(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2019/036484 Al 21 February 2019 (21.02.2019) W !P O PCT

(51) International Patent Classification: TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, C12N 9/22 (2006.01) C12N 15/90 (2006.01) KM, ML, MR, NE, SN, TD, TG). (21) International Application Number: Published: PCT/US2018/046733 — with international search report (Art. 21(3)) (22) International Filing Date: — with sequence listing part of description (Rule 5.2(a)) 14 August 2018 (14.08.2018) (25) Filing Language: English (26) Publication Langi English (30) Priority Data: 62/545,581 15 August 2017 (15.08.2017) US 62/653,630 06 April 2018 (06.04.2018) US (71) Applicant: THE TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA [US/US]; 3160 Chestnut Street, Suite 200, Philadelphia, PA 19104 (US). (72) Inventors: ASHLEY, Scott; 405 Baltimore Ave, Apt C4, Philadelphia, PA 19104 (US). SIDRANE, Jenny, Agnes; 1280 W Evergreen Dr., Phoenixville, PA 19460 (US). WILSON, James, M.; 183 1 Delancey Street, Philadelphia, PA 19103 (US). (74) Agent: KODROFF, Cathy, A. et al; Howson & Howson LLP, 350 Sentry Parkway, Buildong 620, Suite 210, Blue Bell, PA 19422 (US). (81) Designated States (unless otherwise indicated, for every kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP, KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM,

00 (54) Title: COMPOSITIONS AND METHODS FOR TREATMENT OF ARGININOSUCCINIC ACIDURIA (57) Abstract: An engineered nucleic acid sequence encoding a functional argininosuccinate lyase (ASL) protein is provided herein, © which is useful for gene editing and/or delivery via viral vector. A recombinant adeno-associated virus (rAAV) vector is provided herein which has an AAV capsid and an expression cassette packaged therein. The expression cassette comprises the engineered nucleic acid sequences encoding the functional ASL protein and regulatory elements that direct expression of the ASL in a host cell. Also o provided are compositions containing this nucleic acid molecule and/or the rAAV. ASL vector and methods of using same for treatment of argininosuccinic aciduria (ASA) in a patient. COMPOSITIONS AND METHODS FOR TREATMENT OF ARGININOSUCCINIC ACIDURIA

BACKGROUND OF THE INVENTION Argininosuccinic aciduria, also known as argininosuccinate lyase (ASL) deficiency, is an autosomal recessive disorder of the urea acid cycle caused by mutations of the SZ gene that impair arginine synthesis. Genetic testing is important for proper diagnosis due to similarity of ASA to other urea acid cycle disorders, and currently ASA is included in newborn screening programs for all states in the U S (Ganetzky, RD et al. (2016) Argininosuccinic Acid Lyase Deficiency Missed by Newborn Screen. JIMD Reports). The disease has two primary manifestations, neonatal and late onset. Neonatal ASA is characterized by hyperammonemia within days following birth and can be treated by hemodialysis followed by life-time maintenance care to reduce the risk of further episodes (Erez, A (2013) Argininosuccinic aciduria: from a monogenic to a complex disorder. GenetMed 15:251-257; Brusilow, SW et al. (2001) Urea cycle enzymes In: Scriver CR, et al. (ed). The Metabolic and Molecular Bases of Inherited Disease, 8 ed. McGraw-Hill: New York; andNagamani, SCS et al. (1993) Argininosuccinate Lyase

Deficiency. In: Pagon, RA et al. (eds) GeneReviews(R), University of Washington, Seattle). The late onset form of ASA has a less severe phenotype that includes episodic hyperammonemia triggered by acute infection or stress and neurocognitive impairment with associated learning or behavior abnormalities. ASA patients can also manifest neurocognitive deficiencies unrelated to hyperammonemia, cirrhosis of the liver, and systemic hypertension potentially due to lack of nitric oxide production in both forms of the disorder (Erez, A (2013), as cited above; Brusilow, SW et al. (2001), as cited above;

Erez, A et al. (201 1) Requirement of argininosuccinate lyase for systemic nitric oxide production. Nature medicine 17:1619-1626; and Hermann, M et al. (2006) Nitric oxide in hypertension. Journal of clinical hypertension (Greenwich, Conn) 8:17-29). Standard maintenance care includes arginine supplementation and nitrogen scavenging drugs, including sodium benzoate and sodium phenylacetate. Individuals with recurrent hyperammonemia or cirrhosis of the liver can also undergo liver transplant as a curative process, though arginine supplementation is still required (Batshaw, ML et al. (2001) Alternative pathway therapy for urea cycle disorders: twenty years later. The Journal of Pediatrics 138:S46-54; and Ficicioglu, C et al. (2009) Argininosuccinate lyase deficiency: long term outcome of 13 patients detected by newborn screening. Molecular genetics and metabolism 98:273-277). Liver-targeted gene therapy can offer a potential benefit to those with the severe form of the disease, as absence of ASL from hepatocytes is the cause of the hyperammonemia (Robberecht, E, et al. (2006) Successful liver transplantation for argininosuccinate lyase deficiency (ASLD). Journal of inherited metabolic disease 29: 184-1 85; Marble, M et al. (2008) Living related liver transplant in a patient with argininosuccinic aciduria and cirrhosis: metabolic follow-up. Journal of pediatric gastroenterology and nutrition 46:453-456; FSEQand Newnham, T et al. (2008) Liver transplantation for argininosuccinic aciduria: clinical, biochemical, and metabolic outcome. Liver transplantation: official publication of the American Association for the Study of Liver Diseases and the International Liver Transplantation Society 14:41-45). Other urea acid cycle disorders, such as ornithine transcarbamylase deficiency and citrullinemia, have been explored for treatment using AAV vector technology and demonstrated both therapeutic efficacy and avoidance of an immune response (Wang, L et al. (2012). Preclinical evaluation of a clinical candidate AAV8 vector for ornithine transcarbamylase (OTC) deficiency reveals expression of functional enzyme from persisting vector genome. Molecular genetics and metabolism 105: 203-211; and Chandler, RJ et al. (2013) Liver-directed adeno-associated virus serotype 8 gene transfer rescues a lethal murine model of citrullinemia type 1. Gene Ther 20:1188-1191). ASA presents a unique challenge, as the enzyme is not only essential for the urea acid cycle and removal of nitrogen, but also for the synthesis of arginine and removal of argininosuccinic acid, the buildup of which has been thought to cause some of the unique symptoms of this disease (Erez, A (2013), as cited above; and Brusilow, SW et al. (2001), as cited above). Based on results from patients that have received a liver transplant, liver-targeted gene therapy is uniquely positioned to restore the urea acid cycle and increase quality of life without the need for a highly invasive procedure or a continued drug regimen. A continuing need in the art exists for new and effective compositions and methods for successful treatment of ASA.

SUMMARY OF THE INVENTION The embodiments described herein relate to compositions and methods of a gene therapy for treating argininosuccinic aciduria (ASA) via delivering functional human argininosuccinate lyase (ASL) to a subject in need thereof. In one aspect, this application provides an engineered nucleic acid sequence of SEQ ID NO: 3, or a nucleic acid sequence at least about 95% identical thereto, encoding human ASL. In another aspect, provided herein is an expression cassette comprising the engineered nucleic acid sequence of SEQ ID NO: 3, or a nucleic acid sequence at least about 95% identical thereto, encoding human ASL; and regulatory elements which direct expression thereof. In one embodiment, a vector comprising the expression cassette described herein is provided. In yet another aspect, provided herein is a recombinant adeno-associated virus (rAAV) comprising an AAV capsid, and a vector genome packaged therein. Said vector genome comprising: an AAV 5' inverted terminal repeat (ITR); a coding sequence encoding functional argininosuccinate lyase (ASL), wherein the coding sequence is operably linked to regulatory elements which direct expression of ASL; regulatory elements which direct expression of ASL; and an AAV 3' ITR. In one embodiment, the ASL has an amino acid sequence of SEQ ID NO: 2. In another embodiment, the coding sequence is SEQ ID NO: 3, or a nucleic acid sequence at least about 95% identical thereto.

In yet another embodiment, the vector genome is SEQ ID NO: 1. In certain embodiments, the ASL protein is mutated to remove acetylation in order to improve enzyme activity. For examples, positions 2 (Ala), 7 (Lys), 69 (Lys), and 288 (Lys) are known to include acetylation. One, two, three, or four of these positions may be modified to avoid this acetylation. In one aspect, an aqueous suspension suitable for intravenous administration to treat ASA in a subject in need thereof is provided herein. In one embodiment, such a suspension may contain comprising an aqueous suspending liquid and about 1 xlO 10 GC/mL to about 1 xlO 14 GC/mL of rAAV described herein. In another aspect, a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the rAAV described herein is provided. In yet another aspect, provided herein is a method of treating a subject having ASA with a rAAV comprising an AAV capsid, and a vector genome packaged therein. Said vector genome comprising: an AAV 5' inverted terminal repeat (ITR); a coding sequence encoding functional argininosuccinate lyase (ASL), wherein the coding sequence is operably linked to regulatory elements which direct expression of ASL; regulatory elements which direct expression of ASL; and an AAV 3' ITR. In one embodiment, the ASL has an amino acid sequence of SEQ ID NO: 2. In another embodiment, the coding sequence is SEQ ID NO: 3, or a nucleic acid sequence at least about 95% identical thereto.

In yet another embodiment, the vector genome is SEQ ID NO: 1. A rAAV useful as a liver-directed therapeutic for argininosuccinic aciduria (ASA) is provided. In certain embodiments, the rAAV has a vector genome packaged therein which comprises: (a) an AAV 5' inverted terminal repeat (ITR); (b) a coding sequence encoding argininosuccinate lyase (ASL) of SEQ ID NO: 3 or a sequence 95% identical thereto, wherein the coding sequence is operably linked to regulatory elements which direct expression of ASL; (c) regulatory elements which direct expression of ASL or a nucleic acid sequence at least about 95% identical thereto; and (d) an AAV 3' ITR. The vector genome may further comprise a sequence encoding a guide RNA. In certain embodiments, the vector further comprises a CRISPR endonuclease. In other embodiments, the CRISPR endonuclease is delivered via a different vector and/or a different route of delivery. An aqueous suspension suitable for administration to treat ASA in a subject in need thereof is provided. In certain embodiments, such a suspension comprises an aqueous suspending liquid and about 1 xlO 12 GC/mL to about 1 xlO 14 GC/mL of a rAAV as described herein. A gene editing system comprising one or more rAAV vector stocks is provided. In certain embodiments, the system comprises: (a) at least one nucleic acid sequence encoding a CRISPR endonuclease, and (b) at least one nucleic acid sequence encoding a guide RNA; and (c) at least one nucleic acid sequence encoding a donor template comprising an ASL coding sequence comprising SEQ ID NO: 3, or a nucleic acid sequence at least about 95% identical thereto. A method of treating ASA by administrating to a subject in need thereof, a vector or gene editing system as described herein. Other aspects and advantages of these methods and compositions are described further in the following detailed description.

BRI EF DESCRI PTION OF THE FIGU RES FIG. 1 provides a plasmid map of pAAV.TBG.ASLco.BGH(p3796) (also identified as pAAV.ASLco), described herein. The engineered ASL gene is referenced alternatively as coASL or ASLco. FIG. 2A - 2B provide a nucleic acid sequence for the AAV.TBG.ASLco.BGH (AAV.ASLco, AAV8.TBG.hASLco) vector genome, which is also reproduced as SEQ ID

NO: 1. FIG. 2C provides the nucleic acid sequence of an engineered ASL coding sequence (coASL or ASLco), which is also reproduced as nucleotide (nt) 1092 to nt 2483 of SEQ ID

NO: 1. FIG. 3A provides a survival curve of ASA hypomorphic mice. ASA hypomorphic mice bom from heterozygous x heterozygous matings were monitored. FIG. 3B provides a survival curve of untreated ASA hypomorphic mice (solid line), ASA hypomorphic mice injected with lxlO 10 GC of AAV8.TBG.hASLco (dashed line with long strokes), and ASA hypomorphic mice injected with lxlO 11 GC of AAV8.TBG.hASLco (dashed line with short strokes). ASA hypomorphic mice bom from heterozygous x heterozygous matings were injected IV via the temporal vein with lxlO 10 or 1X10 11 GC/mouse of AAV8.TBG.hASLco and monitored for survival. It was observed that AAV8 gene therapy extends survival in an ASA hypomorphic mouse model (**p < 0.01, ***p <0.001). FIG. 3C provides body weights of male ASA hypomorphic mice that received lxlO 11 GC/mouse of AAV8.TBG.hASLco. The tested mice were monitored and compared to wild type (WT) mice and ASA hypomorphic mice that did not receive gene therapy. Error bars = standard error of the mean (SEM). FIG. 3D provides body weights of female ASA hypomorphic mice that received lxlO 11 GC/mouse of AAV8.TBG.hASLco. The tested mice were monitored and compared to wild type (WT) mice ASA hypomorphic mice that did not receive gene therapy. At the last time point, a difference (*P<0.05) between treated and WT was observed. Error bars = standard error of the mean (SEM). FIG. 4A - 4H show representative images of immunohistochemistry to identify ASL protein in the liver of newborn injected mice. ASA hypomorphic mice born from heterozygous x heterozygous matings were injected IV via the temporal vein with lxlO 10 or lxlO 11 GC/mouse of AAV8.TBG.hASLco. Mice were necropsied after cohort-specific median survival was reached. Livers were harvested and immunohistochemistry was performed for detection of the ASL protein. Wild type (WT) and heterozygous (Het) mice served as controls. FIG. 5A - 5G show a survival curve (FIG. 5A), body weights (FIG. 5B), plasma arginine (FIG. 5C), plasma citrulline (FIG. 5D), plasma argininosuccinic aciduria (ASA, FIG. 5E), serum aspartate aminotransferase (AST, FIG. 5F), and serum alanine aminotransferase (ALT, FIG. 5G) of the tested mice. ASA hypomorphic mice born from heterozygous x heterozygous matings were injected IV via the orbital vein on day 30 post birth (P 30) with 6xl0 13 or 10 13 GC/kg of AAV8.TBG.hASLco. Mice were monitored for survival (FIG. 5A) and weight (FIG. 5B). Mice were bled and isolated plasma was analyzed for components of the urea acid cycle, including arginine (FIG. 5C), citrulline (FIG. 5D), and ASA (FIG. 5E). The isolated plasma was also analyzed for the liver transaminases AST (FIG. 5F) and ALT (FIG. 5G). Both female and male mice were evaluated respectively to observe sex difference in survival and efficacy of AAV8 gene therapy in the ASA hypomorph. Error bars = SEM; ns, not significant; *p < 0.05, **p < 0.01, ***p <0.001. FIG. 5H illustrates the timing of harvesting samples as described in the Examples. FIG. 6A - 6F provide representative images of immunohistochemistry for ASL protein in the liver of adult injected mice. ASA hypomorphic mice were injected IV via the orbital vein at day 30 post birth (P30) with 6x1 013 or 10 13 GC/kg of AAV8.TBG.hASLco. Mice were euthanized after 3 months on study. Livers were harvested and immunohistochemistry was performed to detect ASL protein. FIG. 7A - 7B provide measurements of vector genome copies (FIG. 7A) and ASL activity (FIG. 7B) in the liver of adult injected mice. ASA hypomorphic mice were injected IV via the orbital vein at day 30 post birth (P30) with 6xl0 13 or 10 13 GC/kg of AAV8.TBG.hASLco. Mice were euthanized after 3 months on study. Livers were harvested. Following extraction of DNA from liver, vector genome copies (GC) were determined (FIG. 7A). Liver homogenates were assayed for ASL activity compared to wild type levels (data presented as percentage of wild type activity) (FIG. 7B). Analysis of activity was performed by one-way ANOVA with Dunnett's multiple comparison test comparing each group and the ASA hypomorphic control. Error bars = SEM; ***p < 0.001. FIG. 8 provides a plasmid map of pAAV.U6.sg2g6.TBG. PI.hASL.bGH.p4783.gb, described herein (and SEQ ID NO: 9). FIG. 9A - 9D show the survival (FIG. 9A and FIG. 9B) and weights (FIG. 9C and FIG. 9D) of mice treated with SaCas9 hASL gene therapy as neonates. ASA hypomorphic mice, along with wild type and heterozygous littermates, (n=5) from timed matings were injected intravenously via the temporal facial vein on PI with 2xl0 12 GC/mouse of AAV8.U6.2G6.sgR.TBG.PI.ASLco.bGH (guided) or AAV8.U6.NULLsgR.TBG.PI.ASLco.bGH (unguided) and 3x1 011 GC/mouse of AAV8.TBG.hSa.Cas9.bGH. *p < 0.05, **p < 0.01, ***p < 0.001. FIG. 10A - 10D show serum analyses for mice treated with SaCas9 hASL gene therapy as neonates. Mice were bled on the indicated days post injection to measure levels of citrulline (FIG. 10A and FIG. 10B) and argininosuccinic acid (FIG. IOC and FIG. 10D). FIG. 11A - 11H show detection of hASL protein in liver tissue from female mice. Tissue samples were obtained from the lateral left lobe at the termination of the study and sections were labeled for detection of ASL protein by immunohistochemistry. FIG. 12A - 12H show detection of hASL protein in liver tissue from male mice. Tissue samples were obtained from the lateral left lobe at the termination of the study and sections were labeled for detection of ASL protein by immunohistochemistry. FIG. 13A - 13D show levels of hASL gene integration in liver tissue obtained from cohorts of wild type and heterozygous mice sacrificed on day 50.

