic or inorganic, which, when introduced into a cell intended for cell therapy, modifies an activity, a signaling pathway, a signaling outcome and / or an interaction of the cell with the cell’s internal or external environment, including, but not limited to, neighboring cells, extracellular ligands and signaling molecules, the host cell’s immune system or a component thereof, host infection, a diseased host cell (e.g., a cancerous cell), intracellular ligands and signaling molecules, epigenetic regulation, Petition 870260041395, dated 04 / 05 / 2026, page 79 / 185 72 / 145 gene transcription, gene regulation, transcriptome, DNA / RNA translation, protein processing or secretion processes, and proteome. Therapeutic agents include, but are not limited to, recombinant and / or chimeric cell surface receptors, recombinant and / or chimeric transmembrane receptors, recombinant and / or chimeric ion channels, and recombinant and / or chimeric antigen receptors. In certain embodiments, the therapeutic agent is a chimeric antigen receptor (CAR) and, optionally, a chimeric antigen receptor in which the antigen recognition region comprises at least one Centirin. As used throughout the disclosure, a CAR comprising a Centirin is referred to as a CARTirin.

[00105] Disclosure provides inducible proapoptotic polypeptides comprising a ligand-binding region, a ligand, and a proapoptotic peptide, wherein the inducible proapoptotic polypeptide does not comprise a non-human sequence. In certain embodiments, the non-human sequence comprises a restriction site. Disclosure-induced proapoptotic polypeptides dimerize through interaction with an induction agent. Dimerization of a first inducible proapoptotic polypeptide and a second inducible proapoptotic polypeptide facilitates or activates an interaction, cross-linking, cross-activation, or caspase polypeptide activation. The interaction, Petition 870260041395, dated 04 / 05 / 2026, p. 80 / 185 73 / 145 Cross-linking, cross-activation, or caspase polypeptide activation initiates apoptosis in a cell comprising inducible pro-apoptotic dissemination polypeptides or a sequence encoding inducible pro-apoptotic dissemination polypeptides. Induced pro-apoptotic dissemination polypeptides do not initiate apoptosis unless the inducible pro-apoptotic dissemination polypeptide contacts an induction agent. Contact between an induction agent and an inducible pro-apoptotic dissemination polypeptide can occur in vivo, ex vivo, or in vitro. Contact between an induction agent and an inducible pro-apoptotic dissemination polypeptide can occur intracellularly.

[00106] With regard to cell therapies, disclosure provides modified T cells for adoptive cell therapies comprising an inducible pro-apoptotic polypeptide or a sequence encoding an inducible pro-apoptotic polypeptide from disclosure.

[00107] Modified T cells for adoptive cell therapies can be autologous or allogeneic. The term allogeneic, as used herein, refers to HLA or MHC loci that are antigenically distinct. Cells or tissues transferred from the same species can be antigenically distinct. Syngeneic mice may differ in one or more loci (congenic) and allogeneic mice may have Petition 870260041395, dated 04 / 05 / 2026, page 81 / 185 74 / 145 the same precedent.

[00108] Modified T cells for adoptive cell therapies may include activated T cells. T cells (also known as T lymphocytes) belong to a group of white blood cells called lymphocytes. Lymphocytes are generally involved in cell-mediated immunity. OT in T cells refers to cells derived from, or whose maturation is influenced by, the thymus. T cells can be distinguished from other types of lymphocytes, such as B cells and Natural Killer (NK) cells, by the presence of cell surface proteins known as T cell receptors. The term “activated T cells,” as used herein, refers to T cells that have been stimulated to produce an immune response (e.g., clonal expansion of activated T cells) by recognition of an antigenic determinant presented in the context of major histocompatibility marker class II (MHC).T cells are activated by the presence of an antigen determinant, cytokines and / or lymphokines, and clusters of differentiating cell surface proteins (e.g., CD3, CD4, CD8, and similar combinations thereof). Cells expressing a cluster of differentiating proteins are often considered "positive" for the expression of that protein on the surface of T cells (e.g., positive cells). Petition 870260041395, dated 04 / 05 / 2026, page 82 / 185 75 / 145 for CD3 or CD4 expression are referred to as CD3+ or CD4+. CD3 and CD4 proteins are cell surface receptors or co-receptors that may be directly and / or indirectly involved in signal transduction in T cells.

[00109] Modified T cells for adoptive cell therapies may include pan T cells. As used herein, pan T cells include all T lymphocytes isolated from a biological sample, without classification by subtype, activation state, maturation state, or cell surface marker expression.

[00110] Modified T cells for adoptive cell therapies can be obtained and / or prepared from, for example, whole blood, peripheral blood, umbilical cord blood, lymphatic fluid, lymph node tissue, bone marrow, and cerebrospinal fluid (CSF). By "obtained as," or "prepared as," in the case of cells, it is understood that the cells or cell culture are isolated, purified, or partially purified from the source, where the source may be, for example, umbilical cord blood, bone marrow, or peripheral blood. The terms may also be applied to the case where the original source, or a cell culture, has been grown and the cells have replicated, and where progeny cells are now derived from the original source. The term "peripheral blood" as used Petition 870260041395, dated 04 / 05 / 2026, page 83 / 185 76 / 145 here refers to cellular components of blood (e.g., red blood cells, white blood cells, and platelets) that are obtained or prepared from the circulating blood reservoir and not sequestered in the lymphatic system, spleen, liver, or bone marrow. Umbilical cord blood is distinct from peripheral blood and blood sequestered within the lymphatic system, spleen, liver, or bone marrow. The terms “umbilical cord blood,” “umbilical cord blood,” or “umbilical cord blood,” which may be used interchangeably, refer to the blood that remains in the placenta and attached umbilical cord after the birth of the child. Cord blood often contains stem cells, including hematopoietic cells.

[00111] Disclosure provides inducible pro-apoptotic polypeptides comprising a ligand-binding region, a ligand, and a pro-apoptotic peptide, wherein the inducible pro-apoptotic polypeptide does not comprise a non-human sequence. In certain embodiments, the non-human sequence comprises a restriction site. In certain embodiments, the pro-apoptotic peptide is a caspase polypeptide. In certain embodiments, the caspase polypeptide is a caspase 9 polypeptide. In certain embodiments, the caspase 9 polypeptide is a truncated caspase 9 polypeptide. Disclosure's inducible pro-apoptotic polypeptides may be of non-natural occurrence. Petition 870260041395, dated 04 / 05 / 2026, page 84 / 185 77 / 145

[00112] Disclosure caspase polypeptides include, but are not limited to, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11, caspase 12, and caspase 14. Disclosure caspase polypeptides include, but are not limited to, apoptosis-associated caspase polypeptides including caspase 2, caspase 3, caspase 6, caspase 7, caspase 8, caspase 9, and caspase 10. Disclosure caspase polypeptides include, but are not limited to, apoptosis-initiating caspase polypeptides including caspase 2, caspase 8, caspase 9, and caspase 10. Polypeptides of Caspase release molecules include, but are not limited to, caspase polypeptides that perform apoptosis, including caspase 3, caspase 6, and caspase 7.

[00113] Disclosure caspase polypeptides can be encoded by an amino acid or nucleic acid sequence with one or more modifications, compared to a wild-type amino acid or nucleic acid sequence. The nucleic acid sequence encoding a disclosure caspase polypeptide can be codon-optimized. One or more modifications to an amino acid and / or nucleic acid sequence of a disclosure caspase polypeptide can enhance interaction, cross-linking, activation cleavage, or activation of the disclosure caspase polypeptide. Petition 870260041395, dated 04 / 05 / 2026, p. 85 / 185 78 / 145 disclosure compared to a wild-type amino acid or nucleic acid sequence. Alternatively, or in addition, one or more modifications to an amino acid and / or nucleic acid sequence of a disclosure caspase polypeptide may decrease the immunogenicity of the disclosure caspase polypeptide compared to a wild-type amino acid or nucleic acid sequence.

[00114] Disclosure caspase polypeptides can be truncated compared to a wild-type caspase polypeptide. For example, a caspase polypeptide can be truncated to eliminate a sequence encoding a Caspase Recruitment and Activation Domain (CARD) to eliminate or minimize the possibility of activating a local inflammatory response beyond initiating apoptosis in the cell comprising an inducible disclosure caspase polypeptide. The nucleic acid sequence encoding a disclosure caspase polypeptide can be joined to form a variant amino acid sequence of the disclosure caspase polypeptide relative to a wild-type caspase polypeptide. Disclosure caspase polypeptides can be encoded by recombinant and / or chimeric sequences; recombinant and / or chimeric caspase polypeptides of the invention may include sequences from one or more different caspase polypeptides.Alternatively, or in addition, the... Petition 870260041395, dated 04 / 05 / 2026, p. 86 / 185 79 / 145 Recombinant and / or chimeric caspase polypeptides of the invention may include sequences from one or more species (e.g., a human sequence and a non-human sequence). Caspase polypeptides of the disclosure may be of non-natural occurrence.

[00115] The ligand-binding region of an inducible pro-apoptotic dissemination polypeptide may include any polypeptide sequence that facilitates or promotes the dimerization of a first inducible pro-apoptotic dissemination polypeptide with a second inducible pro-apoptotic dissemination polypeptide, whose dimerization activates or induces the cross-linking of the pro-apoptotic polypeptides and initiation of apoptosis in the cell.

[00116] The ligand-binding (dimerization) region may comprise any polypeptide or its functional domain that allows induction using a natural or non-natural ligand (i.e., induction agent), for example, a non-natural synthetic ligand. The ligand-binding region may be internal or external to the cell membrane, depending on the nature of the inducible proapoptotic polypeptide and the choice of ligand (i.e., induction agent). A wide variety of ligand-binding polypeptides and their functional domains are known, including receptors. The ligand-binding regions Petition 870260041395, dated 04 / 05 / 2026, page 87 / 185 80 / 145 ligand disclosure may include one or more sequences of a receptor. Of particular interest are ligand-binding regions for which ligands (e.g., small organic ligands) are known or can be readily produced. These ligand-binding regions or receptors may include, but are not limited to, FKBP and cyclophilin receptors, steroid receptors, tetracycline receptor and the like, as well as non-natural receptors, which may be obtained from antibodies, particularly from the heavy or light chain subunit, their mutated sequences, random amino acid sequences obtained by stochastic procedures, combinatorial syntheses and the like.In certain embodiments, the ligand-binding region is selected from the group consisting of an FKBP ligand-binding region, a cyclophilin receptor ligand-binding region, a steroid receptor ligand-binding region, a cyclophilin receptor ligand-binding region, and a tetracycline receptor ligand-binding region.

[00117] The ligand-binding regions comprising one or more receptor domains may be at least about 50 amino acids and less than about 350 amino acids, usually less than 200 amino acids, either as a natural domain or a truncated active portion thereof. The binding region may, for example, be small (<25 kDa, Petition 870260041395, dated 04 / 05 / 2026, p. 88 / 185 81 / 145 to allow efficient transfection in viral vectors), monomeric, non-immunogenic, have non-toxic, cell-permeable, synthetically accessible ligands that can be configured for dimerization.

[00118] The ligand-binding regions comprising one or more receptor domains may be intracellular or extracellular depending on the design of the inducible pro-apoptotic polypeptide and the availability of an appropriate ligand (i.e., induction agent). For hydrophobic ligands, the binding region may be on either side of the membrane, but for hydrophilic ligands, particularly protein ligands, the binding region will normally be external to the cell membrane unless there is a transport system for internalizing the ligand in a form where it is available for binding. For an intracellular receptor, the inducible pro-apoptotic polypeptide or a transposon or vector comprising the inducible pro-apoptotic polypeptide may encode a signal peptide and a 5' or 3' transmembrane domain of the receptor domain sequence or may have a 5' lipid-binding signal sequence of the receptor domain sequence.When the receptor domain is located between the signal peptide and the transmembrane domain, the receptor domain will be extracellular.

[00119] Antibodies and antibody subunits, by Petition 870260041395, dated 04 / 05 / 2026, page 89 / 185 82 / 145 For example, heavy or light chain, particularly fragments, more particularly all or part of the variable region, or heavy and light chain fusions to create high-affinity binding, can be used as a ligand-binding region of the disclosure. Antibodies contemplated include those that are an ectopically expressed human product, such as an extracellular domain that would not trigger an immune response and is generally not expressed in the periphery (i.e., outside the CNS / brain area). Such examples include, but are not limited to, low-affinity nerve growth factor receptor (LNGFR) and embryonic surface proteins (i.e., carcinoembryonic antigen). Furthermore, antibodies can be prepared against hapten molecules, which are physiologically acceptable, and the individual antibody subunits investigated for binding affinity.The cDNA encoding the subunits can be isolated and modified by deletion of the constant region, parts of the variable region, mutagenesis of the variable region, or similar means, to obtain a protein-binding domain that has the appropriate affinity for the ligand. In this way, almost any physiologically acceptable hapten compound can be used as the ligand or to provide an epitope for the ligand. Instead of antibody subunits, natural receptors can be used, in which the region or domain... Petition 870260041395, dated 04 / 05 / 2026, page 90 / 185 The 83 / 145 linkage is known, and a useful or known linker exists for the linkage.

