Transposases and uses thereof

Abstract

The present disclosure relates generally to fusion proteins comprising a transposase domain and a DNA targeting domain.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Publication Number (43) Publication Date (21) Application Number 202480034977.7 (22) Application Date 2024.04.04 (30) Priority Data 63 / 494,306 2023.04.05 US (85) PCT International Application Entering National Phase Date 2025.11.26 (86) PCT International Application Application Data PCT / US2024 / 022988 2024.04.04 (87) PCT International Application Publication Data WO2024 / 211512 EN 2024.10.10 (71) Applicant: Pocida Therapeutics, Inc. Address: California, USA (72) Inventors: B.B. Madison, J.S. Lucas, J.A. Valderrama, B.R. Molina, O. Batalov (74) Patent Agency: Beijing Kunrui Law Firm, 11494 Patent Attorney: Cen Xiaodong (51) Int.Cl. C12N 9 / 22 (2006.01) C12N 15 / 90 (2006.01) (54) Invention Title: Transposase and its Use (57) Abstract: This disclosure relates in its entirety to a fusion protein comprising a transposase domain and a DNA targeting domain. Claims (3 pages), Description (62 pages), Sequence Listing (electronic publication), Drawings (4 pages) CN 121219409 A 2025.12.26 CN 1 21 21 94 09 A 1. A fusion protein comprising a DNA targeting domain and a transposase domain, said transposase domain comprising the sequence listed in SEQ ID NO: 4, wherein the DNA targeting domain binds to a nucleic acid sequence encoding an LPA repeat sequence element. 2. The fusion protein of claim 1, wherein said DNA targeting domain comprises one, two, or three zinc finger motifs. 3. The fusion protein of claim 1, wherein said DNA targeting domain comprises one or more TAL domains. 4. The method of claim 3, wherein said TAL domain comprises the sequence listed in any one of SEQ ID NO: 35 to 38. 5. The fusion protein according to any one of claims 1 to 4, wherein the DNA targeting domain binds to a nucleic acid sequence encoding a kringle domain repetitive sequence element in the LPA gene or an intron adjacent to the sequence encoding a kringle domain repetitive sequence element. 6. The fusion protein according to any one of claims 1 to 5, wherein the transposase domain and the DNA targeting domain are connected by a linker.7. The fusion protein of claim 6, wherein the linker comprises the sequence GGGGS (SEQ ID NO: 181). 8. The fusion protein of any one of claims 1 to 7, wherein the DNA targeting domain is inserted into the N-terminus of the transposase domain at a position following amino acid 82 and preceding amino acid 105 of SEQ ID NO: 4. 9. The fusion protein of any one of claims 1 to 7, wherein the DNA targeting domain replaces one or more amino acids in the transposase domain located between amino acids 83 and 105 of SEQ ID NO: 4, and including both amino acids. 10. The fusion protein according to any one of claims 1 to 9, wherein the transposase domain comprises an N-terminal deletion of amino acids 1 to 83, 1 to 84, 1 to 85, 186, 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. 11. The fusion protein according to any one of claims 1 to 10, wherein the transposase domain comprises a sequence listed in any one of SEQ ID NO: 7 to 27. 12. The fusion protein according to any one of claims 1 to 11, wherein the transposase domain comprises: (a) at least one mutation selected from the group consisting of: M185R, M185K, D197K, D197R, D198K, D198R, D201K, and D201R; or (b) at least one mutation selected from the group consisting of: L204D, L204E, K500D, K500E, R504E, and R504D. 13. A polynucleotide comprising a nucleic acid sequence encoding the fusion protein according to any one of claims 1 to 12. 14. A vector comprising the polynucleotide according to claim 13. 15. A method for integrating a transgene into a genomic target site in a cell, the method comprising: introducing a fusion protein according to any one of claims 1 to 12 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.16. The method of claim 15, wherein the transposon further comprises an exogenous promoter between the 5' ITR and the transgene. 17. The method of claim 15 or 16, wherein the transgene encodes a detectable biomarker. 18. The method of claim 17, wherein the detectable biomarker is GFP. 19. The method of claim 15 or 16, wherein the transgene is: (a) a gene not expressed by the cell prior to the introduction of the fusion protein and the transposon, or (b) a gene exhibiting reduced, insufficient, and / or altered expression by the cell prior to the introduction of the fusion protein and the transposon. 20. The method of any one of claims 15 to 19, wherein the genomic target site is located on the LPA gene. 21. The method of any one of claims 15 to 19, wherein the genomic target site is located in a repetitive element. 22. The method of claim 21, wherein the repetitive element is an LPA repetitive sequence element. 23. The method of any one of claims 15 to 19, wherein the genomic target site is located in an intron of the gene. 24. The method of claim 23, wherein the genomic target site is located in an intron of the LPA gene. 25. The method of any one of claims 15 to 24, wherein the cell is in vivo. 26. A method of modifying the genome of a cell, the method comprising: providing the cell with a fusion protein of any one of claims 1 to 12, wherein the cell contains a modified binding site comprising, in a 5' to 3' order, a sequence of a target site for a DNA targeting domain, a first spacer region, a TTAA target integration site for SPB, a second spacer region, and an inverse complementary sequence to the sequence of the target site for the DNA targeting domain. 27. The method of claim 26, wherein the target integration site contains the sequence TTAA. 28. The method of claim 26, wherein the target integration site contains a nucleic acid sequence listed in any one of SEQ ID NO: 81 to 88.29. An integration cassette for site-specific transposition of nucleic acid into the genome of a cell, the integration cassette comprising nucleic acid comprising or consisting of: a central transposon ITR integration site TTAA sequence flanked by an upstream TAL array target sequence and a downstream TAL array target sequence, wherein each of the upstream TAL array target sequence and the downstream TAL array target sequence is separated from the TTAA sequence by 12 or 13 base pairs. 30. The integration cassette of claim 29, wherein the integration site comprises the sequence TTAA. 31. The integration cassette of claim 29, wherein the integration site comprises a nucleic acid sequence listed in any one of SEQ ID NO: 81 to 88. 32. The integration cassette of any one of claims 29 to 31, wherein each of the upstream TAL array target sequence and the downstream TAL array target sequence is identical. 33. The integration cassette of any one of claims 29 to 31, wherein each of the upstream TAL array target sequence and the downstream TAL array target sequence is different. 34. The integration cassette according to any one of claims 29 to 33, wherein each of the upstream TAL array target site and the downstream TAL array target site targets a 7 to 30 bp sequence of an LPA repetitive sequence element. 35. A cell comprising an integration cassette according to any one of claims 29 to 34 stably integrated into the genome of said cell. Claims 2 / 3 pages 3 CN 121219409 A 36. A method for site-specific transposition of a DNA molecule into the genome of a cell, said method comprising introducing the following into the cell according to claim 35: a) a nucleic acid encoding a fusion protein comprising a DNA-binding domain and a transposase; wherein said fusion protein is expressed in said cell; and b) a DNA molecule comprising a transposon; wherein the expressed fusion protein integrates said transposon into the TTAA integration site of a stably integrated integration cassette via site-specific transposition. 37. A method for generating engineered cells via site-specific transposition, the method comprising introducing the following into the cell of claim 31: a) a nucleic acid encoding a fusion protein comprising a DNA-binding domain and a transposase; wherein the fusion protein is expressed in the cell; and b) a DNA molecule comprising a transposon; wherein the expressed fusion protein integrates the transposon into a TTAA integration site of a stably integrated integration cassette via site-specific transposition, thereby generating the engineered cell.38. The method of claim 36 or 37, wherein the integration site comprises the sequence TTAA. 39. The method of claim 36 or 37, wherein the integration site comprises a nucleic acid sequence listed in any one of SEQ ID NO: 81 to 88. Claims 3 / 3 Page 4 CN 121219409 A Transposase and Use Therein

[0001] Cross-Reference to Related Applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 494,306, filed April 5, 2023, which is incorporated herein by reference in its entirety.

[0003] Reference to Sequence Listing Filed Electronically

[0004] This application contains a sequence listing that has been filed in XML format via the Patent Center and is incorporated herein by reference in its entirety. The XML copy was created on March 19, 2024, named "POTH-083_001WO_SeqList", and is 237,142 bytes in size. Technical Field

[0005] This disclosure relates throughout to fusion proteins comprising a transposase domain and a DNA targeting domain. Methods for site-specific transposition using fusion proteins are also provided. Background Art

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

[0007] The lipoprotein(a) or LPA gene evolved from a replication event of the adjacent plasminogen (PLG) gene. This replication event occurred approximately 40 million years ago during primate evolution. Both genes contain a circular structure called a kringle domain. In LPA, the kringle domain has been copied in segments, such that each copy of LPA can contain up to 50 copies of the kringle domain (Schmidt et al., J Lipid Res. Aug 2016; 57(8):1339-59). At the genomic DNA level, each kringle domain repetitive sequence spans approximately 5.5 kb of DNA and consists of two exons and two introns. The intronic portion of the repetitive sequence contains several potential target sites for site-specific transposase-mediated integration.

[0008] LPA is an attractive target for site-specific transposition because it contains multiple copies of the same target site, thereby increasing the chance of integrating the transposon into at least one copy.Furthermore, LPA is highly expressed in hepatocytes, meaning it may possess an open chromosomal landscape suitable for editing and supporting high expression of integrated transgenes. It is a non-essential gene, and knockout is associated with lower cholesterol levels. Taken together, these traits make it a potential target for gene therapy. Summary of the Invention

[0009] In one aspect, this document provides a fusion protein comprising a DNA targeting domain and a transposase domain comprising the sequence listed in SEQ ID NO: 4, wherein the DNA targeting domain binds to a nucleic acid sequence encoding a repetitive sequence element of LPA. In some embodiments, the DNA targeting domain comprises one, two, or three zinc finger motifs. In some embodiments, the DNA targeting domain comprises one or more TAL domains. In some embodiments, the TAL domain comprises the sequence listed in any of SEQ ID NO: 35 to 38. In some embodiments, the DNA targeting domain binds to a nucleic acid sequence encoding a kringle domain repeat sequence element in the LPA gene or an intron adjacent to the sequence encoding the kringle domain repeat sequence element.

[0010] In some embodiments, the transposase domain and the DNA targeting domain are connected by a adapter. In some embodiments, the adapter comprises the sequence GGGGS (SEQ ID NO: 181).

[0011] In some embodiments, the DNA targeting domain is inserted into the N-terminus of the transposase domain at a position following the 82nd amino acid and preceding the 105th amino acid of SEQ ID NO: 4. In some embodiments, the DNA targeting domain replaces one or more amino acids in the transposase domain between and including the 83rd and 105th amino acids of SEQ ID NO: 4. In some embodiments, the transposase domain comprises an N-terminal deletion of amino acids 1 to 83, 1 to 84, 1 to 85, 186, 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.

[0012] In some embodiments, the transposase domain comprises a sequence listed in any of SEQ ID NO: 7 to 27.In some embodiments, the transposase domain comprises: (a) at least one mutation selected from the group consisting of M185R, M185K, D197K, D197R, D198K, D198R, D201K, and D201R; or (b) at least one mutation selected from the group consisting of L204D, L204E, K500D, K500E, R504E, and R504D.

[0013] In another aspect, this document provides a polynucleotide comprising a nucleic acid sequence encoding the fusion protein described herein.

[0014] In another aspect, this document provides a vector comprising the polynucleotide described herein.

[0015] In another aspect, this document provides a method for integrating a transgene into a genomic target site of a cell, the method comprising: introducing the fusion protein described herein 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. In some embodiments, the transposon further comprises an exogenous promoter located between the 5' ITR and the transgene. In some embodiments, the transgene encodes a detectable biomarker. In some embodiments, the detectable biomarker is GFP. In some embodiments, the transgene is: (a) a gene not expressed by the cell prior to the introduction of the fusion protein and the transposon, or (b) a gene exhibiting reduced, insufficient, and / or altered expression by the cell prior to the introduction of the fusion protein and the transposon.

[0016] In some embodiments, the genomic target site is located on the LPA gene. In some embodiments, the genomic target site is located in a repetitive element. In some embodiments, the repetitive element is an LPA repetitive sequence element. In some embodiments, the genomic target site is located in an intron of the gene. In some embodiments, the genomic target site is located in the intron of the LPA gene. In some embodiments, the cell is in vivo.

[0017] In another aspect, this document provides a method for modifying the genome of a cell, the method comprising: providing the cell with the fusion protein described herein, wherein the cell contains a modified binding site comprising, in a 5' to 3' sequence, a sequence of a target site for the DNA targeting domain, a first spacer region, a TTAA target integration site for SPB, a second spacer region, and an inverse complementary sequence to the target site sequence for the DNA targeting domain. In some embodiments, the target integration site contains the sequence TTAA. In some embodiments, the target integration site contains a nucleic acid sequence listed in any one of SEQ ID NO: 81 to 88.

[0018] In another aspect, this document provides an integration cassette for site-specific transposition of nucleic acids into the genome of a cell, the integration cassette comprising a nucleic acid comprising or consisting of: a central transposon ITR integration site TTAA sequence flanked by an upstream TAL array target sequence and a downstream TAL array target sequence, wherein each of the upstream TAL array target sequence and the downstream TAL array target sequence is separated from the TTAA sequence by 12 or 13 base pairs. In some embodiments, the integration site comprises the TTAA sequence. In some embodiments, the integration site comprises a nucleic acid sequence listed in any of SEQ ID NO: 81 to 88 on page 2 / 62 of this specification, CN 121219409 A. In some embodiments, each of the upstream TAL array target sequence and the downstream TAL array target sequence is identical. In some embodiments, each of the upstream TAL array target sequence and the downstream TAL array target sequence is different. In some embodiments, each of the upstream TAL array target site and the downstream TAL array target site targets a 7 to 30 bp sequence of an LPA repetitive sequence element.

[0019] In another aspect, this document provides a cell comprising the integration cassette described herein stably integrated into the genome of the cell.

[0020] In another aspect, this document provides a method for site-specific transposition of a DNA molecule into the genome of a cell, comprising introducing the following into a cell comprising the integration cassette described herein: a nucleic acid encoding a fusion protein comprising a DNA-binding domain and a transposase; wherein the fusion protein is expressed in the cell; and a DNA molecule comprising a transposon; wherein the expressed fusion protein integrates the transposon into the TTAA integration site of the stably integrated integration cassette via site-specific transposition.

[0021] In another aspect, this document provides a method for generating engineered cells via site-specific transposition, comprising introducing into a cell comprising an integration cassette as described herein: a nucleic acid encoding a fusion protein comprising a DNA-binding domain and a transposase; wherein the fusion protein is expressed in the cell; and a DNA molecule comprising a transposon; wherein the expressed fusion protein integrates the transposon into a TTAA integration site of a stably integrated integration cassette via site-specific transposition, thereby generating the engineered cell. In some embodiments, the sequence is TTAA. In some embodiments, the integration site comprises a nucleic acid sequence listed in any one of SEQ ID NO: 81 to 88.

[0022] Figures 1A to 1D illustrate the introduction of a DNA-binding domain into a transposase using a specific heterodimer.

[0023] Figure 2 is a schematic diagram illustrating the splitting of a GFP splice site-specific reporter gene.

[0024] Figure 3 is a schematic diagram illustrating a catalytic ssSPB dimer that binds to the excised transposon and recognizes its genomic integration target site. Detailed Description

[0025] This document provides fusion proteins comprising a transposase domain and a DNA-targeting domain. In particular, the DNA-targeting domain can target the lipoprotein A (LPA) gene. Methods for preparing the transposase domain and the fusion protein, cells modified with the fusion protein provided herein, and therapeutic methods using such cells are also provided.

[0026] In some embodiments, this document provides fusion proteins comprising an SPB or PBx domain and a DNA-targeting domain. The DNA-targeting domain is further described below.

[0027] Transposase Domain

[0028] In one aspect, this document provides fusion proteins comprising one or more transposase domains. In some embodiments, the transposase domain is a piggyBac transposase domain. In some embodiments, the piggyBac transposase domain is a super-active piggyBac transposase domain. In a preferred embodiment, the transposase domain is a Super piggyBac™ transposase domain (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, as examples of transposase domains that may be used in the fusion proteins described herein, each of which is incorporated herein by reference in its entirety.

