Treatment of ornithine transcarbamylase (OTC) deficiency

Abstract

A dual vector system for treating ornithine transcarbamylase deficiency is provided. The system includes (a) a gene-edited AAV, the gene-edited AAV comprising a first AAV rh79 capsid and a first vector genome comprising a 5'ITR, a sequence encoding a meganuclease, the sequence targeting PCSK9 under the control of a regulatory sequence directing expression of the meganuclease in a target cell comprising a PCSK9 gene, and a 3'ITR; and (b) a donor AAV vector, the donor AAV vector comprising a second AAV capsid and a second AAV rh79 vector genome comprising a 5'ITR, a 5' homologous recombination (HDR) arm, a transgene encoding ornithine transcarbamylase (OTC) and a regulatory sequence directing expression of the transgene in a target cell, a 3'HDR arm, and a 3'ITR.

Description

[Background technology]

[0001] Ornithine transcarbamylase (OTC) deficiency (OTCD) is an X-linked urea cycle disorder associated with high mortality. Adeno-associated virus (AAV) neonatal gene therapy is a promising treatment for late-onset OTC deficiency, but it may only provide short-term therapeutic benefit because unintegrated genome is lost during hepatocyte proliferation.

[0002] Meganucleases generate double-strand breaks (DSBs) in chromosomes, leading to DNA repair: in the presence of donor DNA, homology-directed repair (HDR) occurs, replacing the genetic information of the chromosome with new information from the donor gene.

[0003] Safe harbor sites (SHSs) are genomic loci where genes or other genetic elements can be safely inserted and expressed. These SHSs are important for effective human disease gene therapy, for investigating gene structure, function and regulation, and for labeling and tracking cells.

[0004] Nuclease-mediated site-specific integration of a transgene cassette in the genomic safe harbor would provide long-term therapeutic benefit to patients with OTC deficiency.

[0005] What is needed are improved compositions and methods for the treatment of OTC. Summary of the Invention

[0006] Provided herein are compositions, methods, systems, and kits for the treatment of OTC in a subject in need thereof that allow for knockdown or ablation of the native PCSK9 gene, and insertion and / or expression of an exogenous OTC transgene at the PCSK9 locus.

[0007] In one aspect, a dual vector system for treating ornithine transcarbamylase deficiency is provided. The system includes (a) a gene-edited AAV, the gene-edited AAV comprising a first AAV capsid and a first vector genome comprising a 5'ITR, a sequence encoding a meganuclease, the sequence targeting PCSK9 under the control of a regulatory sequence directing the expression of the meganuclease in a target cell comprising a PCSK9 gene, and a 3'ITR, and (b) a donor AAV vector, the donor AAV vector comprising a second AAV capsid and a second vector genome comprising a 5'ITR, a 5' homologous recombination (HDR) arm, a transgene encoding ornithine transcarbamylase (OTC) and a regulatory sequence directing the expression of the transgene in a target cell, a 3'HDR arm, and a 3'ITR. In a particular embodiment, the meganuclease is an ARCUS meganuclease having the sequence of SEQ ID NO: 3. In certain embodiments, the sequence encoding the meganuclease comprises nucleotides (nt) 1089 to 2183 of SEQ ID NO: 2, or a sequence at least 90% identical to nucleotides (nt) 1089 to 2183 of SEQ ID NO: 2. In certain embodiments, the transgene encoding the OTC comprises SEQ ID NO: 5, or a sequence at least 90% identical to SEQ ID NO: 5. In certain embodiments, the first and second AAV capsids are AAVrh79 capsids of SEQ ID NO: 16.

[0008] In another aspect, a method for treating OTC deficiency in a subject in need thereof is provided, comprising administering to a subject with OTC (a) a gene-edited AAV comprising a first AAV capsid, a 5'ITR, a sequence encoding a meganuclease, the sequence being under the control of regulatory sequences directing expression of the meganuclease in a target cell comprising the PCSK9 gene; The method includes co-administering a gene-edited AAV comprising: (a) a first vector genome comprising a CSK9 targeting sequence, and a 3'ITR; and (b) a donor AAV vector comprising a second AAV capsid, and a second vector genome comprising a 5'ITR, a 5' homologous recombination (HDR) arm, a transgene encoding ornithine transcarbamylase (OTC) and a regulatory sequence directing expression of the transgene in a target cell, a 3'HDR arm, and a 3'ITR. In certain embodiments, i) the first vector genome comprises nt 211-2964 of SEQ ID NO:2, or a sequence that shares at least 90% identity with nt 211-2964 of SEQ ID NO:2, and ii) the second vector genome comprises nt 178-3281 of SEQ ID NO:6, or a sequence that shares at least 90% identity with nt 178-3281 of SEQ ID NO:6.

[0009] Other aspects and advantages of the present invention will become apparent from the following detailed description of the invention. [Brief description of the drawings]

[0010] [Figure 1A] 1 shows a timeline of studies involving hOTC minigene knock-in at the PCSK9 locus with ARCUS2. [Figure 1B] Shown is the study design for groups (G) 1 to 7. Animals 21-111, 21-122, and 21-113 were positive for AAV-binding antibodies (BAbs) prior to dosing. [Diagram 2] Figure 1 shows a schematic diagram of the dual AAV vector system for ARCUS2-mediated gene correction, where the AAV donor vector contains the hOTC donor template sequence used in the study shown in Figure 1A-1B. Different HDR arms are used as indicated. [Diagram 3] 1A-2 are diagrams showing experimental results of the experiments described in Figures 1A-2, with details provided in Figures 4A-4K. [Figure 4A] 1A-2 show the results of the experiment described in Figures 1A-2. PCSK9 levels, shown as a percentage of day 0 for each group, are shown. [Figure 4B] 1A-2 show the results of the experiment described in. ALT levels, shown as U / L, for each group are shown. Liver biopsies were taken at the indicated time points and dual in situ hybridization (ISH) was performed using specific probes to detect hOTC and ARCUS. [Figure 4C] 1A-2 show the results of the experiment described in Figure 1. Transduction efficiency of the OTC transgene, quantified by ISH and plotted as the percentage of transduced hepatocytes. [Figure 4D] 1A-2 show the results of the experiment described in Figures 1A-2, showing the transduction efficiency of the OTC transgene as quantified by IF. [Figure 4E] 1A to 2 show the results of the experiment. The body weights of NHPs are shown. [Figure 4F] 1A-2 show the results of the experiment described in Figures 1A-2, showing GC of the vector in the liver by quantitative PCR analysis. [Figure 4G] 1A-2 show the results of the experiment described above, showing the expression of hOTC and nuclease in macaque liver, measured by quantitative PCR on total RNA isolated from liver biopsy samples followed by reverse transcription, and expressed as relative expression levels normalized by GAPDH levels. [Figure 4H] 1A-2 show the results of the experiment described in FIG. 1A-2, indel analysis of rhPCSK9 target loci performed by amplicon-seq at the indicated time points. [Figure 4I]

[0023] Figures 1A-2 show the results of the experiment described above, showing on-target indels in the indicated tissues. The only non-liver tissue with indels was the pancreas. [Figure 4J] 1A-2 show the results of the experiment described above, comparing LFTs (IU / mL) of neonatal and infant NHPs treated with 4×10 13 GC / kg with adults treated with AAV.Arcus alone. [Figure 4K]1A-2 show the results of the experiment described in. OTC enzyme activity staining at necropsy at 1 year in some group 2 and group 3 animals. Abbreviations: GAPDH: glyceraldehyde-3-phosphate dehydrogenase, GC: genomic copy, hOTC: human ornithine transcarbamylase, OT: off-target, PCR: polymerase chain reaction, rhPCSK9: proprotein convertase subtilisin / kexin type 9 (rhesus gene), RNA: ribonucleic acid. [Figure 5A] 1 shows a schematic diagram of the PCSK9-hE7-KI mouse model. 2 shows a schematic diagram of mouse pcsk9 exon 7 replaced with human pcsk9 exon 7 (hE7 contains the ARCUS targeting sequence). [Figure 5B] Schematic diagram of PCSK9-hE7-KI mouse model. Schematic diagram of crossing PCSK9-hE7-KI mouse model with other disease mouse models such as OTC spfash, KI-spfash model. PCSK9-hE7-KI knock-in mouse model was first generated by replacing the region including exon 7 of mouse Pcsk9 gene with the region of human PCSK9 gene containing exon 7. PCSK9-hE7-KI mice were then crossed with sparse fur ash (spfash) mice that show 20-fold reduction in OTC expression due to a G to A point mutation at the splice donor site at the end of exon 4 of Otc gene. Mice from this cross were named PCSK9-hE7-KI.spfash mice and utilized as described herein. Abbreviations: bp: base pair, E6: exon 6, E7: exon 7, E8: exon 8, HDR: homology-dependent recombination, PCSK9: proprotein convertase subtilisin / kexin type 9 (gene, human), Pcsk9: proprotein convertase subtilisin / kexin type 9 (gene, mouse). [Figure 5C] 1 shows the sequence of a portion of the swapped human exon 7 region and adjacent intron sequences in the mouse Pcsk9 locus (SEQ ID NO: 17). [Figure 6]1 shows a sequence alignment of 265 bp sequences representing human PCSK9 sequence, mouse PCSK9 (mPCSK9) and rhesus monkey PCSK9 (rhPCSK9) of the pcsk9-hE7 knock-in allele. [Figure 7] Schematic diagram of donor construct for dual AAV vector system for ARCUS2-mediated gene correction. The AAV donor vector contains hOTC donor template sequence. Homology of HDR arms in the construct with target regions of knock-in mouse model (FIG. 5), NHP, and human is shown. [Figure 8A] FIG. 1 shows a timeline of studies involving hOTC minigene knock-in at the PCSK9 locus with ARCUS2, performed in PCSK9-hE7-KI.spf-ash pups (partial OTC deficiency model). [Figure 8B] Figure 8A shows the vector and dosage each group received for the study. [Figure 9A] 8A-8B show the results of a study of mice shown in Figures 8A-8B that were treated with the vectors shown in Figure 7 or not treated (KI WT) and placed on a high protein (HP) diet for 10 days. Survival probability is shown. [Figure 9B] 8A-8B show the results of a study of mice shown in Figures 8A-8B that were treated with the vectors shown in Figure 7 or not treated (KI WT) and fed a high protein (HP) diet for 10 days. Body weight is shown as a percentage of body weight before introduction of the HP diet. [Figure 9C] 8A-8B show the results of a study of mice treated with the vectors shown in FIG 7 or not treated (KI WT) and fed a high protein (HP) diet for 10 days. Plasma NH3 levels on day 10 of the HP diet are shown. [Figure 9D] 8A-8B show the results of a study of mice shown in Figures 8A-8B that were treated with the vectors shown in Figure 7 or not treated (KI WT) and fed a high protein (HP) diet for 10 days. mPCSK9 protein levels at day 48 are shown. [Figure 9E]8A-8B show the results of a study of mice shown in Figures 8A-8B that were treated with the vectors shown in Figure 7 or not treated (KI WT) and fed a high protein (HP) diet for 10 days. Indels (%) measured by amplicon-seq at day 59 are shown. [Figure 9F] 8A-8B show the results of a study of mice shown in Figures 8A-8B that were either treated with the vectors shown in Figure 7 or not (KI WT) and fed a high protein (HP) diet for 10 days. Vector transduction levels in liver biopsy samples are shown plotted as AAV genome copies (GC) per diploid cell measured on day 59. [Figure 10] Schematic diagram of a two-vector approach for the treatment of OTC deficiency. Both vectors use the clade E capsid, AAVrh79, and the liver-specific TBG promoter. The first vector is a nuclease, ARCUS, and the second vector is a hOTC donor gene cassette flanked by 500 bp arms of homology to PCSK9 exon 7. [Figure 11] Plasmid map of the donor construct for the two-vector approach described in Figure 10. The sequence of the plasmid from ITR to ITR is shown in SEQ ID NO:6. [Figure 12] Figure 11 is a plasmid map of the nuclease construct of the two-vector approach described in Figure 10. The sequence of the plasmid from ITR to ITR is shown in SEQ ID NO:2. [Figure 13] 1 is a table showing exemplary HDR sequences used in the donor constructs provided herein. [Figure 14A] Figure 4 shows the results of amplicon-seq validation of off-target editing for the experiments described in Figures 4A-4K. A list of off-target sites is provided along with their location on the chromosome and the best match to the off-target consensus sequence. [Figure 14B]4A-4K show the results of amplicon-seq validation of off-target editing for the experiments described in FIG. 4A-4K. Graph showing indel percentages for OT1-OT10. Editing at OT1, OT4, and OT5 was significantly higher in ARCUS+ donor animals than in non-nuclease controls. [Figure 15A] 1 shows a timeline of the MED study involving hOTC minigene knock-in at the PCSK9 locus with ARCUS2, performed in PCSK9-hE7-KI.spf-ash pups, as described in Example 7. [Figure 15B] The study design of the study depicted in Figure 15A is shown. [Figure 16] As discussed in Example 9, a partial study design of a 1-year toxicity study conducted in NHPs is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Provided herein are compositions, kits, and methods that provide a stable, long-term therapeutic effect to patients with certain genetic disorders, including liver metabolic disorders. The compositions, kits, and methods utilize a nuclease that targets the PCSK9 locus of a target cell, a donor vector provides a template containing an exogenous product for integration into and expression from the PCSK9 locus, and the inserted nucleic acid sequence does not code for PCSK9, disrupting the expression of endogenous PCSK9 and reducing expression levels.

[0012] In one embodiment, the test sample described herein consists of two vectors, both using a clade E capsid, AAVrh79, and a liver-specific TBG promoter. The first vector is a nuclease, ARCUS, and the second vector is a hOTC donor gene cassette flanked by 500 bp arms of homology to PCSK9 exon 7.

[0013] In Pcsk9-hE7-KI.spf-ash mice, the test sample reduced mPCSK9 levels and improved both weight loss and survival after high protein diet challenge. hOTC was successfully transduced and achieved better indels (%) compared to untreated and GFP control treated mice.

[0014] In neonatal non-human primates, the test materials resulted in 18.6% and 11.9% hOTC transduction as assessed by liver biopsy at day 84, both of which conferred substantial benefit to patients. This is higher than the threshold for OTC expression (approximately 5% of OTC-expressing cells).

[0015] No test article-related findings were observed, and ALT and PCSK9 levels (as a percentage of day 0) remained low and stable for at least 3 months after vector administration.

[0016] In certain embodiments, the test sample consists of two non-replicating recombinant adeno-associated virus (AAV) rh79 vectors: AAVrh79.TBG.M2PCSK9.WPRE.bGH (nuclease vector) and AAVrh79.hHDR.TBG.hOTCco.bGH (donor vector), which are mixed in a ratio determined by genome copies (GC) immediately before administration. According to non-clinical studies, the ratio can be a 1:3 ratio of nuclease vector to donor vector. The test sample, in certain embodiments, is administered as a single dose as an intravenous (IV) infusion, and the dose administered is based on GC / kg subject's body weight.