[00120] To multimerize the receptor, the ligand for the ligand-binding region / receptor domains of inducible pro-apoptotic polypeptides may be multimeric in the sense that the ligand may have at least two binding sites, with each of the binding sites capable of binding to a ligand-binding receptor region (i.e., a ligand possessing a first binding site capable of binding to the ligand-binding region of a first inducible pro-apoptotic polypeptide and a second binding site capable of binding to the ligand-binding region of a second inducible pro-apoptotic polypeptide, wherein the ligand-binding regions of the first and second inducible pro-apoptotic polypeptides are identical or distinct). Thus, as used herein, the term "multimeric ligand-binding region" refers to a ligand-binding region of an inducible pro-apoptotic dissemination polypeptide that binds to a multimeric ligand.Multimeric disclosure ligands include dimeric ligands. A dimeric disclosure ligand may have two binding sites capable of binding to the ligand receptor domain. In certain embodiments, multimeric disclosure ligands are a dimer or higher-order oligomer, generally not larger than about tetrameric, of small size. Petition 870260041395, dated 04 / 05 / 2026, p. 91 / 185 84 / 145 synthetic organic molecules, with individual molecules typically at least about 150 Da and less than about 5 kDa, usually less than about 3 kDa. A variety of synthetic ligand and receptor pairs can be used. For example, in modalities involving natural receptors, dimeric FK506 can be used with an FKBP12 receptor, dimerized cyclosporine A can be used with a cyclophilin receptor, dimerized estrogen with an estrogen receptor, dimerized glucocorticoids with a glucocorticoid receptor, dimerized tetracycline with a tetracycline receptor, dimerized vitamin D with a vitamin D receptor, and the like. Alternatively, higher orders of ligands, e.g., trimeric, can be used.For modalities involving non-natural receptors, for example, antibody subunits, modified antibody subunits, single-chain antibodies consisting of variable regions of heavy and light chains joined together, separated by a flexible ligand, or modified receptors and their mutated sequences, and the like, any of a wide variety of compounds can be used. A significant feature of the units comprising a multimeric ligand of the disclosure is that each binding site is capable of binding to the receptor with high affinity and, preferably, that they are capable of being chemically dimerized. Petition 870260041395, dated 04 / 05 / 2026, p. 92 / 185 85 / 145 Furthermore, methods are available to balance the hydrophobicity / hydrophilicity of ligands so that they are able to dissolve in serum at functional levels, yet diffuse across plasma membranes for most applications.

[00121] The activation of inducible pro-apoptotic dissemination polypeptides can be achieved through, for example, chemically induced dimerization (CID) mediated by an induction agent to produce a conditionally controlled protein or polypeptide. Pro-apoptotic dissemination polypeptides are not only inducible, but the induction of these polypeptides is also reversible, due to degradation of the labile dimerizing agent or administration of a monomeric competitive inhibitor.

[00122] In certain embodiments, the ligand-binding region comprises an FK506 12-binding protein polypeptide (FKBP12). In certain embodiments, the ligand-binding region comprises an FKBP12 polypeptide having a valine (V) to phenylalanine (F) substitution at position 36 (F36V). In certain embodiments, wherein the ligand-binding region comprises an FKBP12 polypeptide having a valine (V) to phenylalanine (F) substitution at position 36 (F36V), the inducing agent may comprise AP1903 (Rimiducid), a synthetic drug (CAS Index Name: 2-piperidinecarboxylic acid, 1-[(2S)-1-oxo-2-(3,4,5 Petition 870260041395, dated 04 / 05 / 2026, page 93 / 185 - (9Cl) CAS registration number: 195514-63-7; Molecular Formula: C78H98N4O20; Molecular Weight: 1411.65)). In certain embodiments, where the ligand-binding region comprises an FKBP12 polypeptide having a valine (V) to phenylalanine (F) substitution at position 36 (F36V), the inducing agent may comprise AP20187 (CAS Registry Number: 195514-80-8 and Molecular Formula: C82H107N5O20). In certain embodiments, the inducing agent is an AP20187 analogue, such as, for example, AP1510. As used herein, the inducing agents AP20187, AP1903 (Rimiducid), and AP1510 may be used interchangeably.

[00123] API AP1903 (Rimiducid) is manufactured by Alphora Research Inc. and the pharmaceutical product AP1903 (Rimiducid) for Injection is manufactured by Formatech Inc. It is formulated as a 5 mg / mL solution of AP1903 (Rimiducid) in a 25% solution of the non-ionic solubilizer Solutol HS 15 (250 mg / mL, BASF). At room temperature, this formulation is a clear, slightly yellow solution. Upon refrigeration, this formulation undergoes a reversible phase transition, resulting in a milky solution. This phase transition is reversed upon reheating to room temperature. The fill volume is 2.33 mL in a glass vial. Petition 870260041395, dated 04 / 05 / 2026, page 94 / 185 87 / 145 of 3 mL (approximately 10 mg AP1903 (Rimiducid) for injection total per vial). After determining the need to administer AP1903 (Rimiducid), patients may, for example, be administered a single fixed dose of AP1903 (Rimiducid) for injection (0.4 mg / kg) by IV infusion over 2 hours, using a sterile infusion set not containing ethylene oxide, non-DEHP. The dose of AP1903 (Rimiducid) is calculated individually for all patients and is not recalculated unless body weight fluctuates by ± 10%. The calculated dose is diluted in 100 mL of 0.9% normal saline solution before infusion. In a previous Phase I study of AP1903 (Rimiducid), 24 healthy volunteers were treated with single doses of AP1903 (Rimiducid) for injection at dosage levels of 0.01, 0.05, 0.1, 0.5, and 1.0 mg / kg IV infusion over 2 hours.Plasma levels of AP1903 (Rimiducid) were directly proportional to the dose, with mean Cmax values ​​ranging from approximately 10-1275 ng / mL over the dose range of 0.01-1.0 mg / kg. After the initial infusion period, blood concentrations demonstrated a rapid distribution phase, with plasma levels reduced to approximately 18, 7, and 1% of the maximum concentration at 0.5, 2, and 10 hours post-dose, respectively. AP1903 (Rimiducid) for Injection proved to be safe and well-tolerated at all dose levels and demonstrated a profile. Petition 870260041395, dated 04 / 05 / 2026, page 95 / 185 88 / 145 favorable pharmacokinetics. Luliucci JD, et al., J Clin Pharmacol. 41: 870-9, 2001.

[00124] The fixed dose of AP1903 (Rimiducid) for injection used, for example, may be 0.4 mg / kg infused intravenously over 2 hours. The amount of AP1903 (Rimiducid) required in vitro for effective cell signaling is 10-100 nM (1600 Da MW). This is equivalent to 16-160 pg / L or -0.016-1.6 pg / kg (1.6-160 pg / kg). Doses up to 1 mg / kg were well tolerated in the Phase I study of AP1903 (Rimiducid) described above. Therefore, 0.4 mg / kg may be a safe and effective dose of AP1903 (Rimiducid) for this Phase I study in combination with therapeutic cells.

[00125] The amino acid and / or nucleic acid sequence encoding the ligand binding of the disclosure may contain one or more sequence modifications compared to a wild-type amino acid or nucleic acid sequence. For example, the amino acid and / or nucleic acid sequence encoding the disclosure ligand-binding region may be a codon-optimized sequence. One or more modifications may increase the binding affinity of a ligand (e.g., an induction agent) to the disclosure region relative to a wild-type ligand-binding polypeptide. Alternatively, or in addition, one or more modifications may decrease the immunogenicity of the binding region. Petition 870260041395, dated 04 / 05 / 2026, p. 96 / 185 89 / 145 ligand of disclosure in relation to a wild-type polypeptide. The binding regions of disclosure ligands and / or disclosure-inducing agents may be of non-natural occurrence.

[00126] Inducible pro-apoptotic disclosure polypeptides comprise a ligand-binding region, a ligand, and a pro-apoptotic peptide, wherein the inducible pro-apoptotic polypeptide does not comprise a non-human sequence. In certain embodiments, the non-human sequence comprises a restriction site. The ligand may comprise any organic or inorganic material that permits, by dimerization of the ligand-binding region, the interaction, cross-linking, cross-activation, or activation of the pro-apoptotic polypeptides such that the interaction or activation of the pro-apoptotic polypeptides initiates apoptosis in the cell. In certain embodiments, the ligand is a polypeptide. In certain embodiments, the ligand is a polypeptide comprising a G / S-rich amino acid sequence (a “GS” ligand). In certain embodiments, the ligand is a polypeptide comprising the amino acid sequence GGGGS (SEQ ID NO: 5).In preferred embodiments, the ligand is a polypeptide and the nucleic acid encoding the polypeptide does not contain a restriction site for a restriction endonuclease. The disclosure ligands may be of non-natural occurrence. Petition 870260041395, dated 04 / 05 / 2026, page 97 / 185 90 / 145

[00127] Inducible pro-apoptotic disclosure polypeptides can be expressed in a cell under the transcriptional regulation of any promoter capable of initiating and / or regulating the expression of an inducible pro-apoptotic disclosure polypeptide in that cell. The term promoter, as used herein, refers to a promoter that acts as the initial RNA polymerase binding site for transcribing a gene. For example, induced pro-apoptotic disclosure polypeptides can be expressed in a mammalian cell under the transcriptional regulation of any promoter capable of initiating and / or regulating the expression of an inducible pro-apoptotic disclosure polypeptide in a mammalian cell, including but not limited to native, endene, exene, and heterologous promoters. Preferred mammalian cells include human cells.Thus, inducible pro-apoptotic disclosure polypeptides can be expressed in a human cell under the transcriptional regulation of any promoter capable of initiating and / or regulating the expression of an inducible pro-apoptotic disclosure polypeptide in a human cell, including, but not limited to, a human promoter or a viral promoter. Exemplary promoters for expression in human cells include, but are not limited to, an immediate early gene promoter of human cytomegalovirus (CMV), an early promoter of SV40, a long terminal repeat of... Petition 870260041395, dated 04 / 05 / 2026, page 98 / 185 91 / 145 Rous sarcoma virus, a β-actin promoter, a mouse insulin promoter, and a glyceraldehyde-3-phosphate dehydrogenase promoter, each of which can be used to achieve high-level expression of an inducible pro-apoptotic disclosure polypeptide. The use of other cellular or bacterial phage or bacterial viral promoters that are well known in the art to achieve the expression of an inducible pro-apoptotic disclosure polypeptide is also contemplated, provided that the expression levels are sufficient to initiate apoptosis in a cell. By employing a promoter with well-known properties, the level and pattern of expression of the protein of interest after transfection or transformation can be optimized.

[00128] The selection of a promoter that is regulated in response to specific physiological or synthetic signals can allow the inducible expression of the inducible proapoptotic polypeptide of the dissemination. The ecdysone system (Invitrogen, Carlsbad, California) is one such system. This system is designed to allow the regulated expression of a gene of interest in mammalian cells. It consists of a well-regulated expression mechanism that allows virtually no basal-level expression of a transgene, but more than 200 times the inducibility. The system is based on the heterodimeric ecdysone receptor of Petition 870260041395, dated 04 / 05 / 2026, page 99 / 185 92 / 145 In Drosophila, when ecdysone or an analog such as muristerone A binds to the receptor, the receptor activates a promoter to enable the expression of the downstream transgene. High levels of mRNA transcripts are achieved. In this system, both monomers of the heterodimeric receptor are constitutively expressed from one vector, while the ecdysone-sensitive promoter, which drives the expression of the gene of interest, is on another plasmid. Engineering this type of system into a vector of interest can therefore be useful. Another inducible system that may be useful is the Tet-Off™ or Tet-On™ system (Clontech, Palo Alto, California) originally developed by Gossen and Bujard (Gossen and Bujard, Proc. Natl. Acad. Sci. USA, 89: 5547-5551, 1992; Gossen et al., Science, 268: 1766-1769, 1995). This system also allows high levels of gene expression to be regulated in response to tetracycline or tetracycline derivatives, such as doxycycline.In the Tet-On™ system, gene expression is activated in the presence of doxycycline, while in the Tet-Off™ system, gene expression is activated in the absence of doxycycline. These systems are based on two regulatory elements derived from the tetracycline resistance operation of E. coli: the tetracycline operator sequence (to which the tetracycline repressor binds) and the tetracycline repressor protein. The gene of interest is cloned into a plasmid behind a promoter that... Petition 870260041395, dated 04 / 05 / 2026, pages 100 / 185 93 / 145 has tetracycline-responsive elements present in it. A second plasmid contains a regulatory element called a tetracycline-controlled transactivator, which, in the Tet-Off™ system, is composed of the VP16 domain of the herpes simplex virus and the wild-type tetracycline repressor. Thus, in the absence of doxycycline, transcription is constitutively activated. In the Tet-On™ system, the tetracycline repressor is not wild-type and, in the presence of doxycycline, activates transcription. For gene therapy vector production, the Tet-Off™ system can be used so that producer cells can be cultured in the presence of tetracycline or doxycycline and avoid the expression of a potentially toxic transgene, but when the vector is introduced into the patient, gene expression would be constitutively activated.

[00129] In some circumstances, it is desirable to regulate the expression of a transgene in a gene therapy vector. For example, different viral promoters with varying strengths of activity are used depending on the desired level of expression. In mammalian cells, the immediate early CMV promoter is frequently used to provide strong transcriptional activation. The CMV promoter is reviewed in Donnelly, JJ, et al., 1997. Annu. Rev. Immunol. 15: 617-48. Modified versions of the CMV promoter that are less potent have also been used when reduced levels of transgene expression are desired. When Petition 870260041395, dated 04 / 05 / 2026, page 101 / 185 94 / 145 When the expression of a transgene in hematopoietic cells is desired, retroviral promoters such as MLV or MMTV LTRs are frequently used. Other viral promoters that are used depending on the desired effect include SV40, RSV LTR, HIV-1 and HIV-2 LTR, adenovirus promoters such as E1A, E2A or MLP region, AAV LTR, HSV-TK and avian sarcoma virus.

[00130] In other examples, promoters can be selected that are regulated by development and are active in particular differentiated cells. Thus, for example, a promoter may not be active in a pluripotent stem cell, but, for example, when the pluripotent stem cell differentiates into a more mature cell, the promoter may then be activated.

[00131] Similarly, tissue-specific promoters are used to target transcription in specific tissues or cells, in order to reduce potential toxicity or undesirable effects in non-target tissues. These promoters may result in reduced expression compared to a stronger promoter, such as the CMV promoter, but may also result in more limited expression and immunogenicity (Bojak, A., et al., 2002. Vaccine 20: 197579; Cazeaux N., et al., 2002. Vaccine 20: 3322-31). For example, tissue-specific promoters such as the PSA-associated promoter or kallikrein-associated promoter may be used. Petition 870260041395, dated 04 / 05 / 2026, page 102 / 185 95 / 145 prostate-specific glandular, or the muscle creatine kinase gene, when appropriate.

