Chimeric transposase, transposons, polynucleotides, vectors, fusion protein, methods of integrating a transgene, for site-specific transposition of a DNA molecule and for generating a cell engineered by site-specific transposition.

Description

1 / 102 “CHIMERIC TRANSPOSASE, TRANSPOSONS, POLYNUCLEOTIDES, VECTORS, FUSION PROTEIN, METHODS OF TRANSGENE INTEGRATION, FOR SITE-SPECIFIC TRANSPOSITION OF A DNA MOLECULE AND FOR GENERATING AN ENGINEERED CELL BY SITE-SPECIFIC TRANSPOSITION” Cross-reference to related orders

[001] This application claims the benefit of Provisional Patent Application No. US 63 / 494,297, filed April 5, 2023, which is incorporated herein by reference in its entirety. Reference to the Sequence Listing Submitted Electronically

[002] The instant request contains a Listing of The sequence was sent in XML format via the Patent Office and is incorporated into this document in its entirety by reference. The XML copy, created on March 27, 2024, is named “POTH080_001WO_SeqList” and is 233,942 bytes in size. Field

[003] This disclosure refers generally to chimeric transposases, in particular chimeric transposases, chimeric site-specific transposase fusion proteins comprising chimeric transposases and DNA targeting domains, and chimeric transposon inverted repeat (ITR) polynucleotides. Methods of using chimeric site-specific transposase fusion proteins for site-specific transposition are also provided. Background

[004] Transposases can be used to introduce non-endogenous DNA sequences into genomic DNA and are, in many ways, advantageous compared to other gene editing methods. However, there is still an unmet need for site transposases. Petition 870250091089, dated 06 / 10 / 2025, page 89 / 198 2 / 102 specifically for use, for example, in gene editing.

[005] In their natural form, the PiggyBac transposon and related family members harbor a single open reading frame that encodes their respective transposase. Inverted terminal repeat (ITR) sequences are present at both ends of the transposons and act as binding sites for transposases. Immediately outside the ITRs, a four-nucleotide sequence, TTAA, acts as the cleavage site, allowing transposons to jump from one site in the genome to another TTAA site via a cut-and-paste mechanism.

[006] When used for genome editing purposes, the transposase is often removed from the transposon and expressed from a separate plasmid or mRNA. This leaves an empty transposon where a “payload” or “cargo” of interest, flanked by the ITRs and TTAA sequences, can be encoded. This synthetic transposon can be delivered to cells along with the transposase, where the transposase catalyzes the transposition of the transposon to a TTAA site in the cell's genome.

[007] The PiggyBac transposase and related family members contain two distinct DNA-binding domains that interact with the Inverted Terminal (ITR) sequences of the transposon. The DNA-binding and dimerizing domain (DDBD) binds to the sequence proximal to the TTAA sites flanking the transposon, while the Cysteine-Rich Domain (CRD) at the C-terminus of the protein binds to the Repeat (ITR) sequence distal to the TTAA sites. After transposase dimerization, the DDBDs bind symmetrically to the ITRs, with the DDBD of one monomer binding to the left end (LE) of the ITR and the DDBD of the second monomer binding to the right end (RE) of the ITR. Interestingly, the two CRDs of the dimer bind asymmetrically, both binding to the LE ITR. Based on the identity of the ITR sequence, it was proposed that a second dimer of the piggyBac transposase Petition 870250091089, dated 06 / 10 / 2025, p. 90 / 198 3 / 102 can bind distally to the first dimer. Both CRDs of this second dimer have predicted binding sites within the ITR ER. While the proximal dimer catalyzes the transposition reaction, the role of the distal dimer remains unclear.

[008] The asymmetric linkage of the two CRD sequences of piggyBac transposase is not a feature of all transposase families. For example, Mosl, a member of the mariner / Td transposase family, interacts with its ITRs as a symmetric dimer, as observed in its crystal structure (see, for example, Morris et al., eLife May 25, 2016;5:e15537. doi: 10.7554 / eLife.15537). A transposase that functions as a symmetric dimer may have several beneficial features compared to a transposase that functions as an asymmetric dimer pair (e.g., piggyBac transposase), including shorter ITR sequences and fewer transposase molecules needed to complete the transposition. Brief Description

[009] In one aspect, a chimeric transposase is provided in this document comprising, in N-terminal to C-terminal order: (i) a target-specific DNA-binding domain, (ii) a truncated Super PiggyBac (SPB) transposase comprising a C-terminal Cysteine-Rich Domain (CRD) deletion within amino acid residues 535–594 of the SPB sequence comprising the sequence established in SEQ ID NO: 1, and (iii) one or more Mosl DNA-binding domains. In some embodiments, the truncated PB transposase of (i) comprises the sequence established in any of the SEQ ID NOs: 68–84. In some embodiments, the truncated PB transposase comprises one or more hyperactive mutations. In some embodiments, one or more hyperactive mutations are a selected amino acid substitution from I30V, S103P, G165S, M226F, M282V, S509G, N538K, or N571S of SEQ ID NO: 1. Petition 870250091089, dated 06 / 10 / 2025, page 91 / 198 4 / 102

[0010] In some embodiments, the PB transposase comprising the CRD deletion additionally comprises an in-frame N-terminal nuclear localization (NLS) sequence comprising the amino acid sequence at SEQ ID NO: 39. In some embodiments, the chimeric transposase comprises two MosI DNA-binding domains. In some embodiments, the two MosI DNA-binding domains comprise the amino acid sequence established at SEQ ID NO: 6. In some embodiments, the chimeric transposase comprises one MosI DNA-binding domain. In some embodiments, the MosI DNA-binding domain comprises the amino acid sequence established at SEQ ID NO: 8.

[0011] In some embodiments, the PB transposase and one or more MosI DNA-binding domains are connected by a linker sequence. In some embodiments, the linker sequence is GGGGS (SEQ ID NO: 86). In some embodiments, the target-specific DNA-binding domain is a zinc finger domain or a TAL array.

[0012] In another aspect, a polynucleotide comprising a nucleic acid sequence encoding a chimeric transposase described in this document is provided in this document.

[0013] In another aspect, a vector comprising a polynucleotide provided in this document is provided in this document.

[0014] In another aspect, a chimeric transposon inverted terminal repeat (ITR) polynucleotide is provided in this document, comprising in the 5' to 3' direction: (i) a polynucleotide comprising nucleotides 1-16 of the nucleic acid sequence established in SEQ ID NO: 12 and (ii) a polynucleotide comprising two MosI DNA binding sites. In some embodiments, the polynucleotide comprising two MosI DNA binding sites is fused in the reverse orientation. In some embodiments, the polynucleotide comprising two MosI DNA binding sites Petition 870250091089, dated 06 / 10 / 2025, p. 92 / 198 5 / 102 comprises the nucleic acid sequence with SEQ ID NOs: 13 or 14. In some embodiments, the ITR polynucleotide of the chimeric transposon comprises the nucleic acid sequence with SEQ ID NOs: 15 or 16.

[0015] In some embodiments, the ITR polynucleotide of the chimeric transposon additionally comprises one or two additional nucleotides between the polynucleotide comprising nucleotides 1-16 of the nucleic acid sequence established in SEQ ID NO: 12 and the polynucleotide comprising two MosI binding sites. In some embodiments, the ITR polynucleotide of the chimeric transposon comprises the nucleic acid sequence of SEQ ID NO: 17. In some embodiments, the ITR polynucleotide of the chimeric transposon comprises the nucleic acid sequence of SEQ ID NO: 18.

[0016] In another aspect, a vector comprising a chimeric transposon ITR polynucleotide is provided in this document.

[0017] In another aspect, a transposon comprising a chimeric transposon ITR polynucleotide described in this document is provided herein.

[0018] In another aspect, a chimeric transposon ITR polynucleotide is provided in this document, comprising in the 5' to 3' direction: (i) a polynucleotide comprising nucleotides 1-16 of the nucleic acid sequence established in SEQ ID NO: 65 and (ii) a polynucleotide comprising two MosI DNA binding sites. In some embodiments, the polynucleotide comprising two MosI DNA binding sites is fused in the reverse orientation. In some embodiments, the polynucleotide comprising two MosI DNA binding sites comprises the nucleic acid sequence of SEQ ID NOs: 13 or 14. In some embodiments, the chimeric transposon ITR polynucleotide Petition 870250091089, dated 06 / 10 / 2025, p. 93 / 198 6 / 102 comprises the nucleic acid sequence of SEQ ID NOs: 19 or 20. In some embodiments, the ITR polynucleotide of the chimeric transposon additionally comprises one or two additional nucleotides between the polynucleotide comprising nucleotides 1-16 of the nucleic acid sequence established in SEQ ID NO: 65 and the polynucleotide comprising two MosI DNA binding sites. In some embodiments, the ITR polynucleotide of the chimeric transposon comprises the nucleic acid sequence of SEQ ID NOs: 21 or 22.

[0019] In another aspect, a vector comprising a chimeric transposon ITR polynucleotide described in this document is provided in this document.

[0020] In another aspect, a transposon comprising a chimeric ITR transposon polynucleotides described in this document is provided herein.

[0021] In another aspect, a chimeric site-specific transposase fusion protein is provided in this document comprising, in N-terminal to C-terminal order: (i) a target-specific DNA-binding domain, (ii) a piggyBac transposase comprising an N-terminal deletion relative to SEQ ID NO: 1, one or more integration-deficient mutations relative to SEQ ID NO: 1, and a C-terminal Cysteine-Rich Domain (CRD) deletion at residues 535–594 of the amino acid sequence SEQ ID NO: 1; and (iii) one or more MosI DNA-binding domains. In some embodiments, the target-specific DNA-binding domain is a TAL or a zinc finger motif (ZFM).

[0022] In some embodiments, the truncated PB transposase comprises the sequence established in any of the SEQ ID NOs: 6884. In some embodiments, the N-terminal deletion of the truncated PB transposase is a deletion of amino acid residues 1-85, 1-86, 1-87, 1-88, 1-89, 1-90, Petition 870250091089, dated 06 / 10 / 2025, pp. 94 / 198 7 / 102 1-91, 1-92, 1-93, 1-94, 1-95, 1-96, 1-97, 1-98, 1-99, 1-100, 1-101, 1-102 or 1-103 of SEQ ID NO: 1. In some embodiments, the N-terminal deletion of the truncated PB transposase is a deletion of amino acid residues 1-93. In some embodiments, the PB transposase comprises one or more hyperactive mutations. In some embodiments, one or more hyperactive mutations are an amino acid substitution selected from G165S, M226F, M282V, and N538K of SEQ ID NO: 1 with numbering starting at residue 5. In some embodiments, one or more mutations deficient in the integration of the truncated PB transposase are an amino acid substitution selected from R372A, K375A, and D450N of SEQ ID NO: 1. In some embodiments, the N-terminal deleted truncated PB transposase comprises the amino acid sequence of SEQ ID NO: 85.

[0023] In some embodiments, the PB transposase comprising a C-terminal Cysteine-Rich Domain (CRD) deletion additionally comprises an N-terminal in-frame nuclear localization sequence (NLS), wherein the NLS comprises the amino acid sequence set forth in SEQ ID NO: 39. In some embodiments, the chimeric site-specific transposase fusion protein comprises two MosI DNA-binding domains. In some embodiments, the two MosI DNA-binding domains comprise the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, the chimeric site-specific transposase fusion protein comprises a MosI DNA-binding domain. In some embodiments, the MosI DNA-binding domain comprises the amino acid sequence set forth in SEQ ID NO: 8.

[0024] In some embodiments, the PB transposase and one or more MosI DNA-binding domains are connected by a linker sequence. In some embodiments, the linker sequence is GGGGS (SEQ ID NO: 86). Petition 870250091089, dated 06 / 10 / 2025, pp. 95 / 198 8 / 102

[0025] In some embodiments, the target-specific DNA-binding domain is a zinc finger domain or a TAL array.

[0026] In another aspect, a chimeric site-specific transposase fusion protein targeting LINE1 is provided in this document, comprising, in the N-terminal to C-terminal direction, (i) a DNA-binding domain targeting left or right LINE1 TAL, (ii) a binding sequence, and (iii) a chimeric PB:MosI transposase. In some embodiments, the chimeric PB:MosI transposase comprises the amino acid sequence set forth in SEQ ID NOs: 9 or 7. In some embodiments, the DNA-binding domain targeting left or right LINE1 TAL comprises the amino acid sequence set forth in SEQ ID NOs: 66 or 67.

[0027] In another aspect, a polynucleotide comprising a nucleic acid sequence encoding a chimeric site-specific transposase fusion protein described in this document is provided herein.

[0028] In another aspect, a vector comprising a nucleic acid sequence encoding a chimeric site-specific transposase fusion protein described in this document is provided herein.

[0029] In another aspect, a transposon is provided in this document, comprising (i) a chimeric polynucleotide LE PB:MosI ITR and (ii) a chimeric polynucleotide RE PB:MosI ITR, wherein the polynucleotide LE PB:MosI ITR and a polynucleotide RE PB:MosI ITR comprise the same nucleic acid sequence. In some embodiments, the chimeric polynucleotide LE PB:MosI ITR and the chimeric polynucleotide RE PB:MosI ITR each comprise the nucleic acid sequence of any of the SEQ ID NOs: 15-22.

[0030] In another aspect, this document provides a Petition 870250091089, dated 06 / 10 / 2025, p. 96 / 198 9 / 102 transposon, comprising (i) a chimeric polynucleotide LE PB:MosI ITR comprising the nucleic acid sequence SEQ ID NO: 54 and (ii) a chimeric polynucleotide RE PB:MosI ITR comprising the nucleic acid sequence SEQ ID NO: 55.

[0031] In another aspect, a transposon comprising the nucleic acid sequence of SEQ ID NO: 56 is provided in this document.

[0032] In another aspect, a method for integrating a transgene into a genomic target site of a cell is provided in this document, the method comprising introducing into the cell a chimeric transposase disclosed in this document and a transposon, wherein the transposon comprises, in the order of 5' to 3': a 5'ITR, the transgene and a 3'ITR, wherein the 5'ITR is a terminal inverted repeat (ITR) of a chimeric transposon described in this document and the 3'UTR is a terminal inverted repeat (ITR) of a chimeric transposon described in this document. In some embodiments, the inverted terminal repeat of the chimeric transposon 5'ITR comprises the nucleic acid sequence established at SEQ ID NO: 15. In some embodiments, the inverted terminal repeat of the chimeric transposon 3'ITR comprises the nucleic acid sequence established at SEQ ID NO: 21. In some embodiments, the transposon additionally comprises an exogenous promoter between ITR 5' and the transgene.

[0033] In some embodiments, the transgene encodes a detectable marker. In some embodiments, the detectable marker is GFP.

[0034] In some embodiments, the genomic target site is located on a repetitive element. In some embodiments, the repetitive element is a LINE element.

[0035] In another aspect, a method for site-specific transposition of a DNA molecule into the genome of a cell is provided in this document, comprising the introduction into the cell of: (a) a nucleic acid Petition 870250091089, dated 06 / 10 / 2025, pp. 97 / 198 10 / 102 encoding a chimeric site-specific transposase fusion protein comprising a DNA-binding domain and a chimeric transposase comprising, in N-terminal to C-terminal order: a piggyBac transposase comprising a C-terminal Cysteine-Rich Domain (CRD) deletion and one or more MosI DNA-binding domains; wherein the fusion protein is expressed in the cell; and (b) a DNA molecule comprising a transposon comprising a chimeric transposon ITR polynucleotide LE and a chimeric transposon ITR polynucleotide RE; wherein the expressed chimeric site-specific transposase fusion protein integrates the transposon by site-specific transposition into the TTAA sequence of the cellular genome.

[0036] In another aspect, a method is provided in this document for generating a site-specific transposition engineered cell, comprising introducing into the cell: (a) a nucleic acid encoding a chimeric site-specific transposase fusion protein comprising a DNA-binding domain and a chimeric transposase comprising, in N-terminal to C-terminal order: a piggyBac transposase comprising a C-terminal Cysteine-Rich Domain (CRD) deletion and one or more MosI DNA-binding domains; wherein the chimeric site-specific transposase fusion protein is expressed in the cell; and (b) a DNA molecule comprising a transposon comprising a chimeric transposon ITR polynucleotide LE and a chimeric transposon ITR polynucleotide RE;in which the chimeric site-specific transposase fusion protein integrates the transposon via site-specific transposition into the TTAA sequence in the cell genome, thus generating the engineered cell. Detailed Description

[0037] This document provides chimeric transposases and site-specific chimeric fusion proteins, in particular piggyBac:MosI (PB:MosI) chimeric transposases and fusion proteins. Petition 870250091089, dated 06 / 10 / 2025, pp. 98 / 198 11 / 102 site-specific chimeric PB:MosI proteins, polynucleotides encoding chimeric transposases and site-specific chimeric fusion proteins, and vectors and transposons comprising the polynucleotides. Methods for producing chimeric transposases and chimeric fusion proteins, cells that are modified using chimeric transposases or site-specific chimeric fusion proteins provided in this document, and methods using such cells are also provided.

[0038] Chimeric transposon inverted terminal repeat (ITR) sequences are also provided in this document. In some embodiments, the chimeric transposon inverted terminal repeat (ITR) sequences are left-end chimeric PB:MosI transposon inverted terminal repeat (ITR) sequences. In some embodiments, the chimeric transposon inverted terminal repeat (ITR) sequences are right-end chimeric PB:MosI transposon inverted terminal repeat (ITR) sequences. Transposons comprising the left-end and right-end chimeric ITRs and methods for using the transposons comprising the left-end and right-end chimeric ITR sequences provided in this document are also provided in methods for transposing cells and using them. piggyBac:MosI chimeric transposases

[0039] In one aspect, chimeric transposases are provided in this document comprising, in N-terminal to C-terminal order: a piggyBac transposase comprising a C-terminal Cysteine-Rich Domain (CRD) deletion and one or more MosI DNA-binding domains.

[0040] An exemplary sequence of a wild-type PB transposase is shown in SEQ ID NO: 1: Petition 870250091089, dated 06 / 10 / 2025, pp. 99 / 198 12 / 102 MGSSLDDEHILSALLQSDDELVGEDSDSEISDHVSEDDVQSDTE EAFIDEVHEVQPTSSGSEILDEQNVIEQPGSSLASNRILTLPQRTIRGKNKHCW STSKSTRRSRVSALNIVRSQRGPTRMCRNIYDPLLCFKLFFTDEIISEIVKWTNA EISLKRRESMTGATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLSM VYVSVMSRDRFDFLIRCLRMDDKSIRPTLRENDVFTPVRKIWDLFIHQCIQNYT PGAHLTIDEQLLGFRGRCPFRMYIPNKPSKYGIKILMMCDSGTKYMINGMPYL GRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSIPLAKNLLQEPYKLT IVGTVRSNKREIPEVLKNSRSRPVGTSMFCFDGPLTLVSYKPKPAKMVYLLSS CDEDASINESTGKPQMVMYYNQTKGGVDTLDQMCSVMTCSRKTNRWPMAL LYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMSLTSSFMRKRLEAPT LKRYLRDNISNILPNEVPGTSDDSTEEPVMKKRTYCTYCPSKIRRKANASCKK CKKVICREHNIDMCQSCF (SEQ ID NO: 1)

[0041] In one aspect, chimeric transposases are provided in this document comprising, in N-terminal to C-terminal order: (i) a piggyBac transposase comprising a deletion of the C-terminal Cysteine-Rich Domain (CRD) and (ii) one or more MosI DNA-binding domains. In some embodiments, the PB CRD consists of residues 553–594 of the 594-amino acid PB transposase protein (i.e., in some embodiments, the PB CRD comprises the amino acid sequence set forth in SEQ ID NO: 2). The CRD domain may be attached to the remainder of the N-terminal transposase via a linker sequence. In some embodiments, the linker sequence spans residues 535–552 of the PB transposase sequence (including residues 535 and 552). In some embodiments, the linker comprises the amino acid sequence ILPNEVPGTSDDSTEEPV (SEQ ID NO: 171). In some embodiments, the linker comprises the amino acid sequence ILP KEVPGTS DDSTEE PV (SEQ ID NO: 174).

[0042] In some embodiments, the C-terminal CRD deletion of piggyBac transposase is a deletion of C-terminal amino acid residues. Petition 870250091089, dated 06 / 10 / 2025, pages 100 / 198 13 / 102 545-594 of the amino acid sequence of the PB transposase established in SEQ ID NO: 1. In such embodiments, the resulting truncated piggyBac transposase comprises the amino acid sequence: MGSSLDDEHILSALLQSDDELVGEDSDSEISDHVSEDDVQSDTE EAFIDEVHEVQPTSSGSEILDEQNVIEQPGSSLASNRILTLPQRTIRGKNKHCW STSKSTRRSRVSALNIVRSQRGPTRMCRNIYDPLLCFKLFFTDEIISEIVKWTNA EISLKRRESMTGATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLSM VYVSVMSRDRFDFLIRCLRMDDKSIRPTLRENDVFTPVRKIWDLFIHQCIQNYT PGAHLTIDEQLLGFRGRCPFRMYIPNKPSKYGIKILMMCDSGTKYMINGMPYL GRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSIPLAKNLLQEPYKLT IVGTVRSNKREIPEVLKNSRSRPVGTSMFCFDGPLTLVSYKPKPAKMVYLLSS CDEDASINESTGKPQMVMYYNQTKGGVDTLDQMCSVMTCSRKTNRWPMAL LYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMSLTSSFMRKRLEAPT LKRYLRDNISNILPNEVPGTS (SEQ ID NO: 3).

[0043] In some embodiments, a PB transposition comprising a CRD deletion described herein comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence established in SEQ ID NO: 3. In some embodiments, a PB transposition comprising a CRD deletion described herein comprises the amino acid sequence established in SEQ ID NO: 3 with one, two, three, four, or five conservative amino acid substitutions. In some embodiments, a PB transposition comprising a CRD deletion described herein comprises the amino acid sequence established in SEQ ID NO: 3.

