Compositions and methods for in vivo nuclease-mediated gene targeting for the treatment of genetic disorders - Patents.com

Abstract

A dual vector system for treating a genetic disorder is provided, the system comprising: (a) a gene editing vector comprising an expression cassette comprising a nucleic acid sequence encoding a nuclease and a regulatory sequence directing expression of the nuclease in a target cell comprising a PCSK9 gene; and (b) a donor vector comprising a nucleic acid sequence encoding an exogenous product for expression from the PCSK9 locus, the inserted nucleic acid sequence not encoding PCSK9, the system further comprising a sequence directing the nuclease to specifically target a native PCSK9 locus, and the native PCSK9 in the target cell is optionally ablated or reduced after administration of the dual vector system.

Description

[Background technology]

[0001] Site-specific nucleases (such as CRISPR-Cas9 or 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.

[0002] Homologous recombination repair (HDR) is a process in which DNA double-strand breaks (DSBs) are repaired by homologous recombination using a DNA template. This template can originate from within the cell during the late S or G2 phase of the cell cycle, when sister chromatids are available before the completion of mitosis. In addition, exogenous repair templates, most often in the form of synthetic single-stranded DNA donor oligos or donor plasmids, can be delivered into cells to generate precise changes in the genome.

[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] What is needed are improved compositions and methods for gene editing. Summary of the Invention

[0005] Provided herein are compositions, methods, systems, and kits for gene editing that allow for knockdown or ablation of the native PCSK9 gene, as well as the insertion and / or expression of exogenous transgenes at the PCSK9 locus.

[0006] In a first aspect, a system for treating a genetic disorder is provided herein. The system includes gene editing components, including an expression cassette, comprising a nucleic acid sequence encoding a nuclease that targets the PCSK9 gene and a regulatory sequence that directs expression of the nuclease in a target cell that includes the PCSK9 gene. The system further includes a donor vector, comprising a transgene cassette, comprising a nucleic acid sequence encoding a transgene and a regulatory sequence that directs expression of the transgene in a target cell, the donor vector further comprising homology-directed recombination (HDR) arms on the 5' and 3' sides of the transgene cassette, and the transgene is not PCSK9. The nuclease targets the PCSK9 gene. In some embodiments, the nuclease targets PCSK9 exon 7. In some embodiments, the meganuclease is an ARCUS meganuclease.

[0007] In some embodiments, the gene editing components comprise a sequence encoding Cas9. In certain embodiments, the gene editing vector further comprises a sequence encoding an sgRNA comprising a seed region of at least 20 nucleotides, where the sgRNA specifically binds to a target site in the PCSK9 gene, the target site being 5' to a protospacer adjacent motif (PAM) that is specifically recognized by Cas9.

[0008] In other embodiments, the donor vector further comprises a sequence encoding an sgRNA that includes a seed region of at least 20 nucleotides, where the sgRNA specifically binds to a target site in the PCSK9 gene, where the target site is 5' to a protospacer adjacent motif (PAM) that is specifically recognized by Cas9.

[0009] In another aspect, a system for treating genetic disorders is provided herein. The system includes a gene editing component comprising a nucleic acid sequence encoding a nuclease that targets the PCSK9 gene. The system further includes a donor vector comprising a transgene cassette comprising a nucleic acid sequence encoding a transgene and a regulatory sequence directing the expression of the transgene in a target cell, the donor vector further comprising homology-directed recombination (HDR) arms on the 5' and 3' sides of the transgene cassette, and the transgene is not PCSK9. The nuclease targets the PCSK9 gene. In certain embodiments, the gene editing component is provided in a lipid nanoparticle.

[0010] In some embodiments, the gene editing components comprise a sequence encoding Cas9. In certain embodiments, the gene editing vector further comprises a sequence encoding an sgRNA comprising a seed region of at least 20 nucleotides, where the sgRNA specifically binds to a target site in the PCSK9 gene, the target site being 5' to a protospacer adjacent motif (PAM) that is specifically recognized by Cas9.

[0011] In other embodiments, additional components of the donor vector include a sequence encoding an sgRNA comprising a seed region of at least 20 nucleotides, where the sgRNA specifically binds to a target site in the PCSK9 gene, the target site being 5' to a protospacer adjacent motif (PAM) that is specifically recognized by Cas9.

[0012] In certain embodiments, the transgene is associated with a liver metabolic disorder, hi certain embodiments, the transgene is OTC, PKU, CTLN1, or LDLR.

[0013] In certain embodiments, the vector is an adeno-associated virus (AAV) vector, wherein the vector comprises an AAV 5'ITR and an AAV 3'ITR.

[0014] In another embodiment, a dual vector system for treating a genetic disorder comprises a gene-edited AAV comprising an AAV capsid, 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 a PCSK9 gene, and a 3'ITR, and a donor AAV vector comprising an AAV capsid, a 5'ITR, a 5' homologous recombination (HDR) arm, a transgene and a regulatory sequence that directs expression of the transgene in a target cell, a 3'HDR arm, and a 3'ITR, wherein the transgene does not encode PCSK9.

[0015] In another embodiment, a dual vector system for treating a genetic disorder includes a gene-editing AAV comprising an AAV capsid and a first vector genome comprising a 5'ITR, a 5' nuclear localization signal (NLS), a regulatory sequence that directs expression of saCas9 in a target cell comprising a sequence encoding Cas9 and a PCSK9 gene, a 3'NLS, and a 3'ITR, and a donor AAV vector comprising an AAV capsid, a 5'ITR, a 5' homologous recombination (HDR) arm, a transgene and a regulatory sequence that directs expression of the transgene in a target cell, a 3'HDR arm, a U6 promoter, an sgRNA comprising at least 20 nucleotides that specifically binds to a target site in the PCSK9 gene, where the target site is 5' to a protospacer adjacent motif (PAM) that is specifically recognized by Cas9, and a 3'ITR, where the transgene does not encode PCSK9.

[0016] In yet another embodiment, a dual vector system for treating a genetic disorder includes a gene-editing AAV vector comprising an AAV capsid, a first vector genome comprising a 5'ITR, a U6 promoter, an sgRNA comprising at least 20 nucleotides that specifically binds to a target site in a PCSK9 gene, the target site being 5' to a protospacer adjacent motif (PAM) specifically recognized by Cas9, an sgRNA, a 5' nuclear localization signal (NLS), a sequence encoding Cas9 and a regulatory sequence that directs expression of Cas9 in a target cell comprising the PCSK9 gene, a 3'NLS, and a 3'ITR, and a donor AAV vector comprising a second vector genome comprising an AAV capsid, a 5'ITR, a 5' homologous recombination (HDR) arm, a transgene and a regulatory sequence that directs expression of the transgene in a target cell, a 3'HDR arm, and a 3'ITR.

[0017] In some embodiments, the gene-edited AAV vector and the donor AAV vector have the same AAV capsid. In other embodiments, the gene-edited AAV vector and the donor AAV vector have different AAV capsids. In some embodiments, the AAV capsid is selected from AAV8, AAV9, rh10, AAV6.2, AAV3B, hu37, rh79, and rh64.

[0018] In another aspect, a method of treating a disorder in a human by co-administering the dual vector system described herein is provided.

[0019] In another aspect, a method for treating a liver metabolic disorder in a subject is provided, the method comprising co-administering to a subject having a liver metabolic disorder a gene editing AAV vector, the gene editing AAV vector comprising a sequence encoding a nuclease and a regulatory sequence directing expression of the nuclease in a target cell comprising a PCSK9 gene, and a donor AAV vector, the donor AAV vector comprising a transgene and a regulatory sequence directing expression of the transgene in a target cell, the donor vector further comprising homologous recombination (HDR) arms on the 5' and 3' sides of the transgene cassette, the transgene not being PCSK9. In certain embodiments, the liver metabolic disorder is ornithine transcarbamylase. In other embodiments, familial hypercholesterolemia or phenylketonuria. In one embodiment, the subject is a newborn.

[0020] In another aspect, a system for treating genetic disorders is provided. The system includes a lipid nanoparticle (LNP) comprising an mRNA sequence encoding a nuclease targeting the PCSK9 gene, and a donor AAV vector, the donor AAV vector comprising a transgene and a regulatory sequence directing its expression in a target cell, the donor vector further comprising homologous recombination (HDR) arms on the 5' and 3' sides of the transgene, the transgene not being PCSK9. In some embodiments, the nuclease targets PCSK9 exon 7. In some embodiments, the meganuclease is an ARCUS meganuclease.

[0021] In other embodiments, the gene editing vector encodes Cas9. In certain embodiments, the gene editing vector further encodes an sgRNA comprising at least 20 nucleotides that specifically binds to a target site in the PCSK9 gene, the target site being 5' to a protospacer adjacent motif (PAM) that is specifically recognized by Cas9. In some embodiments, the system comprises an LNP, the LNP comprising both a Cas9 coding sequence and a gRNA.

[0022] In other embodiments, the donor vector further encodes a sgRNA comprising a seed region of at least 20 nucleotides, the sgRNA being specific for a target site in the PCSK9 gene. The target site is 5' to a protospacer adjacent motif (PAM) that is specifically recognized by Cas9.

[0023] In yet another aspect, a dual vector system for treating a genetic disorder is provided. The system includes a gene editing vector comprising an expression cassette, the expression cassette comprising a nucleic acid sequence encoding a nuclease and a regulatory sequence directing expression of the nuclease in a target cell comprising a PCSK9 gene, and a donor vector, the donor vector comprising a nucleic acid sequence encoding an exogenous product for expression from the PCSK9 locus, the inserted nucleic acid sequence not encoding PCSK9, the system further comprising a sequence directing the nuclease to specifically target a native PCSK9 locus, and the native PCSK9 in the target cell is optionally ablated or reduced after administration of the dual vector system.

[0024] In yet another aspect, methods are provided for treating a patient using the systems described herein, wherein the patient's native PCSK9 expression level is reduced and the patient expresses an exogenous product.

[0025] In yet another aspect, engineered coding sequences for ornithine transcarbamylase are provided, as well as vectors, expression cassettes, and recombinant viruses containing the same.

[0026] Other aspects and advantages of the present invention will become apparent from the following detailed description of the invention. [Brief description of the drawings]

