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WO2019036484A1 - Compositions et procédés pour le traitement de l'acidurie argininosuccinique - Google Patents

Compositions et procédés pour le traitement de l'acidurie argininosuccinique Download PDF

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WO2019036484A1
WO2019036484A1 PCT/US2018/046733 US2018046733W WO2019036484A1 WO 2019036484 A1 WO2019036484 A1 WO 2019036484A1 US 2018046733 W US2018046733 W US 2018046733W WO 2019036484 A1 WO2019036484 A1 WO 2019036484A1
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vector
asl
enhancer
raav
aav
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Scott ASHLEY
Jenny Agnes SIDRANE
James M. Wilson
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University of Pennsylvania Penn
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination

Definitions

  • Argininosuccinic aciduria also known as argininosuccinate lyase (ASL) deficiency
  • ASL argininosuccinate lyase
  • 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 US (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
  • Esrez A (2013) Argininosuccinic aciduria: from a monogenic to a complex disorder. Genet Med 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; and Nagamani, SCS et al. (1993) Argininosuccinate Lyase Deficiency. In: Pagon, RA et al.
  • ASA 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. (2011) 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.
  • arginine supplementation includes 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
  • urea acid cycle disorders such as ornithine transcarbamylase deficiency and citrullinemia
  • OTC ornithine transcarbamylase
  • ASA 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).
  • 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.
  • ASA argininosuccinic aciduria
  • ASL functional human argininosuccinate lyase
  • 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.
  • 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.
  • a vector comprising the expression cassette described herein is provided.
  • a recombinant adeno-associated virus 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.
  • the ASL has an amino acid sequence of SEQ ID NO: 2.
  • the coding sequence is SEQ ID NO: 3, or a nucleic acid sequence at least about 95% identical thereto.
  • the vector genome is SEQ ID NO: 1.
  • the ASL protein is mutated to remove acetylation in order to improve enzyme activity.
  • 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.
  • an aqueous suspension suitable for intravenous administration to treat ASA in a subject in need thereof is provided herein.
  • a suspension may contain comprising an aqueous suspending liquid and about 1 xlO 10 GC/mL to about 1 xlO 14 GC/mL of r AAV described herein.
  • composition comprising a pharmaceutically acceptable carrier and the rAAV described herein is provided.
  • a method of treating a subject having ASA with a rAAV comprising an AAV capsid, and a vector genome packaged therein 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.
  • the ASL has an amino acid sequence of SEQ ID NO: 2.
  • the coding sequence is SEQ ID NO: 3, or a nucleic acid sequence at least about 95% identical thereto.
  • the vector genome is SEQ ID NO: 1.
  • the rAAV useful as a liver-directed therapeutic for argininosuccinic aciduria (ASA) is provided.
  • 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.
  • the vector further comprises a CRISPR endonuclease.
  • the CRISPR endonuclease is delivered via a different vector and/or a different route of delivery.
  • aqueous suspension suitable for administration to treat ASA in a subject in need thereof 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.
  • 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.
  • 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
  • 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. 3D provides body weights of female ASA hypomorphic mice that received lxlO 11 GC/mouse of AAV8.TBG.hASLco.
  • 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.
  • 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 6x10 13 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.
  • FIG. 8 provides a plasmid map of pAAV.U6.sg2g6.TBG. PI.hASL.bGH.p4783.gb, described herein (and SEQ ID NO: 9).
  • FIG. 9 A - 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.
  • 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.
  • Argininosuccinic aciduria 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.
  • 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).
  • ASA argininosuccinic aciduria
  • ASL human argininosuccinate lyase
  • 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.
  • 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.
  • 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.
  • AAV8 was administered by retro-orbital injection and resulted in increased survival and body weight, and a correction of metabolites associated with the disease.
  • ASA argininosuccinic aciduria
  • 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.
  • the subject is a male.
  • the subject is a female.
  • the subject of these methods and compositions is a human.
  • the subject of these methods and compositions is an adult.
  • the subject of these methods and compositions is an adolescent.
  • the subject of these methods and compositions is a newborn.