[0044] MosI DNA-binding domain(s) used to generate Petition 870250091089, dated 06 / 10 / 2025, p. 101 / 198 14 / 102 chimeric PB:MosI transposases comprising this C-terminal CRD deletion can be joined to the S544 amino acid of the PB transposase sequence established in SEQ ID NO: 1. The resulting chimeric transposase is referred to in this document as a “chimeric S544 PB:MosI transposase”. In some embodiments, the S544 PB transposase sequence and the MosI DNA-binding domain(s) are connected by a linker sequence. In some embodiments, the linker sequence is GGGGS (SEQ ID NO: 86).

[0045] In some embodiments, the deletion of the C-terminal Cysteine-Rich Domain (CRD) of the PB transposase is a deletion of amino acid residues 553–594 of the PB transposase amino acid sequence established at SEQ ID NO: 1. In such embodiments, the resulting truncated piggyBac transposase comprises the amino acid sequence: MGSSLDDEHILSALLQSDDELVGEDSDSEISDHVSEDDVQSDTE EAFIDEVHEVQPTSSGSEILDEQNVIEQPGSSLASNRILTLPQRTIRGKNKHCW STSKSTRRSRVSALNIVRSQRGPTRMCRNIYDPLLCFKLFFTDEIISEIVKWTNA EISLKRRESMTGATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLSM VYVSVMSRDRFDFLIRCLRMDDKSIRPTLRENDVFTPVRKIWDLFIHQCIQNYT PGAHLTIDEQLLGFRGRCPFRMYIPNKPSKYGIKILMMCDSGTKYMINGMPYL GRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSIPLAKNLLQEPYKLT IVGTVRSNKREIPEVLKNSRSRPVGTSMFCFDGPLTLVSYKPKPAKMVYLLSS CDEDASINESTGKPQMVMYYNQTKGGVDTLDQMCSVMTCSRKTNRWPMAL LYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMSLTSSFMRKRLEAPT LKRYLRDNISNILPNEVPGTSDDSTEEPV (SEQ ID NO: 4).

[0046] In some embodiments, a PB transposition comprising a CRD deletion described in this document comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, by Petition 870250091089, dated 06 / 10 / 2025, page 102 / 198 15 / 102 less 98% or at least 99% identical to the amino acid sequence established in SEQ ID NO: 4. In some embodiments, a PB transposition comprising a CRD deletion described in this document comprises the amino acid sequence established in SEQ ID NO: 4 with one, two, three, four, or five conservative amino acid substitutions. In some embodiments, a PB transposition comprising a CRD deletion described in this document comprises the amino acid sequence established in SEQ ID NO: 4.

[0047] Alternatively, the MosI DNA-binding domain(s) used to generate chimeric PB:MosI transposases comprising this C-terminal CRD deletion may be joined to the V552 amino acid of the piggyBac transposase sequence established in SEQ ID NO: 1. The resulting chimeric transposase is referred to in this document as a “V552 PB:MosI chimeric transposase”. In some embodiments, the V552 PB transposase sequence and the MosI DNA-binding domain(s) are connected by a linking sequence. In some embodiments, the linking sequence is GGGGS (SEQ ID NO: 86).

[0048] In some embodiments, the truncated PB transposase comprises one or more hyperactive mutations. Examples of hyperactive mutations include I30V, S103P, G165S, M226F, M282V, S509G, N538K, and N571S of SEQ ID NO: 1. In some embodiments, the truncated piggyBac transposase comprises at least 4 hyperactive mutations selected from I30V, G165S, M226F, M282V, and N538K of SEQ ID NO: 1.

[0049] In some embodiments, a truncated PB transposase described in this document comprises the sequence SEQ ID NO: 1 and one of the following mutations: I30V, S103P, G165S, M226F, M282V, S509G, N538K, and N571S. In some embodiments, a truncated PB transposase described in this document comprises the sequence SEQ ID NO: 1 and two of the following Petition 870250091089, dated 06 / 10 / 2025, page 103 / 198 16 / 102 mutations: I30V, S103P, G165S, M226F, M282V, S509G, N538K, and N571S. In some embodiments, a truncated PB transposase described in this document comprises the sequence with SEQ ID NO: 1 and three of the following mutations: I30V, S103P, G165S, M226F, M282V, S509G, N538K, and N571S. In some embodiments, a truncated PB transposase described in this document comprises the sequence with SEQ ID NO: 1 and four of the following mutations: I30V, S103P, G165S, M226F, M282V, S509G, N538K, and N571S. In some embodiments, a truncated PB transposase described in this document comprises the SEQ ID NO: 1 sequence and five of the following mutations: I30V, S103P, G165S, M226F, M282V, S509G, N538K, and N571S. In some embodiments, a truncated PB transposase described in this document comprises the SEQ ID NO: 1 sequence and six of the following mutations: I30V, S103P, G165S, M226F, M282V, S509G, N538K, and N571S.In some embodiments, a truncated PB transposase described in this document comprises the SEQ ID NO: 1 sequence and seven of the following mutations: I30V, S103P, G165S, M226F, M282V, S509G, N538K, and N571S.

[0050] In some embodiments, a truncated PB transposase described in this document comprises the SEQ ID NO: 3 sequence and one of the following mutations: I30V, S103P, G165S, M226F, M282V, S509G, N538K, and N571S. In some embodiments, a truncated PB transposase described in this document comprises the SEQ ID NO: 3 sequence and two of the following mutations: I30V, S103P, G165S, M226F, M282V, S509G, N538K, and N571S. In some embodiments, a truncated PB transposase described in this document comprises the SEQ ID NO: 3 sequence and three of the following mutations: I30V, S103P, G165S, M226F, M282V, S509G, N538K, and N571S. In some Petition 870250091089, dated 06 / 10 / 2025, page 104 / 198 In 17 / 102 embodiments, a truncated PB transposase described in this document comprises the sequence with SEQ ID NO: 3 and four of the following mutations: I30V, S103P, G165S, M226F, M282V, S509G, N538K, and N571S. In some embodiments, a truncated PB transposase described in this document comprises the sequence with SEQ ID NO: 3 and five of the following mutations: I30V, S103P, G165S, M226F, M282V, S509G, N538K, and N571S. In some embodiments, a truncated PB transposase described in this document comprises the sequence with SEQ ID NO: 3 and six of the following mutations: I30V, S103P, G165S, M226F, M282V, S509G, N538K, and N571S. In some embodiments, a truncated PB transposase described in this document comprises the SEQ ID NO: 3 sequence and seven of the following mutations: I30V, S103P, G165S, M226F, M282V, S509G, N538K, and N571S.In some embodiments, a truncated PB transposase described in this document comprises the sequence SEQ ID NO: 3 and the following mutations: I30V, S103P, G165S, M226F, M282V, S509G, N538K, and N571S.

[0051] In some embodiments, a truncated PB transposase described in this document comprises the sequence SEQ ID NO: 4 and one of the following mutations: I30V, S103P, G165S, M226F, M282V, S509G, N538K, and N571S. In some embodiments, a truncated PB transposase described in this document comprises the sequence SEQ ID NO: 4 and two of the following mutations: I30V, S103P, G165S, M226F, M282V, S509G, N538K, and N571S. In some embodiments, a truncated PB transposase described in this document comprises the sequence SEQ ID NO: 4 and three of the following mutations: I30V, S103P, G165S, M226F, M282V, S509G, N538K, and N571S. In some embodiments, a truncated PB transposase described in this document comprises the SEQ ID NO: 4 sequence and four of the following mutations: I30V, S103P, G165S, M226F, M282V, S509G, N538K, and N571S. Petition 870250091089, dated 06 / 10 / 2025, page 105 / 198 18 / 102 comprises the sequence with SEQ ID NO: 4 and five of the following mutations: I30V, S103P, G165S, M226F, M282V, S509G, N538K, and N571S. In some embodiments, a truncated PB transposase described in this document comprises the sequence with SEQ ID NO: 4 and six of the following mutations: I30V, S103P, G165S, M226F, M282V, S509G, N538K, and N571S. In some embodiments, a truncated PB transposase described in this document comprises the sequence with SEQ ID NO: 4 and seven of the following mutations: I30V, S103P, G165S, M226F, M282V, S509G, N538K, and N571S. In some embodiments, a truncated PB transposase described in this document comprises the sequence SEQ ID NO: 4 and the following mutations: I30V, S103P, G165S, M226F, M282V, S509G, N538K, and N571S.

[0052] In some embodiments, the truncated domain of the piggyBac transposase is a Super piggyBac® (SPB) transposase. Non-limiting examples of SPB transposases are described in detail in U.S. Patent No. 6,218,182; U.S. Patent No. 6,962,810; U.S. Patent No. 8,399,643 and Publication No. PCT WO 2010 / 099296, each of which is incorporated herein by reference in its entirety, for examples of SPB transposases that may be used in the fusion proteins described herein.

[0053] The chimeric transposases illustrating the Super piggyBac - MosI transposase are described in more detail in Example 6.

[0054] In some embodiments, the truncated PB transposase sequence additionally comprises an in-frame N-terminal nuclear localization (NLS) sequence. An illustrative wild-type SPB sequence comprising an NLS is shown in SEQ ID NO: 5 with the NLS shown in italics, hyperactive mutations shown in bold, and the deleted Cysteine-Rich Domain (CRD) in the truncated PB transposase sequence underlined. The SPB transposase domain sequence numbering for the purpose of describing deletions and mutations begins at residue 12 of SEQ ID NO: 5. Petition 870250091089, dated 06 / 10 / 2025, page 106 / 198 19 / 102 MA PKKKRKVGGGGSSLDDEHILSALLQSDDELVGEDSDSEVSDHVSEDDVQS DTEEAFIDEVHEVQPTSSGSEILDEQNVIEQPGSSLASNRILTLPQRTIRGKNKH CWSTSKSTRRSRVSALNIVRSQRGPTRMCRNIYDPLLCFKLFFTDEIISEIVKW TNAEISLKRRESMTSATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSL SMVYVSVMSRDRFDFLIRCLRMDDKSIRPTLRENDVFTPVRKIWDLFIHQCIQN YTPGAHLTIDEQLLGFRGRCPFRVYIPNKPSKYGIKILMMCDSGTKYMINGMPY LGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSIPLAKNLLQEPYKL TIVGTVRSNKREIPEVLKNSRSRPVGTSMFCFDGPLTLVSYKPKPAKMVYLLS SCDEDASINESTGKPQMVMYYNQTKGGVDTLDQMCSVMTCSRKTNRWPMA LLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMSLTSSFMRKRLEAP TLKRYLRDNISNILPKEVPGTSDDSTEEPVMKKRTYCTYCPSKIRRKANASCKK CKKVICREHNIDMCQSCF (SEQ ID NO: 5).

[0055] The transposases described in this document may be isolated from or derived from an insect, a vertebrate, a crustacean, or a urochordate, as described in more detail in Publication No. PCT WO 2019 / 173636 and PCT / US2019 / 049816. In preferred aspects, the SPB transposase is isolated from or derived from the insect Trichoplusia ni (GenBank accession number AAA87375), Macdunnoughia crassisigna (ABZ85926.1), or Bombyx mori (GenBank accession number BAD11135).

[0056] In some embodiments, the chimeric transposase comprises two MosI DNA-binding domains. In some embodiments, each of the two MosI DNA-binding domains comprises amino acid residues 3-111 of the MosI transposase (i.e., comprises the amino acid sequence established in SEQ ID NO: 6): SFVPNKEQTRTVLIFCFHLKKTAAESHRMLVEAFGEQVPTVKTCERWFQRFKS GDFDVDDKEHGKPPKRYEDAELQALLDEDDAQTQKQLAEQLEVSQQAVSNR LREMG (SEQ ID NO: 6). In some embodiments, each of the two MosI DNA-binding domains comprises an amino acid sequence that is at least Petition 870250091089, dated 06 / 10 / 2025, p. 107 / 198 20 / 102 minus 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence established in SEQ ID NO: 6. In some embodiments, each of the two MosI DNA-binding domains comprises the amino acid sequence established in SEQ ID NO: 3 with one, two, three, four, or five conservative amino acid substitutions.

[0057] In some embodiments, the chimeric transposase comprising a truncated piggyBac transposase domain comprising the amino acid sequence established in SEQ ID NO: 3 connected to the two MosI DNA-binding domains, wherein each of the two MosI DNA-binding domains comprises the sequence established in SEQ ID NO: 6. The two MosI DNA-binding domains can be attached to the truncated PB transposase domain via a linker sequence, resulting in the chimeric PB:MosI S544-111 transposase comprising the amino acid sequence established in SEQ ID NO: 7, with residues 3-111 of the MosI transposase highlighted in bold, the PB domain shown underlined, and the linker sequence highlighted in italics: MAPKKKRKVGGGGSSLDDEHILSALLQSDDELVGEDSDSEVSD HVSEDDVQSDTEEAFIDEVHEVQPTSSGSEILDEQNVIEQPGSSLASNRILTLP QRTIRGKNKHCWSTSKSTRRSRVSALNIVRSQRGPTRMCRNIYDPLLCFKLFF TDEIISEIVKWTNAEISLKRRESMTSATFRDTNEDEIYAFFGILVMTAVRKDNHM STDDLFDRSLSMVYVSVMSRDRFDFLIRCLRMDDKSIRPTLRENDVFTPVRKI WDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRVYIPNKPSKYGIKILMMCDS GTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSIP LAKNLLQEPYKLTIVGTVRSNKREIPEVLKNSRSRPVGTSMFCFDGPLTLVSYK PKPAKMVYLLSSCDEDASINESTGKPQMVMYYNQTKGGVDTLDQMCSVMTC SRKTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMSLTS Petição 870250091089, de 06 / 10 / 2025, pág. 108 / 198 21 / 102 SFMRKRLEAPTLKRYLRDNISNILPKEVPGTS GGGGS SFVP NKEQTRTVLIFCF HLKKTAAESHRMLVEAFGEQVPTVKTCERWFQRFKSGDFDVDDKEHGKPP KRYEDAELQALLDEDDAQTQKQLAEQLEVSQQAVSNRLREMG (SEQ ID NO: 7).

[0058] In some embodiments, a chimeric PB:MosI transposase comprising a CRD deletion described herein comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence established in SEQ ID NO: 7. In some embodiments, a chimeric PB:MosI transposase comprising a CRD deletion described herein comprises the amino acid sequence established in SEQ ID NO: 7 with one, two, three, four, or five conservative amino acid substitutions. In some embodiments, a chimeric PB:MosI transposase comprising a CRD deletion described in this document comprises the amino acid sequence established in SEQ ID NO: 7.

[0059] In some embodiments, the chimeric transposase comprises a MosI DNA-binding domain. In some embodiments, the MosI DNA-binding domain comprises amino acid residues 3-58 of the MosI transposase, that is, it comprises the amino acid sequence set forth in SEQ ID NO: 8: SFVPNKEQTRTVLIFCFHLKKTAAESHRMLVEAFGEQVPTVKTC ERWFQRFKSGDF (SEQ ID NO: 8).

[0060] In some embodiments, the chimeric transposase comprising a truncated piggyBac transposase domain comprising the amino acid sequence set forth in SEQ ID NO: 3 is connected to a MosI DNA-binding domain comprising the amino acid sequence Petition 870250091089, dated 06 / 10 / 2025, p. 109 / 198 22 / 102 established in SEQ ID NO: 8. The truncated PB transposase domain can be attached to the MosI DNA-binding domain via a linker sequence, resulting in the chimeric PB:MosI transposase S544-58 comprising the amino acid sequence established in SEQ ID NO: 9, with residues 3-58 of the MosI transposase highlighted in bold, the PB domain underlined, and the linker sequence highlighted in italics: MAPKKKRKVGGGGSSLDDEHILSALLQSDDELVGEDSDSEVSD HVSEDDVQSDTEEAFIDEVHEVQPTSSGSEILDEQNVIEQPGSSLASNRILTLP QRTIRGKNKHCWSTSKSTRRSRVSALNIVRSQRGPTRMCRNIYDPLLCFKLFF TDEIISEIVKWTNAEISLKRRESMTSATFRDTNEDEIYAFFGILVMTAVRKDNHM STDDLFDRSLSMVYVSVMSRDRFDFLIRCLRMDDKSIRPTLRENDVFTPVRKI WDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRVYIPNKPSKYGIKILMMCDS GTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSIP LAKNLLQEPYKLTIVGTVRSNKREIPEVLKNSRSRPVGTSMFCFDGPLTLVSYK PKPAKMVYLLSSCDEDASINESTGKPQMVMYYNQTKGGVDTLDQMCSVMTC SRKTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMSLTS SFMRKRLEAPTLKRYLRDNISNILPKEVPGTS GGGGS SFVPNKEQTRTVLIFCF HLKKTAAESHRMLVEAFGEQVPTVKTCERWFQRFKSGDF (SEQ ID NO: 9).

[0061] In some embodiments, a chimeric PB:MosI transposase comprising a CRD deletion described herein comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence established in SEQ ID NO: 9. In some embodiments, a chimeric PB:MosI transposase comprising a CRD deletion described herein comprises the amino acid sequence established in SEQ ID NO: 9 with one, two, three, four, or five conservative substitutions. Petition 870250091089, dated 06 / 10 / 2025, p. 110 / 198 23 / 102 of amino acids. In some embodiments, a chimeric PB:MosI transposase comprising a CRD deletion described in this document comprises the amino acid sequence set forth in SEQ ID NO: 9.

[0062] In some embodiments, the chimeric transposase comprises truncated piggyBac transposase residues comprising the amino acid sequence established in SEQ ID NO: 3 and is connected to the two MosI DNA-binding domains comprising the sequence of SEQ ID NO: 6. The truncated PB transposase domain can be attached to the MosI DNA-binding domain via a linker sequence, resulting in the chimeric transposase PB:MosI V552-111 comprising the amino acid sequence established in SEQ ID NO: 10, with residues 3-11 of the MosI transposase highlighted in bold, the PB domain underlined, and the linker sequence highlighted in italics: MAPKKKRKVGGGGSSLDDEHILSALLQSDDELVGEDSDSEVSD HVSEDDVQSDTEEAFIDEVHEVQPTSSGSEILDEQNVIEQPGSSLASNRILTLP QRTIRGKNKHCWSTSKSTRRSRVSALNIVRSQRGPTRMCRNIYDPLLCFKLFF TDEIISEIVKWTNAEISLKRRESMTSATFRDTNEDEIYAFFGILVMTAVRKDNHM STDDLFDRSLSMVYVSVMSRDRFDFLIRCLRMDDKSIRPTLRENDVFTPVRKI WDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRVYIPNKPSKYGIKILMMCDS GTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSIP LAKNLLQEPYKLTIVGTVRSNKREIPEVLKNSRSRPVGTSMFCFDGPLTLVSYK PKPAKMVYLLSSCDEDASINESTGKPQMVMYYNQTKGGVDTLDQMCSVMTC SRKTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMSLTS SFMRKRLEAPTLKRYLRDNISNILPKEVPGTSDDSTEEPVGGGGSSFVPNKEQ TRTVLIFCFHLKKTAAESHRMLVEAFGEQVPTVKTCERWFQRFKSGDFDVD DKEHGKPPKRYEDAELQALLDEDDAQTQKQLAEQLEVSQQAVSNRLREMG (SEQ ID NO: 10).

[0063] Em algumas realizações, uma quimérica transposase Petição 870250091089, de 06 / 10 / 2025, pág. 111 / 198 24 / 102 A PB:MosI transposase comprising a CRD deletion described herein comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence established in SEQ ID NO: 10. In some embodiments, a chimeric PB:MosI transposase comprising a CRD deletion described herein comprises the amino acid sequence established in SEQ ID NO: 10 with one, two, three, four, or five conservative amino acid substitutions. In some embodiments, a chimeric PB:MosI transposase comprising a CRD deletion described herein comprises the amino acid sequence established in SEQ ID NO: 10.

[0064] In some embodiments, the chimeric transposase comprises a truncated piggyBac transposase domain comprising the amino acid sequence at SEQ ID NO: 4 connected to a MosI DNA-binding domain comprising the amino acid sequence set at SEQ ID NO: 8. The truncated PB transposase domain can be attached to the MosI DNA-binding domain via a linker sequence, resulting in the chimeric transposase PB:MosI V552-58 comprising the amino acid sequence set at SEQ ID NO: 11, with residues 358 of the MosI transposase highlighted in bold, the PB domain underlined, and the linker sequence highlighted in italics: MAPKKKRKVGGGSSLDEHILSALLQSDDELVGEDSDSEVSD HVSEDDVQSDTEAFIDEVHEVQPTSSGSEILDEQNVIEKPGSSLASNRILTLP QRTIRGKNKHCWSTSKSTRRSRVSALNIVRSQRGPTRMCRNIYDPLLCFKLFF TDEIISEIVKWTNAEISLKRRESMTSATFRDTNEDEIYAFFGILVMTAVRKDNHM STDDLFDRSLSMVYVSVMSRDRFDFLIRCLRMDKSIRPTLRENDVVRFTPKI WDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRVYIPNKPSKYGIKILMMCDS Petition 870250091089, de 06 / 10 / 2025, pág. 112 / 198 25 / 102 GTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSIP LAKNLLQEPYKLTIVGTVRSNKREIPEVLKNSRSRPVGTSMFCDFDGPLTLVSYK PKPAKMVYLLSSCDEDASINESTGKPQMVMYYNQTKGGVDTLDQMCSVMTC SRKTNRWPMALLYGMINIACINSFIIYSHNVSSKGEVQSRKKFMRNLYMSLTS SFMRKRLEAPTLKRYLRDNISNILPKEVPGTSDSTEEPVGGGGSSFVPNKEQ TRTVLICFHLKKTAAESHRMLVEAFGEQVPTVKTCERWFQRFKSGDF (SEQ ID NO: 11).