[0027] [Figure 1] Schematic diagram of the rhPCSK9 locus showing the donor splice site within exon 7 and the HDR donor vector containing the donor template of interest (e.g., hFIX, hOTC). [Diagram 2] 1 shows the timeline of a pilot study involving hFIX minigene knock-in at the PCSK9 locus with ARCUS2 or SaCas9 in neonatal NHPs. [Figure 3A-3C] Schematic diagram of dual AAV vector system for SaCas9 or ARCUS-mediated gene correction. Figure 3A shows a schematic diagram of dual AAVhu37 vector system for ARCUS2-mediated gene correction, where the AAVhu37 donor vector contains the hOTC donor template sequence. Figure 3B shows a schematic diagram of dual AAVhu37 vector system for SaCas9-mediated gene correction (trans; AAVhu37-SaCas9), where the expression cassettes of SaCas9 and sgRNA are in two separate vectors, and the AAVhu37.sgRNA donor vector contains the hOTC donor template sequence and the U6.sgRNA cassette. Figure 3C shows a schematic diagram of dual AAVhu37 vector system for SaCas9-mediated gene correction (cis; AAVhu37.PCSK9-sgRNA.SaCaS9), where the expression cassettes of SaCas9 and sgRNA are in the same vector, and the hOTC donor vector is in a separate vector. [Fig. 4A-4H]Figure 4 shows an in vivo study of nuclease-mediated gene targeting in neonatal NHPs. Animals were administered 1x1013GC / kg AAVhu37.ARCUS2.WPRE and 3x1013GC / kg AAVhu37.hFIXco-HDR, or 1x1013GC / kg AAVhu37.SaCas9.WPRE and 3x1013GC / kg AAVhu37.hFIXco-HDR.U6.sgR, or 1x1013GC / kg AAVhu37.GFP.WPRE and 3x1013GC / kg AAVhu37.hFIXco-HDR.U6.sgR, as shown in Figures 4A, 4B, and 5G. Figure 4C shows hFIX levels (plotted as ng / mL) in neonatal NHPs at the indicated time points. FIG. 4D shows PCSK9 levels (plotted as percentage of baseline on day 0) in neonatal NHPs at the indicated time points. FIG. 4E shows ALT (alanine aminotransferase) levels (plotted as U / L) in neonatal NHPs at the indicated time points. FIG. 4F shows anti-FIX IgG levels (plotted as dilution factor, 1 / dilution) in neonatal NHPs at the indicated time points. FIG. 4G shows PCSK9 levels (plotted as ng / mL) in neonatal NHPs at the indicated time points. FIG. 4H shows body weight (plotted as g) measured in neonatal NHPs. [Figure 5A-5H]The results of the in vivo study described in FIG. 4 are shown, administered to 3-month-old infant NHPs. FIG. 5A shows hFIX levels (plotted as ng / mL) in infant NHPs at the indicated time points. FIG. 5B shows PCSK9 levels (plotted as percentage of baseline on day 0) in infant NHPs at the indicated time points. FIG. 5C shows ALT (alanine aminotransferase) levels in infant NHPs at the indicated time points (plotted as U / L). FIG. 5D shows anti-FIX IgG levels (plotted as dilution factor, 1 / dilution) in infant NHPs at the indicated time points. FIG. 5E shows PCSK9 levels (plotted as ng / mL) in infant NHPs at the indicated time points. FIG. 5F shows body weights measured at the indicated time points (plotted as g) in infant NHPs. FIG. 5G is a summary table showing data from the experiment described in FIGS. 4A-5G. FIG. 5H shows a comparison of various data between neonatal and infant NHPs tested. [Figures 6A-6G] Figure 6 shows vector transduction (GC) and transgene expression in liver biopsy samples taken at various days after treatment in NHPs treated as described in Figures 4A-4H. Figure 6A shows the level of vector transduction in liver biopsy samples plotted as AAV genome copies (GC) per diploid cell. Figure 6B shows the relative expression of transgene RNA in liver biopsy samples. Figure 6C shows dual in situ hybridization (ISH) using specific probes to detect FIX and ARCUS in liver biopsies. Figure 6D shows digitized ISH images used for quantification of the percentage of transduction. Figure 6E shows the transduction efficiency of the FIX transgene quantified by ISH and plotted as percent transduction. [Figure 7A-7L]Dual in situ hybridization (ISH) using specific probes to detect FIX and ARCUS in liver biopsies taken 84 days after treatment in NHPs shown in various magnification fields (NHPs treated with AAVhu37.ARCUS2 and AAVhu37.Donor-HDR-hFIX). FIG. 7A shows ISH-detected ARCUS in liver biopsies viewed at 4x magnification. FIG. 7B shows ISH-detected hFIX in liver biopsies viewed at 4x magnification. FIG. 7C shows an overlay image of ISH-detected ARCUS and hFIX viewed at 4x magnification. FIG. 7D shows ISH-detected ARCUS and hFIX viewed at 4x magnification as an overlay image with DAPI (nuclear stain). FIG. 7E shows ISH-detected ARCUS in liver biopsies viewed at 10x magnification. FIG. 7F shows ISH-detected hFIX in liver biopsies viewed at 10x magnification. FIG. 7G shows an overlay image of ISH-detected ARCUS and hFIX viewed at 10x magnification. FIG. 7H shows an overlay image of ISH-detected ARCUS and hFIX viewed at 10x magnification with DAPI (nuclear stain). FIG. 7I shows ISH-detected ARCUS expression in a liver biopsy viewed at 20x magnification. FIG. 7J shows ISH-detected hFIX in a liver biopsy viewed at 20x magnification. FIG. 7K shows an overlay image of ISH-detected ARCUS and hFIX viewed at 20x magnification. FIG. 7L shows ISH-detected ARCUS and hFIX viewed at 20x magnification with DAPI (nuclear stain). [Fig. 8A-8M]Dual in situ hybridization (ISH) using specific probes to detect FIX and ARCUS in liver biopsies taken 84 days after treatment in NHPs shown in various magnification fields (NHPs treated with AAVhu37.EGFP and AAVhu37.Donor-HDR-hFIX.U6.sgR). FIG. 8A shows ISH-detected GFP-WRPE in liver biopsies viewed at 4x magnification. FIG. 8B shows ISH-detected hFIX in liver biopsies viewed at 4x magnification. FIG. 8C shows overlaid images of ISH-detected GFP-WRPE and hFIX viewed at 4x magnification. FIG. 8D shows ISH-detected GFP-WRPE and hFIX viewed at 4x magnification as overlaid with DAPI (nuclear stain). FIG. 8E shows ISH-detected GFP-WRPE in liver biopsies viewed at 10x magnification. FIG. 8F shows ISH-detected hFIX in liver biopsies viewed at 10x magnification. FIG. 8G shows an overlay of ISH-detected GFP-WRPE and hFIX viewed at 10x magnification. FIG. 8H shows ISH-detected GFP-WRPE and hFIX viewed at 10x magnification overlaid with DAPI (nuclear stain). FIG. 8I shows ISH-detected GFP-WRPE expression in liver biopsies viewed at 20x magnification. FIG. 8J shows ISH-detected hFIX in liver biopsies viewed at 20x magnification. FIG. 8K shows an overlay of ISH-detected GFP-WRPE and hFIX viewed at 20x magnification. FIG. 8L shows ISH-detected GFP-WRPE and hFIX viewed at 20x magnification overlaid with DAPI (nuclear stain). FIG. 8M shows ISH-detected GFP-WRPE and hFIX in untreated controls viewed at 20x magnification, overlaid with DAPI (nuclear stain). [Figure 9]

[0036] Figure 2 shows ARCUS-mediated on-target editing in NHPs treated with AAVhu37.ARCUS2 and AAVhu37.Donor-HDR-hFIX. After 84 days of treatment, liver biopsies were taken and the percentage of total indels in the targeted regions present therein was calculated based on amplicon-seq. [Figure 10A-10B] Schematic diagram of PCSK9-hE7-KI mouse model. Figure 10A shows a schematic diagram of mouse pcsk9 exon 7 replaced with human pcsk9 exon 7 (hE7 contains ARCUS targeting sequence). The sequence of human PCSK9 exon 7 is shown in SEQ ID NO: 44. Figure 10B shows a schematic diagram of mating 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 mated with sparse fur ash (spfash) mice that show a 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 designated 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). [Figures 11A-11I]Figure 11I shows an in vivo study of nuclease-mediated gene targeting in neonatal NHPs for the vectors shown in Figure 11A. Figure 11A is a diagram showing the experimental design of the in vivo study of nuclease-mediated gene targeting in neonatal NHPs for the vectors described in Example 3. Animals 21-111, 21-122, and 21-113 were positive for AAV-binding antibody (BAb) before administration. Day 0 samples of 21-178 were taken after vector administration that prevented the Bab assay. c: The number of OT sites identified in independent ITRseq assays is listed. Figure 11B shows PCSK9 levels shown as ng / mL (top row) or % of day 0 (bottom row) for the groups shown in Figure 11A. Figure 11C shows ALT levels shown as U / L (top row) or AST shown as U / L (bottom row) for the groups shown in Figure 11A. Figure 11D shows the transduction efficiency of the OTC transgene, quantified by ISH or IF and plotted as the percentage of transduced hepatocytes. Figure 11E shows the body weight of the mice. Figure 11F shows the GC of the vector in the liver by quantitative PCR analysis at day 84. Figure 11G shows the expression of hOTC and nuclease in the liver of macaque monkeys at day 84, 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 11H shows the indel analysis of the rhPCSK9 target locus by amplicon-seq. Figure 11I is a schematic diagram of the timeline of the in vivo test of nuclease-mediated gene targeting in neonatal NHPs, including the vectors tested for the experiments described in Example 3. [Figure 12]Sequence alignment of 265 bp sequences representing human PCSK9 sequence, mouse PCSK9 (mPCSK9) and rhesus PCSK9 (rhPCSK9) of pcsk9-hE7 knock-in allele. 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 13] 1 shows a schematic diagram of the donor construct for the dual AAV vector system for ARCUS2-mediated gene correction. The AAV donor vector contains the hOTC donor template sequence. The homology of the HDR arms in the construct with the target regions in knock-in mouse models (FIGS. 10A-10B), NHP, and humans is shown. [Figure 14A] FIG. 1 shows a timeline of studies involving knock-in of hOTC minigene at the PCSK9 locus with ARCUS2, performed in PCSK9-hE7-KI.spf-ash pups (partial OTC deficiency model), as described in Example 5. [Figure 14B] The vector and dosage each group received for the study is shown in Figure 14A. [Fig. 14C-14I] 14 shows the results of a study of mice treated with the vectors shown in FIG. 7 or untreated (KI WT) mice fed a high protein (HP) diet for 10 days. FIG. 14C shows the survival probability. FIG. 14D shows the body weight as a percentage of the body weight before the introduction of the HP diet. FIG. 14E shows the plasma NH3 levels on day 10 of the HP diet. FIG. 14F shows the mPCSK9 protein levels on day 48. FIG. 14G shows the indels (%) measured by amplicon-seq on day 59. FIG. 14H shows the level of vector transduction in liver biopsy samples plotted as AAV genome copies (GC) per diploid cell measured on day 59. FIG. 14I shows the IF of OTC at week 8. [Figure 15]FIG. 13 is a schematic diagram of the experimental design described in Example 10 for generating a hLDLR minigene knock-in at the PCSK9 locus by SaCas9 in PCSK9-hE7-KI.ldlr- / ldlr-.apobec- / apobec- pups (hoFH model). [Figure 16] FIG. 1 is a schematic diagram showing the vector used in Example 10. [Figure 17] 1 shows the experimental design of Example 10. [Figures 18A-18D] The results of the experiment of Example 10 are shown. Figure 18A shows serum LDL levels of shHDR+saCas9, mhHDR+saCas9, shHDR only, and untreated mice. Figure 18B shows the percentage of indels in mice treated with shHDR+saCas9, mhHDR+saCas9, and shHDR only. Figure 18C shows the hLDLR genome copies per diploid genome measured in liver at day 63. Figure 18D shows serum LDL levels of shHDR+saCas9, mhHDR+saCas9, shHDR only, and untreated mice at day 63. [Figure 19] 1 shows immunohistochemistry data for liver samples taken on day 63 for mice in Example 10. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] 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 in a target cell and delivers the nuclease to a donor vector. This provides a template containing an exogenous product for integration into and expression from the PCSK9 locus, such that the inserted nucleic acid sequence does not encode PCSK9, resulting in disruption of expression of endogenous PCSK9 and reduced expression levels.

[0029] 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. Human PCSK9 has a protein sequence of NP_777596.2, as shown in SEQ ID NO:23, and a coding sequence as shown in SEQ ID NO:22.

[0030] 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.

[0031] The compositions, kits, and methods provided herein include gene editing components (in some embodiments, vectors) and a donor vector (which provides a therapeutic transgene for expression in a host cell).

[0032] Gene editing components The compositions, kits, and methods provided herein include a gene editing component, which includes a nuclease (or coding sequence accordingly) and a sequence that directs the nuclease to specifically target the native PCSK9 locus on chromosome 1. As used herein, a "target PCSK9 locus" or "PCSK9 locus" is any site in the PCSK9 coding region where insertion of a heterologous transgene is desired. In certain embodiments, the target PCSK9 locus is in exon 7 of the PCSK9 coding sequence. Figure 12 provides an alignment of human (h), rhesus (rh), and mouse (m) PCSK9 exon 7 splice sites exemplified herein using SaCas9 and a PCSK9-targeting meganuclease (referred to as ARCUS).

[0033] 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 nucleases are naturally occurring. In other embodiments, the nucleases are not naturally occurring, i.e., engineered in DNA binding and / or cleavage domains. For example, the DNA binding domain of a naturally occurring nuclease can be modified to bind to a selected target site (e.g., meganucleases engineered to bind to a site different from its cognate binding site). In other embodiments, the nuclease comprises a heterologous DNA binding domain and a cleavage domain (e.g., zinc finger nucleases, TAL effector nucleases, meganuclease DNA binding domains with heterologous cleavage domains).

[0034] In certain embodiments, the nuclease is a meganuclease that targets PCSK9. Meganucleases have large recognition sites (double-stranded DNA sequences of 12-40 base pairs), e.g. For example, an endodeoxyribonuclease characterized by I-SceI. When combined with a nuclease, it can cleave DNA at a specific position. Restriction enzymes can be introduced into cells for use in gene editing or for in situ genome editing. In certain embodiments, the nuclease is a member of the LAGLIDADG (SEQ ID NO: 31) family of homing endonucleases. In certain embodiments, the nuclease is a member of the I-CreI family of homing endonucleases, which recognizes and cleaves a 22-base pair recognition sequence (SEQ ID NO: 32-CAAAACGTCGTGAGACAGTTTG). See, for example, WO2009 / 059195. A method for rationally designing mono-LAGLIDADG (SEQ ID NO: 32) homing endonucleases was described (WO2007 / 047859) that comprehensively redesigns I-CreI and other homing endonucleases to target widely divergent DNA sites, including sites in mammalian, yeast, plant, bacterial, and viral genomes. In one embodiment, the nuclease is encoded by the sequence set forth in SEQ ID NO: 19 (nt 330-1424), or a sequence sharing at least 95%, 98%, or 99% identity therewith. In one embodiment, the protein sequence of the nuclease is the sequence set forth in SEQ ID NO: 20, or a sequence sharing at least 95%, 98%, or 99% identity therewith. Such nucleases are sometimes referred to herein as ARCUS nucleases. The term "homing endonuclease" is synonymous with the term "meganuclease." See WO2018 / 195449, which describes certain PCSK9 meganucleases, the entirety of which is incorporated herein by reference.

