Ass1 gene insertion for the treatment of citrullinemia type i

Abstract

Nucleic acid constructs and compositions that allow insertion of an argininosuccinate synthase 1 (ASS1) coding sequence into a target genomic locus such as an endogenous ASS1 locus and / or expression of the ASS1 coding sequence are provided. Also provided are nuclease agents (e.g., targeting an endogenous ASS1 locus) or nucleic acids encoding nuclease agents to facilitate integration of the nucleic acid constructs into a target genomic locus such as an endogenous ASS1 locus. The nucleic acid constructs and compositions can be used in methods of introducing an ASS1 nucleic acid into a cell, methods of integration of an ASS1 nucleic acid into a target genomic locus, methods of expression of ASS1 in a cell, and in methods of treating citrullinemia type I or ASS1 deficiency in a subject.

Description

ASS1 GENE INSERTION FOR THE TREATMENT OF CITRULLINEMIA TYPE ICROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of US Application No. 63 / 662,169, filed June 20, 2024, and US Application No. 63 / 710,120, filed October 22, 2024, each of which is herein incorporated by reference in its entirety for all purposes.REFERENCE TO A SEQUENCE LISTING SUBMITTED AS AN XML FILE

[0002] The Sequence Listing written in file 633157SEQLIST.xml is 734,134 bytes, was created on June 18, 2025, and is hereby incorporated by reference in its entirety.BACKGROUND

[0003] Citrullinemia type I (CTLN1) is a life-threatening urea cycle disorder impacting approximately 1 in 57,000 live births. It is a monogenic condition arising from deficiency of argininosuccinate synthetase (AS J). When left untreated, CTLN1 can lead to serious metabolic decompensation, marked elevation in blood ammonia, and early death. Today, newborn screening in the perinatal period helps to rapidly identify individuals with CTLN1, enabling prompt intervention in the form of dietary protein restriction and nitrogen scavenger therapy. However, lifelong dietary protein restriction poses significant adherence challenges, and frequent administration of nitrogen scavenger medication is difficult to administer to young children. Therefore, many patients receiving standard of care continue to suffer from periodic bouts of hyperammonemia and metabolic decompensation that can progressively lead to irreversible neurological, behavioral, and cognitive sequelae. Liver transplantation is often curative but poses its own set of limitations and complications. The monogenic nature and curative potential of liver transplantation make CTLN1 an ideal indication for gene therapy. However, episome-based hepatic AAV gene therapies are often ineffective when administered early in life due to vector dilution. This poses a significant obstacle to episome-based liver-directed gene therapies for CTLN1 where early intervention is critically important to prevent irreversible neurological manifestations of the disease. Accordingly, better methods to treat CTLN1 are required.SUMMARY

[0004] Provided are compositions comprising a nuclease agent that targets a nuclease target site in an argininosuccinate synthase 1 (ASS / ) gene, compositions comprising a nucleic acid construct comprising a first argininosuccinate synthase protein coding sequence, combinations comprising a composition comprising the nuclease agent that targets the nuclease target site in the .4AS7 gene and a composition comprising the nucleic acid construct comprising the first argininosuccinate synthase protein coding sequence, cells comprising any of the above compositions or combinations, methods of introducing an argininosuccinate synthase nucleic acid into a cell, methods of integrating an argininosuccinate synthase nucleic acid construct into a target gene in a cell, methods of expressing argininosuccinate synthase in a cell, methods of treating an argininosuccinate synthase deficiency in a subject, methods of treating citrullinemia type I in a subject, and methods of preventing or inhibiting hyperammonemia in a subject having citrullinemia type I.

[0005] In one aspect, provided are compositions comprising a nuclease agent that targets a nuclease target site in an argininosuccinate synthase 1 (ASS J) gene. In some such compositions, the ANS7 gene is a human ASS J gene. In some such compositions, the nuclease target site is in intron 1 or intron 2 of the ASS1 gene. In some such compositions, the nuclease target site is in intron 2 of the ASS1 gene. In some such compositions, the nuclease agent comprises: (a) a zinc finger nuclease (ZFN); (b) a transcription activator-like effector nuclease (TALEN); or (c) (i) a Cas protein or a nucleic acid encoding the Cas protein; and (ii) a guide RNA or one or more DNAs encoding the guide RNA, wherein the guide RNA comprises a DNA-targeting segment that targets a guide RNA target sequence, and wherein the guide RNA binds to the Cas protein and targets the Cas protein to the guide RNA target sequence.

[0006] In some such compositions, the nuclease agent comprises: (a) a Cas protein or a nucleic acid encoding the Cas protein; and (b) a guide RNA or one or more DNAs encoding the guide RNA, wherein the guide RNA comprises a DNA-targeting segment that targets a guide RNA target sequence in an intron of an ASS I gene, optionally wherein the intron is intron 1 or intron 2, and wherein the guide RNA binds to the Cas protein and targets the Cas protein to the guide RNA target sequence. Similarly, in another aspect, provided are compositions comprising a guide RNA or one or more DNAs encoding the guide RNA, wherein the guide RNA comprises a DNA-targeting segment that targets a guide RNA target sequence in an intron of an ASS I gene,optionally wherein the intron is intron 1 or intron 2, and wherein the guide RNA binds to a Cas protein and targets the Cas protein to the guide RNA target sequence.

[0007] In some such compositions, the ASS1 gene is a human ASS1 gene, and the guide RNA target sequence is in intron 2 of the human ASS1 gene. In some such compositions, the DNA- targeting segment comprises at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of the sequence set forth in any one of SEQ ID NOS: 31-118, optionally wherein the DNA-targeting segment comprises at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of the sequence set forth in any one of SEQ ID NOS: 58, 67, 60, 73, 78, 89, 92, and 114, optionally wherein the DNA-targeting segment comprises at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of the sequence set forth in any one of SEQ ID NOS: 78, 60, and 89, optionally wherein the DNA-targeting segment comprises at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of the sequence set forth in SEQ ID NO: 78. In some such compositions, the DNA-targeting segment is at least 90% or at least 95% identical to the sequence set forth in any one of SEQ ID NOS: 31-118, optionally wherein the DNA- targeting segment is at least 90% or at least 95% identical to the sequence set forth in any one of SEQ ID NOS: 58, 67, 60, 73, 78, 89, 92, and 114, optionally wherein the DNA-targeting segment is at least 90% or at least 95% identical to the sequence set forth in any one of SEQ ID NOS: 78, 60, and 89, optionally wherein the DNA-targeting segment is at least 90% or at least 95% identical to the sequence set forth in SEQ ID NO: 78. In some such compositions, the DNA-targeting segment comprises any one of SEQ ID NOS: 31-118, optionally wherein the DNA-targeting segment comprises any one of SEQ ID NOS: 58, 67, 60, 73, 78, 89, 92, and 114, optionally wherein the DNA-targeting segment comprises any one of SEQ ID NOS: 78, 60, and 89, optionally wherein the DNA-targeting segment comprises SEQ ID NO: 78. In some such compositions, the DNA-targeting segment consists of any one of SEQ ID NOS: 31-118, optionally wherein the DNA-targeting segment consists of any one of SEQ ID NOS: 58, 67, 60, 73, 78, 89, 92, and 114, optionally wherein the DNA-targeting segment consists of any one of SEQ ID NOS: 78, 60, and 89, optionally wherein the DNA-targeting segment consists of SEQ ID NO: 78. In some such compositions, the guide RNA comprises any one of SEQ ID NOS: 340- 427, optionally wherein the guide RNA comprises any one of SEQ ID NOS: 367, 376, 369, 382, 387, 398, 401, and 423, optionally wherein the guide RNA comprises any one of SEQ ID NOS: 387, 369, and 398, optionally wherein the guide RNA comprises SEQ ID NO: 387. In some suchcompositions, the guide RNA comprises any one of SEQ ID NOS: 252-339, optionally wherein the guide RNA comprises any one of SEQ ID NOS: 279, 288, 281, 294, 299, 310, 313, and 335, optionally wherein the guide RNA comprises any one of SEQ ID NOS: 299, 281, and 310, optionally wherein the guide RNA comprises SEQ ID NO: 299. In some such compositions, the DNA-targeting segment comprises at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of the sequence set forth in any one of SEQ ID NOS: 31-118, optionally wherein the DNA-targeting segment comprises at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of the sequence set forth in any one of SEQ ID NOS: 58, 67, 60, 73, 78, 89, 92, and 114, optionally wherein the DNA-targeting segment comprises at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of the sequence set forth in any one of SEQ ID NOS: 58 and 67. In some such compositions, the DNA-targeting segment is at least 90% or at least 95% identical to the sequence set forth in any one of SEQ ID NOS: 31-118, optionally wherein the DNA-targeting segment is at least 90% or at least 95% identical to the sequence set forth in any one of SEQ ID NOS: 58, 67, 60, 73, 78, 89, 92, and 114, optionally wherein the DNA-targeting segment is at least 90% or at least 95% identical to the sequence set forth in any one of SEQ ID NOS: 58 and 67. In some such compositions, the DNA-targeting segment comprises any one of SEQ ID NOS: 31-118, optionally wherein the DNA-targeting segment comprises any one of SEQ ID NOS: 58, 67, 60, 73, 78, 89, 92, and 114, optionally wherein the DNA-targeting segment comprises any one of SEQ ID NOS: 58 and 67. In some such compositions, the DNA- targeting segment consists of any one of SEQ ID NOS: 31-118, optionally wherein the DNA- targeting segment consists of any one of SEQ ID NOS: 58, 67, 60, 73, 78, 89, 92, and 114, optionally wherein the DNA-targeting segment consists of any one of SEQ ID NOS: 58 and 67. In some such compositions, the guide RNA comprises any one of SEQ ID NOS: 340-427, optionally wherein the guide RNA comprises any one of SEQ ID NOS: 367, 376, 369, 382, 387, 398, 401, and 423, optionally wherein the guide RNA comprises any one of SEQ ID NOS: 367 and 376. In some such compositions, the guide RNA comprises any one of SEQ ID NOS: 252- 339, optionally wherein the guide RNA comprises any one of SEQ ID NOS: 279, 288, 281, 294, 299, 310, 313, and 335, optionally wherein the guide RNA comprises any one of SEQ ID NOS: 279 and 288.

[0008] In some such compositions, the DNA-targeting segment comprises at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of SEQ ID NO: 78. In some suchcompositions, the DNA-targeting segment is at least 90% or at least 95% identical to SEQ ID NO: 78. In some such compositions, the DNA-targeting segment comprises SEQ ID NO: 78. In some such compositions, the DNA-targeting segment consists of SEQ ID NO: 78. In some such compositions, the guide RNA comprises SEQ ID NO: 387. In some such compositions, the guide RNA comprises SEQ ID NO: 299. In some such compositions, the DNA-targeting segment comprises at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of SEQ ID NO: 58. In some such compositions, the DNA-targeting segment is at least 90% or at least 95% identical to SEQ ID NO: 58. In some such compositions, the DNA-targeting segment comprises SEQ ID NO: 58. In some such compositions, the DNA-targeting segment consists of SEQ ID NO: 58. In some such compositions, the guide RNA comprises SEQ ID NO: 367. In some such compositions, the guide RNA comprises SEQ ID NO: 279.

[0009] In some such compositions, the ASS1 gene is a human ASS1 gene, and the guide RNA target sequence is in intron 1 of the human ASS1 gene. In some such compositions, the DNA- targeting segment comprises at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of the sequence set forth in any one of SEQ ID NOS: 430-517. In some such compositions, the DNA-targeting segment is at least 90% or at least 95% identical to the sequence set forth in any one of SEQ ID NOS: 430-517. In some such compositions, the DNA- targeting segment comprises any one of SEQ ID NOS: 430-517. In some such compositions, the DNA-targeting segment consists of any one of SEQ ID NOS: 430-517. In some such compositions, the guide RNA comprises any one of SEQ ID NOS: 606-693.

[0010] In some such compositions, the composition comprises the guide RNA in the form of RNA. In some such compositions, the guide RNA comprises at least one modification. In some such compositions, the at least one modification comprises a 2’-O-methyl-modified nucleotide. In some such compositions, the at least one modification comprises a phosphorothioate bond between nucleotides. In some such compositions, the at least one modification comprises a modification at one or more of the first five nucleotides at the 5’ end of the guide RNA. In some such compositions, the at least one modification comprises a modification at one or more of the last five nucleotides at the 3’ end of the guide RNA. In some such compositions, the at least one modification comprises phosphorothioate bonds between the first four nucleotides at the 5’ end of the guide RNA. In some such compositions, the at least one modification comprises phosphorothioate bonds between the last four nucleotides at the 3’ end of the guide RNA. Insome such compositions, the at least one modification comprises 2’-O-methyl-modified nucleotides at the first three nucleotides at the 5’ end of the guide RNA. In some such compositions, the at least one modification comprises 2’-O-methyl-modified nucleotides at the last three nucleotides at the 3’ end of the guide RNA. In some such compositions, the at least one modification comprises: (i) phosphorothioate bonds between the first four nucleotides at the 5’ end of the guide RNA; (ii) phosphorothioate bonds between the last four nucleotides at the 3’ end of the guide RNA; (iii) 2’-O-methyl-modified nucleotides at the first three nucleotides at the 5’ end of the guide RNA; and (iv) 2’-O-methyl-modified nucleotides at the last three nucleotides at the 3’ end of the guide RNA. In some such compositions, the guide RNA is a single guide RNA (sgRNA). In some such compositions, the guide RNA has the modification pattern set forth in SEQ ID NO: 429. In some such compositions, the guide RNA has the modification pattern set forth in SEQ ID NO: 30.

[0011] In some such compositions, the composition comprises the guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 387, and the guide RNA comprises: (i) phosphorothioate bonds between the first four nucleotides at the 5’ end of the guide RNA; (ii) phosphorothioate bonds between the last four nucleotides at the 3’ end of the guide RNA; (iii) 2’-O-methyl-modified nucleotides at the first three nucleotides at the 5’ end of the guide RNA; and (iv) 2’-O-methyl-modified nucleotides at the last three nucleotides at the 3’ end of the guide RNA. In some such compositions, the composition comprises the guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 387, and the guide RNA has the modification pattern set forth in SEQ ID NO: 429. In some such compositions, the composition comprises the guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 299, and the guide RNA comprises: (i) phosphorothioate bonds between the first four nucleotides at the 5’ end of the guide RNA; (ii) phosphorothioate bonds between the last four nucleotides at the 3’ end of the guide RNA; (iii) 2’-O-methyl-modified nucleotides at the first three nucleotides at the 5’ end of the guide RNA; and (iv) 2’-O-methyl-modified nucleotides at the last three nucleotides at the 3’ end of the guide RNA. In some such compositions, the composition comprises the guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 299, and the guide RNA has the modification pattern set forth in SEQ ID NO: 30. In some such compositions, the composition comprises the guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 367, and the guide RNA comprises: (i) phosphorothioate bonds between the first four nucleotides at the 5’end of the guide RNA; (ii) phosphorothioate bonds between the last four nucleotides at the 3’ end of the guide RNA; (iii) 2’-O-methyl-modified nucleotides at the first three nucleotides at the 5’ end of the guide RNA; and (iv) 2’-O-methyl-modified nucleotides at the last three nucleotides at the 3’ end of the guide RNA. In some such compositions, the composition comprises the guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 367, and the guide RNA has the modification pattern set forth in SEQ ID NO: 429. In some such compositions, the composition comprises the guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 279, and the guide RNA comprises: (i) phosphorothioate bonds between the first four nucleotides at the 5’ end of the guide RNA; (ii) phosphorothioate bonds between the last four nucleotides at the 3’ end of the guide RNA; (iii) 2’-O-methyl-modified nucleotides at the first three nucleotides at the 5’ end of the guide RNA; and (iv) 2’-O-methyl-modified nucleotides at the last three nucleotides at the 3’ end of the guide RNA. In some such compositions, the composition comprises the guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 279, and the guide RNA has the modification pattern set forth in SEQ ID NO: 30.

[0012] In some such compositions, the Cas protein is a Cas9 protein. In some such compositions, the Cas protein is, or is derived from, a Streptococcus pyogenes Cas9 protein. In some such compositions, the Cas protein comprises the sequence set forth in SEQ ID NO: 12.

[0013] In some such composition, the composition further comprises the Cas protein or a nucleic acid encoding the Cas protein. In some such compositions, the nucleic acid encoding the Cas protein is codon-optimized for expression in a mammalian cell or a human cell. In some such compositions, the composition comprises the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein. In some such compositions, the mRNA encoding the Cas protein comprises at least one modification. In some such compositions, the mRNA encoding the Cas protein is modified to comprise a modified uridine at one or more or all uridine positions. In some such compositions, the modified uridine is Nl-methyl-pseudouridine. In some such compositions, the mRNA encoding the Cas protein is fully substituted with Nl-methyl-pseudouridine. In some such compositions, the mRNA encoding the Cas protein comprises a 5’ cap. In some such compositions, the mRNA encoding the Cas protein comprises a poly(A) tail. In some such compositions, the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 250 or 249. In some such compositions, the composition comprises the nucleic acid encoding the Cas protein, wherein thenucleic acid comprises an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 250 or 249, and the mRNA encoding the Cas protein is fully substituted with Nl-methyl-pseudouri dine, comprises a 5’ cap, and comprises a poly(A) tail.

[0014] In some such compositions, the composition comprises the guide RNA in the form of RNA, and the guide RNA comprises SEQ ID NO: 387, and the composition comprises the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, and the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 250 or 249. In some such compositions, the composition comprises the guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 387, and the guide RNA comprises: (i) phosphorothioate bonds between the first four nucleotides at the 5’ end of the guide RNA; (ii) phosphorothioate bonds between the last four nucleotides at the 3’ end of the guide RNA; (iii) 2’-O-methyl-modified nucleotides at the first three nucleotides at the 5’ end of the guide RNA; and (iv) 2’-O-methyl-modified nucleotides at the last three nucleotides at the 3’ end of the guide RNA, and the composition comprises the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 250 or 249, and the mRNA encoding the Cas protein is fully substituted with Nl-methyl-pseudouri dine, comprises a 5’ cap, and comprises a poly(A) tail. In some such compositions, the composition comprises the guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 387, and the guide RNA has the modification pattern set forth in SEQ ID NO: 429, and the composition comprises the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 250 or 249, and the mRNA encoding the Cas protein is fully substituted with Nl-methyl-pseudouri dine, comprises a 5’ cap, and comprises a poly(A) tail. In some such compositions, the composition comprises the guide RNA in the form of RNA, and the guide RNA comprises SEQ ID NO: 299, and the composition comprises the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, and the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 250 or 249. In some such compositions, the composition comprises the guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 299, and the guide RNA comprises: (i) phosphorothioate bonds between the first fournucleotides at the 5’ end of the guide RNA; (ii) phosphorothioate bonds between the last four nucleotides at the 3’ end of the guide RNA; (iii) 2’-O-methyl-modified nucleotides at the first three nucleotides at the 5’ end of the guide RNA; and (iv) 2’-O-methyl-modified nucleotides at the last three nucleotides at the 3’ end of the guide RNA, and the composition comprises the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 250 or 249, and the mRNA encoding the Cas protein is fully substituted with Nl-methyl- pseudouridine, comprises a 5’ cap, and comprises a poly(A) tail. In some such compositions, the composition comprises the guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 299, and the guide RNA has the modification pattern set forth in SEQ ID NO: 30, and the composition comprises the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 250 or 249, and the mRNA encoding the Cas protein is fully substituted with Nl-methyl-pseudouridine, comprises a 5’ cap, and comprises a poly(A) tail. In some such compositions, the composition comprises the guide RNA in the form of RNA, and the guide RNA comprises SEQ ID NO: 367, and the composition comprises the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, and the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 250 or 249. In some such compositions, the composition comprises the guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 367, and the guide RNA comprises: (i) phosphorothioate bonds between the first four nucleotides at the 5’ end of the guide RNA; (ii) phosphorothioate bonds between the last four nucleotides at the 3’ end of the guide RNA; (iii) 2’-O-methyl-modified nucleotides at the first three nucleotides at the 5’ end of the guide RNA; and (iv) 2’-O-methyl-modified nucleotides at the last three nucleotides at the 3’ end of the guide RNA, and the composition comprises the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 250 or 249, and the mRNA encoding the Cas protein is fully substituted with Nl-methyl-pseudouridine, comprises a 5’ cap, and comprises a poly(A) tail. In some such compositions, the composition comprises the guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 367, and the guide RNA has the modification pattern set forth in SEQ ID NO: 429, and the composition comprises the nucleic acid encodingthe Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 250 or 249, and the mRNA encoding the Cas protein is fully substituted with Nl-methyl-pseudouri dine, comprises a 5’ cap, and comprises a poly(A) tail. In some such compositions, the composition comprises the guide RNA in the form of RNA, and the guide RNA comprises SEQ ID NO: 279, and the composition comprises the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, and the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 250 or 249. In some such compositions, the composition comprises the guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 279, and the guide RNA comprises: (i) phosphorothioate bonds between the first four nucleotides at the 5’ end of the guide RNA; (ii) phosphorothioate bonds between the last four nucleotides at the 3’ end of the guide RNA; (iii) 2’-O-methyl-modified nucleotides at the first three nucleotides at the 5’ end of the guide RNA; and (iv) 2’-O-methyl-modified nucleotides at the last three nucleotides at the 3’ end of the guide RNA, and the composition comprises the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 250 or 249, and the mRNA encoding the Cas protein is fully substituted with N1 -methyl - pseudouridine, comprises a 5’ cap, and comprises a poly(A) tail. In some such compositions, the composition comprises the guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 279, and the guide RNA has the modification pattern set forth in SEQ ID NO: 30, and the composition comprises the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 250 or 249, and the mRNA encoding the Cas protein is fully substituted with Nl-methyl-pseudouri dine, comprises a 5’ cap, and comprises a poly(A) tail.

[0015] In some such compositions, the Cas protein or the nucleic acid encoding the Cas protein and the guide RNA or the one or more DNAs encoding the guide RNA are associated with a lipid nanoparticle. In some such compositions, the lipid nanoparticle comprises a cationic lipid, a neutral lipid, a helper lipid, and a stealth lipid. In some such compositions, the cationic lipid is Lipid A. In some such compositions, the neutral lipid is DSPC. In some such compositions, the helper lipid is cholesterol. In some such compositions, the stealth lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG2k-DMG). In some such compositions, the cationic lipid is Lipid A, the neutral lipid is DSPC, the helper lipid is cholesterol, and the stealth lipid is PEG2k-DMG. In some such compositions, the lipid nanoparticle comprises four lipids at the following molar ratios: about 50 mol% Lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG.

[0016] In some such compositions, the ASS1 gene is a human ASS1 gene, the composition comprises the guide RNA in the form of RNA, and the guide RNA comprises SEQ ID NO: 387, the composition comprises the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, and the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 250 or 249, and the guide RNA and the mRNA encoding the Cas protein are associated with a lipid nanoparticle comprising Lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally at the following molar ratios: about 50 mol% Lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG. In some such compositions, the ASS1 gene is a human ASS1 gene, the composition comprises the guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 387, and the guide RNA comprises: (i) phosphorothioate bonds between the first four nucleotides at the 5’ end of the guide RNA; (ii) phosphorothioate bonds between the last four nucleotides at the 3’ end of the guide RNA; (iii) 2’-O-methyl-modified nucleotides at the first three nucleotides at the 5’ end of the guide RNA; and (iv) 2’-O-methyl-modified nucleotides at the last three nucleotides at the 3’ end of the guide RNA, the composition comprises the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 250 or 249, and the mRNA encoding the Cas protein is fully substituted with Nl-methyl-pseudouridine, comprises a 5’ cap, and comprises a poly(A) tail, and the guide RNA and the mRNA encoding the Cas protein are associated with a lipid nanoparticle comprising Lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally at the following molar ratios: about 50 mol% Lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG. In some such compositions, the ASS1 gene is a human ASS1 gene, the composition comprises the guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 387, and the guide RNA has the modification pattern set forth in SEQ ID NO: 429, the composition comprises the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, the mRNA encoding the Casprotein comprises the sequence set forth in SEQ ID NO: 250 or 249, and the mRNA encoding the Cas protein is fully substituted with Nl-methyl-pseudouri dine, comprises a 5’ cap, and comprises a poly(A) tail, and the guide RNA and the mRNA encoding the Cas protein are associated with a lipid nanoparticle comprising Lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally at the following molar ratios: about 50 mol% Lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG. In some such compositions, the ASS J gene is a human ASS1 gene, the composition comprises the guide RNA in the form of RNA, and the guide RNA comprises SEQ ID NO: 367, the composition comprises the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, and the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 250 or 249, and the guide RNA and the mRNA encoding the Cas protein are associated with a lipid nanoparticle comprising Lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally at the following molar ratios: about 50 mol% Lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG. In some such compositions, the ASS1 gene is a human ASS I gene, the composition comprises the guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 367, and the guide RNA comprises: (i) phosphorothioate bonds between the first four nucleotides at the 5’ end of the guide RNA; (ii) phosphorothioate bonds between the last four nucleotides at the 3’ end of the guide RNA; (iii) 2’-O-methyl-modified nucleotides at the first three nucleotides at the 5’ end of the guide RNA; and (iv) 2’-O-methyl- modified nucleotides at the last three nucleotides at the 3’ end of the guide RNA, the composition comprises the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 250 or 249, and the mRNA encoding the Cas protein is fully substituted with Nl-methyl-pseudouri dine, comprises a 5’ cap, and comprises a poly(A) tail, and the guide RNA and the mRNA encoding the Cas protein are associated with a lipid nanoparticle comprising Lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally at the following molar ratios: about 50 mol% Lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG. In some such compositions, the ASS1 gene is a human ASS I gene, the composition comprises the guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 367, and the guide RNA has the modification pattern set forth in SEQ ID NO: 429, the composition comprises the nucleic acid encoding the Cas protein, whereinthe nucleic acid comprises an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 250 or 249, and the mRNA encoding the Cas protein is fully substituted with Nl-methyl-pseudouri dine, comprises a 5’ cap, and comprises a poly(A) tail, and the guide RNA and the mRNA encoding the Cas protein are associated with a lipid nanoparticle comprising Lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally at the following molar ratios: about 50 mol% Lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG.

[0017] In some such compositions, the ASS1 gene is a human ASS1 gene, the composition comprises the guide RNA in the form of RNA, and the guide RNA comprises SEQ ID NO: 299, the composition comprises the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, and the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 250 or 249, and the guide RNA and the mRNA encoding the Cas protein are associated with a lipid nanoparticle comprising Lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally at the following molar ratios: about 50 mol% Lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG. In some such compositions, the ASS1 gene is a human ASS J gene, the composition comprises the guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 299, and the guide RNA comprises: (i) phosphorothioate bonds between the first four nucleotides at the 5’ end of the guide RNA; (ii) phosphorothioate bonds between the last four nucleotides at the 3’ end of the guide RNA; (iii) 2’-O-methyl-modified nucleotides at the first three nucleotides at the 5’ end of the guide RNA; and (iv) 2’-O-methyl-modified nucleotides at the last three nucleotides at the 3’ end of the guide RNA, the composition comprises the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 250 or 249, and the mRNA encoding the Cas protein is fully substituted with Nl-methyl-pseudouri dine, comprises a 5’ cap, and comprises a poly(A) tail, and the guide RNA and the mRNA encoding the Cas protein are associated with a lipid nanoparticle comprising Lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally at the following molar ratios: about 50 mol% Lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG. In some such compositions, the &SV gene is a human ASS1 gene, the composition comprises the guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 299, and the guide RNA has the modification pattern set forth inSEQ ID NO: 30, the composition comprises the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 250 or 249, and the mRNA encoding the Cas protein is fully substituted with Nl-methyl-pseudouri dine, comprises a 5’ cap, and comprises a poly(A) tail, and the guide RNA and the mRNA encoding the Cas protein are associated with a lipid nanoparticle comprising Lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally at the following molar ratios: about 50 mol% Lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG. In some such compositions, the ASS J gene is a human ASS1 gene, the composition comprises the guide RNA in the form of RNA, and the guide RNA comprises SEQ ID NO: 279, the composition comprises the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, and the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 250 or 249, and the guide RNA and the mRNA encoding the Cas protein are associated with a lipid nanoparticle comprising Lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally at the following molar ratios: about 50 mol% Lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG. In some such compositions, the ASS I gene is a human ASS1 gene, the composition comprises the guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 279, and the guide RNA comprises: (i) phosphorothioate bonds between the first four nucleotides at the 5’ end of the guide RNA; (ii) phosphorothioate bonds between the last four nucleotides at the 3’ end of the guide RNA; (iii) 2’-O-methyl-modified nucleotides at the first three nucleotides at the 5’ end of the guide RNA; and (iv) 2’-O-methyl- modified nucleotides at the last three nucleotides at the 3’ end of the guide RNA, the composition comprises the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 250 or 249, and the mRNA encoding the Cas protein is fully substituted with Nl-methyl-pseudouri dine, comprises a 5’ cap, and comprises a poly(A) tail, and the guide RNA and the mRNA encoding the Cas protein are associated with a lipid nanoparticle comprising Lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally at the following molar ratios: about 50 mol% Lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG. In some such compositions, the ASS1 gene is a human ASS I gene, the composition comprises the guide RNA in the form of RNA, the guideRNA comprises SEQ ID NO: 279, and the guide RNA has the modification pattern set forth in SEQ ID NO: 30, the composition comprises the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 250 or 249, and the mRNA encoding the Cas protein is fully substituted with Nl-methyl-pseudouri dine, comprises a 5’ cap, and comprises a poly(A) tail, and the guide RNA and the mRNA encoding the Cas protein are associated with a lipid nanoparticle comprising Lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally at the following molar ratios: about 50 mol% Lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG.