[0017] PCSK9 Proprotein convertase subtilisin / kexin 9 (PCSK9) is a serine protease that reduces both hepatic and extrahepatic low-density lipoprotein (LDL) receptor (LDLR; 606945) levels and increases plasma LDL cholesterol. PCSK9 is important in regulating plasma cholesterol homeostasis. PCSK9 binds to low-density lipid receptor family members low-density lipoprotein receptor (LDLR), very-low-density lipoprotein receptor (VLDLR), apolipoprotein E receptor (LRP1 / APOER), and apolipoprotein receptor 2 (LRP8 / APOER2) and promotes their degradation in intracellular acidic compartments.

[0018] Although the PCSK9 gene has been targeted for the treatment of cholesterol-related diseases, it is demonstrated herein that the PSCK9 locus is a safe harbor for gene targeting for the insertion of other non-PCSK9 transgenes. Thus, the compositions, kits, and methods provided herein utilize nucleases that target the PCSK9 locus and use donor templates to insert therapeutic transgenes into the targeted PCSK9 locus.

[0019] The compositions, kits, and methods provided herein include a gene editing vector and a donor vector (which provides a therapeutic OTC transgene for expression in a host cell).

[0020] Gene editing components The compositions, kits, and methods provided herein include a gene editing component, which includes a nuclease (or coding sequence, therefore) and a sequence that directs the nuclease to specifically target the native PCSK9 locus on chromosome 1. As used herein, the "target PCSK9 locus" or "PCSK9 locus" is in exon 7 of the PCSK9 coding sequence. Figure 6 provides an alignment of human (h), rhesus (rh), and mouse (m) PCSK9 exon 7 splice sites, as exemplified herein using SaCas9 and a PCSK9-targeting meganuclease (referred to as ARCUS).

[0021] Described herein are compositions, particularly nucleases, that are useful for targeting genes for insertion of transgenes (e.g., nucleases specific for PCSK9). In certain embodiments, the nuclease is a meganuclease that targets PCSK9. Meganucleases are endodeoxyribonucleases that feature a large recognition site (a double-stranded DNA sequence of 12-40 base pairs), e.g., I-SceI. When combined with a nuclease, DNA can be cleaved at a specific location. Restriction enzymes can be introduced into cells for use in gene editing or for in situ genome editing. In certain embodiments, the nuclease targets a 22-base pair recognition sequence (SEQ ID NO: 1-C). PCSK9 is a member of the I-CreI family of homing endonucleases that recognizes and cleaves the nucleotide sequence AAACGTCGTGAGACAGTTTG. See, e.g., WO2009 / 059195. In one embodiment, the nuclease is encoded by the sequence set forth in SEQ ID NO:2 (nt 1089-2183), or a sequence sharing at least 95%, 98%, or 99% identity therewith. In one embodiment, the nuclease protein sequence is the sequence set forth in SEQ ID NO:3. Such nucleases may be referred to herein as ARCUS nucleases. The term "homing endonuclease" is synonymous with the term "meganuclease." See, e.g., WO2018 / 195449, which describes certain PCSK9 meganucleases, the entirety of which is incorporated herein by reference.

[0022] In certain embodiments, the compositions, kits, and methods of the nuclease coding sequence are contained in a gene editing vector. The gene editing vector comprises an expression cassette comprising a nucleic acid sequence encoding a nuclease and a regulatory sequence directing the expression of the nuclease in a target cell comprising the PCSK9 gene. As used herein, a "vector" is a biological or chemical moiety that comprises a nucleic acid sequence and can be introduced into a suitable host cell for the replication or expression of the nucleic acid sequence. The vector comprising the nuclease coding sequence is an adeno-associated virus (AAV) vector.

[0023] As used herein, "expression cassette" refers to a nucleic acid molecule that includes a biologically useful nucleic acid sequence and operably linked regulatory sequences that direct or regulate the transcription, translation, and / or expression of the nucleic acid sequence (e.g., a gene cDNA encoding a protein, enzyme, or other useful gene product, mRNA, etc.) and the gene product. As used herein, "operably linked" sequences include both regulatory sequences adjacent to the nucleic acid sequence and regulatory sequences that act in trans or at a distance to control the sequence. Such regulatory sequences typically include, for example, one or more of a promoter, enhancer, intron, Kozak sequence, polyadenylation sequence, and TATA signal. An expression cassette may contain, among other elements, regulatory sequences upstream (5') of the gene sequence, e.g., one or more of a promoter, enhancer, intron, etc., and enhancer, or regulatory sequences downstream (3') of the gene sequence, e.g., one or more of a 3' untranslated region including a polyadenylation site. In other embodiments, the term "transgene" refers to one or more DNA sequences from an exogenous source that are inserted into a target cell. Typically, such expression cassettes for producing viral vectors include coding sequences for gene products described herein flanked by packaging signals of the viral genome, and other expression control sequences, such as those described herein.

[0024] In addition to the coding sequence of the nuclease, the gene editing vector includes a regulatory sequence that directs the expression of the nuclease in the host cell. The regulatory element includes a promoter, for example, the thyroxine-binding globulin (TBG) promoter, which is a liver-specific promoter. In a particular embodiment, the TBG promoter has the sequence of nucleotides 211 to 907 of SEQ ID NO: 2, which includes an enhancer sequence.

[0025] In addition to a promoter, the gene editing cassette, expression cassette, and / or vector can contain one or more suitable "regulatory elements" or "regulatory sequences," including, but not limited to, enhancers, transcription factors, transcription terminators, efficient RNA processing signals such as splicing signals and polyadenylation signals (polyA), sequences that stabilize cytoplasmic mRNA (e.g., Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE)), sequences that improve translation efficiency (i.e., Kozak consensus sequences), sequences that improve protein stability, and, if necessary, sequences that improve secretion of the encoded product. In certain embodiments, the vector is Suitable enhancers include the bGH polyA, such as that set forth at nucleotides 2750-2964 of SEQ ID NO:2. Suitable enhancers include the α1-microglobulin / bikunin enhancer. Suitable WPREs include those set forth at nucleotides 2202-2743 of SEQ ID NO:2. These control or regulatory sequences are operably linked to the nuclease coding sequence and the transgene coding sequence. In certain embodiments, an SV40 intron is included, such as that set forth at nucleotides 939-1071 of SEQ ID NO:2.

[0026] In a particular embodiment, the nuclease vector genome comprises the following components: Inverted Terminal Repeat (ITR): The ITRs are identical reverse complement sequences derived from AAV2 (145 base pairs [bp], GenBank: NC_001401) that flank all components of the vector genome. The ITRs function as both an origin of vector DNA replication and a packaging signal for the vector genome when AAV and adenovirus helper functions are provided in trans. Thus, the ITR sequences represent the only cis sequences required for vector genome replication and packaging. Human thyroxine-binding globulin (TBG) promoter: This regulatory element confers tissue-specific transgene expression in the liver (410 bp, GenBank: L13470.1). Coding sequence: The transgene is an engineered meganuclease (ARCUS; 1095 bp, 365 amino acids). It is derived from a variant of the homing endonuclease I-CreI isolated from Chlamydomonas reinhardtii and recognizes and edits the PCSK9 gene with high efficiency and specificity. WPRE (Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element): A cis-acting RNA element (GenBank: MT612432.1) derived from Woodchuck Hepatitis Virus (WHV) is inserted into the 3' untranslated region of the coding sequence upstream of the polyA signal. WPRE is a hepadnavirus-derived sequence that has been used previously as a cis-acting regulatory module in viral gene vectors to achieve sufficient levels of transgene product expression and improve viral titers during production. WPRE is believed to increase transgene product expression by improving transcript termination and enhancing 3'-end transcript processing, thereby increasing the amount of polyadenylated transcript and the size of the polyA tail, resulting in more transgene mRNA available for translation.The WPRE contained in the vector is a mutated version that contains five point mutations in the putative promoter region of the Woodchuck Hepatitis Virus X protein (WHX) protein open reading frame (ORF), together with an additional point mutation (ATG to TTG) in the start codon of the WHX protein ORF. This mutated WPRE (designated mut6) appears to be sufficient to eliminate expression of a truncated WHX protein, based on sensitive flow cytometric analysis of various human cell lines transduced with lentiviruses containing a WPRE mut6-GFP fusion construct (Zanta-Boussif et al., 2009).

[0027] Bovine growth hormone polyA (bGH polyA): The bGH polyA signal (208 bp, GenBank: MT267334) promotes efficient polyadenylation of the transgene mRNA in cis. This element functions as a signal for transcription termination, a specific cleavage event at the 3' of the nascent transcript, and the addition of a long polyadenylation tail.

[0028] In certain embodiments, the gene editing vector further comprises one or more nuclear localization signals (NLS). See, for example, Lu et al. Types of nuclear localization signals and mechanisms of protein import into the nucleus, Cell Commun Signal (May 2021) 19:60. In one embodiment, the vector comprises an NLS set forth at nt 1095-1115 of SEQ ID NO:2.

[0029] Donor Vector The compositions, kits, and methods include a donor vector that provides a coding sequence for an OTC therapeutic transgene. The donor vector includes an expression cassette that includes a nucleic acid sequence that encodes the transgene and a regulatory sequence that directs expression of the transgene in a target cell.

[0030] Compositions, kits and methods for nuclease-mediated site-specific integration of an OTC transgene cassette in the PCSK9 safe harbor of the genome, providing long-term therapeutic benefit to patients with OTC deficiency. Provided is an engineered coding sequence of OTC, herein referred to as hOTCco2 and set forth in SEQ ID NO:4. Provided is a nucleic acid having the sequence of SEQ ID NO:4 or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.9% identity with SEQ ID NO:4. In one embodiment, the nucleic acid shares less than 80%, less than 79%, less than 78%, less than 77%, less than 76%, less than 75%, less than 74%, less than 73%, less than 72%, less than 71%, or less than 70% identity with the native OTC coding sequence set forth in SEQ ID NO:5.

[0031] In some embodiments, the transgene cassette comprises a TBG promoter, a transgene coding sequence, and a polyA sequence.

[0032] In addition to a promoter, the transgene cassette, expression cassette, and / or (editing or donor) vector may include one or more suitable "regulatory elements" or "regulatory sequences" including, but not limited to, enhancers, transcription factors, transcription terminators, efficient RNA processing signals such as splicing and polyadenylation signals (polyA), sequences that stabilize cytoplasmic mRNA, e.g., Woodchuck Hepatitis Virus (WHP) posttranscriptional regulatory element (WPRE), sequences that improve translation efficiency (i.e., Kozak consensus sequence), sequences that improve protein stability, and, if necessary, sequences that improve secretion of the encoded product. Examples of suitable polyA sequences include, for example, SV40, bovine growth hormone (bGH), and TK polyA. Examples of suitable enhancers include, for example, alpha fetoprotein enhancer, TTR minimal promoter / enhancer, LSP (TH binding globulin promoter / alpha1-microglobulin / bikunin enhancer), among others. These control or regulatory sequences are operably linked to the nuclease coding sequence or the transgene coding sequence.

[0033] In a particular embodiment, the donor vector genome comprises: Inverted Terminal Repeat (ITR): The ITR is an identical reverse complement sequence derived from AAV2 (145 bp, GenBank: NC_001401) that flanks all components of the vector genome. The ITR functions as both an origin of vector DNA replication and a packaging signal for the vector genome when AAV and adenovirus helper functions are provided in trans. Thus, the ITR sequence represents the only cis sequence required for vector genome replication and packaging. 5' and 3' homology arms: The homology-dependent recombination arm (also called hHDR) consists of sequences flanking the cleavage site of exon 7 of the endogenous human PCSK9 locus. The homology arms comprise sequences 500 bp upstream (5' homology arm) and 500 bp downstream (3' homology arm) of the ARCUS meganuclease cleavage site in exon 7 of the PCSK9 gene. Human thyroxine-binding globulin (TBG) promoter: This regulatory element confers tissue-specific transgene expression in the liver (434 bp, GenBank: L13470.1). Coding sequence: The transgene is a codon-optimized version of the human ornithine transcarbamylase (OTC) gene (1068 bp, 356 amino acids). Bovine growth hormone polyA (BGH polyA): The bGH polyA signal (215 bp, GenBank: MT267334) promotes efficient polyadenylation of the transgene mRNA in cis. This element regulates transcription. It functions as a signal for termination, a specific cleavage event 3' of the nascent transcript and addition of a long polyadenylated tail.

[0034] In addition to the transgene cassette, the donor vector also contains homologous recombination (HDR) arms at the 5' and 3' sides of the transgene cassette to facilitate homologous recombination of the transgene into the endogenous genome. The homology arms are directed to the target PCSK9 locus and can be of various lengths. In another embodiment, the HDR arms are about 500 bp. The HDR arms ideally share a high level of complementarity with the target PCSK9 locus, although they do not necessarily have to be 100% complementary. In some embodiments, each HDR arm is allowed to have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more mismatches. Suitable HDR arm sequences for targeting PCSK9 exon 7 are shown in FIG. 14 and SEQ ID NOs: 7-12. In one embodiment, the HDR arm sequences are those shown in SEQ ID NOs: 13 and 14.

[0035] AAV viral vectors The gene editing vector and the donor vector are provided as recombinant AAV. "Recombinant AAV" or "rAAV" is a DNAse-resistant viral particle that includes two elements, an AAV capsid, and a vector genome that includes at least a non-AAV coding sequence packaged within the AAV capsid. Unless otherwise specified, this term can be used interchangeably with the phrase "rAAV vector" or "AAV vector". rAAV is a "replication-defective virus" or "viral vector" because it lacks any functional AAV rep gene or functional AAV cap gene and cannot generate progeny. In certain embodiments, the only AAV sequence is the AAV inverted terminal repeat (ITR), which is typically located at the extreme 5' and 3' ends of the vector genome to allow the genes and regulatory sequences located between the ITRs to be packaged within the AAV capsid.

[0036] Adeno-associated virus (AAV) viral vectors are AAV DNase-resistant particles with an AAV protein capsid into which a nucleic acid sequence is packaged for delivery to a target cell. The AAV capsid is composed of 60 capsid protein subunits, VP1, VP2, and VP3, arranged in icosahedral symmetry in a ratio of approximately 1:1:10 to 1:1:20, depending on the AAV selected.

[0037] The expression cassette is located in the vector genome for packaging into the viral capsid. For example, in the case of an AAV vector genome, the expression cassette components are flanked at the extreme 5' and 3' ends by AAV inverted terminal repeat sequences. For example, 5'AAV ITR, expression cassette, 3'AAV ITR.