[0035] Zinc finger nucleases (ZFNs) are artificial restriction enzymes that are generated by fusing a zinc finger DNA binding domain to a DNA cleavage domain. The zinc finger domain can be engineered to target specific desired DNA sequences, allowing zinc finger nucleases to target unique sequences within complex genomes. By utilizing endogenous DNA repair mechanisms, these reagents can be used to precisely modify the genomes of higher organisms and serve as outstanding tools in the field of genome editing. Transcription activator-like effector nucleases (TALENs) are restriction enzymes that can be engineered to cleave specific sequences of DNA. They are created by fusing a TAL effector DNA binding domain to a DNA cleavage domain (a nuclease that cleaves DNA strands). In another embodiment, the coding sequence encodes a zinc finger nuclease or a transcription activator-like (TAL) effector nuclease (TALEN).

[0036] In certain embodiments, the nuclease is a CRISPR-associated nuclease (Cas), optionally Cas9. "Cas9" (CRISPR-associated protein 9) refers to a family of RNA-guided DNA endonucleases characterized by two signature nuclease domains RuvC (cleaves the non-coding strand) and HNH (coding strand). Suitable bacterial sources of Cas9 include Staphylococcus aureus (SaCas9), Streptococcus pyogenes (SpCas9), and Neisseria meningitides [KM Estelt et al, Nat Meth, 10:1116-1121 (2013)]. Wild-type coding sequences may be utilized in the constructs described herein. Alternatively, bacterial codons are optimized for expression in humans, for example, using any of a variety of known human codon optimization algorithms. Alternatively, these sequences may be produced synthetically, in whole or in part. Other endonucleases with similar properties may optionally be substituted, see, for example, the public CRISPR database (db), accessible at http: / / crispr.u-psud.fr / crispr.

[0037] In certain embodiments, the compositions, kits, and methods of the nuclease coding sequence include Contained in an editing vector. The gene editing vector contains an expression cassette that includes a nucleic acid sequence encoding a nuclease and a regulatory sequence that directs expression of the nuclease in a target cell that contains the PCSK9 gene.

[0038] As used herein, a "vector" is a biological or chemical moiety that contains a nucleic acid sequence and can be introduced into a suitable host cell for replication or expression of the nucleic acid sequence. Common vectors include non-viral and viral vectors. As used herein, non-viral systems can be selected from nanoparticles, electroporation systems, and novel biomaterials, naked DNA, phages, transposons, plasmids, cosmids (Phillip McClean, www.ndsu.edu / pubweb / ~mcclean / -plsc731 / cloning / cloning4.htm), and artificial chromosomes (Gong, Shiaoching, et al. "A gene expression atlas of the central nervous system based on bacterial artificial chromosomes." Nature 425.6961 (2003): 917-925).

[0039] 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 contain coding sequences for gene products described herein adjacent to packaging signals of the viral genome, and other expression control sequences such as those described herein. In certain embodiments, a vector genome may contain two or more expression cassettes.

[0040] In addition to the coding sequence for the nuclease, in certain embodiments, the gene editing vector includes a regulatory sequence that directs the expression of the nuclease in the host cell. In certain embodiments, the regulatory element includes a promoter. In certain embodiments, the system is designed for the treatment of a metabolic disorder characterized by a mutation or phenotype in liver cells, and the gene editing vector can be designed such that the nuclease is expressed under the control of a liver-specific promoter. Exemplary plasmids and vectors described herein use the liver-specific promoter thyroxine-binding globulin (TBG), characterized by the sequence of SEQ ID NO: 41. In other embodiments, a shortened version of TBG (a variant referred to herein as TBG-S1), characterized by the sequence of SEQ ID NO: 11, is useful. In another embodiment, a hybrid liver promoter (HLP) having the sequence of SEQ ID NO: 12 is utilized.

[0041] In some embodiments, it is desirable to utilize a promoter with low transcriptional activity, or a weak promoter. In one embodiment, the promoter is a weakened version of the liver-specific thyroxine-binding globulin (TBG) promoter. In one embodiment, the weak promoter is truncated at the 5' or 3' end of the native promoter or the TBG-S1 sequence. In another embodiment, the promoter is truncated at the 3' end 113 nt from the TBG-S1 promoter. Only the 1444 sequence was retained and referred to as F113 (also referred to as TBG-S1-F113) (SEQ ID NO: 19, nt 206-318). U.S. Provisional Patent Application Nos. 63 / 016,145, filed April 27, 2020, 63 / 033,738, filed June 2, 2020, 63 / 089,796, filed October 9, 2020, PCT / US21 / 29386 and PCT / US21 / 29403, both filed April 27, 2021, are each entitled "Compositions and Methods for Reducing Nuclease Expression and Off-Target Activity Using an A Promoter with Low Transcriptional Activity," and are incorporated herein by reference in their entireties.

[0042] Alternatively, other liver-specific promoters, such as α1 antitrypsin (A1AT), human albumin (Miyatake et al., J. Virol., 71:5124-32 (1997)), and the hepatitis B virus core promoter (Sandig et al. al., Gene Ther.,3:1002 9(1996), TTR minimal enhancer / promoter, alpha-antitrypsin promoter, LSP (845nt) may be used. See, for example, Liver-Specific Gene Promoter Database, Cold Spring Harbor, http: / / rulai.schl.edu / LSPD. Alternatively, other tissue-specific promoters may be used, such as muscle-specific promoters, such as muscle creatine kinase (MCK) promoter, or muscle hybrid (MH) promoter. Alternatively, other promoters may be utilized in the vectors described herein, such as constitutive promoters (CMV, CBG, CB7, etc.), regulatable (inducible) promoters [see, for example, WO2011 / 126808 and WO2013 / 049493 (incorporated herein by reference)], or promoters that respond to physiological cues. Optionally, if a regulatable system is selected, a third vector may be required to provide the regulatable function.

[0043] In addition to a promoter, the gene editing cassette, expression cassette, and / or vector may 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. 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 and the transgene coding sequence.

[0044] In certain embodiments, the gene editing vector comprises a TBG promoter, one or more αmic / bik enhancers, a coding sequence for ARCUS meganuclease, optionally a WPRE, and a poly A. In certain embodiments, the expression cassette comprises nt 211 to nt 2964 of SEQ ID NO:42.

[0045] In some embodiments, the gene editing components further comprise a sequence that directs the nuclease to a target site in the PCSK9 target locus. In certain embodiments, such as meganucleases specific for PCSK9, no additional sequence is required to direct the nuclease to the target site. However, for example, in the case of Cas9, an additional sequence specific for the target sequence is provided, called a "single guide RNA" or "sgRNA." The sgRNA can be provided on the same vector as Cas9 (cis) or on a different vector (trans). As used herein, the sgRNA, in combination with the gRNA scaffold, has at least 20 bases of sequence (or about 24-28 bases, sometimes referred to as the seed region) for specific DNA binding (i.e., homology to the target DNA). Transcription of the sgRNA must be initiated precisely at its 5' end. When targeting a template DNA strand, the base-paired region of the sgRNA has the same sequence identity as the transcribed sequence. When targeting a non-template DNA strand, the base-paired region of the sgRNA is the reverse complement of the transcribed sequence. Optionally, the gene editing vector may contain two or more sgRNAs. The sgRNA is 5' to a protospacer adjacent motif (PAM) that is specifically recognized by the Cas9 (or Cpf1) enzyme. Typically, the sgRNA is "immediately" 5' to the PAM sequence, i.e., there is no spacer or intervening sequence. In one embodiment, the sgRNA "seed" coding sequence is AAGTTGGTCCCAAAGTCCC (SEQ ID NO: 8), which is useful for SaCas9 to target exon 7 of human and macaque PCSK9. However, other sgRNAs can be designed by one of skill in the art.

[0046] In certain embodiments, the sgRNA comprises at least 20 nucleotides and specifically binds to a target site in the PCSK9 gene, the target site being 5' to a protospacer adjacent motif (PAM) that is specifically recognized by Cas9. In some embodiments, the seed region shares 100% complementarity with the target site in the PCSK9 gene. In other embodiments, the seed region contains 1, 2, 3, 4, or 5 mismatches compared to the target site.

[0047] The sgRNA is under the control of an RNA polymerase promoter and / or terminator. In certain embodiments, the RNA polymerase promoter is a PolIII promoter, such as a U6 promoter. In another embodiment, the promoter is an H1 promoter. An exemplary sequence of a U6 promoter can be found in SEQ ID NO: 10. In other embodiments, the sgRNA and the RNA polymerase promoter are located in the donor vector.

[0048] In other embodiments, for example when the nuclease is Cas9, the gene editing components further comprise one or more nuclear localization signals (NLS). In one embodiment, the NLS is adjacent to the coding sequence of Cas9. In certain embodiments, the NLS has the sequence of nt 4241-4288 of SEQ ID NO:5. See, e.g., Lu et al. Types of nuclear localization signals and mechanisms of protein import into the nucleus, Cell Commun Signal (May 2021) 19:60 (incorporated herein by reference).

[0049] In certain embodiments, the coding sequence for the nuclease is provided as a messenger RNA (mRNA). The mRNA may include a 5' untranslated region, a 3' untranslated region, and / or a coding or translation sequence. In certain embodiments, the coding sequence for Cas9 is provided as an mRNA.

[0050] The mRNA may be naturally occurring or non-naturally occurring. The mRNA may contain one or more modified nucleobases, nucleosides, or nucleotides. In some embodiments, the mRNA in the compositions of the invention contains at least one modification that confers improved or enhanced stability to the nucleic acid, including, for example, improved resistance to nuclease digestion in vivo. The mRNA may contain any number of base pairs, including tens, hundreds, or thousands of base pairs. Any number (e.g., all, some, or none) of the nucleobases, nucleosides, or nucleotides may be substituted, modified, or otherwise analogous to non-naturally occurring canonical species. In certain embodiments, all of a particular nucleobase type may be modified. For example, For example, all cytosines in an mRNA may be 5-methylcytosines. As used herein, the terms "modified" and "modified", when such terms relate to the nucleic acids provided herein, preferably include at least one modification that improves stability and renders the mRNA more stable (e.g., resistant to nuclease digestion) than wild-type or naturally occurring versions of the mRNA. As used herein, the terms "stable" and "stability", when such terms relate to the nucleic acids of the invention (particularly with respect to mRNA), refer to, for example, improved or enhanced resistance to degradation by nucleases (i.e., endonucleases or exonucleases) that would normally degrade such mRNA. Improved stability may include, for example, reduced susceptibility to hydrolysis or other destruction by endogenous enzymes (e.g., endonucleases or exonucleases) or conditions within a target cell or tissue, thereby increasing or enhancing the presence of such mRNA in a target cell, tissue, subject, and / or cytoplasm. The stabilized mRNA molecules provided herein exhibit a longer half-life compared to their naturally occurring unmodified counterparts (e.g., wild-type versions of the mRNA). The terms "modification" and "modified", as such terms relate to the mRNAs of the present invention, also contemplate alterations that improve or enhance translation of the mRNA nucleic acid, including, for example, the inclusion of sequences that function in the initiation of protein translation (e.g., Kozak consensus sequences).

[0051] In some embodiments, the mRNAs described herein are chemically or biologically modified to make them more stable. Exemplary modifications to mRNA include base depletion (e.g., by deleting one nucleotide or replacing it with another nucleotide) or base modification (e.g., chemical modification of a base). As used herein, the phrase "chemical modification" includes modifications that introduce chemicals different from those found in naturally occurring mRNA, such as covalent modifications such as the introduction of modified nucleotides (e.g., nucleotide analogs, or the inclusion of pendant groups not naturally found in such mRNA molecules).

[0052] In some embodiments, the number of C and / or U residues in the mRNA sequence is reduced. In another embodiment, the number of C and / or U residues is reduced by replacing one codon encoding a particular amino acid with another codon encoding the same or a related amino acid. Contemplated modifications to the mRNA nucleic acid of the present invention also include the incorporation of pseudouridine, pseudouridine (ψ) or 5-methylcytosine (m5C). Substitutions and modifications to the mRNA of the present invention can be made by methods readily known to those skilled in the art.

[0053] In certain embodiments, the mRNA comprises a 5' cap structure, a chain terminating nucleotide, a stem loop, and / or a polyadenylation signal. The cap structure or cap species is a compound comprising two nucleoside moieties joined by a linker and may be selected from a naturally occurring cap, a non-naturally occurring cap or cap analog, or an anti-reverse cap analog. The mRNA may alternatively or additionally comprise a chain terminating nucleoside.

