Gene Therapy Composition and Method of Use Thereof

Abstract

Compositions and methods for gene therapy are presented herein. In part, the disclosure encompasses the recognition and observation that optimized vector designs and preferred cleavage sites can vary between species. As demonstrated herein, the optimal design in a human or humanized system can be significantly different from the optimal design in another species or model system (e.g., wild-type mouse). In some embodiments, a human or humanized system may allow for increased flexibility in homologous recombination and thus in the selection of cleavage sites to enhance the effectiveness of a particular gene therapy.

Description

Background Art

[0001] There is a subset of human diseases that can be traced back to changes in DNA that are inherited or acquired early in embryonic development. Of particular interest to developers of gene therapy are diseases caused by mutations in a single gene, which are known as single-gene diseases. It is thought that there are over 6,000 single-gene diseases. Typically, specific genetic diseases caused by genetic mutations are relatively rare, but taken together, the toll of gene-related diseases is high. Well-known genetic diseases include cystic fibrosis, Duchenne muscular dystrophy, Huntington's disease, and sickle cell anemia. Other classes of genetic diseases include metabolic disorders such as organic acidemias, and lysosomal storage diseases in which malfunctioning genes result in defects in metabolic processes and the accumulation of toxic by-products, leading to significant morbidity and mortality both short- and long-term.

Summary of the Invention

Means for Solving the Problems

[0002] Genetic diseases caused by malfunctioning genes account for a large portion of diseases worldwide. Gene therapy has emerged as a promising form of treatment aimed at reducing the impact of genetic diseases.

[0003] Prior to the present disclosure, certain AAV gene therapies using homologous recombination introduced the transgene of interest at specific sites using a viral vector composition in the absence of nucleases. In particular, the present disclosure recognizes that a combination of such vectors with one or more nucleases (e.g., provided in a single composition or as separate compositions) can improve the transgene integration rate and / or efficiency in humans.

[0004] Alternatively, or in addition, the present disclosure further encompasses the recognition that a combination of a vector as described herein and one or more nucleases can result in surprising and unexpected improvements in transgene integration rate and / or efficiency in humans, for example, when the target cleavage site is distal to the transgene integration site. In part, the present disclosure encompasses the recognition and observation that optimized vector designs and preferred cleavage sites can vary between species. As demonstrated herein, the optimal design in a human or humanized system can be significantly different from the optimal design in another species or model system (e.g., wild-type mouse). In some embodiments, a human or humanized system can allow for increased flexibility in the choice of cleavage sites to enhance homologous recombination and thus the effectiveness of certain gene therapies.

[0005] In some embodiments, the present disclosure provides a composition comprising: (i) a nuclease or a polynucleotide sequence encoding a nuclease; (ii) an expression cassette comprising a first nucleic acid sequence and a second nucleic acid sequence, wherein the first nucleic acid sequence encodes a transgene, the second nucleic acid sequence is located 5' or 3' relative to the first nucleic acid sequence, and promotes the production of two independent gene products upon integration into a target integration site within the genome of the cell; a third nucleic acid sequence located 5' relative to the expression cassette and comprising a sequence substantially homologous to the 5' human genomic sequence of the target integration site within the genome of the cell; and a fourth nucleic acid sequence located 3' relative to the expression cassette and comprising a sequence substantially homologous to the 3' human genomic sequence of the target integration site within the genome of the cell, wherein the polynucleotide cassette does not comprise 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 present disclosure provides a method for integrating a transgene into the genome of a cell, comprising administering a composition 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 the transgene, the second nucleic acid sequence is located 5' or 3' relative to the first nucleic acid sequence, and the integration into a target integration site within the genome of the cell promotes the production of two independent gene products, a third nucleic acid sequence located 5' relative to the expression cassette and comprising a sequence substantially homologous to the 5' human genomic sequence of the target integration site within the genome of the cell, and a fourth nucleic acid sequence located 3' relative to the expression cassette and comprising a sequence substantially homologous to the 3' human genomic sequence of the target integration site within the genome of the cell, wherein the polynucleotide cassette does not comprise a promoter sequence, the nuclease is capable of inducing a double-strand break and / or a single-strand break at a cleavage site in the genome of the cell, and the cleavage site is distal from the target integration site, and after administering the composition, the transgene is integrated into the genome of the cell population. In some embodiments, the cell is edited in vivo. In some embodiments, the integration of the transgene is performed ex vivo.

[0007] Any of a variety of delivery systems are contemplated, and in some embodiments, the composition further comprises a recombinant viral vector. Any of a variety of viral vectors are contemplated, and in some embodiments, the recombinant viral vector is a recombinant AAV vector. In some embodiments, the recombinant viral vector is 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, or comprises the same. In some embodiments, the composition further comprises AAV2 ITR sequences.

[0008] According to various embodiments, any of various lengths and / or ratios of homologous arms are contemplated. In some embodiments, the third and fourth nucleic acid sequences are each 50 nt to 1600 nt in length. In some embodiments, the third and fourth nucleic acid sequences are of the same length. In some embodiments, the third and fourth nucleic acid sequences are of different lengths.

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

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

[0011] According to various embodiments, a cleavage site suitable for any application can be used. As non-limiting examples, 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 14 of the albumin locus. In some embodiments, the cleavage site is from 1 to 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 useful in the provided methods and in combination with the provided compositions. In some embodiments, the nuclease is selected from meganucleases, TALENs, TALE nickases, ZFNs, ZF nickases, Cas enzymes, or variants thereof.

[0013] According to various embodiments, various second nucleic acids can be used to promote the production of two independent gene products upon integration into a target integration site within the genome of a cell. For example, in some embodiments, the second nucleic acid sequence can be or can 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 splicing region and a C-terminal intein splicing 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] This disclosure encompasses the recognition that any of a variety of transgenes can be used in compositions provided with and / or in methods provided. For example, in some embodiments, the transgene can be selected from CBS, UGT1A1, MUT, FAH, ATP7B, A1AT, ASL, LIPA, factor IX, or variants thereof.

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

[0016] According to various embodiments, the provided methods and compositions can be useful in connection with organ and / or tissue transplantation. In some embodiments, the cells are administered in autotransplantation after transgene integration. In some embodiments, the cells are administered in allotransplantation after transgene integration.

[0017] In one aspect, the present invention provides a nuclease or a polynucleotide sequence encoding a nuclease, wherein the nuclease is selected from a clustered regularly interspaced short palindromic repeat (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; a polynucleotide sequence encoding a nuclease; a first nucleic acid sequence and a second nucleic acid sequence, wherein the first nucleic acid sequence encodes a transgene, the second nucleic acid sequence is located 5' or 3' relative to the first nucleic acid sequence, and promotes the production of two independent gene products upon integration into a target integration site within the genome of a human cell; an expression cassette comprising the first nucleic acid sequence and the second nucleic acid sequence; a third nucleic acid sequence located 5' relative to the expression cassette and comprising a sequence substantially homologous to the 5' human genomic sequence of the target integration site within the human genome; and a fourth nucleic acid sequence located 3' relative to the expression cassette and comprising a sequence substantially homologous to the 3' human genomic sequence of the target integration site within the human genome. The polynucleotide cassette does not contain a promoter sequence, and the nuclease is capable of inducing a double-strand break and / or a single-strand break at a cleavage site within the genome of a human cell.

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

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

[0020] In some embodiments of any one of the foregoing aspects, the composition or set of compositions 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 a capsid polypeptide having at least 95% sequence identity with the amino acid sequence of sL65, LK03, AAV8, AAV-DJ, AAV-LK03, or AAVNP59, or comprises the same. In some embodiments, the recombinant viral vector comprises a capsid polypeptide, and the polynucleotide sequence encoding the nuclease and / or the polynucleotide cassette is encapsulated in the recombinant viral vector. In some embodiments, the polynucleotide cassette is encapsulated in the recombinant viral vector.

[0021] In some embodiments of any one of the foregoing aspects, the composition or set of compositions further comprises an AAV2 inverted terminal repeat (ITR) sequence. In some embodiments, the AAV2 ITR sequence flanks the 5' and 3' ends of a polynucleotide sequence encoding a nuclease and / or a polynucleotide cassette.

[0022] In some embodiments of any one of the foregoing aspects, the third and fourth nucleic acid sequences are each 50 nt to 1600 nt in length.

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

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

[0025] In some embodiments of any one of the foregoing aspects, when the polynucleotide cassette is integrated into a target integration site within the genome of a cell, the transgene is expressed under the control of an endogenous promoter at the target integration site.

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

[0027] In some embodiments of any one 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 one of the foregoing 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 one of the foregoing aspects, the nuclease is a Cas enzyme or a TALEN.

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

[0031] In some embodiments of any one 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 set of compositions further comprises a guide RNA (gRNA). In some embodiments, the gRNA comprises any one nucleic acid sequence 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 with any one of SEQ ID NOs: 27-45, 71-86, or 93-98.

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

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

[0034] In some embodiments of any one of the foregoing aspects, the second nucleic acid sequence is 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 splice donor and a splice acceptor, or comprises them.

[0035] In some embodiments of any one of the foregoing aspects, the second nucleic acid sequence is a nucleic acid sequence encoding a 2A peptide or comprises it.

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

[0037] In some embodiments of any one of the foregoing aspects, the cleavage site is 1 to 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 one of the foregoing aspects, the transgene is selected from CBS, UGT1A1, MUT, FAH, ATP7B, A1AT, ASL, LIPA, PAH, G6PC, Factor IX or variants thereof.

[0039] In some embodiments of any one of the foregoing aspects, the composition or set of compositions is a set of compositions in which a nuclease or polynucleotide sequence encoding a nuclease is formulated in lipid nanoparticles (LNP) and the polynucleotide cassette is encapsulated in a recombinant AAV vector.

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

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

[0042] In another aspect, the present invention provides a method for integrating a transgene into the genome of a human cell, 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; a polynucleotide sequence encoding a nuclease; a first nucleic acid sequence and a second nucleic acid sequence, wherein the first nucleic acid sequence encodes the transgene, the second nucleic acid sequence is located 5' or 3' relative to the first nucleic acid sequence, and promotes the production of two independent gene products upon integration into a target integration site within the genome of the human cell; an expression cassette comprising the first nucleic acid sequence and the second nucleic acid sequence; a third nucleic acid sequence located 5' relative to the expression cassette and comprising a sequence substantially homologous to the 5' human genomic sequence of the target integration site in the genome of the human cell; and a fourth nucleic acid sequence located 3' relative to the expression cassette and comprising a sequence substantially homologous to the 3' human genomic sequence of the target integration site in the genome of the human cell, wherein the polynucleotide cassette does not comprise a promoter sequence, the nuclease is capable of inducing a double-strand break and / or a single-strand break at a cleavage site within the genome of the cell, and after administration of the composition or set of compositions, the transgene is integrated into the genome of the human cell. In some embodiments, the target integration site is an albumin locus comprising an endogenous albumin promoter and an endogenous albumin gene.

[0043] In another aspect, the present invention is a method for integrating a transgene into the genome of a human cell, the method comprising contacting a human cell with a composition or set of compositions comprising a nuclease or 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 first nucleic acid sequence and a second nucleic acid sequence, wherein the first nucleic acid sequence encodes the transgene and the second nucleic acid sequence is located 5' or 3' relative to the first nucleic acid sequence and promotes the production of two independent gene products upon integration into a target integration site within the genome of the human cell, an expression cassette comprising the first nucleic acid sequence and the second nucleic acid sequence, a third nucleic acid sequence located 5' relative to the expression cassette and comprising a sequence substantially homologous to the 5' human genomic sequence of the target integration site in the genome of the human cell, and a fourth nucleic acid sequence located 3' relative to the expression cassette and comprising a sequence substantially homologous to the 3' human genomic sequence of the target integration site in the genome of the human cell, wherein the polynucleotide cassette does not comprise a promoter sequence, the nuclease is capable of inducing a double-strand break and / or a single-strand break at the cleavage site within the genome of the cell, and after contacting the human cell with the composition or set of compositions, the transgene is integrated into the genome of the human cell. In some embodiments, the target integration site is the albumin locus comprising the endogenous albumin promoter and the endogenous albumin gene.

[0044] In another aspect, the present invention provides a method for integrating a transgene into the genome of a human cell, the method comprising administering to a composition or set of compositions comprising a nuclease or polynucleotide sequence encoding a nuclease, a first nucleic acid sequence and a second nucleic acid sequence, wherein the first nucleic acid sequence encodes the transgene, the second nucleic acid sequence is located 5' or 3' relative to the first nucleic acid sequence and promotes the production of two independent gene products upon integration into a target integration site within the genome of the human cell, the target integration site being the albumin locus comprising the endogenous albumin promoter and the endogenous albumin gene, an expression cassette comprising the first nucleic acid sequence and the second nucleic acid sequence, a third nucleic acid sequence located 5' relative to the expression cassette and comprising a sequence substantially homologous to the 5' human genomic sequence of the target integration site in the genome of the human cell, and a fourth nucleic acid sequence located 3' relative to the expression cassette and comprising a sequence substantially homologous to the 3' human genomic sequence of the target integration site in the genome of the human cell, wherein the polynucleotide cassette does not comprise a promoter sequence, the nuclease is capable of inducing a double-strand break and / or a single-strand break at a cleavage site in the genome of the cell, and the cleavage site is distal to the target integration site, and wherein the transgene is integrated into the genome of the human cell after administering the composition or set of compositions.

[0045] In another aspect, the present invention provides a method for integrating a transgene into the genome of a human cell, the method comprising contacting the human cell with a nuclease or a polynucleotide sequence encoding a nuclease, and a first nucleic acid sequence and a second nucleic acid sequence, wherein the first nucleic acid sequence encodes the transgene, the second nucleic acid sequence is located 5' or 3' relative to the first nucleic acid sequence, and promotes the production of two independent gene products upon integration into a target integration site within the genome of the human cell, and the target integration site is an albumin locus comprising an endogenous albumin promoter and an endogenous albumin gene, an expression cassette comprising the first nucleic acid sequence and the second nucleic acid sequence, a third nucleic acid sequence located 5' relative to the expression cassette and comprising a sequence substantially homologous to the 5' human genomic sequence of the target integration site in the human cell genome, and a fourth nucleic acid sequence located 3' relative to the expression cassette and comprising a sequence substantially homologous to the 3' human genomic sequence of the target integration site in the human cell genome, a polynucleotide cassette, wherein the polynucleotide cassette does not contain a promoter sequence, the nuclease is capable of inducing a double-strand break and / or a single-strand break at a cleavage site in the genome of the cell, and the cleavage site is distal to the target integration site, and after contacting the human cell with the composition or set of compositions, the transgene is integrated into the genome of the human cell.

[0046] In some embodiments of any one of the foregoing aspects, the composition or set of compositions 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 a capsid polypeptide having at least 95% sequence identity with the amino acid sequence of LK03, AAV8, AAV-DJ, AAV-LK03, or AAVNP59, or comprises the same. In some embodiments, the recombinant viral vector comprises a capsid polypeptide, and the polynucleotide sequence encoding the nuclease and / or the polynucleotide cassette is encapsulated in the recombinant viral vector. In some embodiments, the polynucleotide cassette is encapsulated in the recombinant viral vector.

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

[0048] In some embodiments of any one of the foregoing aspects, the third and fourth nucleic acid sequences are from 50 nt to 1600 nt in length.

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

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

[0051] In some embodiments of any one of the foregoing aspects, when the polynucleotide cassette is integrated into a target integration site within the genome of a cell, the transgene is expressed under the control of an endogenous promoter at the target integration site.

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

[0053] In some embodiments of any one 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 one of the foregoing 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 one of the foregoing aspects, the nuclease is a Cas enzyme or a TALEN.

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

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

[0058] In some embodiments of any one of the foregoing aspects, the method further comprises a guide RNA (gRNA). In some embodiments, the gRNA comprises any one nucleic acid sequence 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 with any one of SEQ ID NOs: 27-45, 71-86, or 93-98.

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

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

[0061] In some embodiments of any one of the foregoing aspects, the second nucleic acid sequence is 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 splice donor and a splice acceptor, or comprises them.

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

[0063] In some embodiments of any one of the foregoing aspects, the cleavage site is 1 to 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 one of the foregoing aspects, the transgene is selected from CBS, UGT1A1, MUT, FAH, ATP7B, A1AT, ASL, LIPA, PAH, G6PC, factor IX or variants thereof.

[0065] In some embodiments of any one of the foregoing aspects, a method is a set of compositions in which a nuclease or a polynucleotide sequence encoding a nuclease is formulated into lipid nanoparticles and the polynucleotide cassette is encapsulated in a recombinant AAV vector.

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

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

[0068] In some embodiments of any one of the foregoing aspects, the cells are blood, liver, muscle, or CNS cells. In some embodiments, the cells are administered in autotransplantation after integration of the transgene. In some embodiments, the cells are administered in allotransplantation after integration of the transgene.

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

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

[0071] In some embodiments of any one of the foregoing aspects, the nuclease or the polynucleotide sequence encoding a nuclease and the polynucleotide cassette are brought into contact with human cells on the same day.

[0072] In some embodiments of any one of the foregoing aspects, the nuclease or the polynucleotide sequence encoding a nuclease and the polynucleotide cassette are brought into contact with human cells on different days.

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

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

[0075] In other embodiments of any of the foregoing aspects, the cleavage site overlaps the integration site.

[0076] As used in this application, the terms "about" and "approximately" are used as equivalents. Any citation to a publication, patent, or patent application herein is incorporated by reference in its entirety. Any numbers used in this application are meant to cover any normal variations understood by one of ordinary skill in the art, with or without "about / approximately".

[0077] Other features, objects, and advantages of the present invention will be apparent from the following detailed description. However, it should be understood that the detailed description is given by way of illustration only and not limitation, showing embodiments of the present invention. Various changes and modifications within the scope of the present invention will become apparent to those skilled in the art from this detailed description.

[0078] Definitions About: As used herein with respect to a value, the term "about" refers to a value similar in context to the referenced value. In general, one of ordinary skill in the art, having knowledge of the context, will understand the relevant degree of dispersion encompassed by "about" in that context. For example, in some embodiments, the term "about" may encompass values within a range of 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 recited value.

[0079] Adult: As used herein, the term "adult" refers to a human 18 years of age or older. In some embodiments, a human adult has a body weight in the range of about 90 pounds to about 250 pounds.

[0080] Related: Two events or entities are "related" to each other as the term is used herein if the presence, level, and / or form of one correlates with the presence, level, and / or form of the other. For example, a particular entity (e.g., a polypeptide, gene signature, metabolite, microorganism, etc.) is considered related to a particular disease, disorder, or condition if its presence, level, and / or form correlates with the incidence and / or susceptibility of the disease, disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically "related" to each other if they interact directly or indirectly such that as a result, they are physically proximate to and / or remain physically proximate to each other. In some embodiments, two or more entities that are physically related to each other are covalently bonded to each other, and in some embodiments, two or more entities that are physically associated with each other are not covalently bonded to each other but are non-covalently bonded to each other by, for example, hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0081] Biological sample: As used herein, the term "biological sample" typically refers to a sample obtained from or derived from a biological source of interest (e.g., tissue or organism or cell culture), as described herein. In some embodiments, the source of interest includes an organism such as an animal or a human. In some embodiments, the biological sample is or includes a biological tissue or a body fluid. In some embodiments, the biological sample is bone marrow; blood; blood cells; ascites; tissue or fine needle biopsy sample; cell-containing body fluid; cell-free floating nucleic acid; sputum; saliva; urine; cerebrospinal fluid, peritoneal fluid; pleural fluid; feces; lymph; gynecological fluid; skin swab; vaginal swab; oral swab; nasal swab; lavage or lavage fluid such as bronchial lavage fluid; aspirate; scraping; bone marrow specimen; tissue biopsy specimen; surgical sample; feces, other body fluids, secretions and / or excretions; and / or cells therefrom, etc., or may include them. In some embodiments, the biological sample is or includes cells obtained from an individual. In some embodiments, the obtained cells are or include cells derived from the individual from whom the sample was obtained. In some embodiments, the sample is a "primary sample" directly obtained from the source of interest by any suitable 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 fluid (e.g., blood, lymph, feces, etc.). In some embodiments, as is apparent from the context, the term "sample" refers to a preparation obtained by processing the primary sample (e.g., by removing one or more components and / or by adding one or more agents). For example, filtration using a semipermeable membrane. Such a "processed sample" may include, for example, nucleic acids or proteins extracted from the sample or obtained by subjecting the primary sample to techniques such as mRNA amplification or reverse transcription, isolation and / or purification of specific components.

[0082] Biomarker: As used herein, the term “biomarker” is used to refer to an entity whose presence, level, or form correlates with a particular biological event or a state of interest, consistent with its use in the art, and is thus considered a “marker” of that event or state. In particular, the present disclosure encompasses biomarkers for gene therapy (e.g., useful for evaluating one or more properties or characteristics of a gene therapy treatment such as the degree, level, and / or persistence of payload expression). In some embodiments, the biomarker is a cell surface marker. In some embodiments, the biomarker is intracellular. In some embodiments, the biomarker is found outside the cell (e.g., secreted or otherwise produced and present outside the cell in a body fluid such as blood, urine, tears, saliva, cerebrospinal fluid, etc.). In certain embodiments, the present disclosure demonstrates the effectiveness of a biomarker that can be detected in a sample obtained from a subject who has received gene therapy for use in evaluating one or more properties or characteristics of the gene therapy, and 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 was delivered and / or other than the cells, tissues, and / or fluids in which the payload is active.

[0083] Codon optimization: As used herein, the term “codon optimization” refers to the process of changing the codons of a given gene such that the polypeptide sequence encoded by the gene remains the same, but the changed codons improve the expression process of the polypeptide sequence. For example, when a polypeptide is a human protein sequence and is expressed in Escherichia coli (E. coli), codon optimization of the DNA sequence to change human codons to codons more effective for expression in E. coli often results in improved expression.

