Evolved integrases and methods for their use in genome editing

Abstract

Disclosed are evolved Bxb1 and PhiC31 integrases, which exhibit increased insertion activity and efficiency as compared to their wild type counterparts, as well as a method for producing the same. And the use of the evolved integrase for modifying any sequence within the genome of a cell or subject.

Description

[0001] introduction This invention was made with government support under the approval numbers GM103457, GM132779, GM125526, and EB031124 granted by the National Institutes of Health (NIH). The government holds certain rights to this invention.

[0002] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 510,171, filed June 26, 2023, and U.S. Provisional Patent Application Serial No. 63 / 592,945, filed October 25, 2023, the contents of which are incorporated herein by reference in their entirety.

[0003] Statement regarding electronic submission of sequence lists The XML sequence list entitled "UH0010WO_ST26.XML" with a size of 342,786 bytes, which was generated on June 3, 2024 and submitted with this specification, is incorporated herein by reference. Background Technology

[0004] Many genetic diseases are caused by different mutations that vary among different patients. For conventional therapies to be effective, complete replacement of the coding sequence is often required. With the treatment of increasingly complex diseases, multi-gene insertions will be necessary, requiring much larger payloads that exceed the limitations of current virus-based methods. While many gene editing techniques are now possible using CRISPR nuclease-induced non-homologous end joining (NHEJ; deletion) and homologous directed repair (HDR; insertion), basal editing (single-base-pair substitution), or leader editing (small insertions, substitutions, or deletions), it is noteworthy that HDR insertion efficiency decreases significantly for larger payloads, and HDR is inefficient or ineffective in non-dividing cells, which constitute most treatment-related cell types. NHEJ-based insertion methods exist, but are generally inefficient and, like HDR, rely on unprotected double-strand breaks, which can lead to uncontrolled large deletions or chromosomal rearrangements. Leader editing works in both dividing and non-dividing cells and efficiently inserts sequences of <50 base pairs while minimizing double-strand breaks. An improved version of leader editing, called dual leader editing, has been developed, which uses an attachment (att) site from Bxb1 to insert a payload of up to 5.6 kb at the att site (Anzalone et al. (2022)). Nat. Biotechnol (40:731–740). This approach overcomes many obstacles of current gene insertion tools and offers great hope for correcting hereditary diseases.

[0005] However, there remains a need in the art to improve the efficiency of integrase-mediated integration. This disclosure addresses this need in the art. Summary of the Invention

[0006] In one aspect, an evolved Bxb1 integrase is provided, the evolved Bxb1 integrase comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:1, wherein the amino acid sequence of the evolved Bxb1 integrase contains one or more mutations and exhibits at least a 2-fold increase in the integration of exogenous nucleic acids into genomic target sites compared to wild-type Bxb1 integrase.

[0007] In another aspect, an evolved PhiC31 integrase is provided, the evolved PhiC31 integrase comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:2, wherein the amino acid sequence of the evolved PhiC31 integrase contains at least one mutation as described herein.

[0008] This document also provides a method for altering a cell genome sequence by contacting a cell with an evolved Bxb1 or PhiC31 integrase of the present disclosure, wherein the cell includes at least one integrase recognition sequence in its genome that is recognized by the evolved integrase, such that the evolved integrase binds to the integrase recognition sequence and alters the cell genome sequence. Attached Figure Description

[0009] Figure 1 In HEK293 cells, amplicon sequencing comparisons were performed using dual-leader RNA editing at target sites AAVS1, Xq22.1, and human ROSA26. Mean and standard deviation bars represent two biological replicates transfected on different dates.

[0010] Figure 2 An evolved integrase helper plasmid was co-transfected with a 6.6 kb donor plasmid into a cloned HEK293 cell line containing a pre-installed attP landing pad. An un-evolved control is shown for comparison. Target integration was measured using droplet digital PCR. n=3.

[0011] Figure 3 Integration of the PhiC31 mutant into HEK293 cells via PACE evolution at ROSA using double-leader editing, as measured by droplet digital PCR. A non-evolved control is also shown. n=3.

[0012] Figure 4Comparison of dual-leader editing and integration frequencies of the 6.6 kb donor plasmid with PhiC31, evolved PhiC31, Pa01, and Bxb1 integrases in HEK293T cells, measured by droplet digital PCR. n=3.

[0013] Figure 5A and Figure 5B Frequency of dual-leader editing and integration of PACE-evolved Bxb1 mutants in HEK293 cells, measured by droplet digital PCR. n≥4, multi-day.

[0014] Figure 6 Comparison of combined mutants on the HEK293 ROSA attB cell line. Integration of the 6.6 kb donor plasmid as measured by droplet digital PCR. n=4.

[0015] Figure 7 Integration and double PE frequencies of selected PACE evolutionary clones and combinatorial mutants in HEK293T cells. n=4, two separate days.

[0016] Figure 8 Optimized dual-leader editing and integration in HEK293T. Cells were transfected and sorted for dsRed expression 3 days post-transfection, then lysed and measured by droplet digital PCR. n=3.

[0017] Figure 9 Optimized dual-leader editing and integration in K562. Cells were transfected and sorted for dsRed expression 3 days post-transfection, then lysed and measured by droplet digital PCR. n=3.

[0018] Figure 10 Optimized dual-leader editing and integration in HeLa. Cells were transfected and sorted for dsRed expression 3 days post-transfection, then lysed and measured by droplet digital PCR. n=3.

[0019] Figure 11 Detection of droplet digital PCR for integration of human factor VIII (FVIII) and von Willebrand factor (VWF) donors at ROSA26.

[0020] Figure 12 The secretion of FVIII in the culture medium was detected by ELISA and factor X activation assay.

[0021] Figure 13 Detection of VWF secreted in culture medium by ELISA. Detailed Implementation

[0022] Serine integrases, such as PhiC31 and Bxb1, are capable of inserting large DNA payloads at attachment (att) sites. By targeting the att site to the genome using techniques such as leader editing (PE), integrases can deliver payloads to secure loci while avoiding double-strand breaks. Phage-assisted sequential evolution (PACE) was used to evolve PhiC31 and Bxb1 serine integrases to increase their activity at their respective native att sequences. The resulting improved integrase sequences were subcloned into mammalian expression vectors, and their activity in inserting large transgenes into human cell lines was tested. First, dual leader editing was used to insert each integrase into the genome at the designated att site. Next, each integrase inserted its payload DNA into the att site at a specific location in the genome. Efficiency was accurately measured using droplet digital PCR. Under pre-optimized transfection conditions without sorting transfected cells, wild-type integrases inserted their payload DNA into 3.7% of the haploid genome. In contrast, the evolved integrases inserted their payload DNA into up to 21.9% of the haploid genome. Under optimized transfection conditions using fully transfected and sorted cells, wild-type integrase inserts its payload DNA into 32% of the haploid genome. In contrast, integrase that evolved after sorting inserts its payload DNA into up to 80% of the haploid genome. The improved integrase can be used to replace defective genes in the genome. By directing the insertion to specific sites in the genome, the risks of off-target insertion and insertional mutagenesis are significantly reduced. The increased efficiency provided by the improved integrase described herein will significantly enhance the effectiveness of this technology as a therapy for diseases or conditions, and altering gene sequences will provide benefits for treating these diseases or conditions.

[0023] Therefore, some aspects provide evolved integrases that exhibit increased insertion activity and efficiency compared to their wild-type counterparts, and the uses of these evolved integrases for modifying any sequence within the genome of, for example, a cell or a subject. In particular, evolved Bxb1 or PhiC31 integrases are described that exhibit increased insertion efficiency compared to wild-type Bxb1 or PhiC31 integrases. The term "wild-type (or natural) integrase" refers to an integrase that is naturally occurring and / or has not been modified by human intervention.

[0024] The evolved Bxb1 and PhiC31 integrases described herein have at least one mutation or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mutations. Mutations may include substitutions, deletions, or insertions that do not eliminate recombinase activity.

[0025] Wild-type Bxb1 integrase may have the following amino acid sequence: MRALVVIRLSRVTDATTSPERQLESCQQLCAQRGWDVVGVAEDLDVSGAVDPFDRKRRPNLARWLAFEEQPFDVIVAYRVDRLTRSIRHLQQLVHWAEDHKKLVVSATEAHFDTTTPFAAVVIAL MGTVAQMELEAIKERNRSAAHFNIRAGKYRGSLPPWGYLPTRVDGEWRLVPDPVQRERILEVYHRVVDNHEPLHLVAHDLNRRGVLSPKDYFAQLQGREPQGREWSATALKRSMISEAMLGYATL NGKTVRDDDGAPLVRAEPILTREQLEALRAELVKTSRAKPAVSTPSLLLRVLFCAVCGEPAYKFAGGGRKHPRYRCRSMGFPKHCGNGTVAMAEWDAFCEEQVLDLLGDAERLEKVWVAGSDSAV ELAEVNAELVDLTSLIGSPAYRAGSPQREALDARIAALAARQEELEGLEARPSGWEWRETGQRFGDWWREQDTAAKNTWLRSMNVRLTFDVRGGLTRTIDFGDLQEYEQHLRLGSVVERLHTGMS (SEQ ID NO:1).

[0026] In one aspect, an evolved Bxb1 integrase is provided, the evolved Bxb1 integrase having an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO:1, wherein the evolved Bxb1 integrase has at least one mutation and exhibits increased insertion efficiency compared to wild-type Bxb1 integrase. In some aspects, compared to wild-type Bxb1 integrase (e.g., SEQ ID NO:1), the evolved Bxb1 integrase has one to eight mutations, for example, one to three mutations, two to four mutations, three to five mutations, four to six mutations, five to seven mutations, six to eight mutations, two to seven mutations, or four to eight mutations. In other respects, compared with wild-type Bxb1 integrase (e.g., SEQ ID NO:1), evolved Bxb1 integrase has at least one mutation, at least two mutations, at least three mutations, at least four mutations, at least five mutations, at least six mutations, at least seven mutations, or at least eight mutations.