[0054] In certain embodiments, the mRNA comprises a stem loop, such as a histone stem loop. The stem loop may comprise 1, 2, 3, 4, 5, 6, 7, 8, or more nucleotide base pairs. The stem loop may be located in any region of the mRNA. For example, the stem loop may be located in an untranslated region (5' untranslated region or 3' untranslated region), a coding region, or in, before, or after a polyA sequence or polyA tail.

[0055] In certain embodiments, the mRNA comprises a polyA sequence, which may be composed entirely or predominantly of adenine nucleotides or analogs or derivatives thereof. In the , the polyA sequence is a tail located adjacent to the 3' untranslated region of the mRNA.

[0056] The mRNA can encode any polypeptide of interest (e.g., a nuclease), including any naturally occurring or non-naturally occurring or otherwise modified polypeptide. The polypeptide encoded by the mRNA can be of any size and have any secondary structure or activity. In some embodiments, the polypeptide encoded by the mRNA can have a therapeutic effect when expressed in a cell.

[0057] Donor Vector The compositions, kits, and methods include a donor vector that provides a coding sequence for a therapeutic transgene. In certain embodiments, the donor vector includes an expression cassette that includes a nucleic acid sequence that encodes the transgene and a regulatory sequence that directs the expression of the transgene in a target cell. In certain embodiments, the transgene encodes a protein that is abnormally expressed in a liver metabolic disorder or other genetic disorder. The transgene encodes a protein other than PCSK9. Such proteins include, but are not limited to, OTC, low density lipoprotein receptor (LDLr), Factor IX, e.g., the sequence set forth in SEQ ID NO: 55 or 56, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity therewith, and Factor VIII, e.g., the sequence set forth in SEQ ID NO: 53 or 54, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity therewith.

[0058] Further exemplary genes that can be delivered via the donor vector include glucose-6-phosphatase associated with glycogen storage disease or type 1A deficiency (GSD1), phosphoenolpyruvate-carboxykinase (PEPCK) associated with PEPCK deficiency, cyclin-dependent kinase-like 5 (CDKL5), also known as serine / threonine kinase 9 (STK9), associated with seizures and severe neurodevelopmental disorders, galactose-1-phosphate uridyltransferase associated with galactosemia (e.g., as set forth in SEQ ID NO: 63 or 64), and galactose-1-phosphate uridyltransferase associated with galactosemia (e.g., as set forth in SEQ ID NO: 64). phenylalanine hydroxylase (PAH) associated with phenylketonuria (PKU), hydroxyacid oxidase 1 (GO / HAO1) ​​(e.g., a sequence set forth in SEQ ID NO: 49 or 50, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity therewith), gene products associated with primary hyperoxaluria type 1, including AGXT (e.g., a sequence set forth in SEQ ID NO: 47 or 48, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto); branched-chain α-keto acid dehydrogenases including BCKDH, BCKDH-E2, BAKDH-E1a, and BAKDH-E1b associated with maple syrup urine disease; fumarylacetoacetate hydrolase associated with tyrosinemia type 1; Methylmalonyl-CoA mutase associated with tauric acidemia, medium-chain acyl-CoA dehydrogenase associated with medium-chain acetyl-CoA deficiency, ornithine transcarbamylase (OTC) associated with ornithine transcarbamylase deficiency, argininosuccinate synthetase (ASS1) associated with citrullinemia (e.g., a sequence set forth in SEQ ID NO: 69 or 70, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto),Lecithin-cholesterol acyltransferase (LCAT) deficiency, methylmalonic acidemia (MMA), NPC1 associated with Niemann-Pick disease type C1, propionic acidemia (PA), low density lipoprotein receptor (LDLR) protein associated with familial hypercholesterolemia (FH) (e.g., as set forth in SEQ ID NO: 73 or 74, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity therewith (e.g., LDLR variants such as those described in WO2015 / 164778), or a sequence set forth in SEQ ID NO: or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity therewith), ApoE and ApoC proteins associated with dementia, Crigler-Nagy lipoprotein lipase (LPL) associated with leukemia (e.g., a sequence set forth in SEQ ID NO: 67 or 68, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto), UDP-glucuronosyltransferase, adenosine deaminase associated with severe combined immunodeficiency, hypoxanthine guanine phosphoribosyltransferase associated with gout and Lesch-Nyhan syndrome, biotinylase associated with biotinylase deficiency, Fabry-Perot syndrome, α-galactosidase A (α-GalA) associated with Wilson's disease (e.g., a sequence set forth in SEQ ID NO: 75 or 76, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto), β-galactosidase (GLB1) associated with GM1 gangliosidosis, ATP7B associated with Wilson's disease, β-glucocerebrosidase associated with Gaucher disease types 2 and 3 (e.g., a sequence set forth in SEQ ID NO: 51 or 52, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto), , 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto), peroxisomal membrane protein 70 kDa associated with Zellweger syndrome, arylsulfatase A (ARSA) associated with metachromatic leukodystrophy, galactocerebrosidase (GALC) enzyme associated with Krabbe disease, alpha-glucosidase (GAA) associated with Pompe disease (e.g., a sequence set forth in SEQ ID NO: 79 or 80, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto).93%, 94%, 95%, 96%, 97%, 98%, 99% identity), the sphingomyelinase (SMPD1) gene associated with Niemann-Pick disease type A, carnosinase (CN1), hypoxanthine-guanine phosphoribosyltransferase (HGPRT), erythropoietin (EPO), carbamyl phosphate synthetase (CPS1), N-acetylglutamate synthetase (NAGS), argininosuccinate lyase (ASL) (argininosuccinic aciduria) (e.g., sequences set forth in SEQ ID NO: 57 or 58, or sequences with at least one sequence thereof). argininosuccinate synthase associated with adult-onset type II citrullinemia (CTLN2) (WO 2018 / 144709, incorporated herein by reference); carbamoyl phosphate synthase 1 (CPS1) associated with urea cycle disorders; survival of motor neuron (SMN) protein associated with spinal muscular atrophy; ceramidase associated with Farber lipogranulomatosis; GM2 ganglion protein (GM2G) associated with mitochondrial fibrosis; b-hexosaminidase associated with urothelial stenosis and Tay-Sachs disease and Sandhoff disease, aspartylglucosaminidase associated with aspartylglucosaminuria, alpha-fucosidase associated with fucosidosis, alpha-mannosidase associated with alpha-mannosidosis, porphobilinogen deaminase associated with acute intermittent porphyria (AIP), alpha-1 alpha-1 antitrypsin for the treatment of alpha-1 antitrypsin deficiency (emphysema) (e.g., a sequence as set forth in SEQ ID NO: 77 or 78, or a sequence having at least 70%, 75% , 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity), erythropoietin for the treatment of anemia due to thalassemia or renal failure, vascular endothelial growth factor, angiopoietin-1, and fibroblast growth factor for the treatment of ischemic disease, thrombomodulin and tissue factor pathway inhibitor for the treatment of blocked blood vessels such as those found in atherosclerosis, thrombosis, or embolism, aromatic amino acid decarboxylase (AADC) and tyrosine hydroxylase (TYH) for the treatment of Parkinson's disease. These include, but are not limited to, antibodies against phospholamban (TH), beta adrenergic receptors for the treatment of congestive heart failure, antisense to or mutant forms of phospholamban, sarcoplasmic reticulum adenosine triphosphatase-2 (SERCA2), and cardiac adenylyl cyclase, tumor suppressor genes such as p53 for the treatment of various cancers, cytokines (e.g., one of the various interleukins) for the treatment of inflammatory disorders, immune disorders, and cancer, dystrophin or mini-dystrophin and utrophin or mini-utrophin for the treatment of muscular dystrophies, and insulin or GLP-1 for the treatment of diabetes.

[0059] Examples of suitable transgenes for delivery can include those associated with familial hypercholesterolemia (e.g., VLDLr, LDLr, ApoE, see, e.g., WO2020 / 132155, WO2018 / 152485, WO2017 / 100682, which are incorporated by reference herein), muscular dystrophy, cystic fibrosis, and rare or orphan diseases. Examples of such rare diseases may include, among others, spinal muscular atrophy (SMA), Huntington's disease, Rett syndrome (e.g., methyl-CpG binding protein 2 (MeCP2), UniProtKB-P51608), amyotrophic lateral sclerosis (ALS), Duchenne muscular dystrophy, Friedreich's ataxia (e.g., frataxin), progranulin (PRGN) (associated with non-Alzheimer's brain degeneration, including frontotemporal dementia (FTD), progressive non-fluent aphasia (PNFA), and semantic dementia).Other useful gene products include carbamoyl synthetase I, ornithine transcarbamylase (OTC), argininosuccinate synthetase, argininosuccinate lyase (ASL) for the treatment of argininosuccinate lyase deficiency, arginase, fumaroacetate hydrolase, phenylalanine hydroxylase, alpha-1 antitrypsin, rhesus alpha-fetoprotein (AFP), rhesus chorionic gonadotropin (CG), glucose-6 - phosphatase (e.g., a sequence as set forth in SEQ ID NO: 59 or 60, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto), hereditary angioedema-associated plasma protease C1 inhibitor (SERPING1) (e.g., a sequence as set forth in SEQ ID NO: 61 or 62, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity), porphobilinogen deaminase, cystatin beta synthase associated with homocystinuria (e.g., a sequence set forth in SEQ ID NO: 65 or 66, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity therewith), branched-chain keto acid decarboxylase, albumin, isoforms, Valeryl-CoA dehydrogenase, propionyl-CoA carboxylase, methylmalonyl-CoA mutase, glutaryl-CoA dehydrogenase, insulin, β-glucosidase, pyruvate carboxylate, hepatic phosphorylase, phosphorylase kinase, glycine decarboxylase, protein H, protein T, cystic fibrosis transmembrane regulator (CFTR) sequence, and dystrophin gene product (e.g., mini- or micro-dystrophin). Still other useful gene products include enzymes that may be useful in enzyme replacement therapy, which is useful in various conditions caused by insufficient enzyme activity. For example, enzymes that contain mannose-6-phosphate can be used to treat lysosomal storage diseases (e.g., suitable genes include the gene encoding β-glucuronidase (GUSB)).Examples of suitable transgenes for delivery may include human frataxin delivered with an AAV vector, as described in PCT / US20 / 66167, filed December 18, 2020, U.S. Provisional Patent Application No. 62 / 950,834, filed December 19, 2019, and U.S. Provisional Patent Application No. 63 / 136,059, filed January 11, 2021, which are incorporated by reference herein. Another example of a suitable transgene for delivery may be, for example, the human frataxin delivered with an AAV vector, as described in PCT / US20 / 66167, filed December 18, 2020, U.S. Provisional Patent Application No. 62 / 950,834, filed December 19, 2019, and U.S. Provisional Patent Application No. 63 / 136,059, filed January 11, 2021, which are incorporated by reference herein. No. 62 / 840,911, filed April 30, 2019, U.S. Provisional Patent Application No. 62 / 913,401, filed October 10, 2019, U.S. Provisional Patent Application No. 63 / 024,941, filed May 14, 2020, and U.S. Provisional Patent Application No. 63 / 109,677, filed November 4, 2020. Further examples of suitable transgenes for delivery may include human alpha-L-iduronidase (IDUA) delivered by an AAV vector, e.g., as described in PCT / US2014 / 025509, filed March 13, 2014, now published as WO2014 / 151341, and U.S. Provisional Patent Application No. 61 / 788,724, filed March 15, 2013, which are incorporated herein by reference.

[0060] Other useful therapeutic products include those expressed in muscle, including cardiac muscle. Other useful therapeutic products encoded by a transgene include hormones and growth and differentiation factors, including, but not limited to, insulin, glucagon, glucagon-like peptide-1 (GLP-1), growth hormone (GH), parathyroid hormone (PTH), growth hormone releasing factor (GRF), follicle stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiopoietin, angiostatin, granulocyte colony stimulating factor (GCSF), erythropoietin (EPO), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), transforming growth factor alpha (TGFα), platelet derived growth factor (PDGF), insulin growth factor (IGF), insulin-like ... These include: innate growth factor I and II (IGF-I and IGF-II), any one of the transforming growth factor beta superfamily (TGFβ, activin, inhibin), or any one of the bone morphogenetic proteins (BMPs) BMP1-15, any one of the heregluin / neuregulin / ARIA / neu differentiation factor (NDF) family of growth factors, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophins (NT-3 and NT-4 / 5), ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), neurturin, agrin, any one of the semaphorin / collapsin family, netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrin, noggin, sonic hedgehog, and tyrosine hydroxylase. Other transgenes useful herein include those for treating mucopolysaccharidosis types I-VII (IDUA, IDS, GNA, HGSNAT, NAGLU, SGSH, GALNS, GLB1, ARSB, GUSB). Exemplary sequences useful for treating MPS I can be found in WO2019 / 010335 (incorporated herein by reference). Exemplary sequences useful for treating MPS II can be found in WO2019 / 060662 (incorporated herein by reference).Exemplary sequences useful for treating MPSIIIa can be found in WO2019 / 108857 (herein incorporated by reference). Exemplary sequences useful for treating MPSIIIb can be found in WO2019 / 108856 (herein incorporated by reference).