[0084] Detectable moiety: As used herein, the term "detectable moiety" refers to any entity (e.g., a molecule, complex, or a part or component thereof). In some embodiments, the detectable moiety is provided and / or utilized as a separate molecular entity, and in some embodiments, it is part of another molecular entity and / or associated with another molecular entity. Examples of detectable moieties include, but are not limited to, the following. 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 (see below for specific exemplary fluorescent dyes), chemiluminescent agents (e.g., acridinium esters, stabilized 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 (see below for specific examples of enzymes), colorimetric labels (e.g., dyes, gold colloids, etc.), biotin, digoxigenin, haptens, antibodies, and / or proteins for which antiserum or monoclonal antibodies are available.

[0085] Pediatric: As used herein, the term "pediatric" refers to a human between two years and 18 years of age. Body weight can vary widely depending on age and the particular child, with typical ranges being 30 pounds to 150 pounds.

[0086] Combination therapy: As used herein, the term "combination therapy" refers to a situation where a subject is simultaneously exposed to two or more treatment regimens (e.g., two or more therapeutic agents, e.g., gene therapy and non-gene therapy modalities). In some embodiments, two or more regimens can be administered simultaneously, and in some embodiments, such regimens can be administered sequentially (e.g., all "doses" of a first regimen are administered before any dose of a second regimen), and in some embodiments, such agents are administered in overlapping dosing regimens. In some embodiments, "administration" of combination therapy can include administering one or more agents or modalities to a subject who is receiving other agents or modalities in combination. For the sake of clarity, combination therapy does not require that the individual agents be administered together (or necessarily simultaneously) in a single composition.

[0087] Composition: One of ordinary skill in the art will understand that the term "composition" as used herein can be used to refer to an individual physical entity that includes one or more specific components. Generally, unless otherwise specified, a composition can be in any form, e.g., gaseous, gel, liquid, or solid.

[0088] Determine: Many of the methodologies described herein include a step of "determining." One of ordinary skill in the art, upon reading this specification, will understand that such "determination" can be achieved by using, or by the use of, any of a variety of techniques available to one of ordinary skill in the art, including, for example, the specific techniques explicitly referenced herein. In some embodiments, a determination includes manipulation of a physical sample. In some embodiments, a determination includes consideration and / or manipulation of data or information, e.g., using a computer or other processing unit adapted to perform a relevant analysis. In some embodiments, a determination includes receiving relevant information and / or materials from a source. In some embodiments, determining includes comparing one or more characteristics of a sample or entity to an equivalent reference.

[0089] Distal: As used herein, the term "distal" with respect to the position of the cleavage site relative to the target integration site means that the cleavage site is not the same as and / or does not overlap with the target integration site. In some embodiments, the cleavage site is within about 2 kB of the integration site (e.g., 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1 kB, 1.1 kB, 1.2 kB, 1.3 kB, 1.4 kB, 1.5 kB, 1.6 kB, 1.7 kB, 1.8 kB, 1.9 kB or 2.0 kB). In some embodiments, the cleavage site is within the boundaries of the homologous arm. It should be understood that in other embodiments, the cleavage site may be the same as or overlap 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 that encodes a particular gene product), and in some embodiments, a gene includes non-coding sequences. In some particular embodiments, a gene may include both coding (e.g., exon) sequences and non-coding (e.g., intron) sequences. In some embodiments, a gene may include one or more regulatory elements (e.g., a promoter, an enhancer, a silencer, a termination signal), and may 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 found (or has the same nucleotide sequence as that which is located or found) in the 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 (before and / or after processing) transcribed from a gene or a polypeptide (before and / or after modification) encoded by RNA transcribed from a gene.

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

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

[0094] Infant: As used herein, the term "infant" refers to a human less than 2 years of age. The typical weight of an infant ranges from 3 pounds to 20 pounds.

[0095] Newborn: As used herein, the term "newborn" refers to a newborn human.

[0096] Nucleic acid: As used herein, in its broadest sense, it refers to any compound and / or substance that can be incorporated or can be incorporated into an oligonucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that can be incorporated or can be incorporated into an oligonucleotide chain via a phosphodiester bond. As will be apparent from the context, in some embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides), and in some embodiments, "nucleic acid" refers to an oligonucleotide chain containing individual nucleic acid residues. In some embodiments, "nucleic acid" is RNA or contains RNA, and in some embodiments, "nucleic acid" is DNA or contains DNA. In some embodiments, a nucleic acid is one or more natural nucleic acid residues, contains one or more natural nucleic acid residues, or consists of one or more natural nucleic acid residues. In some embodiments, a nucleic acid is one or more nucleic acid analogs, contains one or more nucleic acid analogs, 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 one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), contains the same, or consists of the same. In some embodiments, a nucleic acid is 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-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, intercalating bases, and combinations thereof), contains the same, or consists of the same. In some embodiments, a nucleic acid has a nucleotide sequence encoding a functional gene product such as 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 by polymerization based on a complementary template (in vivo or in vitro), regeneration in a recombinant cell or recombinant 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 residues or longer. In some embodiments, the nucleic acid is partially or wholly single-stranded, and in some embodiments, the nucleic acid is partially or completely double-stranded. In some embodiments, the nucleic acid has a nucleotide sequence comprising at least one element that is a sequence encoding a polypeptide or a complement of a sequence encoding a polypeptide. In some embodiments, the nucleic acid has enzymatic activity.

[0097] Peptide: As used herein, the terms "peptide" or "polypeptide" refer to any polymer chain of amino acids. In some embodiments, the peptide has a naturally occurring amino acid sequence. In some embodiments, the peptide has an amino acid sequence that does not occur naturally. In some embodiments, the peptide has an amino acid sequence that has been engineered to be designed and / or produced by the hand of man. In some embodiments, the peptide may comprise or consist of natural amino acids, non-natural amino acids, or both. In some embodiments, the peptide may comprise or consist of only natural amino acids or only non-natural amino acids. In some embodiments, the peptide may comprise D-amino acids, L-amino acids, or both. In some embodiments, the peptide may comprise only D-amino acids. In some embodiments, the peptide may comprise only L-amino acids. In some embodiments, the peptide is linear. In some embodiments, the term "peptide" may be appended to the name of a reference peptide, activity, or structure, and in such cases, is used herein to refer to peptides that share the associated activity or structure, and can thus be considered members of the same class or family of peptides. For each such class, this specification provides exemplary peptides within the class whose amino acid sequence and / or function are known, and / or will be recognized by those of ordinary skill in the art. In some embodiments, such exemplary peptides are reference peptides for a peptide class or peptide family. In some embodiments, members of a peptide class or family share a common sequence motif (e.g., characteristic sequence elements) that exhibits significant sequence homology or identity with the reference peptide of that class, and / or share a common activity (in some embodiments, at an equivalent level or within a specified range) (in some embodiments, including all peptides within the class).For example, in some embodiments, the member peptide exhibits an overall sequence homology or sequence identity to a reference peptide of at least about 30-40%, often about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, and / or exhibits very high sequence identity, often 90% or even 95%, 96%, 97%, 98% or 99% in at least one region (e.g., a conserved region that may be a characteristic sequence element in some embodiments or that may contain a characteristic sequence element). Such conserved regions typically encompass at least 3-4, often up to 20 or more amino acids, and in some embodiments, the conserved region encompasses at least one stretch of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids.

[0098] Safe harbor: As used herein, the terms "safe harbor", "safe harbor locus", "safe harbor gene" or "safe harbor site" refer to one or more regions of the subject or cell genome (e.g., mouse genome, human genome, humanized animal genome, chimeric animal genome) that allow for stable expression of an integrated transgene without adversely affecting the subject and / or cell. In some embodiments, integration at a safe harbor allows for predictable function of the integrated transgene. In some embodiments, integration at a safe harbor does not cause unintended modification of the subject or cell genome that poses a risk to the subject or cell. In some embodiments, a safe harbor gene can be disrupted (e.g., via NHEJ) without integration of a transgene without adversely 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, and in some embodiments, including a prenatal human form). In some embodiments, the subject has a disease, disorder, or condition. 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 exhibits no symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject is a person having one or more characteristics that are characteristic of susceptibility or risk for a disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual who has been diagnosed and / or treated and / or has had treatment administered. In a preferred embodiment, the subject is a human.

[0100] Substantially: As used herein, the term "substantially" refers to a qualitative condition indicating the whole or nearly the whole range or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, complete and / or proceed completely, or achieve or avoid absolute results. Thus, 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 connection with a molecule, e.g., a nucleic acid, protein, or small molecule, the term "variant" refers to a molecule that exhibits significant structural identity with a reference molecule, but is structurally different from the reference molecule in the presence, absence, or level of one or more chemical moieties, for example, as compared to the reference entity. In some embodiments, the variant is also functionally different from its reference molecule. Generally, whether a particular molecule is properly regarded as a "variant" of a reference molecule is based on the degree of its structural identity with the reference molecule. As will be understood by those of skill in the art, any biological or chemical reference molecule has certain characteristic structural elements. By definition, a variant shares one or more such characteristic structural elements, but is a distinct molecule that differs from the reference molecule in at least one aspect. By way of just a few examples, a polypeptide can have characteristic sequence elements consisting of a plurality of amino acids that have positions specified relative to each other in linear or three-dimensional space and / or that contribute to a particular structural motif and / or biological function, and a nucleic acid can have characteristic sequence elements consisting of a plurality of nucleotide residues that have positions specified relative to another in linear or three-dimensional space. In some embodiments, a variant polypeptide or nucleic acid can differ from a reference polypeptide or nucleic acid as a result of one or more differences in the amino acid or nucleotide sequence and / or one or more differences in chemical moieties (e.g., carbohydrates, lipids, phosphate groups) that are covalently attached components of the polypeptide or nucleic acid (e.g., those attached to the polypeptide or nucleic acid backbone). In some embodiments, a variant polypeptide or nucleic acid exhibits overall sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% with a reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid does not share at least one characteristic sequence element with a reference polypeptide or nucleic acid. In some embodiments, a reference polypeptide or nucleic acid has one or more biological activities. In some embodiments, a variant polypeptide or nucleic acid shares one or more of the biological activities of a reference polypeptide or nucleic acid.In some embodiments, the variant polypeptide or nucleic acid lacks one or more of the biological activities of the reference polypeptide or nucleic acid. In some embodiments, the variant polypeptide or nucleic acid exhibits a reduced level of one or more biological activities as compared to the reference polypeptide or nucleic acid. In some embodiments, a polypeptide or nucleic acid of interest is considered a "variant" of a reference polypeptide or nucleic acid if it is identical to the reference amino acid or nucleotide sequence but has an amino acid or nucleotide sequence for a few sequence changes at specific positions. 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 as compared to the reference. In some embodiments, the variant polypeptide or nucleic acid contains about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 substituted residue as compared to an appropriate reference. Often, the variant polypeptide or nucleic acid contains a very small number (e.g., less than about 5, about 4, about 3, about 2, or less than about 1) of substituted, inserted, or deleted functional residues (i.e., residues involved in a particular biological activity) as compared to the reference. In some embodiments, the variant polypeptide or nucleic acid contains about 5, about 4, about 3, about 2, or about 1 or fewer additions or deletions as compared to the reference, and in some embodiments, contains no additions or deletions. In some embodiments, the variant polypeptide or nucleic acid contains 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, generally less than about 5, about 4, about 3, or about 2 additions or deletions as compared to the reference. In some embodiments, the reference polypeptide or nucleic acid is one 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

Figure 1B

Figure 2A

Figure 2B

Figure 2C

Figure 2D

Figure 2E

Figure 2F

Figure 3A

Figure 3B

Figure 4A

Figure 4B

Figure 4C

Figure 5A

Figure 5B

Figure 6

Figure 7

Figure 8

Figure 9A

Figure 9B

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15A

Figure 15B

Figure 16A

Figure 16B

Figure 17A

Figure 17B

Figure 18A

Figure 18B

Figure 19A

Figure 19B

Figure 19C

Figure 20A

Figure 20B

Figure 21

Figure 22A

Figure 22B

Figure 22C

Figure 22D

Figure 22E

Figure 23A

Figure 23B

Figure 23C

Figure 23D

Figure 23E

Figure 23F

Figure 24A

Figure 24B

Figure 25

Figure 26

Mode for Carrying Out the Invention

[0103] Gene Therapy Gene therapy alters the gene expression profile of a patient's cells by gene transfer, which is a process of delivering an exogenous therapeutic gene called a transgene. Various delivery vehicles are known to be used as vectors for transporting the transgene into the cell nucleus to alter or enhance the cell's capabilities (e.g., proteome, functionality, etc.). Developers have made great progress in introducing genes into cells in tissues such as the liver, the retina of the eye, and hematopoietic cells of the bone marrow using various vectors. These approaches have, in some cases, led to approved therapies and, in other cases, shown very promising results in clinical trials.

[0104] There are multiple gene therapy approaches. In conventional AAV gene therapy (also described herein as conventional or traditional gene therapy), the transgene is introduced into the nucleus of the host cell but is not intended to be integrated into chromosomal DNA. The transgene is expressed from a non-integrating genetic element called an episome that resides in the nucleus. A second type of gene therapy uses the use of different types of viruses such as lentiviruses that insert themselves at random sites into chromosomal DNA along with the transgene.

[0105] Episomal expression of a gene must be driven by an exogenous promoter and results in the production of a protein that modifies or improves the disease state.

[0106] Previously Known Limitations of Gene Therapy In the case of gene therapy based on episomal expression, when cells divide during the process of growth or tissue regeneration, the transgene is not intended to be integrated into the host chromosome and is not replicated during cell division, so the therapeutic benefit typically decreases. Thus, each new generation of cells further reduces the proportion of cells expressing the transgene in the target tissue, leading to a decrease or elimination of the therapeutic benefit over time.

[0107] The use of some gene therapies that employ viral-mediated insertion can potentially provide long-term benefits because the gene is inserted into the host chromosome, but there is no ability to control where the gene inserts, presenting the risk of disrupting essential genes or inserting in locations that can promote unwanted effects such as tumor formation. For this reason, these integrative gene therapy approaches are mainly limited to ex vivo approaches, in which cells are treated outside the body, screened for integration success, and then transplanted into the subject.

[0108] A common feature of many previously used gene therapy approaches is that the transgene is introduced into cells along with an exogenous promoter. The promoter is necessary to initiate the transcription and amplification of DNA into messenger RNA or mRNA that is ultimately translated into protein. High-level expression of therapeutic proteins from transgenes using previous gene therapy methods required strong, engineered promoters. These promoters were a necessary component of previous gene therapy systems for producing protein expression, but studies conducted in animal models have shown that non-specific integration of gene therapy vectors can lead to a significant increase in tumor development. Promoter strength can play an important role in this increased tumor development using previous gene therapy methods. Thus, attempts to drive high-level expression using strong exogenous promoters can lead to harmful results in the long term.

[0109] A. Gene Editing Gene editing is typically used to refer to the deletion, alteration, or enhancement of genes by introducing breaks into the DNA of cells using an exogenously delivered gene editing machinery. The effectiveness of many current gene editing approaches is limited because they have a high rate of unwanted off-target modifications, which arise in part from cells attempting to rapidly repair the introduced DNA breaks, and low efficiency of gene correction. Many gene editing techniques focus on disabling malfunctioning genes or correcting or skipping individual harmful mutations within a gene. These previous approaches often do not fully treat or prevent genetic diseases because of the number of possible mutations within a gene, as compared to methods that involve the insertion of a fully corrected gene.

[0110] Unlike gene therapy approaches that utilize episomal expression, gene editing enables the repaired gene region to be propagated to new generations of cells via normal cell division. Furthermore, the desired protein can be expressed using the cell's own regulatory machinery. Traditional approaches for gene editing are nuclease-based and use nuclease enzymes to introduce single-stranded or double-stranded breaks in the DNA at specific cleavage sites in order to cause deletions, make modifications, or apply correction sequences. These methods are designed to provide gene editing (e.g., deletion, alteration, insertion of a correction sequence, etc.) at the location of the cleavage site.

[0111] When a nuclease cuts the DNA, conventional gene editing techniques use two pathways: homologous recombination repair (HDR) and non-homologous end joining (NHEJ) to modify the DNA. HDR in these previous methods involves the very precise integration of a DNA sequence complementary to the integration site located at the same position as the cleavage site. HDR has the important advantage in that it can repair DNA with high fidelity and avoid the introduction of unwanted mutations into the integration site. NHEJ is a less selective and more error-prone process that rapidly joins the ends of the cut DNA, resulting in a high frequency of insertions or deletions at the cleavage site.

[0112] 1. Conventional nuclease-based gene editing Nuclease-based gene editing uses nucleases, which are endogenous or engineered enzymes that generate single- or double-strand breaks in DNA at the cleavage site. Nuclease-based gene editing is a two-step process. First, an exogenous nuclease capable of cleaving one or both strands of double-stranded DNA is directed to a desired site (e.g., via the use of guide RNA, site-specific residues within the nuclease, etc.) to make a specific cut at the cleavage site. Next, the endogenous cellular DNA repair machinery is activated to complete the editing process either by NHEJ or, less commonly, HDR.

[0113] NHEJ can occur in the absence of a DNA template for the cell to copy when repairing DNA breaks. The proportion of NHEJ compared to other repair mechanisms (e.g., HDR) is species-dependent, but in the absence of exogenous compounds (e.g., repair template sequences, enzymes, etc.), NHEJ is often preferentially used by the cell to repair double-strand breaks. Gene editing via NHEJ can be used to introduce small insertions or deletions known as indels, which can lead to knockout or reduction of gene function. NHEJ, due to its repair pattern, results in insertions and deletions in DNA and can also lead to the introduction of off-target unwanted mutations, including chromosomal abnormalities.

[0114] Nuclease-mediated HDR occurs by delivery of a nuclease and a DNA template that has partial or complete complementarity to the target integration site. In previous methods, the target integration site and the cleavage site either coincided or overlapped. The cell uses the DNA template to construct repaired DNA, resulting in the insertion of modified and / or alternative gene sequences.

[0115] Traditional gene editing (also described herein as conventional gene editing) has frequently used three different classes of nucleases for nuclease-based approaches: transcription activator-like effector nucleases (TALENs), clustered, regularly interspaced short palindromic repeat (CRISPR / Cas) nucleases; and zinc finger nucleases (ZFNs).

[0116] 2. Limitations of Conventional Nuclease-Based Gene Editing Previous nuclease-based gene editing approaches were limited by the use of nuclease enzymes that generate overlapping cleavage and integration sites. Further, these traditional methods relied on exogenous promoters for transgene expression. Nucleases can cause 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 result in genotoxicity, including chromosomal changes, based on the error-prone NHEJ process or can disrupt natural gene expression by targeting exons.

[0117] GENERIDE (trademark) Technology Platform 3. GENERIDE (trademark) Function GENERIDE (TM) is a novel AAV-based genome editing technology that precisely inserts a therapeutic transgene into the cell genome via homologous recombination. GENERIDE (TM) provides sustained transgene expression regardless of cell proliferation and tissue growth, and GENERIDE (TM)-corrected cells exhibit selective proliferation within diseased tissues (e.g., selective proliferation of hepatocytes in the diseased liver of an affected subject). Without wishing to be bound by any particular theory, GENERIDE (TM) is thought to be a genome editing technology that utilizes homologous recombination (HR), a naturally occurring HDR process that maintains genome fidelity. In some embodiments, GENERIDE (TM) enables the insertion of a transgene into a specific target genomic location (e.g., via HR) without exogenous nucleases. In some embodiments, GENERIDE (TM) enables the insertion of a transgene into a specific target genomic location (e.g., via HR) in combination with one or more exogenous nucleases. GENERIDE (TM)-directed transgene integration is designed to drive high levels of tissue-specific gene expression without causing the detrimental problems associated with the use of exogenous promoters in conventional gene therapy by leveraging the endogenous promoter at the target integration site.

[0118] 4. Advantages of the GENERIDE (TM) Technology The GENERIDE (TM) technology is designed to precisely integrate the corrective gene into the patient's genome to provide a stable and durable therapeutic effect. In particular, GENERIDE (TM) can be applied to target rare disorders in pediatric patients (e.g., disorders of the liver, CNS, muscle, blood, etc.), where it is important to provide treatment early in the patient's life before irreversible disease pathology can occur.

[0119] In some embodiments, the GENERIDE™ technology provides improvements over certain important limitations of both conventional gene therapy and conventional gene editing approaches, in a manner suitable for treating genetic diseases (e.g., genetic diseases in pediatric patients). In some embodiments, GENERIDE™ uses an AAV vector to deliver a transgene to the nucleus of a cell, followed by insertion of the transgene into a target integration site within the cell genome, and transgene expression is regulated by one or more endogenous promoters. In some embodiments, GENERIDE™ may enable lifelong protein production even as the body grows and changes over time, which is not achievable with conventional AAV gene therapy.

[0120] In particular, the GENERIDE™ technology can also provide surprising and unexpected improvements when combined with a target nuclease. In some embodiments, GENERIDE™ is combined with one or more nucleases that target specific cleavage sites within the cell genome, where the cleavage sites are distal from the target integration site (e.g., the cleavage site and the target integration site are not the same and / or do not overlap). As demonstrated herein, the use of such targeted nucleases in combination with additional GENERIDE™ components can result in an improvement in the integration rate and / or efficiency of a transgene (e.g., an improvement in the level of modified DNA, modified mRNA, and / or protein expression). In particular, the targeted nuclease may be designed to introduce a single-stranded or double-stranded break at the cleavage site of the DNA sequence (e.g., alone or in combination with a targeting molecule including, but not limited to, a guide RNA), where the cleavage site is distal from the target integration site. The introduction of a cleavage site distal from the target site for transgene integration has been demonstrated to result in improvements in mice (see Caneva et al., JCI Insight, 2019), but these improvements have been shown to be highly dependent on the exact location of the cleavage site. For example, Caneva showed that cleavage within adjacent intron 14 of the mouse albumin resulted in a significant improvement in cleavage and HDR efficiency compared to limited levels of integration when the cleavage site was introduced into intron 13 of the mouse albumin (Caneva's Figure S1). Caneva demonstrated that specific “hot spot” cleavage sites distal from the integration site can potentially increase HDR efficiency and transgene integration, but there was no demonstration that these improvements resulted in site-specific integration and expression of the transgene of interest. Furthermore, Caneva limited all experiments to mouse cells containing wild-type mouse albumin. As will be appreciated by those skilled in the art, significant testing and research are required to determine whether such systems can be modified for functionality in different species.For example, the high degree of sequence variability in certain genes (e.g., albumin) across species such as mouse and human makes it known that it is not possible to determine whether appropriate cleavage or integration sites can be found in other species and, in some cases, whether those skilled in the art can use results from one species to find them in other species. Further, it is known in the art that mouse and human genes (e.g., albumin) can contain significant sequence, epigenetic, and structural differences that can vary the efficiency of nuclease cleavage and / or homologous recombination at specific sites.