[0018] In another aspect, provided are compositions comprising a nucleic acid construct comprising a first argininosuccinate synthase protein coding sequence. In some such compositions, the first argininosuccinate synthase protein coding sequence is a human argininosuccinate synthase protein coding sequence. In some such compositions, the first argininosuccinate synthase protein coding sequence comprises human argininosuccinate synthase exons 2-14 or exons 3-14. In some such compositions, the first argininosuccinate synthase protein coding sequence comprises human argininosuccinate synthase exons 3-14. In some such compositions, the first argininosuccinate synthase protein coding sequence encodes a protein at last 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 230. In some such compositions, the first argininosuccinate synthase protein coding sequence encodes a protein comprising the sequence set forth in SEQ ID NO: 230. In some such compositions, the first argininosuccinate synthase protein coding sequence encodes a protein consisting of the sequence set forth in SEQ ID NO: 230. In some such compositions, the first argininosuccinate synthase protein coding sequence is at least 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOS: 705, 709, and 713. In some such compositions, the first argininosuccinate synthase protein coding sequence is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOS: 706 and 710. In some such compositions, the first argininosuccinate synthase protein coding sequence is at least 99% identical to any one of SEQ ID NOS: 705, 706, 709, 710, and 713. In some such compositions, the first argininosuccinate synthase protein coding sequence comprises, consists essentially of, or consists of any one of SEQ ID NOS: 705, 706, 709, 710, and 713. In some such compositions, the first argininosuccinate synthase protein coding sequence is at least 96%, 97%, 98%, or 99%identical to SEQ ID NO: 705. In some such compositions, the first argininosuccinate synthase protein coding sequence is at least 99% identical to SEQ ID NO: 705. In some such compositions, the first argininosuccinate synthase protein coding sequence comprises, consists essentially of, or consists of SEQ ID NO: 705. In some such compositions, the first argininosuccinate synthase protein coding sequence is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 706. In some such compositions, the first argininosuccinate synthase protein coding sequence is at least 99% identical to SEQ ID NO: 706. In some such compositions, the first argininosuccinate synthase protein coding sequence comprises, consists essentially of, or consists of SEQ ID NO: 706.

[0019] In some such compositions, the first argininosuccinate synthase protein coding sequence comprises human argininosuccinate synthase exons 2-14. In some such compositions, the first argininosuccinate synthase protein coding sequence encodes a protein at last 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 227. In some such compositions, the first argininosuccinate synthase protein coding sequence encodes a protein comprising the sequence set forth in SEQ ID NO: 227. In some such compositions, the first argininosuccinate synthase protein coding sequence encodes a protein consisting of the sequence set forth in SEQ ID NO: 227.

[0020] In some such compositions, the nucleic acid construct comprises a splice acceptor upstream of the first argininosuccinate synthase protein coding sequence. In some such compositions, the nucleic acid construct comprises a polyadenylation signal downstream of the first argininosuccinate synthase protein coding sequence. In some such compositions, the nucleic acid construct comprises a splice acceptor upstream of the first argininosuccinate synthase protein coding sequence, and the nucleic acid construct comprises a polyadenylation signal downstream of the first argininosuccinate synthase protein coding sequence. In some such compositions, the nucleic acid construct does not comprise homology arms. In some such compositions, the nucleic acid construct comprises homology arms. In some such compositions, the nucleic acid construct does not comprise a promoter that drives the expression of the argininosuccinate synthase protein.

[0021] In some such compositions, the nucleic acid construct is a bidirectional construct. In some such compositions, the nucleic acid construct comprises the first argininosuccinate synthase protein coding sequence and a reverse complement of a second argininosuccinatesynthase protein coding sequence. In some such compositions, the first argininosuccinate synthase protein coding sequence and the second argininosuccinate synthase protein coding sequence are different but encode the same argininosuccinate synthase protein sequence. In some such compositions, the first argininosuccinate synthase protein coding sequence and the second argininosuccinate synthase protein coding sequence each encode a protein at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 230. In some such compositions, the first argininosuccinate synthase protein coding sequence and the second argininosuccinate synthase protein coding sequence each encode a protein comprising the sequence set forth in SEQ ID NO: 230. In some such compositions, the first argininosuccinate synthase protein coding sequence and the second argininosuccinate synthase protein coding sequence each encode a protein consisting of the sequence set forth in SEQ ID NO: 230. In some such compositions, the first argininosuccinate synthase protein coding sequence is at least 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOS: 705, 709, and 713. Optionally, the first argininosuccinate synthase protein coding sequence is at least 96%, 97%, 98%, or 99% identical to SEQ ID NO: 705. In some such compositions, the first argininosuccinate synthase protein coding sequence is at least 99% identical to any one of SEQ ID NOS: 705, 709, and 713. Optionally, the first argininosuccinate synthase protein coding sequence is at least 99% identical to SEQ ID NO: 705. In some such compositions, the first argininosuccinate synthase protein coding sequence comprises, consists essentially of, or consists of any one of SEQ ID NOS: 705, 709, and 713. Optionally, the first argininosuccinate synthase protein coding sequence comprises, consists essentially of, or consists of SEQ ID NO: 705. In some such compositions, the second argininosuccinate synthase protein coding sequence is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOS: 706 and 710. Optionally, the second argininosuccinate synthase protein coding sequence is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 706. In some such compositions, the second argininosuccinate synthase protein coding sequence is at least 99% identical to any one of SEQ ID NOS: 706 and 710. Optionally, the second argininosuccinate synthase protein coding sequence is at least 99% identical to SEQ ID NO: 706. In some such compositions, the second argininosuccinate synthase protein coding sequence comprises, consists essentially of, or consists of any one of SEQ ID NOS: 706 and 710. Optionally, the second argininosuccinate synthase protein coding sequence comprises, consists essentially of, or consists of SEQ ID NO: 706. Insome such compositions, the first argininosuccinate synthase protein coding sequence is at least 96%, 97%, 98%, or 99% identical to SEQ ID NO: 705, and the second argininosuccinate synthase protein coding sequence is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 706. In some such compositions, the first argininosuccinate synthase protein coding sequence is at least 99% identical to SEQ ID NO: 705, and the second argininosuccinate synthase protein coding sequence is at least 99% identical to SEQ ID NO: 706. In some such compositions, the first argininosuccinate synthase protein coding sequence comprises, consists essentially of, or consists of SEQ ID NO: 705, and the second argininosuccinate synthase protein coding sequence comprises, consists essentially of, or consists of SEQ ID NO: 706. In some such compositions, the first argininosuccinate synthase protein coding sequence is at least 96%, 97%, 98%, or 99% identical to SEQ ID NO: 709, and the second argininosuccinate synthase protein coding sequence is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 710. In some such compositions, the first argininosuccinate synthase protein coding sequence is at least 99% identical to SEQ ID NO: 709, and the second argininosuccinate synthase protein coding sequence is at least 99% identical to SEQ ID NO: 710. In some such compositions, the first argininosuccinate synthase protein coding sequence comprises, consists essentially of, or consists of SEQ ID NO: 709, and the second argininosuccinate synthase protein coding sequence comprises, consists essentially of, or consists of SEQ ID NO: 710. In some such compositions, the first argininosuccinate synthase protein coding sequence is at least 96%, 97%, 98%, or 99% identical to SEQ ID NO: 713, and the second argininosuccinate synthase protein coding sequence is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 706. In some such compositions, the first argininosuccinate synthase protein coding sequence is at least 99% identical to SEQ ID NO: 713, and the second argininosuccinate synthase protein coding sequence is at least 99% identical to SEQ ID NO: 706. In some such compositions, the first argininosuccinate synthase protein coding sequence comprises, consists essentially of, or consists of SEQ ID NO: 713, and the second argininosuccinate synthase protein coding sequence comprises, consists essentially of, or consists of SEQ ID NO: 706.

[0022] In some such compositions, the first argininosuccinate synthase protein coding sequence and the second argininosuccinate synthase protein coding sequence each encode a protein at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 227. In some such compositions, the first argininosuccinatesynthase protein coding sequence and the second argininosuccinate synthase protein coding sequence each encode a protein comprising the sequence set forth in SEQ ID NO: 227. In some such compositions, the first argininosuccinate synthase protein coding sequence and the second argininosuccinate synthase protein coding sequence each encode a protein consisting of the sequence set forth in SEQ ID NO: 227. In some such compositions, the nucleic acid construct comprises from 5’ to 3’: a first splice acceptor, the first argininosuccinate synthase protein coding sequence, a first polyadenylation signal, a reverse complement of a second polyadenylation signal, the reverse complement of the second argininosuccinate synthase protein coding sequence, and a reverse complement of a second splice acceptor, or wherein the nucleic acid construct comprises from 5’ to 3’ : a first splice acceptor, the second argininosuccinate synthase protein coding sequence, a first polyadenylation signal, a reverse complement of a second polyadenylation signal, the reverse complement of the first argininosuccinate synthase protein coding sequence, and a reverse complement of a second splice acceptor. In some such compositions, the first argininosuccinate synthase protein coding sequence and the second argininosuccinate synthase protein coding sequence are different but encode the same argininosuccinate synthase protein sequence, and wherein the first polyadenylation signal and the second polyadenylation signal are different.

[0023] In some such compositions, the nucleic acid construct is a bidirectional construct comprising the first argininosuccinate synthase protein coding sequence and a reverse complement of a second argininosuccinate synthase protein coding sequence, the first argininosuccinate synthase protein coding sequence and the second argininosuccinate synthase protein coding sequence are different and each encode a protein comprising or consisting of the sequence set forth in SEQ ID NO: 230, the nucleic acid construct comprises from 5’ to 3’ : a first splice acceptor, the first argininosuccinate synthase protein coding sequence, a first polyadenylation signal, a reverse complement of a second polyadenylation signal, the reverse complement of the second argininosuccinate synthase protein coding sequence, and a reverse complement of a second splice acceptor, the nucleic acid construct does not comprise a promoter that drives the expression of the argininosuccinate synthase protein, and the nucleic acid construct does not comprise homology arms. In some such compositions, the nucleic acid construct is a bidirectional construct comprising the first argininosuccinate synthase protein coding sequence and a reverse complement of a second argininosuccinate synthase proteincoding sequence, the first argininosuccinate synthase protein coding sequence and the second argininosuccinate synthase protein coding sequence are different and each encode a protein comprising or consisting of the sequence set forth in SEQ ID NO: 230, the first argininosuccinate synthase protein coding sequence comprises SEQ ID NO: 705, the second argininosuccinate synthase protein coding sequence comprises SEQ ID NO: 706, the nucleic acid construct comprises from 5’ to 3’: a first splice acceptor, the first argininosuccinate synthase protein coding sequence, a first polyadenylation signal, a reverse complement of a second polyadenylation signal, the reverse complement of the second argininosuccinate synthase protein coding sequence, and a reverse complement of a second splice acceptor, the nucleic acid construct does not comprise a promoter that drives the expression of the argininosuccinate synthase protein, and the nucleic acid construct does not comprise homology arms. In some such compositions, the nucleic acid construct is a bidirectional construct comprising the first argininosuccinate synthase protein coding sequence and a reverse complement of a second argininosuccinate synthase protein coding sequence, the first argininosuccinate synthase protein coding sequence and the second argininosuccinate synthase protein coding sequence are different and each encode a protein comprising or consisting of the sequence set forth in SEQ ID NO: 230, the first argininosuccinate synthase protein coding sequence comprises SEQ ID NO: 709, the second argininosuccinate synthase protein coding sequence comprises SEQ ID NO: 710, the nucleic acid construct comprises from 5’ to 3’: a first splice acceptor, the first argininosuccinate synthase protein coding sequence, a first polyadenylation signal, a reverse complement of a second polyadenylation signal, the reverse complement of the second argininosuccinate synthase protein coding sequence, and a reverse complement of a second splice acceptor, the nucleic acid construct does not comprise a promoter that drives the expression of the argininosuccinate synthase protein, and the nucleic acid construct does not comprise homology arms. In some such compositions, the nucleic acid construct is a bidirectional construct comprising the first argininosuccinate synthase protein coding sequence and a reverse complement of a second argininosuccinate synthase protein coding sequence, the first argininosuccinate synthase protein coding sequence and the second argininosuccinate synthase protein coding sequence are different and each encode a protein comprising or consisting of the sequence set forth in SEQ ID NO: 230, the first argininosuccinate synthase protein coding sequence comprises SEQ ID NO: 713, the second argininosuccinate synthase protein coding sequence comprises SEQ ID NO: 706, thenucleic acid construct comprises from 5’ to 3’: a first splice acceptor, the first argininosuccinate synthase protein coding sequence, a first polyadenylation signal, a reverse complement of a second polyadenylation signal, the reverse complement of the second argininosuccinate synthase protein coding sequence, and a reverse complement of a second splice acceptor, the nucleic acid construct does not comprise a promoter that drives the expression of the argininosuccinate synthase protein, and the nucleic acid construct does not comprise homology arms. In some such compositions, the nucleic acid construct is a bidirectional construct comprising the first argininosuccinate synthase protein coding sequence and a reverse complement of a second argininosuccinate synthase protein coding sequence, the first argininosuccinate synthase protein coding sequence and the second argininosuccinate synthase protein coding sequence are different and each encode a protein comprising or consisting of the sequence set forth in SEQ ID NO: 227, the nucleic acid construct comprises from 5’ to 3’: a first splice acceptor, the first argininosuccinate synthase protein coding sequence, a first polyadenylation signal, a reverse complement of a second poly adenylation signal, the reverse complement of the second argininosuccinate synthase protein coding sequence, and a reverse complement of a second splice acceptor, the nucleic acid construct does not comprise a promoter that drives the expression of the argininosuccinate synthase protein, and the nucleic acid construct does not comprise homology arms.

[0024] In some such compositions, the nucleic acid construct is a unidirectional construct. In some such compositions, the nucleic acid construct is a unidirectional construct comprising the first argininosuccinate synthase protein coding sequence, the first argininosuccinate synthase protein coding sequence encodes a protein comprising or consisting of the sequence set forth in SEQ ID NO: 230, the nucleic acid construct comprises from 5’ to 3’: a splice acceptor, the first argininosuccinate synthase protein coding sequence, and a polyadenylation signal, the nucleic acid construct does not comprise a promoter that drives the expression of the argininosuccinate synthase protein, and the nucleic acid construct does not comprise homology arms. In some such compositions, the nucleic acid construct is a unidirectional construct comprising the first argininosuccinate synthase protein coding sequence, the first argininosuccinate synthase protein coding sequence encodes a protein comprising or consisting of the sequence set forth in SEQ ID NO: 227, the nucleic acid construct comprises from 5’ to 3’ : a splice acceptor, the first argininosuccinate synthase protein coding sequence, and a polyadenylation signal, the nucleicacid construct does not comprise a promoter that drives the expression of the argininosuccinate synthase protein, and the nucleic acid construct does not comprise homology arms.

[0025] In some such compositions, the nucleic acid construct is single- stranded DNA or double-stranded DNA. In some such compositions, the nucleic acid construct is single-stranded DNA. In some such compositions, the nucleic acid construct is in a nucleic acid vector or a lipid nanoparticle. In some such compositions, the nucleic acid construct is in the nucleic acid vector, optionally wherein the nucleic acid vector is a viral vector. In some such compositions, the nucleic acid vector is an adeno-associated viral (AAV) vector, optionally wherein the nucleic acid construct is flanked by inverted terminal repeats (ITRs) on each end, optionally wherein the ITR on at least one end comprises, consists essentially of, or consists of SEQ ID NO: 198, and optionally wherein the ITR on each end comprises, consists essentially of, or consists of SEQ ID NO: 198, or optionally wherein the ITR on at least one end comprises, consists essentially of, or consists of SEQ ID NO: 196, and optionally wherein the ITR on each end comprises, consists essentially of, or consists of SEQ ID NO: 196. In some such compositions, the AAV vector is a single-stranded AAV (ssAAV) vector. In some such compositions, the AAV vector is derived from an AAV8 vector, an AAV3B vector, an AAV5 vector, an AAV6 vector, an AAV7 vector, an AAV9 vector, an AAVrh.74 vector, or an AAVhu.37 vector. In some such compositions, the AAV vector is a recombinant AAV8 (rAAV8) vector. In some such compositions, the AAV vector is a single-stranded rAAV8 vector. In some such compositions, the AAV vector is a recombinant AAV5 (rAAV5) vector. In some such compositions, the AAV vector is a singlestranded rAAV5 vector.

[0026] In some such compositions, the nucleic acid construct is a bidirectional construct comprising the first argininosuccinate synthase protein coding sequence and a reverse complement of a second argininosuccinate synthase protein coding sequence, the first argininosuccinate synthase protein coding sequence and the second argininosuccinate synthase protein coding sequence are different and each encode a protein comprising or consisting of the sequence set forth in SEQ ID NO: 230, the nucleic acid construct comprises from 5’ to 3’ : a first splice acceptor, the first argininosuccinate synthase protein coding sequence, a first polyadenylation signal, a reverse complement of a second polyadenylation signal, the reverse complement of the second argininosuccinate synthase protein coding sequence, and a reverse complement of a second splice acceptor, the nucleic acid construct does not comprise a promoterthat drives the expression of the argininosuccinate synthase protein, the nucleic acid construct does not comprise homology arms, and the nucleic acid construct is in a single-stranded rAAV8 vector, optionally wherein the nucleic acid construct is flanked by inverted terminal repeats (ITRs) on each end, optionally wherein the ITR on at least one end comprises, consists essentially of, or consists of SEQ ID NO: 198, and optionally wherein the ITR on each end comprises, consists essentially of, or consists of SEQ ID NO: 198, or optionally wherein the ITR on at least one end comprises, consists essentially of, or consists of SEQ ID NO: 196, and optionally wherein the ITR on each end comprises, consists essentially of, or consists of SEQ ID NO: 196. In some such compositions, the nucleic acid construct is a bidirectional construct comprising the first argininosuccinate synthase protein coding sequence and a reverse complement of a second argininosuccinate synthase protein coding sequence, the first argininosuccinate synthase protein coding sequence and the second argininosuccinate synthase protein coding sequence are different and each encode a protein comprising or consisting of the sequence set forth in SEQ ID NO: 230, the nucleic acid construct comprises from 5’ to 3’ : a first splice acceptor, the first argininosuccinate synthase protein coding sequence, a first polyadenylation signal, a reverse complement of a second polyadenylation signal, the reverse complement of the second argininosuccinate synthase protein coding sequence, and a reverse complement of a second splice acceptor, the nucleic acid construct does not comprise a promoter that drives the expression of the argininosuccinate synthase protein, the nucleic acid construct does not comprise homology arms, and the nucleic acid construct is in a single-stranded rAAV5 vector, optionally wherein the nucleic acid construct is flanked by inverted terminal repeats (ITRs) on each end, optionally wherein the ITR on at least one end comprises, consists essentially of, or consists of SEQ ID NO: 198, and optionally wherein the ITR on each end comprises, consists essentially of, or consists of SEQ ID NO: 198, or optionally wherein the ITR on at least one end comprises, consists essentially of, or consists of SEQ ID NO: 196, and optionally wherein the ITR on each end comprises, consists essentially of, or consists of SEQ ID NO: 196. In some such compositions, the nucleic acid construct is a bidirectional construct comprising the first argininosuccinate synthase protein coding sequence and a reverse complement of a second argininosuccinate synthase protein coding sequence, the first argininosuccinate synthase protein coding sequence and the second argininosuccinate synthase protein coding sequence are different and each encode a protein comprising or consisting of thesequence set forth in SEQ ID NO: 230, the first argininosuccinate synthase protein coding sequence comprises SEQ ID NO: 705, the second argininosuccinate synthase protein coding sequence comprises SEQ ID NO: 706, the nucleic acid construct comprises from 5’ to 3’: a first splice acceptor, the first argininosuccinate synthase protein coding sequence, a first polyadenylation signal, a reverse complement of a second polyadenylation signal, the reverse complement of the second argininosuccinate synthase protein coding sequence, and a reverse complement of a second splice acceptor, the nucleic acid construct does not comprise a promoter that drives the expression of the argininosuccinate synthase protein, the nucleic acid construct does not comprise homology arms, and the nucleic acid construct is in a single-stranded rAAV8 vector, optionally wherein the nucleic acid construct is flanked by inverted terminal repeats (ITRs) on each end, optionally wherein the ITR on at least one end comprises, consists essentially of, or consists of SEQ ID NO: 198, and optionally wherein the ITR on each end comprises, consists essentially of, or consists of SEQ ID NO: 198, or optionally wherein the ITR on at least one end comprises, consists essentially of, or consists of SEQ ID NO: 196, and optionally wherein the ITR on each end comprises, consists essentially of, or consists of SEQ ID NO: 196. In some such compositions, the nucleic acid construct is a bidirectional construct comprising the first argininosuccinate synthase protein coding sequence and a reverse complement of a second argininosuccinate synthase protein coding sequence, the first argininosuccinate synthase protein coding sequence and the second argininosuccinate synthase protein coding sequence are different and each encode a protein comprising or consisting of the sequence set forth in SEQ ID NO: 230, the first argininosuccinate synthase protein coding sequence comprises SEQ ID NO: 709, the second argininosuccinate synthase protein coding sequence comprises SEQ ID NO: 710, the nucleic acid construct comprises from 5’ to 3’: a first splice acceptor, the first argininosuccinate synthase protein coding sequence, a first polyadenylation signal, a reverse complement of a second polyadenylation signal, the reverse complement of the second argininosuccinate synthase protein coding sequence, and a reverse complement of a second splice acceptor, the nucleic acid construct does not comprise a promoter that drives the expression of the argininosuccinate synthase protein, the nucleic acid construct does not comprise homology arms, and the nucleic acid construct is in a single-stranded rAAV8 vector, optionally wherein the nucleic acid construct is flanked by inverted terminal repeats (ITRs) on each end, optionally wherein the ITR on at least one end comprises, consistsessentially of, or consists of SEQ ID NO: 198, and optionally wherein the ITR on each end comprises, consists essentially of, or consists of SEQ ID NO: 198, or optionally wherein the ITR on at least one end comprises, consists essentially of, or consists of SEQ ID NO: 196, and optionally wherein the ITR on each end comprises, consists essentially of, or consists of SEQ ID NO: 196. In some such compositions, the nucleic acid construct is a bidirectional construct comprising the first argininosuccinate synthase protein coding sequence and a reverse complement of a second argininosuccinate synthase protein coding sequence, the first argininosuccinate synthase protein coding sequence and the second argininosuccinate synthase protein coding sequence are different and each encode a protein comprising or consisting of the sequence set forth in SEQ ID NO: 230, the first argininosuccinate synthase protein coding sequence comprises SEQ ID NO: 713, the second argininosuccinate synthase protein coding sequence comprises SEQ ID NO: 706, the nucleic acid construct comprises from 5’ to 3’: a first splice acceptor, the first argininosuccinate synthase protein coding sequence, a first polyadenylation signal, a reverse complement of a second polyadenylation signal, the reverse complement of the second argininosuccinate synthase protein coding sequence, and a reverse complement of a second splice acceptor, the nucleic acid construct does not comprise a promoter that drives the expression of the argininosuccinate synthase protein, the nucleic acid construct does not comprise homology arms, and the nucleic acid construct is in a single-stranded rAAV8 vector, optionally wherein the nucleic acid construct is flanked by inverted terminal repeats (ITRs) on each end, optionally wherein the ITR on at least one end comprises, consists essentially of, or consists of SEQ ID NO: 198, and optionally wherein the ITR on each end comprises, consists essentially of, or consists of SEQ ID NO: 198, or optionally wherein the ITR on at least one end comprises, consists essentially of, or consists of SEQ ID NO: 196, and optionally wherein the ITR on each end comprises, consists essentially of, or consists of SEQ ID NO: 196. In some such compositions, the nucleic acid construct is a bidirectional construct comprising the first argininosuccinate synthase protein coding sequence and a reverse complement of a second argininosuccinate synthase protein coding sequence, the first argininosuccinate synthase protein coding sequence and the second argininosuccinate synthase protein coding sequence are different and each encode a protein comprising or consisting of the sequence set forth in SEQ ID NO: 227, the nucleic acid construct comprises from 5’ to 3’ : a first splice acceptor, the first argininosuccinate synthase protein coding sequence, a firstpolyadenylation signal, a reverse complement of a second polyadenylation signal, the reverse complement of the second argininosuccinate synthase protein coding sequence, and a reverse complement of a second splice acceptor, the nucleic acid construct does not comprise a promoter that drives the expression of the argininosuccinate synthase protein, the nucleic acid construct does not comprise homology arms, and the nucleic acid construct is in a single-stranded rAAV8 vector, optionally wherein the nucleic acid construct is flanked by inverted terminal repeats (ITRs) on each end, optionally wherein the ITR on at least one end comprises, consists essentially of, or consists of SEQ ID NO: 198, and optionally wherein the ITR on each end comprises, consists essentially of, or consists of SEQ ID NO: 198, or optionally wherein the ITR on at least one end comprises, consists essentially of, or consists of SEQ ID NO: 196, and optionally wherein the ITR on each end comprises, consists essentially of, or consists of SEQ ID NO: 196.

[0027] In another aspect is a combination comprising: (I) any of the above compositions comprising the nuclease agent that targets the nuclease target site in the 4557 gene; and (II) any of the above compositions comprising the nucleic acid construct comprising the first argininosuccinate synthase protein coding sequence.

[0028] In another aspect, provided are any of the above compositions or any of the above combinations for use in a method of introducing an argininosuccinate synthase (ASS1) nucleic acid into a cell, a method of integrating an 24557 nucleic acid construct into a target gene in a cell, or a method of expressing argininosuccinate synthase in a cell. In some such compositions or combinations for use, the cell is a neonatal cell. In some such compositions or combinations for use, the neonatal cell is from a human neonatal subject within 24 weeks after birth, is from a human neonatal subject within 12 weeks after birth, is from a human neonatal subject within 8 weeks after birth, is from a human neonatal subject within 4 weeks after birth, is from a human neonatal subject within 2 weeks after birth, or is from a human neonatal subject within 1 week after birth. In some such compositions or combinations for use, the cell is not a neonatal cell.

[0029] In another aspect, provided are uses any of the above compositions or any of the above combinations in the preparation of a reagent for introducing an argininosuccinate synthase (ASS J) nucleic acid into a cell, integrating an ASS I nucleic acid construct into a target gene in a cell, or expressing argininosuccinate synthase in a cell. In some such uses, the cell is a neonatal cell. In some such uses, the neonatal cell is from a human neonatal subject within 24 weeks afterbirth, is from a human neonatal subject within 12 weeks after birth, is from a human neonatal subject within 8 weeks after birth, is from a human neonatal subject within 4 weeks after birth, is from a human neonatal subject within 2 weeks after birth, or is from a human neonatal subject within 1 week after birth. In some such uses, the cell is not a neonatal cell.

[0030] In another aspect, provided are any of the above compositions or any of the above combinations for use in a method of treating an argininosuccinate synthase deficiency in a subject. In another aspect, provided are any of the above compositions or any of the above combinations for use in a method of treating citrullinemia type I in a subject. In some such compositions or combinations for use, the subject is a neonatal subject. In some such compositions or combinations for use, the neonatal subject is a human neonatal subject within 24 weeks after birth, is a human neonatal subject within 12 weeks after birth, is a human neonatal subject within 8 weeks after birth, is a human neonatal subject within 4 weeks after birth, is a human neonatal subject within 2 weeks after birth, or is a human neonatal subject within 1 week after birth. In some such compositions or combinations for use, the subject is not a neonatal subject.

[0031] In another aspect, provided are uses of any of the above compositions or any of the above combinations in the preparation of a medicament treating an argininosuccinate synthase deficiency in a subject. In another aspect, provided are uses of any of the above compositions or any of the above combinations in the preparation of a medicament treating citrullinemia type I in a subject. In some such uses, the subject is a neonatal subject. In some such uses, the neonatal subject is a human neonatal subject within 24 weeks after birth, is a human neonatal subject within 12 weeks after birth, is a human neonatal subject within 8 weeks after birth, is a human neonatal subject within 4 weeks after birth, is a human neonatal subject within 2 weeks after birth, or is a human neonatal subject within 1 week after birth. In some such uses, the subject is not a neonatal subject.

[0032] In another aspect, provided are cells comprising any of the above compositions or any of the above combinations. In some such cells, the nucleic acid construct is integrated into an endogenous target gene locus, and wherein argininosuccinate synthase protein is expressed from the endogenous target gene locus, or wherein the nucleic acid construct is integrated into intron 1 or intron 2 of an endogenous argininosuccinate synthase (ASS1) locus, and wherein argininosuccinate synthase protein is expressed from the endogenous ASSJ locus. In some suchcells, the cell is a human cell, optionally wherein the nucleic acid construct is integrated into intron 2 of the endogenous ASS1 locus. In some such cells, the cell is a liver cell. In some such cells, the liver cell is a hepatocyte. In some such cells, the cell is a neonatal cell. In some such cells, the neonatal cell is from a human neonatal subject within 24 weeks after birth, is from a human neonatal subject within 12 weeks after birth, is from a human neonatal subject within 8 weeks after birth, is from a human neonatal subject within 4 weeks after birth, is from a human neonatal subject within 2 weeks after birth, or is from a human neonatal subject within 1 week after birth. In some such cells, the cell is not a neonatal cell. In some such cells, the cell is ex vivo or in vitro. In some such cells, the cell is in vivo.