[0065] In some embodiments, a chimeric PB:MosI transposase comprising a CRD deletion described herein comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence established in SEQ ID NO: 11. In some embodiments, a chimeric PB:MosI transposase comprising a CRD deletion described herein comprises the amino acid sequence established in SEQ ID NO: 11 with one, two, three, four, or five conservative amino acid substitutions. In some embodiments, a chimeric PB:MosI transposase comprising a CRD deletion described in this document comprises the amino acid sequence established in SEQ ID NO: 11.

[0066] Chimeric transposases can be used to transpose cells using transposons comprising chimeric transposon inverted repeat (ITR) polynucleotides described in this document. Chimeric Transposon Inverted Terminal Repeat (ITR) Polynucleotides

[0067] Also provided in this document are chimeric transposon inverted terminal repeat (ITR) polynucleotides, comprising in the 5' to 3' direction: (i) a left ITR sequence of Petition 870250091089, dated 06 / 10 / 2025, p. 113 / 198 26 / 102 truncated piggyBac without the CRD binding site and (ii) an ITR MosI sequence comprising binding sites for two MosI DNA binding domains.

[0068] In some embodiments, the left-end (LE) ITR sequence of the piggyBac comprises the sequence established at SEQ ID NO: 12. In some embodiments, the 35 bp LE PB ITR sequence is truncated after position 16, excluding the 19 bp CRD binding site. In some embodiments, the resulting truncated PB LE ITR sequence without the CRD binding site comprises only nucleotides 1-16 (highlighted in bold) of the full-length PB LE ITR sequence established at SEQ ID NO: 12: CCCTAGAAAGATAGTCTGCGTAAAATTGACGCATG (SEQ ID NO: 12). In some embodiments, the PB LE ITR sequence without the CRD binding site comprises the sequence CCCTAGAAAGATAGTC (SEQ ID NO: 172).

[0069] In some embodiments, the MosI ITR sequence comprising binding sites for two MosI DNA-binding domains comprises the sequence: TGTACAAGTATGAAATGTCGTTT (SEQ ID NO: 13).

[0070] In some embodiments, the 23 bp MosI sequence (SEQ ID NO: 13) containing the binding sites for the two MosI DNA-binding domains is fused in the reverse orientation to the truncated PB LE ITR (SEQ ID NO: 172), resulting in the established LE PiggyBac:MosI ITR sequence at SEQ ID NO: 15: CCCTAGAAAGATAGTCAAACGACATTTCATACTTGTACA (SEQ ID NO: 15).

[0071] In some embodiments, a single nucleotide is deleted from the 3' end of the 23 bp MosI sequence containing the binding sites for the two MosI DNA-binding domains (i.e., from the sequence established in SEQ ID NO: 13), leaving only residues 1-22, i.e., the sequence established in SEQ ID NO: 14: TGTACAAGTATGAAATGTCGTT (SEQ ID NO: 14).

[0072] In some realizations, the fragment ITR MosI 1-22 Petition 870250091089, dated 06 / 10 / 2025, p. 114 / 198 27 / 102 nucleotides is fused in reverse orientation to the truncated ITR PB LE sequence established at SEQ ID NO: 12, resulting in the LE sequence PiggyBac:MosI ITR-1 established at SEQ ID NO: 16: CCCTAGAAAGATAGTCAACGACATTTCATACTTGTACA (SEQ ID NO: 16).

[0073] In some embodiments, the ITR polynucleotides of the chimeric transposon further comprise one or two additional nucleotides between the left ITR sequence of the truncated piggyBac and the ITR sequence of MosI. In some embodiments, a nucleotide is added to the 3' end of the MosI ITR sequence to create the LE PiggyBac:MosI ITR 1 sequence established in SEQ ID NO: 17: CCCTAGAAAGATAGTCTAAACGACATTTCATACTTGTACA (SEQ ID NO: 17).

[0074] Similarly, two nucleotides can be added to the 3' end of the MosI ITR sequence to create the LE PiggyBac:MosI ITR 2 sequence established in SEQ ID NO: 18: CCCTAGAAAGATAGTCTGAAACGACATTTCATACTTGTACA (SEQ ID NO: 18).

[0075] In some embodiments, the right-end (RE) ITR sequence of the piggyBac comprises the sequence established at SEQ ID NO: 65. In some embodiments, the 63 bp RE PB ITR sequence is truncated after position 16, excluding the 19 bp CRD binding site and the remaining ITR sequence. The truncated PB RE ITR sequence without the CRD binding site comprises only nucleotides 1-16 (highlighted in bold) of the complete PB RE ITR sequence established at SEQ ID NO: 65: CCCTAGAAAGATAATCATATTGTGACGTACGTTAAAGATAATCATGCGTAA AATTGACGCATG (SEQ ID NO: 65). In some embodiments, the truncated sequence PB RE ITR without the CRD binding site comprises the sequence CCCTAGAAAGATAATC (SEQ ID NO: 173).

[0076] In some realizations, the ITR sequence of MosI that Petition 870250091089, dated 06 / 10 / 2025, p. 115 / 198 28 / 102 comprises binding sites for two DNA-binding domains. MosI comprises the sequence: TGTACAAGTATGAAATGTCGTTT (SEQ ID NO: 13).

[0077] In some embodiments, the 23 bp MosI sequence (shown in SEQ ID NO: 13) containing the binding sites for the two MosI DNA-binding domains is merged in reverse orientation to the truncated PB RE ITR (SEQ ID NO: 173) to create the ITR RE PB:MosI sequence set out in SEQ ID NO: 19: CCCTAGAAAGATAATCAAACGACATTTCATACTTGTACA (SEQ ID NO: 19).

[0078] In some embodiments, a single nucleotide is deleted from the 3' end of the 23 bp MosI sequence containing the binding sites for the two MosI DNA-binding domains (established in SEQ ID NO: 13) resulting in the sequence: TGTACAAGTATGAAATGTCGTT (SEQ ID NO: 14).

[0079] In some embodiments, the 1-22 nucleotide ITR MosI fragment (SEQ ID NO: 14) is fused in reverse orientation to the truncated PB RE ITR (SEQ ID NO: 173) to create the established PiggyBac:MosI ITR-1 RE at SEQ ID NO: 20: CCCTAGAAAGATAATCAACGACATTTCATACTTGTACA (SEQ ID NO: 20).

[0080] In some embodiments, the polynucleotides further comprise one or two additional nucleotides between the right-hand ITR sequence of the truncated piggyBac and the ITR MosI sequence. In some embodiments, a nucleotide is added to the 3' end of the ITR MosI sequence to create the RE PB:MosI ITR + 1 sequence set out in SEQ ID NO: 21: CCCTAGAAAGATAATCAAAACGACATTTCATACTTGTACA (SEQ ID NO: 21).

[0081] Similarly, two nucleotides can be added to the 3' end of the MosI ITR sequence to create the RE PiggyBac:MosI ITR 2 sequence established in SEQ ID NO: 22: CCCTAGAAAGATAATCATAAACGACATTTCATACTTGTACA (SEQ ID NO: 22) Petition 870250091089, dated 06 / 10 / 2025, p. 116 / 198 29 / 102

[0082] The ITR polynucleotides of the chimeric transposon can be incorporated into vectors or transposons (as described below) for use in conjunction with chimeric transposases, chimeric site-specific transposase fusion proteins, and in the methods described in this document. Site-Specific PB:MosI Transposase Chimeric Fusion Proteins

[0083] Also provided in this document are chimeric site-specific transposase fusion proteins comprising, in N-terminal to C-terminal order: (i) a target-specific DNA-binding domain, (ii) a truncated, N-terminal, integration-deficient piggyBac transposase comprising a C-terminal Cysteine-Rich Domain (CRD) deletion, and (iii) one or more MosI DNA-binding domains. In some embodiments, the target-specific DNA-binding domain is a TAL or a zinc finger motif (ZFM). N-terminal Pb Transposase Deletions

[0084] N-terminal deleted PB transposase sequences and N-terminal piggyBac transposase with defective integration were previously described in International Patent Application No. PCT / 2022 / 22549 and No. PCT / US2022 / 077549, each of which is incorporated herein by reference in its entirety for examples of N-terminal deleted piggyBac transposases with deficient integration that may be used in the fusion protein constructs and proteins described herein. In some embodiments, the N-terminal deletion of the truncated PB transposase is a deletion of amino acid residues 1-85, 1-86, 1-87, 1-88, 1-89, 1-90, 1-91, 1-92, 1-93, 1-94, 1-95, 1-96, 1-97, 1-98, 1-99, 1-100, 1-101, 1-102, or 1-103 of SEQ ID NO: 1. In some embodiments, the N-terminal deletion of the truncated PB transposase is a deletion of amino acid residues 1-93 of SEQ ID NO: 1, or corresponding residues of SEQ ID NOs: 3 or 4. Petition 870250091089, dated 06 / 10 / 2025, p. 117 / 198 30 / 102 Transfers with Integration Disabilities

[0085] In some embodiments, the chimeric site-specific transposase fusion protein comprises a chimeric transposase that is integration-deficient. An integration-deficient transposase domain is a transposase that can excise its corresponding transposon but integrates the excised transposon at a lower frequency than a corresponding wild-type transposase. Examples of integration-deficient transposases are disclosed in U.S. Patent No. 6,218,185; U.S. Patent No. 6,962,810, U.S. Patent No. 8,399,643 and International Patent Application Publication No. WO 2019 / 173636, each of which is incorporated herein by reference in its entirety for examples of integration-deficient transposases that may be used in the constructs described herein. A list of integration-deficient amino acid substitutions is disclosed in U.S. Patent No. 10,041.077, which is incorporated herein by reference in its entirety for examples of integration-deficient transposases that can be used in the constructs described herein. A wild-type SPB can be considered integration-deficient by the introduction of mutations, for example, K93A, R372A, K375A, R376A, and / or D450N (relative to SEQ ID NOs: 1, 3, or 4). The introduction of the R372A, K375A, R376A, and D450N mutations is believed to render the transposase integration-deficient but retain its excision function.

[0086] Thus, in some embodiments, the chimeric site-specific transposase fusion protein comprises a chimeric transposase domain comprising the SEQ ID NO:1 sequence with one of the following mutations: R372A, K375A, R376A, and D450N. In some embodiments, the chimeric site-specific transposase fusion protein comprises a chimeric transposase domain comprising the SEQ ID NO:1 sequence. Petition 870250091089, dated 06 / 10 / 2025, pp. 118 / 198 31 / 102 with two of the following mutations: R372A, K375A, R376A, and D450N. In some embodiments, the chimeric site-specific transposase fusion protein comprises a chimeric transposase domain comprising the SEQ ID NO:1 sequence with three of the following mutations: R372A, K375A, R376A, and D450N. In some embodiments, the chimeric site-specific transposase fusion protein comprises a chimeric transposase domain comprising the SEQ ID NO:1 sequence with the following mutations: R372A, K375A, R376A, and D450N.

[0087] Thus, in some embodiments, the chimeric site-specific transposase fusion protein comprises a chimeric transposase domain comprising the SEQ ID NO: 3 sequence with one of the following mutations: R372A, K375A, R376A, and D450N. In some embodiments, the chimeric site-specific transposase fusion protein comprises a chimeric transposase domain comprising the SEQ ID NO: 3 sequence with two of the following mutations: R372A, K375A, R376A, and D450N. In some embodiments, the chimeric site-specific transposase fusion protein comprises a chimeric transposase domain comprising the SEQ ID NO: 3 sequence with three of the following mutations: R372A, K375A, R376A, and D450N. In some embodiments, the chimeric site-specific transposase fusion protein comprises a chimeric transposase domain comprising the SEQ ID NO: 3 sequence with the following mutations: R372A, K375A, R376A, and D450N.

[0088] Thus, in some embodiments, the chimeric site-specific transposase fusion protein comprises a chimeric transposase domain comprising the SEQ ID NO:4 sequence with one of the following mutations: R372A, K375A, R376A, and D450N. In some embodiments, the chimeric site-specific transposase fusion protein comprises a chimeric transposase domain comprising the SEQ ID NO: Petition 870250091089, dated 06 / 10 / 2025, pp. 119 / 198 32 / 102 with two of the following mutations: R372A, K375A, R376A, and D450N. In some embodiments, the chimeric site-specific transposase fusion protein comprises a chimeric transposase domain comprising the SEQ ID NO:4 sequence with three of the following mutations: R372A, K375A, R376A, and D450N. In some embodiments, the chimeric site-specific transposase fusion protein comprises a chimeric transposase domain comprising the SEQ ID NO:4 sequence with the following mutations: R372A, K375A, R376A, and D450N.

[0089] An illustrative sequence of a truncated, N-terminal deleted, and integration-deficient transposase, comprising a deletion of amino acids 1-93 from the N-terminal PB transposase sequence and comprising three integration-deficient mutations, is shown in SEQ ID NO: 85: NKHCWSTSKSTRRSRVSALNIVRSQRGPTRMCRNIYDPLLCFKL FFTDEIISEIVKWTNAEISLKRRESMTSATFRDTNEDEIYAFFGILVMTAVRKDN HMSTDDLFDRSLSMVYVSVMSRDRFDFLIRCLRMDDKSIRPTLRENDVFTPVR KIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRVYIPNKPSKYGIKILMMCD SGTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSI PLAKNLLQEPYKLTIVGTVASNAREIPEVLKNSRSRPVGTSMFCFDGPLTLVSY KPKPAKMVYLLSSCDEDASINESTGKPQMVMYYNQTKGGVDTLNQMCSVMT CSRKTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMSLT SSFMRKRLEAPTLKRYLRDNISNILPKEVPGTSDDSTEEPVMKKRTYCTYCPS KIRRKANASCKKCKKVICREHNIDMCQSCF (SEQ ID NO: 85).

[0090] In some embodiments, a truncated, N-terminal deleted, and integration-deficient transposase described in this document comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, by Petition 870250091089, dated 06 / 10 / 2025, pp. 120 / 198 33 / 102 minus 97%, at least 98%, or at least 99% identical to the amino acid sequence established in SEQ ID NO: 85. In some embodiments, a truncated, N-terminal deletion, integration-deficient transposase described in this document comprises the amino acid sequence established in SEQ ID NO: 85 with one, two, three, four, or five conservative amino acid substitutions. In some embodiments, a truncated, N-terminal deletion, integration-deficient transposase described in this document comprises the amino acid sequence established in SEQ ID NO: 85.In some embodiments, a truncated, N-terminal deleted, and integration-deficient transposase described in this document comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence established in SEQ ID NO: 89. In some embodiments, a truncated, N-terminal deleted, and integration-deficient transposase described in this document comprises the amino acid sequence established in SEQ ID NO: 89 with one, two, three, four, or five conservative amino acid substitutions. In some embodiments, a truncated, N-terminal deleted, and integration-deficient transposase described in this document comprises the amino acid sequence established in SEQ ID NO: 89.In some embodiments, a truncated, N-terminal deleted, and integration-deficient transposase described in this document comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence established in SEQ ID NO: 97. In some. Petition 870250091089, dated 06 / 10 / 2025, pp. 121 / 198 In some embodiments, a truncated, N-terminal deletion, integration-deficient transposase described in this document comprises the amino acid sequence established in SEQ ID NO: 97 with one, two, three, four, or five conservative amino acid substitutions. In some embodiments, a truncated, N-terminal deletion, integration-deficient transposase described in this document comprises the amino acid sequence established in SEQ ID NO: 97. In some embodiments, a truncated, N-terminal deletion, integration-deficient transposase described in this document comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence established in SEQ ID NO: 110.In some embodiments, a truncated, N-terminal deletion-deficient, integration-deficient transposase described in this document comprises the amino acid sequence established in SEQ ID NO: 110 with one, two, three, four, or five conservative amino acid substitutions. In some embodiments, a truncated, N-terminal deletion-deficient, integration-deficient transposase described in this document comprises the amino acid sequence established in SEQ ID NO: 110. In some embodiments, a truncated, N-terminal deletion-deficient, integration-deficient transposase described in this document comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence established in SEQ ID NO: 118.In some embodiments, a truncated transposase with an N-terminal deletion and integration deficiency described in this document comprises the amino acid sequence. Petition 870250091089, dated 06 / 10 / 2025, page 122 / 198 35 / 102 established in SEQ ID NO: 118 with one, two, three, four, or five conservative amino acid substitutions. In some embodiments, a truncated, N-terminal deletion-deficient, integration-deficient transposase described in this document comprises the amino acid sequence established in SEQ ID NO: 118. In some embodiments, a truncated, N-terminal deletion-deficient, integration-deficient transposase described in this document comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence established in SEQ ID NO: 131.In some embodiments, a truncated, N-terminal deletion-deficient, integration-deficient transposase described in this document comprises the amino acid sequence established in SEQ ID NO: 131 with one, two, three, four, or five conservative amino acid substitutions. In some embodiments, a truncated, N-terminal deletion-deficient, integration-deficient transposase described in this document comprises the amino acid sequence established in SEQ ID NO: 131. In some embodiments, a truncated, N-terminal deletion-deficient, integration-deficient transposase described in this document comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence established in SEQ ID NO: 139.In some embodiments, a truncated, N-terminal deletion-deficient, integration-deficient transposase described in this document comprises the amino acid sequence established in SEQ ID NO: 139 with one, two, three, four, or five conservative amino acid substitutions. In some embodiments, one. Petition 870250091089, dated 06 / 10 / 2025, p. 123 / 198 The truncated, N-terminal, integration-deficient transposase described in this document comprises the amino acid sequence established in SEQ ID NO: 139. In some embodiments, a truncated, N-terminal, integration-deficient transposase described in this document comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence established in SEQ ID NO: 152. In some embodiments, a truncated, N-terminal, integration-deficient transposase described in this document comprises the amino acid sequence established in SEQ ID NO: 152 with one, two, three, four, or five conservative amino acid substitutions.In some embodiments, a truncated, N-terminal deleted, integration-deficient transposase described in this document comprises the amino acid sequence established in SEQ ID NO: 152. In some embodiments, a truncated, N-terminal deleted, integration-deficient transposase described in this document comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence established in SEQ ID NO: 160. In some embodiments, a truncated, N-terminal deleted, integration-deficient transposase described in this document comprises the amino acid sequence established in SEQ ID NO: 160 with one, two, three, four, or five conservative amino acid substitutions.In some embodiments, a truncated transposase with an N-terminal deletion and integration deficiency described in this document comprises the amino acid sequence. Petition 870250091089, dated 06 / 10 / 2025, pp. 124 / 198 37 / 102 established in SEQ ID NO: 160.

[0091] In some embodiments, a truncated, N-terminal deleted, integration-deficient transposase described in this document comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence established in any of the SEQ ID NOs: 87-170. In some embodiments, a truncated, N-terminal deleted, integration-deficient transposase described in this document comprises the amino acid sequence established in any of the SEQ ID NOs: 87-170 with one, two, three, four, or five conservative amino acid substitutions. In some embodiments, a truncated, N-terminal deletion, integration-deficient transposase described in this document comprises the amino acid sequence established in any of the SEQ ID NOs: 87-170. DNA Targeting Domains

[0092] The chimeric site-specific transposase fusion proteins of the present disclosure may further comprise one or more DNA targeting domains. A DNA targeting domain may be attached to the C-terminus or the N-terminus of the chimeric site-specific transposase fusion protein. In preferred embodiments, the DNA targeting domain is attached to the N-terminus of the chimeric site-specific transposase fusion protein. Without wishing to be limited by theory, it is believed that the addition of a DNA targeting domain to a chimeric site-specific transposase fusion protein enhances site-specific transposase activity by directing the fused chimeric site-specific transposase fusion protein to the DNA targeting domain to the target site. In some embodiments, the insertion of a domain of Petition 870250091089, dated 06 / 10 / 2025, page 125 / 198 38 / 102 DNA targeting improves site-specific transposase activity by at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold compared to the same chimeric site-specific transposase that does not comprise a DNA targeting domain.

[0093] Any DNA targeting domain known in the art may be used in the context of the chimeric transposases and chimeric site-specific transposase fusion proteins provided in this document described, including, without limitation, CRISPR, zinc finger motifs, TALE, and transcription factors.

[0094] Engineering methods for zinc finger nucleases that bind to specific targets are described, for example, in Sander et al., Nat. Methods January 2011; 8(1): 67-69. In some embodiments, the DNA targeting domain comprises three zinc finger motifs. In some embodiments, the three zinc finger motifs are flanked by GGGGS ligands (SEQ ID NO: 86). In some embodiments, the three zinc finger motifs (ZFM) and the flanking GGGGS ligands (SEQ ID NO: 86) cumulatively comprise the sequence established in SEQ ID NO: 23: GGGGSERPYACPVESCDRRFSRSDELTRHIRIHTGQKPFQCRICMRNFSRSD HLTTHIRTHTGEKPFACDICGRKFARSDERKRHTKIHLRQKDGGGGS (SEQ ID NO: 23); or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with it.