[0121] The present disclosure provides a surprising and unexpected insight that combining a target nuclease with a GENERIDE™ component, where the nuclease cleavage site is distal from the target integration site (e.g., the cleavage site and the integration site are not the same and / or do not overlap), can result in an unexpected improvement in the transgene integration rate and / or expression efficiency in human cells and tissues. In some embodiments, such improvement can be observed for multiple cleavage sites within different non-coding sequences in a human DNA sequence of interest (e.g., both intron 13 and 14 of human albumin).

[0122] The modular approach disclosed herein can be applied to enable GENERIDE™ to deliver reproducible and robust tissue-specific gene expression across different therapeutic agents delivered to tissues (e.g., liver, muscle, CNS, blood).

[0123] Previous studies on non - destructive gene targeting are described in WO 2013 / 158309 pamphlet, which is incorporated herein by reference. Previous studies on genome editing without using nucleases are described in WO 2015 / 143177 pamphlet, which is incorporated herein by reference. Previous studies on non - destructive gene therapy for the treatment of MMA are described in WO 2020 / 032986 pamphlet, which is incorporated herein by reference. Previous studies on monitoring gene therapy are described in WO 2020 / 214582 pamphlet, which is incorporated herein by reference.

[0124] B.GENERIDE (trademark) component Delivery vehicle There are multiple gene therapy approaches understood in the art. Accordingly, there are multiple delivery mechanisms understood in the art. In some embodiments, the transgene is provided using a delivery vehicle. In some embodiments, the compositions of the present disclosure include a delivery vehicle. In some embodiments, the delivery vehicle is a viral particle (e.g., a viral vector) or includes it. In some embodiments, the delivery vehicle is a non - viral particle and / or a non - particulate payload (e.g., a nuclease) or includes it. 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, for example, those described in WO 2015184256 pamphlet; WO 2013149140 pamphlet; WO 2014089486A1 pamphlet; WO 2009127060 pamphlet; WO 2011071860 pamphlet; WO 2020219941 pamphlet (the content of each of which is incorporated herein by reference).

[0125] In some embodiments, the delivery vehicle is an exosome or comprises exosomes. Those skilled in the art will recognize various methods of exosome production and use. Examples of such methods and uses are described in Luan et al., Acta Pharmacologica Sinica volume 38, pages 754 - 763 (2017).

[0126] In some embodiments, combinations of one or more different payloads can be delivered by one or more of the delivery systems described herein (e.g., viral vectors, lipid nanoparticles, etc.). In some embodiments, a delivery system can deliver one payload (e.g., one or more nucleases (e.g., Cas protein, endonuclease, TALEN, ZFN)) in combination with a second distinct payload (e.g., a polynucleotide sequence comprising a transgene). In some embodiments, a first delivery system (e.g., a lipid nanoparticle (LNP)) can deliver one payload (e.g., one or more nucleases (e.g., Cas protein, endonuclease, TALEN, ZFN)) in combination with a second distinct delivery system (e.g., a viral vector such as a recombinant AAV vector) that delivers a second distinct payload (e.g., a polynucleotide sequence comprising a transgene).

[0127] Viral vector In some embodiments, the delivery vehicle is a viral vector or comprises a viral vector. A viral vector contains a virus or viral chromosomal material into which a heterologous nucleic acid sequence can be inserted for transfer to a target sequence of interest (e.g., for transfer into genomic DNA within a cell). For example, various viruses including single - stranded DNA (ssDNA), double - stranded DNA (dsDNA) viruses, and / or RNA viruses having a DNA stage in their life cycle can be used as viral vectors. In some embodiments, the viral vector is or comprises an adeno - associated virus (AAV) or an AAV variant.

[0128] In some embodiments, the vector particle is a single unit of a virus that includes a capsid encapsulating a virus-based polynucleotide (e.g., a wild-type virus genome or a recombinant viral vector). In some embodiments, the vector particle is an AAV vector particle or includes 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 encapsulated AAV vector. In some embodiments, the vector particle (also referred to as a viral vector) includes at least one AAV capsid protein and an encapsulated AAV vector, wherein the vector further includes one or more heterologous polynucleotide sequences.

[0129] Capsid protein In some embodiments, the expression construct comprises a polynucleotide sequence encoding a capsid protein derived from one or more AAV subtypes, including naturally occurring AAV and recombinant AAV. In some embodiments, the expression construct is 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 (alternatively 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, AAV13, 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 serotypes of AAV), or a polynucleotide sequence encoding a capsid protein derived from a variant thereof.

[0130] In some embodiments, the viral vector is packaged within a capsid protein (e.g., a capsid protein from one or more AAV subtypes). In some embodiments, the capsid protein provides an increase or enhancement of transduction of cells (e.g., human or mouse cells) compared to a reference capsid protein. In some embodiments, the capsid protein provides an increase or enhancement of transduction of a particular cell or tissue type (e.g., liver tropism, muscle tropism, CNS tropism, lung tropism) compared to a reference capsid protein. In some embodiments, the capsid protein increases or enhances transduction of cells or tissues (e.g., liver, muscle, and / or CNS) 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 compared to a reference capsid protein. In some embodiments, the capsid protein increases or enhances transduction of cells or tissues (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 compared to a reference capsid protein.

[0131] In some embodiments, the sequence encoding the capsid protein can be, or can include, a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity to the corresponding wild-type capsid protein. In some embodiments, the sequence encoding the capsid protein can be, or can include, a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity to the corresponding sequence encoding an engineered capsid protein (e.g., a chimeric capsid protein, a codon-optimized capsid protein, etc.). In some embodiments, the sequence encoding the capsid protein can be, or can include, a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity to the exemplary sequences in Table 1 below.

[0132]

Table 1-1

[0133]

Table 1-2

[0134]

Table 1-3

[0135]

Table 1-4

[0136]

Table 1-5

[0137]

Table 1-6

[0138]

Table 1-7

[0139]

Table 1-8

[0140]

Table 1-9

[0141]

Table 1-10

[0142]

Table 1-11

[0143]

Table 1-12

[0144] Structure and Function of AAV Adeno-associated virus (AAV) is a parvovirus composed 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 at the ends of the genomic sequence. The ITR functions as an origin of replication and plays a role in viral packaging. The viral genome also contains the rep and cap genes, which are related to replication and capsid packaging, respectively. In most wild-type AAVs, the rep gene encodes four proteins required for viral replication, Rep78, Rep68, Rep52, and Rep40. The cap gene encodes the capsid subunits and an assembly activation protein (AAP) that promotes the assembly of viral particles. AAV is generally replication-deficient and requires the presence of a helper virus or helper virus functions (e.g., herpes simplex virus (HSV) and / or adenovirus (AdV)) to replicate in infected cells. For example, in some embodiments, AAV requires the E1A, E2A, E4, and VA RNA genes of adenovirus to replicate in host cells.

[0145] Recombinant AAV Generally, a recombinant AAV (rAAV) vector can contain many of the same elements found in wild-type AAV, including a similar capsid sequence and structure, and a polynucleotide sequence that is not of AAV origin (e.g., a polynucleotide that is heterologous to AAV). In some embodiments, the rAAV replaces the natural wild-type AAV sequence with a polynucleotide sequence encoding a payload. For example, in some embodiments, the rAAV will contain a polynucleotide sequence encoding one or more genes intended for a therapeutic purpose (e.g., for gene therapy). The rAAV can be modified to remove one or more wild-type viral coding sequences. For example, the rAAV can be engineered to contain only one ITR and / or one or more genes necessary for packaging (e.g., the rep and cap genes) than are found in wild-type AAV. Gene expression by rAAV is generally limited to one or more genes totaling 5 kb or less because larger sequences are not efficiently packaged within the viral capsid. In some embodiments, two or more rAAVs can be used to provide portions of a larger payload, for example, to provide the entire coding sequence of a gene that is normally too large to fit in a single AAV.

[0146] In particular, the present disclosure provides viral vectors comprising one or more polypeptides described herein. In some embodiments, the rAAV may comprise an AAV comprising one or more capsid proteins (e.g., 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 (referred to herein interchangeably 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, AAV13, 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 a mutant AAV capsid protein or chimeric AAV capsid (e.g., a capsid having polynucleotide sequences derived from two or more different serotypes of AAV). In some embodiments, the rAAV may comprise one or more polynucleotide sequences encoding a gene or nucleic acid of interest (e.g., a gene for the treatment of a genetic disease / disorder and / or an inhibitory nucleic acid sequence).

[0147] In some embodiments, the recombinant AAV vector may contain at least one ITR. In some embodiments, the recombinant AAV vector contains two ITRs. In some embodiments, the recombinant AAV vector contains a 5’ ITR. In some embodiments, the recombinant AAV vector contains a 3’ ITR. In some embodiments, the recombinant AAV vector contains an AAV2 ITR. In some embodiments, the recombinant AAV vector contains a portion of the AAV2 ITR. In some embodiments, the recombinant AAV vector contains an ITR having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the AAV2 ITR. In some embodiments, the recombinant AAV vector contains an ITR having 90%, 95%, 99%, or 100% sequence identity to one of the exemplary sequences in Table 2 below.

[0148]

Table 2

[0149] The AAV vector may be able to replicate (replication competent) in an infected host cell or may not be able to replicate (replication incompetent) in an infected host cell. Replication-competent AAV (rcAAV) requires the presence of one or more functional AAV packaging genes. Recombinant AAV vectors are generally designed to be non-replicable in mammalian cells to reduce the likelihood that rcAAV is generated by recombination with sequences encoding AAV packaging genes. In some embodiments, the rAAV vector preparations described herein are designed to contain little, if any, rcAAV vector. In some embodiments, the rAAV vector preparation contains less than about 1 rcAAV per 10 2 rAAV vectors. In some embodiments, the rAAV vector preparation contains less than about 1 rcAAV per 10 4 rAAV vectors. In some embodiments, the rAAV vector preparation contains less than about 1 rcAAV per 10 8contains less than about 1 rcAAV per rAAV vector. In some embodiments, the rAAV vector preparation is 10 12 contains less than about 1 rcAAV per rAAV vector. In some embodiments, the rAAV vector preparation does not contain rcAAV vectors.

[0150] Lipid nanoparticles (LNPs) Lipid nanoparticles (LNPs) are delivery systems that can, among other things, achieve intracellular delivery of intact nucleic acids and enable biological changes including therapeutic effects. In one aspect, the lipid nanoparticles are lipid compositions comprising at least one lipid. In some embodiments, the lipid nanoparticles may further comprise at least one nucleic acid (e.g., DNA, RNA, etc.). In some embodiments, the lipid nanoparticles may comprise a therapeutic nucleic acid (e.g., DNA, RNA, etc.) encapsulated in the lipid portion of the nanoparticles.

[0151] 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 of the compounds described herein. In particular, the present disclosure describes that the selection and combination of one or more of the components described herein can affect the properties of the lipid nanoparticles such as diameter, pKa, stabilization, and ionizability.

[0152] In particular, the present disclosure describes that the selection and combination of one or more of the components described herein can affect the functional activities of the lipid nanoparticles such as tropism, stabilization, and delivery efficacy. For example, the present disclosure describes that a combination of components may be better adapted for delivery of a particular payload (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.).

[0153] In some embodiments, the lipid nanoparticles can be, or can include, structures produced by available methods (International Ed. In English, 2012; Li et al. PLOS One, 2013; Kulkarni et al. Nanomedicine: Nanotechnology, Biology, and Medicine, 2017; U.S. Patent Application Publication No. 2019 / 0240345; International Publication No. 2019 / 089828 Pamphlet; International Publication No. 2021 / 102411 Pamphlet; International Publication No. 2019 / 046809 Pamphlet; International Publication No. 2018089540 Pamphlet, each of which is incorporated herein by reference in its entirety). In light of the teachings provided herein, one of ordinary skill in the art will recognize the possibility that alternative available lipid nanoparticles can be substituted for those described herein.

[0154] Ionizable lipid In some embodiments, the lipid nanoparticles include one or more ionizable lipids described herein. In some embodiments, the ionizable lipid can include an amine-containing group on the head group. In some embodiments, the ionizable lipid is, or includes, a compound described herein (e.g., a compound of Formula II, Formula III, or Formula IV). In some embodiments, the ionizable lipid is present in the lipid nanoparticle (LNP) preparation at about 30 mol% to about 70 mol% based on the total moles of the components of the lipid nanoparticles. In some embodiments, the ionizable lipid is present at about 33 mol% to about 60 mol% based on the total moles of the components of the lipid nanoparticles. In some embodiments, the ionizable lipid is present at about 34 mol% to about 55 mol% based on the total moles of the components of the lipid nanoparticles. In some embodiments, the ionizable lipid is present at about 33 mol% to about 51 mol% based on the total moles of the components of the lipid nanoparticles. In some embodiments, the ionizable lipid is present at about 34.7 mol%. In some embodiments, the ionizable lipid is present at about 50 mol% based on the total moles of the components of the lipid nanoparticles.

[0155] Sterol In some embodiments, the lipid nanoparticles comprise one or more sterols described herein. In some embodiments, the sterol is cholesterol, or a variant or derivative thereof. In some embodiments, cholesterol is modified. In some embodiments, cholesterol is oxidized cholesterol. In some embodiments, cholesterol is esterified cholesterol. Unmodified cholesterol can be acted upon by an enzyme to form variants in which the side chain or ring is oxidized. In some embodiments, cholesterol can be oxidized on the beta-ring structure or on the hydrocarbon tail structure. In some embodiments, the sterol is a phytosterol. Exemplary sterols contemplated for use in the disclosed lipid nanoparticles include, but are not limited to, 25-hydroxy cholesterol (25-OH), 20α-hydroxy cholesterol (20α-OH), 27-hydroxy cholesterol, 6-keto-5α-hydroxy cholesterol, 7-ketocholesterol, 7β-hydroxy cholesterol, 7α-hydroxy cholesterol, 7β-25-dihydroxy cholesterol, beta-sitosterol, stigmasterol, brassicasterol, campesterol, or combinations thereof. In some embodiments, side chain oxidized cholesterol can enhance cargo delivery compared to other cholesterol variants. In some embodiments, cholesterol is unmodified cholesterol.

[0156] In some embodiments, the LNP composition comprises from about 20 mole percent to about 50 mole percent sterol. In some embodiments, the LNP composition comprises about 38 mole percent sterol. In some embodiments, the LNP composition comprises about 38.5 mole percent sterol. In some embodiments, the LNP composition comprises about 33.8 mole percent cholesterol.

[0157] Conjugate linker lipid In some embodiments, the lipid nanoparticles comprise one or more conjugate linker lipids described herein. In some embodiments, the conjugate-linker lipid is, or comprises, a polyethylene glycol (PEG)-lipid or PEG-modified lipid. In some embodiments, PEG or PEG-modified lipid may alternatively be referred to as a PEGylated lipid or PEG-lipid. The inclusion of PEGylated lipid can be used to enhance lipid nanoparticle colloidal stability in vitro and circulation time in vivo. In some embodiments, the PEGylation is reversible in that the PEG moiety is gradually released during blood circulation. Exemplary PEG-lipids include, but are not limited to, PEG conjugated to a saturated or unsaturated alkyl chain having a length of C6-C20. PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide (PEG-CER), PEG-modified dialkylamine, PEG-modified diacylglycerol (PEG-DAG), PEG-modified dialkylglycerol, and mixtures thereof. For example, in some embodiments, the PEG lipid can be a PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPE, PEG-DSG or PEG-DSPE lipid.

[0158] In some embodiments, the conjugate-linker lipid comprises a polyethylene glycol lipid. In some embodiments, the conjugate-linker lipid comprises dimyristoyl glycerol (DMG), 1,2-dipalmitoyl-rac-glycerol, methoxypolyethylene glycol (DPG-PEG), or 1,2-distearoyl-rac-glycerol-3-methylpolyoxyethylene (DSG-PEG). In some embodiments, the conjugate-linker lipid has an average molecular weight of about 500 Da to about 5000 Da. In some embodiments, the conjugate-linker lipid has an average molecular weight of about 2000 Da. In some embodiments, the LNP composition comprises from about 0 mole percent to about 5 mole percent of the conjugate-linker lipid. In some embodiments, the LNP composition comprises about 1.5 mole percent of the conjugate-linker lipid. In some embodiments, the LNP composition comprises about 3 mole percent of the conjugate-linker lipid.

[0159] Phospholipid In some embodiments, the lipid nanoparticles comprise one or more phospholipids described herein. In some embodiments, the one or more phospholipids can assemble into one or more lipid bilayers. In some embodiments, the one or more phospholipids can comprise a phospholipid moiety. In some embodiments, the one or more phospholipids can comprise one or more fatty acid moieties. In some embodiments, the one or more phospholipids can comprise a phospholipid moiety and one or more fatty acid moieties. In some embodiments, the phospholipid is or comprises a compound described herein (e.g., a compound of Formula I). In some embodiments, the phospholipid moiety includes, but is not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lyso-phosphatidylcholine, and sphingomyelin. In some embodiments, the fatty acid moiety includes, but is not limited to, lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. Also contemplated are unnatural species, including natural species having modifications and substitutions including branches, oxidations, cyclizations, and alkynes. For example, the phospholipid can be functionalized with or crosslinked to one or more alkynes (e.g., an alkenyl group in which one or more double bonds are replaced with triple bonds). Under appropriate reaction conditions, the alkyne group may undergo copper-catalyzed cycloaddition upon exposure to an azide. Such reactions can be useful for functionalizing the lipid bilayer of the nanoparticle composition to promote membrane permeation or cell recognition, or for conjugating the nanoparticle composition with useful components such as targeting or imaging moieties (e.g., dyes).

[0160] 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-doundecanoyl-sn-glycerophosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesteryl hemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 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-docosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), 1-stearoyl-2 oleoyl phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), or a combination thereof. In some embodiments, the phospholipid is DSPC. In some embodiments, the phospholipid is DMPC..

[0161] In some embodiments, the phospholipids include 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 combinations thereof.

[0162] Diameter In particular, the present disclosure describes compositions having an average hydrodynamic diameter of about 30 to about 220 nm. In some embodiments, the lipid nanoparticles described herein can have an average hydrodynamic diameter of about 30 to about 220 nm. In some embodiments, the lipid nanoparticles described herein can have an average hydrodynamic diameter that is about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 155 nm, 160 nm, 165 nm, 170 nm, 175 nm, 180 nm, 185 nm, 190 nm, 195 nm, 200 nm, 205 nm, 210 nm, 215 nm, 220 nm, or any range defined by any two of the foregoing values. For example, in some embodiments, the lipid nanoparticles described herein have an average hydrodynamic diameter of 50 nm to 200 nm.

[0163] pKa In particular, the present disclosure describes compositions, preparations, nanoparticles, and / or nanomaterials having a pKa of about 5 to about 9.

[0164] In some embodiments, the lipid nanoparticles described herein have a pKa of 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 of pKa having endpoints defined by any two of the foregoing values. In some embodiments, the lipid nanoparticles described herein have a pKa that is any range 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 two of the foregoing values defining the endpoints.

[0165] Exemplary LNP compositions The present invention provides a composition comprising lipid nanoparticles. In some embodiments, the lipid nanoparticle composition comprises from about 30 mole percent to about 70 mole percent ionizable lipid, from about 5 mole percent to about 25 mole percent phospholipid, from about 25 mole percent to about 45 mole percent cholesterol, and from about 0 mole percent to about 5 mole percent conjugate-linker lipid. In some embodiments, the lipid nanoparticle composition comprises about 50 mole percent ionizable lipid, about 20 mole percent phospholipid, about 39 mole percent cholesterol, and about 1 mole percent conjugate-linker lipid.

[0166] In some embodiments, the lipid nanoparticle composition comprises, based on the total moles of these components, from about 30 mole percent to about 70 mole percent ionizable lipid of Formula II, Formula III, or Formula IV, from about 5 mole percent to about 25 mole percent DSPC, from about 25 mole percent to about 45 mole percent cholesterol, and from about 0 mole percent to about 5 mole percent lipid PEG.

[0167] In some embodiments, the lipid nanoparticle (LNP) preparation contains DNA at a mass ratio (total of one or more ionizable lipids, sterols, conjugate linker lipids, and phospholipids):DNA of about 2:1 and 50:1. In some embodiments, the LNP preparation has a mass ratio (total of one or more ionizable lipids, sterols, conjugate linker lipids, and phospholipids):DNA 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, 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.

[0168] Exemplary compounds In particular, the present disclosure describes compositions and / or nanoparticles comprising one or more of the compounds described herein.

[0169] In some embodiments, the present disclosure provides lipid nanoparticles comprising a compound comprising one or more partially unsaturated lipid groups. In some embodiments, the provided lipid nanoparticles comprise a compound comprising one or more trimethylated amine groups. In some embodiments, the provided lipid nanoparticles comprise a compound comprising one or more dimethylated amine groups.

[0170] In some embodiments, the present disclosure provides a compound of formula I:

Chemical formula

[0171] In some embodiments, the present disclosure provides a compound of formula II:

Chemical formula

[0172] In some embodiments, the present disclosure provides a compound of Formula III:

Chemical formula

[0173] In some embodiments, the present disclosure provides a compound of Formula IV:

Chemical formula

[0174] Target nuclease There are multiple DNA-targeting nucleases understood in the art. In some embodiments, the nucleases described herein can include any polypeptide or protein sequence capable of inducing single-stranded or double-stranded DNA breaks. In some embodiments, the nucleases described herein can be wild-type proteins, fusion proteins, engineered proteins, or variants thereof. In some embodiments, the nuclease can be selected from the class of enzymes including 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 nuclease is combined with other GENERIDE™ components to generate targeted integration of a transgene at a target integration site. In some embodiments, the nuclease is combined with other GENERIDE™ components to generate targeted integration of a transgene at a target integration site distal from the nuclease cleavage site.