[0033] In another aspect, provided are methods of introducing an argininosuccinate synthase nucleic acid into a cell, comprising administering any of the above combinations to the cell. In another aspect, provided are methods of integrating an argininosuccinate synthase nucleic acid construct into a target gene in a cell, comprising administering any of the above combinations to the cell, wherein the nuclease agent cleaves the nuclease target site in the target gene to create a cleavage site, the nucleic acid construct is inserted into the cleavage site to create a modified target gene, and argininosuccinate synthase protein is expressed from the modified target gene. In another aspect, provided are methods of expressing argininosuccinate synthase in a cell, comprising administering any of the above combinations to the cell, wherein the nuclease agent cleaves the nuclease target site in the target gene to create a cleavage site, the nucleic acid construct is inserted into the cleavage site to create a modified target gene, and argininosuccinate synthase protein is expressed from the modified target gene.

[0034] In some such methods, the nuclease agent comprises: (a) a Cas protein or a nucleic acid encoding the Cas protein; and (b) a guide RNA or one or more DNAs encoding the guide RNA, wherein the guide RNA comprises a DNA-targeting segment that targets a guide RNA target sequence, and wherein the guide RNA binds to the Cas protein and targets the Cas protein to the guide RNA target sequence. In some such methods, the nucleic acid construct, the Cas protein or the nucleic acid encoding the Cas protein, and the guide RNA or the one or more DNAs encoding the guide RNAs are administered simultaneously. In some such methods, the nucleic acid construct is not administered simultaneously with the Cas protein or the nucleic acid encoding the Cas protein and the guide RNA or the one or more DNAs encoding the guide RNAs.

[0035] In some such methods, the cell is a liver cell. In some such methods, the cell is a hepatocyte. In some such methods, the cell is a human cell. In some such methods, the cell is a neonatal cell. In some such methods, the neonatal cell is from a human neonatal subject within 24 weeks after birth, a human neonatal subject within 12 weeks after birth, a human neonatal subject within 8 weeks after birth, a human neonatal subject within 4 weeks after birth, a human neonatal subject within 2 weeks after birth, or a human neonatal subject within 1 week after birth. In some such methods, the cell is not a neonatal cell. In some such methods, the cell is in vivo. In some such methods, the cell is in vitro or ex vivo.

[0036] In another aspect, provided are methods of treating an argininosuccinate synthase deficiency in a subject, comprising administering any of the above combinations to the subject. In another aspect, provided are methods of treating citrullinemia type I in a subject, comprising administering any of the above combinations to the subject. In another aspect, provided are methods of preventing or inhibiting hyperammonemia in a subject having citrullinemia type I, comprising administering any of the above combinations to the subject.

[0037] In some such methods, the nuclease agent comprises: (a) a Cas protein or a nucleic acid encoding the Cas protein; and (b) a guide RNA or one or more DNAs encoding the guide RNA, wherein the guide RNA comprises a DNA-targeting segment that targets a guide RNA target sequence, and wherein the guide RNA binds to the Cas protein and targets the Cas protein to the guide RNA target sequence. In some such methods, the nucleic acid construct, the Cas protein or the nucleic acid encoding the Cas protein, and the guide RNA or the one or more DNAs encoding the guide RNAs are administered simultaneously. In some such methods, the nucleic acid construct is not administered simultaneously with the Cas protein or the nucleic acid encoding the Cas protein and the guide RNA or the one or more DNAs encoding the guide RNAs.

[0038] In some such methods, the subject is a neonatal subject. In some such methods, the neonatal subject is a human neonatal subject within 24 weeks after birth, a human neonatal subject is within 12 weeks after birth, a human neonatal subject is within 8 weeks after birth, a human neonatal subject is within 4 weeks after birth, a human neonatal subject is within 2 weeks after birth, or a human neonatal subject is within 1 week after birth. In some such methods, the subject is not a neonatal subject. In some such methods, the subject is a human subject.

[0039] In some such methods, the method decreases plasma ammonia and / or plasma citrulline levels in the subject. In some such methods, the method reduces plasma ammonia levels to less than 200 pmol / L, less than 175 pmol / L, less than 150 pmol / L, less than 125 pmol / L, or less than 100 pmol / L, optionally wherein the reduced plasma ammonia levels are at 2 weeks, 4 weeks, 6 weeks, 8 weeks, 12 weeks, 16 weeks, 20 weeks, 6 months, 1 year, or 2 years after administering the combination. In some such methods, the method reduces plasma citrulline levels to less than 2000 pmol / L, less than 1750 pmol / L, less than 1500 pmol / L, less than 1250 pmol / L, less than 1000 pmol / L, less than 900 pmol / L, less than 800 pmol / L, less than 700 pmol / L, less than 600 pmol / L, or less than 500 pmol / L, optionally wherein the reduced plasma citrulline levels are at 2 weeks, 4 weeks, 6 weeks, 8 weeks, 12 weeks, 16 weeks, 20 weeks, 6 months, 1 year, or 2 years after administering the combination. In some such methods, the decreased ammonia and / or plasma citrulline levels are sustained for at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 1 year, or at least 2 years after administering the combination.

[0040] In some such methods, the method further comprises assessing preexisting AAV immunity in the subject prior to administering the composition to the subject. In some such methods, the preexisting AAV immunity is preexisting AAV8 immunity. In some such methods, the preexisting AAV immunity is preexisting AAV5 immunity. In some such methods, assessing preexisting AAV immunity comprises assessing immunogenicity using a total antibody immune assay or a neutralizing antibody assay.BRIEF DESCRIPTION OF THE FIGURES

[0041] Figure 1 shows the experimental setup for an ASS1 episomal AAV efficacy study in adolescent P28 Asslf',ld ,',ldcitrullinemia mice.

[0042] Figure 2 shows body weight changes after treatment of AssIMd'Mmice with episomal human- S',S7-containing virus at various doses or control (GFP) virus.

[0043] Figure 3 shows plasma ammonia changes after treatment of AsslMd d"dmice with episomal human- SlSV-containing virus at various doses or control (GFP) virus.

[0044] Figure 4 shows plasma citrulline changes after treatment of Ass ]f°ld / f°ldmice with episomal human-^AS -containing virus at various doses or control (GFP) virus.

[0045] Figure 5 shows a mouse Assl intron 1 native locus gene insertion strategy in Ass lf°ld'f°,dmice.

[0046] Figure 6 shows the experimental setup for an ASS 1 gene insertion efficacy study in adolescent P28 Asslfold'oldcitrullinemia mice.

[0047] Figure 7 shows body weight changes after treatment of P28 Assld d dddmice with AAV comprising human-.LS'AV insertion template, alone or together with LNP comprising Cas9 mRNA and gRNA targeting mouse Assl intron 1.

[0048] Figures 8A and 8B shows plasma ammonia (Figure 8A) and plasma citrulline (Figure 8B) changes after treatment of P28 Asslt°ldf°ldmice with AAV comprising human-dW insertion template, alone or together with LNP comprising Cas9 mRNA and gRNA targeting mouse Assl intron 1. Measurements were performed the day before dosing and every 2 weeks after dosing.

[0049] Figure 9 shows liver RT-qPCR for hybrid (mouse Assl exonl / human ASS1 exons 2- 14) ASS1 gene expression after treatment of P28 Ass[ddd dddmice with AAV comprising human- ASS1 insertion template, alone or together with LNP comprising Cas9 mRNA and gRNA targeting mouse Assl intron 1.

[0050] Figure 10 shows droplet digital PCR (ddPCR) for hybrid DNA junctions of mouse Assl intron 1 / insertion template derived Alb splice acceptor DNA in mouse liver genomic DNA, relative to the number of total mouse Assl alleles after treatment of P28 Assl'rdd dddmice with AAV comprising human-AASV insertion template, alone or together with LNP comprising Cas9 mRNA and gRNA targeting mouse Assl intron 1.

[0051] Figure 11 shows quantification of hepatocytes positive for hybrid (mouse / human fusion) ASS1 mRNA in situ hybridization (ISH) staining (BaseScope) after treatment of P28 AsslfoldfiMmice with AAV comprising human-4 SSI insertion template, alone or together with LNP comprising Cas9 mRNA and gRNA targeting mouse Assl intron 1.

[0052] Figure 12 shows the experimental setup for an ASS1 gene insertion efficacy study in neonatal P7 fold citrullinemia mice.

[0053] Figure 13 shows body weight changes after treatment of P7 Assl, ld!',ldmice with AAV comprising hu an-4AS7 insertion template, alone or together with LNP comprising Cas9 mRNA and gRNA targeting mouse Assl intron 1.

[0054] Figures 14A and 14B shows plasma ammonia (Figure 14A) and plasma citrulline (Figure 14B) changes after treatment of P7 Assl'ldd dddmice with AAV comprising human-24557 insertion template, alone or together with LNP comprising Cas9 mRNA and gRNA targeting mouse Assl intron 1.

[0055] Figure 15 shows liver RT-qPCR for hybrid (mouse Assl exonl / human ASS1 exons 2-14) 24557 gene expression after treatment of P7 Asslddd dddmice with AAV comprising human- 24557 insertion template, alone or together with LNP comprising Cas9 mRNA and gRNA targeting mouse Assl intron 1.

[0056] Figure 16 shows droplet digital PCR (ddPCR) for hybrid DNA junctions of mouse Assl intron 1 / insertion template derived Alb splice acceptor DNA in mouse liver genomic DNA, relative to the number of total mouse Assl alleles after treatment of P7 Assl'oId,oldmice with AAV comprising human-4557 insertion template, alone or together with LNP comprising Cas9 mRNA and gRNA targeting mouse Assl intron 1.

[0057] Figure 17 shows a schematic showing the insertion scheme for inserting a bidirectional human 24557 insertion template (coding exons 3-14) into intron 2 of the human ASS1 genomic locus (between coding exons 2 and 3).

[0058] Figure 18 shows the experimental setup for an ASSl-HiBiT gene insertion gRNA screen in primary human hepatocytes.

[0059] Figure 19 shows human ASS1 intron 2 gRNAs display a range of insertion efficiencies in a primary human hepatocyte screen.

[0060] Figure 20 shows human ASS1 intron 2 gRNAs display a range of editing efficiencies that do not always correlate to insertion efficiency in a primary human hepatocyte screen.

[0061] Figure 21 shows that luminescence correlates between LNP doses within human ASS1 intron 2 guide RNA screens 1 and 2 in primary human hepatocytes.

[0062] Figure 22 shows that luminescence correlates at tested LNP concentrations between human ASS1 intron 2 guide RNA screens 1 and 2 in primary human hepatocytes.

[0063] Figure 23 shows that guide RNA editing correlates between human ASS1 intron 2 guide RNA screens 1 and 2 in primary human hepatocytes.

[0064] Figure 24 shows a schematic showing the insertion scheme for inserting a bidirectional human 24557 insertion template (coding exons 2-14) into intron 1 of the human ASS1 genomic locus (between coding exons 1 and 2).

[0065] Figure 25 shows the experimental setup for an ASSl-HiBiT gene insertion gRNA screen in primary human hepatocytes.

[0066] Figure 26 shows human ASS1 intron 1 gRNAs display a range of insertion efficiencies in a primary human hepatocyte screen.

[0067] Figure 27 shows human ASS I intron 1 gRNAs display a range of editing efficiencies that do not always correlate to insertion efficiency in a primary human hepatocyte screen.

[0068] Figure 28 shows that luminescence correlates between LNP doses within human ASS1 intron 1 guide RNA screens 1 and 2 in primary human hepatocytes.

[0069] Figure 29 shows that luminescence correlates at tested LNP concentrations between human ASS I intron 1 guide RNA screens 1 and 2 in primary human hepatocytes.

[0070] Figure 30 shows that guide RNA editing correlates between human ASS1 intron 1 guide RNA screens 1 and 2 in primary human hepatocytes.

[0071] Figure 31 shows body weight changes, plasma ammonia changes, and plasma citrulline changes after treatment of neonatal Ass[Md^dmice with episomal human-d.S'AV- containing virus at various doses or control (GFP) virus.

[0072] Figure 32 shows rates of native locus insertion in humanized ASS J wild type mice treated with AAV comprising human-4 AST insertion template together with 0.3 mpk LNP comprising Cas9 mRNA and G035840, G035869, or G035858 gRNA targeting human ASS1 intron 2.

[0073] Figures 33A-33B show all tested CpG-depleted ASS1 insertion templates yielded comparable ASSl-HiBiT expression in a primary human hepatocyte insertion assay when administered together with LNP comprising Cas9 mRNA and G035838 (Figure 33A) or G035847 (Figure 33B).

[0074] Figure 34 shows all tested CpG-depleted ASS1 insertion templates yielded comparable ASSl-HiBiT expression in a primary human hepatocyte insertion assay when administered together with LNP comprising Cas9 mRNA and G035838.

[0075] Figure 35 shows cryptic splicing analysis of 3 fully CpG-depleted ASS1 insertion templates compared to the original ASS1 insertion template for insertion into human ASS1 intron 2.DEFINITIONS

[0076] The terms “protein,” “polypeptide,” and “peptide,” used interchangeably herein, include polymeric forms of amino acids of any length, including coded and non-coded amino acids and chemically or biochemically modified or derivatized amino acids. The terms also include polymers that have been modified, such as polypeptides having modified peptide backbones. The term “domain” refers to any part of a protein or polypeptide having a particular function or structure.

[0077] Proteins are said to have an “N-terminus” and a “C-terminus.” The term “N- terminus” relates to the start of a protein or polypeptide, terminated by an amino acid with a free amine group (-NH2). The term “C-terminus” relates to the end of an amino acid chain (protein or polypeptide), terminated by a free carboxyl group (-COOH).

[0078] The terms “nucleic acid” and “polynucleotide,” used interchangeably herein, include polymeric forms of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, or analogs or modified versions thereof. They include single-, double-, and multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, and polymers comprising purine bases, pyrimidine bases, or other natural, chemically modified, biochemically modified, non-natural, or derivatized nucleotide bases.

[0079] Nucleic acids are said to have “5’ ends” and “3’ ends” because mononucleotides are reacted to make oligonucleotides in a manner such that the 5’ phosphate of one mononucleotide pentose ring is attached to the 3’ oxygen of its neighbor in one direction via a phosphodiester linkage. An end of an oligonucleotide is referred to as the “5’ end” if its 5’ phosphate is not linked to the 3’ oxygen of a mononucleotide pentose ring. An end of an oligonucleotide is referred to as the “3’ end” if its 3’ oxygen is not linked to a 5’ phosphate of another mononucleotide pentose ring. A nucleic acid sequence, even if internal to a larger oligonucleotide, also may be said to have 5’ and 3’ ends. In either a linear or circular DNA molecule, discrete elements are referred to as being “upstream” or 5’ of the “downstream” or 3’ elements.

[0080] The term “genomically integrated” refers to a nucleic acid that has been introduced into a cell such that the nucleotide sequence integrates into the genome of the cell. Any protocol may be used for the stable incorporation of a nucleic acid into the genome of a cell.

[0081] The term “viral vector” refers to a recombinant nucleic acid that includes at least one element of viral origin and includes elements sufficient for or permissive of packaging into a viral vector particle. The vector and / or particle can be utilized for the purpose of transferring DNA, RNA, or other nucleic acids into cells in vitro, ex vivo, or in vivo. Numerous forms of viral vectors are known.

[0082] The term “isolated” with respect to cells, tissues (e.g., liver samples), proteins, and nucleic acids includes cells, tissues (e.g., liver samples), proteins, and nucleic acids that are relatively purified with respect to other bacterial, viral, cellular, or other components that may normally be present in situ, up to and including a substantially pure preparation of the cells, tissues (e.g., liver samples), proteins, and nucleic acids. The term “isolated” also includes cells, tissues (e.g., liver samples), proteins, and nucleic acids that have no naturally occurring counterpart, have been chemically synthesized and are thus substantially uncontaminated by other cells, tissues (e.g., liver samples), proteins, and nucleic acids, or has been separated or purified from most other components (e.g., cellular components) with which they are naturally accompanied (e.g., other cellular proteins, polynucleotides, or cellular components).

[0083] The term “wild type” includes entities having a structure and / or activity as found in a normal (as contrasted with mutant, diseased, altered, or so forth) state or context. Wild type genes and polypeptides often exist in multiple different forms (e.g., alleles).

[0084] The term “endogenous sequence” refers to a nucleic acid sequence that occurs naturally within a cell or animal. For example, an endogenous ASS1 sequence of a human refers to a native ASS I sequence that naturally occurs at h ASS 1 locus in the human.

[0085] “Exogenous” molecules or sequences include molecules or sequences that are not normally present in a cell in that form. Normal presence includes presence with respect to the particular developmental stage and environmental conditions of the cell. An exogenous molecule or sequence, for example, can include a mutated version of a corresponding endogenous sequence within the cell, such as a humanized version of the endogenous sequence, or can include a sequence corresponding to an endogenous sequence within the cell but in a different form (i.e., not within a chromosome). In contrast, endogenous molecules or sequences include molecules or sequences that are normally present in that form in a particular cell at a particular developmental stage under particular environmental conditions.

[0086] The term “heterologous” when used in the context of a nucleic acid or a protein indicates that the nucleic acid or protein comprises at least two segments that do not naturally occur together in the same molecule. For example, the term “heterologous,” when used with reference to segments of a nucleic acid or segments of a protein, indicates that the nucleic acid or protein comprises two or more sub-sequences that are not found in the same relationship to each other (e.g., joined together) in nature. As one example, a “heterologous” region of a nucleic acid vector is a segment of nucleic acid within or attached to another nucleic acid molecule that is not found in association with the other molecule in nature. For example, a heterologous region of a nucleic acid vector could include a coding sequence flanked by sequences not found in association with the coding sequence in nature. Likewise, a “heterologous” region of a protein is a segment of amino acids within or attached to another peptide molecule that is not found in association with the other peptide molecule in nature (e.g., a fusion protein, or a protein with a tag). Similarly, a nucleic acid or protein can comprise a heterologous label or a heterologous secretion or localization sequence.

[0087] Codon optimization” (i.e., “codon optimized” sequences) takes advantage of the degeneracy of codons, as exhibited by the multiplicity of three-base pair codon combinations that specify an amino acid, and generally includes a process of modifying a nucleic acid sequence for enhanced expression in particular host cells by replacing at least one codon of the native sequence with a codon that is more frequently or most frequently used in the genes of the host cell while maintaining the native amino acid sequence. For example, a nucleic acid encoding an argininosuccinate synthase protein can be modified to substitute codons having a higher frequency of usage in a given prokaryotic or eukaryotic cell, including a bacterial cell, a yeast cell, a human cell, a non-human cell, a mammalian cell, a rodent cell, a mouse cell, a rat cell, a hamster cell, or any other host cell, as compared to the naturally occurring nucleic acid sequence. Codon usage tables are readily available, for example, at the “Codon Usage Database.” These tables can be adapted in a number of ways. See Nakamura et al. (2000) Nucleic Acids Res. 28(1):292, herein incorporated by reference in its entirety for all purposes. Computer algorithms for codon optimization of a particular sequence for expression in a particular host are also available (see, e.g., Gene Forge).

[0088] The term “locus” refers to a specific location of a gene (or significant sequence), DNA sequence, polypeptide-encoding sequence, or position on a chromosome of the genome ofan organism. For example, an “ASS1 locus” may refer to the specific location of an ASS I gene, ASS I DNA sequence, argininosuccinate-synthase-encoding sequence, or ASS1 position on a chromosome of the genome of an organism that has been identified as to where such a sequence resides. An “ASS I locus” may comprise a regulatory element of an ASS I gene, including, for example, an enhancer, a promoter, 5’ and / or 3’ untranslated region (UTR), or a combination thereof.

[0089] The term “gene” refers to DNA sequences in a chromosome that may contain, if naturally present, at least one coding and at least one non-coding region. The DNA sequence in a chromosome that codes for a product (e.g., but not limited to, an RNA product and / or a polypeptide product) can include the coding region interrupted with non-coding introns and sequence located adjacent to the coding region on both the 5’ and 3’ ends such that the gene corresponds to the full-length mRNA (including the 5’ and 3’ untranslated sequences).Additionally, other non-coding sequences including regulatory sequences (e.g., but not limited to, promoters, enhancers, and transcription factor binding sites), polyadenylation signals, internal ribosome entry sites, silencers, insulating sequence, and matrix attachment regions may be present in a gene. These sequences may be close to the coding region of the gene (e.g., but not limited to, within 10 kb) or at distant sites, and they influence the level or rate of transcription and translation of the gene.

[0090] The term “allele” refers to a variant form of a gene. Some genes have a variety of different forms, which are located at the same position, or genetic locus, on a chromosome. A diploid organism has two alleles at each genetic locus. Each pair of alleles represents the genotype of a specific genetic locus. Genotypes are described as homozygous if there are two identical alleles at a particular locus and as heterozygous if the two alleles differ.

[0091] A “promoter” is a regulatory region of DNA usually comprising a TATA box capable of directing RNA polymerase II to initiate RNA synthesis at the appropriate transcription initiation site for a particular polynucleotide sequence. A promoter may additionally comprise other regions which influence the transcription initiation rate. The promoter sequences disclosed herein modulate transcription of an operably linked polynucleotide. A promoter can be active in one or more of the cell types disclosed herein (e.g., a mouse cell, a rat cell, a pluripotent cell, a one-cell stage embryo, a differentiated cell, or a combination thereof). A promoter can be, for example, a constitutively active promoter, a conditional promoter, an inducible promoter, atemporally restricted promoter (e.g., a developmentally regulated promoter), or a spatially restricted promoter (e.g., a cell-specific or tissue-specific promoter). Examples of promoters can be found, for example, in WO 2013 / 176772, herein incorporated by reference in its entirety for all purposes.

[0092] “Operable linkage” or being “operably linked” includes juxtaposition of two or more components (e.g., a promoter and another sequence element) such that both components function normally and allow the possibility that at least one of the components can mediate a function that is exerted upon at least one of the other components. For example, a promoter can be operably linked to a coding sequence if the promoter controls the level of transcription of the coding sequence in response to the presence or absence of one or more transcriptional regulatory factors. Operable linkage can include such sequences being contiguous with each other or acting in trans (e.g., a regulatory sequence can act at a distance to control transcription of the coding sequence).

[0093] The methods and compositions provided herein employ a variety of different components. Some components throughout the description can have active variants and fragments. The term “functional” refers to the innate ability of a protein or nucleic acid (or a fragment or variant thereof) to exhibit a biological activity or function. The biological functions of functional fragments or variants may be the same or may in fact be changed (e.g., with respect to their specificity, selectivity, or efficacy) in comparison to the original molecule, but with retention of the molecule’s basic biological function.

[0094] The term “variant” refers to a nucleotide sequence differing from the sequence most prevalent in a population (e.g., by one nucleotide) or a protein sequence different from the sequence most prevalent in a population (e.g., by one amino acid).

[0095] The term “fragment,” when referring to a protein, means a protein that is shorter or has fewer amino acids than the full-length protein. The term “fragment,” when referring to a nucleic acid, means a nucleic acid that is shorter or has fewer nucleotides than the full-length nucleic acid. A fragment can be, for example, when referring to a protein fragment, an N- terminal fragment (i.e., removal of a portion of the C-terminal end of the protein), a C-terminal fragment (i.e., removal of a portion of the N-terminal end of the protein), or an internal fragment (i.e., removal of a portion of each of the N-terminal and C-terminal ends of the protein). A fragment can be, for example, when referring to a nucleic acid fragment, a 5’ fragment (i.e., removal of a portion of the 3’ end of the nucleic acid), a 3’ fragment (i .e., removal of a portion ofthe 5’ end of the nucleic acid), or an internal fragment (i.e., removal of a portion each of the 5’ and 3’ ends of the nucleic acid).

[0096] “Sequence identity” or “identity” in the context of two polynucleotides or polypeptide sequences refers to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins, residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have “sequence similarity” or “similarity.” Means for making this adjustment are well known. Typically, this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC / GENE (Intelligenetics, Mountain View, California).

[0097] “Percentage of sequence identity” includes the value determined by comparing two optimally aligned sequences (greatest number of perfectly matched residues) over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity. Unless otherwise specified (e.g., the shorter sequence includes a linked heterologous sequence), the comparison window is the full length of the shorter of the two sequences being compared.

[0098] Unless otherwise stated, sequence identity / similarity values include the value obtained using GAP Version 10 using the following parameters: % identity and % similarity fora nucleotide sequence using GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for an amino acid sequence using GAP Weight of 8 and Length Weight of 2, and the BLOSUM62 scoring matrix; or any equivalent program thereof. “Equivalent program” includes any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by GAP Version 10.

[0099] The term “conservative amino acid substitution” refers to the substitution of an amino acid that is normally present in the sequence with a different amino acid of similar size, charge, or polarity. Examples of conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, valine, or leucine for another non-polar residue.Likewise, examples of conservative substitutions include the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, or between glycine and serine. Additionally, the substitution of a basic residue such as lysine, arginine, or histidine for another, or the substitution of one acidic residue such as aspartic acid or glutamic acid for another acidic residue are additional examples of conservative substitutions. Examples of non-conservative substitutions include the substitution of a non-polar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine, or methionine for a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid or lysine and / or a polar residue for a non-polar residue. Typical amino acid categorizations are summarized below.

[0100] Table 1. Amino Acid Categorizations.Alanine Ala A Nonpolar Neutral 1.8Arginine Arg R Polar Positive -4.5Asparagine Asn N Polar Neutral -3.5Aspartic acid Asp D Polar Negative -3.5Cysteine Cys C Nonpolar Neutral 2.5Glutamic acid Glu E Polar Negative -3.5Glutamine Gin Q Polar Neutral -3.5Glycine Gly G Nonpolar Neutral -0.4Histidine His H Polar Positive -3.2Isoleucine He I Nonpolar Neutral 4.5Leucine Leu L Nonpolar Neutral 3.8Lysine Lys K Polar Positive -3.9Methionine Met M Nonpolar Neutral 1.9Phenylalanine Phe F Nonpolar Neutral 2.8Proline Pro P Nonpolar Neutral -1.6Serine Ser S Polar Neutral -0.8Threonine Thr T Polar Neutral -0.7Tiyptophan Trp W Nonpolar Neutral -0.9Tyrosine Tyr Y Polar Neutral -1.3Valine Vai V Nonpolar Neutral 4.2

[0101] A “homologous” sequence (e.g., nucleic acid sequence) includes a sequence that is either identical or substantially similar to a known reference sequence, such that it is, for example, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the known reference sequence. Homologous sequences can include, for example, orthologous sequence and paralogous sequences. Homologous genes, for example, typically descend from a common ancestral DNA sequence, either through a speciation event (orthologous genes) or a genetic duplication event (paralogous genes). “Orthologous” genes include genes in different species that evolved from a common ancestral gene by speciation. Orthologs typically retain the same function in the course of evolution. “Paralogous” genes include genes related by duplication within a genome. Paralogs can evolve new functions in the course of evolution.

[0102] The term “in vitro11includes artificial environments and to processes or reactions that occur within an artificial environment (e.g., a test tube or an isolated cell or cell line). The term “in vivo11includes natural environments (e.g., a cell, organism, or body) and to processes orreactions that occur within a natural environment. The term “ex vivo” includes cells that have been removed from the body of an individual and processes or reactions that occur within such cells.

[0103] As used herein, the term “neonatal” in the context of humans covers human subjects up to or under the age of 1 year (52 weeks), preferably up to or under the age of 24 weeks, more preferably up to or under the age of 12 weeks, more preferably up to or under the age of 8 weeks, even more preferably up to or under the age of 4 weeks, even more preferably up to or under the age of 2 weeks, and even more preferably up to or under the age of 1 week. In certain embodiments, a neonatal human subject is up to 1 week of age. In certain embodiments, a neonatal human subject is up to 2 weeks of age. In certain embodiments, a neonatal human subject is up to 4 weeks of age. In certain embodiments, a neonatal human subject is up to 8 weeks of age. In another embodiment, a neonatal human subject is within 3 weeks after birth. In another embodiment, a neonatal human subject is within 2 weeks after birth. In another embodiment, a neonatal human subject is within 1 week after birth. In another embodiment, a neonatal human subject is within 7 days after birth. In another embodiment, a neonatal human subject is within 6 days after birth. In another embodiment, a neonatal human subject is within 5 days after birth. In another embodiment, a neonatal human subject is within 4 days after birth. In another embodiment, a neonatal human subject is within 3 days after birth. In another embodiment, a neonatal human subject is within 2 days after birth. In another embodiment, a neonatal human subject is within 1 day after birth. The time windows disclosed above are for human subjects and are also meant to cover the corresponding developmental time windows for other animals. As used herein, a “neonatal cell” is a cell of a neonatal subject, and a population of neonatal cells is a population of cells of a neonatal subject.

[0104] As used herein, a “control” as in a control sample or a control subject is a comparator for a measurement, e.g., a diagnostic measurement of a sign or symptom of a disease. In certain embodiments, a control can be a subject sample from the same subject an earlier time point, e.g., before a treatment intervention. In certain embodiments, a control can be a measurement from a normal subject, i.e., a subject not having the disease of the treated subject, to provide a normal control, e.g., argininosuccinate-synthase activity in a subject sample. In certain embodiments, a normal control can be a population control, i.e., the average of subjects in the general population. In certain embodiments, a control can be an untreated subject with the same disease. In certainembodiments, a control can be a subject treated with a different therapy, e g., the standard of care. In certain embodiments, a control can be a subject or a population of subjects from a natural history study of subjects with the disease of the subject being compared. In certain embodiments, the control is matched for certain factors to the subject being tested, e.g., age, gender. In certain embodiments, a control may be a control level for a particular lab, e.g., a clinical lab. Selection of an appropriate control is within the ability of those of skill in the art.