  • the subject of these methods and compositions is an infant.
  • operably linked 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.
  • 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.
  • 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.
  • the ASL enzyme sequence is derived from the same mammal that the composition is intended to treat.
  • 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_000039.2, 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).
  • 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_001024944.1, which is incorporated by reference herein in its entirety) is reproduced in SEQ ID NO: 7.
  • 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_001024946.1, 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.
  • an engineered coding sequence which encodes a functional
  • the amino acid sequence of the functional ASL is that of the wild type ASL protein.
  • 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.
  • 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.
  • the wild type ASL protein has a sequence of SEQ ID NO: 2.
  • 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.
  • 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).
  • the wild type ASL coding sequence is SEQ ID NO: 6.
  • the wild type ASL coding sequence is SEQ ID NO: 7.
  • the wild type ASL coding sequence is SEQ ID NO: 8.
  • 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.
  • the engineered sequence has improved production, transcription, expression or safety in a subject.
  • the engineered sequence has increased efficacy of the resulting therapeutic compositions or treatment.
  • the engineered ASL coding sequence is characterized by improved translation rate as compared to wild type ASL coding sequences.
  • 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 6
  • 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.
  • the engineered nucleic acid sequence encoding ASL is a sequence of SEQ ID NO: 3.
  • 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.
  • 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.
  • the genetic element is a vector.
  • the genetic element is a plasmid.
  • nucleotides 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.
  • nucleic acid sequences 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 FastaTM, a program in GCG Version 6.1. FastaTM 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 FastaTM with its default parameters (a word size of 6 and the NOP AM factor for the scoring matrix) as provided in GCG Version 6.1, herein incorporated by reference.
  • FastaTM provides alignments and percent sequence
  • 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.
  • identity is determined in reference to "aligned” sequences.
  • 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).
  • compositions in the ASL functional protein and ASL coding sequence described herein are intended to be applied to other
  • an expression cassette comprising the ASL coding sequence as described herein.
  • the ASL coding sequence is an engineered sequence as described herein.
  • 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.
  • the expression cassette further comprises regulatory elements which direct expression of the sequence encoding functional ASL.
  • the regulatory elements comprise a promoter.
  • the promoter is a TBG promoter, a TBG-S1 promoter, an Al AT promoter, a LSP promoter, a TTR promoter, or a CMV promoter.
  • the regulatory elements comprise an enhancer.
  • 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.
  • the regulatory elements comprise an intron.
  • the intron is selected from CBA, human beta globin, IVS2, SV40, bGH, alpha-globulin, beta-globulin, collagen, ovalbumin, or p53.
  • the regulatory elements comprise a poly A.
  • the polyA is a synthetic poly A or from bovine growth hormone (bGH), human growth hormone (hGH), SV40, rabbit ⁇ -globin (RGB), or modified RGB (mRGB).
  • the regulatory elements may comprise a WPRE sequence.
  • the regulatory elements comprise a Kozak sequence.
  • RNA Ribonucleic acid
  • expression is used herein in its broadest meaning and comprises the production of RNA, of protein, or of both RNA and protein.
  • expression or “translation” relates in particular to the production of peptides or proteins. Expression may be transient or may be stable.
  • an "expression cassette” refers to a nucleic acid molecule which comprises the ASL coding sequences, promoter, and may include other regulatory elements therefor.
  • the expression cassette may be packaged into the capsid of a viral vector (e.g., a viral particle).
  • a viral vector e.g., a viral particle.
  • 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.
  • the packaging signals are the 5' inverted terminal repeat (ITR) and the 3' ITR.
  • regulatory element 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.
  • regulatory elements comprise but not limited to: promoter; enhancer; transcription factor; transcription terminator; efficient RNA processing signals such as splicing and polyadenylation signals (poly A); 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.
  • WPRE Woodchuck Hepatitis Virus
  • a promoter as a regulatory element.
  • 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.