[0061] In some embodiments, the transgene cassette comprises a promoter, a transgene coding sequence, and a polyA sequence. In some embodiments, the promoter is a liver-specific promoter (e.g., TBG promoter, TBG-S1 promoter, HLP promoter) or other promoters described herein. In other embodiments, the transgene is provided without a promoter and is inserted into the genome downstream of the native PSCK9 promoter.

[0062] The transgene cassette, expression cassette, and / or (editing or donor) vector may be The vector may 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 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 / alpha 1-microglobulin / bikunin enhancer), among others. These control or regulatory sequences are operably linked to the nuclease coding sequence or the transgene coding sequence.

[0063] In addition to the transgene cassette, in certain embodiments, the donor vector also includes 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 some embodiments, the HDR arms are each about 100 bp to about 1000 bp in length. In other embodiments, the HDR arms are each about 130 bp to about 500 bp. In other embodiments, the HDR arms are each about 100 bp to about 300 bp. In other embodiments, the HDR arms are each about 100 bp to about 400 bp. In other embodiments, the HDR arms are each about 250 bp to about 500 bp. In other embodiments, the HDR arms are each about 300 bp to about 500 bp. In certain embodiments, the HDR arms are each about 100bp, 125bp, 150bp, 175bp, 200bp, 225bp, 250bp, 275bp, 300bp, 325bp, 350bp, 375bp, 400bp, 425bp, 450bp, 450bp, 475bp, or 500bp. In one embodiment, the HDR arm is 130bp. In another embodiment, the HDR arm is 137bp. In another embodiment, the HDR arm is about 130bp-140bp. In another embodiment, the HDR arm is about 500bp. In another embodiment, the HDR arm is absent. The HDR arms ideally share a high level of complementarity with the target PCSK9 locus, although they do not need to be 100% complementary. In some embodiments, each HDR arm is allowed 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 SEQ ID NOs: 24-29. In one embodiment, the HDR arm sequence is selected from SEQ ID NOs: 24-29.

[0064] Also provided herein are 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. The engineered coding sequence of OTC is referred to herein as hOTCco2 and is set forth in SEQ ID NO: 17. Nucleic acids are provided having the sequence of SEQ ID NO: 17, or a sequence that shares at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.9% identity. 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: 30.

[0065] Other sequences useful for the treatment of OTC are described in WO2015 / 138348 and WO2015 / 138357, which are incorporated herein by reference. Exemplary sequences useful for the treatment of PKU are described in WO2018 / 126112, which are incorporated herein by reference. (The disclosure of this application is incorporated by reference in its entirety.) Other sequences are those set forth in SEQ ID NO: 71 or 72, or sequences sharing at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto.

[0066] Viral and non-viral vectors The (gene editing and donor) expression cassettes or coding sequences described herein can be engineered into any suitable genetic element (e.g., vector) for delivery to target cells (e.g., hepatocytes). As used herein, a "vector" is a biological or chemical moiety that contains a nucleic acid sequence and can be introduced into a suitable host cell for replication or expression of the nucleic acid sequence. Common vectors include non-viral and viral vectors. As used herein, non-viral systems can be selected from nanoparticles, electroporation systems, and novel biomaterials, naked DNA, phages, transposons, plasmids, cosmids (Phillip McClean, www.ndsu.edu / pubweb / ~mcclean / -plsc731 / cloning / cloning4.htm), and artificial chromosomes (Gong, Shiaoching, et al. "A gene expression atlas of the central nervous system based on bacterial artificial chromosomes." Nature 425.6961 (2003): 917-925). In one embodiment, the nucleic acid is delivered via a non-viral vector or lipid nanoparticle described herein or known in the art.

[0067] In certain embodiments, the gene editing components are encapsulated in lipid nanoparticles (LNPs). See, e.g., Conway et al, Non-viral Delivery of Zinc Finger Nuclease mRNA Enables Highly Efficient In Vivo Genome Editing of Multiple Therapeutic Gene Targets,Molecular See Therapy, 27(4):866-877 (April 2019), incorporated herein by reference. As used herein, the phrase "lipid nanoparticle" refers to a transfer vehicle comprising one or more lipids (e.g., cationic lipids, non-cationic lipids, and PEG-modified lipids). Preferably, the lipid nanoparticles are formulated to deliver one or more mRNAs to one or more target cells (e.g., liver and / or muscle). Examples of suitable lipids include, for example, phosphatidyl compounds (e.g., phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides). Also contemplated is the use of polymers as transfer vehicles, alone or in combination with other transfer vehicles. Suitable polymers may include, for example, polyacrylates, polyalkylcyanoacrylates, polylactides, polylactide-polyglycolide copolymers, polycaprolactones, dextran, albumin, gelatin, alginates, collagen, chitosan, cyclodextrin, dendrimers, and polyethyleneimines. In one embodiment, the transfer vehicle is selected based on its ability to facilitate transfection of mRNA into target cells. Lipid nanoparticles useful for mRNA include cationic lipids to encapsulate and / or enhance delivery of mRNA to target cells that function as depots for protein production. As used herein, the phrase "cationic lipid" refers to any of several lipid species that carry a net positive charge at a selected pH, e.g., physiological pH. Contemplated lipid nanoparticles may be prepared by including a multi-component lipid mixture in varying ratios using one or more cationic lipids, non-cationic lipids, and PEG-modified lipids. Several cationic lipids have been described in the literature, many of which are commercially available. See, e.g., WO2014 / 089486, US2018 / 0353616A1, and US8,853,377B2, which are incorporated by reference. In certain embodiments, the LNP formulation comprises cholesterol, an ionizable lipid, a helper lipid, a PEG lipid, and a carboxylate lipid. This is carried out using routine procedures involving polymers that form lipid bilayers around encapsulated nucleic acids (Kowalski et al., 2019, Mol. Ther. 27(4):710-728). In some embodiments, the LNPs include cationic lipids (i.e., N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) or 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)) with the helper lipid DOPE. In some embodiments, the LNPs include the ionizable lipid Dlin-MC3-DMA ionizable lipid or the diketopiperazine-based ionizable lipid (cKK-E12). In some embodiments, the polymer includes polyethyleneimine (PEI) or poly(β-amino)ester (PBAE). See, e.g., WO2014 / 089486, US2018 / 0353616A1, US2013 / 0037977A1, WO2015 / 074085A1, US9670152B2, and US8,853,377B2, which are incorporated by reference. In certain embodiments, the gene editing components include Cas9 mRNA and the LNP also includes a gRNA.

[0068] Particular LNPs useful herein include those described in WO2021 / 077066 and WO2021 / 055892, each of which is incorporated herein by reference in its entirety. Useful LNPs include those that exhibit enhanced delivery to the liver. LNP formulations can be varied to enhance liver delivery. For example, the type and ionizable lipid:mRNA ratio, mRNA:sgRNA ratio, molar ratio of ionizable lipids, phospholipids, cholesterol, and PEG lipids can be varied. In one embodiment, the LNPs are prepared using the methods described in Kauffman, KJ; Dorkin, JR; Yang, JH; Heartlein, MW; DeRosa, F.; Mir, FF; Fenton, OS; Anderson, DG, Optimization of lipid delivery. nanoparticle formulations for mRNA delivery in vivo with fractional factorial and definitive screening designs. Nano letters 2015, 15(11), 7300-7306 (incorporated herein by reference). In certain embodiments, LNPs are designed with ionizable lipid:mRNA weight ratios that vary between 5:1 and 25:1. In certain embodiments, the ionizable lipid:mRNA weight ratio is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 12.5:1, 15:1, 20:1, or 25:1. In certain embodiments, the mRNA:sgRNA weight ratio is 1:1, 1:2, 2:1, 1:4, 1:5, 5:1, 4:1, 3:1, or 2:1.

[0069] Other LNPs have been described and are useful herein, see, e.g., WO2016 / 118724, US10,413,618B2, US10,723,692B2, and US8754062B2, each of which is incorporated herein by reference.

[0070] Particular examples herein illustrate the use of AAV vectors containing gene editing components (nuclease) coding sequences and transgene coding sequences in the AAV vector genome. However, the use of the constructs described herein is not limited to AAV constructs and can be used for other vectors. In certain embodiments, the vector genome can be packaged in a different vector (e.g., recombinant bocavirus). In certain embodiments, the expression cassette can be packaged in a different viral vector, a non-viral vector, and / or a different delivery system. In certain embodiments, the gene editing components are provided in the LNP.

[0071] "Plasmids" or "plasmid vectors" are generally designated herein by a lower case p preceding and / or following the vector name. Plasmids that can be used in accordance with the present invention The nucleic acid sequences described herein, other cloning and expression vectors, their properties, and methods of their construction / manipulation will be readily apparent to those skilled in the art. In one embodiment, the nucleic acid sequences described herein or the expression cassettes described herein are engineered into suitable genetic elements (vectors) useful for generating viral vectors and / or delivery to host cells, e.g., naked DNA, phages, transposons, cosmids, episomes, etc., and transcribing the nuclease sequences carried thereon. The vector selected can be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high-speed DNA-coated pellets, viral infection, and protoplast fusion. Methods used to create such constructs are known to those skilled in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY.

[0072] In certain embodiments, the expression cassette is located in the vector genome for packaging into viral capsid.For example, for an AAV vector genome, the components of the expression cassette 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.In other embodiments, self-complementary AAV may be selected.In other embodiments, retroviral systems, lentiviral vector systems, or adenoviral systems may be used.

[0073] AAV vectors In certain embodiments, the gene editing vector and / or 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.

[0074] The source of the AAV capsid can be any one of dozens of naturally occurring and available adeno-associated viruses and engineered AAVs. The source of the AAV capsid for the gene editing vector and / or donor vector is the same in one embodiment. In another embodiment, the source of the AAV capsid for the gene editing vector and / or donor vector is different. The adeno-associated virus (AAV) viral vector is an AAV DNase-resistant particle with an AAV protein capsid in which a nucleic acid sequence for delivery to a target cell is packaged. 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. Various AAVs can be selected as the source of the capsid for the AAV viral vector described above. See, e.g., U.S. Patent Application Publication No. 2007 / 0036760-A1, U.S. Patent Application Publication No. 2009 / 0197338-A1, EP 1310571. See also WO 2003 / 042397 (AAV7 and other simian AAVs), U.S. Patent No. 7,790,449 and U.S. Patent No. 7,282,199 (AAV8), WO 2005 / 033321 and U.S. Patent No. 7,906,111 (AAV9), as well as WO 2006 / 110689, WO 2003 / 042397 (rh.10) and WO 2018 / 160582 (AAVhu68). These references also describe other AAVs that can be selected to generate AAVs and are incorporated by reference.

[0075] Unless otherwise specified, the AAV capsids, ITRs, and other selected AAV components described herein can be readily selected from among any AAV, including, but not limited to, AAVs commonly identified as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV8bp, AAV7M8, AAVAnc80, AAVrhlO, AAVrh79, and AAVPHP.B, as well as variants of any of the known or mentioned AAVs, or as yet undiscovered AAVs or variants thereof, or mixtures thereof. See, e.g., WO2005 / 033321, incorporated herein by reference. In one embodiment, the AAV capsid is an AAV1 capsid or a variant thereof, an AAV8 capsid or a variant thereof, an AAV9 capsid or a variant thereof, an AAVhu.68 capsid or a variant thereof, an AAVrh.10 capsid or a variant thereof, an AAVrh64R1 capsid or a variant thereof, an AAVhu.37 capsid or a variant thereof, or an AAV3B or a variant thereof. In one aspect, the capsid is an AAVhu.37 capsid. See, for example, WO2019 / 168961 and WO2019 / 169004 (incorporated herein by reference). In another embodiment, the AAV capsid is an AAVrh79 capsid or a variant thereof. In another embodiment, the AAV capsid is an AAVrh.90 or a variant thereof.

[0076] In certain embodiments, the rAAV comprises an AAVhu37 capsid, which comprises a heterogeneous population of vp1 proteins that are the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:38, a heterogeneous population of vp2 proteins that are the product of a nucleic acid sequence encoding the amino acid sequence at least about amino acids 138-738 of SEQ ID NO:38, 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:38, wherein the vp1, vp2, and vp3 proteins include subpopulations having amino acid modifications including at least two highly deasparagine (N) in the asparagine-glycine pair of SEQ ID NO:38, and optionally further subpopulations including other deamidated amino acids, wherein the deamidation results in an amino acid change. AAVhu37 is characterized by having highly deamidated residues at, for example, positions N57, N263, N385, and / or N514, based on the numbering of AAVhu37 VP1 (SEQ ID NO:38).

