Gene therapy compositions and methods of use thereof

Abstract

Compositions and methods for gene therapy are presented herein.

Description

Background Art

[0001] There are subsets of human diseases that can be traced back to changes in DNA that are either inherited or acquired early in embryonic development. Of particular interest to developers of gene therapies are diseases that are caused by mutations in a single gene, called monogenic diseases. There are believed to be more than 6,000 monogenic diseases. Typically, any particular genetic disease caused by an inherited mutation is relatively rare, but overall, genetically related diseases have a high mortality rate. Well-known genetic diseases include cystic fibrosis, pseudohypertrophic muscular dystrophy, Huntington's disease, and sickle cell disease. Other categories of genetic diseases include metabolic disorders, such as organic acidemias and lysosomal storage diseases, in which malfunctioning genes lead to defects in metabolic processes and the accumulation of toxic byproducts, which can lead to severe morbidity and mortality in both the short and long term. Summary of the invention

[0002] Genetic diseases caused by malfunctioning genes account for a large proportion of diseases worldwide. Gene therapy is emerging as a promising form of treatment aimed at mitigating the effects of genetic diseases.

[0003] Prior to the present disclosure, certain AAV gene therapies utilizing homologous recombination employed viral vector compositions in the absence of nucleases to introduce a transgene of interest at a specific site. The present disclosure recognizes, among other things, that the combination of such vectors with one or more nucleases (e.g., provided in a single composition or as separate compositions) can improve transgene integration rates and / or efficiencies in humans.

[0004] Alternatively or additionally, the disclosure further encompasses such recognition: the combination of a vector as described herein with one or more nucleases can provide an unexpected and unexpected improvement in transgenic integration rate and / or efficiency of the human, for example, wherein the target cleavage site is away from the site of transgenic integration. In part, the disclosure encompasses the following recognition and observation: optimized vector design and preferred cleavage sites may be different between species. As demonstrated herein, the optimal design in a human or humanized system may be significantly different from the optimal design in other species or model systems (e.g., wild-type mice). In certain embodiments, a human or humanized system may allow for increased flexibility in the selection of cleavage sites to enhance homologous recombination and therefore enhance the effectiveness of certain gene therapies.

[0005] In some embodiments, the present disclosure provides compositions comprising (i) a nuclease or a polynucleotide sequence encoding a nuclease, and (ii) a polynucleotide cassette comprising an expression cassette comprising a first nucleic acid sequence and a second nucleic acid sequence, a third nucleic acid sequence, and a fourth nucleic acid sequence, wherein the first nucleic acid sequence encodes a transgene, and the second nucleic acid sequence is positioned in the 5' or 3' direction of the first nucleic acid sequence and promotes the production of two independent gene products after integration into a target integration site in the genome of a cell, the third nucleic acid sequence is positioned in the 5' direction of the expression cassette and comprises a sequence that is substantially homologous to a human genomic sequence that is 5' of the target integration site in the genome of the cell, and the fourth nucleic acid sequence is positioned in the 3' direction of the expression cassette and comprises a sequence that is substantially homologous to a human genomic sequence that is 3' of the target integration site in the genome of the cell, wherein: the polynucleotide cassette does not include a promoter sequence, the nuclease is capable of inducing double-strand breaks and / or single-strand breaks at a cleavage site in the genome of the cell, and the cleavage site is distal to the target integration site.

[0006] In some embodiments, the disclosure provides a method for integrating a transgene into the genome of a cell, the method comprising administering to a subject a composition comprising: (i) a nuclease or a polynucleotide sequence encoding the nuclease, and (ii) a polynucleotide cassette comprising: an expression cassette comprising a first nucleic acid sequence and a second nucleic acid sequence, a third nucleic acid sequence, and a fourth nucleic acid sequence, wherein the first nucleic acid sequence encodes a transgene, and the second nucleic acid sequence is positioned in the 5' or 3' direction of the first nucleic acid sequence and promotes the production of two independent gene products after integration into a target integration site in the genome of the cell, the third nucleic acid sequence is positioned in the 5' direction of the expression cassette and comprises a sequence that is substantially homologous to a human genomic sequence that is 5' of the target integration site in the genome of the cell, and the fourth nucleic acid sequence is positioned in the 3' direction of the expression cassette and comprises a sequence that is substantially homologous to a human genomic sequence that is 3' of the target integration site in the genome of the cell, wherein: the polynucleotide cassette does not include a promoter sequence, the nuclease is capable of inducing double-strand breaks and / or single-strand breaks at a cleavage site in the genome of the cell, and the cleavage site is distal to the target integration site, wherein, after administration of the composition, the transgene is integrated into the genome of a cell population. In some embodiments, cells are edited in vivo. In some embodiments, integration of the transgene is performed ex vivo.

[0007] Although any of a variety of delivery systems are contemplated, in some embodiments, the composition further comprises a recombinant viral vector. Although any of a variety of viral vectors are contemplated, in some embodiments, the recombinant viral vector is a recombinant AAV vector. In some embodiments, the recombinant viral vector is or comprises a capsid polypeptide comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of: sL65, LK03, AAV8, AAV-DJ; AAV-LK03; or AAVNP59. In some embodiments, the composition further comprises an AAV2 ITR sequence.

[0008] According to various embodiments, any of various homology arm lengths and / or ratios are considered. In certain embodiments, the third nucleic acid sequence and the fourth nucleic acid sequence each have a length between 50nt and 1600nt. In certain embodiments, the third nucleic acid sequence and the fourth nucleic acid sequence have the same length. In certain embodiments, the third nucleic acid sequence and the fourth nucleic acid sequence have different lengths.

[0009] In some embodiments, the polynucleotide cassette does not comprise a promoter sequence.

[0010] According to various embodiments, the composition provided may include integration at the target integration site. In some embodiments, after the polynucleotide box is integrated into the target integration site in the genome of the cell, the transgene is expressed under the control of the endogenous promoter at the target integration site. In some embodiments, the target integration site is an albumin locus comprising an endogenous albumin promoter and an endogenous albumin gene. In some embodiments, the target integration site is a collagen locus comprising an endogenous collagen promoter and an endogenous collagen gene. In some embodiments, the target integration site is an actin locus comprising an endogenous actin promoter and an endogenous actin gene. In some embodiments, the target integration site is within the coding sequence of the albumin locus and adjacent to the stop codon 5'. In some embodiments, the target integration site is adjacent to the stop codon 5' in exon 14 of the albumin locus.

[0011] According to various embodiments, any cleavage site suitable for the application can be used. As a non-limiting example, in some embodiments, the cleavage site is within the non-coding sequence of the albumin locus. In some embodiments, the cleavage site is within an intron, untranslated region, enhancer, promoter, silencer, or insulator of the albumin locus. In some embodiments, the cleavage site is within intron 13 or intron 14 of the albumin locus. In some embodiments, the cleavage site is between 1 and 2000 bp from the target integration site. In some embodiments, the cleavage site is up to 100 bp from the target integration site.

[0012] As described herein, the present disclosure encompasses the recognition that any of a variety of nucleases can be used in the provided methods and in combination with the provided compositions. In some embodiments, the nuclease is selected from a meganuclease, a TALEN, a TALE nickase, a ZFN, a ZF nickase, a Cas enzyme, or a variant thereof.

[0013] According to various embodiments, a variety of second nucleic acids can be used to promote the production of two independent gene products after integration into the target integration site in the genome of the cell, for example, in some embodiments, the second nucleic acid sequence can be or include a) a nucleic acid sequence encoding a 2A peptide, b) a nucleic acid sequence encoding an internal ribosome entry site (IRES), c) a nucleic acid sequence encoding an N-terminal intein splice region and a C-terminal intein splice region, or d) a nucleic acid sequence encoding a splice donor and a splice acceptor. In some embodiments, the second nucleic acid is or includes a nucleic acid sequence encoding a 2A peptide selected from the group consisting of P2A, T2A, E2A and F2A.

[0014] The present disclosure encompasses the recognition that any of a variety of transgenes can be used with the provided compositions and / or in the provided methods. For example, in some embodiments, the transgene can be selected from CBS, UGT1A1, MUT, FAH, ATP7B, A1AT, ASL, LIPA, Factor IX, or a variant thereof.

[0015] Any of a variety of cell types can be modified by applying one or more provided compositions and / or methods. For example, in some embodiments, the cell is a blood, liver, muscle, or CNS cell.

[0016] According to various embodiments, the provided methods and compositions can be used in the context of organ and / or tissue transplantation. In some embodiments, the cells are administered in autologous transplantation after transgenic integration. In some embodiments, the cells are administered in allogeneic transplantation after transgenic integration.

[0017] In one aspect, the invention features a composition or set of compositions, comprising: a nuclease or a polynucleotide sequence encoding a nuclease, wherein the nuclease is selected from a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) enzyme, a transcription activator-like effector (TALE) nuclease (TALEN), a TALE nickase, a zinc finger (ZF) nuclease (ZFN), a ZF nickase, or a meganuclease; and a polynucleotide cassette comprising: an expression cassette comprising a first nucleic acid sequence and a second nucleic acid sequence, a third nucleic acid sequence, and a fourth nucleic acid sequence, wherein the first nucleic acid sequence encodes a transgene; and the second The nucleic acid sequence is positioned in the 5' or 3' direction of the first nucleic acid sequence and promotes the production of two independent gene products after integration into the target integration site in the genome of the human cell, the third nucleic acid sequence is positioned in the 5' direction of the expression cassette and includes a sequence that is substantially homologous to the human genomic sequence 5' of the target integration site in the genome of the human cell, and the fourth nucleic acid sequence is positioned in the 3' direction of the expression cassette and includes a sequence that is substantially homologous to the human genomic sequence 3' of the target integration site in the genome of the human cell; wherein the polynucleotide cassette does not include a promoter sequence; and the nuclease is capable of inducing double-strand breaks and / or single-strand breaks at the cleavage site in the genome of the human cell.

[0018] In some embodiments of the aforementioned aspects, the target integration site is an albumin locus comprising an endogenous albumin promoter and an endogenous albumin gene.

[0019] In another aspect, the invention features a composition or set of compositions comprising: a nuclease or a polynucleotide sequence encoding a nuclease; and a polynucleotide cassette comprising: an expression cassette comprising a first nucleic acid sequence and a second nucleic acid sequence, a third nucleic acid sequence, and a fourth nucleic acid sequence, wherein the first nucleic acid sequence encodes a transgene; and the second nucleic acid sequence is positioned in the 5' or 3' direction to the first nucleic acid sequence and promotes the production of two independent gene products upon integration into a target integration site in the genome of a human cell, wherein the target integration site is an albumin locus comprising an endogenous albumin promoter and an endogenous albumin gene; the third nucleic acid sequence is positioned in the 5' direction to the expression cassette and comprises a sequence substantially homologous to a human genomic sequence 5' to the target integration site in the genome of a human cell, and the fourth nucleic acid sequence is positioned in the 3' direction to the expression cassette and comprises a sequence substantially homologous to a human genomic sequence 3' to the target integration site in the genome of a human cell; wherein the polynucleotide cassette does not include a promoter sequence; and the nuclease is capable of inducing double-strand breaks and / or single-strand breaks at the cleavage site in the genome of the human cell.

[0020] In some embodiments of any of the foregoing aspects, the composition or composition group further comprises a recombinant viral vector. In some embodiments, the recombinant viral vector is a recombinant AAV vector. In some embodiments, the recombinant viral vector is or comprises a capsid polypeptide comprising an amino acid sequence having at least 95% sequence identity with the following amino acid sequence: sL65, LK03, AAV8, AAV-DJ; AAV-LK03; or AAVNP59. In some embodiments, the recombinant viral vector comprises a capsid polypeptide and a polynucleotide sequence encoding a nuclease and / or a polynucleotide box is encapsidated in the recombinant viral vector. In some embodiments, the polynucleotide box is encapsidated in the recombinant viral vector.

[0021] In some embodiments of any of the aforementioned aspects, the composition or set of compositions further comprises an AAV2 inverted terminal repeat (ITR) sequence. In some embodiments, the AAV2 ITR sequence is located at the 5' end and 3' end of the polynucleotide sequence encoding the nuclease and / or the polynucleotide box.

[0022] In some embodiments of any of the aforementioned aspects, the third nucleic acid sequence and the fourth nucleic acid sequence are each between 50 nt and 1600 nt in length.

[0023] In some embodiments of any of the aforementioned aspects, the third nucleic acid sequence and the fourth nucleic acid sequence are the same length.

[0024] In some embodiments of any of the aforementioned aspects, the third nucleic acid sequence and the fourth nucleic acid sequence are different lengths.

[0025] In some embodiments of any of the aforementioned aspects, following integration of the polynucleotide cassette into the target integration site in the genome of the cell, the transgene is expressed under the control of an endogenous promoter at the target integration site.

[0026] In some embodiments of any of the aforementioned aspects, the target integration site is within the coding sequence of the albumin locus and is 5' adjacent to the stop codon. In some embodiments, the target integration site is 5' adjacent to the stop codon in exon 14 of the albumin locus.

[0027] In some embodiments of any of the foregoing aspects, the cleavage site is within a non-coding sequence of the albumin locus. In some embodiments, the cleavage site is within an intron, untranslated region, enhancer, promoter, silencer, or insulator of the albumin locus. In some embodiments, the cleavage site is within intron 12, 13, or 14 of the albumin locus.

[0028] In some embodiments of any of the aforementioned aspects, the nuclease is selected from a Cas enzyme, a TALEN, a TALE nickase, a ZFN, a ZF nickase, or a meganuclease.

[0029] In some embodiments of any of the aforementioned aspects, the nuclease is a Cas enzyme or a TALEN.

[0030] In some embodiments of any of the foregoing aspects, the nuclease is a Cas enzyme.

[0031] In some embodiments of any of the foregoing aspects, the Cas enzyme is selected from Staphylococcus aureus Cas9 (saCas9), Streptococcus pyogenes (spCas9), AZ nuclease, HF1-Cas9, HF2-Cas9, or HiFi-Cas9. In some embodiments, the composition or composition group further includes a guide RNA (gRNA). In some embodiments, the gRNA comprises a nucleic acid sequence of any one of SEQ ID NO: 27-45, 71-86, or 93-98, or a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NO: 27-45, 71-86, or 93-98.

[0032] In some embodiments of any of the aforementioned aspects, the nuclease or a polynucleotide sequence encoding the nuclease and the gRNA are co-formulated.

[0033] In some embodiments of any of the aforementioned aspects, the nuclease or the polynucleotide sequence encoding the nuclease and the gRNA are formulated separately.

[0034] In some embodiments of any of the aforementioned aspects, the second nucleic acid sequence is or includes: a nucleic acid sequence encoding a 2A peptide; a nucleic acid sequence encoding an internal ribosome entry site (IRES); a nucleic acid sequence encoding an N-terminal intein splicing region and a C-terminal intein splicing region; or a nucleic acid sequence encoding a splicing donor and a splicing acceptor.

[0035] In some embodiments of any of the aforementioned aspects, the second nucleic acid sequence is or includes a nucleic acid sequence encoding a 2A peptide.

[0036] In some embodiments of any of the aforementioned aspects, the second nucleic acid is or comprises a nucleic acid sequence encoding a 2A peptide selected from the group consisting of P2A, T2A, E2A, and F2A.

[0037] In some embodiments of any one of the aforementioned aspects, the cleavage site is between 1 and 2000 bp from the target integration site. In some embodiments, the cleavage site is up to 100 bp from the target integration site.

[0038] In some embodiments of any of the preceding aspects, the transgene is selected from CBS, UGT1A1, MUT, FAH, ATP7B, A1AT, ASL, LIPA, PAH, G6PC, Factor IX, or a variant thereof.

[0039] In some embodiments of any of the aforementioned aspects, the composition or the group of compositions is a group of compositions wherein the nuclease or the polynucleotide sequence encoding the nuclease is formulated in a lipid nanoparticle (LNP) and the polynucleotide box is encapsidated in a recombinant AAV vector.

[0040] In some embodiments of any of the aforementioned aspects, the cleavage site is distal to the target integration site.

[0041] In some embodiments of any of the aforementioned aspects, the cleavage site overlaps with the integration site.

[0042] In another aspect, the invention features a method for integrating a transgene into the genome of a human cell, the method comprising administering to a subject a composition or a composition set comprising: a nuclease or a polynucleotide sequence encoding a nuclease, wherein the nuclease is selected from a Cas enzyme, a TALEN, a TALE nickase, a ZFN, a ZF nickase, or a large-range nuclease; and a polynucleotide box comprising: an expression cassette comprising a first nucleic acid sequence and a second nucleic acid sequence, a third nucleic acid sequence, and a fourth nucleic acid sequence, wherein the first nucleic acid sequence encodes a transgene; and the second nucleic acid sequence is positioned in the 5' or 3' direction of the first nucleic acid sequence and is integrated into the human cell. The invention relates to a method for producing two independent gene products after the target integration site in the genome of the human cell is introduced into the target integration site in the genome of the human cell, the third nucleic acid sequence is located in the 5' direction of the expression cassette and includes a sequence that is substantially homologous to the human genomic sequence 5' of the target integration site in the genome of the human cell, and the fourth nucleic acid sequence is located in the 3' direction of the expression cassette and includes a sequence that is substantially homologous to the human genomic sequence 3' of the target integration site in the genome of the human cell; wherein: the polynucleotide cassette does not include a promoter sequence; and the nuclease is capable of inducing double-strand breaks and / or single-strand breaks at the cleavage site in the genome of the cell; wherein, after the composition or the composition group is administered, the transgene is integrated into the genome of the human cell. In some embodiments, the target integration site is an albumin locus that includes an endogenous albumin promoter and an endogenous albumin gene.

[0043] In another aspect, the invention features a method for integrating a transgene into the genome of a human cell, the method comprising contacting the human cell with a composition or a group of compositions, the composition or compositions comprising: a nuclease or a polynucleotide sequence encoding a nuclease, wherein the nuclease is selected from a Cas enzyme, a TALEN, a TALE nickase, a ZFN, a ZF nickase, or a meganuclease; and a polynucleotide box comprising: an expression cassette comprising a first nucleic acid sequence and a second nucleic acid sequence, a third nucleic acid sequence, and a fourth nucleic acid sequence, wherein the first nucleic acid sequence encodes a transgene; and the second nucleic acid sequence is positioned in the 5' or 3' direction of the first nucleic acid sequence and in the genome of the human cell integrated into the human cell. The invention relates to a polynucleotide cassette that is provided to promote the production of two independent gene products after the target integration site in the genome of the human cell is integrated into the target integration site in the genome of the human cell, the third nucleic acid sequence is located in the 5' direction of the expression cassette and includes a sequence that is substantially homologous to the human genomic sequence 5' of the target integration site in the genome of the human cell, and the fourth nucleic acid sequence is located in the 3' direction of the expression cassette and includes a sequence that is substantially homologous to the human genomic sequence 3' of the target integration site in the genome of the human cell; wherein: the polynucleotide cassette does not include a promoter sequence; and the nuclease is capable of inducing double-strand breaks and / or single-strand breaks at the cleavage site in the genome of the cell; wherein, after contacting the human cell with the composition or the combination of compositions, the transgene is integrated into the genome of the human cell. In some embodiments, the target integration site is an albumin locus that includes an endogenous albumin promoter and an endogenous albumin gene.

[0044] In another aspect, the invention features a method of integrating a transgene into the genome of a human cell, the method comprising administering to a subject a composition or set of compositions comprising: a nuclease or a polynucleotide sequence encoding the nuclease; and a polynucleotide cassette comprising: an expression cassette comprising a first nucleic acid sequence and a second nucleic acid sequence, a third nucleic acid sequence, and a fourth nucleic acid sequence, wherein the first nucleic acid sequence encodes the transgene; and the second nucleic acid sequence is positioned in the 5' or 3' direction of the first nucleic acid sequence and promotes production of two independent gene products upon integration into a target integration site in the genome of the human cell, wherein the target integration site is a region comprising an endogenous albumin promoter and the albumin locus of an endogenous albumin gene; the third nucleic acid sequence is positioned in the 5' direction of the expression cassette and includes a sequence that is substantially homologous to a human genomic sequence that is 5' of the target integration site in the genome of a human cell, and the fourth nucleic acid sequence is positioned in the 3' direction of the expression cassette and includes a sequence that is substantially homologous to a human genomic sequence that is 3' of the target integration site in the genome of a human cell; wherein: the polynucleotide cassette does not include a promoter sequence; the nuclease is capable of inducing double-strand breaks and / or single-strand breaks at a cleavage site in the genome of the cell; and the cleavage site is distal to the target integration site; wherein, after administration of the composition or the group of compositions, the transgene is integrated into the genome of the human cell.

[0045] In another aspect, the invention features a method of integrating a transgene into the genome of a human cell, the method comprising contacting the human cell with a composition or combination of compositions comprising: a nuclease or a polynucleotide sequence encoding a nuclease; and a polynucleotide cassette comprising: an expression cassette comprising a first nucleic acid sequence and a second nucleic acid sequence, a third nucleic acid sequence, and a fourth nucleic acid sequence, wherein the first nucleic acid sequence encodes a transgene; and the second nucleic acid sequence is positioned in a 5' or 3' direction to the first nucleic acid sequence and promotes production of two independent gene products upon integration into a target integration site in the genome of the human cell, wherein the target integration site is a region comprising an endogenous albumin promoter and an endogenous albumin promoter. the albumin locus of an albumin gene; the third nucleic acid sequence is positioned in the 5' direction of the expression cassette and includes a sequence that is substantially homologous to a human genomic sequence that is 5' of a target integration site in the genome of a human cell, and the fourth nucleic acid sequence is positioned in the 3' direction of the expression cassette and includes a sequence that is substantially homologous to a human genomic sequence that is 3' of a target integration site in the genome of a human cell; wherein: the polynucleotide cassette does not include a promoter sequence; the nuclease is capable of inducing double-strand breaks and / or single-strand breaks at a cleavage site in the genome of the cell; and the cleavage site is distal to the target integration site; wherein, upon contacting the human cell with the composition or the group of compositions, the transgene is integrated into the genome of the human cell.

[0046] In some embodiments of any of the foregoing aspects, the composition or composition group further comprises a recombinant viral vector. In some embodiments, the recombinant viral vector is a recombinant AAV vector. In some embodiments, the recombinant viral vector is or comprises a capsid polypeptide comprising an amino acid sequence having at least 95% sequence identity with the following amino acid sequence: LK03, AAV8, AAV-DJ; AAV-LK03; or AAVNP59. In some embodiments, the recombinant viral vector comprises a capsid polypeptide and a polynucleotide sequence encoding a nuclease and / or a polynucleotide box is encapsidated in the recombinant viral vector. In some embodiments, the polynucleotide box is encapsidated in the recombinant viral vector.

[0047] In some embodiments of any of the aforementioned aspects, the composition or set of compositions further comprises an AAV2 ITR sequence. In some embodiments, the AAV2 ITR sequence flanks the 5' end and the 3' end of the polynucleotide sequence encoding the nuclease and / or the polynucleotide cassette.

[0048] In some embodiments of any of the aforementioned aspects, the third nucleic acid sequence and the fourth nucleic acid sequence are between 50 nt and 1600 nt in length.

[0049] In some embodiments of any of the aforementioned aspects, the third nucleic acid sequence and the fourth nucleic acid sequence are the same length.

[0050] In some embodiments of any of the aforementioned aspects, the third nucleic acid sequence and the fourth nucleic acid sequence are different lengths.

[0051] In some embodiments of any of the aforementioned aspects, following integration of the polynucleotide cassette into the target integration site in the genome of the cell, the transgene is expressed under the control of an endogenous promoter at the target integration site.

[0052] In some embodiments of any of the aforementioned aspects, the target integration site is within the coding sequence of the albumin locus and is 5' adjacent to the stop codon. In some embodiments, the target integration site is 5' adjacent to the stop codon in exon 14 of the albumin locus.

[0053] In some embodiments of any of the foregoing aspects, the cleavage site is within a non-coding sequence of the albumin locus. In some embodiments, the cleavage site is within an intron, untranslated region, enhancer, promoter, silencer, or insulator of the albumin locus. In some embodiments, the cleavage site is within intron 12, 13, or 14 of the albumin locus.

[0054] In some embodiments of any of the aforementioned aspects, the nuclease is selected from a Cas enzyme, a TALEN, a TALE nickase, a ZFN, a ZF nickase, or a meganuclease.

[0055] In some embodiments of any of the aforementioned aspects, the nuclease is a Cas enzyme or a TALEN.

[0056] In some embodiments of any of the foregoing aspects, the nuclease is a Cas enzyme.

[0057] In some embodiments of any of the aforementioned aspects, the Cas enzyme is selected from Staphylococcus aureus Cas9 (saCas9), Streptococcus pyogenes (spCas9), AZ nuclease, HF1-Cas9, HF2-Cas9, or HiFi-Cas9.

[0058] In some embodiments of any of the foregoing aspects, the method further comprises a guide RNA (gRNA). In some embodiments, the gRNA comprises a nucleic acid sequence of any one of SEQ ID NOs: 27-45, 71-86, or 93-98, or a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 27-45, 71-86, or 93-98.

[0059] In some embodiments of any of the aforementioned aspects, the nuclease or a polynucleotide sequence encoding the nuclease and the gRNA are co-formulated.

[0060] In some embodiments of any of the aforementioned aspects, the nuclease or the polynucleotide sequence encoding the nuclease and the gRNA are formulated separately.

[0061] In some embodiments of any of the aforementioned aspects, the second nucleic acid sequence is or includes: a nucleic acid sequence encoding a 2A peptide; a nucleic acid sequence encoding an internal ribosome entry site (IRES); a nucleic acid sequence encoding an N-terminal intein splicing region and a C-terminal intein splicing region; or a nucleic acid sequence encoding a splicing donor and a splicing acceptor.

[0062] In some embodiments of any of the aforementioned aspects, the second nucleic acid sequence is or includes a nucleic acid sequence encoding a 2A peptide. In some embodiments, the second nucleic acid sequence is or includes a nucleic acid sequence encoding a 2A peptide selected from the group consisting of P2A, T2A, E2A, and F2A.

[0063] In some embodiments of any one of the aforementioned aspects, the cleavage site is between 1 and 1000 bp from the target integration site. In some embodiments, the cleavage site is up to 100 bp from the target integration site.

[0064] In some embodiments of any of the preceding aspects, the transgene is selected from CBS, UGT1A1, MUT, FAH, ATP7B, A1AT, ASL, LIPA, PAH, G6PC, Factor IX, or a variant thereof.

[0065] In some embodiments of any of the aforementioned aspects, the method is a composition set wherein the nuclease or a polynucleotide sequence encoding the nuclease is formulated in a lipid nanoparticle and the polynucleotide cassette is encapsidated in a recombinant AAV vector.

[0066] In some embodiments of any of the aforementioned aspects, the cells are edited in vivo.

[0067] In some embodiments of any of the aforementioned aspects, the method of integrating a transgene is performed ex vivo.

[0068] In some embodiments of any of the foregoing aspects, the cells are blood, liver, muscle, or CNS cells. In some embodiments, the cells are administered in autologous transplantation after transgenic integration. In some embodiments, the cells are administered in allogeneic transplantation after transgenic integration.

[0069] In some embodiments of any of the foregoing aspects, the nuclease or the polynucleotide sequence encoding the nuclease and the polynucleotide cassette are administered to the subject on the same day. In some embodiments of any of the foregoing aspects, the nuclease or the polynucleotide sequence encoding the nuclease and the polynucleotide cassette are administered to the subject on different days.

[0070] In some embodiments, the subject is administered a nuclease or a polynucleotide sequence encoding a nuclease 1 hour to 3 days after the polynucleotide box. In some embodiments, the subject is administered a nuclease or a polynucleotide sequence encoding a nuclease 1 hour to 24 hours after the polynucleotide box. In some embodiments, the subject is administered a nuclease or a polynucleotide sequence encoding a nuclease 1 hour to 3 days before the polynucleotide box. In some embodiments, the subject is administered a nuclease or a polynucleotide sequence encoding a nuclease 1 hour to 4 hours before the polynucleotide box. In some embodiments, the subject is administered a nuclease or a polynucleotide sequence encoding a nuclease 4 hours before the polynucleotide box.

[0071] In some embodiments of any of the aforementioned aspects, the nuclease or the polynucleotide sequence encoding the nuclease and the polynucleotide cassette are contacted with the human cells on the same day.

[0072] In some embodiments of any of the aforementioned aspects, the nuclease or the polynucleotide sequence encoding the nuclease and the polynucleotide cassette are contacted with the human cells on different days.

[0073] In some embodiments, the nuclease or the polynucleotide sequence encoding the nuclease is contacted with the human cell 1 hour to 3 days after the polynucleotide box. In some embodiments, the nuclease or the polynucleotide sequence encoding the nuclease is contacted with the human cell 1 hour to 24 hours after the polynucleotide box. In some embodiments, the nuclease or the polynucleotide sequence encoding the nuclease is contacted with the human cell 1 hour to 3 days before the polynucleotide box. In some embodiments, the nuclease or the polynucleotide sequence encoding the nuclease is contacted with the human cell 1 hour to 4 hours before the polynucleotide box. In some embodiments, the nuclease or the polynucleotide sequence encoding the nuclease is contacted with the human cell 4 hours before the polynucleotide box.

[0074] In some embodiments of any of the aforementioned aspects, the cleavage site is distal to the target integration site.

[0075] In some embodiments of any of the aforementioned aspects, the cleavage site overlaps with the integration site.

[0076] As used in this application, the terms "about" and "approximately" are used as equivalent forms. Any reference to a publication, patent, or patent application herein is incorporated by reference in its entirety. Any numerals used in this application with or without about / approximately are intended to cover any normal fluctuations understood by a person of ordinary skill in the relevant art.

[0077] Other features, purposes and advantages of the present invention are apparent in the following detailed description. However, it should be understood that the detailed description, although indicating embodiments of the present invention, is provided only by way of illustration and not limitation. Based on the detailed description, various changes and modifications within the scope of the present invention will become apparent to those skilled in the art. definition

[0078] About: The term "about", when used herein to refer to a value, refers to a value that is similar to the value referred to in the context. Generally, those skilled in the art who are familiar with the context will understand the relevant degree of variation encompassed by "about" in that context. For example, in some embodiments, the term "about" can encompass a range of values ​​within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the value mentioned.

[0079] Adult: As used herein, the term "adult" refers to a person eighteen years of age or older. In some embodiments, an adult weighs in the range of about 90 pounds to about 250 pounds.

[0080] Associated: As used herein, two events or entities are "associated" with each other if the presence, level, and / or form of one event or entity is associated with the presence, level, and / or form of another event or entity. For example, if the presence, level, and / or form of a particular entity (e.g., a polypeptide, a genetic signature, a metabolite, a microorganism, etc.) is associated with the incidence and / or susceptibility of a particular disease, disorder, or condition (e.g., across a related population), the particular entity is considered to be associated with the particular disease, disorder, or condition. In some embodiments, two or more entities are physically "associated" with each other if they interact directly or indirectly so that they are physically close to each other and / or remain in close proximity to each other. In some embodiments, two or more entities that are physically associated with each other are covalently attached to each other; in some embodiments, two or more entities that are physically associated with each other are not covalently attached to each other, but are non-covalently associated, for example, by hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0081] Biological sample: As used herein, the term "biological sample" generally refers to a sample obtained or derived from a biological source of interest (e.g., a tissue or organism or cell culture), as described herein. In some embodiments, the source of interest comprises an organism, such as an animal or a human. In some embodiments, the biological sample is or comprises a biological tissue or fluid. In some embodiments, the biological sample may be or comprise bone marrow; blood; blood cells; ascites; tissue or fine needle biopsy samples; body fluids containing cells; free floating nucleic acids; sputum; saliva; urine; cerebrospinal fluid, peritoneal fluid; pleural effusion; feces; lymph; gynecological fluid; skin swab; vaginal swab; oral swab; nasal swab; wash or lavage fluid, such as catheter lavage fluid or bronchoalveolar lavage fluid; aspirate; scrapings; bone marrow sample; tissue biopsy sample; surgical sample; other body fluids, secretions and / or excretions; and / or cells therefrom, etc. In some embodiments, the biological sample is or comprises cells obtained from an individual. In some embodiments, the obtained cells are or include cells from the individual from which the sample was obtained. In some embodiments, the sample is a "primary sample" obtained directly from a target source by any appropriate means. For example, in some embodiments, the primary biological sample is obtained by a method selected from the group consisting of: biopsy (e.g., fine needle aspiration or tissue biopsy), surgery, collection of body fluids (e.g., blood, lymph, excreta, etc.), etc. In some embodiments, it is clear from the context that the term "sample" refers to a preparation obtained by treating a primary sample (e.g., by removing one or more components of the primary sample and / or by adding one or more agents to the primary sample). For example, filtration using a semipermeable membrane. Such a "treated sample" may include, for example, nucleic acids or proteins extracted from a sample or obtained by subjecting the primary sample to techniques such as amplification or reverse transcription of mRNA, separation and / or purification of certain components.