  • TBG-S1 thyroxine binding globulin
  • TBG-S1 a shortened version
  • TBG-S1 shortened version
  • TTR transthyretin promoter
  • 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
  • CMV cytomegalovirus
  • A the promoter, the first exon and the first intron of chicken beta-actin gene
  • G the splice acceptor of the rabbit beta-globin gene.
  • CMV CMV early enhancer/chicken ⁇ actin
  • the promoter is an AIAT promoter combined with an ApoE enhancer, sometimes referred to as ApoE.AlAT (full).
  • 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).
  • promoters such as viral promoters, constitutive promoters, inducible promoters, regulatable promoters (see, e.g. , WO 2011/126808 and WO 2013/04943), or a promoter responsive to physiologic cues may be used may be utilized in the vectors described herein.
  • the expression control sequences include one or more enhancer.
  • the En34 enhancer is included (34 bp core enhancer from the human apolipoprotein hepatic control region).
  • 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.
  • the al-microglogulin/bikunin precursor alpha mic/bik, ABP
  • the ABPS (shortened version of the 100 bp distal enhancer from the al-microglogulin/bikunin precursor (ABP) to 42 bp) enhancer is included.
  • the ApoE enhancer is included.
  • the cytomegalovirus (CMV) early enhancer in included.
  • the Rous sarcoma virus (RSV) enhancer is included.
  • 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.
  • 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:
  • Another suitable intron includes the hFIX intron (WO
  • simian virus 40 SV40
  • bovine growth hormone bGH
  • alpha-globulin intron the collagen intron
  • ovalbumin intron or the p53 intron.
  • SV40 simian virus 40
  • bGH bovine growth hormone
  • alpha-globulin intron the collagen intron
  • 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.
  • poly A polyadenylation signal
  • 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).
  • compositions in the expression cassette described herein are intended to be applied to other compositions, regiments, aspects, embodiments and methods described across the Specification.
  • 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.
  • a vector comprising the ASL coding sequence as described herein.
  • a vector comprising the expression cassette as described herein.
  • the vector is a non-viral vector.
  • the non- viral vector is a plasmid.
  • 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.
  • AAV adeno-associated virus
  • the adeno-associated virus is referenced herein as an exemplary virus vector.
  • 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,
  • 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.
  • 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.
  • a suitable genetic element e.g. , naked DNA, phage, transposon, cosmid, episome, etc.
  • 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.
  • 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.
  • replication-defective viruses may be adeno-associated viruses (AAV), adenoviruses, lentiviruses (integrating or non- integrating), or another suitable virus source.
  • AAV adeno-associated viruses
  • adenoviruses adenoviruses
  • lentiviruses integrating or non- integrating
  • 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.
  • the transgene is a human ASL sequence, encoding a functional ASL protein.
  • 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.
  • the coding sequence is 95% identical to SEQ ID NO: 3.
  • exogenous 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.
  • 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.
  • 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.
  • 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.
  • the term “host cell” may refer to any target cell in which expression of the transgene is desired.
  • 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.
  • the term “host cell” refers to cultures of cells of various mammalian species for in vitro assessment of the compositions described 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.
  • AAV adeno-associated virus
  • 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.
  • 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.
  • 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.
  • 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.
  • the AAV supplying the capsid is AAV8 or variant thereof.
  • the AAV supplying the capsid is AAVrh.10 or variant thereof.
  • 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.
  • This clade further includes modified rh. 2;
  • modified rh. 58; and modified rh.64 See, WO 2005/033321, which is incorporated herein by reference.
  • any of a number of rAAV vectors with liver tropism can be used.
  • the rAAV vector has a tropism for kidney.
  • 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.
  • 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.
  • the AAV capsid shares at least 95% identity with an AAV capsid.
  • the comparison may be made over any of the variable proteins (e.g., vpl, vp2, or vp3).
  • the AAV capsid shares at least 95% identity with the AAV8 vp3.
  • 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.
  • AAV may be isolated, engineered, or obtained from academic, commercial, or public sources (e.g. , the American Type Culture Collection, Manassas, VA).
  • 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.
  • 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.
  • 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).
  • 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.
  • a "vector genome” refers to the nucleic acid sequence packaged inside the rAAV capsid which forms a viral particle.