DETAILED DESCRIPTION OF THE INVENTION Argininosuccinic aciduria (ASA), caused by deleterious mutations in the gene encoding argininosuccinate lyase (ASL), is the second most common genetic disorder affecting the urea acid cycle. Total loss of ASL activity results in severe neonatal onset of the disease characterized by hyperammonemia within few days of birth, which can rapidly progress to coma and death. Current treatments for ASA are limited to dietary restriction, arginine supplementation, and nitrogen scavenging drugs, with treatment-resistant disease currently being managed by orthotropic liver transplant. The methods and compositions described herein are useful for the treatment of argininosuccinic aciduria (ASA) caused by a mutation, defect, or deficiency in the gene encoding human argininosuccinate lyase (ASL). In one embodiment, the compositions and methods described herein involve expression cassettes, vectors, recombinant viruses, other compositions and methods for delivery of the nucleic acid sequence encoding a functional ASL to a mammalian subject for the treatment of ASA. Such compositions may involve both at least the engineered sequence provided herein and multiple and additional, different versions of ASL in the same expression cassette, vector, or recombinant virus. These features not only increase the efficacy of the functional ASL protein being expressed, but may also permit a lower dose of a therapeutic reagent that delivers the functional protein to increase safety. In some embodiments, the adeno- associated viral (AAV) vector-based gene therapy described herein helps to alleviate the symptoms associated with urea acid cycle disruption by providing stable expression of ASL protein in the liver. As described in the Examples, a murine hypomorphic model of ASA with a mean survival of 22 days was used to determine the efficacy of AAV8 gene therapy in newborns and adolescents. The inventors developed an engineered human ASL gene and packaged it in an AAV8 vector for targeted delivery of to the liver of an ASA hypomorphic mouse. Increases in both survival and body weight were observed in mice treated with the AAV8 vector compared to untreated mice. In adolescent ASA hypomorphic mice, AAV8 was administered by retro-orbital injection and resulted in increased survival and body weight, and a correction of metabolites associated with the disease. These results indicate that gene therapy with an AAV8 vector is a viable approach for the treatment of ASA and delivery of the ASL gene by AAV gene therapy is a therapeutic for ASA. Unless defined otherwise in this specification, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and by reference to published texts, which provide one skilled in the art with a general guide to many of the terms used in the present application. As used herein, "disease", "disorder" and "condition" are used interchangeably, to indicate an abnormal state in a subject. In one embodiment, the disease is argininosuccinic aciduria (ASA). "Patient" or "subject" as used herein interchangeably means a mammalian animal, including a human, a veterinary or farm animal, a domestic animal or pet, and animals normally used for clinical research. In one embodiment, the subject is a male. In another embodiment, the subject is a female. In one embodiment, the subject of these methods and compositions is a human. In one embodiment, the subject of these methods and compositions is an adult. In another embodiment, the subject of these methods and compositions is an adolescent. In another embodiment, the subject of these methods and compositions is a newborn. In another embodiment, the subject of these methods and compositions is an infant. It should be understood that while various embodiments in the specification are presented using "comprising" language, under various circumstances, a related embodiment is also described using "consisting of or "consisting essentially of language. "Comprising" is a term meaning inclusive of other components or method steps. When "comprising" is used, it is to be understood that related embodiments include descriptions using the "consisting of terminology, which excludes other components or method steps, and "consisting essentially of terminology, which excludes any components or method steps that substantially change the nature of the embodiment or invention. It is to be noted that the term "a" or "an", refers to one or more, for example, "an enhancer", is understood to represent one or more enhancer(s). As such, the terms "a" (or "an"), "one or more," and "at least one" is used interchangeably herein. As used herein, the term "about" means a variability of plus or minus 10 % from the reference given, unless otherwise specified. The terms "first" and "second" or "additional" are used throughout this specification as reference terms to distinguish between various forms and components of the compositions and methods. As used herein, the term "operably linked" or "operatively associated" refers to both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. I. Engineered ASL Coding Sequence As used herein, the term "argininosuccinate lyase" or "ASL" includes any isoform of ASL which restores a desired function, ameliorate a symptom, or improve a patient's condition when delivered a composition or method provided herein. As used herein, the term "functional ASL" means an enzyme having the amino acid sequence of the full-length wild type (native) ASL, a fragment thereof, a variant thereof, or a polymorph thereof, which provides at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, or about the same, or greater than 100% of the biological activity level of normal human ASL. In one embodiment, the ASL enzyme sequence is derived from the same mammal that the composition is intended to treat. In one embodiment, the ASL has a human sequence. The examples provided herein utilize the longest human isoform, Isoform 1. Isoform 1 is a 464 amino acid protein (see, e.g., NCBI accession NP_00003 9.2, UniProtKB P04424, UniProt P04424-1, each of which is incorporated by reference herein in its entirety), and is reproduced in SEQ ID NO: 2. The coding sequence of Isoform 1 ASL is reproduced in SEQ ID NO: 6 (transcript variant 1, NCBI Reference Sequence: NM_001024943; and transcript variant 2, NCBI Reference Sequence: NM_000048.3; each of which is incorporated by reference herein in its entirety). However, another isoform may be selected, e.g. Isoform 2 and Isoform 3. The amino acid sequence of ASL Isoform 2 (UniProt P04424-2) is reproduced in SEQ ID NO: 4. The nucleic acid sequence of ASL Isoform 2 (transcript variant 3, NCBI Reference Sequence: NM_00 1024944.1, which is incorporated by reference herein in its entirety) is reproduced in SEQ ID NO: 7. The amino acid sequence of ASL Isoform 3 (UniProt P04424-3) is reproduced in SEQ ID NO: 5. The nucleic acid sequence of ASL Isoform 3 (transcript variant 4, NCBI Reference Sequence: NM_00 1024946.1, which is incorporated by reference herein in its entirety) is reproduced in SEQ ID NO: 8. Functional ASL may also include the mutants made to remove mutate one or more of the amino acids at the positions identified above which are characterized by acetylation. In one aspect, an engineered coding sequence which encodes a functional ASL protein is provided. In one embodiment, the amino acid sequence of the functional ASL is that of the wild type ASL protein. In another embodiment, the amino acid sequence of the functional ASL is a sequence sharing at least about 80%, at least about 85%, at least about 90%, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, or at least about 99% identity with the wild type ASL protein. In another embodiment, the amino acid sequence of the functional ASL is a sequence sharing about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity with the wild type ASL protein. In one embodiment, the wild type ASL protein has a sequence of SEQ ID NO: 2. In another embodiment, the wild type ASL protein has a sequence of SEQ ID NO: 4. In yet another embodiment, the wild type ASL protein has a sequence of SEQ ID NO: 5. In one embodiment, the coding sequence is a nucleic acid sequence reproduced in SEQ ID NO: 6, or a nucleic acid sequence at least about 80% identical thereto. In one embodiment, the coding sequence is a nucleic acid sequence reproduced in SEQ ID NO: 7, or a nucleic acid sequence at least about 80% identical thereto. In one embodiment, the coding sequence is a nucleic acid sequence reproduced in SEQ ID NO: 8, or a nucleic acid sequence at least about 80% identical thereto. In one aspect, the ASL coding sequence is an engineered nucleic acid sequence. A nucleic acid refers to a polymeric form of nucleotides and includes RNA, mRNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. A nucleotide refers to a ribonucleotide, deoxynucleotide or a modified form of either type of nucleotide. The term also includes single- and double-stranded forms of DNA. Unless otherwise specified, a "nucleic acid sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleic acid sequences can be cloned using routine molecular biology techniques, or generated de novo by DNA synthesis, which can be performed using routine procedures by service companies having business in the field of DNA synthesis and/or molecular cloning (e.g. GeneArt, GenScript, Life Technologies, Eurofins). By "engineered" is meant that the nucleic acid sequences encoding a functional ASL protein described herein differ from the coding sequence found in nature. The wild type ASL coding sequence is SEQ ID NO: 6. In another embodiment, the wild type ASL coding sequence is SEQ ID NO: 7. In yet another embodiment, the wild type ASL coding sequence is SEQ ID NO: 8. In certain embodiments, an engineered cDNA sequence of SEQ ID NO: 3, or a sequence at least 95% identical thereto, encoding a functional human argininosuccinate lyase (ASL) is provided. Also provided are the complement to this sequence, and its corresponding RNA, mRNA, genomic DNA, and synthetic forms and mixed polymers of these sequences. Such nucleic acid sequences, synthetic forms, and mixed polymers may be useful in generating expression cassettes and vector genomes. In one embodiment, the engineered sequence has improved production, transcription, expression or safety in a subject. In another embodiment, the engineered sequence has increased efficacy of the resulting therapeutic compositions or treatment. In one embodiment, the engineered ASL coding sequence is characterized by improved translation rate as compared to wild type ASL coding sequences. In one embodiment, the ASL coding sequence has about 83% identity to the full- length wild type coding sequence. In one embodiment, the ASL coding sequence shares less than about 99%, less than about 98%, less than about 97%, less than about 96%, less than about 95%, less than about 94%, less than about 93%, less than about 92%, less than about 91%, less than about 90%, less than about 89%, less than about 88%, less than about 87%, less than about 86%, less than about 85%, less than about 84%, less than about 83%, less than about 82%, less than about 81%, less than about 80%, less than about 79%, less than about 78%, less than about 77%, less than about 76%, less than about 75%, less than about 74%, less than about 73%, less than about 72%, less than about 71%, less than about 70%, less than about 69%, less than about 68%, less than about 67%, less than about 66%, less than about 65%, less than about 64%, less than about 63%, less than about 62%, less than about 61% or less identity to the wild type ASL coding sequence. In another embodiment, the ASL coding sequence shares about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 89%, about 88%, about 87%, about 86%, about 85%, about 84%, about 83%, about 82%, about 81%, about 80%, about 79%, about 78%, about 77%, about 76%, about 75%, about 74%, about 73%, about 72%, about 71%, about 70%, about 69%, about 68%, about 67%, about 66%, about 65%, about 64%, about 63%, about 62%, about 61% or less identity to the wild type ASL coding sequence. In another embodiment, the engineered nucleic acid sequence encoding ASL is a sequence of SEQ ID NO: 3. In one embodiment, provided herein is an engineered nucleic acid sequence of SEQ ID NO: 3, or a nucleic acid sequence at least about 95% identical thereto, encoding a functional human ASL. In another embodiment, the ASL coding sequence is less than about 90% identity, less than about 87% identity, or less than about

95% identity, or about 83% identity to SEQ ID NO: 6 or 7. In another embodiment, the

ASL coding sequence is a sequence about 83% identical with SEQ ID NO: 6 or 7. In other embodiments, a different ASL coding sequence is selected. The nucleic acid sequences encoding ASL described herein are assembled and placed into any suitable genetic element, e.g., naked DNA, phage, transposon, cosmid, episome, etc., which transfers the ASL sequences carried thereon to a host cell, e.g., for generating non-viral delivery systems (e.g., RNA-based systems, naked DNA, or the like), or for generating viral vectors in a packaging host cell, and/or for delivery to a host cells in a subject. In one embodiment, the genetic element is a vector. In one embodiment, the genetic element is a plasmid. The methods used to make such engineered constructs are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012). The term "percent (%) identity", "sequence identity", "percent sequence identity", or "percent identical" in the context of nucleic acid sequences refers to the residues in the two sequences which are the same when aligned for correspondence. The length of sequence identity comparison may be over the full-length of the genome, the full-length of a gene coding sequence, or a fragment of at least about 500 to 5000 nucleotides, is desired. However, identity among smaller fragments, e.g. of at least about nine nucleotides, usually at least about 20 to 24 nucleotides, at least about 28 to 32 nucleotides, at least about 36 or more nucleotides, may also be desired. Multiple sequence alignment programs are also available for nucleic acid sequences. Examples of such programs include, "Clustal Omega", "Clustal W", "CAP Sequence Assembly", "BLAST", "MAP", and "MEME", which are accessible through Web Servers on the internet. Other sources for such programs are known to those of skill in the art. Alternatively, Vector NTI utilities are also used. There are also a number of algorithms known in the art that can be used to measure nucleotide sequence identity, including those contained in the programs described above. As another example, polynucleotide sequences can be compared using Fasta™, a program in GCG Version 6.1. Fasta™ provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences. For instance, percent sequence identity between nucleic acid sequences can be determined using Fasta™ with its default parameters (a word size of 6 and the NOPAM factor for the scoring matrix) as provided in

