Chimeric transposases and uses thereof

Abstract

The present disclosure relates generally to chimeric transposases, chimeric site-specific transposase fusion proteins, chimeric transposon inverted repeat polynucleotides, and methods of use thereof.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202480034973.9 (22) Application Date 2024.04.04 (30) Priority Data 63 / 494,297 2023.04.05 US (85) PCT International Application Entering National Phase Date 2025.11.25 (86) PCT International Application Application Data PCT / US2024 / 022978 2024.04.04 (87) PCT International Application Publication Data WO2024 / 211505 EN 2024.10.10 (71) Applicant: Pocida Therapeutics Company Address: California, USA (72) Inventors: J.S. Lucas, B.B. Madison, O. Batalov (74) Patent Agency: Beijing Kunrui Law Firm 11494 Patent Attorney Cen Xiaodong (51) Int.Cl. C12N 9 / 12 (2006.01) C12N 9 / 22 (2006.01) C12N 15 / 90 (2006.01) (54) Invention Title: Chimeric Transposase and Its Use (57) Abstract: This disclosure relates in whole to chimeric transposase, chimeric site-specific transposase fusion protein, chimeric transposon inverted repeat polynucleotide, and its use. Claims (3 pages), Description (45 pages), Sequence Listing (Electronic Publication) CN 121195063 A 2025.12.23 CN 1 21 19 50 63 A 1. A chimeric transposase comprising, in order from N-terminus to C-terminus: (i) a target-specific DNA-binding domain; (ii) a truncated SuperpiggyBac (SPB) transposase comprising a C-terminal cysteine-rich domain (CRD) deletion within amino acid residues 535 to 594 of the sequence of SPB comprising the sequence shown in SEQ ID NO: 1; and (iii) one or more MosI DNA-binding domains. 2. The chimeric transposase according to claim 1, wherein the truncated SPB transposase of (ii) comprises the sequence shown in any one of SEQ ID NO.: 68 to 84. 3. The chimeric transposase according to claim 1 or 2, wherein the truncated PB transposase comprises one or more highly active mutations selected from SEQ ID NO: 1, namely I30V, S103P, G165S, M226F, M282V, S509G, N538K, or N571S. 4. The chimeric transposase according to any one of claims 1 to 3,The PB transposase containing CRD deletion further comprises an in-frame N-terminal nuclear localization sequence (NLS) comprising the amino acid sequence of SEQ ID NO: 39. 5. The chimeric transposase according to any one of claims 1 to 4, wherein the chimeric transposase comprises two MosI DNA-binding domains comprising the amino acid sequence shown in SEQ ID NO: 6. 6. The chimeric transposase according to any one of claims 1 to 4, wherein the chimeric transposase comprises one MosI DNA-binding domain comprising the amino acid sequence shown in SEQ ID NO: 8. 7. The chimeric transposase according to any one of claims 1 to 6, wherein the target-specific DNA-binding domain is a zinc finger domain or a TAL array. 8. A polynucleotide comprising a nucleic acid sequence encoding the chimeric transposase according to any one of claims 1 to 7. 9. A vector comprising the polynucleotide according to claim 13. 10. A chimeric transposon inverted terminal repeat (ITR) polynucleotide comprising, in the 5' to 3' direction: (i) a polynucleotide comprising nucleotides 1 to 16 of the nucleic acid sequence shown in SEQ ID NO: 12; and (ii) a polynucleotide comprising two MosI DNA binding sites. 11. The chimeric transposon ITR polynucleotide of claim 10, wherein the polynucleotide comprising two MosI DNA binding sites comprises the nucleic acid sequence of SEQ ID NO: 13 or 14. 12. The chimeric transposon ITR polynucleotide of claim 10 or 11, wherein the chimeric transposon ITR polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 15 or 16. 13. The chimeric transposon ITR polynucleotide of any one of claims 10 to 12, wherein the chimeric transposon ITR polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 17 or 18. 14. A vector comprising the chimeric transposon ITR polynucleotide according to any one of claims 10 to 13. 15. A chimeric transposon ITR polynucleotide.It comprises, in the 5' to 3' direction: (i) a polynucleotide comprising nucleotides 1 to 16 of the nucleic acid sequence shown in SEQ ID NO: 65; and (ii) a polynucleotide comprising two MosI DNA binding sites. 16. The chimeric transposon ITR polynucleotide of claim 15, wherein the polynucleotide comprising two MosI DNA binding sites comprises the nucleic acid sequence of SEQ ID NO: 13 or 14. Claims 1 / 3 page 2 CN 121195063 A 17. The chimeric transposon ITR polynucleotide of claim 15, wherein the chimeric transposon ITR polynucleotide comprises the nucleic acid sequence of any one of SEQ ID NO: 19 to 22. 18. A vector comprising the chimeric transposon ITR polynucleotide of any one of claims 15 to 17. 19. A LINE1-targeting chimeric site-specific transposase fusion protein comprising, in the direction from the N-terminus to the C-terminus: (i) a left or right LINE1 TAL-targeting DNA-binding domain; (ii) a linker sequence; and (iii) a chimeric PB:MosI transposase. 20. The LINE1-targeting chimeric site-specific transposase fusion protein of claim 19, wherein the chimeric PB:MosI transposase comprises the amino acid sequence shown in SEQ ID NO: 9 or 7. 21. The LINE1-targeting chimeric site-specific transposase fusion protein of claim 20, wherein the left or right LINE1 TAL-targeting DNA-binding domain comprises the amino acid sequence shown in SEQ ID NO: 66 or 67. 22. A transposon comprising (i) a chimeric LE PB:MosI ITR polynucleotide and (ii) a chimeric RE PB:MosI ITR polynucleotide, wherein the LE PB:MosI ITR polynucleotide and the RE PB:MosI ITR polynucleotide comprise the same nucleic acid sequence. 23. The transposon of claim 22, wherein the chimeric LE PB:MosI ITR polynucleotide and the chimeric RE PB:MosI ITR polynucleotide each comprise the nucleic acid sequence of any one of SEQ ID NO: 15 to 22. 24. A transposon comprising: (i) a chimeric LE PB:MosI ITR polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 54; and (ii) a chimeric RE PB:MosI ITR polynucleotide,It contains the nucleic acid sequence of SEQ ID NO: 55. 25. A transposon containing the nucleic acid sequence of SEQ ID NO: 56. 26. A method of integrating a transgene into a genomic target site in a cell, the method comprising introducing a chimeric transposon according to any one of claims 1 to 7 and a transposon into the cell, wherein the transposon comprises, in a 5' to 3' sequence: a 5' ITR, the transgene, and a 3' ITR, wherein the 5' ITR is a chimeric transposon inverted terminal repeat (ITR) according to any one of claims 10 to 13, and the 3' ITR is a chimeric transposon inverted terminal repeat (ITR) according to any one of claims 15 to 17. 27. The method of claim 26, wherein the 5' ITR chimeric transposon inverted terminal repeat contains the nucleic acid sequence shown in SEQ ID NO: 15, and / or wherein the 3' ITR chimeric transposon inverted terminal repeat contains the nucleic acid sequence shown in SEQ ID NO: 21. 28. The method of any one of claims 26 or 27, wherein the genomic target site is located in a repeating element. 29. The method of claim 28, wherein the repeating element is a LINE element. 30. A method for site-specific transposition of a DNA molecule into the genome of a cell, the method 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, the chimeric transposase comprising, in N-terminus to C-terminus, 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 claim 2 / 3 page 3 CN 121195063 A c) a DNA molecule comprising a transposon comprising a chimeric LE transposon ITR polynucleotide and a chimeric RE transposon ITR polynucleotide; wherein the expressed chimeric site-specific transposase fusion protein integrates the transposon into a TTAA sequence of the cell genome via site-specific transposition. 31. A method for generating engineered cells via site-specific transposition, the method comprising: introducing into the cells: a) a nucleic acid encoding a chimeric site-specific transposase fusion protein comprising a DNA-binding domain and a chimeric transposase,The chimeric transposase comprises, in order from N-terminus to C-terminus, a piggyBac transposase containing 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 containing a transposon comprising a chimeric LE transposon ITR polynucleotide and a chimeric RE transposon ITR polynucleotide; wherein the expressed chimeric site-specific transposase fusion protein integrates the transposon into a TTAA sequence in the genome of the cell via site-specific transposition, thereby producing the engineered cell. Claims 3 / 3 Page 4 CN 121195063 A Chimeric transposase and its use

[0001] Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 494,297, filed April 5, 2023, which is incorporated herein by reference in its entirety.

[0003] Reference to the electronically submitted sequence listing

[0004] This application contains a sequence listing that has been submitted in XML format via the Patent Center and incorporated herein by reference in its entirety. The XML copy was created on March 27, 2024, named “POTH-080_001WO_SeqList”, and is 233,942 bytes in size. Technical Field

[0005] This disclosure relates throughout to chimeric transposases, particularly chimeric transposases, chimeric site-specific transposase fusion proteins comprising a chimeric transposase and a DNA targeting domain, and chimeric transposon inverted repeat (ITR) polynucleotides. Methods for site-specific transposition using chimeric site-specific transposase fusion proteins are also provided. Background Art

[0006] Transposases can be used to introduce non-endogenous DNA sequences into genomic DNA and are superior to other gene editing methods in many respects. However, there remains an unmet need for site-specific transposases for use in, for example, gene editing.

[0007] In their natural state, the piggyBac transposon and related family members carry a single open reading frame encoding their respective transposases. Inverted terminal repeats (ITRs) are present at both ends of the transposon and act as binding sites for the transposases. Immediately outside the ITR, the tetranucleotide sequence TTAA acts as a cleavage site, allowing the transposon to jump from one location in the genome to another TTAA site via a cut-and-paste mechanism.

[0008] When used for genome editing purposes, the transposase is typically removed from the transposon and expressed from a separate plasmid or mRNA. This leaves an empty transposon.This can encode a target "cargo" or "payload" flanked by ITR and TTAA sequences. The synthetic transposon can be delivered to the cell along with a transposase, which catalyzes the transposon's transposition to the TTAA site in the cell's genome.

[0009] The piggyBac transposase and related family members contain two distinct DNA-binding domains that interact with the inverted terminal repeat (ITR) sequence of the transposon. The DNA-binding and dimerizing domain (DDBD) binds to the sequence near the TTAA site flanked by the transposon, while the cysteine-rich domain (CRD) at the C-terminus of the protein binds to the inverted terminal repeat (ITR) sequence distal to the TTAA site. After transposase dimerization, the DDBD binds symmetrically to the ITR, with one monomeric DDBD binding to the left-hand (LE) ITR and the second monomeric DDBD binding to the right-hand (RE) ITR. Interestingly, both CRDs of the dimer bind asymmetrically to the LE ITR. Based on ITR sequence identity, it has been proposed that a second dimer of the piggyBac transposase can bind distal to the first dimer. The two CRDs of this second dimer have predicted binding sites within the RE ITR. While the proximal dimer catalyzes the transposition reaction, the role of the distal dimer remains unclear.

[0010] The asymmetric binding of the two CRD sequences of the piggyBac transposase is not characteristic of all transposase families. For example, MosI, a member of the mariner / Tc1 transposase family, interacts with its ITR as a symmetric dimer, as observed in its crystal structure on page 1 / 45 of its specification (see, for example, Morris et al., ELife May 25, 2016; 5:e15537. doi: 10.7554 / eLife.15537). Transposases used as symmetrical dimers may have several advantageous features compared to transposases used as asymmetrical dimer pairs (e.g., piggyBac transposases), including shorter ITR sequences and fewer transposase molecules required to complete transposition.

[0011] In one aspect, this invention provides a chimeric transposase comprising, in N-terminus to C-terminus,: (i) a target-specific DNA-binding domain; (ii) a truncated Super piggyBac (SPB) transposase.It comprises a C-terminal cysteine-rich domain (CRD) deletion within amino acid residues 535 to 594 of the SPB sequence containing the sequence shown in SEQ ID NO: 1; and (iii) one or more MosI DNA-binding domains. In some embodiments, the truncated PB transposase of (i) comprises the sequence shown in any one of SEQ ID NO: 68 to 84. In some embodiments, the truncated PB transposase comprises one or more hyperactive mutations. In some embodiments, the one or more hyperactive mutations are amino acid substitutions selected from I30V, S103P, G165S, M226F, M282V, S509G, N538K, or N571S of SEQ ID NO: 1.

[0012] In some embodiments, the PB transposase containing the CRD deletion further comprises an in-frame N-terminal nuclear localization sequence (NLS) containing the amino acid sequence of 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 shown in SEQ ID NO: 6. In some embodiments, the chimeric transposase comprises one MosI DNA-binding domain. In some embodiments, the one MosI DNA-binding domain comprises the amino acid sequence shown in SEQ ID NO: 8.

[0013] In some embodiments, the PB transposase and one or more MosI DNA-binding domains are linked 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.

[0014] In another aspect, this document provides a polynucleotide comprising a nucleic acid sequence encoding the chimeric transposase described herein.

[0015] In another aspect, this document provides a vector comprising the polynucleotide provided herein.

[0016] In another aspect, this document provides a chimeric transposon inverted terminal repeat (ITR) polynucleotide comprising, in the 5' to 3' direction: (i) a polynucleotide comprising nucleotides 1 to 16 of the nucleic acid sequence shown in SEQ ID NO: 12; and (ii) a polynucleotide comprising two MosI DNA binding sites. In some embodiments, the polynucleotide comprising the two MosI DNA binding sites is fused in the inverted orientation. In some embodiments,The polynucleotide containing two MosI DNA binding sites comprises the nucleic acid sequence of SEQ ID NO: 13 or 14. In some embodiments, the chimeric transposon ITR polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 15 or 16.

[0017] In some embodiments, the chimeric transposon ITR polynucleotide further comprises one or two additional nucleotides between the polynucleotide comprising nucleotides 1 to 16 of the nucleic acid sequence shown in SEQ ID NO: 12 and the polynucleotide comprising two MosI binding sites. In some embodiments, the chimeric transposon ITR polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 17. In some embodiments, the chimeric transposon ITR polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 18.

[0018] In another aspect, this document provides a vector comprising the chimeric transposon ITR polynucleotide provided herein. Specification 2 / 45 pages 6 CN 121195063 A

[0019] In another aspect, this document provides a transposon comprising the chimeric transposon ITR polynucleotide described herein.

[0020] In another aspect, this document provides a chimeric transposon ITR polynucleotide comprising, in the 5' to 3' direction: (i) a polynucleotide comprising nucleotides 1 to 16 of the nucleic acid sequence shown 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 a reverse orientation. In some embodiments, the polynucleotide comprising two MosI DNA binding sites comprises the nucleic acid sequence of SEQ ID NO: 13 or 14. In some embodiments, the chimeric transposon ITR polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 19 or 20. In some embodiments, the chimeric transposon ITR polynucleotide further comprises one or two additional nucleotides between the polynucleotide comprising nucleotides 1 to 16 of the nucleic acid sequence shown in SEQ ID NO: 65 and the polynucleotide comprising two MosI DNA binding sites. In some embodiments, the chimeric transposon ITR polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 21 or 22.

[0021] In another aspect, a vector comprising the chimeric transposon ITR polynucleotide described herein is provided.

[0022] In another aspect, a transposon is provided herein.The transposon comprises the chimeric transposon ITR polynucleotide described herein.

[0023] In another aspect, this document provides a chimeric site-specific transposase fusion protein 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 deletion of a C-terminal cysteine-rich domain (CRD) within residues 535 to 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 zinc finger motif (ZFM).

[0024] In some embodiments, the truncated PB transposase comprises the sequence shown in any one of SEQ ID NO: 68 to 84. In some embodiments, the N-terminal deletion of the truncated PB transposase is a deletion of amino acid residues 1 to 85, 1 to 86, 1 to 87, 1 to 88, 1 to 89, 1 to 90, 1 to 91, 1 to 92, 1 to 93, 1 to 94, 1 to 95, 1 to 96, 1 ​​to 97, 1 to 98, 1 to 99, 1 to 100, 1 to 101, 1 to 102, or 1 to 103. In some embodiments, the N-terminal deletion of the truncated PB transposase is a deletion of amino acid residues 1 to 93. In some embodiments, the PB transposase contains one or more hyperactive mutations. In some embodiments, the one or more hyperactive mutations are amino acid substitutions selected from G165S, M226F, M282V, and N538K (numbered starting at residue 5) of SEQ ID NO: 1. In some embodiments, one or more integration-deficient mutations in the truncated PB transposase are replaced by amino acid substitutions 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.

[0025] In some embodiments, the PB transposase comprising a C-terminal cysteine ​​enrichment domain (CRD) deletion further comprises an in-frame N-terminal nuclear localization sequence (NLS), wherein the NLS comprises the amino acid sequence shown 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 shown in SEQ ID NO: 6. In some embodiments, the chimeric site-specific transposase fusion protein comprises one MosI DNA-binding domain. In some embodiments, the one MosI DNA-binding domain comprises the amino acid sequence shown in SEQ ID NO: 8.

[0026] In some embodiments, the PB transposase and one or more MosI DNA-binding domains are linked by an adapter sequence as described on page 3 / 45 of the specification, CN 121195063 A. In some embodiments, the adapter sequence is GGGGS (SEQ ID NO: 86).

[0027] In some embodiments, the target-specific DNA-binding domain is a zinc finger domain or a TAL array.

[0028] In another aspect, this document provides a LINE1-targeted chimeric site-specific transposase fusion protein comprising, in the direction from the N-terminus to the C-terminus: (i) a left or right LINE1 TAL-targeted DNA-binding domain; (ii) a linker sequence; and (iii) a chimeric PB:MosI transposase. In some embodiments, the chimeric PB:MosI transposase comprises the amino acid sequence shown in SEQ ID NO: 9 or 7. In some embodiments, the left or right LINE1 TAL-targeted DNA-binding domain comprises the amino acid sequence shown in SEQ ID NO: 66 or 67.

[0029] In another aspect, this document provides a polynucleotide comprising a nucleic acid sequence encoding the chimeric site-specific transposase fusion protein described herein.

[0030] In another aspect, this document provides a vector comprising a nucleic acid sequence encoding the chimeric site-specific transposase fusion protein described herein.

[0031] In another aspect, this document provides a transposon comprising (i) a chimeric LE PB:MosI ITR polynucleotide and (ii) a chimeric RE PB:MosI ITR polynucleotide, wherein the LE PB:MosI ITR polynucleotide and the RE PB:MosI ITR polynucleotide comprise the same nucleic acid sequence. In some embodiments, the chimeric LE PB:MosI ITR polynucleotide and the chimeric RE PB:MosI ITR polynucleotide each comprise the nucleic acid sequence of any one of SEQ ID NO: 15 to 22.

[0032] In another aspect, this document provides a transposon,The transposon comprises: (i) a chimeric LE PB:MosI ITR polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 54; and (ii) a chimeric RE PB:MosI ITR polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 55.

[0033] In another aspect, this document provides a transposon comprising the nucleic acid sequence of SEQ ID NO: 56.

[0034] In another aspect, this document provides a method for integrating a transgene into a genomic target site in a cell, the method comprising introducing a chimeric transposon disclosed herein and a transposon into a cell, wherein the transposon comprises, in 5' to 3' order: a 5' ITR, a transgene, and a 3' ITR, wherein the 5' ITR is the chimeric transposon inverted terminal repeat (ITR) sequence described herein, and the 3' ITR is the chimeric transposon inverted terminal repeat (ITR) sequence described herein. In some embodiments, the 5'ITR chimeric transposon inverted terminal repeat sequence comprises the nucleic acid sequence shown in SEQ ID NO: 15. In some embodiments, the 3'ITR chimeric transposon inverted terminal repeat sequence comprises the nucleic acid sequence shown in SEQ ID NO: 21. In some embodiments, the transposon further comprises an exogenous promoter between the 5' ITR and the transgene.

[0035] In some embodiments, the transgene encodes a detectable biomarker. In some embodiments, the detectable biomarker is GFP.