[0095] In some embodiments, a chimeric site-specific transposase fusion protein comprising three ZFM domains joined via a binding sequence to a chimeric PBx:MosI transposase of the present disclosure is provided in this document. An illustrative ZFM-PB:MosI chimeric transposase fusion protein comprises three Petition 870250091089, dated 06 / 10 / 2025, pp. 126 / 198 39 / 102 ZFM domains joined to a chimeric transposase S544:PB:MosI-58 to create a site-specific chimeric transposase ZFM-S544:PB:MosI-58 comprising the nucleic acid sequence: MAPKKKRKVGGGGSERPYACPVESCDRRFSRSDELTRHIRIHT GQKPFQCRICMRNFSRSDHLTTHIRTHTGEKPFACDICGRKFARSDERKRHTK IHLRQKDGGGGSNKHCWSTSKSTRRSRVSALNIVRSQRGPTRMCRNIYDPLL CFKLFFTDEIISEIVKWTNAEISLKRRESMTSATFRDTNEDEIYAFFGILVMTAVR KDNHMSTDDLFDRSLSMVYVSVMSRDRFDFLIRCLRMDDKSIRPTLRENDVFT PVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRVYIPNKPSKYGIKILM MCDSGTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNW FTSIPLAKNLLQEPYKLTIVGTVASNAREIPEVLKNSRSRPVGTSMFCFDGPLTL VSYKPKPAKMVYLLSSCDEDASINESTGKPQMVMYYNQTKGGVDTLNQMCS VMTCSRKTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLY MSLTSSFMRKRLEAPTLKRYLRDNISNILPKEVPGTSGGGGSSFVPNKEQTRT VLIFCFHLKKTAAESHRMLVEAFGEQVPTVKTCERWFQRFKSGDF (SEQ ID NO: 24).

[0096] In some embodiments, a chimeric ZFM-PB:MosI transposase fusion protein described herein comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least less than 98%, or at least 99% identical to the amino acid sequence established in SEQ ID NO: 24. In some embodiments, a chimeric ZFM-PB:MosI transposase fusion protein described herein comprises the amino acid sequence established in SEQ ID NO: 24 with one, two, three, four, or five conservative amino acid substitutions. In some embodiments, a chimeric ZFM-PB:MosI transposase fusion protein described herein comprises the amino acid sequence Petition 870250091089, dated 06 / 10 / 2025, p. 127 / 198 40 / 102 established in SEQ ID NO: 24.

[0097] Another illustrative chimeric transposase fusion protein ZFMPB:MosI comprises three ZFM domains joined to a chimeric transposase S544 PB:MosI-111 to create a site-specific chimeric transposase ZFM-S544:PB:MosI-111 comprising the amino acid sequence: MAPKKKRKVGGGGSERPYACPVESCDRRFSRSDELTRHIRIHT GQKPFQCRICMRNFSRSDHLTTHIRTHTGEKPFACDICGRKFARSDERKRHTK IHLRQKDGGGGSNKHCWSTSKSTRRSRVSALNIVRSQRGPTRMCRNIYDPLL CFKLFFTDEIISEIVKWTNAEISLKRRESMTSATFRDTNEDEIYAFFGILVMTAVR KDNHMSTDDLFDRSLSMVYVSVMSRDRFDFLIRCLRMDDKSIRPTLRENDVFT PVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRVYIPNKPSKYGIKILM MCDSGTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNW FTSIPLAKNLLQEPYKLTIVGTVASNAREIPEVLKNSRSRPVGTSMFCFDGPTLL VSYKPKPAKMVYLLSSCDEDASINESTGKPQMVMYYNQTKGGVDTLNQMCS VMTCSRKTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLY MSLTSSFMRKRLEAPTLKRYLRDNISNILPKEVPGTSGGGGSSFVPNKEQTRT VLIFCFHLKKTAAESHRMLVEAFGEQVPTVKTCERWFQRFKSGDFDVDDKEH GKPPKRYEDAELQALLDEDDAQTQKQLAEQLEVSQQAVSNRLREMG (SEQ ID NO: 25).

[0098] In some embodiments, a chimeric ZFM-PB:MosI transposase fusion protein described in this document comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence established in SEQ ID NO: 25. In some embodiments, a chimeric ZFM-PB:MosI transposase fusion protein described in this document comprises Petition 870250091089, dated 06 / 10 / 2025, p. 128 / 198 41 / 102 the amino acid sequence established in SEQ ID NO: 25 with one, two, three, four, or five conservative amino acid substitutions. In some embodiments, a chimeric ZFM-PB:MosI transposase fusion protein described in this document comprises the amino acid sequence established in SEQ ID NO: 25.

[0099] Another illustrative chimeric transposase fusion protein ZFMPB:MosI comprises three ZFM domains joined to a chimeric transposase V552 PB:MosI-58 to create a site-specific chimeric transposase ZFM-PB:MosI-58 comprising the nucleic acid sequence: MAPKKKRKVGGGGSERPYACPVESCDRRFSRSDELTRHIRIHT GQKPFQCRICMRNFSRSDHLTTHIRTHTGEKPFACDICGRKFARSDERKRHTK IHLRQKDGGGGSNKHCWSTSKSTRRSRVSALNIVRSQRGPTRMCRNIYDPLL CFKLFFTDEIISEIVKWTNAEISLKRRESMTSATFRDTNEDEIYAFFGILVMTAVR KDNHMSTDDLFDRSLSMVYVSVMSRDRFDFLIRCLRMDDKSIRPTLRENDVFT PVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRVYIPNKPSKYGIKILM MCDSGTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNW FTSIPLAKNLLQEPYKLTIVGTVASNAREIPEVLKNSRSRPVGTSMFCFDGPTLL VSYKPKPAKMVYLLSSCDEDASINESTGKPQMVMYYNQTKGGVDTLNQMCS VMTCSRKTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLY MSLTSSFMRKRLEAPTLKRYLRDNISNILPKEVPGTSDDSTEEPVGGGGSSFV PNKEQTRTVLIFCFHLKKTAAESHRMLVEAFGEQVPTVKTCERWFQRFKSGD F (SEQ ID NO: 26).

[00100] In some embodiments, a chimeric ZFM-PB:MosI transposase fusion protein described herein comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence Petition 870250091089, dated 06 / 10 / 2025, p. 129 / 198 42 / 102 established at SEQ ID NO: 26. In some embodiments, a chimeric ZFM-PB:MosI transposase fusion protein described in this document comprises the amino acid sequence established at SEQ ID NO: 26 with one, two, three, four, or five conservative amino acid substitutions. In some embodiments, a chimeric ZFM-PB:MosI transposase fusion protein described in this document comprises the amino acid sequence established at SEQ ID NO: 26.

[00101] Another illustrative chimeric transposase fusion protein ZFMPB:MosI comprises three ZFM domains joined to a chimeric transposase V552:PB:MosI-111 to create a site-specific chimeric transposase ZFM-V552:PB:MosI-111 comprising the nucleic acid sequence: MAPKKKRKVGGGGSERPYACPVESCDRRFSRSDELTRHIRIHT GQKPFQCRICMRNFSRSDHLTTHIRTHTGEKPFACDICGRKFARSDERKRHTK IHLRQKDGGGGSNKHCWSTSKSTRRSRVSALNIVRSQRGPTRMCRNIYDPLL CFKLFFTDEIISEIVKWTNAEISLKRRESMTSATFRDTNEDEIYAFFGILVMTAVR KDNHMSTDDLFDRSLSMVYVSVMSRDRFDFLIRCLRMDDKSIRPTLRENDVFT PVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRVYIPNKPSKYGIKILM MCDSGTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNW FTSIPLAKNLLQEPYKLTIVGTVASNAREIPEVLKNSRSRPVGTSMFCFDGPTLL VSYKPKPAKMVYLLSSCDEDASINESTGKPQMVMYYNQTKGGVDTLNQMCS VMTCSRKTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLY MSLTSSFMRKRLEAPTLKRYLRDNISNILPKEVPGTSDDSTEEPVGGGGSSFV PNKEQTRTVLIFCFHLKKTAAESHRMLVEAFGEQVPTVKTCERWFQRFKSGD FDVDDKEHGKPPKRYEDAELQALLDEDDAQTQKQLAEQLEVSQQAVSNRLR EMG (SEQ ID NO: 27).

[00102] In some embodiments, a chimeric ZFM-PB:MosI transposase fusion protein described in this document comprises Petition 870250091089, dated 06 / 10 / 2025, pp. 130 / 198 43 / 102 an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence established in SEQ ID NO: 27. In some embodiments, a chimeric ZFM-PB:MosI transposase fusion protein described in this document comprises the amino acid sequence established in SEQ ID NO: 27 with one, two, three, four, or five conservative amino acid substitutions. In some embodiments, a chimeric ZFM-PB:MosI transposase fusion protein described in this document comprises the amino acid sequence established in SEQ ID NO: 27.

[00103] In some respects, the DNA targeting domain is a TAL array. Xanthomonas TALEs (transcription activator-like effectors) generally contain a 288-amino acid N-terminus followed by an array of a variable number of repetitive sequences of about 34 amino acids (repeat) followed by a 278-amino acid C-terminus (SEQ ID NO: 29); However, truncated versions have been described in the literature (e.g., see Miller et al., Nat Biotechnol 29, 143-148 (2011)). TALs fused to a FokI nuclease (an effector TAL nuclease; TALEN) generally contain truncations of the N and C termini. For example, the first 152 amino acids of the N terminus may be removed (called delta 152; the sequence of the N terminus after removal of the first 152 amino acids is shown in SEQ ID NO: 30) and the C terminus may be truncated, leaving 63 amino acids (called 63; the sequence is shown in SEQ ID NO: 31).

[00104] TALs generally contain sets of 34 amino acids repeated a variable number of times. The two amino acids at positions 12 and 13 are usually varied and determine which nucleotide the TAL repeat will recognize. Thus, NG amino acids recognize T, NI Petition 870250091089, dated 06 / 10 / 2025, p. 131 / 198 44 / 102 recognize A, NN recognize G or A, HD recognize C, NK recognize G, NS recognize A, C, G, or T. Other amino acids within the 34-residue repeat can also be varied. For example, position 11 is often changed to N for repeats that recognize G. Additionally, positions 4 and 32 are often varied to reduce the repeatability of the array, but not to determine binding specificity. The number of 34 amino acid repeats in an array determines the length of the recognized DNA sequence (one protein repeat binds to one DNA base). Furthermore, the last base is recognized by a “half array” which is composed of 20 amino acids instead of 34. This feature allows a TAL array to be programmed to bind a specific DNA sequence. One skilled in the technique will be able to modify the TALEN sequences to achieve the desired target specificity.

[00105] Furthermore, an N-terminal domain of TALs (e.g., an N-terminal domain comprising the sequence established in SEQ ID NO: 30) recognizes and requires for binding a T that is located immediately 5' from the target DNA sequence. Mutations of N-terminal TAL domains that no longer require a 5' T have been described in the literature (see, for example, Lamb et al., Nucleic Acids Res. November 2013;41(21):9779-85). For example, the NT-G mutant comprising the amino acid sequence established in SEQ ID NO: 32 requires a 5'G instead of a 5'T, while the NT^N mutant comprising the amino acid sequence established in SEQ ID NO: 33 does not require any specific 5' nucleotide. These mutated N-terminal domain sequences can be used to provide additional options for developing fusion protein sequences described in this document that can be targeted using TAL arrays.

[00106] In some embodiments, a TAL matrix provided in this document comprises nine repetitions of 34 amino acids followed by Petition 870250091089, dated 06 / 10 / 2025, p. 132 / 198 45 / 102 “half” repeats of 20 amino acids with flanking BsmBI-type IIS restriction sites. In one embodiment, individual TAL modules containing 34-amino acid repeats or a “half” repeat of 20 amino acids can be designed and synthesized flanked by BsmBI-type IIS restriction sites. In some embodiments, the complete set of TAL modules contains 4 modules capable of recognizing A, C, G, T for each of the 10 bp positions (i.e., 40 TAL modules for a recognized 10 bp target) and one half-repeat TAL module. Illustrative TAL modules are shown in SEQ ID NOs: 34-37, where X is any amino acid: • TAL Module Version 1: LTPDQVVAIAXXXGGKQALETVQRLLPVLCQDHG (SEQ ID NO: 34) • TAL Module Version2: LTPEQVVAIAXXXGGKQALETVQRLLPVLCQAHG (SEQ ID NO: 35) • TAL Module Version 3: LTPDQVVAIAXXXGGKQALETVQRLLPVLCQAHG (SEQ ID NO: 36) • TAL Module Version4: LTPAQVVAIAXXXGGKQALETVQRLLPVLCQDHG (SEQ ID NO: 37).

[00107] An illustrative TAL module is shown in SEQ ID NO: 38, where X is any amino acid: LTPEQVVAIAXXXGGRPALE (SEQ ID NO: 38).

[00108] Pairs of TAL arrays targeting sequences in the desired gene can be designed and the corresponding modules selected and grouped using “Golden Gate Assembly”, to assemble each TAL array into a frame. The DNA sequence encoding the TAL arrays generated in this document can be further optimized codon-wise using GeneArt algorithms (Thermo Fisher).

[00109] TAL arrays can be used to target TTAA sites in the genome. When designing left and right TAL arrays comprising a Petition 870250091089, dated 06 / 10 / 2025, p. 133 / 198 46 / 102 N-terminal domain recognizing a T domain and a C-terminal TAL domain being fused to a site-specific chimeric transposase, one TAL array recognizes a 5' sequence of the TTAA sequence, and the other TAL array recognizes a 3' sequence of the TTAA sequence. Because the 5' sequence of TTAA is more often different from the 3' sequence of TTAA in genomic DNA targets, site-specific TAL Super piggyBac transposase fusion proteins (“TAL-ssSPB” or “TAL-PBx”) will most often be used as a heterodimer consisting of two transposase fusion proteins, each comprising a different TAL domain that recognizes two different DNA sequences. Furthermore, the sequence recognized by the TAL array is not necessarily directly adjacent to the TTAA. Instead, this can be separated from the TTAA by a spacer of a specified bp length, for example, 12 bp, 13 bp, or 14 bp spacers.

[00110] A TAL array can target any DNA sequence (e.g., genomic DNA sequence) of interest. It will be evident to one skilled in the art that any left TAL array for a given target can be combined with any right TAL array for the same target.

[00111] In some embodiments, a TAL matrix targets green fluorescent protein (GFP). TALpiggyBac transposase fusion proteins comprising N-terminal deleted piggyBac transposase sequences and GFP-targeted N-terminal integration-defective piggyBac transposase were described in International Patent Application No. PCT / 2022 / 22549, which is incorporated herein in its entirety by way of example of TAL-piggyBac transposase fusion proteins that may be used in the constructs described herein.

[00112] Illustrative sequences of right TAL arrays that can be incorporated into site-specific transposase fusion proteins. Petition 870250091089, dated 06 / 10 / 2025, p. 134 / 198 47 / 102 chimeric variables that target GFP are shown in SEQ ID Nos: 41-44.

[00113] In some embodiments, a TAL array targets a LINE1 repeat element. TALpiggyBac transposase fusion proteins comprising deleted N-terminal piggyBac transposase sequences and defective N-terminal piggyBac transposase-targeting LINE1 elements were described in International Patent Application No. PCT / 2022 / 22549, which is incorporated herein in its entirety by way of example of TAL-piggyBac transposase fusion proteins that may be used in the constructs described herein.

[00114] Illustrative sequences of left and right TAL matrices directed to LINE1 are presented in SEQ ID NOs: 66 and 67, respectively. Nuclear Location Signals

[00115] In some embodiments, the chimeric transposases and chimeric site-specific transposase fusion proteins provided in this document may comprise an in-frame nuclear localization sequence (NLS). Examples of transposases fused to a nuclear localization signal are disclosed in U.S. Patent No. 6,218,185; U.S. Patent No. 6,962,810, U.S. Patent No. 8,399,643 and International Patent Application Publication No. WO 2019 / 173636, each of which is incorporated herein in its entirety for examples of NLS-fused transposases that may be used in the constructs described in this document. In some embodiments, the NLS comprises the PKKKRKV sequence (SEQ ID NO: 39). In certain aspects, the in-frame NLS is located upstream (N-terminal) of the transposase domain comprising an N-terminal deletion. In certain aspects, the in-frame NLS is located downstream (C-terminal) of the transposase domain, which comprises an N-terminal deletion.

[00116] In general, the NLS is preferably located in Petition 870250091089, dated 06 / 10 / 2025, p. 135 / 198 48 / 102 N-terminal end of a chimeric transposase or chimeric site-specific transposase fusion protein.

[00117] In certain aspects, the in-frame NLS is fused directly to the amino terminus of a chimeric transposase or a chimeric site-specific transposase fusion protein. In some embodiments, a methionine primer is introduced before the NLS. In some embodiments, additional adenine residues are introduced before and / or after the NLS to ensure in-frame translation. Thus, residue numbering in SEQ ID NO: 5 begins at the 12th residue of SEQ ID NO: 5 for the purpose of identifying deleted and mutated residues. Nucleic Acids

[00118] Also provided in this document are polynucleotides comprising nucleic acid sequences encoding the chimeric transposases and chimeric site-specific transposase fusion proteins described in this document. In some embodiments, the polynucleotides are isolated or purified.

[00119] The polynucleotides isolated from the disclosure can be produced using (a) recombinant methods, (b) synthetic techniques, (c) purification techniques and / or (d) combinations thereof, which are well known in the art. Methods for constructing nucleic acids encoding the chimeric transposases and chimeric site-specific transposase fusion proteins described in this document are well known in the art or described in this document, for example, PCR-based mutagenesis.

[00120] The fusion of the present invention can be generated using any suitable method known in the art or described in this document.

[00121] The isolated polynucleotides from this disclosure, such as RNA, cDNA, genomic DNA, or any combination thereof, may be Petition 870250091089, dated 06 / 10 / 2025, pp. 136 / 198 49 / 102 obtained from biological sources using any number of cloning methodologies known to those skilled in the art. In some respects, oligonucleotide probes that selectively hybridize, under stringent conditions, to the polynucleotides of the present disclosure are used to identify the desired sequence in a cDNA or genomic DNA library.

[00122] 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 teaching and guidance presented in this document. 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; 5,066,584 to Gyllensten et al.; 4,889,818 to Gelfand et al.; 4,656,134 to Ringold) and RNA-mediated amplification that uses antisense RNA for the target sequence as a template for DNA synthesis. double-stranded tape (US Patent No. 5,130,238 for Malek, et al., under the trade name NASBA), the content of which is incorporated herein by reference.

[00123] For example, polymerase chain reaction (PCR) technology can be used to amplify dissemination polynucleotide sequences and related genes directly from genomic DNA or cDNA libraries. PCR and other in vitro amplification methods can also be useful, for example, for cloning nucleic acid sequences encoding proteins to be expressed, for producing nucleic acids to be used as probes to detect the presence of the desired mRNA in samples, for nucleic acid sequencing, or for other purposes. Examples of techniques sufficient to guide qualified individuals by Petition 870250091089, dated 06 / 10 / 2025, pp. 137 / 198 50 / 102 methods of in vitro amplification are found in Berger, supra, Sambrook, supra, and Ausubel, supra, as well as Mullis, et al., US Patent No. 4,683,202 (1987); and Innis, et al., PCR Protocols: A Guide to Methods and Applications, Eds., Academic Press Inc., San Diego, Calif. (1990). Commercially available kits for genomic amplification by PCR are known in the art. See, for example, the Advantage-GC Genomic PCR kit (Clontech). Additionally, for example, the T4 32 gene protein (Boehringer Mannheim) can be used to improve the yield of long PCR products.

[00124] Disclosure polynucleotides can also be prepared by direct chemical synthesis using known methods (see, for example, Ausubel, et al., supra). Chemical synthesis generally produces a 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 the single strand as a template. One skilled in the art will recognize that, although chemical DNA synthesis may be limited to sequences of about 100 or more bases, longer sequences can be obtained by ligating shorter sequences. Expression Vectors and Host Cells

[00125] Disclosure also refers to vectors that include disclosure polynucleotides, host cells that are genetically engineered with recombinant vectors, and the production of at least one protein structure by recombinant techniques, as is well known in the art. See, for example, Sambrook, et al., supra; Ausubel, et al., supra, each incorporated herein by reference in its entirety.

[00126] Polynucleotides can optionally be attached to a vector containing a selectable marker for propagation in a host. Typically, a plasmid vector is introduced into a precipitate, Petition 870250091089, dated 06 / 10 / 2025, pp. 138 / 198 51 / 102 as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it can be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.

[00127] In some embodiments, the DNA insertion is operatively linked to an appropriate promoter. In some embodiments, the promoter is an EF-Iα promoter comprising the sequence established in SEQ ID NO: 45. The expression constructs may further contain transcription initiation and termination sites and, in the transcript region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs will preferentially include a translation start codon (e.g., ATG) at the beginning and a termination codon (e.g., UAA, UGA, or UAG) appropriately positioned at the end of the mRNA to be translated, with UAA and UAG preferred for expression in mammalian or eukaryotic cells.

[00128] Expression vectors may also include one or more selectable markers. Examples of selectable markers include, without limitation, ampicillin, zeocin (Sh bla gene), puromycin (pac gene), hygromycin B (hygB gene), G418 / Geneticin (neo gene), DHFR (which encodes dihydrofolate reductase and confers methotrexate resistance), mycophenolic acid or glutamine synthetase (GS, U.S. Patents Nos. 5,122,464; 5,770,359; 5,827,739), blasticidin (bsd gene), resistance genes for eukaryotic cell culture, as well as ampicillin, zeocin (Sh bla gene), puromycin (pac gene), hygromycin B (hygB gene), G418 / Geneticin (neo gene), resistance genes to kanamycin, spectinomycin, streptomycin, carbenicillin, bleomycin, erythromycin, polymyxin B, or tetracycline for Cultivation in E. coli and other bacteria or prokaryotes. Culture media and conditions appropriate for the host cells described above are known in the art. Vectors Petition 870250091089, dated 06 / 10 / 2025, pp. 139 / 198 52 / 102 suitable methods will be readily apparent to those skilled in the art. The 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.