GCG Version 6.1, herein incorporated by reference. Percent identity may be readily determined for amino acid sequences over the full- length of a protein, polypeptide, about 32 amino acids, about 330 amino acids, or a peptide fragment thereof or the corresponding nucleic acid sequence coding sequences. A suitable amino acid fragment may be at least about 8 amino acids in length, and may be up to about 700 amino acids. Generally, when referring to "identity", "homology", or "similarity" between two different sequences, "identity", "homology" or "similarity" is determined in reference to "aligned" sequences. "Aligned" sequences or "alignments" refer to multiple nucleic acid sequences or protein (amino acids) sequences, often containing corrections for missing or additional bases or amino acids as compared to a reference sequence. Identity may be determined by preparing an alignment of the sequences and through the use of a variety of algorithms and/or computer programs known in the art or commercially available (e.g., BLAST, ExPASy; Clustal Omega; FASTA; using, e.g., Needleman-Wunsch algorithm, Smith-Waterman algorithm). Alignments are performed using any of a variety of publicly or commercially available Multiple Sequence Alignment Programs. Sequence alignment programs are available for amino acid sequences, e.g., the "Clustal Omega", "Clustal X", "MAP", "PIMA", "MSA", "BLOCKMAKER", "MEME", and "Match-Box" programs. Generally, any of these programs are used at default settings, although one of skill in the art can alter these settings as needed. Alternatively, one of skill in the art can utilize another algorithm or computer program which provides at least the level of identity or alignment as that provided by the referenced algorithms and programs. See, e.g., J. D . Thomson et al, Nucl. Acids. Res., "A comprehensive comparison of multiple sequence alignments", 27(13):2682-2690 (1999). A variety of assays exist for measuring ASL expression and activity levels by conventional methods. See, e.g., Example 1 as described herein; Stephenne, Xavier, et al. "Sustained engraftment and tissue enzyme activity after liver cell transplantation for argininosuccinate lyase deficiency." Gastroenterology 130.4 (2006): 1317-1323; and Nagamani SCS, Erez A, Lee B. Argininosuccinate Lyase Deficiency. 2011 Feb 3 (Updated 2012 Feb 2). In: Pagon RA, Adam MP, Ardinger HH, et al, editors. GeneReviews® (Internet). Seattle (WA): University of Washington, Seattle; 1993-2017. Available from: www.ncbi.nlm.nih.gov/books/NBK51784/; each of which is incorporated by reference herein in its entirety. It should be understood that the compositions in the ASL functional protein and ASL coding sequence described herein are intended to be applied to other compositions, regiments, aspects, embodiments and methods described across the Specification. II. Expression Cassette In one aspect, an expression cassette comprising the ASL coding sequence as described herein is provided. In one embodiment, the ASL coding sequence is an engineered sequence as described herein. In one embodiment, the expression cassette comprises the engineered nucleic acid sequence of SEQ ID NO: 3, or a nucleic acid sequence at least about 95% identical thereto, encoding functional human ASL. In one embodiment, the expression cassette further comprises regulatory elements which direct expression of the sequence encoding functional ASL. In one embodiment, the regulatory elements comprise a promoter. In a further embodiment, the promoter is a TBG promoter, a TBG-S1 promoter, an Al AT promoter, a LSP promoter, a TTR promoter, or a CMV promoter. In another embodiment, the regulatory elements comprise an enhancer. In a further embodiment, the enhancer(s) is selected from one or more of an APB enhancer, an ABPS enhancer, an alpha mic/bik enhancer, a TTR enhancer, an en34 enhancer, an ApoE enhancer, a CMV enhancer, or an RSV enhancer. In yet another embodiment, the regulatory elements comprise an intron. In a further embodiment, the intron is selected from CBA, human beta globin, IVS2, SV40, bGH, alpha-globulin, beta-globulin, collagen, ovalbumin, or p53. In one embodiment, the regulatory elements comprise a polyA . In a further embodiment, the polyA is a synthetic polyA or from bovine growth hormone (bGH), human growth hormone (hGH), SV40, rabbit β-globin (RGB), or modified RGB (mRGB). In another embodiment, the regulatory elements may comprise a WPRE sequence. In yet another embodiment, the regulatory elements comprise a Kozak sequence. The term "expression" is used herein in its broadest meaning and comprises the production of RNA, of protein, or of both RNA and protein. With respect to RNA, the term "expression" or "translation" relates in particular to the production of peptides or proteins. Expression may be transient or may be stable. As used herein, an "expression cassette" refers to a nucleic acid molecule which comprises the ASL coding sequences, promoter, and may include other regulatory elements therefor. In one embodiment, the expression cassette may be packaged into the capsid of a viral vector (e.g., a viral particle). In one embodiment, such an expression cassette for generating a viral vector contains the ASL coding sequences described herein flanked by packaging signals of the viral genome and other expression control sequences such as those described herein. For example, for an AAV viral vector, the packaging signals are the 5' inverted terminal repeat (ITR) and the 3' ITR. The term "regulatory element" or "regulatory sequence" refers to expression control sequences which are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. As described herein, regulatory elements comprise but not limited to: promoter; enhancer; transcription factor; transcription terminator; efficient RNA processing signals such as splicing and polyadenylation signals (polyA); sequences that stabilize cytoplasmic mRNA, for example Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE); sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product. In one embodiment, described herein is a promoter as a regulatory element. In another embodiment, expression of the ASL coding sequence is driven from a liver- specific promoter. See, e.g. WO 2015/138348, which is incorporated by reference herein in its entirety. An illustrative expression cassette and vector described herein uses the thyroxine binding globulin (TBG) promoter (nucleotide 431 to nucleotide 907 of SEQ ID NO: 1), or a modified version thereof. One modified version of the TBG promoter is a shortened version, termed TBG-S1. Alternatively, other liver-specific promoters may be used such as the transthyretin promoter (TTR). Another suitable promoter is the alpha 1 anti-trypsin (AIAT), or a modified version thereof. Other suitable promoter includes CAGGS promoter also named as CAG promoter, which comprises (C) the cytomegalovirus (CMV) early enhancer element, (A) the promoter, the first exon and the first intron of chicken beta-actin gene, and (G) the splice acceptor of the rabbit beta-globin gene. See, e.g., Alexopoulou, AnnikaN., et al. "The CMV early enhancer/chicken β actin (CAG) promoter can be used to drive transgene expression during the differentiation of murine embryonic stem cells into vascular progenitors." BMC cell biology 9.1 (2008): 2. In one embodiment, the promoter is an AIAT promoter combined with an ApoE enhancer, sometimes referred to as ApoE.AlAT (full). Another suitable promoter is the Liver specific promoter LSP (TH-binding globulin promoter/alphal-microglobulin/bikunin enhancer). Other suitable promoters include human albumin (Miyatake et al, J. Virol., 71:5124 32 (1997)), humAlb; and hepatitis B virus core promoter, (Sandig et al., Gene Ther., 3:1002-9 (1996). See, e.g., The Liver Specific Gene Promoter Database, Cold Spring Harbor, rulai.schl.edu/LSPD, which is incorporated by reference). Although less desired, other promoters, such as viral promoters, constitutive promoters, inducible promoters, regulatable promoters (see, e.g., WO 201 1/126808 and WO 2013/04943), or a promoter responsive to physiologic cues may be used may be utilized in the vectors described herein. In one embodiment, described herein is an enhancer as a regulatory element. See, e.g. WO 2015/138348, which is incorporated by reference herein in its entirety. In one embodiment, the expression control sequences include one or more enhancer. In one embodiment, the En34 enhancer is included (34 bp core enhancer from the human apolipoprotein hepatic control region). In another embodiment, the EnTTR (100 bp enhancer sequence from transthyretin) is included. See, Wu et al, Molecular Therapy, 16(2):280-289, Feb. 2008, which is incorporated herein by reference. In yet another embodiment, the al-microglogulin/bikunin precursor (alpha mic/bik, ABP) enhancer is included. In yet another embodiment, the ABPS (shortened version of the 100 bp distal enhancer from the al-microglogulin/bikunin precursor (ABP) to 42 bp) enhancer is included. In yet another embodiment, the ApoE enhancer is included. In one embodiment, the cytomegalovirus (CMV) early enhancer in included. In another embodiment, the Rous sarcoma virus (RSV) enhancer is included. In another embodiment, more than one enhancer is present. Such combination may include more than one copy of any of the enhancers described herein, and/or more than one type of enhancer. In one embodiment, described herein is an intron as a regulatory element. Suitable introns include the human beta globin, IVS2. See, Kelly et al, Nucleic Acids Research, 43(9):4721-32 (2015), which is incorporated herein by reference. Another suitable promoter includes the Promega chimeric intron. See, Almond, B. and Schenborn, E . T. A Comparison of pCI-neo Vector and pcDNA4/HisMax Vector. 2000, which is incorporated herein by reference. Available from: www.promega.com/resources/pubhub/enotes/a-comparison-of-pcineo-vector-and- pcdna4hismax-vector/. Another suitable intron includes the hFIX intron (WO 2015/138348); the simian virus 40 (SV40) intron; the bovine growth hormone (bGH) intron; the alpha-globulin intron; the collagen intron; the ovalbumin intron; or the p53 intron. Various introns suitable herein are known in the art and include, without limitation, those found at bpg.utoledo.edu/~afedorov/lab/eid.html, which is incorporated herein by reference. See also, Shepelev V., Fedorov A. Advances in the Exon-Intron Database. Briefings in Bioinformatics 2006, 7 : 178-185, which is incorporated herein by reference. In one embodiment, described herein is a polyadenylation signal (polyA) as a regulatory element. Suitable polyA sequences may be derived from many species and sources, e.g., bovine growth hormone, human growth hormone (hGH), SV40, rabbit beta globin, modified RGB (mRGB) or thymidine kinase (TK). It should be understood that the compositions in the expression cassette described herein are intended to be applied to other compositions, regiments, aspects, embodiments and methods described across the Specification. III. Vector In certain embodiments of this invention, the ASL nucleic acid sequence, is delivered to the liver cells in need of treatment by means of a vector or a viral vector, of which many are known and available in the art. In one embodiment, provided is a vector comprising the ASL coding sequence as described herein. In one embodiment, provided is a vector comprising the expression cassette as described herein. In one embodiment, the vector is a non-viral vector. In a further embodiment, the non-viral vector is a plasmid. In another embodiment, the vector is a viral vector. Viral vectors may include any virus suitable for gene therapy, including but not limited to bocavirus, adenovirus; adeno- associated virus (AAV); herpes virus; lentivirus; retrovirus; parvovirus, etc. However, for ease of understanding, the adeno-associated virus is referenced herein as an exemplary virus vector. Thus, in one embodiment, an adeno-associated viral vector comprising a nucleic acid sequence encoding a functional ASL operatively linked to regulatory elements therefor is provided. A "vector" as used herein is a biological or chemical moiety comprising a nucleic acid sequence which can be introduced into an appropriate host cell for replication or expression of said nucleic acid sequence. Common vectors include naked DNA, phage, transposon, plasmids, viral vectors, cosmids (Phillip McClean, www.ndsu.edu/pubweb/~mcclean/plsc73 l/cloning/cloning4.htm) and artificial chromosomes (Gong, Shiaoching, et al. A gene expression atlas of the central nervous system based on bacterial artificial chromosomes." Nature 425.6961 (2003): 917-925). "Plasmid" or "plasmid vector" generally is designated herein by a lower case p preceded and/or followed by a vector name. Plasmids, other cloning and expression vectors, properties thereof, and constructing/manipulating methods thereof that can be used in accordance with the present invention are readily apparent to those of skill in the art. In one embodiment, the ASL coding sequences as described herein or the expression cassette as described herein are engineered into a suitable genetic element (a vector) useful for generating viral vectors and/or for delivery to a host cell, e.g. , naked DNA, phage, transposon, cosmid, episome, etc. , which transfers the ASL sequences carried thereon. The selected vector may be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high velocity DNA-coated pellets, viral infection and protoplast fusion. The methods used to make such constructs are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g. , Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY. A "replication-defective virus" or "viral vector" refers to a synthetic or artificial viral particle in which an expression cassette containing a gene of interest is packaged in a viral capsid or envelope, where any viral genomic sequences also packaged within the viral capsid or envelope are replication-deficient; i.e., they cannot generate progeny virions but retain the ability to infect target cells. In one embodiment, the genome of the viral vector does not include genes encoding the enzymes required to replicate (the genome can be engineered to be "gutless" - containing only the gene of interest flanked by the signals required for amplification and packaging of the artificial genome), but these genes may be supplied during production. Therefore, it is deemed safe for use in gene therapy since replication and infection by progeny virions cannot occur except in the presence of the viral enzyme required for replication. Such replication-defective viruses may be adeno-associated viruses (AAV), adenoviruses, lentiviruses (integrating or non- integrating), or another suitable virus source. The term "transgene" or "gene of interest" as used interchangeably herein means an exogenous and/or engineered protein-encoding nucleic acid sequence that is under the control of a promoter and/or other regulatory elements in an expression cassette, rAAV genome, recombinant plasmid or production plasmid, vector, or host cell described in this specification. In certain embodiments, the transgene is a human ASL sequence, encoding a functional ASL protein. In some embodiments, the transgene is an engineered nucleic acid ASL of SEQ ID NO: 3 encoding the ASL amino acid sequence set forth in SEQ ID NO: 2. In certain embodiments, the coding sequence is 95% identical to SEQ ID