[00132] Examples of tissue-specific or differentiation-specific promoters include, but are not limited to, the following: B29 (B cells); CD14 (monocytic cells); CD43 (leukocytes and platelets); CD45 (hematopoietic cells); CD68 (macrophages); desmin (muscle); elastase-1 (pancreatic acinar cells); endoglin (endothelial cells); fibronectin (differentiating cells, healing tissues); and Flt-1 (endothelial cells); GFAP (astrocytes).

[00133] In certain indications, it is desirable to activate transcription at specific times after administration of the gene therapy vector. This is done with promoters such as those that are regulable by hormones or cytokines. Cytokine- and inflammatory protein-responsive promoters that can be used include Kininogen K and T (Kageyama et al., (1987) J. Biol. Chem., 262, 2345-2351), c-fos, TNFalpha, C-reactive protein. (Arcone, et al., (1988) Nucl. Acids Res., 16 (8), 3195-3207), haptoglobin (Oliviero et al., (1987) EMBO J., 6, 1905-1912), serum amyloid A2, C / EBP alpha, IL-1, IL-6 (Poli and Cortese, (1989) Proc. Natl Acad. Sci. USA, 86, 8202-8206), Complement C3 (Wilson et al., (1990)). Mol. Cell. Biol., 6181-6191), IL-8, alpha-1 acid glycoprotein (Prowse and Baumann, (1988) Mol Cell Biol, 8, 42-51), Petition 870260041395, dated 04 / 05 / 2026, page 103 / 185 96 / 145 alpha-1 antitrypsin, lipoprotein lipase (Zechner et al., Mol. Cell. Biol., 2394-2401, 1988), angiotensinogen (Ron, et al. (1991) Mol. Cell. Biol., 2887-2895), fibrinogen, c-jun (inducible by phorbol esters, TNF-alpha, UV radiation, retinoic acid and hydrogen peroxide), collagenase (induced by phorbol esters and retinoic acid), metallothionein (inducible by heavy metals and glucocorticoids), stromelysin (inducible by phorbol ester, interleukin-1 and EGF), alpha-2 macroglobulin and alpha1 antichymotrypsin. Other promoters include, for example, SV40, MMTV, Human Immunodeficiency Virus (MV), Moloney virus, ALV, Epstein-Barr virus, Rous Sarcoma virus, human actin, myosin, hemoglobin, and creatine.

[00134] It is anticipated that any of the above promoters, alone or in combination with another, may be useful depending on the desired action. Promoters, and other regulatory elements, are selected in such a way that they are functional in the desired cells or tissues. Furthermore, this list of promoters should not be interpreted as exhaustive or limiting; other promoters are used in conjunction with the promoters and methods disclosed herein. Nucleic Acid Molecules

[00135] Disclosure of nucleic acid molecules, including sequences that encode a polypeptide Petition 870260041395, dated 04 / 05 / 2026, page 104 / 185 97 / 145 inducible disclosure, may be in the form of RNA, such as mRNA, nRNA, tRNA or any other form, or in the form of DNA, including, but not limited to, cDNA and genetic DNA obtained by cloning or produced synthetically, or any combination thereof. DNA may be triple-stranded, double-stranded or single-stranded, or any combination thereof. Any portion of at least one strand of DNA or RNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the antisense strand.

[00136] Isolated nucleic acid molecules from disclosure may include nucleic acid molecules comprising an open reading frame (ORF), optionally with one or more introns, for example, but not limited to, at least a specified portion of at least one sequence encoding an inducible disclosure polypeptide; nucleic acid molecules comprising the coding sequence for an inducible disclosure polypeptide; and nucleic acid molecules comprising a nucleotide sequence substantially different from those described above but which, due to the degeneracy of the genetic code, still encode an inducible disclosure polypeptide as described herein and / or as known in the art. Naturally, the genetic code is well known in the art. Thus, it would be routine for a person skilled in the art to generate Petition 870260041395, dated 04 / 05 / 2026, p. 105 / 185 98 / 145 such degenerate nucleic acid variants encoding an inducible disclosure polypeptide. See, for example, Ausubel, et al., Supra, and such nucleic acid variants are included in disclosure.

[00137] As indicated herein, disclosure nucleic acid molecules comprising a nucleic acid encoding an inducible disclosure polypeptide may include, but are not limited to, those encoding the amino acid sequence of an inducible polypeptide or fragment, by itself; the coding sequence for the entire inducible polypeptide or a portion thereof;The coding sequence for an inducible polypeptide of the dissemination, fragment, or portion, as well as additional sequences, such as the coding sequence of at least one signal leader or fusion peptide, with or without the aforementioned additional coding sequences, such as at least one intron, together with additional non-coding sequences, including but not limited to 5' and 3' non-coding sequences, such as transcribed, untranslated sequences that play a role in transcription, mRNA processing, including signal processing and polyadenylation (e.g., ribosome binding and mRNA stability); an additional coding sequence that encodes additional amino acids, such as those that provide additional functionalities. Thus; Petition 870260041395, dated 04 / 05 / 2026, page 106 / 185 99 / 145 The sequence encoding an inducible disclosure polypeptide can be fused with a marker sequence, such as a sequence encoding a peptide that facilitates the purification of the fused protein structure comprising a fragment or portion of a protein structure. Nucleic Acid Construction

[00138] Nucleic acids isolated from disclosure can be made using (a) recombinant methods, (b) synthesis techniques, (c) purification techniques, and / or (d) combinations thereof, as is well known in the art.

[00139] Nucleic acids may conveniently comprise sequences in addition to a sequence encoding an inducible disclosure polypeptide. For example, a multicloning site comprising one or more endonuclease restriction sites may be inserted into the nucleic acid to aid in the isolation of the sequence encoding an inducible disclosure polypeptide. Translatable sequences may also be inserted to aid in the isolation of the translated disclosure polynucleotide. For example, a hexahistidine marker sequence provides a convenient means for purifying disclosure caspase proteins. The disclosure nucleic acid, excluding the coding sequence, is optionally a vector, adapter, or ligand for cloning and / or expression of a sequence encoding an inducible disclosure polypeptide. Petition 870260041395, dated 04 / 05 / 2026, p. 107 / 185 100 / 145 disclosure or an inducible polypeptide of disclosure.

[00140] Additional sequences may be added to such cloning and / or expression sequences to optimize their function in cloning and / or expression, to assist in the isolation of the polynucleotide sequence encoding an inducible disclosure polypeptide, or to improve the introduction of a polynucleotide sequence encoding an inducible disclosure polypeptide into a cell. The use of cloning vectors, expression vectors, adapters, and ligands is well known in the art. (See, for example, Ausubel, supra; or Sambrook, supra). Recombinant Methods for Nucleic Acid Construction

[00141] Isolated nucleic acid compositions of this description, such as RNA, cDNA, genomic DNA, or any combination thereof, can be obtained from biological sources using any number of cloning methodologies known to those skilled in the art. In some embodiments, oligonucleotide probes that selectively hybridize, under restrictive conditions, with the polynucleotides of the present invention are used to identify the desired sequence in a cDNA or gene DNA library. RNA isolation, cDNA construction, and genomic library construction are well known to those skilled in the art. (See, for example, Ausubel, supra; or Sambrook, Petition 870260041395, dated 04 / 05 / 2026, pages 108 / 185 101 / 145 above). Nucleic Acid Screening and Isolation Methods

[00142] A cDNA or genomic library can be searched using a probe based on the sequence of a polynucleotide of the disclosure or a fragment thereof. Probes can be used to hybridize with gene DNA or cDNA sequences to isolate homologous genes in the same organism or in different organisms. Experts in the technique will appreciate that varying degrees of hybridization rigor can be used in the assay; and the hybridization or washing medium can be rigorous. As the conditions for hybridization become more rigorous, there must be a greater degree of complementarity between the probe and the target for duplex formation. The degree of restriction can be controlled by one or more of temperatures, ionic strength, pH, and the presence of a partially denaturing solvent, such as formamide.For example, the rigor of hybridization is conveniently varied by altering the polarity of the reagent solution through, for example, manipulating the concentration of formamide within the range of 0% to 50%. The degree of complementarity (sequence identity) required for detectable binding will vary according to the rigor of the hybridization medium and / or washing medium. The degree of complementarity will ideally be 100%, or 70-100%, or any range or value within that. However, Petition 870260041395, dated 04 / 05 / 2026, p. 109 / 185 102 / 145 it should be understood that minor sequence variations in probes and primers can be compensated for by reducing the rigor of the hybridization and / or washing medium.

[00143] RNA or DNA amplification methods are well known in the art and can be used in accordance with disclosure without undue experimentation, based on the teachings and guidance presented here.

[00144] Known methods of DNA or RNA amplification include, but are not limited to, polymerase chain reaction (PCR) and related amplification processes (see, for example, U.S. Patents Nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188 to Mullis et al.; 4,795,699 and 4,921,794 to Tabor et al.; 5,142,033 to Innis; 5,122,464 to Wilson et al.; 5,091,310 to Innis; Biswas; 4,656,134 to Ringold) and RNA-mediated amplification that uses antisense RNA for the target sequence as a template for double-stranded DNA synthesis (U.S. Patent No. 5,130,238 to Malek et al.) al., trading as NASBA), All content from which references are taken is incorporated herein by reference. (See, for example, Ausubel, supra; or Sambrook, supra.)

[00145] For example, polymerase chain reaction (PCR) technology can be used to amplify the dissemination polynucleotide sequences and genes. Petition 870260041395, dated 04 / 05 / 2026, p. 110 / 185 103 / 145 directly related from genomic DNA or cDNA libraries. PCR and other in vitro amplification methods can also be useful, for example, to clone nucleic acid sequences encoding proteins to be expressed, to produce nucleic acids to use as probes to detect the presence of the desired mRNA in samples, for nucleic acid sequencing, or for other purposes. Examples of techniques sufficient for direct skill people through in vitro amplification methods are found in Berger, supra, Sambrook, supra, and Ausubel, supra, as well as in Mullis, et al., U.S. Pat. No. 4,683,202 (1987); and Innis, et al., PCR Protocols, A Guide for Methods and Applications, Eds., Academic Press Inc., San Diego, California (1990). Commercially available kits for genomic PCR amplification are known in the art. See, for example, Advantage-GC Genomic PCR kit (Clontech). Additionally, for example, protein 32 of the T4 gene (Boehringer Mannheim) can be used to improve the yield of long PCR products. Synthetic Methods for the Construction of Nucleic Acids

[00146] Nucleic acids isolated from the dissemination can also be prepared by direct chemical synthesis using known methods (see, for example, Ausubel, et al., Supra). Chemical synthesis generally produces a Petition 870260041395, dated 04 / 05 / 2026, page 111 / 185 104 / 145 single-stranded oligonucleotide, which can be converted into double-stranded DNA by hybridization with a complementary sequence or by polymerization with a DNA polymerase using a single strand as a template. An expert in the technique will recognize that while the chemical synthesis of DNA may be limited to sequences of about 100, 500, and 1000 or more bases, longer sequences can be obtained by linking shorter sequences. Recombinant Expression Cassettes

[00147] Disclosure also provides recombinant expression cassettes comprising a Disclosure nucleic acid. A Disclosure nucleic acid sequence, for example, a cDNA or a gene sequence encoding a portion of an inducible Disclosure polypeptide, can be used to construct a recombinant expression cassette that can be introduced into at least one desired host cell. A recombinant expression cassette will typically comprise a transcript Disclosure polynucleotide operatively linked to initiation regulatory sequences that will direct transcription of the polynucleotide in the intended host cell. Both heterologous and non-heterologous (i.e., endogenous) promoters can be employed to direct the expression of Disclosure nucleic acids.

[00148] In some forms, nucleic acids Petition 870260041395, dated 04 / 05 / 2026, page 112 / 185 105 / 145 isolates that serve as promoters, enhancers, or other elements can be introduced at the appropriate position (upstream, downstream, or in the intron) of a non-heterologous dissemination polynucleotide in order to up- or down-regulate the expression of a dissemination polynucleotide. For example, endogenous promoters can be altered in vivo or in vitro by mutation, deletion, and / or substitution. Vectors and Host Cells

[00149] The description also refers to vectors that include a sequence encoding an inducible disclosure polypeptide, host cells that are genetically modified with the recombinant vectors, and the production of at least one inducible disclosure polypeptide by recombinant techniques, as is well known in the art. See, for example, Sambrook, et al., Supra; Ausubel, et al., Supra, each fully incorporated herein by reference.

[00150] Polynucleotides, including a sequence encoding an inducible disclosure polypeptide, may optionally be attached to a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced into a precipitate, such as a calcium phosphate precipitate, or into a complex with a loaded lipid. If the vector is a virus, it may be packaged in vitro using a cell line of Petition 870260041395, dated 04 / 05 / 2026, page 113 / 185 106 / 145 appropriate packaging and then transduced into host cells.

[00151] The disclosure provides a composition comprising the disclosure transposon. In certain embodiments, the composition may further comprise a plasmid comprising a sequence encoding a transposase enzyme. The sequence encoding a transposase enzyme may be an mRNA sequence.

[00152] Disclosure transposons can be periodically maintained or integrated into the genome of the recombinant / modified cell. The transposon can be part of a two-component piggyBac system that utilizes a transposon and transposase for enhanced nonviral gene transfer. In certain embodiments of the present method, the transposon is a plasmid DNA transposon with a sequence encoding the inducible disclosure caspase polypeptide flanked by two cis-regulatory isolator elements. In certain embodiments, the transposon is a piggyBac transposon. In certain embodiments, and in particular in those embodiments where the transposon is a piggyBac transposon, the transposase is a piggyBac™ or Super piggyBac™ (SPB) transposase.