[00129] Expression vectors may comprise at least one selectable cell surface marker for isolating cells modified by 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. In some embodiments, the selectable cell surface marker distinguishes cells modified by a disclosure composition or method from those cells that are not modified by a disclosure composition or method. Examples of cell surface markers include, without limitation, “designation cluster” or “classification determinants” proteins (often abbreviated as “CD”), such as a truncated or complete form of CD19, CD271, CD34, CD22, CD20, CD33, CD52, or any combination thereof.Cell surface markers also include the RQR8 suicide gene marker (Philip B et al. Blood. August 21, 2014; 124(8):1277-87).

[00130] Expression vectors may further comprise at least one selectable drug resistance marker for isolation from cells modified by the disclosure compositions and methods. Disclosure selectable drug resistance markers may comprise wild-type or mutant Neo, DHFR, TYMS, FRANCF, RAD51C, GCS, MDR1, ALDH1, NKX2.2, or any combination thereof. Petition 870250091089, dated 06 / 10 / 2025, p. 140 / 198 53 / 102

[00131] Those skilled in the art are aware of the numerous expression systems available for expressing a nucleic acid encoding a release protein. Release nucleic acids can be expressed in a host cell by activation (by manipulation) in a host cell containing endogenous DNA encoding a release protein structure. Such methods are well known in the art, for example, as described in U.S. Patents Nos. 5,580,734, 5,641,670, 5,733,746 and 5,733,761, each of which is incorporated herein by reference in its entirety.

[00132] Cell cultures useful for the production of protein scaffolds, specified portions or variants thereof, are bacterial, yeast and mammalian cells, as known in the art. Mammalian cell systems will generally be in the form of cell monolayers, although mammalian cell suspensions or bioreactors may also be used. Several suitable host cell lines capable of expressing intact glycosylated proteins have been developed using this technique and include COS-1 (e.g., ATCC CRL 1650), COS-7 (e.g., ATCC CRL-1651), HEK293, BHK21 (e.g., ATCC CRL-10), CHO (e.g., ATCC CRL 1610), and BSC-1 (e.g., ATCC CRL-26 cell lines), Cos-7 cells, CHO cells, hep G2 cells, P3X63Ag8653 cells, SP2 / 0-Ag14 cells, 293 cells, HeLa cells, and the like, which are readily available, for example, from the American Type Culture Collection, Manassas, Virginia (www.atcc.org).Preferred host cells include cells of lymphoid origin, such as myeloma and lymphoma cells. Particularly preferred host cells are P3X63Ag8.653 cells (ATCC accession number CRL-1580) and SP2 / O-Ag14 cells (ATCC accession number CRL1851). In a preferred aspect, the recombinant cell is either a P3X63Ab8.653 cell or an SP2 / O-Ag14 cell. Petition 870250091089, dated 06 / 10 / 2025, pp. 141 / 198 54 / 102

[00133] Expression vectors for these cells may include one or more of the following expression control sequences: An origin of replication; a promoter (e.g., late or early SV40 promoters, the CMV promoter (U.S. Patents 5,168,062; 5,385,839), an HSV tk promoter, a pgk (phosphoglycerate kinase) promoter, an EF-1 alpha promoter (U.S. Patent 5,266,491)), at least one human promoter; an enhancer and / or information processing sites, such as ribosome binding sites, RNA splicing sites, polyadenylation sites (e.g., a large SV40 polyAT Ag addition site), and transcriptional 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 disclosure are known and available, for example, in the Catalogue of Cell Lines and Hybridomas of the American Type Culture Collection (www.atcc.org) or in other known or commercial sources.

[00134] When eukaryotic host cells are employed, 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. In some embodiments, the polyA sequence is a polyA SV40 sequence comprising the sequence established in SEQ ID NO: 47.

[00135] Sequences for precise splicing of the transcript may also be included. An example of a splicing sequence is the VP1 intron of SV40 (Sprague, et al., J. Virol. 45:773-781 (1983)). In addition, genetic sequences to control replication in the host cell may be incorporated into the vector, as known in the art.

[00136] The plasmid constructs described in this document can be used to deliver nucleic acids encoding the transposase domains or fusion proteins described in this document to a cell. Petition 870250091089, dated 06 / 10 / 2025, page 142 / 198 55 / 102

[00137] The transposase domains and fusion proteins described in this document can also be delivered to a cell using mRNA constructs. Thus, in one embodiment, an mRNA sequence encoding a transposase domain or a fusion protein described in this document is provided herein. These mRNA sequences can be delivered to a cell using a nanoparticle, for example, a lipid nanoparticle. Examples of lipid nanoparticles are described, for example, in International Patent Application Publications No. WO 2022 / 087148, No. WO 2022 / 182792, No. WO 2023 / 141576, No. WO 2024 / 035783 and No. WO 2023 / 141576, each of which is incorporated herein by reference in its entirety as examples of lipid nanoparticles that can be used to provide mRNA constructs encoding the fusion proteins or transposase domains described herein.An mRNA construct can also be delivered to a cell by electroporation or nucleofection. The mRNA can be encapsulated or otherwise modified. Transposons, Cells and Modified Cells

[00138] The chimeric transposases and chimeric site-specific transposase fusion proteins described in this document can be used in conjunction with a transposon to modify cells. The transposon can be a piggyBac™ (PB) transposon. In some embodiments, when the transposon is a PB transposon, the transposase is a piggyBac™ (PB) transposase, a piggyBac-like (PBL) transposase, or a Super piggyBac™ (SPB) transposase. Non-limiting examples of PB transposons are described in detail in U.S. Patent No. 6,218,182; U.S. Patent No. 6,962,810; US Patent No. 8,399,643 and PCT Publication No. WO 2010 / 099296, each of which is incorporated herein by reference in its entirety by way of example of transposons that may be used in the methods disclosed herein. Transposons may comprise a nucleic acid that encodes Petition 870250091089, dated 06 / 10 / 2025, p. 143 / 198 56 / 102 a therapeutic protein or a therapeutic agent. Examples of therapeutic proteins include those disclosed in PCT Publication No. WO 2019 / 173636 and No. WO 2020 / 051374, each of which is incorporated herein by reference in its entirety as an example of therapeutic proteins that may be used in the chimeric transposases and chimeric site-specific transposase fusion proteins described herein.

[00139] In some embodiments, the transposon may be a transposon comprising one or more chimeric PB:MosI ITR polynucleotides described in this document. In some embodiments, the transposon comprises a chimeric LE PB:Mos1 ITR polynucleotide and a chimeric RE PB:MosI ITR polynucleotide. In some embodiments, the LE PB:MosI ITR polynucleotide is PB:MosI ITR comprising the sequence established in SEQ ID NO: 19. In some embodiments, the LE PB:MosI ITR polynucleotide is PB:MosI ITR-1 comprising the sequence established in SEQ ID NO: 20. In some embodiments, the LE PB:MosI ITR polynucleotide is PB:MosI ITR 1 comprising the sequence of SEQ ID NO: 21. In some embodiments, the LE PB:MosI ITR polynucleotide is PB:MosI ITR +2 comprising the sequence of SEQ ID NO: 22.

[00140] In some embodiments, the polynucleotide RE PB:MosI ITR is PB:MosI ITR comprising the sequence with SEQ ID NO: 15. In some embodiments, the polynucleotide RE PB:MosI ITR is PB:MosI ITR -1 comprising the sequence with SEQ ID NO: 16. In some embodiments, the polynucleotide RE PB:MosI ITR is PB:MosI ITR 1 comprising the sequence with SEQ ID NO: 17. In some embodiments, the polynucleotide RE PB:MosI ITR is PB:MosI ITR +2 comprising the sequence with SEQ ID NO: 18.

[00141] In some embodiments, the PB:MosI ITR transposon comprises the nucleotide sequence: Petition 870250091089, dated 06 / 10 / 2025, pp. 144 / 198 57 / 102 TTAACCCTAGAAAGATAGTCAAACGACATTTCATACTTGTACA GACGCATGCATTCTTGAAATATTGCTCTCTCTTTCTAAATAGCGCGAATCC GTCGCTGTGCATTTAGGACATCTCAGTCGCGAGCTGAAGGGCATCGACTT CAAGGAGGACGGCAACATCCTGGGCACAAAGCTGGAGTACAACTACAACA GCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTG AACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGA CCACTACCAGCAGAAATGTACAAGTATGAAATGTCGTTTGATTATCTTTCTA GGGTTAA (SEQ ID NO: 50). In some embodiments, a PB:MosI ITR transposon described in this document comprises a nucleic acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence established in SEQ ID NO: 50.

[00142] In some embodiments, the PB:MosI ITR-1 transposon comprises the nucleotide sequence: TTAACCCTAGAAAGATAGTCAACGACATTTCATACTTGTACAG ACGCATGCATTCTTGAAATATTGCTCTCTCTTTCTAAATAGCGCGAATCCGT CGCTGTGCATTTAGGACATCTCAGTCGCGAGCTGAAGGGCATCGACTTCA AGGAGGACGGCAACATCCTGGGCACAAGCTGGAGTACAACTACAACAG CCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGA ACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGA CCACTACCAGCAGAAATGTACAAGTATGAAATGTCGTTGATTATCTTTCTAG GGTTAA (SEQ ID NO: 51). In some embodiments, a PB:MosI ITR transposon described in this document comprises a nucleic acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence established in SEQ ID NO: 51. Petition 870250091089, dated 06 / 10 / 2025, p. 145 / 198 58 / 102

[00143] In some embodiments, the PB:MosI ITR+1 transposon comprises the nucleotide sequence: TTAACCCTAGAAAGATAGTCTAAACGACATTTCATACTTGTAC AGACGCATGCATTCTTGAAATATTGCTCTCTCTTTCTAAATAGCGCGAATCC GTCGCTGTGCATTTAGGACATCTCAGTCGCGAGCTGAAGGGCATCGACTT CAAGGAGGACGGCAACATCCTGGGCACAAAGCTGGAGTACAACTACAACA GCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTG AACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGA CCACTACCAGCAGAAATGTACAAGTATGAAATGTCGTTTTGATTATCTTTCT AGGGTTAA (SEQ ID NO: 52). In some embodiments, a PB:MosI ITR transposon described in this document comprises a nucleic acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence established in SEQ ID NO: 52.

[00144] In some embodiments, the PB:MosI ITR+2 transposon comprises the nucleotide sequence: TTAACCCTAGAAAGATAGTCTGAAACGACATTTCATACTTGTA CAGACGCATGCATTCTTGAAATATTGCTCTCTCTTTCTAAATAGCGCGAATC CGTCGCTGTGCATTTAGGACATCTCAGTCGCGAGCTGAAGGGCATCGACT TCAAGGAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAAC AGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGT GAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCC GACCACTACCAGCAGAAATGTACAAGTATGAAATGTCGTTTATGATTATCTT TCTAGGGTTAA (SEQ ID NO: 53). In some embodiments, a PB:MosI ITR transposon described in this document comprises a nucleic acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, Petition 870250091089, dated 06 / 10 / 2025, p. 146 / 198 59 / 102 at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the nucleic acid sequence established in SEQ ID NO: 53.

[00145] In some embodiments, a transposon disclosed in this document comprises a shortened PB:MosI ITR polynucleotide. In some embodiments, the left-end shortened PB:MosI ITR comprises the nucleic acid sequence established in SEQ ID NO: 54. In some embodiments, the right-end shortened PB:MosI ITR comprises the nucleic acid sequence established in SEQ ID NO: 55. In some embodiments, the transposons comprise a left-end shortened PB:MosI ITR and a right-end shortened PB:MosI ITR to generate a short PB:MosI transposon comprising the nucleic acid sequence established in SEQ ID NO: 56.In some embodiments, a short PB:MosI transposon comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence established in SEQ ID NO: 56.

[00146] In another aspect, modified cells comprising one or more transposons and one or more chimeric site-specific transposase fusion proteins or chimeric transposases described in this document are provided herein. Disclosure cells and modified cells may be mammalian cells. Preferably, the cells and modified cells are human cells.

[00147] A cell modified using a chimeric transposase or chimeric site-specific transposase fusion protein described in this document may be a germline cell or a somatic cell. Disclosure cells and modified cells may be Petition 870250091089, dated 06 / 10 / 2025, pp. 147 / 198 60 / 102 immune cells, for example, lymphoid progenitor cells, natural killer (NK) cells, T lymphocytes (T cells), memory stem cells (scm T cells), central memory T cells (cm T cells), stem cell-like T cells, B lymphocytes (B cells), antigen-presenting cells (APCs), cytokine-induced killer (CIK) cells, myeloid progenitor cells, neutrophils, basophils, eosinophils, monocytes, macrophages, platelets, erythrocytes, red blood cells (RBCs), megakaryocytes, or osteoclasts. The modified cell may be differentiated, undifferentiated, or immortalized. The undifferentiated modified cell may be a stem cell. The undifferentiated modified cell may be an induced pluripotent stem cell. The modified cell can be a hepatocyte, a T cell, a hematopoietic stem cell, a natural killer cell, a macrophage, a dendritic cell, a monocyte, a megakaryocyte, or an osteoclast.The modified cell can be modified while quiescent, activated, resting, in interphase, prophase, metaphase, anaphase, or telophase. The modified cell can be fresh, cryopreserved, bulk, subpopulated, derived from whole blood, leukapheresis, or an immortalized cell line. A detailed description for isolating cells from a leukapheresis or blood product is provided in PCT Publications WO 2019 / 173636 and WO 2020 / 051374, each of which is incorporated herein by reference in its entirety.

[00148] The disclosure methods can be used to modify or produce a population of modified T cells, wherein at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, Petition 870250091089, dated 06 / 10 / 2025, pages 148 / 198 61 / 102 at least 99% or 100% or any percentage among the plurality of modified T cells in the population express one or more cell surface markers of a memory stem cell (MST) or a MST-like cell; and wherein the one or more cell surface markers comprise CD45RA and CD62L. The cell surface markers of an MST or a MST-like cell may also comprise one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2Rp. The cell surface markers of an MST or a MST-like cell may also comprise one or more of CD45RA, CD95, IL-2Rp, CCR7 and CD62L.

[00149] This disclosure provides methods for expressing a CAR on the surface of a cell. The method comprises (a) obtaining a cell population; (b) contacting the cell population with a composition comprising a CAR or a sequence encoding the CAR under conditions sufficient to transfer the CAR or the sequence encoding the CAR across a cell membrane of at least one cell in the cell population, thereby generating a modified cell population; (c) culturing the modified cell population under conditions suitable for integration of the sequence encoding the CAR; and (d) expanding and / or selecting at least one cell from the modified cell population that expresses the CAR on the cell surface. A more detailed description of the methods for expressing a CAR on the surface of a cell is disclosed in PCT Publication No. WO 2019 / 049816 and No. WO 2020 / 051374.

[00150] This disclosure further provides a cell or a population of cells wherein the cell comprises a composition comprising (a) an inducible transgenic construct comprising a sequence encoding an inducible promoter and a sequence encoding a transgene, and (b) a receptor construct, which Petition 870250091089, dated 06 / 10 / 2025, pp. 149 / 198 62 / 102 comprises a sequence encoding a constitutive promoter and a sequence encoding an exogenous receptor, such as a CAR, wherein, after integration of construct (a) and construct (b) into a genomic sequence of a cell, the exogenous receptor is expressed, and wherein the exogenous receptor, upon binding to a ligand or antigen, transduces an intracellular signal that directly or indirectly targets the inducible promoter regulating the expression of the inducible transgene (a) to modify gene expression.

[00151] The disclosure further provides a composition comprising the modified, expanded and selected cell population from the methods described in this document.

[00152] Modified disseminating cells (e.g., CAR T cells) can be further modified to increase their therapeutic potential. Alternatively or additionally, the modified cells can be further modified to make them less sensitive to immunological and / or metabolic checkpoints, for example, by blocking and / or diluting specific checkpoint signals delivered to the cells (e.g., checkpoint inhibition) naturally within the immunosuppressive tumor microenvironment.

[00153] Disclosure-modified cells (e.g., CAR T cells) may be further modified to silence or reduce the expression of (i) one or more genes encoding inhibitory checkpoint signaling receptors; (ii) one or more genes encoding intracellular proteins involved in checkpoint signaling; (iii) one or more genes encoding a transcription factor that hinders the effectiveness of a therapy; (iv) one or more genes encoding a cell death or apoptosis receptor; (v) one or more genes encoding a metabolic sensing protein; (vi) one or more genes encoding proteins that confer sensitivity to a cancer therapy, including a monoclonal antibody; Petition 870250091089, dated 06 / 10 / 2025, pp. 150 / 198 63 / 102 and / or (vii) one or more genes encoding a growth advantage factor. The modified cells may also be modified to silence or reduce the expression of the endogenous T cell receptor. Non-limiting examples of genes that may be modified to silence or reduce expression or to repress a function thereof include, without limitation, genes encoding inhibitory checkpoint signals, intracellular proteins, transcription factors, cell death or apoptosis receptors, metabolic sensing protein, proteins that confer sensitivity to a cancer therapy, and growth advantage factors that are disclosed in PCT Publication WO 2019 / 173636, which is incorporated herein by reference in its entirety for examples of genes that may be silenced in the cells of the disclosure.

[00154] Modified disclosure cells (e.g., CAR T cells) can be modified to express a modified / chimeric checkpoint receptor. The modified / chimeric checkpoint receptor may comprise a null receptor, a decoy receptor, or a negative-dominant receptor. Exemplary null, decoy, or negative-dominant intracellular receptors / proteins include, without limitation, downstream signaling components of an inhibitory checkpoint signal, a transcription factor, a cytokine or cytokine receptor, a chemokine or chemokine receptor, a cell death or apoptosis receptor / ligand, a metabolic sensing molecule, a protein that confers sensitivity to a cancer therapy, and an oncogene or tumor suppressor gene.Non-limiting examples of cytokines, cytokine receptors, chemokines, and chemokine receptors are disclosed in PCT Publication WO 2019 / 173636, which is incorporated herein by reference in its entirety for examples of modified / chimeric checkpoint receptors that may be expressed in cells. Petition 870250091089, dated 06 / 10 / 2025, pp. 151 / 198 64 / 102 described in this document.

[00155] Genome modification may involve the introduction of a nucleic acid sequence, transgene, and / or a genome editing construct into a cell ex vivo, in vivo, in vitro, or in situ to stably integrate a nucleic acid sequence, transiently integrate a nucleic acid sequence, produce site-specific integration of a nucleic acid sequence, or produce biased integration of a nucleic acid sequence. The nucleic acid sequence may be a transgene.

[00156] Stable chromosomal integration can be random integration, site-specific integration, or biased integration. Without wanting to be limited by theory, it is believed that the addition of DNA-binding domains to chimeric transposases and site-specific chimeric transposase fusion proteins described in this document improves the site specificity of the transposases.

[00157] Site-specific integration can occur at a safe harbor site. Genomic safe harbor sites have the capacity to accommodate the integration of new genetic material in a way that ensures that the newly inserted genetic elements function reliably (e.g., are expressed at a therapeutically effective expression level) and do not cause deleterious alterations to the host genome that pose a risk to the host organism. Non-limiting examples of potential genomic safe harbors include intronic sequences of the human albumin gene, adeno-associated virus site 1 (AAVS1), a natural AAV virus integration site on chromosome 19, the chemokine receptor 5 (CC motif) gene site (CCR5), and the mouse Rosa26 locus human ortholog site.

[00158] Site-specific transgenic integration can Petition 870250091089, dated 06 / 10 / 2025, p. 152 / 198 65 / 102 occur at a site that interrupts the expression of a target gene. Interruption of target gene expression can occur through site-specific integration into introns, exons, promoters, genetic elements, enhancers, suppressors, start codons, stop codons, and response elements. Non-limiting examples of target genes subject to site-specific integration include TRAC, TRAB, PD1, any gene encoding an immunosuppressive protein, genes encoding endogenous T-cell receptors, and genes encoding proteins involved in allorejection.

[00159] Site-specific transgenic integration can occur at a site that results in increased expression of a target gene. Increased expression of the target gene can occur through site-specific integration in introns, exons, promoters, genetic elements, enhancers, suppressors, start codons, stop codons, and response elements.

[00160] The integration site of the specific transgene may be an unstable chromosomal insertion. The unstable integration may be a transient nonchromosomal integration, a semi-stable nonchromosomal integration, a semi-persistent nonchromosomal insertion, or an unstable chromosomal insertion. The transient nonchromosomal insertion may be epichromosomal or cytoplasmic. In one aspect, the transient nonchromosomal insertion of a transgene does not integrate into a chromosome, and the modified genetic material is not replicated during cell division.

[00161] The site-specific transgene integration site may be a modified binding site for the DNA targeting domain in a chimeric transposase or a chimeric site-specific transposase fusion protein described in this document. For example, the TTAA target DNA integration site for SPB may be modified to insert flanking DNA binding sites for the DNA targeting domain comprising three zinc finger motifs (e.g., a Petition 870250091089, dated 06 / 10 / 2025, p. 153 / 198 66 / 102 DNA targeting domain comprising or consisting of the sequence with SEQ ID NO: 23 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity therewith). For example, a DNA targeting domain comprising three zinc finger motifs is believed to bind to the DNA sequence GCGTGGGCG. Therefore, the introduction of two copies of the GCGTGGGCG DNA sequence flanking the TTAA target integration site for site-specific chimeric transposase fusion proteins is believed to enhance the site-specific integration of the chimeric transposase comprising a DNA targeting domain comprising three zinc finger motifs. In such embodiments, the two copies of the GCGTGGGCG DNA sequence are in the reverse (5') and complementary (3') orientation.

[00162] In some embodiments, a polynucleotide is provided in this document comprising, in the order of 5' to 3', the reverse complement of the sequence of a target site for a DNA targeting domain, a first spacer, the target TTAA integration site for SPB, a second spacer, and the sequence of the target site for a DNA targeting domain. In some embodiments, the first spacer and the second spacer are the same length. In some embodiments, the first and / or the second spacer are 3 bp long. In some embodiments, the first and / or the second spacer are 4 bp long. In some embodiments, the first and / or the second spacer are 5 bp long. In some embodiments, the first and / or the second spacer are 6 bp long. In some embodiments, the first and / or the second spacer are 7 bp long. In some embodiments, the first and / or the second spacer are 8 bp long.In some embodiments, the first and / or second spacer is 9 bp long. In some embodiments, the first and / or... Petition 870250091089, dated 06 / 10 / 2025, pp. 154 / 198 67 / 102 the second spacer is 10 bp long.