NO: 3. The term "exogenous" as used to describe a nucleic acid sequence or protein means that the nucleic acid or protein does not naturally occur in the position in which it exists in a chromosome, or host cell. An exogenous nucleic acid sequence also refers to a sequence derived from and inserted into the same host cell or subject, but which is present in a non-natural state, e.g. a different copy number, or under the control of different regulatory elements. The term "heterologous" as used to describe a nucleic acid sequence or protein means that the nucleic acid or protein was derived from a different organism or a different species of the same organism than the host cell or subject in which it is expressed. The term "heterologous" when used with reference to a protein or a nucleic acid in a plasmid, expression cassette, or vector, indicates that the protein or the nucleic acid is present with another sequence or subsequence with which the protein or nucleic acid in question is not found in the same relationship to each other in nature. As used herein, the term "host cell" may refer to the packaging cell line in which a vector (e.g., a recombinant AAV) is produced from a production plasmid. In the alternative, the term "host cell" may refer to any target cell in which expression of the transgene is desired. Thus, a "host cell," refers to a prokaryotic or eukaryotic cell that contains exogenous or heterologous DNA that has been introduced into the cell by any means, e.g., electroporation, calcium phosphate precipitation, microinjection, transformation, viral infection, transfection, liposome delivery, membrane fusion techniques, high velocity DNA-coated pellets, viral infection and protoplast fusion. In certain embodiments herein, the term "host cell" refers to cultures of cells of various mammalian species for in vitro assessment of the compositions described herein. In other embodiments herein, the term "host cell" refers to the cells employed to generate and package the viral vector or recombinant virus. Still in other embodiment, the term "host cell" is intended to reference the target cells of the subject being treated in vivo for ASA. In a further embodiment, the term "host cell" is a liver cell. The term "AAV" as used herein refers to the dozens of naturally occurring and available adeno-associated viruses, as well as artificial AAVs. An adeno-associated virus (AAV) viral vector is an AAV DNase-resistant particle having an AAV protein capsid into which is packaged nucleic acid sequences for delivery to target cells. An AAV capsid is composed of 60 capsid (cap) protein subunits, VP1, VP2, and VP3, that are arranged in an icosahedral symmetry in a ratio of approximately 1:1:10 to 1:1:20, depending upon the selected AAV. Various AAVs may be selected as sources for capsids of AAV viral vectors as identified above. See, e.g., US Published Patent Application No. 2007-0036760-A1; US Published Patent Application No. 2009-0197338-A1; EP 1310571. See also, WO 2003/042397 (AAV7 and other simian AAV), US Patent 7790449 and US Patent 7282199 (AAV8), WO 2005/033321 and US 7,906,111 (AAV9), and WO 2006/1 10689, and WO 2003/042397 (rh. 10). These documents also describe other AAV which may be selected for generating AAV and are incorporated by reference. Among the AAVs isolated or engineered from human or non-human primates (NHP) and well characterized, human AAV2 is the first AAV that was developed as a gene transfer vector; it has been widely used for efficient gene transfer experiments in different target tissues and animal models. Unless otherwise specified, the AAV capsid, ITRs, and other selected AAV components described herein, may be readily selected from among any AAV, including, without limitation, the AAVs commonly identified as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV8bp, AAV7M8 and AAVAnc80, variants of any of the known or mentioned AAVs or AAVs yet to be discovered or variants or mixtures thereof. See, e.g., WO 2005/033321, which is incorporated herein by reference. In one embodiment, the AAV capsid is an AAV8 capsid or variant thereof, an AAV9 capsid or variant thereof, an AAVrh. 10 capsid or variant thereof, an AAVrh64Rl capsid or variant thereof, an AAVhu.37 capsid or variant thereof, or an AAV3B or variant thereof. In certain embodiments, the capsid protein is designated by a number or a combination of numbers and letters following the term "AAV" in the name of the rAAV vector. Methods of generating the capsid, coding sequences therefore, and methods for production of rAAV viral vectors have been described. See, e.g., Gao, et al, Proc. Natl. Acad. Sci. U.S.A. 100 (10), 6081-6086 (2003) and US 2015/0315612. Because ASL is natively expressed in the liver, it is desirable to use an AAV which shows tropism for liver. In one embodiment, the AAV supplying the capsid is AAV8 or variant thereof. In another embodiment, the AAV supplying the capsid is AAVrh. 10 or variant thereof. In yet another embodiment, the AAV supplying the capsid is a Clade E AAV or variant thereof. Such AAV include rh.2; rh.lO; rh. 25; bb.l, bb.2, pi.l, pi.2, pi.3, rh.38, rh.40, rh.43, rh.49, rh.50, rh.51, rh.52, rh.53, rh.57, rh.58, rh.61, rh.64, hu.6, hu.17, hu.37, hu.39, hu.40, hu.41, hu.42, hu.66, and hu.67. This clade further includes modified rh. 2; modified rh. 58; and modified rh.64. See, WO 2005/033321, which is incorporated herein by reference. However, any of a number of rAAV vectors with liver tropism can be used. In another embodiment, the rAAV vector has a tropism for kidney. As used herein, relating to AAV, the term "variant" means any AAV sequence which is derived from a known AAV sequence, including those sharing at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or greater sequence identity over the amino acid or nucleic acid sequence. In another embodiment, the AAV capsid includes variants which may include up to about 10% variation from any described or known AAV capsid sequence. That is, the AAV capsid shares about 90% identity to about 99.9 % identity, about 95% to about 99% identity or about 97% to about 98% identity to an AAV capsid provided herein and/or known in the art. In one embodiment, the AAV capsid shares at least 95% identity with an AAV capsid. When determining the percent identity of an AAV capsid, the comparison may be made over any of the variable proteins (e.g., vpl, vp2, or vp3). In one embodiment, the AAV capsid shares at least 95% identity with the AAV8 vp3. In another embodiment, a self-complementary AAV is used. The ITRs or other AAV components may be readily isolated or engineered using techniques available to those of skill in the art from an AAV. Such AAV may be isolated, engineered, or obtained from academic, commercial, or public sources (e.g., the American Type Culture Collection, Manassas, VA). Alternatively, the AAV sequences may be engineered through synthetic or other suitable means by reference to published sequences such as are available in the literature or in databases such as, e.g., GenBank, PubMed, or the like. AAV viruses may be engineered by conventional molecular biology techniques, making it possible to optimize these particles for cell specific delivery of nucleic acid sequences, for minimizing immunogenicity, for tuning stability and particle lifetime, for efficient degradation, for accurate delivery to the nucleus, etc. As used herein, "artificial AAV" means, without limitation, an AAV with a non-naturally occurring capsid protein. Such an artificial capsid may be generated by any suitable technique, using a selected AAV sequence (e.g., a fragment of a vpl capsid protein) in combination with heterologous sequences which may be obtained from a different selected AAV, non-contiguous portions of the same AAV, from a non-AAV viral source, or from a non-viral source. An artificial AAV may be, without limitation, a pseudotyped AAV, a chimeric AAV capsid, a recombinant AAV capsid, or a "humanized" AAV capsid. Pseudotyped vectors, wherein the capsid of one AAV is replaced with a heterologous capsid protein, are useful in the invention. In one embodiment, AAV2/5 and AAV2/8 are exemplary pseudotyped vectors. The selected genetic element may be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high velocity DNA-coated pellets, viral infection and protoplast fusion. The methods used to make such constructs are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012). As used herein, the term "treatment" or "treating" is defined encompassing administering to a subject one or more compounds or compositions described herein for the purposes of amelioration of one or more symptoms of argininosuccinic aciduria (ASA). "Treatment" can thus include one or more of reducing onset or progression of argininosuccinic aciduria (ASA), preventing disease, reducing the severity of the disease symptoms, retarding their progression, removing the disease symptoms, delaying progression of disease, or increasing efficacy of therapy in a given subject. Treatment may include treatment of subjects having severe neonatal-onset disease of ASA (males or females), and late-onset (partial) disease of ASA in males and females, which may present from infancy to later childhood, adolescence, or adulthood. As used herein, a "vector genome" refers to the nucleic acid sequence packaged inside the rAAV capsid which forms a viral particle. Such a nucleic acid sequence contains AAV inverted terminal repeat sequences (ITRs). In the examples herein, a vector genome contains, at a minimum, from 5' to 3', an AAV2 5' ITR, a coding sequence encoding a functional ASL, and an AAV2 3' ITR. However, ITRs from a different source AAV other than AAV2 may be selected. Further, other ITRs may be used. Further, the vector genome contains regulatory sequences which direct expression of the gene of interest. Thus, in one aspect, an adeno-associated viral vector is provided which comprises an AAV capsid and at least one expression cassette, wherein the at least one expression cassette comprises nucleic acid sequences encoding ASL and regulatory elements that direct expression of the ASL sequences in a host cell. The AAV vector also comprises AAV ITR sequences. The ITRs are the genetic elements responsible for the replication and packaging of the genome during vector production and are the only viral cis elements required to generate rAAV. In one embodiment, the ITRs are from an AAV different than that supplying a capsid. In a preferred embodiment, the ITR sequences from AAV2, or the deleted version thereof (AITR), which may be used for convenience and to accelerate regulatory approval. However, ITRs from other AAV sources may be selected. Where the source of the ITRs is from AAV2 and the AAV capsid is from another AAV source, the resulting vector may be termed pseudotyped. Typically, AAV vector genome comprises an AAV 5' ITR, the ASL coding sequences and any regulatory sequences, and an AAV 3' ITR. However, other configurations of these elements may be suitable. A shortened version of the 5' ITR, termed AITR, has been described in which the D- sequence and terminal resolution site (trs) are deleted. In other embodiments, the full- length AAV 5' and 3' ITRs are used. In one embodiment, the regulatory sequences are selected such that the total rAAV vector genome is about 2.0 to about 5.5 kilobases in size. In one embodiment, the regulatory sequences are selected such that the total rAAV vector genome is about 2.9 to about 5.5 kilobases in size. In one embodiment, the regulatory sequences are selected such that the total rAAV vector genome is about 2.9 kb in size. In one embodiment, it is desirable that the rAAV vector genome approximate the size of the native AAV genome. Thus, in one embodiment, the regulatory sequences are selected such that the total rAAV vector genome is about 4.7 kb in size. In another embodiment, the total rAAV vector genome is less about 5.2 kb in size. The size of the vector genome may be manipulated based on the size of the regulatory sequences including the promoter, enhancer, intron, poly A, etc. See, Wu et al, Mol Ther, Jan 2010, 18(l):80-6, which is incorporated herein by reference. In one embodiment, provided herein is a recombinant adeno-associated virus (rAAV) useful as a liver-directed therapeutic for argininosuccinic aciduria (ASA), said rAAV comprising an AAV capsid, and a vector genome packaged therein, said vector genome comprising: (a) an AAV 5' inverted terminal repeat (ITR); (b) a coding sequence encoding argininosuccinate lyase (ASL), wherein the coding sequence is operably linked to regulatory elements which direct expression of ASL; (c) regulatory elements which direct expression of ASL; and (d) an AAV 3' ITR. In one embodiment, the coding sequence comprises SEQ ID NO: 3., or a nucleic acid sequence at least about 95% identical thereto. An exemplary rAAV genome is shown in SEQ ID NO: 1. In one embodiment, the recombinant AAV vector (rAAV) used for delivering an ASL coding sequence has atropism for the liver (e.g., an rAAV bearing an AAV8 capsid), and/or the ASL transgene is controlled by liver-specific expression control elements. In one embodiment, the expression control elements include one or more of the following: an enhancer; a promoter; an intron; an optional WPRE; and a polyA signal. In one aspect, a construct is provided which is a vector (e.g., a plasmid) useful for generating viral vectors. In one embodiment, the AAV 5' ITR is an AAV2 ITR. In another embodiment, the AAV 3'ITR is an AAV2 ITR. In one embodiment, the rAAV comprises an AAV capsid as described herein. In one embodiment, the rAAV comprises an AAV8 capsid. In other embodiments, the rAAV comprises an AAV capsid provided that it is not AAV8. An illustrative plasmid and vector described herein uses the TBG promoter and alpha mic/bik (ABP) enhancer. In yet another embodiment, the engineered sequences described herein are useful in a genome editing system, such as the Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR) system. In certain embodiments, a viral vector is used to deliver the components of the genome editing system. While the examples below describe use of AAV vectors and the following discussion focuses on AAV vectors, it will be understood that a different, partially or wholly integrating vector or virus may be used in the system in place of the gene editing vector and/or the vector carrying template. See, e.g., Jinek, M.; Chilynksi, K.; Fonfara, I.,; Hauer, M.,; Doudna, J.,; Charpentier, E., (August 17, 2012). "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity". Science. 337 (6069): 816-821. Bibcode:2012 Sc 337. .816 doi: 10. 1126/science. 1225829. PMID 22745249; US Patent 8,697,359; US 9,909,122, US 2017/0051312; US 2017/0137801; US 2017/0166893; US2017/0360048; US 2018/0002682, which are incorporated by reference in their entirety. In certain embodiments, the vector delivers one or more components (e.g., the guide RNA, donor template, and endonuclease) of the genome editing system, such as CRISPR-Cas9. In another embodiment, a combination or dual AAV vector system is provided to deliver one or more components of the CRISPR system when co-administered to a subject (see, e.g. WO 2016/176191, which is incorporated by reference herein in its entirety). The vectors may be formulated together or separately and delivered essentially simultaneously, preferably by the same route. In certain embodiments, one or more corrections may be made to a target gene (e.g., ASL) using the system gene editing system described herein. Suitably, the vectors delivering donor template which are gene fragments are designed such that the donor template is inserted upstream of the gene mutation or phenotype to be corrected. Alternatively, a vector includes a full-length sequence that can replace the defective gene (e.g., ASL). Thus, in one embodiment, the inserted sequence may be a full-length gene, or a gene encoding a functional protein or enzyme. Where a full-length gene is being delivered, there is more flexibility within the target genome for targeting. As another alternative, a single exon may be inserted upstream of the defective exon. In another alternative, gene deletion or insertion can be corrected. In certain embodiments, the target gene or gene to be replaced or corrected is ASL and the encoding CRISPR system provides an ASL encoding sequence. Preferably, the ASL encoding sequence is an engineered nucleic acid sequence of SEQ ID NO: 3, or a nucleic acid sequence at least about 95% identical thereto. In one aspect, dual vector system is provided which comprises (a) a gene editing vector which comprises a gene for an editing enzyme under control of regulatory sequences which direct its expression in a target cell (e.g., a hepatocyte) comprising a targeted gene which has one or more mutations resulting in a disorder (e.g., ASA) and (b) a targeting vector comprising a sequence specifically recognized by the editing enzyme and donor template, wherein the donor template comprises nucleic acid sequences which replaces at least one of the mutations in the targeted gene (e.g., ASL). In one embodiment, the gene editing vector comprises a Cas9 gene as the editing enzyme and the targeting vector comprises sgRNA which is at least 20 nucleotides in length which specifically bind to a selected site in the targeted genes and is 5 ' to a protospacer- adjacent motif (PAM) which is specifically recognized by the Cas9. Typically, the PAM sequence to the corresponding sgRNA is mutated on the donor template. However, in another embodiment, the gene editing vector may contain a different Crispr. "Cas9" (CRISPR associated protein 9) refers to family of RNA-guided DNA endonucleases which is characterized by two signature nuclease domains, RuvC (cleaves non-coding strand) and HNH (coding strand). Suitable bacterial sources of Cas9 include Staphylococcus aureus (SaCas9), Stapylococcus pyogenes (SpCas9), and Neisseria meningitides (KM Estelt et al, Nat Meth, 10: 11 16- 1121 (2013)). The wild-type coding sequences may be utilized in the constructs described herein. Alternatively, these bacterial codons are optimized for expression in humans, e.g. using any of a variety of known human codon optimizing algorithms. Alternatively, these sequences may be produced synthetically, either in full or in part. In the examples below, the Staphylococcus aureus (SaCas9) and the Stapylococcus pyogenes (SpCas9) versions of cas9 were compared. SaCas9 has a shorter sequence. Other endonucleases with similar properties may optionally be substituted. See, e.g., the public CRISPR database (db) accessible at crispr.u-psud.fr/crispr. In another embodiment, the CRISPR system selected may be Cpfl (CRISPR from Prevotella and Francisella), which may be substituted for Class 2 CRISPR, type II Cas9- based system in the methods described herein. In contrast, Cpfl 's preferred PAM is 5 '-TTN; this contrasts with that of SpCas9 (5'-NGG) and SaCas9 (5 '-NNGRRT; N=any nucleotide; R=adenine or guanine) in both genomic location and GC-content. While at least 16 Cpfl nucleases, two humanized nucleases (AsCpfl and LbCpfl) are particularly useful. See, www.addgene.Org/69982/sequences/#depositor-full (AsCpfl sequences; and www.addgene.Org/69988/sequences/#depositor-full (LbCpfl sequences), which are incorporated herein by reference. Further, Cpfl does not require a tracrRNA; allowing use of shorter guide RNAs (about 42 nucleotides) as compared to Cas9. Plasmids may be obtained from Addgene, a public plasmid database. While the system can be effective if the ratio of gene editing vector to template vector is about 1 to about 1, it may be desirable for the template vector to be present in excess of the gene editing vector. In one embodiment, the ratio of editing vector (a) to targeting vector (b) is about 1:3 to about 1:100, or about 1:10. This ratio of gene editing enzyme (e.g., Cas9 or Cpf) to donor template may be maintained even if the enzyme is additionally or alternatively supplied by a source other than the AAV vector. Such embodiments are discussed in more detail below. A variety of conventional vector elements may be used for delivery of the editing vector to the target cells. A system designed for treatment of a metabolic disorder such as ASA characterized by a mutation or phenotype in hepatocytes may be designed such that the enzyme is expressed under the control of a liver-specific promoter (e.g., TBG). It should be understood that the compositions in the vector described herein are intended to be applied to other compositions, regiments, aspects, embodiments and methods described across the Specification. IV. Composition Provided is an aqueous suspension suitable for administration to treat ASA in a subject in need thereof, said suspension comprising an aqueous suspending liquid and vector comprising a nucleic acid sequence encoding a functional ASL operatively linked to regulatory elements therefor as described herein. In one embodiment, a therapeutically effective amount of said vector is included in the suspension. In one embodiment, the suspension further comprises a surfactant, preservative, excipients, and/or buffer dissolved in the aqueous suspending liquid. In one embodiment, the buffer is PBS. Various suitable solutions are known including those which include one or more of: buffering saline, a surfactant, and a physiologically compatible salt or mixture of salts adjusted to an ionic strength equivalent to about 100 mM sodium chloride (NaCl) to about 250 mM sodium chloride, or a physiologically compatible salt adjusted to an equivalent ionic concentration. The pH may be in the range of 6.5 to 8.5, or 7 to 8.5, or 7.5 to 8. A suitable surfactant, or combination of surfactants, may be selected from among Poloxamers, i.e., nonionic triblock copolymers composed of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)), SOLUTOL HS 15 (Macrogol-15 Hydroxystearate), LABRASOL (Polyoxy capryllic glyceride), polyoxy 10 oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid esters), ethanol and polyethylene glycol. In one embodiment, the formulation contains a poloxamer. Additionally provided is a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a vector comprising a nucleic acid sequence encoding a functional ASL operatively linked to regulatory elements therefor as described herein. As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Supplementary active ingredients can also be incorporated into the compositions. The phrase "pharmaceutically-acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a host. Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, may be used for the introduction of the compositions of the present invention into suitable host cells. In particular, the rAAV vector delivered trangenes or rAAV vectors expressing genes for components of a CRISPR-Cas9 or other genome editing system may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like. In one embodiment, a therapeutically effective amount of said vector is included in the pharmaceutical composition. Suitable carriers may be readily selected by one of skill in the art in view of the indication for which the vector is directed. For example, one suitable carrier includes saline, which may be formulated with a variety of buffering solutions (e.g., phosphate buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The selection of the carrier is not a limitation of the present invention. Other conventional pharmaceutically acceptable carrier, such as preservatives, or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin. In certain embodiments, therapeutic regimens or co-therapies may include use of pharmaceutical compositions comprising arginine or L-arginine, or derivatives thereof. "L-arginine" as used herein is intended to include all biochemical equivalents (i.e., salts, precursors, and its basic form) of L-arginine. Other equivalents of L-arginine may include arginase inhibitors, citrulline, ornithine, and hydralazine. As used herein a "biochemical equivalent" is an agent or composition, or combination thereof, which has a similar biological function or effect as the agent or composition to which it is being deemed equivalent. In one embodiment of the present invention, arginine is in a controlled release formulation or sustained or extended release dosage that supplies a relatively constant amount of arginine and overcomes the large spiking present in instant release formulations. As used herein, the term "dosage" or "amount" can refer to the total dosage or amount delivered to the subject in the course of treatment, or the dosage or amount delivered in a single unit (or multiple unit or split dosage) administration. According to the present invention, a "therapeutically effective amount" of the ASL is delivered as described herein to achieve a desired result or to reach a therapeutic goal. In one embodiment, the desired result is defined herein, e.g. Section V Methods of the Specification. In one embodiment, therapeutic goals for treating ASA are to restore the ASL functional level in a patient to the normal range or to the non-ASA level. In another embodiment, therapeutic goals for ASA are to increase the ASL functional level in a patient to at least about 99%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 45%, about 40%, about 35%, about 30% about 25%, about 20%, about 15%, about 10%, about 5%, about 2%, about 1% of the normal or non-ASA level. Patients rescued by delivering ASL function to less than 100% activity levels, and may optionally be subject to further treatment subsequently. Suitable volumes of the aqueous suspension or pharmaceutical compositions for delivery of these doses may be determined by one of skill in the art. For example, volumes of about 1 to about 1000 µ , about ImL to about 150 mL, including all numbers within the range, may be selected. In one embodiment, the volumes of the aqueous suspension or pharmaceutical compositions is about 0.1 mL to about 10 mL. The dosage is adjusted to balance the therapeutic benefit against any side effects and such dosages may vary depending upon the therapeutic application for which the recombinant vector is employed. The aqueous suspension or pharmaceutical compositions described herein are designed for delivery to subjects in need thereof by any suitable route or a combination of different routes. Direct or intrahepatic delivery to the liver is desired and may optionally be performed via intravascular delivery, e.g., via the portal vein, hepatic vein, bile duct, or by transplant. In one embodiment, the aqueous suspension or pharmaceutical compositions described herein are designed for delivery to subjects in need thereof by intravenous injection. Alternatively, other routes of administration may be selected (e.g., oral, inhalation, intranasal, intratracheal, intraarterial, intraocular, intramuscular, and other parenteral routes). The ASL delivery constructs described herein may be delivered in a single composition or multiple compositions. Optionally, two or more different AAV may be delivered [see, e.g., WO 2011/126808 and WO 2013/049493]. In another embodiment, such multiple viruses may contain different replication-defective viruses (e.g., AAV, adenovirus, and/or lentivirus). Alternatively, delivery may be mediated by non-viral constructs, e.g., "naked DNA", "naked plasmid DNA", RNA, and mRNA; coupled with various delivery compositions and nano particles, including, e.g., micelles, liposomes, cationic lipid - nucleic acid compositions, poly-glycan compositions and other polymers, lipid and/or cholesterol-based - nucleic acid conjugates, and other constructs such as are described herein. See, e.g., X . Su et al, Mol. Pharmaceutics, 201 1, 8 (3), pp 774-787; web publication: March 21, 2011; WO2013/182683, WO 2010/053572 and WO 2012/170930, both of which are incorporated herein by reference. Such non-viral ASL delivery constructs may be administered by the routes described previously. In one embodiment, the aqueous suspension or pharmaceutical compositions is suitable for use in human subjects and is administered intravenously. In one embodiment, the aqueous suspension or pharmaceutical compositions is delivered via a peripheral vein by bolus injection. In one embodiment, the aqueous suspension or pharmaceutical compositions is delivered via a peripheral vein by infusion over about 10 minutes (±5 minutes), over about 20 minutes (±5 minutes), 30 minutes (±5 minutes), 60 minutes (±5 minutes) or 90 minutes (±5 minutes). However, this time may be adjusted as needed or desired. Any suitable method or route can be used to administer a composition of the gene therapy as described herein, and optionally, to co-administer other active drugs or therapies in conjunction with the gene therapy of ASL described herein. In the case of AAV viral vectors, quantification of the genome copies ("GC") may be used as the measure of the dose contained in the aqueous suspension or pharmaceutical compositions. Any method known in the art can be used to determine the genome copy (GC) number of the replication-defective virus compositions of the invention. One method for performing AAV GC number titration is as follows: Purified AAV vector samples are first treated with DNase to eliminate un-encapsidated AAV genome DNA or contaminating plasmid DNA from the production process. The DNase resistant particles are then subjected to heat treatment to release the genome from the capsid. The released genomes are then quantitated by real-time PCR or quantitative PCR using primer/probe sets targeting specific region of the viral genome (usually poly A signal). The replication-defective virus compositions can be formulated in dosage units to contain an amount of replication-defective virus that is in the range of about 1.0 x 109 GC to about 1.0 x 10 15 GC, and preferably 1.0 x 10 12 GC to 1.0 x 10 14 GC for a human patient. Preferably, the concentration of replication-defective virus in the formulation is about 1.0 x 109 GC, about 5.0 x 109 GC, about 1.0 x 10 10 GC, about 5.0 x 10 10 GC, about 1.0 x 10 11 GC, about 5.0 x 10 11 GC, about 1.0 x 10 12 GC, about 5.0 x 10 12 GC, about 1.0 x 10 13 GC, about 5.0 x 10 13 GC, about 1.0 x 10 14 GC, about 5.0 x 10 14 GC, or about 1.0 x 10 15 GC. Alternative or additional method for performing AAV GC number titration is via oqPCR or digital droplet PCR (ddPCR) as described in, e.g., M . Lock et al, Hum Gene Ther Methods. 2014 Apr;25(2): 115-25. doi: 10.1089/hgtb.2013.131. Epub 2014 Feb 14, which is incorporated herein by reference. V. Methods One goal of therapies described herein would provide functional ASL to achieve a desired result, i.e., treatment of argininosuccinic aciduria (ASA) or one or more symptoms thereof. Such symptoms may include but not limit to one of more of the following: lethargy; loss of appetite; erratic breathing; poorly controlled body temperature; seizures; coma; hepatomegaly; hypotonia; delays in physical developmental; intellectual disability; ataxia; liver damage; skin lesions; brittle hair; a decreased ability for arteries to dilate; ammonia accumulation in the bloodstream; elevated levels of argininosuccinic acid; hyperammonemia; and Apnea. As described herein, a desired result may also include improving vascular endothelial function, improving liver function, reducing hyperammonemia and/or minimizing or eliminating one or more of the neurophysical complications including developmental delay, learning disabilities, memory disabilities, intellectual disability, attention deficit hyperactivity disorder, and executive function deficits. Other suitable desired result may include less restrictive diet, reduction in the use of arginine supplementation, reduction in the use of alternative pathway therapy or nitrogen scavenging therapy (e.g., sodium benzoate, sodium phenylbutyrate, and glycerol triphenylbutyrate), or no need for liver transplant. In certain embodiments, the invention provides a method of treating ASA in a subject in need by administering the ASL coding sequence in an expression cassette, in a vector, in a rAAV, in an aqueous suspension, or in a pharmaceutical composition as described herein. In alternative embodiments, the invention provides a method of treating ASA in a subject by delivering the ASL coding sequence in conjunction with components of a CRISPR-Cas9 or other genome editing system. The gene therapy described herein, whether viral or non-viral, may be used in conjunction with other treatments (secondary therapy), i.e., the standard of care for the subject's (patient's) diagnosis and condition. As used herein, the term "secondary therapy" refers to the therapy that could be combined with the gene therapy described herein for the treatment of ASA. In some embodiments, the gene therapy described herein is administered in combination with one or more secondary therapies for the treatment of ASA, such as a restricted diet, arginine supplementation, administration nitrogen scavenger therapy, or dialysis. The secondary therapy may be any therapy which helps prevent, arrest or ameliorate these mutations or defects or any of the effects associated therewith. The secondary therapy can be administered before, concurrent with, or after administration of the compositions described above. Subjects may be permitted to continue their standard of care treatment(s) (e.g., protein restricted diet, and/or medications (including nitrogen scavenger therapy)) prior to and concurrently with the gene therapy treatment at the discretion of their caring physician. In the alternative, the physician may prefer to stop standard of care therapies prior to administering the gene therapy treatment and, optionally, resume standard of care treatments as a co-therapy after administration of the gene therapy. In another embodiment, the gene therapy described herein may be combined with genotypic analysis or genetic screening, which is routine in the art and may include the use of PCR to identify one or more mutations in the nucleic acid sequence of the ASL gene. See, e.g., Ganetzky, RD, et al (2016), cited above. As discussed above, both of the subject having ASA upon birth and the subject having late- onset ASA are the intended recipients of the compositions and methods described herein. By "administering" or "route of administration" is delivery of composition described herein, with or without a pharmaceutical carrier or excipient, of the subject. Routes of administration may be combined, if desired. In some embodiments, the administration is repeated periodically. Sequential administration may imply a time gap of multi-administration from intervals of days, weeks, months or years. In one embodiment, the compositions described herein are administered to a subject in need for one or more times. In one embodiment, the administrations are days, weeks, months or years apart. In one embodiment, one, two, three or more re-administrations are permitted. Such re- administration may be with the same type of vector, or a different vector. In a further embodiment, the ASL vectors may be used alone, or in combination with the standard of care for the patient's diagnosis and condition. The nucleic acid molecules and/or vectors described herein may be delivered in a single composition or multiple compositions. Optionally, two or more different AAV may be delivered, or multiple viruses [see, e.g., WO20 201 1/126808 and WO 2013/049493]. In one embodiment, the nucleic acid sequence, the expression cassette, the vector, or the composition of the gene therapy described herein is delivered as a single dose per patient. In one embodiment, the subject is delivered a therapeutically effective amount of the vectors described herein. In one embodiment, the dosage of the vector is about lxl 09 genome copies (GC)/kg body weight to about lxl 014 GC/kg body weight, including all integers or fractional amounts within the range and the endpoints. In one embodiment, the dosage is 6.0 x 10 13 GC/kg body weight. In another embodiment, the dosage is 1.0 x 10 13 GC/kg body weight. In specific embodiments, the dose of the vector administered to a patient is at least about 1.0 x 109 GC/kg, about 1.5 x 109 GC/kg, about 2.0 x 109 GC/kg, about 2.5 x 109 GC/kg, about 3.0 x 109 GC/kg, about 3.5 x 109 GC/kg, about 4.0 x 109 GC/kg, about 4.5 x 109 GC/kg, about 5.0 x 109 GC/kg, about 5.5 x 109 GC/kg, about 6.0 x 109 GC/kg, about 6.5 x 109 GC/kg, about 7.0 x 109 GC/kg, about 7.5 x 109 GC/kg, about 8.0 x 109 GC/kg, about 8.5 x 109 GC/kg, about 9.0 x 109 GC/kg, about 9.5 x 109 GC/kg, about 1.0 x 10 10 GC/kg, about 1.5 x 10 10 GC/kg, about 2.0 x 10 10 GC/kg, about 2.5 x 10 10 GC/kg, about 3.0 x 10 10 GC/kg, about 3.5 x 10 10 GC/kg, about 4.0 x 10 10 GC/kg, about 4.5 x 10 10 GC/kg, about 5.0 x 10 10 GC/kg, about 5.5 x 10 10 GC/kg, about 6.0 x 1002 GC/kg, about 6.5 x 10 10 GC/kg, about 7.0 x 10 10 GC/kg, about 7.5 x 10 10 GC/kg, about 8.0 x 10 10 GC/kg, about 8.5 x 10 10 GC/kg, about 9.0 x 10 10 GC/kg, about 9.5 x 10 10 GC/kg, about 1.0 x 10 11 GC/kg, about 1.5 x 10 11 GC/kg, about 2.0 x 10 11 GC/kg, about 2.5 x 10 11 GC/kg, about 3.0 x 10 11 GC/kg, about 3.5 x 10 11 GC/kg, about 4.0 x 10 11 GC/kg, about 4.5 x 10 11 GC/kg, about 5.0 x 10 11 GC/kg, about 5.5 x 10 11 GC/kg, about 6.0 x 10 11 GC/kg, about 6.5 x 10 11 GC/kg, about 7.0 x 10 11 GC/kg, about 7.5 x 10 11 GC/kg, about 8.0 x 10 11 GC/kg, about 8.5 x 10 11 GC/kg, about 9.0 x 10 11 GC/kg, about 9.5 x 10 11 GC/kg, about 1.0 x 10 12 GC/kg, about 1.5 x 10 12 GC/kg, about 2.0 x 10 12 GC/kg, about 2.5 x 10 12 GC/kg, about 3.0 x 10 12 GC/kg, about 3.5 x 10 12 GC/kg, about 4.0 x 10 12 GC/kg, about 4.5 x 10 12 GC/kg, about 5.0 x 10 12 GC/kg, about 5.5 x 10 12 GC/kg, about 6.0 x 10 12 GC/kg, about 6.5 x 10 12 GC/kg, about 7.0 x 10 12 GC/kg, about 7.5 x 10 12 GC/kg, about 8.0 x 10 12 GC/kg, about 8.5 x 10 12 GC/kg, about 9.0 x 10 12 GC/kg, about 9.5 x 10 12 GC/kg, about 1.0 x 10 13 GC/kg, about 1.5 x 10 13 GC/kg, about 2.0 x 10 13 GC/kg, about 2.5 x 10 13 GC/kg, about 3.0 x 10 13 GC/kg, about 3.5 x 10 13 GC/kg, about 4.0 x 10 13 GC/kg, about 4.5 x 10 13 GC/kg, about 5.0 x 10 13 GC/kg, about 5.5 x 10 13 GC/kg, about 6.0 x 10 13 GC/kg, about 6.5 x 10 13 GC/kg, about 7.0 x 10 13 GC/kg, about 7.5 x 10 13 GC/kg, about 8.0 x 10 13 GC/kg, about 8.5 x 10 13 GC/kg, about 9.0 x 10 13 GC/kg, about 9.5 x 10 13 GC/kg, or about 1.0 x 10 14 GC/kg body weight. Also, the replication-defective virus compositions can be formulated in dosage units to contain an amount of replication-defective virus that is in the range of about 1.0 x 109 GC to about 1.0 x 10 16 GC (to treat an average subject of 70 kg in body weight) including all integers or fractional amounts within the range, and preferably 1.0 x 10 12 GC to 1.0 x 10 14 GC for a human patient. In one embodiment, the compositions are formulated to contain at least lxlO 9, 2xl0 9, 3xl0 9, 4xl0 9, 5xl0 9, 6xl0 9, 7xl0 9, 8xl0 9, or 9xl0 9 GC per dose including all integers or fractional amounts within the range. In another embodiment, the compositions are formulated to contain at least lxl 010, 2x1 010, 3xl0 10, 4xl0 10, 5xl0 10, 6xl0 10, 7xl0 10, 8xl0 10, or 9xl0 10 GC per dose including all integers or fractional amounts within the range. In another embodiment, the compositions are formulated to contain at least lxlO 11, 2xlO , 3xl0 , 4xlO , 5xl0 , 6xlO , 7xlO , 8xl0 , or 9xlO GC per dose including all integers or fractional amounts within the range. In another embodiment, the compositions are formulated to contain at least lxl 012, 2xl0 12, 3xl0 12, 4xl0 12, 5xl0 12, 6xl0 12, 7xl0 12, 8xl0 12, or 9xl0 12 GC per dose including all integers or fractional amounts within the range. In another embodiment, the compositions are formulated to contain at least lxl 013, 2x1 013, 3x1 013, 4x1 013, 5x1 013, 6x1 013, 7x1 013, 8x1 013, or 9x1 013 GC per dose including all integers or fractional amounts within the range. In another embodiment, the compositions are formulated to contain at least lxlO 14, 2xl0 14, 3xl0 14, 4xl0 14, 5xl0 14, 6xl0 14, 7xl0 14, 8xl0 14, or 9xl0 14 GC per dose including all integers or fractional amounts within the range. In another embodiment, the compositions are formulated to contain at least lxlO 15, 2xl0 15, 3xl0 15, 4xl0 15, 5xl0 15, 6x1 015, 7x1 015, 8x1 015, or 9x1 015 GC per dose including all integers or fractional amounts within the range. In one embodiment, for human application the dose can range from lxl0 10 to about lxlO 12 GC per dose including all integers or fractional amounts within the range. As used herein, the term "dosage" can refer to the total dosage delivered to the subject in the course of treatment, or the amount delivered in a single (of multiple) administration. In one embodiment, the vector is a rAAV vector as described herein. In yet another embodiment, the invention provides a method of rescuing and/or treating a neonatal subject having ASA comprising the step of delivering an ASL coding sequence to the liver of a newborn subject (e.g., a human patient). This method may utilize any nucleic acid sequence encoding a functional ASL as described previously. In one embodiment, neonatal treatment is defined as being administered a composition as described herein within 8 hours, the first 12 hours, the first 24 hours, or the first 48 hours of delivery. In another embodiment, particularly for a primate, neonatal delivery is within the period of about 12 hours to about 1 week, 2 weeks, 3 weeks, or about 1 month, or after about 24 hours to about 48 hours. To address dilution due to the rapid turnover of liver cells in a growing mammal (e.g., a non-human or human primate), neonatal therapy is desirably followed by re-administration at about 3 months of age, about 6 months, about 9 months, or about 12 months. Optionally, more than one re-administration is permitted. In certain embodiments, the present invention provides a method of rescuing and/or treating a neonatal subject having ASA comprising the step of delivering an ASL coding sequence in conjunction with a CRISPR/enzyme editing system. In one embodiment, the ASL coding sequence comprises the engineered nucleic acid sequence of SEQ ID NO: 3, or a nucleic acid sequence at least about 95% identical thereto. Gene editing-mediated correction as described herein in neonates may not require readministration. However, optionally, a second or subsequent additional treatments involving co-administration of the CRISPR/enzyme system provided herein may be pursued. Such subsequent treatment may utilize vectors having different caspids than were utilized for the initial treatment. For example, if initial treatment was by AAV8, a second treatment may utilize rhlO. In another example, if initial treatment utilized rhlO, a subsequent treatment may utilize AAV8. Still other combinations of AAV caspids may be selected by one skilled in the art. It is desirable that the lowest effective concentration of virus or other delivery vehicle be utilized in order to reduce the risk of undesirable effects, such as toxicity. Still other dosages in these ranges may be selected by the attending physician, taking into account the physical state of the subject, preferably human, being treated, the age of the subject, and the degree to which the disorder, if progressive, has developed. Generally, the methods include administering to a mammalian subject in need thereof, a pharmaceutically effective amount of a composition comprising a recombinant adeno-associated virus (AAV) carrying a nucleic acid sequence encoding a functional ASL protein, or fragment thereof, under the control of regulatory sequences which express the product of the gene in the subject's liver cells, and a pharmaceutically acceptable carrier. In one embodiment, such a method is designed for treating, retarding or halting progression of ASA in a mammalian subject. In certain embodiments, methods that include administration of arginine or L-arginine or pharmaceutical compositions comprising arginine or L-arginine to a subject (i.e., arginine supplementation) may be used in combination with an rAAV therapy provided herein. In one embodiment, the method includes administering a sustained release formulation of L-arginine or a biochemical equivalent. In some embodiments, the present invention includes administering arginine supplementation to subject that is being treated for ASA (i.e., co-therapy). In certain embodiments, methods of arginine supplementation or co-therapy may eliminate the need for a subject to otherwise be required to be treated with a low-protein or protein-restricted diet. In certain embodiments, the dosage of arginine supplementation is determined by the age, sex and/or weight of the subject. In some aspects, only female subjects receive arginine supplementation. In other aspects, only male subjects are administered arginine supplementation. In certain embodiments, the methods include administering arginine supplementation or co-therapy at a higher dosage to one sex relative to the amount administered to the other sex. Thus, in certain embodiments a female subject being treated for ASA is administered a higher dosage of arginine supplementation relative to the dosage that would be administered to a male subject that is being treated for ASA. In certain embodiments, the subject being treated for ASA is also administered a composition comprising an AAV vector as set forth herein. Arginine supplementation or co-therapy may be administered before, at the same time, or following other treatments for ASA, such as delivery of an AAV gene-therapy vector. In certain embodiments, arginine supplementation is reduced in frequency and/or dose prior to delivery of gene therapy. For example, an arginine dose for an ASA patient who has not undergone gene therapy may be in the range of about 50 mg/kg/day to about 500 mg/kg/day, or about 10 grams/m2, or about 100 mg/kg/day to about 500 mg/kg/day. In certain embodiments, an arginine supplement is delivered at a dose of about 1000 mg/day to about 35,000 mg/day for an adult. In certain embodiments, doses of arginine are combined with nitrogen scavenging therapy, e.g., by co-administration with sodium phenyl butyrate. Arginine supplementation or co-therapy and other treatments for ASA (such as a composition comprising an AAV vector) may be provided to a subject via the same or different routes of administration. Specifically for human subjects, following administration of a dosage of a composition described in this specification, the subject is tested for efficacy of treatment via assessing the desired results as described above by conventional methods, including but not limited to: measurement of urea production rate; blood test revealing amounts of urea, ammonia, citrulline, glutamine, urine creatinine, bilirubin, hemoglobin, argininosuccinic acid and arginine; measures of liver function, such as AST, ALT, prothrombin time (PT), partial thromboplastin time (PTT), international normalized ratio (INR), plasma levels of coagulation factors I and IX; measurement of blood pressure, vascular endothelial function as assessed by flow mediated dilatation (FMD) of brachial artery measured by Doppler ultrasound; Delis-Kaplan Executive Function System, e.g., Tower subtest; Stanford-Binet - 4th Edition: Bead Memory and Sentence Memory subtests; Grip Strength; Grooved Pegboard; Wechsler Intelligence Scale for Children OR Wechsler Adult Intelligence Scale; Tower of London Test; Conners Continuous Performance Test - 3rd Edition; and blood and urine measurements of isotopic [13C]-urea concentration and blood measurements of isotopic [13-C02] concentration after an intravenous bolus infusion of [13C]-urea. In certain embodiments, the efficacy of treatment is determined by measuring disease markers or metabolites (e.g. citrulline or argininosuccinic aciduria) in a sample obtained from a subject using tandem mass spectrometry. In one embodiment, provided is a method of treating ASA by administrating to a subject in need the vector, the rAAV, the aqueous suspension, or the pharmaceutical composition as described in the present specification. In one embodiment, the rAAV is delivered about 1 x 10 10 to about 1 x 10 15 genome copies (GC)/kg body weight. In one embodiment, the subject is human. In one embodiment, the rAAV is administered at more than one times. In a further embodiment, the rAAV is administered days, weeks, months or years apart. EXAMPLES The invention is now described with reference to the following examples. These examples are provided for the purpose of illustration only and the invention should in no way be construed as being limited to these examples but rather should be construed to encompass any and all variations that become evident as a result of the teaching provided herein. In the examples below, a novel AAV vector encoding sequence for ASL protein and demonstrate efficacy in a mouse model is described.