[0105] Compositions or methods “comprising” or “including” one or more recited elements may include other elements not specifically recited. For example, a composition that “comprises” or “includes” a protein may contain the protein alone or in combination with other ingredients. The transitional phrase “consisting essentially of’ means that the scope of a claim is to be interpreted to encompass the specified elements recited in the claim and those that do not materially affect the basic and novel character! stic(s) of the claimed invention. Thus, the term “consisting essentially of’ when used in a claim of this invention is not intended to be interpreted to be equivalent to “comprising.”

[0106] “Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur and that the description includes instances in which the event or circumstance occurs and instances in which the event or circumstance does not.

[0107] Designation of a range of values includes all integers within or defining the range, and all subranges defined by integers within the range. For example, 5-10 nucleotides is understood as 5, 6, 7, 8, 9, or 10 nucleotides, whereas 5-10% is understood to contain 5% and all possible values through 10%.

[0108] At least 17 nucleotides of a 20 nucleotide sequence is understood to include 17, 18, 19, or 20 nucleotides of the sequence provided, thereby providing an upper limit even if one is not specifically provided as it would be clearly understood. Similarly, up to 3 nucleotides would be understood to encompass 0, 1, 2, or 3 nucleotides, providing a lower limit even if one is not specifically provided. When “at least,” “up to,” or other similar language modifies a number, it can be understood to modify each number in the series.

[0109] As used herein, “no more than” or “less than” is understood as the value adjacent to the phrase and logical lower values or integers, as logical from context, to zero. For example, a duplex region of “no more than 2 nucleotide base pairs” has a 2, 1, or 0 nucleotide base pairs.When “no more than” or “less than” is present before a series of numbers or a range, it is understood that each of the numbers in the series or range is modified.

[0110] Unless otherwise apparent from the context, the term “about” encompasses values ± 5% of a stated value. In certain embodiments, the term “about” is understood to encompass tolerated variation or error within the art, e.g., 2 standard deviations from the mean, or the sensitivity of the method used to take a measurement, or a percent of a value as tolerated in the art, e.g., with age. When “about” is present before the first value of a series, it can be understood to modify each value in the series.

[0111] The term “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0112] The term “or” refers to any one member of a particular list and also includes any combination of members of that list.

[0113] The singular forms of the articles “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a protein” or “at least one protein” can include a plurality of proteins, including mixtures thereof.

[0114] Statistically significant means p <0.05.

[0115] In the event of a conflict between a sequence in the application and an indicated accession number or position in an accession number, the sequence in the application predominates.DETAILED DESCRIPTIONI. Overview

[0116] Provided herein are nucleic acid constructs and compositions that allow insertion of an argininosuccinate synthase 1 (ASS 1 ) coding sequence into a target genomic locus such as an endogenous ASS I locus and / or expression of the ASS1 coding sequence. The nucleic acid constructs and compositions can be used in methods of introducing an ASS J nucleic acid into a cell, in methods of integration of an ASS1 nucleic acid into a target genomic locus, in methods of expression of AS SI in a cell, and in methods of treating citrullinemia type I or AS SI deficiency in a subject. In some cases, the cells or subjects can be neonatal cells or neonatal subjects as defined herein. In other cases, the cells are not neonatal cells, and the subjects are not neonatalsubjects. Also provided are nuclease agents (e.g., targeting an endogenous ASS1 locus) or nucleic acids encoding nuclease agents to facilitate integration of the nucleic acid constructs into a target genomic locus such as an endogenous ASS1 locus.

[0117] Also provided are compositions, combinations, or kits comprising an ASS1 nucleic acid construct in combination with a nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent targets a nuclease target site in a target genomic locus (e.g., an endogenous ASS1 locus). As used herein, the term “in combination with” means that additional component(s) may be administered prior to, concurrent with, or after the administration of the nucleic acid construct. The different components of the combination can be formulated into a single composition, e.g., for simultaneous delivery, or formulated separately into two or more compositions (e.g., a kit including each component, for example, wherein the further agent is in a separate formulation).

[0118] More specifically, described herein in some embodiments is a therapeutic product based on the CRISPR / Cas9 gene editing technology and optionally contained in a lipid nanoparticle (LNP) delivery system, associated with an .45’57 DNA gene insertion template (e.g., a unidirectional or bidirectional ASS1 DNA gene insertion template) optionally contained in a recombinant adeno-associated virus serotype 8 (rAAV8) or recombinant adeno-associated virus serotype 5 (rAAV5). The CRISPR / Cas9 component has been designed to target and cut the double stranded DNA at a target gene locus (e.g., an endogenous ASS1 gene locus in hepatocytes), allowing for the ASS1 DNA template to be inserted in the genome at the target genomic locus. Transgene insertion provides a functional ASS1 gene that when expressed provides functional ASS1 to rescue the defective endogenous, genomic ASS1 in citrullinemia type I patients.

[0119] In some cases, the ASS1 coding sequences in the constructs disclosed herein are optimized for expression as compared to native ASS1 coding sequence. In other cases, the ASS1 coding sequences in the constructs disclosed herein can comprise native ASS1 coding sequences. For example, the ASS1 coding sequences in the constructs disclosed herein may include one or more modifications such as codon optimization (e.g., to human codons), depletion of CpG dinucleotides, mutation of cryptic splice sites, or any combination thereof.

[0120] In particular, provided herein are bidirectional constructs comprising two different ASS1 coding sequences (i.e., a first ASS1 coding sequence and a reverse complement of asecond ASS1 coding sequence). Also provided herein are unidirectional constructs comprising a single AS SI coding sequence.

[0121] In some embodiments, the gene insertion platform described herein also has advantages over existing episomal ASSl platforms. For example, there are no concerns regarding integration of promoter-containing constructs because the ASSl insertion templates used were promoterless. In some embodiments, because there are no promoter / regulatory elements in the AAV cassettes used, they are less likely to influence expression of neighboring loci if randomly inserted. Likewise, in some embodiments, because there is no in-frame ATG / methionine at the 5’ end of the cassettes used, no protein is produced from the template alone. In addition, there is an extremely low likelihood of producing protein from off-target insertion because it would require intronic insertion leading to splicing into the correct reading frame. Moreover, integration of the coding sequence as in the compositions and methods disclosed herein is advantageous over nonintegrating episomal vectors because transgene retention over time can be problematic with nonreplicating episomal vectors, making it necessary to administer more virus for continued therapeutic response. However, these subsequent exposures may result in rapid neutralization of the virus and, therefore, decreased transgene expression. In some embodiments, no redosing is required because the compositions and methods result in integration into the genome and permanent expression. A major advantage that ASSl insertion affords over episomal expression is that it enables durable expression in the face of cellular division, whereas episomal expression is largely lost upon cell division. This is particularly relevant for citrullinemia type I where there is a need for early life intervention. Episome expression approaches are largely ineffective when delivered early in life because the liver is still growing and there is significant cell division. In short, ASSl insertion enables treating young individuals with citrullinemia, whereas ASSl episomal expression approaches do not. This is medically very important as cognitive and motor deficits that accumulate over time in citrullinemia due to insufficient control of plasma ammonia levels are largely irreversible. Another advantage associated with ASSl native locus insertion is that the insertion template packaged in the AAV capsid lacks a promoter. It is increasingly accepted that AAV treatment leads to a low level of random genomic integration of the recombinant AAV genome. AAV episome gene expression cassettes invariably contain promoter / enhancer elements to enable transgene expression. When such promoter / enhancer elements randomly integrate into the genome, there is a possibility that they can lead to mis-regulation of a neighboring gene, which can have unintended negative consequences. The promoter-less nature of the ASS1 insertion template provides an added safety feature in that it is devoid of such promoter / enhancer elements.

[0122] The native locus gene insertion platform described herein also has advantages over insertion platforms in which an ASS J transgene is inserted into a non -4 AS' / genomic locus. Native locus insertion ensures ASS1 insertion transgene is expressed in the proper amount because expression is regulated by the native locus promoter. Insertion into another locus such as an ALB locus would lead to dramatic overexpression of AS SI protein, with unknown consequences. Native locus insertion may also ensure proper cytoplasmic localization of ASS1 protein. Insertion of ASS J into another locus such as an ALB locus would likely lead to some fraction of ASS1 protein being mis-localized into unintended cellular compartments, such as endoplasmic reticulum, secretory vesicles, and extracellular space, because ALB exon 1 encodes an ALB signal peptide. Native locus insertion may also ensure proper regulation of ASS I in response to physiological queues. Insertion into another locus such as an ALB locus would subject ASS7 expression to the acute phase response, which would lead to downregulation of ASS1 expression under inflammatory and stress responses. Insertion into a non-AS',S7 genomic locus would also be difficult to achieve without significantly impacting expression of the endogenous protein from the locus. For instance, insertion into an early intron of a non -AS / genomic locus would likely lead to ablation of expression of the endogenous protein from the targeted allele. Insertion into a downstream intron would likely lead to epitope tagging of endogenous protein.II. Compositions for Expressing Argininosuccinate Synthase

[0123] Provided herein are nucleic acid constructs and compositions that allow insertion of an argininosuccinate synthase (ASS1) coding sequence into a target genomic locus such as an endogenous ASS1 locus and / or expression of the ASS1 coding sequence. The nucleic acid constructs and compositions can be used in methods for integration into a target genomic locus and / or expression in a cell or in methods of treating citrullinemia type I (CTLN1) or ASS1 deficiency. Also provided are nuclease agents (e.g., targeting an endogenous ASS1 locus) or nucleic acids encoding nuclease agents to facilitate integration of the nucleic acid constructs into a target genomic locus such as an endogenous ASS J locus.A. Argininosuccinate Synthase Nucleic Acid Constructs

[0124] The compositions and methods described herein include the use of a nucleic acid construct that comprises an ASS1 protein coding sequence (an 4557 nucleic acid) or a reverse complement of the ASS1 protein coding sequence (e.g., a heterologous ASS1 protein coding sequence (a heterologous ASS1 nucleic acid) or a reverse complement of the heterologous AS SI protein coding sequence). For example, the nucleic acid construct can comprise an ASS1 protein coding sequence an ASS J nucleic acid), such as a heterologous ASS1 protein coding sequence (e g., a heterologous ASS J nucleic acid). For example, the ASS1 protein coding sequence can comprise a human ASS1 (hASSl) protein coding sequence comprising exons 3-14 of hASSl (e.g., encoding the amino acid sequence set forth in SEQ ID NO: 230). As another example, the AS SI protein coding sequence can comprise a hASSl protein coding sequence comprising exons 2-14 of hASSl (e g., encoding the amino acid sequence set forth in SEQ ID NO: 227). As another example, the ASS1 protein coding sequence can comprise the complete hASSl protein coding sequence (e.g., encoding the amino acid sequence set forth in SEQ ID NO: 1). Such nucleic acid constructs can be for insertion into a target genomic locus following cleavage by a nuclease agent or CRISPR / Cas system, as disclosed elsewhere herein, or can be for expression of ASS1 without insertion into a target genomic locus (e.g., in an episome). For example, such nucleic acid constructs can be for insertion into a cleavage site created by a nuclease agent or CRISPR / Cas system, as disclosed elsewhere herein, or can be for expression of ASS1 without insertion into a cleavage site (e.g., in an episome). The term cleavage site includes a DNA sequence at which a nick or double-strand break is created by a nuclease agent (e.g., a Cas9 protein complexed with a guide RNA). A “heterologous” ASS1 protein coding sequence can refer to a coding sequence that has been introduced as an exogenous source to a site within a host cell genome (e g., at a genomic locus such as the endogenous ASS I locus, including human ASS1 intron 2, human ASS1 intron 1, or mouse Assl intron 1). That is, the heterologous protein coding sequence can be heterologous with respect to its insertion site, and the polypeptide expressed from such a heterologous coding sequence is referred to as a heterologous polypeptide.Additional sites within a host cell genome for introduction of a heterologous ASS1 protein coding sequence can comprise a genomic safe harbor locus, including human albumin (ALB) or mouse Alb. The heterologous coding sequence can be naturally-occurring or engineered, and canbe wild type or a variant. The heterologous coding sequence may include nucleotide sequences other than the sequence that encodes the heterologous polypeptide (e.g., an internal ribosomal entry site). The heterologous coding sequence can be a coding sequence that occurs naturally in the host genome, as a wild type or a variant (e.g., mutant). For example, although the host cell contains the coding sequence of interest (as a wild type or as a variant), the same coding sequence or variant thereof can be introduced as an exogenous source (e.g., for expression at a locus that is highly expressed). The heterologous coding sequence can also be a coding sequence that is not naturally occurring in the host genome, or that expresses a heterologous polypeptide that does not naturally occur in the host genome. A heterologous coding sequence can include an exogenous nucleic acid sequence (e.g., a nucleic acid sequence is not endogenous to the recipient cell), or may be heterologous with respect to its insertion site and / or with respect to its recipient cell.

[0125] The length of the ASS I nucleic acid constructs disclosed herein can vary. The construct can be, for example, from about 1 kb to about 5 kb, such as from about 1 kb to about 4.5 kb or about 1 kb to about 4 kb. An exemplary nucleic acid construct is between about 1 kb to about 5 kb in length or between about 1 kb to about 4 kb in length. Alternatively, a nucleic acid construct can be between about 1 kb to about 1.5 kb, about 1.5 kb to about 2 kb, about 2 kb to about 2.5 kb, about 2.5 kb to about 3 kb, about 3 kb to about 3.5 kb, about 3.5 kb to about 4 kb, about 4 kb to about 4.5 kb, or about 4.5 kb to about 5 kb in length. Alternatively, a nucleic acid construct can be, for example, no more than 5 kb, no more than 4.5 kb, no more than 4 kb, no more than 3.5 kb, no more than 3 kb, or no more than 2.5 kb in length. In a specific example, the nucleic acid construct is no more than 3.5 kb in length.

[0126] The constructs can comprise deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), can be single- stranded, double-stranded, or partially single- stranded and partially double-stranded, and can be introduced into a host cell in linear or circular (e.g., minicircle) form. See, e.g., US 2010 / 0047805, US 2011 / 0281361, and US 2011 / 0207221, each of which is herein incorporated by reference in their entirety for all purposes. If introduced in linear form, the ends of the construct can be protected (e.g., from exonucleolytic degradation) by known methods. For example, one or more dideoxynucleotide residues can be added to the 3' terminus of a linear molecule and / or self-complementary oligonucleotides can be ligated to one or both ends. See, e.g., Chang et al. (1987) Proc. Natl. Acad. Sei. U.S.A. 84:4959-4963 and Nehls et al.(1996) Science 272:886-889, each of which is herein incorporated by reference in their entirety for all purposes. Additional methods for protecting exogenous polynucleotides from degradation include, but are not limited to, addition of terminal amino group(s) and the use of modified internucleotide linkages such as, for example, phosphorothioates, phosphoramidates, and O- methyl ribose or deoxyribose residues. A construct can be introduced into a cell as part of a vector molecule having additional sequences such as, for example, replication origins, promoters, and genes encoding antibiotic resistance. A construct may omit viral elements. Moreover, constructs can be introduced as a naked nucleic acid, can be introduced as a nucleic acid complexed with an agent such as a liposome or poloxamer, or can be delivered by viruses (e.g., adenovirus, adeno-associated virus (AAV), herpesvirus, retrovirus, or lentivirus).

[0127] The constructs disclosed herein can be modified on either or both ends to include one or more suitable structural features as needed and / or to confer one or more functional benefit. For example, structural modifications can vary depending on the method(s) used to deliver the constructs disclosed herein to a host cell (e.g., use of viral vector delivery or packaging into lipid nanoparticles for delivery). Such modifications include, for example, terminal structures such as inverted terminal repeats (ITR), hairpin, loops, and other structures such as toroids. For example, the constructs disclosed herein can comprise one, two, or three ITRs or can comprise no more than two ITRs. Various methods of structural modifications are known.

[0128] Some constructs may be inserted so that their expression is driven by the endogenous promoter at the insertion site (e.g., the endogenous ASS1 promoter when the construct is integrated into the host cell’s ASS J locus). Such constructs may not comprise a promoter that drives the expression of ASS1. For example, the expression of ASS1 can be driven by a promoter of the host cell (e.g., the endogenous ASS1 promoter when the transgene is integrated into a host cell’s ASA / locus). In such cases, the construct may lack control elements (e.g., promoter and / or enhancer) that drive its expression (e.g., a promoterless construct). Nonetheless, in other cases the construct may comprise a promoter and / or enhancer, for example, a constitutive promoter or an inducible or tissue-specific (e.g., liver-specific) promoter that drives expression of the ASS1 in an episome or upon integration. Non-limiting exemplary constitutive promoters include cytomegalovirus immediate early promoter (CMV), simian virus (SV40) promoter, adenovirus major late (MLP) promoter, Rous sarcoma virus (RSV) promoter, mouse mammary tumor virus (MMTV) promoter, phosphoglycerate kinase (PGK) promoter, elongationfactor-alpha (EFla) promoter, ubiquitin promoters, actin promoters, tubulin promoters, immunoglobulin promoters, a functional fragment thereof, or a combination of any of the foregoing. For example, the promoter may be a CMV promoter or a truncated CMV promoter. In another example, the promoter may be an EFla promoter. Non-limiting exemplary inducible promoters include those inducible by heat shock, light, chemicals, peptides, metals, steroids, antibiotics, or alcohol. The inducible promoter may be one that has a low basal (non-induced) expression level, such as the Tet-On® promoter (Clontech). Although not required for expression, the constructs may comprise transcriptional or translational regulatory sequences such as promoters, enhancers, insulators, internal ribosome entry sites, additional sequences encoding peptides, and / or polyadenylation signals. In some examples, the nucleic acid construct works in homology-independent insertion of a nucleic acid that encodes an ASS1 protein. Such nucleic acid constructs can work, for example, in non-dividing cells (e.g., cells in which non- homologous end joining (NHEJ), not homologous recombination (HR), is the primary mechanism by which double-stranded DNA breaks are repaired). Such constructs can be, for example, homology-independent donor constructs. Such nucleic acid constructs can work, for example, in dividing cells (e.g., actively dividing cells).

[0129] The constructs disclosed herein can be modified to include or exclude any suitable structural feature as needed for any particular use and / or that confers one or more desired function. For example, some constructs disclosed herein do not comprise a homology arm. Some constructs disclosed herein are capable of insertion into a cut site in a target DNA sequence for a nuclease agent (e.g., capable of insertion into the endogenous ASS1 locus, such as into human ASS1 intron 2, human ASS1 intron 1, or mouse Assl intron 1) by non-homologous end joining. Some such constructs do not comprise homology arms. For example, such constructs can be inserted into a blunt end double-strand break following cleavage with a nuclease agent (e.g., CRISPR / Cas system) as disclosed herein. In a specific example, the construct can be delivered via AAV and can be capable of insertion by non-homologous end joining (e.g., the construct can be one that does not comprise homology arms).

[0130] In a particular example, the construct can be inserted via homology-independent targeted integration. For example, the heterologous ASS1 nucleic acid in the construct can be flanked on each side by a target site for a nuclease agent (e.g., the same target site as in the target DNA sequence for targeted insertion (e g., in the endogenous ASS I locus, such as in humanASS1 intron 2, human ASS1 intron 1, or mouse Assl intron 1), and the same nuclease agent being used to cleave the target DNA sequence for targeted insertion). The nuclease agent can then cleave the target sites flanking the heterologous ASS1 nucleic acid. In a specific example, the construct is delivered AAV-mediated delivery, and cleavage of the target sites flanking the heterologous ASS1 nucleic acid can remove the inverted terminal repeats (ITRs) of the AAV. In some instances, the target DNA sequence for targeted insertion (e.g., target DNA sequence in a safe harbor locus such as a gRNA target sequence including the flanking protospacer adjacent motif) is no longer present if the heterologous ASS J nucleic acid is inserted into the cut site or target DNA sequence in the correct orientation but it is reformed if the heterologous ASS1 nucleic acid is inserted into the cut site or target DNA sequence in the opposite orientation. This can help ensure that the heterologous ASS1 nucleic acid is inserted in the correct orientation for expression.

[0131] The constructs disclosed herein can comprise a polyadenylation tail sequence (e g., downstream or 3’ of an AS SI coding sequence). Methods of designing a suitable polyadenylation tail sequence are well-known. The polyadenylation tail sequence can be encoded, for example, as a “poly-A” stretch downstream of the ASS1 coding sequence. A poly- A tail can comprise, for example, at least 20, 30, 40, 50, 60, 70, 80, 90, or 100 adenines, and optionally up to 300 adenines. In a specific example, the poly-A tail comprises 95, 96, 97, 98, 99, or 100 adenine nucleotides. Methods of designing a suitable polyadenylation tail sequence and / or polyadenylation signal sequence are well known. For example, the polyadenylation signal sequence AAUAAA is commonly used in mammalian systems, although variants such as UAUAAA or AU / GUAAA have been identified. See, e.g., Proudfoot (2011) Genes & Dev. 25(17): 1770-82, herein incorporated by reference in its entirety for all purposes. The term polyadenylation signal sequence refers to any sequence that directs termination of transcription and addition of a poly-A tail to the mRNA transcript. In eukaryotes, transcription terminators are recognized by protein factors, and termination is followed by polyadenylation, a process of adding a poly(A) tail to the mRNA transcripts in presence of the poly(A) polymerase. The mammalian poly(A) signal typically consists of a core sequence, about 45 nucleotides long, that may be flanked by diverse auxiliary sequences that serve to enhance cleavage and polyadenylation efficiency. The core sequence consists of a highly conserved upstream element (AATAAA or AAUAAA) in the mRNA, referred to as a poly A recognition motif or poly Arecognition sequence), recognized by cleavage and polyadenylation-specificity factor (CPSF), and a poorly defined downstream region (rich in Us or Gs and Us), bound by cleavage stimulation factor (CstF). Examples of transcription terminators that can be used include, for example, the human growth hormone (HGH) poly adenylation signal, the simian virus 40 (SV40) late polyadenylation signal, the rabbit beta-globin polyadenylation signal, the bovine growth hormone (BGH) polyadenylation signal, the phosphoglycerate kinase (PGK) polyadenylation signal, an A0X1 transcription termination sequence, a CYC1 transcription termination sequence, or any transcription termination sequence known to be suitable for regulating gene expression in eukaryotic cells. In one example, the polyadenylation signal is a simian virus 40 (SV40) late polyadenylation signal. For example, the polyadenylation signal can comprise, consist essentially of, or consist of SEQ ID NO: 239 or SEQ ID NO: 240. In another example, the polyadenylation signal is a bovine growth hormone (BGH) polyadenylation signal. For example, the polyadenylation signal can comprise, consist essentially of, or consist of SEQ ID NO: 236, SEQ ID NO: 237, or SEQ ID NO: 238. In another example, the polyadenylation signal is a human growth hormone (HGH) polyadenylation signal. For example, the polyadenylation signal can comprise, consist essentially of, or consist of SEQ ID NO: 235.

[0132] The constructs disclosed herein may also comprise splice acceptor sites (e.g., operably linked to the AS SI coding sequence, such as upstream or 5’ of the AS SI coding sequence). The splice acceptor site can, for example, comprise NAG or consist of NAG. In a specific example, the splice acceptor is an ASS1 splice acceptor (e.g., an ASS1 splice acceptor used in the splicing together of exons 2 and 3 of ASS1 (i.e., ASS1 exon 3 splice acceptor) or an ASS1 splice acceptor used in the splicing together of exons 1 and 3 of ASS1 (i.e., ASS1 exon 2 splice acceptor). For example, such a splice acceptor can be derived from the human ASS1 gene (e.g., an ASS1 splice acceptor used in the splicing together of exons 2 and 3 of human ASS J (i.e., human ASS1 exon 3 splice acceptor)). In another example, the splice acceptor can be derived from the mouse Assl gene (e.g., an ASS J splice acceptor used in the splicing together of exons 1 and 2 of mouse Assl (i.e., mouse Assl exon 2 splice acceptor)). In another example, the splice acceptor is an ALB splice acceptor (e.g., an ALB splice acceptor used in the splicing together of exons 1 and 2 of ALB (i.e., ALB exon 2 splice acceptor)). For example, such a splice acceptor can be derived from the human ALB gene. In another example, the splice acceptor can be derived from the mouse Alb gene (e.g., an ALB splice acceptor used in the splicing together of exons 1and 2 of mouse Alb (i.e., mouse Alb exon 2 splice acceptor)). Additional suitable splice acceptor sites useful in eukaryotes, including artificial splice acceptors, are well-known. See, e.g., Shapiro et al. (1987) Nucleic Acids Res. 15:7155-7174 and Burset et al. (2001) Nucleic Acids Res .29:255-259, each of which is herein incorporated by reference in its entirety for all purposes. In a specific example, the splice acceptor is a human ASS1 exon 3 splice acceptor. In another example, the splice acceptor is a mouse Assl exon 2 splice acceptor. In another example, the splice acceptor is a mouse Alb exon 2 splice acceptor. In a specific example, the splice acceptor can comprise, consist essentially of, or consist of SEQ ID NO: 248.

[0133] In some examples, the nucleic acid constructs disclosed herein can be bidirectional constructs, which are described in more detail below. In some examples, the nucleic acid constructs disclosed herein can be unidirectional constructs, which are described in more detail below. Likewise, in some examples, the nucleic acid constructs disclosed herein can be in a vector (e.g., viral vector, such as AAV, or rAAV8 or rAAV5) and / or a lipid nanoparticle as described in more detail elsewhere herein.(1) Argininosuccinate Synthase (ASS1

[0134] Argininosuccinate synthase 1 (ASS1) is encoded by ASS1 and is a 412-amino acid enzyme present in the cytoplasm of most tissues. ASS1 is one of the enzymes of the urea cycle, the metabolic pathway responsible for transforming neurotoxic ammonia produced by protein catabolism into urea in the liver of ureotelic animals. Specifically, ASS1 catalyzes the formation of arginosuccinate from aspartate, citrulline, and ATP. Together with argininosuccinate lyase, it is responsible for the biosynthesis of arginine in most body tissues.

[0135] The ASS1 expressed from the compositions and methods disclosed herein can be any wild type or variant ASSl. In one example, the ASS1 is a human ASS1 protein. Human ASS1 is assigned UniProt reference number P00966. An exemplary amino acid sequence for human ASS1 is assigned NCBI Accession No. NP_000041.2 and is set forth in SEQ ID NO: 1. An exemplary human ASS I mRNA (cDNA) sequence is assigned NCBI Accession No.NM_000050.4 and is set forth in SEQ ID NO: 2. An exemplary human ASS1 coding sequence is assigned CCDS ID CCDS6933.1 and is set forth in SEQ ID NO: 3.

[0136] In some examples, the ASS1 (e.g., human ASS1) is a wild type ASS1 (e.g., wild type human ASS1) sequence or a fragment thereof. In a specific example, the AS SI encoded by thenucleic acid constructs disclosed herein can comprise SEQ ID NO: 230, can consist of SEQ ID NO: 230, or can be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to SEQ ID NO: 230 (encoded by exons 3-14 of human ASS1). In another example, the ASS1 encoded by the nucleic acid constructs disclosed herein can comprise SEQ ID NO: 227, can consist of SEQ ID NO: 227, or can be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to SEQ ID NO: 227 (encoded by exons 2-14 of human ASS1). In another example, the ASS1 encoded by the nucleic acid constructs disclosed herein can comprise SEQ ID NO: 1, can consist of SEQ ID NO: 1, or can be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to SEQ ID NO: 1 (encoded by complete human ASS1 CDS).

[0137] The ASS1 coding sequences in the constructs disclosed herein may include wild type ASS 1 coding sequences without any modifications. Alternatively, the ASS1 coding sequences may be optimized. The ASS1 coding sequences in the constructs disclosed herein may include one or more modifications such as codon optimization (e.g., to human codons), depletion of CpG dinucleotides, mutation of cryptic splice sites, addition of one or more glycosylation sites, or any combination thereof. For example, the ASS1 coding sequences can be CpG-depleted (e.g., fully CpG depleted) and / or codon optimized and / or modified to remove cryptic splice sites.

[0138] In one example, the ASS1 coding sequence is (or comprises a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 229. In another example, the ASS1 coding sequence is (or comprises a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 229 and encodes an ASS 1 protein (or an ASS1 protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230. In another example, the ASS1 coding sequence is (or comprises a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 229 and encodes an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230. In another example, the ASS1 coding sequence is (or comprises a sequence) at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 229. In another example, the ASS1 coding sequence is (or comprises a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 229 and encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230. In another example, the ASS1 coding sequence is (or comprises a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 229 and encodes an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230. In another example, the ASS1 coding sequence is (or comprises a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 229. In another example, the ASS1 coding sequence is (or comprises a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 229 and encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230. In another example, the ASS1 coding sequence is (or comprises a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 229 and encodes an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230. In another example, the ASS1 coding sequence comprises the sequence set forth in SEQ ID NO: 229. In another example, the ASS1 coding sequence consists essentially of the sequence set forth in SEQ ID NO: 229. In another example, the ASS1 coding sequence consists of the sequence set forth in SEQ ID NO: 229. Optionally, the ASS1 coding sequence encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the ASS1 coding sequence encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the ASS1 coding sequence in the above examples encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the ASS1 coding sequence in the above examples encodes an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230. Optionally, the ASS1 coding sequence in the above examples encodes an ASS1 protein consisting essentially of the sequence set forth in SEQ ID NO: 230. Optionally, the ASS1 coding sequence in the above examplesencodes an ASS1 protein consisting of the sequence set forth in SEQ ID NO: 230.