[0175] TALEN (transcription activator-like effector nuclease) TALEN is a restriction enzyme that contains a DNA-binding domain (transcription activator-like (TAL) effector domain) directly or indirectly fused to a nuclease. TAL effectors (TALEs) contain a set of highly conserved 33-34 amino acid sequence repeats that can be engineered to bind to specific DNA sequences within the cellular genome. Fusion of engineered TALEs to a nuclease cleavage domain (e.g., FokI nuclease) can generate TALE nucleases (TALENs) that can target specific DNA sequences to generate DNA cleavage. Combinations of TALENs (e.g., TALEN pairs) can be used to generate double-strand breaks (DSBs) targeted to DNA sequences.

[0176] In some embodiments, TALENs can be engineered to target specific DNA sequences and induce DSBs. In some embodiments, one or more TALENs (e.g., pairs of TALENs) can be combined to induce DSBs by binding to target DNA sequences on strands in genomic DNA. In some embodiments, specific features of TALENs (e.g., DNA-binding regions, linker regions, nuclease fusion regions) can be designed or optimized to result in improved DNA binding and / or cleavage efficiency. In some embodiments, specific features of TALENs (e.g., DNA-binding regions, linker regions, nuclease fusion regions) can be designed or optimized to reduce off-target binding.

[0177] TALE nickase TALE contains a set of highly conserved 33 - 34 amino acid sequence repeats that can be engineered to bind to specific DNA sequences within the cell genome. Combinations of TALENs (e.g., TALEN pairs) can be used to generate targeted DNA breaks in a DNA sequence. TALE nickases can be designed by introducing a mutation (e.g., D450A) into the cleavage domain (e.g., FokI nuclease) of one of the TALEN monomers in an engineered TALEN (e.g., TALEN pair). TALE nickases can recognize a specific region of a target DNA sequence and generate a targeted single - strand break (SSB) in the DNA sequence.

[0178] In some embodiments, TALE nickases can be engineered to target a specific DNA sequence and induce an SSB. In some embodiments, specific features of TALE nickases (e.g., DNA - binding region, linker region, nuclease fusion region) can be designed or optimized to result in improved DNA - binding and / or cleavage efficiency. In some embodiments, specific features of TALE nickases (e.g., DNA - binding region, linker region, nuclease fusion region) can be designed or optimized to reduce off - target binding.

[0179] ZFN (Zinc Finger Nuclease) ZFN is a restriction enzyme that contains a DNA - binding domain (zinc finger protein (ZFP)) directly or indirectly fused to a nuclease. ZFP contains 3 - 6 individual zinc finger repeats and can recognize a target DNA sequence of 9bp - 18bp. Fusion of an engineered ZFP to a nuclease cleavage domain (e.g., FokI nuclease) can generate a ZFN that can target a specific DNA sequence to generate a DNA cleavage. Combinations of ZFNs (e.g., ZFN pairs) can be used to generate targeted double - strand breaks (DSBs) in a DNA sequence.

[0180] In some embodiments, the ZFN can be engineered to target a specific DNA sequence and induce a DSB. In some embodiments, one or more ZFNs (e.g., a pair of ZFNs) can be combined to induce a DSB by binding to a target DNA sequence on a strand in genomic DNA. In some embodiments, specific features of the ZFN (e.g., the DNA binding region, the linker region, the nuclease fusion region) can be designed or optimized to result in improved DNA binding and / or cleavage efficiency. In some embodiments, specific features of the ZFN (e.g., the DNA binding region, the linker region, the nuclease fusion region) can be designed or optimized to reduce off-target binding.

[0181] Zinc finger nickase ZFP contains 3 - 6 individual zinc finger repeats and can recognize a target DNA sequence of 9bp - 18bp. Combinations of ZFNs (e.g., ZFN pairs) can be used to generate a target DNA cleavage in a DNA sequence. ZF nickase can be designed by introducing a mutation (e.g., D450A) into the cleavage domain (e.g., FokI nuclease) of one ZFN monomer in the engineered ZFN. ZF nickase can recognize a specific region of the target DNA sequence and generate a target SSB in the DNA sequence.

[0182] In some embodiments, the ZF nickase can be engineered to target a specific DNA sequence and induce an SSB. In some embodiments, specific features of the ZF nickase (e.g., the DNA binding region, the linker region, the nuclease fusion region) can be designed or optimized to result in improved DNA binding and / or cleavage efficiency. In some embodiments, specific features of the ZF nickase (e.g., the DNA binding region, the linker region, the nuclease fusion region) can be designed or optimized to reduce off-target binding.

[0183] Meganuclease Meganucleases (also called homing endonucleases) are sequence-specific endonucleases that can recognize target DNA sequences of 12 bp to 40 bp. 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. The re-engineering of these homing endonucleases can generate homing endonucleases that can target specific DNA sequences to generate DNA cleavage. Chimeric proteins, including fusions of meganucleases, ZFPs, and TALs, have been engineered to generate novel enzymes that utilize the binding affinities of ZFs and TALEs and the cleavage specificities of meganucleases.

[0184] In some embodiments, the meganuclease can be engineered to target a specific DNA sequence and induce a DSB. In some embodiments, one or more meganucleases can be combined to induce a DSB by binding to a target DNA sequence on a strand in genomic DNA. In some embodiments, one or more meganucleases can be combined with ZFPs and / or TALs to induce a DSB by binding to a target DNA sequence on a strand in genomic DNA. In some embodiments, certain features of the meganuclease (e.g., the DNA binding region, the linker region, the nuclease fusion region) can be designed or optimized to result in improved DNA binding and / or cleavage efficiency. In some embodiments, certain features of the meganuclease (e.g., the DNA binding region, the linker region, the nuclease fusion region) can be designed or optimized to reduce off-target binding.

[0185] CRISPR-related system CRISPR-related systems typically include a Cas nuclease (e.g., Cas9, Cas13, Cas12a, Cas9 nickase, etc.) or a variant thereof and an engineered guide RNA (gRNA) sequence. The gRNA sequence is designed to have partial complementarity to the target genomic DNA sequence within a certain distance from the protospacer adjacent motif (PAM). The PAM sequence and the distance from the Cas nuclease cleavage site are often specific to a particular enzyme type (e.g., Cas9, Cas13, Cas12a, Cas9 nickase). A specific Cas nuclease or a variant thereof can generate a DSB in the target DNA sequence.

[0186] Cas9 Cas9 is an RNA-guided DNA endonuclease enzyme that acts via base-pair complementarity between the first 17 - 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., NGG or NAG) adjacent to the region of gRNA complementarity. Once bound to the appropriate target sequence, Cas9 induces a DSB in the target DNA sequence. Cas9 nucleases can be found in several different species, including, for example, Streptococcus pyogenes (SpCas9), Staphylococcus aureus, and Neisseria meningitidis. Engineered Cas9 nucleases can result in enhanced cleavage activity or reduced off-target cleavage effects.

[0187] Cas12a / Cpf1 Cas12a (Cpf1) is an RNA-guided DNA endonuclease enzyme that acts via 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) adjacent to the region of gRNA complementarity. Once bound to the appropriate target sequence, Cas12a induces staggered double-strand breaks in the target DNA sequence.

[0188] Cas9 nickase A mutation in one of the two wild-type Cas9 nuclease domains (e.g., D10A and / or H840A) results in a Cas9 variant (Cas9 nickase). Similar to wild-type Cas9, the nickase acts 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, the Cas9 nickase cleaves only one strand of DNA to generate an SSB that can be repaired without inducing indels.

[0189] gRNA (guide RNA) The guide RNA molecule can be, or can include, a nucleic acid that facilitates specific targeting or homing of the gRNA / Cas complex to the target. In some embodiments, the gRNA incorporates the functions and structures of crispr RNA (crRNA) and / or trans-activating crispr RNA (tracrRNA). In some embodiments, the gRNA can be chimeric and can include the features of both crRNA and tracrRNA in a single nucleic acid sequence (e.g., 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 complementarity domain, a linker domain, a second complementarity domain (complementary to the first complementarity domain), a proximal domain, and / or a tail domain.

[0190] In some embodiments, the gRNA molecule can include a targeting domain that includes a nucleic acid sequence that is complementary to the 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 can include a targeting domain that includes a nucleic acid sequence that is complementary to a single strand of the 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 targeting domain can be 5 to 50 nucleotides in length (e.g., 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 35, 35 to 40, 40 to 45, 45 to 50, etc.). In some embodiments, the targeting domain is 16 nucleotides in length. In some embodiments, the targeting domain is 16 nucleotides in length. In some embodiments, the targeting domain is 17 nucleotides in length. In some embodiments, the targeting domain is 18 nucleotides in length. In some embodiments, the targeting domain is 19 nucleotides in length. In some embodiments, the targeting domain is 20 nucleotides in length. In some embodiments, the targeting domain is 21 nucleotides in length. In some embodiments, the targeting domain is 22 nucleotides in length. In some embodiments, the targeting domain is 23 nucleotides in length. In some embodiments, the targeting domain is 24 nucleotides in length. In some embodiments, the targeting domain is 25 nucleotides in length. In some embodiments, the targeting domain is 26 nucleotides in length.

[0191] In some embodiments, the gRNA molecule can be designed to reduce off-targeting within the cellular genome. In some embodiments, the gRNA molecule can be designed by use of software for a particular target DNA sequence that has limited sequence similarity (e.g., homology, identity, etc.) to another region of the genome.

[0192] In some embodiments, the gRNA molecule is designed to target a non-coding DNA sequence (e.g., an intron, untranslated region, enhancer, promoter, silencer or insulator sequence). In some embodiments, the gRNA molecule is designed to target a non-coding sequence within a human gene. In some embodiments, the gRNA molecule is designed to target a non-coding sequence within a human safe harbor gene (e.g., albumin, collagen, actin, CCR5, etc.). In some embodiments, the gRNA molecule is designed to target a non-coding sequence in the human albumin gene. In some embodiments, the gRNA molecule is designed to target intron 13 or intron 14 of the human albumin gene.

[0193] In some embodiments, the gRNA molecule can be designed to target a sequence and / or to include a sequence selected from the sequences of Tables 3 and 4 below. In some examples, the sequences of Table 3 or Table 4 can be the spacer sequences of the gRNA.

[0194]

Table 3

[0195]

Table 4

[0196] Heterologous nucleic acid Payload In some embodiments, one or more vectors or constructs described herein may include a polynucleotide sequence encoding one or more payloads. According to various aspects, any of a variety of payloads may be used alone or in combination (e.g., those having diagnostic and / or therapeutic purposes). In some embodiments, the payload may be or may include a polynucleotide sequence encoding a peptide or polypeptide. In some embodiments, the payload is a peptide having an intracellular or extracellular activity that promotes a biological process for treating a medical condition. In some embodiments, the payload may be or may include a transgene (also referred to herein as a gene of interest (GOI)). In some embodiments, the payload may be or may include one or more inverted terminal repeat (ITR) sequences (e.g., one or more AAV ITRs). In some embodiments, the payload may be or may include one or more transgenes having adjacent ITR sequences. In some embodiments, the payload may be or may include one or more heterologous nucleic acid sequences encoding a reporter gene (e.g., a fluorescent or luminescent reporter). In some embodiments, the payload may be or may include one or more biomarkers (e.g., a proxy for payload expression). In some embodiments, the payload may include a sequence for polycistronic expression (e.g., a 2A peptide or an intron sequence, including an internal ribosome entry site). In some embodiments, the 2A peptide is a small (e.g., about 18 - 22 amino acids) peptide sequence that enables the co-expression of two or more distinct protein products within a single coding sequence. In some embodiments, the 2A peptide enables the co-expression of two or more distinct protein products regardless of the arrangement of the protein coding sequences. In some embodiments, the 2A peptide is a consensus motif (e.g., DVEXNPGP) or includes a consensus motif. In some embodiments, the 2A peptide promotes protein cleavage.In some embodiments, the 2A peptide is or comprises a viral sequence (e.g., foot-and-mouth disease virus (F2A), equine rhinitis A virus, porcine teschovirus-1 (P2A) or Thosea asigna virus (T2A)).

[0197] In some embodiments, the payload can 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, the first nucleic acid sequence encodes a transgene, the second nucleic acid sequence is located 5' or 3' to the first nucleic acid sequence, and promotes the production of two independent gene products (e.g., a sequence encoding a 2A peptide).

[0198] 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 (Tian et al., FurinDB: A Database of 20-Residue Furin Cleavage Site Motifs, Substrates and Their Associated Drugs (2011), Int. J. Mol. Sci., 12, 1060-1065). In some embodiments, the biomarker is or comprises a tag (e.g., an immunological tag). In some embodiments, the payload can comprise one or more functional nucleic acids (e.g., one or more siRNAs or miRNAs). In some embodiments, the payload can comprise one or more inhibitory nucleic acids (e.g., including, inter alia, ribozymes, miRNAs, siRNAs or shRNAs). In some embodiments, the payload can comprise one or more nucleases (e.g., Cas proteins, endonucleases, TALENs, ZFNs).

[0199] In some embodiments, the sequence encoding the 2A peptide can have at least 80%, 85%, 90%, 95%, 99%, or 100% identity with the corresponding wild-type reference nucleotide sequence (e.g., wild-type P2A sequence). In some embodiments, the sequence encoding the P2A peptide can be, or can include, a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity to a portion of the corresponding wild-type reference nucleotide sequence (e.g., wild-type gene sequence). In some embodiments, the sequence encoding the P2A peptide can be, or can include, a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with the exemplary sequences in Table 5 below.

[0200]

Table 5

[0201] Transgene In some embodiments, the transgene is a corrective gene selected to ameliorate one or more signs and / or symptoms of a disease, disorder, or condition. In some embodiments, the transgene can be integrated into the host cell genome by use of a vector included in the present disclosure. In some embodiments, the transgene is a functional version of a disease-related gene (i.e., a gene isoform associated with the expression or exacerbation of a disease, disorder, or condition) found in the host cell. In some embodiments, the transgene is an optimized version (e.g., a codon-optimized variant or an expression-optimized variant) of a disease-related gene found in the host cell. In some embodiments, the transgene is a variant (e.g., a functional gene fragment or a variant thereof) of a disease-related gene found in the host cell. In some embodiments, the transgene is a gene that causes the expression of a peptide that is normally expressed in one or more healthy tissues. In some embodiments, the transgene is a gene that causes the expression of a peptide that is normally expressed in hepatocytes. In some embodiments, the transgene is a gene that causes the expression of a peptide that is normally expressed in muscle cells. In some embodiments, the transgene is a gene that causes the expression of a peptide that is normally expressed in central nervous system cells.

[0202] In some embodiments, the transgene can be, or can include, a gene that causes the expression of a peptide that is not normally expressed in one or more healthy tissues (e.g., a heterologously expressed peptide). In some embodiments, the transgene is a gene that causes the expression of a peptide that is heterologously expressed in one or more healthy tissues (e.g., the liver, muscle, central nervous system (CNS), lung). In some embodiments, the transgene is a gene that causes the expression of a peptide that is heterologously expressed in one or more healthy tissues and that is normally expressed in one or more healthy tissues (e.g., the liver, muscle, central nervous system (CNS), lung).

[0203] In some embodiments, the transgene can be or can include a gene encoding a functional nucleic acid. In some embodiments, the therapeutic agent is or includes an agent having a therapeutic effect on a host cell or subject (including, for example, ribozymes, guide RNAs (gRNAs), antisense oligonucleotides (ASOs), miRNAs, siRNAs, and / or shRNAs). For example, in some embodiments, the therapeutic agent promotes a biological process for treating at least one symptom of a medical condition, such as a disease, disorder, or condition.

[0204] In some embodiments, transgene expression in a subject substantially results from integration at a 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 transgene expression in the subject is derived from transgene 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 transgene expression in the subject is derived from sources other than transgene integration at the target integration site (e.g., episomal expression, integration at non-target integration sites).

[0205] In some embodiments, the transgene is transiently expressed in a 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 in the subject is derived 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 in the subject is derived from sources other than transient expression (e.g., integration at non-target integration sites). In some embodiments, the transgene is transiently expressed in a subject (e.g., episomal expression from a plasmid, minicircle DNA, virus, etc.) for 1 week or more after treatment. In some embodiments, the transgene is transiently expressed in a subject (e.g., episomal expression from a plasmid, minicircle DNA, virus, etc.) for 1 month or more after treatment.

[0206] In some embodiments, the transgene is transiently expressed in a subject (e.g., episomal expression from a plasmid, minicircle DNA, virus, etc.) at a level comparable to the level observed 1 day or several days after treatment, 1 week or several weeks after treatment. In some embodiments, the transgene is transiently expressed in a subject (e.g., episomal expression from a plasmid, minicircle DNA, virus, etc.) at a level comparable to the level observed within 1 day or several days after treatment, 1 month or several months after treatment.

[0207] In some embodiments, the transgene is transiently expressed in a subject (e.g., episomal expression from a plasmid, minicircle DNA, virus, etc.) at a level lower than the level observed 1 day or more after treatment, 1 week or more after treatment. In some embodiments, the transgene is transiently expressed in a subject (e.g., episomal expression from a plasmid, minicircle DNA, virus, etc.) at a level lower than the level observed 1 day or more after treatment, 1 month or more after treatment.

[0208] In some embodiments, the transgene is transiently expressed in a subject (e.g., episomal expression from a plasmid, minicircle DNA, virus, etc.) within 1 month after treatment. In some embodiments, the transgene is transiently expressed in a subject (e.g., episomal expression from a plasmid, minicircle DNA, virus, etc.) within 2 months or less after treatment. In some embodiments, the transgene is transiently expressed in a subject (e.g., episomal expression from a plasmid, minicircle DNA, virus, etc.) within 3 months or less after treatment. In some embodiments, the transgene is transiently expressed in a subject (e.g., episomal expression from a plasmid, minicircle DNA, virus, etc.) within 4 months or less after treatment. In some embodiments, the transgene is transiently expressed in a subject (e.g., episomal expression from a plasmid, minicircle DNA, virus, etc.) within 5 months or less after treatment. In some embodiments, the transgene is transiently expressed in a subject (e.g., episomal expression from a plasmid, minicircle DNA, virus, etc.) within 6 months or less after treatment.

[0209] In some embodiments, the size of the combination of the transgene and the homologous arm is optimized to increase the likelihood that these transgenes are packaged efficiently into the delivery vehicle and to increase the likelihood that the transgenes are ultimately delivered appropriately to the patient.

[0210] 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 particular cell type (e.g., mammalian, insect, bacterial, fungal, 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 particular tissue type (e.g., liver, muscle, CNS, lung).

[0211] In certain embodiments, the nucleotide sequence encoding the transgene can 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%.

[0212] In some embodiments, the transgene can be, or can include, a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with the corresponding wild-type reference nucleotide sequence (e.g., a wild-type gene sequence). In some embodiments, the transgene can be, or can include, a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with a portion of the corresponding wild-type reference nucleotide sequence (e.g., a wild-type gene sequence). In some embodiments, the transgene can be, or can include, a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with the exemplary sequences in Table 6 below.

[0213] [Table 6-1]

[0214] [Table 6-2]

[0215] [Table 6-3]

[0216]

Table 6-4

[0217]

Table 6-5

[0218]

Table 6-6

[0219]

Table 6-7

[0220]

Table 6-8

[0221]

Table 6-9

[0222]

Table 6-10

[0223]

Table 6-11

[0224]

Table 6-12

[0225]

Table 6-13

[0226] Identical arm In some embodiments, the viral vectors described herein include one or more flanking polynucleotide sequences having significant sequence homology to a target integration site (e.g., an identical arm). In some embodiments, the identical arm flanks a polynucleotide sequence encoding a payload (e.g., one identical arm is 5' to the payload (also referred to herein as the 5' identical arm) and one identical arm is 3' to the payload (also referred to herein as the 3' identical arm)). In some embodiments, the identical arm directs site-specific integration of the payload.

[0227] In some embodiments, the homologous arms are of the same length (also referred to herein as balanced homologous arms or homologous arms). In some embodiments, a viral vector that includes homologous arms of the same length and the homologous arms are at least a certain length provides an improved effect (e.g., an improved target integration rate). In some embodiments, the homologous arms are 50 nt to 1600 nt in length. In some embodiments, the homologous arms are 100 nt to 1000 nt in length. In some embodiments, the homologous arms are 200 nt to 1000 nt in length. In some embodiments, the homologous arms are 500 nt to 1500 nt in length. In some embodiments, the homologous arms are 1000 nt to 2000 nt in length. In some embodiments, the homologous arms are greater than 2000 nt in length. In some embodiments, each homologous arm is at least 50 nt in length. In some embodiments, each homologous arm is at least 750 nt in length. In some embodiments, each homologous arm is at least 1000 nt in length. In some embodiments, each homologous arm is at least 1250 nt in length. In some embodiments, the homologous arms are less than 1000 nt in length. In some embodiments, the homologous arms include at least 70% homology to the target integration site. In some embodiments, the homologous arms include at least 80% homology to the target integration site. In some embodiments, the homologous arms include at least 90% homology to the target integration site. In some embodiments, the homologous arms include at least 95% homology to the target integration site. In some embodiments, the homologous arms include at least 99% homology to the target integration site. In some embodiments, the homologous arms include 100% homology to the target integration site.

[0228] In some embodiments, the homologous arms are of different lengths (also referred to herein as unbalanced homologous arms or non-uniform homologous arms). In some embodiments, a viral vector comprising unbalanced homologous arms of different lengths provides an improved effect (e.g., an increase in the rate of target site integration) compared to an appropriate reference sequence. In some embodiments, a viral vector comprising homologous arms of different lengths, each homologous arm being at least a certain length, provides an improved effect (e.g., an increase in the rate of target site integration) compared to an appropriate reference sequence (e.g., a viral vector comprising homologous arms of the same length, or a viral vector comprising one or more homologous arms of a length less than 1000 nt).