[0027] In other respects, the evolved Bxb1 integrase has at least one mutation relative to SEQ ID NO:1 at positions 4, 5, 18, 24, 34, 36, 40, 42, 45, 46, 49, 50, 51, 57, 61, 62, 63, 67, 69, 70, 74, 75, 76, 79, 85, 87, 88, 89, 90, 92, 95, 99, 100, 105, 106, 110, 111, 119, 122, 130, 133. 137, 140, 145, 153, 156, 160, 164, 166, 168, 174, 175, 178, 179, 181, 187, 189, 191, 194, 203, 209, 218, 223, 229, 231, 239, 248, 251, 254, 261, 263, 264, 268, 272, 278 280, 281, 282, 283, 285, 287, 288, 292, 295, 302, 306, 307, 311, 313, 319, 321, 328, 331, 332, 333, 334, 341, 347, 353, 355, 359, 360, 361, 362, 366, 369, 370, 375, 380, 3 87, 388, 397, 398, 405, 409, 411, 414, 415, 416, 419, 425, 426, 428, 434, 435, 444, 445, 453, 461, 463, 466, 468, 476, 479, 480, 483, 484, 487, 488, 489, 494, 496 and / or 499. In some aspects, the evolved Bxb1 integrase has at least one mutation, said at least one mutation relative to SEQ IDNO:1 is L4I, V5V, S18S, E24E, G34D, D36A, D36N, V40I, V40A, V40V, E42K, D45G, V46V, A49S, V50I, D51D, D51N, D51G, D51E, R57K, L61F, A62A, R63K, F67S, E69A, E69E, Q70H, V74I, I75V, V76I, R79R, R85R, I87L, I87V, R88R, H89G, H89Y, L90L, Q92H, Q92Q, H95Y, D99N, H100N, H100H, V105A, S106S, A110A, H十一年1P, A119S, V122M, A130A, E133E, I137I, R140R, A145T, K153K, G156G, P160P, L164L, T1六十六I, V168I, L174L, V175V, V175A, P178P, V179A, V179I, R181K, R181R, V187I, V187V, H189N, H189Y, Q191Q, N194D, H203Y, G209G, A218A, R223R, E229K, S231S, M239I, A248T, N251K, N251N, T254S, A261T, A二一S, L263I, V264A, P268P, R272Q, L278L, A280T, A280A, E281E, L282L, V283V, T285A, R287R, R287H, A288V, A288T, P292P, P295P, L302L, V306V, V306A, C307C, A311V, K313R, K313K, R319K, R319G, H321P, H321N, S328S, F331S, P332H, K333K, H334R, A341D, A347E, V353I, D355N, D359N, D359D, D359A, A360V, A360T, E361E, R362K, V366I, A369P, A369E, A369T, A369S, G370G, V375V, V380I, L387L, T388M, R397R, A398S, A405A, R409H, A411V, A414A, A414V, A415S, R416K, E419E, A425T, A425A, R426K It should be noted that there seems to be an error in the original text where "H十一年1P" and "A二一S" are likely incorrect notations. This might affect the accuracy of the translation if the original meaning is not clear. You may want to double-check the original text for accuracy.S428S, E434G, T435T, R444L, E445Q, T453I, R461R, T463I, V466M, V466V, V466L, G468D, G468G, F476F, L479I, Q480STOP, E483E, Q484K, R487R, del L488, G489G, R494S, R494R, H496N and / or M499T. In some aspects, evolved Bxb1 integrase have mutant combinations as described herein, for example with reference to SEQ ID NO:1 (wild-type Bxb1), which are any of the combinations listed in Table 1.

[0028] Table 1

[0029] X can be any amino acid.

[0030] Wild-type PhiC31 integrase may have the following amino acid sequence: MDTYAGAYDRQSRERENSSAASPATQRSANEDKAADLQREVERDGGRFRFVGHFSEAPGTSAFGTAERPEFERILNECRAGRLNMIIVYDVSRFSRLKVMDAIPIVSELLALGVTIVSTQEGVFRQGNVMDLIHLIMRLDASHKESSLKSAKILDTKNLQRELGGYVGGKAPYGFELVSETKEITRNGRMVNVVINKLAHSTTPLTGPFEFEPDVIRWWWREIKTHKHLPFKPGSQAAIHPGSITGLCKRMDADAVPTRGETIGKKTASSAWDPATVMRILRDPRIAGFAAEVIYKKKPDGTPTTKIEGYRIQRDPITLRPVELDCGPIIEPAEWYELQAWLDGRGRGKGLSRGQAILSAMDKLYCECGAVMTSKRGEESIKDSYRCRRRKVVDPSAPGQHEGTCNVSMAALDKFVAERIFNKIRHAEGDEETLALLWEAARRFGKLTEAPEKSGERANLVAERADALNALEELYEDRAAGAYDGPVGRKHFRKQQAALTLRQQGAEERLAELEAAEAPKLPLDQWFPEDADADPTGPKSWWGRASVDDKRVFVGLFVDKIVVTKSTTGRGQGTPIEKRASITWAKPPTDDDEDDAQDGTEDVAA (SEQ ID NO:2) In one aspect, an evolved PhiC31 integrase is provided, the evolved PhiC31 integrase having an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO:2, wherein the evolved PhiC31 integrase has at least one mutation and exhibits increased insertion efficiency compared to wild-type PhiC31 integrase. In some aspects, compared to wild-type PhiC31 integrase (e.g., SEQ ID NO:2), the evolved PhiC31 integrase has one to 13 mutations, for example, one to three mutations, two to four mutations, three to five mutations, four to six mutations, five to seven mutations, six to eight mutations, seven to nine mutations, eight to ten mutations, nine to eleven mutations, ten to twelve mutations, or eleven to thirteen mutations. In other respects, compared with wild-type PhiC31 integrase (e.g., SEQ ID NO:2), evolved PhiC31 integrase has at least one mutation, at least two mutations, at least three mutations, at least four mutations, at least five mutations, at least six mutations, at least seven mutations, at least eight mutations, at least nine mutations, at least ten mutations, at least eleven mutations, at least twelve mutations, or at least thirteen mutations.

[0031] In other respects, the evolved PhiC31 integrase has at least one mutation relative to SEQ ID NO:2 at positions 1, 2, 12, 14, 18, 24, 32, 36, 41, 43, 44, 45, 49, 51, 55, 58, 74, 77, 96, 103, 107, 117, 153, 176, 196, 197, 199, 200, 228, 229, 230, 231, 235, 238, 240, 252, 254, 255, 260, 262, 264, 266, 269, 274, 278, 302, 319, 320, 322, 331, 333, 340, 344, 345, 346, 347, 351, 353, 355, 35 9, 362, 364, 378, 382, ​​393, 396, 397, 399, 406, 410, 424, 429, 431, 436, 438, 445, 448, 449, 450, 452, 457, 468, 472, 478, 498, 499, 501, 505, 512, 514, 516, 517, 520, 527, 535, 536, 549, 551, 552, 553, 563, 568, 580, 585, 586, 587, 590, 592, 594, 600, 603, 604, 609, 612 and / or 616.In some respects, the evolved PhiC31 integrase has at least one mutation, said at least one mutation relative to SEQ ID NO:2 is M1E, M1V, D2V, D2M, S12N, E14G, S18N, A24A, D32A, D36A, V41I, R43R, D44A, G45G, R49R, V51M, S55S, P58P, I74I, E77E, R96R, I103I, S107S, V117V, I153I, I153V, E176D, N196N, K197K, A199T, H200H, H228Y, L229L, P230S, F231L, S2 35S, A238A, H240R, D252G, D254D, A255G, G260G, T262T, G264G, K266K, S269N, P274S, M278L, T302A, L319L, R320R, V3 22V, E331E, A333T, A333S, A333D, A340V, G344V, G344D, R345R, G346S, R347K, L351V, L351Q, R353R, Q355Q, S359S, D3 62N, D362G, L364M, E378K, K382K, V393V, S396N, S396R, A397T, G399G, N406S, A410T, I424I, G429S, E431V, L436L, W4 38R, G445G, W448R, E449D, A450D, E452K, R457R, L468L, E472D, R478R, A498A, L499L, L501L, G505V, G505G, G505S, E5 12K, E514E, A516T, E517E, K520K, F527F, P535L, P535P, T536A, D549D, R551R, V552V, F553F, V563V, T568T, A580V, A585A, K586Q, P587P, D590G, D592G, D594N, D594D, T600S, T600T, V603I, V603A, V603V, A604S, P609S, V612V and / or A616V. In some aspects, evolved PhiC31 integrase have mutant combinations, with wild-type PhiC31 (SEQ ID NO:2) as a reference, said mutant combinations being any of the combinations listed in Table 2.

[0032] Table 2

[0033] X can be any amino acid.

[0034] In some respects, the evolved PhiC31 integrase may also include one or more of the following mutations relative to the wild-type sequence (SEQ ID NO:2): M1E, D2V, D32A, D36A, V41I, and / or D44A. In other respects, the evolved PhiC31 also includes V41I; D32A, D36A, and D44A; or combinations of D32A, D36A, V41I, and D44A.

[0035] In other respects, the evolved PhiC31 integrase may have an N-terminal sequence as shown in SEQ ID NO:2, or may also include an N-terminal amino acid sequence listed in Table 3, which replaces the methionine at position 1 of SEQ ID NO:2.

[0036] Table 3

[0037] In some respects, one or more mutations in the evolved Bxb1 and PhiC31 integrases are amino acid substitutions. Substitutions (such as those specified herein, for example, in Tables 1 and 2) can be conserved or non-conserved, meaning the amino acid residue designated as “X” can be a conserved or non-conserved substitution of a wild-type amino acid residue at the specified position. Conserved amino acid substitutions, in contrast to non-conserved amino acid substitutions, refer to the substitution of one amino acid for another with similar properties, such that the protein's folding or activity is not significantly affected. Interchangeable aromatic amino acids are phenylalanine, tryptophan, and tyrosine; interchangeable hydrophobic amino acids are leucine, isoleucine, methionine, and valine; interchangeable polar amino acids are glutamine and asparagine; interchangeable basic amino acids are arginine, lysine, and histidine; interchangeable acidic amino acids are aspartic acid and glutamic acid; and interchangeable small amino acids are alanine, serine, threonine, cysteine, and glycine. Mutations within the context of this disclosure may also include “silent mutations,” which do not cause changes in the amino acid sequence.

[0038] In some aspects, the evolved Bxb1 integrase or evolved PhiC31 integrase (commonly referred to as "evolved integrase") described herein recognizes (i.e., binds to) an integrase recognition sequence comprising a naturally occurring sequence, i.e., its canonical integrase recognition sequence. In some aspects, the evolved integrase recognizes an integrase recognition sequence in the mammalian genome. In some aspects, the evolved integrase recognizes an integrase recognition sequence in the human genome. In some aspects, the evolved integrase recognizes an integrase recognition sequence that occurs only once in the mammalian genome. In some aspects, the evolved integrase recognizes an integrase recognition sequence in the mammalian genome that differs from any other site in the genome by at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 nucleotides. Variants may share at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the naturally recognized sequence, wherein the variant retains biological activity and thus promotes recombination events in the presence of a suitable integrase. Assays measuring the biological activity of recombination sites are known in the art. See, for example, Senecoll et al. (1988). J. Mol. Biol. 201:406-421; Voziyanov et al. (2002) Nucleic Acid Research 30:7; U.S. Patent No. 6,187,994; and Albert et al. (1995). The Plant Journal 7:649-659.

[0039] In some aspects, the evolved integrase recognition (i.e., binding) is located at the integrase recognition sequence in the safe harbor genome locus. In some aspects, the safe harbor genome locus is the Rosa26 locus. In other aspects, the safe harbor genome locus is the AAVS1 locus. In other aspects, the safe harbor genome locus is the Xq22.1 locus. In some aspects, the evolved integrase recognition is located at the integrase recognition sequence in a genome locus associated with a disease or condition. In other aspects, this disclosure provides the use of lead editing (e.g., dual lead editing) for installing one or more integrase recognition sequences into a designated location in the genome (such as a safe harbor locus) according to the desired application. Introducing two integrase recognition sequences may result in intermediate sequence deletion, intermediate sequence inversion, chromosomal translocation, or cassette exchange, depending on the identity and orientation of the target. By selecting an endogenous sequence that is already very similar to the integrase recognition sequence, the scope of editing required to introduce the complete integrase recognition sequence is reduced. Examples of integrase recognition sequences used in conjunction with the evolved Bxb1 integrase described herein include, but are not limited to, those integrase recognition sequences having the nucleotide sequences of SEQ ID NO: 13-14. Examples of integrase recognition sequences used in conjunction with the evolved PhiC31 integrase described herein include, but are not limited to, those integrase recognition sequences having the nucleotide sequences of SEQ ID NO: 15-16.