[0038] The source of AAV capsid for both the donor vector and the nuclease vector is AAVrh79, as described in WO2019 / 169004 published September 6, 2019, which is incorporated herein by reference. In one embodiment, the AAVrh79 capsid comprises a heterogeneous population of AAVrh79 vp1, vp2, and vp3 proteins. In one embodiment, the AAVrh79 capsid is produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of 1-738 of SEQ ID NO: 16. Optionally, a sequence for co-expressing a vp3 protein from a nucleic acid sequence excluding the vp1 unique region (about aa 1-137) or the vp2 unique region (about aa 1-203), a vp1 protein produced from SEQ ID NO: 15, or a vp1 protein produced from a nucleic acid sequence at least 70% identical to SEQ ID NO: 15 that encodes the predicted amino acid sequence of at least about amino acids 138-738 of SEQ ID NO: 16. In other embodiments, an AAVrh79 vp2 protein produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of at least about amino acids 138-738 of SEQ ID NO: 16, or a sequence for co-expressing a vp3 protein from a nucleic acid sequence excluding the vp1 unique region (about aa 1-137) or the vp2 unique region (about aa 1-203), a vp1 protein produced from a nucleic acid sequence at least 70% identical to SEQ ID NO: 15 that encodes the predicted amino acid sequence of at least about amino acids 138-738 of SEQ ID NO: 16, or a vp1 protein produced from a nucleic acid sequence at least 70% identical to SEQ ID NO: 15 that encodes the predicted amino acid sequence of at least about amino acids 138-738 of SEQ ID NO: 16. vp2 protein produced from a sequence that includes at least nucleotides 412-2214 of SEQ ID NO:15, or a nucleic acid sequence that is at least 70% identical to at least nucleotides 412-2214 of SEQ ID NO:15, which encodes the predicted amino acid sequence of at least about amino acids 138-738 of SEQ ID NO:16; AAVrh79 vp3 protein produced by expression from a nucleic acid sequence that encodes the predicted amino acid sequence of at least about amino acids 204-738 of SEQ ID NO:16; vp3 protein produced from a sequence that includes at least nucleotides 610-2214 of SEQ ID NO:15, or a nucleic acid sequence that encodes the predicted amino acid sequence of at least about amino acids 204-738 of SEQ ID NO:16, which is at least 70% identical to at least nucleotides 610-2214 of SEQ ID NO:15.

[0039] In certain embodiments, the AAVrh79 capsid comprises a heterogeneous population of vp1 proteins that are the product of a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO:16, a heterogeneous population of vp2 proteins that are the product of a nucleic acid sequence encoding an amino acid sequence at least about amino acids 138-738 of SEQ ID NO:16, and a heterogeneous population of vp3 proteins that are the product of a nucleic acid sequence encoding at least amino acids 204-738 of SEQ ID NO:16.

[0040] AAVrh79 vp1, vp2, and vp3 proteins include subpopulations with amino acid modifications that include at least two highly deamidated asparagines (N) in the asparagine-glycine pair in SEQ ID NO: 16, and optionally further include subpopulations that include other deamidated amino acids, where the deamidation results in an amino acid change. High levels of deamidation are observed at NG pairs N57, N263, N385, and / or N514, relative to the numbers in SEQ ID NO: 16. Deamidation has been observed at other residues, as shown in the table and examples below. In certain embodiments, AAVrh79 may be deamidated at other residues, for example, typically less than 10%, and / or methylated (e.g., at up to R487) (typically less than 5% at a given residue, more typically less than 1%), isomerized (e.g., at D97) (typically less than 5% at a given residue, more typically less than 1%), phosphorylated (e.g., if present, at about 10 to about 60%, or about 10 to about 30%, or about 20 to about 60% range) (e.g., at one or more of S149, -S153, -S474, -T570, -S665), or oxidation (e.g., at one or more of W248, W307, W307, M405, M437, M473, W480, W480, W505, M526, M544, M561, W621, M637, and / or W697). Optionally, W can be oxidized to kynurenine. [Table 1]

[0041] In certain embodiments, the AAVrh79 capsid is modified at one or more of the positions identified in the table above, in the ranges shown below, as determined using mass spectrometry with trypsin enzyme. In certain embodiments, one or more of the following positions, or glycines following an N, are modified as described herein. Residue numbers are based on the AAVrh79 sequence provided herein. See SEQ ID NO: 16.

[0042] In certain embodiments, the nucleic acid sequence encoding the AAVrh79 vp1 capsid protein is provided in SEQ ID NO: 15. In other embodiments, the nucleic acid sequence is 70% to 99.9% identical to SEQ ID NO: 15. A nucleic acid sequence that is at least about 75% identical, at least 80% identical, at least 85%, at least 90%, at least 95%, at least 97% identical, or at least 99%, or 99.9% identical to SEQ ID NO: 15 may be selected to express the AAVrh79 capsid protein. In certain other embodiments, the nucleic acid sequence is at least about 75% identical, at least 80% identical, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO: 15. However, other nucleic acid sequences that encode the amino acid sequence of SEQ ID NO: 16 may be selected for use in producing rAAV capsids. In certain embodiments, the nucleic acid sequence has at least 70% to 99% identical, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 15, or a sequence that encodes SEQ ID NO: 16. In certain embodiments, the nucleic acid sequence has a sequence at least 70% to 99%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identical to the nucleic acid sequence of SEQ ID NO: 15, or from about nt 412 to about nt 2214 of SEQ ID NO: 15, and encodes the vp2 capsid protein of SEQ ID NO: 16 (about aa 138 to 738). In certain embodiments, the nucleic acid sequence has a sequence at least 70% to 99%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identical to the nucleic acid sequence of SEQ ID NO: 15 from about nt 610 to about nt 2214 of SEQ ID NO: 15, or from about nt 610 to about nt 2214 of SEQ ID NO: 15, and encodes the vp3 capsid protein of SEQ ID NO: 16 (about aa a204~738).

[0043] In certain embodiments, the rAAV79 vector has an AAVrh79 capsid containing a vector genome, the vector genome comprising a nucleic acid molecule comprising an AAV inverted terminal repeat and a non-AAV nucleic acid sequence encoding a product operably linked to a sequence that directs expression of the product. In certain embodiments, the AAVrh79 capsid comprises an AAVrh79 vp1 protein, an AAVrh79 vp2 protein, and an AAVrh79 vp3 protein selected from a vp1 protein produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of 1-738 of SEQ ID NO: 16, a vp1 protein produced from SEQ ID NO: 15, or a vp1 protein produced from a nucleic acid sequence at least 70%-100% identical to SEQ ID NO: 15 encoding the predicted amino acid sequence of 1-738 of SEQ ID NO: 16. a heterologous population of vp3 proteins and a vp2 protein produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of at least about amino acids 138-738 of SEQ ID NO:16, a vp2 protein produced from a sequence comprising at least nucleotides 412-2214 of SEQ ID NO:15, or a vp2 protein produced from a nucleic acid sequence that is at least 70%-100% identical to at least nucleotides 412-2214 of SEQ ID NO:15 that encodes the predicted amino acid sequence of at least about amino acids 138-738 of SEQ ID NO:16. and a heterogeneous population of AAVrh79 vp3 proteins selected from: vp3 proteins produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of at least about amino acids 204-738 of SEQ ID NO:16; vp3 proteins produced from a sequence including at least nucleotides 610-2214 of SEQ ID NO:15; or vp3 proteins produced from a nucleic acid sequence that is at least 70% identical to at least nucleotides 610-2214 of SEQ ID NO:15 encoding the predicted amino acid sequence of at least about amino acids 204-738 of SEQ ID NO:16.In certain embodiments, the rAAVrh79 capsid is characterized by a heterogeneous population of AAVrh79 vp1, vp2, and vp3 proteins that are the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:16; a heterogeneous population of vp2 proteins that are the product of a nucleic acid sequence encoding an amino acid sequence of at least about amino acids 138-738 of SEQ ID NO:16; and a heterogeneous population of vp3 proteins that are the product of a nucleic acid sequence encoding at least amino acids 204-738 of SEQ ID NO:16. In certain embodiments, the AAVrh79 capsid features AAVrh79 vp1, vp2, and vp3 proteins, which include heterogeneous populations for amino acids 1-738 (vp1), 138-738 (vp2), and 204-738 (vp3), respectively, and the heterogeneous populations of AAVrh79 vp1, AAVrh79 vp2, and AAVrh79 vp3 proteins include subpopulations having amino acid modifications comprising at least 50%-100% of two highly deamidated asparagines (N) in asparagine-glycine pairs at at least two positions relative to SEQ ID NO: 16, and optionally further including subpopulations comprising other deamidated amino acids, wherein the deamidation results in an amino acid change. In certain embodiments, the highly deamidated positions are N57, N263, N385, and N514, based on SEQ ID NO: 16 and as determined using mass spectrometry. In certain embodiments, the AAVrh79 capsid protein is individually deamidated at positions N57, 60% to about 100% at N263, 60% to about 100% at N385, and 60% to about 100% at N514, each based on SEQ ID NO: 16 and measured using mass spectrometry. Other suitable techniques for measuring deamidation or other post-translational modifications can also be selected.

[0044] In certain embodiments, the rAAV79 capsid has about 80% to about 85% deamidation at position N57 of SEQ ID NO:16, about 82% to about 88% deamidation at position N263 of SEQ ID NO:16, about 90% to about 96% deamidation at position N385 of SEQ ID NO:3, and / or about 85% to about 90% deamidation at position N514 of SEQ ID NO:16, as determined using mass spectrometry. The capsid protein comprises an AAVrh79 VP1 protein, optionally with further post-translational modifications at other positions. Optionally, there is deamidation at positions N94, N254, N305, N410, N479, Q601, and N653, and deamidation is generally found at these positions in less than 10%, less than 5%, less than 3%, or less than 2% of the population of AAVrh79 VP1, VP2, and VP3 proteins. Optionally, phosphorylation is observed at position S149, based on residues in SEQ ID NO: 16, and in certain embodiments, 0% or less of the capsid proteins have phosphorylation at this position. In certain embodiments, oxidation is observed at positions W248, W307, M437, M473, M480, W505, M637, and / or W697, and in certain embodiments, less than 10% of the capsid protein is oxidized at any one of these positions. Post-translational modifications may be determined using mass spectrometry or another suitable technique.

[0045] The invention also encompasses nucleic acid sequences encoding mutant AAVrh79 in which one or more residues have been altered to reduce deamidation or other modifications as identified herein, and such nucleic acid sequences can be used in the production of mutant rAAVrh79 capsids.

[0046] As used herein, "vector genome" refers to a nucleic acid sequence that is packaged inside the rAAV capsid that forms the viral particle. Such nucleic acid sequences include AAV inverted terminal repeats (ITRs). In the examples herein, the vector genome includes at least, from 5' to 3', the AAV 5'ITR, an expression cassette containing a transgene or coding sequence(s) operably linked to a regulatory sequence that directs its expression, and the AAV 3'ITR. The ITRs are genetic elements involved in genome replication and packaging during vector production and are the only viral cis elements required to generate rAAV. In a preferred embodiment, the ITR sequence from AAV2, or a deleted version thereof (ΔITR), may be used for convenience. In a particular embodiment, the ITRs are those set forth in nucleotides 1-130 and 3052-3181 of SEQ ID NO:2 and nucleotides 1-130 and 3345-3474 of SEQ ID NO:6.

[0047] A shortened version of the 5'ITR (called ΔITR) has been described in which the D sequence and terminal separation sites (trs) are deleted. In certain embodiments, the vector genome comprises a shortened AAV2 ITR of 130 base pairs, where the external "a" element is deleted. The shortened ITR is restored to the wild-type length of 145 base pairs during amplification of the vector DNA using the internal A element as a template. In other embodiments, full-length AAV 5' and 3' ITRs are used. In other embodiments, full-length or engineered ITRs can be selected. ITRs from AAV2, a source AAV different from the capsid, or other than full-length ITRs can be selected. The ITRs are from the same AAV source as the AAV that provides the rep function or trans-complementing AAV during production.

[0048] For use in producing AAV viral vectors (e.g., recombinant (r)AAV), the expression cassette can be carried on any suitable vector, e.g., a plasmid, that is delivered to a packaging host cell. Plasmids useful in the present invention can be engineered to be suitable for replication and packaging in prokaryotic, insect, or mammalian cells in vitro, among others. Suitable transfection techniques and packaging host cells are known and / or can be readily designed by one of skill in the art.

[0049] Methods for generating and isolating AAV suitable for use as a vector are known in the art. See, generally, for example, Grieger & Samulski, 2005, "Adeno-associated virus as a gene therapy vector: Vector development, production and nd clinical applications,” Adv. Biochem. Engin / Biotechnol. 99:119-145; Buning et al., 2008, “Recent developments in adeno-associated viral vector technology,” J. Gene Med. 10:717-733, and the references cited below, each of which is incorporated by reference in its entirety. If the transgene is to be packaged into virions, the ITRs are the only AAV components required in cis in the same construct as the nucleic acid molecule containing the expression cassette. The cap and rep genes can be supplied in trans.

[0050] The term "AAV intermediate" or "AAV vector intermediate" refers to assembled rAAV capsids that lack the desired genomic sequence packaged therein. These may be referred to as "empty" capsids. Such capsids may contain no detectable genomic sequence of the expression cassette or may contain only partially packaged genomic sequence that is insufficient to achieve expression of the gene product. These empty capsids are non-functional for introducing a gene of interest into a host cell.

[0051] The recombinant adeno-associated virus (AAV) described herein can be produced using known techniques. See, for example, WO2003 / 042397, WO2005 / 033321, WO2006 / 110689, US7588772B2. Such methods include culturing a host cell that contains an expression cassette consisting of a nucleic acid sequence encoding an AAV capsid protein, a functional rep gene, at least an AAV inverted terminal repeat (ITR) and a transgene, and sufficient helper functions to allow packaging of the expression cassette into an AAV capsid protein. Methods for generating capsids, coding sequences therefor, and methods for producing rAAV viral vectors have been described. See, for example, Gao, et al, Proc. Natl. Acad. Sci. USA100(10), 6081-6086(2003) and US 2013 / 0045186A1.

[0052] In one embodiment, a producer cell culture is provided that is useful for producing recombinant AAV. Such cell culture contains a vector genome that contains a nucleic acid that expresses an AAV capsid protein in a host cell, a nucleic acid molecule suitable for packaging into an AAV capsid, such as a non-AAV nucleic acid sequence that encodes a gene product operably linked to an AAV ITR and a sequence that directs expression of the product in a host cell, and sufficient AAV rep function and adenovirus helper function to allow packaging of the nucleic acid molecule into a recombinant AAV capsid. In one embodiment, the cell culture is composed of mammalian cells (e.g., human embryonic kidney 293 cells, among others) or insect cells (e.g., baculovirus).

[0053] Optionally, the rep function is provided by an AAV other than the AAV that provides the capsid. For example, rep can be, but is not limited to, AAV1 rep protein, AAV2 rep protein, AAV3 rep protein, AAV4 rep protein, AAV5 rep protein, AAV6 rep protein, AAV7 rep protein, AAV8 rep protein, or rep 78, rep 68, rep 52, rep 40, rep68 / 78, and rep40 / 52, or fragments thereof, or another source. Optionally, the rep and cap sequences are on the same genetic element in the cell culture. There may be a spacer between the rep sequence and the cap gene. Any of these AAV or mutant AAV capsid sequences may be under the control of exogenous regulatory control sequences that direct their expression in the host cell.