[0077] Deamidation has been observed at other residues, as shown in the table below, and in, for example, WO2019 / 168961 (published September 6, 2019, incorporated herein by reference). In certain embodiments, the AAVhu37 capsid is modified at one or more of the following positions, with the ranges provided 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. For example, in certain embodiments, a G can be modified to an S or A, for example, at positions 58, 264, 386, or 515. In one embodiment, the AAVhu37 capsid is modified at positions N57 / G58 to N57Q or G58A, resulting in a capsid with reduced deamidation at this position. In another embodiment, N57 / G58 is altered to NS57 / 58 or NA57 / 58. However, in certain embodiments, when NG is altered to NS or NA, increased deamidation is observed. In certain embodiments, the N of the NG pair is modified to Q while retaining the G. In certain embodiments, both amino acids of the NG pair are modified. In certain embodiments, N385Q results in a significant reduction in deamidation at that position. In certain embodiments, N499Q results in a significant increase in deamidation at that position.

[0078] In certain embodiments, AAVhu37 is characterized in that these or other residues are, for example, typically It may be deamidated in 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., when present, in the range of about 10 to about 60%, or about 10 to about 30%, or about 20 to about 60%) (e.g., S149, Optionally, W may have other modifications including, for example, at one or more of -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 may be oxidized to kynurenine. [Table 1]

[0079] Still other positions may have these or other modifications (e.g., acetylation or further deamidation). In certain embodiments, the nucleic acid sequence encoding the AAVhu37 vp1 capsid protein is provided in SEQ ID NO:37. In other embodiments, a nucleic acid sequence that is 70% to 99.9% identical to SEQ ID NO:37 may be selected to express the AAVhu37 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% identical, or at least 99% identical to SEQ ID NO:37. However, other nucleic acid sequences that encode the amino acid sequence of SEQ ID NO:38 may be selected for use in producing rAAVhu37 capsids. In certain embodiments, the nucleic acid sequence has a sequence at least 70% to 99% identical, 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:37, or SEQ ID NO:37 that encodes SEQ ID NO:38. In certain embodiments, the nucleic acid sequence is at least 70% to 99%, at least 75%, at least 80%, at least 85%, at least identical to the nucleic acid sequence of SEQ ID NO: 37, or from about nt 412 to about nt 2214 of SEQ ID NO: 37. In certain embodiments, the nucleic acid sequence has a sequence from about nt 610 to about nt 2214 of SEQ ID NO: 37, or 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 nt of SEQ ID NO: 37, and encodes the vp3 capsid protein (about aa 204 to 738) of SEQ ID NO: 38. See EP2345731B1 and SEQ ID NO: 88 therein, which are incorporated by reference.

[0080] In certain embodiments, the rAAV comprises an AAV8 capsid. The AAV8 capsid comprises a heterogeneous population of VP isoforms that are deamidated as defined in the table below based on the total amount of VP protein in the capsid, as determined using mass spectrometry. Suitable modifications include those described in the paragraph above that are labeled with deamidation, and are incorporated herein by reference. In certain embodiments, the AAV capsid is modified at one or more of the following positions, as determined using mass spectrometry, to the extent provided below. In certain embodiments, one or more of the following positions, or glycines following N, are modified as described herein. In certain embodiments, an artificial NG is introduced at a position different from one of the positions shown below. In certain embodiments, one or more of the following positions, or glycines following N, are modified as described herein. For example, in certain embodiments, G can be modified to S or A, for example, at positions 58, 67, 95, 216, 264, 386, 411, 460, 500, 515, or 541. When NG57 / 58 is modified to NS57 / 58 or NA57 / 58, a significant decrease in deamidation is observed. However, in certain embodiments, when NG is modified to NS or NA, an increase in deamidation is observed. In certain embodiments, the N of the NG pair is modified to Q while retaining the G. In certain embodiments, both amino acids of the NG pair are modified. In certain embodiments, N385Q results in a significant decrease in deamidation at that position. In certain embodiments, N499Q results in a significant increase in deamidation at that position. In certain embodiments, the NG mutation is made in the pair located at N263 (e.g., N263A). In certain embodiments, an NG mutation is made in the pair located at N514 (e.g., N514A). In certain embodiments, an NG mutation is made in the pair located at N540 (e.g., N540A). In certain embodiments, an AAV mutation is engineered that includes multiple mutations and at least one of the mutations at these positions. In certain embodiments, no mutation is made at position N57. In certain embodiments, no mutation is made at position N94. In certain embodiments, no mutation is made at position N305. In certain embodiments, no mutation is made at position G386.In certain embodiments, no mutations are made at position Q467. In certain embodiments, no mutations are made at position N479. In certain embodiments, no mutations are made at position N653. In certain embodiments, the capsid is modified to reduce an "N" or a "Q" at a position other than the "NG" pair. Residue numbers are based on the published AAV8 sequence reproduced in SEQ ID NO:36. [Table 2-1] [Table 2-2]

[0081] In certain embodiments, the rAAV comprises an AAVrh79 capsid as described in WO2019 / 169004 published September 6, 2019 (hereby incorporated 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 expressing from a nucleic acid sequence encoding the predicted amino acid sequence of 1-738 of SEQ ID NO:34. Optionally, 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:33, or a sequence that co-expresses a vp1 protein produced from a nucleic acid sequence at least 70% identical to SEQ ID NO:33 encoding the predicted amino acid sequence of 1-738 of SEQ ID NO:34. 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:34; a vp2 protein produced from a sequence that includes at least nucleotides 412-2214 of SEQ ID NO:33; or a vp2 protein produced from a nucleic acid sequence that encodes the predicted amino acid sequence of at least about amino acids 138-738 of SEQ ID NO:34 and that is at least 70% identical to at least nucleotides 412-2214 of SEQ ID NO:33; an 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:34; a vp3 protein produced from a sequence that includes at least nucleotides 610-2214 of SEQ ID NO:33; or a vp3 protein produced from a nucleic acid sequence that encodes the predicted amino acid sequence of at least about amino acids 204-738 of SEQ ID NO:34 and that is at least 70% identical to at least nucleotides 610-2214 of SEQ ID NO:33.

[0082] In certain embodiments, the AAVrh79 capsid comprises a heterogeneous population of vp1 proteins that are the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:34, vp2 proteins that are the product of a nucleic acid sequence encoding the amino acid sequence at least about amino acids 138-738 of SEQ ID NO:34, and and a heterogeneous population of vp3 protein that is the product of a nucleic acid sequence encoding at least amino acids 204-738 of SEQ ID NO:34.

[0083] 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: 34, 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: 34. 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 3]

[0084] In certain embodiments, the AAVrh79 capsid is modified at one or more of the positions identified in the table above, as determined using mass spectrometry with trypsin enzyme, to the ranges shown below. In certain embodiments, one or more of the following positions, or glycines following an N, are modified as described herein. The residue numbers are the same as those of the AAVrh79 capsids provided herein. Based on sequence 9. See SEQ ID NO:34.

[0085] In certain embodiments, the nucleic acid sequence encoding the AAVrh79 vp1 capsid protein is provided in SEQ ID NO:33. In other embodiments, a nucleic acid sequence that is 70% to 99.9% identical to SEQ ID NO:33 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% identical, or at least 99%, or 99.9% identical to SEQ ID NO:33. However, other nucleic acid sequences that encode the amino acid sequence of SEQ ID NO:34 may be selected for use in producing rAAV capsids. In certain embodiments, the nucleic acid sequence has a sequence 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:33, or SEQ ID NO:33 that encodes SEQ ID NO:34. In certain embodiments, the nucleic acid sequence has 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: 33, or to about nt 412 to about nt 2214 of SEQ ID NO: 33, and encodes the vp2 capsid protein (about aa 138 to 738) of SEQ ID NO: 34. In certain embodiments, the nucleic acid sequence has 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: 33, or to the nt of SEQ ID NO: 33 that encodes the vp3 capsid protein (about aa 204 to 738) of SEQ ID NO: 34.

[0086] 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.

[0087] In certain embodiments, the rAAV comprises the AAVrh.90 capsid described in WO2020 / 223232, published on November 5, 2020 (incorporated by reference herein). In a further aspect, a recombinant adeno-associated virus (rAAV) is provided, comprising: (A) an AAVrh.90 capsid comprising: (1) a heterogeneous population of AAVrh.90 vp1 proteins 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:40, a vp1 protein produced from SEQ ID NO:39, or a vp1 protein produced from a nucleic acid sequence that encodes the predicted amino acid sequence of 1-738 of SEQ ID NO:40 that is at least 70% identical to SEQ ID NO:39; a vp2 protein produced by expression from a nucleic acid sequence encoding at least about amino acid 138-738 of SEQ ID NO:40, a vp2 protein produced from a sequence that includes at least nucleotides 412-2214 of SEQ ID NO:39, or a vp2 protein produced from a nucleic acid sequence that encodes the predicted amino acid sequence of at least about amino acid 138-738 of SEQ ID NO:40 that is at least 70% identical to at least nucleotides 412-2214 of SEQ ID NO:39 that encodes the predicted amino acid sequence of at least about amino acid 138-738 of SEQ ID NO:40; a heterogeneous population of AAVrh.90 vp3 proteins selected from (1) a heterogeneous population of vp2 proteins, a vp3 protein 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:40, a vp3 protein produced from a sequence that includes at least nucleotides 610-2214 of SEQ ID NO:39, or a vp3 protein produced from a nucleic acid sequence that is at least 70% identical to at least nucleotides 610-2214 of SEQ ID NO:39 that encodes the predicted amino acid sequence of at least about amino acids 204-738 of SEQ ID NO:40, and / or (2) a heterogeneous population of vp1 proteins that are the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:40, and (B) a heterogeneous population of vp2 proteins that are the product of a nucleic acid sequence encoding an amino acid sequence from at least about amino acids 138 to 738 of SEQ ID NO:40, and a heterogeneous population of vp3 proteins that are the product of a nucleic acid sequence encoding at least amino acids 204 to 738 of SEQ ID NO:40, wherein the vp1, vp2, and vp3 proteins include a subpopulation having an amino acid modification that includes at least two highly deamidated asparagines (N) in the asparagine-glycine pair of SEQ ID NO:40, and optionally further subpopulations that include other deamidated amino acids, wherein the deamidation results in an amino acid change in one or more of the AAVrh.90 capsid proteins.

[0088] In certain embodiments, the vp1, vp2, and vp3 proteins of AAVrh.90 comprise a subpopulation having an amino acid modification comprising at least two highly deamidated asparagines (N) in the asparagine-glycine pair of SEQ ID NO:40, and optionally further comprising subpopulations comprising other deamidated amino acids, where the deamidation results in an amino acid change. High levels of deamidation are observed at ∼N57, ∼N263, ∼N385, and / or ∼N514 of the N-G pair, relative to the numbers in SEQ ID NO:40. Deamidation has been observed at other residues, as shown in the table below. In certain embodiments, AAVrh.90 may have other residues deamidated (e.g., at -N305, -N499, and / or -N599, typically less than 20%) and / or other modifications including phosphorylation (e.g., at S149) (e.g., in a range of about 2 to about 30%, or about 2 to about 20%, or about 2 to about 10%, if present) or oxidation (e.g., at one or more of -W23, -M204, -M212, W248, W282, M405, M473, W480, W505, M526, -N544, M561, and / or -M607). Optionally, W may be oxidized to kynurenine. [Table 4]

[0089] In certain embodiments, the AAVrh.90 capsid is modified at one or more positions identified in the table above, in the ranges provided, as determined using mass spectrometry with trypsin enzyme. In certain embodiments, one or more positions, or the glycine following the N, is modified as described herein. Residue numbers are based on the AAVrh.90 sequence provided herein. See SEQ ID NO:40.

[0090] In certain embodiments, the AAVrh.90 capsid comprises the amino acid sequence of SEQ ID NO:40. a heterogeneous population of vp1 proteins that are the product of a nucleic acid sequence encoding an amino acid sequence at least about amino acids 138 to 738 of SEQ ID NO:40; 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 204 to 738 of SEQ ID NO:40; and a heterogeneous population of vp3 proteins that are the product of a nucleic acid sequence encoding an amino acid sequence at least about amino acids 204 to 738 of SEQ ID NO:40.

[0091] In certain embodiments, the parvovirus vector capsid is selected for hepatic tropism and the patient being treated has a liver metabolic disorder. In certain embodiments, the parvovirus vector capsid is selected for cardiac tropism and the patient being treated has a cardiac disorder. In certain embodiments, the parvovirus vector capsid is selected for skeletal muscle cell tropism and the patient being treated has a muscle disorder.