[0036] In some embodiments, the genomic target site is located in a repeat element. In some embodiments, the repeat element is a LINE element.

[0037] In another aspect, this document provides a method for site-specific transposition of a DNA molecule into the genome of a cell, the method 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 fusion protein is expressed in the cell; and (b) a DNA molecule comprising a chimeric LE transposon ITR polynucleotide and a chimeric RE transposon ITR polynucleotide; wherein the expressed chimeric site-specific transposase fusion protein integrates the transposon into the TTAA sequence of the cell genome via site-specific transposition.

[0038] In another aspect, this document provides a method for generating engineered cells via site-specific transposition.The method comprises: (a) a nucleic acid encoding a chimeric site-specific transposase fusion protein comprising a DNA-binding domain and a chimeric transposase, the chimeric transposase comprising, in N-terminal to C-terminal order: a piggyBac transposase containing 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 a cell; and (b) a DNA molecule containing a transposon comprising a chimeric LE transposon ITR polynucleotide and a chimeric RE transposon ITR polynucleotide; wherein the expressed chimeric site-specific transposase fusion protein integrates the transposon into a TTAA sequence of the cell genome via site-specific transposition, thereby producing engineered cells. Detailed Description

[0039] This document provides chimeric transposases and chimeric site-specific fusion proteins (particularly chimeric piggyBac:MosI (PB:MosI) transposases and chimeric PB:MosI site-specific fusion proteins), polynucleotides encoding chimeric transposases and chimeric site-specific fusion proteins, and vectors and transposons comprising the polynucleotides. Methods for preparing chimeric transposases and chimeric fusion proteins are also provided, as well as cells modified with the chimeric transposases or chimeric site-specific fusion proteins provided herein, and methods for using such cells.

[0040] This document also provides chimeric transposon inverted terminal repeat (ITR) sequences. In some embodiments, the chimeric transposon inverted terminal repeat (ITR) sequence is a chimeric PB:MosI transposon left-handed inverted terminal repeat (ITR). In some embodiments, the chimeric transposon inverted terminal repeat (ITR) sequence is a chimeric PB:MosI transposon right-handed inverted terminal repeat (ITR). Transposons comprising chimeric left and right ITRs are also provided, as well as methods for transposing cells using transposons comprising the left and right ITR sequences of the chimeric transposons provided herein, and their uses.

[0041] Chimeric piggyBac:MosI transposase

[0042] In one aspect, chimeric transposases are provided herein,These chimeric transposases, in N-terminus to C-terminus sequence, comprise: a piggyBac transposase containing a C-terminal cysteine ​​enrichment domain (CRD) deletion and one or more MosI DNA-binding domains.

[0043] An exemplary sequence of the wild-type PB transposase is shown in SEQ ID NO: 1. MGSSLDDEHILSALLQSDDELVGEDSDSEISDHVSEDDVQSDTEEAFIDEVHEVQPTSSGSEILDEQN VIEQPGSSLASNRILTLPQRTIRGKNKHCWSTSKSTRRSRVSALNIVRSQRGPTRMCRNIYDPLLCFKLFFTDEII SEIVKWTNAEISLKRRESMTGATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLSMVYVSVMSRDRFDFL IRCLRMDDKSIRPTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRMYIPNKPSKYGIKIL MMCDSGTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSIPLAKNLLQEPYKLTIVGTVR SNKREIPEVLKNSRSRPVGTSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINESTGKPQMVMYYNQTKGGVDT LDQMCSVMTCSRKTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMSLTSSFMRKRLEAPTL KRYLRDNISNILPNEVPGTSDDSTEEPVMKKRTYCTYCPSKIRRKANASCKKCKKVICREHNIDMCQSCF (SEQ ID NO: 1)

[0045] In one aspect, this document provides chimeric transposases comprising, in order from N-terminus to C-terminus: (i) a piggyBac transposase containing a C-terminal cysteine ​​enrichment domain (CRD) deletion; and (ii) one or more MosI DNA-binding domains. In some embodiments, the PB CRD consists of residues 553 to 594 of the 594 amino acid PB transposase protein (i.e., in some embodiments,The PB CRD comprises the amino acid sequence shown in SEQ ID NO: 2. The CRD domain can be linked to the remainder of the N-terminus of the transposase via a linker sequence. In some embodiments, the linker sequence spans residues 535 to 552 (including residues 535 and 552) of the PB transposase sequence. 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 ILPKEVPGTSDDSTEEPV (SEQ ID NO: 174).

[0046] In some embodiments, the C-terminal CRD deletion of the piggyBac transposase is the deletion of C-terminal amino acid residues 545 to 594 of the PB transposase amino acid sequence shown in SEQ ID NO: 1. In such embodiments, the resulting truncated piggyBac transposase contains the following amino acid sequence:

[0047] MGSSLDDEHILSALLQSDDELVGEDSDSEISDHVSEDDVQSDTEEAFIDEVHEVQPTSSGSEILDEQN VIEQPGSSLASNRILTLPQRTIRGKNKHCWSTSKSTRRSRVSALNIVRSQRGPTRMCRNIYDPLLCFKLFFTDEII SEIVKWTNAEISLKRRESMTGATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLSMVYVSVMSRDRFDFL IRCLRMDDKSIRPTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRMYIPNKPSKYGIKIL MMCDSGTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSIPLAKNLLQEPYKLTIVGTVR SNKREIPEVLKNSRSRPVGTSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINESTGKPQMVMYYNQTKGGVDT LDQMCSVMTCSRKTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMSLTSSFMRKRLEAPTL KRYLRDNISNILPNEVPGTS (SEQ ID NO:3)

[0048] In some embodiments,The PB transposon containing the CRD deletion described herein contains 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% of the same amino acid sequence as shown in SEQ ID NO: 3. In some embodiments, the PB transposon containing the CRD deletion described herein contains the amino acid sequence shown in SEQ ID NO: 3 having one, two, three, or four of the five conserved amino acid substitutions. In some embodiments, the PB transposase containing the CRD deletion described herein contains the amino acid sequence shown in SEQ ID NO: 3.

[0049] The MosI DNA-binding domain used to generate the chimeric PB:MosI transposase containing the C-terminal CRD deletion may be linked to amino acid S544 of the PB transposase sequence shown in SEQ ID NO: 1. The resulting chimeric transposase is referred to herein as "chimeric S544 PB:MosI transposase". In some embodiments, the S544 PB transposase sequence and the MosI DNA-binding domain are linked by an adapter sequence. In some embodiments, the adapter sequence is GGGGS (SEQ ID NO: 86).

[0050] In some embodiments, the C-terminal cysteine ​​enrichment domain (CRD) deletion of the PB transposase is the deletion of amino acid residues 553 to 594 of the PB transposase amino acid sequence shown in SEQ ID NO: 1. In such embodiments,The resulting truncated piggyBac transposase contains the amino acid sequence:

[0051] MGSSLDDEHILSALLQSDDELVGEDSDSEISDHVSEDDVQSDTEEAFIDEVHEVQPTSSGSEILDEQN VIEQPGSSLASNRILTLPQRTIRGKNKHCWSTSKSTRRSRVSALNIVRSQRGPTRMCRNIYDPLLCFKLFFTDEII SEIVKWTNAEISLKRRESMTGATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLSMVYVSVMSRDRFDFL IRCLRMDDKSIRPTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRMYIPNKPSKYGIKIL MMCDSGTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSIPLAKNLLQEPYKLTIVGTVR SNKREIPEVLKNSRSRPVGTSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINESTGKPQMVMYYNQTKGGVDT LDQMCSVMTCSRKTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMSLTSSFMRKRLEAPTL KRYLRDNISNILPNEVPGTSDDSTEEPV (SEQ ID NO:4) Specification 6 / 45 pages 10 CN 121195063 A

[0052] In some embodiments, the PB transposon containing the CRD deletion described herein contains 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% of the amino acid sequence shown in SEQ ID NO: 4. In some embodiments, the PB transposon containing the CRD deletion described herein comprises the amino acid sequence shown in SEQ ID NO: 4 having one, two, three, or four of the five conserved amino acid substitutions. In some embodiments, the PB transposase containing the CRD deletion described herein comprises the amino acid sequence shown in SEQ ID NO: 4.

[0053] Alternatively,The MosI DNA-binding domain used to generate a chimeric PB:MosI transposase containing the C-terminal CRD deletion can be linked to amino acid V552 of the piggyBac transposase sequence shown in SEQ ID NO: 1. The resulting chimeric transposase is referred to herein as "chimeric V552 PB:MosI transposase". In some embodiments, the V552 PB transposase sequence and the MosI DNA-binding domain are linked by an adapter sequence. In some embodiments, the adapter sequence is GGGGS (SEQ ID NO: 86).

[0054] In some embodiments, the truncated PB transposase contains 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 four hyperactive mutations selected from SEQ ID NO: 1, namely I30V, G165S, M226F, M282V, and N538K.

[0055] In some embodiments, the truncated PB transposase described herein comprises the sequence of SEQ ID NO: 1 and one of the following mutations: I30V, S103P, G165S, M226F, M282V, S509G, N538K, and N571S. In some embodiments, the truncated PB transposase described herein comprises the sequence of SEQ ID NO: 1 and two of the following mutations: I30V, S103P, G165S, M226F, M282V, S509G, N538K, and N571S. In some embodiments, the truncated PB transposase described herein comprises the sequence of SEQ ID NO: 1 and three of the following mutations: I30V, S103P, G165S, M226F, M282V, S509G, N538K, and N571S. In some embodiments, the truncated PB transposase described herein comprises the sequence of SEQ ID NO: 1 and four of the following mutations: I30V, S103P, G165S, M226F, M282V, S509G, N538K, and N571S. In some embodiments, the truncated PB transposase described herein comprises the sequence of SEQ ID NO: 1 and five of the following mutations: I30V, S103P, G165S, M226F, M282V, S509G, N538K, and N571S. In some embodiments,The truncated PB transposase described herein comprises the sequence of SEQ ID NO: 1 and six of the following mutations: I30V, S103P, G165S, M226F, M282V, S509G, N538K, and N571S. In some embodiments, the truncated PB transposase described herein comprises the sequence of SEQ ID NO: 1 and seven of the following mutations: I30V, S103P, G165S, M226F, M282V, S509G, N538K, and N571S. In some embodiments, the truncated PB transposase described herein comprises the sequence of SEQ ID NO: 1 and the following mutations: I30V, S103P, G165S, M226F, M282V, S509G, N538K, and N571S.

[0056] In some embodiments, the truncated PB transposase described herein comprises the sequence of SEQ ID NO: 3 and one of the following mutations: I30V, S103P, G165S, M226F, M282V, S509G, N538K, and N571S. In some embodiments, the truncated PB transposase described herein comprises the sequence of SEQ ID NO: 3 and two of the following mutations: I30V, S103P, G165S, M226F, M282V, S509G, N538K, and N571S. In some embodiments, the truncated PB transposase described herein comprises the sequence of SEQ ID NO: 3 and three of the following mutations: I30V, S103P, G165S, M226F, M282V, S509G, N538K, and N571S. In some embodiments, the truncated PB transposase described herein comprises the sequence of SEQ ID NO: 3 and four of the following mutations: I30V, S103P, G165S, M226F, M282V, S509G, N538K, and N571S. In some embodiments, the truncated PB transposase described herein comprises the sequence of SEQ ID NO: 3 and five of the following mutations: I30V, S103P, G165S, M226F, M282V, S509G, N538K, and N571S (page 7 / 45 of specification, CN 121195063 A). In some embodiments, the truncated PB transposase described herein comprises the sequence of SEQ ID NO: 3 and six of the following mutations: I30V, S103P, G165S, M226F, M282V, S509G, N538K, and N571S. In some embodiments,The truncated PB transposase described herein comprises the sequence of SEQ ID NO: 3 and seven of the following mutations: I30V, S103P, G165S, M226F, M282V, S509G, N538K, and N571S. In some embodiments, the truncated PB transposase described herein comprises the sequence of SEQ ID NO: 3 and the following mutations: I30V, S103P, G165S, M226F, M282V, S509G, N538K, and N571S.

[0057] In some embodiments, the truncated PB transposase described herein comprises the sequence of SEQ ID NO: 4 and one of the following mutations: I30V, S103P, G165S, M226F, M282V, S509G, N538K, and N571S. In some embodiments, the truncated PB transposase described herein comprises the sequence of SEQ ID NO: 4 and two of the following mutations: I30V, S103P, G165S, M226F, M282V, S509G, N538K, and N571S. In some embodiments, the truncated PB transposase described herein comprises the sequence of SEQ ID NO: 4 and three of the following mutations: I30V, S103P, G165S, M226F, M282V, S509G, N538K, and N571S. In some embodiments, the truncated PB transposase described herein comprises the sequence of SEQ ID NO: 4 and four of the following mutations: I30V, S103P, G165S, M226F, M282V, S509G, N538K, and N571S. In some embodiments, the truncated PB transposase described herein comprises the sequence of SEQ ID NO: 4 and five of the following mutations: I30V, S103P, G165S, M226F, M282V, S509G, N538K, and N571S. In some embodiments, the truncated PB transposase described herein comprises the sequence of SEQ ID NO: 4 and six of the following mutations: I30V, S103P, G165S, M226F, M282V, S509G, N538K, and N571S. In some embodiments, the truncated PB transposase described herein comprises the sequence of SEQ ID NO: 4 and seven of the following mutations: I30V, S103P, G165S, M226F, M282V, S509G, N538K, and N571S. In some embodiments,The truncated PB transposase described herein comprises the sequence of SEQ ID NO: 4 and the following mutations: I30V, S103P, G165S, M226F, M282V, S509G, N538K, and N571S.

[0058] In some embodiments, the truncated piggyBac transposase domain is a Super piggyBac® transposase (SPB). Non-limiting examples of SPB transposases are described in detail in U.S. Patent Nos. 6,218,182, 6,962,810, 8,399,643, and PCT Publication No. WO 2010 / 099296, each of which is incorporated herein by reference in its entirety as an example of an SPB transposase that may be used in the fusion protein described herein.

[0059] An exemplary SuperpiggyBac transposase – MosI chimeric transposase is further described in Example 6.

[0060] In some embodiments, the truncated PB transposase sequence further includes an in-frame N-terminal nuclear localization sequence (NLS). An exemplary wild-type SPB sequence containing an NLS is shown in SEQ ID NO: 5, wherein the NLS is shown in italics and the hyperactive mutation is shown in bold.Furthermore, the cysteine ​​enrichment domain (CRD) missing in the truncated PB transposase sequence is underlined. The sequence numbering used to describe the deleted and mutated SPB transposase domains begins at residue 12 of SEQ ID NO: 5. MAPKKKRKVGGGGSSLDDEHILSALLQSDDELVGEDSDSEVSDHVSEDDVQSDTEEAFIDEVHEVQPT SSGSEILDEQNVIEQPGSSLASNRILTLPQRTIRGKNKHCWSTSKSTRRSRVSALNIVRSQRGPTRMCRNIYDPLL CFKLFFTDEIISEIVKWTNAEISLKRRESMTSATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLSMVYV SVMSRDRFDFLIRCLRMDDKSIRPTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRVYIP NKPSKYGIKILMMCDSGTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSIPLAKNLLQE PYKLTIVGTVRSNKREIPEVLKNSRSRPVGTSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINESTGKPQMVM YYNQTKGGVDTLDQMCSVMTCSRKTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMSLTSS Instruction manual 8 / 45 pages 12 CN 121195063 A FMRKRLEAPTLKRYLRDNISNILPKEVPGTSDDSTEEPVMKKRTYCTYCPSKIRRKANASCKKCKKVICREHNIDM CQSCF (SEQ ID NO: 5).

[0062] The transposases described herein may be isolated from or derived from insects, vertebrates, crustaceans or chordates, as described in more detail in PCT Publication Nos. WO 2019 / 173636 and PCT / US2019 / 049816. In terms of preference,SPB transposases were isolated from or derived from insects such as *Trichoplusia ni* (GenBank accession number AAA87375), *Macdunnoughia crassisigna* (ABZ85926.1), or *Bombyx mori* (GenBank accession number BAD11135).

[0063] 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 to 111 of the MosI transposase (i.e., the amino acid sequence shown in SEQ ID NO: 6): SFVPNKEQTRTVLIFCFHLKKTAAESHRMLVEAFGEQVPT VKTCERWFQRFKSGDFDVDDKEHGKPPKRYEDAELQALLDEDDAQTQKQLAEQLEVSQQAVSNRLREMG (SEQ ID NO: 6). In some embodiments, each of the two MosI DNA-binding domains 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 shown in SEQ ID NO: 6. In some embodiments, each of the two MosI DNA-binding domains comprises an amino acid sequence shown in SEQ ID NO: 3 having one, two, three, or four of the five conserved amino acid substitutions.

[0064] In some embodiments, the chimeric transposase comprises a truncated piggyBac transposase domain comprising the amino acid sequence shown in SEQ ID NO: 3 linked to the two MosI DNA-binding domains, wherein each of the two MosI DNA-binding domains comprises the sequence shown in SEQ ID NO: 6. Two MosI DNA-binding domains can be linked to a truncated PB transposase domain via an adapter sequence to obtain a chimeric PB:MosI S544-111 transposase containing the amino acid sequence shown in SEQ ID NO: 7, wherein residues 3 to 111 of the MosI transposase are highlighted in bold, and the PB domain shown is underlined.And the connector sequence is highlighted in italics:

[0065] MAPKKKRKVGGGGSSLDDEHILSALLQSDDELVGEDSDSEVSDHVSEDDVQSDTEEAFIDEVHEVQPT SSGSEILDEQNVIEQPGSSLASNRILTLPQRTIRGKNKHCWSTSKSTRRSRVSALNIVRSQRGPTRMCRNIYDPLL CFKLFFTDEIISEIVKWTNAEISLKRRESMTSATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLSMVYV SVMSRDRFDFLIRCLRMDDKSIRPTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRVYIP NKPSKYGIKILMMCDSGTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSIPLAKNLLQE PYKLTIVGTVRSNKREIPEVLKNSRSRPVGTSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINESTGKPQMVM YYNQTKGGVDTLDQMCSVMTCSRKTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMSLTSS FMRKRLEAPTLKRYLRDNISNILPKEVPGTSGGGGSSFVPNKEQTRTVLIFCFHLKKTAAESHRMLVEAFGEQVPT VKTCERWFQRFKSGDFDVDDKEHGKPPKRYEDAELQALLDEDDAQTQKQLAEQLEVSQQAVSNRLREMG (SEQ ID NO: 7)

[0066] In some embodiments, the chimeric PB:MosI transposon containing the CRD deficiency described herein comprises the same as SEQ ID NO: 7 The amino acid sequence shown 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. In some embodiments, the chimeric PB:MosI transposon containing the CRD deletion described herein comprises the amino acid sequence shown in SEQ ID NO: 7 having one, two, three, or four of the five conserved amino acid substitutions. In some embodiments,The chimeric PB:MosI transposon containing the CRD deletion described herein contains the amino acid sequence shown in SEQ ID NO: 7. Specification 9 / 45 pages 13 CN 121195063 A

[0067] In some embodiments, the chimeric transposon contains a MosI DNA-binding domain. In some embodiments, the MosI DNA-binding domain contains amino acid residues 3 to 58 of the MosI transposon, that is, contains the amino acid sequence shown in SEQ ID NO: 8:

[0068] SFVPNKEQTRTVLIFCFHLKKTAAESHRMLVEAFGEQVPTVKTCERWFQRFKSGDF (SEQ ID NO: 8).