  • a nucleic acid sequence contains AAV inverted terminal repeat sequences (ITRs).
  • ITRs AAV inverted terminal repeat sequences
  • 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.
  • ITRs from a different source AAV other than AAV2 may be selected. Further, other ITRs may be used.
  • the vector genome contains regulatory sequences which direct expression of the gene of interest.
  • an adeno-associated viral vector 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.
  • the ITRs are from an AAV different than that supplying a capsid.
  • the ITR sequences from AAV2, or the deleted version thereof (AITR), which may be used for convenience and to accelerate regulatory approval.
  • 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.
  • AAV vector genome comprises an AAV 5' ITR, the ASL coding sequences and any regulatory sequences, and an AAV 3' ITR.
  • 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.
  • 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.
  • a recombinant adeno-associated virus 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.
  • ITR AAV 5' inverted terminal repeat
  • ASL coding sequence encoding argininosuccinate lyase
  • 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.
  • the recombinant AAV vector (rAAV) used for delivering an ASL coding sequence has a tropism for the liver (e.g. , an rAAV bearing an AAV8 capsid), and/or the ASL transgene is controlled by liver-specific expression control elements.
  • the expression control elements include one or more of the following: an enhancer; a promoter; an intron; an optional WPRE; and a polyA signal.
  • a construct which is a vector (e.g., a plasmid) useful for generating viral vectors.
  • the AAV 5' ITR is an AAV2 ITR.
  • the AAV 3 'ITR is an AAV2 ITR.
  • the rAAV comprises an AAV capsid as described herein.
  • the rAAV comprises an AAV8 capsid.
  • 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.
  • the engineered sequences described herein are useful in a genome editing system, such as the Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR) system.
  • 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).
  • 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.
  • 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.
  • one or more corrections may be made to a target gene (e.g. , ASL) using the system gene editing system described herein.
  • 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.
  • a vector includes a full-length sequence that can replace the defective gene (e.g., ASL).
  • 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.
  • a single exon may be inserted upstream of the defective exon.
  • gene deletion or insertion can be corrected.
  • the target gene or gene to be replaced or corrected is ASL and the encoding CRISPR system provides an ASL encoding sequence.
  • 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.
  • dual vector system 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).
  • 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)
  • 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
  • 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.
  • sgRNA 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.
  • 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.
  • these bacterial codons are optimized for expression in humans, e.g. using any of a variety of known human codon optimizing algorithms.
  • these sequences may be produced synthetically, either in full or in part.
  • 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.
  • 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.
  • SpCas9 5'-NGG
  • SaCas9 5 '-N GRRT
  • N any nucleotide
  • R adenine or guanine
  • 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.
  • 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.
  • 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).
  • a liver-specific promoter e.g. , TBG
  • compositions in the vector described herein are intended to be applied to other compositions, regiments, aspects, embodiments and methods described across the Specification.
  • an aqueous suspension suitable for administration to treat ASA in a subject in need thereof 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.
  • a therapeutically effective amount of said vector is included in the suspension.
  • the suspension further comprises a surfactant, preservative, excipients, and/or buffer dissolved in the aqueous suspending liquid.
  • the buffer is PBS.
  • suitable solutions 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
  • the formulation contains a poloxamer.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • one suitable carrier includes saline, which may be formulated with a variety of buffering solutions (e.g. , phosphate buffered saline).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a “therapeutically effective amount” of the ASL is delivered as described herein to achieve a desired result or to reach a therapeutic goal.
  • the desired result is defined herein, e.g. Section V Methods of the
  • 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.
  • 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.
  • 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 ⁇ , 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.
  • two or more different AAV may be delivered [see, e.g. , WO 2011/126808 and WO 2013/049493].
  • multiple viruses may contain different replication-defective viruses (e.g. , AAV, adenovirus, and/or lentivirus).
  • delivery may be mediated by non-viral constructs, e.g.
  • Non-viral ASL delivery constructs may be administered by the routes described previously.
  • the aqueous suspension or pharmaceutical compositions is suitable for use in human subjects and is administered intravenously.