[0082] Biomarker: The term "biomarker" is used herein (consistent with its use in the art) to refer to an entity whose presence, level, or form is associated with a specific biological event or state of interest such that it is considered a "marker" for that event or state. Among other things, the present disclosure covers biomarkers for gene therapy (e.g., which can be used to assess one or more characteristics or properties of a gene therapy treatment, such as, for example, the extent, level, and / or persistence of load expression). In some embodiments, the biomarker is a cell surface marker. In some embodiments, the biomarker is intracellular. In some embodiments, the biomarker is present outside the cell (e.g., secreted or otherwise generated or present outside the cell, for example, in a body fluid (such as blood, urine, tears, saliva, cerebrospinal fluid, etc.)). In certain embodiments, the present disclosure demonstrates that biomarkers that can be detected in a sample obtained from a subject who has received gene therapy are used to assess the effectiveness of one or more features or characteristics of the gene therapy; in some such embodiments, the sample is a sample of cells, tissues and / or fluids other than the cells, tissues and / or fluids to which the gene therapy is delivered and / or in which the cells, tissues and / or fluids having activity are loaded.

[0083] Codon optimization: As used herein, the term "codon optimization" refers to the process of changing the codons of a given gene in such a way that the polypeptide sequence encoded by the gene remains the same, while the altered codons improve the expression process of the polypeptide sequence. For example, if the polypeptide belongs to a human protein sequence and is expressed in E. coli, expression will generally be improved if codon optimization is performed on the DNA sequence to change the human codons to codons that are more efficient for expression in E. coli.

[0084] Detectable moiety: As used herein, the term "detectable moiety" refers to any entity (e.g., a molecule, a complex, or a portion or component thereof). In some embodiments, the detectable moiety is provided and / or utilized as a discrete molecular entity; in some embodiments, it is part of and / or associated with another molecular entity. Examples of detectable moieties include, but are not limited to, various ligands, radionuclides (e.g., 3 H. 14 C. 18 F. 19 F. 32 P. 35 S. 135 I. 125 I. 123 I. 64 Cu, 187 Re, 111 In, 90 Y.99m Tc, 177 Lu, 89 Zr, etc.), fluorescent dyes (for specific exemplary fluorescent dyes, see below), chemiluminescent agents (such as, for example, acridinum esters, stable dioxetanes, etc.), bioluminescent agents, spectrally resolvable inorganic fluorescent semiconductor nanocrystals (i.e., quantum dots), metal nanoparticles (e.g., gold, silver, copper, platinum, etc.) nanoclusters, paramagnetic metal ions, enzymes (for specific examples of enzymes, see below), colorimetric labels (such as, for example, dyes, colloidal gold, etc.), biotin, dioxigenin, haptens, antibodies and / or proteins (for which antisera or monoclonal antibodies can be obtained).

[0085] Child: As used herein, the term "child" refers to a person between the ages of two and eighteen. The weight of children of different ages and of a particular child varies widely, with a typical range of 30 pounds to 150 pounds.

[0086] Combination therapy: As used herein, the term "combination therapy" refers to those situations in which a subject is simultaneously exposed to two or more treatment regimens (e.g., two or more therapeutic agents, such as gene therapy and non-gene therapy treatment modalities). In some embodiments, two or more regimens can be administered simultaneously; in some embodiments, such regimens can be administered sequentially (e.g., all "doses" of the first regimen are administered before the administration of any doses of the second regimen); in some embodiments, such agents are administered in overlapping dosing regimens. In some embodiments, the "administration" of a combination therapy may involve administering one or more other agents or modalities to a subject receiving one or more agents or modalities in the combination. For clarity, combination therapy does not require that the separate agents be administered together in a single composition (or even necessarily at the same time).

[0087] Composition: Those skilled in the art will appreciate that, as used herein, the term "composition" can be used to refer to a discrete physical entity comprising one or more specified components. Generally, unless otherwise specified, a composition can be in any form - for example, a gas, gel, liquid or solid.

[0088] Determine: Many of the methods described herein include a "determine" step. Those of ordinary skill in the art who read this specification will understand that such "determine" may utilize any of a variety of techniques available to those of skill in the art (including, for example, specific techniques explicitly mentioned herein), or may be accomplished using any of the various techniques. In some embodiments, determination involves manipulation of a physical sample. In some embodiments, determination involves consideration and / or manipulation of data or information, such as using a computer or other processing unit suitable for performing relevant analysis. In some embodiments, determination involves receiving relevant information and / or material from a source. In some embodiments, determination involves comparing one or more features of a sample or entity to a comparable reference.

[0089] Distal end: As used herein, the term "distal end" with respect to the position of the cleavage site relative to the target integration site means that the cleavage site and the target integration site are not identical and / or do not overlap. In some embodiments, the cleavage site is within the range of about 2kB (e.g., 100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp, 1kB, 1.1kB, 1.2kB, 1.3kB, 1.4kB, 1.5kB, 1.6kB, 1.7kB, 1.8kB, 1.9kB or 2.0kB) of the integration site. In some embodiments, the cleavage site is within the boundaries of the homology arms. It should be understood that in other embodiments, the cleavage site may be identical or overlapping with the target integration site.

[0090] Gene: As used herein, the term "gene" refers to a DNA sequence that encodes a gene product (e.g., an RNA product and / or a polypeptide product). In some embodiments, a gene includes a coding sequence (e.g., a sequence encoding a specific gene product); in some embodiments, a gene includes a non-coding sequence. In some specific embodiments, a gene may include a coding sequence (e.g., an exon) and a non-coding (e.g., an intron) sequence. In some embodiments, a gene may include one or more regulatory elements (e.g., a promoter, an enhancer, a silencer, a termination signal), which may, for example, control or affect one or more aspects of gene expression (e.g., cell type-specific expression, inducible expression). In some embodiments, a gene is located or present in a genome (or has a nucleotide sequence identical to a nucleotide sequence located or present in a genome) (e.g., in or on a chromosome or other replicable nucleic acid).

[0091] Gene product or expression product: As used herein, the term "gene product" or "expression product" generally refers to RNA transcribed from a gene (before and / or after processing) or a polypeptide encoded by RNA transcribed from a gene (before and / or after modification).

[0092] Identity: As used herein, the term "identity" refers to the overall relatedness between polymer molecules, for example, between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, if the sequences of polymer molecules are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical, they are considered to be "substantially identical" to each other. For example, the calculation of the percent identity of two nucleic acid or polypeptide sequences can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first sequence and the second sequence to achieve optimal alignment, and for comparison purposes, non-identical sequences can be ignored). In certain embodiments, the length of the sequence compared for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or substantially 100% of the length of the reference sequence. The nucleotides at the corresponding positions are then compared. When the position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the two sequences, taking into account the number of gaps, the length of each gap, which need to be introduced to perform the best comparison of the two sequences. The determination of the percent identity between the sequence comparison and the two sequences can be completed using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using the Meyers and Miller algorithm (CABIOS, 1989, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0). In some exemplary embodiments, the nucleic acid sequence comparison performed using the ALIGN program uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package, using the NWSgapdna.CMP matrix.

[0093] "Improve", "increase", "inhibit", or "decrease": As used herein, the terms "improve", "increase", "inhibit", "decrease", or their grammatical equivalents indicate a value relative to a baseline or other reference measurement. In some embodiments, an appropriate reference measurement may be or include a measurement in a specific system (e.g., in a single individual) under otherwise comparable conditions in the absence of a particular agent or treatment (e.g., before and / or after a particular agent or treatment), or in the presence of an appropriate comparable reference agent. In some embodiments, an appropriate reference measurement may be or include a measurement in a comparable system that is known or expected to respond in a particular manner in the presence of a relevant agent or treatment.

[0094] Infant: As used herein, the term "infant" refers to a human under the age of two. The typical weight range for an infant is 3 pounds to 20 pounds.

[0095] Neonate: As used herein, the term "neonatal" refers to a human who has just been born.

[0096] Nucleic acid: As used herein, in its broadest sense, refers to any compound and / or substance that is or can be incorporated into an oligonucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester linkage. As is clear from the context, in some embodiments, "nucleic acid" refers to a single nucleic acid residue (e.g., a nucleotide and / or nucleoside); in some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising a single nucleic acid residue. In some embodiments, "nucleic acid" is or comprises RNA; in some embodiments, "nucleic acid" is or comprises DNA. In some embodiments, a nucleic acid is, comprises, or consists of: one or more natural nucleic acid residues. In some embodiments, a nucleic acid is, comprises, or consists of: one or more nucleic acid analogs. In some embodiments, a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone. In some embodiments, a nucleic acid is, comprises, or consists of: one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine). In some embodiments, the nucleic acid is, comprises, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxyadenosine, 8-oxyguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, embedded bases, and combinations thereof). In some embodiments, the nucleic acid has a nucleotide sequence encoding a functional gene product (e.g., RNA or protein). In some embodiments, the nucleic acid comprises one or more introns. In some embodiments, the nucleic acid is prepared by one or more of isolation from a natural source, enzymatic synthesis (in vivo or in vitro) by polymerization based on a complementary template, replication in a recombinant cell or system, and chemical synthesis. In some embodiments, the nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, or more residues in length.In some embodiments, the nucleic acid is partially or entirely single-stranded; in some embodiments, the nucleic acid is partially or entirely double-stranded. In some embodiments, the nucleic acid has a nucleotide sequence that comprises at least one element that encodes a polypeptide or is the complement of a sequence that encodes a polypeptide. In some embodiments, the nucleic acid has enzymatic activity.

[0097] Peptide: As used herein, the term "peptide" or "polypeptide" refers to any polymer chain of amino acids. In some embodiments, a peptide has an amino acid sequence that occurs in nature. In some embodiments, a peptide has an amino acid sequence that does not occur in nature. In some embodiments, a peptide has an engineered amino acid sequence because it is designed and / or produced by the action of man. In some embodiments, a peptide may comprise or consist of: natural amino acids, non-natural amino acids, or both. In some embodiments, a peptide may comprise or consist of: only natural amino acids or only non-natural amino acids. In some embodiments, a peptide may comprise D-amino acids, L-amino acids, or both. In some embodiments, a peptide may comprise only D-amino acids. In some embodiments, a peptide may comprise only L-amino acids. In some embodiments, a peptide is linear. In some embodiments, the term "peptide" may be appended to the name of a reference peptide, activity, or structure; in this case, it is used herein to refer to peptides having a related activity or structure and therefore can be considered to be members of the same class or family of peptides. For each such class, the present specification provides and / or those skilled in the art will know exemplary peptides within the class whose amino acid sequence and / or function is known; in some embodiments, such exemplary peptides are reference peptides for a class or family of peptides. In some embodiments, members of a class or family of peptides exhibit significant sequence homology or identity with a reference peptide of that class (in some embodiments, with all peptides within that class), share common sequence motifs (e.g., characteristic sequence elements) with a reference peptide of that class (in some embodiments, with all peptides within that class), and / or share common activities (in some embodiments, at comparable levels or within a specified range) with a reference peptide of that class (in some embodiments, with all peptides within that class). For example, in some embodiments, the member peptides exhibit an overall sequence homology or identity with the reference peptide of at least about 30% to 40% and typically greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more and / or contain at least one region (e.g., in some embodiments, a conserved region that may be or contain a characteristic sequence element) that exhibits very high sequence identity (typically greater than 90% or even 95%, 96%, 97%, 98% or 99%). Such conserved regions typically include at least 3-4, typically up to 20 or more amino acids; in some embodiments, the conserved regions encompass at least a stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more consecutive amino acids.

[0098] Safe harbor: As used herein, the term "safe harbor", "safe harbor locus", "safe harbor gene" or "safe harbor site" refers to one or more regions of a subject or cell genome (e.g., a mouse genome, a human genome, a humanized animal genome, a chimeric animal genome) that enable stable expression of an integrated transgene without negatively affecting the subject and / or cell. In some embodiments, integration at a safe harbor allows predictable function of the integrated transgene. In some embodiments, integration at a safe harbor does not result in unexpected changes to the genome of a subject or cell that pose a risk to the subject or cell. In some embodiments, a safe harbor gene can be destroyed (e.g., by NHEJ) without integrating a transgene without negatively affecting the subject and / or cell (e.g., without causing tumorigenesis).

[0099] Subject: As used herein, the term "subject" refers to an organism, typically a mammal (e.g., a human, including prenatal humanoids in some embodiments). In some embodiments, the subject suffers from a disease, disorder, or condition of interest. In some embodiments, the subject is susceptible to a disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject is a person having one or more characteristics characterized by susceptibility or risk to a disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual who is receiving and / or has received diagnosis and / or therapy. In a preferred embodiment, the subject is a human.

[0100] Substantially: As used herein, the term "substantially" refers to the qualitative condition of exhibiting an overall or near overall range or degree of a characteristic or property of interest. It should be understood by those of ordinary skill in the biological arts that biological and chemical phenomena rarely, if ever, reach completeness and / or proceed to perfection or achieve or avoid an absolute result. Therefore, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0101] Variant: As used herein, in the context of a molecule (e.g., a nucleic acid, a protein, or a small molecule), the term "variant" refers to a molecule that exhibits significant structural identity to a reference molecule but is structurally different from a reference molecule, for example, in the presence or absence of one or more chemical moieties or at the level of one or more chemical moieties, compared to a reference entity. In some embodiments, the variant is also functionally different from its reference molecule. Generally, whether a particular molecule is properly considered a "variant" of a reference molecule is based on the degree of structural identity with the reference molecule. As will be appreciated by those skilled in the art, any biological or chemical reference molecule has certain characteristic structural elements. By definition, a variant is a unique molecule that shares one or more such characteristic structural elements, but is different from a reference molecule in at least one aspect. To name just a few examples, a polypeptide may have a characteristic sequence element consisting of a plurality of amino acids that have a specified position relative to each other in a linear or three-dimensional space and / or contribute to a specific structural motif and / or biological function; a nucleic acid may have a characteristic sequence element consisting of a plurality of nucleotide residues that have a specified position relative to another in a linear or three-dimensional space. In some embodiments, variant polypeptides or nucleic acids may differ from reference polypeptides or nucleic acids due to one or more differences in amino acid or nucleotide sequence and / or one or more differences in chemical moieties (e.g., carbohydrates, lipids, phosphate groups) as covalent components of polypeptides or nucleic acids (e.g., attached to polypeptide or nucleic acid backbones). In some embodiments, variant polypeptides or nucleic acids exhibit an overall sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 99% with reference polypeptides or nucleic acids. In some embodiments, variant polypeptides or nucleic acids do not share at least one characteristic sequence element with reference polypeptides or nucleic acids. In some embodiments, reference polypeptides or nucleic acids have one or more biological activities. In some embodiments, variant polypeptides or nucleic acids share one or more biological activities of reference polypeptides or nucleic acids. In some embodiments, variant polypeptides or nucleic acids lack one or more biological activities of reference polypeptides or nucleic acids. In some embodiments, variant polypeptides or nucleic acids exhibit reduced levels of one or more biological activities compared to the reference polypeptides or nucleic acids. In some embodiments, if the target polypeptide or nucleic acid has the same amino acid or nucleotide sequence as the reference but has a small amount of sequence changes at a specific position, the target polypeptide or nucleic acid is considered to be a "variant" of the reference polypeptide or nucleic acid. Typically, less than about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3% or about 2% of the residues in the variant are substituted, inserted or deleted compared to the reference.In some embodiments, the variant polypeptide or nucleic acid comprises about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 substituted residues compared to an appropriate reference. Typically, the variant polypeptide or nucleic acid comprises a very small amount (e.g., less than about 5, about 4, about 3, about 2, or about 1) of substituted, inserted, or deleted functional residues (i.e., residues involved in a specific biological activity) relative to the reference. In some embodiments, the variant polypeptide or nucleic acid comprises no more than about 5, about 4, about 3, about 2, or about 1 additions or deletions compared to the reference, and in some embodiments does not comprise additions or deletions. In some embodiments, the variant polypeptide or nucleic acid comprises less than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and usually less than about 5, about 4, about 3, or about 2 additions or deletions compared to the reference. In some embodiments, the reference polypeptide or nucleic acid is a polypeptide or nucleic acid found in nature. In some embodiments, the reference polypeptide or nucleic acid is a human polypeptide or nucleic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0102] Figure 1A An example map of potential cleavage sites in the human albumin gene (ALB), eg, by one or more nucleases, is shown. Figure 1B The human ALB sequence surrounding the stop codon in exon 14 is shown, with the underlined sequence indicating an exemplary region targeted for cleavage.

[0103] Figure 2A It was shown that a double-strand break (DSB) induced in intron 14 of the human ALB gene enhances integration efficiency. Figure 2B It was shown that DSB induced in intron 13 of the human ALB gene also enhanced integration efficiency. Figure 2C Shown is the degree of enhancement of integration efficiency relative to the position of the DSB introduced at different positions in intron 13 or intron 14 of the ALB gene. Figure 2D Representative fluorescence images are shown, depicting that Cas9 protein together with guide RNA can increase GFP-positive cells. Figure 2E It was shown that DSBs induced in intron 13 and / or intron 14 of human ALB enhanced integration efficiency (measured as the percentage of GFP-positive cells). Figure 2F It was shown that DSBs induced in intron 13 and / or intron 14 of human ALB enhanced integration efficiency (measured as fusion mRNA levels).

[0104] Figure 3ARepresentative fluorescence images are shown, depicting that the use of Cas9 and appropriate guide RNA can increase integration efficiency. Figure 3B Different ratios of Cas9 mRNA to gRNA are shown, as well as integration efficiency (measured as the percentage of GFP-positive cells).

[0105] Figure 4A GENERIDE is shown TM Schematic representation of the construct before integration (AAV) and after HR-mediated integration into the genome of a targeted locus (e.g., albumin). Figure 4B It was shown that longer homology alignments predict higher integration efficiency (measured as fusion mRNA levels). Figure 4C It was shown that longer homology alignments can predict higher integration efficiency (measured as the percentage of GFP-positive cells).

[0106] Figure 5A It was shown that various Cas9s (e.g., HF1-Cas9, HF2-Cas9) can enhance on-target integration. Figure 5B High-fidelity versions of Cas9 (e.g., HiFi-Cas9) were shown to enhance on-target integration.

[0107] Figure 6 Shown that Staphylococcus aureus Cas9 (saCas9) can improve human GENERIDE TM Integration of vectors.

[0108] Figure 7 An exemplary plasmid designed to express saCas9 and gRNA is shown (upper panel). This plasmid can enhance integration after transfection when packaged into AAV (lower panel).

[0109] Figure 8 Representative fluorescence images are shown, depicting that the use of Cas9 and appropriate guide RNA can increase integration efficiency.

[0110] Fig.9A It was shown that DSBs induced in intron 13 and / or intron 14 of human ALB enhanced integration efficiency (measured as the percentage of GFP-positive cells). Fig. 9B It was shown that DSBs induced in intron 13 and / or intron 14 of human ALB enhanced integration efficiency (measured as fusion mRNA levels).

[0111] Fig.10 GENERIDE is shown TM , Cas9 and appropriate guide RNA in vivo 3 weeks after GENERIDE TM Assessment of biomarkers. D, Day of LNP administration relative to AAV administration.

[0112] Fig.11 Images are shown depicting that in vivo administration of Cas9 and appropriate guide RNA can increase integration efficiency.

[0113] Fig.12 Shown are the results of an experiment in which GENERIDE was administered alone TM Constructs (GR-hATP7B or GR-GFP) or combined with Cas9 with a pilot control. The GR-hATP7b construct includes homology arms of different sizes (0.4 / 0.8 kB; 0.8 / 0.4 kB or 0.6 / 0.6 kB), while the GR-GFP has 1.0 / 1.0 kB homology arms. GOI, gene of interest. Fig.12 A series of graphs are shown measuring the levels of fusion mRNA (copy number / 20 mg) (left panel) and normalized fusion mRNA (normalized to 0.6 / 0.6GR-hATP7B) (right panel). GR-GFP has a higher baseline, and without wishing to be bound by any particular theory, this may be due to longer homology arms or a smaller GOI.

[0114] Fig.13 Shown are the results of an experiment in which the fusion mRNA fold increase between GOIs was measured as the normalized fusion mRNA of the indicated construct (normalized relative to GR alone). The fusion mRNA level is a measure of integration efficiency.

[0115] Fig.14 Shown are the results of experiments in which GENERIDE was administered to human HepG2 cells alone or in combination with spCas9 mRNA TM Construct (GR-hAAT) with naive and H20 controls. Fig.14 A graph measuring fusion mRNA levels (copy number / 100 ng) is shown. spCas9 mRNA increases the integration efficiency of GR-hAAT.

[0116] Fig.15A and Fig. 15B Results of experiments are shown in which the integration efficiency of wild-type (WT) versus mutant plasmids in circular or linear form was measured with or without Cas9. The plasmids have restriction enzyme cleavage sites to linearize the plasmids, and PAM mutations can further enhance integration. Fig.15A Shown are graphs measuring fusion mRNA levels (copies / 100 ng) of WT and mutant plasmids in both circular and linear forms in the presence or absence of Cas9, with a pilot control (left panel). Fig. 15BShown are graphs measuring fusion mRNA levels of WT and mutant plasmids in both circular and linear forms in the presence of Cas9 (normalized to C-WT+Cas9) (right panel).

[0117] Fig.16A and Fig. 16B The results of an experiment in which different donor-to-gRNA ratios were detected using AZ nuclease ( Fig.16A ) or SpCas9( Fig. 16B ) mRNA transfection was followed by measurement of overall editing efficiency (Editing_Efficiency), the percentage of reads with HDR donor integration (KI_HDR), and the percentage of reads with NHEJ donor integration in the forward or reverse orientation (NHEJ_Fw+Rv). Arrows indicate overlapping gRNAs for SpCas9 and AZ nucleases; asterisks indicate gRNAs with the highest KI-HDR for AZ nucleases.

[0118] Fig.17A and 17B Shown are the results of an experiment in which transgene integration efficiency was measured in HEPG2 cells for different human gRNAs. Fig.17A Shown is a graph measuring fusion mRNA levels (copy number / 2.5 ng) in HEPG2 cells in the presence of spCas9, with a GR (PD128) only control. Fig. 17B Shown is a graph measuring normalized fusion mRNA levels (normalized relative to vt alone) in HEPG2 cells in the presence of spCas9, with a GR (PD128) only control.

[0119] Fig.18A and Fig.18B Shown are the results of an experiment evaluating the effect of cleavage position (i.e., cleavage within the GR homology arm (HA)) in which donor HA segments 3' and 5' of the cleavage site participate in HDR; in other words, for spCas9 in human cells, the DSB site is within the boundaries of the homology arm compared to cleavage outside the GR homology arm (where only one end can undergo HDR). Fig.18A Shown is a graph comparing fusion mRNA levels (normalized) when cleavage was performed outside HA (1 arm) compared to when cleavage was performed within HA (2 arms); cleavage within HA resulted in significantly higher integration efficiency of spCas9. Fig.18B Shown are graphs measuring fusion mRNA levels (normalized) as a function of alignment length (introns 12 and 13) when cleavage was performed outside HA as well as within HA.

[0120] Figures 19A-19CShown are the results of an experiment in which the expression of GENERIDE in the presence of AZ nuclease Cas9 was measured for different human gRNAs in HEPG2 cells. TM The transgene integration efficiency. Fig.19A Shown is a graph measuring fusion mRNA levels (copies / 2.5 ng) in HEPG2 cells in the presence of GR-mut and AZ nuclease Cas9, with a PD128 control. Fig.19B Shown is a graph measuring fusion mRNA levels (copies / 2.5 ng) in HEPG2 cells in the presence of GR-GFP and AZ nuclease Cas9, with naive and GR only (vt-0290) controls. Fig.19C Shown is a graph measuring fusion mRNA levels (copies / 2.5 ng) in HEPG2 cells in the presence of GR-ATP7B and AZ nuclease Cas9, with naive and GR (vt-235) only controls.

[0121] Fig. 20A and Fig. 20B Shown are the results of an experiment measuring GENERIDE expression in HEPG2 cells using AZ nuclease Cas9 when cleaving within HA compared to outside HA. TM Integrated efficiency. Fig. 20A Shown is a graph comparing fusion mRNA levels (normalized to vt alone) when cleavage is performed outside the HA (1 arm) compared to when cleavage is performed within the HA (2 arms) (left panel). Fig. 20B Shown are graphs measuring fusion mRNA levels (normalized) as a function of alignment length (introns 12 and 13) when cleavage is performed outside HA as well as within HA (right panel).

[0122] Fig.21 Shown are the results of an experiment in which the overall editing efficiency (Editing_Efficiency), the percentage of reads with HDR donor integration (KI_HDR), and the percentage of reads with NHEJ donor integration in the forward or reverse orientation (NHEJ_Fw+Rv) were measured after transfection with AZ nuclease mRNA for different donor-to-gRNA ratios, respectively. Fig.21A series of graphs are shown in which editing efficiency (%) (upper sub-graph), KI_HDR (%) (middle sub-graph) and NHEJ_Fw+Rv (%) (lower sub-graph) are measured in HEPG2 cells after transfection with AZ nuclease mRNA for the specified gRNA and donor (NoDonor, ssDNA or dsDNA). The average KI_HDR (%) compared to dsDNA is higher for ssDNA, and vice versa for NHEJ_Fw+Rv (%). Arrows indicate overlapping gRNAs of SpCas9 and AZ nucleases; asterisks indicate gRNAs with the highest KI-HDR for AZ nucleases.

[0123] FIG. 22A to FIG. 22E Shown are the results of experiments performed to optimize GENERIDE in hepatocytes TM The dosing strategy of the GenVoy-ILM lipid nanoparticles (LNPs) was determined, the delivery of Cas9 via the GenVoy-ILM LNPs was confirmed, and the timing of peak integration of the components was determined. Different groups of mice were administered different combinations of GR (3e13 vg / kg), GR (3e13 vg / kg) in combination with Cas9 (1 mg / kg), or LNPs containing Cas9+gRNA alone (1 mg / kg) after 1, 3, or 7 days. GENERIDE TM The tool vector is DJ-GFP (LB-Vt-0298-001). Fig.22A Shown are immunohistochemistry (IHC) images of hepatocytes detecting Cas9-Flag, confirming Cas9 expression following delivery of Cas9-containing LNPs, with a naive WT control. Fig. 22B A series of graphs are shown measuring ALB-2A levels (μg / ml) up to 6 weeks after LNP administration (left panel) for GR administered with LNPs at different time points (D1, D3, and D7), and at 3 weeks after LNP administration (right panel), with a GR only control. Fig. 22C IHC images of hepatocytes are shown, demonstrating improved integration of GR2.0(Cas9) following delivery of GR(3e13)+LNPs containing Cas9 (1 mg / kg). For this experiment, FvB / NJ mice were given 3e13 vk GR-GFP(1.0 / 1.0) followed by 1 mg / kg GenVoy-ILM LNP-Cas9 on day 1. Fig.22D Shown are bar graphs measuring ALB-2A levels (μg / ml) at 1 week after administration to demonstrate the efficacy of stored LNPs when the LNPs have been stored for 1 or 2 months. Fig.22E Shown is a series of graphs quantifying indel frequencies via TIDE for D4-1 (upper panel) and D4-2 (lower panel).

[0124] Figures 23A-23F Shown are the results of experiments performed to evaluate RNA from Integrated DNA Technologies (IDT) to ensure the feasibility of AstraZeneca (AZ) LNPs. The GR-HDR donor used was DJ-mHA (1.0 / 1.0) GFP-LB-Vt-0298-001, and the LNP used was AZ LNP. Five different combinations were administered to different groups of WT mice: WT GR only, WT GR+LNP N: P=3 (co-formulated) D1 interlaced, WT GR+LNP N: P=6 (co-formulated) D1 interlaced, WT GR+LNP N: P=6 (separate (Sep) formulation) D1 interlaced, and WT GR+LNP N: P=6 (co-formulated) H4 interlaced. Fig.23A Shown are the results of an experiment performed to measure ALB-2A levels (μg / ml) up to 3 weeks after LNP administration for the 5 different combinations listed above, with GR alone as a control. Fig. 23B is a bar graph measuring the copy number of fusion mRNA / 100 ng RNA for the 5 different combinations administered, with GR alone as a control. Fig.23C is a set of IHC images, where IHC was performed to mark and measure GFP+ cells after administration of the 5 combinations listed above. Fig.23D is a bar graph measuring the % of GFP positive cells after administration of the 5 combinations listed above. Fig.23E is a bar graph measuring ALB-2A levels (μg / ml) after administration of the 5 combinations listed above. Fig.23F Shown are ALB-2A levels (μg / ml) as a function of % GFP-positive cells after administration of the five combinations listed above.

[0125] Fig.24A and 24B Shown are the results of experiments performed to optimize the treatment timeline in which cells were treated according to 2 different regimens to determine which regimen better enhanced integration. Fig.24A Shown are the results of experiments in which cells were treated with GR on the same day as mRNA transfection (original protocol). Seeding was performed at D0, followed by nuclease mRNA transfection and GR-GFP treatment at D1, and harvesting at D4 (upper panels). Fig.24AShown is a bar graph measuring the % of hemagglutinin (HA)+ cells for the 4 different combinations administered: naive (control), GR-GFP, GR-GFP+Cas9, and mRNA only (lower left panel), and another bar graph measuring the % of HA+ cells (normalized) for the same 4 combinations administered (lower right panel). Fig. 24B Results are shown for experiments in which cells were treated with GR on day 1 after mRNA transfection. Seeding was performed at D0, followed by nuclease mRNA transfection at D1, followed by GR-GFP treatment at D2, and harvesting at D5 (upper panel). Fig. 24B Shown is a bar graph measuring the % of HA+ cells for the 4 different combinations administered: naive (control), GR-GFP, GR-GFP+Cas9 and mRNA only (lower left panel), and another bar graph measuring the % of HA+ cells (normalized) for the same 4 combinations administered (lower right panel).

[0126] Fig.25 Shown are the results of experiments performed to compare the transfection efficiency of different reagents via immunofluorescence. Fig.25 Lipofectamine tested for transfection of mCherry and eGFP is shown TM Four immunofluorescence images of the transfection efficiency of the 3000 (upper left panel) (with DAPI channel and eGFP-mCherry merge image) and testing of the MessengerMax for transfection of mCherry and eGFP TM Four immunofluorescence images of transfection efficiency (upper right panel) (with DAPI channel and eGFP-mCherry merged image). Fig.25 Also shown is a bar graph for Lipofectamine TM 3000 and MessengerMax TM The eGFP and mCherry transfection efficiency % were measured, as shown in the lower left and lower right panels, respectively.