[0029] In some embodiments, the transposase domain is a Super PiggyBac transposase (SPB) domain. Compared to the wild-type piggyBac transposase, the SPB contains one or more super-high activity mutations. An exemplary wild-type SPB sequence containing a nuclear localization sequence (NLS) is shown in SEQ ID NO: 1, wherein the NLS is shown in italics and the super-high activity mutation is shown in bold. The sequence numbering of the SPB transposase domain for the purpose of describing deletions and mutations begins at residue 12 of SEQ ID NO: 1.MAPKKKRKVGGGGSSLDDEHILSALLQSDDELVGEDSDSEVSDHVSEDDVQSDTEEAFIDEVHEVQPT SSGSEILDEQNVIEQPGSSLASNRILTLPQRTIRGKNKHCWSTSKSTRRSRVSALNIVRSQRGPTRMCRNIYDPLL CFKLFFTDEIISEIVKWTNAEISLKRRESMTSATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLSMVYV SVMSRDRFDFLIRCLRMDDKSIRPTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRVYIP NKPSKYGIKILMMCDSGTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSIPLAKNLLQE PYKLTIVGTVRSNKREIPEVLKNSRSRPVGTSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINESTGKPQMVM YYNQTKGGVDTLDQMCSVMTCSRKTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMSLTSS FMRKRLEAPTLKRYLRDNISNILPKEVPGTSDDSTEEPVMKKRTYCTYCPSKIRRKANASCKKCKKVICREHNIDM CQSCF (SEQ ID NO: 1).

[0031] In some embodiments, the fusion protein described herein comprises a transposase domain containing 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 listed in SEQ ID NO: 1. In some embodiments, the fusion protein described herein comprises a transposase domain containing the amino acid sequence listed in SEQ ID NO: 1, having one, two, three, four, or five conserved amino acid substitutions. In some embodiments, the fusion protein described herein comprises a transposase domain containing the amino acid sequence listed in SEQ ID NO: 1.

[0032] An exemplary sequence of a wild-type SPB transposase lacking the NLS domain is listed in SEQ ID NO: 2.For purposes of describing deletions and mutations, the sequence numbering of the SPB transposase domain begins with residue 5 of SEQ ID NO: 2. In some embodiments, the fusion protein described herein comprises a transposase domain containing 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 listed in SEQ ID NO: 2. In some embodiments, the fusion protein described herein comprises a transposase domain containing the amino acid sequence listed in SEQ ID NO: 2, having one, two, three, four, or five conserved amino acid substitutions. In some embodiments, the fusion protein described herein comprises a transposase domain containing the amino acid sequence listed in SEQ ID NO: 2.

[0033] The transposase domain used in the fusion proteins described herein can be isolated or derived from insects, vertebrates, crustaceans, or chordates, as described in more detail in PCT Publications WO 2019 / 173636 and WO 2020 / 051374. In a preferred aspect, the SPB transposase domain is isolated or derived from the insects *Trichoplusia ni* (GenBank accession number AAA87375), *Bombyx mori* (GenBank accession number BAD11135), or *Macdunnoughia crassisigna* (GenBank accession number ABZ85926.1).

[0034] In some embodiments, the transposase domain is an integration-deficient type. An integration-deficient transposase domain is a transposase that can cleave its corresponding transposon but 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 CN 121219409 A 8,399,643; and WO 2019 / 173636, each of which is incorporated herein by reference in its entirety as an example of a transposase domain that may be used in the fusion protein described herein. A series of integration-deficient amino acid substitutions are disclosed in U.S. Patent No. 10,041,077, which is incorporated herein by reference in its entirety as an example of mutations that may be introduced into the transposase domain described herein.

[0035] Wild-type SPB can be made integration-deficient by introducing mutations, for example, K93A, R372A, K375A, R376A and / or D450N (relative to SEQ ID NO: 2, where the numbering begins at residue 5). It is believed that the introduction of mutations R372A, K375A, R376A and D450N makes the transposase integration-deficient but retains excision function. An exemplary sequence of an integration-deficient transposase domain is PBx containing NLS, listed in SEQ ID NO: 3. In some embodiments, the fusion protein described herein comprises a transposase domain containing 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 listed in SEQ ID NO: 3. In some embodiments, the fusion protein described herein comprises a transposase domain containing the amino acid sequence listed in SEQ ID NO: 3, having one, two, three, four, or five conserved amino acid substitutions. In some embodiments, the fusion protein described herein comprises a transposase domain containing the amino acid sequence listed in SEQ ID NO: 3.

[0036] The sequence of the integration defect type PBx transposable domain that does not contain NLS is listed in SEQ ID NO: 4:

[0037] GGSSLDDEHILSALLQSDDELVGEDSDSEVSDHVSEDDVQSDTEEAFIDEVHEVQPTSSGSEILDEQN VIEQPGSSLASNRILTLPQRTIRGKNKHCWSTSKSTRRSRVSALNIVRSQRGPTRMCRNIYDPLLCFKLFFTDEII SEIVKWTNAEISLKRRESMTSATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLSMVYVSVMSRDRFDFL IRCLRMDDKSIRPTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRVYIPNKPSKYGIKIL MMCDSGTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSIPLAKNLLQEPYKLTIVGTVA SNAREIPEVLKNSRSRPVGTSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINESTGKPQMVMYYNQTKGGVDT LNQMCSVMTCSRKTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMSLTSSFMRKRLEAPTL KRYLRDNISNILPKEEVPGTSDDSTEEPVMKKRTYCTYCPSKIRRKANASCKKCKKVICREHNIDMCQSCF (SEQ ID NO: 4).

[0038] In some embodiments, the fusion protein described herein comprises a transposase domain containing 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 listed in SEQ ID NO: 4. In some embodiments, the fusion protein described herein comprises a transposase domain containing the amino acid sequence listed in SEQ ID NO: 4, having one, two, three, four, or five conserved amino acid substitutions. In some embodiments, the fusion protein described herein comprises a transposase domain containing the amino acid sequence listed in SEQ ID NO: 4.

[0039] Transposase Domain Containing N-Terminal Deletion

[0040] In some embodiments, this document provides a transposase domain (e.g., an SPB transposase domain or a PBx transposase domain) that contains a deletion of a portion of the amino terminus of the transposase domain (also referred to as the “N-terminus”, “N-terminal domain”, or “NTD”). An SPB transposase domain or PBx transposase domain containing an N-terminal domain deletion has previously been described in International Patent Application Publication No. PCT / US2022 / 77549 as an example of a transposase domain that can be used in the fusion protein described herein, and is incorporated herein by reference in its entirety.

[0041] The exemplary sequences of the SPB transposase domain having the deletion of amino acids 1 to 93 at the N-terminus and the PBx transposase domain having the deletion of amino acids 1 to 93 at the N-terminus are shown in SEQ ID NO: 5 and 6, respectively:

[0042] NKHCWSTSKSTRRSRVSALNIVRSQRGPTRMCRNIYDPLLCFKLFFTDEIISEIVKWTNAEISLKRRE SMTSATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLSMVYVSVMSRDRFDFLIRCLRMDDKSIRPTLRE NDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRVYIPNKPSKYGIKILMMCDSGTKYMINGMPYL GRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSIPLAKNLLQEPYKLTIVGTVRSNKREIPEVLKNSRSRP VGTSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINESTGKPQMVMYYNQTKGGVDTLDQMCSVMTCSRKTNRW PMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMSLTSSFMRKRLEAPTLKRYLRDNISNILPKEVP GTSDDSTEEPVMKKRTYCTYCPSKIRRKANASCKKCKKVICREHNIDMCQSCF (SEQ ID NO: 5).

[0043] In some embodiments, the fusion protein described herein comprises a transposase domain containing 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 listed in SEQ ID NO: 5. In some embodiments, the fusion protein described herein comprises a transposase domain containing the amino acid sequence listed in SEQ ID NO: 5, having one, two, three, four, or five conserved amino acid substitutions. In some embodiments, the fusion protein described herein comprises a transposase domain containing the amino acid sequence listed in SEQ ID NO: 5. NKHCWSTSKSTRRSRVSALNIVRSQRGPTMCRNIYDPLLCFKLFFTDEIISEIVKWTNAEISLKRRE SMTSATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLSMVYVSVMSRDRFDFLIRCLRMDDKSIRPTLRE NDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRVYIPNKPSKYGIKILMMCDSGTKYMINGMPYL GRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSIPLAKNLLQEPYKLTIVGTVASNAREIPEVLKNSRSRP VGTSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINESTGKPQMVMYYNQTKGGVDTLNQMCSVMTCSRKTNRW PMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMSLTSSFMRKRLEAPTLKRYLRDNISNILPKEVP GTSDDSTEEPVMKKRTYCTYCPSKIRRKANASCKKCKKVICREHNIDMCQSCF (SEQ ID NO: 6).

[0045] In some embodiments, the fusion protein described herein comprises a transposase domain containing 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 listed in SEQ ID NO: 6.In some embodiments, the fusion protein described herein comprises a transposase domain comprising the amino acid sequence listed in SEQ ID NO: 6, having one, two, three, four, or five conserved amino acid substitutions. In some embodiments, the fusion protein described herein comprises a transposase domain comprising the amino acid sequence listed in SEQ ID NO: 6.

[0046] Other exemplary sequences comprising an N-terminal deleted PBx transposase domain are listed in SEQ ID NO: 7 to 27 in Table 1.

[0047] In some embodiments, the fusion protein described herein comprises a transposase domain comprising 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 listed in any one of SEQ ID NO: 7 to 27. In some embodiments, the fusion protein described herein comprises a transposase domain containing the amino acid sequence listed in any one of SEQ ID NO: 7 to 27, having one, two, three, four, or five conserved amino acid substitutions. In some embodiments, the fusion protein described herein comprises a transposase domain containing the amino acid sequence listed in any one of SEQ ID NO: 7 to 27.Specification page 6 / 62, 10 CN 121219409 A

[0048] Table 1: Indicative sequences of N-terminal deleted PBx domains

[0049] Specification page 7 / 62, 11 CN 121219409 A Specification page 8 / 62, 12 CN 121219409 A Specification page 9 / 62, 13 CN 121219409 A Specification page 10 / 62, 14 CN 121219409 A Specification page 11 / 62, 15 CN 121219409 A Specification page 12 / 62, 16 CN 121219409 A Specification page 13 / 62, 17 CN 121219409 A Specification page 14 / 62, 18 CN 121219409 A Specification page 15 / 62, 19 CN 121219409 A Specification 16 / 62 pages 20 CN 121219409 A Specification 17 / 62 pages 21 CN 121219409 A Specification 18 / 62 pages 22 CN 121219409 A Specification 19 / 62 pages 23 CN 121219409 A Specification 20 / 62 pages 24 CN 121219409 A

[0050] DNA Targeting Domain

[0051] The transposase domains and fusion proteins provided herein 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 transposase domain or fusion protein. In some embodiments, the DNA targeting domain is attached to the N-terminus of the transposase domain, for example, comprising a transposase domain with an N-terminus missing. Without being bound by theory, it is believed that adding a DNA targeting domain to a transposase domain improves site-specific transposase activity by targeting the transposase fused to the DNA targeting domain to the target 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 transposase domain that does not contain a DNA targeting domain.

[0052] Any DNA targeting domain known in the art can be used in the transposase domains, fusion proteins, and tandem dimeric transposases described herein, including but not limited to CRISPR, zinc finger motifs, TALE, and transcription factors. In some embodiments, the DNA targeting domain contains one, two, or three zinc finger motifs. In some embodiments, the DNA targeting domain contains three zinc finger motifs.In some embodiments, the three zinc finger motifs are side-connected to a GGGGS (SEQ ID NO: 25 CN 121219409 A 181) connector. In some embodiments, the three zinc finger motifs side-connected to the GGGGS (SEQ ID NO: 181) connector collectively comprise the sequence listed in SEQ ID NO: 28: GGGGSERPYACPVESCDRRFSRSDELTRHIRIHTGQKPFQCR ICMRNFSRSDHLTTHIRTHTGEKPFACDICGRKFARSDERKRHTKIHLRQKDGGGGS (SEQ ID NO: 28) 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.

[0053] In a particular embodiment, this document provides a fusion protein comprising a transposase domain comprising an N-terminal deletion, an NLS, and three zinc finger motifs. In some embodiments, the NLS comprises or consists of the sequence listed in SEQ ID NO: 29.

[0054] 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 an array of approximately 34 amino acid repeats of a variable number, followed by a 278-amino acid C-terminus (SEQ ID NO: 30); however, truncated forms have been described in the literature (e.g., see Miller et al., Nat Biotechnol 29, 143–148 (2011)). TAL fused with FokI nuclease (referred to as TALEN) most often contains truncated N-termini and C-termini. For example, the first 152 amino acids of the N-terminus are often removed (referred to as Δ152; SEQ ID No. 31), and the C-terminus is often truncated, leaving 63 amino acids (referred to as +63; SEQ ID NO: 32).

[0055] TAL contains an array of 34 amino acids repeated a variable number of times. Two amino acids at positions 12 and 13 are variable and determine which nucleotide the TAL repeat sequence will recognize. This feature allows the TAL array to be programmed to bind to specific DNA sequences. Amino acids NG recognize T, NI recognize A, NN recognize G or A, HD recognize C, NK recognize G, and NS recognize 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 N that recognizes the repeating sequence of G. Additionally, positions 4 and 32 are often varied to reduce the repetitiveness of the array, but the binding specificity is uncertain. The number of 34 amino acid repeating sequences in the array determines the length of the DNA sequence recognized (one protein repeating sequence binds one DNA bp). Furthermore, the last bp is recognized by a “half-array” of 20 amino acids instead of 34 amino acids.

[0056] Furthermore, the N-terminal domain of TAL (e.g., SEQ ID NO: 31) recognizes and requires a T immediately grounded at the 5' end of the target DNA sequence. Mutations that no longer require the 5' T N-terminal domain of TAL have been described in the literature (Lamb et al., Nucleic Acids Res. 2013 Nov;41(21):9779-85). For example, the NT-G mutant requires 5'G instead of 5'T (SEQ ID NO: 33), while the NT-βN mutant does not require any specific 5' nucleotide (SEQ ID NO: 34). These mutated N-terminal domain sequences can be used to provide additional sequence selections that can be targeted using TAL arrays.

[0057] Generally, each TAL array contains nine 34-amino acid repeat sequences followed by 20-amino acid "half" repeat sequences. TAL arrays can be synthesized with BsmBI IIS type restriction sites flanked. In one embodiment, individual TAL modules containing either 34-amino acid or 20-amino acid "half" repeat sequences flanked by BsmBI IIS type restriction sites can be designed and synthesized. The entire TAL module set contains four modules capable of recognizing A, C, G, and T at each of the 10 bp positions (40 modules / 10 bp target), and one TAL half repeat sequence module.Exemplary TAL modules are listed in SEQ ID NO: 35 to 38, where X is any amino acid:

[0058] ·TAL module type 1: LTPDQVVAIAXXXGGKQALETVQRLLPVLCQDHG (SEQ ID NO: 35)

[0059] ·TAL module type 2: LTPEQVVAIAXXXGGKQALETVQRLLPVLCQAHG (SEQ ID NO: 36)

[0060] ·TAL module type 3'' LTPDQVVAIAXXXGGKQALETVQRLLPVLCQAHG (SEQ ID NO: 37)

[0061] ·TAL module type 4: LTPAQVVAIAXXXGGKQALETVQRLLPVLCQDHG (SEQ ID NO: 38). Specification page 22 / 62 26 CN 121219409 A

[0062] An exemplary TAL half-module is listed in SEQ ID NO: 39, where X is any amino acid: LTPEQVVAIAXXXGGRPALE (SEQ ID NO: 39).

[0063] TAL array pairs targeting sequences in desired genes can be designed, and “Golden Gate Assembly” can be used to select the corresponding modules and assemble them together to assemble each TAL array in a frame. The GeneArt algorithm (Thermo Fisher) can be used to further codon optimize the DNA sequences encoding the TAL arrays generated herein.

[0064] When designing left and right TAL arrays (i.e., TAL-ssSPB or TAL-PBx; as described below) that include the N-terminal domain of the transposase sequence to be fused to the N-terminal deletion transposase, recognizing the T-terminal domain, and the C-terminal domain of the TAL, one TAL array recognizes the 5' sequence of TTAA, and the other TAL array recognizes the 3' sequence of TTAA. Because the 5' sequence of TTAA is most often different from the 3' sequence of TTAA in the genomic DNA target, TAL-ssSPB is most often used as a heterodimer consisting of two different TAL domains recognizing two different DNA sequences. Furthermore, the sequence recognized by the TAL array is not directly adjacent to TTAA. Instead, it is separated from TTAA by a spacer region of a given bp length (e.g., a 12bp, 13bp, or 14bp spacer region).

[0065] The TAL array can target any DNA sequence of interest (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.

[0066] 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 commonly owned international patent application publication number PCT / 2022 / 22549.