[0069] In some embodiments, the chimeric transposon containing a truncated piggyBac transposon domain containing the amino acid sequence shown in SEQ ID NO: 3 is linked to a MosI DNA-binding domain containing the amino acid sequence shown in SEQ ID NO: 8. The truncated PB transposase domain can be linked to the MosI DNA-binding domain via an adapter sequence to obtain a chimeric PB:MosI S544-58 transposase containing the amino acid sequence shown in SEQ ID NO: 9, wherein residues 3 to 58 of the MosI transposase are highlighted in bold, and the PB domain is underlined.And the connector sequence is highlighted in italics:

[0070] MAPKKKRKVGGGGSSLDDEHILSALLQSDDELVGEDSDSEVSDHVSEDDVQSDTEEAFIDEVHEVQPT SSGSEILDEQNVIEQPGSSLASNRILTLPQRTIRGKNKHCWSTSKSTRRSRVSALNIVRSQRGPTRMCRNIYDPLL CFKLFFTDEIISEIVKWTNAEISLKRRESMTSATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLSMVYV SVMSRDRFDFLIRCLRMDDKSIRPTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRVYIP NKPSKYGIKILMMCDSGTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSIPLAKNLLQE PYKLTIVGTVRSNKREIPEVLKNSRSRPVGTSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINESTGKPQMVM YYNQTKGGVDTLDQMCSVMTCSRKTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMSLTSS FMRKRLEAPTLKRYLRDNISNILPKEVPGTSGGGGSSFVPNKEQTRTVLIFCFHLKKTAAESHRMLVEAFGEQVPT VKTCERWFQRFKSGDF (SEQ ID NO: 9).

[0071] In some embodiments, the chimeric PB:MosI transposon containing the 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 shown in SEQ ID NO: 9. In some embodiments, the chimeric PB:MosI transposon containing the CRD deletion described herein comprises the amino acid sequence shown in SEQ ID NO: 9 having one, two, three, or four of the five conserved amino acid substitutions. In some embodiments,The chimeric PB:MosI transposon containing the CRD deletion described herein contains the amino acid sequence shown in SEQ ID NO: 9.

[0072] In some embodiments, a chimeric transposon containing truncated piggyBac transposase residues containing the amino acid sequence shown in SEQ ID NO: 3 is linked to two MosI DNA-binding domains containing the following sequence: SEQ ID NO: 6. The truncated PB transposase domain can be linked to the MosI DNA-binding domain via a linker sequence to obtain a chimeric PB:MosI V552-111 transposon containing the amino acid sequence shown in SEQ ID NO: 10, wherein residues 3 to 11 of the MosI transposase are highlighted in bold and the PB domain is underlined.And the connector sequence is highlighted in italics:

[0073] MAPKKKRKVGGGGSSLDDEHILSALLQSDDELVGEDSDSEVSDHVSEDDVQSDTEEAFIDEVHEVQPT SSGSEILDEQNVIEQPGSSLASNRILTLPQRTIRGKNKHCWSTSKSTRRSRVSALNIVRSQRGPTRMCRNIYDPLL CFKLFFTDEIISEIVKWTNAEISLKRRESMTSATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLSMVYV SVMSRDRFDFLIRCLRMDDKSIRPTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRVYIP NKPSKYGIKILMMCDSGTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSIPLAKNLLQE PYKLTIVGTVRSNKREIPEVLKNSRSRPVGTSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINESTGKPQMVM Manual 10 / 45 Page 14 CN 121195063 A YYNQTKGGVDTLDQMCSVMTCSRKTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMSLTSS FMRKRLEAPTLKRYLRDNISNILPKEVPGTSDDSTEEPVGGGGSSFVPNKEQTRTVLIFCFHLKKTAAESHRMLVE AFGEQVPTVKTCERWFQRFKSGDFDVDDKEHGKPPKRYEDAELQALLDEDDAQTQKQLAEQLEVSQQAVSNRLREMG (SEQ ID NO: 10).

[0074] In some embodiments, the chimeric PB:MosI transposon containing the CRD deletion described herein comprises 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% of the amino acid sequence shown in SEQ ID NO: 10. In some embodiments,The chimeric PB:MosI transposon containing the CRD deletion described herein comprises the amino acid sequence shown in SEQ ID NO: 10, having one, two, three, or four of the five conserved amino acid substitutions. In some embodiments, the chimeric PB:MosI transposon containing the CRD deletion described herein comprises the amino acid sequence shown in SEQ ID NO: 10.

[0075] In some embodiments, a chimeric transposon comprising a truncated piggyBac transposase domain containing the amino acid sequence of SEQ ID NO: 4 is linked to a MosI DNA-binding domain comprising the amino acid sequence shown in SEQ ID NO: 8. The truncated PB transposase domain may be linked to the MosI DNA-binding domain via a linker sequence to obtain a chimeric PB:MosI V552-58 transposon comprising the amino acid sequence shown in SEQ ID NO: 11, wherein residues 3 to 58 of the MosI transposase are highlighted in bold, and the PB domain is underlined.And the connector sequence is highlighted in italics:

[0076] MAPKKKRKVGGGGSSLDDEHILSALLQSDDELVGEDSDSEVSDHVSEDDVQSDTEEAFIDEVHEVQPT SSGSEILDEQNVIEQPGSSLASNRILTLPQRTIRGKNKHCWSTSKSTRRSRVSALNIVRSQRGPTRMCRNIYDPLL CFKLFFTDEIISEIVKWTNAEISLKRRESMTSATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLSMVYV SVMSRDRFDFLIRCLRMDDKSIRPTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRVYIP NKPSKYGIKILMMCDSGTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSIPLAKNLLQE PYKLTIVGTVRSNKREIPEVLKNSRSRPVGTSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINESTGKPQMVM YYNQTKGGVDTLDQMCSVMTCSRKTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMSLTSS FMRKRLEAPTLKRYLRDNISNILPKEEVPGTSDDSTEEPVGGGGSSFVPNKEQTRTVLIFCFHLKKTAAESHRMLVE AFGEQVPTVKTCERWFQRFKSGDF (SEQ ID NO: 11).

[0077] In some embodiments, the chimeric PB:MosI transposon containing the 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 shown in SEQ ID NO: 11. In some embodiments, the chimeric PB:MosI transposon containing the CRD deletion described herein comprises the amino acid sequence shown in SEQ ID NO: 11 having one, two, three, or four of the five conserved amino acid substitutions. In some embodiments,The chimeric PB:MosI transposon containing the CRD deletion described herein contains the amino acid sequence shown in SEQ ID NO: 11.

[0078] Chimeric transposases can be used to transposon cells using transposons containing chimeric transposon inverted terminal repeat (ITR) polynucleotides described herein.

[0079] Chimeric transposon inverted terminal repeat (ITR) polynucleotides

[0080] Chimeric transposon inverted terminal repeat (ITR) polynucleotides are also provided herein, which contain in the 5' to 3' direction: (i) a truncated piggyBac ITR sequence lacking a CRD binding site and (ii) a MosI ITR sequence containing binding sites for two MosI DNA binding domains. (See specification 11 / 45 pages 15 CN 121195063 A)

[0081] In some embodiments, the piggyBac left-end (LE) ITR sequence comprises the sequence shown in SEQ ID NO: 12. In some embodiments, the 35bp LE PB ITR sequence is truncated after position 16, lacking a 19bp CRD binding site. In some embodiments, the resulting truncated PB LE ITR sequence lacking the CRD binding site comprises only nucleotides 1 to 16 (highlighted in bold) of the full-length PB LE ITR sequence shown in SEQ ID NO: 12: CCCTAGAAAGATAGT CTGCGTAAAATTGACGCATG (SEQ ID NO: 12). In some embodiments, the PB LE ITR sequence lacking the CRD binding site comprises the sequence CCCTAGAAAGATAGTC (SEQ ID NO: 172).

[0082] In some embodiments, the MosI ITR sequence comprising binding sites for both MosI DNA binding domains comprises the sequence:

[0083] TGTACAAGTATGAAATGTCGTTT (SEQ ID NO: 13).

[0084] In some embodiments, a 23bp MosI sequence (SEQ ID NO: 13) containing binding sites for two MosI DNA binding domains is fused with a truncated PBLE ITR (SEQ ID NO: 172) in reverse orientation.The LE PiggyBac:MosI ITR sequence shown in SEQ ID NO: 15 is obtained as follows: CCCTAGAAAGATAGTCAAACGACATT TCATACTTGTACA (SEQ ID NO: 15)

[0085] In some embodiments, a single nucleotide is deleted from the 3' end of the 23bp MosI sequence containing binding sites for the two MosI DNA binding domains (i.e., from the sequence shown in SEQ ID NO: 13), leaving only residues 1 to 22, i.e., the sequence shown in SEQ ID NO: 14: TGTACAAGTATGAAATGTCGTT (SEQ ID NO: 14).

[0086] In some embodiments, a 1 to 22 nucleotide MosI ITR fragment is fused with the truncated PB LE ITR sequence shown in SEQ ID NO: 12 in reverse orientation to obtain the LE PiggyBac:MosI ITR-1 sequence shown in SEQ ID NO: 16: CCCTAGAAAGATAGTCAACGACATTTCATACTTGTACA (SEQ ID NO: 16).

[0087] In some embodiments, the chimeric transposon ITR polynucleotide further includes one or two additional nucleotides between the truncated piggyBac left ITR sequence and the MosI ITR sequence. In some embodiments, a nucleotide is added to the 3' end of the MosI ITR sequence to form the LE PiggyBac:MosI ITR+1 sequence shown in SEQ ID NO: 17: CCCTAGAAAGATAGTCTAAACGACATTTCATACTTGTACA (SEQ ID NO: 17).

[0088] Similarly, two nucleotides can be added to the 3' end of the MosI ITR sequence to form the LE PiggyBac:MosI ITR+2 sequence shown in SEQ ID NO: 18: CCCTAGAAAGATAGTCTGAAACGACATTTCATACTTGT ACA (SEQ ID NO: 18).

[0089] In some embodiments, the piggyBac right-hand (RE) ITR sequence comprises the sequence shown in SEQ ID NO: 65. In some embodiments, the 63bp RE PB ITR sequence is truncated after position 16.The 19bp CRD binding site and the remaining ITR sequence are missing. The truncated PB RE ITR sequence lacking the CRD binding site contains only nucleotides 1 to 16 (highlighted in bold) of the full-length PB RE ITR shown in SEQ ID NO: 65: CCCTAGAAAGATAATCATA TTGTGACGTACGTTAAAGATAATCATGCGTAAAATTGACGCATG (SEQ ID NO: 65). In some embodiments, the truncated PB RE ITR sequence lacking the CRD binding site contains the sequence CCCTAGAAAGATAATC (SEQ ID NO: 173).

[0090] In some embodiments, the MosI ITR sequence containing binding sites for both MosI DNA binding domains contains the sequence: TGTACAAGTATGAAATGTCGTTT (SEQ ID NO: 13).

[0091] In some embodiments, a 23bp MosI sequence (shown in SEQ ID NO: 13) containing binding sites for two MosI DNA binding domains is fused with a truncated PB RE ITR (SEQ ID NO: 173) in reverse orientation to form the RE PB:MosI ITR sequence shown in SEQ ID NO: 19: CCCTAGAAAGATAATCAAACGAC ATTTCATACTTGTACA (SEQ ID NO: 19)

[0092] In some embodiments, a single nucleotide is deleted from the 3' end of the 23bp MosI sequence containing binding sites for two MosI DNA binding domains (shown in SEQ ID NO: 13) to obtain the following sequence: TGTACAAGTATGAAATGTCGTT (SEQ ID NO: 14).

[0093] In some embodiments, a 1 to 22 nucleotide MosI ITR fragment (SEQ ID NO: 14) is fused with a truncated PB RE ITR (SEQ ID NO: 173) in opposite orientations to form RE PiggyBac:MosI ITR-1:CCCTAGAAAGATAATCAACGACATTTCATACTTGTACA (SEQ ID NO: 20) as shown in SEQ ID NO: 20.

[0094] In some embodiments,The polynucleotide further includes one or two additional nucleotides between the truncated piggyBac right ITR sequence and the MosI ITR sequence. In some embodiments, one nucleotide is added to the 3' end of the MosI ITR sequence to form the RE PB:MosI ITR+1 sequence shown in SEQ ID NO: 21: CCCTAGAA AGATAATCAAAACGACATTTCATACTTGTACA (SEQ ID NO: 21).

[0095] Similarly, two nucleotides can be added to the 3' end of the MosI ITR sequence to form the RE PiggyBac:MosI ITR+2 sequence shown in SEQ ID NO: 22: CCCTAGAAAGATAATCATAAACGACATTTCATACTTGT ACA (SEQ ID NO: 22)

[0096] The chimeric transposon ITR polynucleotide can be incorporated into a vector or transposon (as described below), used in conjunction with a chimeric transposon, a chimeric site-specific transposon fusion protein, and as described herein.

[0097] Chimeric Site-Specific PB:MosI Transposase Fusion Protein

[0098] This document also provides chimeric site-specific transposase fusion proteins comprising, in order from N-terminus to C-terminus: (i) a target-specific DNA-binding domain, (ii) a truncated N-terminus-deficient piggyBac transposase containing a C-terminal cysteine-rich domain (CRD), 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).

[0099] N-terminal Pb transposase deletion

[0100] N-terminal deleted PB transposase sequences and integration-deficient N-terminal piggyBac transposases have been previously described in International Patent Applications PCT / 2022 / 22549 and PCT / US2022 / 077549 as examples of integration-deficient N-terminal deleted piggyBac transposases that can be used in the constructs and fusion proteins described herein, each of which is incorporated herein by reference in its entirety. In some embodiments,The truncated PB transposase has an N-terminal deletion of amino acid residues 1 to 85, 1 to 86, 1 to 87, 1 to 88, 1 to 89, 1 to 90, 1 to 91, 1 to 92, 1 to 93, 1 to 94, 1 to 95, 1 to 96, 1 ​​to 97, 1 to 98, 1 to 99, 1 to 100, 1 to 101, 1 to 102, or 1 to 103. In some embodiments, the truncated PB transposase has an N-terminal deletion of amino acid residues 1 to 93 of SEQ ID NO: 1 or the corresponding residues of SEQ ID NO: 3 or 4.

[0101] Integration-deficient transposase

[0102] In some embodiments, the chimeric site-specific transposase fusion protein comprises an integration-deficient chimeric transposase. The integration-deficient transposase domain is a transposase capable of cleaving its corresponding transposon, but it integrates the cleaved transposon at a lower frequency than the corresponding wild-type transposase. Examples of integration-deficient transposases are disclosed in U.S. Patent Nos. 6,218,185, 6,962,810, 8,399,643, and International Patent Application Publication No. WO 2019 / 173636, each of which is incorporated herein by reference in its entirety as an example of an integration-deficient transposase that can 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 as an example of an integration-deficient transposase that can be used in the constructs described herein. Wild-type SPB can exhibit integration defects by introducing mutations (e.g., K93A, R372A, K375A, R376A and / or D450N (relative to SEQ ID NO: 1, 3 or 4)). It is believed that the introduction of mutations R372A, K375A, R376A and D450N causes the transposase to exhibit integration defects but retains excision function.

[0103] Therefore, in some embodiments, the chimeric site-specific transposase fusion protein comprises a chimeric transposase domain containing the sequence of SEQ ID NO: 1 having 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 containing a sequence of SEQ ID NO: 1 having 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 containing a sequence of SEQ ID NO: 1 having 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 containing a sequence of SEQ ID NO: 1 having the following mutations: R372A, K375A, R376A, and D450N.

[0104] Therefore, in some embodiments, the chimeric site-specific transposase fusion protein comprises a chimeric transposase domain containing a sequence of SEQ ID NO: 3 having 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 containing a sequence of SEQ ID NO: 3 having 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 containing a sequence of SEQ ID NO: 3 having 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 containing a sequence of SEQ ID NO: 3 having the following mutations: R372A, K375A, R376A, and D450N.

[0105] Therefore, in some embodiments, the chimeric site-specific transposase fusion protein comprises a chimeric transposase domain comprising a sequence of SEQ ID NO: 4 having 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 a sequence of SEQ ID NO: 4 having 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 a sequence of SEQ ID NO: 4 having 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,The chimeric transposase domain contains the sequence of SEQ ID NO: 4 with the following mutations: R372A, K375A, R376A, and D450N.

[0106] An exemplary sequence of an integration-deficient transposase with a truncated N-terminus deletion comprises the deletion of amino acids 1 to 93 at the N-terminus of the PB transposase sequence and contains the three integration-deficient mutations shown in SEQ ID NO: 85:

[0107] NKHCWSTSKSTRRSRVSALNIVRSQRGPTRMCRNIYDPLLCFKLFFTDEIISEIVKWTNAEISLKRRE SMTSATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLSMVYVSVMSRDRFDFLIRCLRMDDKSIRPTLRE NDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRVYIPNKPSKYGIKILMMCDSGTKYMINGMPYL GRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSIPLAKNLLQEPYKLTIVGTVASNAREIPEVLKNSRSRP VGTSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINESTGKPQMVMYYNQTKGGVDTLNQMCSVMTCSRKTNRW Instructions 14 / 45 Page 18 CN 121195063 A PMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMSLTSSFMRKRLEAPTLKRYLRDNISNILPKEVP GTSDDSTEEPVMKKRTYCTYCPSKIRRKANASCKKCKKVICREHNIDMCQSCF (SEQ ID NO: 85).

[0108] In some embodiments, the truncated N-terminal deletion integration-deficient transposase 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 shown in SEQ ID NO: 85. In some embodiments, the truncated N-terminal deletion integration-deficient transposase described herein comprises the amino acid sequence shown in SEQ ID NO: 85 having one, two, three, or four of the five conserved amino acid substitutions. In some embodiments,The truncated N-terminal deletion integration-deficient transposase described herein comprises the amino acid sequence shown in SEQ ID NO: 85. In some embodiments, the truncated N-terminal deletion integration-deficient transposase 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 shown in SEQ ID NO: 89. In some embodiments, the truncated N-terminal deletion integration-deficient transposase described herein comprises the amino acid sequence shown in SEQ ID NO: 89 having one, two, three, or four of the five conserved amino acid substitutions. In some embodiments, the truncated N-terminal deletion integration-deficient transposase described herein comprises the amino acid sequence shown in SEQ ID NO: 89. In some embodiments, the truncated N-terminal deletion integration-deficient transposase 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 shown in SEQ ID NO: 97. In some embodiments, the truncated N-terminal deletion integration-deficient transposase described herein comprises the amino acid sequence shown in SEQ ID NO: 97 having one, two, three, or four of the five conserved amino acid substitutions. In some embodiments, the truncated N-terminal deletion integration-deficient transposase described herein comprises the amino acid sequence shown in SEQ ID NO: 97. In some embodiments, the truncated N-terminal deletion integration-deficient transposase 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 shown in SEQ ID NO: 110. In some embodiments, the truncated N-terminal deletion integration-deficient transposase described herein comprises the amino acid sequence shown in SEQ ID NO: 110 having one, two, three, or four of the five conserved amino acid substitutions. In some embodiments, the truncated N-terminal deletion integration-deficient transposase described herein comprises the amino acid sequence shown in SEQ ID NO: 110. In some embodiments,The truncated N-terminal deletion integration-deficient transposase 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 shown in SEQ ID NO: 118. In some embodiments, the truncated N-terminal deletion integration-deficient transposase described herein comprises the amino acid sequence shown in SEQ ID NO: 118 having one, two, three, or four of the five conserved amino acid substitutions. In some embodiments, the truncated N-terminal deletion integration-deficient transposase described herein comprises the amino acid sequence shown in SEQ ID NO: 118. In some embodiments, the truncated N-terminal deletion integration-deficient transposase 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 shown in SEQ ID NO: 131. In some embodiments, the truncated N-terminal deletion integration-deficient transposase described herein comprises the amino acid sequence shown in SEQ ID NO: 131 having one, two, three, or four of the five conserved amino acid substitutions. In some embodiments, the truncated N-terminal deletion integration-deficient transposase described herein comprises the amino acid sequence shown in SEQ ID NO: 131. In some embodiments, the truncated N-terminal deletion integration-deficient transposase described herein (pages 15 / 45, CN 121195063 A) 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 shown in SEQ ID NO: 139. In some embodiments, the truncated N-terminal deletion integration-deficient transposase described herein comprises the amino acid sequence shown in SEQ ID NO: 139 having one, two, three, or four of the five conserved amino acid substitutions. In some embodiments, the truncated N-terminal deletion integration-deficient transposase described herein comprises the amino acid sequence shown in SEQ ID NO: 139. In some embodiments,The truncated N-terminal deletion integration-deficient transposase 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 shown in SEQ ID NO: 152. In some embodiments, the truncated N-terminal deletion integration-deficient transposase described herein comprises the amino acid sequence shown in SEQ ID NO: 152 having one, two, three, or four of the five conserved amino acid substitutions. In some embodiments, the truncated N-terminal deletion integration-deficient transposase described herein comprises the amino acid sequence shown in SEQ ID NO: 152. In some embodiments, the truncated N-terminal deletion integration-deficient transposase 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 shown in SEQ ID NO: 160. In some embodiments, the truncated N-terminal deletion integration-deficient transposase described herein comprises the amino acid sequence shown in SEQ ID NO: 160 having one, two, three, or four of the five conserved amino acid substitutions. In some embodiments, the truncated N-terminal deletion integration-deficient transposase described herein comprises the amino acid sequence shown in SEQ ID NO: 160.