EXAMPLE 1 - Methods A . Animals Two mouse models exist for ASA. A knockout model (Asl-/-) was created by replacement of exons 8 and 9 with a 1,400 bp neomycin cassette resulting in a frame shift in the mRNA beginning with exon 10 (Reid Sutton, V et al. (2003) A mouse model of argininosuccinic aciduria: biochemical characterization. Molecular genetics and metabolism 78: 11-16). All homozygotes from this model have elevated plasma ammonia, argininosuccinic acid, and citrulline as well as low plasma arginine. However, as these mice expire within 48 hours of birth, this model is difficult to use other than for the purpose of treating a non-neonatal cohort. An alternative ASA hypomorphic mouse model has also been developed, where a 1,200 bp neomycin cassette was inserted into intron 9, resulting in reduced, but not ablated, mRNA levels and slightly prolonged survival (Erez, A et al. (2011), as cited above). These mice also have the same characteristic variations in amino acids and liver metabolites, and they also display a sparse fur coat. In this study, we have developed a gene therapy for ASA using an AAV8 vector expressing the human ASL gene under the control of the liver-specific TBG promoter, and examined the therapeutic efficacy in both neonatal and adult ASA hypomorphic mice. ASA hypomorphic mice on a C57B1/6 background were acquired from the Jackson Laboratory (Bar Harbor, ME) Stock and bred at animal facility of Translational Research Laboratories (TRL), University of Pennsylvania, Philadelphia, PA. All mice were housed under specific pathogen-free conditions. All experimental procedures, including the use of mice, were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania. B. Vectors AAV8 vectors expressing engineered human argininosuccinate lyase (AAV8.TBG.hASLco.bGH, AAV8.TBG.hASLco, AAV8.hASLco or AAV8.ASL) were designed under the control of a thyroxine-binding globulin (TBG) promoter with a bovine growth hormone (BGH) poly (A) signal sequence. AAV vectors were produced by the Penn Vector Core at the University of Pennsylvania as previously described (Lock M Alvira et al. (2010). Rapid, simple, and versatile manufacturing of recombinant adeno- associated viral vectors at scale. Hum Gene Ther 21: 1259-1271.). Guide RNAs (gRNAs) compatible with SaCas9 were designed to target intronic regions of ASL using Benchling. These guides were cloned into PX330.Sa.Cas9 using the Bbsl cut sites. Donor plasmid was generated by first cloning PX330.Sa.Cas9 U6.gRNA + RNA scaffold sequence into cis AAV.TBG.bGH backbone. Then the engineered human ASL with flanking arms of homology was cloned into the construct between the TBG promoter and bGH polyA signal sequence (FIG. 8). This plasmid was then used to generate the donor AAV8 vector. AAV8.Sa.Cas9 was generated from a previously used plasmid (Yang Y et al. (2016) Nature Biotechnology 34(3):334-8). AAV vectors were produced by the Penn Vector Core at the University of Pennsylvania, as previously described (Lock M et al. (2010) Hum Gene Ther 21(10):1259-71). C. Cell Culture and Transfection H2.35 cells (ATCC) were maintained in DMEM medium supplemented with 10% FBS and cultured at 32 °C with 5% CO2. For in vitro target and/or donor template testing, plasmids were transfected into H2.35 cells using Lipofectamine 2000 per manufacturer's recommendations. Transfected cells were under puromycin (0.75 µg m 1) selection for 2 days to enrich transfected cells. D . Genomic DNA extraction and SURVEYOR assay Genomic DNA from transfected H2.35 cells was extracted using the Qiagen, QIAamp DNA Mini Kit (Gaithersburg, MD). The efficiency of each individual sgRNA was tested by the IDT SURVEYOR nuclease assay (Coralville, IA) using manufacturer's recommendations. E . Neonatal Gene Therapy Heterozygous mice were set up in timed matings. In Example 2, during the first 24 h following birth, pups were administered intravenously (IV) with lxlO 10 or lxlO 11 GC/mouse of AAV8.TBG.hASLco.bGH by the temporal facial vein. In Example 3, during the first 24 h following birth, pups (n=5) received 50 µΐ IV administration of 2xl0 12 GC/mouse of AAV8.U6.2G6.sgR.TBG.PI.ASL.co.bGH (guided) or AAV8.U6.Null.sgR.TBG.PI.ASL.co.bGH (unguided) mixed with 3xlO GC/mouse of AAV8.TBG.hSa.Cas9.bGH via the temporal facial vein. Mice were genotyped and weighed upon weaning and bled every two weeks. Duplicate cohorts of wild type and heterozygous mice that had received one of the two dosing strategies were sacrificed at day 50 for an early measure of hASL gene integration. The remaining mice were sacrificed upon termination of the short term study at 120 days, and tissues were collected for histology and measurement of hASL gene integration. F. Adult Gene Therapy ASA hypomorphic mice 4-5 weeks of age were administered IV with 6x1 013 or lxl 013 GC/kg of AAV8.TBG.hASLco.bGH. Mice were weighed throughout the study. Sub-mandibular bleeds were performed weekly collecting plasma in order to monitor transaminase and urea acid cycle metabolites, and samples were submitted to Antech Diagnostics (Irvine, CA) and Agilux Laboratories (Worcester, MA) for analysis, respectively. Mice were sacrificed after 3 months and tissues were collected for biodistribution and histology. G. On-Target Integration Genomic DNA was extracted from mouse liver using the Qiagen, QIAamp DNA Mini Kit (Gaithersburg, MD). Briefly, 3 µg of gDNA is digested with 20 units of Dral or, Blpl for 2 hours at 37°C, enzymes were inactivated according to the manufacturer recommendations. Digested DNA was purified, end-repaired, and ligated using the protocol described in literature (Tsai SQ et al. (2015) Nature Biotechnology 33(2): 187-97). Digested DNA was purified with Agencourt AMPure XP beads (Beckman Coulter, Sharon Hill, PA) and suspended in 20µ1of EB buffer. DNA was quantified using Quant-iT Picogreen analysis (Thermo Fisher, Waltham, MA). Purified DNA was end- repaired and ligated to Y-adapters containing unique-molecular indexes to reduce PCR bias, as previously described. Ligated DNA was then purified with AMPure XP beads (0.9X ratio). DNA was amplified by touchdown PCR using Platinum Taq DNA polymerase (Thermo Fisher Scientific, Waltham, MA). DNA was purified with AMPure XP beads and suspended in 15 µΐ of EB. We used a 1:100 dilution of the 1st PCR product and used 1.5 µΐ of this dilution as template for the second PCR with a second round of amplification by touchdown PCR to amplify the internal sequence. PCR product was purified with AMPure XP beads and suspended in 15µ1of EB buffer. DNA libraries were prepared for next-generation sequencing using unique P7 primers (P701 to P734) for each sample. DNA was purified using Ampure beads and eluted in 25 µΐ . The quality of the libraries was checked using an Agilent 2100 Bioanlyzer (Santa Clara, CA) and DNA was quantitated using Picogreen analysis and pool libraries at equal molarity. The concentration of the final pool was measured using Qubit (Thermo Fisher, Waltham, MA), dilute for loading. H . Serum analysis Plasma samples were submitted to Antech Diagnostics (Irvine, CA) for analysis of liver transaminases and Agilux Laboratories (Worcester, MA) for analysis of arginine, citrulline, and argininosuccinic acid. I. ASL Immunohistochemistry Tissues were fixed in formalin for a minimum of 24 h and paraffin embedded. Sections were deparaffinized through an ethanol and xylene series, boiled for 6 min in 10 mM citrate buffer (pH 6.0) for antigen retrieval, and sequentially treated with