[0127] Fig.26 Shown are the results of experiments performed to measure the expression of Cas9 mRNA and MessengerMax TM Protein expression (% of HA+ cells) when used as a transfection reagent. Inoculation was performed at D0, followed by nuclease mRNA transfection at D1, followed by GR-GFP treatment at D2, and finally harvesting at D5 (upper panel). Fig.26Shown are bar graphs measuring the % of HA+ cells for the 8 different combinations administered: primary (control), mRNA only, vehicle + GR-GFP + Cas9 mRNA, vehicle + dsODN + GR-GFP + Cas9 mRNA, #2gRNA + GR-GFP + Cas9 mRNA, #2gRNA + dsODN + GR-GFP + Cas9 mRNA, #10gRNA + GR-GFP + Cas9 mRNA, and #10gRNA + dsODN + GR-GFP + Cas9 mRNA (lower left panel), a bar graph measuring the % of HA+ cells (normalized) for the same 8 combinations administered (lower middle panel), and another bar graph measuring cell counts / images for the same 8 combinations administered (lower right panel). DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS Gene therapy

[0128] Gene therapy changes the gene expression profile of a patient's cells through gene transfer, which is the process of delivering exogenous therapeutic genes (called transgenes). A variety of delivery vehicles are known to be used as carriers to transport transgenes into the nucleus of cells to change or enhance cell capabilities (e.g., proteome, functionality, etc.). Developers have made great progress in using various vectors to introduce genes into tissue cells such as liver, retina of the eye, and hematopoietic cells of the bone marrow. These approaches have led to approved therapies in some cases, and in other cases, have shown very promising results in clinical trials.

[0129] There are multiple gene therapy approaches. In conventional AAV gene therapy (also described herein as traditional or conventional gene therapy), the transgene is introduced into the nucleus of the host cell, but is not intended to be integrated into the chromosomal DNA. The transgene is expressed by a non-integrated genetic element called an episome present in the nucleus. The second type of gene therapy uses different types of viruses (such as lentiviruses) that insert themselves into the chromosomal DNA together with the transgene (but at any site).

[0130] Episomal expression of the gene must be driven by an exogenous promoter in order to produce a protein that corrects or ameliorates the disease symptoms. Previously known limitations of gene therapy

[0131] In the case of gene therapies based on episomal expression, the benefit of the therapy typically declines when cells divide during the process of growth or tissue regeneration, because the transgene is not intended to integrate into the host chromosomes and is not replicated during cell division. Therefore, each new generation of cells further reduces the proportion of cells expressing the transgene in the target tissue, resulting in a reduction or elimination of the therapeutic benefit over time.

[0132] While some gene therapies using viral-mediated insertion have the potential to provide long-term benefits because the gene is inserted into the host chromosome, there is no control over where the gene is inserted, which carries the risk of disrupting essential genes or inserting into a location that may promote undesirable effects, such as tumor formation. Therefore, these integrative gene therapy approaches are primarily limited to ex vivo approaches, in which cells are manipulated outside the body, screened for successful integration, and then transplanted into a subject.

[0133] A common feature of many previously used gene therapy approaches is the introduction of a transgene into cells along with an exogenous promoter. The promoter is required to initiate transcription and amplification of DNA to form messenger RNA or mRNA, which is ultimately translated into protein. Expressing high levels of therapeutic proteins from transgenes using previous gene therapy approaches requires strong, engineered promoters. Although these promoters are necessary components of previous gene therapy systems in order to produce protein expression, studies conducted in animal models have shown that nonspecific integration of gene therapy vectors can cause significant increases in tumor development. Using previous gene therapy approaches, promoter strength may play a crucial role in the increased development of these tumors. Therefore, attempts to drive high levels of expression with strong exogenous promoters may have long-term harmful consequences. A. Gene Editing

[0134] Gene editing is typically used to refer to the introduction of broken gene deletions, alterations or enhancements in the DNA of cells using exogenously delivered gene editing mechanisms. Many current gene editing approaches have been limited in their efficacy due to the high incidence of unwanted off-target modifications and the low efficiency of gene correction (partly because cells attempt to quickly repair the introduced DNA breaks). Many gene editing techniques focus on disabling dysfunctional genes or correcting or skipping individual harmful mutations within genes. Compared to methods including inserting complete corrective genes, these previous approaches are generally not able to adequately treat or prevent genetic diseases due to the number of possible mutations in genes.

[0135] Unlike the gene therapy approach using free expression, gene editing allows the repaired genetic region to be propagated to the new generation of cells through normal cell division. In addition, the regulatory mechanism of the cell itself can be used to express the desired protein. The traditional approach for gene editing is based on nucleases, and nucleases are used to introduce single-strand or double-strand breaks at specific cleavage sites in DNA to cause deletions, changes or application of corrective sequences. These methods are intended to provide gene editing (e.g., deletions, changes, insertions, etc. of corrective sequences) at the location of the cleavage site.

[0136] Once the nuclease has cut the DNA, traditional gene editing techniques have used two pathways to modify the DNA: homology-directed repair (HDR) and non-homologous end joining (NHEJ). HDR in these previous methods involves the highly precise incorporation of a DNA sequence complementary to an integration site that is co-located with the cut site. The main advantage of HDR is that it repairs the DNA with high fidelity and avoids introducing unwanted mutations at the integration site. NHEJ is a less selective, more error-prone process that rapidly joins the ends of broken DNA, resulting in a high frequency of insertions or deletions at the cut site. 1. Traditional gene editing based on nuclease

[0137] Nuclease-based gene editing uses nucleases, which are endogenous enzymes or engineered enzymes that can produce single-strand breaks or double-strand breaks at DNA cleavage sites. Nuclease-based gene editing is a two-step process. First, an exogenous nuclease capable of cutting one or both strands of double-stranded DNA is guided to the desired site (e.g., by using guide RNA, site-specific residues within the nuclease, etc.) and performs specific cutting at the cleavage site. Next, the endogenous cellular DNA repair mechanism is activated, and the editing process is completed by NHEJ or less common HDR.

[0138] NHEJ may occur when cells lack a DNA template for replication when repairing DNA cuts. Although the rate of NHEJ is species-dependent compared to other repair mechanisms (e.g., HDR), NHEJ is often preferentially used by cells to repair double-strand breaks in the absence of exogenous compounds (e.g., repair template sequences, enzymes, etc.). Gene editing by NHEJ can be used to introduce small insertions or deletions (called indels), which may result in knockout or reduction of gene function. NHEJ produces insertions and deletions in DNA due to its repair mode, and may also result in the introduction of off-target, unwanted mutations (including chromosomal aberrations).

[0139] Nuclease-mediated HDR occurs when a nuclease and a DNA template that is partially or completely complementary to the target integration site are delivered. In previous methods, the target integration site and the cleavage site would coincide or overlap. The cell uses the DNA template to construct repair DNA, resulting in the insertion of a corrected and / or alternative gene sequence.

[0140] Traditional gene editing (also referred to herein as conventional gene editing) often employs three different classes of nucleases for nuclease-based approaches: transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats-associated (CRISPR / Cas) nucleases; and zinc finger nucleases (ZFNs). 2. Limitations of traditional nuclease-based gene editing

[0141] Previous nuclease-based gene editing approaches have been limited by the use of nucleases to generate overlapping cleavage and integration sites. Additionally, these traditional methods rely on exogenous promoters for transgene expression. Nucleases can cause both on-target and off-target mutations. A major concern in implementing nuclease-based gene editing approaches is the relatively high frequency of off-target effects. Previous gene editing technologies can cause genotoxicity, including chromosomal alterations based on the error-prone NHEJ process, or interruption of native gene expression by targeting exons. GENERIDE TM Technology Platform 3. GENERIDE TM feature

[0142] GENERIDE TM It is a novel AAV-based genome editing technology that can precisely insert therapeutic transgenes into the genome of cells via homologous recombination. TM Provides durable transgene expression regardless of cell proliferation and tissue growth and is approved by GENERIDE TM The modified cells show selective expansion in tissues affected by the disease (e.g., selective expansion of hepatocytes in the liver of a diseased subject). Without wishing to be bound by any particular theory, it is believed that GENERIDE TM is a genome editing technology that utilizes homologous recombination (HR), a naturally occurring HDR process that maintains genomic fidelity. TM Allows for insertion of transgenes into specific targeted genomic locations (e.g., by HR) without the need for exogenous nucleases. TM Allows for insertion of transgenes into specific targeted genomic locations (e.g., via HR) in combination with one or more exogenous nucleases. TM The directed transgene integration is designed to utilize endogenous promoters at the target integration site to drive high levels of tissue-specific gene expression without the deleterious issues associated with the use of exogenous promoters in traditional gene therapy. 4.GENERIDE TM Advantages of technology

[0143] GENERIDE TMThe technology is designed to precisely integrate the corrective gene into the patient's genome to provide a stable, durable therapeutic effect. GENERIDE can be applied, among other things, for rare disorders (e.g., disorders of the liver, CNS, muscle, blood, etc.) in pediatric patients where it is critical to provide treatment early in the patient's life before irreversible disease pathology can occur. TM .

[0144] In some embodiments, GENERIDE TM The technology provides improvements over certain key limitations of both traditional gene therapy and conventional gene editing approaches in a manner that is fully ready to treat genetic diseases (e.g., genetic diseases in pediatric patients). TM AAV vectors are used to deliver the transgene into the nucleus of a cell, where it is subsequently inserted into a target integration site within the cell genome where transgene expression is regulated by one or more endogenous promoters. TM This could allow for lifelong protein production even as the body grows and changes over time, which is not feasible with conventional AAV gene therapies.

[0145] Among other things, GENERIDE TM The technology can also provide surprising and unexpected improvements when combined with targeted nucleases. TM In combination with one or more nucleases that target a specific cleavage site in the cellular genome, wherein the cleavage site is distal to the target integration site (e.g., the cleavage site and the target integration site are not identical and / or do not overlap). TMThe use of such targeted nucleases in combination of components can produce improved transgenic gene integration rates and / or efficiencies (e.g., improved levels of modified DNA, modified mRNA, and / or protein expression). In particular, targeted nucleases can be designed (e.g., alone or in combination with targeting molecules (including but not limited to guide RNA)) to introduce single-strand or double-strand breaks at cleavage sites in DNA sequences, wherein the cleavage sites are away from the target integration site. Although it has been shown that the introduction of cleavage sites away from the target site for transgenic integration can provide improvements in mice (see Caneva et al., JCI Insight [Journal of Clinical Investigation Insights], 2019), these improvements have been shown to depend largely on the precise location of the cleavage site. For example, Caneva showed that cleavage within the flanking intron 14 of mouse albumin produced significant improvements in cleavage and HDR efficiency compared to the limited integration levels when the cleavage site was introduced into intron 13 of mouse albumin (Figure S1 of Caneva). Although Caneva demonstrated that certain "hotspot" cleavage sites away from the integration site may increase HDR efficiency and transgenic integration, it was not demonstrated that these improvements caused site-specific integration and expression of the transgenic of interest. In addition, Caneva limited all experiments to mouse cells containing wild-type mouse albumin. As will be appreciated by those skilled in the art, considerable testing and research will be required to determine whether such a system can be modified for functionality in different species. For example, the high degree of sequence variability across species (such as mice and humans) in known specific genes (e.g., albumin) does not allow technicians to use results from one species to determine whether and where appropriate cleavage or integration sites can be found in other species. Further, it is known in the art that mouse and human genes (e.g., albumin) can contain significant sequence differences, epigenetic differences, and structural differences that can alter the efficiency of nuclease cleavage and / or homologous recombination at specific sites.

[0146] The present disclosure provides the following surprising and unexpected insights: combining targeted nucleases with GENERIDE TM Combinations of components in which the nuclease cleavage site is distal to the target integration site (e.g., the cleavage site and integration site are not identical and / or do not overlap) can provide unexpected improvements in transgene integration rate and / or expression efficiency in human cells and tissues. In some embodiments, such improvements can be observed for multiple cleavage sites (e.g., multiple cleavage sites within different non-coding sequences in the human DNA sequence of interest (e.g., both introns 13 and 14 of human albumin)).

[0147] The modular approach disclosed herein can be applied to allow GENERIDE TMDelivers robust tissue-specific gene expression that is reproducible across different therapeutics delivered to tissues (e.g., liver, muscle, CNS, blood).

[0148] Previous work on non-disruptive gene targeting is described in WO 2013 / 158309, the contents of which are incorporated herein by reference. Previous work on genome editing without the use of nucleases is described in WO 2015 / 143177, the contents of which are incorporated herein by reference. Previous work on non-disruptive gene therapy for the treatment of MMA is described in WO 2020 / 032986, the contents of which are incorporated herein by reference. Previous work on gene therapy monitoring is described in WO / 2020 / 214582, the contents of which are incorporated herein by reference. B.GENERIDE TM Components Delivery vehicle

[0149] There are multiple gene therapy approaches known in the art. Therefore, there are multiple delivery mechanisms known in the art. In some embodiments, a transgene is provided using a delivery vehicle. In some embodiments, the composition of the present disclosure comprises a delivery vehicle. In some embodiments, the delivery vehicle is or comprises a viral particle (e.g., a viral vector). In some embodiments, the delivery vehicle is or comprises a non-viral particle and / or a non-particle load (e.g., a nuclease). In some embodiments, the delivery vehicle is a lipid particle (e.g., a lipid nanoparticle). Various lipid nanoparticles for delivering nucleic acids are known in the art, such as those described in the following documents: WO 2015184256; WO 2013149140; WO 2014089486 A1; WO2009127060; WO 2011071860; WO 2020219941, the contents of each of these documents are incorporated herein by reference.

[0150] In some embodiments, the delivery vehicle is or comprises exosomes. Those skilled in the art will recognize various methods for the production and use of exosomes. Examples of such methods and uses are described in Luan et al., Acta Pharmacologica Sinica [Chinese Journal of Pharmacology], Vol. 38, pp. 754-763 (2017).

[0151] In some embodiments, a combination of one or more different loads can be delivered using one or more delivery systems described herein (e.g., viral vectors, lipid nanoparticles, etc.). In some embodiments, a delivery system can deliver a load (e.g., one or more nucleases (e.g., Cas proteins, endonucleases, TALENs, ZFNs)) in combination with a second distinct load (e.g., a polynucleotide sequence comprising a transgene). In some embodiments, a first delivery system (e.g., lipid nanoparticles (LNPs)) can deliver a load (e.g., one or more nucleases (e.g., Cas proteins, endonucleases, TALENs, ZFNs)) in combination with a second distinct delivery system (e.g., a viral vector, such as a recombinant AAV vector) that delivers a second distinct load (e.g., a polynucleotide sequence comprising a transgene). Viral vectors

[0152] In some embodiments, the delivery vehicle is or comprises a viral vector. The viral vector comprises a virus or a viral chromosome material, in which a heterologous nucleic acid sequence can be inserted to transfer to a target sequence of interest (e.g., to transfer to the genomic DNA in a cell). Various viruses can be used as viral vectors, including, for example, single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) viruses and / or RNA viruses with a DNA stage in their life cycle. In some embodiments, the viral vector is or comprises an adeno-associated virus (AAV) or an AAV variant.

[0153] In some embodiments, the vector particle is a single unit of a virus comprising a capsid, and the capsid encapsidates a polynucleotide based on the virus (e.g., a wild-type viral genome or a recombinant viral vector). In some embodiments, the vector particle is or comprises an AAV vector particle. In some embodiments, the AAV vector particle refers to a vector particle consisting of at least one AAV capsid protein and an encapsidated AAV vector. In some embodiments, the vector particle (also referred to as a viral vector) comprises at least one AAV capsid protein and an encapsidated AAV vector, wherein the vector further comprises one or more heterologous polynucleotide sequences. Capsid protein

[0154] In some embodiments, the expression construct comprises a polynucleotide sequence encoding a capsid protein from one or more AAV subtypes (including naturally occurring and recombinant AAV). In some embodiments, the expression construct comprises a polynucleotide sequence encoding a capsid protein from: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVC11.01, AAVC11.02, AAVC11.03, AAVC11.04, AAVC11.05, AAVC11.06, AAVC11.07, AAVC11.08, AAVC11.09, AAVC11. 10, AAVC11.11 (interchangeably referred to herein as sL65), AAVC11.12, AAVC11.13, AAVC11.14, AAVC11.15, AAVC11.16, AAVC11.17, AAVC11.18, AAVC11.19, AAV-DJ, AAV-LK03, AAV-LK19, AAVrh.74, AAVrh.10, AAVhu.37, AAVrh.K, AAVrh.39, AAV12, AAV 13, AAVrh.8, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, ovine AAV, hybrid AAV (e.g., AAV comprising one or more sequences of one AAV subtype and one or more sequences of a second subtype) and / or AAV comprising a mutant AAV capsid protein or a chimeric AAV capsid (e.g., a capsid having polynucleotide sequences derived from two or more different AAV serotypes) or variants thereof.

[0155] In some embodiments, the viral vector is packaged in a capsid protein (e.g., a capsid protein from one or more AAV subtypes). In some embodiments, the capsid protein provides increased or enhanced cell (e.g., human cell or mouse cell) transduction relative to a reference capsid protein. In some embodiments, the capsid protein provides increased or enhanced transduction (e.g., liver tropism, muscle tropism, CNS tropism, lung tropism) relative to a reference capsid protein for certain cell or tissue types. In some embodiments, the capsid protein increases or enhances the transduction of cells or tissues (e.g., liver, muscle and / or CNS) relative to a reference capsid protein by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more. In some embodiments, the capsid protein increases or enhances transduction of a cell or tissue (e.g., liver, muscle, lung, and / or CNS) by at least about 1.2x, 1.5x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 11x, 12x, 13x, 14x, 15x, 16x, 17x, 18x, 19x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, or more relative to a reference capsid protein.

[0156] In some embodiments, the sequence encoding the capsid protein may be or include a sequence having at least 80%, 85%, 90%, 95%, 99% or 100% identity with the corresponding wild-type capsid protein. In some embodiments, the sequence encoding the capsid protein may be or include a sequence having at least 80%, 85%, 90%, 95%, 99% or 100% identity with the corresponding sequence encoding the engineered capsid protein (e.g., chimeric capsid protein, codon-optimized capsid protein, etc.). In some embodiments, the sequence encoding the capsid protein may be or include a sequence having at least 80%, 85%, 90%, 95%, 99% or 100% identity with the exemplary sequence in Table 1 below.

[0157] Table 1: Exemplary sequences encoding one or more capsid proteins AAV structure and function

[0158] Adeno-associated virus (AAV) is a small virus consisting of an icosahedral protein capsid and a single-stranded DNA genome. The AAV viral capsid contains three subunits (VP1, VP2 and VP3) and two terminal inverted repeat (ITR) regions located at the ends of the genome sequence. ITR serves as a replication origin and plays a role in viral packaging. The viral genome also contains rep and cap genes, which are associated with replication and capsid packaging, respectively. In most wild-type AAVs, the rep gene encodes four proteins required for viral replication: Rep 78, Rep68, Rep52 and Rep40. The cap gene encodes capsid subunits and assembly activation protein (AAP), which can promote the assembly of viral particles. AAV is usually replication-defective and requires the presence of helper viruses or helper virus functions (e.g., herpes simplex virus (HSV) and / or adenovirus (AdV)) in order to replicate in infected cells. For example, in some embodiments, AAV requires adenovirus E1A, E2A, E4 and VA RNA genes in order to replicate in host cells. Recombinant AAV

[0159] In general, recombinant AAV (rAAV) vectors can contain many of the same elements found in wild-type AAV (including similar capsid sequences and structures) and polynucleotide sequences from non-AAV sources (e.g., polynucleotides heterologous to AAV). In some embodiments, rAAV will replace natural, wild-type AAV sequences with polynucleotide sequences encoding payloads. For example, in some embodiments, rAAV will contain polynucleotide sequences encoding one or more genes intended for therapeutic purposes (e.g., for gene therapy). rAAV can be modified to remove one or more wild-type viral coding sequences. For example, rAAV can be engineered to contain only one ITR and / or one or more genes (e.g., rep and cap genes) necessary for packaging that will be found in wild-type AAV. Gene expression with rAAV is generally limited to one or more genes with a total length of 5kb or less, because larger sequences cannot be effectively packaged in viral capsids. In some embodiments, two or more rAAVs can be used to provide a portion of a larger payload, for example, to provide a complete coding sequence of a gene that would normally be too large to fit in a single AAV.

[0160] Among other things, the present disclosure provides viral vectors comprising one or more polypeptides described herein. In some embodiments, rAAV can comprise one or more capsid proteins (e.g., one or more capsid proteins selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVC11.01, AAVC11.02, AAVC11.03, AAVC11.04, AAVC11.05, AAVC11.06, AAVC11.07, AAVC11.08, AAVC11.09, AAVC11.10, AAVC11.11, AAVC11.12, AAVC11.13, AAVC11.14, AAVC11.15, AAVC11.16, AAVC11.17, AAVC11.18, AAVC11.19, AAVC11.20, AAVC11.21, AAVC11.22, AAVC11.23, AAVC11.24, AAVC11.25, AAVC11.26, AAVC11.27, AAVC11.28, AAVC11.29, AAVC11.30, AAVC11.31, AAVC11.32, AAVC11.33, AAVC11.34, AAVC11.35, AAVC11.36, AAVC11.37, AAVC11.38, AAVC11.39, AAVC11.40, AAVC11.41 VC11.10, AAVC11.11 (interchangeably referred to herein as sL65), AAVC11.12, AAVC11.13, AAVC11.14, AAVC11.15, AAVC11.16, AAVC11.17, AAVC11.18, AAVC11.19, AAV-DJ, AAV-LK03, AAV-LK19, AAVrh.74, AAVrh.10, AAVhu.37, AAVrh.K, AAVrh.39, AAV12, AAV 13, AAVrh.8, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, ovine AAV, hybrid AAV (e.g., AAV comprising one or more sequences of one AAV subtype and one or more sequences of a second subtype), and / or AAV comprising mutant AAV capsid proteins or chimeric AAV capsids (e.g., capsids having polynucleotide sequences derived from two or more different AAV serotypes). In some embodiments, rAAV may comprise one or more polynucleotide sequences encoding a gene or nucleic acid of interest (e.g., a gene and / or inhibitory nucleic acid sequence for treating a genetic disease / disorder).

[0161] In some embodiments, the recombinant AAV vector may comprise at least one ITR. In some embodiments, the recombinant AAV vector comprises two ITRs. In some embodiments, the recombinant AAV vector comprises a 5' ITR. In some embodiments, the recombinant AAV vector comprises a 3' ITR. In some embodiments, the recombinant AAV vector comprises an AAV2 ITR. In some embodiments, the recombinant AAV vector comprises a portion of an AAV2 ITR. In some embodiments, the recombinant AAV vector comprises an ITR having at least 80%, 85%, 90%, 95%, 99% or 100% sequence identity with an AAV2 ITR. In some embodiments, the recombinant AAV vector comprises an ITR having 90%, 95%, 99% or 100% sequence identity with one of the exemplary sequences in Table 2 below.

[0162] Table 2: Exemplary ITR sequences

[0163] AAV vectors may be able to replicate in infected host cells (replication competent) or not (replication incompetent). Replication competent AAV (rcAAV) requires the presence of one or more functional AAV packaging genes. Recombinant AAV vectors are generally designed to be replication incompetent in mammalian cells to reduce the likelihood of producing rcAAV by recombination with sequences encoding AAV packaging genes. In some embodiments, the rAAV vector preparations described herein are designed to contain very few, if any, rcAAV vectors. In some embodiments, the rAAV vector preparations contain less than about 1 rcAAV / 10 2 rAAV vector. In some embodiments, the rAAV vector preparation contains less than about 1rcAAV / 10 4 rAAV vector. In some embodiments, the rAAV vector preparation contains less than about 1rcAAV / 10 8 rAAV vector. In some embodiments, the rAAV vector preparation contains less than about 1 rcAAV / 10 12 rAAV vectors. In some embodiments, the rAAV vector preparation does not comprise a rcAAV vector. Lipid Nanoparticles (LNP)

[0164] Lipid nanoparticles (LNPs) are delivery systems that, among other things, can achieve intracellular delivery of intact forms of nucleic acids, thereby achieving biological changes including therapeutic effects. On the one hand, lipid nanoparticles are lipid compositions comprising at least one lipid. In certain embodiments, lipid nanoparticles may further comprise at least one nucleic acid (e.g., DNA, RNA, etc.). In certain embodiments, lipid nanoparticles may comprise therapeutic nucleic acids (e.g., DNA, RNA, etc.) encapsidated in the lipid portion of the nanoparticle.

[0165] The present disclosure provides compositions comprising lipid nanoparticles. In some embodiments, the lipid nanoparticles comprise one or more components. In some embodiments, the lipid nanoparticles comprise one or more components, such as ionizable lipids, sterols, conjugate-linker lipids and phospholipids. In some embodiments, the lipid nanoparticles may comprise one or more compounds as described herein. Among other things, the present disclosure describes that the selection and combination of one or more components in the components as described herein can affect the properties of the lipid nanoparticles, such as diameter, pKa, stabilization and ionizability.

[0166] Among other things, the present disclosure describes that the selection and combination of one or more components in the components as described herein affect the functional activity of lipid nanoparticles, such as tropism, stabilization and delivery efficacy. For example, the present disclosure describes that the combination of components can be better suited for delivering a specific load (e.g., DNA (e.g., plasmid DNA, linearized DNA (e.g., linearized and dimerized DNA (CELID))) compared to an appropriate reference (e.g., alternative nucleic acid sequences, proteins, etc.).

[0167] In some embodiments, the lipid nanoparticles may be or include structures produced by available methods (see Jayaraman et al., Angewandte Chemie [Applied Chemistry] (English International Edition), 2012; Li et al. PLOS One [Public Library of Science Comprehensive], 2013; Kulkarni et al. Nanomedicine: Nanotechnology, Biology, and Medicine [Nanomedicine: Nanotechnology, Biology, and Medicine], 2017; US2019 / 0240345; WO 2019 / 089828; WO / 2021 / 102411; WO / 2019 / 046809; WO / 2018089540, each of which is incorporated herein by reference in its entirety). Based on the teachings provided herein, those skilled in the art will appreciate the possibility that alternative, available lipid nanoparticles may replace those described herein. Ionizable lipids

[0168] In some embodiments, lipid nanoparticles include one or more ionizable lipids as described herein. In some embodiments, ionizable lipids may include amine-containing groups on the head group. In some embodiments, ionizable lipids are or include compounds as described herein (e.g., compounds with formula II, formula III, or formula IV). In some embodiments, by the total mole of the components of lipid nanoparticles, ionizable lipids are present in lipid nanoparticle (LNP) formulations from about 30 mole percent to about 70 mole percent. In some embodiments, by the total mole of the components of lipid nanoparticles, ionizable lipids are present at about 33 mole percent to about 60 mole percent. In some embodiments, by the total mole of the components of lipid nanoparticles, ionizable lipids are present at about 34 mole percent to about 55 mole percent. In some embodiments, by the total mole of the components of lipid nanoparticles, ionizable lipids are present at about 33 mole percent to about 51 mole percent. In some embodiments, by the total mole of the components of lipid nanoparticles, ionizable lipids are present at about 34.7 mole percent. In some embodiments, by the total mole of the components of lipid nanoparticles, ionizable lipids are present at about 50 mole percent. Sterols

[0169] In some embodiments, lipid nanoparticles include one or more sterols as described herein. In some embodiments, sterols are cholesterol or variants or derivatives thereof. In some embodiments, cholesterol is modified. In some embodiments, cholesterol is oxidized cholesterol. In some embodiments, cholesterol is esterified cholesterol. Unmodified cholesterol can form side chain or epoxidized variants by enzyme action. In some embodiments, cholesterol can be oxidized on a β-ring structure or on a hydrocarbon tail structure. In some embodiments, sterols are plant sterols. Exemplary sterols considered for use in the disclosed lipid nanoparticles include, but are not limited to, 25-hydroxycholesterol (25-OH), 20α-hydroxycholesterol (20α-OH), 27-hydroxycholesterol, 6-keto-5α-hydroxycholesterol, 7-ketocholesterol, 7β-hydroxycholesterol, 7α-hydroxycholesterol, 7β-25-dihydroxycholesterol, β-sitosterol, stigmasterol, brassica sterol, campesterol, or a combination thereof. In some embodiments, cholesterol with side chain oxidation can enhance cargo delivery relative to other cholesterol variants. In some embodiments, cholesterol is unmodified cholesterol.

[0170] In some embodiments, the LNP composition comprises about 20 mole percent to about 50 mole percent of sterol. In some embodiments, the LNP composition comprises about 38 mole percent of sterol. In some embodiments, the LNP composition comprises about 38.5 mole percent of sterol. In some embodiments, the LNP composition comprises about 33.8 mole percent of cholesterol. Conjugate-Linker Lipid

[0171] In some embodiments, lipid nanoparticles include one or more conjugates-linker lipids as described herein. In some embodiments, conjugate-linker lipids are or include polyethylene glycol (PEG) lipids or PEG-modified lipids. In some embodiments, PEG or PEG-modified lipids may be alternatively referred to as PEGylated lipids or PEG lipids. PEGylated lipids may be used to enhance the in vitro colloidal stability and in vivo circulation time of lipid nanoparticles. In some embodiments, PEGization is reversible because the PEG moiety is gradually released in the blood circulation. Exemplary PEG lipids include but are not limited to PEG conjugated with saturated or unsaturated alkyl chains with a length of C6-C20. PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramides (PEG-CER), PEG-modified dialkylamines, PEG-modified diacylglycerols (PEG-DAG), PEG-modified dialkylglycerols, and mixtures thereof. For example, in some embodiments, PEG lipids may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPE, PEG-DSG or PEG-DSPE lipids.

[0172] In some embodiments, conjugate-joint lipids include polyethylene glycol lipids. In some embodiments, conjugate-joint lipids include dimyristoylglycerol (DMG), 1,2-dipalmitoyl-rac-glycerol, methoxypolyethylene glycol (DPG-PEG) or 1,2-distearoyl-rac-glycerol-3-methylpolyoxyethylene (DSG-PEG). In some embodiments, conjugate-joint lipids have an average molecular weight of about 500Da to about 5000Da. In some embodiments, conjugate-joint lipids have an average molecular weight of about 2000Da. In some embodiments, LNP compositions include conjugate-joint lipids of about 0 molar percent to about 5 molar percent. In some embodiments, LNP compositions include conjugate-joint lipids of about 1.5 molar percent. In some embodiments, LNP compositions include conjugate-joint lipids of about 3 molar percent. Phospholipids

[0173] In some embodiments, lipid nanoparticles include one or more phospholipids as described herein. In some embodiments, one or more phospholipids can be assembled into one or more lipid bilayers. In some embodiments, one or more phospholipids may include a phospholipid moiety. In some embodiments, one or more phospholipids may include one or more fatty acid moieties. In some embodiments, one or more phospholipids may include a phospholipid moiety and one or more fatty acid moieties. In some embodiments, phospholipids are or include compounds as described herein (e.g., compounds with formula I). ​​In some embodiments, phospholipid moieties include but are not limited to phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine and sphingomyelin. In some embodiments, fatty acid moieties include but are not limited to lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid and docosahexaenoic acid. Non-natural substances are also envisioned, including natural substances with modifications and substitutions (including branching, oxidation, cyclization and alkynes). For example, phospholipids can be functionalized or cross-linked with one or more alkynes (e.g., alkenyl groups in which one or more double bonds are replaced by triple bonds). Under appropriate reaction conditions, alkyne groups can undergo copper-catalyzed cycloadditions upon exposure to azides. Such reactions can be used to functionalize the lipid bilayer of nanoparticle compositions to facilitate membrane penetration or cell recognition, or to conjugate nanoparticle compositions with useful components such as targeting or imaging moieties (e.g., dyes).

[0174] Exemplary phospholipids include, but are not limited to, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycerophosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero- 3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycerophosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-0-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dialinolenoyl-sn-glycero-3-phosphocholine, 1,2-diacetoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-racem-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG) In some embodiments, the phospholipid is DSPC. In some embodiments, the phospholipid is DMPC.