[0054] In one embodiment, the cells are produced in a suitable cell culture (e.g., HEK293) cells. Methods for producing gene therapy vectors described herein include gene therapy vectors. The methods include those well known in the art, such as the generation of plasmid DNA used in the production of the method vector, the generation of the vector, and the purification of the vector. In some embodiments, the gene therapy vector is an AAV vector, and the generated plasmids are AAV cis-plasmids encoding the AAV genome and the gene of interest, AAV trans-plasmids containing the AAV rep and cap genes, and adenovirus helper plasmids. The vector production process may include method steps such as initiation of cell culture, passaging of cells, seeding of cells, transfection of cells with plasmid DNA, medium exchange with serum-free medium after transfection, and collection of vector-containing cells and culture medium. The harvested vector-containing cells and culture medium are referred to herein as crude cell harvest. In yet another system, the gene therapy vector is introduced into insect cells by infection with a baculovirus-based vector. For a review of these production systems, see generally, e.g., Zhang et al., 2009, "Adenovirus-adeno-associated virus hybrid for large-scale recombinant adeno-associated virus production," Human Gene Therapy 20:922-929, the contents of each of which are incorporated herein by reference in their entireties. Methods of making and using these and other AAV production systems are also described in the following U.S. patents, the contents of each of which are incorporated by reference in their entirety: 5,139,941; 5,741,683; 6,057,152; 6,204,059; 6,268,213; 6,491,907; 6,660,514; 6,951,753; 7,094,604; 7,172,893; 7,201,898; 7,229,823; and 7,439,065.

[0055] The crude cell harvest may then be subjected to process steps such as concentration of the vector harvest, diafiltration of the vector harvest, microfluidization of the vector harvest, nuclease digestion of the vector harvest, filtration of the microfluidized intermediate, crude purification by chromatography, crude purification by ultracentrifugation, buffer exchange by tangential flow filtration, and / or formulation and filtration to prepare bulk vector.

[0056] A two-step affinity chromatography purification at high salt concentration, followed by anion exchange resin chromatography, is used to purify the vector drug product and remove empty capsids. These methods are described in more detail in International Patent Publication No. 2017 / 160360, which is incorporated herein by reference. The purification methods in International Patent Publication No. 2017 / 100676 for AAV8, International Patent Publication No. 2017 / 100704 for rh10, and International Patent Publication No. 2017 / 100674 for AAV1 are all incorporated herein by reference.

[0057] To calculate the content of empty and filled particles, the VP3 band volume for a selected sample (e.g., in the present example, a preparation purified on an iodixanol gradient, number of GC = number of particles) is plotted against the GC particles loaded. The resulting linear equation (y = mx + c) is used to calculate the number of particles in the band volume of the peak of the test sample. The number of particles (pt) per 20 μL loaded is then multiplied by 50 to obtain particles (pt) / mL. Pt / mL is divided by GC / mL to obtain the ratio of particles to genome copies (pt / GC). Pt / mL ~ GC / mL gives the empty pt / mL. The percentage of empty particles is obtained by dividing empty pt / mL by pt / mL and multiplying by 100.

[0058] In general, methods for assaying AAV vector particles, including empty capsids and packaged genomes, are known in the art. See, e.g., Grimm et al., Gene Therapy (1999) 6:1322-1330; Sommer et al., J. Immunol. 1999, 11:1112-1114; See, et al., Molec. Ther. (2003) 7:122-128. To test for denatured capsids, the method involves the separation of the three capsid proteins The method includes subjecting the cured AAV stock to SDS-polyacrylamide gel electrophoresis, consisting of any gel capable of separating the capsid proteins, for example a gradient gel containing 3-8% Tris-acetate in a buffer, then running the gel until the sample material is separated and blotting the gel onto a nylon or nitrocellulose membrane, preferably nylon. An anti-AAV capsid antibody is then used as the primary antibody that binds to the denatured capsid protein, preferably an anti-AAV capsid monoclonal antibody, most preferably a B1 anti-AAV-2 monoclonal antibody (Wobus et al., J. Virol. (2000) 74:9281-9293). A secondary antibody is then used that binds to the primary antibody and includes a means for detecting binding to the primary antibody, more preferably an anti-IgG antibody containing a detection molecule covalently attached to the antibody, most preferably a sheep anti-mouse IgG antibody covalently attached to horseradish peroxidase. To semi-quantitatively determine the binding between the primary and secondary antibodies, a method for detecting binding is used, preferably a detection method capable of detecting radioisotope radiation, electromagnetic radiation, or colorimetric changes, most preferably a chemiluminescence detection kit. For example, in SDS-PAGE, samples from the column fractions can be taken and heated in SDS-PAGE loading buffer containing a reducing agent (e.g., DTT), and capsid proteins were resolved in a precast gradient polyacrylamide gel (e.g., Novex). Silver staining may be performed using SilverXpress (Invitrogen, CA) according to the manufacturer's instructions, or other suitable staining methods, i.e., SYPRO Ruby or Coomassie staining. In one embodiment, the concentration of AAV vector genome (vg) in the column fractions can be measured by quantitative real-time PCR (Q-PCR). Samples are diluted and DNase The AAV vector is digested with 1000 ribosomal nuclease I (or another suitable nuclease) to remove exogenous DNA. After inactivation of the nuclease, the sample is further diluted and amplified using a TaqMan™ fluorogenic probe specific for the primers and the DNA sequence between the primers. The number of cycles required to reach a defined level of fluorescence (threshold cycle, Ct) is measured for each sample on an Applied Biosystems Prism 7700 sequence detection system. Plasmid DNA containing the same sequence as that contained in the AAV vector is used to generate a standard curve in a Q-PCR reaction. The cycle threshold (Ct) value obtained from the sample is used to determine the vector genome titer by normalizing it to the Ct value of the plasmid standard curve. An endpoint assay based on digital PCR can also be used.

[0059] In one embodiment, an optimized q-PCR method utilizing a broad-spectrum serum protease, such as proteinase K (e.g., commercially available from Qiagen), is used. More specifically, the optimized qPCR genomic titer assay is similar to the standard assay, except that after DNase I digestion, the sample is diluted with proteinase K buffer and treated with proteinase K, followed by heat inactivation. Suitably, the sample is diluted with an amount of proteinase K buffer equal to the sample size. The proteinase K buffer can be concentrated 2-fold or more. Typically, the proteinase K treatment is about 0.2 mg / mL, but can vary from 0.1 mg / mL to about 1 mg / mL. The treatment step is generally carried out at about 55° C. for about 15 minutes, but may be carried out at lower temperatures (e.g., about 37° C. to about 50° C.) for longer times (e.g., about 20 minutes to about 30 minutes), or at higher temperatures (e.g., up to about 60° C.) for shorter times (e.g., about 5 to 10 minutes). Similarly, heat inactivation is generally at about 95° C. for about 15 minutes, but may be performed at lower temperatures (e.g., about 70 to about 90° C.) and for longer times (e.g., about 20 minutes to about 30 minutes). The sample is then diluted (e.g., 1000-fold) and subjected to TaqMan analysis as described for standard assays.

[0060] Additionally or alternatively, droplet digital PCR (ddPCR) may be used, for example to determine single-stranded and self-complementary AAV vector genome titers by ddPCR. A method for detecting encapsidated vector genomes has been described. See, for example, M. Lock et al, Hu Gene Therapy Methods, Hum Gene Ther Methods. 2014 Apr;25(2):115-25. doi:10.1089 / hgtb.2013.131. Epub 2014 Feb 14. The ddPCR method directly measures the concentration of encapsidated vector genomes. The sample is treated with DNaseI to digest any non-encapsidated DNA present in the sample, followed by treatment with proteinase K to destroy the capsid. The sample is then diluted to fit the assay range. The sample is mixed with ddPCR Supermix, and detection is achieved using a sequence-specific primer that targets the meganuclease (ARCUS) specific to the PCSK9 gene in combination with a fluorescently labeled probe that hybridizes to this same region. Twenty microliters of the ddPCR reaction mixture is processed through a Bio-Rad droplet generator, which divides the ddPCR reaction mixture into ≥10,000 droplets. After droplet generation, the ddPCR reaction mixture is PCR amplified, and the amplified ddPCR reaction mixture is read using a Bio-Rad Droplet Reader.

[0061] An infectious unit (IU) assay can be used to measure productive uptake and replication of rAAV vectors in RC32 cells (HeLa cells expressing rep2). A 96-well end-point format similar to that previously published was used. Briefly, RC32 cells are co-infected with serial dilutions of rAAV BDS and uniform dilutions of Ad5, with 12 replicates at each dilution of rAAV. 72 hours post-infection, cells are lysed and qPCR is performed to detect rAAV vector amplification relative to input. End-point dilutions of 50% tissue culture infectious dose (TCID 50) calculation (Spearman-Karber) is performed to measure the infectious titer and is expressed as IU / mL. Since "infectivity" values ​​depend on each particle contacting a cell, receptor binding, internalization, transport to the nucleus, and genome replication, they are influenced by the assay geometry and the presence of appropriate receptors and post-binding pathways in the cell line used. Receptors and post-binding pathways are not usually maintained in immortalized cell lines, and therefore the infectious assay titer is not an absolute measure of the number of "infectious" particles present. However, the ratio of GC encapsulated in capsids to "infectious units" (described as the GC / IU ratio) can be used as a measure of product consistency from lot to lot.

[0062] Briefly, a method for separating rAAV particles with packaged genome sequences from genome-defective AAV intermediates includes subjecting a suspension containing recombinant AAV viral particles and AAV capsid intermediates to high performance liquid chromatography, in which the AAV viral particles and AAV intermediates are bound to a strong anion exchange resin equilibrated at high pH and subjected to a salt gradient while monitoring the eluate for UV absorbance at about 260 and about 280. The pH can be adjusted depending on the AAV selected. See, for example, WO2017 / 160360 (AAV9), WO2017 / 100704 (AAVrhlO), WO2017 / 100676 (e.g., AAV8), and WO2017 / 100674 (AAV1), which are incorporated herein by reference. In this method, AAV complete capsids are recovered from the fraction eluted when the ratio of A260 / A280 reaches an inflection point. In one example, for an affinity chromatography step, the diafiltered product may be applied to Capture Select™ Poros-AAV2 / 9 affinity resin (Life Technologies), which efficiently captures AAV2 serotypes. Under these ionic conditions, a significant proportion of residual cellular DNA and proteins flow through the column, while AAV particles are efficiently captured.

[0063] Dual Vector System In another aspect, a dual vector system for treating a genetic disorder is provided, the system comprising: (a) a gene editing component, a meganuclease that targets PCSK9; and a regulatory sequence directing expression of the nuclease in a target cell containing the PCSK9 gene; and (b) a donor vector, comprising a nucleic acid sequence encoding OTC for expression from the PCSK9 locus, the system further comprising a sequence directing the nuclease to specifically target the native PCSK9 locus. The components of the dual vector are the same as those described herein.

[0064] In one embodiment, the expression cassette of the gene editing vector comprises the sequence of nucleotides 211 to 2964 of SEQ ID NO:2, or a sequence sharing at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.9% identity with the sequence of nucleotides 211 to 2964 of SEQ ID NO:2.

[0065] In one embodiment, the expression cassette of the donor vector comprises the sequence of nucleotides 178 to 3281 of SEQ ID NO:6, or a sequence sharing at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.9% identity with the sequence of nucleotides 178 to 3281 of SEQ ID NO:6.

[0066] Although the system may be effective when the ratio of gene editing vector to template vector is about 1 to about 1, it is desirable that the donor template vector is present in excess of the gene editing vector. In one embodiment, the ratio of editing vector (a) to donor vector (b) is about 1:3 to about 1:100, or about 1:10. In certain embodiments, the ratio of editing vector (a) to donor vector (b) is about 1:3. In certain embodiments, the ratio of editing vector (a) to donor vector (b) is about 1:2. In certain embodiments, the ratio of editing vector (a) to donor vector (b) is about 1:2.5. In certain embodiments, the ratio of editing vector (a) to donor vector (b) is about 1:3.5. In certain embodiments, the ratio of editing vector (a) to donor vector (b) is about 1:4. In certain embodiments, the ratio of editing vector (a) to donor vector (b) is about 1:4.5. In certain embodiments, the ratio of editing vector (a) to donor vector (b) is about 1:5.

[0067] In one embodiment, the dual vector system comprises a gene-edited AAV comprising an AAV capsid and a first vector genome comprising a 5'ITR, a sequence encoding a meganuclease that targets PCSK9 under the control of a regulatory sequence that directs expression of the meganuclease in a target cell that contains the PCSK9 gene, and a 3'ITR, and a donor AAV vector comprising an AAV capsid and a second vector genome comprising a 5'ITR, a 5' homologous recombination (HDR) arm, an OTC transgene and a regulatory sequence that directs expression of the transgene in a target cell, a 3'HDR arm, and a 3'ITR.

[0068] Pharmaceutical Compositions In another aspect, a pharmaceutical composition is provided, comprising: a first rAAV stock comprising a rAAV gene editing vector, the rAAV gene editing vector comprising an expression cassette comprising a nucleic acid sequence encoding a meganuclease targeting PCSK9 (e.g., the protein sequence of SEQ ID NO: 3) and a regulatory sequence directing expression of the nuclease in a target cell comprising a PCSK9 gene; and a second rAAV stock comprising a rAAV donor vector, the rAAV donor vector comprising a transgene cassette comprising a nucleic acid sequence encoding an OTC transgene (e.g., the coding sequence of SEQ ID NO: 4) and a regulatory sequence directing expression of the transgene in a target cell. The pharmaceutical composition contains optional carriers, excipients, and / or preservatives. In some embodiments, the donor vector further comprises homologous recombination (HDR) arms 5' and 3' to the transgene cassette. In one embodiment, the AAV capsid of the donor vector, the gene editing vector, or both, is an AAVrh79 capsid.

[0069] As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharma- ceutical active substances is well known in the art. Supplementary active ingredients can also be incorporated into the composition. The phrase "pharmacologically acceptable" refers to molecular entities and compositions that do not produce allergic or similar adverse reactions when administered to a host. Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like can be used to introduce the compositions of the present invention into suitable host cells. In particular, the rAAV vector delivery vector genome can be formulated for delivery either encapsulated in lipid particles, liposomes, vesicles, nanospheres, nanoparticles, or the like.

[0070] In one embodiment, the composition comprises a final formulation suitable for delivery to a subject, for example, an aqueous liquid suspension buffered to a physiologically compatible pH and salt concentration.Optionally, one or more surfactants are present in the formulation.In another embodiment, the composition can be delivered as a concentrate that is diluted for administration to a subject.In other embodiments, the composition can be lyophilized and reconstituted at the time of administration.