2% H2O2 (15 min), avidin/biotin blocking reagents (15 min each; Vector Laboratories), and blocking buffer (1% donkey serum in PBS + 0.2% Triton for 10 min). Sections were then incubated with a rabbit serum against ASL (Sigma HPA016646; lh) and biotinylated secondary anti-rabbit antibodies (45 min; Jackson Immunoresearch) diluted in blocking buffer at the manufacturer's recommended concentration. A Vectastain Elite ABC kit (Vector Laboratories) was used according to the manufacturer's instructions with 3,3'- diaminobenzidine as the substrate to stain bound antibodies. J. Bio-distribution Liver samples were frozen on dry ice at the time of necropsy, and DNA was extracted using the QIAamp DNA Mini Kit (Qiagen, Valencia, CA). Detection and quantification of vector GCs in extracted DNA were performed by real-time PCR, as described previously (Bell, P, et al. (2006). Analysis of Tumors Arising in Male B6C3F1 Mice with and without AAV Vector Delivery to Liver. Molecular therapy: thejournal of the American Society of Gene Therapy 14: 34-44). Briefly, genomic DNA was isolated, and vector GCs were quantified using primers/probes designed against the poly(A) sequence of the vector. Quantification of GCs from liver was performed on one liver sample from each mouse. K . ASL Activity Assay 1. Homogenization

Liver (25-30 mg) was added to 200 µΐ cold homogenizing buffer containing 50 mM phosphate buffer (pH 7.5) and proteinase inhibitors (EDTA-free proteinase inhibitor cocktail (Roche)) by use of an electric homogenizer (Biospec Products) or Tissue Lyser II (Qiagen, Valencia, CA) at a frequency of 30 for 30 seconds. Homogenates were centrifuged at 10,000 x g for 20 min at 4°C, and supernatants were kept frozen at 80°C. 2. Activity Assay

Lysate (2 µΐ) was added to 48 µΐ of 50 mM phosphate buffer (pH 7.3), 3.6 mM argininosuccinic acid (Sigma Aldrich, St. Louis MO). The reaction was incubated at 37°C for 1 h, and stopped by heating at 80°C for 20 min. Fumarate was measured by a kit (Fumarate Assay Kit, Sigma Aldrich, St. Louis MO) per the manufacturer's specifications using 5 µΐ of reaction sample mixture. L . Statistical analysis Comparison of survival was performed by logrank test with each experimental group compared to uninjected control groups, unless otherwise stated. Other parameters were compared by one-way ANOVA with Dunnett's multiple comparison test comparing each group to the untreated wild type control group unless otherwise stated.