[0175] In some embodiments, the phospholipid comprises 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl) (succinyl PE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl) (succinyl DPPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), or a combination thereof. diameter

[0176] Among other things, the present disclosure describes compositions having an average hydrodynamic diameter of from about 30 nm to about 220 nm. In some embodiments, the average hydrodynamic diameter of the lipid nanoparticles described herein may be from about 30 nm to about 220 nm. In some embodiments, lipid nanoparticles as described herein can have following average hydrodynamic diameter: about 30nm, 35nm, 40nm, 45nm, 50nm, 55nm, 60nm, 65nm, 70nm, 75nm, 80nm, 85nm, 90nm, 95nm, 100nm, 105nm, 110nm, 115nm, 120nm, 125nm, 130nm, 135nm, 140nm, 145nm, 150nm, 155nm, 160nm, 165nm, 170nm, 175nm, 180nm, 185nm, 190nm, 195nm, 200nm, 205nm, 210nm, 215nm, 220nm or any scope with endpoints defined by any two of the above values. For example, in some embodiments, the average hydrodynamic diameter of lipid nanoparticles as described herein is between 50nm and 200nm. pKa

[0177] Among other things, the present disclosure describes compositions, formulations, nanoparticles, and / or nanomaterials having a pKa from about 5 to about 9.

[0178] In some embodiments, the lipid nanoparticles described herein have a pKa from about 5 to about 9. In some embodiments, the lipid nanoparticles described herein have a pKa of about 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, or any range having endpoints defined by any two of the aforementioned values. In some embodiments, the lipid nanoparticles described herein have a pKa of about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, or any range having endpoints defined by any two of the aforementioned values. Exemplary LNP Compositions

[0179] The present invention provides compositions comprising lipid nanoparticles. In some embodiments, the lipid nanoparticle composition comprises about 30 mole percent to about 70 mole percent of ionizable lipids, about 5 mole percent to about 25 mole percent of phospholipids, about 25 mole percent to about 45 mole percent of cholesterol, and about 0 mole percent to about 5 mole percent of conjugate-linker lipids. In some embodiments, the lipid nanoparticle composition comprises about 50 mole percent of ionizable lipids, about 20 mole percent of phospholipids, about 39 mole percent of cholesterol, and about 1 mole percent of conjugate-linker lipids.

[0180] In some embodiments, the lipid nanoparticle composition comprises about 30 to about 70 mole percent of an ionizable lipid of Formula II, Formula III, or Formula IV, about 5 to about 25 mole percent of DSPC, about 25 to about 45 mole percent of cholesterol, and about 0 to about 5 mole percent of lipid PEG (based on the total moles of these ingredients).

[0181] In some embodiments, the lipid nanoparticle (LNP) formulation comprises a (total of one or more ionizable lipids, sterols, conjugate-linker lipids, and phospholipids): DNA mass ratio of about 2: 1 to 50: 1. In some embodiments, the LNP formulation comprises a (total of one or more ionizable lipids, sterols, conjugate-linker lipids, and phospholipids): DNA mass ratio of about 2: 1, about 3: 1, about 4: 1, about 5: 1, about 6: 1, about 7: 1, about 8: 1, about 9: 1, about 10: 1, about 11: 1, about 12: 1, about 13: 1, about 14: 1, about 15: 1, about 16: 1, about 17: 1, about 18: 1, about 19: 1, about 20: 1, about 21: 1, about 22: 1 :1, about 23:1, about 24:1, about 25:1, about 26:1, about 27:1, about 28:1, about 29:1, about 30:1, about 31:1, about 32:1, about 33:1, about 34:1, about 35:1, about 36:1, about 37:1, about 38:1, about 39:1, about 40:1, about 41:1, about 42:1, about 43:1, about 44:1, about 45:1, about 46:1, about 47:1, about 48:1, about 49:1, about 50:1. Exemplary compounds

[0182] Among other things, the present disclosure describes compositions and / or nanoparticles comprising one or more compounds as described herein.

[0183] In some embodiments, the present disclosure provides lipid nanoparticles comprising compounds containing one or more partially unsaturated lipid groups. In some embodiments, the lipid nanoparticles provided include compounds containing one or more trimethylated amine groups. In some embodiments, the lipid nanoparticles provided include compounds containing one or more dimethylated amine groups.

[0184] In some embodiments, the present disclosure provides a compound having Formula I: or a pharmaceutically acceptable salt thereof, wherein: L 1 and L 2 are each independently a covalent bond, -C(O)-, -OC-(O)-, or an optionally substituted divalent saturated or unsaturated, linear or branched C1-C 12 hydrocarbon chain; R 1 and R 2 Each of which is independently selected from saturated or unsaturated straight or branched C1-C 20 an optionally substituted group of a hydrocarbon chain in which 1 to 3 methylene units are optionally substituted by -O-; and R3 , R 4 and R 5 are each independently hydrogen or an optionally substituted divalent saturated or unsaturated straight or branched C1-C 10 Hydrocarbon chain.

[0185] In some embodiments, the present disclosure provides a compound having Formula II: or a pharmaceutically acceptable salt thereof, wherein: L 1 and L 2 are each independently a covalent bond, -C(O)-, -OC-(O)-, or an optionally substituted divalent saturated or unsaturated, linear or branched C1-C 12 hydrocarbon chain; R 1 and R 2 Each of which is independently selected from saturated or unsaturated straight or branched C1-C 20 an optionally substituted group of a hydrocarbon chain in which 1 to 3 methylene units are optionally substituted by -O-; and R 3 and R 4 are each independently hydrogen or an optionally substituted divalent saturated or unsaturated straight or branched C1-C 10 Hydrocarbon chain.

[0186] In some embodiments, the present disclosure provides a compound having Formula III: or a pharmaceutically acceptable salt thereof, wherein: L 1 and L 2 are each independently a covalent bond, -C(O)-, -OC-(O)-, or an optionally substituted divalent saturated or unsaturated, linear or branched C1-C 12 hydrocarbon chain; R 1 are independently hydrogen, or optionally substituted selected from saturated or unsaturated straight or branched C1-C 20 A hydrocarbon chain group in which 1 to 3 methylene units are optionally replaced by -O-; and R 2 and R 3 are each independently hydrogen or an optionally substituted divalent saturated or unsaturated straight or branched C1-C 10 Hydrocarbon chain.

[0187] In some embodiments, the present disclosure provides a compound having Formula IV: or a pharmaceutically acceptable salt thereof, wherein: R 1 and R 2 Each of which is independently selected from saturated or unsaturated straight or branched C1-C 20 an optionally substituted group of a hydrocarbon chain in which 1 to 3 methylene units are optionally substituted by -O-; and R 3 and R 4 are each independently hydrogen or an optionally substituted divalent saturated or unsaturated straight or branched C1-C 10 Hydrocarbon chain. Targeted nuclease

[0188] There are a variety of DNA targeting nucleases known in the art. In some embodiments, the nucleases described herein may comprise any polypeptide or protein sequence capable of inducing single-stranded or double-stranded DNA breaks. In some embodiments, the nucleases described herein may be wild-type proteins, fusion proteins, engineered proteins, or variants thereof. In some embodiments, the nuclease may be selected from a class of enzymes comprising TALEN, ZFN, meganuclease, TALE nickase, zinc finger nickase, and Cas nuclease (e.g., Staphylococcus aureus Cas9 (saCas9), Streptococcus pyogenes (spCas9), AZ nuclease, HF1-Cas9, HF2-Cas9, or HiFi-Cas9). In some embodiments, the nucleases are combined with other GENERIDE TM The components are combined to produce targeted integration of the transgene at the target integration site. In some embodiments, the nuclease is combined with other GENERIDE TM The components are combined to produce targeted integration of the transgene at a target integration site distal to the nuclease cleavage site. TALEN (Transcription Activator-Like Effector Nuclease)

[0189] TALEN is a restriction enzyme comprising a DNA binding domain (transcription activator-like (TAL) effector domain) fused directly or indirectly to a nuclease. TAL effectors (TALEs) comprise a set of highly conserved repeats of 33-34 amino acids that can be engineered to bind to specific DNA sequences within the genome of the cell. The fusion of engineered TALEs with a nuclease cleavage domain (e.g., FokI nuclease) can produce TALE nucleases (TALENs) that can target specific DNA sequences to produce DNA breaks. Combinations of TALEN (e.g., TALEN pairs) can be used to produce targeted double-strand breaks (DSBs) in DNA sequences.

[0190] In some embodiments, TALEN can be engineered to target specific DNA sequences and induce DSB. In some embodiments, one or more TALEN (e.g., TALEN pairs) can be combined to induce DSB by binding to a target DNA sequence on a genomic DNA chain. In some embodiments, certain features of TALEN (e.g., DNA binding region, joint region, nuclease fusion region) can be designed or optimized to produce improved DNA binding and / or cutting efficiency. In some embodiments, certain features of TALEN (e.g., DNA binding region, joint region, nuclease fusion region) can be designed or optimized to reduce off-target binding. TALE nickase

[0191] TALE comprises a set of highly conserved repeat sequences with 33-34 amino acids, which can be engineered to bind to a specific DNA sequence in the genome of a cell. The combination of TALEN (e.g., TALEN pairs) can be used to produce targeted DNA breaks in a DNA sequence. TALE nickases can be designed by introducing mutations (e.g., D450A) into the cleavage domain (e.g., FokI nuclease) of a TALEN monomer in an engineered TALEN (e.g., TALEN pair). TALE nickases are able to recognize specific regions of a target DNA sequence and produce targeted single-strand breaks (SSBs) in a DNA sequence.

[0192] In some embodiments, TALE nickases can be engineered to target specific DNA sequences and induce SSBs. In some embodiments, certain features of TALE nickases (e.g., DNA binding regions, linker regions, nuclease fusion regions) can be designed or optimized to produce improved DNA binding and / or cleavage efficiency. In some embodiments, certain features of TALE nickases (e.g., DNA binding regions, linker regions, nuclease fusion regions) can be designed or optimized to reduce off-target binding. ZFN (zinc finger nuclease)

[0193] ZFN is a restriction enzyme comprising a DNA binding domain (zinc finger protein (ZFP)) fused directly or indirectly to a nuclease. ZFP comprises 3-6 separate zinc finger repeats that can recognize between 9bp and 18bp of a target DNA sequence. The fusion of an engineered ZFP with a nuclease cleavage domain (e.g., FokI nuclease) can produce a ZFN that can target a specific DNA sequence to produce DNA breaks. Combinations of ZFNs (e.g., ZFN pairs) can be used to produce targeted double-strand breaks (DSBs) in DNA sequences.

[0194] In some embodiments, ZFNs can be engineered to target specific DNA sequences and induce DSBs. In some embodiments, one or more ZFNs (e.g., ZFN pairs) can be combined to induce DSBs by binding to target DNA sequences on genomic DNA strands. In some embodiments, certain features of ZFNs (e.g., DNA binding regions, linker regions, nuclease fusion regions) can be designed or optimized to produce improved DNA binding and / or cleavage efficiency. In some embodiments, certain features of ZFNs (e.g., DNA binding regions, linker regions, nuclease fusion regions) can be designed or optimized to reduce off-target binding. Zinc finger nickase

[0195] ZFP comprises 3-6 separate zinc finger repeat sequences, and these zinc finger repeat sequences can recognize between 9bp and 18bp of target DNA sequence.Combinations of ZFN (for example, ZFN pairs) can be used to produce targeted DNA breaks in DNA sequences.ZF nickase can be designed by introducing mutation (for example, D450A) into the cleavage domain (for example, FokI nuclease) of a ZFN monomer in engineered ZFN.ZF nickase can recognize specific regions of target DNA sequence and generate targeted SSB in DNA sequence.

[0196] In some embodiments, ZF nickases can be engineered to target specific DNA sequences and induce SSBs. In some embodiments, certain features of ZF nickases (e.g., DNA binding regions, linker regions, nuclease fusion regions) can be designed or optimized to produce improved DNA binding and / or cleavage efficiency. In some embodiments, certain features of ZF nickases (e.g., DNA binding regions, linker regions, nuclease fusion regions) can be designed or optimized to reduce off-target binding. Meganuclease

[0197] Meganucleases (also called homing endonucleases) are sequence-specific endonucleases that can recognize between 12bp and 40bp of a target DNA sequence. The largest class of homing endonucleases is the LAGLIDADG family, which includes but is not limited to the well-characterized and commonly used I-CreI and I-SceI enzymes. Reengineering these homing endonucleases can produce homing endonucleases that can target specific DNA sequences to produce DNA breaks. Chimeric proteins comprising fusions of meganucleases, ZFPs, and TALs have been engineered to generate novel enzymes that exploit the binding affinity of ZFs and TALEs and the cleavage specificity of meganucleases.

[0198] In some embodiments, meganucleases may be engineered to target specific DNA sequences and induce DSBs. In some embodiments, one or more meganucleases may be combined to induce DSBs by binding to target DNA sequences on genomic DNA strands. In some embodiments, one or more meganucleases may be combined with ZFPs and / or TALs to induce DSBs by binding to target DNA sequences on genomic DNA strands. In some embodiments, certain features of meganucleases (e.g., DNA binding regions, linker regions, nuclease fusion regions) may be designed or optimized to produce improved DNA binding and / or cleavage efficiency. In some embodiments, certain features of meganucleases (e.g., DNA binding regions, linker regions, nuclease fusion regions) may be designed or optimized to reduce off-target binding. CRISPR-related systems

[0199] CRISPR related systems generally include Cas nucleases (e.g., Cas9, Cas13, Cas12a, Cas9 nickases, etc.) or variants thereof and engineered guide RNA (gRNA) sequences. The gRNA sequence is designed to have partial complementarity with the target target genomic DNA sequence within a certain distance from the adjacent motif (PAM) of the pre-spacer sequence. The PAM sequence and the distance from the Cas nuclease cleavage site are generally specific for specific enzyme types (e.g., Cas9, Cas13, Cas12a, Cas9 nickases). Some Cas nucleases or variants thereof can produce DSBs in the target DNA sequence. Cas9

[0200] Cas9 is an RNA-guided DNA endonuclease that works through base pair complementarity between the first 17-20 nucleotides of an engineered gRNA and the complementary strand of the target genomic DNA. The target genomic DNA must present an appropriate PAM sequence (e.g., NGG or NAG) near the gRNA complementary region. Once bound to the appropriate target sequence, Cas9 induces DSBs in the target DNA sequence. Cas9 nucleases may be present in many different species, including, for example, Streptococcus pyogenes (SpCas9), Staphylococcus aureus, and Neisseria meningitidis. Engineered Cas9 nucleases may enhance cleavage activity or reduce off-target cleavage effects. Cas12a / Cpf1

[0201] Cas12a (Cpf1) is an RNA-guided DNA endonuclease that works through base pair complementarity between the first 20 nucleotides of the engineered gRNA and the complementary strand of the target genomic DNA. The target genomic DNA must present an appropriate PAM sequence (e.g., TTN / TTTN / TTTV) near the gRNA complementary region. Once bound to the appropriate target sequence, Cas12a induces staggered double-stranded cuts in the target DNA sequence. Cas9 nickase

[0202] Mutations in one of the two wild-type Cas9 nuclease domains (e.g., D10A and / or H840A) result in Cas9 variants (Cas9 nickases). Similar to wild-type Cas 9, nickases work through base pair complementarity between the nucleotides of the engineered gRNA and the complementary strand of the target genomic DNA. Unlike wild-type Cas9, once bound to the appropriate target sequence, Cas9 nickases only cut one strand of DNA, generating repairable SSBs without inducing indels. gRNA (guide RNA)

[0203] The guide RNA molecule can be or include a nucleic acid that promotes gRNA / Cas complex specific targeting or homing to the target. In some embodiments, gRNA will be incorporated into the function and structure of crispr RNA (crRNA) and / or trans-activation crispr RNA (tracrRNA). In some embodiments, gRNA can be chimeric and include crRNA and tracrRNA features in a single nucleic acid sequence (e.g., a single guide RNA or sgRNA). In some embodiments, the gRNA molecule includes multiple domains. In some embodiments, the gRNA molecule includes a targeting domain (complementary to the target nucleic acid), a first complementary domain, a connecting domain, a second complementary domain (complementary to the first complementary domain), a proximal domain, and / or a tail domain.

[0204] In some embodiments, the gRNA molecule may include a targeting domain comprising a nucleic acid sequence complementary to a target DNA sequence (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary). In some embodiments, the gRNA molecule may include a targeting domain comprising a nucleic acid sequence complementary to a single strand of a target DNA sequence (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary). In some embodiments, the length of the targeting domain is 5-50 nucleotides (e.g., 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, etc.). In some embodiments, the length of the targeting domain is 16 nucleotides. In some embodiments, the length of the targeting domain is 16 nucleotides. In some embodiments, the length of the targeting domain is 17 nucleotides. In some embodiments, the length of the targeting domain is 18 nucleotides. In some embodiments, the length of the targeting domain is 19 nucleotides. In some embodiments, the length of the targeting domain is 20 nucleotides. In some embodiments, the length of the targeting domain is 21 nucleotides. In some embodiments, the length of the targeting domain is 22 nucleotides. In some embodiments, the length of the targeting domain is 23 nucleotides. In some embodiments, the length of the targeting domain is 24 nucleotides. In some embodiments, the length of the targeting domain is 25 nucleotides. In some embodiments, the length of the targeting domain is 26 nucleotides.

[0205] In some embodiments, gRNA molecules can be designed to reduce missing targets in the cell genome. In some embodiments, gRNA molecules can be designed by using software for a specific target DNA sequence with limited sequence similarity (e.g., homology, identity, etc.) to another region of the genome.

[0206] In some embodiments, the gRNA molecules are designed to target non-coding DNA sequences (e.g., introns, untranslated regions, enhancers, promoters, silencers, or insulator sequences). In some embodiments, the gRNA molecules are designed to target non-coding sequences in human genes. In some embodiments, the gRNA molecules are designed to target non-coding sequences in human safe harbor genes (e.g., albumin, collagen, actin, CCR5, etc.). In some embodiments, the gRNA molecules are designed to target non-coding sequences in human albumin genes. In some embodiments, the gRNA molecules are designed to target intron 13 or intron 14 of the human albumin gene.

[0207] In some embodiments, the gRNA molecule can be designed to target a sequence and / or a sequence including a sequence selected from the following Tables 3 and 4. In some examples, the sequence in Table 3 or Table 4 can be a spacer sequence of the gRNA.

[0208] Table 3: Exemplary gRNA sequences for spCas9.

[0209] Table 4: Exemplary gRNA sequences for saCas9. Heterologous Nucleic Acid load

[0210] In some embodiments, one or more vectors or constructs described herein may include polynucleotide sequences encoding one or more loads. According to various aspects, any one of the various loads (e.g., those with diagnostic and / or therapeutic purposes) may be used alone or in combination. In some embodiments, the load may be or include a polynucleotide sequence encoding a peptide or polypeptide. In some embodiments, the load is a cell-intrinsic active peptide or a cell-innate active peptide having a biological process that promotes the treatment of a medical condition. In some embodiments, the load may be or include a transgenic (also referred to herein as a gene of interest (GOI)). In some embodiments, the load may be or include one or more terminal inverted repeat (ITR) sequences (e.g., one or more AAV ITRs). In some embodiments, the load may be or include one or more transgenics with flanking ITR sequences. In some embodiments, the load may be or include one or more heterologous nucleic acid sequences encoding a reporter gene (e.g., a fluorescent or luminescent reporter gene). In some embodiments, the load may be or include one or more biomarkers (e.g., agents for load expression). In some embodiments, the load may include sequences for polycistronic expression (including, for example, 2A peptides, or intron sequences, internal ribosome entry sites). In some embodiments, 2A peptides are small (e.g., about 18-22 amino acids) peptide sequences that are capable of co-expressing two or more discrete protein products within a single coding sequence. In some embodiments, 2A peptides allow co-expression of two or more discrete protein products, regardless of the arrangement of the protein coding sequence. In some embodiments, 2A peptides are or comprise a consensus motif (e.g., DVEXNPGP). In some embodiments, 2A peptides promote protein cleavage. In some embodiments, 2A peptides are or comprise viral sequences (e.g., foot-and-mouth disease virus (F2A), equine rhinitis virus type A, porcine Teschovirus-1 (P2A), or Tossa asigna virus (T2A)).

[0211] In some embodiments, the cargo may be or comprise a polynucleotide sequence comprising an expression cassette. In some embodiments, the expression cassette comprises a first nucleic acid sequence and a second nucleic acid sequence, wherein the first nucleic acid sequence encodes a transgene, and the second nucleic acid sequence is positioned in the 5' or 3' direction of the first nucleic acid sequence and promotes the production of two independent gene products (e.g., a sequence encoding a 2A peptide).

[0212] In some embodiments, the biomarker is or comprises a 2A peptide (e.g., P2A, T2A, E2A and / or F2A). In some embodiments, the biomarker is or comprises a furin cleavage motif (see, Tian et al., FurinDB: A Database of 20-Residue Furin Cleavage Site Motifs, Substrates and Their Associated Drugs [FurinDB: A database of 20-residue furin cleavage site motifs, substrates and their associated drugs], (2011), Int. J. Mol. Sci. [International Journal of Molecular Sciences], Vol. 12: 1060-1065). In some embodiments, the biomarker is or comprises a label (e.g., an immunological label). In some embodiments, the load may comprise one or more functional nucleic acids (e.g., one or more siRNAs or miRNAs). In some embodiments, the load may comprise one or more inhibitory nucleic acids (including, for example, ribozymes, miRNAs, siRNAs or shRNAs, etc.). In some embodiments, the load may comprise one or more nucleases (e.g., Cas proteins, endonucleases, TALENs, ZFNs).

[0213] In some embodiments, the sequence encoding the 2A peptide may be at least 80%, 85%, 90%, 95%, 99% or 100% identical to the corresponding wild-type reference nucleotide sequence (e.g., a wild-type P2A sequence). In some embodiments, the sequence encoding the P2A peptide may be or include a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to a portion of the corresponding wild-type reference nucleotide sequence (e.g., a wild-type gene sequence). In some embodiments, the sequence encoding the P2A peptide may be or include a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to the exemplary sequences in Table 5 below.

[0214] Table 5: Exemplary 2A sequences genetically modified

[0215] In some embodiments, transgenic is a corrective gene selected to improve one or more signs and / or symptoms of a disease, disorder or illness. In some embodiments, transgenic can be integrated into the genome of a host cell by using one or more vectors covered by the present disclosure. In some embodiments, transgenic is a functional version of a disease-related gene found in a host cell (i.e., one or more gene isoforms associated with the performance or deterioration of a disease, disorder or illness). In some embodiments, transgenic is an optimized version of a disease-related gene found in a host cell (e.g., a codon-optimized or expression-optimized variant). In some embodiments, transgenic is a variant of a disease-related gene found in a host cell (e.g., a functional gene fragment or its variant). In some embodiments, transgenic is a gene causing a peptide normally expressed in one or more healthy tissues to be expressed. In some embodiments, transgenic is a gene causing a peptide normally expressed in a hepatocyte to be expressed. In some embodiments, transgenic is a gene causing a peptide normally expressed in a muscle cell to be expressed. In some embodiments, transgenic is a gene causing a peptide normally expressed in a central nervous system cell to be expressed.

[0216] In some embodiments, the transgene may be or comprise a gene that causes expression of a peptide that is not normally expressed in one or more healthy tissues (e.g., a peptide that is ectopically expressed). In some embodiments, the transgene is a gene that causes expression of a peptide that is ectopically expressed in one or more healthy tissues (e.g., liver, muscle, central nervous system (CNS), lung). In some embodiments, the transgene is a gene that causes expression of a peptide that is ectopically expressed in one or more healthy tissues and that is normally expressed in one or more healthy tissues (e.g., liver, muscle, central nervous system (CNS), lung).

[0217] In some embodiments, the transgene may be or include a gene encoding a functional nucleic acid. In some embodiments, the therapeutic agent is or includes an agent that has a therapeutic effect on a host cell or subject (including, for example, ribozymes, guide RNA (gRNA), antisense oligonucleotides (ASO), miRNA, siRNA and / or shRNA). For example, in some embodiments, the therapeutic agent promotes a biological process that treats a medical condition (e.g., at least one symptom of a disease, disorder or condition).

[0218] In some embodiments, transgenic expression in a subject is substantially caused by integration at the target integration site. In some embodiments, 75% or more (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 99% or more, 99.5% or more) of the total transgenic expression of a subject is from transgenic integration at the target integration site. In some embodiments, 25% or less (e.g., 20% or less, 15% or less, 10% or less, 5% or less, 1% or less, 0.5% or less, 0.1% or less) of the total transgenic expression of a subject is from sources other than transgenic integration at the target integration site (e.g., episomal expression, integration at a non-target integration site).

[0219] In some embodiments, the transgene is transiently expressed in the subject (e.g., episomal expression from a plasmid, minicircle DNA, virus, etc.). In some embodiments, 75% or more (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 99% or more, 99.5% or more) of the total transgene expression of the subject is from transient expression. In some embodiments, 25% or less (e.g., 20% or less, 15% or less, 10% or less, 5% or less, 1% or less, 0.5% or less, 0.1% or less) of the total transgene expression of the subject is from sources other than transient expression (e.g., integration at a non-target integration site). In some embodiments, the transgene is transiently expressed in the subject (e.g., episomal expression from a plasmid, minicircle DNA, virus, etc.) and lasts for one or more weeks after treatment. In some embodiments, the transgene is transiently expressed in the subject (e.g., episomal expression from a plasmid, minicircle DNA, virus, etc.) and lasts for one or more months after treatment.

[0220] In some embodiments, the transgene is transiently expressed in the subject one or more weeks after treatment at a level comparable to that observed one or more days after treatment (e.g., episomal expression from a plasmid, minicircle DNA, virus, etc.). In some embodiments, the transgene is transiently expressed in the subject one or more months after treatment at a level comparable to that observed one or more days after treatment (e.g., episomal expression from a plasmid, minicircle DNA, virus, etc.).

[0221] In some embodiments, the transgene is transiently expressed in the subject at a reduced level one or more weeks after treatment (e.g., episomal expression from a plasmid, minicircle DNA, virus, etc.) compared to levels observed one or more days after treatment. In some embodiments, the transgene is transiently expressed in the subject at a reduced level one or more months after treatment (e.g., episomal expression from a plasmid, minicircle DNA, virus, etc.) compared to levels observed one or more days after treatment.

[0222] In some embodiments, the transgene is transiently expressed in the subject (e.g., episomal expression from a plasmid, minicircle DNA, virus, etc.) and persists for no more than one month after treatment. In some embodiments, the transgene is transiently expressed in the subject (e.g., episomal expression from a plasmid, minicircle DNA, virus, etc.) and persists for no more than two months after treatment. In some embodiments, the transgene is transiently expressed in the subject (e.g., episomal expression from a plasmid, minicircle DNA, virus, etc.) and persists for no more than three months after treatment. In some embodiments, the transgene is transiently expressed in the subject (e.g., episomal expression from a plasmid, minicircle DNA, virus, etc.) and persists for no more than four months after treatment. In some embodiments, the transgene is transiently expressed in the subject (e.g., episomal expression from a plasmid, minicircle DNA, virus, etc.) and persists for no more than five months after treatment. In some embodiments, the transgene is transiently expressed in the subject (e.g., episomal expression from a plasmid, minicircle DNA, virus, etc.) and persists for no more than six months after treatment.

[0223] In some embodiments, the combined size of the transgene and homology arms can be optimized to increase the likelihood that the transgenes have the appropriate sequence length to be efficiently packaged in the delivery vehicle, which can increase the likelihood that the transgenes will ultimately be properly delivered in the patient.

[0224] In some embodiments, the nucleotide sequence encoding the transgene is codon optimized. In some embodiments, the nucleotide sequence encoding the transgene is codon optimized for a certain cell type (e.g., mammals, insects, bacteria, fungi, etc.). In some embodiments, the nucleotide sequence encoding the transgene is codon optimized for human cells. In some embodiments, the nucleotide sequence encoding the transgene is codon optimized for human cells of a specific tissue type (e.g., liver, muscle, CNS, lung).

[0225] In certain embodiments, the nucleotide sequence encoding the transgene may be codon optimized to have less than 100% nucleotide homology with a reference nucleotide sequence (e.g., a wild-type gene sequence). In certain embodiments, the nucleotide homology between the codon-optimized nucleotide sequence encoding the transgene and the reference nucleotide sequence is less than 100%, less than 99%, less than 98%, less than 97%, less than 96%, less than 95%, less than 94%, less than 93%, less than 92%, less than 91%, less than 90%, less than 89%, less than 88%, less than 87%, less than 86%, less than 85%, less than 84%, less than 83%, less than 82%, less than 81%, less than 80%, less than 78%, less than 76%, less than 74%, less than 72%, less than 70%, less than 68%, less than 66%, less than 64%, less than 62%, less than 60%, less than 55%, less than 50%, and less than 40%.

[0226] In some embodiments, the transgene may be or include a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to a corresponding wild-type reference nucleotide sequence (e.g., a wild-type gene sequence). In some embodiments, the transgene may be or include a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to a portion of a corresponding wild-type reference nucleotide sequence (e.g., a wild-type gene sequence). In some embodiments, the transgene may be or include a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to an exemplary sequence in Table 6 below.

[0227] Table 6: Exemplary transgenic sequences Homology arm

[0228] In some embodiments, the viral vectors described herein comprise one or more flanking polynucleotide sequences (e.g., homology arms) having significant sequence homology to the target integration site. In some embodiments, the homology arms are located on the flanks of the polynucleotide sequence encoding the payload (e.g., one homology arm is in the 5' direction of the payload (also referred to herein as the 5' homology arm) and one homology arm is in the 3' direction of the payload (also referred to herein as the 3' homology arm)). In some embodiments, the homology arms guide site-specific integration of the payload.

[0229] In some embodiments, the homology arms have the same length (also referred to herein as balanced homology arms or uniform homology arms). In some embodiments, viral vectors comprising homology arms of the same length (wherein the homology arms are at least a certain length) provide improved effects (e.g., improved target integration rates). In some embodiments, the length of the homology arms is between 50nt and 1600nt. In some embodiments, the length of the homology arms is between 100nt and 1000nt. In some embodiments, the length of the homology arms is between 200nt and 1000nt. In some embodiments, the length of the homology arms is between 500nt and 1500nt. In some embodiments, the length of the homology arms is between 1000nt and 2000nt. In some embodiments, the length of the homology arms is greater than 2000nt. In some embodiments, the length of each homology arm is at least 50nt. In some embodiments, the length of each homology arm is at least 750nt. In some embodiments, the length of each homology arm is at least 1000nt. In some embodiments, the length of each homology arm is at least 1250nt. In some embodiments, the length of the homology arms is less than 1000nt. In some embodiments, the homology arms contain at least 70% homology to the target integration site. In some embodiments, the homology arms contain at least 80% homology to the target integration site. In some embodiments, the homology arms contain at least 90% homology to the target integration site. In some embodiments, the homology arms contain at least 95% homology to the target integration site. In some embodiments, the homology arms contain at least 99% homology to the target integration site. In some embodiments, the homology arms contain 100% homology to the target integration site.

[0230] In some embodiments, the homology arms have different lengths (also referred to herein as unbalanced homology arms or uneven homology arms). In some embodiments, viral vectors comprising unbalanced homology arms of different lengths provide improved effects (e.g., increased target site integration rate) compared to an appropriate reference sequence. In some embodiments, viral vectors comprising homology arms of different lengths (wherein each homology arm is at least a certain length) provide improved effects (e.g., increased target site integration rate) compared to an appropriate reference sequence (e.g., a viral vector comprising homology arms of the same length or a viral vector comprising one or more homology arms of less than 1000nt in length).