[0040] In other respects, the evolved integrase exhibits at least 2-fold, preferably at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold improvement in the integration of the loaded DNA into the target site compared to wild-type integrase (e.g., SEQ ID NO:1 or SEQ ID NO:2).

[0041] In some aspects, a nucleic acid (RNA, DNA, or a combination thereof) encoding any of the integrases described herein is provided. In some aspects, the nucleic acid encoding the integrase is under the control of a heterologous promoter. In some aspects, the encoding nucleic acid is included in an expression construct (e.g., a plasmid, viral vector, or linear expression construct). In some aspects, the nucleic acid or expression construct is in a cell, tissue, or organism.

[0042] Nucleic acids encoding any of the evolved integrase described herein may be in any number of nucleic acid “vectors” known in the art. As used herein, “vector” means any nucleic acid or particle, cell, or organism carrying nucleic acid that can be used to transfer nucleic acid into a host cell. The term “vector” includes both viral and non-viral products and is a construct used to introduce nucleic acid into a cell. “Vectors” may be used in vitro, ex vivo, or in vivo. Non-viral vectors include plasmids, granules, artificial chromosomes (e.g., bacterial or yeast artificial chromosomes), and may contain, for example, liposomes, charged lipids (cytotransfectins), DNA-protein complexes, and biopolymers. Viral vectors include, for example, retroviruses, lentiviruses, adeno-associated viruses, poxviruses, baculoviruses, reoviruses, vaccinia viruses, herpes simplex viruses, Epstein-Barr viruses, and adenovirus vectors. Vectors may also include the entire genome sequence or recombinant genome sequence of a virus. Vectors may also include a portion of the genome that includes functional sequences for producing a virus capable of infecting, entering, or being introduced into a cell to deliver nucleic acid therein.

[0043] The expression of any integrase described herein can be controlled by any regulatory sequence (e.g., promoter sequence) known in the art. As described herein, a regulatory sequence is a nucleic acid sequence that regulates the expression of a nucleic acid sequence. Regulatory or control sequences may include sequences responsible for expressing a specific nucleic acid (e.g., a nucleic acid encoding an integrase), or may include other sequences, such as heterologous, synthetic, or partially synthetic sequences. These sequences may be eukaryotic, prokaryotic, or viral in origin, stimulating or repressing gene transcription in a specific or non-specific and inducible or non-inducible manner. Regulatory or control regions may include origins of replication, RNA splicing sites, introns, chimeric or heterozygous introns, promoters, enhancers, transcription termination sequences, polyA sites, locus control regions, signal sequences guiding polypeptides into target cell secretion pathways, and introns. Heterologous regulatory regions are not naturally associated with the expressed nucleic acid to which they are linked. Heterologous regulatory regions include regulatory regions from different species, regulatory regions from different genes, heterozygous regulatory sequences, and regulatory sequences not found in nature but designed by those skilled in the art.

[0044] The term "operably linked" refers to an arrangement of sequences or regions in which components are configured to perform their usual or intended function. Thus, a regulatory or control sequence operably linked to a coding sequence can influence the expression of the coding sequence. The regulatory or control sequence does not need to be adjacent to the coding sequence, as long as it serves to guide proper expression or peptide production. Therefore, for example, an untranslated but transcribed sequence may exist between a promoter sequence and a coding sequence, and the promoter sequence can still be considered operably linked to the coding sequence. As described herein, a promoter sequence is a DNA regulatory region located shortly from the 5′ end of a gene, acting as a binding site for RNA polymerase. A promoter sequence can bind to RNA polymerase in the cell and / or initiate transcription of the downstream (3′ direction) coding sequence. A promoter sequence can be a promoter capable of initiating transcription in prokaryotes or eukaryotes. Some non-limiting examples of eukaryotic promoters include the cytomegalovirus (CMV) promoter, the chicken β-actin (CBA) promoter, and a heterozygous form of the CBA promoter (CBh).

[0045] Some aspects of this disclosure provide delivery of the evolved integrase described herein (e.g., as an isolated protein or via a nucleic acid encoding the evolved integrase) to a host cell. Some aspects provide a host cell expressing the evolved integrase provided herein, for example, by means of a nucleic acid having an evolved integrase and / or an expression construct. In some aspects, cells are provided that come into contact with the evolved integrase as described herein (e.g., a protein or nucleic acid encoding the evolved integrase), for example, cells in contact in vitro, in vivo, or ex vivo. In some aspects, the cell is a prokaryotic cell, such as a bacterial cell. In some aspects, the cell is *Escherichia coli* (E. coli). E. coli Cells. In some respects, cells are eukaryotic cells, such as yeast cells, insect cells, or mammalian cells, such as hamster host cells, human host cells, rat host cells, or mouse host cells. In some respects, the host cells are Chinese hamster ovary (CHO) host cells, CHO K1 host cells, CHO K1SV host cells, DG44 host cells, DUKXB-11 host cells, CHOK1S host cells, CHO K1M host cells, monkey kidney CV1 line transformed from SV40 (COS-7), human embryonic kidney line (293 or HEK293T), young hamster kidney cells (BHK), mouse podocytes (TM4 cells), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), Buffalo rat hepatocytes (BRL 3A), human lung cells (W138), human hepatocytes (Hep G2), mouse mammary tumors (MMT 060562), TRI cells, MRC5 cells, FS4 cells, YO cells, NSO cells, Sp2 / 0 cells, or PER.C6® cells.

[0046] In some respects, the host cell is a cell line that has been cultured for a certain number of passages. In some respects, the host cell is a primary cell. In some respects, the host cell is a T cell.

[0047] Some aspects also provide pharmaceutical compositions comprising an evolved integrase as provided herein and a pharmaceutically acceptable carrier. In some aspects, the pharmaceutical composition is formulated for administration to a subject. In some aspects, the pharmaceutical composition comprises an effective amount of an evolved integrase for recombination of an integrase recognition sequence in vivo, in vitro, or in vitro in the cells of a subject. In some aspects, the composition further comprises a nucleic acid molecule comprising at least one integrase recognition sequence adjacent to a sequence at a genetic locus to be inserted into the genome of a subject.

[0048] As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or medium, such as liquid or solid fillers, diluents, excipients, manufacturing aids (e.g., lubricants, talc, magnesium stearate, calcium stearate, zinc stearate, or stearic acid), or solvent encapsulating materials, which participate in the transport or delivery of a compound from one site of the body (e.g., site of delivery) to another site (e.g., organ, tissue, or body part). A pharmaceutically acceptable carrier is "acceptable" in the sense that it is compatible with other components of the formulation and harmless to the tissues of the subject (e.g., physiologically compatible, sterile, physiological pH, etc.). Examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, methyl cellulose, ethyl cellulose, microcrystalline cellulose, and cellulose acetate; (4) powdered tragacanth gum; (5) malt; (6) gelatin; (7) lubricants, such as magnesium stearate, sodium lauryl sulfate, and talc; (8) excipients, such as cocoa butter and suppository waxes; 9) Oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) Diols, such as propylene glycol; (11) Polyols, such as glycerol, sorbitol, mannitol, and polyethylene glycol (PEG); (12) Esters, such as ethyl oleate and ethyl laurate; (13) Agar; (14) Buffers, such as magnesium hydroxide and aluminum hydroxide; (15) Alginate; (16) Atherless water; (17) Isotonic saline; (18) Ringer's solution; (19) Ethanol; (20) pH buffer solution; (21) Polyesters, polycarbonates, and / or polyanhydrides; (22) Compatibilizers, such as peptides and amino acids; (23) Serum components, such as serum albumin, HDL, and LDL; (24) C2-C12 alcohols, such as ethanol; and (25) Other non-toxic and compatible substances used in pharmaceutical preparations. Wetting agents, colorants, releasing agents, coating agents, sweeteners, flavoring agents, aromatizers, preservatives, and antioxidants may also be present in the formulation. Terms such as “excipient,” “carrier,” and “pharmaceuticalally acceptable carrier” are used interchangeably herein. In some respects, pharmaceutical compositions are formulated for delivery to subjects, for example, for gene editing.

[0049] Evolved integrase can be provided or packaged in unit dose form. When referring to the use of a pharmaceutical composition, the term "unit dose" refers to a physical discrete unit suitable as a unit dose for a subject, each unit containing a predetermined amount of active material, which is calculated to produce the desired therapeutic effect when combined with a desired diluent (i.e., a carrier or medium).

[0050] In some aspects, a method for altering the sequence of a cell genome is provided. According to this method, a cell is brought into contact in vivo, in vitro, or in vitro with an evolved integrase (e.g., an evolved Bxb1 or PhiC31 integrase protein or a nucleic acid molecule encoding an evolved Bxb1 or PhiC31 integrase), wherein the cell contains at least one integrase recognition sequence recognized by the evolved integrase in its genome, such that the evolved integrase binds to the integrase recognition sequence and alters the sequence of the cell genome. In some aspects, the alteration or change in the genome is a transversion, conversion, deletion, or insertion. In some aspects, the alteration or change in the genome includes the removal of sequences associated with disease or condition, such as mutated forms of genes or integrated viral genomes. In some aspects, the alteration or change in the genome can be a mutation-correction change in a nucleotide sequence, the insertion of a protein or RNA tag, the insertion of an immunoepitope on a protein of interest, the insertion of an inducible dimerizing domain in a protein, and / or the introduction or removal of sequences to alter the activity of a biomolecule. In some aspects, the contact enables the foreign nucleic acid sequence to be integrated into the cell's genome at, for example, a safe harbor locus (e.g., Rosa26, AAVS1, or Xq22.1 locus) or to replace unwanted sequences.

[0051] In some aspects, evolved integrase recognition sequences are combined with lead editing (PE), dual-lead editing, or multi-lobed lead editing methods for genome editing. See, for example, PCT / US2021 / 31439 and PCT / US2022 / 078655. Dual-lead and multi-lobed lead editing involve installing one or more site-specific integrase recognition sequences into a target genomic locus (e.g., target DNA, target gene, target genome, or a specific gene, exon, intron, or regulatory sequence in a target cell). The integrated site-specific integrase recognition sequences can then undergo site-specific recombination in the presence of integrase to achieve large-scale genetic alterations or changes, such as insertions, deletions, inversions, substitutions, and / or chromosomal translocations.

[0052] In some aspects, the insertion of two or more integrase recognition sites can be accomplished using lead editing with a single PEgRNA (multiple single-lobe lead editing) or dual lead editing. For example, in some aspects, a lead editing system comprising a single PEgRNA guides a lead editing system to introduce two or more integrase recognition sites into the target DNA. In some aspects, the lead editing system comprises two or more PEgRNAs, each of which includes a DNA synthesis template, the DNA synthesis template independently including an integrase recognition site. For example, the lead editing system may include a first PEgRNA and a second PEgRNA, wherein the first PEgRNA includes a first spacer complementary to a first target region in the target DNA and a first DNA synthesis template including a first integrase recognition site, and wherein the second PEgRNA includes a second spacer complementary to a second target region in the target DNA and a second DNA synthesis template including a second integrase recognition site, and wherein the first and second target regions are located at different locations in the target DNA.