[0139] In one example, the ASS1 coding sequence is (or comprises a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 228. In another example, the ASS1 coding sequence is (or comprises a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 228and encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230. In another example, the ASS1 coding sequence is (or comprises a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 228 and encodes an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230. In another example, the ASS1 coding sequence is (or comprises a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 228. In another example, the ASS1 coding sequence is (or comprises a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 228 and encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230. In another example, the ASS1 coding sequence is (or comprises a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 228 and encodes an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230. In another example, the ASS1 coding sequence is (or comprises a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 228. In another example, the ASS1 coding sequence is (or comprises a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 228 and encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230. In another example, the ASS1 coding sequence is (or comprises a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 228 and encodes an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230. In another example, the ASS1 coding sequence comprises the sequence set forth in SEQ ID NO: 228. In another example, the ASS1 coding sequence consists essentially of the sequence set forth in SEQ ID NO: 228. In another example, the ASS1 coding sequence consists of the sequence set forth in SEQ ID NO: 228.Optionally, the ASS1 coding sequence encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the ASS1 coding sequence encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the ASS1 coding sequence in the above examples encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the ASS1 coding sequence in the above examples encodes an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230. Optionally, the ASS1 coding sequence in the above examples encodes an ASS1 protein consisting essentially of the sequence set forth in SEQ ID NO: 230. Optionally, the ASS1 coding sequence in the above examples encodes an ASS1 protein consisting of the sequence set forth in SEQ ID NO: 230.

[0140] In one example, the ASS1 coding sequence is (or comprises a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to any one of SEQ ID NOS: 706 and 710 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence is (or comprises a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to any one of SEQ ID NOS: 706 and 710 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence is (or comprises a sequence) at least 99%, at least 99.5%, or 100% identical to any one of SEQ ID NOS: 706 and 710 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence comprises the sequence set forth in any one of SEQ ID NOS: 706 and 710 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence consists essentially of the sequence set forth in any one of SEQ ID NOS: 706 and 710 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence consists of the sequence set forth in any one of SEQ ID NOS: 706 and 710 (optionally encoding an ASS1 protein comprising the sequence set forth inSEQ ID NO: 230). Optionally, the ASS1 coding sequence encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the AS SI coding sequence encodes an AS SI protein (or an AS SI protein comprising a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the AS SI coding sequence in the above examples encodes an AS SI protein (or an AS SI protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the ASS1 coding sequence in the above examples encodes an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230. Optionally, the ASS1 coding sequence in the above examples encodes an ASS1 protein consisting essentially of the sequence set forth in SEQ ID NO: 230. Optionally, the ASS1 coding sequence in the above examples encodes an ASS1 protein consisting of the sequence set forth in SEQ ID NO: 230 (i.e., protein encoded by exons 3-14 of human ASS1).

[0141] In one example, the ASS1 coding sequence is (or comprises a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 706 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence is (or comprises a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 706 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence is (or comprises a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 706 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence comprises the sequence set forth in SEQ ID NO: 706 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence consists essentially of the sequence set forth in SEQ ID NO: 706 (optionally encoding an AS SI protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence consists of the sequence set forth in SEQ ID NO: 706 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). Optionally,the ASS1 coding sequence encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the ASS1 coding sequence encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the AS SI coding sequence in the above examples encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the ASS1 coding sequence in the above examples encodes an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230. Optionally, the ASS1 coding sequence in the above examples encodes an ASS1 protein consisting essentially of the sequence set forth in SEQ ID NO: 230. Optionally, the ASS1 coding sequence in the above examples encodes an ASS1 protein consisting of the sequence set forth in SEQ ID NO: 230 (i.e., protein encoded by exons 3-14 of human 4, SA / ).

[0142] In one example, the ASS1 coding sequence is (or comprises a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 710 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence is (or comprises a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 710 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence is (or comprises a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 710 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence comprises the sequence set forth in SEQ ID NO: 710 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence consists essentially of the sequence set forth in SEQ ID NO: 710 (optionally encoding an AS SI protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence consists of the sequence set forth in SEQ ID NO: 710 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). Optionally,the ASS1 coding sequence encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the ASS1 coding sequence encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the AS SI coding sequence in the above examples encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the ASS1 coding sequence in the above examples encodes an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230. Optionally, the ASS1 coding sequence in the above examples encodes an ASS1 protein consisting essentially of the sequence set forth in SEQ ID NO: 230. Optionally, the ASS1 coding sequence in the above examples encodes an ASS1 protein consisting of the sequence set forth in SEQ ID NO: 230 (i.e., protein encoded by exons 3-14 of human 4,S1S7).

[0143] In one example, the ASS1 coding sequence is (or comprises a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to any one of SEQ ID NOS: 705, 709, and 713 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence is (or comprises a sequence) at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to any one of SEQ ID NOS: 705, 709, and 713 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence is (or comprises a sequence) at least 99%, at least 99.5%, or 100% identical to any one of SEQ ID NOS: 705, 709, and 713 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence comprises the sequence set forth in any one of SEQ ID NOS: 705, 709, and 713 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence consists essentially of the sequence set forth in any one of SEQ ID NOS: 705, 709, and 713 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence consists of the sequence set forth in any one of SEQ ID NOS: 705, 709, and 713 (optionally encoding an ASS1protein comprising the sequence set forth in SEQ ID NO: 230). Optionally, the ASS1 coding sequence encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the ASS1 coding sequence encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the AS SI coding sequence in the above examples encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the ASS1 coding sequence in the above examples encodes an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230. Optionally, the ASS1 coding sequence in the above examples encodes an ASS1 protein consisting essentially of the sequence set forth in SEQ ID NO: 230. Optionally, the ASS1 coding sequence in the above examples encodes an ASS1 protein consisting of the sequence set forth in SEQ ID NO: 230 (i.e., protein encoded by exons 3-14 of human ASS I .

[0144] In one example, the ASS1 coding sequence is (or comprises a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 705 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence is (or comprises a sequence) at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 705 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence is (or comprises a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 705 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence comprises the sequence set forth in SEQ ID NO: 705 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence consists essentially of the sequence set forth in SEQ ID NO: 705 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence consists of the sequence set forth in SEQ ID NO: 705 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230).Optionally, the ASS1 coding sequence encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the ASS1 coding sequence encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the ASS1 coding sequence in the above examples encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the ASS1 coding sequence in the above examples encodes an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230. Optionally, the ASS1 coding sequence in the above examples encodes an ASS1 protein consisting essentially of the sequence set forth in SEQ ID NO: 230. Optionally, the ASS1 coding sequence in the above examples encodes an ASS1 protein consisting of the sequence set forth in SEQ ID NO: 230 (i.e., protein encoded by exons 3-14 of human ASS J).

[0145] In one example, the ASS1 coding sequence is (or comprises a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 709 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence is (or comprises a sequence) at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 709 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence is (or comprises a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 709 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence comprises the sequence set forth in SEQ ID NO: 709 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence consists essentially of the sequence set forth in SEQ ID NO: 709 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence consists of the sequence set forth in SEQ ID NO: 709 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). Optionally, the ASS1 coding sequence encodes an ASS1 protein (or an AS SI protein comprisinga sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the ASS1 coding sequence encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the ASS1 coding sequence in the above examples encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the ASS1 coding sequence in the above examples encodes an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230. Optionally, the ASS1 coding sequence in the above examples encodes an ASS1 protein consisting essentially of the sequence set forth in SEQ ID NO: 230. Optionally, the ASS1 coding sequence in the above examples encodes an ASS1 protein consisting of the sequence set forth in SEQ ID NO: 230 (i.e., protein encoded by exons 3-14 of human ASS I).

[0146] In one example, the ASS1 coding sequence is (or comprises a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 713 (optionally encoding an AS SI protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence is (or comprises a sequence) at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 713 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence is (or comprises a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 713 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence comprises the sequence set forth in SEQ ID NO: 713 (optionally encoding an AS SI protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence consists essentially of the sequence set forth in SEQ ID NO: 713 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence consists of the sequence set forth in SEQ ID NO: 713 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). Optionally, the ASS1 coding sequence encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the ASS1 coding sequence encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the ASS1 coding sequence in the above examples encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the ASS1 coding sequence in the above examples encodes an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230. Optionally, the ASS1 coding sequence in the above examples encodes an ASS1 protein consisting essentially of the sequence set forth in SEQ ID NO: 230. Optionally, the ASS1 coding sequence in the above examples encodes an ASS1 protein consisting of the sequence set forth in SEQ ID NO: 230 (i.e., protein encoded by exons 3-14 of human ASS J).

[0147] When specific ASS1 nucleic acid constructs sequences are disclosed herein, they are meant to encompass the sequence disclosed or the reverse complement of the sequence. For example, if an ASS J nucleic acid construct disclosed herein consists of the hypothetical sequence 5’-CTGGACCGA-3’, it is also meant to encompass the reverse complement of that sequence (5’-TCGGTCCAG-3’). Likewise, when bidirectional construct elements are disclosed herein in a specific 5’ to 3’ order, they are also meant to encompass the reverse complement of the order of those elements. Likewise, when unidirectional construct elements are disclosed herein in a specific 5’ to 3’ order, they are also meant to encompass the reverse complement of the order of those elements. One reason for this is that, in many embodiments disclosed herein, the ASS1 nucleic acid constructs are part of a single-stranded recombinant AAV vector. Single-stranded AAV genomes are packaged as either sense (plus-stranded) or anti-sense (minus-stranded genomes), and single- stranded AAV genomes of + and - polarity are packaged with equal frequency into mature rAAV virions. See, e.g., Ling et al. (2015) J. Mol. Genet. Med. 9(3): 175, Zhou et al. (2008) Mol. Ther. 16(3):494-499, and Samulski et al. (1987) J. Virol. 61 :3096-3101, each of which is herein incorporated by reference in its entirety for all purposes.(2) Bidirectional Constructs

[0148] The ASS1 nucleic acid constructs disclosed herein can be bidirectional constructs.Such bidirectional constructs can allow for enhanced insertion and expression of encoded ASS1 . When used in combination with a nuclease agent (e.g., CRISPR / Cas system, zinc finger nuclease (ZFN) system; transcription activator-like effector nuclease (TALEN) system) as described herein, the bidirectionality of the nucleic acid construct allows the construct to be inserted in either direction (i.e., is not limited to insertion in one direction) within a target genomic locus, allowing the expression of ASS1 when inserted in either orientation, thereby enhancing expression efficiency, as exemplified herein. For example, when used in combination with a nuclease agent (e.g., CRISPR / Cas system, zinc finger nuclease (ZFN) system; transcription activator-like effector nuclease (TALEN) system) as described herein, the bidirectionality of the nucleic acid construct allows the construct to be inserted in either direction (i.e., is not limited to insertion in one direction) within a cleavage site or target insertion site, allowing the expression of ASS1 when inserted in either orientation, thereby enhancing insertion and expression efficiency, as exemplified herein.

[0149] A bidirectional construct as disclosed herein can comprise at least two nucleic acid segments, wherein a first segment comprises a first ASS1 coding sequence, and a second segment comprises the reverse complement of a second ASS1 coding sequence, or vice versa. However, other bidirectional constructs disclosed herein can comprise at least two nucleic acid segments, wherein the first segment comprises an ASS1 coding sequence, and the second segment comprises the reverse complement of a coding sequence for another protein, or vice versa. A reverse complement refers to a sequence that is a complement sequence of a reference sequence, wherein the complement sequence is written in the reverse orientation. For example, for a hypothetical sequence 5’-CTGGACCGA-3’, the perfect complement sequence is 3’- GACCTGGCT-5’, and the perfect reverse complement is written 5’-TCGGTCCAG-3’. A reverse complement sequence need not be perfect and may still encode the same polypeptide or a similar polypeptide as the reference sequence. Due to codon usage redundancy, a reverse complement can diverge from a reference sequence that encodes the same polypeptide. The coding sequences can optionally comprise one or more additional sequences, such as sequences encoding amino- or carboxy- terminal amino acid sequences such as a signal sequence, label sequence (e.g., HiBit), or heterologous functional sequence (e.g., nuclear localization sequence (NLS) or self-cleaving peptide) linked to the ASS1 or other protein.

[0150] When specific bidirectional construct sequences are disclosed herein, they are meantto encompass the sequence disclosed or the reverse complement of the sequence. For example, if a bidirectional construct disclosed herein consists of the hypothetical sequence 5’- CTGGACCGA-3’, it is also meant to encompass the reverse complement of that sequence (5’- TCGGTCCAG-3’) Likewise, when bidirectional construct elements are disclosed herein in a specific 5’ to 3’ order, they are also meant to encompass the reverse complement of the order of those elements. For example, if a bidirectional construct is disclosed herein that comprises from 5’ to 3’ a first splice acceptor, a first coding sequence, a first terminator, a reverse complement of a second terminator, a reverse complement of a second coding sequence, and a reverse complement of a second splice acceptor, it is also meant to encompass a construct comprising from 5’ to 3’ the second splice acceptor, the second coding sequence, the second terminator, a reverse complement of the first terminator, a reverse complement of the first coding sequence, and a reverse complement of the first splice acceptor. One reason for this is that, in many embodiments disclosed herein, the bidirectional constructs are part of a single-stranded recombinant AAV vector. Single-stranded AAV genomes are packaged as either sense (plus- stranded) or anti-sense (minus-stranded genomes), and single- stranded AAV genomes of + and - polarity are packaged with equal frequency into mature rAAV virions. See, e.g., Ling et al. (2015) J. Mol. Genet. Med. 9(3):175, Zhou et al. (2008)Afo / . Ther. 16(3):494-499, and Samulski et al. (1987) J. Virol. 61:3096-3101, each of which is herein incorporated by reference in its entirety for all purposes.

[0151] When the at least two segments both encode ASS1, the at least two segments can encode the same ASS1 protein or different ASS1 proteins. The different ASS1 proteins can be at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% identical. For example, the first segment can encode a wild type ASS1 protein or fragment thereof, and the second segment can encode a variant ASS1 protein or fragment thereof, or vice versa. Alternatively, the first segment can encode a first variant ASS1 protein, and the second segment can encode a second variant ASS1 protein that is different from the first variant AS SI protein. Preferably, the two segments encode the same ASS1 protein (i.e., 100% identical).

[0152] Even when the two segments encode the same AS SI protein, the AS SI coding sequence in the first segment can differ from the AS SI coding sequence in the second segment. In some bidirectional constructs, the codon usage in the first coding sequence is the same as thecodon usage in the second coding sequence. In other bidirectional constructs, the second coding sequence adopts a different codon usage from the codon usage of the first coding sequence in order to reduce hairpin formation. One or both of the coding sequences can be codon-optimized for expression in a host cell. In some bidirectional constructs, only one of the coding sequences is codon-optimized. In some bidirectional constructs, the first coding sequence is codon- optimized. In some bidirectional constructs, the second coding sequence is codon-optimized. In some bidirectional constructs, both coding sequences are codon-optimized. For example, the second ASS1 coding sequence can be codon optimized or may use one or more alternative codons for one or more amino acids of the same AS SI (i.e., same amino acid sequence) encoded by the AS SI coding sequence in the first segment. An alternative codon, as used herein, refers to variations in codon usage for a given amino acid, and it may or may not be a preferred or optimized codon (codon optimized) for a given expression system. Preferred codon usage, or codons that are well-tolerated in a given system of expression, are known.

[0153] In one example, the second segment comprises a reverse complement of an ASS1 coding sequence that adopts different codon usage from that of the ASS1 coding sequence in the first segment in order to reduce hairpin formation. Such a reverse complement forms base pairs with fewer than all nucleotides of the coding sequence in the first segment, yet it optionally encodes the same polypeptide. In one example, the reverse complement sequence in the second segment is not substantially complementary (e.g., not more than 70% complementary) to the coding sequence in the first segment. In other cases, however, the second segment comprises a reverse complement sequence that is highly complementary (e.g., at least 90% complementary) to the coding sequence in the first segment.

[0154] The second segment (the reverse complement of the second AS SI coding sequence) can have any percentage of complementarity to the first segment (the first ASS1 coding sequence). For example, the second segment sequence can have at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% complementarity to the first segment. As another example, the second segment sequence can have at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at leastabout 75%, at least about 80%, or at least about 85% complementarity to the first segment. As another example, the second segment sequence can have less than about 30%, less than about 35%, less than about 40%, less than about 45%, less than about 50%, less than about 55%, less than about 60%, less than about 65%, less than about 70%, less than about 75%, less than about 80%, less than about 85%, less than about 90%, less than about 95%, less than about 97%, or less than about 99% complementarity to the first segment. As another example, the second segment sequence can have less than about 86%, less than about 87%, less than about 88%, less than about 89%, less than about 90%, less than about 95%, less than about 97%, or less than about 99% complementarity to the first segment. The reverse complement of the second coding sequence can be, in some nucleic acid constructs, not substantially complementary (e.g., not more than 70% complementary) to the first coding sequence, not substantially complementary to a fragment of the first coding sequence, highly complementary (e.g., at least 90% complementary) to the first coding sequence, highly complementary to a fragment of the first coding sequence, about 50% to about 80% identical to the reverse complement of the first coding sequence, or about 60% to about 100% identical to the reverse complement of the first coding sequence. In some embodiments, the second segment can have between about 82% and about 90%, between about 83% and about 89%, between about 84% and about 88%, between about 85% and about 87% complementarity to the first segment.

[0155] The bidirectional constructs disclosed herein can be modified to include any suitable structural feature as needed for any particular use and / or that confers one or more desired function. For example, the bidirectional nucleic acid constructs disclosed herein need not comprise a homology arm and / or can be, for example, homology-independent donor constructs. Owing in part to the bidirectional function of the nucleic acid constructs, the bidirectional constructs can be inserted into a genomic locus in either direction as described herein to allow for efficient insertion and / or expression of ASS1.

[0156] In some cases, the bidirectional nucleic acid construct does not comprise a promoter that drives the expression of ASS1. For example, the expression of AS SI can be driven by a promoter of the host cell (e.g., the endogenous ASS1 promoter when the transgene is integrated into a host cell’s ASS1 locus). In other cases, the bidirectional nucleic acid construct can comprise one or more promoters operably linked to the ASS1 coding sequences. That is, although not required for expression, the constructs disclosed herein may also includetranscriptional or translational regulatory sequences such as promoters, enhancers, insulators, internal ribosome entry sites, additional sequences encoding peptides, and / or polyadenylation signals. Some bidirectional constructs can comprise a promoter that drives expression of the first ASS1 coding sequence and / or the reverse complement of a promoter that drives expression of the reverse complement of the second AS SI coding sequence.

[0157] The bidirectional constructs disclosed herein can be modified to include or exclude any suitable structural feature as needed for any particular use and / or that confers one or more desired functions. For example, some bidirectional nucleic acid constructs disclosed herein do not comprise a homology arm. Owing in part to the bidirectional function of the nucleic acid construct, the bidirectional construct can be inserted into a genomic locus in either direction (orientation) as described herein to allow for efficient insertion and / or expression of a heterologous ASS1.

[0158] The bidirectional constructs can, in some cases, comprise one or more (e g., two) polyadenylation tail sequences or polyadenylation signal sequences. In some bidirectional constructs, the first segment can comprise a polyadenylation signal sequence. In some bidirectional constructs, the second segment can comprise a polyadenylation signal sequence. In some bidirectional constructs, the first segment can comprise a first polyadenylation signal sequence, and the second segment can comprise a second polyadenylation signal sequence (e.g., a reverse complement of a polyadenylation signal sequence). In some bidirectional constructs, the first segment can comprise a first polyadenylation signal sequence located 3’ of the first coding sequence. In some bidirectional constructs, the second segment can comprise a reverse complement of a second poly adenylation signal sequence located 5’ of the reverse complement of the second coding sequence. In some bidirectional constructs, the first segment can comprise a first polyadenylation signal sequence located 3’ of the first coding sequence, and the second segment can comprise a reverse complement of a second polyadenylation signal sequence located 5’ of the reverse complement of the second coding sequence. The first and second polyadenylation signal sequences can be the same or different. In one example, the first and second polyadenylation signals are different. In a specific example, the first polyadenylation signal is a simian virus 40 (SV40) late polyadenylation signal (or a variant thereof), and the second polyadenylation signal is a bovine growth hormone (BGH) polyadenylation signal (or a variant thereof), or vice versa. In a specific example, one polyadenylation signal can comprise,consist essentially of, or consist of any one of SEQ ID NOS: 239-240, and the other polyadenylation signal can comprise, consist essentially of, or consist of any one of SEQ ID NOS: 236-238. In a specific example, one polyadenylation signal can comprise, consist essentially of, or consist of SEQ ID NO: 239, and the other polyadenylation signal can comprise, consist essentially of, or consist of SEQ ID NO: 237.

[0159] In some bidirectional constructs, both the first segment and the second segment comprise a polyadenylation tail sequence. Methods of designing a suitable polyadenylation tail sequence are known. For example, in some bidirectional constructs, one or both of the first and second segment comprises a polyadenylation tail sequence and / or a polyadenylation signal sequence downstream of an open reading frame (i.e., a polyadenylation tail sequence and / or a polyadenylation signal sequence 3’ of a coding sequence, or a reverse complement of a polyadenylation tail sequence and / or a polyadenylation signal sequence 5’ of a reverse complement of a coding sequence). The polyadenylation tail sequence can be encoded, for example, as a “poly-A” stretch downstream of the ASS1 coding sequence (or other protein coding sequence) in the first and / or second segment. A poly-A tail can comprise, for example, at least 20, 30, 40, 50, 60, 70, 80, 90, or 100 adenines, and optionally up to 300 adenines. In a specific example, the poly-A tail comprises 95, 96, 97, 98, 99, or 100 adenine nucleotides. Methods of designing a suitable polyadenylation tail sequence and / or polyadenylation signal sequence are well known. For example, the polyadenylation signal sequence AAUAAA is commonly used in mammalian systems, although variants such as UAUAAA or AU / GUAAA have been identified. See, e.g., Proudfoot (2011) Genes A Dev. 25(17): 1770-82, herein incorporated by reference in its entirety for all purposes. In some bidirectional constructs, a single bidirectional terminator can be used to terminate RNA polymerase transcription in either the sense or the antisense direction (i.e., to terminate RNA polymerase transcription from both the first segment and the second segment). Examples of bidirectional terminators include the AR04, TRP1, TRP4, ADH1, CYC1, GALI, GAL7, and GAL 10 terminators.

[0160] The bidirectional constructs can, in some cases, comprise one or more (e g., two) splice acceptor sites. In some bidirectional constructs, the first segment can comprise a splice acceptor site. In some bidirectional constructs, the second segment can comprise a splice acceptor site. In some bidirectional constructs, the first segment can comprise a first splice acceptor site, and the second segment can comprise a second splice acceptor site (e g., a reversecomplement of a splice acceptor site). In some bidirectional constructs, the first segment comprises a first splice acceptor site located 5’ of the first coding sequence. In some bidirectional constructs, the second segment comprises a reverse complement of a second splice acceptor site located 3’ of the reverse complement of the second coding sequence. In some bidirectional constructs, the first segment comprises a first splice acceptor site located 5’ of the first coding sequence, and the second segment comprises a reverse complement of a second splice acceptor site located 3’ of the reverse complement of the second coding sequence. The first and second splice acceptor sites can be the same or different. In a specific example, both splice acceptors are human ASS1 exon 3 splice acceptors. In another specific example, both splice acceptors are human ASS I exon 2 splice acceptors. In another example, both splice acceptors are mouse Assl exon 2 splice acceptors. In another example, both splice acceptors are mouse Alb exon 2 splice acceptors. In a specific example, both splice acceptors can comprise, consist essentially of, or consist of SEQ ID NO: 248.

[0161] A bidirectional construct may comprise a first coding sequence that encodes a first coding sequence linked to a splice acceptor and a reverse complement of a second coding sequence operably linked to the reverse complement of a splice acceptor. The bidirectional constructs disclosed herein can also comprise a splice acceptor site on either or both ends of the construct, or splice acceptor sites in both the first segment and the second segment (e.g., a splice acceptor site 5’ of a coding sequence, or a reverse complement of a splice acceptor 3’ of a reverse complement of a coding sequence). The splice acceptor site can, for example, comprise NAG or consist of NAG. In a specific example, the splice acceptor is an ASS I splice acceptor (e.g., an ASS1 splice acceptor used in the splicing together of exons 2 and 3 of ASS1 (i.e., ASS1 exon 3 splice acceptor) or an ASS I splice acceptor used in the splicing together of exons 1 and 3 of ASS J (i.e., ASS1 exon 2 splice acceptor). For example, such a splice acceptor can be derived from the human ASS1 gene (e.g., an ASS1 splice acceptor used in the splicing together of exons 2 and 3 of human ASS I (i.e., human ASS I exon 3 splice acceptor)). In another example, the splice acceptor can be derived from the mouse Assl gene (e.g., an ASS I splice acceptor used in the splicing together of exons 1 and 2 of mouse Assl (i.e., mouse Assl exon 2 splice acceptor)). In another example, the splice acceptor is an ALB splice acceptor (e.g., an ALB splice acceptor used in the splicing together of exons 1 and 2 of ALB (i.e., ALB exon 2 splice acceptor)). For example, such a splice acceptor can be derived from the human ALB gene. In another example, the spliceacceptor can be derived from the mouse Alb gene (e.g., an ALB splice acceptor used in the splicing together of exons 1 and 2 of mouse Alb (i.e., mouse d® exon 2 splice acceptor)). Additional suitable splice acceptor sites useful in eukaryotes, including artificial splice acceptors, are known. See, e.g., Shapiro et al. (1987) Nucleic Acids Res. 15:7155-7174 and Burset et al. (2001) Nucleic Acids Res. 29:255-259, each of which is herein incorporated by reference in its entirety for all purposes. The splice acceptors used in a bidirectional construct may be the same or different. In a specific example, both splice acceptors are human ASS1 exon 3 splice acceptors. In another specific example, both splice acceptors are human ASS1 exon 2 splice acceptors. In another example, both splice acceptors are mouse Assl exon 2 splice acceptors. In another example, both splice acceptors are mouse Alb exon 2 splice acceptors. In a specific example, both splice acceptors can comprise, consist essentially of, or consist of SEQ ID NO: 248.

[0162] The bidirectional constructs can be circular or linear. For example, a bidirectional construct can be linear. The first and second segments can be joined in a linear manner through a linker sequence. For example, the 5’ end of the second segment that comprises a reverse complement sequence can be linked to the 3’ end of the first segment. Alternatively, the 5’ end of the first segment can be linked to the 3’ end of the second segment that comprises a reverse complement sequence. The linker can be any suitable length. For example, the linker can be between about 5 to about 2000 nucleotides in length. As an example, the linker sequence can be about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 150, about 200, about 250, about 300, about 500, about 1000, about 1500, about 2000, or more nucleotides in length. Other structural elements in addition to, or instead of, a linker sequence, can also be inserted between the first and second segments.

[0163] The bidirectional constructs disclosed herein can be DNA or RNA, single-stranded, double-stranded, or partially single-stranded and partially double-stranded. For example, the constructs can be single- or double-stranded DNA. In some embodiments, the nucleic acid can be modified (e.g., using nucleoside analogs), as described herein. In a specific example, the bidirectional construct is single-stranded (e.g., single-stranded DNA).

[0164] The bidirectional constructs disclosed herein can be modified on either or both ends to include one or more suitable structural features as needed and / or to confer one or more functional benefit. For example, structural modifications can vary depending on the method(s) used to deliver the constructs disclosed herein to a host cell (e.g., use of viral vector delivery or packaging into lipid nanoparticles for delivery). Such modifications include, for example, terminal structures such as inverted terminal repeats (ITR), hairpin, loops, and other structures such as toroids. For example, the constructs disclosed herein can comprise one, two, or three ITRs or can comprise no more than two ITRs. Various methods of structural modifications are known.

[0165] Similarly, one or both ends of the construct can be protected (e.g., from exonucleolytic degradation) by known methods. For example, one or more dideoxynucleotide residues can be added to the 3' terminus of a linear molecule and / or self-complementary oligonucleotides can be ligated to one or both ends. See, e.g., Chang et al. (1987) Proc. Natl. Acad. Set. U.S.A. 84:4959-4963 and Nehls et al. (1996) Science 272:886-889, each of which is herein incorporated by reference in its entirety for all purposes. Additional methods for protecting the constructs from degradation include, but are not limited to, addition of terminal amino group(s) and the use of modified intemucleotide linkages such as, for example, phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyribose residues.

[0166] As disclosed in more detail herein, the bidirectional constructs disclosed herein can be introduced into a cell as part of a vector having additional sequences such as, for example, replication origins, promoters, and genes encoding antibiotic resistance. The constructs can be introduced as a naked nucleic acid, can be introduced as a nucleic acid complexed with an agent such as a liposome, polymer, or poloxamer, or can be delivered by viral vectors (e.g., adenovirus, AAV, herpesvirus, retrovirus, lentivirus).

[0167] The ASS1 coding sequences in the bidirectional constructs disclosed herein may include one or more modifications such as codon optimization (e.g., to human codons), depletion of CpG dinucleotides, mutation of cryptic splice sites, addition of one or more glycosylation sites, or any combination thereof.