EXAMPLE 2 - Correction of Argininosuccinic Aciduria by AAV Gene Therapy The ASA hypomorphic mouse was acquired from The Jackson Laboratory and a breeding colony mating heterozygous males to heterozygous females was set up. Initial characterization of the model found that homozygous ASA hypomorphic mice had a mean survival of 22 days (FIG. 3A). Homozygous pups were indistinguishable from heterozygous or wild type littermates until five days after birth, at which time the hypomorphic mice failed to begin growing a fur coat and were visibly smaller. Prophylactic AA V8 gene therapy extends survival of neonatal ASA hypomorphic mice AAV therapy was evaluated in neonatal mice. Timed heterozygous matings were initiated, and all mice in the litters were administered vector (AAV8.TBG.hASLco.bGH) through the temporal facial vein within the first 24 hours of birth at a dose of either lxl 010 GC or lxlO 11 GC/mouse. Mice were genotyped after weaning. In both groups, survival and body weight were increased compared to untreated ASA hypomorphic mice over the duration of the study (FIG. 3B - FIG. 3D). The increase in median survival was dose dependent, with a median survival of 165 days for the 10 11 GC/mouse group compared to 136 days for the 10 10 GC/mouse group. Weight gain for vector-administered ASA hypomorphic mice was comparable to wild-type littermates throughout the study, with the exception of the female vector-administered hypomorphic mice at the latest time point evaluated (day 63; FIG. 3C and FIG. 3D). Once mice reached median survival, they were euthanized and necropsied. Liver was collected for immunohistochemistry (IHC) to visualize ASL protein (FIG. 4). IHC revealed a small number of strongly-stained positive cells, with no observable differences between the high- and low-dose groups. The low level of transgene expression observed in these adult mice is expected, as the rapid proliferation of liver cells post vector administration has been shown to significantly dilute non-integrating vector genomes (Cunningham, SC et al. (2009) AAV2/8-mediated correction of OTC deficiency is robust in adult but not neonatal Spf(ash) mice. Mol her 17: 1340-1346; and Wang, L et al. (2012) Hepatic gene transfer in neonatal mice by adeno-associated virus serotype 8 vector. Hum Gene Ther 23:533-539). Despite the low level of transgene expression observed at the time of necropsy, ASL activity was sufficient to allow all vector-treated ASA hypomorphic mice to live beyond the 22 day mean survival of untreated ASA hypomorphic mice. However, as expected without re-administration, we confirmed that vector dilution as the animal grew led to reduced efficacy and resulted in premature death (FIG. 3B). AAV8 gene therapy treatment extends survival, increases weight, and normalizes serum transaminase levels in adult ASA hypomorphic mice In addition to a prophylactic gene therapy model following neonatal administration, we investigated the potential treatment effects of AAV8 gene therapy in adult ASA hypomorphic mice. We administered AAV8 to 30-day-old adult mice via the retro orbital vein; the standard adult mouse IV administration route via the tail vein could not be used for these adult ASA hypomorphic mice as their average weight was 8.8 g. The first cohort of mice was bled to collect plasma for baseline metabolite and transaminase evaluation prior to IV administration with 6x10 1 GC/kg of vector. Survival was less than expected in this cohort (FIG. 5A), likely due to reduced blood volume as a result of the initial bleed; therefore, baseline values were not determined for additional cohorts. Further cohorts of adolescent ASA hypomorphic mice were either untreated or administered IV with 10 13 GC/kg of vector via the retro-orbital vein. Survival for mice in all vector- administered cohorts was increased compared to untreated ASA hypomophic mice (p < 0.001; FIG. 5A). Mean survival was extended to 9 1 days in the low-dose female group, with the study terminated before the high-dose female and low-dose male cohorts had lost enough mice to determine mean survival (FIG. 5A). We observed a sex difference in survival, with untreated female ASA hypomorphic mice having increased mean survival compared to untreated male hypomorphic mice (p < 0.001). However, following low-dose vector administration, male mice showed increased survival and weight gain compared to female mice, which could possibly be due to an androgen-dependent effect on AAV transduction of the liver as previously described (Davidoff, AM et al. (2003) Sex significantly influences transduction of murine liver by recombinant adeno-associated viral vectors through an androgen-dependent pathway. Blood 102:480-488). We observed a dose-dependent increase in weight gain; however, even mice administered 6xl0 13 GC/kg of vector did not match wild-type body weights (FIG. 5B). All vector-treated groups had reductions in plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) compared to untreated controls (FIG. 5G and FIG. 5F). AAV gene therapy normalizes plasma argininosuccinic acid and citrulline levels in ASA hypomorphic mice AAV gene therapy has the unique potential to restore the urea cycle in hepatocytes without the need for a liver transplant. Therefore, the effect of the AAV8 vector on metabolites associated with ASA and other aspects of the urea cycle was evaluated. Argininosucccinic acid is broken down into arginine and fumarate by the ASL enzyme and is uniquely elevated in ASA patients. Wild-type mice had plasma arginine levels in the range of 45-185 µΜ with a mean of 95 µΜ (FIG. 5C); the ASA hypomorphic mouse had similar levels at day 0 (75 µΜ) (FIG. 5C). However, at day 35 we observed significant differences between wild-type mice and the male and female cohorts treated with 10 13 GC/mouse of AAV8 vector (FIG. 5C). Plasma arginine levels in the hypomorphic mouse model are not significantly lower than in wild-type littermates (FIG. 5C); therefore, we only observed a trend towards arginine elevation in the high-dose group (FIG. 5C). However, we did notice that mice in the lower treatment group had depressed arginine levels. We anticipate that this difference is due to treatment allowing these mice to live longer, yet not fully correcting the disorder. Citrulline is an upstream metabolite that feeds into the urea cycle and arginine synthesis pathway, and is elevated in ASA. In the high- dose female and low-dose male groups, we saw a dramatic reduction of citrulline in plasma, although this did not reach statistical significance compared to wild-type littermates (FIG. 5D). We did not observe an effect of the low dose of vector on plasma citrulline levels in the female mice. Finally, we examined argininosuccinic acid, which is the metabolite that differentiates ASA from other urea acid cycle disorders and is thought to play a role in the unique symptoms of the disease (Brusilow, SW et al. (2001), as cited above). Plasma argininosuccinic acid was corrected to normal levels in the high-dose female group (FIG. 5E), but remained elevated in both the male and female low-dose cohorts. This result indicates a steep dose effect. Upon termination of the study, mice were sacrificed and liver was harvested for determination of ASL protein distribution and vector concentration. Female ASA hypomorphic mice administered with the high dose of vector had a strong presence of ASL protein in the liver (FIG. 6A - 6F). Male and female mice dosed with the low vector dose showed similar levels of staining, possibly because female mice with ASL expression below this level would have not survived to this point of the study. Activity of ASL protein in liver lysate of high-dose females was on average 25% of wild-type levels and statistically significantly higher than untreated hypomorphic mice, which had a mean activity of 3% (FIG. 7B). The activity in mice administered with the low vector dose was not statistically higher than in the untreated hypomorphic mice. Vector genome copy analysis on liver samples demonstrated that a ~4 fold higher level of vector was present in hepatocytes in the high dose cohort compared to the low dose groups (FIG. 7A). These results demonstrate that with sufficient transduction of liver by an ASL gene therapy vector, survival in the ASA hypomorphic mouse model can be greatly extended. This is concurrent with a reduction in liver transaminases indicating reduced hepatic toxicity and the correction of metabolite markers to normal levels. Argininosuccinic aciduria, caused by the loss of ASL activity, is characterized by the dysregulation of the urea acid cycle that inhibits arginine synthesis and nitric oxide production (Erez, A (2013), as cited above). Here, we showed that delivery of the ASL gene into hepatocytes increased survival and weight gain of ASA hypomorphic mice. In some of the previous work on urea acid cycle disorders, vectors have been administered to neonatal OTC-deficient mice with some success increasing survival - if left untreated these mice die within the first day of birth. While not as severe, median survival for ASA hypomorphic mice is 22 days post birth due to a failure to thrive that is often marked by an increase in liver transaminases and elevations in urea acid cycle metabolites. The inventors were able to increase survival in a dose-dependent manner via neonatal administration of vector through the facial vein. Treated mice, however, did not gain weight equivalent to their wild type litter mates and liver histology suggests that dilution of vector genomes occurred with growth of the animal consistent to what has been previously reported (Cunningham, SC et al. (2009) AAV2/8-mediated correction of OTC deficiency is robust in adult but not neonatal Spf(ash) mice. Mol Ther 17:1340-1346; and Wang, L et al. (2012) Hepatic gene transfer in neonatal mice by adeno-associated virus serotype 8 vector. Hum Gene Ther 23:533-539). This is a critically important phenomenon in advancing gene therapy for ASA infants, since viable re-administration methods may be necessary. To determine the efficacy of ASA gene therapy in a mature liver, the inventors administered vector via a retro-orbital IV injection to adolescent mice. These mice exhibited both increased survival and weight gain, although only the high dose resulted in survival similar to wild type littermates. Cirrhosis of the liver is one of the hallmarks of ASA, differentiating it from other urea acid cycle disorders, and is a primary reason to undergo liver transplant (Nagamani, SCS et al. (1993) Argininosuccinate Lyase Deficiency. In: Pagon, RA, et al. (eds). GeneReviews(R). University of Washington, Seattle). Liver pathology was not observed in ASA hypomorphic mice, potentially due to affected mice dying before cirrhosis could occur. However, a reduction was observed in transaminase levels in all treated mouse groups at most time points. Furthermore, mice in the high-dose group showed a trend for corrected arginine levels, indicating that AAV gene therapy might correct arginine deficiency. Importantly, this is not corrected sufficiently with liver transplant (Nagamani, SCS et al, as cited above; Erez, A (2013), as cited above). The high-dose group also achieved normalization of plasma argininosuccinic acid. This encouraging result indicates that argininosuccinic acid is being cleared from the liver. Citrulline, a metabolite upstream of ASL, was normalized in both high-dose females and low-dose males. There is evidence that an androgen-dependent mechanism enhances transduction in male mice; however, the inventors did not observe a difference in the vector genome copy number between low dose groups based on sex, suggesting a potential difference in disease severity based on sex (Davidoff, AM et al. (2003), as cited above). Based on the inventor's findings it would seem prudent to consider continuing arginine supplementation after gene therapy, and with early treatment and improved care outcomes it may come to resemble more recent transplant success. In summary, the feasibility of gene therapy for ASA using AAV8 vector technology was demonstrated. Neonatal administration was only able to extend survival compared to untreated ASA hypomorphic mice but not to that of wild type mice, which may be solved with readministration of the gene therapy vector. Adult administration, while able to correct metabolites at the highest dose tested, was less efficacious at a lower dose. EXAMPLE 3- Cas-9-mediated correction of ASA In vivo genome editing of disease-causing mutations is a promising approach for the treatment of genetic disorders. We investigated whether an AAV-mediated CRISPR/Cas9 system can be used for treatment of ASA. A dual vector AAV8-based system was used to deliver an engineered hASL sequence to neonatal ASA hypomorphic mice. Vector 1 expressed the SaCas9 gene from liver-specific TBG promoter, while vector 2 contained a guide RNA sequence targeting ASL intron 2 expressed from the U6 promoter and an engineered ASL donor sequence AAV8.U6.2G6.sgR.TBG.PI.ASL.co.bGH (guided) or an engineered ASL donor sequence without guide RNA (AAV8.U6.NULLsgR.TBG.PI.ASL.co.bGH; unguided). Wildtype, heterozygous, and ASA hypomorphic mice were injected intravenously on postnatal day 1 (PI) with a mixture of vectors 1 and 2. Mice, regardless of sex, had a substantial increase in survival over untreated ASA- hypomorph control cohorts demonstrating the short-term, consequential efficacy of the hASL gene therapy (FIG. 9A and FIG. 9B). Mice that received donor vector guided to the second intron of the mouse ASL gene had a trend toward increased survival; however, the unguided control group that received a donor vector containing a guide that does not have a complete match to a mouse sequence (unguided) did not have sufficient mortality to reach statistical significance. This was the case for both male and female treated cohorts. Weight gain in male ASA-hypomorph mice was dramatically improved by guided vector treatment with only the first time point not being significantly different from unguided controls (FIG. 9D). In female mice, the effect was more subdued with only four of the twelve time points being significantly different (FIG. 9C). Mice were bled three times over the course of the study for analysis of plasma amino acids (FIG. 10A - FIG. 10D). Results of the analyses indicated that guided vector treated ASA-hypomorph mice initially trended toward lower levels of citrulline and argininosuccinic acid compared to unguided vector treated ASA-hypomorph mice. This was especially pronounced in the male cohort. However, at the final time point citrulline levels converged between the guided and unguided vector treated ASA-hypomorph groups in both male and female cohorts, which may be due to the guided vector merely slowing down disease progression but not fully restoring the urea cycle to normal levels. This reversion to the mean was also observed in ASA levels in the female cohort; however, this did not occur in the male group, another indicator that the treatment was more pronounced in male mice. Arginine analysis (data not shown) did not show any difference between wild type littermate and ASA-hypomorph groups. While arginine is expected to be lower in these mice based on the metabolic pathway inhibited by the disease, patients with ASA often do not have abnormal arginine levels. This seems to be reproduced in our mouse model. At the termination of the study, the lateral left lobe liver tissue was harvested for histology and sections were stained for the hASL protein (representative images shown in the FIG. 11A - 11H and FIG. 12A - 12H). The staining showed a strong difference between the guided vector and unguided vector treated groups with more hASL protein present in the guided vector treated cohorts than their respective unguided vector treated genotype controls. We also observed more hASL staining in the ASA-hypomorph cohort compared to their similarly vector-treated wild type littermates. The increased hASL expression in the ASA-hypomorph mice could be due to a survival advantage among the hASL expressing hepatocytes since one of the elevated metabolites, argininosuccinic acid, is believed to be toxic and our work suggests that expression of hASL will reduce argininosuccinic acid levels. We also observed a difference in hASL expression between the male and female guided vector treated ASA-hypomorph mice indicating that a reason for better outcomes in male cohorts may be the coverage and levels of hASL expression. Cohorts of wild type and heterozygous mice that received one of the two dosing strategies were sacrificed at day 50 for measurement of hASL gene integration (FIG. 13A - 13D). Results from the analysis indicated that the integration rate of the full hASL gene by homologous DNA repair (HDR) was -6% of total reads from isolated liver genomic DNA from mice that received guided vector. This was well above the integration rate of -0.3% in mice treated with the unguided vector, demonstrating that having arms of homology flanking the hASL gene alone is not enough to encourage high rates of integration and that a targeted nuclease is necessary for higher rates of genomic integration. While ITR integration was higher than integration by HDR, due to methodology used, we were not able to determine if the hASL gene was located downstream of the ITR sequence. This would happen if the entire donor plasmid integrated 5' ITR to 3' ITR and would likely increase the amount of hASL gene that integrates into the mouse genome.