[0067] In some embodiments, the TAL array targets repetitive sequence elements of the LPA gene. Exemplary sequences of the left TAL array targeting LPA repetitive sequence elements are listed in SEQ ID NO: 116, 118, 121, 124, 125, 127, 129, 131, 133, 135, 137, 139, and 141. Exemplary sequences of the right TAL array targeting LPA are listed in SEQ ID NO: 117, 119, 120, 122, 123, 126, 128, 130, 132, 134, 136, 138, 140, and 142. In some embodiments, the left TAL array targeting LPA repetitive sequence elements binds to nucleic acid molecules comprising sequences listed in SEQ ID NO: 89, 91, 94, 97, 98, 100, 102, 104, 106, 108, 110, 112, and 114. In some embodiments, the right TAL array targeting LPA repetitive sequence elements binds to nucleic acid molecules comprising sequences listed in SEQ ID NO: 90, 92, 93, 95, 96, 99, 101, 103, 105, 107, 109, 111, 113, and 115. It will be apparent to those skilled in the art that any left TAL array disclosed herein can be combined with any right TAL array disclosed herein. Exemplary genomic targets for LPA repetitive sequence elements are listed in SEQ ID NO: 81 to 88.

[0068] This disclosure provides fusion proteins comprising a DNA targeting domain attached in various ways to a transposase domain. In some embodiments, the DNA targeting domain may be fused to or linked to the N-terminus of a transposase domain comprising an N-terminal deletion. For example, the DNA targeting domain may be inserted into the transposase domain at a suitable location in the N-terminal region of the transposase domain.In some embodiments, the DNA targeting domain may replace one or more amino acids in the N-terminal region of the transposase domain. In some embodiments, the DNA targeting domain is inserted into the transposase domain at a suitable position in the N-terminal region of the transposase domain without replacing amino acids.

[0069] The DNA targeting domain may be inserted into the N-terminus of the transposase domain. For example, the DNA targeting domain is inserted into the N-terminus of the transposase domain at a position after the 82nd amino acid and before the 105th amino acid in SEQ ID NO: 4. In some embodiments, the DNA targeting domain is inserted between the 82nd and 83rd amino acids in SEQ ID NO: 2 or 3 (wherein numbering starts from the 5th or 12th amino acid, respectively) or SEQ ID NO: 4. In some embodiments, the DNA targeting domain is inserted between amino acids 83 and 84 of SEQ ID NO: 2 or 3 (numbered starting from the 5th or 12th amino acid, respectively) or SEQ ID NO: 4. In some embodiments, the DNA targeting domain is inserted between amino acids 84 and 85 of SEQ ID NO: 2 or 3 (numbered starting from the 5th or 12th amino acid, respectively) or SEQ ID NO: 4. In some embodiments, the DNA targeting domain is inserted between amino acids 85 and 86 of SEQ ID NO: 2 or 3 (numbered starting from the 5th or 12th amino acid, respectively) or SEQ ID NO: 4. In some embodiments, the DNA targeting domain is inserted between amino acids 86 and 87 of SEQ ID NO: 2 or 3 (numbered starting from the 5th or 12th amino acid, respectively) or SEQ ID NO: 4. In some embodiments, the DNA targeting domain is inserted between amino acids 87 and 88 of SEQ ID NO: 2 or 3 (wherein numbering starts from the 5th or 12th amino acid, respectively) or SEQ ID NO: 4. In some embodiments, the DNA targeting domain is inserted between amino acids 88 and 89 of SEQ ID NO: 2 or 3 (wherein numbering starts from the 5th or 12th amino acid, respectively) or SEQ ID NO: 4.In some embodiments, the DNA targeting domain is inserted between amino acids 89 and 90 of SEQ ID NO: 2 or 3 (numbered starting from the 5th or 12th amino acid, respectively) or SEQ ID NO: 4. In some embodiments, the DNA targeting domain is inserted between amino acids 90 and 91 of SEQ ID NO: 2 or 3 (numbered starting from the 5th or 12th amino acid, respectively) or SEQ ID NO: 4. In some embodiments, the DNA targeting domain is inserted between amino acids 91 and 92 of SEQ ID NO: 2 or 3 (numbered starting from the 5th or 12th amino acid, respectively) or SEQ ID NO: 4. In some embodiments, the DNA targeting domain is inserted between amino acids 92 and 93 of SEQ ID NO: 2 or 3 (numbered starting from the 5th or 12th amino acid, respectively) or SEQ ID NO: 4. In some embodiments, the DNA targeting domain is inserted between amino acids 93 and 94 of SEQ ID NO: 2 or 3 (numbered starting from the 5th or 12th amino acid, respectively) or SEQ ID NO: 4. In some embodiments, the DNA targeting domain is inserted between amino acids 94 and 95 of SEQ ID NO: 2 or 3 (numbered starting from the 5th or 12th amino acid, respectively) or SEQ ID NO: 4. In some embodiments, the DNA targeting domain is inserted between amino acids 95 and 96 of SEQ ID NO: 2 or 3 (numbered starting from the 5th or 12th amino acid, respectively) or SEQ ID NO: 4. In some embodiments, the DNA targeting domain is inserted between amino acids 96 and 97 of SEQ ID NO: 2 or 3 (numbered starting from the 5th or 12th amino acid, respectively) or SEQ ID NO: 4. In some embodiments, the DNA targeting domain is inserted between amino acids 97 and 98 of SEQ ID NO: 2 or 3 (wherein numbering starts from the 5th or 12th amino acid, respectively) or SEQ ID NO: 4. In some embodiments, the DNA targeting domain is inserted between amino acids 98 and 99 of SEQ ID NO: 2 or 3 (wherein numbering starts from the 5th or 12th amino acid, respectively) or SEQ ID NO: 4.In some embodiments, the DNA targeting domain is inserted between the 99th and 100th amino acids of SEQ ID NO: 2 or 3 (numbered starting from the 5th or 12th amino acid, respectively) or SEQ ID NO: 4. In some embodiments, the DNA targeting domain is inserted between the 100th and 101st amino acids of SEQ ID NO: 2 or 3 (numbered starting from the 5th or 12th amino acid, respectively) or SEQ ID NO: 4. In some embodiments, the DNA targeting domain is inserted between the 101st and 102nd amino acids of SEQ ID NO: 2 or 3 (numbered starting from the 5th or 12th amino acid, respectively) or SEQ ID NO: 4. In some embodiments, the DNA targeting domain is inserted between the 102nd and 103rd amino acids of SEQ ID NO: 2 or 3 (numbered starting from the 5th or 12th amino acid, respectively) or SEQ ID NO: 4. In some embodiments, the DNA targeting domain is inserted between the 103rd and 104th amino acids of SEQ ID NO: 2 (page 24 / 62, CN 121219409 A or 3, respectively, numbered starting from the 5th or 12th amino acid) or SEQ ID NO: 4. In some embodiments, the DNA targeting domain is inserted between the 104th and 105th amino acids of SEQ ID NO: 2 or 3 (numbered starting from the 5th or 12th amino acid) or SEQ ID NO: 4. In some embodiments, the DNA targeting domain comprises the sequence of SEQ ID NO: 28 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. The transposase domain may further comprise, for example, an NLS, such as the NLS of SEQ ID NO: 29.

[0070] The DNA targeting domain may replace one or more amino acids in the N-terminal region of the transposase domain. For example, the DNA targeting domain may replace one or more amino acids in the transposase domain between and including the 83rd and 105th amino acids in SEQ ID NO: 2 or 3 (wherein the amino acid numbering starts from the 5th or 12th amino acid, respectively) or SEQ ID NO: 4.In some embodiments, the DNA targeting domain replaces the 83rd amino acid of SEQ ID NO: 2 or 3 (wherein numbering starts from the 5th or 12th amino acid, respectively) or SEQ ID NO: 4. In some embodiments, the DNA targeting domain replaces the 84th amino acid of SEQ ID NO: 2 or 3 (wherein numbering starts from the 5th or 12th amino acid, respectively) or SEQ ID NO: 4. In some embodiments, the DNA targeting domain replaces the 85th amino acid of SEQ ID NO: 2 or 3 (wherein numbering starts from the 5th or 12th amino acid, respectively) or SEQ ID NO: 4. In some embodiments, the DNA targeting domain replaces the 86th amino acid of SEQ ID NO: 2 or 3 (wherein numbering starts from the 5th or 12th amino acid, respectively) or SEQ ID NO: 4. In some embodiments, the DNA targeting domain replaces the 87th amino acid of SEQ ID NO: 2 or 3 (wherein numbering starts from the 5th or 12th amino acid, respectively) or SEQ ID NO: 4. In some embodiments, the DNA targeting domain replaces the 88th amino acid of SEQ ID NO: 2 or 3 (wherein numbering starts from the 5th or 12th amino acid, respectively) or SEQ ID NO: 4. In some embodiments, the DNA targeting domain replaces the 89th amino acid of SEQ ID NO: 2 or 3 (wherein numbering starts from the 5th or 12th amino acid, respectively) or SEQ ID NO: 4. In some embodiments, the DNA targeting domain replaces the 90th amino acid of SEQ ID NO: 2 or 3 (wherein numbering starts from the 5th or 12th amino acid, respectively) or SEQ ID NO: 4. In some embodiments, the DNA targeting domain replaces the 91st amino acid of SEQ ID NO: 2 or 3 (wherein numbering starts from the 5th or 12th amino acid, respectively) or SEQ ID NO: 4. In some embodiments, the DNA targeting domain replaces the 92nd amino acid of SEQ ID NO: 2 or 3 (wherein numbering starts from the 5th or 12th amino acid, respectively) or SEQ ID NO: 4. In some embodiments, the DNA targeting domain replaces the 93rd amino acid of SEQ ID NO: 2 or 3 (wherein numbering starts from the 5th or 12th amino acid, respectively) or SEQ ID NO: 4.In some embodiments, the DNA targeting domain replaces the 94th amino acid of SEQ ID NO: 2 or 3 (wherein numbering starts from the 5th or 12th amino acid, respectively) or SEQ ID NO: 4. In some embodiments, the DNA targeting domain replaces the 95th amino acid of SEQ ID NO: 2 or 3 (wherein numbering starts from the 5th or 12th amino acid, respectively) or SEQ ID NO: 4. In some embodiments, the DNA targeting domain replaces the 96th amino acid of SEQ ID NO: 2 or 3 (wherein numbering starts from the 5th or 12th amino acid, respectively) or SEQ ID NO: 4. In some embodiments, the DNA targeting domain replaces the 97th amino acid of SEQ ID NO: 2 or 3 (wherein numbering starts from the 5th or 12th amino acid, respectively) or SEQ ID NO: 4. In some embodiments, the DNA targeting domain replaces the 98th amino acid of SEQ ID NO: 2 or 3 (wherein numbering starts from the 5th or 12th amino acid, respectively) or SEQ ID NO: 4. In some embodiments, the DNA targeting domain replaces the 99th amino acid of SEQ ID NO: 2 or 3 (wherein numbering starts from the 5th or 12th amino acid, respectively) or SEQ ID NO: 4. In some embodiments, the DNA targeting domain replaces the 100th amino acid of SEQ ID NO: 2 or 3 (wherein numbering starts from the 5th or 12th amino acid, respectively) or SEQ ID NO: 4. In some embodiments, the DNA targeting domain replaces the 101st amino acid of SEQ ID NO: 2 or 3 (wherein numbering starts from the 5th or 12th amino acid, respectively) or SEQ ID NO: 4. In some embodiments, the DNA targeting domain replaces the 102nd amino acid of SEQ ID NO: 2 or 3 (wherein numbering starts from the 5th or 12th amino acid, respectively) or SEQ ID NO: 4. In some embodiments, the DNA targeting domain replaces the 103rd amino acid in SEQ ID NO: 2 or 3 (wherein numbering starts from the 5th or 12th amino acid, respectively) or SEQ ID NO: 4.In some embodiments, the DNA targeting domain replaces the 104th amino acid of SEQ ID NO: 2 or 3 (wherein numbering starts from the 5th or 12th amino acid, respectively) or SEQ ID NO: 4. In some embodiments, the DNA targeting domain replaces the 105th amino acid of SEQ ID NO: 2 or 3 (wherein numbering starts from the 5th or 12th amino acid, respectively) or SEQ ID NO: 4. In some embodiments, the DNA targeting domain comprises the sequence of SEQ ID NO: 28 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. The transposase domain may further comprise an NLS, for example, the NLS of SEQ ID NO: 29.

[0071] An exemplary sequence of a fusion protein comprising a transposase domain (the transposase domain comprising an N-terminal deletion of 93 amino acids, an NLS, and three zinc finger motifs flanked by a GGGGS (SEQ ID NO: 181) linker) is shown in SEQ ID NO: 40, wherein the NLS is shown in italics, the sequence comprising the three zinc finger motifs and the GGGGS linker is underlined, and the transposase domain comprising an N-terminal deletion of 93 amino acids is shown in bold: Specification 26 / 62 pages 30 CN 121219409 A

[0072]

[0073] In some embodiments, the fusion protein described herein comprises a transposase domain comprising 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%, or at least 98% of the amino acid sequence listed in SEQ ID NO: 40. Or at least 99% identical amino acid sequences. In some embodiments, the fusion protein described herein comprises a transposase domain containing the amino acid sequence listed in SEQ ID NO: 40, having one, two, three, four, or five conserved amino acid substitutions. In some embodiments, the fusion protein described herein comprises a transposase domain containing the amino acid sequence listed in SEQ ID NO: 40.An exemplary sequence of a fusion protein containing an integration-deficient transposase domain (which contains a 93-amino acid N-terminal deletion, an NLS, and three zinc finger motifs flanked by a GGGGS (SEQ ID NO: 181) linker) is listed in SEQ ID NO: 180, wherein the NLS is shown in italics, the sequence containing the three zinc finger motifs and the GGGGS linker is underlined, and the N-terminal deletion transposase domain containing 93 amino acids is shown in bold: Specification 27 / 62 pages 31 CN 121219409 A

[0074] .

[0075] In some embodiments, the fusion protein described herein comprises a transposase domain containing 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 listed in SEQ ID NO: 180. In some embodiments, the fusion protein described herein comprises a transposase domain containing the amino acid sequence listed in SEQ ID NO: 180, having one, two, three, four, or five conserved amino acid substitutions. In some embodiments, the fusion protein described herein comprises a transposase domain containing the amino acid sequence listed in SEQ ID NO: 180.

[0076] Nuclear Localization Signal

[0077] In some embodiments, the transposase domain and fusion protein provided herein may comprise a frame nuclear localization sequence (NLS). Examples of transposases fused with nuclear localization signals are disclosed in U.S. Patent Nos. 6,218,185, 6,962,810, 8,399,643, and WO 2019 / 173636, each of which is incorporated herein by reference in its entirety as an example of a transposase domain that may be used in the fusion protein described herein. In some embodiments, the NLS comprises the sequence of PKKKRKV (SEQ ID NO: 29). In some aspects, within the framework, the NLS is located upstream (N-terminus) of a transposase domain containing an N-terminal deletion. Specification 28 / 62 pages 32 CN 121219409 A

[0078] Generally, the NLS is preferably located at the N-terminus of the fusion protein. In some embodiments, the NLS is fused to or linked to the N-terminus of the transposase domain. In some embodiments, the NLS is fused to or linked to the N-terminus of a DNA targeting domain.

[0079] In some aspects, the in-frame NLS is directly fused to the amino terminus of the transposase domain containing the N-terminus deletion. In some embodiments, the NLS is attached to the N-terminus of the transposase domain containing the N-terminus deletion via a linker (e.g., a GGGGS linker or a GGS linker).

[0080] In some embodiments, the initiator methionine is introduced before the NLS. In some embodiments, an additional alanine residue is introduced before and / or after the NLS to ensure in-frame translation. Thus, for the purpose of identifying deleted and mutated residues, the residues in SEQ ID NO: 1 and 3 are numbered starting from the 12th residue of SEQ ID NO: 1 and 3. In SEQ ID NO: 2 (which is the sequence of SPB without NLS), the residues are numbered starting from the 5th residue for the purpose of identifying deleted and mutated residues. In SEQ ID NO: 4, the residues are numbered starting from the first residue for the purpose of identifying deleted and mutated residues.

[0081] In some embodiments, the fusion protein comprises an NLS and an N-terminal-deleted transposase domain comprising 93 amino acids. In some embodiments, the fusion protein comprises at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the amino acid sequence listed in SEQ ID NO: 5. In some embodiments, the fusion protein comprises the amino acid sequence listed in SEQ ID NO: 5.

[0082] Specific heterodimers and tandem dimers

[0083] In another aspect, this document provides tandem dimer transposases comprising two fusion proteins, each comprising a transposase domain, and one or both fusion proteins further comprising a DNA targeting domain. In some embodiments, both fusion proteins comprise a DNA targeting domain. In some embodiments, both fusion proteins comprise a DNA targeting domain, and the DNA targeting domain targets a DNA sequence adjacent to the DNA sequence that serves as the insertion site targeted by the transposase. In some embodiments, only one of the two fusion proteins in the tandem dimeric transposase contains a DNA targeting domain. The DNA targeting domain may be attached to the C-terminus or N-terminus of the fusion protein.