[00153] Disclosure transposons may comprise piggyBac transposons. In certain embodiments of disclosure methods, the transposon is a DNA transposon. Petition 870260041395, dated 04 / 05 / 2026, p. 114 / 185 107 / 145 plasmid with a sequence encoding the inducible disclosure caspase polypeptide flanked by two cis regulatory isolator elements. In certain embodiments, the transposon is a piggyBac transposon. In certain embodiments, and in particular in those embodiments where the transposon is a piggyBac transposon, the transposase is a piggyBac™ or Super piggyBac™ (SPB) transposase. In certain embodiments, and in particular in those embodiments where the transposase is a Super piggyBac™ (SPB) transposase, the sequence encoding the transposase is an mRNA sequence.

[00154] In certain embodiments of the disclosure methods, the transposase enzyme is a piggyBac™ (PB) transposase enzyme. The piggyBac (PB) transposase enzyme may comprise or consist of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or any percentage thereof identical to: MGSSLDDEHI LSALLQSDDE LVGEDSSEI SDHVSEDDVQ SDTEEAFIDE VHEVQPTSSG SEILDEQNVI EQPGSSLASN RILTLPQRTI RGKNKHCWST SKSTRRSRVS ALNIVRSQRG 121 PTRMCRNIYD PLLCFKLFFT DEIISEIVKW TNAEISLKRR ESMTGATFRD TNEDEIYAFF 181 GILVMTAVRK DNHMSTDDLF DRSLSMVYVS VMSRDRFDFL IRCLRMDDKS IRPTLRENDV 241 FTPVRKIWDL FIHQCIQNYT PGAHLTIDEQ LLGFRGRCPF RMYIPNKPSK YGIKILMMCD 301 SGTKYMINGM PYLGRGTQTN GVPLGEYYVK ELSKPVHGSC RNITCDNWFT Petition 870260041395, on 04 / 05 / 2026, page. 115 / 185 108 / 145 SIPLAKNLLQ 361 EPYKLTIVGT VRSNKREIPE VLKNSRSRPV GTSMFCFDGP LTLVSYKPKP AKMVYLLSSC 421 DEDASINEST GKPQMVMYYN QTKGGVDTLD QMCSVMTCSR KTNRWPMALL YGMINIACIN 481 SFIIYSHNVS SKGEKVQSRK KFMRNLYMSL TSSFMRKRLE APTLKRYLRD NISNILPNEV 541 PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV ICREHNIDMC QSCF (SEQ ID NO: 1).

[00155] In certain embodiments of the disclosure methods, the transposase enzyme is a piggyBac™ (PB) transposase enzyme comprising or consisting of an amino acid sequence with an amino acid substitution at one or more of the 30, 165, 282, or 538 positions of the sequence: MGSSLDDEHI LSALLQSDDE LVGEDSDSEI SDHVSEDDVQ SDTEEAFIDE VHEVQPTSSG SEILDEQNVI EQPGSSLASN RILTLPQRTI RGKNKHCWST SKSTRRSRVS ALNIVRSQRG 121 PTRMCRNIYD PLCCFKLFFT DEIISEIVKW TNAEISLKRR ESMTGATFRD TNEDEIYAFF 181 GILVMTAVRK DNHMSTDDLF DRSLSMVYVS VMSRDRFDFL IRCLRMDDKS IRPTLRENDV 241 FTPVRKIWDL FIHQCIQNYT PGAHLTIDEQ LLGFRGRCPF RMYIPNKPSK YGIKILMMCD 301 SGTKYMINGM PYLGRGTQTN GVPLGEYYVK ELSKPVHGSC RNITCDNWFT SIPLAKNLLQ 361 EPYKLTIVGT VRSNKREIPE VLKNSRSRPV GTSMFCFDGP LTLVSYKPKP AKMVYLLSSC 421 DEDASINEST GKPQMVMYYN QTKGGVDTLD QMCSVMTCSR KTNRWPMALL YGMINIACIN Petition 870260041395, dated 04 / 05 / 2026, pages 116 / 185 109 / 145 481 SFIIYSHNVS SKGEKVQSRK KFMRNLYMSL TSSFMRKRLE APTLKRYLRD NISNILPNEV 541 PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV ICREHNIDMC QSCF (SEQ ID NO: 1).

[00156] In certain embodiments, the transposase enzyme is a piggyBac™ (PB) transposase enzyme comprising or consisting of an amino acid sequence having an amino acid substitution at two or more of the 30, 165, 282, or 538 positions of the SEQ ID NO: 1 sequence. In certain 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 the 30, 165, 282, or 538 positions of the SEQ ID sequence. NO: 1. In certain 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 the following positions: 30, 165, 282, and 538 of the SEQ ID NO: 1 sequence. In certain embodiments, the amino acid substitution at position 30 of the SEQ ID NO: 1 sequence is a substitution of a valine (V) for an isoleucine (I).In certain embodiments, the amino acid substitution at position 165 of the sequence SEQ ID NO: 1 is a substitution of a serine (S) for a glycine (G). In certain embodiments, the amino acid substitution at position 282 of the sequence SEQ ID NO: 1 is a substitution of a valine (V) for a methionine (M). Petition 870260041395, dated 04 / 05 / 2026, pp. 117 / 185 110 / 145 In certain modalities, the amino acid substitution at position 538 of the SEQ ID NO: 1 sequence is a substitution of a lysine (K) for an asparagine (N).

[00157] In certain embodiments of the disclosure methods, the transposase enzyme is a Super piggyBac™ transposase enzyme (SPB). In certain embodiments, the Super piggyBac™ transposase enzymes of the disclosure may comprise or consist of the amino acid sequence of SEQ ID NO: 1, wherein the amino acid substitution at position 30 is a substitution of a valine (V) for an isoleucine (I), the amino acid substitution at position 165 is a substitution of a serine (S) for a glycine (G), the amino acid substitution at position 282 is a substitution of a valine (V) for a methionine (M), and the amino acid substitution at position 538 is a substitution of a lysine (K) for an asparagine (N). In certain embodiments, the Super piggyBac™ transposase enzyme (SPB) may comprise or consist of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or any percentage thereof identical to: MGSSLDDEHI LSALLQSDDE LVGEDSDSEV SDHVSEDDVQ SDTEEAFIDE VHEVQPTSSG SEILDEQNVI EQPGSSLASN RILTLPQRTI RGKNKHCWST SKSTRRSRVS ALNIVRSQRG 121 PTRMCRNIYD PLLCFKLFFT DEIISEIVKW TNAEISLKRR ESMTSATFRD TNEDEIYAFF Petition 870260041395, on 04 / 05 / 2026, page. 118 / 185 111 / 145 181 GILVMTAVRK DNHMSTDDLF DRSLSMVYVS VMSRDRFDFL IRCLRMDDKS IRPTLRENDV 241 FTPVRKIWDL FIHQCIQNYT PGAHLTIDEQ LLGFRGRCPF RVYIPNKPSK YGIKILMMCD 301 SGTKYMINGM PYLGRGTQTN GVPLGEYYVK ELSKPVHGSC RNITCDNWFT SIPLAKNLLQ 361 EPYKLTIVGT VRSNKREIPE VLKNSRSRPV GTSMFCFDGP LTLVSYKPKP AKMVYLLSSC 421 DEDASINEST GKPQMVMYYN QTKGGVDTLD QMCSVMTCSR KTNRWPMALL YGMINIACIN 481 SFIIYSHNVS SKGEKVQSRK KFMRNLYMSL TSSFMRKRLE APTLKRYLRD NISNILPKEV 541 PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV ICREHNIDMC QSCF (SEQ ID NO: 2).

[00158] In certain embodiments of the disclosure methods, including those embodiments in which the transposase comprises the mutations described above at positions 30, 165, 282 and / or 538, the piggyBac™ or Super piggyBac™ transposase enzyme may further comprise an amino acid substitution at one or more of the positions 3, 46, 82, 103, 119, 125, 177, 180, 185, 187, 200, 207, 209, 226, 235, 240, 241, 243, 258, 296, 298, 311, 315, 319, 327, 328, 340, 421, 436, 456, 470, 486, 503, 552, 570 and 591 of the SEQ ID NO: 1 or SEQ ID NO: 2 sequence. In certain embodiments, including embodiments in which the transposase comprises the mutations described above at positions 30, 165, 282 and / or 538, the piggyBac™ or Super piggyBac™ transposase enzyme may also comprise a substitution of Petition 870260041395, dated 04 / 05 / 2026, p. 119 / 185 112 / 145 amino acids at one or more of the positions 46, 119, 125, 177, 180, 185, 187, 200, 207, 209, 226, 235, 240, 241, 243, 296, 298, 311, 315, 319, 327, 328, 340, 421, 436, 456, 470, 485, 503, 552 and 570. In certain embodiments, the amino acid substitution at position 3 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of an asparagine (N) for a serine (S). In certain embodiments, the amino acid substitution at position 46 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of a serine (S) for an alanine (A). In certain embodiments, the amino acid substitution at position 46 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of a threonine (T) for an alanine (A). In certain embodiments, the amino acid substitution at position 82 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of a tryptophan (W) for an isoleucine (I). In certain embodiments, the amino acid substitution at position 103 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of a proline (P) for a serine (S).In certain embodiments, the amino acid substitution at position 119 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of proline (P) for arginine (R). In certain embodiments, the amino acid substitution at position 125 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of alanine (A) for cysteine ​​(C). In certain embodiments, the amino acid substitution at position 125 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of leucine (L) for cysteine ​​(C). In certain embodiments, Petition 870260041395, dated 04 / 05 / 2026, pp. 120 / 185 113 / 145 The amino acid substitution at position 177 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of a lysine (K) for a tyrosine (Y). In certain embodiments, the amino acid substitution at position 177 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of a histidine (H) for a tyrosine (Y). In certain embodiments, the amino acid substitution at position 180 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of a leucine (L) for a phenylalanine (F). In certain embodiments, the amino acid substitution at position 180 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of an isoleucine (I) for a phenylalanine (F). In certain embodiments, the amino acid substitution at position 180 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of a valine (V) for a phenylalanine (F). In certain embodiments, the amino acid substitution at position 185 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of a leucine (L) for a methionine (M).In certain embodiments, the amino acid substitution at position 187 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of glycine (G) for alanine (A). In certain embodiments, the amino acid substitution at position 200 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of tryptophan (W) for phenylalanine (F). In certain embodiments, the amino acid substitution at position 207 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of proline (P) for valine (V). In certain embodiments, the substitution... Petition 870260041395, dated 04 / 05 / 2026, pp. 121 / 185 In certain embodiments, the amino acid substitution at position 209 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of a phenylalanine (F) for a valine (V). In certain embodiments, the amino acid substitution at position 226 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of a phenylalanine (F) for a methionine (M). In certain embodiments, the amino acid substitution at position 235 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of an arginine (R) for a leucine (L). In certain embodiments, the amino acid substitution at position 240 of SEQ ID NO: 1 or SEQ ID NO: 1 is a substitution of a lysine (K) for a valine (V). In certain embodiments, the amino acid substitution at position 241 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of leucine (L) for phenylalanine (F). In certain embodiments, the amino acid substitution at position 243 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of lysine (K) for proline (P).In certain embodiments, the amino acid substitution at position 258 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of a serine (S) for an asparagine (N). In certain embodiments, the amino acid substitution at position 296 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of a tryptophan (W) for a leucine (L). In certain embodiments, the amino acid substitution at position 296 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of a tyrosine (Y) for a leucine (L). In certain embodiments, the amino acid substitution at position 296 of... Petition 870260041395, dated 04 / 05 / 2026, pp. 122 / 185 115 / 145 SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of phenylalanine (F) for leucine (L). In certain embodiments, the amino acid substitution at position 298 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of leucine (L) for methionine (M). In certain embodiments, the amino acid substitution at position 298 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of alanine (A) for methionine (M). In certain embodiments, the amino acid substitution at position 298 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of valine (V) for methionine (M). In certain embodiments, the amino acid substitution at position 311 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of isoleucine (I) for proline (P). In certain embodiments, the amino acid substitution at position 311 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of a valine for a proline (P).In certain embodiments, the amino acid substitution at position 315 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of a lysine (K) for an arginine (R). In certain embodiments, the amino acid substitution at position 319 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of a glycine (G) for a threonine (T). In certain embodiments, the amino acid substitution at position 327 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of an arginine (R) for a tyrosine (Y). In certain embodiments, the amino acid substitution at position 328 of SEQ ID NO: 1. Petition 870260041395, dated 04 / 05 / 2026, pp. 123 / 185 116 / 145 or SEQ ID NO: 2 is a substitution of a valine (V) for a tyrosine (Y). In certain embodiments, the amino acid substitution at position 340 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of a glycine (G) for a cysteine ​​(C). In certain embodiments, the amino acid substitution at position 340 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of a leucine (L) for a cysteine ​​(C). In certain embodiments, the amino acid substitution at position 421 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of a histidine (H) for aspartic acid (D). In certain embodiments, the amino acid substitution at position 436 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of an isoleucine (I) for a valine (V). In certain embodiments, the amino acid substitution at position 456 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of a tyrosine (Y) for a methionine (M).In certain embodiments, the amino acid substitution at position 470 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of phenylalanine (F) for leucine (L). In certain embodiments, the amino acid substitution at position 485 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of lysine (K) for serine (S). In certain embodiments, the amino acid substitution at position 503 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of leucine (L) for methionine (M). In certain embodiments, the amino acid substitution at position 503 of SEQ ID NO: 1 or SEQ ID NO: 2. Petition 870260041395, dated 04 / 05 / 2026, pp. 124 / 185 117 / 145 is a substitution of isoleucine (I) for methionine (M). In certain embodiments, the amino acid substitution at position 552 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of lysine (K) for valine (V). In certain embodiments, the amino acid substitution at position 570 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of threonine (T) for alanine (A). In certain embodiments, the amino acid substitution at position 591 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of proline (P) for glutamine (Q). In certain embodiments, the amino acid substitution at position 591 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of arginine (R) for glutamine (Q).