  • the aqueous suspension or pharmaceutical compositions is delivered via a peripheral vein by bolus injection.
  • 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.
  • GC genome copy
  • 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 10 9 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.
  • the concentration of replication-defective virus in the formulation is about 1.0 x 10 9 GC, about 5.0 x 10 9 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.
  • 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.
  • a desired result may also include improving vascular endothelial function, improving liver function, reducing
  • 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.
  • nitrogen scavenging therapy e.g., sodium benzoate, sodium phenylbutyrate, and glycerol triphenylbutyrate
  • 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.
  • 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 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.
  • secondary therapy refers to the therapy that could be combined with the gene therapy described herein for the treatment of ASA.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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, two, three or more re-administrations are permitted. Such re- administration may be with the same type of vector, or a different vector.
  • 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.
  • two or more different AAV may be delivered, or multiple viruses [see, e.g., WO20 2011/126808 and WO 2013/049493].
  • 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.
  • the subject is delivered a therapeutically effective amount of the vectors described herein.
  • the dosage of the vector is about lxl 0 9 genome copies (GC)/kg body weight to about lxl 0 14 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.
  • GC genome copies
  • the dose of the vector administered to a patient is at least about 1.0 x 10 9 GC/kg, about 1.5 x 10 9 GC/kg, about 2.0 x 10 9 GC/kg, about 2.5 x 10 9 GC/kg, about 3.0 x 10 9 GC/kg, about 3.5 x 10 9 GC/kg, about 4.0 x 10 9 GC/kg, about 4.5 x 10 9 GC/kg, about 5.0 x 10 9 GC/kg, about 5.5 x 10 9 GC/kg, about 6.0 x 10 9 GC/kg, about 6.5 x 10 9 GC/kg, about 7.0 x 10 9 GC/kg, about 7.5 x 10 9 GC/kg, about 8.0 x 10 9 GC/kg, about 8.5 x 10 9 GC/kg, about 9.0 x 10 9 GC/kg, about 9.5 x 10 9 GC/kg, about 1.0 x 10 10 GC/kg, about 1.5 x 10 10 GC/kg, about 2.0 x 10
  • 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 10 9 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.
  • 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.
  • the compositions are formulated to contain at least lxl 0 10 , 2x10 10 , 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.
  • compositions are formulated to contain at least lxlO 11 , 2xlO n , 3xl0 n , 4xlO n , 5xl0 n , 6xlO n , 7xlO n , 8xl0 n , or 9xlO n GC per dose including all integers or fractional amounts within the range.
  • compositions are formulated to contain at least lxl 0 12 , 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.
  • compositions are formulated to contain at least lxl 0 13 , 2x10 13 , 3x10 13 , 4x10 13 , 5x10 13 , 6x10 13 , 7x10 13 , 8x10 13 , or 9x10 13 GC per dose including all integers or fractional amounts within the range.
  • 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.
  • compositions are formulated to contain at least lxlO 15 , 2xl0 15 , 3xl0 15 , 4xl0 15 , 5xl0 15 , 6x10 15 , 7x10 15 , 8x10 15 , or 9x10 15 GC per dose including all integers or fractional amounts within the range.
  • the dose can range from lxl0 10 to about lxlO 12 GC per dose including all integers or fractional amounts within the range.
  • 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.
  • the vector is a rAAV vector as described herein.
  • 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.
  • 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.
  • 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.
  • neonatal therapy is desirably followed by re-administration at about 3 months of age, about 6 months, about 9 months, or about 12 months.
  • re-administration is permitted.
  • 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.
  • 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
  • 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.
  • a second treatment may utilize rhlO.
  • a subsequent treatment may utilize AAV8.
  • Still other combinations of AAV caspids may be selected by one skilled in the art.
  • 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.
  • 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.
  • AAV adeno-associated virus
  • such a method is designed for treating, retarding or halting progression of ASA in a mammalian subject.
  • methods that include administration of arginine or
  • L-arginine or pharmaceutical compositions comprising arginine or L-arginine to a subject may be used in combination with an rAAV therapy provided herein.