[0084] Accordingly, in some embodiments, this document provides a complex comprising: (a) a first fusion protein comprising a first transposase domain and a first DNA targeting domain; and (b) a second fusion protein comprising a first transposase domain and a second DNA targeting domain, wherein the first DNA targeting domain and the second DNA targeting domain are different; wherein the transposase domain of the first fusion protein and the transposase domain of the second fusion protein have opposite charges that allow the two fusion proteins to form a complex.

[0085] In some embodiments, this document provides a complex comprising: (a) a first fusion protein comprising, in N-terminal to C-terminal order: a first NLS, a first DNA targeting domain, and a first transposase domain comprising an N-terminal deletion; and (b) a second fusion protein comprising, in N-terminal to C-terminal order: a second NLS, a second DNA targeting domain, and a second transposase domain comprising an N-terminal deletion; wherein the transposase domains of the first and second fusion proteins have opposite charges that allow the two fusion proteins to form a complex. In some embodiments, the first and / or second transposase domains are SPB domains. In some embodiments, the first and / or second transposase domains are PBx transposase domains. In some embodiments, the first transposase domain and / or the second transposase domain comprises an N-terminal deletion of 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, or 103 amino acids. In some embodiments, the first transposase domain and the second transposase domain comprise the sequence of SEQ ID NO: 5 or 6. In some embodiments, the DNA targeting domain and / or the second DNA targeting domain comprise one, two, or three zinc finger motifs. In some embodiments, the first DNA targeting domain and / or the second DNA targeting domain comprise the sequence of SEQ ID NO: 28. In some embodiments, the first DNA targeting domain and / or the second DNA targeting domain comprise a TAL motif.

[0086] In some embodiments, this document provides a complex comprising: (a) a first fusion protein comprising, in N-terminal to C-terminal order: a first NLS and a first transposase domain comprising the sequence of SEQ ID NO: 2, 3, or 4; and (b) a second fusion protein comprising, in N-terminal to C-terminal order: a second NLS and a second transposase domain comprising the sequence of SEQ ID NO: 2, 3, or 4; wherein the first transposase domain and the second transposase domain comprise a DNA-targeting domain, and wherein the transposase domain of the first fusion protein and the transposase domain of the second fusion protein have opposite charges that allow the two fusion proteins to form a complex. In some embodiments, the first DNA-targeting domain and / or the second DNA-targeting domain comprises one, two, or three zinc finger motifs. In some embodiments, the first DNA-targeting domain and / or the second DNA-targeting domain comprises the sequence of SEQ ID NO: 28. In some embodiments, the first DNA targeting domain and / or the second DNA targeting domain comprises a TAL motif. In some embodiments, the first DNA targeting domain replaces one or more amino acids between the 83rd and 105th amino acids of the first transposase domain, wherein the numbering begins at residue 5 or 12 of SEQ ID NO: 2 or 3, respectively. In some embodiments, the first DNA targeting domain replaces residues 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, or 103 of the first transposase domain, wherein the numbering begins at residue 5 or 12 of SEQ ID NO: 2 or 3, respectively. In some embodiments, the first DNA targeting domain replaces one or more amino acids of the second transposase domain between the 83rd and 105th amino acids, including both of these amino acids, wherein the numbering begins at residue 5 or 12 of SEQ ID NO: 2 or 3, respectively.In some embodiments, the second DNA targeting domain replaces residues 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, or 103 of the second transposase domain, wherein residues numbered starting at 5 or 12 of SEQ ID NO: 2 or 3, respectively.

[0087] In another aspect, this document provides fusion proteins comprising a transposase domain that can form a specific heterodimer with another fusion protein comprising a transposase domain. Without wishing to be bound by theory, it is believed that two such fusion proteins assemble into a dimer structure held together through a combination of charge interactions, hydrogen bonds, π-cation pairs, and hydrophobic interactions. Therefore, each specific heterodimeric complex contains two transposase domains. In some embodiments, the two fusion proteins provided herein form a complex comprising: (a) a first fusion protein containing a transposase domain, and (b) a second fusion protein containing a transposase domain; wherein the transposase domain of the first fusion protein and the transposase domain of the second fusion protein have opposite charges that allow the two fusion proteins to form a complex. In non-limiting examples, the assembled complex may be a single dimer (2 protein molecules) or a dimer of dimers (4 protein molecules or a tetramer).

[0088] By introducing charged residues into the amino acids that facilitate dimerization with the second fusion protein, paired fusion proteins that can only associate with each other in a predetermined conformation to form tandem dimers can be designed. By introducing mutations that allow only one conformation of the tandem dimer, it becomes feasible to introduce a DNA-targeting domain into the fusion protein, thereby increasing the specificity of the transposase domain. This is illustrated for SPB in Figures 1A and 1B, and for PBx in Figures 1C and 1D: Introducing a DNA-targeting domain into a fusion protein that can dimerize in any conformation (including homodimerization), as described on page 30 / 62 of the specification 34 CN 121219409 A, results in the presence of four DNA-targeting domains in the tandem dimer transposase. However, only two of the DNA-targeting domains will interact with the DNA, and the remaining two may spatially impede the transposase-DNA interaction. Any suitable DNA-targeting domain described herein or known in the art may be used in the fusion protein described herein.

[0089] Mutations conferring positive or negative charges in the transposase domains can be determined by those skilled in the art.In cases where the fusion protein contains both a first and a second transposase domain, the crystal structure published by Chen et al. (Nat Commun 11, 3446 (2020)) can be used to identify residue pairs in closely adjacent transposase domains in tandem dimers formed from two such fusion proteins. Changing the charge of these residue pairs to produce positively and negatively charged transposase domains can be achieved using standard techniques such as site-directed mutagenesis.

[0090] For example, one or more of M185, R189, K190, D191, H193, M194, D198, D201, S203, L204, S205, V207, K500, R504, K575, K576, R583, N586, I587, D588, M589, C593 and / or F594 may be mutated in the SPB transposase domain (e.g., the SPB listed in SEQ ID NO: 1 or 2, wherein the numbering begins at residue 12 of SEQ ID NO: 1 and residue 5 of SEQ ID NO: 2) to generate an SPB- transposase domain or an SPB+ transposase domain. Similarly, one or more of M185, R189, K190, D191, H193, M194, D198, D201, S203, L204, S205, V207, K500, R504, K575, K576, R583, N586, I587, D588, M589, C593 and / or F594 may be mutated in the PBx transposase domain (e.g., the PBx transposase domain of SEQ ID NO: 3, wherein the numbering begins at the 12th residue of SEQ ID NO: 3, or the PBx transposase domain of SEQ ID NO: 4) to generate a PBx- (minus) transposase domain or a PBx+ (addition) transposase domain.

[0091] In some embodiments, the fusion protein described herein may comprise: (i) an SPB+ transposase domain, or (ii) an SPB- transposase domain.

[0092] To achieve the formation of a specific heterodimer, paired mutations may be introduced into the fusion protein or transposase domain to generate positively and negatively charged fusion protein or transposase domains, which can then interact to form a heterodimer. In some embodiments, the mutated residue pairs are the residue pairs listed in Table 2.For example, one or more of the mutations listed in the column labeled “protein 1” may be introduced into the first SPB or PBx domain, and one or more corresponding mutations listed in the column labeled “protein 2” may be introduced into the second SPB or PBx domain. In some embodiments, the members of the residue pair are mutated to have opposite charges.

[0093] Table 2: Exemplary residue pairs; numbered starting with residue 5 of SEQ ID NO: 2 or residue 12 of SEQ ID NO: 1 or 3. Specification 31 / 62 pages 35 CN 121219409 A

[0094]

[0095] To introduce a positive charge, an amino acid with an uncharged side chain (such as methionine) or an amino acid with a negatively charged side chain (such as aspartic acid) may be changed to a positively charged amino acid (such as lysine or arginine). To introduce a negative charge, amino acids with positively charged side chains (such as arginine or lysine) or with hydrophobic side chains (such as leucine) can be replaced with negatively charged amino acids (such as aspartic acid or glutamic acid).

[0096] In some embodiments, one or more of the following mutations are introduced into the SPB transposase domain of the fusion protein provided herein (e.g., the SPB listed in SEQ ID NO: 1 or 2, wherein the numbering begins at residue 12 of SEQ ID NO: 1 and residue 5 of SEQ ID NO: 2) to generate an SPB+ fusion protein: M185R, M185K, D197K, D197R, D198K, D198R, D201K, and D201R. In some embodiments, the SPB+ transposase domain comprises the M185R mutation and the D198K mutation. In some embodiments, the SPB+ transposase domain contains the M185R mutation and the D201R mutation. In some embodiments, the SPB+ transposase domain contains the D197K mutation and the D201R mutation. In some embodiments, the SPB+ transposase domain contains the D198K mutation and the D201R mutation. In some embodiments, the SPB+ transposase domain contains the M185R mutation, the D198K mutation, and the D201R mutation.

[0097] In some embodiments, one or more of the following mutations are introduced into the transposase domain of the PBx fusion protein described herein (e.g., the PBx transposase domain of SEQ ID NO: 3, wherein the numbering begins at the 12th residue of SEQ ID NO: 3; or the PBx transposase domain of SEQ ID NO: 4) to generate the PBx+ fusion protein: M185R, M185K, D197K, D197R, D198K, D198R, D201K, and D201R. In some embodiments, the PBx+ transposase domain comprises the M185R mutation and the D198K mutation. In some embodiments, the PBx+ transposase domain comprises the M185R mutation and the D201R mutation. In some embodiments, the PBx+ transposase domain contains the D197K mutation and the D201R mutation. In some embodiments, the SPB+ transposase domain contains the D198K mutation and the D201R mutation. In some embodiments, the PBx+ transposase domain contains the M185R mutation, the D198K mutation, and the D201R mutation.

[0098] In some embodiments, one or more of the following mutations are introduced into the SPB transposase domain of the fusion protein provided herein (e.g., the SPB listed in SEQ ID NO: 1 or 2, wherein the numbering begins at residue 12 of SEQ ID NO: 1 and residue 5 of SEQ ID NO: 2) to generate the SPB- fusion protein: L204D, L204E, K500D, K500E, R504E, and R504D. In some embodiments, the SPB-transposase domain contains the L204E mutation and the K500D mutation. In some embodiments, the SPB-transposase domain contains the L204E mutation and the R504D mutation. In some embodiments, the SPB-transposase domain contains the K500 mutation and the R504D mutation. In some embodiments, the SPB-transposase domain contains the L204E mutation, the K500D mutation, and the R504D mutation.

[0099] In some embodiments, one or more of the following mutations are introduced into the PBx transposase of the fusion protein provided herein (e.g., the PBx transposase domain of SEQ ID NO: 3, wherein the numbering begins at the 12th residue of SEQ ID NO: 3, or the PBx transposase domain of SEQ ID NO: 4) to generate the PBx-fusion protein: L204D, L204E, K500D, K500E, R504E, and R504D. In some embodiments, the PBx-transposase domain comprises the L204E mutation and the K500D mutation. In some embodiments, the PBx-transposase domain comprises the L204E mutation and the R504D mutation. In some embodiments, the PBx-transposase domain comprises the K500 mutation and the R504D mutation. In some embodiments, the PBx-transposase domain comprises the L204E mutation, the K500D mutation, and the R504D mutation.

[0100] Exemplary sequences of the SPB+ transposase domain are listed in SEQ ID NO: 42 to 54. Exemplary sequences of the SPB-transposase domain are listed in SEQ ID NO: 55 to 64. In some embodiments, the transposase domain provided herein comprises the amino acid sequence listed in any one of SEQ ID NO: 42 to 64. In some embodiments, the transposase domain provided herein comprises the amino acid sequence listed in any one of SEQ ID NO: 42 to 64, further comprising one or more conserved amino acid sequences.

[0101] In some embodiments, the fusion protein described herein comprises a transposase domain comprising the amino acid sequence listed in any one of SEQ ID NO: 42 to 54. In some embodiments, the transposase domain comprises the amino acid sequence listed in any one of SEQ ID NO: 42 to 54, and further comprises one or more conserved amino acid sequences.

[0102] In some embodiments, the fusion protein described herein comprises a transposase domain comprising the amino acid sequence listed in any one of SEQ ID NO: 55 to 64. In some embodiments, the transposase domain comprises the amino acid sequence listed in any one of SEQ ID NO: 55 to 64, and further comprises one or more conserved amino acid sequences.

[0103] In some embodiments, this document provides a complex comprising: (a) a first fusion protein comprising a transposase domain comprising the amino acid sequence listed in any one of SEQ ID NO: 42 to 54; and (b) a second fusion protein comprising a transposase domain comprising the amino acid sequence listed in any one of SEQ ID NO: 55 to 64.

[0104] The SPB+, SPB-, PBx+, and PBx- fusion proteins and the transposase domain may further comprise an N-terminal deletion of the transposase domain described herein. Thus, in some embodiments, this document provides an SPB+ fusion protein comprising a transposase domain comprising an N-terminal deletion of about 20 amino acids, about 40 amino acids, about 60 amino acids, about 80 amino acids, about 100 amino acids, or about 115 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 83 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 90 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 84 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 85 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 86 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 87 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 88 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 89 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 90 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 91 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 92 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 93 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 94 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 95 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 96 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 97 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 98 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 99 amino acids.In some embodiments, the transposase domain comprises an N-terminal deletion of 100 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 101 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 102 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 103 amino acids.

[0105] In some embodiments, this document provides an SPB- fusion protein comprising a transposase domain comprising an N-terminal deletion of about 20 amino acids, about 40 amino acids, about 60 amino acids, about 80 amino acids, about 81 amino acids, about 82 amino acids, about 83 amino acids, about 84 amino acids, about 85 amino acids, about 86 amino acids, about 87 amino acids, about 88 amino acids, about 89 amino acids, about 90 amino acids, about 91 amino acids, about 92 amino acids, about 93 amino acids, about 94 amino acids, about 95 amino acids, about 96 amino acids, about 97 amino acids, about 98 amino acids, about 99 amino acids, about 100 amino acids, about 101 amino acids, about 102 amino acids, about 103 amino acids, or about 115 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 83 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 84 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 85 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 86 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 87 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 88 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 89 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 90 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 91 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 92 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 93 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 94 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 95 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 96 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 97 amino acids.In some embodiments, the transposase domain comprises an N-terminal deletion of 98 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 99 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 100 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 101 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 102 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 103 amino acids.

[0106] In some embodiments, this document provides a PBx+ fusion protein comprising a transposase domain comprising an N-terminal deletion of about 20 amino acids, about 40 amino acids, about 60 amino acids, about 80 amino acids, about 100 amino acids, or about 115 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 83 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 84 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 85 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 86 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 87 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 88 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 89 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 90 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 91 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 92 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 93 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 94 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 95 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 96 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 97 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 98 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 99 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 100 amino acids.In some embodiments, the transposase domain comprises an N-terminal deletion of 101 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 102 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 103 amino acids.

[0107] In some embodiments, this document provides a PBx- fusion protein comprising a transposase domain comprising an N-terminal deletion of about 20 amino acids, about 40 amino acids, about 60 amino acids, about 80 amino acids, about 81 amino acids, about 82 amino acids, about 83 amino acids, about 84 amino acids, about 85 amino acids, about 86 amino acids, about 87 amino acids, about 88 amino acids, about 89 amino acids, about 90 amino acids, about 91 amino acids, about 92 amino acids, about 93 amino acids, about 94 amino acids, about 95 amino acids, about 96 amino acids, about 97 amino acids, about 98 amino acids, about 99 amino acids, about 100 amino acids, about 101 amino acids, about 102 amino acids, about 103 amino acids, or about 115 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 83 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 84 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 85 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 86 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 87 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 88 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 89 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 90 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 91 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 92 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 93 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 94 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 95 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 96 amino acids. In some embodiments, the transposase domain contains an N-terminal deletion of 97 amino acids.In some embodiments, the transposase domain comprises an N-terminal deletion of 98 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 99 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 100 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 101 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 102 amino acids. In some embodiments, the transposase domain comprises an N-terminal deletion of 103 amino acids.