[00163] The modified target site can be introduced into a cell or cell line to facilitate targeted genomic engineering. For example, a cell line that has been engineered to comprise a modified target site for a chimeric transposase or a chimeric site-specific transposase fusion protein provided in this document can be transfected with said chimeric transposase or a chimeric site-specific transposase fusion protein, as well as a transposon comprising donor DNA, such that the donor DNA is inserted into the modified target site. In some embodiments, the cell line is a T cell line. In some embodiments, the ZFM-PB:MosI fusion proteins comprise an amino acid sequence of SEQ ID Nos. 24-27.

[00164] Genome modification can be an unstable chromosomal integration of a transgene. The integrated transgene can be silenced, removed, excised, or further modified.

[00165] In some embodiments, the chimeric transposases and chimeric site-specific transposase fusion proteins provided in this document have better transposase efficacy than their wild-type equivalents. Transposase activity can be measured by any suitable assay known in the art or described in this document, for example, a Split GFP assay. For example, the chimeric transposases and chimeric site-specific transposase fusion proteins provided in this document may have genome integration activity on the target comparable to their wild-type counterparts, but have decreased genome integration activity off the target compared to their wild-type counterparts.

[00166] In some embodiments, a chimeric PB:MosI transposase comprising a DNA-directed domain provided Petition 870250091089, dated 06 / 10 / 2025, pp. 155 / 198 68 / 102 in this document has an on-target to off-target activity ratio that is increased by at least 50 times, at least about 100 times, at least about 150 times, at least about 200 times, at least about 250 times, at least about 300 times, at least about 350 times, at least about 400 times, at least about 450 times, at least about 500 times, at least about 550 times, at least about 600 times, at least about 650 times, at least about 700 times, at least about 750 times, at least about 800 times, at least about 850 times, at least about 900 times, at least about 950 times, or at least about 1000 times compared to the wild-type transposase domain (e.g., PB or SPB).

[00167] In certain embodiments, modified cells are used therapeutically in adoptive cell therapy.

[00168] Adoptive cell compositions that are “universally” safe for administration to any patient (not just the patient from whom they are derived) require a significant reduction or elimination of alloreactivity. To this end, dissemination cells (e.g., allogeneic cells) can be modified to disrupt the expression or function of a T cell receptor (TCR) and / or a class of major histocompatibility complex (MHC). The TCR mediates graft-versus-host (GvH) reactions, while the MHC mediates host-versus-graft (HvG) reactions. Preferably, any TCR expression and / or function is eliminated in a modified cell disclosed in this document to prevent T cell-mediated GvH that could cause subject death.Thus, in a preferred aspect, the disclosure provides a composite of pure TCR-negative allogeneic T cells (i.e., each cell in the composite expresses the endogenous TCR at such a low level that it may be undetectable or nonexistent).

[00169] The expression and / or function of MHC class I (MHC-I, Petition 870250091089, dated 06 / 10 / 2025, pp. 156 / 198 Specifically, HLA-A, HLA-B, and HLA-C (69 / 102) can be reduced or eliminated to prevent HvG and consequently improve cell engraftment in an individual. It is believed that improved engraftment results in greater cell persistence and therefore a larger therapeutic window for the individual. In some embodiments, the expression and / or function of a structural element of MHC-I, Beta-2-Microglobulin (B2M), is reduced or eliminated. Non-limiting examples of guide RNAs (gRNAs) for targeting and deleting MHC activators are disclosed in PCT Application Publication WO 2020 / 051374, which is incorporated herein by reference in its entirety for examples of gRNAs that may be used in the methods described herein.

[00170] A detailed description of non-natural chimeric stimulatory receptors, genetic modifications of endogenous sequences encoding TCR-alpha (TCR-α), TCR-beta (TCR-β) and / or Beta-2-Microglobulin (β2M) and non-natural polypeptides comprising an HLA class I histocompatibility antigen, alpha chain E polypeptide (HLA-E) is disclosed in PCT Solicitation Publication No. WO 2020 / 051374, which is incorporated herein by reference in its entirety for examples of non-natural receptors and polypeptides and other genetic modifications that may be introduced into cells disclosed herein.

[00171] Under normal conditions, full T cell activation depends on the involvement of the TCR in conjunction with a second signal mediated by one or more co-stimulatory receptors (e.g., CD28, CD2, 4-1BBL) that enhance the immune response. However, when the TCR is not present, T cell expansion is severely reduced when stimulated using standard activation / stimulation reagents, including the anti-CD3 agonist mAb. Thus, the present disclosure provides a non-natural chimeric stimulatory receptor (CSR) comprising: (a) an ectodomain comprising a Petition 870250091089, dated 06 / 10 / 2025, pp. 157 / 198 70 / 102 activation component, wherein the activation component is isolated from or derived from a first protein; (b) a transmembrane domain; and (c) an endodomain comprising at least one signal transduction domain, wherein the at least one signal transduction domain is isolated from or derived from a second protein; wherein the first protein and the second protein are not identical.

[00172] The activation component of a CSR described in this document may comprise a portion of one or more components of a T cell receptor (TCR), a component of a TCR complex, a component of a TCR co-receptor, a component of a TCR co-stimulatory protein, a component of a TCR inhibitory protein, a cytokine receptor, and a chemokine receptor to which an agonist of the activation component binds. The activation component may comprise an extracellular CD2 domain or a portion thereof to which an agonist binds.

[00173] The signal transduction domain of a CSR described in this document may comprise one or more components of a human signal transduction domain, a component of a TCR complex, a component of a TCR co-receptor, a component of a TCR co-stimulatory protein, a component of a TCR inhibitory protein, a cytokine receptor, and a chemokine receptor. The signal transduction domain may comprise a CD3 protein or a portion thereof. The CD3 protein may comprise a CD3Z domain or a portion thereof.

[00174] The endodomain of a CSR described in this document may further comprise a cytoplasmic domain. The ectodomain of a CSR may further comprise a signal peptide. The CSR may additionally comprise a transmembrane domain. The cytoplasmic domain, the signal peptide, and / or the transmembrane domain may be derived from the same protein or from different proteins. For example, the signal peptide and the domain Petition 870250091089, dated 06 / 10 / 2025, pages 158 / 198 71 / 102 transmembrane receptors may be derived from CD8 or CD28 or a combination thereof.

[00175] This disclosure also provides an unnatural CSR, in which the ectodomain comprises a modification. The modification may comprise a mutation or truncation of the amino acid sequence of the activating component when compared to a wild-type sequence of the activating component. The mutation or truncation of the amino acid sequence of the activating component may comprise a mutation or truncation of an extracellular CD2 domain or a portion thereof to which an agonist binds. The mutation or truncation of the extracellular CD2 domain may reduce or eliminate binding to natural CD58.

[00176] This disclosure also provides a nucleic acid sequence encoding any CSR disclosed in this document. This disclosure provides a transposon or vector comprising a nucleic acid sequence encoding any CSR disclosed in this document.

[00177] This disclosure provides a cell comprising any CSR disclosed in this document. This disclosure provides a cell comprising a nucleic acid sequence encoding any CSR disclosed in this document. This disclosure provides a cell comprising a vector comprising a nucleic acid sequence encoding any CSR disclosed in this document. This disclosure provides a cell comprising a transposon comprising a nucleic acid sequence encoding any CSR disclosed in this document.

[00178] This disclosure provides a composition comprising any CSR disclosed in this document. This disclosure provides a composition comprising a nucleic acid sequence that Petition 870250091089, dated 06 / 10 / 2025, pp. 159 / 198 72 / 102 encodes any CSR disclosed in this document. This disclosure provides a composition comprising a vector comprising a nucleic acid sequence encoding any CSR disclosed in this document. This disclosure provides a composition comprising a transposon comprising a nucleic acid sequence encoding any CSR disclosed in this document. This disclosure provides a composition comprising a modified cell disclosed in this document or a composition comprising a plurality of modified cells disclosed in this document.

[00179] Methods for site-specific gene integration are also provided in this document. The chimeric transposase and chimeric site-specific transposase fusion proteins provided in this document can be used to deliver a transgene to a cell and integrate the transgene at a target site. The target site can be, for example, a genomic safe harbor, i.e., a genomic site where a transgene can be integrated in a way that ensures the transgene functions predictably and does not cause alterations in the host's genomic DNA sequence. In some embodiments, the target site is a repetitive element, such as a LINE-1 or ALU sequence. Repetitive elements do not encode essential gene products, making it unlikely that an insertion will lead to detrimental changes in a cell's gene expression profile. There may be one, two, or more target sites within a repetitive element.In some embodiments, the target site is located within an intron.

[00180] Site-specific integration can be used in vitro or in vivo. An example of an in vivo application is gene therapy, which involves administering a transgene to the genomic DNA of a cell. Formulations, Dosages and Methods of Administration

[00181] This disclosure provides formulations, dosages and Petition 870250091089, dated 06 / 10 / 2025, pp. 160 / 198 73 / 102 methods for administering the compositions and cells described in this document. In one aspect, a pharmaceutical composition comprising a chimeric transposase or a chimeric site-specific transposase fusion protein described in this document and a pharmaceutically acceptable carrier is provided in this document. In another aspect, a pharmaceutical composition comprising a modified cell described in this document and a pharmaceutically acceptable carrier is provided in this document.

[00182] The disclosed pharmaceutical compositions and compounds may comprise at least one of any suitable auxiliaries, such as, without limitation, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like. Pharmaceutically acceptable auxiliaries are preferred. Non-limiting examples and methods of preparation of such sterile solutions are well known in the art, such as, without limitation, Gennaro, Ed., Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Co. (Easton, Pa.) 1990 and in the “Physician's Desk Reference”, 52nd ed., Medical Economics (Montvale, NJ) 1998. Pharmaceutically acceptable vehicles may be routinely selected that are suitable for the mode of administration, solubility and / or stability of the protein structure, fragment or variant composition, as well known in the art or as described herein.

[00183] Non-limiting examples of pharmaceutical excipients and additives suitable for use include proteins, peptides, amino acids, lipids and carbohydrates (e.g., sugars, including monosaccharides, di-, tri-, tetra- and oligosaccharides; derivatized sugars, such as alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers), which may be present individually or in combination, comprising alone or in combination 1-99.99% by weight or volume. Non-limiting examples of protein excipients include serum albumin, such as Petition 870250091089, dated 06 / 10 / 2025, pp. 161 / 198 74 / 102 Human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and similar components. Representative amino acid / protein components, which may also function as buffers, include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and similar components. Glycine is a preferred amino acid.

[00184] Non-limiting examples of carbohydrate excipients suitable for use include monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose and the like; disaccharides such as lactose, sucrose, trehalose, cellobiose and the like; polysaccharides such as raffinose, melezitose, maltodextrins, dextrans, starches and the like; and alditols such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), myo-inositol and the like. Preferably, the carbohydrate excipients are mannitol, trehalose and / or raffinose.

[00185] The compositions may also include a buffer or pH adjusting agent; typically, the buffer is a salt prepared from an organic acid or base. Representative buffers include salts of organic acids, such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris, tromethamine hydrochloride, or phosphate buffers. Preferred buffers are salts of organic acids, such as citrate.

[00186] In addition, the disclosed compositions may include polymeric excipients / additives, such as polyvinylpyrrolidones, ficolls (a polymeric sugar), dextrates (e.g., cyclodextrins, such as 2-hydroxypropyl-cyclodextrin), polyethylene glycols, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, surfactants (e.g., polysorbates, such as “TWEEN 20” and “TWEEN 80”), lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol) and chelating agents (e.g., EDTA). Petition 870250091089, dated 06 / 10 / 2025, pp. 162 / 198 75 / 102

[00187] Many known and developed methods can be used to administer therapeutically effective amounts of the compositions or pharmaceutical compositions disclosed in this document. Non-limiting examples of administration methods include bolus, buccal, infusion, intra-articular, intrabronchial, intra-abdominal, intracapsular, intracartilaginous, intracavitary, intracerebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intralesional, intramuscular, intramyocardial, intranasal, intraocular, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intratumoral, intravenous, intravesical, oral, parenteral, rectal, sublingual, subcutaneous, transdermal, or vaginal.In preferred embodiments, a composition comprising a modified cell described in this document is administered intravenously, for example, by intravenous infusion.

[00188] A composition disclosed herein may be prepared for parenteral use (subcutaneous, intramuscular, or intravenous) or any other administration, particularly in the form of liquid solutions or suspensions. For parenteral administration, a composition disclosed herein may be formulated as a solution, suspension, emulsion, particle, powder, or lyophilized powder in combination, or supplied separately, with a pharmaceutically acceptable parenteral vehicle. Formulations for parenteral administration may contain as common excipients sterile water or saline solution, polyalkylene glycols such as polyethylene glycol, vegetable oils, hydrogenated naphthalenes, and the like. Aqueous or oily suspensions for injection may be prepared using an appropriate emulsifier or humidifier and a suspending agent, according to known methods. The agents for injection or infusion may be a non-toxic diluent and not Petition 870250091089, dated 06 / 10 / 2025, pp. 163 / 198 76 / 102 is administered orally as an aqueous solution, a sterile injectable solution, or a suspension in a solvent. Water, Ringer's solution, isotonic saline solution, etc., are permitted as vehicles or solvents; sterile volatile oil may be used as a common solvent or suspension solvent. For these purposes, any type of volatile oil and fatty acid may be used, including natural, synthetic, or semi-synthetic fatty oils or fatty acids; natural, synthetic, or semi-synthetic mono-, di-, or triglycerides. Parenteral administration is known in the art and includes, without limitation, conventional means of injection, a gas-pressurized needleless injection device as described in U.S. Patent No. 5,851,198, and a laser-piercing device as described in U.S. Patent No. 5,839,446.

[00189] It may be desirable to administer the disclosed compounds to the subject over extended periods of time, for example, for periods of one week to one year following a single administration. Various dosage forms such as slow-release, depot, or implant may be used.For example, a dosage form may contain a pharmaceutically acceptable non-toxic salt of compounds that have a low degree of solubility in body fluids, for example, (a) an acid addition salt with a polybasic acid, such as phosphoric acid, sulfuric acid, citric acid, tartaric acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalene mono- or disulfonic acids, polygalacturonic acid and the like; (b) a salt with a polyvalent metallic cation, such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium and the like, or with an organic cation formed from, for example, N,N'-dibenzyl-ethylenediamine or ethylenediamine; or (c) combinations of (a) and (b), for example, a zinc tannate salt. Furthermore, the disclosed compounds, or preferably a relatively insoluble salt such as those we have just described, can be formulated into a gel, for example, an aluminum monostearate gel with... Petition 870250091089, dated 06 / 10 / 2025, pp. 164 / 198 77 / 102 for example, sesame oil, suitable for injection. Particularly preferred salts are zinc salts, zinc tannate salts, pamoate salts and the like. Another type of slow-release depot formulation for injection would contain the compound or salt dispersed for encapsulation in a slow-degrading, non-toxic and non-antigenic polymer, such as a polylactic acid / polyglycolic acid polymer, for example, as described in US Patent No. 3,773,919. The relatively insoluble compounds or, preferably, salts, such as those described above, may also be formulated in cholesterol matrix silastic pellets, particularly for use in animals. Additional slow-release, depot, or implant formulations, for example, gaseous or liquid liposomes, are known in the literature (US Patent No. 5,770,222 and “Sustained and Controlled Release Drug Delivery Systems”, JR Robinson ed., Marcel Dekker, Inc., NY, 1978). Treatment Methods

[00190] In another aspect, methods of treating a disease or disorder in a subject are provided in this document, wherein the method comprises administering to the individual a composition comprising the modified cells described in this document. The terms “individual” and “patient” are used interchangeably in this document. In preferred embodiments, the patient is human.

[00191] The modified cells can be allogeneic or autologous to the patient. In some preferred embodiments, the modified cell is an allogeneic cell. In some embodiments, the modified cell is an autologous T cell or a modified autologous CAR T cell. In some preferred embodiments, the modified cell is an allogeneic T cell or a modified allogeneic CAR T cell.

[00192] In some embodiments, the disease or disorder treated according to the methods described in this document is cancer. In some Petition 870250091089, dated 06 / 10 / 2025, pp. 165 / 198 Based on 78 / 102 findings, a treatment method described in this document may slow cancer progression and / or reduce tumor burden.

[00193] In some embodiments, the disease or disorder treated according to the methods described in this document is an autoimmune disease. In some embodiments, the autoimmune disease is autoimmune neutropenia, Guillain-Barré syndrome, epilepsy, autoimmune encephalitis, Isaacs syndrome, nevus syndrome, pemphigus vulgaris, pemphigus decidua, bullous pemphigoid, acquired epidermolysis bullosa, gestational pemphigoid, mucous membrane pemphigoid, antiphospholipid syndrome, autoimmune anemia, myasthenia gravis, autoimmune Graves' disease, thyroid eye disease (TOD), Goodpasture's syndrome, multiple sclerosis, rheumatoid arthritis, lupus, idiopathic thrombocytopenic purpura (ITP), warm autoimmune hemolytic anemia (AIHA), chronic inflammatory demyelinating polyneuropathy (CIDP), lupus nephritis, or membranous nephropathy.

[00194] The dosage of a pharmaceutical composition to be administered to a subject may vary depending on known factors, such as the pharmacodynamic characteristics of the specific agent and its mode and route of administration; age, health and weight of the recipient; nature and extent of symptoms, type of concomitant treatment, frequency of treatment and the desired effect.

[00195] In aspects where the compositions to be administered to a subject in need thereof are modified cells, as disclosed in this document, between approximately 1x10³ and approximately 1x10⁴ cells; between approximately 1x10⁴ and approximately 1x10⁵ cells; between approximately 1x10⁵ and approximately 1x10⁶ cells; between approximately 1x10⁶ and approximately 1x10⁷ cells; between approximately 1x10⁷ and approximately 1x10⁸ cells; between approximately 1x10⁸ and approximately 1x10⁹ cells; between approximately 1x10⁹ and approximately 1x10¹⁰ cells; between approximately 1x10¹⁰ and approximately 1x10¹¹ cells; between approximately 1x10¹¹ and approximately 1x10¹² cells; between Petition 870250091089, dated 06 / 10 / 2025, pp. 166 / 198 79 / 102 approximately 1x1012 and approximately 1x1013 cells, between approximately 1x1013 and approximately 1x1014 cells, between approximately 1x1014 and approximately 1x1015 cells, between approximately 1x1015 and approximately 1x1016 cells, between approximately 1x1016 and approximately 1x1017 cells, between approximately 1x1017 and approximately 1x1018 cells, between approximately 1x1018 and approximately 1x1019 cells; or between approximately 1x1019 and approximately 1x1020 cells may be administered. In some embodiments, the cells are administered in a dose between approximately 5x106 and approximately 25x106 cells.

[00196] In other embodiments, the cell dosage may depend on the person's body weight, for example, between approximately 1x10³ and approximately 1x10⁴ cells; between approximately 1x10⁴ and approximately 1x10⁵ cells; between approximately 1x10⁵ and approximately 1x10⁶ cells; between approximately 1x10⁶ and approximately 1x10⁷ cells; between approximately 1x10⁷ and approximately 1x10⁸ cells; between approximately 1x10⁸ and approximately 1x10⁹ cells; between about 1x10⁹ and about 1x10¹⁰ cells, between about 1x10¹⁰ and about 1x10¹¹ cells, between about 1x10¹¹ and about 1x10¹² cells, between about 1x10¹² and about 1x10¹³ cells, between about 1x10¹³ and about 1x10¹⁴ cells, between about 1x10¹⁴ and about 1x10¹⁵ cells, between about 1x10¹⁵ and about 1x10¹⁶ cells, between about 1x10¹⁶ and about 1x10¹⁷ cells, between about 1x10¹⁷ and about 1x10¹⁸ cells, between about 1x10¹⁸ and about 1x10¹⁹ cells; Between approximately 1x10¹⁹ and approximately 1x10²⁰ cells can be administered per kg of the individual's body weight.

[00197] A more detailed description of excipients, formulations, dosages and pharmaceutically acceptable methods of administration of the disclosed compositions and pharmaceutical compositions is published in Publication No. PCT WO 2019 / 049816.

[00198] The transposon domains and fusion proteins provided in this document can be used to administer gene therapy. Gene therapy typically involves administering a transgene to the genomic DNA of a cell. Usually, the transgene replaces Petition 870250091089, dated 06 / 10 / 2025, pp. 167 / 198 80 / 102 a gene that has undergone mutation or is not adequately expressed in the cell. The fusion proteins, transposase domains, and complexes described in this document can be used to deliver a therapeutic transgene to a cell and integrate the transgene into a target site. In some embodiments, a treatment method comprises introducing into the cell the fusion protein of any of claims 1 to 13 and a transposon, wherein the transposon comprises, in the order of 5' to 3': a 5'ITR, the transgene, and a 3'ITR. Kits

[00199] In another aspect, a kit is provided in this document comprising a cell line that has been engineered to comprehend a modified target site for an SPB or PBx provided in this document within its genome, preferably in a highly expressed genomic region. The kit may further comprise a composition comprising one or more SPB or PBx transposase domains or fusion proteins described in this document. In some embodiments, the cell line is a T cell line. TERMS

[00200] As used throughout the disclosure, the singular forms “a”, “and”, and “the” include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to “a method” includes a plurality of such methods, and reference to “a dose” includes reference to one or more doses and equivalents thereof known to experts in the field, and so forth.