  • the method includes administering a sustained release formulation of L-arginine or a biochemical equivalent.
  • the present invention includes administering arginine supplementation to subject that is being treated for ASA (i.e., co-therapy).
  • 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.
  • 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.
  • arginine supplementation is reduced in frequency and/or dose prior to delivery of gene therapy.
  • 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/m 2 , or about 100 mg/kg/day to about 500 mg/kg/day.
  • an arginine supplement is delivered at a dose of about 1000 mg/day to about 35,000 mg/day for an adult.
  • 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 may be provided to a subject via the same or different routes of administration.
  • urea production rate a dosage of a composition described in this specification
  • 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.
  • 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.
  • disease markers or metabolites e.g. citrulline or argininosuccinic aciduria
  • 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.
  • the rAAV is delivered about 1 x 10 10 to about 1 x 10 15 genome copies (GC)/kg body weight.
  • the subject is human.
  • the rAAV is administered at more than one times.
  • the rAAV is administered days, weeks, months or years apart.
  • 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.
  • 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.
  • 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.
  • AAV8 vectors expressing engineered human argininosuccinate lyase were designed under the control of a thyroxine-binding globulin (TBG) promoter with a bovine growth hormone (BGH) poly (A) signal sequence.
  • TCG thyroxine-binding globulin
  • BGH bovine growth hormone
  • 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.).
  • gRNAs Guide RNAs 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.
  • gRNAs Guide RNAs
  • 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).
  • H2.35 cells (ATCC) were maintained in DMEM medium supplemented with 10% FBS and cultured at 32 °C with 5% CO2.
  • plasmids were transfected into H2.35 cells using Lipofectamine 2000 per manufacturer's recommendations. Transfected cells were under puromycin (0.75 ⁇ g m 1 ) selection for 2 days to enrich transfected cells.
  • 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.
  • mice Heterozygous mice were set up in timed matings.
  • 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.
  • AAV8.U6.Null.sgR.TBG.PI.ASL.co.bGH (unguided) mixed with 3xlO n 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.
  • ASA hypomorphic mice 4-5 weeks of age were administered IV with
  • mice 6x10 13 or lxl 0 13 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.
  • Genomic DNA was extracted from mouse liver using the Qiagen, QIAamp DNA Mini Kit (Gaithersburg, MD). Briefly, 3 ⁇ g of gDNA is digested with 20 units of Dral or, Blpl for 2 hours at 37°C, enzymes were inactivated according to the
  • 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 20 ⁇ 1 of 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 ⁇ of EB. We used a 1 : 100 dilution of the 1st PCR product and used 1.5 ⁇ 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 15 ⁇ 1 of 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 ⁇ . 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. Thermo Fisher Scientific, Waltham, MA). DNA was purified with AMPure XP
  • concentration of the final pool was measured using Qubit (Thermo Fisher, Waltham, MA), dilute for loading.
  • 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.
  • 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.
  • 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: the journal 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.
  • Liver (25-30 mg) was added to 200 ⁇ 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.
  • Lysate (2 ⁇ ) was added to 48 ⁇ 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 ⁇ of reaction sample mixture.
  • 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
  • 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 0 10 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).
  • mice 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 trans gene 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.
  • AAV8 gene therapy treatment extends survival, increases weight, and normalizes serum transaminase levels in adult ASA hypomorphic mice
  • mice we investigated the potential treatment effects of AAV8 gene therapy in adult ASA hypomorphic mice.
  • AAV8 was 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.
  • 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 91 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).
  • mice 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).
  • ALT plasma alanine aminotransferase
  • AST aspartate aminotransferase
  • 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 ⁇ with a mean of 95 ⁇ (FIG. 5C); the ASA hypomorphic mouse had similar levels at day 0 (75 ⁇ ) (FIG. 5C).
  • the ASA hypomorphic mouse had similar levels at day 0 (75 ⁇ ) (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).
  • 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.
  • mice 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.
  • 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).
  • delivery of the ASL gene into hepatocytes increased survival and weight gain of ASA hypomorphic mice.