[0053] Integrase recognition sites introduced through lead editing can be used to produce the desired edits, including deletions, insertions, integrations, and substitutions by the donor sequence. In some aspects, insertions disrupt the expression of protein-coding genes, where the disruption of expression confers a therapeutic benefit in the cell. In some aspects, insertions disrupt the expression of protein-coding genes, where the protein-coding genes have a detrimental effect on the cell or the subject, such as genes with gain-of-function mutations. In some aspects, insertions are performed at genomic locations that allow the expression of the inserted DNA sequence. For example, in some aspects, insertions are performed directly downstream of the promoter of an endogenous gene.

[0054] In some aspects, the inserted sequence encodes a protein or is part of a gene that encodes a protein. For example, in some aspects, the target gene comprises a 5' portion and a 3' portion, where the 3' portion includes a disease-related mutation. In some aspects, the inserted DNA sequence may be downstream of the 5' portion, for example, at the 3' end of an endogenous exon, where the inserted DNA sequence has a wild-type sequence corresponding to the 3' portion of the gene, thereby restoring the wild-type sequence of the gene. In some aspects, the inserted DNA sequence includes a stop codon at the 3' end. In some aspects, lead editing with an evolved integrase replaces the endogenous sequence in the target DNA with an exogenous nucleic acid sequence, or replaces the target gene with an exogenous nucleic acid sequence.

[0055] For example, in some embodiments, the target DNA in the cell genome is a target gene that includes one or more mutations associated with a disease. In some embodiments, the target gene fragment including one or more mutations can be replaced with a foreign nucleic acid sequence having a wild-type sequence corresponding to an endogenous portion of the same gene containing one or more disease-related mutations.

[0056] In some aspects, the inserted exogenous nucleic acid sequence encodes a therapeutic protein. In some aspects, the inserted exogenous nucleic acid sequence is part of a therapeutic gene or a therapeutic gene encoding a therapeutic protein, wherein the target genome includes one or more endogenous copies of the same gene with a mutation. In some aspects, the mutation is a loss-of-function mutation, and expression of the inserted exogenous nucleic acid sequence restores or partially restores wild-type expression of the protein. For example, the therapeutic protein is factor VIII. In some aspects, the therapeutic protein is adult hemoglobin or fetal hemoglobin. Other therapeutic genes encoding therapeutic proteins include genes encoding, for example, steroids, growth factors, insulin, neurotransmitters, dopamine, norepinephrine, epinephrine, histamine, serotonin, checkpoint inhibitor proteins, antibodies, antigen receptor peptides, cell surface marker recognition peptides, chimeric antigen receptors (CARs), T-cell receptors, and proteins involved in protein processing (e.g., degrons or ubiquitin). Other sequences that can be inserted include tissue-specific promoters, enhancers, repressors, or insulators.

[0057] In some respects, evolved integrases are used to treat diseases or symptoms by performing site-specific recombination via evolved integrases to achieve large-scale genetic alterations or changes that delay the development or progression of a disease or reduce its severity. Treating a disease does not necessarily require a cure. As used herein, “delaying” the development of a disease means postponing, hindering, slowing, delaying, stabilizing, and / or slowing the progression of the disease. This delay can have varying lengths depending on medical history and / or the individual being treated. Methods of “delaying” or mitigating the development of a disease or delaying its onset involve reducing the probability of developing one or more symptoms of the disease and / or reducing the degree of symptoms within a given timeframe, compared to not using the method. Such comparisons are typically based on clinical studies using a large number of subjects sufficient to provide statistically significant results.

[0058] The term "development" or "progression" of a disease refers to the initial manifestations and / or subsequent progression of the disease. The development of a disease can be detected and assessed using standard clinical techniques well known in the art. However, development also refers to progression that may be undetectable. For the purposes of this disclosure, development or progression refers to the biological process of symptoms. "Development" includes occurrence, recurrence, and onset.

[0059] As used herein, the term "onset" or "occurrence" of a disease includes initial onset and / or recurrence. Isolated peptides or pharmaceutical compositions may be administered to a subject using conventional methods known to those skilled in the art, depending on the type or site of the disease to be treated.

[0060] Many large-scale genomic alterations or changes (such as gene insertions, deletions, inversions, substitutions, or chromosomal translocations) are associated with hereditary diseases. For example, microdeletions of chromosomes can cause disease, while replacing these deletions with the insertion of key DNA elements can lead to permanent improvement. Additionally, diseases caused by inversions, changes in gene copy number, or chromosomal translocations can be resolved by restoring the previous gene structure in the affected cells. Examples of hereditary diseases associated with large-scale genomic modifications that can be treated with evolved integrase therapies include, but are not limited to, trisomy 17p (gene duplication), Charcot-Marie-Tooth disease type I (gene duplication), Smith-Magenis syndrome (gene deletion), Williams-Beuren syndrome (gene deletion), De la Chapellesyndrome (chromosomal translocation), some forms of Down syndrome (chromosomal translocation), hemophilia A (gene inversion), and Hunter syndrome (gene inversion).

[0061] The following non-limiting embodiments are provided to further illustrate the invention.

[0062] Example 1: Materials and Methods General method. Oligonucleotides and synthetic DNA fragments were purchased from Integrated DNA Technologies. PCR amplification of the DNA fragments was performed using a KOD One™ PCR Mastermix (Toyobo). Plasmids were constructed using NEBuilder® HiFi DNA Components or USER® Cloning (New England Biolabs) and purified using the Zyppy™ Plasmid Micropreparation Kit (Zymo). DNA for transfection was purified using the Zymopure™ Plasmid Micropreparation Kit (Zymo), Zymopure™ Expression Plasmid Micropreparation Kit (Zymo), or Qiagen® Plasmid Micropreparation Kit (Qiagen).

[0063] Mammalian expression plasmids.Helper plasmids for mammalian expression, named pCMV2 PhiC31 wt, pCMV2 HuOpt Bxb1, and pCMV2 HuOpt Pa01, encode wild-type PhiC31, Bxb1, and Pa01 integrases, respectively. The wild-type protein sequences of Bxb1 and PhiC31 are provided herein as SEQ ID NO:1 and SEQ ID NO:2, respectively. Hyperactive mutations in the PhiC31 and Bxb1 integrases were incorporated into pCMV2 PhiC31 wt and pCMV2 HuOpt Bxb1 for genome targeting experiments in mammalian cells. Originally proposed by Keravala et al. (2009) Mol. Ther. The hyperactive mutation identified in 17(1):112-20) was also incorporated into pCMV2 PhiC31 wt to generate plasmids named pCMV2 PhiC31 P1, pCMV2 PhiC31 P2, and pCMV2 PhiC31 P3. The donor plasmids contain att sites and payload DNA for genomic insertion. The donor plasmids pCMV PhiC31 attB DsRed Hygro, pCMV Bxb1 attP DsRed Hygro, and pCMV Pa01 attPDsRed Hygro contain att sites for PhiC31, Bxb1, and Pa01 integrases, respectively. Each donor plasmid contains DsRed fluorescent protein and a hygromycin resistance gene. In addition to DsRed and hygromycin, the donor plasmids pCDNA4 FVIII Bxb1attP HIR Donor and pCDNA3.1-WT-VWF Bxb1 attP HIR Donor contain the genes for therapeutic factor VIII and von Willebrand factor (vWF), respectively. The PE5max plasmid encodes Streptococcus pyogenes (…). S. pyogenes Cas9 (SpCas9) cleavage enzyme, which is fused with a reverse transcriptase for PE (Chen et al. (2021)). Cell 184:5635-5652). Plasmids used to express epigRNA include spacer sequences that direct the SpCas9 nickase to genomic target sequences (Anzalone et al. (2022)). Nat. Biotechnol. 40:731-740; Nelson et al. Nat. Biotechnol. 402-410). The epegRNA expression plasmid includes a reverse transcription template (RTT) targeting the att site, which is incorporated into the target sequence via PE. The epegRNA expression plasmid is used to perform PE insertion at the att sites of the human genome target sequences AAVS1, Xq22.1, and ROSA26. Helper plasmids, donor plasmids, and epegRNA plasmids are made available via Addgene.

[0064] Mammalian cell culture. HEK293 (ATCC CRL-1573) and HEK293T (Thermo Fisher Scientific) cells were maintained in Durbecco's Modified Eagle Medium (DMEM) + GlutaMAX™ (Gibco), HeLa cells (Sigma) were maintained in Eagle's Minimum Essential Medium (EMEM) (ATCC) containing L-glutamine, and K562 cells (Sigma) were maintained in RPMI-1640 medium (Sigma). All media were supplemented with 10% heat-inactivated fetal bovine serum (FBS; Neuromics), 1x penicillin / streptomycin (Gibco), and 0.75 μg / mL amphotericin B (Gibco). Phosphate-buffered saline (PBS), trypsin, and Opti-Mem™ were purchased from Gibco. Primary hepatocytes (Xenotech) were thawed using the OptiThaw Hepatocyte Kit, plated in OptiPlate medium, and cultured in OptiCulture medium (Xenotech).

[0065] Transfection. HEK293, HEK293T, and K562 cells were transfected with TransIT®-2020 (Mirus Bio). Primary hepatocytes were transfected with TransIT®-X2 dynamic (Mirus Bio). HeLa cells were transfected with HeLaMONSTER® (Mirus Bio).

[0066] Fluorescence-activated cell sorting (FACS). All donor plasmids constitutively expressed DsRed. For experiments requiring sorting of transfected cells and exclusion of untransfected cells, including those transfected with selected HEK293T as well as HeLa and K562, cells were sorted based on DsRed fluorescence intensity (excitation 488 nm, emission 610 nm) using a FACSAria™ IIu flow cytometer (BD Biosciences). Cells were immediately precipitated and lysed for droplet digital PCR analysis.

[0067] Cell lysis.Cell pellet was lysed by adding 90 μL of DirectPCR® cell lysis reagent (Viagen Biotech) and 1.0 mg / mL of freshly prepared proteinase K (Meridian Bioscience), and then incubated by rotation at 55°C for 3 hours in a hybridization oven. The cell lysate was then heat-inactivated at 85°C for 45 minutes and stored at -20°C before assay.

[0068] Cloning the HEK293 cell line. Double-leader editing was used to generate single-cell clones containing the att site at either the adeno-associated virus integration site 1 (AAVS1) locus or the reverse splice acceptor 26 (ROSA26) locus. HEK293 cells were transfected in 24-well plates with 450 ng of PE5max and 150 ng of each epigRNA plasmid (a and b). Cells were serially diluted and visually confirmed to have a single colony per well. The presence of the att site was confirmed by droplet digital PCR. Clones containing the pre-inserted att site were transfected with 360 ng of helper plasmid and 240 ng of donor plasmid. After 3 days, cells were lysed for analysis by droplet digital PCR.