[0109] In some embodiments, the truncated N-terminal deletion integration-deficient transposase 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 shown in any of SEQ ID NO: 87 to 170. In some embodiments, the truncated N-terminal deletion integration-deficient transposase described herein comprises an amino acid sequence having one, two, three, or four of the five conserved amino acid substitutions shown in any of SEQ ID NO: 87 to 170. In some embodiments,The truncated N-terminal-deficient integration-deficient transposase described herein comprises the amino acid sequence shown in any of SEQ ID NO: 87 to 170.

[0110] DNA Targeting Domain

[0111] The chimeric site-specific transposase fusion protein of this disclosure may further comprise one or more DNA targeting domains. The DNA targeting domain may be attached to the C-terminus or N-terminus of the chimeric site-specific transposase fusion protein. In a preferred embodiment, the DNA targeting domain is attached to the N-terminus of the chimeric site-specific transposase fusion protein. It is not desired to be bound by theory, but it is believed that adding a DNA targeting domain to a chimeric site-specific transposase fusion protein improves site-specific transposase activity by targeting the chimeric site-specific transposase fusion protein fused to the DNA targeting domain to the targeted site. In some embodiments, the insertion of a DNA targeting domain 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 without a DNA targeting domain.

[0112] Any DNA targeting domain known in the art can be used in the context of the chimeric transposases and chimeric site-specific transposase fusion proteins described herein, including but not limited to CRISPR, zinc finger motifs, TALE, and transcription factors.

[0113] Methods for engineering zinc finger nucleases that bind to specific targets are described, for example, in the following literature: 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 side-mounted with GGGGS (SEQ ID NO: 86) adapters. In some embodiments, the three zinc finger motifs (ZFM) and flanking GGGGS (SEQ ID NO: 86) linker cumulatively comprise the sequence shown in SEQ ID NO: 23: GGGGSERPYACPVESCDRRFSRSDELTRHIRIHTGQKPFQCRICMRNFSRSDHLT THIRTHTGEKPFACDICGRKFARSDERKRHTKIHLRQKDGGGGS (SEQ ID NO: 23)

[0114] or a sequence having 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.

[0115] In some embodiments, this document provides a chimeric site-specific transposase fusion protein,This chimeric site-specific transposase fusion protein contains three ZFM domains linked to the chimeric PBx:MosI transposase disclosed herein via a linker sequence. An exemplary chimeric ZFM-PB:MosI transposase fusion protein comprises three ZFM domains linked to a chimeric S544:PB:MosI-58 transposase to form a ZFM-S544:PB:MosI-58 chimeric site-specific transposase comprising the following nucleic acid sequence:

[0116] MAPKKKRKVGGGGSERPYACPVESCDRRFSRSDELTRHIRIHTGQKPFQCRICMRNFSRSDHLTTHIR THTGEKPFACDICGRKFARSDERKRHTKIHLRQKDGGGGSNKHCWSTSKSTRRSRVSALNIVRSQRGPTRMCRNIY DPLLCFKLFFTDEIISEIVKWTNAEISLKRRESMTSATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLS MVYVSVMSRDRFDFLIRCLRMDDKSIRPTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFR VYIPNKPSKYGIKILMMCDSGTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSIPLAKN LLQEPYKLTIVGTVASNAREIPEVLKNSRSRPVGTSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINESTGKP QMVMYYNQTKGGVDTLNQMCSVMTCSRKTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMS LTSSFMRKRLEAPTLKRYLRDNISNILPKEVPGTSGGGGSSFVPNKEQTRTVLIFCFHLKKTAAESHRMLVEAFGE QVPTVKTCERWFQRFKSGDF (SEQ ID NO:24)

[0117] In some embodiments, the chimeric ZFM-PB:MosI transposase fusion protein described herein comprises 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% of the amino acid sequence shown in SEQ ID NO: 24. In some embodiments,The chimeric ZFM-PB:MosI transposase fusion protein described herein comprises the amino acid sequence shown in SEQ ID NO: 24 having one, two, three, or four of the five conserved amino acid substitutions. In some embodiments, the chimeric ZFM-PB:MosI transposase fusion protein described herein comprises the amino acid sequence shown in SEQ ID NO: 24.

[0118] Another exemplary chimeric ZFM-PB:MosI transposase fusion protein comprises three ZFM domains linked to the chimeric S544:PB:MosI-111 transposase.To form a ZFM-S544:PB:MosI-111 chimeric site-specific transposase containing the following amino acid sequence:

[0119] MAPKKKRKVGGGGSERPYACPVESCDRRFSRSDELTRHIRIHTGQKPFQCRICMRNFSRSDHLTTHIR THTGEKPFACDICGRKFARSDERKRHTKIHLRQKDGGGGSNKHCWSTSKSTRRSRVSALNIVRSQRGPTRMCRNIY DPLLCFKLFFTDEIISEIVKWTNAEISLKRRESMTSATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLS MVYVSVMSRDRFDFLIRCLRMDDKSIRPTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFR VYIPNKPSKYGIKILMMCDSGTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSIPLAKN LLQEPYKLTIVGTVASNAREIPEVLKNSRSRPVGTSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINESTGKP QMVMYYNQTKGGVDTLNQMCSVMTCSRKTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMS LTSSFMRKRLEAPTLKRYLRDNISNILPKEVPGTSGGGGSSFVPNKEQTRTVLIFCFHLKKTAAESHRMLVEAFGE QVPTVKTCERWFQRFKSGDFDVDDKEHGKPPKRYEDAELQALLDEDDAQTQKQLAEQLEVSQQAVSNRLREMG Manual 17 / 45 Page 21 CN 121195063 A (SEQ ID NO:25)

[0120] In some embodiments, the chimeric ZFM-PB:MosI transposase fusion protein described herein comprises 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% of the amino acid sequence shown in SEQ ID NO: 25. In some embodiments,The chimeric ZFM-PB:MosI transposase fusion protein described herein comprises the amino acid sequence shown in SEQ ID NO: 25, having one, two, three, or four of the five conserved amino acid substitutions. In some embodiments, the chimeric ZFM-PB:MosI transposase fusion protein described herein comprises the amino acid sequence shown in SEQ ID NO: 25.

[0121] Another exemplary chimeric ZFM-PB:MosI transposase fusion protein comprises three ZFM domains linked to a chimeric V552 PB:MosI-58 transposase to form a ZFM-PB:MosI-58 chimeric site-specific transposase comprising the following nucleic acid sequence:

[0122] MAPKKKRKVGGGGSERPYACPVESCDRRFSRSDELTRHIRIHTGQKPFQCRICMRNFSRSDHLTTHIR THTGEKPFACDICGRKFARSDERKRHTKIHLRQKDGGGGSNKHCWSTSKSTRRSRVSALNIVRSQRGPTRMCRNIY DPLLCFKLFFTDEIISEIVKWTNAEISLKRRESMTSATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLS MVYVSVMSRDRFDFLIRCLRMDDKSIRPTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFR VYIPNKPSKYGIKILMMCDSGTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSIPLAKN LLQEPYKLTIVGTVASNAREIPEVLKNSRSRPVGTSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINESTGKP QMVMYYNQTKGGVDTLNQMCSVMTCSRKTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMS LTSSFMRKRLEAPTLKRYLRDNISNILPKEEVPGTSDDSTEEPVGGGGSSFVPNKEQTRTVLIFCFHLKKTAAESHR MLVEAFGEQVPTVKTCERWFQRFKSGDF (SEQ ID NO: 26).

[0123] In some embodiments,The 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 shown in SEQ ID NO: 26. In some embodiments, the chimeric ZFM-PB:MosI transposase fusion protein described herein comprises the amino acid sequence shown in SEQ ID NO: 26 having one, two, three, or four of the five conserved amino acid substitutions. In some embodiments, the chimeric ZFM-PB:MosI transposase fusion protein described herein comprises the amino acid sequence shown in SEQ ID NO: 26.

[0124] Another exemplary chimeric ZFM-PB:MosI transposase fusion protein comprises three ZFM domains linked to the chimeric V552:PB:MosI-111 transposase.To form a ZFM-V552:PB:MosI-111 chimeric site-specific transposase containing the following nucleic acid sequence:

[0125] MAPKKKRKVGGGGSERPYACPVESCDRRFSRSDELTRHIRIHTGQKPFQCRICMRNFSRSDHLTTHIR THTGEKPFACDICGRKFARSDERKRHTKIHLRQKDGGGGSNKHCWSTSKSTRRSRVSALNIVRSQRGPTRMCRNIY DPLLCFKLFFTDEIISEIVKWTNAEISLKRRESMTSATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLS MVYVSVMSRDRFDFLIRCLRMDDKSIRPTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFR VYIPNKPSKYGIKILMMCDSGTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSIPLAKN LLQEPYKLTIVGTVASNAREIPEVLKNSRSRPVGTSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINESTGKP QMVMYYNQTKGGVDTLNQMCSVMTCSRKTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMS LTSSFMRKRLEAPTLKRYLRDNISNILPKEEVPGTSDDSTEEPVGGGGSSFVPNKEQTRTVLIFCFHLKKTAAESHR MLVEAFGEQVPTVKTCERWFQRFKSGDFDVDDKEHGKPPKRYEDAELQALLDEDDAQTQKQLAEQLEVSQQAVSNR Instructions 18 / 45 Page 22 CN 121195063 A LREMG (SEQ ID NO: 27).

[0126] In some embodiments, the chimeric ZFM-PB:MosI transposase fusion protein described herein comprises 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% of the amino acid sequence shown in SEQ ID NO: 27. In some embodiments,The chimeric ZFM-PB:MosI transposase fusion protein described herein comprises the amino acid sequence shown in SEQ ID NO: 27, having one, two, three, or four of the five conserved amino acid substitutions. In some embodiments, the chimeric ZFM-PB:MosI transposase fusion protein described herein comprises the amino acid sequence shown in SEQ ID NO: 27.

[0127] In some aspects, the DNA targeting domain is a TAL array. TALE (transcription activator-like effector) from Xanthomonas typically contains a 288-amino acid N-terminus followed by a variable number of repeat sequences (repetitions) of approximately 34 amino acids in length, followed by a 278-amino acid C-terminus (SEQ ID NO: 29); however, truncated forms have been described in the literature (e.g., see Miller et al., Nat Biotechnol 29, 143–148 (2011)). TAL fused to FokI nuclease (TAL effector nuclease; TALEN) typically contains truncated N-terminus and C-terminus. For example, the first 152 amino acids of the N-terminus can be removed (referred to as δ152; the N-terminal sequence after removing the first 152 amino acids is shown in SEQ ID NO: 30), and the C-terminus can be truncated, leaving 63 amino acids (referred to as +63; the sequence is shown in SEQ ID NO: 31).

[0128] TAL typically contains an array of 34 amino acids repeated a variable number of times. The two amino acids at positions 12 and 13 are usually variable and determine which nucleotide the TAL repeat sequence will recognize. Thus, amino acid NG recognizes T, NI recognizes A, NN recognizes G or A, HD recognizes C, NK recognizes G, and NS recognizes A, C, G, or T. Other amino acids within the 34-residue repeat sequence can also be varied. For example, position 11 is often changed to recognize the repeating N of G. Additionally, positions 4 and 32 are often varied to reduce the repeatability of the array but do not determine the binding specificity. The number of 34 amino acid repeats in the array determines the length of the DNA sequence being recognized (one protein repeat binds one DNA base). Furthermore, the last base is recognized by a "half-array" of 20 amino acids instead of 34 amino acids. This feature allows the TAL array to be programmed to bind specific DNA sequences. Those skilled in the art will be able to modify the TALEN sequence to achieve desired target specificity.

[0129] Furthermore, the N-terminal domain of TAL (e.g.,The N-terminal domain containing the sequence shown in SEQ ID NO: 30 recognizes and requires the binding of the T at the 5' end of the target DNA sequence. Mutations of the TAL N-terminal domain that no longer require the 5' T have been described in the literature (see, for example, Lamb et al., Nucleic Acids Res. 2013 Nov;41(21):9779-85). For example, the NT-G mutant containing the amino acid sequence shown in SEQ ID NO: 32 requires the 5' G instead of the 5' T, while the NT-βN mutant containing the amino acid sequence shown 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 sequences of the fusion proteins described herein that can be targeted using a TAL array.

[0130] In some embodiments, the TAL array provided herein comprises nine 34-amino acid repeat sequences followed by 20-amino acid "half" repeat sequences with flanking BsmBI IIS type restriction sites. In one embodiment, a single TAL module containing a 34-amino acid repeat sequence or a 20-amino acid "half" repeat sequence can be designed and synthesized, with a BsmBI IIS type restriction site attached to its side. In some embodiments, the entire TAL module set contains four modules capable of recognizing A, C, G, and T at each 10 bp position (i.e., 40 TAL modules for a 10 bp identified target) and one TAL half repeat sequence module. Exemplary TAL modules are shown in SEQ ID NO: 34 to 37, where X is any amino acid:

[0131] TAL module version 1: LTPDQVVAIAXXXGGKQALETVQRLLPVLCQDHG (SEQ ID NO: 34) Specification 19 / 45 pages 23 CN 121195063 A

[0132] TAL module version 2: LTPEQVVAIAXXXGGKQALETVQRLLPVLCQAHG (SEQ ID NO: 35)

[0133] TAL module version 3: LTPDQVVAIAXXXGGKQALETVQRLLPVLCQAHG (SEQ ID NO: 36)

[0134] TAL module version 4: LTPAQVVAIAXXXGGKQALETVQRLLPVLCQDHG (SEQ ID NO: 37).

[0135] An exemplary TAL half-module is shown in SEQ ID NO: 38,Where X is any amino acid: LTPEQVVAIAXXXGGRPALE (SEQ ID NO: 38).

[0136] TAL array pairs targeting sequences in desired genes can be designed, and the corresponding modules can be selected and assembled together using “Golden Gate Assembly” to assemble each TAL array in a frame. The DNA sequences encoding the TAL arrays generated herein can be further optimized for codons using the GeneArt algorithm (Thermo Fisher).

[0137] TAL arrays can be used to target TTAA sites in the genome. When designing left and right TAL arrays containing N-terminal domains that recognize the T-terminal and C-terminal domains of the transposase to be fused to the chimeric site-specific transposase, one TAL array recognizes the 5' sequence of the TTAA sequence, and the other TAL array recognizes the 3' sequence of the TTAA sequence. Since the 5' sequence of TTAA is most often different from the 3' sequence of TTAA in a genomic DNA target, TAL site-specific SuperpiggyBac transposase fusion proteins (“TAL-ssSPB” or “TAL-PBx”) are most commonly used as heterodimers composed of two transposase fusion proteins, each containing a different TAL domain that recognizes two different DNA sequences. Furthermore, the sequence recognized by the TAL array is not necessarily directly adjacent to TTAA. Instead, it can be separated from TTAA by a spacer of a given bp length (e.g., a 12 bp, 13 bp, or 14 bp spacer).

[0138] The TAL array can target any target DNA sequence (e.g., a genomic DNA sequence). It will be apparent to those skilled in the art that any left TAL array targeting a given target can be combined with any right TAL array targeting the same target.

[0139] In some embodiments, the TAL array targets green fluorescent protein (GFP). A TAL-piggyBac transposase fusion protein comprising an N-terminal deleted piggyBac transposase sequence and an integration-deficient N-terminal piggyBac transposase targeting GFP has been described in co-owned International Patent Application No. PCT / 2022 / 22549 as an example of a TAL-piggyBac transposase fusion protein that can be used in the constructs described herein.The international patent application is incorporated herein by reference in its entirety.

[0140] An exemplary sequence of the right TAL array that can be incorporated into a chimeric site-specific transposase fusion protein targeting GFP is shown in SEQ ID NO: 41 to 44.

[0141] In some embodiments, the TAL array targets the LINE1 repeat element. A TAL-piggyBac transposase fusion protein comprising an N-terminal deleted piggyBac transposase sequence and an integration-deficient N-terminal piggyBac transposase targeting the LINE1 element has been described in International Patent Application No. PCT / 2022 / 22549 as an example of a TAL-piggyBac transposase fusion protein that can be used in the constructs described herein, and is incorporated herein by reference in its entirety.

[0142] Exemplary sequences of the left and right TAL arrays targeting LINE1 are shown in SEQ ID NO: 66 and 67, respectively.

[0143] Nuclear Localization Signal

[0144] In some embodiments, the chimeric transposases and chimeric site-specific transposase fusion proteins provided herein may include a nuclear localization sequence (NLS) within the frame. Examples of transposases fused to a nuclear localization signal are disclosed in U.S. Patent Nos. 6,218,185, 6,962,810, 8,399,643, and International Patent Application Publication No. WO 2019 / 173636, each of which is incorporated herein in its entirety as an example of transposases fused to an NLS that may be used in the constructs described herein. In some embodiments, the NLS comprises the sequence PKKKRKV (SEQ ID NO: 39). In some aspects, the in-frame NLS is located upstream (N-terminus) of a transposase domain containing an N-terminal deletion. In some aspects, on page 20 / 45 of a specification, CN 121195063 A, the in-frame NLS is located downstream (C-terminus) of the transposase domain containing the N-terminal deletion.

[0145] Generally, the NLS is preferably located at the N-terminus of a chimeric transposase or a chimeric site-specific transposase fusion protein.

[0146] In some aspects, the in-frame NLS is directly fused to the N-terminus of a chimeric transposase or a chimeric site-specific transposase fusion protein. In some embodiments, an initiator methionine 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, the residue numbering in SEQ ID NO: 5 begins at residue 12 of SEQ ID NO: 5 for the purpose of identifying deleted and mutated residues.

[0147] Nucleic Acids

[0148] Polynucleotides are also provided herein.These polynucleotides contain nucleic acid sequences encoding the chimeric transposase and chimeric site-specific transposase fusion protein described herein. In some embodiments, the polynucleotides are isolated or purified.

[0149] The isolated polynucleotides of this disclosure can be prepared using (a) recombinant methods, (b) synthetic techniques, (c) purification techniques and / or (d) combinations thereof well known in the art. Methods for constructing nucleic acids encoding the chimeric transposase and chimeric site-specific transposase fusion protein described herein are well known in the art or as described herein, for example, PCR-based mutagenesis.

[0150] The fusions of the present invention can be generated using any suitable method known in the art or described herein.

[0151] The isolated polynucleotides of this disclosure (such as RNA, cDNA, genomic DNA or any combination thereof) can be obtained from biological sources using any number of cloning methods known to those skilled in the art. In some aspects, oligonucleotide probes that will selectively hybridize with the polynucleotides of this disclosure under stringent conditions are used to identify desired sequences in cDNA or genomic DNA libraries.