[0092] As used herein, "vector genome" refers to a nucleic acid sequence that is packaged into the rAAV capsid that forms the viral particle. Such a nucleic acid sequence includes 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 that includes a transgene or coding sequence operably linked to a regulatory sequence that directs its expression, and the AAV 3'ITR. The ITRs are genetic elements that are involved in genome replication and packaging during vector production, and are the only viral cis elements required to generate rAAV. In one embodiment, the ITRs are from a different AAV than the one that supplies the capsid. In a preferred embodiment, the ITR sequence from AAV2, or a deleted version thereof (ΔITR), may be used for convenience. However, ITRs from other AAV sources may be selected. If the source of the ITRs is from AAV2 and the AAV capsid is from another AAV source, the resulting vector may be referred to as pseudotyped. Typically, an AAV vector genome comprises an AAV 5' ITR, a nucleic acid sequence encoding a gene product, any regulatory sequences, and an AAV 3' ITR. However, other configurations of these elements may be suitable. In one embodiment, a self-complementary AAV is used. A shortened version of the 5' ITR (called ΔITR) has been described, in which the D sequence and terminal separation site (trs) are deleted. In certain embodiments, the vector genome comprises a shortened AAV2 ITR of 130 base pairs, in which the external "a" element is deleted. The shortened ITR is restored to a wild-type length of 145 base pairs during vector DNA amplification 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 ITRs or engineered ITRs may be selected. ITRs from AAV2, a source AAV different from the capsid, or other than full-length ITRs may be selected. The ITRs are from the same AAV source as the AAV that provides the rep function or trans-complementing AAV during production. Additionally, other ITRs may be used. Examples of suitable ITR sequences are shown in the sequence listing, e.g., SEQ ID NO: 42, nt 1-130 and 3052-3181.Additionally, the vector genome comprises regulatory sequences that directly regulate expression of the gene product (e.g., directly or indirectly, by regulating transcription and / or translation). Suitable components of the vector genome are discussed in more detail herein.

[0093] In certain embodiments, the gene editing vector genome comprises a TBG promoter, one or more αMIC / bik enhancers, a coding sequence for ARCUS meganuclease, optionally a WPRE, and a poly A. In certain embodiments, the expression cassette comprises nt 211 to nt 2964 of SEQ ID NO: 42 and is flanked by 5' and 3' ITRs.

[0094] 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, and mammalian cells in vitro, among others. Suitable transfection techniques and packaging host cells are known and may be used in a variety of applications. , and / or can be readily designed by one of ordinary skill in the art.

[0095] Methods for generating and isolating AAV suitable for use as a vector are known in the art. See generally, e.g., Grieger & Samulski, 2005, "Adeno-associated virus as a gene therapy vector: Vector development, production and distribution." and 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. The ITRs are the only AAV components required in cis in the same construct as the nucleic acid molecule containing the expression cassette in order to package the transgene into virions. The cap and rep genes can be supplied in trans.

[0096] The term "AAV intermediate" or "AAV vector intermediate" refers to an assembled rAAV capsid that lacks the desired genomic sequence packaged therein. These may be referred to as "empty" capsids. Such capsids may not contain detectable genomic sequences of an expression cassette, or may contain only partially packaged genomic sequences that are insufficient to achieve expression of a gene product. These empty capsids are non-functional for introducing a gene of interest into a host cell.

[0097] 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 the expression cassette to be packaged 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 US2013 / 0045186A1.

[0098] 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).

[0099] 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 rep78, rep68, rep52, rep40, 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 can be a spacer between the rep sequence and the cap gene. These AAV or mutant AAV capsids can be used in combination with other AAVs. Any of the psyllid sequences may be under the control of exogenous regulatory control sequences which direct their expression in the host cell.

[0100] In one embodiment, the cells are produced in suitable cell culture (e.g., HEK293) cells. Methods for producing gene therapy vectors described herein include methods well known in the art, such as the generation of plasmid DNA used in the production of gene therapy vectors, the generation of vectors, and the purification of vectors. In some embodiments, the gene therapy vector is an AAV vector, and the generated plasmids are AAV cis-plasmids encoding the AAV genome and 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 harvesting 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 reviews of these production systems, see, in general, e.g., Zhang et al., 2009, “Adenovirus-adeno-associated virus hybrid for large-scale See, "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 herein by reference in their entireties: 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.

[0101] 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.

[0102] 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 of 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.

[0103] To calculate the empty and filled particle content, the VP3 band volume for a selected sample (e.g., in the example herein, 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. Dividing Pt / mL by GC / mL gives the ratio of particles to genome copies (pt / GC). Pt / mL-GC / mL gives the empty pt / mL. Dividing empty pt / mL by pt / mL and multiplying by 100 gives the percentage of empty particles. do.

[0104] In general, methods for assaying AAV vector particles containing empty capsids and packaged genomes are known in the art. See, for example, Grimm et al., Gene Therapy (1999) 6:1322-1330; Sommer et al., Molec. Ther. (2003) 7:122-128. To test for denatured capsids, the method involves subjecting the processed AAV stock to SDS-polyacrylamide gel electrophoresis, consisting of any gel capable of separating the three 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. The 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 with the primary antibody, more preferably an anti-IgG antibody that contains a detection molecule covalently bound to the antibody, most preferably a sheep anti-mouse IgG antibody covalently bound 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, for SDS-PAGE, samples from column fractions can be taken and heated in SDS-PAGE loading buffer containing a reducing agent (e.g., DTT), and capsid proteins resolved in precast gradient polyacrylamide gels (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 dye.In one embodiment, the concentration of AAV vector genome (vg) in the column fractions can be measured by quantitative real-time PCR (Q-PCR). The samples are diluted and digested with DNaseI (or another suitable nuclease) to remove exogenous DNA. After inactivation of the nuclease, the samples are 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 contained in the AAV vector is used to generate a standard curve in the 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.

[0105] 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 may be concentrated 2-fold or more. Typically, the proteinase K treatment is about 0.2 mg / mL, but may 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 periods (e.g., about 20 minutes to about 30 minutes), or at higher temperatures (e.g., up to about 60° C.) for shorter periods (e.g., about 5 to 10 minutes). Similarly, heat inactivation is generally carried out at about 95° C. for about 15 minutes, but may be carried out at reduced temperatures (e.g., about 70 to 10 minutes). The incubation time may be extended (e.g., about 90° C.) and the sample is then diluted (e.g., 1000-fold) and subjected to TaqMan analysis as described for standard assays.

[0106] Additionally or alternatively, droplet digital PCR (ddPCR) may be used. For example, a method for determining single-stranded and self-complementary AAV vector genome titer by ddPCR 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 genome. 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 sequence-specific primers targeting the meganuclease specific for the PCSK9 gene (M2PCSK9) in combination with a fluorescently labeled probe hybridizing to this same region. Twenty microliters of the ddPCR reaction mixture is processed in 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.

[0107] 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.

[0108] 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 the affinity chromatography step, the diafiltered product was applied to Capture Select™ Poros-AAV2 / 9 affinity resin (Life Technologies), which efficiently captures the AAV2 serotype. Under these ionic conditions, a significant percentage of the residual cellular DNA and proteins flow through the column and the AAV particles are efficiently captured.

[0109] Dual Vector System In another aspect, a dual vector system for treating genetic disorders is provided. The system includes: (a) a gene editing component, the gene editing component including a nucleic acid sequence encoding a nuclease that targets PCSK9, and optionally a regulatory sequence that directs expression of the nuclease in a target cell that includes the PCSK9 gene; and (b) a donor vector, the donor vector including a nucleic acid sequence encoding an exogenous product for expression from the PCSK9 locus, the inserted nucleic acid sequence not encoding PCSK9, the system further including a sequence that directs the nuclease to specifically target the native PCSK9 locus. The system optionally includes a component that allows native PCSK9 to be ablated or reduced in the target cell after administration with the dual vector system, for example, through the use of an inducer with an inducible promoter. In one embodiment, the gene editing component is included in a gene editing vector that includes an expression cassette including a nucleic acid sequence encoding the nuclease, and a regulatory sequence that directs expression of the nuclease in a target cell that includes the PCSK9 gene. The components of the dual vector are the same as those described herein.

[0110] This system can be effective when the ratio of gene editing components to donor vector is about 1 to about 1, although it is desirable that there is more donor template vector than gene editing components. 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. This ratio of gene editing enzyme (e.g., Cas9 or meganuclease) to donor template can be maintained even if the enzyme is additionally or alternatively provided by a source other than the AAV vector.

[0111] In one embodiment, the dual vector system comprises a gene-editing AAV vector 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 a second vector genome comprising an AAV capsid and a 5' ITR, a 5' homologous recombination (HDR) arm, a transgene and a regulatory sequence that directs expression of the transgene in a target cell, a 3' HDR arm, and a 3' ITR.

[0112] In another embodiment, the dual vector system comprises a gene-editing AAV comprising an AAV capsid and a first vector genome comprising a 5'ITR, a 5' nuclear localization signal (NLS), a regulatory sequence that directs expression of SaCas9 in a target cell comprising a sequence encoding Cas9 and a PCSK9 gene, a 3'NLS, and a 3'ITR, and a donor AAV vector comprising a second vector genome comprising an AAV capsid, a 5'ITR, a 5' homologous recombination (HDR) arm, a transgene and a regulatory sequence that directs expression of the transgene in a target cell, a 3'HDR arm, a U6 promoter, an sgRNA, the sgRNA comprising at least 20 nucleotides that specifically binds to a target site in the PCSK9 gene, the target site being 5' to a protospacer adjacent motif (PAM) that is specifically recognized by Cas9, and the 3'ITR.

[0113] In another embodiment, the dual vector system is a gene editing AAV vector comprising an AAV capsid, a 5'ITR, a U6 promoter, and a sgRNA of at least 20 nucleotides that specifically binds to a target site in the PCSK9 gene; The gene editing AAV vector includes a first vector genome comprising an sgRNA, a 5' nuclear localization signal (NLS), a sequence encoding Cas9 and a regulatory sequence that directs expression of Cas9 in a target cell comprising a PCSK9 gene, a 3' NLS, and a 3' ITR, the target site being 5' to a protospacer adjacent motif (PAM) that is specifically recognized by Cas9, and a donor AAV vector including an AAV capsid and a second vector genome comprising a 5' ITR, a 5' homologous recombination (HDR) arm, a transgene, and a regulatory sequence that directs expression of the transgene in a target cell, a 3' HDR arm, and a 3' ITR.

[0114] In certain embodiments of the system described herein, the gene editing AAV vector and the donor AAV vector have the same AAV capsid. In other embodiments, the gene editing AAV vector and the donor AAV vector have different AAV capsids. In some embodiments, the AAV capsid is selected from AAV8, AAV9, rh10, AAV6.2, AAV3B, hu37, rh79, and rh64.

[0115] In certain embodiments, the nuclease is a Cas9 nuclease, and the Cas9 is Staphylococcus aureus Cas9 or Streptococcus pyogenes Cas9.

[0116] In certain embodiments, the nuclease and / or transgene is under the control of a tissue-specific promoter. In certain embodiments, the nuclease and / or transgene is under the control of a constitutive promoter. In certain embodiments, the nuclease and / or transgene is under the control of an inducible promoter. In certain embodiments, the nuclease and / or transgene is under the control of a liver-specific promoter, optionally a human thyroxine-binding globulin (TBG) promoter, or a hybrid liver promoter (HLP). In certain embodiments, the system further comprises an inducer.

[0117] In another embodiment, the system includes (a) a gene editing component, the gene editing component including a nucleic acid sequence encoding a nuclease that targets PCSK9 and a regulatory sequence that directs expression of the nuclease in a target cell that includes a PCSK9 gene encapsulated in an LNP, and (b) a donor vector, the donor vector including a nucleic acid sequence encoding an exogenous product for expression from the PCSK9 locus encapsulated in the LNP, the inserted nucleic acid sequence not encoding PCSK9, the system further including a sequence that directs the nuclease to specifically target the native PCSK9 locus. The system optionally includes a component that allows native PCSK9 to be ablated or reduced in the target cell following administration with the dual vector system, for example, via the use of an inducer with an inducible promoter.

[0118] In another embodiment, the system includes: (a) a gene editing component, the gene editing component including a nucleic acid sequence encoding a nuclease that targets PCSK9 and a regulatory sequence that directs expression of the nuclease in a target cell that includes a PCSK9 gene, the gene editing component being provided via an AAV vector; and (b) a donor vector, the donor vector including a nucleic acid sequence encoding an exogenous product for expression from the PCSK9 locus encapsulated in the LNP, the inserted nucleic acid sequence not encoding PCSK9, the system further including a sequence that directs the nuclease to specifically target the native PCSK9 locus. The system optionally includes a component that allows native PCSK9 to be ablated or reduced in the target cell following administration with the dual vector system, for example, via the use of an inducer with an inducible promoter.