[00159] In certain embodiments of the disclosure methods, including those embodiments in which the transposase comprises the mutations described above at positions 30, 165, 282 and / or 538, the piggyBac™ transposase enzyme may comprise, or the Super piggyBac™ transposase enzyme may further comprise, an amino acid substitution at one or more positions 103, 194, 372, 375, 450, 509 and 570 of the SEQ ID NO: 1 or SEQ ID NO: 2 sequence. In certain embodiments of the disclosure methods, including embodiments in which the transposase comprises the mutations described above at positions 30, 165, 282 and / or 538, the piggyBac™ transposase enzyme may comprise, or the super piggyBac™ transposase enzyme may further comprise a Petition 870260041395, dated 04 / 05 / 2026, pp. 125 / 185 118 / 145 amino acid substitution at two, three, four, five, six or more positions 103, 194, 372, 375, 450, 509 and 570 of the SEQ ID NO: 1 or SEQ ID NO: 2 sequence. In certain embodiments, including embodiments in which the transposase comprises the mutations described above at positions 30, 165, 282 and / or 538, the piggyBac™ transposase enzyme may comprise or the super piggyBac™ transposase enzyme may further comprise an amino acid substitution at positions 103, 194, 372, 375, 450, 509 and 570 of the SEQ ID NO: 1 or SEQ ID NO: 2 sequence. In certain embodiments, the amino acid substitution at position 103 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of a proline (P) for a serine (S). In certain embodiments, the amino acid substitution at position 194 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of a valine (V) for a methionine (M).In certain embodiments, the amino acid substitution at position 372 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of an alanine (A) for an arginine (R). In certain embodiments, the amino acid substitution at position 375 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of an alanine (A) for a lysine (K). In certain embodiments, the amino acid substitution at position 450 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of an asparagine (N) for an aspartic acid (D). In certain embodiments, the amino acid substitution at position 509 of SEQ ID NO: 1 or SEQ ID NO: Petition 870260041395, dated 04 / 05 / 2026, pp. 126 / 185 119 / 145 is a substitution of a glycine (G) for a serine (S). In certain embodiments, the amino acid substitution at position 570 of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of a serine (S) for an asparagine (N). In certain embodiments, the piggyBac™ transposase enzyme may comprise a substitution of a valine (V) for a methionine (M) at position 194 of SEQ ID NO: 1. In certain embodiments, including those embodiments in which the piggyBac™ transposase enzyme may comprise a substitution of a valine (V) for a methionine (M) at position 194 of SEQ ID NO: 1, the piggyBac™ transposase enzyme may further comprise an amino acid substitution at positions 372, 375, and 450 of the sequence SEQ ID NO: 1 or SEQ ID NO: 2.In certain embodiments, the piggyBac™ transposase enzyme may comprise a valine (V) substitution for a methionine (M) at position 194 of SEQ ID NO: 1, an alanine (A) substitution for an arginine at position 372 of SEQ ID NO: 1, and an alanine (A) substitution for a lysine (K) at position 375 of SEQ ID NO: 1. In certain embodiments, the piggyBac™ transposase enzyme may comprise a valine (V) substitution for a methionine (M) at position 194 of SEQ ID NO: 1, an alanine (A) substitution for an arginine (R) at position 372 of SEQ ID NO: 1, an alanine (A) substitution for a lysine (K) at position 375 of SEQ ID NO: 1 and a... Petition 870260041395, dated 04 / 05 / 2026, p. 127 / 185 120 / 145 replacement of an asparagine (N) by an aspartic acid (D) at position 450 of SEQ ID NO: 1.

[00160] In certain modalities of the dissemination methods, the transposon is a Sleeping Beauty transposon. In certain modalities, and in particular in those modalities in which the transposon is a Sleeping Beauty transposon, the transposase is a Sleeping Beauty transposase or a hyperactive Sleeping Beauty transposase (SB100X).

[00161] In certain embodiments of the disclosure methods, the Sleeping Beauty transposase enzyme comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or any percentage thereof identical to: MGKSKEISQD LRKKIVDLHK SGSSLGAISK RLKVPRSSVQ TIVRKYKHHG TTQPSYRSGR 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 301 HQLCQEEWAK IHPTYCGKLV EGYPKRLTQV KQFKGNATKY (SEQ ID NO: 19).

[00162] In certain embodiments of the disclosure methods, the hyperactive Sleeping Beauty transposase enzyme (SB100X) comprises an amino acid sequence of at least Petition 870260041395, dated 04 / 05 / 2026, pages 128 / 185 121 / 145 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to: MGKSKEISQD LRKRIVDLHK SGSSLGAISK RLAVPRSSVQ TIVRKYKHHG TTQPSYRSGR 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 301 HQLCQEEWAK IHPNYCGKLV EGYPKRLTQV KQFKGNATKY (SEQ ID NO: 20).

[00163] In certain forms of dissemination methods, the transposase is a Helitron transposase. Helitron transposases mobilize the transposon of Helraiser, an ancient element of the bat genome that was active approximately 30 to 36 million years ago. An example of a Helraiser transposon is Helibat1, which comprises a nucleic acid sequence including: 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 Petition 870260041395, dated 04 / 05 / 2026, p. 129 / 185 122 / 145 CCGGGACCTT 241 CGCCAGCAGA GAGCAGAGCG GGAGAGCGGG CGGAGAGCGG GGACTTGGCA 301 GAGCAGGAGG CCGCTGGACA TAGAGCAGAG CGAGAGAGAG AGGGCGTGGC 361 TCCCTCTGTC ACCCCAGCTT CCTCATCACA GCTGTGGAAA GGAGGAGGAA 421 GTCCCACCCC CACAGAATCA GCCAGAATCA GCCGTTGGTC CAGCGGCCTG 481 ACAGCCAGGA CTCTCATTCA CCTGCATCTC AGACCGTGAC TGGGACTATG 541 TCTAAAGAAC AACTGTTGAT ACAACGTAGC TCTGCAGCCG GCGTTATCGA 601 CAGAAAATGT CTGCAGAGCA ACGTGCGTCT GATCTTGAAA CCTGCAACAG 661 AATGTATCTG AAGAGCAGCT ACTGGAAAAA CGTCGCTCTG ACAGCGGCGT 721 CATCGACAGA AAATGTCTAA AGACCAACGT GCCTTTGAAG GCGGTGGCGA 781 CGACAGAATA TGTCTAGAGA ACAGTCATCA ACAAGTACTA TAGGAACTGC 841 CTTCTCAGCA AAAATGGAGT ACATGAGGAT GCAATTCTCG TGGTGGAATG 901 ACTGTTCGAT GTGAATTTTG CCTATCACTA AATTTCTCTG ATCCGATGGG 961 AAATTTACTC GATGTTGTAG CAAAGGGAAA GTCTGTCCAA TTTTCCAGAT 1021 TACCCGGCAT ATTTAAAAAG ATTAATGACA AACGAAGATT AAATTTCATG 1081 GAAATATTC GTTCCATAAA TAGTTCTTTT GCTTTTGCTT GAGGTTTGGA GGTGGCTTGG CTGACAGCAG AGACAGCTCT IT IS BEING TAKEN AAAGATGCCG GAAGGCGGCG AAGCCAAAA TTGAAAGAAG CCAATACCGG AACATAGTTG ATGAAAAACC ATTACK CTSTART CCATGGGTGC Petition 870260041395, dated 04 / 05 / 2026, p. 130 / 185 123 / 145 TCGCCATCAG GATATGGGCC ATACTGTTTT AGAATACACG GACAAGTTTA GGAACTTTAC ATCCTTCGGA TGGTGTTTCT CGGAAGTTTG CTCAACTCTA ACAGCCGAAG CTACAAGTAA AAGATTAGCA ATGCCAGAAA ACCAGGGCTG CTCATGATCA COMPETITION CCTCATGCAT COMPETITION GATTACAAA ATGCTACATG AGGTAAAA GGAAGCCCAA TCTGAAGCAG CAGCAAAAGG ACTIONS HELPGGCGAT TAAATACGAT CGTAACTG ACCCAGGTAG CCCCGTGTAA CCGAGGTTGC TGTCATATTC AGAAACGAAG ATGGAGAACC AGGGACTTGC TCATTCATTG TAAACCAGAT CCCAATAATC CAAATGCCAC SEQUENCE TCCTGTTTCC SEQUENCE GCHAPTER ATCCTATTCT GGTGAAAAAG GCTGGGGAAC NATIONTTGCA TTAGACTCA GGAGAAC AATATACTTA CHAPTERAAATGT CHANGE GTCAXAA TGCHAPTER CTCTCTGTGC GGGACACGTT CAATCCTATT TTAAATGCAG GAAAATTAAC ATTGTGGATT CATATTCAAA AATGGAGGCC AATCGTAA ATTTCATCAA TCTAAGTTGA GAGTTGAAAA ATATAGTGGT TTGATGGATT ATCTCAAATC AATGACAATG TGCCGATTGG TAAAATGATA ATACTTCCAT CATCTTTTGA Petition 870260041395, dated 04 / 05 / 2026, p. 131 / 185 124 / 145 GGGTAGTCCC 2041 AGAAATATGC AGCAGCGATA TGGCAAGCCC 2101 TACKETTATTCA TACCATGAC ASSISTANT 2161 CCGCTGGCAAAA AAGTTGAAAAA TATTAAGCTG 2221 AATGCTCTTT TAAATGAT AGCTAAATT 2281 CATGTCATTG AATTTCAGAA SUDDENLY 2341 AGTGAGTCCA AATTACGTTC AGAAATTCCA 2401 CONTINUOUS CC AGTGTTCCTCG ACATGGACCA 2461 TGTGGAATAC AAAATCCAAAA AAAGGTA 2521 CCAAAAGAAT TTCAAAATGC ATTACK 2581 AGATCTGGTA GCACCATGTC GATTGTCCCT 2641 TATAACCCGT ATTTGTGCCT CTGTGCATCA 2701 ATTAAAAGTG TCAAATATTT TGCAAATATT 2761 CAAATTTCTG AAAAAAATAT TGACTCCAGG 2821 TATGTGAGCG CTCCTGAGGC TGACCAATCT 2881 CATGCAATCA CAAGATTAGC TCAGGATGCT ATGGCAATTG TAACGAAGTA ATGCAACCCC AAATGGGCAG ATTACTCAAA CAGACCTGAC TTGGTAGCCA GAGTTTTTAA ATGTAAATTC CATTTATTTG GCAAAGTAAT ACGCGGACTG CCTCACGCTC ACATATTATT AGAAGATGAC ATTGACCGTA TAGTTAAGGC ACTTTTCAA ATTGTAAAAT CAAATATGGT TAGTCCATGT ATGGAAAATG GAAAATGTTC GACCATTGGA AATATTGATG GATATCCCAA TATTGGAAAT AAAGTTGTCG ATAACACTTG TAAATAAC TGTCATATAA ATGTTGAAGT ATTTAAATAC ATCTATAAAG GGCACGATTG TATCAATCAT GACGAAGTAC AGGACTTCAT TGTTTGGAGA CTTTTTGCAA TGCGAATGCA TATTCATTTG CCAAATGATC AGAATTTGTA Petition 870260041395, dated 04 / 05 / 2026, p. 132 / 185 125 / 145 TTTTCATACC 2941 GATGATTTTG CTGAAGTTTT AGATAGGGCT AAAAGGCATA GATGGCTTGG 3001 TTCTTATTGA ATAGAGAAGA TTCTGATGCA CGTAATTATT GATTCCACAG 3061 CATTATGTGT TTAATAATTC TTTGTGGACA AAACGCCGAA ACTCGACTTT ATTATTGGGA AGGGTGGGAA WINDOW 3121 GGTAGACTGT TCACTGTGAG CTTTAGAGAA CCAGAACGAT ATTACK ACTTTTGCTT 3181 CTGCATGTAA AAGGTGCGAT AAGTTTTGAG GATCTGCGAA CTGTGAGG TGTAACTTAT 3241 GATACATTTC ATGAAGCTGC TAAACACCGA GGATTATTAC TTGATGACAC TATCTGGAAA 3301 GATACGATTG ACGATGCAAT CATCCTTAAT ATGCCCAAAC ACTTTTTGCA 3361 TATATATGTG TGTTTGGATG TCCTTCTGCT GCAGACAAAT AACTACGGCA FATHER GAATAAATCT 3421 CATTTTATTG AAGATTTCTG TTGGAAATTA CACCGAAGAG TGTGAACTGT 3481 GAAATGCATG CCCTTAACGA AATTCAGGAG GTATTCACAT GAAATGTTCA 3541 CATTTCAAAC TTCCGGACTA TCCTTTATTA ATGAATGCAA TCAATTGTAC 3601 GAGCAACAAC AGGCAGAGGT TTTGATAAAT TCTCTGAATG GGCAGCCTTT 3661 CAGACTATAA CTTCAGCCAT CGAAGATCAA ACTGTACACC TTTCTTGGAT 3721 GGTCCAGGTG GTAGTGGAAA AACATATCTG TATAAAGTTT TATTAGAGGT 3781 CGTGGTGGTA CTGTTTTACC CACAGCATCT ACAGGAATTG AAGGTGCCTG TGCATGGAAT ATACATGTGA ATGAACAGTT CCAAATGCTT TAACACATTA CTGCAAATTT Petition 870260041395, of 04 / 05 / 2026, p. 133 / 185 126 / 145 GGAAGAACCT TTCATTCCCA ATATAAATTA CCAATTCCAT TAAATGAAAC AGACTCGATA TAAAGAGTGA AGTTGCTAAA ACCATTAAAA AGGCCCAACT GATGAATGCA CCATGGCATC CAGTCATGCT ATAAACGCCA TAGATAGATT ATTATGAATT TGAATGTTGC ATTTGGTGGG AAAGTTCTCC TTCTCGGAGG CAATGTCTCA GTATTGTACC ACATGCTATG CGATCGGCCA TAGTACAAAC TACTGTAATG TTTGGGGATG TTTCAGAAAG TTGTCTCTTA AAACAAATAT GATTCTGCTT ATAGTGAATG GTTAGTAAAA CTTGGAGATG GCAAACTTGA CATTTAGGAA TGGATATTAT TGAAATCCCC CATGAAATGA TTTGTAACGG GAAGCTACCT TTGGAAATAG TATATCTATA GATAATATTA AAAATATATC ATTCTTTGTC CAAAAAATGA GCATGTTCAA AAATTAAATG AAGAAATTTT GATGGAGATT TTCACACATA TTTGAGTGAT GATTCCATTG ATTCAACAGA AAGGAAAATT TTCCCATCGA ATTTCTTAAT AGTATTACTC CTTCGGGAAT AAATTAAAAT TGAAAGTGGG TGCAATCATC ATGCTATTGA GAAATCTTAA GGTCTTTGTA ATGGTACTAG ATTTATTATC AAAAGATTAC GACCTAACAT GAAGTATTAA CAGGATCTGC AGAGGGAGAG GTTGTTCTGA TTCCAAGAAT Petition 870260041395, of 04 / 05 / 2026, p. 134 / 185 127 / 145 TGATTTGTCC 4741 CCATCTGACA CTGGCCTCCC ATTACK ATTCGAAGAC AGTTTCCCGT GATGCCAGCA 4801 TTTGCGATGA CTATTAATAA ATCACAAGGA CAAACTCTAG ACAGAGTAGG AATATTCCTA 4861 CCTGAACCCG TTTTCGCACA TGGTCAGTTA TATGTTGCTT TCTCTCGAGT TCGAAGAGCA 4921 TGTGACGTTA AAGTTAAAGT TGTAAATACT TCATCACAAG GGAAATTAGT CAAGCACTCT 4981 GAAAGTGTTT TTACTCTTAA TGTGGTATAC AGGGAGATAT TAGAATAAGT TTAATCACTT 5041 TATCAGTCAT TGTTTGCATC AATGTTGTTT TTATATCATG TTTTTGTTGT TTTTATATCA 5101 TGTCTTTGTT GTTGTTATAT CATGTTGTTA TTGTTTATTT ATTAATAAAT TTATGTATTA 5161 TTTTCATATA CATTTTACTC ATTTCCTTTC ATCTCTCACA CTTCTATTAT AGAGAAAGGG 5221 CAAATAGCAA TATTAAAATA TTTCCTCTAA TTAATTCCCT TTCAATGTGC ACGAATTTCG 5281 TGCACCGGGC CACTAG (SEQ ID NO: 21).