[0071] Methods and agents well known in the art for making formulations are described, for example, in "Remington's Pharmaceutical Sciences," Mack Publishing Company, Easton, Pa. Formulations may contain, for example, excipients, carriers, stabilizers, or diluents, such as sterile water, saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated naphthalenes, preservatives (octadecyldimethylbenzyl, ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight polypeptides, proteins, such as serum albumin, gelatin, or the like. or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine, and lysine, monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, and dextrins, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose, or sorbitol, salt-forming counterions such as sodium, metal complexes (e.g., Zn-protein complexes), and / or non-ionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0072] The active ingredient may also be encapsulated in microcapsules, e.g., hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacylate) microcapsules, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions, respectively, prepared, for example, by coacervation techniques or by interfacial polymerization. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0073] A suitable surfactant, or combination of surfactants, may be selected from among non-ionic surfactants that are non-toxic. In one embodiment, for example, a surfactant having a neutral pH and an average pH of 8400 is used. A primary hydroxyl terminated bifunctional block copolymer surfactant, such as Pluronic® F68 [BASF], also known as Poloxamer 188, with an average molecular weight, is selected. Other surfactants and other poloxamers may be selected, i.e., non-ionic triblock copolymers consisting of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)), SOLUTOL HS 15 (macrogol-15 hydroxystearate), LABRASOL (polyoxycaprylic acid glyceride), polyoxy 10 oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid ester), ethanol, and polyethylene glycol. In one embodiment, the formulation contains a poloxamer. These copolymers are generally named using the letter "P" (for poloxamer) followed by three digits, the first two digits x 100 indicating the approximate molecular mass of the polyoxypropylene core and the last digit x 10 indicating the percentage polyoxyethylene content. In one embodiment, poloxamer 188 is selected. The surfactant may be present in an amount up to about 0.0005% to about 0.001% of the suspension.

[0074] The vector is administered in an amount sufficient to transfect cells and provide sufficient levels of gene transfer and expression to provide a therapeutic effect without undue adverse effects or with medically acceptable physiological effects, which can be determined by one skilled in the art. Conventional and pharmacologic acceptable routes of administration include, but are not limited to, direct delivery to the desired organ (e.g., liver (optionally via the hepatic artery), lung, heart, eye, kidney), oral, inhalation, intranasal, intrathecal, intratracheal, intraarterial, intraocular, intravenous, intramuscular, subcutaneous, intradermal, and other parent routes of administration. In certain embodiments, the route of administration is IV. Routes of administration may be combined if desired.

[0075] The dosage of the viral vector depends primarily on factors such as the condition being treated, the age, weight, and health of the patient, and may therefore vary between patients. For example, a therapeutically effective human dosage of a viral vector is generally about 1×10 9 ~1×10 16 The range of solutions containing the concentration of genomic viral vectors.Dosage is adjusted to balance therapeutic effect against any side effects, and such dosage may vary depending on the therapeutic application for which the recombinant vector is utilized.The level of expression of the transgene product can be monitored to determine the frequency of dosage that results in viral vector, preferably AAV vector containing minigene.Optionally, the same dosage regimen as described for therapeutic purposes can be utilized for immunization using the composition of the present invention.

[0076] The replication-defective virus composition may be administered to a human patient in a dose range of about 1.0×10 (to treat an average subject weighing 70 kg), including all integers or fractions within the range. 9 GC~approx. 1.0×10 16 GC range, preferably 1.0 x 10 12 GC~1.0×10 14 The composition can be formulated in dosage units containing an amount of replication-defective virus in the range of GC. In one embodiment, the composition contains at least 1 x 10 per dose, including all integers or decimals within the range. 9, 2×10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , or 9 x 10 9 In another embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, including all integers or decimals within the range. 10 , 2×10 10 , 3×10 10 , 4×10 10 , 5×10 10 , 6×10 10 , 7×10 10 , 8×10 10 , or 9 x 10 10 In another embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, including all integers or decimals within the range. 11 , 2×10 11 , 3×10 11 , 4×10 11 , 5×10 11 , 6×10 11 , 7×10 11 , 8×10 11 , or 9 x 10 11 In another embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, including all integers or decimals within the range. 12 , 2×10 12 , 3×10 12 , 4×10 12 , 5×10 12 , 6×10 12 , 7×10 12 , 8×10 12 , or 9× 10 12 In another embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, including all integers or decimals within the range. 13 , 2×10 13 , 3×10 13 , 4×10 13 , 5×10 13 , 6×10 13 , 7×10 13, 8×10 13 , or 9 x 10 13 In another embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, including all integers or decimals within the range. 14 , 2×10 14 , 3×10 14 , 4×10 14 , 5×10 14 , 6×10 14 , 7×10 14 , 8×10 14 , or 9 x 10 14 In another embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, including all integers or decimals within the range. 15 , 2×10 15 , 3×10 15 , 4×10 15 , 5×10 15 , 6×10 15 , 7×10 15 , 8×10 15 , or 9 x 10 15 In one embodiment, for human applications, the dose is formulated to contain 1×10 per dose, including all integers or fractions within the range. 10 ~Approx. 1×10 12 It may be in the GC range.

[0077] These above-mentioned doses may be administered in various volumes of the carrier, excipient, or buffer formulation, including from about 25 to about 1000 microliters, or larger volumes, or any number within that range, depending on the size of the area to be treated, the viral titer used, the route of administration, and the desired effect of the method.

[0078] Any suitable route of administration may be selected. Thus, the pharmaceutical composition may be formulated for any suitable route of administration, for example, in the form of a liquid solution or suspension (e.g., for intravenous administration, oral administration, etc.). Alternatively, the pharmaceutical composition may be in solid form (e.g., in the form of a tablet or capsule, for oral administration, etc.). In some embodiments, the pharmaceutical composition may be in the form of a powder, a drip, an aerosol, etc.

[0079] In one aspect, provided herein is a pharmaceutical composition comprising at least a parvovirus vector comprising at least one gene editing vector as described herein and at least one donor vector in a formulation buffer. In certain embodiments, the pharmaceutical composition comprises a combination of different vector populations. In one embodiment, provided herein is a pharmaceutical composition comprising a single rAAV population as described herein in a formulation buffer. The method provided herein provides for the simultaneous administration of two separate vector-containing suspensions.

[0080] method The compositions provided herein are useful for treating ornithine transcarbamylase deficiency. Methods for treating disorders in humans by co-administering the dual vector systems described herein are provided herein. In certain embodiments, compositions are provided herein comprising non-replicating recombinant adeno-associated virus serotype rh79 vectors: AAVrh79.TBG.M2PCSK9.WPRE.bGH and AAVrh79.hHDR.TBG.hOTCco.bGH for treating ornithine transcarbamylase deficiency.

[0081] In one embodiment, a method for treating OTC deficiency in a subject is provided.The method comprises co-administering a gene editing AAV vector comprising a sequence encoding a nuclease that targets PCSK9 and a regulatory sequence that directs the expression of the nuclease in target cells that comprise PCSK9 gene, and a donor AAV vector comprising a transgene and a regulatory sequence that directs the expression of the OTC transgene in target cells to a subject with OTC deficiency.In one embodiment, the subject is a newborn.

[0082] In certain embodiments, the gene-edited AAV vector and the donor vector are delivered essentially simultaneously via the same route. In other embodiments, the gene-editing vector is administered first. In other embodiments, the donor vector is administered first.

[0083] In one embodiment, the dosage of rAAV is about 1×10 per dose (to treat an average subject weighing 70 kg). 9 GC~approx. 1×10 15 Genome copies (GC), preferably 1.0×10 for human patients 12 GC~2.0×10 15 In another embodiment, the dose is about 1×10 14 In certain embodiments, the dose administered to a patient is at least about 1.0×10 9 GC / kg, approx. 1.5×10 9 GC / kg, approx. 2.0×10 9 GC / g, approximately 2.5 x 10 9 GC / kg, approx. 3.0×10 9 GC / kg, approx. 3.5×10 9 GC / kg, approx. 4.0×10 9 GC / kg, approx. 4.5×10 9 GC / kg, approx. 5.0×10 9 GC / kg, approx. 5.5×10 9 GC / kg, approx. 6.0×10 9 GC / kg, approx. 6.5×10 9 GC / kg, approx. 7.0×10 9 GC / kg, approx. 7.5×10 9 GC / kg, approx. 8.0×10 9 GC / kg, approx. 8.5×10 9 GC / kg, approx. 9.0×10 9 GC / kg, approx. 9.5×10 9 GC / kg, approximately 1.0×10 10 GC / kg, approx. 1.5×10 10 GC / kg, approx. 2.0×10 10 GC / kg, approx. 2.5×10 10 GC / kg, approx. 3.0×10 10 GC / kg, approx. 3.5×10 10 GC / kg, approx. 4.0×10 10 GC / kg, approx. 4.5×10 10 GC / kg, approx. 5.0×10 10 GC / kg, approx. 5.5×10 10 GC / kg, approx. 6.0×10 10 GC / kg, approx. 6.5×1010 GC / kg, about 7.0×10 10 GC / kg, about 7.5×10 10 GC / kg, about 8.0×10 10 GC / kg, about 8.5×10 10 GC / kg, about 9.0×10 10 GC / kg, about 9.5×10 10 GC / kg, about 1.0×10 11 GC / kg, about 1.5×10 11 GC / kg, about 2.0×10 11 GC / kg, about 2.5×10 11 GC / kg, about 3.0×10 11 GC / kg, about 3.5×10 11 GC / kg, about 4.0×10 11 GC / kg, about 4.5×10 11 GC / kg, about 5.0×10 11 GC / kg, about 5.5×10 11 GC / kg, about 6.0×10 11 GC / kg, about 6.5×10 11 GC / kg, about 7.0×10 11 GC / kg, about 7.5×10 11 GC / kg, about 8.0×10 11 GC / kg, about 8.5×10 11 GC / kg, about 9.0×10 11 GC / kg, about 9.5×10 11 GC / kg, about 1.0×10 12 GC / kg, about 1.5×10 12 GC / kg, about 2.0×10 12 GC / kg, about 2.5×10 12 GC / kg, about 3.0×10 12 GC / kg, about 3.5×10 12 GC / kg, about 4.0×10 12 GC / kg, about 4.5×10 12 GC / kg, about 5.0×10 12 GC / kg, about 5.5×10 12 GC / kg, about 6.0×10 12 GC / kg, about 6.5×10 12 GC / kg, about 7.0×10 12 GC / kg, about 7.5×10 12GC / kg, approx. 8.0×10 12 GC / kg, approx. 8.5×10 12 GC / kg, approx. 9.0×10 12 GC / kg, approx. 9.5×10 12 GC / kg, approximately 1.0×10 13 GC / kg, approx. 1.5×10 13 GC / kg, approx. 2.0×10 13 GC / kg, approx. 2.5×10 13 GC / kg, approx. 3.0×10 13 GC / kg, approx. 3.5×10 13 GC / kg, approx. 4.0×10 13 GC / kg, approx. 4.5×10 13 GC / kg, approx. 5.0×10 13 GC / kg, approx. 5.5×10 13 GC / kg, approx. 6.0×10 13 GC / kg, approx. 6.5×10 13 GC / kg, approx. 7.0×10 13 GC / kg, approx. 7.5×10 13 GC / kg, approx. 8.0×10 13 GC / kg, approx. 8.5×10 13 GC / kg, approx. 9.0×10 13 GC / kg, approx. 9.5×10 13 GC / kg, or approximately 1.0 x 10 14 GC / kg body weight or subject.

[0084] The vector is administered in an amount sufficient to transfect the cells and provide sufficient levels of gene transfer and expression to provide a therapeutic effect without undue adverse effects or with a medically acceptable physiological effect, as can be determined by one of skill in the art. In certain embodiments, IV administration is used.

[0085] The systems described herein may be therapeutically useful if a sufficient amount of functional enzyme or protein is produced to improve a patient's condition. In certain embodiments, gene expression levels as low as 5% of the levels in healthy patients will provide sufficient therapeutic benefit to the patient. In other embodiments, gene expression levels are greater than or equal to the normal levels observed in humans (or other veterinary subjects). At least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 109%, 109%, 108% %, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. For example, a "functional enzyme" refers to an enzyme that has at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 109%, 109% or more of the biological activity of a wild-type enzyme or a naturally occurring variant or polymorph thereof that is not associated with a disease. By this definition is meant a gene encoding a wild-type enzyme (e.g., OTCase) that provides 2%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or substantially the same, or 100% or more. Similarly, patients who do not have detectable amounts of enzyme can be rescued by delivering enzyme function to less than 100% activity levels, and optionally can then undergo further treatment.In certain embodiments where gene function is delivered by a donor template, the patient may express higher levels than found in a "normal" healthy subject. As described herein, the therapies described herein may be used in conjunction with other treatments (i.e., standard of care for the subject's (patient's) diagnosis).

[0086] In one embodiment, the method further comprises administering an immunosuppressive combination therapy to the subject. Such immunosuppressive combination therapy may be initiated prior to delivery of the rAAV or the disclosed composition, for example, if undesirably high neutralizing antibody levels against the AAV capsid are detected. In certain embodiments, the combination therapy may be initiated prior to delivery of the rAAV as a preventative measure. In certain embodiments, the immunosuppressive combination therapy is initiated after delivery of the rAAV, for example, if an undesirable immune response is observed after treatment.

[0087] Immunosuppressants for such combination therapy include, but are not limited to, glucocorticoids, steroids, antimetabolites, T-cell inhibitors, macrolides (e.g., rapamycin or rapalogs), and cytostatic agents (including alkylating agents, antimetabolites, cytotoxic antibiotics, antibodies, or agents active against immunophilins). Immunosuppressants may include prednisolone, nitrogen mustards, nitrosoureas, platinum compounds, methotrexate, azathioprine, mercaptopurine, fluorouracil, dactinomycin, anthracyclines, mitomycin C, bleomycin, mithramycin, IL-2 receptor (CD25)-specific antibodies or CD3-specific antibodies, anti-IL-2 antibodies, cyclosporine, tacrolimus, sirolimus, IFN-β, IFN-γ, opioids, or TNF-α (tumor necrosis factor-α) binding agents. In certain embodiments, immunosuppressive therapy may be initiated 0, 1, 2, 7 or more days prior to rAAV administration, or 0, 1, 2, 3, 7 or more days after rAAV administration. Such therapy may involve the simultaneous administration of a single drug (e.g., prednisolone) or two or more drugs (e.g., prednisolone, mycophenolate mofetil (MMF), and / or sirolimus (i.e., rapamycin)) on the same day. Among these drugs, One or more of the following may be continued at the same dose or at an adjusted dose after administration of gene therapy. Such therapy may be for about 1 week (7 days), 2 weeks, 3 weeks, about 60 days, or longer, as needed. In certain embodiments, a tacrolimus-free regimen is selected.