[0231] In some embodiments, the length of each homology arm is greater than 50nt. In some embodiments, the length of each homology arm is greater than 100nt. In some embodiments, the length of each homology arm is greater than 400nt. In some embodiments, the length of each homology arm is at least 750nt. In some embodiments, the length of each homology arm is at least 1000nt. In some embodiments, the length of a homology arm is at least 750nt, and the length of another homology arm is at least 1000nt. In some embodiments, the length of a homology arm is at least 750nt, and the length of another homology arm is at least 1100nt. In some embodiments, the length of a homology arm is at least 750nt, and the length of another homology arm is at least 1200nt. In some embodiments, the length of a homology arm is at least 750nt, and the length of another homology arm is at least 1300nt. In some embodiments, the length of a homology arm is at least 750nt, and the length of another homology arm is at least 1400nt. In some embodiments, the length of a homology arm is at least 750nt, and the length of another homology arm is at least 1500nt. In some embodiments, the length of a homology arm is at least 750nt, and the length of another homology arm is at least 1600nt. In some embodiments, the length of a homology arm is at least 750nt, and the length of another homology arm is at least 1700nt. In some embodiments, the length of a homology arm is at least 750nt, and the length of another homology arm is at least 1800nt. In some embodiments, the length of a homology arm is at least 750nt, and the length of another homology arm is at least 1900nt. In some embodiments, the length of a homology arm is at least 750nt, and the length of another homology arm is at least 2000nt. In some embodiments, the length of a homology arm is at least 1000nt, and the length of another homology arm is at least 1100nt. In some embodiments, the length of a homology arm is at least 1000nt, and the length of another homology arm is at least 1200nt. In some embodiments, the length of a homology arm is at least 1000nt, and the length of another homology arm is at least 1300nt. In certain embodiments, the length of a homology arm is at least 1000nt, and the length of another homology arm is at least 1400nt. In certain embodiments, the length of a homology arm is at least 1000nt, and the length of another homology arm is at least 1500nt. In certain embodiments, the length of a homology arm is at least 1000nt, and the length of another homology arm is at least 1600nt. In certain embodiments, the length of a homology arm is at least 1000nt, and the length of another homology arm is at least 1700nt.In some embodiments, the length of a homology arm is at least 1000nt, and the length of another homology arm is at least 1800nt. In some embodiments, the length of a homology arm is at least 1000nt, and the length of another homology arm is at least 1900nt. In some embodiments, the length of a homology arm is at least 1000nt, and the length of another homology arm is at least 2000nt. In some embodiments, the length of a homology arm is at least 1300nt, and the length of another homology arm is at least 1400nt. In some embodiments, the length of a homology arm is at least 1600nt, and the length of another homology arm is at least 1000nt. In some embodiments, the length of a homology arm is at least 1250nt, and the length of another homology arm is at least 1250nt. In some embodiments, the length of a homology arm is at least 400nt, and the length of another homology arm is at least 800nt. In some embodiments, the length of a homology arm is at least 600nt, and the length of another homology arm is at least 600nt.

[0232] In some embodiments, the 5' homology arm is longer than the 3' homology arm. In some embodiments, the 3' homology arm is longer than the 5' homology arm. For example, in some embodiments, the length of the 5' homology arm is about 1600nt, and the length of the 3' homology arm is about 1000nt. In some embodiments, the length of the 5' homology arm is about 1000nt and the length of the 3' homology arm is about 1600nt. In some embodiments, the viral vector comprising the homology arm provides an improved effect (e.g., an increased target site integration rate) compared to an appropriate reference sequence (e.g., a viral vector lacking a homology arm). In some embodiments, the viral vector comprising a homology arm provides a target site integration rate of 0.01% or more (e.g., 0.05% or more, 0.1% or more, 0.2% or more, 0.3% or more, 0.4% or more, 0.5% or more, 0.6% or more, 0.7% or more, 0.8% or more, 0.9% or more, 1% or more, 1.5% or more, 2% or more, 5% or more, 10% or more, 20% or more, 30% or more). In some embodiments, the viral vector comprising a homology arm provides a target site integration rate that increases over time. In some embodiments, the target site integration rate increases over time relative to the initial measurement of target site integration. In some embodiments, the target site integration rate over time is at least 1.5X (e.g., 1.5X, 2X, 3X, 4X, 5X, 10X, 20X, 30X, 40X, 50X, 60X, 70X, 80X, 90X, 100X, 200X) higher than the initial measurement of target site integration. In some embodiments, the target site integration rate is measured after one or more days. In some embodiments, the target site integration rate is measured after one or more weeks. In some embodiments, the target site integration rate is measured after one or more months. In some embodiments, the target site integration rate is measured after one or more years.

[0233] In some embodiments, viral vectors comprising homology arms of different lengths provide improved effects (e.g., increased target site integration rates) relative to reference sequences (e.g., viral vectors having homology arms of the same length, viral vectors having at least one homology arm of less than 500nt). In some embodiments, viral vectors comprising homology arms of different lengths provide at least 1.1X, at least 1.2X, at least 1.3X, at least 1.4X, at least 1.5X, at least 1.6X, at least 1.7X, at least 1.8X, at least 1.9X, at least 2.0X, at least 2.5X, at least 3.0X, at least 3.5X, or at least 4.0X improved editing activity relative to reference compositions (e.g., viral vectors having homology arms of the same length, viral vectors having at least one homology arm of less than 500nt).

[0234] In some embodiments, viral vectors comprising homology arms of different lengths provide a target site integration rate of 0.01% or more (e.g., 0.05% or more, 0.1% or more, 0.2% or more, 0.3% or more, 0.4% or more, 0.5% or more, 0.6% or more, 0.7% or more, 0.8% or more, 0.9% or more, 1% or more, 1.5% or more, 2% or more, 5% or more, 10% or more, 20% or more, 30% or more). In some embodiments, viral vectors comprising homology arms of different lengths provide a target site integration rate that increases over time. In some embodiments, the target site integration rate increases over time relative to the initial measurement of target site integration. In some embodiments, the target site integration rate over time is at least 1.5X (e.g., 1.5X, 2X, 3X, 4X, 5X, 10X, 20X, 30X, 40X, 50X, 60X, 70X, 80X, 90X, 100X, 200X) greater than the initial measurement of target site integration.

[0235] In some embodiments, viral vectors comprising homology arms of different lengths can provide improved gene editing in a species or a model system of a species (e.g., mice, people, or its model). In some embodiments, when optimizing expression in a model system for a particular species or a particular species (e.g., mice, people, or its model), viral vectors can include different combinations of homology arm lengths. In some embodiments, viral vectors comprising a specific combination of homology arm lengths can provide improved gene editing compared to a second species or a model system of a second species (e.g., mice, pure mouse models) in a species or a model system of a species (e.g., people, humanized mouse models). In some embodiments, viral vectors comprising a specific combination of homology arm lengths can be optimized in a species or a model system of a species (e.g., people, humanized mouse models) to perform high-level gene editing compared to a second species or a model system of a second species (e.g., mice, pure mouse models).

[0236] In some embodiments, the homology arms direct integration of the transgene behind a highly expressed endogenous gene. In some embodiments, the homology arms direct integration of the transgene without disrupting endogenous gene expression (non-disruptive integration).

[0237] In some embodiments, one or more homology arm sequences may have at least 80%, 85%, 90%, 95%, 99% or 100% identity with a corresponding wild-type reference nucleotide sequence (e.g., a wild-type genomic sequence). In some embodiments, one or more homology arm sequences may be or include a sequence having at least 80%, 85%, 90%, 95%, 99% or 100% identity with a portion of a corresponding wild-type reference nucleotide sequence (e.g., a wild-type genomic sequence). In some embodiments, one or more homology arm sequences may be or include a sequence having at least 80%, 85%, 90%, 95%, 99% or 100% identity with an exemplary sequence in Table 7 below. In some embodiments, the compositions described herein may include one or more homology arm sequences selected from Table 7 below.

[0238] Table 7: Exemplary homology arm sequences

[0239] In some embodiments, the viral vectors provided herein may comprise a 5' homology arm and a 3' homology arm designed to target the albumin locus. In some embodiments, the viral vectors provided herein may comprise a 5' homology arm sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to SEQ ID NO: 62 and a 3' homology arm sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to SEQ ID NO: 67. In some embodiments, the viral vector comprises a 5' homology arm comprising the sequence of SEQ ID NO: 62 and a 3' homology arm comprising the sequence of SEQ ID NO: 67.

[0240] In some embodiments, the viral vectors provided herein can comprise a 5' homology arm sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to SEQ ID NO: 63 and a 3' homology arm sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to SEQ ID NO: 66. In some embodiments, the viral vector comprises a 5' homology arm comprising the sequence of SEQ ID NO: 63 and a 3' homology arm comprising the sequence of SEQ ID NO: 66.

[0241] In some embodiments, the viral vectors provided herein can comprise a 5' homology arm sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 64 and a 3' homology arm sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 65. In some embodiments, the viral vector comprises a 5' homology arm comprising the sequence of SEQ ID NO: 64 and a 3' homology arm comprising the sequence of SEQ ID NO: 65.

[0242] In some embodiments, the viral vectors provided herein can comprise a 5' homology arm sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 68 and a 3' homology arm sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 69. In some embodiments, the viral vector comprises a 5' homology arm comprising the sequence of SEQ ID NO: 68 and a 3' homology arm comprising the sequence of SEQ ID NO: 69. In some embodiments, the viral vectors provided herein can comprise a 5' homology arm sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to SEQ ID NO:70 and a 3' homology arm sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to SEQ ID NO:69. Measurement of target site integration

[0243] As described elsewhere herein, one of the problems with the traditional use of nucleases to introduce nucleic acid material into cells is the significant potential for off-target integration. Therefore, as described below, it is important to verify correct integration by one or more specific targeting assays.

[0244] According to various embodiments, the integration rate can be measured at any time point in various time points. In some embodiments, the target site integration rate is measured after one or more days. In some embodiments, the target site integration rate is measured after one week or more weeks. In some embodiments, the target site integration rate is measured after one month or more months. In some embodiments, the target site integration rate is measured after one year or more years. In some embodiments, the target site integration rate is measured by the assessment of one or more biomarkers (e.g., biomarkers comprising 2A peptides). In some embodiments, the target site integration rate is measured by the assessment of one or more isolated nucleic acids (e.g., mRNA, gDNA). In some embodiments, the target site integration rate is measured by the assessment of gene expression (e.g., by immunohistochemical staining).

[0245] Table 8: Exemplary methods for assessment of target site integration Treatment

[0246] The compositions and constructs disclosed herein can be used in any in vitro or in vivo application to cause or enhance expression of a payload (e.g., a transgene) from a specific target integration site in a cell while maintaining expression of endogenous genes at and around the target integration site. For example, the compositions and constructs disclosed herein can be used to treat a disorder, disease, or medical condition in a subject (e.g., by gene therapy).

[0247] In some embodiments, treatment includes obtaining or maintaining a desired pharmacological and / or physiological effect. In some embodiments, the desired pharmacological and / or physiological effect may include preventing disease completely or in part (e.g., preventing disease symptoms). In some embodiments, the desired pharmacological and / or physiological effect may include curing disease completely or in part (e.g., curing the side effects associated with the disease). In some embodiments, the desired pharmacological and / or physiological effect may include preventing the recurrence of the disease. In some embodiments, the desired pharmacological and / or physiological effect may include slowing down the progress of the disease. In some embodiments, the desired pharmacological and / or physiological effect may include alleviating the symptoms of the disease. In some embodiments, the desired pharmacological and / or physiological effect may include preventing the disappearance of the disease. In some embodiments, the desired pharmacological and / or physiological effect may include stabilizing and / or reducing the symptoms associated with the disease.

[0248] In some embodiments, treatment comprises administering the composition before, during, or after the onset of the disease (e.g., before, during, or after the onset of symptoms associated with the disease). In some embodiments, treatment comprises combination therapy (e.g., in combination with one or more therapies, including different types of therapies). Target disease

[0249] In some embodiments, the compositions and constructs disclosed herein can be used to treat any disease of interest that includes a genetic defect or abnormality as a component of the disease.

[0250] By way of specific example, in some embodiments, compositions and constructs (such as those disclosed herein) can be used to treat branched-chain organic aciduria (e.g., maple syrup urine disease (MSUD), methylmalonic acidemia (MMA), propionic acidemia (PA), isovaleric acidemia (IVA), argininosuccinic aciduria). In some embodiments, treatment comprises introducing a polynucleotide sequence encoding one or more transgenes of interest (e.g., BCKDH complex (E1a, E1b, and E2 subunits), methylmalonyl-CoA mutase, propionyl-CoA carboxylase (α and β subunits), isovaleryl-CoA dehydrogenase, argininosuccinate lyase (ASL), and / or variants thereof). In some embodiments, treatment comprises reducing an abnormal protein (e.g., a non-functional protein) associated with a branched-chain organic aciduria. In some embodiments, treatment includes reducing the signs and / or symptoms associated with branched-chain organic aciduria (e.g., hypotonia, developmental delay, seizures, optic atrophy, acute encephalopathy, hyperventilation, respiratory distress, temperature instability, recurrent vomiting, ketoacidosis, pancreatitis, constipation, neutropenia, pancytopenia, secondary erythrophagocytosis, cardiac arrhythmias, cardiomyopathy, chronic renal failure, dermatitis, hearing loss).

[0251] In some embodiments, the compositions and constructs disclosed herein can be used to treat fatty acid oxidation disorders (e.g., trifunctional protein deficiency, long-chain L-3 hydroxyacyl-CoA dehydrogenase (LCAD) deficiency, medium-chain acyl-CoA dehydrogenase (MCHAD) deficiency, very long-chain acyl-CoA dehydrogenase (VLCHAD) deficiency). In some embodiments, treatment comprises introducing a polynucleotide sequence encoding one or more transgenes of interest (e.g., HADHA, HADHB, LCHAD, ACADM, ACADVL and / or variants thereof). In some embodiments, treatment comprises reducing abnormal proteins (e.g., non-functional proteins) associated with fatty acid oxidation disorders. In some embodiments, treatment comprises reducing signs and / or symptoms associated with fatty acid oxidation disorders (e.g., hepatomegaly, intellectual and physical retardation, myocardial weakness, arrhythmias, neurological damage, abnormal liver function, rhabdomyolysis, myoglobinuria, hypoglycemia, metabolic acidosis, respiratory distress, hepatomegaly, hypotonia, cardiomyopathy).

[0252] In some embodiments, the compositions and constructs disclosed herein can be used to treat glycogen storage diseases (e.g., glycogen storage disease type 1 (GSD1), glycogen storage disease type 2 (Pompe disease, GSD2)). In some embodiments, treatment comprises introducing a polynucleotide sequence encoding one or more transgenes of interest (e.g., G6PC (GSD1a), G6PT1 (GSD1b), SLC17A3, SLC37A4 (GSD1c), acid alpha-glucosidase and / or variants thereof). In some embodiments, treatment comprises reducing abnormal proteins (e.g., non-functional proteins) associated with glycogen storage diseases. In some embodiments, treatment comprises reducing signs and / or symptoms associated with glycogen storage diseases (e.g., hepatomegaly, hypoglycemia, muscle weakness, muscle cramps, fatigue, developmental delay, obesity, bleeding disorders, abnormal liver function, abnormal kidney function, abnormal respiratory function, abnormal heart function, oral ulcers, gout, cirrhosis, fibrosis, liver tumors).

[0253] In some embodiments, the compositions and constructs disclosed herein can be used to treat carnitine cycle disorders. In some embodiments, treatment includes introducing a polynucleotide sequence encoding one or more target transgenes (e.g., OCTN2, CPT1, CACT, CPT2 and / or variants thereof). In some embodiments, treatment includes reducing abnormal proteins (e.g., non-functional proteins) associated with carnitine cycle disorders. In some embodiments, treatment includes reducing signs and / or symptoms associated with carnitine cycle disorders (e.g., hypoketotic hypoglycemia, cardiomyopathy, myasthenia, fatigue, motor retardation, edema).

[0254] In some embodiments, the compositions and constructs disclosed herein can be used to treat urea cycle disorders. In some embodiments, treatment includes introducing a polynucleotide sequence encoding one or more target transgenes (e.g., CPS1, ARG1, ASL, OTC and / or variants thereof). In some embodiments, treatment includes reducing abnormal proteins (e.g., non-functional proteins) associated with urea cycle disorders. In some embodiments, treatment includes reducing signs and / or symptoms associated with urea cycle disorders (e.g., vomiting, nausea, abnormal behavior, fatigue, coma, psychosis, lethargy, cyclical vomiting, myopia, hyperammonemia, elevated ornithine levels).

[0255] In some embodiments, the compositions and constructs disclosed herein can be used to treat homocystinuria (HCU). In some embodiments, treatment includes the introduction of a polynucleotide sequence encoding one or more target transgenes (e.g., cystathionine beta synthase (CBS) and / or variants thereof). In some embodiments, treatment includes reducing abnormal proteins (e.g., non-functional proteins) associated with HCU. In some embodiments, treatment includes reducing signs and / or symptoms associated with HCU (e.g., ectopia lentis, myopia, iris tremor, cataracts, optic atrophy, glaucoma, retinal detachment, retinal damage, developmental delay milestones, intellectual disability, depression, anxiety, obsessive-compulsive disorder, slender fingers / toes, genu valgum, high arched feet, scoliosis, pectus carinatum, pectus excavatum, osteoporosis, increased thrombosis, thromboembolism, pulmonary embolism, skin fragility, hypopigmentation, malar flushing, inguinal hernia, pancreatitis, kyphosis, spontaneous pneumothorax).

[0256] In some embodiments, the compositions and constructs disclosed herein can be used to treat Crigler-Najjar syndrome. In some embodiments, treatment comprises introducing a polynucleotide sequence encoding one or more transgenes of interest (e.g., UGT1A1 and / or variants thereof). In some embodiments, treatment comprises reducing abnormal proteins (e.g., non-functional proteins) associated with Crigler-Najjar syndrome. In some embodiments, treatment comprises reducing signs and / or symptoms associated with Crigler-Najjar syndrome (e.g., jaundice, kernicterus, lethargy, vomiting, fever, dysreflexia, muscle cramps, opisthotonos, spasms, hypotonia, athetosis, elevated bilirubin levels, diarrhea, slurred speech, confusion, dysphagia, epilepsy).

[0257] In some embodiments, the compositions and constructs disclosed herein can be used to treat hereditary tyrosinemia. In some embodiments, treatment includes introducing a polynucleotide sequence encoding one or more target transgenes (e.g., FAH and / or variants thereof). In some embodiments, treatment includes reducing abnormal proteins (e.g., non-functional proteins) associated with hereditary tyrosinemia. In some embodiments, treatment includes reducing signs and / or symptoms associated with hereditary tyrosinemia (e.g., hepatomegaly, jaundice, liver disease, cirrhosis, liver cancer, fever, diarrhea, melena, vomiting, splenomegaly, edema, coagulopathy, renal dysfunction, rickets, weakness, hypertonia, intestinal obstruction, tachycardia, hypertension, neurological crisis, respiratory failure, cardiomyopathy).

[0258] In some embodiments, the compositions and constructs disclosed herein can be used to treat epidermolysis bullosa. In some embodiments, treatment comprises introducing a polynucleotide sequence encoding one or more transgenes of interest (e.g., COL7A1, COL17A1, MMP1, KRT5, LAMA3, LAMB3, LAMC2, ITGB4, and / or variants thereof). In some embodiments, treatment comprises reducing abnormal proteins (e.g., non-functional proteins) associated with epidermolysis bullosa. In some embodiments, treatment comprises reducing signs and / or symptoms associated with epidermolysis bullosa (e.g., skin fragility, abnormal nail growth, blisters, thickened skin, scarring alopecia, atrophic scars, milia, dental problems, difficulty swallowing, itchy skin, and pain).

[0259] In some embodiments, the compositions and constructs disclosed herein can be used to treat alpha-1 antitrypsin deficiency (A1ATD). In some embodiments, treatment comprises introducing a polynucleotide sequence encoding one or more transgenes of interest (e.g., alpha-1 antitrypsin deficiency (A1AT) and / or variants thereof). In some embodiments, treatment comprises reducing abnormal proteins (e.g., non-functional proteins) associated with alpha-1 antitrypsin deficiency. In some embodiments, treatment comprises reducing signs and / or symptoms associated with A1ATD (e.g., emphysema, chronic cough, sputum production, wheezing, chronic respiratory infections, jaundice, hepatomegaly, bleeding, abnormal effusions, elevated liver enzymes, abnormal liver function, portal hypertension, fatigue, edema, chronic active hepatitis, cirrhosis, liver cancer, panniculitis).

[0260] In some embodiments, the compositions and constructs disclosed herein can be used to treat Wilson's disease. In some embodiments, treatment includes introducing a polynucleotide sequence encoding one or more target transgenes (e.g., ATP7B and / or variants thereof). In some embodiments, treatment includes reducing abnormal proteins associated with Wilson's disease (e.g., non-functional proteins). In some embodiments, treatment includes reducing signs and / or symptoms associated with Wilson's disease (e.g., fatigue, loss of appetite, abdominal pain, jaundice, Kay-Fer rings, edema, speech problems, swallowing problems, loss of body coordination, uncontrolled movements, muscle stiffness, liver disease, anemia, depression, schizophrenia, amenorrhea, infertility, kidney stones, tubular damage, arthritis, osteoporosis, osteophytes).

[0261] In some embodiments, the compositions and constructs disclosed herein can be used to treat blood diseases (e.g., hemophilia A, hemophilia B). In some embodiments, treatment includes introducing a polynucleotide sequence encoding one or more transgenes of interest (e.g., factor IX (FIX), factor VIII (FVIII) and / or variants thereof). In some embodiments, treatment includes reducing abnormal proteins (e.g., non-functional proteins) associated with blood diseases. In some embodiments, treatment includes reducing signs and / or symptoms associated with blood diseases (e.g., heavy bleeding, abnormal bruising, joint pain and swelling, hematuria, bloody stools, abnormal epistaxis, headache, drowsiness, vomiting, diplopia, weakness, convulsions, epilepsy).

[0262] In some embodiments, the compositions and constructs disclosed herein can be used to treat hereditary angioedema. In some embodiments, treatment includes introducing a polynucleotide sequence encoding one or more transgenes of interest (e.g., C1 esterase inhibitor (C1-inh)). In some embodiments, treatment includes reducing abnormal proteins (e.g., non-functional proteins) associated with hereditary angioedema. In some embodiments, treatment includes reducing signs and / or symptoms associated with hereditary angioedema (e.g., edema, itching, urticaria, nausea, vomiting, acute abdominal pain, dysphagia, dysphonia, wheezing).

[0263] In some embodiments, the compositions and constructs disclosed herein can be used to treat Parkinson's disease. In some embodiments, treatment includes introducing a polynucleotide sequence encoding one or more target transgenes (e.g., dopamine decarboxylase (DDC)). In some embodiments, treatment includes reducing abnormal proteins associated with Parkinson's disease (e.g., non-functional proteins). In some embodiments, treatment includes reducing signs and / or symptoms associated with Parkinson's disease (e.g., tremors, bradykinesia, muscle stiffness, impaired posture and balance, loss of automatic movements, changes in speech, changes in writing).

[0264] In some embodiments, the compositions and constructs disclosed herein can be used to treat muscle diseases. In some embodiments, treatment includes introducing a polynucleotide sequence encoding one or more transgenics of interest (e.g., muscular dystrophy, Duchenne muscular dystrophy (DMD), limb-girdle muscular dystrophy, X-linked myotubular myopathy). In some embodiments, treatment includes reducing abnormal proteins associated with muscle diseases (e.g., non-functional proteins). In some embodiments, treatment includes reducing signs and / or symptoms associated with muscle diseases (e.g., dyskinesia, enlarged calf muscles, muscle pain and stiffness, developmental delay, learning disabilities, gait abnormalities, scoliosis, respiratory problems, dysphagia, arrhythmias, cardiomyopathy, joint dysfunction, hypotonia, respiratory distress, loss of reflexes).

[0265] In some embodiments, the compositions and constructs disclosed herein can be used to treat mucopolysaccharidosis (MPS) (e.g., MPS IH, MPS IH / S, MPS IS, MPS II, MPS IIIA, MPS IIIB, MPS IIIC, MPS IIID, MPS IVA, MPS IVB, MPS V, MPS VI, MPS VII, MPS IX). In some embodiments, treatment comprises introducing a polynucleotide sequence encoding one or more transgenes of interest (e.g., IDUA, IDS, SGSH, NAGLU, HGSNAT, GNS, GALNS, GLB1, ARSB, GUSB, HYAL1). In some embodiments, treatment comprises reducing an abnormal protein (e.g., a non-functional protein) associated with mucopolysaccharidosis. In some embodiments, treatment includes reducing the signs and / or symptoms associated with MPS (e.g., heart abnormalities, irregular breathing, hepatomegaly, spleen enlargement, neurological abnormalities, developmental delay, recurrent infections, persistent nasal discharge, noisy breathing, corneal cloudiness, tongue enlargement, spinal deformities, joint stiffness, carpal tunnel, aortic regurgitation, progressive hearing loss, seizures, gait instability, heparan sulfate accumulation, enzyme deficiencies, bone and muscle tissue abnormalities, heart disease, cysts, soft tissue masses).

[0266] In certain embodiments, compositions and constructs disclosed herein can be used to treat lysosomal acid lipase deficiency. In certain embodiments, treatment includes introducing a polynucleotide sequence encoding one or more target transgenes (e.g., LIPA and / or its variants). In certain embodiments, treatment includes reducing abnormal proteins (e.g., non-functional proteins) associated with lysosomal acid lipase deficiency. In certain embodiments, treatment includes reducing signs and / or symptoms associated with lysosomal acid lipase deficiency (e.g., vomiting, diarrhea, abdominal swelling, and weight gain failure, weight loss, jaundice, fever, calcification, anemia, liver dysfunction or failure, cachexia, malabsorption, bile duct problems, heart disease, stroke).

[0267] In some embodiments, the compositions and constructs disclosed herein can be used to treat disorders associated with bile acid metabolism, transport and / or cholestasis. In some embodiments, treatment includes the introduction of a polynucleotide sequence encoding one or more target transgenes (e.g., PFIC1, PFIC2, PFIC3, ABCB4 and / or variants thereof). In some embodiments, treatment includes reducing abnormal proteins (e.g., non-functional proteins) associated with bile acid metabolism, transport and / or cholestasis. In some embodiments, treatment includes reducing signs and / or symptoms associated with bile acid metabolism, transport and / or cholestasis (e.g., pruritus, jaundice, growth retardation, portal hypertension, hepatosplenomegaly, diarrhea, pancreatitis, hepatocellular carcinoma).

[0268] In some embodiments, the compositions and constructs disclosed herein can be used to treat phenylketonuria. In some embodiments, treatment includes introducing a polynucleotide sequence encoding one or more target transgenes (e.g., phenylalanine hydroxylase (PAH) and / or variants thereof). In some embodiments, treatment includes reducing abnormal proteins (e.g., non-functional proteins) associated with phenylketonuria. In some embodiments, treatment includes reducing signs and / or symptoms associated with phenylketonuria (e.g., musty odor in breath, skin, and / or urine, epilepsy, rash, microcephaly, hyperactivity, intellectual disability, asthma, eczema, anemia, weight gain, renal insufficiency, osteoporosis, gastritis, esophageal and kidney defects, kidney stones, hypertension, mental problems, dizziness).

[0269] In some embodiments, the compositions and constructs disclosed herein can be used to treat primary hyperoxaluria. In some embodiments, treatment includes introducing a polynucleotide sequence encoding one or more target transgenes (e.g., AGT, AGXT, GRHPR, HOGA1 and / or its variants). In some embodiments, treatment includes reducing abnormal proteins (e.g., non-functional proteins) associated with primary hyperoxaluria. In some embodiments, treatment includes reducing signs and / or symptoms associated with phenylketonuria (e.g., flank pain, oxalate deposition, kidney stones and / or other parts of the urinary tract (such as bladder or urethra) stones, nephrocalcinosis, hematuria, dysuria, frequent urination, renal colic, urinary tract obstruction, repeated urinary tract infections, kidney damage, renal failure, growth retardation).

[0270] In some embodiments, the compositions and constructs disclosed herein can be used to treat porphyria. In some embodiments, treatment comprises introducing a polynucleotide sequence encoding one or more target transgenes (e.g., ALAD, HMBS, UROS, UROD, CPOX, PPOCX, FECH, ALAS2 and / or variants thereof). In some embodiments, treatment comprises reducing abnormal proteins (e.g., non-functional proteins) associated with porphyria. In some embodiments, treatment comprises reducing signs and / or symptoms associated with porphyria (e.g., abdominal pain, pain in the arms and legs, general weakness, vomiting, mental confusion, constipation, tachycardia, blood pressure fluctuations, urinary retention, psychosis, hallucinations, epilepsy, abrasions, blisters, skin erosions, skin lesions, nausea, elevated blood pressure, mental confusion).

[0271] In some embodiments, the compositions and constructs disclosed herein can be used to treat disorders associated with antibody production (e.g., autoimmune disorders). In some embodiments, treatment includes introducing polynucleotide sequences encoding one or more target transgenes (e.g., POLB, HLA-DRB1, IL7R, CYP27B1, TNFRSF1A, HLA-B, HLA-DPB1, HLA-DRB1, IRF5, PTPN22, RBPJ, RUNX1, STAT4 and / or variants thereof). In some embodiments, treatment includes reducing abnormal proteins (e.g., non-functional proteins) associated with antibody production. In some embodiments, treatment includes reducing signs and / or symptoms associated with antibody production (e.g., joint swelling, joint stiffness, fatigue, fever, loss of appetite, vision problems, tremor, unsteady gait, dizziness, rash, lesions, hyperalgesia).

[0272] In some embodiments, the compositions and constructs disclosed herein can be used to treat disorders associated with secretory protein production. In some embodiments, treatment comprises introducing a polynucleotide sequence encoding one or more transgenes of interest. In some embodiments, treatment comprises reducing abnormal proteins (e.g., non-functional proteins) associated with secretory protein production. In some embodiments, treatment comprises reducing signs and / or symptoms associated with secretory protein production. Targeted integration

[0273] In some embodiments, the compositions and constructs provided herein guide the integration of a load (e.g., a transgenic and / or a functional nucleic acid) at a target integration site (e.g., an endogenous gene). In some embodiments, the compositions and constructs provided herein guide the integration of a load (e.g., a transgenic and / or a functional nucleic acid) at a target integration site (e.g., an endogenous gene) found only in a specific tissue. In some embodiments, the compositions and constructs provided herein guide the integration of a load (e.g., a transgenic and / or a functional nucleic acid) at a target integration site (e.g., an endogenous gene) found in cells present in more than one tissue (e.g., 2, 3, 4, 5 or all tissues) of a subject. In some embodiments, the compositions and constructs provided herein guide the integration of a load (e.g., a transgenic and / or a functional nucleic acid) at a target integration site (e.g., an endogenous gene) that can allow the inducible expression of the load. In some embodiments, inducible expression can be controlled via artificial means (e.g., administration of drugs or other exogenous signals) or via naturally occurring means (e.g., IgH expression from B cells). In some embodiments, the compositions and constructs provided herein direct the integration of a load at a target integration site (e.g., a tissue-specific locus) in a specific cell type. In some embodiments, the integration of a load occurs in a specific tissue (e.g., liver, central nervous system (CNS), muscle, kidney, blood vessel, lung). In some embodiments, the integration of a load occurs in multiple tissues (e.g., liver, central nervous system (CNS), muscle, kidney, blood vessel, lung).

[0274] In some embodiments, the compositions and constructs provided herein direct the integration of a payload at a target integration site that is considered a safe harbor site (e.g., albumin, apolipoprotein A2 (ApoA2), haptoglobin, IgH (e.g., B cells), beta-2 microglobulin, beta-actin (β-actin), GAPDH). In some embodiments, the target integration site can be selected from any genomic site suitable for use with the methods and compositions provided herein. In some embodiments, the target integration site encodes a polypeptide. In some embodiments, the target integration site encodes a polypeptide that is highly expressed in a subject (e.g., a subject that does not suffer from a disease, disorder, or condition, or a subject that suffers from a disease, disorder, or condition). In some embodiments, integration of the payload occurs at the 5' or 3' end of one or more endogenous genes (e.g., a gene encoding a polypeptide). In some embodiments, integration of the payload occurs between the 5' or 3' ends of one or more endogenous genes (e.g., a gene encoding a polypeptide).