[00164] Unlike other transposases, Helitron transposase does not contain an RNaseH catalytic domain, but instead comprises a RepHel motif composed of a replication initiator domain (Rep) and a DNA helicase domain. The Rep domain is a nuclease domain of the HUH superfamily of nucleases.

[00165] An exemplary helitron transposase from disclosure comprises an amino acid sequence Petition 870260041395, dated 04 / 05 / 2026, pp. 135 / 185 128 / 145 comprising: KRRSEAEKQR RHRQKMSKDQ RAFEVERRRW 121 MTVRCEFCLS LNFSDEKPSD 181 MENIRSINSS FAFASMGANI 241 DTAEATSKRL AMPENQGCSE 301 PTEVTMAIKY DRNSDPGRYN 361 KQISILFPTL DAMTYPILFP 421 HLSVRDTFNP ILNAGKLTQQ 481 ENDNVPIGKM IILPSSFEGS 541 QRWQKVENRP DLVARVFNIK 601 DSESKLRSED DIDRIVKAEI 661 YPKEFQNATI GNIDGYPKYK 721 SIKSVKYLFK YIYKGHDCAN 781 SHAITRLAIH LPNDQNLYFH 841 QHYVFNNSLW TKRRKGGNKV 901 YDTFHEAAKH RGLLLDDTIW 961 SHFIEDFCWK LHRREGACVN 1021 YEQQQAEVLI NSLNDEQLAA 1081 GRGGTVLPTA STGIAANLLL 1141 IDECTMASSH AINAIDRLLR 1201 KYCNVWGCFR KLSLKTNMRS 1261 IEATFGNSIS IDNIKNISKR 1321 EKENFPIEFL NSITPSGMPC 1381 AEVLTGSAEG EVVLIPRIDL 1441 LPEPVFAHGQ LYVAFSRVRR (SEQ ID NO: 22).

[00166] MSKEQLLIQR SSAAERCRRY RQKMSAEQRA SDLERRRRLQ QNVSEEQLLE RRQNMSREQS STSTTNTGRN GKFTRCCSKG KVCPNDIHFP ASPSGYGPYC FRIHGQVYHR RLMININNLM HEINELTKSY SPRVTEVAVI FRNEDGEPPF HGEKGWGTDI ALRLRDNSVI FIVDSYSKME ANRINFIKAN PRNMQQRYQD AMAIVTKYGK LNALLNDICK FHLFGKVIAK PDEDQCPRLF QIVKSNMVHG RRSGSTMSIG NKVVDNTWIV IQISEKNIIN HDEVQDFIDS TDDFAEVLDR AKRHNSTLMA LGRLFTVSFR EPERYYLRLL KDTIDDAIIL NMPKQLRQLF CEMHALNEIQ EVFTLHGMKC FQTITSAIED QTVHPKCFFL GGRTFHSQYK LPIPLNETSI EIMNLNVAFG GKVLLLGGDF EDSAYSEWLV KLGDGKLDSS AILCPKNEHV QKLNEEILDI HKLKLKVGAI IMLLRNLNSK SPSDTGLPFK LIRRQFPVMP ACDVKVKVVN TSSQGKLVKH Helitron, In transpositions CLLSKNGVHE DAILEHSCGG DYPAYLKRLM TNEDSDSKNF TGTLHPSDGV SRKFAQLYIL KMLHEVEKEA QSEAAAKGIA ERDLLIHCKP DPNNPNATKM DNNTRQNVRT RVTQMQYYGF QSKLRVEKYS GLMDYLKSRS PDLFITMTCN PKWADITNNL IHVIEFQKRG LPHAHILLIL PCGIQNPNSP CMENGKCSKG PYNPYLCLKY NCHINVEVCA RYVSAPEAVW RLFAMRMHDQ WFLLNREDSD ARNYYYWEIP LLHVKGAISF EDLRTVGGVT AYICVFGCPS AADKLWDENK SHFKLPDYPL LMNANTCDQL DGPGGSGKTY LYKVLTHYIR SRLDIKSEVA KTIKKAQLLI RQCLSIVPHA MRSAIVQTSL FHLGMDIIEI PHEMICNGSI LDGDFHTYLS DDSIDSTDDA WGLCNGTRFI IKRLRPNIIE AFAMTINKSQ GQTLDRVGIF SESVFTLNVV YREILE a staple near the Petition 870260041395, dated 04 / 05 / 2026, pages 136 / 185 The 129 / 145 3' end of the transposon functions as a terminator. However, this hairpin can be bypassed by the transposase, resulting in the transduction of flanking sequences. Furthermore, Helraiser transpositions generate covalently closed circular intermediates. Additionally, Helitron transpositions may lack target site duplications. In the Helraiser sequence, the transposase is flanked by left and right terminal sequences called LTS and RTS. These sequences terminate with a conserved 5'-TC / CTAG-3' motif. A 19 bp palindritic sequence with the potential to form the hairpin termination structure is located 11 nucleotides upstream of the RTS and consists of the GTGCACGAATTTCGTGCACCGGGCCACTAG sequence (SEQ ID NO: 23).

[00167] In certain embodiments of the disclosure methods, the transposase is a Tol2 transposase. Tol2 transposons can be isolated from or derived from the medaka fish genome and may be similar to hAT family transposons. Exemplary disclosure Tol2 transposons are encoded by a sequence comprising approximately 4.7 kilobases and contain a gene encoding the Tol2 transposase, which contains four exons. An exemplary disclosure Tol2 transposase comprises an amino acid sequence comprising the following: MEEVCDSSAA ASSTVQNQPQ DQEHPWPYLR EFFSLSGVNK DSFKMKCVLC LPLNKEISAF Petition 870260041395, dated 04 / 05 / 2026, pp. 137 / 185 130 / 145 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 601 NTPLPASAAC ERLFSTAGLL FSPKRARLDT NNFENQLLLK LNLRFYNFE (SEQ ID NO: 24).

[00168] An example of a disclosure of Tol2 transposon, including inverted repeats, subterminal sequences, and the Tol2 transposase, is encoded by a nucleic acid sequence comprising the following: CAGAGGTGTA AAGTACTTGA GTAATTTTAC TTGATTACTG TACTTAAGTA TTATTTTTGG GGATTTTTAC TTTACTTGAG TACAATTAAA AATCAATACT TTTACTTTTA CTTAATTACA 121 TTTTTTTAGA AAAAAAAGTA CTTTTTACTC CTTACAATTT TATTTACAGT CAAAAAGTAC 181 TTATTTTG GAGATCAATT TCCCTTCATTGCATTATT 241 CGCTGATGCC CAGTTTAATT TAAATGTTAT TTATTCTGCC TATGAAAATC GTTTCATTGG TCCTTTGGAA GTGACGTCAT GTCAGCAGTCTA 361 TTACCTAGACGAAT ATGACTTACCAGCAAT TCAATCAGTG 421 GAAGAAAATG GAGGAAGTAT GTGATTCATC AGCAGCTGCG AGCAGCACAG TCCAAAATCA 481 GCCACAGGAT CAAGAGCACC CGTGGCCGTA TCTTCGCGAA TTCTTTGTTCTT TAAGTGGTGT 541 CGAATTCAAAGAT TCGATTCAAGAT CCGCTTAATA AAGAAATATC 601 GGCCTTCAAA AGTTCGCCAT CAAACCTAAG GAAGCATATT GAGGTAAGTA CATTAAGTAT 661 TTTGTTTTAC TGATAGTTTT TTTTTTTTTT TTGTTTTTT TTTTTGGGTG TGCATGATGTGCGCTT 721 GTAGGCCTAT TTTCACTAAT GCATGCGATT 781 GACAATAA GGCTCACGTA ATAAAATGCT AAAATGCATT TGTAATTGGT AACGTTAGGT Petition 870260041395, dated 04 / 05 / 2026, p. 138 / 185 131 / 145CCACGGGAAA TTTGGCGCCT ATTGCAGCTT TGAATAATCA TTATCATTCC GTGCTCTCAT TGTGTTTGAA TTCATGCAAA ACACAAGAAA ACCAAGCGAG AAAATTTTTTT CCAAACATGT TGTATTGTCA AAACGGTAAC ACTTTACAAT GAGGTTGATT AGTACTCATTAACTACCATACCATTA ATGAGCAATA CATTTGTTAC TGTATCTGTT AATCTTTGTT AACGTTAGTT AATAGAAATA CAGATGTCCA TTGTTTGTTC ATGTTAGTTC ACAGTGCATT AACTAATGTT AACAAGATAT AAAGTATTAG TAAATGTTGA AATTAACATG TATACGTGTA AGTTCATTTATGTTGTT GTTAACTAAC GAACCTTATT GTAAAAGTGT TACCATCAAA ACTAATGTAA TGAAATCAAT TCACCCTGTC ATGTCAGCCT TACAGTCCTG TGTTTTTGTC AATATAATCA GAAATAAAAT TAATGTTTGA TTGTCACTAA ATGCTACTGT ATTTCTAAAA TCAAAGTA TACCATACTAGTATTA AATTGGCTGC AAATGTCAGT TTTATTAAAG GGTTAGTTCA CCCAAAAATG AAAATAATGT CATTAATGAC TCGCCCTCAT GTCGTTCCAA GCCCGTAAGA CCTCCGTTCA TCTTCAGAAC ACAGTTTAAG ATATTTTAGA TTTAGTCCGA GAGCTTTCTG TGACGCTCCATTAGTAGTATATAT CTGTCCATGT CCAGAAAGGT AATAAAAACA TCAAAGTAGT CCATGTGACA TCAGTGGGTT AGTTAGAATT TTTTGAAGCA TCGAATACAT TTTGGTCCAA AAATAACAAA ACCTACGACT TTATTCGGCA TTGTATTCTC TTCCGGGTCTGTTGTCAATC CGCGTTCACG ACTTCGCAGT GACGCTACAA TGCTGAATAA AGTCGTAGGT TTTGTTATTT TTGGACCAAA ATGTATTTTC GATGCTTCAA ATAATTCTAC CTAACCCACT GATGTGTCACAT GGACTACTTT GATGTTGATTTA GATACATCCTTCT TCAGTGGAGG GACAGAAAGC TCTCGGACTA AATCTAAAAT ATCTTAAACT GTGTTCCGAA GATGAACGGA GGTGTTACGG GCTTGGAACG ACATGAGGGT GAGTCATTAA TGACATCTTT TCATTTTTGG GTGAACTAAC CCTTATTAGAATTAGC GTATT ATACAATATA AATATTTATT TGTTGTTTTT ACAGAGAATG CACCCAAATT ACCTCAAAAA CTACTCTAAA TTGACAGCAC AGAGAGAAA GATCGGGACC TCCACCCATG CTTCCAGCAG TAAGCAACTG AAAGTTGACT CAGTTTTCCC AGTCTAAGCCAGACAT TCAGTTAA TCATTCAAGG ACTTCATCCT TTCAGCACTG TTGATCTGCC ATCATTTAAA GAGCTGATTA GTACACTGCA GCCTGGCATT TCTGTCATTA CAAGGCCTAC TTTACGCTCC AAGATAGCTG AAGCTGCTCT GATCATGAAA CAGAAAGTG CTGACGACTGACTTG GGATTGTTGG ACTGCACGTA GAAAGTCATT CATTGGTGTA ACTGCTCACT GGATCAACCC TGGAAGTCTT GAAAGACATT CCGCTGCACT TGCCTGCAAAA AGATTAATGG GCTCTCATAC Petition 870260041395, dated 04 / 05 / 2026, pages 139 / 185 132 / 145 TTTTGAGGTA GGTTGTTTGC TGTGGAAAAC TGAAGGCTG CCAAGACGAT TAACCTAGTC CTACAGATCT AGCAGCTGAA GTGGAATTCA AGGGCCATT TATCGATGTA CCTGTAGGTT CAGTTGCAAA TGCCTAGTGT GTGACCCACT AAGATCCTGA ATGATGAAAC TAATCTGGGC TTTTAGGAAT ATTCTTGCAG ATATGTGGTT GATGTAGAT CAGAAGCCCC ACATCAGAGT ATGATGAAGA ATGGATATCT GCAGCCTCTC TCAGCACTGC AGAATCAGCT CATTAGATTG CTGGCCAGTG CCATGAATGA TATCCACTCA GAGTATGAAA TACGTGACAA ACAACCACAG ACAGTGGTTC CAACTTTATG AAGGCTTTCA GAGTTTTTGG AATGATATCG AGACTGAGGC AAGAAGGTGT GAAAGTGATG ACACTGATTC GGTGAGGAGGATTGAGGAGGAGTCGAGTA GATGCCTCAC GAGTCCTGGA GGCTTCGAAT TCCAGCTACC AAAACATCAA AAGTGTGCCT GTCACTTACT TCAAGCGTTG ATGCCCAAAA AGCTCTCTCA AATGAACACT ACAAGAAACT GTCTTTGGCA AATGCCAAGC TTTATGGAAT AAAAGCAGCC GATCGCTGATTGATTGATTGATT GCTTCAGCTT TTAAGGCCAA ACCAAACGCG ACTTTTATGG CTGTTGACAG AATTCTTCAA ATTTGCAAAG AAGCAGGAGA CGGAATATAT GCACCTCTCT TGAGGTTCCA ATGTAAGTGT TTTTCCCCTC AACAAATGTG GGTTGTTTTT GTTTAATACT GCTGATTCATTATGTTATGTT GAAATGCTGT TCTTGACAGA GTGGGCCAAC ACAATGCGTC AGTACTCGAC ATCTTGCAAG CGGAAACGAA TACACAGCTG GGGTGGCTGC CCATCAGTTA AGCTTGAAAC TTCAGCGACT CCACCATTCT CTCAGGTACT TGTGGATGCC CTACCATCAAGGATTGATTCAAG GATCATAGCA GCTGCCATCC TTCTCCCTAA ATTTCGGACC TCTTGGACAA CATCATAAAA CGAGGTAAAT GAATGCAAGC AACATACACT TGACGAATTC AACCTTTGAG CCATACCAAA ATTATTCTTT TATTTATTTA TTTTTGCACT GTTATATCCCATCTTTGGCT GTGATCTCAA TATGAATATT GATGTAAAGT CAGGTTGTAG TTATCCCTCA GTGTTTCTTG AAACCAAACT CATATGTATC TGGAAATGCA GTTAGATTTT ATGCTAAAAT AAGGGATTTG CATGATTTTA ACTGCACGTA AATGTAGTTA ATGACAAAAT CCATAAAATT TGTTCCCAGT TCAACCAAAC TTTTCTTTGT GTCTGCTCAC TGTGCTTGTA GGCATGGACT GCATCTGGAG CCTTTGGACC ACAAGAAGGA ATTGGCCAAC AGTTCATCTG TTTTTTCGCT TCTTTGAAAC CGACAACACA TGAAGCCAGC AAAGAGTTGG GGCCTGTGTT TCAGACACCA GGGAGTCTCT GCTCACGTTT CCTGCTATTT TATCAAGACT AATACACCTC TTCCCGCATC GGCTGCCTGT GAGAGGCTTT AGGATTGCTT TTCAGCCCCA AAAGAGCTAG GCTTGACACT AACAATTTTG TCTACTGAAG TTAAATCTGA GGTTTTACAA CTTTGAGTAG CGTGTACTGG TCTGTCTTAT AGTTTGATAA TTAAATACAA ACAGTTCTAA AGCAGGATAA Petition 870260041395, dated 04 / 05 / 2026, pages 140 / 185 133 / 145 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: 25).