[0088] In another embodiment, the method includes concurrent treatment with standard OTC therapy. Treatment of OTC deficiency is primarily focused on dietary management of blood ammonia levels to avoid hyperammonemia or to remove excess ammonia from the blood during hyperammonemic episodes (NORD, 2021). Individuals with OTC deficiency are followed to dietary restrictions that limit protein intake to control blood ammonia levels. Dietary restrictions must be carefully balanced in infants who need to consume enough protein to ensure proper growth while avoiding excess protein intake that can cause hyperammonemic episodes (Berry and Steiner, 2001). Thus, infants are fed a high-calorie, low-protein diet supplemented with essential amino acids. In hyperammonemic episodes, all protein may be removed from the patient's diet for 24 hours (NORD, 2021).

[0089] There are several drugs designed to stimulate the removal of nitrogen from the bloodstream. Sodium phenylbutyrate (Buphenyl) is approved by the U.S. Food and Drug Administration (FDA) for the treatment of chronic hyperammonemia in patients with OTC deficiency. Buphenyl, when metabolized, is converted to phenylacetate. This is conjugated with glutamine to form phenylacetylglutamine, which is excreted by the kidney, providing an alternative route for nitrogen excretion. Glycerol phenylbutyrate (Ravicti) is FDA approved for the treatment of chronic hyperammonemia in patients with urea cycle disorders. Similar to Buphenyl, Ravicti is converted to phenylacetate and follows the same mechanism for excreting nitrogen (Lichter-Konecki et al., 1993; Gordon, 2003; Magellan, 2021). Finally, Ammonul (sodium phenylacetate and sodium benzoate) is approved by the FDA as an adjunctive therapy for the treatment of acute hyperammonemia in patients with urea cycle disorders. The sodium phenylacetate component of Ammonul follows the same nitrogen excretion mechanism as the phenylacetate metabolites produced by Buphenyl and Ravicti. The sodium benzoate component of Ammonul is conjugated with glycine to form hippuric acid, which is excreted by the kidney, removing nitrogen through this process. Sodium benzoate is also given as an oral preparation for long-term maintenance of OTC deficiency and is often preferred over Buphenyl and Ravicti because it is thought to have fewer side effects (Lichter-Konecki et al., 1993).

[0090] Many people with OTC deficiency are treated with arginine or citrulline, which are needed to ensure that protein synthesis proceeds at a normal rate. Citrulline is often chosen over arginine for chronic treatment because it incorporates aspartate into the urea cycle, meaning it contributes one additional nitrogen molecule to this pathway (Lichter-Konecki et al., 1993; Magellan, 2021).

[0091] In hyperammonemic episodes progressing to vomiting and lethargy, hospitalization may be required and the individual may be treated with IV infusions containing arginine and Ammonul. If these treatments are unsuccessful, hemofiltration or hemodialysis may be required to rapidly lower blood ammonia levels (Lichter-Konecki et al., 1993).

[0092] Liver transplantation may be an effective treatment for the development of hyperammonemia in patients with severe neonatal-onset OTC deficiency. It is the most effective strategy to prevent the deterioration of neurogenesis and disease. In patients with mild or well-controlled OTC deficiency, stress factors can trigger life-threatening hyperammonemic episodes at any age. The fear of such episodes, in addition to the reduced quality of life these patients face because of dietary restrictions and chronic treatment with nitrogen scavengers that can have significant side effects, motivates many patients to seek liver transplantation, even when their condition is well controlled (Lichter-Konecki et al., 1993).

[0093] In one aspect, a method is provided for treating a patient with ornithine transcarbamylase (OTC) deficiency using a nuclease expression cassette comprising a meganuclease that recognizes a site in the human PCSK9 gene under the control of a TBG promoter as described herein. The method further comprises administering an expression cassette carrying an OTC transgene of SEQ ID NO: 4 or a sequence sharing at least 90% identity therewith, as described herein.

[0094] There are various assays to measure the expression and activity levels of OTC in vitro. See, for example, X. Ye, et al., 1996 Prolonged metabolic See correction in adult ornithine transcarbamylase-deficient mice with adenoviral vectors.J Biol Chem 271:3639-3646) or in vivo.For example, OTC enzyme activity can be measured using liquid chromatography mass spectrometry stable isotope dilution method to detect the formation of citrulline normalized to [1,2,3,4,5-13C5] citrulline (98% 13C).This method is adapted from an assay previously developed for the detection of N-acetylglutamate synthase activity [Morizono H, et al, Mammalian N-acetylglutamate synthase.Mol Genet Metab.2004;81(Suppl 1):S4-11.]. Fresh frozen liver debris is weighed and briefly homogenized in a buffer containing 10 mM HEPES, 0.5% TritonX-100, 2.0 mM EDTA, and 0.5 mM DTT. The volume of homogenization buffer is adjusted to obtain 50 mg / ml of tissue. Enzyme activity is measured using 250 μg of liver tissue in 50 mM Tris-acetate, 4 mM ornithine, 5 mM carbamyl phosphate, pH 8.3. Enzyme activity is initiated by the addition of freshly prepared 50 mM carbamyl phosphate dissolved in 50 mM Tris-acetate, pH 8.3, allowed to proceed for 5 min at 25°C, and quenched by the addition of an equal volume of 5 mM 13C5-citrulline in 30% TCA. Debris is separated by microcentrifugation for 5 min, and the supernatant is transferred to a vial for mass spectrometry analysis. 10 μL of sample is injected into an Agilent 1100 Series LC-MS under isocratic conditions with a mobile phase of 93% solvent A (1 ml trifluoroacetic acid in 1 L water): 7% solvent B (1 ml trifluoroacetic acid in 1 L water / acetonitrile (1:9)). Peaks corresponding to citrulline [176.1 mass:charge ratio (m / z)] and 13C5 citrulline (181.1 m / z) are quantified and their ratios compared to those obtained for a standard curve of citrulline run with each assay.Samples are normalized to either total liver tissue or protein concentration determined using a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA). Other assays that do not require a liver biopsy may also be used. One such assay is the plasma amino acid assay, where the ratio of glutamine to citrulline is evaluated; if glutamine is high (>800 micromoles / liter) and citrulline is low (e.g., single digits), a urea cycle defect is suspected. Plasma ammonia levels may be measured; a concentration of approximately 100 micromoles per liter indicates OTCD. Blood gases can be evaluated if the patient is hyperventilating, and respiratory alkalosis frequently occurs in OTCD. Urinary orotic acid, e.g., greater than approximately 20 micromoles / mmol creatine, is indicative of allopurinol. The diagnostic criteria for OTCD are as follows: Tuchman et al, 2008, Urea Cycle Disorders Consortium (UCDC) of the Rare Disease Clinical Research Network (RDCRN). Tuchman M, et al., Consortium of the Rare Diseases Clinical Research Network. Cross-sectional multicenter study of patients with urea cycle disorders in the United States. Mol Genet Metab. 2008;94:397-402, incorporated herein by reference. Also see http: / / www.ncbi.nlm.nih.gov / books / NBK154378 / , which provides a discussion of the current standard of care for OTCD.

[0095] In certain embodiments, any of the nuclease expression cassettes, viral vectors (e.g., rAAV), or pharmaceutical compositions described herein can be administered for gene editing in a patient. In certain embodiments, the methods are useful for non-embryonic gene editing. In certain embodiments, the patient is an infant (e.g., birth to about 4 months of age). In certain embodiments, the patient is a toddler or older, e.g., 12 months or older.

[0096] As used herein, "a," "an," or "the" can mean one or more than one. For example, "a" cell can mean a single cell or multiple cells.

[0097] As used herein, the term "specificity" refers to the ability of a nuclease to recognize and cleave a double-stranded DNA molecule only at a specific sequence of base pairs, referred to as a recognition sequence, or only at a specific set of recognition sequences. A set of recognition sequences share certain conserved positions or sequence motifs, but may be degenerate at one or more positions. A highly specific nuclease can cleave only one or very few recognition sequences. Specificity can be determined by any method known in the art.

[0098] The (gene editing and donor) expression cassettes described herein can be engineered into any suitable genetic element (e.g., vector) for delivery to a target cell. As used herein, a "vector" is a biological or chemical moiety that contains a nucleic acid sequence and can be introduced into an appropriate host cell for replication or expression of the nucleic acid sequence.

[0099] "Plasmids" or "plasmid vectors" are generally designated herein by a lower case p preceding and / or following the vector name. Plasmids and other cloning and expression vectors that can be used in accordance with the present invention, their properties, and methods of their construction / manipulation will be readily apparent to those of skill in the art.

[0100] As used herein, the term "operably linked" refers both to expression control sequences that are contiguous with a gene of interest, and to expression control sequences that act in trans or at a distance to control the gene of interest.

[0101] The term "exogenous" as used to describe a nucleic acid sequence or protein means that the nucleic acid or protein does not naturally occur in the chromosome or at the location present in the host cell. Exogenous nucleic acid sequences also refer to sequences that are derived from and inserted into the same expression cassette or host cell, but are present in a non-native state (e.g., in different copy number or under the control of different regulatory elements).

[0102] The term "heterologous" when used with reference to a protein or nucleic acid indicates that the protein or nucleic acid comprises two or more sequences or subsequences that are not found in the same relationship to each other in nature. For example, nucleic acids that have two or more sequences from unrelated genes arranged to create a new functional nucleic acid are typically produced recombinantly. For example, in one embodiment, a nucleic acid has a promoter from one gene arranged to direct expression of a coding sequence from a different gene.

[0103] As used herein, the term "host cell" may refer to a packaging cell line in which a vector (e.g., recombinant AAV) is produced from a production plasmid. Alternatively, the term "host cell" may refer to any target cell in which expression of a transgene is desired. Thus, "host cell" refers to a prokaryotic or eukaryotic cell that contains an exogenous or heterologous nucleic acid sequence that has been introduced into the cell by any means, such as electroporation, calcium phosphate precipitation, microinjection, transformation, viral infection, transfection, liposome delivery, membrane fusion techniques, high-speed DNA-coated pellets, viral infection, and protoplast fusion. In certain embodiments herein, the term "host cell" refers to cultures of cells of various mammalian species for in vitro evaluation of the compositions described herein. In other embodiments herein, the term "host cell" refers to cells used to generate and package viral vectors or recombinant viruses. In still other embodiments, the term "host cell" is intended to refer to a target cell of a subject to be treated in vivo for a disease or condition described herein. In certain embodiments, the term "host cell" is a liver cell or liver cell.

[0104] A "subject" is a mammal (e.g., a human, a mouse, a rat, a guinea pig, a dog, a cat, a horse, a cow, a pig) or a non-human primate (e.g., a monkey, a chimpanzee, a baboon, or a gorilla). A patient refers to a human. A veterinary subject refers to a non-human mammal. In certain embodiments, the subject does not have a defect in the PCSK9 gene.

[0105] In certain embodiments, the subject has or is at risk of developing OTC deficiency. In certain embodiments, the subject has documented genetic confirmation of OTCD mutation. In certain embodiments, the subject has or has previously had a hyperammonemic crisis (HAC). In certain embodiments, the subject is currently undergoing treatment for OTC deficiency, for example, with at least one nitrogen scavenger therapy and / or a protein-restricted diet.

[0106] In certain embodiments, the subject is male. In certain embodiments, it is desirable to treat the subject within hours of birth, for example, within at least 12, 24, 36, or 48 hours of birth. In other embodiments, it is desirable to treat the subject within days of birth, for example, within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days of birth. In other embodiments, it is desirable to treat the subject within weeks of birth, for example, within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks of birth. In other embodiments, it is desirable to treat the subject within months of birth, for example, within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months of birth. In certain embodiments, subjects are treated at 24 hours to 4 months of age. In some embodiments, subjects are male infants at 24 hours to 4 months of age with documented genetic confirmation of OTCD mutations consistent with neonatal-onset OTC deficiency with or without current or past hyperammonemic crisis (HAC) and currently being treated with at least one nitrogen scavenger therapy and a protein-restricted diet. The proposed study population includes subjects with high unmet need who may be observed to have a stabilization or improvement in the frequency and severity of recurrent hyperammonemic events with reduced morbidity and mortality, and increased survival, while delaying the need for liver transplantation within the 52-week protocol time frame.

[0107] "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, and any viral genomic sequences also packaged in the viral capsid or envelope are replication-defective, i.e., they are unable to generate progeny virions, but retain the ability to infect target cells. In one embodiment, the genome of the viral vector does not contain genes encoding enzymes required for replication (the genome can be engineered to be "gutless," containing only the gene of interest flanked by signals required for amplification and packaging of the artificial genome), although these genes can be supplied during production. It is therefore considered safe for use in gene therapy, since replication and infection by progeny virions cannot occur except in the presence of viral enzymes required for replication.

[0108] The terms "sequence identity," "percent sequence identity," or "percent identical" in the context of nucleic acid sequences refer to residues in two sequences that are the same when aligned for maximum correspondence. The length of sequence identity comparison may span the entire length of a genome, the entire length of a gene coding sequence, or a fragment of at least about 500-5000 nucleotides, which is preferred. However, identity between smaller fragments, e.g., at least about 9 nucleotides, usually at least about 20-24 nucleotides, at least about 28-32 nucleotides, at least about 36 nucleotides or more, may also be desired. Similarly, "percent sequence identity" can be readily determined for amino acid sequences over the entire length of a protein or fragments thereof. Preferably, the fragments are at least about 8 amino acids in length and can be up to about 700 amino acids in length. Examples of suitable fragments are described herein.

[0109] The terms "substantial homology" or "substantial similarity", when referring to an amino acid or a fragment thereof, when optimally aligned with another amino acid (or its complementary strand) with appropriate amino acid insertions or deletions, there is amino acid sequence identity in at least about 95-99% of the aligned sequence. Preferably, the homology is over the full length sequence, or the protein thereof, e.g., cap protein, rep protein, or fragments thereof that are at least 8 amino acids in length, or more preferably, at least 15 amino acids in length. Examples of suitable fragments are described herein.

[0110] The term "highly conserved" means at least 80% identity, preferably at least 90% identity, more preferably greater than 97% identity. Identity is easily determined by those skilled in the art by resorting to algorithms and computer programs known to those skilled in the art.