  • 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.
  • 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
  • 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.
  • 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
  • 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
  • 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.
  • 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.
  • the lateral left lobe liver tissue was harvested for histology and sections were stained for the hASL protein (representative images shown in the FIG. 11 A - 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.
  • 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.
  • 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.

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Abstract

L'invention concerne une séquence modifiée d'acide nucléique codant pour une protéine d'argininosuccinate lyase (ASL) fonctionnelle, qui est utile pour l'édition et/ou la distribution de gènes par l'intermédiaire d'un vecteur viral. L'invention concerne un vecteur de virus adéno-associé recombinant (rAAV) qui présente une capside d'AAV et une cassette d'expression encapsulée en son sein. La cassette d'expression comprend les séquences d'acide nucléique modifiées codant pour la protéine ASL fonctionnelle et des éléments régulateurs qui dirigent l'expression de l'ASL dans une cellule hôte. L'invention concerne également des compositions contenant cette molécule d'acide nucléique et/ou le rAAV. L'invention concerne également un vecteur ASL et des procédés d'utilisation de celui-ci pour le traitement de l'acidurie argininosuccinique (ASA) chez un patient.
PCT/US2018/046733 2017-08-15 2018-08-14 Compositions et procédés pour le traitement de l'acidurie argininosuccinique Ceased WO2019036484A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022165313A1 (fr) 2021-02-01 2022-08-04 Regenxbio Inc. Thérapie génique de céroïdes-lipofuscinoses neuronales
WO2024038287A1 (fr) * 2022-08-19 2024-02-22 Ucl Business Ltd Thérapie génique pour le traitement d'une déficience en argininosuccinate lyase

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WO2002068579A2 (fr) * 2001-01-10 2002-09-06 Pe Corporation (Ny) Kits tels que des dosages d'acides nucleiques comprenant une majorite d'exons ou de transcrits humains, destines a detecter l'expression et pouvant avoir d'autres applications
US20030198620A1 (en) * 2002-04-16 2003-10-23 Keiya Ozawa Method of treating amino acid metabolic disorders using recombinant adeno-associated virus virions
US20130259924A1 (en) * 2012-04-02 2013-10-03 modeRNA Therapeutics Modified polynucleotides for the production of biologics and proteins associated with human disease
US20150278904A1 (en) * 2004-07-14 2015-10-01 Life Technologies Corporation Collections of Matched Biological Reagents and Methods for Identifying Matched Reagents
WO2016176191A1 (fr) * 2015-04-27 2016-11-03 The Trustees Of The University Of Pennsylvania Système de vecteur aav double pour la correction médiée par crispr/cas9 d'une maladie humaine

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Publication number Priority date Publication date Assignee Title
WO2002068579A2 (fr) * 2001-01-10 2002-09-06 Pe Corporation (Ny) Kits tels que des dosages d'acides nucleiques comprenant une majorite d'exons ou de transcrits humains, destines a detecter l'expression et pouvant avoir d'autres applications
US20030198620A1 (en) * 2002-04-16 2003-10-23 Keiya Ozawa Method of treating amino acid metabolic disorders using recombinant adeno-associated virus virions
US20150278904A1 (en) * 2004-07-14 2015-10-01 Life Technologies Corporation Collections of Matched Biological Reagents and Methods for Identifying Matched Reagents
US20130259924A1 (en) * 2012-04-02 2013-10-03 modeRNA Therapeutics Modified polynucleotides for the production of biologics and proteins associated with human disease
WO2016176191A1 (fr) * 2015-04-27 2016-11-03 The Trustees Of The University Of Pennsylvania Système de vecteur aav double pour la correction médiée par crispr/cas9 d'une maladie humaine

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022165313A1 (fr) 2021-02-01 2022-08-04 Regenxbio Inc. Thérapie génique de céroïdes-lipofuscinoses neuronales
WO2024038287A1 (fr) * 2022-08-19 2024-02-22 Ucl Business Ltd Thérapie génique pour le traitement d'une déficience en argininosuccinate lyase

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