[0069] Simultaneous dual-leader editing and integration assays in HEK293, HEK293T, HeLa, K562, and primary hepatocyte cell lines. HEK293, HEK293T, and HeLa cells (1 x 10⁻⁶) were used. 5 K562 cells (4 x 10⁻⁶ cells) were seeded in 24-well plates. The next day, the wells were transfected with 300 ng of PE5max, 30 ng of each epeRNA, 120 ng of helper plasmid (integrase), and 120 ng of donor plasmid. 6 (Number of cells) were seeded in 6-well plates. After 3 days, cells were lysed for analysis. All donor plasmids expressed DsRed. To minimize the impact of transfection efficiency, HEK293T, K562, and HeLa cells were sorted for DsRed to ensure DNA uptake.

[0070] Sample preparation for amplicon sequencing.HEK293 cells were transfected with 600 ng of PE5max and 60 ng of each epigRNA plasmid (a and b). Three days later, cells were lysed by incubating the pellet with 90 μL of freshly prepared 1.0 mg / mL proteinase K (Viagen Biotech) direct lysis buffer and then rotating at 55°C for 3 hours. The cell lysates were heat-inactivated at 85°C for 45 minutes. To confirm the editing results using amplicon sequencing, a first round of PCR (PCR1) was performed using KOD One™ blue polymerase (Toyobo), 0.3 μM of locus-specific barcoded primers containing Nextera™ P5-tags and P7-tags (Table 4), and 1 μL of cell lysates in a total volume of 20 μL. An 8 bp barcoded sequence was inserted between the Nextera™ tag sequence and the primer annealing sequence. PCR cycling conditions were 98°C for 2 min, followed by 30 cycles of 98°C for 10 sec, 60°C for 5 sec, and 68°C for 5 sec, with a final extension at 68°C for 30 sec. Products from PCR1 were separated on a 2% gel in TAE (Tris-acetate-EDTA) buffer, and fragments were extracted using the Zymo™ Gel DNA Recovery Kit (Zymo). Equimolar amounts of the eluted products were pooled and sent to the UC Davis DNA Technologies and Expression Analysis Core laboratories for a second round of PCR (PCR2) and amplicon sequencing.

[0071] Table 4

[0072] Analysis of amplicon sequencing data. First, we use Ultraplex (Wilkins et al. (2021)). Welcome Open Res. 6:141) Barcode-based demultiplexing of amplicon sequencing results allows for one barcode mismatch. Barcode sequences are pruned, and low-quality reads are discarded using a q-score of 20 (default is 30). To sort forward and reverse reads in the same order, the sequences are re-paired using the repair function in BBtools. Edit and insertion / deletion frequencies are determined as previously described (Doman et al. (2022)). Nat. Protoc(17:2431–2468). In short, run CRISPResso2 in HDR mode, enable the discard_indel_reads flag, and set the quantification window coordinates to 10 base pairs upstream of each nick site. Input unedited template and PE sequences. Use data from the "CRISPResso_quantification_of_editing_frequency" file to determine the editing frequency by dividing "Read_aligned" (in the HDR tab) by "Reads_aligned_all_amplicons". Determine the insertion / deletion frequency by dividing the total discarded reads (reference + HDR) by "Reads_aligned_all_amplicons". Examples of leader edited sequences and primers are provided in Table 5.

[0073] Table 5

[0074] Droplet digital PCR (ddPCR) protocol. Perform ddPCR according to the manufacturer's instructions (BioRad Laboratories). In short, each reaction (22 μL) contains 1.0 μL of cell lysate (1:1 diluted in water), 11 μL of 2X probe ddPCR premix (ddPCR Supermix for Probes) (dUTP-free), and 10 U... Xho I (New England Biolabs), 900 nM primers for the target and reference (Table 6), and 250 nM probe. The plate was sealed with a PX1 PCR sealing film (BioRad), vortexed thoroughly to mix, and centrifuged at 1000 rpm for 1 min. Droplets were then generated using a QX200 AutoDG droplet generator system (BioRad). PCR was performed as follows: 45 cycles of denaturation at 95°C for 10 min, followed by 30 sec at 95°C, 2 min at 60°C, and 2 min at 72°C, followed by a final incubation at 98°C for 10 min. Droplets were read using a QX200 droplet reader (BioRad) and analyzed using QX Manager 1.2 Standard Edition software (BioRad).

[0075] Table 6

[0076] Bacteriophage-assisted continuous evolution. PACE achieves selection by associating the desired activity of a gene of interest with the production of infectious phage progeny. Phage gene III (gIII) expression is proportional to proliferation. Using helper plasmids (AP), gIII expression is driven solely by the desired activity of the evolving gene. Each selected phage (SP) encodes the gene to be evolved. Phages encoding active variants of the gene produce infectious progeny, while phages encoding less active variants do not produce as many progeny and are competitively eliminated. Beneficial mutations multiply with the lifespan of successful phages. Inducible mutagenic plasmids (MP) increase the mutation rate by reducing proofreading during replication and increasing error-prone bypasses.

[0077] During PACE, host cells containing AP, XP, and MP are sequentially added to fixed-volume flasks ("lagoons") where they encounter phages. Following infection, host cells begin expressing integrase proteins from SPs. Successfully evolved integrases carry beneficial mutations that enhance their activity at the attP site, aligning the gIII promoter on AP. Activated gIII expression causes SPs encoding the improved integrase to proliferate. These SPs are mutated again in the next cycle of DNA replication, efficiently generating new libraries from the most suitable variants of the previous library. Fresh host cells are then infected with this new library from the lagoon, and the process is repeated. Phage proliferation increases with increasing gIII expression levels, ranging from two orders of magnitude. This establishes competition among the mutant library members, allowing the most active variants to outperform the others.

[0078] PACE strategy and selection rigor. In addition to the MP used for mutagenesis, the host cell also contains AP and CP to enable selection of integrase with improved activity. AP contains an attP site upstream of the C-terminus of gIII. CP contains a promoter aligned with the N-terminus (leader sequence) of gIII and an attB site. The att sites on AP and CP form a single plasmid with intact gIII through successful crossover of the active integrase variant. The resulting attL site is encoded and located in the linker region between the leader sequence and the N1 domain to prevent disruption of gene III activity. During each PACE experiment, strictness is periodically increased by reducing promoter strength, decreasing plasmid copy number, or increasing flow rate.

[0079] Phage preparation and mutagenesis. The SP backbone was modified by replacing the VI promoter with RecA to reduce the production of recombinant wild-type M13 phages. This resulted in the reaction pool being infected with 10... 8The starting phage for PFU was used. Mutagenesis was induced via the MP6 plasmid at a concentration of 100 mM arabinose. The reaction chamber was sampled every 1–4 hours in each PACE experiment.

[0080] PACE procedures, such as Miller et al. (2020) Nat. Protoc. As described in 15(12):4101-4127), and modified. In particular, the modified PACE setup includes two chemostats maintained in 2 L flasks at a volume of 300-400 mL, with the host cell culture being changed every 2 days to ensure arabinose sensitivity.

[0081] S2060 cells were transformed with AP, XP, and MP6 plasmids and cultured in Davis RichMedia (DRM) (Harvard-customized media A and C-US Biological) supplemented with trace metals, 0.1% polysorbate 80, and appropriate antibiotics for plasmid maintenance (AP - 50 μg / mL carbenicillin, CP - 50 μg / mL kanamycin, MP - 25 μg / mL chloramphenicol, and 10 μg / mL tetracycline, 10 μg / mL fluconazole, and 10 μg / mL amphotericin B). The host cell colonies were tested for arabinose sensitivity before amplification to a chemostat. The two cultures were mixed using a fixed-volume 100 mL mixer in a 500 mL flask, and then dispensed into 2–6 reaction chambers, which were typically maintained at 40 mL in the 100 mL flask (except when using extremely high flow rates to increase stringency), with flow rates ranging from 1–10 V / h. L-arabinose was supplied to the reaction chambers at a constant rate of 0.5 μL / min (concentration depending on flow rate to maintain 100 mM L-arabinose in the reaction chambers). In experiments with 6 reaction chambers, a second chemostat was added along with the mixer maintained at 100 mL before dispensing into the reaction chambers (Tables 7 and 8).

[0082] Table 7

[0083] Table 8

[0084] bacteriophage preparation and mutagenesis The SP backbone was modified by replacing the VI promoter with RecA to reduce the production of recombinant wild-type phages. The reaction pool was then infected with 10... 8The starting phage for PFU was used. Mutagenesis was induced via the MP6 plasmid at a concentration of 100 mM arabinose. The reaction chamber was sampled every 1–4 hours in each PACE experiment.

[0085] Phage enrichment assay. Use 10 per milliliter 5 Purified M13 phage of PFU was inoculated into an E. coli culture containing both AP and CP (2 mL, OD). 600 = 0.3-0.6), and incubated at 37°C and 180 rpm for 18 hours. The culture was centrifuged at 12,000 xg for 2 minutes, and the supernatant was collected. Phage counts were obtained by non-activity-dependent plaque assay in S2208 cells (Badran et al. (2016)). Nature 533:58–63).

[0086] Flipper Flow Cytometry Assay. A helper plasmid (100 ng) encoding an integrase variant and a flipped plasmid (500 ng; Table 9) containing ZsGreen flanking attB and attP sites were transfected into 24-well plates seeded with HEK293T cells as described above. After 3 days of culture, the cells were resuspended in Live Cell Imaging Solution (Thermo Fisher Scientific), and fluorescein-5-isothiocyanate (FITC) emission was assessed by flow cytometry (excitation 488 nm, emission 530 nm (BL1)) of >20,000 live single cells under each condition.

[0087] Table 9

[0088] 1 Groth et al. (2009) Proc. Natl. Acad. Sci. USA 97:5995–6000; 2 Ghosh et al. (2003) Mol. Cell 12(5):1101-11; 3 Durrant et al. (2023) Nat. Biotechnol .41:488–499.

[0089] The basic principle of standardization using the product / reference ratio (PRR).There are two options for target probe localization: (1) the probe can be placed in the donor, with the forward primer in the donor and the reverse primer in the genome, or (2) the probe can be placed in the genome, with the forward primer in the donor and the reverse primer in the genome. The problem with donor-localized probes is the insertion at off-target sequences, placing the probe and reverse primer downstream of an unknown genomic sequence. Incorrect initiation from the genome can produce spurious signals. A second problem is that donor-localized probes cannot be used to determine the ratio of potential target sequences to reference sequences. If there are more target sequences than reference sequences, there are a greater number of targeting opportunities, and the final targeting efficiency will be overreported. This is especially important to consider when testing cell lines with abnormal chromosome numbers where the reference sequence is often located on a different chromosome than the target sequence. To prevent overreporting or underreporting of ddPCR, the ratio of target product to reference signal is defined as the product / reference ratio (PRR). The targeting efficiency of unknown samples is divided by the PRR to normalize for the difference between available targets and references. To determine the PRR, untransfected cell lines were assayed using FAM probes designed to bind genomic target sequences. A reverse primer was located in the genome, and a second PRR-optimized primer was also located in the genome and flanked by the probe. ddPCR was run using a standard HEX probe designed to bind a genomic reference sequence, flanked by genomic primers. The PRR values ​​for the target sequences used in this study are provided in Table 10. The template was lysate (n=3) isolated from wells of untransfected cells from the specified cell lines. The HEK293 clone was first edited using a leader to insert into the specified att site. The probes were designed to bind the genomic target sequence. Primers for the HEK293 clone included a forward primer at the att site and a reverse primer in the genome. Primers for the remaining cell lines included both a forward and reverse primer in the genome.