[00169] The DNA insertion must be operatively linked to an appropriate promoter. The expression constructs will also contain transcription initiation sites, termination sites, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs will preferably include early-initiated translation and a termination codon (e.g., UAA, UGA, or UAG) appropriately positioned at the end of the mRNA to be translated, with UAA and UAG preferred for mammalian or eukaryotic cell expression.

[00170] Expression vectors will preferably, but optionally, include at least one selectable marker. Such markers include, for example, but are not limited to, ampicillin, zeocin (Sh bla gene), puromycin (pac gene), hygromycin B (hygB gene), G418 / Geneticin (neo gene), mycophenolic acid or glutamine synthetase (GS, US 5,124,464, 5,770,359, 5,827,739), blasticidin (bsd gene), resistance genes for eukaryotic cell culture, as well as ampicillin, zeocin (gene Petition 870260041395, dated 04 / 05 / 2026, pp. 141 / 185 134 / 145 Sh bla), puromycin (pac gene), hygromycin B (hygB gene), G418 / Geneticin genes (neo gene), kanamycin, spectinomycin, streptomycin, carbenicillin, bleomycin, erythromycin, polymyxin B or tetracycline for culture in E. coli and other bacteria or prokaryotes (the above patents are incorporated herein in their entirety by reference). The appropriate culture media and conditions for the host cells described above are known in the art. Suitable vectors will be readily apparent to those skilled in the art. Introduction of a vector construct into a host cell can be effected by transfection with calcium phosphate, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other known methods. Such methods are described in the art, such as Sambrook, supra, Chapters 1-4 and 16-18; Ausubel, supra, chapters 1, 9, 13, 15, 16.

[00171] Expression vectors will preferably, but optionally, include at least one selectable cell surface marker for isolating cells modified by the disclosure compositions and methods. Selectable disclosure cell surface markers comprise surface proteins, glycoproteins, or groups of proteins that distinguish one cell or subset of cells from another defined subset of cells. From Petition 870260041395, dated 04 / 05 / 2026, pages 142 / 185 135 / 145 preference, the selectable cell surface marker distinguishes cells modified by a composition or method of disclosure from those cells that are not modified by a composition or method of disclosure. Such cell surface markers include, for example, but are not limited to, “designation cluster” or “classification determinant” proteins (often abbreviated as “CD”) such as a truncated or full-length form of CD19, CD271, CD34, CD22, CD20, CD33, CD52 or any combination thereof. Cell surface markers also include the suicide gene marker RQR8 (Philip B et al. Blood. 2014, Aug 21; 124(8): 1277-87).

[00172] Expression vectors will preferably, but optionally, include at least one selectable drug resistance marker for isolation of cells modified by the compositions and methods of disclosure. Selectable drug resistance markers of disclosure may comprise Neo, DHFR, TYMS, FRANCF, RAD51C, GCS, MDR1, ALDH1, NKX2.2, or any combination thereof.

[00173] At least one inducible disclosure polypeptide can be expressed in a modified form, such as a fusion protein, and may include additional heterologous functional regions. For example, a region of additional amino acids, particularly amino acids Petition 870260041395, dated 04 / 05 / 2026, pp. 143 / 185 136 / 145 charged, can be added to the N-terminus of a protein structure to improve stability and persistence in the host cell, during purification or during subsequent handling and storage. Furthermore, peptide fractions can be added to a protein structure to facilitate purification. Such regions can be removed before the final preparation of an inducible polypeptide or at least a fragment thereof. Such methods are described in many standard laboratory manuals, such as Sambrook, supra, Chapters 17.29-17.42 and 18.1-18.74; Ausubel, supra, Chapters 16, 17 and 18.

[00174] Skilled in the art are knowledgeable of the numerous expression systems available for expressing a nucleic acid encoding a release protein. Alternatively, release nucleic acids can be expressed in a host cell by ligation (by manipulation) into a host cell containing a sequence encoding an inducible release polypeptide. Such methods are well known in the art, for example, as described in Pat. 5,580,734, 5,641,670, 5,733,746 and 5,733,761, which are incorporated herein by reference in their entirety.

[00175] Expression vectors for modified cells may include one or more of the following Petition 870260041395, dated 04 / 05 / 2026, pages 144 / 185 137 / 145 expression control sequences, such as, but not limited to, an origin of replication; a promoter (e.g., late or early SV40 promoters, the CMV promoter (US Patents Nos. 5,168,062; 5,385,839), an HSV tk promoter, a pgk (phosphoglycerate kinase) promoter, an EF-1 alpha promoter (Pat. 5267491), at least one human promoter, a stimulator, and / or processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites (e.g., a TPA poly A large A addition site and transcription terminator sequences). See, for example, Ausubel et al., Supra, Sambrook, et al., Supra. Other cells useful for the production of nucleic acids or proteins of the present invention are known and / or available, for example, in the American Type Culture Collection Catalogue Of Cell Lines and Hybridomes (www.atcc.org) or other known or commercial sources.

[00176] When eukaryotic host cells are used, polyadenylation or transcription termination sequences are typically incorporated into the vector. An example of a termination sequence is the polyadenylation sequence of the bovine growth hormone gene. Sequences for precise transcript splicing may also be included. An example of a processing sequence is the VP1 intron of SV40 (Sprague, et al., J. Virol. 45: 773-781 (1983)). Additionally, Petition 870260041395, dated 04 / 05 / 2026, pages 145 / 185 138 / 145 genetic sequences to control replication in the host cell can be incorporated into the vector, as is known in the technique. Amino Acid Codes

[00177] The amino acids that form disclosure protein structures are frequently abbreviated. Amino acid designations may be indicated by designating the amino acid by its single-letter code, its three-letter code, name, or three nucleotide codons as is well known in the art (see Alberts, B., et al., Molecular Biology of the Cell, Third Ed., Garland Publishing, Inc., New York, 1994). An inducible disclosure polypeptide may include one or more amino acid substitutions, deletions, or additions, whether from natural mutations or human manipulation, as specified herein. The amino acids in an inducible disclosure polypeptide that are essential for function may be identified by modes known in the specialty, such as site-directed mutagenesis or alanine scavenging mutagenesis (e.g., Ausubel, supra, Chapters 8, 15; Cunningham and Wells, Science 244: 1081-1085 (1989)).This latter procedure introduces unique alanine mutations at each residue in the molecule. The resulting mutant molecules are then tested for biological activity, such as, but not limited to, at least one neutralizing activity. Critical sites for... Petition 870260041395, dated 04 / 05 / 2026, pp. 146 / 185 139 / 145 function (i.e., induction of apoptosis) can also be identified by structural analyses, such as crystallization, resonance, or photoaffinity labeling (Smith, et al., J. Mol. Biol. 224: 899-904 (1992) and de Vos et al., Science 255: 306-312 (1992)).

[00178] A biologically active inducible disclosure polypeptide includes one or more proteins or enzymes (e.g., a caspase such as caspase 9) capable of inducing apoptosis in a cell with an efficacy that is at least 20%, 30%, or 40%, and preferably at least 50%, 60%, or 70%, and more preferably at least 80%, 90%, or 95%–99% or more effective as expression or induction of a known, cell-native (non-synthetic), endogenous or related protein or enzyme. The assay and quantification methods for protein binding and enzymatic activity are well known to those skilled in the art. Infusion of Modified Cells as Adoptive Cell Therapy

[00179] Disclosure provides modified cells expressing one or more inducible disclosure polypeptides that have been selected for administration to an individual in need thereof. Modified disclosure cells can be formulated for storage at any temperature, including room temperature and body temperature. Modified disclosure cells Petition 870260041395, dated 04 / 05 / 2026, pages 147 / 185 140 / 145 can be formulated for cryopreservation and subsequent thawing. Modified disclosure cells can be formulated in a pharmaceutically acceptable carrier for direct administration to a sterile packaged individual. EXAMPLES Example 1: Expression and Function of the iC9 safety switch integrated into piggyBac in human pan T cells

[00180] Human pan T cells were nucleofected using an Amaxa 4D nucleofector with one of four piggyBac transposons. Modified T cells that received the mock condition were nucleofected with an empty piggyBac transposon. Modified T cells received either a piggyBac transposase containing a therapeutic agent alone (a sequence encoding a CARTirin) or a piggyBac transposase containing an integrated iC9 sequence and a therapeutic agent (a sequence encoding a CARTirin).