[0108] Integration cassette

[0109] An integration cassette is also provided herein for site-specific transposition of DNA molecules into the cellular genome. In some embodiments, the integration cassette comprises an integration site of the sequence TTAA. In some embodiments, the integration cassette for site-specific transposition of nucleic acid into the cellular genome comprises a nucleic acid containing or consisting of: a central transposon ITR integration site CTTAAA sequence flanked by an upstream TAL array target sequence and a downstream TAL array target sequence, wherein each of the upstream and downstream TAL array target sequences is separated from the CTTAAA sequence by 12 or 13 base pairs. In some embodiments, each of the at least one upstream TAL array target sequence and the downstream TAL array target sequence is identical. In some embodiments, each of the at least one upstream TAL array target sequence and the downstream TAL array target sequence is distinct. In some embodiments, each of the at least one upstream TAL array target site and the downstream TAL array target site targets a 10 bp sequence of an LPA repeat sequence element.

[0110] A method is also provided for site-specific transposition of a DNA molecule into the genome of a cell containing a stably integrated integration cassette, comprising introducing into the cell: a) a nucleic acid encoding a fusion protein comprising a DNA-binding domain and a transposase; wherein the fusion protein is expressed in the cell; and b) a DNA molecule containing a transposon; wherein the expressed fusion protein integrates the transposon into the CTTAAA sequence of the stably integrated integration cassette via site-specific transposition.

[0111] A method for generating engineered cells via site-specific transposition is also provided, comprising: introducing into a cell containing a stably integrated integration cassette: a) a nucleic acid encoding a fusion protein comprising a DNA-binding domain and a transposase; wherein the fusion protein is expressed in the cell; and b) a DNA molecule containing a transposon; wherein the expressed fusion protein integrates the transposon into the CTTAAA sequence of the stably integrated integration cassette via site-specific transposition, thereby generating engineered cells.

[0112] Nucleic Acids

[0113] Polynucleotides comprising nucleic acid sequences encoding the fusion proteins described herein are also provided herein. In some embodiments, the polynucleotides are isolated.

[0114] 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.

[0115] Methods for constructing nucleic acids encoding a transposase domain containing an N-terminal deletion as described herein are well known in the art or described herein, for example, PCR-based mutagenesis.

[0116] The fusions of the present invention can be generated using any suitable method known in the art or described herein.

[0117] The isolated polynucleotides (such as RNA, cDNA, genomic DNA, or any combination thereof) of the present disclosure 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 selectively hybridize with the polynucleotides of the present disclosure under stringent conditions will be used to identify desired sequences in cDNA or genomic DNA libraries.

[0118] Methods for amplifying RNA or DNA are well known in the art and can be used according to the present 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 entire contents of which are incorporated herein by reference. (See, for example, Ausubel, ibid.; or Sambrook, ibid.)

[0119] For example, polymerase chain reaction (PCR) technology can be used to directly amplify the polynucleotide sequences and related genes disclosed herein 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 samples, for nucleic acid sequencing, or for other purposes. Examples of techniques sufficient to guide technicians 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.

[0120] 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.

[0121] Expression Vectors and Host Cells

[0122] 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.

[0123] The polynucleotide may optionally be ligated to a vector containing a selective marker for replication in a host. Generally, the plasmid vector is introduced into a precipitate (such as a calcium phosphate precipitate) or into a complex 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 a host cell.

[0124] The DNA insert should be operatively ligable to a suitable promoter. In some embodiments, the promoter is the EF-1α promoter. The expression construct will further contain sites for transcription initiation and termination, as well as ribosome binding sites in the transcribed region for translation. The coding portion of the mature transcript expressed by the construct will preferably include a translation initiation codon (e.g., ATG) 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 expression in mammalian or eukaryotic cells.

[0125] The expression carrier may include at least one optional marker.Such biomarkers include, but are not limited to, ampicillin, zeocin (Shbla gene), puromycin (pac gene), hygromycin B (hygB gene), G418 / geneticin (neo gene), DHFR (encoding dihydrofolate reductase and conferring resistance to methotrexate), mycophenolic acid or glutamine synthase (GS, US patents 5,122,464; 5,770,359; 5,827,739), blastomycin (bsd gene), eukaryotic cell culture resistance genes, and ampicillin, zeocin (Shbla gene), puromycin (pac gene), hygromycin B (hygB gene), G418 / geneticin (neo gene), kanamycin, spectinomycin, streptomycin, carbenicillin, bleomycin, erythromycin, and polymyxin B. Or for use in the culture of tetracycline resistance genes in Escherichia coli and other bacteria or prokaryotes (the above patents are incorporated herein by reference in their entirety). 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 to 4 and 16 to 18; Ausubel, ibid., Chapters 1, 9, 13, 15, and 16.

[0126] The expression vector may include at least one selective cell surface marker for the isolation of 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. Preferably, 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. Such cell surface markers include, for example, but not limited to, “designated cluster” or “taxonomic determinant 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 et al. Blood. 21 Aug 2014; 124(8):1277-87).

[0127] The expression vector may include 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.

[0128] Those skilled in the art are familiar with many expression systems that can be used to express nucleic acids encoding the proteins of this disclosure. Alternatively, the nucleic acids of this disclosure may 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, which are incorporated herein by reference in their entirety.

[0129] Examples of cell cultures that can be used to produce protein scaffolds, specific portions thereof, or variants are bacterial, yeast, and mammalian cells 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-derived cells, such as myeloma and lymphoma cells. Particularly preferred host cells are P3X63Ag8.653 cells (ATCC accession number CRL-1580) and SP2 / 0-Ag14 cells (ATCC accession number CRL-1851). In one preferred aspect, the recombinant cells are P3X63Ab8.653 or SP2 / 0-Ag14 cells.

[0130] The expression vectors of these cells may include one or more of the following expression control sequences, such as, but not limited to, origin of replication; promoters (e.g., late or early SV40 promoters, CMV promoters (US Patent Nos. 5,168,062; 5,385,839), HSV tk promoters, pgk (glycerol phosphate kinase) promoters, EF-1α promoters (US Patent No. 5,266,491), at least one human promoter); enhancers and / or processing information sites, such as ribosome binding sites, RNA splicing sites, polyadenylation sites (e.g., SV40 large T Ag polyA addition sites), 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.

[0131] When using eukaryotic host cells, polyadenylated or transcription terminator sequences are typically incorporated into the vector. An example of a terminator sequence is a polyadenylated sequence from the bovine growth hormone gene. In some embodiments, the polyA sequence is an SV40 polyA sequence.

[0132] 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.

[0133] The plasmid constructs described herein can be used to deliver nucleic acids encoding the transposase domains or fusion proteins described herein to cells.

[0134] mRNA constructs can also be used to deliver the transposase domains and fusion proteins described herein to cells. Therefore, in one embodiment, an mRNA sequence encoding the transposase domains or fusion proteins described herein is provided herein. Such mRNA sequences can be delivered to cells using nanoparticles (e.g., lipid nanoparticles). Examples of lipid nanoparticles are described, for example, in International Patent Applications PCT / US2021 / 055876, PCT / US2022 / 017570, U.S. Provisional Applications 63 / 397,268, 63 / 301,855, and 63 / 348,614, as examples of lipid nanoparticles that can be used to deliver mRNA constructs encoding the fusion protein or transposase domains described herein, each of which is incorporated herein by reference in its entirety. mRNA constructs can also be delivered to cells via electroporation or nuclear transfection.mRNA may be capped or otherwise modified.

[0135] Cells and Modified Cells

[0136] The transposases and fusion proteins described herein may 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 transposon may be a piggyBac™ (PB) transposon, a piggyBac-like (PBL) transposon, or a Super piggyBac™ (SPB) transposon. Non-limiting examples of PB transposons are disclosed 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 transposons that may be used in conjunction with the transposases and fusion proteins described herein. Transposons may contain nucleic acids encoding therapeutic proteins or therapeutic agents. Examples of therapeutic proteins include those disclosed in PCT Publications WO 2019 / 173636 and WO 2020 / 051374, each of which is incorporated herein by reference in its entirety as an example of therapeutic proteins encoded by transposons that can be used in conjunction with the transposases and fusion proteins described herein.

[0137] Therefore, this document provides modified cells comprising one or more transposons and one or more tandem dimeric transposases or fusion proteins described herein. The cells and modified cells of this disclosure may be mammalian cells. Optionally, the cells and modified cells are human cells.

[0138] Cells modified with the site-specific transposase fusion proteins described herein may be germ cells or somatic cells. The cells disclosed herein, and the modified cells, can be immune cells, such as lymphoprogenitor cells, natural killer (NK) cells, T lymphocytes (T cells), memory T stem cells (TSCM cells), central memory T cells (TCM), stem cell-like T cells, B lymphocytes (B cells), antigen-presenting cells (APCs), cytokine-induced killer (CIK) cells, myeloid progenitor cells, neutrophils, basophils, eosinophils, monocytes, macrophages, platelets, erythrocytes (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 T cells, hematopoietic stem cells, natural killer cells, macrophages, dendritic cells, monocytes, megakaryocytes, or osteoclasts.Modified cells can be modified when they are in a dormant, activated, quiescent, interphase, prophase, metaphase, anaphase, or telophase state. Modified cells can be fresh, cryopreserved, bulk, subpopulation-classified, derived from whole blood, from leukocyte removal, or from immortalized cell lines. Detailed descriptions of cells isolated from leukocyte removal products or blood are published in PCT Publications WO 2019 / 173636 and WO 2020 / 051374, each of which is incorporated herein by reference in its entirety.

[0139] The methods of this disclosure can modify and / or generate a population of modified T cells, wherein at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% or any percentage of the modified T cells in the population express one or more cell surface markers of stem memory T cells (TSCM) or TSCM-like cells; and wherein the one or more cell surface markers include CD45RA and CD62L. The cell surface markers may include one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2Rβ. Cell surface markers may include one or more of CD45RA, CD95, IL-2Rβ, CCR7, and CD62L.

[0140] 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 a sequence encoding CAR; and (d) expanding and / or selecting at least one cell from the modified cell population expressing CAR on a 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, each of which is incorporated herein by reference in its entirety.

[0141] This disclosure 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 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.

[0142] This disclosure further provides compositions comprising modified, expanded, and selected cell populations comprising the methods described herein.

[0143] The modified cells of this disclosure (e.g., CAR T cells) may be further modified to enhance their therapeutic potential. Alternatively or additionally, 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).

[0144] The modified cells of this disclosure (e.g., CAR T cells) may be further modified to silence or reduce the expression of: (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 the efficacy of therapies; (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 advantage factors. Non-limiting examples of genes that can be modified to silence or reduce or inhibit their function include, but are not limited to, exemplary inhibitory checkpoint signals, intracellular proteins, transcription factors, cell death or apoptosis receptors, metabolic sensing proteins, proteins that confer sensitivity to cancer therapies, and growth advantage factors disclosed in PCT Publication No. WO 2019 / 173636.

[0145] 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.Examples of empty receptors / proteins, decoy receptors / proteins, or dominant-negative intracellular receptors / proteins include, but are not limited to: signal transduction components downstream of 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 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.

[0146] Genome modification may include introducing nucleic acid sequences, transgenes, and / or genome editing constructs into cells, either ex vivo, in vivo, in vitro, or in situ, to stably integrate nucleic acid sequences, transiently integrate nucleic acid sequences, site-specific integration to produce nucleic acid sequences, or biased integration to produce nucleic acid sequences. The nucleic acid sequences may be transgenes.

[0147] Stable chromosomal integration may be random integration, site-specific integration, or biased integration. Without being bound by theory, it is believed that adding a DNA-binding domain to the tandem dimeric transposase described herein improves the site specificity of the transposase.

[0148] Site-specific integration can occur at safe harbor sites. Genomic 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 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.

[0149] 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, PDI, any gene encoding an immunosuppressive protein, and genes encoding proteins involved in allo-rejection.

[0150] Site-specific transgene integration can occur at sites that cause enhanced target gene expression. Enhancement 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.

[0151] 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 insertion can be epichromosomal or cytoplasmic. In one respect, the transient nonchromosomal insertion of the transgene does not integrate into the chromosome, and the modified genetic material does not replicate during cell division.

[0152] Site-specific transgene integration sites can be modified binding sites targeting DNA-targeting domains in transposon domains, fusion proteins, or tandem dimers as described herein. For example, the TTAA target DNA integration site for SPB can be modified to insert a side-linked DNA binding site targeting a DNA-targeting domain comprising three zinc finger motifs (e.g., a DNA-targeting domain comprising or consisting of the sequence of SEQ ID NO: 28 or 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, it is believed that a DNA targeting domain containing three zinc finger motifs binds to the DNA sequence GCGTGGGCG. Therefore, it is believed that the introduction of two copies of the sequence GCGTGGGCG, which is laterally attached to the TTAA target integration site for SPB, can improve site-specific integration of the SPB transposase domain containing the DNA targeting domain containing three zinc finger motifs. In some embodiments, the two copies of the sequence GCGTGGGCG are in reverse (5') and complementary (3') orientations.

[0153] In some embodiments, this document provides a polynucleotide comprising, in 5' to 3' order, a reverse complementary sequence to the target site sequence for the DNA targeting domain, a first spacer region, a TTAA target integration site for SPB, a second spacer region, and a target site sequence for the DNA targeting domain. In some embodiments, the first spacer region and the second spacer region have the same length. In some embodiments, the length of the first spacer region and / or the second spacer region is 3 bp. In some embodiments, the length of the first spacer region and / or the second spacer region is 4 bp. In some embodiments, the length of the first and / or second spacing region is 5 bp. In some embodiments, the length of the first and / or second spacing region is 6 bp. In some embodiments, the length of the first and / or second spacing region is 7 bp. In some embodiments, the length of the first and / or second spacing region is 8 bp. In some embodiments, the length of the first and / or second spacing region is 9 bp. In some embodiments, the length of the first and / or second spacing region is 10 bp.

[0154] Modified target sites can be introduced into cells or cell lines to facilitate targeted genome engineering. For example, cell lines engineered to contain modified target sites for SPB or PBx provided herein can be transfected with said SPB or PBx 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 modified target sequence is introduced into a highly expressed genomic region. In some embodiments, the cell is an in vitro cell, for example, a cell in a cell culture.

[0155] For DNA-binding domains containing TAL, the target site is determined by the sequence of TAL. Those skilled in the art will be able to modify the TAL sequence to achieve desired target specificity.

[0156] Genome modification can be for the unstable chromosomal integration of transgenes. The integrated transgene can be silenced, removed, excised, or further modified.

[0157] In some embodiments, the transposase domains, fusion proteins, and tandem dimer complexes 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 transposase domains, fusion proteins, and tandem dimer complexes provided herein may have target genome integration activity comparable to their wild-type counterparts, but with reduced off-target genome integration activity compared to their wild-type counterparts.

[0158] In some embodiments, the transposase domain and DNA targeting domain provided herein have an increased ratio of on-target to off-target activity compared to unmodified SPB transposase, by a factor of 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.

[0159] In some embodiments, the transposase domain comprising a DNA-targeting domain inserted into the N-terminal region of the transposase domain provided herein has an increased ratio of on-target to off-target activity compared to the wild-type transposase domain, by a factor of 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.

[0160] In some embodiments, the modified cells are used therapeutically in adoptive cell therapy.

[0161] For the administration of a “universally” safe adoptive cell composition to any patient (not just those from whom it is derived), there is a need to significantly reduce or eliminate allogeneic reactivity. To this end, the cells of this disclosure (e.g., allogeneic cells) may be modified to disrupt the expression or function of the T cell receptor (TCR) and / or a major histocompatibility complex (MHC). TCR mediates graft-versus-host (GvH) responses, while MHC mediates host-versus-graft (HvG) responses. In a preferred aspect, any expression and / or function of the TCR is eliminated to prevent T cell-mediated GvH that could lead to the death of the subject. Therefore, in a preferred aspect, this disclosure provides a purely TCR-negative allogeneic T cell composition (e.g., each cell in the composition is expressed at such a low level that it is undetectable or absent).

[0162] Reducing or eliminating the expression and / or function of MHC class I (MHC-I, specifically HLA-A, HLA-B, and HLA-C) prevents HvG and thus improves cell engraftment in the subject. Improved engraftment results in longer cell persistence and thus a larger therapeutic window for the subject. Specifically, reducing or eliminating the expression and / or function of the structural element β-2-microglobulin (B2M) of MHC-I. Non-limiting examples of guide RNAs (gRNAs) for targeting and deleting MHC activators are disclosed in PCT application PCT / US2019 / 049816.

[0163] Detailed descriptions of non-naturally occurring chimeric stimulatory receptors, genetic modifications encoding endogenous sequences of TCR-α (TCR-α), TCR-β (TCR-β), and / or β-2-microglobulin (β2M), and non-naturally occurring polypeptides comprising HLA class I histocompatibility antigen 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.

[0164] Under normal conditions, complete T cell activation depends on the binding of TCRs to a second signal mediated by one or more co-stimulatory receptors that enhance the immune response (e.g., CD28, CD2, 4-1BBL). However, when TCRs are absent, T cell expansion is severely reduced when stimulated with standard activating / stimulating 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 activating component, wherein the activating 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, wherein the at least one signal transduction domain is isolated from or derived from a second protein; wherein the first protein and the second protein are not identical.