[0090] Table 10

[0091] Off-target analysis. Genomic PCR primers were generated for the previously predicted Bxb1 pseudosite (Table 11) (Anzalone et al. (2022)). Nat. Biotechnol. 40:731–740; Yarnall et al. (2023) Nat. Biotechnol(41:500–512). For each site, two primer sets were designed. The first set (control) was used to amplify the genome to verify that sufficient template was used. The second set was used to amplify the ligation between the genome and the donor. Additional primers were generated to amplify the successful insertion at ROSA26. Lysates from HEK293T transfections performed on two separate days were used as templates for genomic PCR using Q5 polymerase (NEB) and visualized on 2% agarose gels.

[0092] Table 11

[0093] Factor VIII and von Willebrand factor. To measure the expression and output of recombinant proteins, HEK293T cells were transfected with 300 ng of PE5max, 30 ng of each epigRNA plasmid, 120 ng of helper plasmid (integrase), 120 ng of donor plasmid, and 40 ng of DsRed expression plasmid (for sorting). Culture medium was collected three days post-transfection, and factor VIII protein expression was measured using the Human Coagulation Factor VIII Total Antigen ELISA kit (Innovative Research), while factor VIII protein activity was measured using the Chromogenix Coamatic® Factor VIII Kit (DiaPharma). Von von Willebrand factor was detected using the VWF Human ELISA Kit (Invitrogen).

[0094] Example 2: Directed Evolution of Serine Integrases Improves Site-Specific Insertion of Transgenes Using Leader Editing Previously, directed evolution was used to improve the activity of PhiC31 integrase at its native att site (Keravala et al. (2009)). Mol. Ther 17(1):112-20). It was hypothesized that PhiC31 and its improved variants could increase the insertion efficiency of targeted integrases. Initially, previously characterized safe harbor loci were selected, which are distant from cancer-related genes and showed permission for the expression of the inserted genes. These sites included AAVS1 (Oceguera-Yanez et al. (2016)). Methods 101:43-55), Xq22.1 (Chalberg et al. (2006) J. Mol. Biol . 357(1):28-48) and ROSA26 (Irion et al. (2007) Nat. Biotechnol.25:1477–1482). PE uses a fusion protein consisting of a Cas9 nicking enzyme and a reverse transcriptase called PE5, as well as a lead editing guide RNA (pegRNA). The pegRNA is a modified version of a single guide RNA (sgRNA) that both directs PE5 to the target genomic sequence and encodes the desired editing information. The pegRNA contains a 5' spacer sequence that guides PE5 to form a nick at the target sequence. A primer binding site (PBS) located at the 3' end of the pegRNA anneals to the non-target DNA strand after the nick in the target strand. The pegRNA contains a reverse transcriptase template (RTT), which is reverse transcribed to produce a 3' single-stranded DNA flap encoding the inserted DNA (such as an att site). During double PE, a second pegRNA guides the insertion of adjacent flaps, including overlapping homologous sequences, such that the two flaps form a double-stranded intermediate. After the intermediate is resolved, the new sequence becomes a permanent insertion.

[0095] Because shorter inserts are generally more efficient at leading edits, pegRNAs are designed for insertion into very small PhiC31 attB sites (35 bp) or attP sites (41 bp). To maximize efficiency, pegRNAs containing structured RNA motifs are used to prevent degradation (Nelson et al. (2022)). Nat. Biotechnol. 40(3):402-410). Furthermore, an optimized CR772 stent was used (Jost et al. (2020)). Nat. Biotechnol . 38(3):355-364) and PE5max, PE5max includes dominant-negative mutants of human mutL homolog 1 (MLH1) involved in DNA mismatch repair and optimized editor architecture (Chen et al. (2021)). Cell 184(22):5635-5652.e29) (Tables 15 to 16). PE5max and two biepegRNA expression plasmids for each target site were transfected into HEK293 cells. Droplet digital PCR (ddPCR) is a form of quantitative PCR that reports highly accurate copy numbers of genomic inserts at a wide range of template concentrations.

[0096] As measured by amplicon sequencing using genomic primers with side-insertion inserts ( Figure 1Furthermore, ddPCR confirmed that the lead editing insertion at the att site ranged from 0.3% to 62.1%. A cloned HEK293 cell line was generated, comprising the most efficient and accurate epegRNA pairs targeting AAVS1 and ROSA26, which were used to compare the integration efficiency of PhiC31 integrase variants. Wild-type PhiC31 integrase was compared with three highly active variants designated P1, P2, and P3 (Keravala et al. (2009)). Mol. Ther . 17(1):112-20). By ddPCR, two of the three variants (P1 and P3) showed increased activity of integrase relative to wild-type at two loci, with P3 achieving integration efficiencies of 11.1% and 3.0% at ROSA26 and AAVS1, respectively. Figure 1 Instead of using cell lines containing pre-inserted att sites, PE and integrase expression plasmids were combined in a single transfection. This strategy allowed for insertion of the att site using PE, followed by insertion of the donor plasmid at the target sequence via PhiC31 integrase. PE5max and epigRNA plasmids were co-transfected to insert a 41 bp PhiC31 attP site at ROSA26, along with a 6.6 kb donor plasmid containing the attB site and an auxiliary plasmid expressing PhiC31 integrase. Notably, PhiC31 insertion of the donor plasmid was detected in all four integrase variants, with a favorable efficiency of 1.9% (P3) compared to 0.54% in the wild-type. This indicates that the hyperactive variants outperformed the wild-type PhiC31 integrase during PE-mediated gene insertion. Based on these results, it is inferred that directed evolution of PhiC31 variants could further improve gene insertion efficiency.

[0097] Previous directed evolution experiments that produced P1, P2, and P3 required cumbersome steps of mutagenesis, gene expression, selection, and replication. Alternatively, PACE can rapidly and iteratively select, replicate, and mutate genes at a rate of >70 generations per day without human intervention (Esvelt et al. (2011)). Nature 472:499–503). PACE achieves selection by associating the desired activity of the gene of interest with the production of infectious phage progeny.

[0098] Gene III (gIII) expression in M13 phage is proportional to proliferation. Using helper plasmids (APs), gIII expression is driven solely by the desired activity of the evolving gene. Each selected phage (SP) encodes a gene to evolve. Phages encoding active variants of the gene produce infectious progeny, while phages encoding less active variants do not produce as many progeny and are competitively eliminated. Beneficial mutations multiply with the lifespan of successful phages. Inducible mutagenic plasmids (MPs) increase the mutation rate by reducing proofreading during replication and increasing error-prone bypasses. During PACE, host cells containing APs, CPs, and MPs are sequentially added to fixed-volume flasks (called “reaction pools”) where they encounter phages. After infection, host cells begin expressing integrase proteins from the SPs. Successfully evolved integrases carry beneficial mutations that enhance their activity at the attP site, aligning the gIII promoter on the AP. Activated gIII expression enables the SPs encoding the improved integrase to proliferate. These SPs mutate again in the next cycle of DNA replication, effectively generating a new library from the most suitable variant of the previous library. Fresh host cells are then infected with this new library in the reaction pool, and the process is repeated. Phage proliferation increases with increasing gIII expression levels, ranging from two orders of magnitude. This establishes competition among the mutant library members, allowing the most active variant to outperform the others.

[0099] To adapt PACE for improved integrase activity, an integrase is encoded on a selected phage (SP), and pIII expression is associated with a successful recombination event between the attB and attP sites. The promoter and the amino terminus of gene III (gIII encodes pIII) (called the leader sequence), followed by the attP site, are placed on a complementary plasmid (CP). The helper plasmid (AP) contains the attB site and the remainder of gIII (the N1-C domain). During the attB-attP recombination event, the promoter and leader sequence align with gIII, subsequently inducing expression. The resulting attL site is encoded within a flexible loop to avoid disrupting pIII function. A successful event requires precise and directed recombination at the attL site.

[0100] PACE experiments were initiated with SPs encoding PhiC31, P1, P2, and P3, with progressively increased stringency by decreasing gIII promoter strength and increasing flow rate, and run for 212 hours (222 evolutionary cycles). Different host cells containing CPs with four gIII promoters were sequentially added to reaction chambers. Evolved integrases were challenged using promoters ProB, ProA, Pro3, and Pro1, in order of increasing strength from strongest to weakest. Because each recombination event produces low levels of pIII when using weak gIII promoters, only highly active integrase variants capable of recombinating more plasmids than less active variants could produce sufficient pIII to generate enough infectious progeny to maintain in the system. Less active integrase variants could not undergo enough recombination events to produce a sufficient number of progeny and were expelled from the system. To further challenge the evolved integrase, the flow rate was increased from 2 reaction chamber volumes per hour to 3, reducing the time available for integrase variants to generate progeny. The presence of phages in each reaction chamber was monitored by collecting culture effluent and performing an inactivity-dependent plaque assay. Diluted phage-containing medium was plated onto *E. coli* strain S2208, producing blue plaques. Phage concentration was calculated by counting these plaques. For each of the four host cell transfers, effluxed medium was collected at time points 0, 1, 2, and 4 hours, and every 4 hours thereafter. The expected pattern of an initial decrease in titer was observed as many early phages were effluxed. The titer then gradually increased, presumably due to the accumulation of beneficial mutations in the integrase variants. Subsequently, the phage number stabilized, possibly because the integrase variants reached an overactivity point, rendering them no longer challenged by their current stringency level, thus requiring increased stringency to continue productive evolution. Phages isolated at various time points throughout the InterPACE experiment were selected, and recombinant activity was measured individually using an overnight phage enrichment assay. The titer was calculated using 10-1 phages per mL. 5Purified phages isolated from IntePACE were inoculated into gIII cells containing AP and CP using the ProA promoter. Cells were cultured overnight in the presence of phages, and phage concentrations were measured the following day using a non-activity-dependent plaque assay similar to that described above. It was expected that phages containing a variant encoding a highly active integrase would recombine more AP and CP and produce more phage progeny compared to phages encoding an unevolved integrase. This method allows for direct comparison of individual time points. As measured by the overnight phage enrichment assay, the evolved library showed 15.4-fold, 70.2-fold, and 16.4-fold increased efficiency compared to P1, P2, and P3, respectively. To test activity in human cells, evolved integrase variants were isolated from single plaques and cloned into expression plasmids. A plasmid-based 'flipper' assay was designed where a recombination event between the attP and attB sites flips the orientation of ZsGreen, aligning it with the H1 promoter and activating expression. Consistent with the results for E. coli, an increase in the number of cells expressing the evolved integrase ZsGreen was observed, with the greatest improvement induced by P2, from 9.9% to 44.9%.

[0101] Evolved variants were compared in cloned HEK293 cell lines with pre-inserted PhiC31 attP sites at the AAVS1 or ROSA26 locus (Table 12). Notably, the evolved integrase gene insertions outperformed their initiating variants. The greatest improvement was between P2 (1.3%) and the 36-hour evolved variant P2-L2-3 (11.8%). The highest integration rate came from the P3 variant (L1-2), which integrated 18.4% at the ROSA26 site. Figure 2 Similarly, evolved integrases co-transfected with the lead editing component used for attP insertion yielded higher gene targeting efficiencies. Specifically, the 36-hour variant evolved from P2 (L1-10) increased by 10.5-fold, and the 184-hour variant from P3 (L1-13) achieved 4.2% efficiency at the ROSA26 target sequence (an increase from 1.9% for the P3 initiating variant). Figure 3 ).