[0111] Generally, when referring to "identity", "homology", or "similarity" between two different adeno-associated viruses, the "identity", "homology", or "similarity" is determined with reference to "aligned" sequences. An "aligned" sequence or "alignment" refers to multiple nucleic acid or protein (amino acid) sequences, often including corrections for missing or additional bases or amino acids, as compared to a reference sequence. In an example, an AAV alignment is performed using the published AAV9 sequence as a reference point. The alignment is performed using any of a variety of publicly or commercially available multiple sequence alignment programs. Examples of such programs include "Clustal Omega", "Clustal W", "CAP Sequence Assembly", "MAP", and "MEME", which are accessible through web servers on the Internet. Other sources of such programs are known to those of skill in the art. Alternatively, the Vector NTI utility is also used. There are also several algorithms known in the art that can be used to measure nucleotide sequence identity, including those included in the programs described above. As another example, a polynucleotide sequence alignment program is performed using a polynucleotide sequence alignment program. The sequences of nucleic acids can be compared using Fasta™, a program in GCG version 6.1. Fasta™ provides alignment and percent sequence identity of the best overlapping regions between the query sequence and the search sequence. For example, percent sequence identity between nucleic acid sequences can be determined using Fasta™ with its default parameters (word size 6 and NOPAM factor for the scoring matrix) as provided in GCG version 6.1 (hereby incorporated by reference). Multiple sequence alignment programs such as "Clustal Omega", "Clustal X", "MAP", "PIMA", "MSA", "BLOCKMAKER", "MEME" and "Match-Box" programs are also available for amino acid sequences. Generally, any of these programs are used with default settings, but if necessary, the skilled artisan can change these settings. Alternatively, the skilled artisan can utilize another algorithm or computer program, providing at least the level of identity or alignment as provided by the referenced algorithm and program. See, e.g., JD Thomson et al, Nucl. Acids. Res., "A comprehensive comparison of multiple sequence alignments", 27(13):2682-2690 (1999).

[0112] As used herein, the term "about" refers to a ±10% variation from the reference integer and values ​​therebetween. For example, "about" 40 base pairs includes ±4 (i.e., 36 to 44, which includes integers 36, 37, 38, 39, 40, 41, 42, 43, 44). For other values, particularly when referring to percentages (e.g., 90% identity, about 10% variance, or about 36% mismatch), the term "about" includes all values ​​within the range, including both integers and fractions.

[0113] As used throughout this specification and claims, the terms "comprising," "containing," "including," and variations thereof are inclusive of other components, elements, integers, steps, etc. Conversely, the term "consisting" and variations thereof exclude other components, elements, integers, steps, etc.

[0114] Unless otherwise defined herein, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art and by reference to the published literature, which provides general guidance to those of ordinary skill in the art for many of the terms used herein. EXAMPLES

[0115] We describe a genome editing approach for the treatment of ornithine transcarbamylase deficiency (OTCD), which can cause fatal hyperammonemia episodes in infancy. The goal of genome editing is for the therapeutic effect to be sustained and achieved in all OTCD patients, regardless of mutation. We achieve this by treating surviving newborns with two AAV vectors: the first to deliver a nuclease to create a double-strand break at the safe harbor site, and the second to deliver an OTC minigene for knock-in at this site. Our hypothesis is that dividing hepatocytes in the newborn liver will facilitate efficient knock-in of the OTC gene and eliminate unintegrated input vector genomes by dilution. We decided to use the PCSK9 gene as a safe harbor site and a meganuclease called ARCUS to target it, based on our previous work in adult macaques that showed a safe, efficient, and stable reduction of PCSK9 following AAV delivery of ARCUS. Our initial study of genome editing for OTCD involved the deletion of the endogenous PCSK9 gene. This was done in OTC-deficient mice that were rendered susceptible to PCSK9 ARCUS nuclease via germline modification of exon 7. Injection of the two vectors into neonatal mice resulted in efficient knock-in of the human OTC minigene and provided protection against lethal hyperammonemia when challenged with a high protein diet. In preparation for clinical studies, we evaluated important safety and efficacy parameters in neonatal and infant macaques. A total of 24 animals were treated with AAV vectors and analyzed, including examination of liver biopsies at 3 and 12 months. In these studies, we evaluated the impact of the following parameters on editing efficiency and toxicity: transgenes (human factor IX and human OTC), promoter driving ARCUS, clade E capsid, donor length flanking the transgene, and age of the macaque at the time of dosing. We found that injection of AAV vectors was very safe, with no evidence of transaminase elevation or liver histopathology in any of the ARCUS-treated animals. An important measure of efficacy in primate models is transduction efficiency, measured by in situ hybridization and immunostaining to detect cells expressing human OTC mRNA and protein, respectively. The highest and most consistent results were obtained with vectors using a novel clade E capsid driving ARCUS with a TBG promoter in the first vector and 500 bp of flanking homology arms on the donor vector. This combination achieved transduction of 10.0±6.4% (N=6). This is higher than the threshold (approximately 5% of OTC-expressing cells) that is considered to be of substantial benefit to patients. Preliminary data suggest that editing levels are stable over a year and efficient targeted insertion can be achieved when injected into macaques up to 3 months of age. Molecular analysis of the PCSK9 target locus suggested that the majority of knock-in of the vector genome was via non-homologous end joining (NHEJ) rather than homology-directed repair (HDR). In summary, the substantial unmet need for neonatal forms of OTCD justifies consideration of experimental therapies, such as genome editing, as described in this report.

[0116] Example 1 - Materials and Methods AAV vectors were constructed according to previously established procedures and manufacturer's instructions. AAVhu37 or AAVrh79 capsids were used in the experiments described herein where indicated.

[0117] All animal procedures were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania.

[0118] Example 2 - ARCUS2-mediated hOTC gene targeting in neonatal NHPs Neonatal (1-16 days old) or infant (3-4 months old) rhesus macaques were used for non-GLP compliant POC pharmacology studies. ARCUS meganuclease targets a 22 bp sequence present in the human and rhesus macaque PCSK9 genes. Thus, rhesus macaques can be used to evaluate on-target editing (pharmacology) and safety / toxicology. Furthermore, neonatal and infant rhesus macaques have similar anatomical and physiological characteristics to human infants, which will allow for the use of the intended clinical ROA (IV). The similarity of anatomy and ROA is expected to result in representative vector distribution and transduction profiles, which will allow for a more accurate assessment of the pharmacology and toxicity of the test samples, including on-target and off-target editing, as well as clinical pathology that is not possible in neonatal mice.

[0119] FIG. 1A shows the timeline of the study provided herein. FIG. 1B provides information on dosing groups, while a schematic diagram of the vectors is shown in FIG. 2. Newborn NHPs (1-16 days) were administered the ARCUS2 nuclease vector and donor vectors with HDR arms of various lengths (500 bp arms or short HDR arms). FIG. 3 shows the results from the experiment. Summary table showing the data. All 14 neonatal macaques tolerated the vector injections well (i.e., no obvious clinical sequelae) and gained weight over time (Figure 4E). Liver enzyme levels were within normal ranges, except for a transient and mild elevation of ALT levels in some animals on day 14 (Figure 4B). Liver inflammation in neonatal and infant NHPs was substantially reduced compared to adult NHPs administered AAV.Arcus alone (Figure 4J). Pancreas was the only non-liver tissue in which any indels were identified (Figure 4I).

[0120] Analysis of day 0 plasma samples taken from neonates prior to dosing showed that three animals (21-111, 21-113, 21-122) had high levels (>400) of binding antibodies to AAVrh79 (Figure 3). These pre-existing anti-AAVrh79 antibodies would block AAV gene transfer.

[0121] PCSK9 levels were followed over time in all newborn animals, including the donor-only control animals. PCSK9 levels on day 0 differed among the newborns (Figure 4A). Nine animals showed a trend toward decreased PCSK9 levels after vector administration, including one donor-only control animal, whereas the remaining five animals showed a sustained or transient increase in PCSK9 levels after administration (Figure 4A).

[0122] Liver biopsies were performed at day 84 and one year. Transduction efficiency of hOTC in the liver was assessed by dual ISH using hOTC-specific and ARCUS-specific probes to detect transgene mRNA and OTC immunofluorescence to detect human OTC protein followed by quantification on scanned slides (Figure 4C-ISH, Figure 4D-4F). Three animals (21-111, 21-113, and 21-122) with pre-existing anti-AAVrh79 binding antibodies at the time of dosing showed no OTC-positive hepatocytes by both methods. Two donor-only control animals continued to show low levels of hOTC transduction (2% or less). The highest transduction efficiency (27.8% and 23.6% by ISH) was detected in two animals co-administered with AAVrh79.TBG.PI.ARCUS.WPRE.bGH and AAVrh79.rhHDR.TBG.hOTCco.bGH donor vectors (GTP-506). Positive hOTC-expressing hepatocytes were also found to be present in clusters. These levels are above the threshold (approximately 5% of OTC-expressing cells) that would provide substantial benefit to patients. The data demonstrate that driving ARCUS from the strong TBG promoter and long homology arms in the donor vector resulted in the greatest editing. In addition, similarly high levels of editing were observed with the same ARCUS vector and the same homology arms in the F9 donor vector in the NB study. Mouse OTCD pharmacology studies demonstrated consistent high editing with donors containing different homology arms, including the mouse correlates of the long homology arm vector in this study. The level of editing in NB NHPs by GTP-506 is sufficient to show a strong therapeutic effect.

[0123] Molecular analysis of day 84 liver biopsy samples from each animal was performed to measure transgene copy number per diploid genome, mRNA expression levels, on-target editing, and off-target editing (Figure 4H). Consistent with the transduction efficiency analysis, two animals in group 6 (21-157 and 21-175) had the highest GC of hOTC vector (Figure 4F), hOTC mRNA (Figure 4G), and on-target indels (%) (Figure 4H). The GC of ARCUS vector in animals was 2- to 7-fold lower than the GC of hOTC vector, while ARCUS mRNA levels were 23- and 765-fold lower than hOTC mRNA levels (Figures 4F and 4G).

[0124] Off-target (OT) activity assessed by ITR-seq identified 2–40 potential off-targets in the day 84 liver biopsy samples of this study. Off-target sites were detected in multiple animals, including animals administered human factor IX (hfIX) donor vector. Off-target editing was further characterized by amplicon-seq of potential off-target sites. Figure 14A provides a list of off-target sites with their chromosomal location and best match to the off-target consensus sequence. Figure 14B is a graph showing the indel percentages of OT1-OT10. Editing at OT1, OT4, and OT5 was significantly higher in ARCUS+ donor animals than in non-nuclease controls. Off-target editing was lower in neonates / infants than in adult NHPs.

[0125] The developing liver of neonatal / infant primates is highly receptive to site-specific insertion of donor genes. OTC levels consistently exceed the 5% therapeutic threshold, and the effect appears to be highly durable. Furthermore, neonatal / infant primates are resistant to systemic AAV toxicity. There was an excellent correlation between dosing and site-specific integration when comparing NHP and mouse models of OTCD.

[0126] In summary, we have identified a combination of ARCUS vector and hOTC co-donor vector that, when co-administered to neonatal macaques, can achieve transduction efficiencies of 12-18.6% in the liver at 3 months post-administration, both of which are substantially higher than the patient threshold for benefit (i.e., approximately 5% OTC-expressing hepatocytes). Animals in this study are being followed for long-term efficiency and safety evaluation. A second liver biopsy will be performed 1 year post-administration to assess the stability of hOTC transduction, histopathology in the liver, and on- and off-targeting in the liver.

[0127] Example 3 - PCSK9-hE7-KI Mouse Model The ARCUS targeting sequences in human and macaque PCSK9 genes are not conserved in the mouse Pcsk9 gene, so ARCUS cannot be used for genome editing in mouse genomic loci. Therefore, we asked The Jackson Laboratory to generate a knock-in mouse model (named PCSK9-hE7-KI mice) in which a region of the mouse Pcsk9 gene containing exon 7 is replaced with a region of the human PCSK9 gene containing exon 7 (Figures 5A-5C). This model can be used for evaluation of in vivo genome editing and gene targeting efficiency. PCSK9-hE7-KI mice were then transfected with sparse fur ash (spf ash ) mice. ash Mice carry a G to A point mutation at the splice donor site at the end of exon 4 of the Otc gene, which results in aberrant splicing of Otc mRNA, resulting in a 20-fold reduction in both OTC mRNA and protein expression (Hodges and Rosenberg, 1989). Affected animals have 5-10% residual OTC activity and can survive on a chow diet, but develop hyperammonia that can be lethal on a high-protein diet (Yang et al., 2016).

[0128] PCSK9-hE7-KI.spfash The mouse model can be used to evaluate the efficacy of in vivo gene targeting of human OTC and to demonstrate the correlation between targeting efficiency and efficacy. However, due to the small size of neonatal mice, evaluation of blood clinical pathology and clinical efficacy of gene targeting can only be performed as a terminal procedure after the mice have reached sufficient weight after weaning.

[0129] Figure 6 shows a sequence alignment of 265 bp sequences representing human PCSK9 sequence, mouse PCSK9 (mPCSK9) and rhesus monkey PCSK9 (rhPCSK9) of the PCSK9-hE7 knock-in allele. In this 265 bp region, there are six mismatches between the human and rhesus monkey sequences. The rodent and primate sequences are consistent with the various LINE and LTR insertions. It diverges beyond this window to account for the differences in the genomic structure of mPCSK9 between humans and mice. There are two amino acid differences in exon 7 between humans and mice. hE7-KI mice express normal levels of mPCSK9 by ELISA.

[0130] Example 4 - Pcsk9-hE7-KI.spf ash In vivo OTC gene targeting to the Pcsk9 locus in offspring This non-GLP compliant pharmacology study was conducted on neonatal PCSK9-hE7-KI.spf ash To evaluate whether ARCUS meganuclease-mediated knock-in of the human OTC gene in mice can achieve expression of therapeutic human OTC in target tissues for the treatment of OTC deficiency (liver) after a single co-administration of ARCUS nuclease expression vector and human OTC donor vector via the intended clinical ROA (IV). A schematic of the experimental design is shown in Figure 8A.

[0131] On day 0, newborn (PND1-2) male PCSK9-hE7-KI.spf ash Mouse, 1.0 x 10 13 AAVrh79 vector expressing ARCUS meganuclease (AAVrh79.TBG.PI.ARCUS.WPRE.bGH) at a dose of GC / kg was administered at 3.0 × 10 13The ARCUS meganuclease expression vector evaluated in this study (AAVrh79.TBG.PI.ARCUS.WPRE.bGH) was identical to the lead clinical candidate, while each hOTCco donor vector was identical to the lead clinical candidate, except for the HDR arm. Specifically, the clinical candidate contains a long version of the human HDR sequence (AAVrh79.hHDR.TBG.hOTCco.bGH), whereas the hOTCco donor vectors evaluated in this study contain a mouse-human hybrid HDR sequence (AAVrh79.mhHDR.TBG.hOTCco.bGH), a shorter version of the human HDR sequence (AAVrh79.shHDR.TBG.hOTCco.bGH), or no HDR sequence (AAVrh79.TBG.hOTCco.bGH). Figure 7 shows a comparison of the homology of the HDR arms with the human sequence, the knock-in mouse sequence, and the NHP sequence. As a negative control, an additional age-matched PCSK9-hE7-KI.spf ash Mice were administered an AAVrh79 vector that does not express the meganuclease (AAVrh79.TBG.PI.EGFP.WPRE.bGH) in combination with AAVrh79.mhHDR.TBG.hOTCco.bGH.