[00201] The term “about” or “approximately” means within an acceptable range of error for the specific value, as determined by one skilled in the art, which will depend in part on how the value is measured or determined, for example, the limitations of the system of Petition 870250091089, dated 06 / 10 / 2025, pp. 168 / 198 81 / 102 measurement. For example, “approximately” may mean within 1 or more standard deviations. Alternatively, “approximately” may mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given value. Alternatively, particularly with regard to biological systems or processes, the term may mean within an order of magnitude, preferably within 5 times, and more preferably within 2 times, of a value. When specific values ​​are described in the application and claims, unless otherwise indicated, the term “approximately”, meaning within an acceptable error range for the specific value, shall be assumed.

[00202] Disclosure provides compositions of isolated or substantially purified polynucleotides or proteins. An isolated or purified polynucleotide or protein, or biologically active portion thereof, is substantially or essentially free of components that normally accompany or interact with the polynucleotide or protein as found in its natural environment. Thus, an isolated or purified polynucleotide or protein is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Ideally, an isolated polynucleotide is free of sequences (preferably protein-coding sequences) that naturally flank the polynucleotide (i.e., sequences located at the 5' and 3' ends of the polynucleotide) in the genomic DNA of the organism from which the polynucleotide is derived.For example, in several respects, the isolated polynucleotide may contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequence that naturally flanks the polynucleotide in the genomic DNA of the cell from which the polynucleotide is derived. A protein that is substantially free of cellular material includes protein preparations with less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of protein. Petition 870250091089, dated 06 / 10 / 2025, pp. 169 / 198 82 / 102 contaminant. When the disclosure protein or its biologically active portion is produced recombinantly, the ideal culture medium contains less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or non-protein chemicals of interest.

[00203] The disclosure provides fragments and variants of the disclosed DNA sequences and proteins encoded by those DNA sequences. As used throughout the disclosure, the term fragment refers to a portion of the DNA sequence or a portion of the amino acid sequence and therefore to the protein encoded by it. Fragments of a DNA sequence comprising coding sequences may encode protein fragments that retain the biological activity of the native protein and therefore DNA recognition or binding activity to a target DNA sequence, as described in this document. Alternatively, fragments of a DNA sequence that are useful as hybridization probes generally do not encode proteins that retain biological activity or do not retain promoter activity.Thus, fragments of a DNA sequence can range from at least about 20 nucleotides, to about 50 nucleotides, to about 100 nucleotides, and up to the full-length polynucleotide of the fragment.

[00204] The term "comprising" is intended to mean that the compositions and methods include the elements described, but do not exclude others. "Consist essentially of" when used to define compositions and methods means excluding other elements of any essential significance to the combination when used for the intended purpose. Thus, a composition that essentially consists of the elements as defined in this document would not exclude residual contaminants or inert carriers. "Consist of" means excluding more than trace elements from other substantial ingredients and steps of the method. Aspects defined by each of these transitional terms are within the scope of this disclosure. Petition 870250091089, dated 06 / 10 / 2025, pp. 170 / 198 83 / 102

[00205] As used in this document, expression refers to the process by which polynucleotides are transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, the expression may include mRNA splicing in a eukaryotic cell.

[00206] “Gene expression” refers to the conversion of the information contained in a gene into a gene product. A gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, shRNA, microRNA, structural RNA, or any other type of RNA) or a protein produced by the translation of an mRNA. Gene products also include RNAs that are modified by processes such as encapsulation, polyadenylation, methylation, and editing, and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristylation, and glycosylation.

[00207] The term “operationally linked” or its equivalents (e.g., “operatively linked”) means that two or more molecules are positioned relative to each other in such a way that they have the ability to interact to affect a function attributable to one or both molecules or a combination thereof. In the context of nucleic acids, a promoter may be operatively linked to a nucleotide sequence encoding a transposable domain or fusion protein described in this document, placing the expression of the nucleotide sequence under the control of the promoter.

[00208] Non-covalently linked components and methods of manufacturing and using non-covalently linked components are disclosed. The various components can take a variety of different forms, as described in this document. For example, non-proteins Petition 870250091089, dated 06 / 10 / 2025, pp. 171 / 198 84 / 102 covalently linked (i.e., operatively linked) components can be used to allow temporary interactions that avoid one or more problems in the technique. The ability of non-covalently linked components, such as proteins, to associate and dissociate allows functional association only or primarily in circumstances where such association is necessary for the desired activity. The linkage can last long enough to allow the desired effect.

[00209] A method is disclosed for targeting proteins to a specific locus in an organism's genome. The method may comprise the steps of providing a DNA localization component and providing an effector molecule, wherein the DNA localization component and the effector molecule have the ability to bind operatively via a non-covalent bond.

[00210] A “target site” or “target sequence” is a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule will bind, provided there are sufficient conditions for binding.

[00211] The terms nucleic acid or oligonucleotide or polynucleotide refer to at least two nucleotides covalently linked together. The representation of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid may also encompass the complementary strand of a single strand shown. A nucleic acid disclosure also encompasses substantially identical nucleic acids and their complements that retain the same structure or encode the same protein.

[00212] The nucleic acids in the disclosure can be single-stranded or double-stranded. The nucleic acids in the disclosure can contain double-stranded sequences, even when most of the molecule is single-stranded. The nucleic acids in the disclosure can contain single-stranded sequences, even Petition 870250091089, dated 06 / 10 / 2025, pp. 172 / 198 85 / 102 when the majority of the molecule is double-stranded. Disclosure nucleic acids may include genomic DNA, cDNA, RNA, or a hybrid thereof. Disclosure nucleic acids may contain combinations of deoxyribonucleotides and ribonucleotides. Disclosure nucleic acids may contain combinations of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. Disclosure nucleic acids may be synthesized to comprise non-natural amino acid modifications. Disclosure nucleic acids may be obtained by chemical synthesis methods or by recombinant methods.

[00213] The nucleic acids in the disclosure, whether their entire sequence or any part thereof, may not occur naturally. The nucleic acids in the disclosure may contain one or more mutations, substitutions, deletions, or insertions that do not occur naturally, making the entire nucleic acid sequence unnatural. The nucleic acids in the disclosure may contain one or more duplicated, inverted, or repeated sequences, the resulting sequence of which does not occur naturally, making the entire nucleic acid sequence unnatural. The nucleic acids in the disclosure may contain modified, artificial, or synthetic nucleotides that do not occur naturally, making the entire nucleic acid sequence unnatural.

[00214] Given the redundancy in the genetic code, a plurality of nucleotide sequences can code for any specific protein. All such nucleotide sequences are covered in this document.

[00215] As used throughout the disclosure, the term promoter refers to a synthetic or naturally derived molecule that has the ability to confer, activate, or enhance the expression of a nucleic acid in a cell. A promoter may comprise one or more specific transcriptional regulatory sequences to further enhance expression. Petition 870250091089, dated 06 / 10 / 2025, pp. 173 / 198 86 / 102 and / or alter its spatial and / or temporal expression. A promoter may also comprise distal enhancer or repressor elements, which may be located up to several thousand base pairs from the transcription start site. A promoter can be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. A promoter may regulate the expression of a genetic component constitutively or differentially with respect to the cell, tissue, or organ in which the expression occurs, or with respect to the developmental stage at which the expression occurs, or in response to external stimuli such as physiological stresses, pathogens, metal ions, or inducing agents.Representative examples of promoters include the bacteriophage T7 promoter, the bacteriophage T3 promoter, the SP6 promoter, the lac operator-promoter, the tac promoter, the SV40 late promoter, the SV40 early promoter, the RSV-LTR promoter, the CMV IE promoter, the EF-1 Alpha promoter, the CAG promoter, the SV40 early promoter or the SV40 late promoter, and the CMV IE promoter.

[00216] As used throughout the disclosure, the term vector refers to a nucleic acid sequence containing an origin of replication. A vector may be a viral vector, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. A vector may be a DNA or RNA vector. A vector may be a self-replicating extrachromosomal vector and, preferably, is a DNA plasmid. A vector may comprise a combination of an amino acid with a DNA sequence, an RNA sequence, or a DNA and RNA sequence.

[00217] A conservative amino acid substitution, that is, the substitution of an amino acid for a different amino acid with similar properties (e.g., hydrophilicity, degree and distribution of charged regions), is recognized in the art as typically involving a small change. These small changes can be identified, in Petition 870250091089, dated 06 / 10 / 2025, pp. 174 / 198 87 / 102 part, considering the hydropathic index of amino acids, as understood in the art. Kyte et al., J. Mol. Biol. 157: 105-132 (1982). The hydropathic index of an amino acid is based on consideration of its hydrophobicity and charge. Amino acids with similar hydropathic indices can be substituted and still maintain protein function. In one aspect, amino acids with hydropathic indices of ±2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that would result in proteins retaining biological function. A consideration of the hydrophilicity of amino acids in the context of a peptide allows the calculation of the highest local average hydrophilicity of that peptide, a useful measure that correlates well with antigenicity and immunogenicity. US Patent No. 4,554,101, incorporated in its entirety herein by reference.

[00218] The substitution of amino acids with similar hydrophilicity values ​​can result in peptides that retain biological activity, for example, immunogenicity. Substitutions can be made with amino acids with hydrophilicity values ​​within ±2 of each other. Both the hydrophobicity index and the hydrophilicity value of amino acids are influenced by the specific side chain of that amino acid. According to this observation, it is understood that amino acid substitutions that are compatible with biological function depend on the relative similarity of the amino acids and, particularly, on the side chains of these amino acids, as revealed by hydrophobicity, hydrophilicity, charge, size, and other properties.

[00219] As used in this document, “conservative” amino acid substitutions can be defined as set out in Tables 1, 2, and 3 below. In some respects, fusion polypeptides and / or nucleic acids encoding such fusion polypeptides include conservative substitutions that have been introduced by modification of polynucleotides that Petition 870250091089, dated 06 / 10 / 2025, pp. 175 / 198 88 / 102 encode disseminating polypeptides. Amino acids can be classified according to their physical properties and their contribution to the secondary and tertiary structure of the protein. A conservative substitution is the replacement of one amino acid with another amino acid that has similar properties. Exemplary conservative substitutions are presented in Table 1. Table 1: Conservative Substitutions I Side chain characteristics: Aliphatic amino acid: Nonpolar; GAPILVF: Polar - uncharged; CSTMNQ: Polar - charged; DEKR: Aromatic; HFWY: Other; NQDE

[00220] Alternatively, the conservative amino acids can be grouped as described in Lehninger, (Biochemistry, Second Edition; Worth Publishers, Inc. NY, NY (1975), pages 71-77) as set out in Table 2. Table 2: Conservative Substitutions II Side chain characteristic Amino acid Nonpolar (hydrophobic) Aliphatic: ALIVP Aromatic: FWY Sulfur-containing: M Limiting: GY Polar uncharged Hydroxyl: STY Amides: NQ Sulfhydryl: C Limiting: GY Positively charged (Basic): KRH Negatively charged (Acidic): DE

[00221] Alternatively, exemplary conservative substitutions are shown in Table 3. Table 3: Conservative Substitutions III Original Residue Exemplary Substitution Ala (A) Val Leu Ile Met Arg (R) Lys His Asn (N) Gln Petition 870250091089, of 06 / 10 / 2025, p. 176 / 198 89 / 102 Original Residue Substitution Exemplar Asp (D) Glu Cys (C) Ser Thr Gln (Q) Asn Glu (E) Asp Gly (G) Ala Val Leu Pro His (H) Lys Arg Ile (I) Leu Val Met Ala Phe Leu (L) Ile Val Met Ala Phe Lys (K) Arg Leu Met Alar (K) Tyr Ile Pro (P) Gly Ala Val Leu Ile Ser(S) Thr Thr (T) Ser Trp (W) Tyr Phe Ile Tyr (Y) Trp Phe Thr Ser Val (V) Ile Leu Met Ala

[00222] Disclosure polypeptides and proteins, whether their entire sequence or any part thereof, may not occur naturally. Disclosure polypeptides and proteins may contain one or more mutations, substitutions, deletions, or insertions that do not occur naturally, making the entire amino acid sequence unnatural. Disclosure polypeptides and proteins may contain one or more duplicated, inverted, or repeated sequences, the resulting sequence of which does not occur naturally, making the entire amino acid sequence unnatural. Disclosure polypeptides and proteins may contain modified, artificial, or synthetic amino acids that do not occur naturally, making the entire amino acid sequence unnatural.

[00223] As used throughout the disclosure, the identity between two sequences can be determined using the standalone executable program of the BLAST mechanism to detonate two sequences (bl2seq), which can be retrieved from the National Center for Biotechnology Information (NCBI) FTP site, using the default parameters (Tatusova and Madden, FEMS). Petition 870250091089, dated 06 / 10 / 2025, pp. 177 / 198 90 / 102 Microbiol Lett., 1999, 174, 247-250; which is incorporated herein by reference in its entirety). The terms identical or identity, when used in the context of two or more nucleic acids or polypeptide sequences, refer to a specified percentage of residues that are the same in a specified region of each of the sequences. In some embodiments, sequence identity is determined over the entire length of a sequence. The percentage can be calculated by ideally aligning the two sequences, comparing them in the specified region, determining the number of positions where the identical residue occurs in both sequences to yield the number of matching positions, dividing the number of matching positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the sequence identity percentage.In cases where the two sequences have different lengths or the alignment produces one or more staggered ends and the specified comparison region includes only a single sequence, the residues of the single sequence are included in the denominator but not in the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity can be determined manually or using a computer sequence algorithm such as BLAST or BLAST 2.0.

[00224] In certain embodiments, if a sequence has a certain sequence identity (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) for a given SEQ ID NO, the sequence and the sequence of the SEQ ID NO will have the same length. In certain embodiments, if a sequence has a certain sequence identity (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) with a given SEQ ID NO, the sequence and the sequence of the SEQ ID NO differ only due to conservative amino acid substitutions. Petition 870250091089, dated 06 / 10 / 2025, pp. 178 / 198 91 / 102

[00225] As used throughout the disclosure, the term endogenous refers to the nucleic acid or protein sequence naturally associated with a target gene or a host cell into which it is introduced.

[00226] As used throughout the disclosure, the term exogenous refers to a nucleic acid or protein sequence not naturally associated with a target gene or a host cell into which it is introduced, including unnatural multiple copies of a natural nucleic acid, for example, a DNA sequence or a natural nucleic acid sequence located at a site in the genome that does not occur naturally.

[00227] Disclosure provides methods for introducing a polynucleotide construct comprising a DNA sequence into a host cell. By introduction is meant presenting the polynucleotide construct to the cell in such a way that the construct gains access to the interior of the host cell. Disclosure methods do not depend on a specific method for introducing a polynucleotide construct into a host cell, only that the polynucleotide construct gains access to the interior of a host cell. Methods for introducing polynucleotide constructs into bacteria, plants, fungi, and animals are known in the art, including, without limitation, stable transformation methods, transient transformation methods, and virus-mediated methods. Examples

[00228] The examples in this section are provided for illustration purposes only and are not intended to limit the invention. Example 1: PiggyBac (“PB”)-MosI Chimeric Transposase Construct

[00229] Chimeric PB:MosI transposases were created by deleting the CRD domain from the PB transposase and replacing it with DNA-binding domain(s) from the MosI transposase. PB CRD consists of residues 553594 of the 594-amino acid PB transposase protein (see SEQ ID NO: 2). The Petition 870250091089, dated 06 / 10 / 2025, pp. 179 / 198 The 92 / 102 CRD domain was attached to the remainder of the transposase via a linker sequence spanning residues 535-552 of the PB transposase sequence. The PB CRD domain can be truncated from the C-terminal of the PB transposase anywhere within the linker sequence. Two fusion points within the linker sequence were selected as exemplary sites. In the first fusion design, the C-terminal of the PB transposase was truncated from residues 545-594 (SEQ ID NO: 3) resulting in the S544 fusion point (S544 PB) to construct the S544 PB-MosI chimera. In the second fusion design, the C-terminal of the PB transposase was truncated from residues 553-594 (SEQ ID NO: 4) resulting in the V552 fusion point (V552 PB) to construct the chimeric transposases V552 PB-MosI.

[00230] The C-terminal truncated S544PB and V552PB sequences were fused to the DNA-binding domain(s) of MosI. MosI binds with its C-terminal proximal to the transposon ends, unlike PB, which binds with its N-terminal proximal to the transposon ends. The N-terminal of MosI contains two DNA-binding domains, which are fused to the catalytic domain of the transposase. The first DNA-binding domain spans the first approximately 58 residues of the protein (SEQ ID NO: 8) while the two DNA-binding domains span the first approximately 111 residues of the protein (SEQ ID NO: 6).

[00231] TAL-piggyBac transposase fusion proteins comprising N-terminal deleted piggyBac transposase sequences and N-terminal integration-defective piggyBac transposase targeting GFP were produced as described in International Patent Application Publication No. PCT / US2022 / 77549. Briefly, two pairs of TAL arrays targeting sequences in the GFP coding sequence were designed, and each TAL array containing nine 34-amino acid repeats followed by a 20-amino acid “half” repeat was synthesized flanked by sites of Petition 870250091089, dated 06 / 10 / 2025, pages 180 / 198 93 / 102 BsmBI type IIS restriction. This allowed cloning of each TAL inframe array with the remainder of the open reading frame with the piggyBac transposase sequence deleted at the N-terminus in the expression plasmid to generate Right GFP1R TAL-PBx (SEQ ID NO: 40). The PBx transposase sequence was replaced with chimeric PB:MosI transposase sequences to construct chimeric site-specific transposase fusion proteins, specifically targeting GFP.

[00232] Chimeric sequences of the PB:MosI transposase were constructed by joining residues 3-58 of MosI to the S544 PBx transposase using a GGGGS linker sequence (SEQ ID NO: 86) and inserting it in place of the PBx transposase sequence to create GFP1R TAL-PB:S544-MosI58 (SEQ ID NO: 41). Similarly, residues 3-58 of MosI were fused to the V552 PBx transposase sequence using a GGGGS linker (SEQ ID NO: 86) and inserting it in place of the PBx transposase sequence to create GFP1R TAL-PB:V552-MosI-58 (SEQ ID NO: 42). Furthermore, residues 3-111 of MosI were fused to S544 PB using a GGGGS linker (SEQ ID NO: 86) and inserted in place of the PBx transposase sequence to create GFP1R TALPB:S544-MosI-111 (SEQ ID NO: 43). Finally, residues 3-111 of MosI were fused to PiggyBac in V552 PBx using a GGGGS linker (SEQ ID NO: 86) and inserted in place of the PBx transposase sequence to create GFP1R TAL-ssSPB:V552-MosI-111 (SEQ ID NO: 44). Example 2: Construct of Chimeric PB:MosI Inverted Terminal Repeats (ITRs)

[00233] This example illustrates exemplary methods for constructing chimeric ITR PB:MosI sequences.

[00234] The chimeric PB:MosI transposases prepared in Example 1 each comprise the PB DNA-binding and dimerization domain (DDBD) fused to the MosI DNA-binding domain(s). Thus, Petition 870250091089, dated 06 / 10 / 2025, pp. 181 / 198 94 / 102 chimeric PB:MosI ITR sequences were constructed comprising the PB DDBD binding site fused to the MosI DNA binding domain binding site.

[00235] The 35 bp LE PB ITR sequence (SEQ ID NO: 12) was truncated after position 16, deleting the 19 bp CRD binding site. To this end, a 23 bp MosI sequence (SEQ ID NO: 13) containing the binding sites for the two MosI DNA-binding domains was fused in reverse orientation to create LE PB:MosI ITR (SEQ ID NO: 15). In addition, alternative versions of the chimeric ITR comprising various lengths of spacers between the PB and MosI ITR domains in the chimeric protein were designed to test the effect of ligand length. In one case, one bp of the MosI binding site was deleted adjacent to the DDBD binding site to create LE PB:MosI ITR-1 (SEQ ID NO: 16). In addition, one or two bp of DNA were added between the MosI binding site and the DDBD binding site to create LE PB:MosI ITR+1 (SEQ ID NO: 17) and LE PB:MosI ITR+2 (SEQ ID NO: 18).The 63bp RE ITR (SEQ ID NO: 65) was similarly modified to create RE PB:MosI ITR (SEQ ID NO: 19), RE PB:MosI ITR-1 (SEQ ID NO: 20), RE PB:MosI ITR+1 (SEQ ID NO: 21), RE PB:MosI ITR+2 (SEQ ID NO: 22).

[00236] Chimeric PB:MosI transposases and chimeric ITR sequences were analyzed for their relative excision activity. Example 3: Methods for Measuring the Excision Activity of Chimeric PB-MosI Transposases Using Chimeric PB:MosI ITRs

[00237] This example describes an assay designed to measure the excision activity of chimeric PB-MosI transposases and chimeric ITRs. In this assay, each of the chimeric PB-MosI transposases prepared in Example 1 was co-administered to cells along with a reporter transposon construct, in which the transposon comprises a DNA nucleotide sequence encoding a non-functional GFP in which the sequence Petition 870250091089, dated 06 / 10 / 2025, pp. 182 / 198 95 / 102 of the coding sequence was interrupted by an intervening piece of DNA flanked by TTAA sequences and a set of inverted terminal repeat (ITR) sequences prepared in Example 2. The TTAA and ITR sequences serve as recognition sites for the chimeric transposase PB:MosI, and if the chimeric transposase possesses excision activity, the intervening DNA will be excised, restoring the intact and complete coding sequence of the GFP gene. Thus, chimeric transposases that have transposase activity for constructs comprising the cognate chimeric ITR sequence produce GFP-positive cells in that assay that can be identified and quantified by FACS.