[0102] Table 12

[0103] 1 Keravala et al. (2009) Mol. Ther. 17(1):112-120.

[0104] 2The amino acid position is referenced to the wild-type PhiC31 protein (SEQ ID NO:2) with amino acid 605.

[0105] Using optimized lead editing parameters for inserting the att site, the integration rate of evolved PhiC31 was compared with that of the previously described integrase Bxb1 (Anzalone et al. (2022)). Nat. Biotechnol . 40:731–740) and Pa01 (Durrant et al. (2023) Nat. Biotechnol A comparison was made between wild-type Bxb1 and the evolved PhiC31 mutant (41:488–499). Surprisingly, wild-type Bxb1 showed greater efficiency compared to the evolved PhiC31 mutant. Figure 4 There is reason to believe that Bxb1 may serve as a good starting point for evolution. Inspired by the results of PhiC31, the same PACE strategy was adopted to increase the activity of the Bxb1 integrase at its native att site. The complete Bxb1 attP site and the minimal attB site (Ghosh et al. (2003)). Mol. Cell 12(5):1101-11; Anzalone et al. (2022) Nat. Biotechnol. 40:731–740) replaced the PhiC31 att site in AP and CP, respectively (Tables 7 and 8). The phage evolved for a total of 9 days (approximately 276 mutagenesis cycles) (Esvelt et al. (2011)). Nature 472:499–503).

[0106] Similar to PACE in the case of PhiC31, enrichment assays confirmed that evolutionary variants of Bxb1 were significantly improved in bacterial systems. The new variants (Table 13) were cloned into mammalian expression plasmids and then co-transfected with donor plasmids into the cloned HEK293 cell line containing a pre-inserted Bxb1 attB site at ROSA26. The highest-activity variant (Bxb1-L8-5) was observed to have an efficiency of up to 21%, 2.5 times that of wild-type Bxb1 integrase. Simultaneous lead editing and evolutionary Bxb1 integrase-mediated gene insertion produced efficiencies ranging from 2.1% to 35.4%, representing a 3.1-fold increase in efficiency compared to wild-type Bxb1. Figure 5A and Figure 5B Several additional rounds of evolution did not produce variants with increased efficiency.

[0107] Table 13

[0108] Five mutants of the best-performing Bxb1 integrase variant isolated from PACE were assembled in all possible combinations to generate new mutants. To compare the activity between PE-independent mutants, helper plasmids encoding the combined mutants were co-transfected with donor plasmids into the cloned HEK293 cell line with a pre-inserted attB site at ROSA26. Significantly, the combined mutants increased activity by up to 9.2-fold compared to wild-type Bxb1 integrase. Figure 6 Multiple synergistic and antagonistic effects were observed. Among the five mutations, I87L and H95Y had the greatest individual impact on integration efficiency (6.4 and 7.1 times higher than wild-type Bxb1 integrase, respectively), but their combined effect was poor (4.7 times higher than wild-type Bxb1 integrase). The individual mutations A369P and E434G had a moderate effect on integration activity, but showed a synergistic effect with each of the other mutations tested (including V122M), with V122M alone reducing activity relative to wild-type Bxb1 integrase.

[0109] Subsequently, by co-transfecting the PE plasmid with a combinatorial mutant helper plasmid and a donor plasmid, PE was used to insert the attB site at ROSA26 in HEK293 cells. When combined with PE, the combinatorial mutant exhibited up to a 6-fold increase in activity compared to the wild-type Bxb1 integrase. Figure 7 Different activity patterns of the combined mutants were observed between experiments using and without PE (Table 14). For example, Bxb1-c25 exhibited high activity in clonal cell lines but low activity when paired with PE, indicating that the mutation negatively impacts PE, thus reducing the integrase insertion efficiency despite being hyperactive. Similarly, in all cases of the H95Y and V122M combination, PE efficiency was significantly reduced (2.3 to 4-fold relative to wild-type Bxb1 integrase). It has been previously shown that the att site found on the epigRNA expression plasmid can recombine with the att site on the donor plasmid, rendering both plasmids ineffective. This was expected to reduce the amount of epigRNA available for PE and the number of donor plasmids available for integrase insertion. To mitigate the impact of Bxb1 integrase recombination of these plasmids, the epigRNA expression plasmids used in this study contain a partial att site. During double PE, although each inserted 3' single-stranded lobe contains only a portion of the att site, sufficient overlapping sequences exist to form a double-stranded intermediate. The remaining single-stranded sequence at the at site can then be padded to encourage insertion into the complete att site. While this strategy appears sufficient to prevent unwanted wild-type Bxb1 integrase recombination, it is speculated that certain hyperactive integrase mutants acquire activity at partial att site sequences and recombine epegRNA expression plasmids, which could explain the reduced PE efficiency.

[0110] AlphaFold modeling of the Bxb1 integrase as a dimer revealed that the three mutants I87L, H95Y, and V122M are very close to each other in terms of their catalytic domains. The mutants A369P and E434G are located together near the predicted leucine zipper motif, suggesting that they may affect dimerization.

[0111] Compared to HEK293 cells, HEK293T cells showed a higher transfection rate. Re-testing of the optimal variant using HEK293T cells yielded an insertion efficiency as high as 44.1%, with the combinatorial mutant outperforming the parental variant. Figure 7 Furthermore, no off-target insertions were detected at the predicted Bxb1 spurious site by genomic PCR (Anzalone et al. (2022)). Nat. Biotechnol .40:731–740; Yarnall et al. (2023) Nat. Biotechnol. (41:500–512). PACE evolutionary combination mutants were used, but no variants with further increased efficiency were recovered.

[0112] Programmable addition via a site-specific targeting element (PASTE) system has been described, which uses a single PEG leader edit and the fusion of Bxb1 with nCas9-RT (Yarnall et al. (2023)). Nat. Biotechnol. 41:500–512). Similar to others, low lead editing efficiency was observed using a single pegRNA (Wang et al. (2022)). Nat. Methods 19(3):331-340; Anzalone et al. (2022) Nat. Biotechnol (40:731–740), where PASTE leader editing and integration into the ROSA26 locus reached 11.2% and 2.4%, respectively. PASTE was improved by incorporating dual-leader editing pegRNAs, and the fused integrase architecture was indeed superior to conventional dual-leader editing using wild-type Bxb1. However, neither conventional PASTE nor dual-leader edited PASTE reached the level of evolved Bxb1 integrase. Several hyperactive mutations were introduced into PASTE and dual-leader edited PASTE, but no further increase in efficiency was observed.

[0113] To eliminate the influence of incomplete transfection efficiency, the optimal evolutionary variant was determined in HEK293T cells, and sorting was performed based on DsRed expression. These optimized conditions resulted in an insertion rate of greater than 80% at the target site. Figure 8 In a therapeutic context, considering that normal cells contain target sites on each of the two chromosomes, this efficiency means that the probability of a cell accepting at least one insertion is >96%.

[0114] The evolved Bxb1 variant was active in HeLa and K562 cells. Surprisingly, while the lead editing was effective (>40%) in HeLa cells, integration was not detected using wild-type Bxb1 integrase. However, the evolved integrase successfully integrated, albeit at low levels. In K562 cells, the evolved integrase performed 11.2-fold better than wild-type Bxb1 (2.7% vs. 30.3%). Figure 9 and Figure 10 ).

[0115] Large-load integrase insertion will enable therapies that are currently impossible with conventional methods. Specifically, full-length human factor VIII (FVIII) gene (7kb cDNA) and von Willebrand factor (VWF) gene (8.5kb cDNA) are unsuitable for HDR, AAV, or lentiviral delivery. Lead editing and integrase insertion of 14kb FVIII (50.5%) and 15.7kb VWF (36.4%) expression loads were measured, and expression was validated by ELISA and factor X activation. Figures 11 to 13 ).

[0116] The active insertion induced by the integrase is independent of host repair and highly specific for att sequences not present in the unmodified human genome. Importantly, the efficiency of targeted insertion using evolved integrase and lead editing has been demonstrated to surpass current methods. A key finding is that, under pre-optimized transfection conditions, the evolved integrase significantly outperforms wild-type Bxb1 (>6-fold) (Table 14), suggesting that this technique is suitable for in vivo gene delivery, where tissue transfection efficiency may be challenging.

[0117] Table 14

[0118] Table 15

[0119] Table 16

[0120] cr772 stent: GTTTAAGAGCTAAGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTCGAAAGAGTGGCA CCGAGTCGGTGC (SEQ ID NO:371) TevopreQ1:CGCGGTTCTATCTAGTTACGCGTTAAAACCAACTAGAA (SEQ ID NO:372) Example 3: Further Directed Evolution of Bxb1 As described herein, the Bxb1 integrase of SEQ ID NO:1 was further evolved to produce additional mutant Bxb1 integrase combinations including amino acid substitutions (Table 17).

[0121] Table 17

[0122] As demonstrated elsewhere in this article, the evolved Bxb1 integrase outperforms the wild-type Bxb1 (Tables 18 and 19).

[0123] Table 18

[0124] Table 19

[0125] Additional amino acid substitutions were introduced at positions 87, 91, 95, and 98 of Bxb1 in SEQ ID NO:1. The lead editing efficiency and integration of these mutants were analyzed (Table 20).

[0126] Table 20

[0127] Example 4: Combinations of Bxb1 mutations In some respects, evolved Bxb1 integrase may include mutations (e.g., amino acid substitutions) at one or more of positions I87 to H100 in SEQ ID NO:1 or at any position or range thereof. In some respects, the evolved Bxb1 integrase includes amino acid substitutions at position I87, H89, Q91, Q92, H95, E98, D99 and / or H100 in SEQ ID NO:1, such as I87L, I87V, I87A, I87S, I87D, I87K, I87H, I87Y, H89Y, H89G, Q91E, Q91K, Q91Y, Q91L, Q91A, Q91S, Q92H, H95Y, H95D, H95K, H95L, H95A, H95S, E98D, E98K, E98H, E98Y, E98L, E98A, E98S, D99N and / or H100N.

[0128] In some aspects, the evolved Bxb1 integrase may include mutations (e.g., amino acid substitutions) at one or more of positions K101 to P117 in SEQ ID NO:1 or at any position or range thereof. In some aspects, the evolved Bxb1 integrase includes amino acid substitutions at positions V105, A110, and / or H111 in SEQ ID NO:1, such as V105A, A110T, and / or H111P.

[0129] In some aspects, the evolved Bxb1 integrase may include mutations (e.g., amino acid substitutions) at one or more of positions F118 to I137 or any position or range thereof in SEQ ID NO:1. In some aspects, the evolved Bxb1 integrase includes amino acid substitutions at positions A119 and / or V122 in SEQ ID NO:1, such as A119S and / or V122M.