[0229] In some embodiments, each homologous arm is longer than 50 nt. In some embodiments, each homologous arm is longer than 100 nt. In some embodiments, each homologous arm is longer than 400 nt. In some embodiments, each homologous arm is at least 750 nt in length. In some embodiments, each homologous arm is at least 1000 nt in length. In some embodiments, one homologous arm is at least 750 nt in length and the other homologous arm is at least 1000 nt in length. In some embodiments, one homologous arm is at least 750 nt in length and the other homologous arm is at least 1100 nt in length. In some embodiments, one homologous arm is at least 750 nt in length and the other homologous arm is at least 1200 nt in length. In some embodiments, one homologous arm is at least 750 nt in length and the other homologous arm is at least 1300 nt in length. In some embodiments, one homologous arm is at least 750 nt in length and the other homologous arm is at least 1400 nt in length. In some embodiments, one homologous arm is at least 750 nt in length and the other homologous arm is at least 1500 nt in length. In some embodiments, one homologous arm is at least 750 nt in length and the other homologous arm is at least 1600 nt in length. In some embodiments, one homologous arm is at least 750 nt in length and the other homologous arm is at least 1700 nt in length. In some embodiments, one homologous arm is at least 750 nt in length and the other homologous arm is at least 1800 nt in length. In some embodiments, one homologous arm is at least 750 nt in length and the other homologous arm is at least 1900 nt in length. In some embodiments, one homologous arm is at least 750 nt in length and the other homologous arm is at least 2000 nt in length. In some embodiments, one homologous arm is at least 1000 nt in length and the other homologous arm is at least 1100 nt in length.In some embodiments, one of the homologous arms is at least 1000 nt in length and the other homologous arm is at least 1200 nt in length. In some embodiments, one of the homologous arms is at least 1000 nt in length and the other homologous arm is at least 1300 nt in length. In some embodiments, one of the homologous arms is at least 1000 nt in length and the other homologous arm is at least 1400 nt in length. In some embodiments, one of the homologous arms is at least 1000 nt in length and the other homologous arm is at least 1500 nt in length. In some embodiments, one of the homologous arms is at least 1000 nt in length and the other homologous arm is at least 1600 nt in length. In some embodiments, one of the homologous arms is at least 1000 nt in length and the other homologous arm is at least 1700 nt in length. In some embodiments, one of the homologous arms is at least 1000 nt in length and the other homologous arm is at least 1800 nt in length. In some embodiments, one of the homologous arms is at least 1000 nt in length and the other homologous arm is at least 1900 nt in length. In some embodiments, one of the homologous arms is at least 1000 nt in length and the other homologous arm is at least 2000 nt in length. In some embodiments, one of the homologous arms is at least 1300 nt in length and the other homologous arm is at least 1400 nt in length. In some embodiments, one of the homologous arms is at least 1600 nt in length and the other homologous arm is at least 1000 nt in length. In some embodiments, one of the homologous arms is at least 1250 nt in length and the other homologous arm is at least 1250 nt in length. In some embodiments, one of the homologous arms is at least 400 nt in length and the other homologous arm is at least 800 nt in length. In some embodiments, one of the homologous arms is at least 600 nt in length and the other homologous arm is at least 600 nt in length.

[0230] In some embodiments, the 5' homologous arm is longer than the 3' homologous arm. In some embodiments, the 3' homologous arm is longer than the 5' homologous arm. For example, in some embodiments, the 5' homologous arm is about 1600 nt in length and the 3' homologous arm is about 1000 nt in length. In some embodiments, the 5' homologous arm is about 1000 nt in length and the 3' homologous arm is about 1600 nt in length. In some embodiments, a viral vector comprising a homologous arm provides an improved effect (e.g., an increased rate of target site integration) compared to a suitable reference sequence (e.g., a viral vector lacking the homologous arm). In some embodiments, a viral vector comprising a homologous 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, a viral vector comprising a homologous arm increases the target site integration rate over time. In some embodiments, the rate of target site integration increases over time compared to an initial measurement of target site integration. In some embodiments, the rate of target site integration over time is at least 1.5 times higher (e.g., 1.5 times, 2 times, 3 times, 4 times, 5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 200 times) than the initial measurement of target site integration. In some embodiments, the target site integration rate is measured 1 day or multiple days later. In some embodiments, the target site integration rate is measured 1 week or multiple weeks later. In some embodiments, the target site integration rate is measured 1 month or multiple months later. In some embodiments, the target site integration rate is measured 1 year or multiple years later.

[0231] In some embodiments, a viral vector comprising homology arms of different lengths provides an improved effect (e.g., an increased rate of target site integration) compared to a reference sequence (e.g., a viral vector having homology arms of the same length, a viral vector having at least one homology arm less than 500 nt). In some embodiments, a viral vector comprising homology arms of different lengths provides an improvement in editing activity of at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2.0-fold, at least 2.5-fold, at least 3.0-fold, at least 3.5-fold, or at least 4.0-fold compared to a reference composition (e.g., a viral vector having homology arms of the same length, a viral vector having at least one homology arm less than 500 nt).

[0232] In some embodiments, a viral vector comprising homology arms of different lengths 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, a viral vector comprising homology arms of different lengths increases the target site integration rate over time. In some embodiments, the rate of target site integration increases over time compared to an initial measurement of target site integration. In some embodiments, the rate of target site integration over time is at least 1.5-fold (e.g., 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold) higher than the initial measurement value of target site integration.

[0233] In some embodiments, viral vectors comprising homologous arms of different lengths can provide improved gene editing in a species or species model system (e.g., mouse, human, or a model thereof). In some embodiments, a viral vector can include different combinations of homologous arm lengths when optimized for expression in a particular species or particular species model system (e.g., mouse, human, or a model thereof). In some embodiments, a viral vector comprising a particular combination of homologous arm lengths can provide improved gene editing in one species or one species model system (e.g., human, humanized mouse model) as compared to a second species or second species model system (e.g., mouse, pure mouse model). In some embodiments, a viral vector comprising a particular combination of homologous arm lengths can be optimized for high-level gene editing in one species or one species model (e.g., human, humanized mouse model) as compared to a second species or second species model system (e.g., mouse, pure mouse model).

[0234] In some embodiments, the homologous arms direct integration of the transgene immediately downstream of a highly expressed endogenous gene. In some embodiments, the homologous arms direct integration of the transgene without disrupting endogenous gene expression (non-disruptive integration).

[0235] In some embodiments, one or more homologous arm sequences can have at least 80%, 85%, 90%, 95%, 99%, or 100% identity with a corresponding wild-type reference nucleotide sequence (e.g., wild-type genomic sequence). In some embodiments, one or more homologous arm sequences can be, or can include, a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity to a portion of a corresponding wild-type reference nucleotide sequence (e.g., wild-type genomic sequence). In some embodiments, one or more homologous arm sequences can be, or can include, a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity to an exemplary sequence in Table 7 below. In some embodiments, the compositions described herein can include one or more homologous arm sequences selected from Table 7 below.

[0236]

Table 7-1

[0237]

Table 7-2

[0238]

Table 7-3

[0239] In some embodiments, the viral vectors provided herein may include a 5' homology arm and a 3' homology arm designed to target the albumin locus. In some embodiments, the viral vectors provided herein have 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 SEQ ID NO: 62 for the 5' homology arm sequence and 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 SEQ ID NO: 67 for the 3' homology arm sequence. In some embodiments, the viral vector includes 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 may include 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 with 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 with SEQ ID NO: 66. In some embodiments, the viral vector includes 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 may include 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 includes 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 may include 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 includes 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 may include 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.

[0243] Measurement of Target Site Integration As described elsewhere in this specification, one of the problems associated with the conventional use of nucleases to introduce nucleic acid materials into cells is the high potential for off-target integration. Thus, as described below, it is important to verify correct integration by one or more specific target assays.

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

[0245]

Table 8

[0246] Therapeutic methods The compositions and constructs disclosed herein can be used for any in vitro or in vivo application to cause or enhance the expression of a payload (e.g., a transgene) from a specific target integration site within a cell while maintaining the expression of the 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., via 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 completely or partially preventing a disease (e.g., preventing the symptoms of a disease). In some embodiments, the desired pharmacological and / or physiological effect may include completely or partially curing a disease (e.g., curing the detrimental effects associated with a disease). In some embodiments, the desired pharmacological and / or physiological effect may include preventing recurrence of a disease. In some embodiments, the desired pharmacological and / or physiological effect may include slowing the progression of a disease. In some embodiments, the desired pharmacological and / or physiological effect may include reducing the symptoms of a disease. In some embodiments, the desired pharmacological and / or physiological effect may include preventing regression of a disease. In some embodiments, the desired pharmacological and / or physiological effect may include stabilizing and / or reducing the symptoms associated with a disease.

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

[0249] Disease of interest 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] As a specific example, in some embodiments, the compositions and constructs disclosed herein can be used to treat branched-chain organic acidurias (e.g., maple syrup urine disease (MSUD), methylmalonic acidemia (MMA), propionic acidemia (PA), isovaleric acidemia (IVA), argininosuccinic aciduria). In some embodiments, the treatment involves the introduction of a polynucleotide sequence encoding one or more therapeutic transgenes (e.g., the 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, the treatment involves the reduction of abnormal proteins (e.g., non-functional proteins) associated with branched-chain organic acidurias. In some embodiments, the treatment involves the alleviation of signs and / or symptoms associated with branched-chain organic acidurias (e.g., hypotension, developmental delay, seizures, optic atrophy, acute encephalopathy, hyperventilation, respiratory distress, temperature instability, recurrent vomiting, ketoacidosis, pancreatitis, constipation, neutropenia, pancytopenia, secondary hemophagocytosis, cardiac arrhythmia, 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, the treatment includes the introduction of a polynucleotide sequence encoding one or more target transgenes (e.g., HADHA, HADHB, LCHAD, ACADM, ACADVL, and / or variants thereof). In some embodiments, the treatment includes the reduction of abnormal proteins (e.g., non-functional proteins) associated with fatty acid oxidation disorders. In some embodiments, the treatment includes the alleviation of symptoms and / or signs associated with fatty acid oxidation disorders (e.g., liver enlargement, delayed mental and physical development, myocardial weakness, cardiac arrhythmia, nerve damage, abnormal liver function, rhabdomyolysis, myoglobinuria, hypoglycemia, metabolic acidosis, respiratory distress, hepatomegaly, hypotension, 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, the treatment includes the introduction of a polynucleotide sequence encoding one or more target transgenes (e.g., G6PC (GSD1a), G6PT1 (GSD1b), SLC17A3, SLC37A4 (GSD1c), acid alpha-glucosidase, and / or variants thereof). In some embodiments, the treatment includes the reduction of abnormal proteins (e.g., non-functional proteins) associated with glycogen storage diseases. In some embodiments, the treatment includes the alleviation of symptoms and / or signs associated with glycogen storage disorders (e.g., hepatomegaly, hypoglycemia, muscle weakness, muscle cramps, fatigue, developmental delay, obesity, bleeding disorders, abnormal liver function, abnormal kidney function, abnormal respiratory function, abnormal cardiac function, oral prolapse, 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, the treatment includes the introduction of a polynucleotide sequence encoding one or more target transgenes (e.g., OCTN2, CPT1, CACT, CPT2, and / or variants thereof). In some embodiments, the treatment includes the reduction of abnormal proteins (e.g., non-functional proteins) associated with carnitine cycle disorders. In some embodiments, the treatment includes the alleviation of signs and / or symptoms associated with carnitine cycle disorders (e.g., hypoketotic hypoglycemia, cardiomyopathy, muscle weakness, fatigue, delayed motor development, edema).

[0254] In some embodiments, the compositions and constructs disclosed herein can be used to treat urea cycle disorders. In some embodiments, the treatment includes the introduction of a polynucleotide sequence encoding one or more target transgenes (e.g., CPS1, ARG1, ASL, OTC, and / or variants thereof). In some embodiments, the treatment includes the reduction of abnormal proteins (e.g., non-functional proteins) associated with urea cycle disorders. In some embodiments, the treatment includes the alleviation of signs and / or symptoms associated with urea cycle disorders (e.g., vomiting, nausea, behavioral abnormalities, fatigue, lethargy, psychosis, somnolence, periodic 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, the 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, the treatment includes a reduction in abnormal proteins (e.g., non-functional proteins) associated with HCU. In some embodiments, the treatment includes the alleviation of signs and / or symptoms associated with HCU (e.g., ectopia lentis, myopia, iridodonesis, cataracts, optic atrophy, glaucoma, retinal detachment, retinal damage, indicators of developmental delay, intellectual disability, depression, anxiety, obsessive-compulsive disorder, dolichostenomelia, genu valgum, pes cavus, scoliosis, temporal muscle, pectus excavatum, osteoporosis, increased blood clot formation, thromboembolism, pulmonary embolism, fragile skin, hypopigmentation, 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, the treatment includes the introduction of a polynucleotide sequence encoding one or more target transgenes (e.g., UGT1A1 and / or variants thereof). In some embodiments, the treatment includes a reduction in abnormal proteins (e.g., non-functional proteins) associated with Crigler-Najjar syndrome. In some embodiments, the treatment includes the alleviation of signs and / or symptoms associated with Crigler-Najjar syndrome (e.g., jaundice, corneal clouding, lethargy, vomiting, fever, abnormal reflexes, muscle contractions, opisthotonus, convulsions, hypotonia, athetoid movements, elevated bilirubin levels, diarrhea, slurred speech, disorientation, dysphagia, seizures).

[0257] In some embodiments, the compositions and constructs disclosed herein can be used to treat hereditary tyrosinemia. In some embodiments, the treatment comprises the introduction of a polynucleotide sequence encoding one or more transgenes of interest (e.g., FAH and / or variants thereof). In some embodiments, the treatment comprises the reduction of abnormal proteins (e.g., non-functional proteins) associated with hereditary tyrosinemia. In some embodiments, the treatment comprises the alleviation of signs and / or symptoms associated with hereditary tyrosinemia (e.g., hepatomegaly, jaundice, liver disease, cirrhosis, hepatocellular carcinoma, fever, diarrhea, hematochezia, vomiting, splenomegaly, edema, coagulation disorder, renal dysfunction, kuru disease, weakness, muscle hypertonia, ileum, tachycardia, hypertension, neurological seizures, respiratory failure, cardiomyopathy).

[0258] In some embodiments, the compositions and constructs disclosed herein can be used to treat epidermolysis bullosa. In some embodiments, the treatment comprises the introduction of 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, the treatment comprises the reduction of abnormal proteins (e.g., non-functional proteins) associated with epidermolysis bullosa. In some embodiments, the treatment comprises the alleviation of signs and / or symptoms associated with epidermolysis bullosa (e.g., fragile skin, abnormal nail growth, blisters, thickened skin, cicatricial alopecia, atrophic scars, milia, dental problems, dysphagia, skin pruritus 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 includes the introduction of a polynucleotide sequence encoding one or more transgenes of interest (e.g., alpha-1 antitrypsin (A1AT) and / or variants thereof). In some embodiments, treatment includes the reduction of abnormal proteins (e.g., non-functional proteins) associated with alpha-1 antitrypsin deficiency. In some embodiments, treatment includes the alleviation of symptoms and / or signs associated with A1ATD (e.g., emphysema, chronic cough, sputum production, wheezing, chronic respiratory infections, jaundice, liver enlargement, bleeding, abnormal fluid retention, elevated liver enzymes, liver dysfunction, portal hypertension, fatigue, edema, chronic active hepatitis, cirrhosis, hepatocellular carcinoma, sebaceous gland inflammation).

[0260] In some embodiments, the compositions and constructs disclosed herein can be used to treat Wilson's disease. In some embodiments, treatment includes the introduction of a polynucleotide sequence encoding one or more transgenes of interest (e.g., ATP7B and / or variants thereof). In some embodiments, treatment includes the reduction of abnormal proteins (e.g., non-functional proteins) associated with Wilson's disease. In some embodiments, treatment includes the reduction of symptoms and / or signs associated with Wilson's disease (e.g., fatigue, loss of appetite, abdominal pain, jaundice, Kayser-Fleischer ring, edema, speech problems, swallowing problems, loss of physical coordination, uncontrolled movements, muscle rigidity, liver disease, anemia, depression, dystonia, menstrual difficulties, infertility, kidney stones, renal tubular damage, arthritis, osteoporosis, bone spurs).

[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, the treatment includes the introduction of a polynucleotide sequence encoding one or more target transgenes (e.g., factor IX (FIX), factor VIII (FVIII), and / or variants thereof). In some embodiments, the treatment includes the reduction of abnormal proteins (e.g., non-functional proteins) associated with the blood disease. In some embodiments, the treatment includes the alleviation of signs and / or symptoms associated with the blood disease (e.g., excessive bleeding, abnormal bruising, joint pain and swelling, bloody urine, bloody stools, abnormal nosebleeds, headache, drowsiness, vomiting, double vision, weakness, seizures, fainting).

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

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

[0264] In some embodiments, the compositions and constructs disclosed herein can be used to treat muscle diseases. In some embodiments, the treatment includes the introduction of a polynucleotide sequence encoding one or more therapeutic transgenes (e.g., for muscular dystrophy, Duchenne muscular dystrophy (DMD), limb-girdle muscular dystrophy, X-linked myotubular myopathy). In some embodiments, the treatment includes the reduction of abnormal proteins (e.g., non-functional proteins) associated with muscle diseases. In some embodiments, the treatment includes the alleviation of symptoms and / or signs associated with muscle diseases (e.g., difficulty in movement, hypertrophy of calf muscles, muscle pain and stiffness, developmental delay, learning disabilities, abnormal gait, scoliosis, respiratory disorders, dysphagia, arrhythmia, cardiomyopathy, abnormal joint function, hypotonia, respiratory distress, lack of reflex).

[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, the treatment includes the introduction of a polynucleotide sequence encoding one or more therapeutic transgenes (e.g., IDUA, IDS, SGSH, NAGLU, HGSNAT, GNS, GALNS, GLB1, ARSB, GUSB, HYAL1). In some embodiments, the treatment includes the reduction of abnormal proteins (e.g., non-functional proteins) associated with MPS. In some embodiments, the treatment includes the alleviation of symptoms and / or signs associated with MPS (e.g., cardiac abnormalities, respiratory irregularities, hepatomegaly, splenomegaly, neurological abnormalities, developmental delay, recurrent infections, persistent nasal discharge, noisy breathing, corneal clouding, macroglossia, spinal deformities, joint stiffness, carpal tunnel, aortic valve regurgitation, progressive hearing loss, seizures, unsteady gait, heparan sulfate accumulation, enzyme deficiency, skeletal and muscle tissue abnormalities, heart disease, cysts, soft tissue masses).

[0266] In some embodiments, the compositions and constructs disclosed herein can be used to treat lysosomal acid lipase deficiency. In some embodiments, the treatment comprises introduction of a polynucleotide sequence encoding one or more desired transgenes (e.g., LIPA and / or variants thereof). In some embodiments, the treatment comprises reduction of an abnormal protein (e.g., a non-functional protein) associated with lysosomal acid lipase deficiency. In some embodiments, the treatment comprises alleviation of symptoms and / or signs associated with lysosomal acid lipase deficiency (e.g., vomiting, diarrhea, abdominal distension, and failure to thrive, weight loss, jaundice, fever, calcification, anemia, liver dysfunction or failure, cachexia, malabsorption, biliary 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, the treatment comprises introduction of a polynucleotide sequence encoding one or more desired transgenes (e.g., PFIC1, PFIC2, PFIC3, ABCB4, and / or variants thereof). In some embodiments, the treatment comprises reduction of an abnormal protein (e.g., a non-functional protein) associated with bile acid metabolism, transport, and / or cholestasis. In some embodiments, the treatment comprises alleviation of symptoms and / or signs associated with bile acid metabolism, bile transport, and / or cholestasis (e.g., pruritus, jaundice, failure to thrive, 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, the treatment comprises the introduction of a polynucleotide sequence encoding one or more target transgenes (e.g., phenylalanine hydroxylase (PAH) and / or variants thereof). In some embodiments, the treatment comprises the reduction of an abnormal protein (e.g., a non-functional protein) associated with phenylketonuria. In some embodiments, the treatment comprises the reduction of signs and / or symptoms associated with phenylketonuria (e.g., musty odor in breath, skin, and / or urine, seizures, skin rash, microcephaly, hyperactivity, intellectual disability, asthma, eczema, anemia, weight gain, renal insufficiency, osteoporosis, gastritis, esophageal and renal deficiencies, kidney stones, hypertension, psychiatric problems, dizziness).

[0269] In some embodiments, the compositions and constructs disclosed herein can be used to treat primary hyperoxaluria. In some embodiments, the treatment comprises the introduction of a polynucleotide sequence encoding one or more target transgenes (e.g., AGT, AGXT, GRHPR, HOGA1, and / or variants thereof). In some embodiments, the treatment comprises the reduction of an abnormal protein (e.g., a non-functional protein) associated with primary hyperoxaluria. In some embodiments, the treatment comprises the reduction of signs and / or symptoms associated with phenylketonuria (e.g., flank pain, oxalosis, kidney stones and / or stones in other locations of the urinary tract such as the bladder or urethra, nephrocalcinosis, hematuria, dysuria, frequent urge to urinate, renal colic, urinary tract obstruction, recurrent urinary tract infections, kidney damage, renal failure, hypoplasia).

[0270] In some embodiments, the compositions and constructs disclosed herein can be used to treat porphyria. In some embodiments, the treatment includes the introduction of 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, the treatment includes a reduction in an abnormal protein (e.g., a non-functional protein) associated with porphyria. In some embodiments, the treatment includes a reduction in the signs and / or symptoms associated with porphyria (e.g., abdominal pain, pain in the arms and legs, general weakness, vomiting, confusion, constipation, rapid pulse, fluctuating blood pressure, urinary retention, psychosis, hallucinations, seizures, abrasions, blisters, skin erosions, skin lesions, nausea, elevated blood pressure, confusion).

[0271] In some embodiments, the compositions and constructs disclosed herein can be used to treat disorders associated with the production of antibodies (e.g., autoimmune disorders). In some embodiments, the treatment includes the introduction of a polynucleotide sequence 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, the treatment includes a reduction in an abnormal protein (e.g., a non-functional protein) associated with the production of antibodies. In some embodiments, the treatment includes a reduction in the signs and / or symptoms associated with the production of antibodies (e.g., joint swelling, joint stiffness, fatigue, fever, loss of appetite, vision problems, tremors, unsteady gait, dizziness, skin rash, lesions, hyperalgesia).