[0152] Methods for amplifying RNA or DNA are well known in the art and can be used in accordance with this disclosure based on the teachings and guidance provided herein, without requiring excessive experimentation. Known DNA or RNA amplification methods include, but are not limited to, polymerase chain reaction (PCR) and related amplification processes (see, for example, U.S. Patent Nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188 to Mullis et al.; U.S. Patent Nos. 4,795,699 and 4,921,794 to Tabor et al.; U.S. Patent No. 5,142,033 to Innis; U.S. Patent No. 5,122,464 to Wilson et al.; U.S. Patent No. 5,091,310 to Innis; U.S. Patent No. 5,066,584 to Gyllensten et al.; U.S. Patent No. 4,889,818 to Gelfand et al.; U.S. Patent No. 4,994,370 to Silver et al.; U.S. Patent No. 4,766,067 to Biswas; and U.S. Patent No. 4,066 to Ringold et al.). 656,134) and RNA-mediated amplification, which uses antisense RNA targeting the target sequence as a template for double-stranded DNA synthesis (US Patent No. 5,130,238 to Malek et al., trade name NASBA), the contents of which are incorporated herein by reference.

[0153] For example, polymerase chain reaction (PCR) technology can be used,The disclosed polynucleotide sequences and related genes are amplified directly from genomic DNA or cDNA libraries. PCR and other in vitro amplification methods can also be used, for example, to clone nucleic acid sequences encoding proteins to be expressed, to prepare nucleic acids for use as probes to detect the presence of desired mRNA in a sample, for nucleic acid sequencing, or for other purposes. Examples of techniques sufficient to guide a technician through in vitro amplification methods can be found in Berger (ibid.); Sambrook (ibid.); Ausubel (ibid.); Mullis et al., U.S. Patent No. 4,683,202 (1987); and Innis et al. (ed.), PCR Protocols: A Guide to Methods and Applications, Academic Press Inc., San Diego, Calif. (1990). Commercially available kits for genomic PCR amplification are known in the art. See, for example, the Advantage-GC Genomic PCR Kit (Clontech). Additionally, for example, the T4 gene 32 protein (Boehringer Mannheim) can be used to improve the yield of long PCR products as described on pages 21 / 45 of the specification, CN 121195063 A.

[0154] The polynucleotides of this disclosure can also be prepared by direct chemical synthesis using known methods (see, for example, Ausubel et al., ibid.). Chemical synthesis generally produces single-stranded oligonucleotides, which can be converted into double-stranded DNA by hybridization with complementary sequences or by polymerization with DNA polymerase using single strands as templates. Those skilled in the art will recognize that although the chemical synthesis of DNA may be limited to sequences of about 100 or more bases, longer sequences can be obtained by linking shorter sequences.

[0155] Expression Vectors and Host Cells

[0156] This disclosure also relates to vectors comprising the polynucleotides of this disclosure, host cells genetically engineered with recombinant vectors, and the production of at least one protein scaffold by recombinant techniques, as is well known in the art. See, for example, Sambrook et al., ibid.; Ausubel et al., ibid., each incorporated herein by reference in its entirety.

[0157] Polynucleotides may optionally be ligated into a vector containing a selective marker for replication in a host. Generally, plasmid vectors are introduced into precipitates (such as calcium phosphate precipitates) or into complexes containing charged lipids. If the vector is a virus, it may be packaged in vitro using a suitable packaging cell line and then transduced into host cells.

[0158] In some embodiments,The DNA insert is operatively ligated to a suitable promoter. In some embodiments, the promoter is an EF-1α promoter comprising the sequence shown in SEQ ID NO: 45. The expression construct may further contain sites for transcription initiation and termination, as well as ribosome binding sites in the transcription region for translation. The coding portion of the mature transcript expressed by the construct will preferably include a translation start codon (e.g., ATG) appropriately positioned at the start of the mRNA to be translated and a stop codon (e.g., UAA, UGA, or UAG) at the end, wherein UAA and UAG are preferred for mammalian or eukaryotic cell expression.

[0159] The expression vector may further comprise one or more selectable markers. Examples of selective biomarkers include, but are not limited to, ampicillin, zeocin (Shbla gene), puromycin (pac gene), hygromycin B (hygB gene), G418 / genemycin (neo gene), DHFR (encoding dihydrofolate reductase and conferring resistance to methotrexate), mycophenolic acid or glutamine synthase (GS, US Patent Nos. 5,122,464; 5,770,359; 5,827,739), blastomycin (bsd gene), resistance genes from eukaryotic cell culture, and ampicillin, zeocin (Shbla gene), puromycin (pac gene), hygromycin B (hygB gene), G418 / genemycin (neo gene), kanamycin, spectinomycin, streptomycin, carbenicillin, bleomycin, erythromycin, polymyxin B, or tetracycline resistance genes used in Escherichia coli and other bacteria or prokaryotes. Appropriate culture media and conditions for the host cells described above are known in the art. Suitable vectors will be apparent to those skilled in the art. Vector constructs can be introduced into host cells by calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other known methods. Such methods are described in the art, such as Sambrook, ibid., Chapters 1-4 and 16-18; Ausubel, ibid., Chapters 1, 9, 13, 15, and 16.

[0160] The expression vector may contain at least one selective cell surface marker for isolating cells modified by the compositions and methods of this disclosure. Selective cell surface markers of this disclosure include surface proteins, glycoproteins, or a proteome that distinguishes one cell or cell subpopulation from another defined cell subpopulation. In some embodiments,Selective cell surface markers distinguish cells modified by the compositions or methods of this disclosure from those not modified by the compositions or methods of this disclosure. Examples of cell surface markers include, but are not limited to, “designated cluster” or “taxonomic cluster” proteins (generally abbreviated as “CD”), such as truncated or full-length forms of CD19, CD271, CD34, CD22, CD20, CD33, CD52, or any combination thereof. Cell surface markers further include the suicide gene marker RQR8 (Philip B, specification 22 / 45 pages 26 CN 121195063 A et al. Blood. 21 Aug 2014; 124(8):1277-87).

[0161] The expression vector may further comprise at least one selective drug resistance marker for isolating cells modified by the compositions and methods of this disclosure. The selective drug resistance markers disclosed herein may include wild-type or mutant Neo, DHFR, TYMS, FRANCF, RAD51C, GCS, MDR1, ALDH1, NKX2.2, or any combination thereof.

[0162] Those skilled in the art are aware of many expression systems that can be used to express nucleic acids encoding the proteins of this disclosure. The nucleic acids of this disclosure can be expressed in host cells by opening (by manipulation) in host cells containing endogenous DNA encoding the protein scaffold of this disclosure. Such methods are well known in the art, for example, as described in U.S. Patent 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.

[0163] Cell cultures that can be used to produce protein scaffolds, specific portions thereof, or variants thereof are bacteria, yeast, and mammalian cells as known in the art. Mammalian cell systems are typically in the form of a cell monolayer, although mammalian cell suspensions or bioreactors may also be used. Many suitable host cell lines capable of expressing fully glycosylated proteins have been developed in this art, including 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, P3X63Ag8.653, SP2 / 0-Ag14, 293 cells, HeLa cells, etc., which are readily available from, for example, the American Center for Type Culture Collection (www.atcc.org) in Manassas, Virginia. Preferred host cells include lymphoid cells.Cells 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 CRL-1851). In a preferred aspect, the recombinant cells are P3X63Ab8.653 or SP2 / O-Ag14 cells.

[0164] The expression vectors of these cells may include one or more of the following expression control sequences: origin of replication; promoter (e.g., late or early SV40 promoter, CMV promoter (US Patent Nos. 5,168,062; 5,385,839), HSV tk promoter, pgk (glycerol phosphate kinase) promoter, EF-1α promoter (US Patent No. 5,266,491), at least one human promoter); enhancer and / or processing information sites, such as ribosome binding sites, RNA splicing sites, polyadenylation sites (e.g., SV40 large T Ag poly A addition site), and transcription terminator sequences. See, for example, Ausubel et al., ibid.; Sambrook et al., ibid. Other cells that can be used to produce the nucleic acids or proteins of this disclosure are known and / or available, for example, from the U.S. Type Culture Collection's Cell Lines and Hybridoma Catalogue (www.atcc.org) or other known or commercial sources.

[0165] When using eukaryotic host cells, polyadenylation or transcription terminator sequences are typically incorporated into the vector. An example of a terminator sequence is a polyadenylation sequence from the bovine growth hormone gene. In some embodiments, the polyA sequence is an SV40 polyA sequence comprising the sequence shown in SEQ ID NO: 47.

[0166] Sequences for accurate transcript splicing may also be included. An example of a splicing sequence is the VP1 intron from SV40 (Sprague et al., J. Virol. 45:773-781 (1983)). Furthermore, as is known in the art, gene sequences controlling replication in host cells may be incorporated into the vector.

[0167] The plasmid constructs described herein can be used to deliver nucleic acids encoding the transposase domains or fusion proteins described herein to cells.

[0168] The transposase domains and fusion proteins described herein can also be delivered to cells using mRNA constructs. Therefore, in one embodiment, an mRNA sequence encoding the transposase domains or fusion proteins described herein is provided herein. Such mRNAs The sequence can be made using nanoparticles (e.g.,Lipid nanoparticles) are delivered to cells. Examples of lipid nanoparticles are described, for example, in International Patent Application Publications WO 2022 / 087148, WO 2022 / 182792, WO 2023 / 141576, CN 121195063 A, WO 2024 / 035783, and WO 2023 / 141576, as examples of lipid nanoparticles that can be used to deliver mRNA constructs encoding the fusion protein or transposase domain described herein, each of which is incorporated herein by reference in its entirety. The mRNA constructs can also be delivered to cells by electroporation or nuclear transfection. The mRNA may be capped or otherwise modified.

[0169] Transposons, Cells, and Modified Cells

[0170] The chimeric transposases and chimeric site-specific transposase fusion proteins described herein can be used in conjunction with transposons to modify cells. The transposon may 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 transposases are described in detail in U.S. Patent Nos. 6,218,182, 6,962,810, 8,399,643, and PCT Publication No. WO 2010 / 099296, each of which is incorporated herein by reference in its entirety as an example of a transposon that may be used in the methods described herein. The transposon may contain a nucleic acid encoding a therapeutic protein or therapeutic agent. Examples of therapeutic proteins include those disclosed in PCT Publications WO 2019 / 173636 and WO 2020 / 051374, as examples of therapeutic proteins that can be used in the chimeric transposases and chimeric site-specific transposase fusion proteins described herein, each of which is incorporated herein by reference in its entirety.

[0171] In some embodiments, the transposon may be a transposon comprising one or more chimeric PB:MosI ITR polynucleotides described herein. In some embodiments, the transposon comprises chimeric LE PB:MosI ITR polynucleotides and chimeric RE PB:MosI ITR polynucleotides. In some embodiments, the LE PB:MosI ITR polynucleotide is a PB:MosI ITR comprising the sequence shown in SEQ ID NO: 19. In some embodiments,LE PB:MosI ITR polynucleotide is PB:MosI ITR-1 containing the sequence shown in SEQ ID NO: 20. In some embodiments, LE PB:MosI ITR polynucleotide is PB:MosI ITR+1 containing the sequence shown in SEQ ID NO: 21. In some embodiments, LE PB:MosI ITR polynucleotide is PB:MosI ITR+2 containing the sequence shown in SEQ ID NO: 22.

[0172] In some embodiments, RE PB:MosI ITR polynucleotide is PB:MosI ITR containing the sequence shown in SEQ ID NO: 15. In some embodiments, RE PB:MosI ITR polynucleotide is PB:MosI ITR-1 containing the sequence shown in SEQ ID NO: 16. In some embodiments, RE PB:MosI ITR polynucleotide is PB:MosI ITR+1 containing the sequence shown in SEQ ID NO: 17. In some embodiments, the RE PB:MosI ITR polynucleotide is PB:MosI ITR+2 containing the sequence of SEQ ID NO: 18.

[0173] In some embodiments, the PB:MosI ITR transposon contains the nucleotide sequence:

[0174] TTAACCCTAGAAAGATAGTCAAACGACATTTCATACTTGTACAGACGCATGCATTCTTGAAATATTGC TCTCTCTTTCTAAATAGCGCGAATCCGTCGCTGTGCATTTAGGACATCTCAGTCGCGAGCTGAAGGGCATCGACTT CAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGAC AAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACC ACTACCAGCAGAAATGTACAAGTATGAAATGTCGTTTGATTATCTTTCTAGGGTTAA (SEQ ID NO: 50). In some embodiments,The PB:MosI ITR transposon described herein contains 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% of the nucleic acid sequence shown in SEQ ID NO: 50.

[0175] In some embodiments, the PB:MosI ITR-1 transposon contains the nucleotide sequence:

[0176] TTAACCCTAGAAAGATAGTCAACGACATTTCATACTTGTACAGACGCATGCATTCTTGAAATATTGCT CTCTCTTTCTAAATAGCGCGAATCCGTCGCTGTGCATTTAGGACATCTCAGTCGCGAGCTGAAGGGCATCGACTTC Specification 24 / 45 pages 28 CN 121195063 A AAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACA AGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCA CTACCAGCAGAAATGTACAAGTATGAAATGTCGTTGATTATCTTTCTAGGGTTAA (SEQ ID NO: 51). In some embodiments, the PB:MosI ITR transposon described herein contains 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% of the nucleic acid sequence shown in SEQ ID NO: 51.

[0177] In some embodiments,PB: MosI ITR+1 transposon contains the nucleotide sequence:

[0178] TTAACCCTAGAAAGATAGTCTAAACGACATTTCATACTTGTACAGACGCATGCATTCTTGAAATATTG CTCTCTCTTTCTAAATAGCGCGAATCCGTCGCTGTGCATTTAGGACATCTCAGTCGCGAGCTGAAGGGCATCGACT TCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGA CAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACATCGAGGACGGCAGCGTGCAGCTCGCCGAC CACTACCAGCAGAAATGTACAAGTATGAAATGTCGTTTTGATTATCTTTCTAGGGTTAA (SEQ ID NO: 52). In some embodiments, the PB:MosI ITR transposon described herein contains 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% of the nucleic acid sequence shown in SEQ ID NO: 52.

[0179] In some embodiments, the PB:MosI ITR+2 transposon contains the nucleotide sequence:

[0180] TTAACCCTAGAAAGATAGTCTGAAACGACATTTCATACTTGTACAGACGCATGCATTCTTGAAATATT GCTCTCTCTTTTTAAATAGCGCGAATCCGTCGCTGTGCATTTAGGACATCTCAGTCGCGAGCTGAAGGGCATCGAC TTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCG ACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGA CCACTACCAGCAGAAATGTACAAGTATGAAATGTCGTTTATGATTATCTTTCTAGGGTTAA (SEQ ID NO: 53). In some embodiments,The PB:MosI ITR transposon described herein contains 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 nucleic acid sequence to the nucleic acid sequence shown in SEQ ID NO: 53.

[0181] In some embodiments, the transposon disclosed herein contains a shortened PB:MosI ITR polynucleotide. In some embodiments, the left-terminal shortened PB:MosI ITR contains the nucleic acid sequence shown in SEQ ID NO: 54. In some embodiments, the right-terminal shortened PB:MosI ITR contains the nucleic acid sequence shown in SEQ ID NO: 55. In some embodiments, the transposon contains both a left-terminal shortened PB:MosI ITR and a right-terminal shortened PB:MosI ITR to produce a short PB:MosI transposon containing the nucleic acid sequence shown in SEQ ID NO: 56. In some embodiments, the PB:MosI short transposon comprises 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 shown in SEQ ID NO: 56.

[0182] In another aspect, this document provides modified cells comprising one or more transposons and one or more chimeric transposases or chimeric site-specific transposase fusion proteins described herein. The cells of this disclosure and the modified cells may be mammalian cells. Preferably, the cells and the modified cells are human cells.

[0183] Cells modified using chimeric transposases or chimeric site-specific transposase fusion proteins described herein may be germ cells or somatic cells. The cells of this disclosure and the modified cells may be immune cells, for example,Lymphoid progenitor cells, natural killer (NK) cells, T lymphocytes (T cells), stem memory T cells (TSCM cells), central memory T cells (TCM), stem cell-like T cells, B lymphocytes (B cells), antigen-presenting cells (APCs), cytokine-induced killer cells (CIK cells), myeloid progenitor cells, neutrophils, basophils, eosinophils, monocytes, macrophages, platelets, erythrocytes, red blood cells (RBCs), megakaryocytes, or osteoclasts. Modified cells can be differentiated, undifferentiated, or immortalized. Modified undifferentiated cells can be stem cells. Modified undifferentiated cells can be induced pluripotent stem cells. Modified cells can be hepatocytes, T cells, hematopoietic stem cells, natural killer cells, macrophages, dendritic cells, monocytes, megakaryocytes, or osteoclasts. The modified cells can be modified when they are in a dormant state, an activated state, a quiescent state, an interphase, a prophase, a metaphase, an anaphase, or a telophase. The modified cells can be fresh cells, cryopreserved cells, large numbers of cells, cells classified into subpopulations, cells derived from whole blood, cells derived from apheresis, or cells derived from immortalized cell lines. Detailed descriptions of cell isolation from apheresis products or blood are disclosed in PCT Publications WO 2019 / 173636 and WO 2020 / 051374, each of which is incorporated herein by reference in its entirety.

[0184] The methods of this disclosure can be used to modify or generate populations of modified T cells.In this population, 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%, at least 99%, or 100%, or any percentage thereof, of the modified T cells express one or more cell surface markers of stem memory T cells (TSCM) or TSCM-like cells; and said one or more cell surface markers include CD45RA and CD62L. The cell surface markers of TSCM or TSCM-like cells may also include one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95, and IL-2Rβ. Cell surface markers of TSCM or TSCM-like cells may also include one or more of CD45RA, CD95, IL-2Rβ, CCR7, and CD62L.

[0185] This disclosure provides a method for expressing CAR on a cell surface. The method includes: (a) obtaining a cell population; (b) contacting the cell population with a composition containing CAR or a sequence encoding CAR under conditions sufficient to transfer CAR across the 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 integrating the sequence encoding CAR; and (d) expanding and / or selecting at least one cell from the modified cell population expressing CAR on the cell surface. A more detailed description of the method for expressing CAR on a cell surface is disclosed in PCT Publications WO 2019 / 049816 and WO 2020 / 051374.

[0186] This disclosure further provides a cell or population of cells comprising a composition comprising: (a) an inducible transgene construct comprising a sequence encoding an inducible promoter and a sequence encoding a transgene; and (b) a receptor construct comprising a sequence encoding a constitutive promoter and a sequence encoding an exogenous receptor (such as a CAR), wherein the exogenous receptor is expressed when the construct of (a) and the construct of (b) are integrated into the genomic sequence of the cell, and wherein the exogenous receptor transduces intracellular signals that directly or indirectly target the inducible promoter upon binding to a ligand or antigen, thereby regulating the expression of the inducible transgene (a), thereby modifying gene expression.

[0187] This disclosure further provides a composition,The composition comprises a modified, expanded, and selected cell population of the methods described herein.

[0188] The modified cells of this disclosure (e.g., CAR T cells) may be further modified to enhance their therapeutic potential. Alternatively or otherwise, the modified cells may be further modified to make them less sensitive to immune and / or metabolic checkpoints, for example by blocking and / or diluting specific checkpoint signals that are naturally delivered to the cells in the tumor immunosuppressive microenvironment (e.g., checkpoint inhibition).

[0189] The modified cells of this disclosure (e.g., CAR T cells) may be further modified to silence or reduce (i) one or more genes encoding receptors for inhibitory checkpoint signals; (ii) one or more genes encoding intracellular proteins involved in checkpoint signaling; (iii) one or more genes encoding transcription factors that inhibit therapeutic efficacy; (iv) one or more genes encoding receptors for cell death or apoptosis; (v) one or more genes encoding metabolic sensing proteins; (vi) one or more genes encoding proteins that confer sensitivity to cancer therapies (including monoclonal antibodies); and / or (vii) one or more genes encoding growth dominance factors. The modified cells may also be modified to silence or reduce the expression of endogenous T cell receptors. Non-limiting examples of genes that can be modified to silence or reduce expression or inhibit function include, but are not limited to, genes encoding inhibitory checkpoint signals, intracellular proteins, transcription factors, cell death or apoptosis receptors, metabolic sensing proteins, proteins conferring sensitivity to cancer therapies, and growth advantage factors disclosed in PCT Publication No. WO 2019 / 173636, which is incorporated herein by reference in its entirety as an example of genes that can be silenced in the cells of this disclosure.