[0130] In some aspects, the evolved Bxb1 integrase may include mutations (e.g., amino acid substitutions) at one or more of positions A3 to I7, V74 to V76 and / or L103 to V105 in SEQ ID NO:1, or at any position or range thereof. In some aspects, the evolved Bxb1 integrase includes amino acid substitutions (e.g., conserved amino acid substitutions) at positions L4, V74, I75, V76 and / or V105 in SEQ ID NO:1, such as L4I, V74I, I75V, V76I and / or V105A.

[0131] In some aspects, the evolved Bxb1 integrase may include mutations (e.g., amino acid substitutions) at one or more of positions P19 to Q32, P59 to A66, and / or V80 to L83 in SEQ ID NO:1, or at any position or range thereof. In some aspects, the evolved Bxb1 integrase includes amino acid substitutions at positions L61 and / or R63 in SEQ ID NO:1, such as L61F and / or R63K.

[0132] In some aspects, the evolved Bxb1 integrase may include mutations (e.g., amino acid substitutions) at one or more of positions G39 to L44 or any position or range thereof in SEQ ID NO:1. In some aspects, the evolved Bxb1 integrase includes amino acid substitutions at positions V40 and / or E42 in SEQ ID NO:1, such as V40I, V40A, and / or E42K.

[0133] In some aspects, the evolved Bxb1 integrase may include mutations (e.g., amino acid substitutions) at one or more of positions R33 to V38, F67 to D73 and / or S106 to P117 in SEQ ID NO:1, or at any position or range thereof. In some aspects, the evolved Bxb1 integrase includes amino acid substitutions at positions G34, D36, F67, E69, Q70, V105 and / or S106, A110 and / or H111 in SEQ ID NO:1, such as G34D, D36A, D36N, F67S, E69A, Q70H, V105A, A110T and / or H111P.

[0134] In some aspects, the evolved Bxb1 integrase may include mutations (e.g., amino acid substitutions) at one or more of positions A374 to G422 or any position or range thereof in SEQ ID NO:1. In some aspects, the evolved Bxb1 integrase includes amino acid substitutions at positions V380, T388, A398, R409, A411, A414, A415 and / or R416 in SEQ ID NO:1, such as V380I, T388M, A398S, R409H, A411V, A414V, A415S and / or R416K.

[0135] In some aspects, the evolved Bxb1 integrase may include mutations (e.g., amino acid substitutions) at one or more of positions D359 to A374 and / or L423 to R438 or any position or range thereof in SEQ ID NO:1. In some aspects, the evolved Bxb1 integrase includes amino acid substitutions at positions D359, A360, R362, A369, A425, R426, R433 and / or E434 in SEQ ID NO:1, such as D359A, D359N, A360T, A360V, R362K, V366I, A369E, A369P, A369T, A369S, A425T, R426K, R433K and / or E434G.

Claims

1. An evolved Bxbl integrase comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 1, wherein the amino acid sequence of the evolved Bxbl integrase comprises one or more mutations and exhibits at least a 2-fold increase in integration of a foreign nucleic acid into a genomic target site compared to wild-type Bxbl integrase.

2. The evolved Bxbl integrase of claim 1, wherein the one or more mutations are at positions 4, 5, 18, 24, 34, 36, 40, 42, 46, 51, 61, 62, 63, 67, 69, 79, 85, 87, 88, 89, 90, 92, 95, 99, 100, 105, 106, 110, 111, 119, 122, 130, 133, 137, 140, 145, 153, 156, 160, 164, 166, 174, 175, 178, 179, 181, 187, 189, 191, 203, 209, 218, 223, 229, 231, 239, 248, 251, 254, 261, 264, 268, 272, 278, 280, 281, 282, 283, 285, 287, 288, 292, 295, 302, 306, 307, 311, 313, 319, 321, 328, 331, 332, 333, 334, 347, 353, 355, 359, 360, 361, 362, 369, 370, 375, 380, 388, 397, 398, 405, 409, 411, 414, 415, 416, 419, 425, 428, 434, 435, 444, 453, 461, 463, 466, 468, 476, 479, 480, 483, 484, 487, 488, 489, 494, 496, and / or 499.

3. The evolved Bxbl integrase of claim 1 or claim 2, wherein the one or more mutations comprise L4I, V5V, S18S, E24E, G34D, D36A, V40I, V40A, V40V, E42K, V46V, D51D, D51N, L61F, A62A, R63K, F67S, E69A, E69E, R79R, R85R, I87L, I87V, R88R, H89G, L90L, Q92H, Q92Q, H95Y, D99N, H100N, V105A, S106S, A110A, H111P, A119S, V122M, A130A, E133E, I137I, R140R, A145T, K153K, G156G, P160P, L164L, T166I, L174L, V175V, P178P, V179A, V179I, R181K, V187I, V187V, H189N, Q191Q, H203Y, G209G, A218A, R223R, E229K, S231S, M239I, A248T, N251K, N251N, T254S, A261T, V264A, P268P, R272Q, L278L, A280T, A280A, E281E, L282L, V283V, T285A, R287R, A288V, A288T, P292P, P295P, L302L, V306V, C307C, A311V, K313R, K313K, R319K, R319G, H321P, H321N, S328S, F331S, P332H, K333K, H334R, A347E, V353I, D355N, D359N, D359D, A360T, E361E, R362K, A369P, A369E, A369T, A369S, G370G, V375V, V380I, T388M, R397R, A398S, A405A, R409H, A411V, A414A, A414V, A415S, R416K, E419E, A425T, A425A, S428S, E434G, T435T, R444L, T453I, R461R, T463I, V466M, V466V, G468D, G468G, F476F, L479I, Q480STOP, E483E, Q484K, R487R, del L488, G489G, R494S, R494R, H496N, and / or M499T.

4. The evolved Bxbl integrase of any of the preceding claims, wherein the one or more mutations comprise D36N, D51G, D51E, H89Y, H100H, V175A, R181R, H189Y, A261S, R287H, V306A, D359A, A360V, and / or V466L.

5. The evolved Bxbl integrase of any of the preceding claims, wherein the one or more mutations comprise I87D, I87K, I87H, I87Y, I87A, I87S, H95D, H95K, H95L, H95A, and / or H95S.

6. The evolved Bxbl integrase of any of the preceding claims, wherein the one or more mutations are at positions 45, 49, 50, 57, 70, 74, 75, 76, 168, 194, 263, 341, 366, 387, 426, and / or 445.

7. The evolved Bxbl integrase of any of the preceding claims, wherein the one or more mutations comprise D45G, A49S, V50I, R57K, Q70H, V74I, I75V, V76I, V168I, N194D, L263I, A341D, V366I, L387L, R426K, and / or E445Q.

8. The evolved Bxbl integrase of any of the preceding claims, wherein the one or more mutations are at positions 91 and / or 98.

9. The evolved Bxbl integrase of any of the preceding claims, wherein the one or more mutations comprise Q91E, Q91K, Q91Y, Q91L, Q91A, Q91S, E98D, E98K, E98H, E98Y, E98L, E98A, and / or E98S.

10. The evolved Bxbl integrase of any of the preceding claims, wherein the one or more mutations are selected from one or more mutations of Table 1, Table 13, Table 17, and Table 20.

11. An evolved PhiC31 integrase comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 2, wherein the amino acid sequence of the evolved PhiC31 integrase comprises at least one mutation at position 1, 2, 12, 14, 18, 24, 32, 36, 41, 43, 44, 45, 49, 51, 55, 58, 74, 77, 96, 103, 107, 117, 153, 176, 196, 197, 199, 200, 228, 229, 230, 231, 235, 238, 240, 252, 254, 255, 260, 262, 264, 266, 269, 274, 278, 302, 319, 320, 322, 331, 333, 340, 344, 345, 346, 347, 351, 353, 355, 359, 362, 364, 378, 382, 393, 396, 397, 399, 406, 410, 424, 429, 431, 436, 438, 445, 448, 449, 450, 452, 457, 468, 472, 478, 498, 499, 501, 505, 512, 514, 516, 517, 520, 527, 535, 536, 549, 551, 552, 553, 563, 568, 580, 585, 586, 587, 590, 592, 594, 600, 603, 604, 609, 612, and / or 616.

12. The evolved PhiC31 integrase of claim 11, wherein the at least one mutation comprises M1E, M1V, D2V, D2M, S12N, E14G, S18N, A24A, D32A, D36A, V41I, R43R, D44A, G45G, R49R, V51M, S55S, P58P, I74I, E77E, R96R, I103I, S107S, V117V, I153I, I153V, E176D, N196N, K197K, A199T, H200H, H228Y, L229L, P230S, F231L, S235S, A238A, H240R, D252G, D254D, A255G, G260G, T262T, G264G, K266K, S269N, P274S, M278L, T302A, L319L, R320R, V322V, E331E, A333T, A333S, A333D, A340V, G344V, G344D, R345R, G346S, R347K, L351V, L351Q, R353R, Q355Q, S359S, D362N, D362G, L364M, E378K, K382K, V393V, S396N, S396R, A397T, G399G, N406S, A410T, I424I, G429S, E431V, L436L, W438R, G445G, W448R, E449D, A450D, E452K, R457R, L468L, E472D, R478R, A498A, L499L, L501L, G505V, G505G, G505S, E512K, E514E, A516T, E517E, K520K, F527F, P535L, P535P, T536A, D549D, R551R, V552V, F553F, V563V, T568T, A580V, A585A, K586Q, P587P, D590G, D592G, D594N, D594D, T600S, T600T, V603I, V603A, V603V, A604S, P609S, V612V, and / or A616V.

13. The evolved PhiC31 integrase of claim 10 or claim 12, wherein the evolved PhiC31 integrase comprises at least one of the following mutations: M1E, D2V, D32A, D36A, V41I, and / or D44A.

14. The evolved PhiC31 integrase of any one of claims 11-13, wherein the evolved PhiC31 integrase further comprises an amino-terminal sequence comprising any one of SEQ ID NOs: 3-12.

15. The evolved PhiC31 integrase of any one of claims 11-14, wherein the one or more mutations are selected from one or more mutations of Table 2 and Table 12.

16. An isolated nucleic acid encoding the evolved Bxbl or PhiC31 integrase of any one of claims 1-15.

17. A vector comprising the isolated nucleic acid of claim 16.

18. A cell comprising the evolved Bxbl or PhiC31 integrase of any one of claims 1-15, the isolated nucleic acid of claim 16, or the vector of claim 17.

19. The cell of claim 18, wherein the cell is a human cell.

20. A method of altering a sequence of a genome of a cell, the method comprising contacting a cell with the evolved Bxbl or PhiC31 integrase of any one of claims 1-14, wherein the cell comprises at least one integrase recognition sequence recognized by the evolved Bxbl or PhiC31 integrase in its genome, such that the evolved Bxbl or PhiC31 integrase binds to the integrase recognition sequence and alters the sequence of the genome of the cell.

21. The method of claim 20, wherein the alteration of the genome comprises a transversion, transition, deletion, insertion, inversion, substitution, or chromosomal translocation.

22. The method of claim 21, wherein the insertion comprises integration of an exogenous nucleic acid sequence into the genome of the cell at a safe harbor locus.

23. The method of claim 22, wherein the exogenous nucleic acid sequence encodes a therapeutic protein.

24. The method of claim 20, wherein the alteration delays development or progression of a disease or disorder or reduces disease severity.

Images