[0272] In some embodiments, the compositions and constructs disclosed herein can be used to treat disorders associated with the production of secreted proteins. In some embodiments, the treatment includes the introduction of a polynucleotide sequence encoding one or more transgenes of interest. In some embodiments, the treatment includes the reduction of abnormal proteins (e.g., non-functional proteins) associated with the production of secreted proteins. In some embodiments, the treatment includes the alleviation of signs and / or symptoms associated with the production of secreted proteins.

[0273] Targeted integration In some embodiments, the compositions and constructs provided herein direct the integration of a payload (e.g., a transgene 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 direct the integration of a payload (e.g., a transgene and / or a functional nucleic acid) at a target integration site (e.g., an endogenous gene) that is found only in a particular tissue. In some embodiments, the compositions and constructs provided herein direct the integration of a payload (e.g., a transgene and / or a functional nucleic acid) at a target integration site (e.g., an endogenous gene) that is found in cells present in two or more tissues of a subject (e.g., 2, 3, 4, 5, or all tissues). In some embodiments, the compositions and constructs provided herein direct the integration of a payload (e.g., a transgene and / or a functional nucleic acid) at a target integration site (e.g., an endogenous gene) that enables inducible expression of the payload. In some embodiments, inducible expression can be controlled via artificial means (e.g., administration of a drug or other exogenous signal) 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 payload at a target integration site of a particular cell type (e.g., a tissue-specific locus). In some embodiments, payload integration occurs in a particular tissue (e.g., liver, central nervous system (CNS), muscle, kidney, vasculature, lung). In some embodiments, payload integration occurs in multiple tissues (e.g., liver, central nervous system (CNS), muscle, kidney, vasculature, lung).

[0274] In some embodiments, the compositions and constructs provided herein direct the integration of a payload at a target integration site that may be considered a safe harbor site (e.g., albumin, apolipoprotein A2 (ApoA2), haptoglobin, IgH (e.g., B cells), β-2 microglobulin, 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 not suffering from a disease, disorder, or condition, or a subject suffering 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., genes encoding a polypeptide). In some embodiments, integration of the payload occurs between the 5' and 3' ends of one or more endogenous genes (e.g., genes encoding a polypeptide).

[0275] In some embodiments, the compositions and constructs provided herein direct the integration of a payload at a target integration site with minimal or no off-target integration (e.g., integration at non-target loci). In some embodiments, the compositions and constructs provided herein direct the integration of a payload at a target integration site with reduced off-target integration compared to a reference composition or construct (e.g., compared to a composition or construct having no adjacent homologous sequences).

[0276] In some embodiments, integration of the transgene at the target integration site enables expression of the payload without disrupting endogenous gene expression. In some embodiments, integration of the transgene into the target integration site enables expression of the payload from an endogenous promoter. In some embodiments, integration of the transgene at the target integration site disrupts endogenous gene expression. In some embodiments, integration of the transgene at the target integration site disrupts endogenous gene expression without adversely affecting the target cell and / or subject (e.g., by targeting a safe harbor site). In some embodiments, integration of the transgene at the target integration site does not require the use of a nuclease (e.g., a Cas nuclease, TALEN, ZFN). In some embodiments, integration of the transgene at the target integration site is assisted by the use of a nuclease (e.g., a Cas nuclease, TALEN, ZFN).

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

[0278] Cleavage site In some embodiments, integration of the transgene at the target integration site is enhanced by the use of a nuclease (e.g., Cas nuclease, TALEN, ZFN) that targets a specific cleavage site for single-stranded or double-stranded DNA cleavage. In some embodiments, integration of the transgene at the target integration site is enhanced by the use of a nuclease that targets a cleavage site in a DNA sequence distal to the target integration site. In some embodiments, integration of the transgene at the target integration site is assisted by the use of a nuclease that targets a cleavage site in a 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 the use of a nuclease that targets a cleavage site in a 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 the use of a nuclease that targets a cleavage site in a DNA sequence that is 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., intron, untranslated region, enhancer, promoter, silencer or 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 at 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, cleavage sites within one or more non-coding sequences of a human gene (e.g., introns 13 and 14 of the albumin gene) may provide improved transgene integration when combined with a GENERIDE™ component and one or more suitable nucleases. In some embodiments, the selection of the cleavage site can be based on the predicted off-target effects (e.g., reduction of off-target effects) by using external databases or software prediction / calculation 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 within a safe harbor locus to reduce the possible impact of disruptive integration.

[0281] Composition In some embodiments, the composition can be manufactured using the methods and constructs (e.g., viral vectors) provided herein. In some embodiments, the composition includes liquid, solid, and gaseous compositions. In some embodiments, the composition includes additional components (e.g., diluents, stabilizers, excipients, adjuvants). In some embodiments, the additional components include, among other things, 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., polyvinylpyrrolidone), amino acids (e.g., glycine, glutamine, asparagine, arginine, lysine), carbohydrates (e.g., monosaccharides, disaccharides, glucose, mannose, dextrin), 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™, Pluronics™, polyethylene glycol (PEG)). In some embodiments, the aqueous carrier is a pH buffered aqueous solution.

[0282] In some embodiments, the compositions provided herein can be provided at a range of dosages. In some embodiments, the compositions provided herein can be provided as a single dosage. In some embodiments, the compositions provided herein can be provided in multiple dosages. In some embodiments, the composition is provided over a period of time. In some embodiments, the composition is provided at specific intervals (e.g., variable intervals, set intervals). In some embodiments, the dosage can vary depending on the dosage form and route of administration. In some embodiments, the compositions provided herein can be provided at a dosage of 1e11 to 1e14 vg / kg. In some embodiments, the compositions provided herein can be provided at a dosage of 1e12 to 1e13 vg / kg. In some embodiments, the compositions provided herein can be provided at a dosage of 1e12 to 1e14 vg / kg. In some embodiments, the compositions provided herein can be provided at a dosage of 1e14 to 1e15 vg / kg. In some embodiments, the compositions provided herein can be provided at a dosage of 1e14 vg / kg or less. In some embodiments, the compositions provided herein can be provided at a dosage of 1e15 vg / kg or less.

[0283] In some embodiments, one of ordinary skill in the art can devise appropriate dosage levels and regimens using the pharmaceutical compositions described herein to treat various conditions of various patients. For example, in some embodiments, the dosage selected depends on the desired therapeutic effect, the route of administration, and the desired duration of treatment. In some embodiments, dosage levels of nucleic acid from about 0.001 mg to about 6 mg per kg of body weight are administered to a subject (e.g., an animal, a human) during each administration. In some embodiments, the dosage level of nucleic acid within the disclosed lipid nanoparticles is from about 0.1 mg / kg to about 1.0 mg / kg. In some embodiments, the dosage level of nucleic acid within the disclosed lipid nanoparticles is from about 0.1 mg / kg to about 3.0 mg / kg. In some embodiments, the dosage level of the disclosed lipid nanoparticles is about 0.2 mg to about 100 mg per kg of body weight of one or more total components (e.g., ionizable lipid, sterol, conjugate linker lipid, phospholipid) administered 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 one or more total components (e.g., ionizable lipid, sterol, conjugate linker lipid, phospholipid) per kg of body weight administered to a subject.

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

[0285] In some embodiments, the compositions provided herein can be administered to a subject at a particular 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 a 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 a tissue or organ is about 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 a tissue or organ is about 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 a tissue or organ is about 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, which are incorporated herein by reference in their entirety).

[0286] In some embodiments, the compositions described herein include (i) a nuclease or polynucleotide sequence encoding a nuclease that can induce double-strand breaks and / or single-strand breaks at a cleavage site distal from the target integration site, and (ii) 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 the sequence of Table 6 (e.g., SEQ ID NO: 49), a second nucleic acid sequence located 5' or 3' to the 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 the sequences of Table 5 (e.g., SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48), an expression cassette, a third nucleic acid sequence located 5' to the expression cassette and including 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 to the sequences of Table 7 (SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 68, or SEQ ID NO: 70), or a fourth nucleic acid sequence located 3' to the expression cassette and including 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 to the sequences of Table 7 (e.g., SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, or SEQ ID NO: 69), a polynucleotide cassette. In some embodiments, (i) and (ii) can be administered as separate compositions.

[0287] In some embodiments, the compositions described herein comprise: (i) a nuclease or a polynucleotide sequence encoding a nuclease that can induce double-strand breaks and / or single-strand breaks at a cleavage site distal to the target integration site; and (ii) 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 the sequence of Table 6 (e.g., SEQ ID NO: 50), a second nucleic acid sequence located in a 5' or 3' sequence relative to the 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 the sequences of Table 5 (e.g., SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48), an expression cassette comprising the first nucleic acid sequence, a third nucleic acid sequence located 5' to 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 to the sequences in Table 7 (SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 68, or SEQ ID NO: 70), or a fourth nucleic acid sequence located 3' to 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 to the sequences in Table 7 (e.g., SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, or SEQ ID NO: 69), a polynucleotide cassette. In some embodiments, (i) and (ii) can be administered as separate compositions.

[0288] In some embodiments, the compositions described herein 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 the target integration site; and (ii) 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 the sequence of Table 6 (e.g., SEQ ID NO: 51), a second nucleic acid sequence located 5' or 3' to the 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 the sequences of Table 5 (e.g., SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48), an expression cassette; a third nucleic acid sequence located 5' to 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 to the sequences of Table 7 (SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 68, or SEQ ID NO: 70), or a fourth nucleic acid sequence located 3' to 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 to the sequences of Table 7 (e.g., SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, or SEQ ID NO: 69), a polynucleotide cassette. In some embodiments, (i) and (ii) can be administered as separate compositions.

[0289] In some embodiments, the compositions described herein include: (i) a nuclease or a polynucleotide sequence encoding a nuclease that can induce double-strand breaks and / or single-strand breaks at a cleavage site distal to the target integration site; and (ii) 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 the sequence of Table 6 (e.g., SEQ ID NO: 52), a second nucleic acid sequence located 5' or 3' to the 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 the sequences of Table 5 (e.g., SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48), an expression cassette; a third nucleic acid sequence located 5' to the expression cassette and 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 the sequences of Table 7 (SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 68, or SEQ ID NO: 70), or a fourth nucleic acid sequence located 3' to the expression cassette and 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 the sequences of Table 7 (e.g., SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, or SEQ ID NO: 69), a polynucleotide cassette. In some embodiments, (i) and (ii) can be administered as separate compositions.

[0290] In some embodiments, the compositions described herein comprise: (i) a nuclease or a polynucleotide sequence encoding a nuclease that can induce double-strand breaks and / or single-strand breaks at a cleavage site distal to the target integration site; and (ii) 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 the sequence of Table 6 (e.g., SEQ ID NO: 53), a second nucleic acid sequence located in the 5' or 3' sequence relative to the 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 the sequences of Table 5 (e.g., SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48), an expression cassette; a third nucleic acid sequence located 5' to 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 to the sequences of Table 7 (SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 68, or SEQ ID NO: 70), or a fourth nucleic acid sequence located 3' to 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 to the sequences of 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, the compositions described herein 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 from a target integration site; and (ii) 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 the sequence of Table 6 (e.g., SEQ ID NO: 54), a second nucleic acid sequence located in a sequence 5' or 3' to the 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 the sequences of Table 5 (e.g., SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48), an expression cassette; a third nucleic acid sequence located 5' to 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 to the sequences of Table 7 (SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 68, or SEQ ID NO: 70), or a fourth nucleic acid sequence located 3' to 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 to the sequences of Table 7 (e.g., SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, or SEQ ID NO: 69), a polynucleotide cassette. In some embodiments, (i) and (ii) can be administered as separate compositions.

[0292] In some embodiments, the compositions described herein 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 from the target integration site; and (ii) 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 the sequence of Table 6 (e.g., SEQ ID NO: 55), a second nucleic acid sequence located 5' or 3' to the 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 the sequences of Table 5 (e.g., SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48), an expression cassette comprising the second nucleic acid sequence, a third nucleic acid sequence located 5' to 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 to the sequences of Table 7 (SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 68, or SEQ ID NO: 70), or a fourth nucleic acid sequence located 3' to 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 to the sequences of Table 7 (e.g., SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, or SEQ ID NO: 69), a polynucleotide cassette. In some embodiments, (i) and (ii) can be administered as separate compositions.

[0293] In some embodiments, the compositions described herein include: (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 from the target integration site; and (ii) 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 the sequence of Table 6 (e.g., SEQ ID NO: 56), a second nucleic acid sequence located 5' or 3' to the 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 the sequences of Table 5 (e.g., SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48), an expression cassette; a third nucleic acid sequence located 5' to the expression cassette and including 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 to the sequences of Table 7 (SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 68, or SEQ ID NO: 70), or a fourth nucleic acid sequence located 3' to the expression cassette and including 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 to the sequences of Table 7 (e.g., SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, or SEQ ID NO: 69), a polynucleotide cassette. In some embodiments, (i) and (ii) can be administered as separate compositions.

[0294] In some embodiments, the compositions described herein include (i) a nuclease or a polynucleotide sequence encoding a nuclease that can induce double-strand breaks and / or single-strand breaks at a cleavage site distal from the target integration site, and (ii) 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 the sequence of Table 6 (e.g., SEQ ID NO: 57), a second nucleic acid sequence located in a 5' or 3' sequence relative to the 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 the sequences of Table 5 (e.g., SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48), an expression cassette, a third nucleic acid sequence located 5' to the expression cassette and including 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 to the sequences of Table 7 (SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 68, or SEQ ID NO: 70), or a fourth nucleic acid sequence located 3' to the expression cassette and including 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 to the sequences of Table 7 (e.g., SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, or SEQ ID NO: 69), a polynucleotide cassette. In some embodiments, (i) and (ii) can be administered as separate compositions.

[0295] In some embodiments, the compositions described herein comprise: (i) a nuclease or polynucleotide sequence encoding a nuclease that can induce double-strand breaks and / or single-strand breaks at a cleavage site distal from the target integration site; and (ii) 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 the sequence of Table 6 (e.g., SEQ ID NO: 58), a second nucleic acid sequence located 5' or 3' to the 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 the sequences of Table 5 (e.g., SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48), an expression cassette comprising the second nucleic acid sequence, a third nucleic acid sequence located 5' to the expression cassette and 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 the sequences of Table 7 (SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 68, or SEQ ID NO: 70), or a fourth nucleic acid sequence located 3' to the expression cassette and 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 the sequences of Table 7 (e.g., SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, or SEQ ID NO: 69), a polynucleotide cassette. In some embodiments, (i) and (ii) can be administered as separate compositions.

[0296] In some embodiments, the compositions described herein include: (i) a nuclease or a polynucleotide sequence encoding a nuclease that can induce double-strand breaks and / or single-strand breaks at a cleavage site distal to the target integration site; and (ii) 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 the sequence of Table 6 (e.g., SEQ ID NO: 59), a second nucleic acid sequence located 5' or 3' to the 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 the sequences of Table 5 (e.g., SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48), an expression cassette comprising the second nucleic acid sequence, a third nucleic acid sequence located 5' to the expression cassette and 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 the sequences in Table 7 (SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 68, or SEQ ID NO: 70), or a fourth nucleic acid sequence located 3' to the expression cassette and 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 the sequences in Table 7 (e.g., SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, or SEQ ID NO: 69), a polynucleotide cassette. In some embodiments, (i) and (ii) can be administered as separate compositions.

[0297] In some embodiments, the compositions described herein include (i) a nuclease or a polynucleotide sequence encoding a nuclease that can induce double-strand breaks and / or single-strand breaks at a cleavage site distal from the target integration site, and (ii) 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 the sequence of Table 6 (e.g., SEQ ID NO: 60), a second nucleic acid sequence located 5' or 3' to the 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 the sequences of Table 5 (e.g., SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48), a third nucleic acid sequence located 5' to the expression cassette and 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 the sequences of Table 7 (SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 68, or SEQ ID NO: 70), or a fourth nucleic acid sequence located 3' to the expression cassette and 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 the sequences of Table 7 (e.g., SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, or SEQ ID NO: 69), a polynucleotide cassette. In some embodiments, (i) and (ii) can be administered as separate compositions.

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

[0299] In some embodiments, genome editing by the GENERIDE™ platform is different from conventional gene therapy because it uses HR to deliver a corrective gene to one specific location within the genome. In some embodiments, GENERIDE™ inserts the corrective gene in an accurate manner, resulting in site-specific integration within the genome.

[0300] In some embodiments, the provided compositions 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., 2A peptide). In some embodiments, the present disclosure provides an expression cassette that includes the first nucleic acid sequence and the second nucleic acid sequence 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 splice donor and a splice acceptor. In some embodiments, the compositions and methods of the present disclosure comprise a polynucleotide cassette 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 located 5' relative to the expression cassette and comprises a sequence substantially homologous to the genomic sequence 5' of a target integration site within the genome of the cell. In some embodiments, the fourth nucleic acid sequence is located 3' relative to the expression cassette and comprises a sequence substantially homologous to the genomic sequence 3' of a target integration site within the genome of the cell.

[0302] In some embodiments, one or more of the 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 minutes, hours, days, weeks, or months of each other). In some embodiments, one or more of the compositions can be administered via the same route (e.g., parenteral, subcutaneous, intravenous, intracranial, intraspinal, intraocular, intramuscular, intravaginal, intraperitoneal, topical, intradermal, rectal, pulmonary, intraosseous, oral, buccal, intraportal, intraarterial, intratracheal, or nasal). In some embodiments, one or more of the compositions can be administered via different routes (e.g., parenteral, subcutaneous, intravenous, intracranial, intraspinal, intraocular, intramuscular, intravaginal, intraperitoneal, topical, intradermal, rectal, pulmonary, intraosseous, oral, buccal, intraportal, intraarterial, intratracheal, or nasal).

[0303] In some embodiments, one or more compositions administered in combination may include: (i) a first composition comprising one 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 nuclease, HF1-Cas9, HF2-Cas9 or HiFi-Cas9), endonucleases, meganucleases, TALENs, ZFNs)); (ii) a second composition comprising a second distinct payload (e.g., a polynucleotide sequence comprising a transgene coding region). In some embodiments, the first and second compositions are delivered by the same delivery system (e.g., viral vector, lipid nanoparticle, etc.). In some embodiments, the first and second compositions are delivered by different delivery systems (e.g., viral vector, lipid nanoparticle, etc.). In some embodiments, the first composition is administered prior to the second composition (e.g., by a difference in minutes, hours, days, weeks, or months). In some embodiments, the second composition is administered prior to the first composition (e.g., by a difference in minutes, hours, days, weeks, or months). In some embodiments, the first and second compositions are administered simultaneously. In some embodiments, the first and second compositions are combined prior to administration.

[0304] In some embodiments, the first and / or second composition is administered only once at a specific dose (e.g., a fixed dose or a weight-based dose). In some embodiments, the first and / or second composition is administered two or more times at a specific dose (e.g., a fixed dose or a weight-based dose). In some embodiments, when two or more doses are administered (e.g., a fixed dose or a weight-based dose), the first and / or second composition may be administered simultaneously, substantially simultaneously, or sequentially. In some embodiments, multiple doses (e.g., a fixed dose or a weight-based dose) are administered within a specified period (e.g., within a few minutes, hours, days, weeks, or months).

[0305] In some embodiments, the first and / or second composition is administered in response to a biomarker (e.g., a circulating biomarker as described in WO 2020 / 214582 A1). For example, the first and / or second composition is administered at a specific dose (e.g., a fixed dose or a weight-based dose), and the level of the biomarker (e.g., as described in WO 2020 / 214582 A1) is monitored within a specific period (e.g., within minutes, hours, days, weeks, or months). If the level of the biomarker (e.g., as described in WO 2020 / 214582 A1) is low (e.g., compared to an appropriate reference (e.g., the level of the biomarker before administration)), the first and / or second composition is administered at a specific dose (e.g., a fixed dose or a weight-based dose). If the level of the biomarker (e.g., as described in WO 2020 / 214582 A1) is high (e.g., compared to an appropriate reference (e.g., the level of the biomarker after the first administration)), subsequent administrations of the first and / or second composition (e.g., a fixed dose or a weight-based dose) can be re-evaluated (e.g., a therapeutic suspension, a reduced fixed dose or a weight-based dose).

[0306] Method for producing a viral vector Production of a viral vector In some embodiments, the production of a viral vector (e.g., an AAV viral vector) can include both an upstream process for generating the viral vector (e.g., cell-based culture) and a downstream process for processing the viral vector (e.g., purification, formulation, etc.). In some embodiments, the upstream process can include one or more of cell growth, cell culture, cell transfection, cell lysis, viral vector production, and / or viral vector recovery.

[0307] In some embodiments, the downstream process can 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 yields (e.g., the yield of AAV vectors), decrease the levels of replication-competent viral vectors (e.g., replication-competent AAV (rcAAV)), improve viral vector packaging efficiency (e.g., AAV vector capsid packaging), and / or any combination thereof, compared to reference constructs or methods, such as those of Xiao et al. 1998 and Grieger et al. 2015, which are incorporated herein by reference in their entirety.

[0309] Cell Lines and Transfection Reagents In some embodiments, the production of viral vectors involves the use of cells (e.g., cell culture). In some embodiments, the production of viral vectors involves the use of cell cultures of one or more cell lines (e.g., mammalian cell lines). In some embodiments, the production of viral vectors involves the use of the HEK293 cell line or a variant thereof (e.g., HEK293T, HEK293F cell lines). In some embodiments, the cells can be grown in suspension. In some embodiments, the cells consist of adherent cells. In some embodiments, the cells can be grown in a medium that does not contain animal components (e.g., animal serum). In some embodiments, the cells can be grown in a serum-free medium (e.g., F17 medium, Expi293 medium). In some embodiments, the production of viral vectors involves the transfection of cells with an expression construct (e.g., a plasmid). In some embodiments, the cells are selected for high expression of a viral vector (e.g., an AAV vector). In some embodiments, the cells are selected for high packaging efficiency of a viral vector (e.g., capsid packaging of an AAV vector). In some embodiments, the cells are selected (e.g., using a chemical transfection reagent containing a cationic molecule) to improve transfection efficiency. In some embodiments, the cells are engineered for high expression of a viral vector (e.g., an AAV vector). In some embodiments, the cells are engineered for high packaging efficiency of a viral vector (e.g., capsid packaging of an AAV vector). In some embodiments, the cells are engineered to improve transfection efficiency (e.g., using a chemical transfection reagent containing a cationic molecule). In some embodiments, the cells can be engineered or selected for two or more of the above attributes. In some embodiments, the cells are contacted with one or more expression constructs (e.g., plasmids). In some embodiments, the cells are contacted with one or more transfection reagents (e.g., lipid, polymer, and chemical transfection reagents containing 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 PEI MAX reagent and one or more expression constructs. In some embodiments, cells are contacted with FectoVir AAV reagent 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 reagent to expression construct improves the production of viral vectors (e.g., improved vector yield, improved packaging efficiency, and / or improved transfection efficiency).