[0168] In an exemplary bidirectional construct, the second segment is located 3’ of the first segment, the first ASS1 coding sequence and the second ASS1 coding sequence both encode the same human ASS1 protein, the second ASS1 coding sequence adopts a different codon usagefrom the codon usage of the first ASS1 coding sequence, the first segment comprises a first polyadenylation signal sequence located 3’ of the first ASS1 coding sequence, the second segment comprises a reverse complement of a second polyadenylation signal sequence located 5’ of the reverse complement of the second ASS1 coding sequence, the first segment comprises a first splice acceptor site located 5’ of the first AS SI coding sequence, the second segment comprises a reverse complement of a second splice acceptor site located 3’ of the reverse complement of the second ASS1 coding sequence, the nucleic acid construct does not comprise a promoter that drives expression of the first ASS1 protein or the second ASS1 protein, and optionally the nucleic acid construct does not comprise a homology arm.

[0169] In one example, the one of the ASS1 coding sequences is (or comprises a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 229. In another example, the one of the ASS1 coding sequences is (or comprises a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 229 and encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230. In another example, the one of the ASS1 coding sequences is (or comprises a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 229 and encodes an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230. In another example, the one of the ASS1 coding sequences is (or comprises a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 229. In another example, the one of the ASS1 coding sequences is (or comprises a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 229 and encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230. In another example, the one of the ASS1 coding sequences is (or comprises a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 229 and encodes an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230. In another example, the one of the ASS1 coding sequences is (or comprises a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 229. Inanother example, the one of the ASS1 coding sequences is (or comprises a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 229 and encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230. In another example, the one of the ASS1 coding sequences is (or comprises a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 229 and encodes an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230. In another example, the one of the ASS1 coding sequences comprises the sequence set forth in SEQ ID NO: 229. In another example, the one of the ASS1 coding sequences consists essentially of the sequence set forth in SEQ ID NO: 229. In another example, the one of the ASS1 coding sequences consists of the sequence set forth in SEQ ID NO: 229. Optionally, the one of the AS SI coding sequences encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the one of the ASS1 coding sequences encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the one of the AS SI coding sequences in the above examples encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the one of the ASS1 coding sequences in the above examples encodes an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230. Optionally, the one of the ASS1 coding sequences in the above examples encodes an ASS1 protein consisting essentially of the sequence set forth in SEQ ID NO: 230. Optionally, the one of the ASS1 coding sequences in the above examples encodes an ASS1 protein consisting of the sequence set forth in SEQ ID NO: 230.

[0170] In one example, the one of the ASS1 coding sequences is (or comprises a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 228. In another example, the one of the ASS1 coding sequences is (or comprises a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 228 andencodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230. In another example, the one of the ASS1 coding sequences is (or comprises a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 228 and encodes an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230. In another example, the one of the ASS1 coding sequences is (or comprises a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 228. In another example, the one of the ASS1 coding sequences is (or comprises a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 228 and encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230. In another example, the one of the ASS1 coding sequences is (or comprises a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 228 and encodes an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230. In another example, the one of the ASS1 coding sequences is (or comprises a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 228. In another example, the one of the ASS1 coding sequences is (or comprises a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 228 and encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230. In another example, the one of the ASS1 coding sequences is (or comprises a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 228 and encodes an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230. In another example, the one of the ASS1 coding sequences comprises the sequence set forth in SEQ ID NO: 228. In another example, the one of the ASS1 coding sequences consists essentially of the sequence set forth in SEQ ID NO: 228. In another example, the one of the ASS1 coding sequences consists of the sequence set forth in SEQ ID NO: 228. Optionally, the one of the AS SI coding sequences encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the one of the ASS1 coding sequences encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e ., retaining the activity of native ASS1). Optionally, the one of the ASS1 coding sequences in the above examples encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the one of the ASS1 coding sequences in the above examples encodes an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230. Optionally, the ASS1 coding sequence in the above examples encodes an ASS1 protein consisting essentially of the sequence set forth in SEQ ID NO: 230. Optionally, the one of the ASS1 coding sequences in the above examples encodes an ASS1 protein consisting of the sequence set forth in SEQ ID NO: 230.

[0171] In one example, the one of the ASS1 coding sequences is (or comprises a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 228. In another example, the one of the ASS1 coding sequences is (or comprises a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 228 and encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230. In another example, the one of the ASS1 coding sequences is (or comprises a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 228 and encodes an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230. In another example, the one of the ASS1 coding sequences is (or comprises a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 228. In another example, the one of the ASS1 coding sequences is (or comprises a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 228 and encodes an ASS 1 protein (or an ASS1 protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230. In another example, the one of the ASS1 coding sequences is (or comprises a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 228 and encodes an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230. In another example, the one of the ASS1 coding sequences is (or comprises a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 228. Inanother example, the one of the ASS1 coding sequences is (or comprises a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 228 and encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230. In another example, the one of the ASS1 coding sequences is (or comprises a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 228 and encodes an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230. In another example, the one of the ASS1 coding sequences comprises the sequence set forth in SEQ ID NO: 228. In another example, the one of the ASS1 coding sequences consists essentially of the sequence set forth in SEQ ID NO: 228. In another example, the one of the ASS1 coding sequences consists of the sequence set forth in SEQ ID NO: 228. In one example, the other ASS1 coding sequence is (or comprises a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 229. In another example, the other ASS1 coding sequence is (or comprises a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 229 and encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230. In another example, the other ASS1 coding sequence is (or comprises a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 229 and encodes an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230. In another example, the other AS SI coding sequence is (or comprises a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 229. In another example, the other ASS1 coding sequence is (or comprises a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 229 and encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230. In another example, the other ASS1 coding sequence is (or comprises a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 229 and encodes an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230. In another example, the other ASS1 coding sequence is (or comprises a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 229. In anotherexample, the other ASS1 coding sequence is (or comprises a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 229 and encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230. In another example, the other ASS1 coding sequence is (or comprises a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 229 and encodes an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230. In another example, the other ASS1 coding sequence comprises the sequence set forth in SEQ ID NO: 229. In another example, the other ASS1 coding sequence consists essentially of the sequence set forth in SEQ ID NO: 229. In another example, the other ASS1 coding sequence consists of the sequence set forth in SEQ ID NO: 229. Optionally, one or both of the ASS1 coding sequence encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, one or both of the ASS1 coding sequence encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, one or both of the ASS1 coding sequence in the above examples encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, one or both of the ASS1 coding sequence in the above examples encodes an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230. Optionally, one or both of the ASS1 coding sequence in the above examples encodes an ASS1 protein consisting essentially of the sequence set forth in SEQ ID NO: 230. Optionally, one or both of the ASS1 coding sequence in the above examples encodes an ASS1 protein consisting of the sequence set forth in SEQ ID NO: 230.

[0172] In one example, one of the ASS1 coding sequences is (or comprises a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to any one of SEQ ID NOS: 706 and 710 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, one of the ASS1 coding sequences is (or comprises a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to any one of SEQ ID NOS: 706 and 710 (optionally encoding an ASS1 proteincomprising the sequence set forth in SEQ ID NO: 230). In another example, one of the ASS1 coding sequences is (or comprises a sequence) at least 99%, at least 99.5%, or 100% identical to any one of SEQ ID NOS: 706 and 710 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, one of the ASS1 coding sequences comprises the sequence set forth in any one of SEQ ID NOS: 706 and 710 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, one of the ASS1 coding sequences consists essentially of the sequence set forth in any one of SEQ ID NOS: 706 and 710 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, one of the AS SI coding sequences consists of the sequence set forth in any one of SEQ ID NOS: 706 and 710 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). Optionally, the one of the ASS1 coding sequences encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the one of the ASS1 coding sequences encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the one of the ASS1 coding sequences in the above examples encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e g., retaining the activity of native AS SI). Optionally, the one of the AS SI coding sequences in the above examples encodes an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230. Optionally, the one of the ASS1 coding sequences in the above examples encodes an ASS1 protein consisting essentially of the sequence set forth in SEQ ID NO: 230. Optionally, the one of the ASS1 coding sequences in the above examples encodes an ASS1 protein consisting of the sequence set forth in SEQ ID NO: 230 (i.e., protein encoded by exons 3-14 of human 4AS7).

[0173] In one example, one of the ASS1 coding sequences is (or comprises a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 706 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, one of the ASS1 coding sequences is (or comprises a sequence) at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 706 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, one of the ASS1 coding sequences is (or comprises a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 706 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, one of the ASS1 coding sequences comprises the sequence set forth in SEQ ID NO: 706 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, one of the ASS1 coding sequences consists essentially of the sequence set forth in SEQ ID NO: 706 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, one of the ASS1 coding sequences consists of the sequence set forth in SEQ ID NO: 706 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). Optionally, the one of the ASS1 coding sequences encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the one of the ASS1 coding sequences encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the one of the ASS1 coding sequences in the above examples encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the one of the ASS1 coding sequences in the above examples encodes an AS SI protein comprising the sequence set forth in SEQ ID NO: 230. Optionally, the one of the ASS1 coding sequences in the above examples encodes an ASS1 protein consisting essentially of the sequence set forth in SEQ ID NO: 230. Optionally, the one of the ASS1 coding sequences in the above examples encodes an ASS1 protein consisting of the sequence set forth in SEQ ID NO: 230 (i.e., protein encoded by exons 3-14 of human ASS!).

[0174] In one example, one of the ASS1 coding sequences is (or comprises a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 710 (optionally encoding an AS SI protein comprising the sequence set forth in SEQ ID NO: 230). Inanother example, one of the ASS1 coding sequences is (or comprises a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 710 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, one of the ASS1 coding sequences is (or comprises a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 710 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, one of the ASS1 coding sequences comprises the sequence set forth in SEQ ID NO: 710 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, one of the ASS1 coding sequences consists essentially of the sequence set forth in SEQ ID NO: 710 (optionally encoding an AS SI protein comprising the sequence set forth in SEQ ID NO: 230). In another example, one of the ASS1 coding sequences consists of the sequence set forth in SEQ ID NO: 710 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). Optionally, the one of the ASS1 coding sequences encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the one of the ASS1 coding sequences encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the one of the ASS1 coding sequences in the above examples encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the one of the AS SI coding sequences in the above examples encodes an AS SI protein comprising the sequence set forth in SEQ ID NO: 230. Optionally, the one of the ASS1 coding sequences in the above examples encodes an ASS1 protein consisting essentially of the sequence set forth in SEQ ID NO: 230. Optionally, the one of the ASS1 coding sequences in the above examples encodes an ASS1 protein consisting of the sequence set forth in SEQ ID NO: 230 (i.e., protein encoded by exons 3-14 of human ASS1).

[0175] In one example, one of the ASS1 coding sequences is (or comprises a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to any one of SEQ ID NOS: 705, 709, and 713 (optionally encoding an ASS1 proteincomprising the sequence set forth in SEQ ID NO: 230). In another example, one of the ASS1 coding sequences is (or comprises a sequence) at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to any one of SEQ ID NOS: 705, 709, and 713 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, one of the ASS1 coding sequences is (or comprises a sequence) at least 99%, at least 99.5%, or 100% identical to any one of SEQ ID NOS: 705, 709, and 713 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, one of the ASS1 coding sequences comprises the sequence set forth in any one of SEQ ID NOS: 705, 709, and 713 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, one of the ASS1 coding sequences consists essentially of the sequence set forth in any one of SEQ ID NOS: 705, 709, and 713 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, one of the ASS1 coding sequences consists of the sequence set forth in any one of SEQ ID NOS: 705, 709, and 713 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). Optionally, the one of the ASS1 coding sequences encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the one of the ASS1 coding sequences encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the one of the ASS1 coding sequences in the above examples encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the one of the ASS1 coding sequences in the above examples encodes an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230. Optionally, the one of the ASS1 coding sequences in the above examples encodes an ASS1 protein consisting essentially of the sequence set forth in SEQ ID NO: 230. Optionally, the one of the ASS1 coding sequences in the above examples encodes an ASS1 protein consisting of the sequence set forth in SEQ ID NO: 230 (i.e., protein encoded by exons 3-14 of human ASS1).

[0176] In one example, one of the ASS1 coding sequences is (or comprises a sequence) atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 705 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, one of the ASS1 coding sequences is (or comprises a sequence) at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 705 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, one of the ASS1 coding sequences is (or comprises a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 705 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, one of the AS SI coding sequences comprises the sequence set forth in SEQ ID NO: 705 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, one of the ASS1 coding sequences consists essentially of the sequence set forth in SEQ ID NO: 705 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, one of the ASS1 coding sequences consists of the sequence set forth in SEQ ID NO: 705 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). Optionally, the one of the ASS1 coding sequences encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the one of the ASS1 coding sequences encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the one of the ASS1 coding sequences in the above examples encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native AS SI). Optionally, the one of the AS SI coding sequences in the above examples encodes an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230. Optionally, the one of the ASS1 coding sequences in the above examples encodes an ASS1 protein consisting essentially of the sequence set forth in SEQ ID NO: 230. Optionally, the one of the ASS1 coding sequences in the above examples encodes an ASS1 protein consisting of the sequence set forth in SEQ ID NO: 230 (i.e., protein encoded by exons 3-14 of human ASS1).

[0177] In one example, one of the ASS1 coding sequences is (or comprises a sequence) atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 709 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, one of the ASS1 coding sequences is (or comprises a sequence) at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 709 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, one of the ASS1 coding sequences is (or comprises a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 709 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, one of the AS SI coding sequences comprises the sequence set forth in SEQ ID NO: 709 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, one of the ASS1 coding sequences consists essentially of the sequence set forth in SEQ ID NO: 709 (optionally encoding an AS SI protein comprising the sequence set forth in SEQ ID NO: 230). In another example, one of the ASS1 coding sequences consists of the sequence set forth in SEQ ID NO: 709 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). Optionally, the one of the ASS1 coding sequences encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the one of the ASS1 coding sequences encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the one of the ASS1 coding sequences in the above examples encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native AS SI). Optionally, the one of the AS SI coding sequences in the above examples encodes an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230. Optionally, the one of the ASS1 coding sequences in the above examples encodes an ASS1 protein consisting essentially of the sequence set forth in SEQ ID NO: 230. Optionally, the one of the ASS1 coding sequences in the above examples encodes an ASS1 protein consisting of the sequence set forth in SEQ ID NO: 230 (i.e., protein encoded by exons 3-14 of human ASS1).

[0178] In one example, one of the ASS1 coding sequences is (or comprises a sequence) atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 713 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, one of the ASS1 coding sequences is (or comprises a sequence) at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 713 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, one of the ASS1 coding sequences is (or comprises a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 713 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, one of the AS SI coding sequences comprises the sequence set forth in SEQ ID NO: 713 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, one of the ASS1 coding sequences consists essentially of the sequence set forth in SEQ ID NO: 713 (optionally encoding an AS SI protein comprising the sequence set forth in SEQ ID NO: 230). In another example, one of the ASS1 coding sequences consists of the sequence set forth in SEQ ID NO: 713 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). Optionally, the one of the ASS1 coding sequences encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the one of the ASS1 coding sequences encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the one of the ASS1 coding sequences in the above examples encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native AS SI). Optionally, the one of the AS SI coding sequences in the above examples encodes an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230. Optionally, the one of the ASS1 coding sequences in the above examples encodes an ASS1 protein consisting essentially of the sequence set forth in SEQ ID NO: 230. Optionally, the one of the ASS1 coding sequences in the above examples encodes an ASS1 protein consisting of the sequence set forth in SEQ ID NO: 230 (i.e., protein encoded by exons 3-14 of human ASS1).

[0179] In one example, the first ASS1 coding sequence is (or comprises a sequence) at least95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 705 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the first ASS1 coding sequence is (or comprises a sequence) at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 705 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the first ASS1 coding sequence is (or comprises a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 705 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the first ASS1 coding sequence comprises the sequence set forth in SEQ ID NO: 705 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the first ASS1 coding sequence consists essentially of the sequence set forth in SEQ ID NO: 705 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the first ASS1 coding sequence consists of the sequence set forth in SEQ ID NO: 705 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). Optionally, the first ASS1 coding sequence encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the first ASS1 coding sequence encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the first ASS1 coding sequence in the above examples encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the first ASS1 coding sequence in the above examples encodes an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230. Optionally, the first ASS1 coding sequence in the above examples encodes an ASS1 protein consisting essentially of the sequence set forth in SEQ ID NO: 230. Optionally, the first ASS1 coding sequence in the above examples encodes an ASS1 protein consisting of the sequence set forth in SEQ ID NO: 230 (i.e., protein encoded by exons 3- 14 of human ASS / ). In one example, the second ASS1 coding sequence is (or comprises a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to any one of SEQ ID NOS: 706 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the second ASS1 coding sequence is (or comprises a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to any one of SEQ ID NOS: 706 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the second ASS1 coding sequence is (or comprises a sequence) at least 99%, at least 99.5%, or 100% identical to any one of SEQ ID NOS: 706 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the second ASS1 coding sequence comprises the sequence set forth in any one of SEQ ID NOS: 706 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the second ASS1 coding sequence consists essentially of the sequence set forth in any one of SEQ ID NOS: 706 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the second ASS1 coding sequence consists of the sequence set forth in any one of SEQ ID NOS: 706 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). Optionally, the second AS SI coding sequence encodes an AS SI protein (or an ASS1 protein comprising a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the second ASS1 coding sequence encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the second ASS1 coding sequence in the above examples encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the second ASS1 coding sequence in the above examples encodes an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230. Optionally, the second ASS1 coding sequence in the above examples encodes an ASS1 protein consisting essentially of the sequence set forth in SEQ ID NO: 230. Optionally, the second ASS1 coding sequence in the above examples encodes an ASS1 protein consisting of the sequence set forth in SEQ ID NO: 230 (i.e., protein encoded by exons 3-14 of human ASS1).

[0180] In one example, the first ASS1 coding sequence is (or comprises a sequence) at least95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 709 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the first ASS1 coding sequence is (or comprises a sequence) at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 709 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the first ASS1 coding sequence is (or comprises a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 709 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the first ASS1 coding sequence comprises the sequence set forth in SEQ ID NO: 709 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the first ASS1 coding sequence consists essentially of the sequence set forth in SEQ ID NO: 709 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the first ASS1 coding sequence consists of the sequence set forth in SEQ ID NO: 709 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). Optionally, the first ASS1 coding sequence encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the first ASS1 coding sequence encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the first ASS1 coding sequence in the above examples encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the first ASS1 coding sequence in the above examples encodes an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230. Optionally, the first ASS1 coding sequence in the above examples encodes an ASS1 protein consisting essentially of the sequence set forth in SEQ ID NO: 230. Optionally, the first ASS1 coding sequence in the above examples encodes an ASS1 protein consisting of the sequence set forth in SEQ ID NO: 230 (i.e., protein encoded by exons 3- 14 of human ASS / ). In one example, the second ASS1 coding sequence is (or comprises a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to any one of SEQ ID NOS: 710 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the second ASS1 coding sequence is (or comprises a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to any one of SEQ ID NOS: 710 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the second ASS1 coding sequence is (or comprises a sequence) at least 99%, at least 99.5%, or 100% identical to any one of SEQ ID NOS: 710 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the second ASS1 coding sequence comprises the sequence set forth in any one of SEQ ID NOS: 710 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the second ASS1 coding sequence consists essentially of the sequence set forth in any one of SEQ ID NOS: 710 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the second ASS1 coding sequence consists of the sequence set forth in any one of SEQ ID NOS: 710 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). Optionally, the second AS SI coding sequence encodes an AS SI protein (or an ASS1 protein comprising a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the second ASS1 coding sequence encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the second ASS1 coding sequence in the above examples encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the second ASS1 coding sequence in the above examples encodes an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230. Optionally, the second ASS1 coding sequence in the above examples encodes an ASS1 protein consisting essentially of the sequence set forth in SEQ ID NO: 230. Optionally, the second ASS1 coding sequence in the above examples encodes an ASS1 protein consisting of the sequence set forth in SEQ ID NO: 230 (i.e., protein encoded by exons 3-14 of human ASS1).

[0181] In one example, the first ASS1 coding sequence is (or comprises a sequence) at least95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 713 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the first ASS1 coding sequence is (or comprises a sequence) at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 713 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the first ASS1 coding sequence is (or comprises a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 713 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the first ASS1 coding sequence comprises the sequence set forth in SEQ ID NO: 713 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the first ASS1 coding sequence consists essentially of the sequence set forth in SEQ ID NO: 713 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the first ASS1 coding sequence consists of the sequence set forth in SEQ ID NO: 713 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). Optionally, the first ASS1 coding sequence encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the first ASS1 coding sequence encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the first ASS1 coding sequence in the above examples encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the first ASS1 coding sequence in the above examples encodes an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230. Optionally, the first ASS1 coding sequence in the above examples encodes an ASS1 protein consisting essentially of the sequence set forth in SEQ ID NO: 230. Optionally, the first ASS1 coding sequence in the above examples encodes an ASS1 protein consisting of the sequence set forth in SEQ ID NO: 230 (i.e., protein encoded by exons 3- 14 of human ASS / ). In one example, the second ASS1 coding sequence is (or comprises a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to any one of SEQ ID NOS: 706 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the second ASS1 coding sequence is (or comprises a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to any one of SEQ ID NOS: 706 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the second ASS1 coding sequence is (or comprises a sequence) at least 99%, at least 99.5%, or 100% identical to any one of SEQ ID NOS: 706 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the second ASS1 coding sequence comprises the sequence set forth in any one of SEQ ID NOS: 706 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the second ASS1 coding sequence consists essentially of the sequence set forth in any one of SEQ ID NOS: 706 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the second ASS1 coding sequence consists of the sequence set forth in any one of SEQ ID NOS: 706 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). Optionally, the second AS SI coding sequence encodes an AS SI protein (or an ASS1 protein comprising a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the second ASS1 coding sequence encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the second ASS1 coding sequence in the above examples encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the second ASS1 coding sequence in the above examples encodes an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230. Optionally, the second ASS1 coding sequence in the above examples encodes an ASS1 protein consisting essentially of the sequence set forth in SEQ ID NO: 230. Optionally, the second ASS1 coding sequence in the above examples encodes an ASS1 protein consisting of the sequence set forth in SEQ ID NO: 230 (i.e., protein encoded by exons 3-14 of human ASS1).

[0182] In a particular example, an exemplary bidirectional construct comprises a sequence atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to any one of SEQ ID NOS: 703, 704, 707, 708, 711, and 712. In another particular example, an exemplary bidirectional construct comprises a sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to any one of SEQ ID NOS: 703, 704, 707, 708, 711, and 712. In another particular example, an exemplary bidirectional construct comprises a sequence at least 99%, at least 99.5%, or 100% identical to any one of SEQ ID NOS: 703, 704, 707, 708, 711, and 712. In another particular example, an exemplary bidirectional construct comprises any one of SEQ ID NOS: 703, 704, 707, 708, 711, and 712. In another particular example, an exemplary bidirectional construct consists essentially of any one of SEQ ID NOS: 703, 704, 707, 708, 711, and 712. In another particular example, an exemplary bidirectional construct consists of any one of SEQ ID NOS: 703, 704, 707, 708, 711, and 712.

[0183] In a particular example, an exemplary bidirectional construct comprises a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to any one of SEQ ID NOS: 703 and 704. In another particular example, an exemplary bidirectional construct comprises a sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to any one of SEQ ID NOS: 703 and 704. In another particular example, an exemplary bidirectional construct comprises a sequence at least 99%, at least 99.5%, or 100% identical to any one of SEQ ID NOS: 703 and 704. In another particular example, an exemplary bidirectional construct comprises any one of SEQ ID NOS: 703 and 704. In another particular example, an exemplary bidirectional construct consists essentially of any one of SEQ ID NOS: 703 and 704. In another particular example, an exemplary bidirectional construct consists of any one of SEQ ID NOS: 703 and 704.

[0184] In a particular example, an exemplary bidirectional construct comprises a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO 703. In another particular example, an exemplary bidirectional construct comprises a sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO 703. In another particular example, an exemplary bidirectional construct comprises a sequence at least 99%, at least 99.5%, or 100% identical to SEQ ID NO 703. In anotherparticular example, an exemplary bidirectional construct comprises SEQ ID NO 703. In another particular example, an exemplary bidirectional construct consists essentially of SEQ ID NO 703. In another particular example, an exemplary bidirectional construct consists of SEQ ID NO 703.

[0185] In a particular example, an exemplary bidirectional construct comprises a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 704. In another particular example, an exemplary bidirectional construct comprises a sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 704. In another particular example, an exemplary bidirectional construct comprises a sequence at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 704. In another particular example, an exemplary bidirectional construct comprises SEQ ID NO: 704. In another particular example, an exemplary bidirectional construct consists essentially of SEQ ID NO: 704. In another particular example, an exemplary bidirectional construct consists of SEQ ID NO: 704.

[0186] In a particular example, an exemplary bidirectional construct comprises a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to any one of SEQ ID NOS: 707 and 708. In another particular example, an exemplary bidirectional construct comprises a sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to any one of SEQ ID NOS: 707 and 708. In another particular example, an exemplary bidirectional construct comprises a sequence at least 99%, at least 99.5%, or 100% identical to any one of SEQ ID NOS: 707 and 708. In another particular example, an exemplary bidirectional construct comprises any one of SEQ ID NOS: 707 and 708. In another particular example, an exemplary bidirectional construct consists essentially of any one of SEQ ID NOS: 707 and 708. In another particular example, an exemplary bidirectional construct consists of any one of SEQ ID NOS: 707 and 708.

[0187] In a particular example, an exemplary bidirectional construct comprises a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 707. In another particular example, an exemplary bidirectional construct comprises a sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 707. In another particular example, an exemplary bidirectional construct comprisesa sequence at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 707. In another particular example, an exemplary bidirectional construct comprises SEQ ID NO: 707. In another particular example, an exemplary bidirectional construct consists essentially of SEQ ID NO: 707. In another particular example, an exemplary bidirectional construct consists of SEQ ID NO: 707.

[0188] In a particular example, an exemplary bidirectional construct comprises a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 708. In another particular example, an exemplary bidirectional construct comprises a sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 708. In another particular example, an exemplary bidirectional construct comprises a sequence at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 708. In another particular example, an exemplary bidirectional construct comprises SEQ ID NO: 708. In another particular example, an exemplary bidirectional construct consists essentially of SEQ ID NO: 708. In another particular example, an exemplary bidirectional construct consists of SEQ ID NO: 708.

[0189] In a particular example, an exemplary bidirectional construct comprises a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to any one of SEQ ID NOS: 711 and 712. In another particular example, an exemplary bidirectional construct comprises a sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to any one of SEQ ID NOS: 711 and 712. In another particular example, an exemplary bidirectional construct comprises a sequence at least 99%, at least 99.5%, or 100% identical to any one of SEQ ID NOS: 711 and 712. In another particular example, an exemplary bidirectional construct comprises any one of SEQ ID NOS: 711 and 712. In another particular example, an exemplary bidirectional construct consists essentially of any one of SEQ ID NOS: 711 and 712. In another particular example, an exemplary bidirectional construct consists of any one of SEQ ID NOS: 711 and 712.

[0190] In a particular example, an exemplary bidirectional construct comprises a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 711. In another particular example, an exemplary bidirectional construct comprises a sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical toSEQ ID NO: 711 . In another particular example, an exemplary bidirectional construct comprises a sequence at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 711. In another particular example, an exemplary bidirectional construct comprises SEQ ID NO: 711. In another particular example, an exemplary bidirectional construct consists essentially of SEQ ID NO: 711. In another particular example, an exemplary bidirectional construct consists of SEQ ID NO: 711.

[0191] In a particular example, an exemplary bidirectional construct comprises a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 712. In another particular example, an exemplary bidirectional construct comprises a sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 712. In another particular example, an exemplary bidirectional construct comprises a sequence at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 712. In another particular example, an exemplary bidirectional construct comprises SEQ ID NO: 712. In another particular example, an exemplary bidirectional construct consists essentially of SEQ ID NO: 712. In another particular example, an exemplary bidirectional construct consists of SEQ ID NO: 712.(3) Unidirectional Constructs

[0192] The ASS1 nucleic acid constructs disclosed herein can be unidirectional constructs.

[0193] When specific unidirectional construct sequences are disclosed herein, they are meant to encompass the sequence disclosed or the reverse complement of the sequence. For example, if a unidirectional construct disclosed herein consists of the hypothetical sequence 5’- CTGGACCGA-3’, it is also meant to encompass the reverse complement of that sequence (5’- TCGGTCCAG-3’). Likewise, when unidirectional construct elements are disclosed herein in a specific 5’ to 3’ order, they are also meant to encompass the reverse complement of the order of those elements. One reason for this is that, in many embodiments disclosed herein, the unidirectional constructs are part of a single-stranded recombinant AAV vector. Single-stranded AAV genomes are packaged as either sense (plus-stranded) or anti-sense (minus-stranded genomes), and single-stranded AAV genomes of + and - polarity are packaged with equal frequency into mature rAAV virions. See, e.g., Ling et al. (2015) J. Mol. Genet. Med. 9(3): 175, Zhou et al. (2008) Mol. Ther. 16(3):494-499, and Samulski et al. (1987) J. Virol. 61 :3096-3101, each of which is herein incorporated by reference in its entirety for all purposes.

[0194] In the unidirectional constructs, the ASS1 coding sequence can be a wild type ASS1 coding sequence without further modification. In the unidirectional constructs, the ASS1 coding sequence can be codon-optimized for expression in a host cell. For example, the ASS1 coding sequence can be codon optimized or may use one or more alternative codons for one or more amino acids of the ASS1 (i.e., same amino acid sequence). An alternative codon, as used herein, refers to variations in codon usage for a given amino acid, and it may or may not be a preferred or optimized codon (codon optimized) for a given expression system. Preferred codon usage, or codons that are well-tolerated in a given system of expression, are known.