[0275] In some embodiments, the compositions and constructs provided herein direct integration of cargo at the target integration site with minimal or no off-target integration (e.g., integration at a non-target locus). In some embodiments, the compositions and constructs provided herein direct integration of cargo at the target integration site with reduced off-target integration compared to a reference composition or construct (e.g., relative to a composition or construct without flanking homologous sequences).

[0276] In some embodiments, the integration of transgenic at the target integration site allows the expression of load without destroying endogenous gene expression. In some embodiments, the integration of transgenic at the target integration site allows the expression of load from an endogenous promoter. In some embodiments, the integration of transgenic at the target integration site destroys endogenous gene expression. In some embodiments, the integration of transgenic at the target integration site destroys endogenous gene expression, but does not adversely affect target cells and / or subjects (e.g., by targeting safe harbor sites). In some embodiments, the integration of transgenic at the target integration site does not require the use of nucleases (e.g., Cas nucleases, TALEN, ZFN). In some embodiments, the integration of transgenic at the target integration site is assisted by the use of nucleases (e.g., Cas nucleases, TALEN, ZFN).

[0277] In some embodiments, integration of a transgene at a target integration site confers a selective advantage (e.g., increased survival of a plurality of cells relative to other cells in a tissue). In some embodiments, the selective advantage can result in an increased percentage of cells in one or more tissues expressing the transgene. Cleavage site

[0278] In some embodiments, the integration of the transgene at the target integration site is enhanced by using nucleases (e.g., Cas nucleases, TALENs, ZFNs) that target specific cleavage sites of single-stranded or double-stranded DNA breaks. In some embodiments, the integration of the transgene at the target integration site is enhanced by using nucleases that target a cleavage site in the DNA sequence that is away from the target integration site. In some embodiments, the integration of the transgene at the target integration site is assisted by using nucleases that target a cleavage site in the DNA sequence that does not overlap with the target integration site. In some embodiments, integration of the transgene at the target integration site is enhanced by using a nuclease that targets a cleavage site in the DNA sequence that is at least about 100 bp (e.g., about 100 bp, 150 bp, 200 bp, 250 bp, 300 bp, 350 bp, 400 bp, 450 bp, 500 bp, 550 bp, 600 bp, 650 bp, 700 bp, 750 bp, 800 bp, 850 bp, 900 bp, 950 bp, 1000 bp, 1050 bp, 1100 bp, 1150 bp, 1200 bp, 1250 bp, 1300 bp, 1350 bp, 1400 bp, 1450 bp, 1500 bp, 1550 bp, 1600 bp, 1650 bp, 1700 bp, 1800 bp, 1850 bp, 1900 bp, 1950 bp, 2000 bp) away from the target integration site. In some embodiments, integration of the transgene at the target integration site is enhanced by using a nuclease that targets a cleavage site in the DNA sequence within about 100 bp (e.g., about 1 bp, 3 bp, 5 bp, 10 bp, 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp) of the target integration site.

[0279] In some embodiments, the cleavage site is within a non-coding sequence of DNA (e.g., an intron, an untranslated region, an enhancer, a promoter, a silencer, or an insulator). In some embodiments, the cleavage site is within a non-coding sequence of a human gene. In some embodiments, the cleavage site is within an intron of a human gene (e.g., collagen, actin, albumin, beta-2 microglobulin, IgH, GAPDH, G6PC). In some embodiments, the cleavage site is within intron 13 or intron 14 of the human albumin gene. In some embodiments, the cleavage site is within intron 5 of the human actin gene or a site after exon 6. In some embodiments, the cleavage site is within intron 2 or intron 3 of the human beta-2 microglobulin gene. In some embodiments, the cleavage site is within IGHJ6 of the human IgH gene or between IGHJ6 and IGHM.

[0280] In some embodiments, when used with GENERIDE TMWhen the components and one or more suitable nucleases are combined, the cleavage site within one or more non-coding sequences of a human gene (e.g., introns 13 and 14 of the albumin gene) can provide improved transgenic integration. In some embodiments, the selection of the cleavage site can be based on off-target effects (e.g., reduced off-target effects) predicted by using external databases or software prediction / computational tools known in the art (e.g., Benchling, CHOPCHOP, IDT, CRISPOR, E-CRISP, TureDesign, CRISPick). In some embodiments, the cleavage site can reduce unwanted off-target effects (e.g., cytotoxicity, carcinogenicity, immunogenicity, etc.). In some embodiments, the cleavage site can be located within a safe harbor locus to reduce the possible effects of destructive integration. Composition

[0281] In some embodiments, compositions can be produced using the methods and constructs provided herein (e.g., viral vectors). In some embodiments, compositions include liquid, solid and gaseous compositions. In some embodiments, compositions include additional ingredients (e.g., diluents, stabilizers, excipients, adjuvants). In some embodiments, among other things, additional ingredients may include buffers (e.g., phosphates, citrates, organic acid buffers), antioxidants (e.g., ascorbic acid), low molecular weight polypeptides (e.g., less than 10 residues), various proteins (e.g., serum albumin, gelatin, immunoglobulins), hydrophilic polymers (e.g., polyvinyl pyrrolidone), amino acids (e.g., glycine, glutamine, asparagine, arginine, lysine), carbohydrates (e.g., monosaccharides, disaccharides, glucose, mannose, dextrins), chelating agents (e.g., EDTA), sugar alcohols (e.g., mannitol, sorbitol), salt-forming counterions (e.g., sodium, potassium) and / or nonionic surfactants (e.g., Tween TM 、Pluronics TM , polyethylene glycol (PEG)). In some embodiments, the aqueous carrier is an aqueous pH buffered solution.

[0282] In some embodiments, the compositions provided herein can be provided in a certain dosage range. In some embodiments, the compositions provided herein can be provided in a single dose. In some embodiments, the compositions provided herein can be provided in multiple doses. In some embodiments, the compositions are provided over a period of time. In some embodiments, the compositions are provided at specific intervals (e.g., variable intervals, set intervals). In some embodiments, the dosage can vary according to the dosage form and route of administration. In some embodiments, the compositions provided herein can be provided at a dosage between 1e11 and 1e14 vg / kg. In some embodiments, the compositions provided herein can be provided at a dosage between 1e12 and 1e13 vg / kg. In some embodiments, the compositions provided herein can be provided at a dosage between 1e12 and 1e14 vg / kg. In some embodiments, the compositions provided herein can be provided at a dosage between 1e14 and 1e15 vg / kg. In some embodiments, the compositions provided herein can be provided at a dosage not exceeding 1e14 vg / kg. In some embodiments, the compositions provided herein can be provided at a dosage not exceeding 1e15 vg / kg.

[0283] In some embodiments, those of ordinary skill in the art will be able to use the pharmaceutical compositions described herein to conceive suitable dosage levels and dosage regimens for treating various conditions of different patients. For example, in some embodiments, the dosage selected depends on the desired therapeutic effect, route of administration, and the duration of the desired treatment. In some embodiments, a dosage level of about 0.001 mg to about 6 mg nucleic acid / kg body weight is applied to a subject (e.g., an animal, a human) during each administration. In some embodiments, the dosage level of the nucleic acid in the disclosed lipid nanoparticle is about 0.1 mg / kg to about 1.0 mg / kg. In some embodiments, the dosage level of the nucleic acid in the disclosed lipid nanoparticle is about 0.1 mg / kg to about 3.0 mg / kg / . In some embodiments, the dosage level of the disclosed lipid nanoparticle is to apply about 0.2 mg to about 100 mg of one or more components (e.g., ionizable lipids, sterols, conjugates-linker lipids, phospholipids) total amount / kg body weight to a subject (e.g., an animal, a human). In some embodiments, the dosage level of the disclosed lipid nanoparticles is about 0.5 mg / kg to about 6 mg / kg of the total amount of one or more components (e.g., ionizable lipids, sterols, conjugate-linker lipids, phospholipids) per kg body weight.

[0284] In some embodiments, the compositions provided herein can be applied to a subject at a specific time point (e.g., the age of the subject). In some embodiments, the compositions provided herein can be applied to a newborn subject. In some embodiments, the compositions provided herein can be applied to a newborn subject. In some embodiments, the age of a newborn mouse subject is between 0 and 7 days. In some embodiments, the age of a newborn human subject is between 0 days and 1 month. In some embodiments, the compositions provided herein can be applied to a subject between 7 days old and 30 days old. In some embodiments, the compositions provided herein can be applied to a subject between 3 months old and 1 year old. In some embodiments, the compositions provided herein can be applied to a subject between 1 year old and 5 years old. In some embodiments, the compositions provided herein can be applied to a subject between 4 years old and 7 years old. In some embodiments, the compositions provided herein can be applied to a subject 5 years old or older.

[0285] In some embodiments, the compositions provided herein can be administered to a subject at a specific time point based on the growth stage of a particular tissue or organ (e.g., percentage of estimated / average adult size or weight). In some embodiments, the compositions provided herein can be administered to a subject in which the tissue or organ (e.g., liver, muscle, CNS, lung, etc.) is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99% of the estimated / average adult size or weight. In some embodiments, the compositions provided herein can be administered to a subject in which the tissue or organ is approximately 20% (+ / -5%) of the estimated / average adult size or weight. In some embodiments, the compositions provided herein can be administered to a subject in which the tissue or organ is approximately 50% (+ / -5%) of the estimated / average adult size or weight. In some embodiments, the compositions provided herein can be administered to a subject in which the tissue or organ is approximately 60% (+ / -5%) of the estimated / average adult size or weight. In some embodiments, the estimated / average adult size or weight of a particular tissue or organ can be determined as described in the art (see Noda et al. Pediatric radiology, 1997; Johnson et al. Liver transplantation, 2005; and Szpinda et al. Biomed research international, 2015, each of which is incorporated herein by reference in its entirety).

[0286] In some embodiments, a composition as described herein can comprise (i) a nuclease or a polynucleotide sequence encoding a nuclease capable of inducing double-strand breaks and / or single-strand breaks at a cleavage site distal to a target integration site; and (ii) a polynucleotide cassette comprising an expression cassette comprising a first nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence in Table 6 (e.g., SEQ ID NO: 49), a second nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence in Table 5 (e.g., SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48) positioned in the 5' or 3' sequence direction of the first nucleic acid sequence, a second nucleic acid sequence positioned in the 5' direction of the expression cassette and comprising a sequence in Table 7 (e.g., SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64), a second nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence in Table 7 (e.g., SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68), a second nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence in Table 8 (e.g., SEQ ID NO: NO:64, SEQ ID NO:68 or SEQ ID NO:70) having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity, and a fourth nucleic acid sequence positioned in the 3' direction of the expression cassette and comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity with a sequence in Table 7 (e.g., SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67 or SEQ ID NO:69). In some embodiments, (i) and (ii) can be administered as separate compositions.

[0287] In some embodiments, a composition as described herein can comprise (i) a nuclease or a polynucleotide sequence encoding a nuclease capable of inducing double-strand breaks and / or single-strand breaks at a cleavage site distal to a target integration site; and (ii) a polynucleotide cassette comprising an expression cassette comprising a first nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence in Table 6 (e.g., SEQ ID NO: 50), a second nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence in Table 5 (e.g., SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48) positioned in the 5' or 3' sequence direction of the first nucleic acid sequence, a second nucleic acid sequence positioned in the 5' direction of the expression cassette and comprising a sequence in Table 7 (e.g., SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64), a second nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence in Table 7 (e.g., SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68), a second nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence in Table 8 (e.g., SEQ ID NO: NO:64, SEQ ID NO:68 or SEQ ID NO:70) having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity, and a fourth nucleic acid sequence positioned in the 3' direction of the expression cassette and comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity with a sequence in Table 7 (e.g., SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67 or SEQ ID NO:69). In some embodiments, (i) and (ii) can be administered as separate compositions.

[0288] In some embodiments, a composition as described herein can comprise (i) a nuclease or a polynucleotide sequence encoding a nuclease capable of inducing double-strand breaks and / or single-strand breaks at a cleavage site distal to a target integration site; and (ii) a polynucleotide cassette comprising an expression cassette comprising a first nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence in Table 6 (e.g., SEQ ID NO: 51), a second nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence in Table 5 (e.g., SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48) positioned in the 5' or 3' sequence direction of the first nucleic acid sequence, a second nucleic acid sequence positioned in the 5' direction of the expression cassette and comprising a sequence in Table 7 (e.g., SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64), a second nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence in Table 7 (e.g., SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68), a second nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence in Table 8 (e.g., SEQ ID NO: NO:64, SEQ ID NO:68 or SEQ ID NO:70) having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity, and a fourth nucleic acid sequence positioned in the 3' direction of the expression cassette and comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity with a sequence in Table 7 (e.g., SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67 or SEQ ID NO:69). In some embodiments, (i) and (ii) can be administered as separate compositions.

[0289] In some embodiments, a composition as described herein can comprise (i) a nuclease or a polynucleotide sequence encoding a nuclease capable of inducing double-strand breaks and / or single-strand breaks at a cleavage site distal to a target integration site; and (ii) a polynucleotide cassette comprising an expression cassette comprising a first nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence in Table 6 (e.g., SEQ ID NO: 52), a second nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence in Table 5 (e.g., SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48) positioned in the 5' or 3' sequence direction of the first nucleic acid sequence, a second nucleic acid sequence positioned in the 5' direction of the expression cassette and comprising a sequence in Table 7 (e.g., SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64), a second nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence in Table 7 (e.g., SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68), a second nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence in Table 8 (e.g., SEQ ID NO: NO:64, SEQ ID NO:68 or SEQ ID NO:70) having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity, and a fourth nucleic acid sequence positioned in the 3' direction of the expression cassette and comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity with a sequence in Table 7 (e.g., SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67 or SEQ ID NO:69). In some embodiments, (i) and (ii) can be administered as separate compositions.

[0290] In some embodiments, a composition as described herein can comprise (i) a nuclease or a polynucleotide sequence encoding a nuclease capable of inducing double-strand breaks and / or single-strand breaks at a cleavage site distal to a target integration site; and (ii) a polynucleotide cassette comprising an expression cassette comprising a first nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence in Table 6 (e.g., SEQ ID NO: 53), a second nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence in Table 5 (e.g., SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48) positioned in the 5' or 3' sequence direction of the first nucleic acid sequence, a second nucleic acid sequence positioned in the 5' direction of the expression cassette and comprising a sequence in Table 7 (e.g., SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64), a second nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence in Table 7 (e.g., SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68), a second nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence in Table 8 (e.g., SEQ ID NO: NO:64, SEQ ID NO:68 or SEQ ID NO:70) having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity, and a fourth nucleic acid sequence positioned in the 3' direction of the expression cassette and comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity with a sequence in Table 7 (e.g., SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67 or SEQ ID NO:69). In some embodiments, (i) and (ii) can be administered as separate compositions.

[0291] In some embodiments, a composition as described herein can comprise (i) a nuclease or a polynucleotide sequence encoding a nuclease capable of inducing double-strand breaks and / or single-strand breaks at a cleavage site distal to a target integration site; and (ii) a polynucleotide cassette comprising an expression cassette comprising a first nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence in Table 6 (e.g., SEQ ID NO: 54), a second nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence in Table 5 (e.g., SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48) positioned in the 5' or 3' sequence direction of the first nucleic acid sequence, a second nucleic acid sequence positioned in the 5' direction of the expression cassette and comprising a sequence in Table 7 (e.g., SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64), a second nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence in Table 7 (e.g., SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68), a second nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence in Table 8 (e.g., SEQ ID NO: NO:64, SEQ ID NO:68 or SEQ ID NO:70) having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity, and a fourth nucleic acid sequence positioned in the 3' direction of the expression cassette and comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity with a sequence in Table 7 (e.g., SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67 or SEQ ID NO:69). In some embodiments, (i) and (ii) can be administered as separate compositions.

[0292] In some embodiments, a composition as described herein can comprise (i) a nuclease or a polynucleotide sequence encoding a nuclease capable of inducing double-strand breaks and / or single-strand breaks at a cleavage site distal to a target integration site; and (ii) a polynucleotide cassette comprising an expression cassette comprising a first nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence in Table 6 (e.g., SEQ ID NO: 55), a second nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence in Table 5 (e.g., SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48) positioned in the 5' or 3' sequence direction of the first nucleic acid sequence, a second nucleic acid sequence positioned in the 5' direction of the expression cassette and comprising a sequence in Table 7 (e.g., SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64), a second nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence in Table 7 (e.g., SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68), a second nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence in Table 8 (e.g., SEQ ID NO: NO:64, SEQ ID NO:68 or SEQ ID NO:70) having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity, and a fourth nucleic acid sequence positioned in the 3' direction of the expression cassette and comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity with a sequence in Table 7 (e.g., SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67 or SEQ ID NO:69). In some embodiments, (i) and (ii) can be administered as separate compositions.

[0293] In some embodiments, a composition as described herein can comprise (i) a nuclease or a polynucleotide sequence encoding a nuclease capable of inducing double-strand breaks and / or single-strand breaks at a cleavage site distal to a target integration site; and (ii) a polynucleotide cassette comprising an expression cassette comprising a first nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence in Table 6 (e.g., SEQ ID NO: 56), a second nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence in Table 5 (e.g., SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48) positioned in the 5' or 3' sequence direction of the first nucleic acid sequence, a second nucleic acid sequence positioned in the 5' direction of the expression cassette and comprising a sequence in Table 7 (e.g., SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64), a second nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence in Table 7 (e.g., SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68), a second nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence in Table 8 (e.g., SEQ ID NO: NO:64, SEQ ID NO:68 or SEQ ID NO:70) having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity, and a fourth nucleic acid sequence positioned in the 3' direction of the expression cassette and comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity with a sequence in Table 7 (e.g., SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67 or SEQ ID NO:69). In some embodiments, (i) and (ii) can be administered as separate compositions.

[0294] In some embodiments, a composition as described herein can comprise (i) a nuclease or a polynucleotide sequence encoding a nuclease capable of inducing double-strand breaks and / or single-strand breaks at a cleavage site distal to a target integration site; and (ii) a polynucleotide cassette comprising an expression cassette comprising a first nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence in Table 6 (e.g., SEQ ID NO: 57), a second nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence in Table 5 (e.g., SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48) positioned in the 5' or 3' sequence direction of the first nucleic acid sequence, a second nucleic acid sequence positioned in the 5' direction of the expression cassette and comprising a sequence in Table 7 (e.g., SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64), a second nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence in Table 7 (e.g., SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68), a second nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence in Table 8 (e.g., SEQ ID NO: NO:64, SEQ ID NO:68 or SEQ ID NO:70) having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity, and a fourth nucleic acid sequence positioned in the 3' direction of the expression cassette and comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity with a sequence in Table 7 (e.g., SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67 or SEQ ID NO:69). In some embodiments, (i) and (ii) can be administered as separate compositions.

[0295] In some embodiments, a composition as described herein can comprise (i) a nuclease or a polynucleotide sequence encoding a nuclease capable of inducing double-strand breaks and / or single-strand breaks at a cleavage site distal to a target integration site; and (ii) a polynucleotide cassette comprising an expression cassette comprising a first nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence in Table 6 (e.g., SEQ ID NO: 58), a second nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence in Table 5 (e.g., SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48) positioned in the 5' or 3' sequence direction of the first nucleic acid sequence, a second nucleic acid sequence positioned in the 5' direction of the expression cassette and comprising a sequence in Table 7 (e.g., SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64), a second nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence in Table 7 (e.g., SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68), a second nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence in Table 8 (e.g., SEQ ID NO: NO:64, SEQ ID NO:68 or SEQ ID NO:70) having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity, and a fourth nucleic acid sequence positioned in the 3' direction of the expression cassette and comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity with a sequence in Table 7 (e.g., SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67 or SEQ ID NO:69). In some embodiments, (i) and (ii) can be administered as separate compositions.

[0296] In some embodiments, a composition as described herein can comprise (i) a nuclease or a polynucleotide sequence encoding a nuclease capable of inducing double-strand breaks and / or single-strand breaks at a cleavage site distal to a target integration site; and (ii) a polynucleotide cassette comprising an expression cassette comprising a first nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence in Table 6 (e.g., SEQ ID NO: 59), a second nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence in Table 5 (e.g., SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48) positioned in the 5' or 3' sequence direction of the first nucleic acid sequence, a second nucleic acid sequence positioned in the 5' direction of the expression cassette and comprising a sequence in Table 7 (e.g., SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64), a second nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence in Table 7 (e.g., SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68), a second nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence in Table 8 (e.g., SEQ ID NO: NO:64, SEQ ID NO:68 or SEQ ID NO:70) having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity, and a fourth nucleic acid sequence positioned in the 3' direction of the expression cassette and comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity with a sequence in Table 7 (e.g., SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67 or SEQ ID NO:69). In some embodiments, (i) and (ii) can be administered as separate compositions.

[0297] In some embodiments, a composition as described herein can comprise (i) a nuclease or a polynucleotide sequence encoding a nuclease capable of inducing double-strand breaks and / or single-strand breaks at a cleavage site distal to a target integration site; and (ii) a polynucleotide cassette comprising an expression cassette comprising a first nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence in Table 6 (e.g., SEQ ID NO: 60), a second nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence in Table 5 (e.g., SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48) positioned in the 5' or 3' sequence direction of the first nucleic acid sequence, a second nucleic acid sequence positioned in the 5' direction of the expression cassette and comprising a sequence in Table 7 (e.g., SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64), a second nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence in Table 7 (e.g., SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70 NO:64, SEQ ID NO:68 or SEQ ID NO:70) having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity, and a fourth nucleic acid sequence positioned in the 3' direction of the expression cassette and comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity with a sequence in Table 7 (e.g., SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67 or SEQ ID NO:69). In some embodiments, (i) and (ii) can be administered as separate compositions. Route of administration

[0298] In some embodiments, the compositions provided herein can be administered to a subject via any (or multiple) of a variety of approaches known in the art (e.g., parenteral, subcutaneous, intravenous, intracranial, intraspinal, intraocular, intramuscular, intravaginal, intraperitoneal, epidermal, intradermal, rectal, pulmonary, intraosseous, oral, buccal, intraportal, intraarterial, intratracheal or nasal). In some embodiments, the compositions provided herein can be introduced into cells, which are then introduced into the subject (e.g., liver, muscle, central nervous system (CNS), lung, blood cells). In some embodiments, the compositions provided herein can be introduced via delivery methods known in the art (e.g., injection, catheter).

[0299] In some embodiments, GENERIDE TMGenome editing performed by the GENERIDE platform differs from conventional gene therapy in that it uses HR to deliver the corrective gene to a specific location in the genome. TM The corrective gene is inserted in a precise manner, resulting in site-specific integration in the genome.

[0300] In some embodiments, the compositions provided include one or more homology arms, a transgene, and a nucleic acid that promotes the production of two independent gene products. In some embodiments, the compositions and methods of the present disclosure include a first nucleic acid sequence encoding a transgene. In some embodiments, the compositions and methods of the present disclosure include a second nucleic acid that promotes the production of two independent gene products (e.g., a 2A peptide). In some embodiments, the present disclosure provides an expression cassette comprising a first nucleic acid sequence and a second nucleic acid sequence as described herein.

[0301] In some embodiments, the second nucleic acid comprises a nucleic acid sequence encoding a 2A peptide; a nucleic acid sequence encoding an internal ribosome entry site (IRES); a nucleic acid sequence encoding an N-terminal intein splicing region and a C-terminal intein splicing region; and / or a nucleic acid sequence encoding a splicing donor and a splicing acceptor. In some embodiments, the compositions and methods of the present disclosure comprise a polynucleotide box comprising an expression cassette comprising the first nucleic acid and the second nucleic acid. In some embodiments, the compositions and methods of the present disclosure comprise a third nucleic acid sequence comprising a sequence substantially homologous to a genomic sequence. In some embodiments, the compositions and methods of the present disclosure comprise a fourth nucleic acid sequence comprising a sequence substantially homologous to a genomic sequence. In some embodiments, the third nucleic acid sequence is positioned in the 5' direction of the expression cassette and comprises a sequence substantially homologous to a genomic sequence of a target integration site 5' in the genome of the cell. In some embodiments, the fourth nucleic acid sequence is positioned in the 3' direction of the expression cassette and comprises a sequence substantially homologous to a genomic sequence of a target integration site 3' in the genome of the cell.

[0302] In some embodiments, one or more compositions described herein are administered in combination. In some embodiments, the first composition can be administered simultaneously with the second composition. In some embodiments, the first composition and the second composition can be administered sequentially (e.g., within a few minutes, hours, days, weeks, or months of each other). In some embodiments, one or more compositions can be administered via the same approach (e.g., parenteral, subcutaneous, intravenous, intracranial, intraspinal, intraocular, intramuscular, intravaginal, intraperitoneal, epidermal, intradermal, rectal, pulmonary, intraosseous, oral, cheek, intraportal, intraarterial, intratracheal, or nasal). In some embodiments, one or more compositions can be administered via different approaches (e.g., parenteral, subcutaneous, intravenous, intracranial, intraspinal, intraocular, intramuscular, intravaginal, intraperitoneal, epidermal, intradermal, rectal, pulmonary, intraosseous, oral, cheek, intraportal, intraarterial, intratracheal, or nasal).

[0303] In some embodiments, one or more compositions administered in combination may include: (i) a first composition comprising a payload (e.g., one or more nuclease mRNAs and / or proteins (e.g., Cas proteins (e.g., Staphylococcus aureus Cas9 (saCas9), Streptococcus pyogenes (spCas9), AZ nucleases, HF1-Cas9, HF2-Cas9, or HiFi-Cas9), endonucleases, meganucleases, TALENs, ZFNs)); and (ii) a second composition comprising a second, different payload (e.g., a polynucleotide sequence comprising a transgene coding region). In some embodiments, the first and second compositions are delivered with the same delivery system (e.g., viral vectors, lipid nanoparticles, etc.). In some embodiments, the first composition is administered before the second composition (e.g., a difference of minutes, hours, days, weeks, or months). In some embodiments, the second composition is administered before the first composition (e.g., a difference of minutes, hours, days, weeks, or months). In some embodiments, the first composition and the second composition are administered simultaneously. In some embodiments, the first composition and the second composition are combined prior to administration.

[0304] In some embodiments, the first composition and / or the second composition are administered only once at a specific dose (e.g., a fixed dose or a dose based on body weight). In some embodiments, the first composition and / or the second composition are administered more than once at a specific dose (e.g., a fixed dose or a dose based on body weight). In some embodiments, in the case of administering more than one dose (e.g., a fixed dose or a dose based on body weight), the first and / or second composition may be administered simultaneously, substantially simultaneously, or continuously. In some embodiments, multiple doses (e.g., fixed doses or doses based on body weight) are administered within a specified time period (e.g., within a few minutes, hours, days, weeks, or months).

[0305] In some embodiments, the first composition and / or the second composition are administered in response to a biomarker (e.g., a circulating biomarker as described in WO 2020214582 A1). For example, the first and / or second compositions are administered at a specific dose (e.g., a fixed dose or a dose based on body weight), and the level of the biomarker is monitored over a specified time period (e.g., within minutes, hours, days, weeks, or months) (e.g., as described in WO 2020214582 A1). If the level of the biomarker (e.g., as described in WO 2020214582 A1) is low (e.g., compared to an appropriate reference (e.g., the level of the biomarker before administration)), the first and / or second compositions are administered at a specific dose (e.g., a fixed dose or a dose based on body weight). If the level of a biomarker (e.g., as described in WO 2020214582 A1) is high (e.g., compared to an appropriate reference (e.g., the level of the biomarker after an initial administration)), subsequent dosing (e.g., fixed dose or weight-based dose) of the first and / or second composition can be re-evaluated (e.g., a therapeutic suspension, a reduced fixed dose or weight-based dose). Methods of producing viral vectors Production of viral vectors

[0306] In some embodiments, the production of viral vectors (e.g., AAV viral vectors) can include both upstream steps of generating viral vectors (e.g., cell-based culture) and downstream steps of processing viral vectors (e.g., purification, formulation, etc.). In some embodiments, the upstream steps can include one or more of cell expansion, cell culture, cell transfection, cell lysis, viral vector production, and / or viral vector harvesting.

[0307] In some embodiments, the downstream steps may include one or more of separation, filtration, concentration, clarification, purification, chromatography (e.g., affinity, ion exchange, hydrophobic, mixed mode), centrifugation (e.g., ultracentrifugation), and / or formulation.

[0308] In some embodiments, the constructs and methods described herein are designed to increase viral vector yield (e.g., AAV vector yield), reduce the level of replication-competent viral vector (e.g., replication-competent AAV (rcAAV)), improve viral vector packaging efficiency (e.g., AAV vector capsid packaging), and / or any combination thereof relative to reference constructs or methods (e.g., those in Xiao et al. 1998 and Grieger et al. 2015, each of which is incorporated herein by reference in its entirety). Cell lines and transfection reagents

[0309] In some embodiments, the production of viral vectors includes the use of cells (e.g., cell culture). In some embodiments, the production of viral vectors includes the use of cell culture of one or more cell lines (e.g., mammalian cell lines). In some embodiments, the production of viral vectors includes the use of HEK293 cell lines or variants thereof (e.g., HEK293T, HEK293F cell lines). In some embodiments, cells can grow in suspension. In some embodiments, cells are composed of adherent cells. In some embodiments, cells can grow in a culture medium that does not contain animal components (e.g., animal serum). In some embodiments, cells can grow in serum-free culture medium (e.g., F17 culture medium, Expi293 culture medium). In some embodiments, the production of viral vectors includes transfecting cells with an expression construct (e.g., plasmid). In some embodiments, cells are selected for high expression of viral vectors (e.g., AAV vectors). In some embodiments, cells are selected for high packaging efficiency of viral vectors (e.g., capsid packaging of AAV vectors). In some embodiments, cells are selected for improving transfection efficiency (e.g., using chemical transfection reagents, including cationic molecules). In some embodiments, cells are engineered for high expression of viral vectors (e.g., AAV vectors). In some embodiments, cells are engineered for high packaging efficiency of viral vectors (e.g., capsid packaging of AAV vectors). In some embodiments, cells are engineered for improving transfection efficiency (e.g., using chemical transfection reagents, including cationic molecules). In some embodiments, cells can be engineered or selected for two or more of the above attributes. In some embodiments, cells are contacted with one or more expression constructs (e.g., plasmids). In some embodiments, cells are contacted with one or more transfection reagents (e.g., chemical transfection reagents, including lipids, polymers and cationic molecules) and one or more expression constructs. In some embodiments, cells are contacted with one or more cationic molecules (e.g., cationic lipids, PEI reagents) and one or more expression constructs. In some embodiments, cells are contacted with PEIMAX reagents and one or more expression constructs. In some embodiments, cells are contacted with FectoVir-AAV reagents and one or more expression constructs. In some embodiments, cells are contacted with one or more transfection reagents and one or more expression constructs at a specific ratio. In some embodiments, the ratio of transfection reagents to expression constructs improves the production of viral vectors (e.g., improves vector yield, improves packaging efficiency, and / or improves transfection efficiency). Expression constructs

[0310] In some embodiments, the expression construct is or comprises one or more polynucleotide sequences (e.g., plasmid). In some embodiments, the expression construct comprises specific polynucleotide sequence elements (e.g., load, promoter, viral gene, etc.). In some embodiments, the expression construct comprises a polynucleotide sequence encoding a viral gene (e.g., rep or cap gene or gene variant, one or more helper virus genes or gene variant). In some embodiments, a specific type of expression construct comprises a specific combination of polynucleotide sequence elements. In some embodiments, a specific type of expression construct does not comprise a specific combination of polynucleotide sequence elements. In some embodiments, a specific expression construct does not comprise polynucleotide sequence elements encoding rep and cap genes and / or gene variants.