[0190] The modified cells of this disclosure (e.g., CAR T cells) may be further modified to express modified / chimeric checkpoint receptors. Modified / chimeric checkpoint receptors may comprise empty receptors, decoy receptors, or dominant-negative receptors. Exemplary empty, decoy, or dominant / negative intracellular receptors / proteins include, but are not limited to: inhibitory checkpoint signals, transcription factors, cytokines or cytokine receptors, chemokines or chemokine receptors, cell death or apoptosis receptors / ligands, metabolic sensing molecules, proteins conferring sensitivity to cancer therapies, and expression of signaling components downstream of oncogenes or tumor suppressor genes. Non-limiting examples of cytokines, cytokine receptors, chemokines, and chemokine receptors are disclosed in PCT Publication No. WO 2019 / 173636, which is incorporated herein by reference in its entirety.For example, modified / chimeric checkpoint receptors that can be expressed in the cells described herein.

[0191] Genome modification may include introducing nucleic acid sequences, transgenes, and / or genome editing constructs into cells, either in vitro, in vivo, in vitro, or in situ, to stably integrate nucleic acid sequences, transiently integrate nucleic acid sequences, generate site-specific integration of nucleic acid sequences, or generate biased integration of nucleic acid sequences. The nucleic acid sequences may be transgenes.

[0192] Stable chromosomal integration may be random integration, site-specific integration, or biased integration. Without wishing to be bound by theory, it is believed that adding DNA-binding domains to chimeric site-specific transposases and chimeric site-specific transposase fusion proteins described herein improves the site specificity of transposases.

[0193] Site-specific integration may occur at safe harbor sites. Genome safe harbor sites are capable of accommodating the integration of new genetic material in a manner that ensures the newly inserted genetic element functions reliably (e.g., expressed at therapeutically effective expression levels) and does not cause harmful alterations to the host genome, thereby posing a risk to the host organism. Non-limiting examples of potential genomic safe harbors include intron sequences of the human albumin gene, adeno-associated virus site 1 (AAVS1), naturally occurring integration sites of AAV virus on chromosome 19, sites of the chemokine (C-C motif) receptor 5 (CCR5) gene, and sites of human orthologs of the mouse Rosa26 locus.

[0194] Site-specific transgene integration can occur at sites that disrupt target gene expression. Disruption of target gene expression can occur through site-specific integration at introns, exons, promoters, genetic elements, enhancers, repressors, start codons, stop codons, and response elements. Non-limiting examples of target genes targeted by site-specific integration include TRAC, TRAB, PD1, any gene encoding immunosuppressive proteins, genes encoding endogenous T cell receptors, and genes encoding proteins involved in allogeneic rejection responses.

[0195] Site-specific transgene integration can occur at sites that cause enhanced expression of target genes. Enhanced expression of target genes can occur through site-specific integration at introns, exons, promoters, genetic elements, enhancers, repressors, start codons, stop codons, and response elements.

[0196] Site-specific transgene integration sites can be unstable chromosomal insertions. Instable integration can be transient nonchromosomal integration, semi-stable nonchromosomal integration, semi-persistent nonchromosomal insertion, or unstable chromosomal insertion. Transient nonchromosomal insertions can be epichromosomal or cytoplasmic. On the one hand, transient nonchromosomal insertions of transgenes do not integrate into chromosomes.Furthermore, the modified genetic material does not replicate during cell division.

[0197] The site-specific transgene integration site may be a modified binding site of the DNA-targeting domain in a chimeric transposase or chimeric site-specific transposase fusion protein as described herein. For example, the TTAA target DNA integration site for SPB may be modified to insert into a flanking DNA binding site of a DNA-targeting domain comprising three zinc finger motifs (e.g., a DNA-targeting domain comprising or consisting of the following sequence: SEQ ID NO: 23 or a sequence having 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). For example, a DNA-targeting domain comprising three zinc finger motifs is believed to bind to the DNA sequence GCGTGGGCG. Therefore, it is believed that introducing two copies of the DNA sequence GCGTGGGCG, which is side-attached to the TTAA target integration site of a chimeric site-specific transposase fusion protein containing a DNA targeting domain with three zinc finger motifs, can improve site-specific integration of the chimeric transposase. In these embodiments, the two copies of the DNA sequence GCGTGGGCG are in reverse (5') and complementary (3') orientations.

[0198] In some embodiments, this document provides a polynucleotide comprising, in 5' to 3' order: an inverse complement of the sequence for the target site of the DNA targeting domain; a first spacer; a TTAA target integration site for SPB; a second spacer region; and the sequence for the target site of the DNA targeting domain. In some embodiments, the first spacer and the second spacer have the same length. In some embodiments, the length of the first spacer and / or the second spacer is 3 bp. In some embodiments, the length of the first spacer and / or the second spacer is 4 bp. In some embodiments, the length of the first spacer and / or the second spacer is 5 bp. In some embodiments, the length of the first spacer and / or the second spacer is 6 bp. In some embodiments, the length of the first spacer and / or the second spacer is 7 bp. In some embodiments, the length of the first spacer and / or the second spacer is 8 bp. In some embodiments, the length of the first spacer and / or the second spacer is 9 bp. In some embodiments, the length of the first spacer and / or the second spacer is 10 bp.

[0199] Modified target sites can be introduced into cells or cell lines to facilitate targeted genome engineering. For example,Cell lines engineered to contain a modified target site for a chimeric transposase or chimeric site-specific transposase fusion protein provided herein can be transfected with the chimeric transposase or chimeric site-specific transposase fusion protein and a transposon containing donor DNA, such that the donor DNA is inserted at the modified target site. In some embodiments, the cell line is a T cell line. In some embodiments, the ZFM-PB:MosI fusion protein contains one amino acid sequence of SEQ ID No. 24-27.

[0200] Genomic modification can be the unstable chromosomal integration of a transgene. The integrated transgene can be silenced, removed, excised, or further modified.

[0201] In some embodiments, the chimeric transposase and chimeric site-specific transposase fusion protein provided herein have better transposase efficacy than their wild-type equivalents. Transposase activity can be measured by any suitable assay known in the art or described herein (e.g., splitting GFP assay). For example, the chimeric transposase and chimeric site-specific transposase fusion proteins provided in this paper may have targeted genome integration activity comparable to their wild-type counterparts, but have reduced off-target genome integration activity compared to their wild-type counterparts.

[0202] In some embodiments, the chimeric PB:MosI transposase containing the DNA targeting domain provided herein has a targeting / off-target activity ratio that is at least 50-fold, at least about 100-fold, at least about 150-fold, at least about 200-fold, at least about 250-fold, at least about 300-fold, at least about 350-fold, at least about 400-fold, at least about 450-fold, at least about 500-fold, at least about 550-fold, at least about 600-fold, at least about 650-fold, at least about 700-fold, at least about 750-fold, at least about 800-fold, at least about 850-fold, at least about 900-fold, at least about 950-fold, or at least about 1000-fold increased compared to the wild-type transposase domain (e.g., PB or SPB).

[0203] In some embodiments, the modified cells are used therapeutically for adoptive cell therapy.

[0204] Adoptive cell compositions that are “universally” and safely administered to any patient (not just those from which they originate) require significantly reduced or eliminated allogeneic reactivity. For this purpose, the cells of this disclosure (e.g., allogeneic cells) may be modified to interrupt the expression or function of T cell receptors (TCRs) and / or a class of major histocompatibility complexes (MHCs). TCRs mediate graft-versus-host (GvH) responses, while MHCs mediate host-versus-graft (HvG) responses. In a preferred aspect, any expression and / or function of TCRs is eliminated in the modified cells disclosed herein.To prevent T-cell-mediated GvH that could lead to subject death. Therefore, in a preferred aspect, this disclosure provides a pure TCR-negative allogeneic T-cell composition (e.g., each cell in the composition expresses endogenous TCR at levels so low as to be undetectable or absent).

[0205] The expression and / or function of MHC class I (MHC-I, specifically HLA-A, HLA-B, and HLA-C) can be reduced or eliminated to prevent HvG, thereby improving cell engraftment in the subject. It is believed that the improved engraftment results in longer cell persistence, and therefore a larger therapeutic window for the subject. In some embodiments, the expression and / or function of the MHC-I structural element β-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 number WO 2020 / 051374, which is incorporated herein by reference in its entirety as an example of gRNAs that can be used in the methods described herein.

[0206] Detailed descriptions of non-naturally occurring chimeric stimulatory receptors, genetic modifications of endogenous sequences encoding TCR-α (TCR-α), TCR-β (TCR-β), and / or β-2-microglobulin (β2M), and non-naturally occurring polypeptides comprising HLA class I histocompatibility antigens, α-chain E (HLA-E) polypeptides are disclosed in PCT application publication number WO 2020 / 051374, which is incorporated herein by reference in its entirety as an example of non-naturally occurring receptors and polypeptides and other genetic modifications that can be introduced into the cells disclosed herein.

[0207] Under normal circumstances, complete T cell activation depends on the binding of the TCR to 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 absent, T cell expansion is severely reduced when stimulated with standard activation / stimulation agents (including the agonist antiCD3 mAb). Therefore, this disclosure provides a non-naturally occurring chimeric stimulatory receptor (CSR) comprising: (a) an extracellular domain containing an activation component, wherein the activation component is isolated from or derived from a first protein; (b) a transmembrane domain; and (c) an intracellular domain containing at least one signal transduction domain.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.

[0208] The activating component of the CSR described herein may include a component 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 repressor protein, a cytokine receptor, and a portion thereof of an agonist-binding chemokine receptor of the activating component. The activating component may include an agonist-binding CD2 extracellular domain or a portion thereof.

[0209] The signal transduction domain of the CSR described herein may include a component 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 repressor protein, a cytokine receptor, and a chemokine receptor, or a portion thereof. The signal transduction domain may include a CD3 protein or a portion thereof. The CD3 protein may include a CD3ζ domain or a portion thereof.

[0210] The intracellular domain of the CSR described herein may further include a cytoplasmic domain. The extracellular domain of the CSR may further include a signal peptide. The CSR may further include a transmembrane domain. The cytoplasmic domain, signal peptide, and / or transmembrane domain may be derived from the same protein or different proteins. For example, the signal peptide and transmembrane domain may be derived from CD8 or CD28 or a combination thereof. Specification 29 / 45 pages 33 CN 121195063 A

[0211] This disclosure also provides non-naturally occurring CSRs in which the extracellular domain contains modifications. The modifications may include mutations or truncations of the amino acid sequence of the activating component compared to the wild-type sequence of the activating component. Mutations or truncations of the amino acid sequence of the activating component may include mutations or truncations of the agonist-binding CD2 extracellular domain or a portion thereof. Mutations or truncations of the CD2 extracellular domain may reduce or eliminate binding to naturally occurring CD58.

[0212] This disclosure also provides a nucleic acid sequence encoding any CSR disclosed herein. This disclosure provides a transposon or vector comprising a nucleic acid sequence encoding any CSR disclosed herein.

[0213] This disclosure provides a cell comprising any CSR disclosed herein. This disclosure provides a cell comprising a nucleic acid sequence encoding any CSR disclosed herein. This disclosure provides a cell comprising a vector containing a nucleic acid sequence encoding any CSR disclosed herein. This disclosure provides a cell comprising a transposon containing a nucleic acid sequence encoding any CSR disclosed herein.

[0214] This disclosure provides a composition comprising any CSR disclosed herein. This disclosure provides a composition,The composition comprises a nucleic acid sequence encoding any CSR disclosed herein. This disclosure provides a composition comprising a vector containing a nucleic acid sequence encoding any CSR disclosed herein. This disclosure provides a composition comprising a transposon containing a nucleic acid sequence encoding any CSR disclosed herein. This disclosure provides compositions comprising modified cells disclosed herein or compositions comprising multiple modified cells disclosed herein.

[0215] This disclosure also provides methods for site-specific gene integration. The chimeric transposases and chimeric site-specific transposase fusion proteins provided herein can be used to deliver transgenes to cells and integrate transgenes into target sites. Target sites can be, for example, genomic safe harbors, i.e., genomic sites where the transgene is integrated in a manner that ensures predictable transgene function and does not cause changes in the host genomic DNA sequence. In some embodiments, target sites are repetitive elements, such as LINE-1 or ALU sequences. Repetitive elements do not encode essential gene products, making insertion unlikely to cause harmful changes in the gene expression profile of the cell. One, two, or more target sites may be present in a repetitive element. In some embodiments, target sites are located within introns.

[0216] Site-specific integration can be used in vitro or in vivo. One example of in vivo application is gene therapy, which involves delivering transgenes into genomic DNA in cells.

[0217] Formulation, Dosage, and Administration

[0218] This disclosure provides formulations, dosages, and administration methods for the compositions and cells described herein. In one aspect, a pharmaceutical composition is provided herein comprising the chimeric transposase or chimeric site-specific transposase fusion protein described herein and a pharmaceutical carrier. In another aspect, a pharmaceutical composition is provided herein comprising the modified cells described herein and a pharmaceutical carrier.

[0219] The disclosed compositions and pharmaceutical compositions may comprise at least one of any suitable adjuvants, such as, but not limited to, diluents, binders, stabilizers, buffers, salts, lipophilic solvents, preservatives, adjuvants, etc. Pharmaceutical adjuvants are preferred. Non-limiting examples and methods of preparing such sterile solutions are well known in the art.Examples include, but are not limited to, Gennaro, Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Co. (Easton, Pa.) 1990 and "Physician's Desk Reference", 52nd edition, Medical Economics (Montvale, NJ) 1998. Pharmaceutical carriers suitable for the administration method, solubility, and / or stability of protein scaffolds, fragments, or variant compositions well known in the art or as described herein can be conventionally selected.

[0220] Non-limiting examples of suitable pharmaceutical excipients and additives include proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides; derived sugars, such as sugar alcohols, aldonic acids, esterified sugars, etc.; and polysaccharides or sugar polymers), which may be present alone or in combination, and may be present alone or in combination from 1% to 99.99% by weight or volume. Non-limiting examples of protein excipients include serum albumin, such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, etc. Representative amino acid / protein components that can also be used for buffering capacity include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, etc. A preferred amino acid is glycine.

[0221] Non-limiting examples of carbohydrate excipients suitable for use include monosaccharides, such as fructose, maltose, galactose, glucose, D-mannose, sorbitol, etc.; disaccharides, such as lactose, sucrose, trehalose, cellobiose, etc.; polysaccharides, such as raffinose, mesotriose, maltodextrin, dextran, starch, etc.; and sugar alcohols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucosol), inositol, etc. Preferably, the carbohydrate excipient is mannitol, trehalose, and / or raffinose.

[0222] The composition may also include a buffer or pH adjuster; typically, the buffer is a salt prepared from an organic acid or base. Representative buffers include organic acid salts, such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; tris(hydroxymethyl)aminomethane, tromethamine hydrochloride, or phosphate buffers. Preferred buffers are organic acid salts, such as citrate.

[0223] Additionally, the disclosed composition may include polymeric excipients / additives, such as polyvinylpyrrolidone, sucrose (polymeric sugar), glucose dextrorate (e.g., cyclodextrin, ...Examples of ingredients include 2-hydroxypropyl-β-cyclodextrin, polyethylene glycol, 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).

[0224] Therapeutic amounts of the compositions or pharmaceutical compositions disclosed herein can be administered using many known and developed methods. Non-limiting examples of administration methods include bolus, buccal, infusion, intra-articular, intrabronchial, intra-abdominal, intra-cystic, intra-cartilaginous, intracavitary, intra-body cavity, intracerebellum, intravenous, intracolonic, intracervical, intragastric, intrahepatic, intralesional, intramuscular, intracardiac, intranasal, intraocular, intraosseous, intrapelvic, intrapelvic cavity, intraperitoneal, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intratumoral, intravenous, intravesical, intrabladder, oral, parenteral, rectal, sublingual, subcutaneous, transdermal, or vaginal administration. In preferred embodiments, for example, intravenous administration of a composition comprising the modified cells described herein via intravenous infusion.

[0225] The compositions of this disclosure can be prepared for parenteral (subcutaneous, intramuscular, or intravenous) or any other administration, particularly in the form of a liquid solution or suspension. For parenteral administration, any composition disclosed herein may be formulated in combination with or alone with a pharmaceutical parenteral carrier as a solution, suspension, emulsion, granules, powder, or lyophilized powder. Formulations for parenteral administration may contain sterile water or saline, polyalkylene glycols (such as polyethylene glycol), plant-derived oils, hydrogenated naphthalene, etc., as common excipients. Aqueous or oily suspensions for injection may be prepared using appropriate emulsifiers or humectants and suspending agents according to known methods. Injectable or infusion formulations may be non-toxic, non-oral diluents, such as aqueous solutions, sterile injectable solutions, or suspensions in solvents. Water, Ringer's solution, isotonic saline, etc., may be used as suitable carriers or solvents; sterile non-volatile oils may be used as common solvents or suspension solvents. For these purposes, any kind of non-volatile oil and fatty acid may be used, including natural or synthetic or semi-synthetic fatty oils or fatty acids; natural or synthetic or semi-synthetic monoglycerides, diglycerides, or triglycerides. Parenteral administration is known in the art and includes, but is not limited to, conventional injection methods, pneumatic needleless injection devices as described in U.S. Patent No. 5,851,198, and laser perforation devices as described in U.S. Patent No. 5,839,446.

[0226] It is desirable to deliver the disclosed compounds to subjects over a longer period of time (e.g., from one week to one year via a single administration, as per the specification, page 31 / 45, CN 121195063 A). Various sustained-release, reservoir, or implantable dosage forms are available. For example, dosage forms may contain pharmaceutically nontoxic salts of compounds that have low solubility in bodily fluids.For example, (a) an acid addition salt with a polybasic acid such as phosphoric acid, sulfuric acid, citric acid, tartaric acid, tannic acid, dihydroxynaphthyl acid, alginic acid, polyglutamic acid, naphthalene monosulfonic acid or naphthalene disulfonic acid, polygalacturonic acid, etc.; (b) a salt with a polyvalent metal cation such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, etc., or a salt with an organic cation formed, for example, N,N'-dibenzyl-ethylenediamine or ethylenediamine; or (c) a combination of (a) and (b), such as zinc tannate salt. Additionally, the disclosed compounds or preferably relatively insoluble salts, such as those described above, can be formulated in gels suitable for injection, such as aluminum monostearate gel containing, for example, sesame oil. Particularly preferred salts are zinc salts, zinc tannate salts, dihydroxynaphthyl acid salts, etc. Another type of slow-release reservoir formulation for injection will contain a compound or salt dispersed in a slowly degrading, non-toxic, non-antigenic polymer, such as the polylactic acid / polyglycolic acid polymer described in U.S. Patent No. 3,773,919. The compound or preferably a relatively insoluble salt, such as those described above, can also be formulated in cholesterol-based silicone rubber pellets, particularly for use in animals. Other slow-release, reservoir, or implantable formulations, such as gaseous or liquid liposomes, are known in the literature (U.S. Patent No. 5,770,222 and “Sustained and Controlled Release Drug Delivery Systems”, edited by J.R. Robinson, Marcel Dekker, Inc., N.Y., 1978).

[0227] Treatment Methods

[0228] In another aspect, methods for treating a disease or ailment of a subject are provided herein, comprising administering to the subject a composition comprising the modified cells described herein. The terms “subject” and “patient” are used interchangeably herein. In a preferred embodiment, the patient is a person.

[0229] The modified cells can be allogeneic or autologous for the patient. In some preferred embodiments, the modified cells are allogeneic cells. In some embodiments, the modified cells are autologous T cells or modified autologous CAR T cells. In some preferred embodiments, the modified cells are allogeneic T cells or modified allogeneic CAR T cells.