[0195] The unidirectional constructs disclosed herein can be modified to include any suitable structural feature as needed for any particular use and / or that confers one or more desired functions. For example, the unidirectional nucleic acid constructs disclosed herein need not comprise a homology arm and / or can be, for example, homology-independent donor constructs.

[0196] In some cases, the unidirectional nucleic acid construct does not comprise a promoter that drives the expression of ASS1. For example, the expression of ASS1 can be driven by a promoter of the host cell (e.g., the endogenous ASS I promoter when the transgene is integrated into a host cell’s ASS J locus). In other cases, the unidirectional nucleic acid construct can comprise one or more promoters operably linked to the ASS1 coding sequence. That is, although not required for expression, the constructs disclosed herein may also include transcriptional or translational regulatory sequences such as promoters, enhancers, insulators, internal ribosome entry sites, additional sequences encoding peptides, and / or polyadenylation signals. Some unidirectional constructs can comprise a promoter that drives expression of the ASS1 coding sequence.

[0197] The unidirectional constructs can, in some cases, comprise one or more polyadenylation tail sequences or polyadenylation signal sequences. Some unidirectional constructs can comprise a polyadenylation signal sequence located 3’ of the ASS1 coding sequence. In one example, the polyadenylation signal is a simian virus 40 (SV40) late polyadenylation signal. For example, the polyadenylation signal can comprise, consist essentially of, or consist of SEQ ID NO: 239 or SEQ ID NO: 240. In another example, the polyadenylation signal is a bovine growth hormone (BGH) polyadenylation signal. For example, the polyadenylation signal can comprise, consist essentially of, or consist of SEQ ID NO: 236, SEQ ID NO: 237, or SEQ ID NO: 238. In another example, the polyadenylation signal is a humangrowth hormone (HGH) polyadenylation signal. For example, the polyadenylation signal can comprise, consist essentially of, or consist of SEQ ID NO: 235.

[0198] Methods of designing a suitable polyadenylation tail sequence are known. For example, some unidirectional constructs comprise a polyadenylation tail sequence and / or a polyadenylation signal sequence downstream of an open reading frame (i.e., a polyadenylation tail sequence and / or a polyadenylation signal sequence 3’ of a coding sequence). The polyadenylation tail sequence can be encoded, for example, as a “poly-A” stretch downstream of the AS SI coding sequence (or other protein coding sequence) in the first and / or second segment. A poly-A tail can comprise, for example, at least 20, 30, 40, 50, 60, 70, 80, 90, or 100 adenines, and optionally up to 300 adenines. In a specific example, the poly-A tail comprises 95, 96, 97, 98, 99, or 100 adenine nucleotides. Methods of designing a suitable polyadenylation tail sequence and / or polyadenylation signal sequence are well known. For example, the polyadenylation signal sequence AAUAAA is commonly used in mammalian systems, although variants such as UAUAAA or AU / GUAAA have been identified. See, e.g., Proudfoot (2011) Genes & Dev. 25(17): 1770-82, herein incorporated by reference in its entirety for all purposes.

[0199] The unidirectional constructs can, in some cases, comprise one or more splice acceptor sites. Some unidirectional constructs comprise a splice acceptor site located 5’ of the ASS1 coding sequence. In a specific example, the splice acceptor is a human ASS1 exon 3 splice acceptor. In another specific example, the splice acceptor is a human ASS1 exon 2 splice acceptor. In another example, the splice acceptor is a mouse Assl exon 2 splice acceptor. In another specific example, the splice acceptor is a mouse Alb exon 2 splice acceptor. In a specific example, the splice acceptor can comprise, consist essentially of, or consist of SEQ ID NO: 248.

[0200] The splice acceptor site can, for example, comprise NAG or consist of NAG. In a specific example, the splice acceptor is an ASS J splice acceptor (e.g., an ASS I splice acceptor used in the splicing together of exons 2 and 3 of ASS1 (i.e., ASS1 exon 3 splice acceptor) or an ASS I splice acceptor used in the splicing together of exons 1 and 3 of ASS1 (i.e., ASS1 exon 2 splice acceptor). For example, such a splice acceptor can be derived from the human ASS1 gene (e.g., an ASS1 splice acceptor used in the splicing together of exons 2 and 3 of human ASS1 (i.e., human ASS I exon 3 splice acceptor)). In another example, the splice acceptor can be derived from the mouse Assl gene (e.g., an ASS1 splice acceptor used in the splicing together of exons 1 and 2 of mouse Assl (i.e., mouse Assl exon 2 splice acceptor)). In another example, the spliceacceptor is an ALB splice acceptor (e.g., an ALB splice acceptor used in the splicing together of exons 1 and 2 of ALB (i.e., ALB exon 2 splice acceptor)). For example, such a splice acceptor can be derived from the human ALB gene. In another example, the splice acceptor can be derived from the mouse Alb gene (e.g., an ALB splice acceptor used in the splicing together of exons 1 and 2 of mouse d / / ) (i.e., moused / / ) exon 2 splice acceptor)). Additional suitable splice acceptor sites useful in eukaryotes, including artificial splice acceptors, are known. See, e.g., Shapiro et al. (1987) Nucleic Acids Res. 15:7155-7174 and Burset et al. (2001) Nucleic Acids Res. 29:255-259, each of which is herein incorporated by reference in its entirety for all purposes.

[0201] The unidirectional constructs can be circular or linear. For example, a unidirectional construct can be linear.

[0202] The unidirectional constructs disclosed herein can be DNA or RNA, single-stranded, double-stranded, or partially single-stranded and partially double-stranded. For example, the constructs can be single- or double-stranded DNA. In some embodiments, the nucleic acid can be modified (e.g., using nucleoside analogs), as described herein. In a specific example, the unidirectional construct is single-stranded (e.g., single-stranded DNA).

[0203] The unidirectional constructs disclosed herein can be modified on either or both ends to include one or more suitable structural features as needed and / or to confer one or more functional benefit. For example, structural modifications can vary depending on the method(s) used to deliver the constructs disclosed herein to a host cell (e.g., use of viral vector delivery or packaging into lipid nanoparticles for delivery). Such modifications include, for example, terminal structures such as inverted terminal repeats (ITR), hairpin, loops, and other structures such as toroids. For example, the constructs disclosed herein can comprise one, two, or three ITRs or can comprise no more than two ITRs. Various methods of structural modifications are known.

[0204] Similarly, one or both ends of the construct can be protected (e.g., from exonucleolytic degradation) by known methods. For example, one or more dideoxynucleotide residues can be added to the 3' terminus of a linear molecule and / or self-complementary oligonucleotides can be ligated to one or both ends. See, e.g., Chang et al. (1987) Proc. Natl. Acad. Sci. U.S.A. 84:4959-4963 and Nehls et al. (1996) Science 272:886-889, each of which is herein incorporated by reference in its entirety for all purposes. Additional methods for protecting the constructs from degradation include, but are not limited to, addition of terminalamino group(s) and the use of modified intemucleotide linkages such as, for example, phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyribose residues.

[0205] As disclosed in more detail herein, the unidirectional constructs disclosed herein can be introduced into a cell as part of a vector having additional sequences such as, for example, replication origins, promoters, and genes encoding antibiotic resistance. The constructs can be introduced as a naked nucleic acid, can be introduced as a nucleic acid complexed with an agent such as a liposome, polymer, or poloxamer, or can be delivered by viral vectors (e.g., adenovirus, AAV, herpesvirus, retrovirus, lentivirus).

[0206] The ASS1 coding sequences in the unidirectional constructs disclosed herein may include one or more modifications such as codon optimization (e.g., to human codons), depletion of CpG dinucleotides, mutation of cryptic splice sites, addition of one or more glycosylation sites, or any combination thereof.

[0207] In an exemplary unidirectional construct, the construct comprises a polyadenylation signal sequence located 3’ of the ASS1 coding sequence, the construct comprises a splice acceptor site located 5’ of the ASS1 coding sequence, and the nucleic acid construct does not comprise a promoter that drives expression of the ASS1 protein, and optionally the nucleic acid construct does not comprise a homology arm.

[0208] In one example, the ASS1 coding sequence is (or comprises a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 229. In another example, the ASS1 coding sequence is (or comprises a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 229 and encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230. In another example, the ASS1 coding sequence is (or comprises a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 229 and encodes an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230. In another example, the ASS1 coding sequence is (or comprises a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 229. In another example, the ASS1 coding sequence is (or comprises a sequence) at least 95%, at least96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 229 and encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230. In another example, the ASS1 coding sequence is (or comprises a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 229 and encodes an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230. In another example, the ASS1 coding sequence is (or comprises a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 229. In another example, the ASS1 coding sequence is (or comprises a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 229 and encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230. In another example, the ASS1 coding sequence is (or comprises a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 229 and encodes an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230. In another example, the ASS1 coding sequence comprises the sequence set forth in SEQ ID NO: 229. In another example, the ASS1 coding sequence consists essentially of the sequence set forth in SEQ ID NO: 229. In another example, the one of the ASS1 coding sequences consists of the sequence set forth in SEQ ID NO: 229. Optionally, the ASS1 coding sequence encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the AS SI coding sequence encodes an AS SI protein (or an AS SI protein comprising a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the AS SI coding sequence in the above examples encodes an AS SI protein (or an AS SI protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the ASS1 coding sequence in the above examples encodes an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230. Optionally, the ASS1 coding sequence in the above examples encodes an ASS1 protein consisting essentially of the sequence set forth in SEQ ID NO: 230. Optionally, the ASS1 coding sequence in the above examples encodes an ASS1 protein consisting of the sequence set forth in SEQ ID NO: 230.

[0209] In one example, the ASS1 coding sequence is (or comprises a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 228. In another example, the ASS1 coding sequence is (or comprises a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 228 and encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230. In another example, the ASS1 coding sequence is (or comprises a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 228 and encodes an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230. In another example, the ASS1 coding sequence is (or comprises a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 228. In another example, the ASS1 coding sequence is (or comprises a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 228 and encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230. In another example, the ASS1 coding sequence is (or comprises a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 228 and encodes an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230. In another example, the ASS1 coding sequence is (or comprises a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 228. In another example, the ASS1 coding sequence is (or comprises a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 228 and encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230. In another example, the ASS1 coding sequence is (or comprises a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 228 and encodes an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230. In another example, the ASS1 coding sequence comprises the sequence set forth in SEQ ID NO: 228. In another example, the ASS1 coding sequence consists essentially of the sequence set forth in SEQ ID NO: 228. In another example, the one of the ASS1 coding sequences consists of the sequence set forth in SEQ ID NO: 228. Optionally, the ASS1 coding sequence encodes an ASS1 protein (or an ASS1 proteincomprising a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the ASS1 coding sequence encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the AS SI coding sequence in the above examples encodes an AS SI protein (or an AS SI protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the ASS1 coding sequence in the above examples encodes an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230. Optionally, the ASS1 coding sequence in the above examples encodes an ASS1 protein consisting essentially of the sequence set forth in SEQ ID NO: 230. Optionally, the ASS1 coding sequence in the above examples encodes an ASS1 protein consisting of the sequence set forth in SEQ ID NO: 230.

[0210] In one example, the ASS1 coding sequence is (or comprises a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to any one of SEQ ID NOS: 706 and 710 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence is (or comprises a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to any one of SEQ ID NOS: 706 and 710 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence is (or comprises a sequence) at least 99%, at least 99.5%, or 100% identical to any one of SEQ ID NOS: 706 and 710 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence comprises the sequence set forth in any one of SEQ ID NOS: 706 and 710 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence consists essentially of the sequence set forth in any one of SEQ ID NOS: 706 and 710 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence consists of the sequence set forth in any one of SEQ ID NOS: 706 and 710 (optionally encoding an ASS1 protein comprising the sequence set forth inSEQ ID NO: 230). Optionally, the ASS1 coding sequence encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the AS SI coding sequence encodes an AS SI protein (or an AS SI protein comprising a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the AS SI coding sequence in the above examples encodes an AS SI protein (or an AS SI protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the ASS1 coding sequence in the above examples encodes an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230. Optionally, the ASS1 coding sequence in the above examples encodes an ASS1 protein consisting essentially of the sequence set forth in SEQ ID NO: 230. Optionally, the ASS1 coding sequence in the above examples encodes an ASS1 protein consisting of the sequence set forth in SEQ ID NO: 230 (i.e., protein encoded by exons 3-14 of human ASS1).

[0211] In one example, the ASS1 coding sequence is (or comprises a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 706 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence is (or comprises a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 706 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence is (or comprises a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 706 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence comprises the sequence set forth in SEQ ID NO: 706 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence consists essentially of the sequence set forth in SEQ ID NO: 706 (optionally encoding an AS SI protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence consists of the sequence set forth in SEQ ID NO: 706 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). Optionally,the ASS1 coding sequence encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the ASS1 coding sequence encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the AS SI coding sequence in the above examples encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the ASS1 coding sequence in the above examples encodes an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230. Optionally, the ASS1 coding sequence in the above examples encodes an ASS1 protein consisting essentially of the sequence set forth in SEQ ID NO: 230. Optionally, the ASS1 coding sequence in the above examples encodes an ASS1 protein consisting of the sequence set forth in SEQ ID NO: 230 (i.e., protein encoded by exons 3-14 of human 4,S1S7).

[0212] In one example, the ASS1 coding sequence is (or comprises a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 710 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence is (or comprises a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 710 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence is (or comprises a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 710 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence comprises the sequence set forth in SEQ ID NO: 710 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence consists essentially of the sequence set forth in SEQ ID NO: 710 (optionally encoding an AS SI protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence consists of the sequence set forth in SEQ ID NO: 710 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). Optionally,the ASS1 coding sequence encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the ASS1 coding sequence encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the AS SI coding sequence in the above examples encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the ASS1 coding sequence in the above examples encodes an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230. Optionally, the ASS1 coding sequence in the above examples encodes an ASS1 protein consisting essentially of the sequence set forth in SEQ ID NO: 230. Optionally, the ASS1 coding sequence in the above examples encodes an ASS1 protein consisting of the sequence set forth in SEQ ID NO: 230 (i.e., protein encoded by exons 3-14 of human 4,S1S7).

[0213] In one example, the ASS1 coding sequence is (or comprises a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to any one of SEQ ID NOS: 705, 709, and 713 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence is (or comprises a sequence) at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to any one of SEQ ID NOS: 705, 709, and 713 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence is (or comprises a sequence) at least 99%, at least 99.5%, or 100% identical to any one of SEQ ID NOS: 705, 709, and 713 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence comprises the sequence set forth in any one of SEQ ID NOS: 705, 709, and 713 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence consists essentially of the sequence set forth in any one of SEQ ID NOS: 705, 709, and 713 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence consists of the sequence set forth in any one of SEQ ID NOS: 705, 709, and 713 (optionally encoding an ASS1protein comprising the sequence set forth in SEQ ID NO: 230). Optionally, the ASS1 coding sequence encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the ASS1 coding sequence encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the AS SI coding sequence in the above examples encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the ASS1 coding sequence in the above examples encodes an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230. Optionally, the ASS1 coding sequence in the above examples encodes an ASS1 protein consisting essentially of the sequence set forth in SEQ ID NO: 230. Optionally, the ASS1 coding sequence in the above examples encodes an ASS1 protein consisting of the sequence set forth in SEQ ID NO: 230 (i.e., protein encoded by exons 3-14 of human ASS I .

[0214] In one example, the ASS1 coding sequence is (or comprises a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 705 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence is (or comprises a sequence) at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 705 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence is (or comprises a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 705 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence comprises the sequence set forth in SEQ ID NO: 705 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence consists essentially of the sequence set forth in SEQ ID NO: 705 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence consists of the sequence set forth in SEQ ID NO: 705 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230).Optionally, the ASS1 coding sequence encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the ASS1 coding sequence encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the ASS1 coding sequence in the above examples encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the ASS1 coding sequence in the above examples encodes an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230. Optionally, the ASS1 coding sequence in the above examples encodes an ASS1 protein consisting essentially of the sequence set forth in SEQ ID NO: 230. Optionally, the ASS1 coding sequence in the above examples encodes an ASS1 protein consisting of the sequence set forth in SEQ ID NO: 230 (i.e., protein encoded by exons 3-14 of human ASS J).

[0215] In one example, the ASS1 coding sequence is (or comprises a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 709 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence is (or comprises a sequence) at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 709 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence is (or comprises a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 709 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence comprises the sequence set forth in SEQ ID NO: 709 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence consists essentially of the sequence set forth in SEQ ID NO: 709 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). In another example, the ASS1 coding sequence consists of the sequence set forth in SEQ ID NO: 709 (optionally encoding an ASS1 protein comprising the sequence set forth in SEQ ID NO: 230). Optionally, the ASS1 coding sequence encodes an ASS1 protein (or an AS SI protein comprisinga sequence) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the ASS1 coding sequence encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the ASS1 coding sequence in the above examples encodes an ASS1 protein (or an ASS1 protein comprising a sequence) at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 230 (and, e.g., retaining the activity of native ASS1). Optionally, the ASS1 coding sequence in the ...

Claims

We claim:

1. A composition comprising a nuclease agent that targets a nuclease target site in an argininosuccinate synthase 1 (ASS / ) gene.

2. The composition of claim 1, wherein the ASS1 gene is a human ASS1 gene.

3. The composition of claim 1 or 2, wherein the nuclease target site is in intron 1 or intron 2 of the ASS1 gene.

4. The composition of any one of claims 1-3, wherein the nuclease target site is in intron 2 of the ASS1 gene.

5. The composition of any one of claims 1-4, wherein the nuclease agent comprises:(a) a zinc finger nuclease (ZFN);(b) a transcription activator-like effector nuclease (TALEN); or(c) (i) a Cas protein or a nucleic acid encoding the Cas protein; and(ii) a guide RNA or one or more DNAs encoding the guide RNA, wherein the guide RNA comprises a DNA-targeting segment that targets a guide RNA target sequence, and wherein the guide RNA binds to the Cas protein and targets the Cas protein to the guide RNA target sequence.

6. A composition comprising a guide RNA or one or more DNAs encoding the guide RNA, wherein the guide RNA comprises a DNA-targeting segment that targets a guide RNA target sequence in an intron of an ASS I gene, and wherein the guide RNA binds to a Cas protein and targets the Cas protein to the guide RNA target sequence.

7. The composition of claim 6, wherein the intron is intron 1 or intron 2.

8. The composition of claim 6 or 7, wherein the ASS J gene is a human ASS I gene, and the guide RNA target sequence is in intron 2 of the human ASS I gene.

9. The composition of any one of claims 6-8, wherein:(I) the DNA-targeting segment comprises at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of the sequence set forth in any one of SEQ ID NOS: 78, 60, 89, 31-59, 61-77, 79-88, and 90-118, optionally wherein the DNA-targeting segment comprises at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of the sequence set forth in any one of SEQ ID NOS: 78, 60, 89, 58, 67, 73, 92, and 114, optionally wherein the DNA-targeting segment comprises at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of the sequence set forth in any one of SEQ ID NOS: 78, 60, and 89, optionally wherein the DNA-targeting segment comprises at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of the sequence set forth in SEQ ID NO: 78; and / or(II) the DNA-targeting segment is at least 90% or at least 95% identical to the sequence set forth in any one of SEQ ID NOS: 78, 60, 89, 31-59, 61-77, 79-88, and 90-118, optionally wherein the DNA-targeting segment is at least 90% or at least 95% identical to the sequence set forth in any one of SEQ ID NOS: 78, 60, 89, 58, 67, 73, 92, and 114, optionally wherein the DNA-targeting segment is at least 90% or at least 95% identical to the sequence set forth in any one of SEQ ID NOS: 78, 60, and 89, optionally wherein the DNA-targeting segment is at least 90% or at least 95% identical to the sequence set forth in SEQ ID NO: 78.

10. The composition of any one of claims 6-9, wherein the DNA-targeting segment comprises any one of SEQ ID NOS: 78, 60, 89, 31-59, 61-77, 79-88, and 90-118, optionally wherein the DNA-targeting segment comprises any one of SEQ ID NOS: 78, 60, 89, 58, 67, 73, 92, and 114, optionally wherein the DNA-targeting segment comprises any one of SEQ ID NOS: 78, 60, and 89, optionally wherein the DNA-targeting segment comprises SEQ ID NO: 78.

11. The composition of any one of claims 6-10, wherein the DNA-targeting segment consists of any one of SEQ ID NOS: 78, 60, 89, 31-59, 61-77, 79-88, and 90-118, optionally wherein the DNA-targeting segment consists of any one of SEQ ID NOS: 78, 60, 89, 58, 67, 73, 92, and 114, optionally wherein the DNA-targeting segment consists of any one of SEQ ID NOS: 78, 60, and 89, optionally wherein the DNA-targeting segment consists of SEQ ID NO: 78.

12. The composition of any one of claims 6-11 , wherein the guide RNA comprises any one of SEQ ID NOS: 387, 369, 398, 340-368, 370-386, 388-397, and 399-427, optionally wherein the guide RNA comprises any one of SEQ ID NOS: 387, 369, 398, 367, 376, 382, 401, and 423, optionally wherein the guide RNA comprises any one of SEQ ID NOS: 387, 369, and 398, optionally wherein the guide RNA comprises SEQ ID NO: 387.

13. The composition of any one of claims 6-12, wherein:(I) the DNA-targeting segment comprises at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of SEQ ID NO: 78; and / or(II) the DNA-targeting segment is at least 90% or at least 95% identical to SEQ ID NO: 78.

14. The composition of any one of claims 6-13, wherein the DNA-targeting segment comprises SEQ ID NO: 78.

15. The composition of any one of claims 6-14, wherein the DNA-targeting segment consists of SEQ ID NO: 78.

16. The composition of any one of claims 6-11 and 13-15, wherein the guide RNA comprises SEQ ID NO: 387.

17. The composition of claim 6 or 7, wherein the ASS1 gene is a human ASS1 gene, and the guide RNA target sequence is in intron 1 of the human ASS I gene.

18. The composition of any one of claims 6, 7, and 17, wherein:(I) the DNA-targeting segment comprises at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of the sequence set forth in any one of SEQ ID NOS: 430- 517; and / or(II) the DNA-targeting segment is at least 90% or at least 95% identical to the sequence set forth in any one of SEQ ID NOS: 430-517.

19. The composition of any one of claims 6, 7, 17, and 18, wherein the DNA- targeting segment comprises any one of SEQ ID NOS: 430-517.

20. The composition of any one of claims 6, 7, and 17-19, wherein the DNA- targeting segment consists of any one of SEQ ID NOS: 430-517.

21. The composition of any one of claims 6, 7, and 17-20, wherein the guide RNA comprises any one of SEQ ID NOS: 606-693.

22. The composition of any one of claims 6-21, wherein the composition comprises the guide RNA in the form of RNA.

23. The composition of any one of claims 6-22, wherein the guide RNA comprises at least one modification.

24. The composition of claim 23, wherein the at least one modification comprises a 2’-O-methyl-modified nucleotide.

25. The composition of claim 23 or 24, wherein the at least one modification comprises a phosphorothioate bond between nucleotides.

26. The composition of any one of claims 23-25, wherein the at least one modification comprises a modification at one or more of the first five nucleotides at the 5’ end of the guide RNA.

27. The composition of any one of claims 23-26, wherein the at least one modification comprises a modification at one or more of the last five nucleotides at the 3’ end of the guide RNA.

28. The composition of any one of claims 23-27, wherein the at least one modification comprises phosphorothioate bonds between the first four nucleotides at the 5’ end of the guide RNA.

29. The composition of any one of claims 23-28, wherein the at least one modification comprises phosphorothioate bonds between the last four nucleotides at the 3’ end of the guide RNA.

30. The composition of any one of claims 23-29, wherein the at least one modification comprises 2’-O-methyl-modified nucleotides at the first three nucleotides at the 5’ end of the guide RNA.

31. The composition of any one of claims 23-30, wherein the at least one modification comprises 2’-O-methyl-modified nucleotides at the last three nucleotides at the 3’ end of the guide RNA.

32. The composition of any one of claims 23-31, wherein the at least one modification comprises: (i) phosphorothioate bonds between the first four nucleotides at the 5’ end of the guide RNA; (ii) phosphorothioate bonds between the last four nucleotides at the 3’ end of the guide RNA; (iii) 2’-O-methyl-modified nucleotides at the first three nucleotides at the 5’ end of the guide RNA; and (iv) 2’-O-methyl-modified nucleotides at the last three nucleotides at the 3’ end of the guide RNA.

33. The composition of any one of claims 6-32, wherein the guide RNA is a single guide RNA (sgRNA).

34. The composition of claim 33, wherein the guide RNA has the modification pattern set forth in SEQ ID NO: 429.

35. The composition of any one of claims 6-16 and 22-34, wherein the composition comprises the guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 387, and the guide RNA comprises: (i) phosphorothioate bonds between the first four nucleotides at the 5’ end of the guide RNA; (ii) phosphorothioate bonds between the last four nucleotides at the 3’ end of the guide RNA; (iii) 2’-O-methyl-modified nucleotides at the first three nucleotides at the 5’ end of the guide RNA; and (iv) 2’-O-methyl-modified nucleotides at the last three nucleotides at the 3’ end of the guide RNA.

36. The composition of claim 35, wherein the composition comprises the guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 387, and the guide RNA has the modification pattern set forth in SEQ ID NO: 429.

37. The composition of any one of claims 6-36, wherein the Cas protein is aCas9 protein.

38. The composition of claim 37, wherein the Cas protein is derived from a Streptococcus pyogenes Cas9 protein.

39. The composition of any one of claims 6-38, wherein the Cas protein comprises the sequence set forth in SEQ ID NO: 12.

40. The composition of any one of claims 6-39, further comprising the Cas protein or a nucleic acid encoding the Cas protein.

41. The composition of claim 40, wherein the nucleic acid encoding the Cas protein is codon-optimized for expression in a mammalian cell or a human cell.

42. The composition of claim 40 or 41, wherein the composition comprises the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein.

43. The composition of claim 42, wherein the mRNA encoding the Cas protein comprises at least one modification.

44. The composition of claim 42 or 43, wherein the mRNA encoding the Cas protein is modified to comprise a modified uridine at one or more or all uridine positions.

45. The composition of claim 44, wherein the modified uridine is N1 -methylpseudouridine.

46. The composition of claim 44 or 45, wherein the mRNA encoding the Cas protein is fully substituted with Nl-m ethyl-pseudouri dine.

47. The composition of any one of claims 42-46, wherein the mRNA encoding the Cas protein comprises a 5’ cap.

48. The composition of any one of claims 42-47, wherein the mRNA encoding the Cas protein comprises a poly(A) tail.

49. The composition of any one of claims 42-48, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 250 or 249.

50. The composition of any one of claims 40-49, wherein the composition comprises the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 250 or 249, and the mRNA encoding the Cas protein is fully substituted with Nl-methyl-pseudouri dine, comprises a 5’ cap, and comprises a poly(A) tail.

51. The composition of any one of claims 40-50, wherein the composition comprises the guide RNA in the form of RNA, and the guide RNA comprises SEQ ID NO: 387, and wherein the composition comprises the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, and the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 250 or 249.

52. The composition of any one of claims 40-51, wherein the composition comprises the guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 387, and the guide RNA comprises: (i) phosphorothioate bonds between the first four nucleotides at the 5’ end of the guide RNA; (ii) phosphorothioate bonds between the last four nucleotides at the 3’ end of the guide RNA; (iii) 2’-O-methyl-modified nucleotides at the first three nucleotides at the 5’ end of the guide RNA; and (iv) 2’-O-methyl-modified nucleotides at the last three nucleotides at the 3’ end of the guide RNA, and wherein the composition comprises the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 250 or 249, and the mRNA encoding the Cas protein is fully substituted with Nl-methyl-pseudouri dine, comprises a 5’ cap, and comprises a poly(A) tail.

53. The composition of any one of claims 40-52, wherein the composition comprises the guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 387, and the guide RNA has the modification pattern set forth in SEQ ID NO: 429, andwherein the composition comprises the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 250 or 249, and the mRNA encoding the Cas protein is fully substituted with Nl-methyl-pseudouri dine, comprises a 5’ cap, and comprises a poly(A) tail.

54. The composition of any one of claims 40-53, wherein the Cas protein or the nucleic acid encoding the Cas protein and the guide RNA or the one or more DNAs encoding the guide RNA are associated with a lipid nanoparticle.

55. The composition of claim 54, wherein the lipid nanoparticle comprises a cationic lipid, a neutral lipid, a helper lipid, and a stealth lipid.

56. The composition of claim 55, wherein the cationic lipid is Lipid A.

57. The composition of claim 55 or 56, wherein the neutral lipid is DSPC.

58. The composition of any one of claims 55-57, wherein the helper lipid is cholesterol.

59. The composition of any one of claims 55-58, wherein the stealth lipid is l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol -2000 (PEG2k-DMG).

60. The composition of any one of claims 55-59, wherein the cationic lipid is Lipid A, the neutral lipid is DSPC, the helper lipid is cholesterol, and the stealth lipid is PEG2k- DMG.

61. The composition of any one of claims 55-60, wherein the lipid nanoparticle comprises four lipids at the following molar ratios: about 50 mol% Lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG.

62. The composition of any one of claims 40-61, wherein the ASS J gene is a human ASS1 gene, wherein the composition comprises the guide RNA in the form of RNA, and the guide RNA comprises SEQ ID NO: 387,wherein the composition comprises the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, and the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 250 or 249, and wherein the guide RNA and the mRNA encoding the Cas protein are associated with a lipid nanoparticle comprising Lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally at the following molar ratios: about 50 mol% Lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG.