All patents, patent applications, and publications, and references to GenBank or another publicly available sequences database cited throughout the disclosure, are expressly incorporated herein by reference in their entirety. Also incorporated by reference are US Patent Application No. 62/545,581 , filed August 15, 2017, US Patent Application No. 62/653,630, filed April 6, 201 8, and the Sequence Listing (filed named 17-8287PCT_ST25.txt) filed herewith. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention are devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims include such embodiments and equivalent variations.

(Sequence Listing Free Text)

The following information is provided for sequences containing free text under numeric identifier <223>. SEQ ID NO: Free text under <223> (containing free text)

<220>

<221> enhancer

<222> (211)..(310)

<223> alpha mic/bik

<220>

<221> enhancer

<222> (317)..(416)

<223> alpha mic/bik

<220>

<221> promoter

<222> (431)..(907)

<223> TBG promoter

<220>

<221> Intron SEQ ID NO: Free text under <223> (containing free text)

<222> (939)..(1071)

<223> SV40 misc intron (Promega)

<220>

<221> CDS

<222> (1092)..(2483)

<223> engineered ASL coding sequence (ALSco)

<220>

<221> polyA signal

<222> (2502)..(27 16)

<223> BGH polyA

<220>

<221> mutation

<222> (2742)..(2742)

<223> T to A mutation SEQ ID NO: Free text under <223> (containing free text)

<220>

<221> misc_feature

<222> (2758)..(2803)

<223> Additional AAV sequences

<220>

<221> repeat_region

<222> (2766)..(2933)

<223> 3'ITR

2 <223> Synthetic Construct

3 <223> Engineered ASL coding sequence

9 <223> synthetic construct

<220>

<221> repeat_region

<222> (1)..(168)

<223> AAV ITR(l) SEQ ID NO: Free text under <223> (containing free text)

<220>

<221> promoter

<222> (187)..(435)

<223> U6 promoter

<220>

<221> misc_RNA

<222> (436)..(456)

<223> 2G6

<220>

<22 1> misc_feature

<222> (46 1)..(533)

<223> gRNA scaffold(hSACas9)

<220>

<22 1> terminator

<222> (533)..(539) SEQ ID NO: Free text under <223> (containing free text)

<223> U6 terminator

<220>

<221> misc_feature

<222> (630)..(1379)

<223> 5'Arm

<220>

<221> enhancer

<222> (1380)..(1479)

<223> alpha mic/bik

<220>

<221> enhancer

<222> (1486)..(1585)

<223> alpha mic/bik

<220> SEQ ID NO: Free text under <223> (containing free text)

<221> promoter

<222> (1600)..(2076)

<223> TBG promoter

<220>

<221> Intron

<222> (2108)..(2240)

<223> SV40 misc intron (Promega)

<220>

<221> CDS

<222> (2261)..(3652)

<223> ASLco

<220>

<221> polyA_signal

<222> (3671)..(3885)

<223> BGH pA SEQ ID NO: Free text under <223> (containing free text)

<220>

<221> misc_feature

<222> (3886)..(4635)

<223> 3'Arm

<220>

<221> repeat_region

<222> (4644)..(4807)

<223> AAV ITR

<220>

<221> CDS

<222> (5570)..(6427)

<223> Amp-R

<220>

<221> misc_feature SEQ ID NO: Free text under <223> (containing free text)

<222> (6601)..(7189)

<223> Origin

10 <223> Synthetic Construct

1 1 <223> Synthetic Construct WHAT IS CLAIMED IS:

1. An engineered nucleic acid sequence of SEQ ID NO: 3, or a nucleic acid sequence at least about 95% identical thereto, encoding a functional human argininosuccinate lyase (ASL).

2. A vector comprising an expression cassette which comprises nucleic acid sequence SEQ ID NO: 3 or a nucleic acid sequence at least about 95% identical thereto which encodes a functional human ASL, and one or more regulatory elements which direct expression of the sequence encoding human ASL.

3. The vector according to claim 2, wherein the vector further comprises a sequence encoding a guide RNA.

4. The vector according to claim 2 or 3, wherein the vector is a non-viral vector or a viral vector.

5. The vector according to claim 4, wherein the non-viral vector is a plasmid.

6. The vector according to claim 4, wherein the viral vector is an adeno- associated virus (AAV), bocavirus, an adenovirus, a lentivirus, or a retrovirus.

7. The vector according to claim any one of claims 2 to 6, wherein the regulatory elements comprise a TBG promoter, a TBG-Sl promoter, an AIAT promoter, a LSP promoter, a TTR promoter, or a CMV promoter.

8. The vector according to any one of claims 2 to 7, wherein the regulatory elements further comprise an enhancer. 9. The vector according to claim 8, wherein the enhancer is an APB enhancer, an ABPS enhancer, an alpha mic/bik enhancer, a TTR enhancer, an en34 enhancer, an ApoE enhancer, a CMV enhancer, or an RSV enhancer.

10. The vector according to any one of claims 2 to 9, wherein the regulatory elements further comprise an intron.

11. The vector according to claim 10, wherein the intron is from CBA, human beta globin, IVS2, SV40, bGH, alpha-globulin, beta-globulin, collagen, ovalbumin, or p53.

12. The vector according to any of claims 2 to 11, wherein the regulatory elements comprise a polyA which is a synthetic polyA or from bovine growth hormone (bGH), human growth hormone (hGH), SV40, rabbit β-globin (RGB), or modified RGB (mRGB).

13. The vector according to any one of claims 2 to 12, wherein the regulatory elements comprise a Kozak sequence.

14. The vector according to any one of claims 1 to 13 for use in a method for treating ASL.

15. Use of a vector according to any one of claims 1 to 14 in the manufacture of a medicament for the treatment of ASL.

16. A recombinant adeno-associated virus (rAAV) useful as a liver-directed therapeutic for argininosuccinic aciduria (ASA), said rAAV comprising an AAV capsid, and a vector genome packaged therein, said vector genome comprising: (a) an AAV 5' inverted terminal repeat (ITR); (b) a nucleic acid sequence of SEQ ID NO:3 or a sequence 95% identical thereto which encodes functional argininosuccinate lyase (ASL), wherein the sequence is operably linked to regulatory elements which direct expression of ASL; (c) regulatory elements which direct expression of ASL; and (d) an AAV 3' ITR.

17. The rAAV according to claim 16, said vector genome further comprising a sequence encoding a guide RNA.

18. The rAAV according to claim 16 or claim 17, said vector further comprising a sequence encoding a CRISPR endonuclease.

19. The rAAV according to claim 18, wherein the CRISPR endonuclease is Cas9.

20. The rAAV according to any one of claims 16 to 19, wherein the capsid is an AAV8 capsid or variant thereof, an AAV9 capsid or variant thereof, an AAVrh. 10 capsid or variant thereof, an AAVrh64Rl capsid or variant thereof, an AAVhu.37 capsid or variant thereof, an AAV3B or variant thereof.

21. The rAAV according to any one of claims 16 to 19, wherein the regulatory elements comprise a promoter which is a TBG promoter, a TBG-Sl promoter, an Al AT promoter, a LSP promoter, a TTR promoter, or a CMV promoter.

22. The rAAV according to any one of claims 16 to 21, wherein the regulatory elements comprise an enhancer.

23. The rAAV according to claim 22, wherein the enhancer is an APB enhancer, an ABPS enhancer, an alpha mic/bik enhancer, a TTR enhancer, an en34 enhancer, an ApoE enhancer, a CMV enhancer, or an RSV enhancer. 24. The rAAV according to any one of claims 16 to 23, wherein the regulatory elements comprise an intron.

25. The rAAV according to claim 24, wherein the intron is from CBA, human beta globin, IVS2, SV40, bGH, alpha-globulin, beta-globulin, collagen, ovalbumin, or p53.

26. The rAAV according to any one of claims 16 to 25, wherein the 5' ITR and/or 3' ITR is from AAV2.

27. The rAAV according to any one of claims 16 to 26, wherein the regulatory elements comprise a polyA which is a synthetic polyA or from bovine growth hormone (bGH), human growth hormone (hGH), SV40, rabbit β-globin (RGB), or modified RGB (mRGB).

28. The rAAV according to any one of claims 16 to 27, wherein the regulatory elements comprise a Kozak sequence.

29. The rAAV according to any one of claims 16 to 28, wherein the vector genome is about 2.9 kilobases to about 5.5 kilobases in size.

30. The rAAV according to claim 16, wherein the vector genome comprises

SEQ ID NO: 1.

31. The rAAV according to any one of claims 16 to 30 for use in a method for treating ASL.

32. Use of a rAAV according to any one of claims 16 to 30 in the manufacture of a medicament for treatment of ASL. 33. An aqueous suspension suitable for administration to treat ASA in a subject in need thereof, said suspension comprising an aqueous suspending liquid and about 1 xlO 12 GC/mL to about 1 xlO 14 GC/mL of a rAAV according to any of claims 16 to 30.

34. The aqueous suspension according to claim 33, wherein the suspension is suitable for intravenous injection.

35. The aqueous suspension according to claim 33 or 34, wherein the suspension further comprises a surfactant, preservative, excipients, and/or buffer dissolved in the aqueous suspending liquid.

36. A gene editing system comprising one or more rAAV vector stocks, said system comprising: (a) at least one nucleic acid sequence encoding a CRISPR endonuclease, and (b) at least one nucleic acid sequence encoding a guide RNA; and (c) at least one nucleic acid sequence encoding a donor template comprising an ASL coding sequence which comprises SEQ ID NO: 3, or a nucleic acid sequence at least about 95% identical thereto, or fragment thereof.

37. The gene editing system according to claim 36, wherein the system comprises at least two AAV stocks, each of which has the same AAV capsid.

38. A method of treating ASA by administrating to a human patient in need the vector according to any one of claims 2 to 13, the rAAV according to any one of claims 16 to 30, the aqueous suspension according to any one of claims 33 to 35, or the gene editing system according to claim 36 or 37.

39. The method according to claim 38, wherein the rAAV is delivered about 1 x 10 10 to about 1 x 10 15 genome copies (GC)/kg. 40. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and an rAAV according to any one of claims 16 to 30.

INTERNATIONAL SEARCH REPORT International application No.

PCT/US 18/46733

Box No. I Nucleotide and/or amino acid sequence(s) (Continuation of item l.c of the first sheet)

1. With regard to any nucleotide and/or amino acid sequence disclosed in the international application, the international search was carried out on the basis of a sequence listing:

a. forming part of the international application as filed: in the form of an Annex C/ST.25 text file. I I on paper or in the form of an image file.

b. furnished together with the international application under PCT Rule \ 3ler. 1(a) for the purposes of international search only in the form of an Annex C/ST 25 text file.

c. I furnished subsequent to the international filing date for the purposes of international search only: I I in the form of an Annex C/ST.25 text file (Rule I3ler. 1(a)). I on paper or in the form of an image file (Rule \T>ter. 1(b) and Administrative Instructions, Section 713).

In addition, in the case that more than one version or copy of a sequence listing has been filed or furnished, the required statements that the information in the subsequent or additional copies is identical to that forming part of the application as filed or does not go beyond the application as filed, as appropriate, were furnished.

3. Additional comments:

Form PCT/ISA/210 (continuation of first sheet (1)) (January 201 5) INTERNATIONAL SEARCH REPORT International application No. PCT/US 18/46733

Box No. II Observations where certain claims were found unsearchable (Continuation of item 2 of first sheet)

This international search report has not been established in respect of certain claims under Article 17(2)(a) for the following reasons:

1. I Claims Nos.: because they relate to subject matter not required to be searched by this Authority, namely:

□ Claims Nos.: because they relate to parts of the international application that do not comply with the prescribed requirements to such an extent that no meaningful international search can be carried out, specifically:

Claims Nos.: 7-15, 20-29, 31-35, 38-40 because they are dependent claims and are not drafted in accordance with the second and third sentences of Rule 6.4(a).

Box No. Ill Observations where unity of invention is lacking (Continuation of item 3 of first sheet)

This International Searching Authority found multiple inventions in this international application, as follows:

As all required additional search fees were timely paid by the applicant, this international search report covers all searchable claims.

As all searchable claims could be searched without effort justifying additional fees, this Authority did not invite payment of additional fees.

□ As only some of the required additional search fees were timely paid by the applicant, this international search report covers only those claims for which fees were paid, specifically claims Nos.:

No required additional search fees were timely paid by the applicant. Consequently, this international search report restricted to the invention first mentioned in the claims; it is covered by claims Nos.:

Remark on Protest I The additional search fees were accompanied by the applicant's protest and, where applicable, the . . payment of a protest fee. I I The additional search fees were accompanied by the applicant's protest but the applicable protest fee was not paid within the time limit specified in the invitation. □ No protest accompanied the payment of additional search fees. Form PCT/lSA/2 10 (continuation of first sheet (2)) (January 201 5) INTERNATIONAL SEARCH REPORT International application No. PCT/US 18/46733

A . CLASSIFICATION O F SUBJECT MATTER IPC(8) - C 12N 9/22, C 12N 15/90 (201 8.01 ) CPC - C 12N 9/22, C07K 14/32, C 12N 15/907, C 12N 9/2497

According to International Patent Classification (IPC) or to both national classification and IPC B. FIELDS SEARCHED Minimum documentation searched (classification system followed by classification symbols) See Search History Document Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched See Search History Document Electronic data base consulted during the international search (name of data base and, where practicable, search terms used) See Search History Document

C. DOCUMENTS CONSIDERED TO BE RELEVANT Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.

US 2013/0259924 A 1 (MODERNA THERAPEUTICS) 3 October 2013 (03.10.2013) Table 6; 1-6, 16-19, 30, 36-37 SEQ ID NO: 1732

WO 2016/176191 A 1 (THE TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA) 3 1-6, 16-19, 30, 36-37 November 2016 (03.1 1.2016) pg 30, In 5-6, pg 1, In 3 1 - pg 2, In 1, pg 2, In 1-3, pg 10, In 6-9, pg 24, In 3-16

US 2003/0198620 A 1 (OZAWA et al.) 23 October 2003 (23.10.2003) Abstract, [0024], [0026], 1-6, 16-19, 30, 36-37 [0043], [0031], [0038], [0066], [0082]

US 2015/0278904 A 1 (Life Technologies Corporation) 1 October 2015 (01 .10.2015) para 1-6, 16-19, 30, 36-37 [0120]; SEQ ID NO: 7993

WO 02/068579 A2 (PE CORPORATION NY) 6 September 2002 (06.09.2002) Claim 4 ; SEQ ID 1-6, 16-19, 30, 36-37 NO: 3157

I Further documents are listed in the continuation of Box C. | | See patent family annex.

* Special categories of cited documents: "T" later document published after the international filing date or priority "A" document defining the general state of the art which is not considered date and not in conflict with the application but cited to understand to be of particular relevance the principle or theory underlying the invention "E" earlier application or patent but published n u after the international "X" document of particular relevance; the claimed invention cannot be filing date considered novel or cannot be considered to involve an inventive "L" document which may throw doubts on priority claim(s) or which is step when the document is taken alone cited to establish the publication date of another citation or other special reason "Y" document of particular relevance; the claimed invention cannot be (as specified) considered to involve an inventive step when the document is "O" document referring to an oral disclosure, use, exhibition or other combined with one or more other such documents, such combination means being obvious to a person skilled in the art "P" document published prior to the international filing date but later than "&" document member of the same patent family the priority date claimed Date of the actual completion o f the international search Date of mailing of the international search report 30 September 2013 2 OCT 2018 Name and mailing address of the ISA/US Authorized officer: Mail Stop PCT, Attn: ISA/US, Commissioner for Patents Lee W. Young P.O. Box 1450, Alexandria, Virginia 22313-1450 Facsimile No. 571-273-8300 Form PCT/ISA/210 (second sheet) (January 2015)