[0165] The activating component may comprise a portion of one or more of the following: a T cell receptor (TCR) component, 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 to which an agonist of the activating component binds. The activating component may comprise an extracellular domain of CD2 to which an agonist binds, or a portion thereof.

[0166] The signal transduction domain may include one or more of the following: components of a human signal transduction domain, a T cell receptor (TCR), components of a TCR complex, components of a TCR co-receptor, components of a TCR co-stimulatory protein, components of a TCR repressor protein, cytokine receptors, and chemokine receptors. The signal transduction domain may include a CD3 protein or a portion thereof. The CD3 protein may include a CD3ζ protein or a portion thereof.

[0167] The intracellular domain may further include a cytoplasmic domain. The cytoplasmic domain may be isolated from or derived from a third protein. The first and third proteins may be the same. The extracellular domain may further include a signal peptide. The signal peptide may be derived from a fourth protein. The first and fourth proteins may be the same. The transmembrane domain may be isolated from or derived from a fifth protein. The first and fifth proteins may be the same.

[0168] This disclosure also provides a non-naturally occurring chimeric stimulatory receptor (CSR) wherein the extracellular domain contains modifications. Modifications may include mutations or truncations of the amino acid sequence of the activating component or the first protein when compared to a wild-type sequence of the activating component or the first protein. Mutations or truncations of the amino acid sequence of the activating component may include mutations or truncations of the CD2 extracellular domain to which the agonist binds, or a portion thereof. Mutations or truncations of the CD2 extracellular domain may reduce or eliminate binding to naturally occurring CD58.

[0169] This disclosure provides a nucleic acid sequence encoding any CSR disclosed herein. This disclosure also provides a transposon or vector comprising a nucleic acid sequence encoding any CSR disclosed herein.

[0170] 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.

[0171] This disclosure provides a composition comprising any CSR disclosed herein. This disclosure provides a composition comprising 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 a composition comprising the modified cells disclosed herein or a composition comprising a plurality of modified cells disclosed herein.

[0172] This disclosure also provides a method for site-specific gene integration. The transposase domains and 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 alteration of the host genomic DNA sequence. In some embodiments, the target site is a repetitive element, such as an LPA sequence. One, two, or more target sites may be present within a repetitive element. In some embodiments, the target site is located within an intron (e.g., an intron of the LPA gene).

[0173] Site-specific integration can be used in vitro or in vivo. An example of in vivo application is gene therapy, which involves delivering transgenes into genomic DNA in cells.

[0174] Formulations, Dosages, and Methods of Administration

[0175] This disclosure provides formulations, dosages, and methods of administration for the compositions and cells described herein.In one aspect, this document provides a pharmaceutical composition comprising the tandem dimer transposase or fusion protein described herein and a pharmaceutical carrier. In another aspect, this document provides a pharmaceutical composition comprising the modified cells described herein and a pharmaceutical carrier.

[0176] 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, such as, but not limited to, Gennaro (ed.), 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 on pages 44 / 62 of this specification, CN 121219409 A, can be conventionally selected.

[0177] 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 as 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 may 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.

[0178] Non-limiting examples of suitable carbohydrate excipients include monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbitol, etc.; disaccharides such as lactose, sucrose, trehalose, cellobiose, etc.; polysaccharides such as raffinose, melitriose, maltodextrin, dextran, starch, etc.; and sugar alcohols such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucol), inositol, etc. Preferably, the carbohydrate excipient is mannitol, trehalose, and / or raffinose.

[0179] 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 citrates.

[0180] Additionally, the disclosed composition may include polymeric excipients / additives, such as polyvinylpyrrolidone, sucrose (polymeric sugar), glucose dextrorates (e.g., cyclodextrins, such as 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).

[0181] 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, intra-nasal, 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, extra-gastric, rectal, sublingual, subcutaneous, transdermal, or vaginal administration. In a preferred embodiment, intravenous administration (e.g., by intravenous infusion) is used to administer the compositions comprising modified cells described herein.

[0182] The compositions disclosed herein can be prepared for parenteral (subcutaneous, intramuscular, or intravenous) or any other administration, particularly in the form of liquid solutions or suspensions. For parenteral administration, the compositions disclosed herein can be formulated in combination with or alone with a pharmaceutical parenteral carrier as solutions, suspensions, emulsions, granules, powders, or lyophilized powders. 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 can be prepared using appropriate emulsifiers or humectants and suspending agents according to known methods. Injectable or infusion formulations can be non-toxic, non-oral diluents, such as aqueous solutions, sterile injectable solutions, or suspensions in solvents. Water, Ringer's solution, isotonic saline, etc., can be used as suitable carriers or solvents; sterile non-volatile oils can 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 US Patent No. 45 / 62, page 49, CN 121219409 A 5,851,198, and laser perforation devices as described in US Patent No. 5,839,446.

[0183] It is desirable to deliver the disclosed compound to the subject over a longer period of time (e.g., from one week to one year by a single administration). Various sustained-release, reservoir, or implantable dosage forms are available. For example, the dosage form may contain pharmaceutically acceptable non-toxic salts of compounds with low solubility in bodily fluids, such as (a) acid addition salts of polybasic acids, 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) salts of polyvalent metal cations, such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, etc., or salts of organic cations, such as N,N'-dibenzyl-ethylenediamine or ethylenediamine; or (c) combinations of (a) and (b), such as zinc tannate salts. Additionally, the disclosed compounds or preferably relatively insoluble salts (such as those described above) may be formulated in gels, for example, aluminum monostearate gel containing, for example, sesame oil, suitable for injection. 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 JR Robinson, Marcel Dekker, Inc., NY, 1978).

[0184] Treatment Methods

[0185] In another aspect, methods for treating a disease or ailment of a subject are provided herein, comprising: administering to the subject a composition comprising modified cells as described herein. The terms “subject” and “patient” are used interchangeably herein. In a preferred embodiment, the patient is a person.

[0186] 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.

[0187] In some embodiments, the disease or ailment treated according to the methods described herein is cancer. Non-limiting examples of cancer include leukemia, acute leukemia, acute lymphoblastic leukemia (ALL), acute lymphoblastic leukemia, B-cell, T-cell or FAB ALL, acute myeloid leukemia (AML), acute myeloid leukemia, chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, myelodysplastic syndrome (MDS), lymphoma, Hodgkin's disease, malignant lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, multiple myeloma, Kaposi's sarcoma, colorectal carcinoma, pancreatic cancer, nasopharyngeal carcinoma, malignant histiocytosis, paraneoplastic syndrome / malignant hypercalcemia, solid tumors, bladder cancer, breast cancer, and colorectal cancer. Cancer), endometrial cancer, head cancer, neck cancer, hereditary nonpolyposis cancer, Hodgkin's lymphoma, liver cancer, lung cancer, non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, testicular cancer, adenocarcinoma, sarcoma, malignant melanoma, hemangioma, metastatic disease, cancer-related bone resorption, cancer-related bone pain, etc.

[0188] In some embodiments, the disease or ailment treated according to the method described herein is a liver disease or ailment, urea cycle disorder, metabolic liver disorder, or hemophilia. In some aspects, a metabolic liver disorder may be ornithine transcarbamate (OTC) deficiency. In some aspects, a metabolic liver disorder may be methylmalonic acidemia (MMA).

[0189] In a non-limiting example, this disclosure provides a method for treating a subject with hemophilia. In some aspects, hemophilia A may be hemophilia A. In some aspects, hemophilia may be hemophilia B.

[0190] In a non-limiting example, this disclosure provides a method for treating phenylketonuria (PKU) in a subject.

[0191] In some embodiments, this disclosure provides a method for treating an autoimmune disease.In some embodiments, the autoimmune disease is autoimmune neutropenia, Guillain-Barré syndrome, epilepsy, autoimmune encephalitis, Isaacs syndrome, nevus syndrome, pemphigus vulgaris, pemphigus foliaceus, bullous pemphigoid, acquired epidermolysis bullosa, pemphigoid of pregnancy, mucosal pemphigoid, antiphospholipid syndrome, autoimmune anemia, myasthenia gravis, autoimmune Graves' disease, thyroid ophthalmopathy (TED), Goodpasture 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.

[0192] The dosage of the pharmaceutical composition 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.

[0193] In the aspect where the composition to be administered to a subject in need is a modified cell as disclosed herein, 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 and about 1x10¹³; cells between about 1x10¹³ and about 1x10¹⁴; and cells between about 1x10¹⁴ and about 1x10¹⁴. Cells between approximately 1 x 10¹⁵ 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 approximately 25 x 10⁶.

[0194] In other embodiments, the cell dosage may depend on the person's weight, for example, about 1x10³ to about 1x10⁴ cells per kg of body weight; about 1x10⁴ to about 1x10⁵ cells; about 1x10⁵ to about 1x10⁶ cells; about 1x10⁶ to about 1x10⁷ cells; about 1x10⁷ to about 1x10⁸ cells; about 1x10⁸ to about 1x10⁹ cells; about 1x10⁹ to about 1x10¹⁰ cells; about 1x10¹⁰ to about 1x10¹¹ cells; about 1x10¹¹ to about 1x10¹² cells; about 1x10¹² to about 1x10¹³ cells; about 1x10¹³ to about 1x10¹⁴ cells; about 1x10¹⁴ cells per kg of body weight. Cells between approximately 1 x 10¹⁵ 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.

[0195] 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 2020 / 051374.

[0196] The transposase 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. For example, the transgene may replace a gene that exhibits reduced, insufficient, and / or altered expression in the cell. In some embodiments, such reduced, insufficient, and / or altered expression can directly or indirectly lead to diseases or disorders, such as liver diseases or disorders, urea cycle disorders, metabolic liver disorders, or hemophilia. The fusion protein, transposase domain, and complex described herein (pages 47 / 62, CN 121219409 A) can be used to deliver therapeutic transgenes into cells and integrate transgenes into target sites. In some embodiments, the treatment method includes introducing the fusion protein provided in this disclosure and a transposon into cells, wherein the transposon comprises, in a 5' to 3' sequence: a 5' ITR, a transgene, and a 3' ITR.

[0197] In some embodiments, the therapeutic transgene is a gene expressed at a lower level and whose lower expression leads to disease or disorder.In some embodiments, a therapeutic transgene is a gene expressed in an altered pattern compared to a wild-type gene, and the altered expression results in a disease or disorder. Therefore, this document provides a method for treating a disease or disorder caused by or associated with altered gene expression, comprising administering the transposon and transposase described herein to a subject in need of it.

[0198] Therapeutic transgenes delivered to cells via the fusion protein, transposase domain, and complex described herein may encode a therapeutic polypeptide. In some embodiments, the therapeutic polypeptide is a factor VIII polypeptide, a factor IX polypeptide, a phenylalanine hydroxylase (PAH), an ornithine transcarbamate (OTC) polypeptide, or a methylmalonyl-CoA mutase (MUT1) polypeptide.

[0199] In a non-limiting example, the transposase domain and fusion protein provided herein may be used for the delivery of liver-directed gene therapy. In some aspects, liver-directed gene therapy may be used to treat ornithine transcarbamate (OTC) deficiency, and the therapeutic polypeptide encoded by the therapeutic transgene may comprise an ornithine transcarbamate (OTC) polypeptide. In some aspects, liver-directed gene therapy can be used to treat methylmalonic acidemia (MMA), and at least one therapeutic protein encoded by a therapeutic transgene may contain a methylmalonyl-CoA mutase (MUT1) polypeptide.

[0200] In some aspects, liver-directed gene therapy can be used to treat hemophilia A, and at least one therapeutic protein encoded by a therapeutic transgene may contain factor VIII. In some aspects, liver-directed gene therapy can be used to treat hemophilia B, and at least one therapeutic protein encoded by a therapeutic transgene may contain factor IX.

[0201] In some aspects, liver-directed gene therapy can be used to treat phenylketonuria (PKU), and at least one therapeutic protein encoded by a therapeutic transgene may contain phenylalanine hydroxylase (PAH).

[0202] Kits

[0203] In another aspect, a kit comprising a cell line engineered to contain, preferably in a highly expressed genomic region, the modified target site for SPB or PBx provided herein. The kit may further comprise a composition containing one or more SPB or PBx transposase domains or fusion proteins described herein. In some embodiments, the cell line is a T cell line.

[0204] Definitions

[0205] As used throughout this disclosure, unless the context clearly indicates 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 dosages and their equivalents known to those skilled in the art, etc.

[0206] The terms “about” or “approximately” refer to an acceptable error range for a particular value as determined by one of ordinary skill in the art, said acceptable error range 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 standard deviations. 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 a particular value is described in this application and claims, unless otherwise stated, the term “about” should be considered to mean within an acceptable error range for that particular value.

[0207] This disclosure provides isolated or substantially purified polynucleotide or protein compositions. "Isolated" or "pure" polynucleotides or proteins, or their biologically active portions, are substantially or substantially free of components that typically accompany or interact with polynucleotides or proteins as seen in their natural environment. Therefore, when produced by recombinant technology, the isolated or purified polynucleotides or proteins are substantially free of other cellular material or culture medium, or when chemically synthesized, are substantially free of chemical precursors or other chemical substances. Ideally, the "isolated" polynucleotides do not contain sequences (preferably protein-coding sequences) naturally flanking the polynucleotide in the genomic DNA of the organism from which the polynucleotide is derived (i.e., sequences located at the 5' and 3' ends of the polynucleotide). For example, in various respects, isolated polynucleotides may contain nucleotide sequences of less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb, which are naturally flanked by polynucleotides 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, the culture medium preferably represents less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or non-target proteins.

[0208] 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 a portion of an amino acid sequence, and therefore to a portion of a protein encoded therefrom.DNA sequence fragments containing coding sequences may encode protein fragments that retain the biological activity of native proteins 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.

[0209] Nucleic acids or proteins of this disclosure may be constructed using a modular approach that includes pre-assembling monomeric units and / or repeating units in a target vector, which may subsequently be assembled into a final target vector. Polypeptides of this disclosure may contain repeating monomers of this disclosure and may be constructed using a modular approach by pre-assembling repeating units in a target vector (which may subsequently be assembled into a final target vector). This disclosure provides polypeptides produced by this method and nucleic acid sequences encoding these polypeptides. This disclosure provides host organisms and cells containing nucleic acid sequences encoding polypeptides produced by this modular approach.

[0210] The term “comprising” is intended to mean that a composition and method comprises the listed elements, but does not exclude other elements. When used to define a composition and method, “consisting substantially of” should mean excluding other elements that are of any significance to the composition when used for the intended purpose. Thus, 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.

[0211] 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.

[0212] “Gene expression” means the conversion of information contained in a gene into a gene product. Gene products can be direct transcription products of genes (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozymes, shRNA, microRNA, structural RNA, or any other type of RNA) or proteins 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.

[0213] “Regulation” or “control” of gene expression refers to changes in gene activity. Regulation of expression may include, but is not limited to, gene activation and gene repression.Specification 49 / 62 pages 53 CN 121219409 A

[0214] "Operationally linked" or its equivalents (e.g., "operationally linked") means that two or more molecules are positioned relative to each other such that they can interact to affect the function of one or two molecules or a combination thereof. In the context of nucleic acids, a promoter can be operably linked to a nucleotide sequence encoding a transposon domain or fusion protein as described herein, thereby bringing the expression of the nucleotide sequence under the control of the promoter.

[0215] The present invention discloses non-covalently linked components and methods for preparing and using non-covalently linked components. Various components can 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 allows functional association to occur only or primarily when such association is required to achieve the desired activity. The bond can last long enough to achieve the desired effect.

[0216] This 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.

[0217] A “target site” or “target sequence” is a nucleic acid sequence that defines the portion of the nucleic acid to which the binding molecule will bind, provided that sufficient binding conditions are present.

[0218] The terms “nucleic acid” or “oligonucleotide” or “polynucleotide” refer to at least two nucleotides covalently linked together. The depiction of a single strand also defines the sequence of the complementary strand. Therefore, nucleic acids may also encompass the complementary strand of the depicted 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.

[0219] 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. The nucleic acids disclosed herein may include genomic DNA, cDNA, RNA, or hybrids thereof. The nucleic acids disclosed herein may contain combinations of deoxyribonucleotides and ribonucleotides. The nucleic acids disclosed herein may contain combinations of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. The nucleic acids disclosed herein can be synthesized to include non-natural amino acid modifications. The nucleic acids disclosed herein can be obtained by chemical synthesis or by recombinant methods.