[00238] In summary, the GFP reporter system comprises an EF1a promoter (SEQ ID NO: 45) that triggers expression of a GFP reporter (SEQ ID NO: 46) followed by an SV40 polyadenylation sequence (SEQ ID NO: 47). The GFP reporter is interrupted by a transposon in a TTAA sequence, breaking it into the first GFP section (SEQ ID NO: 48) and the second GFP section (SEQ ID NO: 49). The interrupted GFP reporters for each chimeric ITR of Example 2 were constructed containing either a PB:MosI ITR transposon (SEQ ID NO: 50), a PiggyBac:MosI ITR-1 transposon (SEQ ID NO: 51), a PiggyBac:MosI ITR+1 transposon (SEQ ID NO: 52), or a PiggyBac:MosI ITR 2 transposon (SEQ ID NO: 53).

[00239] Using the GFP reporter system, the excision activity of the GFPR1 TAL-PB:S544-MosI-58, GFP1R TALPB:V552-MosI-58, GFPR1 TAL-PB:S544-MosI-111, and GFP1R TAL-ssSPB:V552MosI-111 transposases described in Example 1 was determined using each of the chimeric PB:MosI ITRs prepared in Example 2. On Day 0, each reporter was cotransfected into HEK293T cells with each chimeric transposase. Briefly, 120,000 cells were placed in 24-well plates in 500 µl of DMEM medium supplemented with 10% by volume of FBS one day prior. Petition 870250091089, dated 06 / 10 / 2025, pp. 183 / 198 96 / 102 of the transfection. On day 1, 50 ng of the transposase expression vector were combined with 450 ng of the reporter and transfected using 1 μl of JetPrime transfection reagent (Polyplus Transfection) according to the manufacturer's instructions. One day after transfection, the percentage of GFP-positive cells was measured by flow cytometry. The results are shown in Table 4. Table 4 Percentage of Cells Positive for GFP (Transposon Excision) PB:MosI ITR-1 PB:MosI ITR PB:MosI ITR+1 PB:MosI ITR+2 Rep 1 Rep 2 Average Rep 1 Rep 2 Average Rep 1 Rep 2 Average Rep 1 Rep 2 Average S544MosI58 1.40 1.44 1.42 7.71 6.98 7.35 2.84 2.78 2.81 2.82 2.85 2.84 S544MosI111 1.07 0.84 0.96 6.44 6.64 6.54 2.75 2.36 2.56 2.42 2.75 2.59 V552MosI58 0.96 0.77 0.87 4.92 4.87 4.90 2.34 2.83 2.59 4.00 3.76 3.88 V552MosI111 0.86 0.69 0.78 4.38 4.35 4.37 3.23 3.10 3.17 2.49 2.65 2.57 Without Transposase 0.16 0.08 0.12 0.06 0.08 0.07 0.13 0.13 0.13 0.05 0.07 0.06

[00240] As shown in Table 4, chimeric transposases with MosI inserted at the S544 melting point resulted in greater transposon excision activity than those with MosI inserted at the V552 melting point. Furthermore, greater transposon excision activity was observed with reporters containing PB:MosI ITRs instead of ITRs with -1, 1, or 2 spacing.

[00241] The chimeric ITRs tested in Table 4 contain a 23 bp sequence comprising the binding sites for both MosI DNA-binding domains. Since the first 58 MosI residues contain a single DNA-binding domain, a series of truncated ITR sequences were constructed in which the MosI portion of the ITR comprises a binding site for only the single domain.

[00242] In a second experiment, the MosI binding site Petition 870250091089, dated 06 / 10 / 2025, pp. 184 / 198 97 / 102 was shortened from 23 bp to 14 bp to create the left-end short LE PB:MosI ITR (SEQ ID NO: 54) and the right-end short RE PB:MosI ITR (SEQ ID NO: 55). These shorter ITRs were used to create a disrupted GFP reporter, as described in Example 1, using the short PB:MosI ITR transposon (SEQ ID NO: 56). The original short chimeric ITR reporters were tested along with the S544, S544-MosI-58, and S544-MosI-111 chimeric melting point transposases using the GFP system. Reporter above. The results are shown in Table 5. Table 5 Percentage of GFP-Positive Cells (Transposon Excision) PiggyBac:MosI ITR PiggyBac:MosI ITR short Rep1 Rep2 Average Rep1 Rep2 Average S544-MosI-58 3.57 4.10 3.84 2.08 2.29 2.19 S544-MosI-111 3.97 3.80 3.89 2.36 1.81 2.09 No Transposase 0.07 0.05 0.06 0.08 0.03 0.05

[00243] As shown in Table 5, the original and short ITRs resulted in functional transposon excision activity; however, the original 23 bp version resulted in higher activity for chimeric transposases comprising the MosI fusions of residue 3-58 or residue 3-111, as observed by the percentage of GFP-positive cells. Example 4: Construction and Analysis of Chimeric TAL-PB:MosI Transposases Designed for Site-Specific Transposition of a Small Transposon into LINE-1 Elements

[00244] This example illustrates the construction of chimeric TAL-PB:MosI transposase fusion protein compositions that are useful in methods to achieve site-specific transposition at a specific target locus, LINE1.

[00245] LINE1-specific TAL DNA-binding domains have been previously linked to piggyBac transposase sequences Petition 870250091089, dated 06 / 10 / 2025, pages 185 / 198 98 / 102 deleted at the N-terminus to construct LINE1 TAL-ssSPB fusion proteins (e.g., left; SEQ ID NO: 57; right SEQ ID NO: 58). Similar to the construction of GFP-targeted chimeric TAL-PB:MosI transposase fusion proteins, the left and right LINE1 TAL-ssSPB fusion proteins were converted into site-specific chimeric PB:MosI transposases by replacing the PB transposase sequence with a PB:MosI transposase, as described above in Example 1. Two versions of the left LINE1 L2 TAL-PB:MosI transposase were created using the S544 fusion point and the 3-58 residue MosI fragment (SEQ ID NO: 59) or the 3-111 residue MosI fragment (SEQ ID NO: 60). Similarly, two versions of the correct LINE1 R2.2 TAL-PB:MosI transposases were created using the S544 melting point and the 3-58 residue MosI fragment (SEQ ID NO: 61) or the 3-111 residue MosI fragment (SEQ ID NO: 62).A small 365 bp transposon comprising minimal PB ITRs (SEQ ID NO: 63) and a small 353 bp transposon comprising chimeric PB:MosI ITRs (23 bp) (SEQ ID NO: 50) from Example 2 were cloned into a 4.5 kb donor vector. On day 0, 450 ng of each transposon donor were cotransfected into 120,000 HEK293T cells (plated one day prior) along with a total of 50 ng of the LINE1 TAL-PB pair using 1 pl of JetPrime transfection reagent (Polyplus Transfection) according to the manufacturer's instructions. On day 3, genomic DNA was collected from transposed cells, and specific transposon integration at target sites in direct orientations was quantified by digital droplet PCR (ddPCR) to determine the number of integrations per haploid genome. The results are shown in Table 6. Table 6 Integrations by haploid genome PB:MosI ITR ITR Minimum PB Rep1 Rep2 Average Rep1 Rep2 Average TAL-PB-S544-MosI-58 1.66 1.71 1.69 0.95 1.00 0.98 Petition 870250091089, dated 06 / 10 / 2025, pp. 186 / 198 99 / 102 Integrations by haploid genome PB:MosI ITR ITR Minimum PB Rep1 Rep2 Average Rep1 Rep2 Average TAL-PB-S544-MosI-111 1.99 1.94 1.97 0.74 0.74 0.74 TAL-ssSPB 1.03 1.07 1.05 2.91 2.40 2.66

[00246] As shown in Table 6, the chimeric version of 3-111 MosI residues of site-specific chimeric PB:MosI transposases resulted in higher edit rates than the chimeric version of 3-58 MosI PB:MosI residues. Each of the two chimeric transposases catalyzed greater site-specific transposition of the transposon using chimeric PB:MosI ITRs than of the transposon comprising non-chimeric PB minimal ITRs. The non-chimeric TAL-ssSPB transposase catalyzed greater site-specific transposition of the transposon using non-chimeric PB minimal ITRs than of the transposon comprising chimeric PB:MosI ITRs. These data suggest that chimeric and non-chimeric transposases can be used as site-specific orthogonal transposition systems to simultaneously deliver transposons to two unique genetic loci, one target site with a chimeric PB:MosI ITR target and the other target site with a wild-type PB minimal ITR. Example 5: Site-Specific Transposition of Large-Cargo Transposons at a Specific Genetic Locus Using Transposases PB:MosI Chimerics

[00247] This example illustrates site-specific transposition at a specific target locus, LINE-1, of transposons comprising a large payload of DNA.

[00248] The chimeric transposases TAL-PB:MosI constructed in Example 4 and the control transposase TAL-ssSPB were analyzed for their ability to specifically transpose a transposon comprising a large payload of DNA into LINE1 elements. Petition 870250091089, dated 06 / 10 / 2025, pages 187 / 198 100 / 102

[00249] The transposon donor nanoplasmid comprising a PB transposon containing, from the 5' to 3' direction: a first TTAA sequence, a 309 bp fragment containing the 5' ITR of PB and part of the UTR, a “large payload” consisting of an EF1a promoter (SEQ ID NO: 45), a puromycin resistance gene, a 2A peptide, and a GFP reporter, followed by a 238 bp fragment containing the 3' ITR of PB and part of the UTR, and a second TTAA sequence (SEQ ID NO: 64). This donor plasmid was used to analyze the wild-type PB control TAL-ssSPB and a negative control PB transposase comprising mutations that render transposase integration defective.

[00250] For analysis of chimeric TAL-PB:MosI transposases, the transposon donor nanoplasmid was modified to replace the 309 bp ITR / UTR LE and the 238 bp ITR / UTR RE with the chimeric PB:MosI(23 bp) ITRs of SEQ ID NO: 1 and SEQ ID NO: 19). The results are shown in Table 7. Table 7 Integrations by haploid genome PB:MosI ITR ITR Total PB TAL-PB:MosI-S544-MosI-58 0.87 0.31 TAL-PB:MosI-S544-MosI-111 1.76 0.33 TAL-ssSPB 0.36 0.95 PBx (Defective integration) 0.02 0.01

[00251] As shown in Table 7, the two chimeric and non-chimeric transposases functioned as orthogonal systems, showing a high degree of specificity for their respective ITRs. The chimeric TAL-PB:MosI version of 3-111 residues of the chimeric PB:MosI transposase resulted in higher edit rates than the chimeric TAL-PB:MosI version of 3-58 residues, as well as the non-chimeric version, TAL-ssSPB. As a control Petition 870250091089, dated 06 / 10 / 2025, pages 188 / 198 101 / 102 negative, the PBx transposase was defective in its integration, not comprising TAL DNA targeting domains, resulting only in background integration levels. Example 6: Construction of Chimeric PB:MosI Transposases Comprising Hyperactive PB (“SPB”) Mutations that Increase Transposition Frequency

[00252] This example illustrates methods for constructing chimeric SPB:MosI transposases and analyzing integration and excision activity. In this example, SPB with an N-terminal NLS (SEQ ID NO: 5) was modified as described above to create chimeric SPB:MosI transposases.

[00253] More specifically, residues 3-58 of MosI (SEQ ID NO: 8) were fused to SPB at S544 to create SPB:MosI-S544-58 (SEQ ID NO: 9) and residues 3-111 of MosI (SEQ ID NO: 6) were fused to SPB at S544 to create SPB:MosI-S544-111 (SEQ ID NO: 7). The chimeric SPB:MosI transposases were tested using the interrupted GFP Reporter system containing the PB:MosI ITR transposon (SEQ ID NO: 50), the short PB:MosI ITR transposon (SEQ ID NO: 56), or the minimal PB ITR transposon (SEQ ID NO: 63). On Day 0, each reporter construct was cotransfected into HEK293T cells with each chimeric PB:MosI transposase. Briefly, 120,000 cells were placed in 24-well plates in 500 µl DMEM supplemented with 10% by volume of FBS one day before transfection. On Day 0, 10 ng of the transposase expression vector were combined with 490 ng of the reporter and transfected using 1 µl of JetPrime transfection reagent (PolyPlus Transfection).On day 1, the percentage of GFP-positive cells was measured by flow cytometry. The results are shown in Table 8. Petition 870250091089, dated 06 / 10 / 2025, pages 189 / 198 102 / 102 Table 8 Percentage of Cells Positive for GFP (Transposon Excision) PB:MosI ITR PB:MosI ITR short ITR Minimum PB Rep 1 Rep 2 Rep 3 Average Rep 1 Rep 2 Rep 3 Average Rep 1 Rep 2 Rep 3 Average SPB: MosIS54458 19.2 20.0 18.5 19.2 21.8 22.7 24.3 22.9 1.4 1.0 1.0 1.1 SPB: MosIS544111 17.0 17.0 16.7 16.9 7.2 7.8 7.5 7.5 0.1 0.1 0.1 0.1 SPB 0.1 0.2 0.3 0.2 0.3 0.4 0.5 0.4 52.4 49.2 50.8 50.8

[00254] As shown in Table 8, the chimeric SPB:MosI transposases catalyzed transposon excision, measured by the percentage of GFP-positive cells, for transposons comprising chimeric PB:MosI ITRs, but not for transposons comprising non-chimeric minimal PB ITRs. The non-chimeric SPB transposase catalyzed transposon excision for non-chimeric minimal PB ITRs, but not for transposons with chimeric PB:MosI ITRs, demonstrating the specificity of the PB:MosI transposase / chimeric PB:MosI ITR combination. Petition 870250091089, dated 06 / 10 / 2025, pages 190 / 198

Claims

1 / 6 Claims 1. A chimeric transposase, characterized by comprising, in N-terminal to C-terminal order: (i) a target-specific DNA-binding domain, (ii) a truncated Super PiggyBac (SPB) transposase comprising a C-terminal Cysteine-Rich Domain (CRD) deletion within amino acid residues 535-594 of the SPB sequence comprising the sequence established in SEQ ID NO: 1, and (iii) one or more MosI DNA-binding domains.

2. CHIMERIC TRANSPOSASE, according to claim 1, characterized by the truncated SPB transposase of (ii) comprising the sequence set forth in any of the SEQ ID NOs: 6884.

3. A chimeric transposase, according to any one of claims 1 to 2, characterized by the truncated PB transposase comprising one or more hyperactive mutations selected from I30V, S103P, G165S, M226F, M282V, S509G, N538K or N571S of SEQ ID NO:

1.

4. A chimeric transposase, according to any one of claims 1 to 3, characterized by the PB transposase comprising the CRD deletion further comprising an in-frame N-terminal nuclear localization (NLS) sequence comprising the amino acid sequence of SEQ ID NO:

39.

5. Chimeric transposase, according to any one of claims 1 to 4, characterized by the chimeric transposase comprising two MosI DNA-binding domains comprising the amino acid sequence set forth in SEQ ID NO:

6.

6. CHIMERIC TRANSPOSASE, according to any one of claims 1 to 4, characterized by the chimeric transposase comprising a MosI DNA-binding domain comprising the amino acid sequence Petition 870250091089, dated 06 / 10 / 2025, p. 191 / 198 2 / 6 set forth in SEQ ID NO:

8.

7. Chimeric TRANSPOSON, according to any one of claims 1 to 6, characterized in that the target-specific DNA-binding domain is a zinc finger domain or a TAL array.

8. POLYNUCLEOTIDE, characterized by comprising a nucleic acid sequence encoding the chimeric transposase, as defined in any one of claims 1 to 7.

9. VECTOR, characterized by comprising the polynucleotide, as defined in claim 13.

10. A chimeric transposon inverted terminal repeat (ITR) polynucleotide, characterized by comprising, in the 5' to 3' direction: (i) a polynucleotide comprising nucleotides 1-16 of the nucleic acid sequence established in SEQ ID NO: 12 and (ii) a polynucleotide comprising two MosI DNA binding sites.

11. Chimeric transposon ITR polynucleotide, according to claim 10, characterized by the polynucleotide comprising two MosI DNA binding sites comprising the nucleic acid sequences of SEQ ID NOs: 13 or 14.

12. Chimeric transposon ITR polynucleotide, according to any one of claims 10 to 11, characterized by the chimeric transposon ITR polynucleotide comprising the nucleic acid sequence SEQ ID NOS: 15 or 16.

13. Chimeric transposon ITR polynucleotide, according to any one of claims 10 to 12, characterized by the chimeric transposon ITR polynucleotide comprising the nucleic acid sequence of SEQ ID NOS: 17 or 18.

14. VECTOR, characterized by comprising the chimeric transposon ITR polynucleotide, as defined in any of claims 10 to 13.

15. Chimeric transposon ITR polynucleotide, characterized by comprising, in the 5' to 3' direction: (i) a polynucleotide comprising nucleotides 1-16 of the nucleic acid sequence established in SEQ ID NO: 65 and (ii) a polynucleotide comprising two MosI DNA binding sites.

16. Chimeric transposon ITR polynucleotide, according to claim 15, characterized by the polynucleotide comprising two MosI DNA binding sites comprising the nucleic acid sequence of SEQ IDs: 13 or 14.

17. Chimeric transposon ITR polynucleotide, according to claim 15, characterized in that the chimeric transposon ITR polynucleotide comprises the nucleic acid sequence of any of the SEQ ID Nos: 19-22.

18. VECTOR, characterized by comprising the chimeric transposon ITR polynucleotide, as defined in any one of claims 15 to 17.

19. A chimeric site-specific transposase fusion protein directed towards LINE1, characterized by comprising, in the N-terminal to C-terminal direction, (i) a DNA-binding domain directed towards left or right LINE1 TAL, (ii) a binding sequence, and (iii) a chimeric PB:MosI transposase.

20. A chimeric site-specific transposase fusion protein targeting LINE1, according to claim 19, characterized in that the chimeric PB:MosI transposase comprises the amino acid sequence set forth in SEQ ID NO: 9 or 7.

21. Chimeric site-specific transposase fusion protein targeting LINE1, according to claim 20, characterized by the DNA-binding domain targeting left or right LINE1 comprising the amino acid sequence set forth in SEQ ID Nos: 66 or 67.

22. TRANSPOSON, characterized by comprising (i) a chimeric polynucleotide LE PB:MosI ITR and (ii) a chimeric polynucleotide RE PB:MosI ITR, wherein the polynucleotide LE PB:MosI ITR and a polynucleotide RE PB:MosI ITR comprise the same nucleic acid sequence.

23. TRANSPOSON, according to claim 22, characterized by the chimeric polynucleotide LE PB:MosI ITR and the chimeric polynucleotide RE PB:MosI ITR each comprising the nucleic acid sequence of any of the SEQ ID NOs: 15-22.

24. TRANSPOSON, characterized by comprising (i) a chimeric polynucleotide LE PB:MosI ITR comprising the nucleic acid sequence SEQ ID NO: 54 and (ii) a chimeric polynucleotide RE PB:MosI ITR comprising the nucleic acid sequence SEQ ID NO:

55.

25. TRANSPOSON, characterized by comprising the nucleic acid sequence of SEQ ID NO:

56.

26. METHOD FOR INTEGRATING A TRANSGENE into a genomic target site of a cell, characterized by the method comprising introducing into the cell the chimeric transposase, as defined in any one of claims 1 to 7, and a transposon, wherein the transposon comprises, in the order of 5' to 3': a 5'ITR, the transgene and a 3'ITR, wherein the 5'ITR is a terminal inverted repeat (ITR) of a chimeric transposon, as defined in any one of claims 10 to 13, and the 3'ITR is a terminal inverted repeat (ITR) of a chimeric transposon, as defined in any one of claims 15 to 17.

27. METHOD, according to claim 26, characterized by the inverted terminal repeat of chimeric transposon 5'ITR comprising the nucleic acid sequence established in SEQ ID NO: 15 and / or where the inverted terminal repeat of chimeric transposon 3'ITR comprises the nucleic acid sequence established in SEQ ID NO:

21.

28. METHOD, according to any one of claims 26 to 27, characterized in that the genomic target site is located on a repetitive element.

29. METHOD, according to claim 28, characterized in that the repeating element is a LINE element.

30. METHOD FOR SITE-SPECIFIC TRANSPOSITION OF A DNA MOLECULE into the genome of a cell, characterized by comprising introducing into the cell: a) a nucleic acid encoding a chimeric site-specific transposase fusion protein comprising a DNA-binding domain and a chimeric transposase comprising, in N-terminal to C-terminal order: a piggyBac transposase comprising a C-terminal Cysteine-Rich Domain (CRD) deletion and one or more MosI DNA-binding domains; b) wherein the fusion protein is expressed in the cell; and c) a DNA molecule comprising a transposon comprising a chimeric transposon ITR polynucleotide LE and a chimeric transposon ITR polynucleotide RE; in which the chimeric site-specific transposase fusion protein expressed integrates the transposon via site-specific transposition into the TTAA sequence of the cellular genome.

31. METHOD FOR GENERATING A SITE-SPECIFIC TRANSPOSITION-ENGINEERED CELL, characterized by comprising introducing into the cell: a) a nucleic acid encoding a chimeric site-specific transposase fusion protein comprising a binding domain. Petition 870250091089, dated 06 / 10 / 2025, page 1.195 / 198 6 / 6 to DNA and a chimeric transposase comprising, in N-terminal to C-terminal order: a) a piggyBac transposase comprising a C-terminal Cysteine-Rich Domain (CRD) deletion and one or more MosI DNA-binding domains; b) wherein the chimeric site-specific transposase fusion protein is expressed in the cell; and c) a DNA molecule comprising a transposon comprising a chimeric transposon ITR polynucleotide LE and a chimeric transposon ITR polynucleotide RE; wherein the expressed chimeric site-specific transposase fusion protein integrates the transposon by site-specific transposition into the TTAA sequence in the cell genome, thereby generating the engineered cell. Petition 870250091089, dated 06 / 10 / 2025, p. 196 / 198.