[0132] In-life evaluations include daily viability monitoring, body weight measurement, evaluation of plasma PCSK9, plasma NH3, and urinary orotic acid levels after high protein diet challenge, and partial hepatectomy on day 120 to evaluate the stability of human OTC transduction after two-thirds partial hepatectomy. Subgroups from each cohort are challenged with a 10-day high protein diet on days 49 and 170, and necropsy is performed at the end of the challenge. At necropsy, livers are harvested to evaluate knock-in of the human OTC gene, including evaluation of human OTC mRNA expression (in situ hybridization), OTC protein expression (immunostaining), and OTC enzyme activity assessed by staining and / or enzyme activity assays. Liver DNA is isolated to evaluate on-target editing (amplicon-seq, Oxford nanopore long-read sequencing) and to evaluate vector genome copies.

[0133] Mice administered vectors bearing mhHDR arms showed comparable survival rates to wild-type mice, and shHDR-treated mice achieved 80% survival after a 10-day high-protein diet challenge (Figure 9A). All treated mice maintained their body weight better than KI-spf-ash untreated mice (Figure 9B). Plasma ammonia levels in mHDR-treated mice were significantly reduced compared to untreated mice (Figure 9C).

[0134] mPCSK9 levels were measured on day 48 and all treated mice showed a decrease (Figure 9D ). Indel rates were fairly consistent between HDR types (Figure 9E). hOTC levels were increased in shHDR- and mhHDR-treated mice (Figure 9F).

[0135] Example 5 - Pcsk9-hE7-KI.spf ash Evaluating efficacy in offspring and determining the minimal effective dose This planned GLP-compliant pharmacology study will assess the neonatal PCSK9-hE7-KI.spf ashThe aim is to evaluate the efficacy of IV-administered AAV in a mouse model and determine the MED. The AAVrh79 vector expressing ARCUS meganuclease (AAVrh79.TBG.PI.ARCUS.WPRE.bGH) will be the toxicology vector lot manufactured for the planned GLP-compliant toxicity study. Instead of utilizing a test sample containing the long version of the human HDR sequence (AAVrh79.hHDR.TBG.hOTCco.bGH), this study will utilize a hOTCco donor vector containing a mouse-human hybrid HDR sequence (AAVrh79.mhHDR.TBG.hOTCco.bGH). This vector will be manufactured in a manner equivalent to the toxicology vector lots of the clinical candidates.

[0136] For this study, we used a mouse-human hybrid HDR sequence in a donor vector (AAVrh79.mhHDR.TBG.hOTCco.bGH) (donor sequence is PCSK9-hE7-KI.spf ash We chose to use a sequence similar to that in mouse (which is directly homologous to the sequence in mouse) to allow us to effectively study the pharmacology of this approach.

[0137] In this study, N=60 newborn (PND1-2) neonatal PCSK9-hE7-KI.spf ash Mice and N=15 age-matched male PCSK9-hE7-KI.WT (wild type) as controls will be evaluated. The study will include one necropsy time point (90 days) (see study design in Figure 15A). For efficacy evaluation, mice will be challenged with a 10-day course of high protein diet from day 81 to day 90. Survival rate, body condition, and biomarker changes will be evaluated. Mice will be administered three dose levels (1.0x10 12 GC / kg nuclease vector and 3.0×10 12 GC / kg donor vector, 3.3 × 10 12 GC / kg nuclease vector and 1.0×10 13 GC / kg donor vector or 1.0×10 13GC / kg nuclease vector and 3.0×10 13 Each mouse was administered one test article (100 mg / kg GC / kg, N=20 per dose) or vehicle (phosphate buffered saline [PBS], N=20). Dosages are shown in FIG. 15B.

[0138] In-life evaluations include daily viability checks, viability monitoring, body weight measurement, and evaluation of serum PCSK9, plasma NH3, and urinary orotic acid levels after high protein diet challenge. Necropsy is performed on day 90. At necropsy, blood is collected for CBC / differential and serum clinical chemistry analysis. A list of tissues is collected for histopathological evaluation. Livers are harvested to evaluate knock-in of the human OTC gene, including evaluation of human OTC mRNA expression (in situ hybridization), OTC protein expression (immunostaining), and OTC enzyme activity assessed by staining and / or enzyme activity assays. Liver DNA is also isolated to evaluate on-target editing (amplicon-seq) and to evaluate vector genome copies.

[0139] MED is vehicle-treated neonatal PCSK9-hE7-KI.spf ash Compared to control mice, AAV-treated neonatal PCSK9-hE7-KI.spf ash The results are based on analysis of survival rates after a high-protein diet in mice, plasma NH3 levels at the end of the high-protein diet challenge, human OTC mRNA and protein expression, OTC enzymatic activity, and on-target editing.

[0140] Example 8 - Pcsk9-hE7-KI.spf ash Toxicity studies in offspring Neonatal (PND1~2) PCSK9-hE7-KI.spf ashA 6-month GLP-compliant safety study will be conducted to examine the safety, tolerability, pharmacology, and pharmacokinetics of the test article following IV co-administration in mice. Interim analyses will include on-target editing, off-target editing, transgene expression, and histopathology analyses, which will be performed at days 60 and 180 (these time points allow sufficient time for nuclease-dependent gene insertion to reach stable steady-state levels following administration). Neonatal PCSK9-hE7-KI.spf ash Mice were treated with 3 dose levels (1.0 × 10 12 GC / kg nuclease vector and 3.0×10 12 GC / kg donor vector, 3.3 × 10 12 GC / kg nuclease vector and 1.0×10 13 GC / kg donor vector or 1.0×10 13 GC / kg nuclease vector and 3.0×10 13 Each rat is administered one test article (100 mg / kg GC / kg, N=20 per dose) or vehicle (phosphate buffered saline [PBS], N=20). After administration of the test article or vehicle, in-life evaluations include clinical observations to monitor daily for signs of distress and abnormal behavior, body weight measurements, and clinical serum chemistry of blood (specifically, ALT, AST, and total bilirubin).

[0141] Sixty days after test article administration, cohorts 1, 3, 5, and 7 will be euthanized and histopathological analysis will be performed on a comprehensive list of tissues including, but not limited to, brain, spinal cord, heart, liver, spleen, kidneys, lungs, reproductive organs, adrenal glands, and lymph nodes. Organ weights will be measured, if required.

[0142] Liver samples are collected and analyzed for on-target editing by amplicon-seq and AMP-seq, off-target editing by ITR-seq and amplicon-seq, vector biodistribution, and transgene expression. In liver samples, biodistribution is evaluated by PCR, and meganuclease RNA expression is analyzed by RT-PCR. Highly perfused organs are analyzed for meganuclease RNA, and tissues with detectable expression of meganuclease RNA are evaluated for on-target editing by amplicon-seq. Tissues with detectable on-target editing are further evaluated for off-target editing.

[0143] For vector biodistribution, qPCR detection specific for the transgenes (ARCUS and hOTCco) of the dual vector will be developed. Assay efficiency, linearity, precision, reproducibility, and detection limit will be evaluated using AAV cis plasmid as a surrogate for the target sequence. The lower limit of quantification (LLOQ) of the assay will be determined before assays on test tissues or excreta are initiated. A qualification plan will be implemented to bridge the transgene-specific assays to previously performed qualification studies. Matrices tested include the intended target, liver for biodistribution. Matrix effects will be further evaluated based on recovery of spiked target controls from all samples tested during the course of the biodistribution study as well as data subtracted from previously performed qualification studies.

[0144] Example 9 – Toxicity Study in Infant (6-9 Weeks Old) NHPs A 1-year GLP-compliant safety study will be conducted to examine the safety, tolerability, pharmacology, and pharmacokinetics of the test article following IV co-administration in infant (6-9 weeks old) rhesus monkeys. Interim analyses will include on-target editing, off-target editing, transgene expression, and histopathology analyses, which will be performed at day 90 (these time points allow sufficient time for nuclease-dependent gene insertion to reach stable steady-state levels following administration). Infant rhesus monkeys will receive 1.0 x 10 13GC / kg nuclease vector and 3.0×10 13 Each rat received either GC / kg (N=4) or vehicle (phosphate-buffered saline [PBS] / surfactant (N=2). After administration of the test article or vehicle, in-life evaluations included clinical observations to monitor for signs of distress and abnormal behavior daily, body weight measurements, and clinical serum chemistry of blood. (Specifically, ALT, AST, and total bilirubin) The study design is shown in FIG.

[0145] After 90 days of test article administration, one animal in the vehicle control group and one animal in group 3 are euthanized and histopathological analysis is performed on a comprehensive list of tissues including, but not limited to, brain, spinal cord, heart, liver, spleen, kidneys, lungs, reproductive organs, adrenal glands, and lymph nodes. Organ weights are measured as appropriate. On day 90, the remaining animals undergo liver biopsy.

[0146] Liver samples are collected and analyzed for on-target editing by amplicon-seq and AMP-seq, off-target editing by ITR-seq and amplicon-seq, vector biodistribution, and transgene expression. In liver samples, biodistribution is evaluated by PCR, and meganuclease RNA expression is analyzed by RT-PCR. Highly perfused organs are analyzed for meganuclease RNA, and tissues with detectable expression of meganuclease RNA are evaluated for on-target editing by amplicon-seq. Tissues with detectable on-target editing are further evaluated for off-target editing.

[0147] For vector biodistribution, qPCR detection specific for the transgenes (ARCUS and hOTCco) of the dual vector will be developed. Assay efficiency, linearity, precision, reproducibility, and detection limit will be evaluated using AAV cis plasmid as a surrogate for the target sequence. The lower limit of quantification (LLOQ) of the assay will be determined before assays on test tissues or excreta are initiated. A qualification plan will be implemented to bridge the transgene-specific assays to previously performed qualification studies. Matrices tested include the intended target, liver for biodistribution. Matrix effects will be further evaluated based on recovery of spiked target controls from all samples tested during the course of the biodistribution study as well as data subtracted from previously performed qualification studies.

[0148] All documents cited herein are incorporated herein by reference, and the sequences and text of the sequence listings submitted herewith are also incorporated by reference. U.S. Provisional Patent Application Nos. 63 / 301,917, filed January 21, 2022, 63 / 331,384, filed April 15, 2022, 63 / 364,861, filed May 17, 2022, 63 / 370,049, filed August 1, 2022, and 63 / 379,067, filed October 11, 2022, are each incorporated by reference in their entirety. The present invention has been described with reference to certain embodiments, but it will be understood that modifications can be made without departing from the spirit of the invention. Such modifications are intended to be within the scope of the appended claims. 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Claims

1. 1. A dual vector system for treating ornithine transcarbamylase deficiency, comprising: (a) a gene-edited AAV comprising a first AAV rh79 capsid and a first vector genome comprising a 5′ ITR, a sequence encoding a meganuclease having a sequence of SEQ ID NO:3, wherein the sequence targets PCSK9 under the control of a regulatory sequence that directs expression of the meganuclease in a target cell comprising the PCSK9 gene, and a 3′ ITR; and (b) a donor AAV vector comprising a second AAV rh79 capsid and a second vector genome comprising a 5' ITR, a 5' homologous recombination (HDR) arm, a transgene comprising the sequence of SEQ ID NO:4 encoding ornithine transcarbamylase (OTC), or a sequence at least 90% identical to SEQ ID NO:4, and a regulatory sequence directing expression of the transgene in the target cell, a 3' HDR arm, and a 3' ITR.

2. 2. The dual vector system of claim 1, wherein the sequence encoding the meganuclease comprises nucleotides (nt) 1089 to 2183 of SEQ ID NO:2, or a sequence at least 90% identical to nucleotides (nt) 1089 to 2183 of SEQ ID NO:

2.

3. The dual vector system of claim 1 or 2, wherein the transgene encoding OTC comprises SEQ ID NO:

4.

4. The dual vector system of any one of claims 1 to 3, wherein the first and second AAV capsids are AAVrh79 capsids of SEQ ID NO:

16.

5. The dual vector system according to any one of claims 1 to 3, wherein the ratio of the gene-editing AAV vector (a) to the donor AAV vector (b) is 1:

3.

6. The dual vector system according to any one of claims 1 to 5, wherein the nuclease is under the control of a TBG promoter.

7. The dual vector system according to any one of claims 1 to 6, wherein the transgene is under the control of a TBG promoter.

8. i) the first vector genome comprises nt 211 to 2964 of SEQ ID NO:2, or a sequence sharing at least 90% identity with nt 211 to 2964 of SEQ ID NO:2; ii) the second vector genome comprises nt 178 to 3281 of SEQ ID NO:6, or a sequence sharing at least 90% identity with nt 178 to 3281 of SEQ ID NO:

6. The dual vector system of any one of claims 1 to 7.

9. A method for treating OTC deficiency in humans by co-administering the dual vector system of any one of claims 1 to 8.

10. 1. A method of treating OTC deficiency in a subject, comprising administering to said subject having OTC: (a) a gene-edited AAV comprising a first AAV rh79 capsid and a first vector genome comprising a 5′ ITR, a sequence encoding a meganuclease having a sequence of SEQ ID NO:3, wherein the sequence targets PCSK9 under the control of a regulatory sequence that directs expression of the meganuclease in a target cell comprising the PCSK9 gene, and a 3′ ITR; and (b) co-administering a donor AAV vector comprising a second AAV rh79 capsid and a second vector genome comprising a 5' ITR, a 5' homologous recombination (HDR) arm, a transgene comprising the sequence of SEQ ID NO:4 encoding ornithine transcarbamylase (OTC), or a sequence at least 90% identical to SEQ ID NO:4, and a regulatory sequence directing expression of the transgene in the target cell, a 3' HDR arm, and a 3' ITR.

11. i) the first vector genome comprises nt 211 to 2964 of SEQ ID NO:2, or a sequence sharing at least 90% identity with nt 211 to 2964 of SEQ ID NO:2; ii) the second vector genome comprises nt 178 to 3281 of SEQ ID NO:6, or a sequence sharing at least 90% identity with nt 178 to 3281 of SEQ ID NO:

6.

12. The method of any one of claims 9 to 11, wherein the gene-edited AAV vector of (a) and the donor vector of (b) are delivered essentially simultaneously via IV.

13. The gene-editing AAV vector of (a) is about 1 × 10 １３ The method according to any one of claims 9 to 12, wherein the dose of GC / kg is suspended in a vehicle for injection.

14. (b) the AAV donor vector is about 3×10 １３ The method according to any one of claims 9 to 13, wherein the compound is suspended in a vehicle for injection at a concentration of GC / kg.

15. The method of any one of claims 9 to 14, wherein the subject is between 1 day and 4 months old.

16. The method of any one of claims 9 to 15, wherein the subject is male.

17. Use of the dual vector system according to any one of claims 1 to 8 for the treatment of OTC deficiency in a subject in need thereof.

18. Use of the dual vector system according to any one of claims 1 to 8 in the preparation of a medicament for the treatment of OTC deficiency in a subject in need thereof.

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