[0220] The nucleic acids disclosed herein (whether their complete sequence or any part thereof) may be non-naturally occurring.The nucleic acids disclosed herein may contain one or more non-naturally occurring mutations, substitutions, deletions, or insertions, such that the entire nucleic acid sequence is non-natural. The nucleic acids disclosed herein 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 disclosed herein may contain non-naturally occurring modified, artificial, or synthetic nucleotides, such that the entire nucleic acid sequence is non-natural.

[0221] Due to the redundancy of the genetic code, multiple nucleotide sequences can encode any particular protein. All such nucleotide sequences are contemplated herein.

[0222] 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 can constitutively or differentially regulate the expression of genomic components at the cell, tissue, or organ where expression occurs, at the developmental stage where 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 the CMV IE promoter (page 50 / 62, CN 121219409 A).

[0223] As used throughout this disclosure, the term "vector" refers to a nucleic acid sequence containing an origin of replication. Vectors can be viral vectors, bacteriophages, bacterial artificial chromosomes, or yeast artificial chromosomes. Vectors can be DNA or RNA vectors. The vector may be a self-replicating extrachromosomal vector, and preferably a DNA plasmid. The vector may contain amino acids and a DNA sequence, an RNA sequence, or a combination of both DNA and RNA sequences.

[0224] 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 considerations of its hydrophobicity and charge. Amino acids with similar hydrophilicity indices can be substituted and still retain protein function. In one respect, 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. Considering the hydrophilicity of amino acids in the context of a peptide allows for the calculation of the peptide's maximum local average hydrophilicity, 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.

[0225] Substitution of amino acids with 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 of 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, biocompatible amino acid substitutions are understood to depend on the relative similarity of the amino acids and, in particular, the side chains of these amino acids, as revealed by properties such as hydrophobicity, hydrophilicity, charge, size, and other characteristics.

[0226] As used herein, “conservative” amino acid substitutions may be defined as listed in Tables 3, 4, and 5 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 their physical properties and their contribution to secondary and tertiary protein structures. A conserved substitution is the substitution of one amino acid with another amino acid having similar properties. Exemplary conserved substitutions are listed in Table 3.

[0227] Table 3: Conservative Substitutions I

[0228]

[0229] Alternatively, conserved amino acids may be as described in Lehninger (Biochemistry, 2nd edition; Worth Publishers, Inc. NY, NY (1975), pp. 71–77), as shown in Table 4.

[0230] Table 4: Conservative Substitution II Specification 51 / 62 pages 55 CN 121219409 A

[0231]

[0232] Alternatively, exemplary conservative substitutions are listed in Table 5.

[0233] Table 5: Conservative Substitution III Specification 52 / 62 pages 56 CN 121219409 A

[0234]

[0235] 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 naturally occurring, such that the entire amino acid sequence is not naturally occurring. The polypeptides and proteins of this disclosure may contain modified, artificial, or synthetic amino acids that are not naturally occurring, such that the entire amino acid sequence is not naturally occurring.

[0236] As used throughout this disclosure, the identity between two sequences may be determined by using a separate executable BLAST engine program (bl2seq) for blasting the two sequences, using default parameters, which is available from the National Center for Biotechnology Information (NCBI) ftp site (Tatusova and Madden, FEMS Microbiol Lett., 1999, 174, 247-250; which are incorporated herein by reference in their entirety). When the terms “identical” or “identity” are used in the context of two or more nucleic acid or polypeptide sequences, they mean that a specified percentage of residues 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 two sequences, comparing specified regions of the two sequences, determining the number of positions in the two sequences where the same 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 the two sequences have different lengths or the alignment produces one or more staggered ends and the specified comparison region includes only a single sequence, the residues of the single sequence are included in the denominator of the calculation but not in the numerator. 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.

[0237] 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 and the sequence of that SEQ ID NO have the same length. 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 and the sequence of the SEQ ID NO differ only due to conserved amino acid substitutions.

[0238] As used throughout this disclosure, the term “endogenous” refers to a nucleic acid or protein sequence naturally associated with a target gene or a host cell into which the target gene is introduced.

[0239] As used throughout this disclosure, the term “exogenous” means a nucleic acid or protein sequence that is 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 naturally occurring nucleic acid sequences located at non-natural genomic locations.

[0240] This disclosure provides methods 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 methods of this disclosure are 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.

[0241] Examples

[0242] The examples in this section are provided for illustration and are not intended to limit the invention.

[0243] Example 1: Construction of an N-terminal deletion of Super PiggyBac transposase

[0244] A plasmid containing the nucleotide sequence encoding the full-length wild-type Super PiggyBac transposase (SPB; SEQ ID NO: 2) or the nucleotide sequence encoding an integration-deficient variant of the Super PiggyBac transposase with amino acid substitutions at positions R372A, K375A, and D450N (PBx; SEQ ID NO: 3) was used as a template for PCR mutagenesis to generate N-terminal deletion transposase variants lacking 93 N-terminal amino acids (SPB Δ1-93 and PBx Δ1-93, respectively).

[0245] Briefly, the forward and reverse primers were designed to amplify portions of the SPB and PBx coding sequences corresponding to amino acids 94 to 594. The resulting DNA fragment encoding SPBΔ1-93 or PBxΔ1-93 was used in conjunction with the gBlock gene fragment purchased from page 54 / 62 of the instruction manual (CN 121219409 A) to construct a DNA-binding domain-transposase fusion protein via a prior art two-fragment Gibson Assembly.

[0246] Additional N-terminal deletion transposase variants (SPBΔ1-85 and PBxΔ1-85, respectively) were generated as described herein, lacking 85 amino acids from the N-terminus.

[0247] Example 2: Design and Construction of TAL Arrays Targeting LPA

[0248] This example illustrates the design and construction of a TAL array composition targeting the LPA gene, which can be used in methods for validating the target specificity of TAL arrays. The TAL arrays were constructed using the design criteria listed below.

[0249] The lipoprotein A (LPA) gene contains up to 50 copies of segmental replication elements, making it a potentially attractive target for optimizing the chance of site-specific transposition at the target sequence, thereby resulting in an increased number of transposable cells.

[0250] TAL array pairs containing the N-terminal domain that recognizes T were designed to target four specific 10 bp right and left pairs of sequences within the repetitive sequence elements of the LPA gene. For three of the targets, multiple TAL array pairs were designed using 12 bp or 13 bp spacers.

[0251] The left and right target sequences, as well as the upstream 5'T used to generate the TAL array targeting the LPA gene, are shown in Table 7.

[0252] Table 7: Exemplary TAL arrays targeting LPA

[0253]

[0254] Individual TAL modules containing 34-amino acid or 20-amino acid “half” repeat sequences were synthesized with BsmBI IIS type restriction sites lateralized. The entire module set contains 4 modules that can recognize A, C, G, T at each of the 10 bp positions within the target sequence (40 modules / 10 bp target). TAL array pairs targeting sequences in the LPA gene were designed, and each LPA TAL array was generated by using “Golden Gate Assembly” to select the corresponding modules and assemble them together in a frame. All coding sequences used were codon-optimized for human expression.

[0255] Seven left and right pair combinations were used to design and construct the LPA left TAL arrays LPAL1, LPAL2, LPAL3, LPAL4.1 and LPAL4.2 (SEQ ID Nos. 116, 118, 121, 124 and 125, respectively) and the LPA right TAL arrays LPAR1, LPAR2.1, LPAR2.2, LPA3.1, LPAR3.2 and LPAR4 (SEQ ID Nos. 117, 119, 120, 122, 123 and 126, respectively).

[0256] Example 3: Construction and analysis of a TAL array designed for site-specific transposition at the LPA gene – piggyBac transposase (ss-SPB) composition (TAL-PBx)

[0257] This example illustrates the construction of a TAL array – Super piggyBac transposase fusion protein composition (TAL-ssSPB) that can be used in a method for achieving site-specific transposition at a specific target site.

[0258] The TAL-PBx fusion construct was prepared as follows: an expression plasmid containing the following items from 5' to 3' direction was synthesized: CMV promoter, T7 promoter, Kozak sequence, 3x Flag tag (SEQ ID NO: 65), SV40 NLS (SEQ ID NO: 66), Δ152 TAL N-terminal domain (SEQ ID NO: 31), two BsmBI IIS type restriction enzyme sites, +63 TAL C-terminal domain (SEQ ID NO: 32), GGGS linker, Δ1-93 PBx (containing a 93-amino acid deletion at the N-terminus and mutations at R372A, K375A, and D450N in the Super piggyBac transposase codon sequence; SEQ ID NO: 6), and bGH polyadenylation sequence.

[0259] The left or right TAL array with BsmBI lateralization is cloned into the BsmBI site of the expression plasmid, and in-frame fusion of the TAL array and PBx coding sequence is induced via adapter sequence to generate a full-length TAL-PBx construct. All coding sequences used were codon-optimized for human expression using the GeneArt algorithm (Thermo Fisher).

[0260] The eleven TAL arrays with BsmBI ends designed and constructed in Example 2 were cloned into the BsmBI restriction sites of the above expression plasmids to generate eleven TAL-PBx constructs: LPAL1, LPAL2, LPAL3, LPAL4.1 and LPAL4.2 left TAL-PBx (SEQ ID No. 143, 145, 148, 151 and 152, respectively) and LPAR1, LPAR2.1, LPAR2.2, LPA3.1, LPAR3.2 and LPAR4 right TAL-PBx (SEQ ID No. 144, 146, 147, 149, 150 and 153, respectively).

[0261] Example 4: Demonstration of site-specific transposition using a TAL array-piggyBac transposase (ss-SPB) composition (TAL-PBx) and a free-body splitting GFP splicing reporter gene system

[0262] This example illustrates exemplary compositions and methods for demonstrating site-specific transposition at specific free-body loci using TAL array-SPB transposase fusion proteins.

[0263] The site-specific transposition efficiency of the various TAL array-SPB transposase fusion proteins constructed in Example 3 was evaluated using a free-body splitting GFP splicing reporter gene system. The reporter gene system consisted of two plasmids. The first plasmid, the "reporter gene," was constructed to contain the following from 5' to 3': an EF1a promoter (SEQ ID NO: 67), a Kozak sequence, the first part of the GFP open reading frame (SEQ ID NO: 68), a splice donor (SEQ ID NO: 69), and two BsaI IIS type restriction enzyme sites. The BsaI sites allow cloning of the following: a target TTAA sequence flanked by a variable-length spacer region flanked by a target recognition sequence for the TAL array. The second plasmid "donor" was constructed to contain the following from 5' to 3': TTAA sequence, 35bp PiggyBac minimum 5' ITR (SEQ ID NO: 70), splice acceptor site (SEQ ID NO: 71), second part of GFP open reading frame (SEQ ID NO: 72), synthetic polyadenylated sequence (SEQ ID NO: 73), 63bp PiggyBac minimum 3' ITR (SEQ ID NO: 74), and TTAA sequence. Figure 2 shows a schematic diagram of the split GFP reporter gene plasmid.

[0264] Four different LPA target sequences (SEQ ID No. 81 to 84) naturally occurring in genomic DNA were cloned into the above-mentioned free-body reporter gene plasmid. Complementary oligonucleotides containing LPA genomic DNA sequences (SEQ ID NO. 81 to 84) were synthesized. The complementary oligonucleotides contained 4bp overhangs compatible with the overhangs generated in the split GFP splice reporter gene after digestion with BsaI. The oligonucleotides were annealed and ligated into the digested vector to produce reporter genes compatible with each LPA TAL-PBx pair constructed in Example 3.

[0265] The TAL arrays were designed and constructed to produce heterodimeric pairs of TAL-ssSPB (i.e., a left TAL array - PBx and a right TAL array - PBx). Each TAL-PBx construct pair, along with its corresponding reporter gene plasmid (see page 56 / 62 of the manual, 60 CN 121219409 A), and the donor plasmid, was co-transfected into HEK293T cells. As a negative control, each TAL-PBx construct pair was co-transfected into HEK293T cells with unmatched reporter gene plasmids (i.e., TAL-PBx pair 1 with reporter gene 2, TAL-PBx pair 2 with reporter gene 3, TAL-PBx pair 3 with reporter gene 4, and TAL-PBx pair 4 with reporter gene 1) and the donor plasmid. A transfection mixture containing 26 ng of TAL-ssSPB expression vector, 170 ng of reporter gene plasmid, 117 ng of donor plasmid, and 0.78 μL of Transit-2020 transfection reagent was assembled in 250 μL of serum-free OptiMem medium. 95,000 HEK293T cells were added to 250 μL of DMEM medium supplemented with 10% FBS, and the transfection mixture was plated in 48-well plates and incubated at 37°C and 5% CO2 for four days. On the second day, the cells were divided at a 1:3 ratio.

[0266] When the reporter gene plasmid and donor plasmid were co-transfected into cells with TAL-PBx, TAL-PBx catalyzed the excision of the transposon from the donor plasmid and its site-specific integration into the TTAA target site of the reporter gene plasmid. Figure 3 is a schematic diagram showing the catalytic ssSPB dimer that binds to the excised transposon and recognizes its genomic integration target site. Following site-specific transposition, transcription, splicing, and translation, a reconstructed GFP coding sequence (DNA, SEQ ID NO: 75; amino acid; SEQ ID NO: 76) is generated and fluorescence is detectable. The percentage of target-site specific transposition-positive cells for each TAL-PBx pair was determined by FACS analysis, and the results are shown in Table 8.

[0267] Table 8

[0268]

[0269] As can be seen in Table 8, all TAL-ssSPBs catalyze site-specific transposition of their corresponding target reporter genes, but do not catalyze site-specific transposition of reporter genes containing unmatched off-target genes. In addition, the highest transposition was observed at target 1, which is the only target with the TTTAAA integration site.

[0270] Example 5: Determination of the optimal 5' and 3' nucleotides adjacent to the TTAA integration site

[0271] Previous examples have shown that target 1, with the most robust integration, contains 5' T and 3' A adjacent to the TTAA target site, thereby generating the TTTAA integration site. This example illustrates additional compositions and methods for preparing the optimal target site for site-specific transposition by determining the optimal 5' and 3' nucleotides adjacent to the TTAA integration site.

[0272] The site-specific transposition efficiency of various TAL-PBx fusion proteins targeting the green fluorescent protein (GFP) gene was evaluated using the free-body splitting GFP splicing reporter gene as described in Example 4. TAL arrays targeting a specific 10 bp right and 10 bp left sequence in the coding region of the GFP gene – SPB transposase fusion proteins GFP1 right TAL-PBx and GFP1 left TAL-PBx – were prepared as described in Examples 14 and 18 of International Patent Application Publication No. PCT / US2022 / 77549, the contents of which are incorporated herein by reference in their entirety. Specification 57 / 62 pages 61 CN 121219409 A

[0273] To generate a reporter gene plasmid compatible with GFP1 right TAL-PBx, a complementary oligonucleotide was synthesized containing a target site of GFP1 right TAL downstream of T, followed by a 12 bp spacer, followed by TTAA, followed by a 12 bp spacer, followed by the reverse complementary sequence of the TAL target site, followed by A (SEQ ID No. 172). The spacer sequence is such that the 5' nucleotide immediately following TTAA is C and the 3' nucleotide immediately following TTAA is C. The complementary oligonucleotide contains a 4 bp overhang compatible with the overhang produced in the split GFP splice reporter gene after digestion with BsaI. The oligonucleotide is annealed and ligated into the digested vector to produce a reporter gene compatible with GFP1 right TAL-PBx. Similar oligonucleotides are synthesized using the 12 bp modified spacer sequence to mutate the flanking 5' and 3' nucleotides immediately following the TTAA integration sequence to T and A, respectively, to generate the TTTAAA integration site (SEQ ID No. 173), or to C and A, respectively, to generate the CTTAAA integration site (SEQ ID No. 174).A similar oligonucleotide was synthesized containing a GFP1 right TAL target site downstream of T, followed by a 13 bp spacer, then TTAA, followed by another 13 bp spacer, then the inverse complementary sequence of the TAL target site, and then A (SEQ ID No. 175). The sequence of the spacer is such that the nucleotide immediately following TTAA at the 5' end is C and the nucleotide immediately following TTAA at the 3' end is C. Similarly, using the modified 13bp spacer sequence, similar oligonucleotides were synthesized to mutate the 5' and 3' nucleotides adjacent to the TTAA integration sequence to T and A, respectively, to generate the TTTAAA integration site (SEQ ID No. 176), or to C and A, respectively, to generate the CTTAAA integration site (SEQ ID No. 177), or to T and G, respectively, to generate the TTTAAG integration site (SEQ ID No. 178), or to C and G, respectively, to generate the CTTAAG integration site (SEQ ID No. 179).