[0311] In some embodiments, the expression construct comprises a polynucleotide sequence encoding a wild-type viral gene (e.g., a wild-type rep gene, a cap gene, a viral helper gene, or a combination thereof). In some embodiments, the expression construct comprises a polynucleotide sequence encoding a viral helper gene or a gene variant (e.g., a herpes virus gene or a gene variant, an adenovirus gene or a gene variant). In some embodiments, the expression construct comprises a polynucleotide sequence encoding one or more gene copies (e.g., 1 copy, 2 copies, 3 copies, 4 copies, 5 copies, etc.) expressing one or more wild-type Rep proteins. In some embodiments, the expression construct comprises a polynucleotide sequence encoding a single gene copy expressing one or more wild-type Rep proteins (e.g., Rep68, Rep40, Rep52, Rep78, or a combination thereof). In some embodiments, the expression construct comprises a polynucleotide sequence encoding one or more wild-type Rep proteins (e.g., Rep68, Rep40, Rep52, Rep78, or a combination thereof). In some embodiments, the expression construct comprises a polynucleotide sequence encoding at least four wild-type Rep proteins (e.g., Rep68, Rep40, Rep52, Rep78). In some embodiments, the expression construct comprises a polynucleotide sequence encoding each of Rep68, Rep40, Rep52, and Rep78. In some embodiments, the expression construct comprises a polynucleotide sequence encoding one or more wild-type adenovirus accessory proteins (e.g., E2 and E4).

[0312] In some embodiments, the expression construct comprises a wild-type polynucleotide sequence encoding a wild-type viral gene (e.g., a rep gene, a cap gene, an auxiliary gene). In some embodiments, the expression construct comprises a modified polynucleotide sequence (e.g., codon optimized) encoding a wild-type viral gene (e.g., a rep gene, a cap gene, an auxiliary gene). In some embodiments, the expression construct comprises a modified polynucleotide sequence encoding a modified viral gene (e.g., a rep gene, a cap gene, an auxiliary gene). In some embodiments, the modified viral gene is designed and / or engineered for certain improvements (e.g., improved transduction, tissue specificity, reduced size, reduced immune response, improved packaging, reduced rcAAV levels, etc.).

[0313] According to various embodiments, compared with previous technologies, the expression construct disclosed herein can provide increased flexibility and modularity. In certain embodiments, the expression construct disclosed herein can allow the exchange of various polynucleotide sequences (e.g., different rep genes, cap genes, loads, auxiliary genes, promoters, etc.), while providing certain improvements (e.g., increased viral vector yields, increased packaging, reduced rcAAV levels, etc.). In certain embodiments, the expression construct disclosed herein is compatible with various upstream production processes (e.g., different cell culture conditions, different transfection reagents, etc.), while providing certain improvements (e.g., increased viral vector yields, increased packaging, reduced rcAAV levels, etc.).

[0314] In some embodiments, different types of expression constructs include different combinations of polynucleotide sequences. In some embodiments, one type of expression construct includes one or more polynucleotide sequence elements (e.g., load, promoter, viral gene, etc.) that do not exist in different types of expression constructs. In some embodiments, one type of expression construct includes polynucleotide sequence elements encoding viral genes (e.g., rep or cap genes or gene variants) and polynucleotide sequence elements encoding loads (e.g., transgenic and / or functional nucleic acids). In some embodiments, one type of expression construct includes polynucleotide sequence elements encoding one or more viral genes (e.g., rep or cap genes or gene variants and / or one or more helper virus genes). In some embodiments, one type of expression construct includes polynucleotide sequence elements encoding one or more viral genes, wherein these viral genes are from one or more virus types (e.g., genes or gene variants from AAV and adenovirus). In some embodiments, viral genes from adenovirus are genes and / or gene variants. In some embodiments, the viral gene from an adenovirus is one or more of E2A (e.g., E2A DNA binding protein (DBP)), E4 (e.g., E4 open reading frame (ORF) 2, ORF3, ORF4, ORF6 / 7), VA, and / or variants thereof.

[0315] In some embodiments, the expression construct is used for the production of viral vectors (e.g., by cell culture). In some embodiments, the expression construct is contacted with cells in combination with one or more transfection reagents (e.g., chemical transfection reagents). In some embodiments, the expression construct is contacted with cells in combination with one or more transfection reagents at a specific ratio. In some embodiments, different types of expression constructs are contacted with cells in combination with one or more transfection reagents at a specific ratio (e.g., weight ratio). In some embodiments, different types of expression constructs are contacted with cells in a ratio (e.g., weight ratio) of about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1.5:1, 1:1, 1:1.5, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10. In some embodiments, a first expression construct comprising one or more viral helper genes and a second expression construct comprising one or more cargoes are contacted with a cell at a ratio (e.g., weight ratio) of about 10: 1, 9: 1, 8: 1, 7: 1, 6: 1, 5: 1, 4: 1, 3: 1, 2: 1, 1.5: 1, 1: 1, 1: 1.5, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1: 10 of first expression construct to second expression construct. In some embodiments, a first expression construct comprising one or more loads and a second expression construct comprising one or more viral helper genes are contacted with cells at a ratio (e.g., weight ratio) of about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1.5:1, 1:1, 1:1.5, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10 of the first expression construct to the second expression construct. In some embodiments, the expression construct of a specific ratio improves the production of AAV (e.g., increases viral vector yield, increases packaging efficiency and / or increases transfection efficiency). In some embodiments, cells are contacted with two or more expression constructs (e.g., sequentially contacted, or substantially simultaneously contacted). In some embodiments, three or more expression constructs are contacted with cells. In some embodiments, the expression construct comprises one or more promoters (e.g., one or more exogenous promoters). In some embodiments, the promoter is or comprises CMV, RSV, CAG, EF1α, PGK, A1AT, C5-12, MCK, desmin, p5, p40 or a combination thereof. In some embodiments, the expression construct comprises one or more promoters upstream of a specific polynucleotide sequence element (e.g., rep or cap gene or gene variant). In some embodiments, the expression construct comprises one or more promoters downstream of a specific polynucleotide sequence element (e.g., rep or cap gene or gene variant).

[0316] In some embodiments, the expression construct comprises one or more polynucleotide sequences encoding elements necessary for cell culture (e.g., bacterial cell culture, mammalian cell culture) (e.g., selectable markers, origins of replication). In some embodiments, the expression construct comprises one or more polynucleotide sequences encoding antibiotic resistance genes (e.g., kanamycin resistance genes, ampicillin resistance genes). In some embodiments, the expression construct comprises one or more polynucleotide sequences encoding bacterial origins of replication (e.g., colE1 origin of replication).

[0317] In some embodiments, the expression construct comprises one or more transcription termination sequences (e.g., poly A sequences). In some embodiments, the expression construct comprises one or more of BGH poly A, FIX poly A, SV40 poly A, synthetic poly A, or a combination thereof. In some embodiments, the expression construct comprises one or more transcription termination sequences downstream of a specific sequence element (e.g., a rep or cap gene or a gene variant). In some embodiments, the expression construct comprises one or more transcription termination sequences upstream of a specific sequence element (e.g., a rep or cap gene or a gene variant).

[0318] In some embodiments, the expression construct comprises one or more intron sequences. In some embodiments, the expression construct comprises one or more introns (e.g., known genes) from different sources, including but not limited to FIX introns, albumin introns or combinations thereof. In some embodiments, the expression construct comprises one or more introns of different lengths (e.g., 133bp to 4kb). In some embodiments, the expression construct comprises one or more intron sequences upstream of a specific sequence element (e.g., rep or cap gene or gene variant). In some embodiments, the expression construct comprises one or more intron sequences in a specific sequence element (e.g., rep or cap gene or gene variant). In some embodiments, the expression construct comprises one or more intron sequences downstream of a specific sequence element (e.g., rep or cap gene or gene variant). In some embodiments, the expression construct comprises one or more intron sequences (e.g., p5 promoter) after a promoter. In some embodiments, the expression construct comprises one or more intron sequences before a rep gene or gene variant. In some embodiments, the expression construct comprises one or more intron sequences between a promoter and a rep gene or gene variant. Examples Example 1: Double-strand breaks induced by Cas9 can enhance GENERIDE TM efficiency

[0319] This example demonstrates, among other things, administration of GENERIDE to a target integration site in the coding region of an endogenous human locus (e.g., human albumin (ALB)) in combination with one or more Cas9 enzymes that target a cleavage site within a non-coding sequence of the endogenous human locus (e.g., a human ALB intron). TM The constructs may provide improved transgene integration efficiency.

[0320] A viral vector containing a viral capsid (e.g., LK03), a P2A sequence, a transgenic gene (e.g., UGT1A1), and balanced (1 kb / 1 kb) flanking 5' and 3' homology arm lengths was constructed. The homology arms were designed to be complementary to the human genomic albumin target integration site. The viral vector was used at an MOI of 1E 5 HepG2 cells (a human liver hepatocellular carcinoma cell line) were transduced with a dose of 50 μg / ml. After transduction, Lipofectamine TM CRISPRMAX (ThermoFisher Scientific, Inc) was used to transfect cells with spCas9 and gRNA (designed using the IDT gRNA algorithm). The gRNA was designed to target intron 13 or 14 of the ALB gene (Table 3). 48 hours after transfection, total RNA was isolated from cells using the RNeasy kit (Qiagen) according to the supplier's instructions, and the level of fusion mRNA was assessed (e.g., see Table 8).

[0321] Among other things, this example demonstrates that a viral vector comprising a transgene (e.g., UGT1A1) in combination with spCas9 as described herein can provide improved editing activity. In some embodiments, spCas9 can induce a double-strand break (DSB) at a cleavage site within intron 13 and / or intron 14 of the ALB gene ( Figure 1A and Figure 1B ). In some embodiments, Figure 2A As demonstrated in , induced DSBs in intron 13 of the human ALB gene can enhance transgene integration efficiency by at least 400-fold compared to an appropriate reference (e.g., administration of the vector without induced DSBs). Figure 2B As demonstrated in , induced DSBs in intron 14 of the human ALB gene can enhance transgene integration efficiency by at least 200-fold compared to an appropriate reference (e.g., administration of the vector without induced DSBs). TM Constructs, GENERIDE is administered in combination with DSBs in any noncoding sequence within the endogenous locus TMThe constructs may provide improved transgene integration efficiency. In some embodiments, GENERIDE is administered in combination with DSBs in specific regions of non-coding sequences within the endogenous locus, as compared to DSBs in alternative regions of non-coding sequences. TM Constructs may provide improved or comparable transgene integration efficiency ( Figure 2C ). In some embodiments, the DSB is located in a non-coding sequence distal to the transgene integration site.

[0322] A viral vector containing a viral capsid (e.g., LK03), a P2A sequence, a transgenic gene (e.g., GFP), and balanced (1 kb / 1 kb) flanking 5' and 3' homology arm lengths was constructed. The homology arms were designed to be complementary to the human genomic albumin target integration site. The viral vector was used at an MOI of 1E 5 HepG2 cells (a human liver hepatocellular carcinoma cell line) were transduced with a dose of 50 μg / ml. After transduction, Lipofectamine TM CRISPRMAX (Thermo Fisher Scientific) transfects cells with spCas9 and gRNA (designed using IDT gRNA algorithm). gRNA is designed to target introns 13 or 14 of the ALB gene (Table 3). 48 hours after transfection, total RNA is isolated from cells using RNeasy kit (Qiagen) according to the instructions of the supplier, and the level of fusion mRNA is evaluated. In addition, immunohistochemistry and / or immunocytochemistry (e.g., see Table 8) were performed. Images were evaluated using imaging processing software (e.g., ImageJ). Among other things, this example demonstrates that viral vectors comprising a transgene (e.g., GFP) combined with spCas9 as described herein can provide improved editing activity and protein expression. Figure 2D and Figure 2E As shown in Figure 2, DSBs induced in intron 13 and / or intron 14 can enhance protein expression (e.g., % GFP-positive cells) by at least 5-fold. Figure 2F As shown, the increase in fusion mRNA levels may be associated with the increase in protein expression.

[0323] A viral vector containing a viral capsid (e.g., LK03), a P2A sequence, a transgenic gene (e.g., GFP), and balanced (1 kb / 1 kb) flanking 5' and 3' homology arm lengths was constructed. The homology arms were designed to be complementary to the human genomic albumin target integration site. The viral vector was used at an MOI of 1E 5 HepG2 cells (a human liver hepatocellular carcinoma cell line) were transduced with a dose of 50 μg / ml. After transduction, Lipofectamine TMCRISPRMAX (Thermo Fisher Scientific) was used to transfect cells with a mixture of spCas9 mRNA and gRNA (designed using the IDT gRNA algorithm). The gRNA was designed to target intron 13 or 14 of the ALB gene (Table 8). 72 hours after transfection, cells were fixed and imaged. GFP-positive cells were quantified using software (e.g., Image J or CellProfiler). Figure 3A ).

[0324] Among other things, this example demonstrates that spCas9 mRNA can also enhance the editing activity of viral vectors as described herein ( Figure 3B 4, a ratio of spCas9 mRNA to guide RNA of about 5 can provide improved editing activity of the viral vector. In some embodiments, the ratio of spCas9 mRNA to guide RNA can be optimized to improve transgene integration efficiency.

[0325] A viral vector containing a viral capsid (e.g., LK03), a P2A sequence, a transgene (e.g., GFP, HA-tag conjugated GFP, or UGT1A1), and balanced (1 kb / 1 kb) flanking 5' and 3' homology arm lengths was constructed. The homology arms were designed to be complementary to the human genomic albumin target integration site. The viral vector was used at an MOI of 1E 5 HepG2 cells (a human liver hepatocellular carcinoma cell line) were transduced with a dose of 50 μg / ml. After transduction, Lipofectamine TM CRISPRMAX (Thermo Fisher Scientific) was used to transfect cells with Cas9 (e.g., spCas9-HF1 and / or spCas9-HF2) and gRNA (designed using the IDT gRNA algorithm). The gRNA was designed to target intron 13 or 14 of the ALB gene (Table 3). 48 hours after transfection, immunohistochemistry and / or immunocytochemistry were performed using antibodies. Images were evaluated using image processing software (e.g., ImageJ).

[0326] Among other things, this example demonstrates that viral vectors as described herein combined with different spCas9 (e.g., spCas9-HF1 and / or spCas9-HF2) utilizing gRNAs designed to target introns 13 and / or 14 of albumin can improve GENERIDE TM Editing Activity. As shown in FIG5 , DSBs induced by spCas9-HF1 and / or spCas9-HF2 in intron 14 can enhance protein expression (eg, % GFP-positive cells) compared to DSBs induced in intron 13.

[0327] A viral vector containing a viral capsid (e.g., LK03), a P2A sequence, a HA-conjugated transgene (e.g., GFP, HA-conjugated GFP, and / or UGT1A1), and balanced (1 kb / 1 kb) flanking 5' and 3' homology arm lengths was constructed. The homology arms were designed to be complementary to the human genomic albumin target integration site. The viral vector was used at an MOI of 1E 5 HepG2 cells (a human liver hepatocellular carcinoma cell line) were transduced with a dose of 50 μg / ml. After transduction, Lipofectamine TM CRISPRMAX (Thermo Fisher Scientific) transfected cells with saCas9 and gRNA (designed using the IDT gRNA algorithm). The gRNA was designed to target introns 13 or 14 of the ALB gene (Table 3). Immunohistochemistry and / or immunocytochemistry (e.g., see Table 8) were performed. Images were evaluated using imaging processing software (e.g., ImageJ). Among other things, this example demonstrates that viral vectors as described herein combined with saCas9 utilizing gRNAs designed to target introns 13 and / or 14 of albumin can improve GENERIDE TM Edit activity. Figure 6 As shown, saCas9-induced DSBs can enhance protein expression (e.g., % HA+ positive cells). Example 2: DNA breaks induced by Cas nucleases can enhance GENERIDE TM efficiency

[0328] This example demonstrates, among other things, administration of GENERIDE to a target integration site in the coding region of an endogenous human locus (e.g., human albumin) in combination with one or more Cas enzymes (e.g., Cas9, Cas13, Cas12a, Cas9 nickase, etc.) that target a cleavage site within a non-coding sequence of the endogenous human locus (e.g., a human ALB intron) TM The constructs may provide improved transgene integration efficiency.

[0329] A first composition comprising a viral vector comprising a viral capsid, a P2A sequence, a transgene, and balanced or unbalanced flanking 5' and 3' homology arm lengths is constructed. The homology arms are designed to be complementary to the target integration site (eg, human genomic albumin).

[0330] Constructed comprising Cas enzyme (e.g., Cas9, Cas13, Cas12a, Cas9 nickase, etc.) and designed to target endogenous human locus non-coding sequence (e.g., people ALB intron) of the gRNA second composition. For delivering this composition of several methods are known in the art (e.g., with encoding Cas enzyme (e.g., Cas9, Cas13, Cas12a, Cas9 nickase, etc.) plasmid or mRNA and gRNA transfection, with encoding Cas enzyme (e.g., Cas9, Cas13, Cas12a, Cas9 nickase, etc.) viral vector and gRNA transduction and / or intracellular delivery Cas enzyme (e.g., Cas9, Cas13, Cas12a, Cas9 nickase, etc.) -gRNA ribonucleoprotein (RNP)).

[0331] After constructing each kind of composition, several experimental conditions were tested.In the experiment, cells (e.g., HepG2) were transduced and / or transfected with the first and second compositions in parallel.In another experiment, cells (e.g., HepG2) were transduced with the first composition sequentially, and then transduced and / or transfected with the second composition.In another experiment, cells (e.g., HepG2) were transduced and / or transfected with the second composition sequentially, and then transduced with the first composition.In another experiment, cells (e.g., HepG2) were transduced and / or transfected with the first and second compositions in parallel, and then transduced with the second composition (e.g., at least more than once).In another experiment, cells were transduced with the first composition sequentially, and then transduced and / or transfected with the second composition repeatedly (e.g., at least more than once).After transfection and / or transduction, total RNA was isolated from cells by using RNeasy kit (Qiagen) according to the instructions of the supplier, and the level of fusion mRNA was evaluated.

[0332] Among other things, this example demonstrates that viral vectors in combination with Cas enzymes (e.g., Cas9, Cas13, Cas12a, Cas9 nickase, etc.) as described herein can provide improved editing activity. In some embodiments, Cas enzymes (e.g., Cas9, Cas13, Cas12a, Cas9 nickase, etc.) can induce DNA breaks (e.g., double-strand breaks (DSBs) and / or single-strand breaks (SSBs)) within the non-coding sequence of the human ALB gene. In some embodiments, induced DNA breaks (e.g., DSBs and / or SSBs) within the non-coding sequence of the human ALB gene can enhance transgenic integration efficiency compared to an appropriate reference (e.g., administration of the vector without induced DSBs and / or SSBs). In some embodiments, relative to GENERIDE alone, the efficiency of transgenic integration is significantly improved. TMConstructs, GENERIDE is administered in combination with induced DNA breaks (e.g., DSBs and / or SSBs) in any non-coding sequence within the endogenous locus TM The constructs can provide improved transgene integration efficiency. In some embodiments, GENERIDE is administered in combination with induced DNA breaks (e.g., DSBs and / or SSBs) in specific regions of non-coding sequences within the endogenous locus, compared to induced DNA breaks (e.g., DSBs and / or SSBs) in alternative regions of non-coding sequences. TM The constructs may provide improved or comparable transgene integration efficiency. In some embodiments, the induced DNA breaks (eg, DSBs and / or SSBs) are located in non-coding sequences distal to the transgene integration site. Example 3: DNA breaks induced by ZFNs can enhance GENERIDE TM efficiency

[0333] This example demonstrates, among other things, administration of GENERIDE to a target integration site in the coding region of an endogenous human locus (e.g., human albumin (ALB)) in combination with one or more zinc finger nucleases (ZFNs) (e.g., a ZFN and / or ZFN nickase (ZF nickase) pair) that target a cleavage site within a non-coding sequence of the endogenous human locus (e.g., a human albumin intron). TM The constructs may provide improved transgene integration efficiency.

[0334] A first composition comprising a viral vector comprising a viral capsid, a P2A sequence, a transgene, and balanced or unbalanced flanking 5' and 3' homology arm lengths is constructed. The homology arms are designed to be complementary to the target integration site (eg, human genomic albumin).

[0335] A second composition comprising one or more ZFNs (e.g., ZFNs and / or ZF nickase pairs) designed to target non-coding sequences of endogenous human loci (e.g., human ALB introns) is constructed. The ZFN / ZF nickase can be prepared so that it can bind to a specific region of a gene (e.g., introns 13 and / or 14 of the human albumin gene) and comprises a nuclease domain (e.g., FokI) that can nick (e.g., ZF nickase) or cut (e.g., ZFN pairs) the target sequence. Several methods for delivering such compositions are known in the art (e.g., transfection with plasmids or mRNA encoding one or more ZFNs (e.g., ZFNs and / or ZF nickase pairs), transduction with viral vectors encoding one or more ZFNs (e.g., ZFNs and / or ZF nickase pairs), and / or electroporation).

[0336] After constructing each composition, several experimental conditions were tested.In the experiment, cells (e.g., HepG2) were transduced and / or transfected with the first composition and the second composition in parallel.In subsequent experiments, cells (e.g., HepG2) were transduced with the first composition sequentially, and then transduced and / or transfected with the second composition.In subsequent experiments, cells (e.g., HepG2) were transduced and / or transfected with the second composition sequentially, and then transduced with the first composition.In subsequent experiments, cells (e.g., HepG2) were transduced and / or transfected with the first composition and the second composition in parallel, and then transduced (e.g., at least more than once) transduction and / or transfection were repeated with the second composition.In subsequent experiments, cells were transduced with the first composition sequentially, and then transduced and / or transfected repeatedly (e.g., at least more than once) with the second composition.After transfection and / or transduction, total RNA was isolated from cells according to the instructions of the supplier by using RNeasy kit (Qiagen), and the level of fusion mRNA was evaluated.

[0337] Among other things, this example demonstrates that a viral vector as described herein in combination with one or more ZFNs (e.g., a ZFN and / or a ZF nickase pair) can provide improved editing activity. In some embodiments, one or more ZFNs (e.g., a ZFN and / or a ZFN ZF nickase pair) can induce DNA breaks (e.g., DSBs and / or SSBs) within the non-coding sequence of the human ALB gene. In some embodiments, the induced DNA breaks (e.g., DSBs and / or SSBs) within the non-coding sequence of the human ALB gene can enhance transgenic integration efficiency compared to an appropriate reference (e.g., administration of the vector without induced DSBs and / or SSBs). In some embodiments, relative to GENERIDE alone, the transgenic integration efficiency is improved. TM Constructs, GENERIDE is administered in combination with induced DNA breaks (e.g., DSBs and / or SSBs) in any non-coding sequence within the endogenous locus TM The constructs can provide improved transgene integration efficiency. In some embodiments, GENERIDE is administered in combination with induced DNA breaks (e.g., DSBs and / or SSBs) in specific regions of non-coding sequences within the endogenous locus, compared to induced DNA breaks (e.g., DSBs and / or SSBs) in alternative regions of non-coding sequences. TM The constructs may provide improved or comparable transgene integration efficiency. In some embodiments, the induced DNA breaks (eg, DSBs and / or SSBs) are located in non-coding sequences distal to the transgene integration site. Example 4: DNA fragmentation induced by meganucleases can enhance GENERIDE TM efficiency

[0338] This example demonstrates, among other things, the administration of GENERIDE to a target integration site in the coding region of an endogenous human locus (e.g., human albumin) in combination with one or more meganucleases that target a cleavage site within a non-coding sequence of the endogenous human locus (e.g., a human albumin (ALB) intron) TM The constructs may provide improved transgene integration efficiency.

[0339] A first composition comprising a viral vector comprising a viral capsid, a P2A sequence, a transgene, and balanced or unbalanced flanking 5' and 3' homology arm lengths is constructed. The homology arms are designed to be complementary to the target integration site (eg, human genomic albumin).

[0340] A second composition comprising one or more meganucleases designed to target non-coding sequences of an endogenous human locus (e.g., a human ALB intron) is constructed. Several methods for delivering such compositions are known in the art (e.g., transfection with a plasmid or mRNA encoding one or more meganucleases and / or transduction with a viral vector encoding one or more meganucleases).

[0341] After constructing each composition, several experimental conditions were tested.In the experiment, cells (e.g., HepG2) were transduced and / or transfected with the first composition and the second composition in parallel.In subsequent experiments, cells (e.g., HepG2) were transduced with the first composition sequentially, and then transduced and / or transfected with the second composition.In subsequent experiments, cells (e.g., HepG2) were transduced and / or transfected with the second composition sequentially, and then transduced with the first composition.In subsequent experiments, cells (e.g., HepG2) were transduced and / or transfected with the first composition and the second composition in parallel, and then transduced (e.g., at least more than once) transduction and / or transfection were repeated with the second composition.In subsequent experiments, cells were transduced with the first composition sequentially, and then transduced and / or transfected repeatedly (e.g., at least more than once) with the second composition.After transfection and / or transduction, total RNA was isolated from cells according to the instructions of the supplier by using RNeasy kit (Qiagen), and the level of fusion mRNA was evaluated.

[0342] Among other things, this example demonstrates that a viral vector as described herein in combination with one or more meganucleases can provide improved editing activity. In some embodiments, one or more meganucleases can induce DNA breaks (e.g., DSBs and / or SSBs) within the non-coding sequence of the human ALB gene. In some embodiments, induced DNA breaks (e.g., DSBs and / or SSBs) within the non-coding sequence of the human ALB gene can enhance transgene integration efficiency compared to an appropriate reference (e.g., administration of the vector without induced DSBs and / or SSBs). In some embodiments, relative to GENERIDE alone, the transgenic efficiency of the transgenic vector is significantly improved. TM Constructs, GENERIDE is administered in combination with induced DNA breaks (e.g., DSBs and / or SSBs) in any non-coding sequence within the endogenous locus TM The constructs can provide improved transgene integration efficiency. In some embodiments, GENERIDE is administered in combination with induced DNA breaks (e.g., DSBs and / or SSBs) in specific regions of non-coding sequences within the endogenous locus compared to induced DNA breaks (e.g., DSBs and / or SSBs). TM The constructs may provide improved or comparable transgene integration efficiency. In some embodiments, the induced DNA breaks (eg, DSBs and / or SSBs) are located in non-coding sequences distal to the transgene integration site. Example 5: DNA fragmentation induced by exemplary nucleases can enhance GENERIDE TM efficiency

[0343] This example demonstrates, among other things, the use of GENERIDE to a target integration site in the coding region of an endogenous human locus (e.g., human albumin) in combination with one or more exemplary nucleases as described herein that target a cleavage site within a non-coding sequence of the endogenous human locus (e.g., a human albumin (ALB) intron). TM The constructs may provide improved transgene integration efficiency.

[0344] A first composition comprising a viral vector comprising a viral capsid, a P2A sequence, a transgene, and balanced or unbalanced flanking 5' and 3' homology arm lengths is constructed. The homology arms are designed to be complementary to the target integration site (eg, human genomic albumin).

[0345] A second composition comprising one or more exemplary nucleases as described herein is constructed, which are designed to target non-coding sequences of endogenous human loci (e.g., human ALB introns). Several methods for delivering such compositions are known in the art (e.g., transfection with plasmids or mRNA encoding one or more exemplary nucleases (as described herein), transduction with viral vectors encoding one or more exemplary nucleases (as described herein), and / or intracellular delivery of one or more exemplary nuclease RNPs (as described herein)).

[0346] After constructing each composition, several experimental conditions were tested.In the experiment, cells (e.g., HepG2) were transduced and / or transfected with the first composition and the second composition in parallel.In subsequent experiments, cells (e.g., HepG2) were transduced with the first composition sequentially, and then transduced and / or transfected with the second composition.In subsequent experiments, cells (e.g., HepG2) were transduced and / or transfected with the second composition sequentially, and then transduced with the first composition.In subsequent experiments, cells (e.g., HepG2) were transduced and / or transfected with the first composition and the second composition in parallel, and then transduced (e.g., at least more than once) transduction and / or transfection were repeated with the second composition.In subsequent experiments, cells were transduced with the first composition sequentially, and then transduced and / or transfected repeatedly (e.g., at least more than once) with the second composition.After transfection and / or transduction, total RNA was isolated from cells according to the instructions of the supplier by using RNeasy kit (Qiagen), and the level of fusion mRNA was evaluated.

[0347] Among other things, this example demonstrates that a viral vector as described herein in combination with one or more exemplary nucleases as described herein can provide improved editing activity. In some embodiments, one or more exemplary nucleases as described herein can induce DNA breaks (e.g., DSBs and / or SSBs) within the non-coding sequence of the human ALB gene. In some embodiments, induced DNA breaks (e.g., DSBs and / or SSBs) within the non-coding sequence of the human ALB gene can enhance transgene integration efficiency compared to an appropriate reference (e.g., administration of the vector without induced DSBs and / or SSBs). In some embodiments, relative to GENERIDE alone, the efficiency of transgene integration is enhanced. TM Constructs, GENERIDE is administered in combination with induced DNA breaks (e.g., DSBs and / or SSBs) in any non-coding sequence within the endogenous locus TMThe constructs can provide improved transgene integration efficiency. In some embodiments, GENERIDE is administered in combination with induced DNA breaks (e.g., DSBs and / or SSBs) in specific regions of non-coding sequences within the endogenous locus, compared to induced DNA breaks (e.g., DSBs and / or SSBs) in alternative regions of non-coding sequences. TM The constructs may provide improved or comparable transgene integration efficiency. In some embodiments, the induced DNA breaks (eg, DSBs and / or SSBs) are located in non-coding sequences distal to the transgene integration site. Example 6: Induced DNA breaks can enhance GENERIDE TM In vivo or in vitro efficiency

[0348] This example demonstrates, among other things, the use of GENERIDE to a target integration site in the coding region of an endogenous locus (e.g., human albumin) in combination with one or more endonucleases as described herein that target a cleavage site within a non-coding sequence of the endogenous locus (e.g., a human albumin intron). TM The constructs can provide improved transgene integration efficiency in a subject (eg, a subject suffering from a disease).

[0349] In some embodiments, this example includes using one or more GENERIDE TM Constructs (e.g., viral vectors comprising a viral capsid as described herein, a 2A sequence (e.g., P2A), a transgene as described herein, and flanking human homology arms (e.g., balanced or unbalanced)).

[0350] In some embodiments, GENERIDE TM The construct is administered sequentially or concurrently with one or more endonucleases as described herein. TM The construct is administered intravenously to a subject (e.g., a subject suffering from a disease) in combination with one or more endonucleases. TM The construct is co-administered with one or more endonucleases ex vivo to cells (e.g., hepatocytes), which are then optionally administered to a subject (e.g., a subject suffering from a disease) after transgene integration. TM The construct is administered prior to administration of one or more endonucleases. TM The construct is administered after administration of one or more endonucleases. In some embodiments, the GENERIDE construct is co-administered in vivo to a subject (eg, a subject suffering from a disease) with one or more endonucleases, optionally followed by administration of one or more endonucleases.

[0351] Among other things, this example provides that induced DNA breaks (eg, DSBs and / or SSBs) can enhance GENERIDE TM Insights into in vivo efficacy. In some embodiments, the level of fusion mRNA can be increased in a subject and / or cell and / or tissue compared to an appropriate reference (e.g., the vector is administered in the absence of induced DNA breaks (e.g., DSBs and / or SSBs)). In some embodiments, the level of ALB-2A can be increased in a subject and / or cell and / or tissue compared to an appropriate reference (e.g., the vector is administered in the absence of induced DNA breaks (e.g., DSBs and / or SSBs). In some embodiments, transgene expression can be increased in a subject and / or cell and / or tissue compared to an appropriate reference (e.g., the vector is administered in the absence of induced DNA breaks (e.g., DSBs and / or SSBs).