[0310] expression construct In some embodiments, an expression construct is or comprises one or more polynucleotide sequences (e.g., plasmids). In some embodiments, an expression construct comprises specific polynucleotide sequence elements (e.g., payload, promoter, viral genes, etc.). In some embodiments, an 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 variants). In some embodiments, a particular type of expression construct comprises a particular combination of polynucleotide sequence elements. In some embodiments, a particular type of expression construct does not comprise a particular combination of polynucleotide sequence elements. In some embodiments, a particular expression construct does not comprise polynucleotide sequence elements encoding both the rep gene and the cap gene 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, cap gene, viral helper gene, or a combination thereof). In some embodiments, the expression construct comprises a polynucleotide sequence encoding a viral helper gene or gene variant (e.g., a herpesvirus gene or gene variant, an adenovirus gene or gene variant). In some embodiments, the expression construct comprises a polynucleotide sequence encoding one or more gene copies that express one or more wild-type Rep proteins (e.g., one part, two parts, three parts, four parts, five parts, etc.). In some embodiments, the expression construct comprises a polynucleotide sequence encoding a single gene copy that expresses 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 polynucleotide sequences 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 helper 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., rep gene, cap gene, helper gene). In some embodiments, the expression construct comprises a modified polynucleotide sequence (e.g., codon optimization) encoding a wild-type viral gene (e.g., rep gene, cap gene, helper gene). In some embodiments, the expression construct comprises a modified polynucleotide sequence encoding a modified viral gene (e.g., rep gene, cap gene, helper gene). In some embodiments, the modified viral gene is designed and / or engineered for a particular improvement (e.g., improved transduction, tissue specificity, size reduction, reduced immune response, improved packaging, reduced rcAAV levels, etc.).

[0313] According to various embodiments, the expression constructs disclosed herein can provide high flexibility and modularity compared to prior art. In some embodiments, the expression constructs disclosed herein can allow for the exchange of various polynucleotide sequences (e.g., different rep genes, cap genes, payloads, helper genes, promoters, etc.) while providing a particular improvement (e.g., increased viral vector yield, increased packaging, reduced rcAAV levels, etc.). In some embodiments, the expression constructs disclosed herein are compatible with various upstream production processes (e.g., different cell culture conditions, different transfection reagents, etc.) while providing a particular improvement (e.g., increased viral vector yield, increased packaging, reduced rcAAV levels, etc.).

[0314] In some embodiments, different types of expression constructs contain different combinations of polynucleotide sequences. In some embodiments, one type of expression construct contains one or more polynucleotide sequence elements (e.g., payload, promoter, viral gene, etc.) that are not present in the different types of expression constructs. In some embodiments, one type of expression construct contains a polynucleotide sequence element encoding a viral gene (e.g., a rep or cap gene or gene variant) and a polynucleotide sequence element encoding a payload (e.g., a transgene and / or a functional nucleic acid). In some embodiments, one type of expression construct contains a polynucleotide sequence element encoding one or more viral genes (e.g., a rep or cap gene or gene variant and / or one or more helper virus genes). In some embodiments, one type of expression construct contains a polynucleotide sequence element encoding one or more viral genes, and the viral genes are derived from one or more viral types (e.g., genes or gene variants derived from AAV and adenovirus). In some embodiments, the adenovirus-derived viral genes are genes and / or gene variants. In some embodiments, the adenovirus-derived viral genes are 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., via 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 at a specific ratio in combination with one or more transfection reagents. In some embodiments, different types of expression constructs are contacted with cells at a specific ratio (e.g., weight ratio) in combination with one or more transfection reagents. In some embodiments, different types of expression constructs are contacted with cells at a 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 (e.g., weight ratio). In some embodiments, a first expression construct containing one or more viral helper genes and a second expression construct containing one or more payloads are contacted with cells at a 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 between the first expression construct and the second expression construct (e.g., weight ratio). In some embodiments, a first expression construct containing one or more payloads and a second expression construct containing one or more viral helper genes are contacted with cells at a 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 between the first expression construct and the second expression construct (e.g., weight ratio). In some embodiments, a specific ratio of expression constructs improves the production of AAV (e.g., increased viral vector yield, increased packaging efficiency, and / or increased transfection efficiency). In some embodiments, cells are contacted with two or more expression constructs (e.g., sequentially or substantially simultaneously). 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 combinations thereof. In some embodiments, the expression construct comprises one or more promoters upstream of a particular polynucleotide sequence element (e.g., a rep or cap gene or gene variant). In some embodiments, the expression construct comprises one or more promoters downstream of a particular polynucleotide sequence element (e.g., a 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., a selectable marker, an origin of replication). In some embodiments, the expression construct comprises one or more polynucleotide sequences encoding an antibiotic resistance gene (e.g., a kanamycin resistance gene, an ampicillin resistance gene). In some embodiments, the expression construct comprises one or more polynucleotide sequences encoding a bacterial origin of replication (e.g., the colE1 origin of replication).

[0317] In some embodiments, the expression construct comprises one or more transcription termination sequences (e.g., a polyA sequence). In some embodiments, the expression construct comprises one or more of BGH polyA, FIX polyA, SV40 polyA, synthetic polyA, or combinations thereof. In some embodiments, the expression construct comprises one or more transcription termination sequences downstream of a particular sequence element (e.g., a rep or cap gene or gene variant). In some embodiments, the expression construct comprises one or more transcription termination sequences upstream of a particular sequence element (e.g., a rep or cap gene or 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 of different origins (e.g., known genes), such as, 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., 133 bp to 4 kb). In some embodiments, the expression construct comprises one or more intron sequences upstream of a particular sequence element (e.g., a rep or cap gene or gene variant). In some embodiments, the expression construct comprises one or more intron sequences within a particular sequence element (e.g., a rep or cap gene or gene variant). In some embodiments, the expression construct comprises one or more intron sequences downstream of a particular sequence element (e.g., a rep or cap gene or gene variant). In some embodiments, the expression construct comprises one or more intron sequences after a promoter (e.g., the p5 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.

Example

[0319] Exemplification Example 1: Double-strand breaks induced by Cas9 can enhance GENERIDE (trademark) efficiency This example demonstrates, among other things, that administration of a GENERIDE (trademark) construct to a target integration site within the coding region of an endogenous human gene (e.g., human albumin (ALB)) in combination with one or more Cas9 enzymes targeting cleavage sites within the non-coding sequences of an endogenous human locus (e.g., the human ALB intron) can result in an improvement in transgene integration efficiency.

[0320] A viral vector was constructed that contains a viral capsid (e.g., LK03), a P2A sequence, a transgene (e.g., UGT1A1), and balanced (1 kb / 1 kb) 5' and 3' homologous arm lengths adjacent to it. The homologous arms were designed to be complementary to the human genomic albumin target integration site. The human hepatocellular carcinoma cell line HepG2 cells were transduced with the viral vector at a dose of MOI = 1E 5 . After transduction, Lipofectamine™ CRISPRMAX (ThermoFisher Scientific, Inc) was used according to the manufacturer's instructions to transfect the cells with spCas9 and gRNA (designed using the IDT gRNA algorithm). The gRNA was designed to target the ALB gene in either intron 13 or 14 (Table 3). 48 hours after transfection, total RNA was isolated from the cells using the RNeasy kit (Qiagen) according to the manufacturer's instructions for the production method, and the level of the fusion mRNA was evaluated (see, for example, Table 8).

[0321] In particular, this example demonstrates that the viral vectors described herein, which include a transgene (e.g., UGT1A1) in combination with spCas9, can provide improved editing activity. In some embodiments, spCas9 can induce double-strand breaks (DSBs) at cleavage sites within intron 13 and / or 14 of the ALB gene (Figures 1A and 1B). In some embodiments, as shown in Figure 2A, DSBs induced in intron 13 of the human ALB gene can increase transgene integration efficiency by at least 400-fold compared to an appropriate reference (e.g., administration of a vector without an induced DSB). In some embodiments, as shown in Figure 2B, DSBs induced in intron 14 of the human ALB gene can increase transgene integration efficiency by at least 200-fold compared to an appropriate reference (e.g., administration of a vector without an induced DSB). In some embodiments, administration of the GENERIDE™ construct in combination with a DSB in any non-coding sequence within an endogenous locus can provide an improvement in transgene integration efficiency compared to the GENERIDE™ construct alone. In some embodiments, administration of the GENERIDE™ construct in combination with a DSB in a specific region of a non-coding sequence within an endogenous locus can provide improved or equivalent transgene integration efficiency compared to a DSB in an alternative region of the non-coding sequence (Figure 2C). In some embodiments, the DSB is located in a non-coding sequence distal from the integration site of the transgene.

[0322] A viral vector was constructed that included a viral capsid (e.g., LK03), a P2A sequence, a transgene (e.g., GFP), and balanced (1 kb / 1 kb) 5' and 3' homology arms adjacent to the human genomic albumin target integration site. The homology arms were designed to be complementary to the human genomic albumin target integration site. HepG2 cells, a human hepatocellular carcinoma cell line, were transfected with an MOI = 1E 5The cells were transduced with the viral vector at the indicated dosage. After transduction, Lipofectamine™ CRISPRMAX (ThermoFisher Scientific, Inc) was used according to the manufacturer's instructions to transfect the cells with spCas9 and gRNA (designed using the IDT gRNA algorithm). The gRNA was designed to target the ALB gene in either intron 13 or 14 (Table 3). Forty-eight hours after transfection, total RNA was isolated from the cells using the RNeasy kit (Qiagen) according to the manufacturer's instructions for the production method, and the levels of the fusion mRNA were evaluated. Furthermore, immunohistochemistry and / or immunocytochemistry were performed (see, for example, Table 8). The images were evaluated using image processing software (for example, ImageJ). In particular, this example demonstrates that the viral vectors described herein containing a transgene (for example, GFP) in combination with spCas9 can provide improved editing activity and protein expression. As shown in FIGS. 2D and 2E, the DSBs induced in intron 13 and / or 14 can enhance protein expression (for example, % GFP-positive cells) by at least 5-fold. Furthermore, as shown in FIG. 2F, the increase in the level of the fusion mRNA can be associated with the increase in protein expression.

[0323] A viral vector was constructed that included a viral capsid (for example, LK03), a P2A sequence, a transgene (for example, GFP), and balanced (1 kb / 1 kb) 5' and 3' homology arms adjacent to the human genomic albumin target integration site. The homology arms were designed to be complementary to the human genomic albumin target integration site. The human hepatocellular carcinoma cell line HepG2 cells were transduced at an MOI = 1E 5The cells were transduced with the viral vector at the indicated dose. After transduction, the cells were transfected with spCas9 mRNA, and the gRNA (designed using the IDT gRNA algorithm) was mixed using Lipofectamine(™) CRISPRMAX (ThermoFisher Scientific, Inc) according to the manufacturer's instructions. The gRNA was designed to target the ALB gene in either intron 13 or 14 (Table 8). 72 hours after transfection, the cells were fixed and imaged. GFP-positive cells were quantified using software (e.g., Image J or CellProfiler) (Figure 3A).

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

[0325] A viral vector was constructed that included a viral capsid (e.g., LK03), a P2A sequence, a transgene (e.g., GFP, HA-tagged GFP, or UGT1A1), and balanced (1 kb / 1 kb) 5' and 3' homology arms flanking the transgene. The homology arms were designed to be complementary to the human genomic albumin target integration site. The human hepatocellular carcinoma cell line HepG2 cells were transduced with an MOI = 1E 5The cells were transduced with the viral vector at a dose of . After transduction, Lipofectamine(™) CRISPRMAX (ThermoFisher Scientific, Inc) was used according to the manufacturer's instructions to transfect the 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 the ALB gene in either intron 13 or 14 (Table 3). 48 hours after transfection, immunohistochemistry and / or immunocytochemistry were performed using antibodies. The images were evaluated using image processing software (e.g., ImageJ).

[0326] In particular, this example demonstrates that the viral vectors described herein in combination with different spCas9s (e.g., spCas9-HF1 and / or spCas9-HF2) utilizing gRNAs designed to target intron 13 and / or 14 of albumin can improve GENERIDE(™) editing activity. As shown in Figure 5, spCas9-HF1 and / or spCas9-HF2-induced DSBs in intron 14 can enhance protein expression (e.g., % GFP-positive cells) compared to induced DSBs in intron 13.

[0327] A viral vector was constructed that included a viral capsid (e.g., LK03), a P2A sequence, an HA-conjugated transgene (e.g., GFP, HA-conjugated GFP, and / or UGT1A1), and balanced (1 kb / 1 kb) 5' and 3' homologous arms flanking the target integration site. The homologous arms were designed to be complementary to the human genomic albumin target integration site. The human hepatocellular carcinoma cell line HepG2 cells were transduced at an MOI = 1E 5The cells were transduced with the viral vector at the indicated dosage. After transduction, Lipofectamine™ CRISPRMAX (ThermoFisher Scientific, Inc) was used according to the manufacturer's instructions to transfect the cells with saCas9 and gRNA (designed using the IDT gRNA algorithm). The gRNA was designed to target the ALB gene in either intron 13 or 14 (Table 3). Immunohistochemistry and / or immunocytochemistry was performed (see, e.g., Table 8). Images were evaluated using image processing software (e.g., ImageJ). In particular, this example demonstrates that the viral vectors described herein in combination with saCas9 utilizing gRNAs designed to target intron 13 and / or 14 of albumin can improve GENERIDE™ editing activity. As demonstrated in FIG. 6, saCas9-induced DSBs can enhance protein expression (e.g., %HA+ positive cells).

[0328] Example 2: DNA cleavage induced by Cas nuclease can enhance GENERIDE™ efficiency This example demonstrates, inter alia, that administration of a GENERIDE™ construct to a target integration site within the coding region of an endogenous human locus (human albumin) in combination with one or more Cas enzymes (e.g., Cas9, Cas13, Cas12a, Cas9 nickase, etc.) targeting cleavage sites within non-coding sequences of an endogenous human locus (e.g., human ALB intron) can result in an improvement in transgene integration efficiency.

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

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

[0331] After constructing each composition, test several experimental conditions. In one experiment, simultaneously transduce and / or transfect cells (e.g., HepG2) with the first and second compositions. In another experiment, continuously transduce cells (e.g., HepG2) with the first composition and then transduce and / or transfect with the second composition. In another experiment, continuously transduce and / or transfect cells (e.g., HepG2) with the second composition and then transduce with the first composition. In another experiment, simultaneously transduce and / or transfect cells (e.g., HepG2) with the first and second compositions and then repeatedly (e.g., at least 2 or more times) transduce and / or transfect with the second composition. In another experiment, continuously transduce cells with the first composition and then repeatedly (e.g., at least 2 or more times) transduce and / or transfect with the second composition. After transfection and / or transduction, isolate total RNA from the cells using the RNeasy kit (Qiagen) according to the manufacturer's instructions and evaluate the level of the fusion mRNA.

[0332] In particular, this example demonstrates that the viral vectors described herein in combination with a Cas enzyme (e.g., Cas9, Cas13, Cas12a, Cas9 nickase, etc.) can provide improved editing activity. In some embodiments, a Cas enzyme (e.g., Cas9, Cas13, Cas12a, Cas9 nickase, etc.) can induce DNA cleavage (e.g., double-strand break (DSB) and / or single-strand break (SSB)) within the non-coding sequence of the human ALB gene. In some embodiments, the induced DNA cleavage (e.g., DSB and / or SSB) within the non-coding sequence of the human ALB gene can enhance transgene integration efficiency as compared to an appropriate control (e.g., administration of a vector that does not contain the induced DSB and / or SSB). In some embodiments, administration of a GENERIDE™ construct in combination with induced DNA cleavage (e.g., DSB and / or SSB) in any non-coding sequence within an endogenous locus can provide an improvement in transgene integration efficiency as compared to the GENERIDE™ construct alone. In some embodiments, administration of a GENERIDE™ construct in combination with induced DNA cleavage (e.g., DSB and / or SSB) in a specific region of a non-coding sequence within an endogenous locus can provide improved or equivalent transgene integration efficiency as compared to induced DNA cleavage (e.g., DSB and / or SSB) in an alternative region of the non-coding sequence. In some embodiments, the induced DNA cleavage (e.g., DSB and / or SSB) is located in a non-coding sequence distal to the integration site of the transgene.

[0333] Example 3: Induction of DNA cleavage by ZFN can enhance GENERIDE™ efficiency This example demonstrates, inter alia, that administration of a GENERIDE™ construct to a target integration site within 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 pair of ZFNs and / or ZFN nickases (ZF nickases)) targeting cleavage sites within the non-coding sequences of an endogenous human locus (e.g., the human albumin intron) can result in improved transgene integration efficiency.

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

[0335] Construct a second composition comprising one or more ZFNs (e.g., a pair of ZFNs and / or ZF nickases) designed to target non-coding sequences of an endogenous human locus (e.g., the human ALB intron). The ZFN / ZF nickase can be prepared to include a nuclease domain (e.g., FokI) that can bind to a specific region of the gene (e.g., intron 13 and / or 14 of the human albumin gene) and nick (e.g., ZF nickase) or cleave (e.g., pair of ZFNs) the target sequence. Several methods for delivering such compositions are known in the art (e.g., transfection with a plasmid or mRNA encoding one or more ZFNs (e.g., a pair of ZFNs and / or ZF nickases), transduction with a viral vector encoding one or more ZFNs (e.g., a pair of ZFNs and / or ZF nickases) and / or electroporation).

[0336] After the construction of each composition, several experimental conditions are tested. In one experiment, cells (e.g., HepG2) are simultaneously transduced and / or transfected with a first composition and a second composition. In the following experiments, cells (e.g., HepG2) are continuously transduced with the first composition and then transduced and / or transfected with the second composition. In the following experiments, cells (e.g., HepG2) are continuously transduced and / or transfected with the second composition and then transduced with the first composition. In the following experiments, cells (e.g., HepG2) are simultaneously transduced and / or transfected with the first composition and the second composition and then the second composition is repeatedly (e.g., at least 2 times or more) transduced and / or transfected. In the following experiments, cells are continuously transduced with the first composition and then the second composition is repeatedly (e.g., at least 2 times or more) transduced and / or transfected. After transfection and / or transduction, total RNA is isolated from the cells by using an RNeasy kit (Qiagen) according to the manufacturer's instructions and the level of fusion mRNA is evaluated.

[0337] In particular, this example demonstrates that a viral vector as described herein, in combination with one or more ZFNs (e.g., a pair of ZFNs and / or ZF nickases), can provide improved editing activity. In some embodiments, one or more ZFNs (e.g., a pair of ZFNs and / or ZF nickases) can induce DNA cleavage (e.g., DSB and / or SSB) within the non-coding sequence of the human ALB gene. In some embodiments, the induced DNA cleavage (e.g., DSB and / or SSB) within the non-coding sequence of the human ALB gene can enhance transgene integration efficiency as compared to an appropriate control (e.g., administration of a vector that does not contain the induced DSB and / or SSB). In some embodiments, administration of a GENERIDE™ construct in combination with induced DNA cleavage (e.g., DSB and / or SSB) in any non-coding sequence within an endogenous locus can provide an improvement in transgene integration efficiency as compared to the GENERIDE™ construct alone. In some embodiments, administration of a GENERIDE™ construct in combination with induced DNA cleavage (e.g., DSB and / or SSB) in a specific region of a non-coding sequence within an endogenous locus can provide improved or equivalent transgene integration efficiency as compared to induced DNA cleavage (e.g., DSB and / or SSB) in an alternative region of the non-coding sequence. In some embodiments, the induced DNA cleavage (e.g., DSB and / or SSB) is located in a non-coding sequence distal to the integration site of the transgene.

[0338] Example 4: Induced DNA Cleavage by Meganucleases Can Enhance GENERIDE™ Efficiency This example demonstrates, in particular, that administration of a GENERIDE™ construct to a target integration site within the coding region of an endogenous human gene (e.g., human albumin (ALB)), in combination with one or more meganucleases that target cleavage sites within the non-coding sequence of an endogenous human locus (e.g., the human albumin intron), can result in an improvement in transgene integration efficiency.

[0339] Construct a first composition comprising a viral vector comprising a viral capsid, a P2A sequence, a transgene, and balanced or unbalanced adjacent 5' and 3' homologous arm lengths. The homologous arms are designed to be complementary to a target integration site (e.g., the human genomic albumin).

[0340] Construct a second composition comprising one or more meganucleases designed to target non-coding sequences of an endogenous human locus (e.g., the human ALB intron). 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, test several experimental conditions. In one experiment, simultaneously transduce and / or transfect cells (e.g., HepG2) with the first composition and the second composition. In the following experiment, continuously transduce cells (e.g., HepG2) with the first composition and then transduce and / or transfect with the second composition. In the following experiment, continuously transduce and / or transfect cells (e.g., HepG2) with the second composition and then transduce with the first composition. In the following experiment, simultaneously transduce and / or transfect cells (e.g., HepG2) with the first composition and the second composition and then repeatedly (e.g., at least 2 times or more) transduce and / or transfect with the second composition. In the following experiment, continuously transduce cells with the first composition and then repeatedly (e.g., at least 2 times or more) transduce and / or transfect with the second composition. After transduction and / or transfection, isolate total RNA from the cells using an RNeasy kit (Qiagen) according to the manufacturer's instructions and evaluate the level of fusion mRNA.