[0230] In some embodiments, the disease or ailment treated according to the methods described herein is cancer. In some embodiments, the treatment methods described herein can delay cancer progression and / or reduce tumor burden.

[0231] In some embodiments, the disease or ailment treated according to the methods described herein is an autoimmune disease. In some embodiments,Autoimmune diseases include autoimmune neutropenia, Guillain-Barré syndrome, epilepsy, autoimmune encephalitis, Isaac syndrome, nevus syndrome, pemphigus vulgaris, pemphigus foliaceus, bullous pemphigoid, acquired epidermolysis bullosa, pemphigoid of pregnancy, mucosal pemphigoid, antiphospholipid syndrome, autoimmune anemia, myasthenia gravis, autoimmune Graves' disease, thyroid ophthalmopathy (TED), Goodpasser syndrome, multiple sclerosis, rheumatoid arthritis, lupus, idiopathic thrombocytopenic purpura (ITP), warm autoimmune hemolytic anemia (WAIHA), chronic inflammatory demyelinating polyneuropathy (CIDP), lupus nephritis, or membranous nephropathy.

[0232] The dosage of the pharmaceutical composition to be administered to the subject may vary based on known factors such as the pharmacodynamic characteristics of the particular agent and its administration method and route; the recipient’s age, health status and weight; the nature and severity of symptoms; the type of concurrent treatment, the frequency of treatment and the desired effect.

[0233] In the aspect of the composition to be administered to a subject with this need being a modified cell as disclosed herein, it can be administered to cells between about 1x10³ and about 1x10⁴; cells between about 1x10⁴ and about 1x10⁵; cells between about 1x10⁵ and about 1x10⁶; cells between about 1x10⁶ and about 1x10⁷; cells between about 1x10⁷ and about 1x10⁸; cells between about 1x10⁸ and about 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 Cells between approximately 1 x 10¹³ and approximately 1 x 10¹⁴; cells between approximately 1 x 10¹⁴ and approximately 1 x 10¹⁵; cells between approximately 1 x 10¹⁵ and approximately 1 x 10¹⁶; cells between approximately 1 x 10¹⁶ and approximately 1 x 10¹⁷; cells between approximately 1 x 10¹⁷ and approximately 1 x 10¹⁸; cells between approximately 1 x 10¹⁸ and approximately 1 x 10¹⁹; or cells between approximately 1 x 10¹⁹ and approximately 1 x 10²⁰. In some embodiments, cells are administered at a dose between approximately 5 x 10⁶ and 25 x 10⁶ cells.

[0234] In other embodiments, the cell dose may depend on the person's weight.For example, per kilogram of subject body weight, approximately 1x10³ to 1x10⁴ cells can be administered; approximately 1x10⁴ to 1x10⁵ cells; approximately 1x10⁵ to 1x10⁶ cells; approximately 1x10⁶ to 1x10⁷ cells; approximately 1x10⁷ to 1x10⁸ cells; approximately 1x10⁸ to 1x10⁹ cells; approximately 1x10⁹ to 1x10¹⁰ cells; approximately 1x10¹⁰ to 1x10¹¹ cells; approximately 1x10¹¹ to 1x10¹² cells; approximately 1x10¹² to 1x10¹³ cells; approximately 1x10¹³ to 1x10¹⁴ cells; approximately 1x10¹⁴ to 1x10¹⁵ cells; approximately 1x10¹⁴ to 1x10¹⁵ cells; approximately 1x10¹⁰ ... 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; or cells between approximately 1x10¹⁹ and approximately 1x10²⁰.

[0235] A more detailed description of the pharmaceutical excipients, formulations, dosages, and methods of administration of the disclosed compositions and pharmaceutical compositions is disclosed in PCT Publication No. WO 2019 / 049816.

[0236] The transposon domains and fusion proteins provided herein can be used for the delivery of gene therapies. Gene therapy typically involves the delivery of transgenes to genomic DNA in cells. Typically, the transgene replaces a gene that is mutated or otherwise not properly expressed in the cell. The fusion proteins, transposase domains, and complexes described herein can be used to deliver therapeutic transgenes to cells and to integrate transgenes into target sites. In some embodiments, the treatment method includes introducing a fusion protein and transposon according to any one of claims 1 to 13 into cells, wherein the transposon comprises, in a 5' to 3' sequence: a 5' ITR, a transgene, and a 3' ITR.

[0237] Kit

[0238] In another aspect, this document provides a kit comprising a cell line engineered to contain, within its genome, preferably in a highly expressed genomic region, a modified target site for the SPB or PBx provided herein. The kit may further comprise a composition comprising one or more SPB or PBx transposase domains or fusion proteins described herein. In some embodiments, the cell line is a T cell line.

[0239] Terminology

[0240] As used throughout this disclosure, unless the context clearly indicates otherwise,Otherwise, the singular forms “a,” “and,” and “the / said” include a plural of the referred objects. Thus, for example, reference to “a method” includes multiple such methods, and reference to “dosage” includes reference to one or more doses and their equivalents known to those skilled in the art, and so on.

[0241] The terms “about” or “approximately” mean within an acceptable range of error for a particular value as determined by those skilled in the art, the acceptable range of error depending in part on how the value is measured or determined, such as limitations of the measurement system. For example, “about” may mean within one or more times the standard deviation. Alternatively, “about” 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 respect to biological systems or processes, the term may mean within a certain order of magnitude of a value, preferably within 5 times, and more preferably within 2 times. Where specific values ​​are described in this application and claims, the term “about” should be considered to mean within an acceptable range of error for that specific value, unless otherwise stated.

[0242] This disclosure provides isolated or substantially purified polynucleotide or protein compositions. The “isolated” or “purified” polynucleotide or protein, or its biologically active portion, is substantially or substantially free of components that typically accompany or interact with the polynucleotide or protein as seen in its natural environment. Thus, when produced by recombinant technology, the isolated or purified polynucleotide or protein is substantially free of other cellular material or culture medium, or when chemically synthesized, is substantially free of chemical precursors or other chemical substances. Preferably, the “isolated” polynucleotide does not contain the sequence of naturally side-joined polynucleotides (preferably protein-coding sequences) in the genomic DNA of the organism from which the polynucleotide is derived (i.e., the sequences located at the 5' and 3' ends of the polynucleotide). For example, in various respects, the isolated polynucleotide may contain a nucleotide sequence of less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb, which is naturally side-linked to the polynucleotide in the genomic DNA of the cell from which the polynucleotide is derived. Proteins that are substantially free of cellular material include protein formulations having less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating proteins. When the proteins of this disclosure or their bioactive portions are produced recombinantly, preferably, the culture medium represents less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or non-target proteins.

[0243] This disclosure provides fragments and variants of the disclosed DNA sequences and proteins encoded by these DNA sequences. As used throughout this disclosure,The term “fragment” refers to a portion of a DNA sequence or an amino acid sequence, and therefore a portion of a protein encoded therein. DNA sequence fragments containing coding sequences may encode protein fragments that retain the biological activity of the native protein, and thus retain DNA recognition or binding activity to target DNA sequences as described herein. Alternatively, DNA sequence fragments that can be used as hybridization probes generally do not encode biologically active proteins or do not retain promoter activity. Thus, DNA sequence fragments may range from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, and at most the full-length polynucleotides of this disclosure.

[0244] The term “comprising” is intended to mean that compositions and methods include the listed elements, but do not exclude other elements. When used to define compositions and methods, “consisting substantially of” should mean excluding other elements that are of any significance to the composition when used for the intended purpose. Therefore, a composition consisting substantially of elements as defined herein does not exclude trace contaminants or inert carriers. “Containing” should mean excluding portions and substantial method steps other than trace elements of other components. Each of these transitional terms is defined within the scope of this disclosure.

[0245] As used herein, “expression” means the process by which a polynucleotide is transcribed into mRNA and / or the process by which transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. If the polynucleotide is derived from genomic DNA, expression may include splicing of mRNA in eukaryotic cells.

[0246] “Gene expression” means the conversion of information contained in a gene into a gene product. The gene product may be a direct transcript of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, shRNA, microRNA, structural RNA, or any other type of RNA) or a protein translated from mRNA. Gene products also include RNA modified by processes such as capping, polyadenylation, methylation, and editing, and proteins modified by processes such as methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristylation, and glycosylation.

[0247] "Operably linked" or its equivalents (e.g., "operably linked") mean that two or more molecules are positioned relative to each other such that they can interact to affect the function of one or both molecules or a combination thereof. In the context of nucleic acids, a promoter may be operably linked to a nucleotide sequence encoding a transposon domain or fusion protein as described herein.This brings the expression of the nucleotide sequence under the control of the promoter.

[0248] The present invention discloses non-covalently linked components and methods for preparing and using non-covalently linked components. Various specifications 34 / 45 pages 38 CN 121195063 A Components may take various different forms as described herein. For example, non-covalently linked (i.e., operably linked) proteins can be used to achieve temporary interactions to avoid one or more problems in the art. The ability of non-covalently linked components (such as proteins) to associate and dissociate makes functional association only or primarily occur when such association is required to achieve the desired activity. The bond can last long enough to achieve the desired effect.

[0249] The present invention discloses a method for directing a protein to a specific locus in the genome of an organism. The method may include the steps of providing a DNA positioning component and providing an effector molecule, wherein the DNA positioning component and the effector molecule are operably linked via non-covalent bonding.

[0250] “Target site” or “target sequence” is a nucleic acid sequence that defines the nucleic acid portion to which the binding molecule will bind, provided that sufficient binding conditions are present.

[0251] The terms “nucleic acid” or “oligonucleotide” or “polynucleotide” refer to at least two nucleotides covalently linked together. The description of a single strand also defines the sequence of the complementary strand. Therefore, nucleic acids may also encompass the complementary strand of the described single strand. Nucleic acids of this disclosure also encompass substantially identical nucleic acids and their complements that retain the same structure or encode the same protein.

[0252] Nucleic acids of this disclosure may be single-stranded or double-stranded. Even if the majority of the molecule is single-stranded, the nucleic acids of this disclosure may contain double-stranded sequences. Even if the majority of the molecule is double-stranded, the nucleic acids of this disclosure may contain single-stranded sequences. Nucleic acids of this disclosure may include genomic DNA, cDNA, RNA, or hybrids thereof. Nucleic acids of this disclosure may contain combinations of deoxyribonucleotides and ribonucleotides. Nucleic acids of this disclosure may contain combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. Nucleic acids of this disclosure may be synthesized to include non-natural amino acid modifications. The nucleic acids disclosed herein can be obtained by chemical synthesis or by recombination.

[0253] The nucleic acids of this disclosure (whether their complete sequence or any part thereof) may be non-natural. The nucleic acids of this disclosure may contain one or more non-natural mutations, substitutions, deletions, or insertions, such that the entire nucleic acid sequence is non-natural. The nucleic acids of this disclosure may contain one or more repeating, inverted, or repetitive sequences, the resulting sequences of which are not naturally occurring, such that the entire nucleic acid sequence is non-natural. The nucleic acids of this disclosure may contain non-natural modified, artificial, or synthetic nucleotides.This makes the entire nucleic acid sequence non-natural.

[0254] Due to the redundancy of the genetic code, multiple nucleotide sequences can encode any particular protein. All such nucleotide sequences are envisioned herein.

[0255] As used throughout this disclosure, the term "promoter" refers to a synthetic or naturally derived molecule capable of conferring, activating, or enhancing nucleic acid expression in a cell. A promoter may contain one or more specific transcriptional regulatory sequences to further enhance expression and / or alter its spatial and / or temporal expression. A promoter may also contain distal enhancer or repressor elements, which may be located thousands of base pairs from the transcription start site. Promoters may be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. Promoters may constitutively or differentially regulate the expression of genomic molecules in terms of the cell, tissue, or organ in which expression occurs, or in terms of the developmental stage in which expression occurs, or in response to external stimuli such as physiological stress, pathogens, metal ions, or inducers. Representative examples of promoters include the phage T7 promoter, phage T3 promoter, SP6 promoter, lac operon promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, EF-1α promoter, CAG promoter, SV40 early promoter, or SV40 late promoter and CMV IE promoter.

[0256] As used throughout this disclosure, the term "vector" refers to a nucleic acid sequence containing an origin of replication. A vector can be a viral vector, a phage, a bacterial artificial chromosome, or a yeast artificial chromosome. A vector can be a DNA or RNA vector. A vector can be a self-replicating extrachromosomal vector, and is preferably a DNA plasmid. A vector can contain amino acids and a DNA sequence, an RNA sequence, or a combination of both DNA and RNA sequences. Specification 35 / 45 pages 39 CN 121195063 A

[0257] Conservative substitution of amino acids, i.e., replacing an amino acid with a different amino acid having similar properties (e.g., hydrophilicity, degree, and distribution of charged regions), is generally recognized in the art as typically involving minute variations. As understood in the art, these minute variations can be identified in part by taking into account the hydrophilicity index of the amino acid. Kyte et al., J. Mol. Biol. 157: 105-132 (1982). The hydrophilicity index of an amino acid is based on consideration of its hydrophobicity and charge. Amino acids with similar hydrophilicity indices can be substituted and still retain protein function. In one aspect, amino acids with a hydrophilicity index of ±2 are substituted. The hydrophilicity of an amino acid can also be used to reveal substitutions that will result in the retention of biological function of the protein. Taking into account the hydrophilicity of amino acids in the context of a peptide allows for the calculation of the maximum local average hydrophilicity of the peptide.This is a available metric that has been reported to correlate well with antigenicity and immunogenicity. U.S. Patent No. 4,554,101, which is incorporated herein by reference in its entirety.

[0258] Substitution of amino acids having similar hydrophilicity values ​​can result in the peptide retaining biological activity, such as immunogenicity. Substitution can be made with amino acids having hydrophilicity values ​​within ±2 to each other. Both the hydrophobicity index and the hydrophilicity value of an amino acid are influenced by the specific side chain of that amino acid. Consistent with this observation, biologically compatible amino acid substitutions are understood to depend on the relative similarity of the amino acids and, specifically, the side chains of those amino acids, as revealed by hydrophobicity, hydrophilicity, charge, size, and other properties.

[0259] As used herein, “conservative” amino acid substitutions may be defined as listed in Tables 1, 2, or 3 below. In some respects, fusion polypeptides and / or nucleic acids encoding such fusion polypeptides include conserved substitutions introduced by modifying polynucleotides encoding polypeptides of the present disclosure. Amino acids may be classified according to physical properties and their contribution to secondary and tertiary protein structures. Conservative substitution is the substitution of one amino acid with another amino acid having similar properties. Exemplary conservative substitutions are listed in Table 1.

[0260] Table 1: Conservative Substitution I

[0261]

[0262] Alternatively, the conserved amino acids may be as described in Lehninger (Biochemistry, second edition; Worth Publishers, Inc. NY, NY (1975), pp. 71-77), as shown in Table 2.

[0263] Table 2: Conservative Substitution II Specification 36 / 45 pages 40 CN 121195063 A

[0264]

[0265] Alternatively, exemplary conservative substitutions are listed in Table 3.

[0266] Table 3: Conservative Substitution III Specification 37 / 45 pages 41 CN 121195063 A

[0267] Specification 38 / 45 pages 42 CN 121195063 A

[0268] The polypeptides and proteins of this disclosure (whether their complete sequence or any part thereof) may be non-natural. The polypeptides and proteins of this disclosure may contain one or more non-natural mutations, substitutions, deletions or insertions, such that the entire amino acid sequence is non-natural. The polypeptides and proteins of this disclosure may contain one or more repeating, inverted or repetitive sequences, the resulting sequences of which are not natural, such that the entire amino acid sequence is non-natural. The polypeptides and proteins of this disclosure may contain non-natural modified, artificial or synthetic amino acids, such that the entire amino acid sequence is non-natural.

[0269] As used throughout this disclosure,The identity between two sequences can be determined using a standalone executable BLAST engine program (bl2seq) for blasting the two sequences, with default parameters. This program is available from the National Center for Biotechnology Information (NCBI) FTP site (Tatusova and Madden, FEMS Microbiol Lett., 1999, 174, 247–250; which is incorporated herein by reference in its entirety). When the terms “identical” or “identical” are used in the context of two or more nucleic acid or polypeptide sequences, they refer to the specified percentage of residues that are identical within a specific region of each sequence. In some embodiments, sequence identity is determined over the entire length of the sequence. Percentage comparability is calculated by optimally aligning the two sequences, comparing specified regions of the two sequences, determining the number of positions in the two sequences where identical residues are present to obtain 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 obtain the percentage of sequence identity. In cases where 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 calculated denominator but not in the molecule. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Characterization can be performed manually or using computer sequencing algorithms such as BLAST or BLAST 2.0.

[0270] In some embodiments, if a sequence has a specific sequence identity with a particular SEQ ID NO (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%), then the sequence has the same length as the sequence of SEQ ID NO. In some embodiments, if a sequence has a specific sequence identity with a particular SEQ ID NO (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%), then the sequence differs from the sequence of SEQ ID NO only due to conserved amino acid substitutions.

[0271] As used throughout this disclosure, the term "endogenous" means a nucleic acid or protein sequence naturally associated with a target gene or a host cell into which the target gene is introduced.

[0272] As used throughout this disclosure, the term "exogenous" means a nucleic acid or protein sequence not naturally associated with a target gene or a host cell into which the target gene is introduced, including multiple non-natural copies of naturally occurring nucleic acids, such as DNA sequences.Or a naturally occurring nucleic acid sequence located at a non-naturally occurring genomic location.

[0273] This disclosure provides a method for introducing a polynucleotide construct comprising a DNA sequence into a host cell. The term “introduction” is intended to present the polynucleotide construct to the cell in a manner that allows the construct to enter the interior of the host cell. The method of this disclosure is not dependent on a specific method for introducing the polynucleotide construct into the host cell, as long as the polynucleotide construct can enter the interior of a host cell. Methods for introducing polynucleotide constructs into bacteria, plants, fungi and animals are known in the art, including but not limited to stable transformation, transient transformation and virus-mediated transformation.

[0274] Examples

[0275] The examples in this section are provided for illustrative purposes and are not intended to limit the invention.

[0276] Example 1: Construction of a chimeric PiggyBac (“PB”) – MosI transposase

[0277] The chimeric PB:MosI transposase is formed by deleting the CRD domain from the PB transposase and replacing it with the DNA-binding domain of the MosI transposase. The PB CRD consists of 594 amino acids, residues 553 to 594 of the PB transposase protein (see SEQ ID NO: 2). The CRD domain is connected to the remainder of the transposase via a linker sequence spanning residues 535 to 552 of the PB transposase sequence. The PB CRD domain can be truncated from the C-terminus of the PB transposase at any position within the linker sequence. Two fusion sites within the linker sequence are selected as exemplary sites. In the first fusion design, the C-terminus of the PB transposase is truncated from residues 545 to 594 (SEQ ID NO: 3) to obtain the S544 fusion site (S544 PB) to construct the S544 PB-MosI chimera. In the second fusion design, the C-terminus of the PB transposase was truncated from residues 553 to 594 (SEQ ID NO: 4) to obtain the V552 fusion site (V552 PB) to construct a chimeric V552 PB-MosI transposase.

[0278] The C-terminated S544PB and V552 PB sequences were fused to the DNA-binding domain of MosI. MosI binds to its C-terminus near the transposon end, while PB binds to its N-terminus near the transposon end. The N-terminus of MosI contains two DNA-binding domains 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).The two DNA-binding domains span the first approximately 111 residues of the protein (SEQ ID NO: 6).