[0352] This example provides, among other things, the following insights: GENERIDE administered sequentially or in parallel with one or more endonucleases TM The construct may provide an enhanced selective advantage to cells that have successfully integrated the transgene of interest. Example 7: Using GENERIDE TM Induced DNA breaks can improve Wilson's disease phenotype

[0353] This example demonstrates, among other things, the administration of a GENERIDE comprising a therapeutic transgene encoding ATP7B (e.g., a truncated ATP7B) to a target integration site in the coding region of an endogenous locus (e.g., human albumin) in combination with one or more endonucleases as described herein that target a cleavage site within a non-coding sequence of the endogenous locus (e.g., a human albumin intron). TM The constructs can be used to treat or prevent Wilson's disease in a subject (eg, a subject suffering from Wilson's disease) (eg, by reducing phenotypic effects and / or symptoms in vivo).

[0354] Built GENERIDE TM Constructs (e.g., viral vectors comprising a viral capsid as described herein, a 2A sequence (e.g., P2A), a human ATP7B (e.g., a truncated ATP7B) transgene, and flanking human homology arms (e.g., balanced or unbalanced). GENERIDE TM The construct is administered (e.g., intravenously) to a subject (e.g., a subject with Wilson's disease) at a dose (e.g., a fixed dose or a weight-based dose). TMThe construct is administered sequentially or concurrently with one or more endonucleases as described herein. TM The construct is administered intravenously to a subject (e.g., a subject suffering from Wilson's disease) in combination with one or more endonucleases. TM The construct is co-administered with one or more endonucleases ex vivo to cells (e.g., hepatocytes), which are then optionally administered to a subject (e.g., a subject with Wilson's disease) after transgene integration. TM The construct is co-administered with one or more endonucleases to a subject (eg, a subject suffering from Wilson's disease) in vivo, optionally followed by administration of one or more endonucleases.

[0355] Among other things, this example provides that induced DNA breaks (eg, DSBs and / or SSBs) can enhance GENERIDE TM Insights into in vivo efficacy. In some embodiments, the level of fusion mRNA can be increased in a subject and / or cell and / or tissue compared to an appropriate reference (e.g., the vector is administered in the absence of induced DNA breaks (e.g., DSBs and / or SSBs)). In some embodiments, the level of ALBA-2A can be increased in a subject and / or cell and / or tissue compared to an appropriate reference (e.g., the vector is administered in the absence of induced DNA breaks (e.g., DSBs and / or SSBs). In some embodiments, transgene expression can be increased in a subject and / or cell and / or tissue compared to an appropriate reference (e.g., the vector is administered in the absence of induced DNA breaks (e.g., DSBs and / or SSBs).

[0356] Among other things, this example demonstrates that treatment of a subject (e.g., a subject with Wilson's disease) with a viral vector of the present disclosure can increase integration rate and / or level (e.g., as measured by the percentage of positive cells) relative to an appropriate reference (e.g., untreated or vehicle). In some embodiments, treatment of a subject (e.g., a subject with Wilson's disease) with a viral vector of the present disclosure can allow or restore normal growth (e.g., as measured by the percentage of weight change over time) relative to an appropriate reference (e.g., untreated or vehicle). In some embodiments, treatment of a subject (e.g., a subject with Wilson's disease) with a viral vector of the present disclosure can provide reduced levels of biomarkers associated with Wilson's disease (e.g., copper, hAAT, alanine aminotransferase (ALT)). In some embodiments, treatment of a subject (e.g., a subject with Wilson's disease) with a viral vector of the present disclosure can provide one or more of the following relative to a reference (e.g., untreated or vehicle): improved tissue (e.g., liver) function (e.g., measured by assessing markers of tissue (e.g., liver) function), normal growth (e.g., measured by percent change in body weight over time), decreased levels of biomarkers associated with Wilson's disease (e.g., liver or urine copper, hAAT, alanine aminotransferase (ALT)). Example 8: Using GENERIDE TM Induced DNA breaks can improve the phenotype of hereditary tyrosinemia

[0357] This example demonstrates, among other things, the administration of a GENERIDE comprising a therapeutic transgene encoding FAH and / or variants thereof to a target integration site in the coding region of an endogenous locus (e.g., human albumin) in combination with one or more endonucleases as described herein that target a cleavage site within a non-coding sequence of the endogenous locus (e.g., a human albumin intron). TM The constructs can be used to treat or prevent inherited tyrosinemia in a subject (eg, a subject having inherited tyrosinemia) (eg, by reducing phenotypic effects and / or symptoms in vivo).

[0358] Built GENERIDE TM Constructs (e.g., viral vectors comprising a viral capsid, a 2A sequence (e.g., P2A), a human FAH transgene and / or variants thereof, and flanking human homology arms (e.g., balanced or unbalanced) as described herein). The GENERIDE construct is administered (e.g., intravenously) to a subject (e.g., a subject with hereditary tyrosinemia) at a dose (e.g., a fixed dose or a weight-based dose). In some embodiments, the GENERIDE TMThe construct is administered sequentially or concurrently with one or more endonucleases as described herein. TM The construct is administered intravenously to a subject (e.g., a subject with hereditary tyrosinemia) in combination with one or more endonucleases. TM The construct is co-administered with one or more endonucleases ex vivo to cells (e.g., hepatocytes), which are optionally subsequently administered to a subject (e.g., a subject with hereditary tyrosinemia) after transgene integration. TM The constructs are co-administered with one or more endonucleases to a subject (eg, a subject suffering from hereditary tyrosinemia) in vivo, optionally followed by administration of one or more endonucleases.

[0359] Among other things, this example provides that induced DNA breaks (eg, DSBs and / or SSBs) can enhance GENERIDE TM Insights into in vivo efficacy. In some embodiments, the level of fusion mRNA can be increased in a subject and / or cell and / or tissue compared to an appropriate reference (e.g., the vector is administered in the absence of induced DNA breaks (e.g., DSBs and / or SSBs)). In some embodiments, the level of ALBA-2A can be increased in a subject and / or cell and / or tissue compared to an appropriate reference (e.g., the vector is administered in the absence of induced DNA breaks (e.g., DSBs and / or SSBs). In some embodiments, transgene expression can be increased in a subject and / or cell and / or tissue compared to an appropriate reference (e.g., the vector is administered in the absence of induced DNA breaks (e.g., DSBs and / or SSBs).

[0360] Among other things, this example demonstrates that treatment of a subject (e.g., a subject with inherited tyrosinemia) with a viral vector of the present disclosure can increase integration rate and / or level (e.g., as measured by the percentage of positive cells) relative to an appropriate reference (e.g., untreated or vehicle). In some embodiments, treatment of a subject (e.g., a subject with inherited tyrosinemia) with a viral vector of the present disclosure can allow or restore normal growth (e.g., as measured by the percentage of weight change over time) relative to an appropriate reference (e.g., untreated or vehicle). In some embodiments, treatment of a subject (e.g., a subject with inherited tyrosinemia) with a viral vector of the present disclosure can provide reduced levels of biomarkers associated with inherited tyrosinemia (e.g., ALT, bilirubin, succinylacetone (SUAC), alpha-fetoprotein (AFP)). In some embodiments, treatment of a subject (e.g., a subject with inherited tyrosinemia) with a viral vector of the present disclosure can provide one or more of the following relative to a reference (e.g., untreated or vehicle): improved tissue (e.g., liver) function (e.g., measured by assessing markers of tissue (e.g., liver) function), normal growth (e.g., measured by percent change in body weight over time), decreased levels of biomarkers associated with inherited tyrosinemia (e.g., ALT, bilirubin, succinylacetone (SUAC), alpha-fetoprotein (AFP)). Example 9: Using GENERIDE TM Induced DNA breaks may improve Crigler-Najjar syndrome

[0361] This example demonstrates, among other things, that administration of a GENERIDE comprising a UGT1A1 gene and / or variants thereof to a target integration site in the coding region of an endogenous locus (e.g., human albumin) in combination with one or more endonucleases as described herein that target a cleavage site within a non-coding sequence of the endogenous locus (e.g., a human albumin intron) can be used to TM The constructs can be used to treat or prevent Crigler-Najjar Syndrome in a subject (eg, a subject having Crigler-Najjar Syndrome) (eg, by reducing phenotypic effects and / or symptoms in vivo).

[0362] Built GENERIDE TM Constructs (e.g., viral vectors comprising a viral capsid, a 2A sequence (e.g., P2A), a UGT1A1 gene and / or variants thereof, and flanking human homology arms (e.g., balanced or unbalanced) as described herein). TMThe construct is administered (e.g., intravenously) to a subject (e.g., a subject with Crigler-Najjar syndrome) at a dose (e.g., a fixed dose or a weight-based dose). In some embodiments, GENERIDE TM The construct is administered sequentially or concurrently with one or more endonucleases as described herein. TM The construct is administered intravenously to a subject (e.g., a subject with Crigler-Najjar syndrome) in combination with one or more endonucleases. TM The construct is co-administered with one or more endonucleases ex vivo to cells (e.g., hepatocytes), which are optionally subsequently administered to a subject (e.g., a subject with Crigler-Najjar syndrome) after transgene integration. TM The construct is co-administered with one or more endonucleases to a subject (eg, a subject suffering from Crigler-Najjar Syndrome) in vivo, optionally followed by administration of one or more endonucleases.

[0363] Among other things, this example provides that induced DNA breaks (eg, DSBs and / or SSBs) can enhance GENERIDE TM Insights into in vivo efficacy. In some embodiments, the level of fusion mRNA can be increased in a subject and / or cell and / or tissue compared to an appropriate reference (e.g., the vector is administered in the absence of induced DNA breaks (e.g., DSBs and / or SSBs)). In some embodiments, the level of ALBA-2A can be increased in a subject and / or cell and / or tissue compared to an appropriate reference (e.g., the vector is administered in the absence of induced DNA breaks (e.g., DSBs and / or SSBs). In some embodiments, transgene expression can be increased in a subject and / or cell and / or tissue compared to an appropriate reference (e.g., the vector is administered in the absence of induced DNA breaks (e.g., DSBs and / or SSBs).

[0364] Among other things, this example demonstrates that treatment of a subject (e.g., a subject with Crigler-Najjar syndrome) with a viral vector of the present disclosure can increase integration rate and / or level (e.g., as measured by percentage of positive cells) relative to an appropriate reference (e.g., untreated or vehicle). In some embodiments, treatment of a subject (e.g., a subject with Crigler-Najjar syndrome) with a viral vector of the present disclosure can allow or restore normal growth (e.g., as measured by percentage of weight change over time) relative to an appropriate reference (e.g., untreated or vehicle). In some embodiments, treatment of a subject (e.g., a subject with Crigler-Najjar syndrome) with a viral vector of the present disclosure can provide reduced levels of biomarkers associated with Crigler-Najjar syndrome. In some embodiments, treatment of a subject (e.g., a subject with Crigler-Najjar syndrome) with a viral vector of the present disclosure can provide one or more of: improved tissue (e.g., liver) function (e.g., as measured by assessing markers of tissue (e.g., liver) function), normal growth (e.g., as measured by percent change in body weight over time), reduced levels of biomarkers associated with Crigler-Najjar syndrome, relative to a reference (e.g., untreated or vehicle). Example 10: Using GENERIDE TM Induced DNA fragmentation can improve alpha-1 antitrypsin deficiency

[0365] This example demonstrates, among other things, the administration of a GENERIDE comprising a SERPINA1 transgene and / or variants thereof to a target integration site in the coding region of an endogenous locus (e.g., human albumin) in combination with one or more endonucleases as described herein that target a cleavage site within a non-coding sequence of the endogenous locus (e.g., a human albumin (ALB) intron) TM The constructs can be used to treat or prevent alpha-1 antitrypsin deficiency in a subject (eg, a subject suffering from alpha-1 antitrypsin deficiency) (eg, by reducing phenotypic effects and / or symptoms in vivo).

[0366] Built GENERIDE TM Constructs (eg, viral vectors comprising a viral capsid, a 2A sequence (eg, P2A), a SERPINA1 transgene and / or variants thereof, and flanking human homology arms (eg, balanced or unbalanced) as described herein). GENERIDE TM The construct is administered (e.g., intravenously) to a subject (e.g., a subject suffering from alpha-1 antitrypsin deficiency) at a dose (e.g., a fixed dose or a weight-based dose). In some embodiments, GENERIDE TMThe construct is administered sequentially or concurrently with one or more endonucleases as described herein. TM The construct is administered intravenously to a subject (e.g., a subject with alpha-1 antitrypsin deficiency) in combination with one or more endonucleases. TM The construct is co-administered with one or more endonucleases ex vivo to cells (e.g., hepatocytes and / or lung cells), which are optionally subsequently administered to a subject (e.g., a subject with alpha-1 antitrypsin deficiency) after transgene integration. TM The construct is co-administered in vivo to a subject (eg, a subject suffering from alpha-1 antitrypsin deficiency) with one or more endonucleases, optionally followed by administration of the one or more endonucleases.

[0367] Among other things, this example provides that induced DNA breaks (eg, DSBs and / or SSBs) can enhance GENERIDE TM Insights into in vivo efficacy. In some embodiments, the level of fusion mRNA can be increased in a subject and / or cell and / or tissue compared to an appropriate reference (e.g., the vector is administered in the absence of induced DNA breaks (e.g., DSBs and / or SSBs)). In some embodiments, the level of ALBA-2A can be increased in a subject and / or cell and / or tissue compared to an appropriate reference (e.g., the vector is administered in the absence of induced DNA breaks (e.g., DSBs and / or SSBs). In some embodiments, transgene expression can be increased in a subject and / or cell and / or tissue compared to an appropriate reference (e.g., the vector is administered in the absence of induced DNA breaks (e.g., DSBs and / or SSBs).

[0368] Among other things, this example demonstrates that treatment of a subject (e.g., a subject with alpha-1 antitrypsin deficiency) with a viral vector of the present disclosure can increase integration rate and / or level (e.g., measured by percentage of positive cells) relative to an appropriate reference (e.g., untreated or vehicle). In some embodiments, treatment of a subject (e.g., a subject with alpha-1 antitrypsin deficiency) with a viral vector of the present disclosure can allow or restore lung function relative to an appropriate reference (e.g., untreated or vehicle). In some embodiments, treatment of a subject (e.g., a subject with alpha-1 antitrypsin deficiency) with a viral vector of the present disclosure can provide reduced levels of biomarkers associated with alpha-1 antitrypsin deficiency. In some embodiments, treatment of a subject (e.g., a subject with alpha-1 antitrypsin deficiency) with a viral vector of the present disclosure can provide one or more of: improved tissue (e.g., lung) function (e.g., measured by assessing markers of tissue (e.g., lung) function), normal respiratory function or reduced respiratory function deterioration, reduced levels of biomarkers associated with alpha-1 antitrypsin deficiency. Example 11: Using GENERIDE TM Induced double-strand breaks can improve tissue function

[0369] This example demonstrates, among other things, the administration of a GENERIDE comprising a therapeutic transgene and / or variants thereof to a target integration site in the coding region of an endogenous locus (e.g., human albumin) in combination with one or more endonucleases as described herein that target a cleavage site within a non-coding sequence of the endogenous locus (e.g., a human albumin intron). TM The constructs can be used to treat or prevent a disease in a subject (eg, a subject suffering from the disease) (eg, by reducing phenotypic effects and / or symptoms in vivo).

[0370] Built GENERIDE TM Constructs (e.g., viral vectors comprising a...

Claims

1. A composition or a combination of compositions, comprising: (i) a nuclease or a polynucleotide sequence encoding a nuclease, wherein the nuclease is selected from a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) enzyme, a transcription activator-like effector (TALE) nuclease (TALEN), a TALE nickase, a zinc finger (ZF) nuclease (ZFN), a ZF nickase or a meganuclease; and (ii) a polynucleotide cassette comprising: An expression cassette comprising: a first nucleic acid sequence and a second nucleic acid sequence, wherein the first nucleic acid sequence encodes a transgene; and the second nucleic acid sequence is positioned in a 5' or 3' direction to the first nucleic acid sequence and promotes the production of two independent gene products upon integration into a target integration site in the genome of a human cell; a third nucleic acid sequence located in the 5' direction of the expression cassette and comprising a sequence substantially homologous to a human genomic sequence 5' of the target integration site in the genome of a human cell; and a fourth nucleic acid sequence located in the 3' direction of the expression cassette and comprising a sequence substantially homologous to a human genomic sequence 3' to the target integration site in the genome of the human cell; in: The polynucleotide cassette does not include a promoter sequence; and The nuclease is capable of inducing double strand breaks and / or single strand breaks at the cleavage site in the genome of the human cell.

2. The composition or set of compositions of claim 1, wherein the target integration site is an albumin locus comprising an endogenous albumin promoter and an endogenous albumin gene.

3. A composition or a combination of compositions, comprising: (i) a nuclease or a polynucleotide sequence encoding a nuclease; and (ii) a polynucleotide cassette comprising: An expression cassette comprising: a first nucleic acid sequence and a second nucleic acid sequence, wherein the first nucleic acid sequence encodes a transgene; and the second nucleic acid sequence is positioned in a 5' or 3' direction to the first nucleic acid sequence and promotes production of two independent gene products upon integration into a target integration site in the genome of a human cell, wherein the target integration site is an albumin locus comprising an endogenous albumin promoter and an endogenous albumin gene; a third nucleic acid sequence located in the 5' direction of the expression cassette and comprising a sequence substantially homologous to a human genomic sequence 5' of the target integration site in the genome of a human cell; and a fourth nucleic acid sequence located in the 3' direction of the expression cassette and comprising a sequence substantially homologous to a human genomic sequence 3' to the target integration site in the genome of the human cell; in: The polynucleotide cassette does not include a promoter sequence; and The nuclease is capable of inducing double strand breaks and / or single strand breaks at the cleavage site in the genome of the human cell.

4. The composition or composition set as described in any one of claims 1 to 3, further comprising a recombinant viral vector.

5. The composition or composition set of claim 4, wherein the recombinant viral vector is a recombinant AAV vector.

6. The composition or composition set of claim 5, wherein the recombinant viral vector is or comprises a capsid polypeptide comprising an amino acid sequence having at least 95% sequence identity with the following amino acid sequences: sL65, LK03, AAV8, AAV-DJ; AAV-LK03; or AAVNP59.

7. The composition or composition set as claimed in claim 6, wherein the recombinant viral vector comprises the capsid polypeptide and the polynucleotide sequence encoding the nuclease and / or the polynucleotide box is encapsidated in the recombinant viral vector.

8. The composition or composition set of claim 6, wherein the polynucleotide cassette is encapsidated in the recombinant viral vector.

9. The composition or composition set of any one of claims 1-8, further comprising an AAV2 inverted terminal repeat (ITR) sequence.

10. The composition or set of compositions of claim 9, wherein the AAV2 ITR sequence flanks the 5' end and the 3' end of the polynucleotide sequence encoding the nuclease and / or the polynucleotide cassette.

11. The composition or composition set as described in any one of claims 1-10, wherein the third nucleic acid sequence and the fourth nucleic acid sequence are each between 50 nt and 1600 nt in length.

12. The composition or composition set as described in any one of claims 1-11, wherein the third nucleic acid sequence and the fourth nucleic acid sequence have the same length.

13. The composition or composition set as described in any one of claims 1-12, wherein the third nucleic acid sequence and the fourth nucleic acid sequence have different lengths.

14. The composition or set of compositions of any one of claims 1-13, wherein after the polynucleotide cassette is integrated into the target integration site in the genome of the cell, the transgene is expressed under the control of an endogenous promoter at the target integration site.

15. The composition or set of compositions of any one of claims 2-14, wherein the target integration site is within the coding sequence of the albumin locus and is adjacent to the stop codon 5'.

16. The composition or set of compositions of claim 15, wherein the target integration site is adjacent to the stop codon 5' in exon 14 of the albumin locus.

17. The composition or set of compositions of any one of claims 1-16, wherein the cleavage site is within the non-coding sequence of the albumin locus.

18. The composition or set of compositions of claim 17, wherein the cleavage site is within an intron, untranslated region, enhancer, promoter, silencer or insulator of the albumin locus.

19. The composition or set of compositions of claim 18, wherein the cleavage site is within intron 12, 13 or 14 of the albumin locus.

20. The composition or composition set of any one of 3 to 18, wherein the nuclease is selected from a Cas enzyme, a TALEN, a TALE nickase, a ZFN, a ZF nickase or a meganuclease.

21. The composition or composition set of any one of claims 1-20, wherein the nuclease is a Cas enzyme or a TALEN.

22. The composition or composition set of any one of claims 1-21, wherein the nuclease is a Cas enzyme.

23. The composition or composition group of any one of claims 1-22, wherein the Cas enzyme is selected from Staphylococcus aureus Cas9 (saCas9), Streptococcus pyogenes (spCas9), AZ nuclease, HF1-Cas9, HF2-Cas9 or HiFi-Cas9.

24. The composition or composition set of claims 22 or 23, further comprising a guide RNA (gRNA).

25. The composition or composition set of claim 24, wherein the gRNA comprises a nucleic acid sequence of any one of SEQ ID NOs: 27-45, 71-86, or 93-98, or a nucleic acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 27-45, 71-86, or 93-98.

26. The composition or composition set of claim 24 or 25, wherein the nuclease or the polynucleotide sequence encoding the nuclease is co-formulated with the gRNA.

27. The composition or composition set of claim 24 or 25, wherein the nuclease or the polynucleotide sequence encoding the nuclease is prepared separately from the gRNA.

28. The composition or composition set of any one of claims 1-27, wherein the second nucleic acid sequence is or comprises: a) a nucleic acid sequence encoding a 2A peptide; b) a nucleic acid sequence encoding an internal ribosome entry site (IRES); c) a nucleic acid sequence encoding an N-terminal intein splicing region and a C-terminal intein splicing region; or d) Nucleic acid sequences encoding a splice donor and a splice acceptor.

29. The composition or set of compositions of any one of claims 1-28, wherein the second nucleic acid sequence is or comprises a nucleic acid sequence encoding a 2A peptide.

30. The composition or set of compositions of claim 29, wherein the second nucleic acid is or comprises a nucleic acid sequence encoding a 2A peptide selected from the group consisting of P2A, T2A, E2A and F2A.

31. The composition or set of compositions of claim 1 or 3, wherein the cleavage site is between 1 bp and 2000 bp away from the target integration site.

32. The composition or set of compositions of claim 31 , wherein the cleavage site is up to 100 bp away from the target integration site.

33. The composition or set of compositions of any one of claims 1-32, wherein the transgene is selected from CBS, UGT1A1, MUT, FAH, ATP7B, A1AT, ASL, LIPA, PAH, G6PC, Factor IX or a variant thereof.

34. compositions or composition groups as described in any one in claim 1-33, it is a composition group, wherein this nuclease or this polynucleotide sequence encoding this nuclease is formulated in lipid nanoparticle (LNP), and this polynucleotide box is encapsidated in recombinant AAV vector.

35. A method for integrating a transgene into the genome of a human cell, the method comprising Administering to a subject a composition or a combination of compositions comprising: (i) a nuclease or a polynucleotide sequence encoding a nuclease, wherein the nuclease is selected from a Cas enzyme, a TALEN, a TALE nickase, a ZFN, a ZF nickase or a meganuclease; and (ii) a polynucleotide cassette comprising: An expression cassette comprising: a first nucleic acid sequence and a second nucleic acid sequence, wherein the first nucleic acid sequence encodes a transgene; and the second nucleic acid sequence is positioned in a 5' or 3' direction to the first nucleic acid sequence and promotes the production of two independent gene products upon integration into a target integration site in the genome of a human cell; a third nucleic acid sequence located in the 5' direction of the expression cassette and comprising a sequence substantially homologous to a human genomic sequence 5' of the target integration site in the genome of a human cell; and a fourth nucleic acid sequence located in the 3' direction of the expression cassette and comprising a sequence substantially homologous to a human genomic sequence 3' to the target integration site in the genome of the human cell; in: The polynucleotide cassette does not include a promoter sequence; and The nuclease is capable of inducing double-strand breaks and / or single-strand breaks at the cleavage site in the genome of the cell; Wherein, after administration of the composition or the combination of compositions, the transgene is integrated into the genome of the human cells.

36. The method of claim 35, wherein the target integration site is an albumin locus comprising an endogenous albumin promoter and an endogenous albumin gene.

37. A method for integrating a transgene into the genome of a human cell, the method comprising Administering to a subject a composition or a combination of compositions comprising: (i) a nuclease or a polynucleotide sequence encoding a nuclease; and (ii) a polynucleotide cassette comprising: An expression cassette comprising: a first nucleic acid sequence and a second nucleic acid sequence, wherein the first nucleic acid sequence encodes a transgene; and the second nucleic acid sequence is positioned in a 5' or 3' direction to the first nucleic acid sequence and promotes production of two independent gene products upon integration into a target integration site in the genome of a human cell, wherein the target integration site is an albumin locus comprising an endogenous albumin promoter and an endogenous albumin gene; a third nucleic acid sequence located in the 5' direction of the expression cassette and comprising a sequence substantially homologous to a human genomic sequence 5' of the target integration site in the genome of a human cell; and a fourth nucleic acid sequence located in the 3' direction of the expression cassette and comprising a sequence substantially homologous to a human genomic sequence 3' to the target integration site in the genome of the human cell; in: The polynucleotide cassette does not include a promoter sequence; and The nuclease is capable of inducing double-strand breaks and / or single-strand breaks at the cleavage site in the genome of the cell; Wherein, after administration of the composition or the combination of compositions, the transgene is integrated into the genome of the human cells.

38. The method of any one of claims 35-37, wherein the composition or the set of compositions further comprises a recombinant viral vector.

39. The method of claim 38, wherein the recombinant viral vector is a recombinant AAV vector.

40. The method of claim 39, wherein the recombinant viral vector is or comprises a capsid polypeptide comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of: LK03, AAV8, AAV-DJ; AAV-LK03; or AAVNP59.

41. The method of claim 40, wherein the recombinant viral vector comprises the capsid polypeptide and the polynucleotide sequence encoding the nuclease and / or the polynucleotide cassette is encapsidated in the recombinant viral vector.

42. The method of claim 41, wherein the polynucleotide cassette is encapsidated in the recombinant viral vector.

43. The method of any one of claims 35-42, wherein the composition or the set of compositions further comprises an AAV2 ITR sequence.

44. The method of claim 43, wherein the AAV2 ITR sequence flanks the 5' end and the 3' end of the polynucleotide sequence encoding the nuclease and / or the polynucleotide cassette.

45. The method of any one of claims 35-44, wherein the third nucleic acid sequence and the fourth nucleic acid sequence are between 50 nt and 1600 nt in length.

46. ​​The method of any one of claims 35-45, wherein the third nucleic acid sequence and the fourth nucleic acid sequence are the same length.

47. The method of any one of claims 35-45, wherein the third nucleic acid sequence and the fourth nucleic acid sequence are different lengths.

48. The method of any one of claims 35-47, wherein following integration of the polynucleotide cassette into the target integration site in the genome of the cell, the transgene is expressed under the control of an endogenous promoter at the target integration site.

49. The method of any one of claims 36-48, wherein the target integration site is within the coding sequence of the albumin locus and is 5' adjacent to the stop codon.

50. The method of claim 49, wherein the target integration site is adjacent to the stop codon 5' in exon 14 of the albumin locus.

51. The method of any one of claims 36-50, wherein the cleavage site is within a non-coding sequence of the albumin locus.

52. The method of claim 51, wherein the cleavage site is within an intron, untranslated region, enhancer, promoter, silencer or insulator of the albumin locus.

53. The method of claim 52, wherein the cleavage site is within intron 12, 13 or 14 of the albumin locus.

54. The method of any one of claims 37-53, wherein the nuclease is selected from a Cas enzyme, a TALEN, a TALE nickase, a ZFN, a ZF nickase, or a meganuclease.

55. The method of any one of claims 35-54, wherein the nuclease is a Cas enzyme or a TALEN.

56. The method of any one of claims 35-55, wherein the nuclease is a Cas enzyme.

57. The method of any one of claims 35-56, wherein the Cas enzyme is selected from Staphylococcus aureus Cas9 (saCas9), Streptococcus pyogenes (spCas9), AZ nuclease, HF1-Cas9, HF2-Cas9 or HiFi-Cas9.

58. The method of claim 56 or 57, further comprising a guide RNA (gRNA).

59. The method of claim 58, wherein the gRNA comprises a nucleic acid sequence of any one of SEQ ID NOs: 27-45, 71-86, or 93-98, or a nucleic acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 27-45, 71-86, or 93-98.

60. The method of claim 58 or 59, wherein the nuclease or the polynucleotide sequence encoding the nuclease is co-formulated with the gRNA.

61. The method of claim 58 or 59, wherein the nuclease or the polynucleotide sequence encoding the nuclease is formulated separately from the gRNA.

62. The method of any one of claims 35-61, wherein the second nucleic acid sequence is or comprises: a) a nucleic acid sequence encoding a 2A peptide; b) a nucleic acid sequence encoding an internal ribosome entry site (IRES); c) a nucleic acid sequence encoding an N-terminal intein splicing region and a C-terminal intein splicing region; or d) Nucleic acid sequences encoding a splice donor and a splice acceptor.

63. The method of any one of claims 35-62, wherein the second nucleic acid sequence is or comprises a nucleic acid sequence encoding a 2A peptide.

64. The method of claim 63, wherein the second nucleic acid sequence is or comprises a nucleic acid sequence encoding a 2A peptide selected from the group consisting of P2A, T2A, E2A and F2A.

65. The method of claim 35 or 37, wherein the cleavage site is between 1 bp and 1000 bp away from the target integration site.

66. The method of claim 65, wherein the cleavage site is up to 100 bp away from the target integration site.

67. The method of any one of claims 35-66, wherein the transgene is selected from CBS, UGT1A1, MUT, FAH, ATP7B, A1AT, ASL, LIPA, PAH, G6PC, Factor IX, or a variant thereof.

68. The method of any one of claims 35-67, which is a composition set, wherein the nuclease or the polynucleotide sequence encoding the nuclease is formulated in a lipid nanoparticle, and the polynucleotide box is encapsidated in a recombinant AAV vector.

69. The method of any one of claims 35-68, wherein the cell is edited in vivo.

70. The method of any one of claims 35-69, wherein the method of integrating a transgene is performed ex vivo.

71. The method of claim 69 or 70, wherein the cell is a blood, liver, muscle or CNS cell.

72. The method of claim 70, wherein the cells are administered in an autologous transplant following transgene integration.

73. The method of claim 70, wherein the cells are administered in an allogeneic transplant following transgene integration.

74. The method of any one of claims 35-73, wherein (i) the nuclease or the polynucleotide sequence encoding the nuclease and (ii) the polynucleotide cassette are administered to the subject on the same day.

75. The method of any one of claims 35-73, wherein (i) the nuclease or the polynucleotide sequence encoding the nuclease and (ii) the polynucleotide cassette are administered to the subject on different days.

76. The method of claim 75, wherein the nuclease or the polynucleotide sequence encoding the nuclease is administered to the subject 1 hour to 3 days after the polynucleotide cassette.

77. The method of claim 76, wherein the nuclease or the polynucleotide sequence encoding the nuclease is administered to the subject 1 hour to 24 hours after the polynucleotide cassette.

78. The method of claim 75, wherein the nuclease or the polynucleotide sequence encoding the nuclease is administered to the subject 1 hour to 3 days prior to the polynucleotide cassette.

79. The method of claim 76, wherein the nuclease or the polynucleotide sequence encoding the nuclease is administered to the subject 1 hour to 4 hours before the polynucleotide cassette.

80. The method of claim 79, wherein the nuclease or the polynucleotide sequence encoding the nuclease is administered to the subject 4 hours prior to the polynucleotide cassette.

81. The composition or group of compositions of any one of claims 1-34, wherein the cleavage site is distal to the target integration site.

82. The method of any one of claims 35-80, wherein the cleavage site is distal to the target integration site.

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