63. The composition of any one of claims 40-62, wherein the ASS1 gene is a human ASS I gene, wherein the composition comprises the guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 387, and the guide RNA comprises: (i) phosphorothioate bonds between the first four nucleotides at the 5’ end of the guide RNA; (ii) phosphorothioate bonds between the last four nucleotides at the 3’ end of the guide RNA; (iii) 2’-O-methyl-modified nucleotides at the first three nucleotides at the 5’ end of the guide RNA; and (iv) 2’-O-methyl- modified nucleotides at the last three nucleotides at the 3’ end of the guide RNA, wherein the composition comprises the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 250 or 249, and the mRNA encoding the Cas protein is fully substituted with Nl-methyl-pseudouridine, comprises a 5’ cap, and comprises a poly(A) tail, and wherein the guide RNA and the mRNA encoding the Cas protein are associated with a lipid nanoparticle comprising Lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally at the following molar ratios: about 50 mol% Lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG.

64. The composition of any one of claims 40-63, wherein the ASSJ gene is a human ASS1 gene, wherein the composition comprises the guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 387, and the guide RNA has the modification pattern set forth in SEQ ID NO: 429,wherein the composition comprises the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 250 or 249, and the mRNA encoding the Cas protein is fully substituted with Nl-methyl-pseudouri dine, comprises a 5’ cap, and comprises a poly(A) tail, and wherein the guide RNA and the mRNA encoding the Cas protein are associated with a lipid nanoparticle comprising Lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally at the following molar ratios: about 50 mol% Lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG.

65. A composition comprising a nucleic acid construct comprising a first argininosuccinate synthase protein coding sequence.

66. The composition of claim 65, wherein the first argininosuccinate synthase protein coding sequence is a human argininosuccinate synthase protein coding sequence.

67. The composition of claim 65 or 66, wherein the first argininosuccinate synthase protein coding sequence comprises human argininosuccinate synthase exons 2-14 or exons 3-14.

68. The composition of any one of claims 65-67, wherein the first argininosuccinate synthase protein coding sequence comprises human argininosuccinate synthase exons 3-14.

69. The composition of any one of claims 65-68, wherein the first argininosuccinate synthase protein coding sequence encodes a protein at last 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 230.

70. The composition of any one of claims 65-69, wherein the first argininosuccinate synthase protein coding sequence encodes a protein comprising the sequence set forth in SEQ ID NO: 230.71 . The composition of any one of claims 65-70, wherein the first argininosuccinate synthase protein coding sequence encodes a protein consisting of the sequence set forth in SEQ ID NO: 230.

72. The composition of any one of claims 65-71, wherein the first argininosuccinate synthase protein coding sequence is at least 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOS: 705, 709, and 713.

73. The composition of any one of claims 65-71, wherein the first argininosuccinate synthase protein coding sequence is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOS: 706 and 710.

74. The composition of any one of claims 65-71, wherein the first argininosuccinate synthase protein coding sequence is at least 99% identical to any one of SEQ ID NOS: 705, 706, 709, 710, and 713.

75. The composition of any one of claims 65-71, wherein the first argininosuccinate synthase protein coding sequence comprises, consists essentially of, or consists of any one of SEQ ID NOS: 705, 706, 709, 710, and 713.

76. The composition of any one of claims 65-71, wherein the first argininosuccinate synthase protein coding sequence is at least 96%, 97%, 98%, or 99% identical to SEQ ID NO: 705.

77. The composition of any one of claims 65-71, wherein the first argininosuccinate synthase protein coding sequence is at least 99% identical to SEQ ID NO: 705.

78. The composition of any one of claims 65-71, wherein the first argininosuccinate synthase protein coding sequence comprises, consists essentially of, or consists of SEQ ID NO: 705.

79. The composition of any one of claims 65-71, wherein the first argininosuccinate synthase protein coding sequence is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 706.

80. The composition of any one of claims 65-71, wherein the first argininosuccinate synthase protein coding sequence is at least 99% identical to SEQ ID NO: 706.

81. The composition of any one of claims 65-71, wherein the first argininosuccinate synthase protein coding sequence comprises, consists essentially of, or consists of SEQ ID NO: 706.

82. The composition of any one of claims 65-67, wherein the first argininosuccinate synthase protein coding sequence comprises human argininosuccinate synthase exons 2-14.

83. The composition of any one of claims 65-67 and 82, wherein the first argininosuccinate synthase protein coding sequence encodes a protein at last 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 227.

84. The composition of any one of claims 65-67, 82, and 83, wherein the first argininosuccinate synthase protein coding sequence encodes a protein comprising the sequence set forth in SEQ ID NO: 227.

85. The composition of any one of claims 65-67 and 82-84, wherein the first argininosuccinate synthase protein coding sequence encodes a protein consisting of the sequence set forth in SEQ ID NO: 227.

86. The composition of any one of claims 65-85, wherein the nucleic acid construct comprises a splice acceptor upstream of the first argininosuccinate synthase protein coding sequence.

87. The composition of any one of claims 65-86, wherein the nucleic acid construct comprises a polyadenylation signal downstream of the first argininosuccinate synthase protein coding sequence.

88. The composition of any one of claims 65-85, wherein the nucleic acid construct comprises a splice acceptor upstream of the first argininosuccinate synthase protein coding sequence, and the nucleic acid construct comprises a polyadenylation signal downstream of the first argininosuccinate synthase protein coding sequence.

89. The composition of any one of claims 65-88, wherein the nucleic acid construct does not comprise homology arms.

90. The composition of any one of claims 65-88, wherein the nucleic acid construct comprises homology arms.

91. The composition of any one of claims 65-90, wherein the nucleic acid construct does not comprise a promoter that drives expression of the argininosuccinate synthase protein.

92. The composition of any one of claims 65-91, wherein the nucleic acid construct is a bidirectional construct.

93. The composition of claim 92, wherein the nucleic acid construct comprises the first argininosuccinate synthase protein coding sequence and a reverse complement of a second argininosuccinate synthase protein coding sequence.

94. The composition of claim 93, wherein the first argininosuccinate synthase protein coding sequence and the second argininosuccinate synthase protein coding sequence are different but encode the same argininosuccinate synthase protein sequence.

95. The composition of claim 93 or 94, wherein the first argininosuccinate synthase protein coding sequence and the second argininosuccinate synthase protein coding sequence each encode a protein at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 230.

96. The composition of any one of claims 93-95, wherein the first argininosuccinate synthase protein coding sequence and the second argininosuccinate synthase protein coding sequence each encode a protein comprising the sequence set forth in SEQ ID NO: 230.

97. The composition of any one of claims 93-96, wherein the first argininosuccinate synthase protein coding sequence and the second argininosuccinate synthase protein coding sequence each encode a protein consisting of the sequence set forth in SEQ ID NO: 230.

98. The composition of any one of claims 93-97, wherein the first argininosuccinate synthase protein coding sequence is at least 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOS: 705, 709, and 713, optionally wherein the first argininosuccinate synthase protein coding sequence is at least 96%, 97%, 98%, or 99% identical to SEQ ID NO: 705.

99. The composition of any one of claims 93-98, wherein the first argininosuccinate synthase protein coding sequence is at least 99% identical to any one of SEQ ID NOS: 705, 709, and 713, optionally wherein the first argininosuccinate synthase protein coding sequence is at least 99% identical to SEQ ID NO: 705.

100. The composition of any one of claims 93-99, wherein the first argininosuccinate synthase protein coding sequence comprises, consists essentially of, or consists of any one of SEQ ID NOS: 705, 709, and 713, optionally wherein the first argininosuccinate synthase protein coding sequence comprises, consists essentially of, or consists of SEQ ID NO:705.

101. The composition of any one of claims 93-100, wherein the second argininosuccinate synthase protein coding sequence is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOS: 706 and 710, optionally wherein the second argininosuccinate synthase protein coding sequence is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 706.

102. The composition of any one of claims 93-101, wherein the second argininosuccinate synthase protein coding sequence is at least 99% identical to any one of SEQ ID NOS: 706 and 710, optionally wherein the second argininosuccinate synthase protein coding sequence is at least 99% identical to SEQ ID NO: 706.

103. The composition of any one of claims 93-102, wherein the second argininosuccinate synthase protein coding sequence comprises, consists essentially of, or consists of any one of SEQ ID NOS: 706 and 710, optionally wherein the second argininosuccinate synthase protein coding sequence comprises, consists essentially of, or consists of SEQ ID NO:706.

104. The composition of any one of claims 93-97, wherein the first argininosuccinate synthase protein coding sequence is at least 96%, 97%, 98%, or 99% identical to SEQ ID NO: 705, and wherein the second argininosuccinate synthase protein coding sequence is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 706.

105. The composition of any one of claims 93-97 and 104, wherein the first argininosuccinate synthase protein coding sequence is at least 99% identical to SEQ ID NO: 705, and wherein the second argininosuccinate synthase protein coding sequence is at least 99% identical to SEQ ID NO: 706.

106. The composition of any one of claims 93-97, 104, and 105, wherein the first argininosuccinate synthase protein coding sequence comprises, consists essentially of, or consists of SEQ ID NO: 705, and wherein the second argininosuccinate synthase protein coding sequence comprises, consists essentially of, or consists of SEQ ID NO: 706.

107. The composition of claim 93 or 94, wherein the first argininosuccinate synthase protein coding sequence and the second argininosuccinate synthase protein coding sequence each encode a protein at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 227.

108. The composition of any one of claims 93, 94, and 107, wherein the first argininosuccinate synthase protein coding sequence and the second argininosuccinate synthase protein coding sequence each encode a protein comprising the sequence set forth in SEQ ID NO: 227.

109. The composition of any one of claims 93, 94, 107, and 108, wherein the first argininosuccinate synthase protein coding sequence and the second argininosuccinate synthase protein coding sequence each encode a protein consisting of the sequence set forth in SEQ ID NO: 227.

110. The composition of any one of claim 93-109, wherein the nucleic acid construct comprises from 5’ to 3’: a first splice acceptor, the first argininosuccinate synthase protein coding sequence, a first polyadenylation signal, a reverse complement of a second polyadenylation signal, the reverse complement of the second argininosuccinate synthase proteincoding sequence, and a reverse complement of a second splice acceptor, or wherein the nucleic acid construct comprises from 5’ to 3’ : a first splice acceptor, the second argininosuccinate synthase protein coding sequence, a first polyadenylation signal, a reverse complement of a second polyadenylation signal, the reverse complement of the first argininosuccinate synthase protein coding sequence, and a reverse complement of a second splice acceptor.

111. The composition of claim 110, wherein the first argininosuccinate synthase protein coding sequence and the second argininosuccinate synthase protein coding sequence are different but encode the same argininosuccinate synthase protein sequence, and wherein the first polyadenylation signal and the second polyadenylation signal are different.

112. The composition of any one of claims 93-106, wherein the first argininosuccinate synthase protein coding sequence and the second argininosuccinate synthase protein coding sequence are different and each encode a protein comprising or consisting of the sequence set forth in SEQ ID NO: 230, wherein the nucleic acid construct comprises from 5’ to 3’: a first splice acceptor, the first argininosuccinate synthase protein coding sequence, a first polyadenylation signal, a reverse complement of a second polyadenylation signal, the reverse complement of the second argininosuccinate synthase protein coding sequence, and a reverse complement of a second splice acceptor, wherein the nucleic acid construct does not comprise a promoter that drives expression of the argininosuccinate synthase protein, and wherein the nucleic acid construct does not comprise homology arms.

113. The composition of any one of claims 93-106, wherein the first argininosuccinate synthase protein coding sequence and the second argininosuccinate synthase protein coding sequence are different and each encode a protein comprising or consisting of the sequence set forth in SEQ ID NO: 230, wherein the first argininosuccinate synthase protein coding sequence comprises SEQ ID NO: 705, wherein the second argininosuccinate synthase protein coding sequence comprises SEQ ID NO: 706,wherein the nucleic acid construct comprises from 5’ to 3’: a first splice acceptor, the first argininosuccinate synthase protein coding sequence, a first polyadenylation signal, a reverse complement of a second polyadenylation signal, the reverse complement of the second argininosuccinate synthase protein coding sequence, and a reverse complement of a second splice acceptor, wherein the nucleic acid construct does not comprise a promoter that drives expression of the argininosuccinate synthase protein, and wherein the nucleic acid construct does not comprise homology arms.

114. The composition of any one of claims 93-108 and 110-113, wherein the first argininosuccinate synthase protein coding sequence and the second argininosuccinate synthase protein coding sequence are different and each encode a protein comprising or consisting of the sequence set forth in SEQ ID NO: 227, wherein the nucleic acid construct comprises from 5’ to 3’: a first splice acceptor, the first argininosuccinate synthase protein coding sequence, a first polyadenylation signal, a reverse complement of a second polyadenylation signal, the reverse complement of the second argininosuccinate synthase protein coding sequence, and a reverse complement of a second splice acceptor, wherein the nucleic acid construct does not comprise a promoter that drives expression of the argininosuccinate synthase protein, and wherein the nucleic acid construct does not comprise homology arms.

115. The composition of any one of claims 65-91, wherein the nucleic acid construct is a unidirectional construct.

116. The composition of claim 115, wherein the nucleic acid construct is a unidirectional construct comprising the first argininosuccinate synthase protein coding sequence, wherein the first argininosuccinate synthase protein coding sequence encodes a protein comprising or consisting of the sequence set forth in SEQ ID NO: 230, wherein the nucleic acid construct comprises from 5’ to 3’: a splice acceptor, the first argininosuccinate synthase protein coding sequence, and a polyadenylation signal,wherein the nucleic acid construct does not comprise a promoter that drives expression of the argininosuccinate synthase protein, and wherein the nucleic acid construct does not comprise homology arms.

117. The composition of claim 115, wherein the nucleic acid construct is a unidirectional construct comprising the first argininosuccinate synthase protein coding sequence, wherein the first argininosuccinate synthase protein coding sequence encodes a protein comprising or consisting of the sequence set forth in SEQ ID NO: 227, wherein the nucleic acid construct comprises from 5’ to 3’: a splice acceptor, the first argininosuccinate synthase protein coding sequence, and a polyadenylation signal, wherein the nucleic acid construct does not comprise a promoter that drives the expression of the argininosuccinate synthase protein, and wherein the nucleic acid construct does not comprise homology arms.

118. The composition of any one of claims 65-117, wherein the nucleic acid construct is single-stranded DNA or double-stranded DNA.

119. The composition of claim 118, wherein the nucleic acid construct is single-stranded DNA.

120. The composition of any one of claims 65-119, wherein the nucleic acid construct is in a nucleic acid vector or a lipid nanoparticle.

121. The composition of claim 120, wherein the nucleic acid construct is in the nucleic acid vector, optionally wherein the nucleic acid vector is a viral vector.

122. The composition of claim 121, wherein the nucleic acid vector is an adeno-associated viral (AAV) vector, optionally wherein the nucleic acid construct is flanked by inverted terminal repeats (ITRs) on each end, optionally wherein the ITR on at least one end comprises, consists essentially of, or consists of SEQ ID NO: 198, and optionally wherein the ITR on each end comprises, consists essentially of, or consists of SEQ ID NO: 198, or optionally wherein the ITR on at least one end comprises, consists essentially of, or consists of SEQ ID NO: 196, andoptionally wherein the ITR on each end comprises, consists essentially of, or consists of SEQ ID NO: 196.

123. The composition of claim 122, wherein the AAV vector is a singlestranded AAV (ssAAV) vector.

124. The composition of claim 122 or 123, wherein the AAV vector is derived from an AAV8 vector, an AAV3B vector, an AAV5 vector, an AAV6 vector, an AAV7 vector, an AAV9 vector, an AAVrh.74 vector, or an AAVhu.37 vector, optionally wherein the AAV vector is a recombinant AAV8 (rAAV8) or a recombinant AAV5 (rAAV5) vector.

125. The composition of claim 124, wherein the AAV vector is a recombinant AAV8 (rAAV8) vector.

126. The composition of claim 125, wherein the AAV vector is a singlestranded rAAV8 vector.

127. The composition of any one of claims 93-111, wherein the first argininosuccinate synthase protein coding sequence and the second argininosuccinate synthase protein coding sequence are different and each encode a protein comprising or consisting of the sequence set forth in SEQ ID NO: 230, wherein the nucleic acid construct comprises from 5’ to 3’: a first splice acceptor, the first argininosuccinate synthase protein coding sequence, a first polyadenylation signal, a reverse complement of a second polyadenylation signal, the reverse complement of the second argininosuccinate synthase protein coding sequence, and a reverse complement of a second splice acceptor, wherein the nucleic acid construct does not comprise a promoter that drives expression of the argininosuccinate synthase protein, wherein the nucleic acid construct does not comprise homology arms, and wherein the nucleic acid construct is in a single-stranded rAAV8 vector, optionally wherein the nucleic acid construct is flanked by inverted terminal repeats (ITRs) on each end, optionally wherein the ITR on at least one end comprises, consists essentially of, or consists of SEQ ID NO: 198, and optionally wherein the ITR on each endcomprises, consists essentially of, or consists of SEQ ID NO: 198, or optionally wherein the ITR on at least one end comprises, consists essentially of, or consists of SEQ ID NO: 196, and optionally wherein the ITR on each end comprises, consists essentially of, or consists of SEQ ID NO: 196.

128. The composition of any one of claims 93-111, wherein the first argininosuccinate synthase protein coding sequence and the second argininosuccinate synthase protein coding sequence are different and each encode a protein comprising or consisting of the sequence set forth in SEQ ID NO: 230, wherein the first argininosuccinate synthase protein coding sequence comprises SEQ ID NO: 705, wherein the second argininosuccinate synthase protein coding sequence comprises SEQ ID NO: 706, wherein the nucleic acid construct comprises from 5’ to 3’: a first splice acceptor, the first argininosuccinate synthase protein coding sequence, a first polyadenylation signal, a reverse complement of a second polyadenylation signal, the reverse complement of the second argininosuccinate synthase protein coding sequence, and a reverse complement of a second splice acceptor, wherein the nucleic acid construct does not comprise a promoter that drives expression of the argininosuccinate synthase protein, wherein the nucleic acid construct does not comprise homology arms, and wherein the nucleic acid construct is in a single-stranded rAAV8 vector, optionally wherein the nucleic acid construct is flanked by inverted terminal repeats (ITRs) on each end, optionally wherein the ITR on at least one end comprises, consists essentially of, or consists of SEQ ID NO: 198, and optionally wherein the ITR on each end comprises, consists essentially of, or consists of SEQ ID NO: 198, or optionally wherein the ITR on at least one end comprises, consists essentially of, or consists of SEQ ID NO: 196, and optionally wherein the ITR on each end comprises, consists essentially of, or consists of SEQ ID NO: 196.

129. The composition of any one of claims 93-111, wherein the first argininosuccinate synthase protein coding sequence and the second argininosuccinate synthaseprotein coding sequence are different and each encode a protein comprising or consisting of the sequence set forth in SEQ ID NO: 227, wherein the nucleic acid construct comprises from 5’ to 3’: a first splice acceptor, the first argininosuccinate synthase protein coding sequence, a first polyadenylation signal, a reverse complement of a second polyadenylation signal, the reverse complement of the second argininosuccinate synthase protein coding sequence, and a reverse complement of a second splice acceptor, wherein the nucleic acid construct does not comprise a promoter that drives expression of the argininosuccinate synthase protein, wherein the nucleic acid construct does not comprise homology arms, and wherein the nucleic acid construct is in a single-stranded rAAV8 vector, optionally wherein the nucleic acid construct is flanked by inverted terminal repeats (ITRs) on each end, optionally wherein the ITR on at least one end comprises, consists essentially of, or consists of SEQ ID NO: 198, and optionally wherein the ITR on each end comprises, consists essentially of, or consists of SEQ ID NO: 198, or optionally wherein the ITR on at least one end comprises, consists essentially of, or consists of SEQ ID NO: 196, and optionally wherein the ITR on each end comprises, consists essentially of, or consists of SEQ ID NO: 196.

130. A combination comprising:(I) the composition of any one of claims 1-5 and 40-64; and(II) the composition comprising the nucleic acid construct comprising the first argininosuccinate synthase protein coding sequence of any one of claims 65-129.

131. The composition of any one of claims 1-129 or the combination of claim 130 for use in a method of introducing an argininosuccinate synthase (ASS J) nucleic acid into a cell, a method of integrating an ASS J nucleic acid construct into a target gene in a cell, or a method of expressing argininosuccinate synthase in a cell.

132. Use of the composition of any one of claims 1-129 or the combination of claim 130 in the preparation of a reagent for introducing an argininosuccinate synthase (ASS / )nucleic acid into a cell, integrating an ASS I nucleic acid construct into a target gene in a cell, or expressing argininosuccinate synthase in a cell.

133. The composition or combination for use of claim 131 or the use of claim132, wherein the cell is a neonatal cell.

134. The composition or combination for use of claim 133 or the use of claim133, wherein the neonatal cell is from a human neonatal subject within 24 weeks after birth, is from a human neonatal subject within 12 weeks after birth, is from a human neonatal subject within 8 weeks after birth, is from a human neonatal subject within 4 weeks after birth, is from a human neonatal subject within 2 weeks after birth, or is from a human neonatal subject within 1 week after birth.

135. The composition or combination for use of claim 131 or the use of claim 132, wherein the cell is not a neonatal cell.

136. The composition of any one of claims 1-129 or the combination of claim 130 for use in a method of treating an argininosuccinate synthase deficiency in a subject.

137. The composition of any one of claims 1 -129 or the combination of claim 130 for use in a method of treating citrullinemia type I in a subject.

138. Use of the composition of any one of claims 1-129 or the combination of claim 130 in the preparation of a medicament treating an argininosuccinate synthase deficiency in a subject.

139. Use of the composition of any one of claims 1-129 or the combination of claim 130 in the preparation of a medicament for treating citrullinemia type I in a subject.

140. The composition or combination for use of claim 136 or 137 or the use of claim 138 or 139, wherein the subject is a neonatal subject.

141. The composition or combination for use of claim 140 or the use of claim 140, wherein the neonatal subject is a human neonatal subject within 24 weeks after birth, is a human neonatal subject within 12 weeks after birth, is a human neonatal subject within 8 weeksafter birth, is a human neonatal subject within 4 weeks after birth, is a human neonatal subject within 2 weeks after birth, or is a human neonatal subject within 1 week after birth.

142. The composition or combination for use of claim 136 or 137 or the use of claim 138 or 139, wherein the subject is not a neonatal subject.

143. A cell comprising the composition of any one of claims 1-129 or the combination of claim 130.

144. The cell of claim 143, wherein the nucleic acid construct is integrated into an endogenous target gene locus, and wherein argininosuccinate synthase protein is expressed from the endogenous target gene locus, or wherein the nucleic acid construct is integrated into intron 1 or intron 2 of an endogenous argininosuccinate synthase (ASS / ) locus, and wherein argininosuccinate synthase protein is expressed from the endogenous ASS1 locus.

145. The cell of claim 143 or 144, wherein the cell is a human cell, optionally wherein the nucleic acid construct is integrated into intron 2 of the endogenous ASS1 locus.

146. The cell of any one of claims 143-145, wherein the cell is a liver cell.

147. The cell of claim 146, wherein the liver cell is a hepatocyte.

148. The cell of any one of claims 143-147, wherein the cell is a neonatal cell.

149. The cell of claim 148, wherein the neonatal cell is from a human neonatal subject within 24 weeks after birth, is from a human neonatal subject within 12 weeks after birth, is from a human neonatal subject within 8 weeks after birth, is from a human neonatal subject within 4 weeks after birth, is from a human neonatal subject within 2 weeks after birth, or is from a human neonatal subject within 1 week after birth.

150. The cell of any one of claims 143-147, wherein the cell is not a neonatal cell.

151. The cell of any one of claims 143-150, wherein the cell is ex vivo or in vitro.

152. The cell of any one of claims 143-150, wherein the cell is in vivo.

153. A method of introducing an argininosuccinate synthase nucleic acid into a cell, comprising administering the combination of claim 130 to the cell.

154. A method of integrating an argininosuccinate synthase nucleic acid construct into a target gene in a cell, comprising administering the combination of claim 130 to the cell, wherein the nuclease agent cleaves the nuclease target site in the target gene to create a cleavage site, the nucleic acid construct is inserted into the cleavage site to create a modified target gene, and argininosuccinate synthase protein is expressed from the modified target gene.

155. A method of expressing argininosuccinate synthase in a cell, comprising administering the combination of claim 130 to the cell, wherein the nuclease agent cleaves the nuclease target site in the target gene to create a cleavage site, the nucleic acid construct is inserted into the cleavage site to create a modified target gene, and argininosuccinate synthase protein is expressed from the modified target gene.

156. The method of any one of claims 153-155, wherein the nuclease agent comprises:(a) a Cas protein or a nucleic acid encoding the Cas protein; and(b) a guide RNA or one or more DNAs encoding the guide RNA, wherein the guide RNA comprises a DNA-targeting segment that targets a guide RNA target sequence, and wherein the guide RNA binds to the Cas protein and targets the Cas protein to the guide RNA target sequence.

157. The method of claim 156, wherein the nucleic acid construct, the Cas protein or the nucleic acid encoding the Cas protein, and the guide RNA or the one or more DNAs encoding the guide RNA are administered simultaneously.

158. The method of claim 1 6, wherein the nucleic acid construct is not administered simultaneously with the Cas protein or the nucleic acid encoding the Cas protein and the guide RNA or the one or more DNAs encoding the guide RNA.

159. The method of any one of claims 153-158, wherein the cell is a liver cell.

160. The method of any one of claims 153-159, wherein the cell is a hepatocyte.

161. The method of any one of claims 153-160, wherein the cell is a human cell.

162. The method of any one of claims 153-161, wherein the cell is a neonatal cell.

163. The method of claim 162, wherein the neonatal cell is from a human neonatal subject within 24 weeks after birth, a human neonatal subject within 12 weeks after birth, a human neonatal subject within 8 weeks after birth, a human neonatal subject within 4 weeks after birth, a human neonatal subject within 2 weeks after birth, or a human neonatal subject within 1 week after birth.

164. The method of any one of claims 153-163, wherein the cell is not a neonatal cell.

165. The method of any one of claims 153-164, wherein the cell is in vivo.

166. The method of any one of claims 153-164, wherein the cell is in vitro or ex vivo.

167. A method of treating an argininosuccinate synthase deficiency in a subject, comprising administering the combination of claim 130 to the subject.

168. A method of treating citrullinemia type I in a subject, comprising administering the combination of claim 130 to the subject.

169. A method of preventing or inhibiting hyperammonemia in a subject having citrullinemia type I, comprising administering the combination of claim 130 to the subject.

170. The method of any one of claims 167-169, wherein the nuclease agent comprises:(a) a Cas protein or a nucleic acid encoding the Cas protein; and(b) a guide RNA or one or more DNAs encoding the guide RNA, wherein the guide RNA comprises a DNA-targeting segment that targets a guide RNA target sequence, and wherein the guide RNA binds to the Cas protein and targets the Cas protein to the guide RNA target sequence.

171. The method of claim 170, wherein the nucleic acid construct, the Cas protein or the nucleic acid encoding the Cas protein, and the guide RNA or the one or more DNAs encoding the guide RNA are administered simultaneously.

172. The method of claim 170, wherein the nucleic acid construct is not administered simultaneously with the Cas protein or the nucleic acid encoding the Cas protein and the guide RNA or the one or more DNAs encoding the guide RNA.

173. The method of any one of claims 167-172, wherein the subject is a neonatal subject.

174. The method of claim 173, wherein the neonatal subject is a human neonatal subject within 24 weeks after birth, a human neonatal subject is within 12 weeks after birth, a human neonatal subject is within 8 weeks after birth, a human neonatal subject is within 4 weeks after birth, a human neonatal subject is within 2 weeks after birth, or a human neonatal subject is within 1 week after birth.

175. The method of any one of claims 167-172, wherein the subject is not a neonatal subject.

176. The method of any one of claims 167-175, wherein the subject is a human subject.

177. The method of any one of claims 167-176, wherein the method decreases plasma ammonia and / or plasma citrulline levels in the subject.

178. The method of claim 177, wherein the method reduces plasma ammonia levels to less than 200 pmol / L, less than 175 pmol / L, less than 150 pmol / L, less than 125 pmol / L, or less than 100 pmol / L, optionally wherein the reduced plasma ammonia levels are at 2 weeks, 4 weeks, 6 weeks, 8 weeks, 12 weeks, 16 weeks, 20 weeks, 6 months, 1 year, or 2 years after administering the combination.

179. The method of claim 177 or 178, wherein the method reduces plasma citrulline levels to less than 2000 pmol / L, less than 1750 pmol / L, less than 1500 pmol / L, less than 1250 pmol / L, less than 1000 pmol / L, less than 900 pmol / L, less than 800 pmol / L, less than 700 pmol / L, less than 600 pmol / L, or less than 500 pmol / L, optionally wherein the reduced plasma citrulline levels are at 2 weeks, 4 weeks, 6 weeks, 8 weeks, 12 weeks, 16 weeks, 20 weeks, 6 months, 1 year, or 2 years after administering the combination.

180. The method of any one of claims 177-179, wherein the decreased ammonia and / or plasma citrulline levels are sustained for at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 1 year, or at least 2 years after administering the combination.

181. The method of any one of claim 167-180, wherein the method further comprises assessing preexisting AAV immunity in the subject prior to administering the composition to the subject.

182. The method of claim 181, wherein the preexisting AAV immunity is preexisting AAV8 immunity.

183. The method of claim 181 or 182, wherein assessing preexisting AAV immunity comprises assessing immunogenicity using a total antibody immune assay or a neutralizing antibody assay.

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