[00181] Figure 1 provides a schematic diagram of the iC9 safety switch, which contains a ligand-binding region, a ligand, and a truncated caspase 9 polypeptide. Specifically, the iC9 polypeptide contains a ligand-binding region comprising an FK506-binding protein 12 polypeptide (FKBP12) including a valine (V) to phenylalanine (F) substitution at position 36 (F36V). The FKBP12 polypeptide of the iC9 polypeptide is encoded by an amino acid sequence comprising Petition 870260041395, dated 04 / 05 / 2026, pages 148 / 185 141 / 145 GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGW EEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE (SEQ ID NO: 3). The FKBP12 polypeptide of the iC9 polypeptide is encoded by a nucleic acid sequence comprising GGGGTCCAGGTCGAGACTATTTCACCAGGGGATGGGCGAACATTTCCAAAAAGGGGCCA GACTTGCGTCGTGCATTACACCGGGATGCTGGAGGACGGGAAGAAAGTGGACAGCTCCA GGGATCGCAACAAGCCCTTCAAGTTCATGCTGGGAAAGCAGGAAGTGATCCGAGGATGG GAGGAAGGCGTGGCACAGATGTCAGTCGGCCAGCGGGCCAAACTGACCATTAGCCCTGA CTACGCTTATGGAGCAACAGGCCACCCAGGGATCATTCCCCCTCATGCCACCCTGGTCT TCGAT GTGGAACTGCTGAAGCTGGAG (SEQ ID NO: 4). The iC9 polypeptide binding region is encoded by an amino acid comprising GGGGS (SEQ ID NO: 5) and a nucleic acid sequence comprising GGAGGAGGAGGATCC (SEQ ID NO: 6). The amino acid sequence encoding the IC9 polypeptide is encoded by an amino acid comprising GFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRES GLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVI LSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKD HGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFV SWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKL FFKTS (SEQ ID NO: 7). The nucleic acid sequence encoding the iC9 polypeptide is encoded by a nucleic acid sequence comprising TTTGGGGACGTGGGGGCCCTGGAGTCTCTGCGAGGAAATGCCGATCTGGCTTACATCCT GAGCATGGAACCCTGCGGCCACTGTCTGATCATTAACAATGTGAACTTCTGCAGAGAAA Petition 870260041395, dated 04 / 05 / 2026, pages 149 / 185 142 / 145 GCGGACTGCGAACACGGACTGGCTCCAATATTGACTGTGAGAAGCTGCGGAGAAGGTTC TCTAGTCTGCACTTTATGGTCGAAGTGAAAGGGGATCTGACCGCCAAGAAAATGGTGCT GGCCCTGCTGGAGCTGGCTCAGCAGGACCATGGAGCTCTGGATTGCTGCGTGGTCGTGA TCCTGTCCCACGGGTGCCAGGCTTCTCATCTGCAGTTCCCCGGAGCAGTGTACGGAACA GACGGCTGTCCTGTCAGCGTGGAGAAGATCGTCAACATCTTCAACGGCACTTCTTGCCC TAGTCTGGGGGGAAAGCCAAAACTGTTCTTTATCCAGGCCTGGTGGCGGGGAACAGAAAG ATCACGGCTTCGAGGTGGCCAGCACCAGCCCTGAGGACGAATCACCAGGGAGCAACCCT GAACCAGATGCAACTCCATTCCAGGAGGGACTGAGGACCTTTGACCAGCTGGATGCTAT CTCAAGCCTGCCCACTCCTAGTGACATTTTCGTGTCTTACAGTACCTTCCCAGGCTTTG TCTCATGGCGCGATCCCAAGTCAGGGAGCTGGTACGTGGAGACACTGGACGACATCTTT GAACAGTGGGCCCATTCAGAGGACCTGCAGAGCCTGCTGCTGCGAGTGGCAAACGCTGT CTCTGTGAAGGGCATCTACAAACAGATGCCCGGGTGCTTCAATTTTCTGAGAAAGAAAC TGTTCTTTAAGACTTCC comprising nucleic (SEQ ID NO: 8).

[00182] To test the iC9 security key, each of the four modified T cells was incubated for 24 hours with 0, 0.1 nM, 1 nM, 10 nM, 100 nM, or 1000 nM AP1903 (an induction agent for AP1903, also known as Rimiducid). Viability was assessed by flow cytometry using 7-aminoactinomycin D (7-AAD), a fluorescent intercalator, as a marker for cells that had undergone apoptosis.

[00183] Cell viability was assessed on day 12 (see Figure 2). The data demonstrated a shift in frequency from the upper right quadrant to the upper left quadrant, with a strong influence of the induction agent on the cells. Petition 870260041395, dated 04 / 05 / 2026, pages 150 / 185 143 / 145 containing the iC9 construct; however, this effect is not observed in cells without the iC9 construct (those receiving only CARTirin), however, the cells are uniformly distributed between the two areas, regardless of the concentration of the inducing agent. Furthermore, cell viability was assessed on day 19 (see Figure 3). The data reveal a similar trend shown in Figure 2 (day 12 post-nucleofection); currently, the population momentum towards the upper left quadrant is more pronounced at this later time point (day 19 post-nucleofection).

[00184] A quantification of the aggregated results was performed and is provided in Figure 4, showing the significant impact of the safety switch on a percentage of cell viability as a function of the concentration of the iC9 switch induction agent (AP1903, also known as Rimiducid) for each modified cell type either on day 12 (Figure 2 and graph on the left) or on day 19 (Figure 3 and graph on the right). The presence of the iC9 safety switch induces apoptosis in a significant majority of cells on day 12 and the effect is even more dramatic on day 19.

[00185] The results of this study show that the iC9 safety switch is extremely effective in eliminating live cells by contact with an agent. Petition 870260041395, dated 04 / 05 / 2026, pages 151 / 185 144 / 145 induction (e.g., AP1903, also known as Rimiducid) because AP1903 (Rimiducid) induces apoptosis even at the lowest concentrations in the study (0.1 nM). Furthermore, the iC9 safety switch can be functionally expressed as part of a tricistronic vector. Example 2: Highly efficient killing of cells comprising P-BCMA-101 using the iC9 safety switch in NGS mice in vivo.

[00186] NSG mice were injected IV with MM.1S / luciferase+ cells, prepared on day 8, injected with T cells on day 9, and treated with AP1903 (Rimiducid) on day 12 at the indicated doses. 24 hours later, the mice were euthanized and blood, spleen, and bone marrow cells were collected and stained for the presence of huCD45+ cells (Figure 5). Blood, spleen, and bone marrow cells were analyzed by flow cytometry for the presence of huCD45+ cells. Relative viability was determined by dividing the number of huCD45 cells by the number of msCD45 cells and normalizing the mean huCD45 / msCD45 at the time points of the untreated group, 100% by 1500 counting events for each sample. Each data point represents a different mouse (Figure 6). Incorporation by Reference

[00187] All documents cited herein, including any patent or application referenced or related to Petition 870260041395, dated 04 / 05 / 2026, pages 152 / 185 145 / 145, are incorporated herein by reference in their entirety, unless expressly excluded or otherwise limited. Citation of any document is not an admission that it is prior art with respect to any invention disclosed or claimed herein or that it alone, or in any combination with any other reference or references, teaches, suggests or discloses any such invention. Furthermore, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition ascribed to that term in this document shall prevail. OTHER MODALITIES

[00188] Although particular embodiments of the disclosure have been illustrated and described, various other changes and modifications may be made without departing from the spirit and scope of the disclosure. The scope of the appended claims includes all changes and modifications that are within the scope of this disclosure. Petition 870260041395, dated 04 / 05 / 2026, pp. 153 / 185

Claims

1 / 10 CLAIMS 1. Polynucleotide encoding an inducible caspase polypeptide characterized in that it comprises: (a) a nucleic acid sequence encoding a ligand-binding region, wherein the ligand-binding region comprises a FK506-binding protein 12 (FKBP12) polypeptide, wherein the FKBP12 polypeptide comprises the amino acid sequence of SEQ ID NO: 3; (b) a nucleic acid sequence encoding a ligand, wherein the ligand comprises the amino acid sequence of SEQ ID NO: 5; and (c) a nucleic acid sequence encoding a truncated caspase 9 polypeptide, wherein the truncated caspase 9 polypeptide comprises the amino acid sequence of SEQ ID NO:

7.

2. Polynucleotide, according to claim 1, characterized in that the nucleic acid sequence encoding the ligand-binding region comprises the nucleic acid sequence of SEQ ID NO: 4; Petition 870260041395, dated 04 / 05 / 2026, page 154 / 185 2 / 10 the nucleic acid sequence encoding the ligand comprises the nucleic acid sequence of SEQ ID NO: 6; and the nucleic acid sequence encoding the truncated caspase 9 polypeptide comprises the nucleic acid sequence of SEQ ID NO:

8.

3. Inducible caspase polypeptide characterized in that it comprises: (a) a ligand-binding region, wherein the ligand-binding region comprises an FKB12 ​​polypeptide, wherein the FKB12 ​​polypeptide comprises the amino acid sequence of SEQ ID NO: 3; (b) a linker comprising the amino acid sequence of SEQ ID NO: 5; and (c) a truncated caspase 9 polypeptide comprising the amino acid sequence of SEQ ID NO:

7.

4. Polypeptide, according to claim 3, characterized in that the polypeptide comprises the amino acid sequence of SEQ ID NO:

9.

5. Polynucleotide, according to claim 1, characterized in that the polynucleotide comprises Petition 870260041395, dated 04 / 05 / 2026, page 155 / 185 3 / 10 the nucleic acid sequence of SEQ ID NO:

10.

6. Transposon characterized in that it comprises the polynucleotide as described in any one of claims 1, 2 and 5.

7. Transposon, according to claim 6, characterized in that it further comprises a nucleic acid sequence encoding a therapeutic protein.

8. Transposon, according to claim 7, characterized in that the therapeutic protein comprises an antigen receptor, a cell surface protein, a membrane-bound protein, an extracellular membrane-bound protein, an intracellular membrane-bound protein, an intracellular protein, a nuclear protein, a nuclear protein, a cytoplasmic protein, a cytosolic protein, a secreted protein, a lysosomal protein, an endosomal protein, a vesicle-associated protein, a mitochondrial protein, an endoplasmic reticulum protein, a cytoskeletal protein, a protein involved in intracellular signaling and / or a protein involved in extracellular signaling or any combination thereof.

9. Transposon, according to claim 8, characterized in that the antigen receptor comprises a T-cell receptor (TCR) or a chimeric antigen receptor (CAR).

10. Transposon, according to claim 9, characterized in that CAR comprises one or more Centirin sequences or CAR comprises one or more VHH sequences.

11. Transposon, according to any one of claims 6 to 10, characterized in that the transposon is a piggyBac transposon, a Sleeping Beauty transposon, a Helraiser transposon, or a Tol2 transposon.

12. Composition characterized by comprising the transposon as described in any one of claims 6 to 11, wherein the transposon is a piggyBac transposon, a Sleeping Beauty transposon, a Helraiser transposon or a Tol2 transposon; and wherein the composition further comprises a plasmid Petition 870260041395, dated 04 / 05 / 2026, page 157 / 185 5 / 10 comprising a sequence encoding a transposase enzyme, wherein the transposase is a piggyBac transposase, a Super piggyBac transposase (SPB), a Sleeping Beauty transposase, a hyperactive Sleeping Beauty transposase (SB100X), a Helitron transposase or a Tol2 transposase.

13. Vector characterized by comprising the polynucleotide as described in any one of claims 1, 2 and 5.

14. Vector, according to claim 13, characterized in that the vector is either a viral vector or a nanoparticle vector.

15. Vector, according to claim 14, characterized in that the viral vector comprises an isolated or derived sequence of a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus (AAV), or any combination thereof.

16. Cell characterized by comprising the polynucleotide as described in any one of claims 1, 2 and 5. Petition 870260041395, dated 04 / 05 / 2026, pp. 158 / 185 6 / 10 17. Cell characterized in that it comprises the Transposon as described in any one of claims 6 to 11.

18. A cell characterized by comprising the polypeptide as described in any one of claims 3 to 4.

19. A cell characterized in that it comprises the vector as described in any one of claims 13 to 15.

20. A cell, according to any one of claims 16 to 19, characterized in that the cell is a human cell, an immune cell, an artificial antigen-presenting cell, a stem cell, or any combination thereof.

21. A cell, according to any one of claims 16 to 20, characterized in that the cell is an isolated somatic cell or derived from a human heart; skeletal or smooth muscle; blood vessel, vein or capillary; spleen; thyroid; lymph node or lymphatic vessel; bone or bone marrow; skin or endothelium; adrenal gland; esophagus; larynx; brain or spinal cord; peripheral nervous system; eye; hypothalamus; liver; olfactory tissue; prostate; stomach; large or small intestine; lung or bronchi; kidney; pancreas; thymus gland; ureter or urethra; bladder; auditory tissue; bladder; parathyroid gland; salivary gland; or trachea.

22. Composition characterized by comprising the cell as described in any one of claims 16 to 21.

23. Use of the composition as described in claim 22, characterized by the fact that it is in the manufacture of a medicament for an adoptive cell therapy or for use in an ex vivo gene therapy.

24. Use according to claim 23, characterized in that the cell is autologous.

25. Use according to claim 23, characterized in that the cell is allogeneic.

26. Use of the composition as defined in claim 22 characterized by the fact that it is used in the manufacture of a medicament to modify a cell therapy in an individual in need thereof, wherein apoptosis can be selectively induced in the cell by contact of the cell with an inducing agent.

27. Use according to claim 26, characterized in that the modification is a termination of cell therapy or is a depletion of a portion of the cells supplied in cell therapy.

28. Use, according to claim 25 or 26, characterized in that the medicament further comprises an inhibitor of the induction agent to inhibit the modification of cell therapy, thereby restoring the function and / or efficacy of cell therapy.

29. Use of the composition as defined in claim 22 characterized by being in the manufacture of a medicament for treating a disease or disorder in an individual in need thereof, wherein the cell further comprises a kinetic agent, wherein apoptosis can be selectively induced in the cell by contact of the cell with an inducing agent, and wherein the cell comprising the kinetic agent induces local tissue toxicity within a target tissue of the individual, and wherein apoptosis in the cell is selectively induced in the cell comprising the kinetic agent prior to the induction of significant toxicity in a non-target tissue of the individual, thereby eliminating the target tissue, preserving the non-target tissue and treating the disease or disorder in the individual.

30. Use, according to claim 29, characterized in that the disease or disorder is a proliferative disorder, cancer, an inflammatory disease or disorder, an immune or autoimmune disease or disorder, an infectious disease or disorder, a genetic or epigenetic disease or disorder, a metabolic disorder, a vascular disorder, a respiratory disorder, or a fibrotic disorder.

31. A method of ex vivo genetic modification of a cell therapy characterized in that it comprises contacting the cells of an individual with cells as defined in claims 16 to 21 or the composition as defined in claim 22, wherein the cell as defined in claims 16 to 21 selectively induces apoptosis in target cells before inducing toxicity in non-target cells, thereby treating the target tissue, preserving the non-target tissue, and treating the disease or disorder in the individual.

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