[0274] Each reporter gene plasmid and donor plasmid were co-transfected with the GFP1 right TAL-PBx expression plasmid (SEQ ID No. 77) into HEK293T cells. As a negative control, the GFP1 left TAL-PBx expression plasmid (SEQ ID NO. 78), which does not recognize the GFP1 right target sequence, was transfected instead of the GFP1 right TAL-PBx expression plasmid. HEK293T cells were seeded in 500 µL of DMEM medium supplemented with 10% FBS in 24-well plates. On day 2, a transfection mixture containing 50 µL of JetPrime buffer, 50 ng of the TAL-ssSPB expression vector, 225 ng of the reporter gene plasmid, 225 ng of the donor plasmid, and 1 µL of JetPrime transfection reagent was assembled. The mixture was added to HEK293T cells, and the cells were incubated at 37°C and 5% CO2 for four days. On day 1, the cells were divided at a ratio of 1:6. On day 4, FACS analysis was used to determine the percentage of target-site specific transposable positive cells for each construct.

[0275] When the reporter gene plasmid and the donor plasmid are co-transfected into cells with TAL-PBx, TAL-PBx catalyzes the excision of the transposon from the donor plasmid and its site-specific integration into the TTAA target site of the reporter gene plasmid.Following site-specific transposition, transcription, splicing, and translation, a reconstructed GFP coding sequence (DNA SEQ ID No. 75; amino acid SEQ ID No. 76) is generated and fluorescence is detectable. The percentage of target-site-specific transposition-positive cells for each spacer length construct was determined by FACS analysis, and the results are shown in Table 9.

[0276] As shown in Table 9, the right TAL-PBx catalytic site-specific transposition of GFP1 resulted in a GFP signal higher than the background level for all target sites. The TTTAAA target site produced a larger GFP signal than the CTTAAC and CTTAAG target sites. The CTTAAA and TTTAAG target sites produced the largest GFP signals. When using the right-specific reporter gene of GFP1, the left TAL-PBx of GFP1 did not produce a GFP signal higher than the background.

[0277] Table 9 Specification 58 / 62 pages 62 CN 121219409 A

[0278]

[0279] Example 6: Demonstration of site-specific transposition using TAL array-piggyBac transposase (ss-SPB) composition (TAL-PBx)

[0280] Based on the results in Example 5, four different LPA target sequences naturally occurring in the second group (SEQ ID No. 85 to 88) were cloned into the free-body reporter gene plasmid described in Example 4. As with the first group of targets evaluated in Example 4, each of the target sequences in the second group has a 10bp TAL binding site and a 12bp or 13bp spacer region on both sides of the TTAA. Additionally, each of the target sequences in the second group contains a spacer sequence such that the nucleotide immediately following TTAA at 5' is T and the nucleotide immediately following TTAA at 3' is A to generate a TTTAAA integration site, or such that the nucleotide immediately following TTAA at 5' is C and the nucleotide immediately following TTAA at 3' is A to generate a CTTAAA integration site. Further, since thymidine is not immediately following the 5' of all LPA target sites, the N-terminal domain of TAL is mutated to eliminate the need for any specific nucleotide 5' of the binding site. These mutations are introduced into the wild-type TAL sequence by replacing the amino acid sequence QWS at positions 79 to 81 of SEQ ID NO: 31 with YH to generate the NT-βN variant (SEQ ID NO: 34).

[0281] TAL arrays are constructed to target these TAL binding sites using the design criteria described herein or listed below.

[0282] The TAL array pairs are designed to target four specific 10 bp right and left pairs of sequences within the four LPA target sites of the second group. For each of the targets, multiple TAL array pairs are designed using 12 bp or 13 bp spacers. Specification 59 / 62 pages 63 CN 121219409 A

[0283] The left and right target sequences and the 5' nucleotides used to generate the TAL arrays targeting the LPA genes are shown in Table 10.

[0284] Table 10: Indicative TAL arrays for targeting LPA

[0285]

[0286] As described in Example 2, eight left and right pair combinations were used to design and construct LPA left TAL arrays LPAL5.1, LPAL5.2, LPAL6.1, LPAL6.2, LPAL7.1, LPAL7.2, LPAL8.1 and LPAL8.2 (SEQ ID Nos. 127, 129, 131, 133, 135, 137, 139 and 141, respectively) and LPA right TAL arrays LPAR5.1, LPAR5.2, LPAR6.1, LPAR6.2, LPAR7.1, LPAR7.2, LPAR8.1 and LPAR8.2 (SEQ ID Nos. 128, 130, 132, 134, 136, 138, 140 and 142, respectively).

[0287] The TAL-PBx fusion construct was prepared as follows: an expression plasmid containing the following items from 5' to 3' direction was synthesized: CMV promoter, T7 promoter, Kozak sequence, 3x Flag tag (SEQ ID NO: 65), SV40 NLS (SEQ ID NO: 66), Δ152 TAL N-terminal domain (SEQ ID NO: 31) of TAL NT-BN variant (SEQ ID NO: 34), two BsmBI IIS type restriction enzyme sites, +73 TAL C-terminal domain (SEQ ID NO: 79), GGGS linker, Δ1-85 PBx (containing an 85-amino acid deletion at the N-terminus and mutations at R372A, K375A, and D450N in the Super piggyBac transposase codon sequence; SEQ ID NO: 9), and bGH polyadenylation sequence.

[0288] The left or right TAL array with BsmBI side-linked is cloned into the BsmBI site of the expression plasmid and the TAL array and PBx coding sequence are fused within the framework via the adapter sequence to generate a full-length TAL-PBx construct.All encoded sequences used were codon-optimized for human representation using the GeneArt (Thermo Fisher) algorithm.

[0289] Sixteen TAL arrays with BsmBI ends were cloned into the BsmBI restriction sites of the above expression plasmids to generate sixteen TAL-PBx constructs: LPAL5.1, LPAL5.2, LPAL6.1, LPAL6.2, LPAL7.1, LPAL7.2, LPAL8.1 and LPAL8.2 left TAL-PBx (SEQ ID Nos. 154, 156, 158, 160, 162, 164, 166 and 168 respectively) and LPAR5.1, LPAR5.2, LPAR6.1, LPAR6.2, LPAR7.1, LPAR7.2, LPAR8.1 and LPAR8.2 right TAL-PBx (SEQ ID Nos. 155, 157, 159, 161, 163, 165, 167 and 169 respectively), as in Example 3. As described in

[0290] Additionally, the TAL array LPAL1 (SEQ ID No. 116) and LPAR1 specification (pages 60 / 62, CN 121219409 A, SEQ ID No. 117) described in Example 2 were cloned into the above expression plasmids in this Example 6 to generate the TAL-PBx constructs LPAL1 v2 (SEQ ID No. 170) and LPAR2 v2 (SEQ ID No. 171).

[0291] A. Free-body target site-specific transposition

[0292] The activity of the new mutant TAL-PBx fusion was determined using its corresponding free-body splitting GFP splicing reporter gene. Briefly, each reporter gene plasmid and donor plasmid were co-transfected with the corresponding TAL-PBx expression plasmid into HEK293T cells. Approximately 120,000 HEK293T cell plates were seeded in 500 µl of DMEM medium supplemented with 10% FBS in 24-well plates. The next day, a transfection mixture containing 50 µl of JetPrime buffer, 50 ng of the TAL-PBx expression vector, 225 ng of the reporter gene plasmid, 225 ng of the donor plasmid, and 1 µl of JetPrime transfection reagent was assembled. This mixture was added to HEK293T cells and incubated at 37°C and 5% CO2 for four days. Cells were divided at a 1:6 ratio on day 1. The percentage of GFP-positive cells in each sample was determined. The results are shown in Table 11.

[0293] Table 11

[0294]

[0295] As seen in Table 11, TAL-ssSPB targeting targets 1, 6, and 8 produced the highest transposition. Additionally, TAL-ssSPB utilizing the 13 bp spacer region produced higher editing than those utilizing the 12 bp spacer region.

[0296] B. Genome Target-Specific Transposition Specification 61 / 62 pages 65 CN 121219409 A

[0297] After confirming the functionality of the newly designed LPA TAL and its recognition of its target sequences, the TAL-PBx construct was used to edit endogenous genomic LPA targets in Huh7 (an immortalized hepatocyte cell line). Briefly, 100,000 cell plates were seeded in 24-well plates in RPMI medium + 10% FBS one day prior to transfection. On the second day, 0.5 μg or 1 μg of mRNA encoding the LPA target 1TAL-ssSPB pair (SEQ ID NO: 143 and 144) was mixed with 0.5 μl or 1 μl of Messenger Max reagent to generate ssSPB-mRNA-lipid complexes. Simultaneously, 450 ng of the transposon donor vector (SEQ ID NO: 80) was mixed with 0.5 μl of P3000 reagent and 1 μl of lipofectamine 3000 to generate DNA-lipid complexes. 50 μl of the ssSPB mRNA-lipid complex and 50 μl of the DNA-lipid complex were delivered to cells and incubated at 37°C.

[0298] To assess site-specific integration of the transposon donor into the LPA locus, genomic DNA was extracted from transfected cells two days post-transfection and analyzed using a probe-based detection protocol via digital droplet PCR (ddPCR). A primer binding within the transposon was paired with a primer binding LPA genomic DNA near the TTAA integration site. Therefore, amplicons are generated only after site-specific transposition into the LPA locus. Because integration is not directional, two assays were designed for each LPA target to detect transposon integration in both the forward and reverse directions. As a negative control, genomic DNA was extracted from untransfected cells and used as a template in ddPCR reactions to demonstrate the specificity of the primer / probe set. The results are shown in Table 12.

[0299] Table 12

[0300]

[0301] As shown in Table 12, the detection of amplicon corresponding to integration with forward and / or reverse transposons from genomic DNA isolated from cells transfected with the LPA TAL-PBx construct along with transposons provides direct evidence of genomic integration at the LPA locus. Specification 62 / 62 pages 66 CN 121219409 A Specification Figure 1 / 4 page 67 CN 121219409 A Specification Figure 2 / 4 page 68 CN 121219409 A Specification Figure 3 / 4 page 69 CN 121219409 A Specification Figure 4 / 4 page 70 CN 121219409 A.

Claims

1. A fusion protein comprising a DNA targeting domain and a transposase domain, the transposase domain comprising the sequence set forth in SEQ ID NO: 4, wherein the DNA targeting domain binds to a nucleic acid sequence encoding a LPA repeat element.

2. The fusion protein of claim 1, wherein the DNA targeting domain comprises one, two, or three zinc finger motifs.

3. The fusion protein of claim 1, wherein the DNA targeting domain comprises one or more TAL domains.

4. The method of claim 3, wherein the TAL domain comprises the sequence set forth in any one of SEQ ID NOs: 35-38.

5. The fusion protein of any one of claims 1-4, wherein the DNA targeting domain binds to a nucleic acid sequence in the LPA gene encoding a kringle domain repeat element or an intron adjacent to a sequence encoding a kringle domain repeat element.

6. The fusion protein of any one of claims 1-5, wherein the transposase domain and the DNA targeting domain are linked by a linker.

7. The fusion protein of claim 6, wherein the linker comprises the sequence GGGGS (SEQ ID NO: 181).

8. The fusion protein of any one of claims 1-7, wherein the DNA targeting domain is inserted into the N-terminus of the transposase domain at a position located after amino acid 82 and before amino acid 105 of SEQ ID NO:

4.

9. The fusion protein of any one of claims 1-7, wherein the DNA targeting domain replaces one or more amino acids in the transposase domain between and including amino acids 83 and 105 of SEQ ID NO:

4.

10. The fusion protein of any one of claims 1-9, wherein the transposase domain comprises an N-terminal deletion of amino acids 1-83, 1-84, 1-85, 186, 1-87, 1-88, 1-89, 1-90, 1-91, 1-92, 1-93, 1-94, 1-95, 1-96, 1-97, 1-98, 1-99, 1-100, 1-101, 1-102, or 1-103.

11. The fusion protein of any one of claims 1-10, wherein the transposase domain comprises the sequence set forth in any one of SEQ ID NOs: 7-27.

12. The fusion protein of any one of claims 1-11, wherein the transposase domain comprises: (a) at least one mutation selected from the group consisting of: M185R, M185K, D197K, D197R, D198K, D198R, D201K, and D201R; or (b) at least one mutation selected from the group consisting of: L204D, L204E, K500D, K500E, R504E, and R504D.

13. A polynucleotide comprising a nucleic acid sequence encoding the fusion protein of any one of claims 1-12.

14. A vector comprising the polynucleotide of claim 13.

15. A method of integrating a transgene into a genomic target site of a cell, the method comprising:

15. A method of introducing a fusion protein of any one of claims 1-12 and a transposon into a cell, wherein the transposon comprises, in 5' to 3' order: a 5' ITR, the transgene, and a 3' ITR.

16. The method of claim 15, wherein the transposon further comprises an exogenous promoter between the 5' ITR and the transgene.

17. The method of claim 15 or 16, wherein the transgene encodes a detectable marker.

18. The method of claim 17, wherein the detectable marker is GFP.

19. The method of claim 15 or 16, wherein the transgene is: (a) a gene that is not expressed by the cell prior to introduction of the fusion protein and the transposon, or (b) a gene that exhibits reduced, insufficient, and / or altered expression by the cell prior to introduction of the fusion protein and the transposon.

20. The method of any one of claims 15-19, wherein the genomic target site is on the LPA gene.

21. The method of any one of claims 15-19, wherein the genomic target site is in a repetitive element.

22. The method of claim 21, wherein the repetitive element is an LPA repeat element.

23. The method of any one of claims 15-19, wherein the genomic target site is in an intron of a gene.

24. The method of claim 23, wherein the genomic target site is in an intron of the LPA gene.

25. The method of any one of claims 15-24, wherein the cell is in vivo.

26. A method of modifying the genome of a cell, the method comprising: The cell is provided with the fusion protein of any one of claims 1 to 12, wherein the cell comprises a modified binding site comprising, in 5' to 3' order, a sequence of a target site for a DNA targeting domain, a first spacer, a TTAA target integration site for a SPB, a second spacer, and an anti-sense complement of the sequence of the target site for the DNA targeting domain.

27. The method of claim 26, wherein the target integration site comprises the sequence TTAA.

28. The method of claim 26, wherein the target integration site comprises the nucleic acid sequence set forth in any one of SEQ ID NOs: 81 to 88.

29. An integration cassette for site-specific transposition of a nucleic acid into the genome of a cell, the integration cassette comprising a nucleic acid, the nucleic acid comprising or consisting of: a central transposon ITR integration site TTAA sequence flanked by an upstream TAL array target sequence and a downstream TAL array target sequence, wherein each of the upstream TAL array target sequence and the downstream TAL array target sequence is separated from the TTAA sequence by 12 or 13 base pairs.

30. The integration cassette of claim 29, wherein the integration site comprises the sequence TTAA.

31. The integration cassette of claim 29, wherein the integration site comprises the nucleic acid sequence set forth in any one of SEQ ID NOs: 81 to 88.

32. The integration cassette of any one of claims 29 to 31, wherein each of the upstream TAL array target site sequence and the downstream TAL array target site sequence is the same.

33. The integration cassette of any one of claims 29 to 31, wherein each of the upstream TAL array target site sequence and the downstream TAL array target site sequence is different.

34. The integration cassette of any one of claims 29 to 33, wherein each of the upstream TAL array target site and the downstream TAL array target site targets a 7 to 30 bp sequence of an LPA repeat sequence element.

35. A cell comprising the integration cassette of any one of claims 29 to 34 stably integrated into the genome of the cell.

36. A method for site-specifically transposing a DNA molecule into the genome of a cell, the method comprising introducing into the cell of claim 35: a) a nucleic acid encoding a fusion protein comprising a DNA binding domain and a transposase; wherein the fusion protein is expressed in the cell; and b) a DNA molecule comprising a transposon; wherein the expressed fusion protein integrates the transposon into the TTAA integration site of the stably integrated integration cassette by site-specific transposition.

37. A method for generating an engineered cell by site-specific transposition, the method comprising introducing into the cell of claim 31: a) a nucleic acid encoding a fusion protein comprising a DNA binding domain and a transposase; wherein the fusion protein is expressed in the cell; and b) a DNA molecule comprising a transposon; wherein the expressed fusion protein integrates the transposon into the TTAA integration site of the stably integrated integration cassette by site-specific transposition. a) a nucleic acid encoding a fusion protein comprising a DNA binding domain and a transposase; wherein the fusion protein is expressed in the cell; and b) a DNA molecule comprising a transposon; wherein the expressed fusion protein integrates the transposon into a TTAA integration site of the stably integrated integration cassette by site-specific transposition, thereby generating the engineered cell.

38. The method of claim 36 or 37, wherein the integration site comprises the sequence TTAA.

39. The method of claim 36 or 37, wherein the integration site comprises the nucleic acid sequence set forth in any one of SEQ ID NOs: 81-88.

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