[0342] In particular, this example demonstrates that a viral vector in combination with one or more meganucleases, as described herein, can provide improved editing activity. In some embodiments, one or more meganucleases can induce DNA cleavage (e.g., DSB and / or SSB) within non-coding sequences of the human ALB gene. In some embodiments, the induced DNA cleavage (e.g., DSB and / or SSB) within non-coding sequences of the human ALB gene can enhance transgene integration efficiency as compared to an appropriate control (e.g., administration of a vector that does not contain the induced DSB and / or SSB). In some embodiments, administration of a GENERIDE™ construct in combination with induced DNA cleavage (e.g., DSB and / or SSB) in any non-coding sequence within an endogenous locus can provide an improvement in transgene integration efficiency as compared to the GENERIDE™ construct alone. In some embodiments, administration of a GENERIDE™ construct in combination with induced DNA cleavage (e.g., DSB and / or SSB) in a specific region of a non-coding sequence within an endogenous locus can provide improved or equivalent transgene integration efficiency as compared to the induced DNA cleavage (e.g., DSB and / or SSB). In some embodiments, the induced DNA cleavage (e.g., DSB and / or SSB) is located in a non-coding sequence distal to the integration site of the transgene.

[0343] Example 5: Induced DNA cleavage by exemplary nucleases can enhance GENERIDE™ efficiency This example demonstrates, in particular, that administration of a GENERIDE™ construct to a target integration site within the coding region of an endogenous human locus (e.g., human albumin (ALB)) in combination with one or more exemplary nucleases described herein that target cleavage sites within non-coding sequences of an endogenous human locus (e.g., human albumin intron) can result in an improvement in transgene integration efficiency.

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

[0345] Construct a second composition comprising one or more exemplary nucleases described herein that are designed to target non-coding sequences of an endogenous human locus (e.g., the human ALB intron). Several methods for delivering such compositions are known in the art (e.g., transfection with a plasmid or mRNA encoding one or more exemplary nucleases as described herein, transduction with a viral vector 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, test several experimental conditions. In one experiment, simultaneously transduce and / or transfect cells (e.g., HepG2) with the first composition and the second composition. In the following experiments, continuously transduce cells (e.g., HepG2) with the first composition and then transduce and / or transfect with the second composition. In the following experiments, continuously transduce and / or transfect cells (e.g., HepG2) with the second composition and then transduce with the first composition. In the following experiments, simultaneously transduce and / or transfect cells (e.g., HepG2) with the first composition and the second composition and then repeatedly (e.g., at least 2 times or more) transduce and / or transfect with the second composition. In the following experiments, continuously transduce cells with the first composition and then repeatedly (e.g., at least 2 times or more) transduce and / or transfect with the second composition. After transfection and / or transduction, isolate total RNA from the cells using the RNeasy kit (Qiagen) according to the manufacturer's instructions and evaluate the level of fusion mRNA.

[0347] In particular, this example demonstrates that a viral vector as described herein, in combination with a more exemplary nuclease as described herein, or in combination with a more exemplary nuclease, can provide improved editing activity. In some embodiments, one or more of the exemplary nucleases described herein can induce DNA cleavage (e.g., DSB and / or SSB) within the non-coding sequences of the human ALB gene. In some embodiments, the induced DNA cleavage (e.g., DSB and / or SSB) within the non-coding sequences of the human ALB gene can enhance transgene integration efficiency as compared to an appropriate control (e.g., administration of a vector that does not contain the induced DSB and / or SSB). In some embodiments, administration of a GENERIDE™ construct in combination with induced DNA cleavage (e.g., DSB and / or SSB) in any non-coding sequence within an endogenous locus can provide an improvement in transgene integration efficiency as compared to the GENERIDE™ construct alone. In some embodiments, administration of a GENERIDE™ construct in combination with induced DNA cleavage (e.g., DSB and / or SSB) in a specific region of a non-coding sequence within an endogenous locus can provide improved or equivalent transgene integration efficiency as compared to induced DNA cleavage (e.g., DSB and / or SSB) in an alternative region of the non-coding sequence. In some embodiments, the induced DNA cleavage (e.g., DSB and / or SSB) is located in a non-coding sequence distal from the integration site of the transgene.

[0348] Example 6: Induced DNA cleavage can enhance GENERIDE™ efficiency in vivo or ex vivo This example demonstrates, inter alia, that administration of a GENERIDE™ construct to a target integration site within the coding region of an endogenous locus (e.g., a human albumin intron), in combination with one or more endonucleases described herein that target cleavage sites within the non-coding sequences of an endogenous locus (e.g., human albumin), can result in improved transgene integration efficiency in a subject (e.g., a subject suffering from a disease).

[0349] In some embodiments, this example involves the use of one or more GENERIDE™ constructs, e.g., a viral vector comprising a viral capsid described herein, a 2A sequence (e.g., P2A) described herein, a transgene described herein, and adjacent human homology arms (e.g., balanced or unbalanced).

[0350] In some embodiments, the GENERIDE™ construct is administered continuously or simultaneously with one or more endonucleases as described herein. In some embodiments, the GENERIDE™ construct is co-administered in vivo to a subject (e.g., a subject suffering from a disease) with one or more endonucleases. In some embodiments, the GENERIDE™ construct is co-administered ex vivo to cells (e.g., hepatocytes) with one or more endonucleases, and optionally, the cells are administered to a subject (e.g., a subject suffering from a disease) after transgene integration. In some embodiments, the GENERIDE™ construct is administered before administration with one or more endonucleases. In some embodiments, the GENERIDE™ construct is administered after administration with one or more endonucleases. In some embodiments, the GENERIDE construct is administered to a subject (e.g., a subject suffering from a disease) in vivo together with one or more endonucleases, and optionally, is then administered with one or more endonucleases.

[0351] In particular, this example provides the insight that induced DNA cleavage (e.g., DSB and / or SSB) can enhance the efficacy of GENERIDE™ in vivo. In some embodiments, the level of the fusion mRNA can increase in a subject and / or cell and / or tissue as compared to an appropriate reference (e.g., administration of a vector in which DNA cleavage is not induced (e.g., DSB and / or SSB)). In some embodiments, the level of ALB-2A can increase in a subject, and / or cell, and / or tissue as compared to an appropriate reference (e.g., administration of a vector in which DNA cleavage is not induced (e.g., DSB and / or SSB)). In some embodiments, the transgene expression can increase in a subject and / or cell and / or tissue as compared to an appropriate reference (e.g., administration of a vector without induced DNA cleavage (e.g., DSB and / or SSB)).

[0352] In particular, this example provides the insight that a GENERIDE™ construct administered continuously or simultaneously with one or more endonucleases can provide an improved selective advantage to cells that have successfully integrated the transgene of interest.

[0353] Example 7: Induced DNA cleavage by GENERIDE™ can improve the Wilson's disease phenotype This example, in particular, demonstrates that administering a GENERIDE™ construct comprising a therapeutic transgene encoding ATP7B (e.g., truncated ATP7B) in combination with one or more of the endonucleases described herein that target a cleavage site within the non-coding sequence of an endogenous locus (e.g., human albumin) to a target integration site within the coding region of the endogenous locus (e.g., human albumin intron) can be used to treat or prevent Wilson's disease (e.g., by reducing the phenotypic impact and / or symptoms in vivo) in a subject (e.g., a subject suffering from Wilson's disease).

[0354] A GENERIDE™ construct, such as a viral capsid described herein, a 2A sequence (e.g., P2A), a human ATP7B (e.g., truncated ATP7B) transgene, and adjacent human homology arms (e.g., balanced or unbalanced) is constructed into a viral vector. The GENERIDE™ construct is administered (e.g., intravenously) to a subject (e.g., a subject suffering from Wilson's disease) at a dosage (e.g., a fixed dosage or a weight-based dosage). In some embodiments, the GENERIDE™ construct is administered continuously or simultaneously with one or more endonucleases as described herein. In some embodiments, the GENERIDE™ construct is co-administered in vivo to a subject (e.g., a subject suffering from Wilson's disease) with one or more endonucleases. In some embodiments, the GENERIDE™ construct is co-...

Claims

1. A composition or a set of compositions, (i) A nuclease or a polynucleotide sequence encoding a nuclease, wherein the nuclease is selected from clustered and regularly arranged short palindromic repeat (CRISPR)-related (Cas) enzymes, transcription activator-like effector (TALE) nuclease (TALEN), TALE nickase, zinc finger (ZF) nuclease (ZFN), ZF nickase, or meganuclease, (ii) A polynucleotide cassette, 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 located 5' or 3' relative 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 at 5' relative to the expression cassette and containing a sequence substantially homologous to the human genome sequence 5' of the target integration site in the genome of a human cell; and A fourth nucleic acid sequence located at 3' relative to the expression cassette and containing a sequence substantially homologous to the human genome sequence 3' of the target integration site in the genome of the human cell. A polynucleotide cassette containing, Includes, The aforementioned polynucleotide cassette does not contain a promoter sequence. The nuclease can induce double-strand breaks and / or single-strand breaks at cleavage sites in the genome of the human cell. A composition or a set of compositions.

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

3. A composition or a set of compositions, (i) a nuclease or a polynucleotide sequence encoding a nuclease, (ii) 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 located 5' or 3' relative 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, the target integration site being an albumin locus containing an endogenous albumin promoter and an endogenous albumin gene; A third nucleic acid sequence located at 5' relative to the expression cassette and containing a sequence substantially homologous to the human genome sequence 5' of the target integration site in the genome of a human cell; and A fourth nucleic acid sequence located at 3' relative to the expression cassette and containing a sequence substantially homologous to the human genome sequence 3' of the target integration site in the genome of the human cell. A polynucleotide cassette containing, Includes, The aforementioned polynucleotide cassette does not contain a promoter sequence. The nuclease can induce double-strand breaks and / or single-strand breaks at cleavage sites in the genome of the human cell. A composition or a set of compositions.

4. A composition or set of compositions according to any one of claims 1 to 3, further comprising a recombinant viral vector.

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

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

7. The composition or set of compositions according to claim 6, wherein the recombinant viral vector comprises the capsid polypeptide, and the polynucleotide sequence encoding a nuclease and / or the polynucleotide cassette is capsidized to the recombinant viral vector.

8. The composition or set of compositions according to claim 6, wherein the polynucleotide cassette is capsidized to the recombinant viral vector.

9. A composition or set of compositions according to any one of claims 1 to 3, further comprising an AAV2 terminal inverted repeat (ITR) sequence.

10. The composition or set of compositions according to claim 9, wherein the AAV2 ITR sequence is adjacent to the 5' and 3' ends of the polynucleotide sequence and / or the polynucleotide cassette encoding the nuclease.

11. The composition or set of compositions according to any one of claims 1 to 3, wherein the third and fourth nucleic acid sequences are each 50 nt to 1600 nt in length.

12. The composition or set of compositions according to any one of claims 1 to 3, wherein the third and fourth nucleic acid sequences are of the same length.

13. The composition or set of compositions according to any one of claims 1 to 3, wherein the third and fourth nucleic acid sequences have different lengths.

14. The composition or set of compositions according to any one of claims 1 to 3, wherein when the polynucleotide cassette is incorporated into the target integration site in the genome of the cell, the transgene is expressed at the target integration site under the control of an endogenous promoter.

15. The composition or set of compositions according to claim 2 or 3, wherein the target integration site is located within the coding sequence of the albumin gene locus and is 5' adjacent to the stop codon.

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

17. The composition or set of compositions according to any one of claims 1 to 3, wherein the cleavage site is located within the non-coding sequence of the albumin gene locus.

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

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

20. The composition or set of compositions according to claim 3, wherein the nuclease is selected from Cas enzyme, TALEN, TALEN nickasase, ZFN, ZF nickasase, or meganuclease.

21. The composition or set of compositions according to any one of claims 1 to 3, wherein the nuclease is a Cas enzyme or TALEN.

22. The composition or set of compositions according to any one of claims 1 to 3, wherein the nuclease is a Cas enzyme.

23. The composition or set of compositions according to any one of claims 1 to 3, 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 set of compositions according to claim 22, further comprising guide RNA (gRNA).

25. The composition or set of compositions according to claim 24, wherein the gRNA comprises one nucleic acid sequence of sequence numbers 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 with one of sequence numbers 27-45, 71-86, or 93-98.

26. The composition or set of compositions according to claim 24, wherein the nuclease or a polynucleotide sequence encoding the nuclease and the gRNA are co-formulated.

27. The composition or set of compositions according to claim 24, wherein the nuclease or the polynucleotide sequence encoding the nuclease and the gRNA are formulated separately.

28. The second nucleic acid sequence is a) Nucleic acid sequence encoding peptide 2A; b) Nucleic acid sequences encoding the internal ribosome entry site (IRES); c) Nucleic acid sequences encoding the N-terminal intent splicing region and the C-terminal intent splicing region; or d) Nucleic acid sequences encoding splice donors and splice acceptors A composition or set of compositions according to any one of claims 1 to 3, which is or includes the same.

29. The composition or set of compositions according to claim 28, wherein the second nucleic acid sequence is a nucleic acid sequence encoding a 2A peptide, or comprises the same.

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

31. The composition or set of compositions according to claim 1 or 3, wherein the cutting portion is 1 to 2000 bp from the target integration portion.

32. The composition or set of compositions according to claim 31, wherein the cutting portion is 100 bp from the target integration portion.

33. The composition or set of compositions according to any one of claims 1 to 3, wherein the introduced gene is selected from CBS, UGT1A1, MUT, FAH, ATP7B, A1AT, ASL, LIPA, PAH, G6PC, factor IX, or a variant thereof.

34. A composition or set of compositions according to any one of claims 1 to 3, wherein the nuclease or the polynucleotide sequence encoding the nuclease is formulated into lipid nanoparticles (LNPs), and the polynucleotide cassette is capsidized into a recombinant AAV vector.

35. A composition or set of compositions for incorporating a transgene into the genome of a human cell, comprising: (i) a nuclease or a polynucleotide sequence encoding a nuclease, wherein the nuclease is selected from Cas enzyme, TALEN, TALE niccas, ZFN, ZF niccas, or meganuclease; (ii) 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 located 5' or 3' relative 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 at 5' relative to the expression cassette and containing a sequence substantially homologous to the human genome sequence 5' of the target integration site in the genome of a human cell; and A fourth nucleic acid sequence located at 3' relative to the expression cassette and containing a sequence substantially homologous to the human genome sequence 3' of the target integration site in the genome of the human cell. A polynucleotide cassette containing, Includes, The aforementioned polynucleotide cassette does not contain a promoter sequence. The nuclease can induce double-strand breaks and / or single-strand breaks at cleavage sites in the cell's genome. A composition or set of compositions characterized in that, after administration of the composition or set of compositions to a target, the introduced gene is incorporated into the genome of the human cells.

36. The composition or set of compositions according to claim 35, wherein the target integration site is an albumin locus containing an endogenous albumin promoter and an endogenous albumin gene.

37. A composition or set of compositions for incorporating a transgene into the genome of a human cell, (i) a nuclease or a polynucleotide sequence encoding a nuclease, (ii) 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 located 5' or 3' relative 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, the target integration site being an albumin locus containing an endogenous albumin promoter and an endogenous albumin gene; A third nucleic acid sequence located at 5' relative to the expression cassette and containing a sequence substantially homologous to the human genome sequence 5' of the target integration site in the genome of a human cell; and A fourth nucleic acid sequence located at 3' relative to the expression cassette and containing a sequence substantially homologous to the human genome sequence 3' of the target integration site in the human cell genome. A polynucleotide cassette containing, Includes, The aforementioned polynucleotide cassette does not contain a promoter sequence. The nuclease can induce double-strand breaks and / or single-strand breaks at cleavage sites in the cell's genome. A composition or set of compositions characterized in that, after administration of the composition or set of compositions to a target, the introduced gene is incorporated into the genome of the human cells.

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

39. The composition or set of compositions according to claim 38, wherein the recombinant viral vector is a recombinant AAV vector.

40. The composition or set of compositions according to claim 39, wherein the recombinant viral vector is a capsid polypeptide having 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 composition or set of compositions according to claim 40, wherein the recombinant viral vector comprises the capsid polypeptide, and the polynucleotide sequence encoding a nuclease and / or the polynucleotide cassette is capsidized to the recombinant viral vector.

42. The composition or set of compositions according to claim 41, wherein the polynucleotide cassette is capsidized to the recombinant viral vector.

43. The composition or set of compositions according to any one of claims 35 to 37, wherein the composition or set of compositions further comprises an AAV2 ITR sequence.

44. The composition or set of compositions according to claim 43, wherein the AAV2 ITR sequence is adjacent to the 5' and 3' ends of the polynucleotide sequence and / or the polynucleotide cassette encoding the nuclease.

45. The composition or set of compositions according to any one of claims 35 to 37, wherein the third and fourth nucleic acid sequences have a length of 50 nt to 1600 nt.

46. The composition or set of compositions according to any one of claims 35 to 37, wherein the third and fourth nucleic acid sequences are of the same length.

47. The composition or set of compositions according to any one of claims 35 to 37, wherein the third and fourth nucleic acid sequences are of different lengths.

48. The composition or set of compositions according to any one of claims 35 to 37, wherein when the polynucleotide cassette is incorporated into the target integration site in the genome of the cell, the introduced gene is expressed at the target integration site under the control of an endogenous promoter.

49. The composition or set of compositions according to claim 36 or 37, wherein the target integration site is located within the coding sequence of the albumin gene locus and is 5' adjacent to the stop codon.

50. The composition or set of compositions according to claim 49, wherein the target integration site is 5' adjacent to the stop codon in exon 14 of the albumin gene locus.

51. The composition or set of compositions according to claim 36 or 37, wherein the cleavage site is located within the non-coding sequence of the albumin gene locus.

52. The composition or set of compositions according to claim 51, wherein the cleavage site is located within an intron, untranslated region, enhancer, promoter, silencer, or insulator of the albumin gene locus.

53. The composition or set of compositions according to claim 52, wherein the cleavage site is located within intron 12, 13, or 14 of the albumin gene locus.

54. The composition or set of compositions according to claim 37, wherein the nuclease is selected from Cas enzyme, TALEN, TALEN nickasase, ZFN, ZF nickasase, or meganuclease.

55. The composition or set of compositions according to any one of claims 35 to 37, wherein the nuclease is Cas enzyme or TALEN.

56. The composition or set of compositions according to any one of claims 35 to 37, wherein the nuclease is a Cas enzyme.

57. The composition or set of compositions according to any one of claims 35 to 37, 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 composition or set of compositions according to claim 56, further comprising guide RNA (gRNA).

59. The composition or set of compositions according to claim 58, wherein the gRNA comprises one nucleic acid sequence of sequence numbers 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 with one of sequence numbers 27-45, 71-86, or 93-98.

60. The composition or set of compositions according to claim 58, wherein the nuclease or a polynucleotide sequence encoding the nuclease and the gRNA are co-formulated.

61. The composition or set of compositions according to claim 58, wherein the nuclease or the polynucleotide sequence encoding the nuclease and the gRNA are formulated separately.

62. The second nucleic acid sequence is a) Nucleic acid sequence encoding peptide 2A; b) Nucleic acid sequences encoding the internal ribosome entry site (IRES); c) Nucleic acid sequences encoding the N-terminal intent splicing region and the C-terminal intent splicing region; or d) Nucleic acid sequences encoding splice donors and splice acceptors A composition or set of compositions according to any one of claims 35 to 37, which is or includes them.

63. The composition or set of compositions according to any one of claims 35 to 37, wherein the second nucleic acid sequence is a nucleic acid sequence encoding a 2A peptide, or comprises the same.

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

65. The composition or set of compositions according to claim 35 or 37, wherein the cutting portion is 1 to 1000 bp from the target integration portion.

66. The composition or set of compositions according to claim 65, wherein the cutting portion is 100 bp from the target integration portion.

67. The composition or set of compositions according to any one of claims 35 to 37, wherein the introduced gene is selected from CBS, UGT1A1, MUT, FAH, ATP7B, A1AT, ASL, LIPA, PAH, G6PC, factor IX, or a variant thereof.

68. A composition or set of compositions according to any one of claims 35 to 37, wherein the nuclease or the polynucleotide sequence encoding the nuclease is formulated into lipid nanoparticles, and the polynucleotide cassette is capsidated into a recombinant AAV vector.

69. The composition or set of compositions according to any one of claims 35 to 37, wherein the cells are edited in vivo.

70. A composition or set of compositions according to any one of claims 35 to 37, characterized in that the introduction of the introduced gene is performed ex vivo.

71. The composition or set of compositions according to claim 69, wherein the cells are blood, liver, muscle, or CNS cells.

72. The composition or set of compositions according to claim 70, wherein the cells are administered in autotransplantation after the introduction of the transgene.

73. The composition or set of compositions according to claim 70, wherein the cells are administered in an allogeneic transplant after the introduction of the transgene.

74. (i) the nuclease or the polynucleotide sequence encoding the nuclease and (ii) the polynucleotide cassette are administered to the subject on the same day, characterized in that the composition or set of compositions according to any one of claims 35 to 37.

75. (i) the nuclease or the polynucleotide sequence encoding the nuclease and (ii) the polynucleotide cassette are administered to the subject on different days, characterized in that the composition or set of compositions according to any one of claims 35 to 37.

76. The composition or set of compositions according to claim 75, characterized in that 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 composition or set of compositions according to claim 76, characterized in that the nuclease or the polynucleotide sequence encoding the nuclease is administered to the subject 1 to 24 hours after the polynucleotide cassette.

78. The composition or set of compositions according to claim 75, characterized in that the nuclease or the polynucleotide sequence encoding the nuclease is administered to the subject 1 hour to 3 days before the polynucleotide cassette.

79. The composition or set of compositions according to claim 76, characterized in that the nuclease or the polynucleotide sequence encoding the nuclease is administered to the subject one to four hours before the polynucleotide cassette.

80. The composition or set of compositions according to claim 79, characterized in that the nuclease or the polynucleotide sequence encoding the nuclease is administered to the subject four hours before the polynucleotide cassette.

81. The composition or set of compositions according to any one of claims 1 to 3, wherein the cutting portion is distal to the target integration portion.

82. The composition or set of compositions according to any one of claims 35 to 37, wherein the cutting portion is distal to the target integration portion.

Images