[0279] As described in International Patent Application Publication No. PCT / US2022 / 77549, a TAL-piggyBac transposase fusion protein containing an N-terminal deleted piggyBac transposase sequence and an integration-deficient N-terminal piggyBac transposase targeting GFP are generated. Briefly, two pairs of TAL arrays targeting sequences in the GFP coding sequence are designed, and TAL arrays containing nine 34-amino acid repeat sequences followed by a 20-amino acid “half” repeat sequence are synthesized with BsmBI IIS type restriction sites side-joined. This allows each TAL array to be cloned with the N-terminal deleted piggyBac transposase sequence in the expression plasmid along with the rest of the open reading frame to generate GFP1R right TAL-PBx (SEQ ID NO: 40). The PBx transposase sequence was replaced with a chimeric PB:MosI transposase sequence to construct a chimeric site-specific transposase fusion protein that specifically targets GFP.

[0280] A chimeric PB:MosI transposase sequence was constructed by linking residues 3 to 58 of MosI to the S544 PBx transposase using a GGGGS adapter sequence (SEQ ID NO: 86), and the PBx transposase sequence was inserted in place of it to form GFP1R TAL-PB:S544-MosI-58 (SEQ ID NO: 41). Similarly, residues 3 to 58 of MosI were fused to the V552 PBx transposase sequence using a GGGGS adapter sequence (SEQ ID NO: 86), and the PBx transposase sequence was inserted in place of it to form GFP1R TAL-PB:V552-MosI-58 (SEQ ID NO: 42). Additionally, residues 3 to 111 of MosI were fused to S544 PB using a GGGGS adapter (SEQ ID NO: 86), and inserted in place of the PBx transposase sequence to form GFP1R TAL-PB:S544-MosI-111 (SEQ ID NO: 43). Finally, residues 3 to 111 of MosI were fused to PiggyBac at V552 PBx using a GGGGS adapter (SEQ ID NO: 86).And the PBx transposase sequence was inserted instead to form GFP1R TAL-ssSPB:V552-MosI-111 (SEQ ID NO: 44).

[0281] Example 2: Construction of chimeric PB:MosI inverted terminal repeat (ITR) sequence

[0282] This example illustrates an exemplary method for constructing a chimeric PB:MosI ITR sequence.

[0283] The chimeric PB:MosI transposases prepared in Example 1 each contain a PB DNA binding and dimerization domain (DDBD) fused to a DNA binding domain from MosI. Thus, a chimeric PB:MosI ITR sequence containing a binding site of PB DDBD fused to a binding site of the MosI DNA binding domain was constructed.

[0284] The 35bp LE PB ITR sequence (SEQ ID NO: 12) was truncated after position 16, with a 19bp CRD binding site missing. To this end, a 23 bp MosI sequence (SEQ ID NO: 13) containing binding sites targeting both MosI DNA-binding domains was fused in opposite orientations to form LE PB:MosI ITR (SEQ ID NO: 15). Furthermore, alternative forms of chimeric ITRs containing spacers of various lengths between the PB and MosI ITR domains in the chimeric protein were designed to test the effect of adapter length. In one example, a bp deletion was made of the MosI binding site adjacent to the DDBD binding site to form LE PB:MosI ITR-1 (SEQ ID NO: 16). Additionally, one or two bp of DNA was added between the MosI binding site and the DDBD binding site to form LE PB:MosI ITR+1 (SEQ ID NO: 17) and LE PB:MosI ITR+2 (SEQ ID NO: 18). Similarly, modify 63bp RE ITR (SEQ ID NO: 65),To form 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), and RE PB:MosI ITR+2 (SEQ ID NO: 22).

[0285] The relative excision activities of the chimeric PB:MosI transposase and the chimeric ITR sequence were analyzed.

[0286] Example 3: Method for measuring the excision activity of chimeric PB-MosI transposase using chimeric PB:MosI ITR

[0287] This example describes an assay designed to measure the excision activity of chimeric PB-MosI transposase and the chimeric ITR. In this assay, the chimeric PB:MosI transposase prepared in Example 1 is co-administered to cells with a reporter transposon construct containing a DNA nucleotide sequence encoding nonfunctional GFP, wherein the coding sequence has been disrupted by an insert DNA fragment flanked by a TTAA sequence and a set of inverted terminal repeats (ITRs) prepared in Example 2. The TTAA sequence and ITRs serve as recognition sites for the chimeric PB:MosI transposase, and if the chimeric transposase has excision activity, it will excise the insert DNA, restoring the complete, full-length coding sequence of the GFP gene. Therefore, for constructs containing homologous chimeric ITR sequences, the chimeric transposase with transposase activity produces GFP-positive cells that can be identified and quantified by FACS in this assay.

[0288] In short, the GFP reporter gene system comprises an EF1a promoter (SEQ ID NO: 45) driving the expression of a GFP reporter gene (SEQ ID NO: 46), followed by an SV40 polyadenylated sequence (SEQ ID NO: 47). The GFP reporter gene is disrupted by a transposon at the TTAA sequence, breaking it down into a first GFP segment (SEQ ID NO: 48) and a second GFP segment (SEQ ID NO: 49). A disrupted GFP reporter gene for each chimeric ITR of Example 2 is constructed, carrying 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).

[0289] Using the GFP reporter gene system,The excision activities of the GFPR1 TAL-PB:S544-MosI-58, GFP1R TAL-PB:V552-MosI-58, GFPR1 TAL-PB:S544-MosI-111, and GFP1R TAL-ssSPB:V552-MosI-111 transposases described in Example 1 were determined using each of the chimeric PB:MosI ITRs prepared in Example 2. On Day 0, each reporter gene was co-transfected with each chimeric transposase into HEK293T cells. Briefly, one day prior to transfection, 120,000 cells were seeded in 24-well plates in 500 µL of DMEM medium supplemented with 10% v / v FBS. On day 1, 50 ng of transposase expression vector was combined with 450 ng of reporter gene and transfected using 1 µL 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.

[0290] Table 4 Instructions 41 / 45 pages 45 CN 121195063 A

[0291]

[0292] As shown in Table 4, the chimeric transposase with MosI inserted at the S544 fusion site resulted in higher transposon excision activity compared to the chimeric transposase with MosI inserted at the V552 fusion site. In addition, higher transposon excision activity was observed for reporter genes containing PB:MosI ITR instead of ITRs with a spacing of -1, +1, or +2.

[0293] The chimeric ITRs tested in Table 4 contain a 23 bp sequence that includes binding sites for both MosI DNA-binding domains. Since the first 58 residues of MosI contain a single DNA-binding domain, a series of truncated ITR sequences were constructed, where the MosI portion of the ITR contains binding sites for only a single domain.

[0294] In the second experiment, the MosI binding site was shortened from 23 bp to 14 bp.The left-terminal LE PB:MosI ITR short (SEQ ID NO: 54) and right-terminal RE PB:MosI ITR short (SEQ ID NO: 55) were formed. These shorter ITRs were used to form the disrupted GFP reporter gene as described in Example 1 using the PB:MosI ITR short transposon (SEQ ID NO: 56). The short and original chimeric ITR reporter genes, as well as the S544 fusion site chimeric transposase, S544-MosI-58, and S544-MosI-111, were tested using the above GFP reporter gene system. The results are shown in Table 5.

[0295] Table 5

[0296]

[0297] As shown in Table 5, both the original and short ITRs result in functional transposon excision activity; however, the original 23 bp form results in higher activity of chimeric transposases containing residues 3-58 or 3-111 of the MosI fusion, as observed by the percentage of GFP-positive cells.

[0298] Example 4: Construction and analysis of chimeric TAL-PB:MosI transposases for site-specific transposition of small transposons in LINE-1 elements Specification 42 / 45 pages 46 CN 121195063 A

[0299] This example illustrates the construction of chimeric TAL-PB:MosI transposase fusion protein compositions that can be used in methods for achieving site-specific transposition at a specific target locus LINE1.

[0300] Previously, a LINE1-specific TAL DNA-binding domain was ligated to an N-terminally deleted piggyBac transposase sequence to construct a LINE1 TAL-ssSPB fusion protein (e.g., left SEQ ID NO: 57; right SEQ ID NO: 58). Similar to the construction of a GFP-targeting chimeric TAL-PB:MosI transposase fusion protein, as described in Example 1, the LINE1 left and right TAL-ssSPB fusion proteins were converted into chimeric site-specific PB:MosI transposases by replacing the PB transposase sequence with a PB:MosI transposase. Two forms of the left LINE1 L2 TAL-PB:MosI transposase were formed using the S544 fusion site and a 3-58 residue MosI fragment (SEQ ID NO: 59) or a 3-111 residue MosI fragment (SEQ ID NO: 60). Similarly,Using the S544 fusion site and either a 3-58 residue MosI fragment (SEQ ID NO: 61) or a 3-111 residue MosI fragment (SEQ ID NO: 62), two forms of the right LINE1 R2.2 TAL-PB:MosI transposase were formed. A small 365 bp transposon containing the minimum PB ITR (SEQ ID NO: 63) from Example 2 and a small 353 bp transposon containing the chimeric PB:MosI (23 bp) ITR (SEQ ID NO: 50) were cloned into a 4.5 kb donor vector. On day 0, following the manufacturer's instructions, 450 ng of each transposon donor and a total of 50 ng of LINE1 TAL-PB pairs were co-transfected into 120,000 HEK293T cells (platen the day before) using 1 µL JetPrime transfection reagent (Polyplus transfection). On day 3, genomic DNA was harvested from the transposoned cells, and site-specific integration of transposons to target sites in the forward orientation was quantified by digital droplet PCR (ddPCR) to determine the number of integrations per haploid genome. The results are shown in Table 6.

[0301] Table 6

[0302]

[0303] As shown in Table 6, the editing rate of the chimeric site-specific PB:MosI transposase in the form of chimeric MosI 3-111 residues was higher than that in the form of chimeric MosI 3-58 residues in the site-specific PB:MosI transposase. Compared to transposons containing the non-chimeric PB minimal ITR, both chimeric transposases each use the chimeric PB:MosI ITR to catalyze higher site-specific transposition of the transposon. Compared to transposons containing the chimeric PB:MosI ITR, the non-chimeric TAL-ssSPB transposase uses the non-chimeric PB minimal ITR to catalyze higher site-specific transposition of the transposon. These data suggest that chimeric and non-chimeric transposases can be used as orthogonal site-specific transposition systems to simultaneously deliver transposons to two distinct genetic loci, one targeted by the chimeric PB:MosI ITR and the other by the wild-type PB minimal ITR.

[0304] Example 5: Site-specific transposition of large cargo transposons at a specific locus using chimeric PB:MosI transposase

[0305] This example illustrates the site-specific transposition of a transposon containing a large DNA cargo at a specific target site LINE-1.

[0306] The chimeric TAL-PB:MosI transposase constructed in Example 4 and the control transposase TAL-ssSPB were analyzed.To understand their ability to specifically transpose transposons containing large DNA cargo into LINE1 elements. Specification 43 / 45 pages 47 CN 121195063 A

[0307] The transposon donor nanoparticle contains the PB transposon, which contains, from 5' to 3': a first TTAA sequence; a 309 bp fragment containing the PB 5' ITR and part of the UTR; a "large cargo" consisting of the EF1a promoter (SEQ ID NO: 45), a puromycin resistance gene, a 2A peptide, and a GFP reporter gene; followed by a 238 bp fragment containing the PB 3' ITR and part of the UTR; and a second TTAA sequence (SEQ ID NO: 64). This donor plasmid was used to analyze wild-type PB control TAL-ssSPB and negative control PB transposases containing mutations that give the transposase an integration defect.

[0308] For the chimeric TAL-PB:MosI transposase molecule, the transposon donor nanoparticles were modified to replace the 309 bp LE ITR / UTR and 238 bp RE ITR / UTR with the chimeric PB:MosI (23 bp) ITR of SEQ ID NO: 1 and SEQ ID NO: 19. The results are shown in Table 7.

[0309] Table 7

[0310]

[0311] As shown in Table 7, both chimeric and non-chimeric transposases, used as orthogonal systems, showed high specificity for their respective ITRs. The chimeric TAL-PB:MosI 3-111 residue form of the chimeric PB:MosI transposase had a higher editing rate than the chimeric TAL-PB:MosI 3-58 residue form and the non-chimeric TAL-ssSPB form. As a negative control, the integration-deficient PBx transposase without the TAL DNA targeting domain only resulted in background integration levels.

[0312] Example 6: Construction of a chimeric PB:MosI transposase containing a PB hyperactive mutation (“SPB”) with increased transposition frequency

[0313] This example illustrates a method for constructing a chimeric SPB:MosI transposase and analysis of integration and excision activities. In this example, SPB having an N-terminal NLS (SEQ ID NO: 5) was modified as described above to form an SPB:MosI chimeric transposase.

[0314] More specifically, residues 3 to 58 (SEQ ID NO: 8) of MosI were fused to SPB at S544 to form SPB:MosI-S544-58 (SEQ ID NO: 9),Furthermore, residues 3 to 111 of MosI (SEQ ID NO: 6) were fused to SPB at S544 to form SPB:MosI-S544-111 (SEQ ID NO: 7). The chimeric SPB:MosI transposase was tested using a disrupted GFP reporter gene system carrying the PB:MosI ITR transposon (SEQ ID NO: 50), the PB:MosI ITR short transposon (SEQ ID NO: 56), or the PB minimal ITR transposon (SEQ ID NO: 63). On day 0, each reporter gene construct was co-transfected with each chimeric PB:MosI transposase into HEK293T cells. Briefly, one day prior to transfection, 120,000 cells were seeded in 24-well plates in 500 µL DMEM supplemented with 10% v / v FBS. On day 0, 10 ng of transposase expression vector was combined with 490 ng of reporter gene and transfected using 1 µL 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.

[0315] Table 8 Specification 44 / 45 pages 48 CN 121195063 A

[0316]

[0317] As shown in Table 8, the chimeric SPB:MosI transposase catalyzes the excision of transposons containing chimeric PB:MosI ITR, as measured by the percentage of GFP-positive cells, but does not catalyze the excision of transposons containing non-chimeric minimal PB ITR. Non-chimeric SPB transposases catalyze the cleavage of transposons with the non-chimeric minimum PB ITR, but do not catalyze the cleavage of transposons with chimeric PB:MosI ITR, demonstrating the specificity of the chimeric PB:MosI transposase / PB:MosI ITR transposon combination. (See specification, pages 45 / 45, 49, CN 121195063 A)

Claims

1. A chimeric transposase comprising, in order from N-terminus to C-terminus: (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 to 594 of the sequence of SPB comprising the sequence shown in SEQ ID NO: 1; and (iii) one or more MosI DNA-binding domains.

2. The chimeric transposase according to claim 1, wherein the truncated SPB transposase of (ii) comprises the sequence shown in any one of SEQ ID NO.: 68 to 84.

3. The chimeric transposase according to claim 1 or 2, wherein the truncated PB transposase comprises one or more highly active mutations selected from I30V, S103P, G165S, M226F, M282V, S509G, N538K or N571S of SEQ ID NO:

1.

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

39.

5. The chimeric transposase according to any one of claims 1 to 4, wherein the chimeric transposase comprises two MosI DNA-binding domains, the MosI DNA-binding domains comprising the amino acid sequence shown in SEQ ID NO:

6.

6. The chimeric transposase according to any one of claims 1 to 4, wherein the chimeric transposase comprises a MosI DNA-binding domain, the MosI DNA-binding domain comprising the amino acid sequence shown in SEQ ID NO:

8.

7. The chimeric transposase according to any one of claims 1 to 6, wherein the target-specific DNA binding domain is a zinc finger domain or a TAL array.

8. A polynucleotide comprising a nucleic acid sequence encoding a chimeric transposase according to any one of claims 1 to 7.

9. A vector comprising the polynucleotide according to claim 13.

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

11. The chimeric transposon ITR polynucleotide of claim 10, wherein the polynucleotide containing two MosI DNA binding sites comprises the nucleic acid sequence of SEQ ID NO: 13 or 14.

12. The chimeric transposon ITR polynucleotide according to claim 10 or 11, wherein the chimeric transposon ITR polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 15 or 16.

13. The chimeric transposon ITR polynucleotide according to any one of claims 10 to 12, wherein the chimeric transposon ITR polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 17 or 18.

14. A vector comprising a chimeric transposon ITR polynucleotide according to any one of claims 10 to 13.

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

16. The chimeric transposon ITR polynucleotide of claim 15, wherein the polynucleotide comprising two MosI DNA binding sites comprises the nucleic acid sequence of SEQ ID NO: 13 or 14.

17. The chimeric transposon ITR polynucleotide of claim 15, wherein the chimeric transposon ITR polynucleotide comprises the nucleic acid sequence of any one of SEQ ID NO: 19 to 22.

18. A vector comprising a chimeric transposon ITR polynucleotide according to any one of claims 15 to 17.

19. A LINE1-targeting chimeric site-specific transposase fusion protein comprising, in the direction from the N-terminus to the C-terminus: (i) a left or right LINE1 TAL-targeting DNA-binding domain; (ii) a linker sequence; and (iii) a chimeric PB:MosI transposase.

20. The LINE1-targeted chimeric site-specific transposase fusion protein of claim 19, wherein the chimeric PB:MosI transposase comprises the amino acid sequence shown in SEQ ID NO: 9 or 7.

21. The LINE1-targeting chimeric site-specific transposase fusion protein of claim 20, wherein the left or right LINE1 TAL-targeting DNA-binding domain comprises the amino acid sequence shown in SEQ ID NO: 66 or 67.

22. A transposon comprising (i) a chimeric LE PB:MosI ITR polynucleotide and (ii) a chimeric RE PB:MosI ITR polynucleotide, wherein the LE PB:MosI ITR polynucleotide and the RE PB:MosI ITR polynucleotide contain the same nucleic acid sequence.

23. The transposon of claim 22, wherein the chimeric LE PB:MosI ITR polynucleotide and the chimeric RE PB:MosI ITR polynucleotide each comprise a nucleic acid sequence of any one of SEQ ID NO: 15 to 22.

24. A transposon comprising: (i) a chimeric LE PB:MosI ITR polynucleotide comprising the nucleic acid sequence of SEQ ID NO:54; and (ii) a chimeric RE PB:MosI ITR polynucleotide comprising the nucleic acid sequence of SEQ ID NO:

55.

25. A transposon comprising the nucleic acid sequence of SEQ ID NO:

56.

26. A method for integrating a transgene into a genomic target site in a cell, the method comprising introducing a chimeric transposon according to any one of claims 1 to 7 and a transposon, wherein the transposon comprises, in a 5' to 3' sequence: a 5' ITR, the transgene, and a 3' ITR, wherein the 5' ITR is a chimeric transposon inverted terminal repeat (ITR) according to any one of claims 10 to 13, and the 3' ITR is a chimeric transposon inverted terminal repeat (ITR) according to any one of claims 15 to 17.

27. The method of claim 26, wherein the 5' ITR chimeric transposon inverted terminal repeat sequence comprises the nucleic acid sequence shown in SEQ ID NO: 15, and / or wherein the 3' ITR chimeric transposon inverted terminal repeat sequence comprises the nucleic acid sequence shown in SEQ ID NO:

21.

28. The method according to any one of claims 26 or 27, wherein the genomic target site is located in a repeating element.

29. The method of claim 28, wherein the repeating element is a LINE element.

30. A method for site-specific transposition of DNA molecules into the genome of a cell, the method 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, wherein the chimeric transposase comprises, in order from N-terminus to C-terminus, 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 cells; and c) A DNA molecule containing a transposon comprising a chimeric LE transposon ITR polynucleotide and a chimeric RE transposon ITR polynucleotide; wherein the expressed chimeric site-specific transposase fusion protein integrates the transposon into the TTAA sequence of the cell genome via site-specific transposition.

31. A method for generating engineered cells via site-specific transposition, the method comprising: Introduced into the cells: a) A nucleic acid encoding a chimeric site-specific transposase fusion protein comprising a DNA-binding domain and a chimeric transposase, wherein the chimeric transposase comprises, in order from N-terminus to C-terminus, 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 cells; and c) A DNA molecule containing a transposon comprising a chimeric LE transposon ITR polynucleotide and a chimeric RE transposon ITR polynucleotide; wherein the expressed chimeric site-specific transposase fusion protein integrates the transposon into the TTAA sequence in the genome of the cell via site-specific transposition, thereby producing the engineered cell.