[0119] In another embodiment, the system comprises: (a) a gene editing component that targets PCSK9; The system includes a gene editing component comprising a nucleic acid sequence encoding a nuclease that specifically targets a native PCSK9 locus, the gene editing component comprising a nucleic acid sequence encoding a nuclease that specifically targets a native PCSK9 locus, and a regulatory sequence that directs expression of the nuclease in a target cell comprising a PCSK9 gene encapsulated in an LNP, and (b) a donor vector comprising a nucleic acid sequence encoding an exogenous product for expression from the PCSK9 locus, the donor vector being an AAV vector, and the inserted nucleic acid sequence does not encode PCSK9, the system further comprising a sequence that directs the nuclease to specifically target the native PCSK9 locus. The system optionally includes a component that allows native PCSK9 to be ablated or reduced in the target cell following administration with the dual vector system, for example, via the use of an inducer with an inducible promoter.

[0120] In one embodiment, the dual vector system includes (a) an LNP comprising an mRNA, where the mRNA encodes a meganuclease, where the meganuclease targets PCSK9 under the control of a regulatory sequence that directs expression of the meganuclease in a target cell comprising a PCSK9 gene, and (b) a donor AAV vector, where the donor AAV vector comprises an AAV capsid and a second vector genome comprising a 5'ITR, a 5' homologous recombination (HDR) arm, a transgene and a regulatory sequence that directs expression of the transgene in a target cell, a 3'HDR arm, and a 3'ITR.

[0121] In another embodiment, the dual vector system includes (a) an LNP, the LNP comprising a nucleic acid comprising a sequence encoding Cas9 and an sgRNA, the sgRNA comprising at least 20 nucleotides that specifically binds to a target site in the PCSK9 gene, the target site being 5' to a protospacer adjacent motif (PAM) that is specifically recognized by Cas9, and (b) a donor AAV vector, the donor AAV vector comprising an AAV capsid, a 5'ITR, a 5'homologous recombination (HDR) arm, a transgene and a regulatory sequence that directs expression of the transgene in a target cell, a 3'HDR arm, and a 3'ITR. The sequence encoding Cas9 is provided as an mRNA.

[0122] In another embodiment, the dual vector system comprises a gene-editing AAV vector comprising an AAV capsid, a first vector genome comprising a 5'ITR, a U6 promoter, an sgRNA comprising at least 20 nucleotides that specifically binds to a target site in the PCSK9 gene, the target site being 5' to a protospacer adjacent motif (PAM) specifically recognized by Cas9, an sgRNA, a 5' nuclear localization signal (NLS), a sequence encoding Cas9 and a regulatory sequence that directs expression of Cas9 in a target cell comprising the PCSK9 gene, a 3'NLS, and a 3'ITR, and a donor AAV vector comprising a second vector genome comprising an AAV capsid, a 5'ITR, a 5' homologous recombination (HDR) arm, a transgene and a regulatory sequence that directs expression of the transgene in a target cell, a 3'HDR arm, and a 3'ITR.

[0123] Pharmaceutical Compositions In another aspect, a pharmaceutical composition is provided, comprising: a first rAAV stock comprising an rAAV gene editing vector, the rAAV gene editing vector comprising an expression cassette comprising a nucleic acid sequence encoding a nuclease targeting PCSK9 and a regulatory sequence directing expression of the nuclease in a target cell comprising a PCSK9 gene; and a second rAAV stock comprising an rAAV donor vector, the rAAV donor vector comprising a transgene cassette comprising a nucleic acid sequence encoding a transgene 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 comprises a 5' and 3' homologous to the transgene cassette. Further comprising a recombination (HDR) arm. In one embodiment, the AAV capsid of the donor vector, the gene editing vector, or both is an AAVrh79 capsid. In another embodiment, the AAV capsid of the donor vector, the gene editing vector, or both is an AAVrh.90 capsid. In another embodiment, the AAV capsid of the donor vector, the gene editing vector, or both is an AAVhu.37 capsid. In one embodiment, the AAV capsid of the donor vector, the gene editing vector, or both is an AAV8 capsid. In one embodiment, the AAV capsid of the donor vector, the gene editing vector, or both is an AAVrh.91 capsid. In one embodiment, the AAV capsid of the donor vector, the gene editing vector, or both is an AAVhu.68 capsid.

[0124] 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.

[0125] 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.

[0126] 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).

[0127] The active ingredient can also be administered in microcapsules, e.g., hydroxymethylcellulose or gelatin-macroemulsions, colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or macroemulsions, respectively, prepared, for example, by coacervation techniques or by interfacial polymerization. The pharmaceutical compositions may also be encapsulated in microcapsules and poly-(methyl methacylate) microcapsules. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0128] Suitable surfactants or combinations of surfactants may be selected from non-toxic non-ionic surfactants. In one embodiment, a primary hydroxyl terminated bifunctional block copolymer surfactant is selected, such as Pluronic® F68 [BASF], also known as Poloxamer 188, with neutral pH and average molecular weight of 8400. 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 percent 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.

[0129] 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 a medically acceptable physiological effect, 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. Routes of administration may be combined if desired.

[0130] 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. The dosage is adjusted to balance the therapeutic effect against any side effects, and such dosage may vary depending on the therapeutic application for which the recombinant vector is utilized. The expression level of the transgene can be monitored to determine the dosage frequency that results in the viral vector (preferably the AAV vector containing a minigene). Optionally, the same dosage regimen as that described for therapeutic purposes can be utilized for immunization using the composition of the present invention.

[0131] The vector composition may be administered in a dose range of about 1.0×10 for a human patient (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 1x10 per dose, including all integers or decimals within the range. 9 , 2x10 9 , 3x10 9 , 4x10 9 , 5x10 9 , 6x10 9 , 7x10 9 , 8x10 9 , or 9x10 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 , 7×1010 , 8×10 10 , or 9 ×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 x 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.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] method The compositions provided herein are useful for treating various genetic disorders, including liver metabolic disorders. In certain embodiments, the compositions are useful for treating ornithine transcarbamylase. In other embodiments, the compositions are useful for treating familial hypercholesterolemia. In other embodiments, the compositions are useful for treating phenylketonuria.

[0136] Exemplary liver diseases or disorders that can be treated include, but are not limited to, hepatitis A, hepatitis B, hepatitis C, autoimmune hepatitis, primary biliary cholangitis, primary sclerosing cholangitis, hemochromatosis, Wilson's disease, alpha-1 antitrypsin deficiency, liver cancer, cholangiocarcinoma, hepatocellular adenoma, transthyretin (TTR), proprotein convertase subtilisin / kexin type 9 (PCSK9)-based diseases or disorders, or any combination thereof. Additional disorders include glycogen storage disease or type 1A deficiency (GSD1), PEPCK deficiency, CDKL5 deficiency, galactosemia, phenylketonuria ( PKU), primary hyperoxaluria type 1, maple syrup urine disease, tyrosinemia type 1, methylmalonic acidemia, medium-chain acetyl-CoA deficiency, ornithine transcarbamylase deficiency, citrullinemia, lecithin-cholesterol acyltransferase (LCAT) deficiency, methylmalonic acidemia (MMA), Niemann-Pick disease, propionibacterium deficiency (PA), familial hypercholesterolemia (FH), dementia, lipoprotein lipase deficiency, Crigler-Najjar disease, severe combined immunodeficiency, gout and Lesch-Nyhan syndrome, biotinidase deficiency, Fabry disease, GM1 gangliosidosis, Wilson's disease, Golgi syndrome, These include Shea disease types 2 and 3, Zellweger syndrome, metachromatic leukodystrophy, Krabbe disease, Pompe disease, Niemann-Pick disease type A, argininosuccinic aciduria, adult-onset type II citrullinemia, urea cycle disorders, Farber lipogranulomatosis, aspartylglucosaminuria, fucosidosis, α-mannosidosis, acute intermittent porphyria (AIP), alpha-1 antitrypsin deficiency (emphysema), anemia due to thalassemia or renal failure, ischemic disease, blocked blood vessels (as in atherosclerosis, thrombosis, or embolism), Parkinson's disease, congestive heart failure, muscular dystrophy, and diabetes.

[0137] In certain methods described herein, expression of native PCSK9 is reduced or ablated and the transgene is expressed from an insertion at the native PCSK9 locus. In other embodiments, expression of native PCSK9 is reduced or ablated and the transgene is expressed exogenously, i.e., without integration into the subject's genome.

[0138] Provided herein are methods of treating disorders in humans by co-administering the dual vector systems described herein.

[0139] In one embodiment, a method for treating liver metabolic disorder in a subject is provided.The method comprises co-administering a gene editing AAV vector comprising a sequence encoding a nuclease targeting PCSK9 and a regulatory sequence directing the expression of the nuclease in target cells comprising PCSK9 gene, and a donor AAV vector comprising a transgene and a regulatory sequence directing the expression of the transgene in target cells.In another embodiment, the method comprises co-administering a LNP comprising a sequence encoding a Cas9 nuclease and a sgRNA targeting PCSK9 in target cells comprising PCSK9 gene, and a donor AAV vector comprising a transgene and a regulatory sequence directing the expression of the transgene in target cells, to a subject with liver metabolic disorder.In one embodiment, the subject is a neonate.

[0140] 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.

[0141] 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 9GC / kg, about 5.0×10 9 GC / kg, about 5.5×10 9 GC / kg, about 6.0×10 9 GC / kg, about 6.5×10 9 GC / kg, about 7.0×10 9 GC / kg, about 7.5×10 9 GC / kg, about 8.0×10 9 GC / kg, about 8.5×10 9 GC / kg, about 9.0×10 9 GC / kg, about 9.5×10 9 GC / kg, about 1.0×10 10 GC / kg, about 1.5×10 10 GC / kg, about 2.0×10 10 GC / kg, about 2 .5×10 10 GC / kg, about 3.0×10 10 GC / kg, about 3.5×10 10 GC / kg, about 4.0×10 10 GC / kg, about 4.5×10 10 GC / kg, about 5.0×10 10 GC / kg, about 5.5×10 10 GC / kg, about 6.0×10 10 GC / kg, about 6.5×10 10 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 11GC / 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 12 GC / kg, about 8.0×10 12 GC / kg, about 8.5×10 12 GC / kg, about 9.0×10 12 GC / kg, about 9.5×10 12 GC / kg, about 1.0×10 13 GC / kg, about 1.5×10 13 GC / kg, about 2.0×10 13 GC / kg, about 2.5×10 13 GC / kg, about 3.0×10 13 GC / kg, about 3.5×10 13 GC / kg, about 4.0×10 13 GC / kg, about 4.5×10 13 GC / kg, about 5.0×10 13 GC / kg, about 5.5×10 13 GC / kg, about 6.0×10 13 GC / kg, about 6.5×10 13 GC / kg, about 7.0×1013 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.

[0142] Other examples of suitable diseases that may be treated using the compositions described herein are familial hypercholesterolemia, muscular dystrophy, cystic fibrosis, and rare or orphan diseases. Examples of such rare diseases may include, among others, spinal muscular atrophy, Huntington's disease, Rett syndrome, amyotrophic lateral sclerosis (ALS), Duchenne muscular dystrophy, Friedreich's ataxia, spinocerebellar ataxia type 2 (SCA2) / ALS, progranulin (PRGN) (associated with non-Alzheimer's brain degeneration, including frontotemporal dementia (FTD), progressive non-fluent aphasia (PNFA), and semantic dementia). See, for example, www.orpha.net / consor / cgi-bin / Disease_Search_List.php;rarediseases.info.nih.gov / diseases. Other diseases indicated by the transgenes described herein may also be treated using the methods described herein.

[0143] 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 a medically acceptable physiological effect, which can be determined by one of skill in the art. Desired routes of administration include, but are not limited to, direct delivery to the desired organ (e.g., liver (optionally via the hepatic artery), lungs, heart, eyes, kidneys), oral, inhalation, intranasal, intratracheal, intrathecal, intraarterial, intraocular, intravenous, intramuscular, subcutaneous, intradermal, and other parenteral routes of administration. Routes of administration may be combined, if desired.

[0144] The systems described herein may be therapeutically useful if sufficient amounts of functional enzyme or protein are produced to improve a patient's condition. In certain embodiments, A gene expression level as low as 5% would provide sufficient therapeutic benefit that the patient is treatable with non-gene therapy approaches. In other embodiments, the gene expression level is 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, 104, 105, 106, 107, 108, 109, 109, %, 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%, and up to 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.More specifically, heterozygous patients may have enzyme function levels of about 50% or less, so effective treatment may not require replacing enzyme activity to levels within the range of "normal" or non-deficient patients. Similarly, patients without detectable amounts of enzymes may be rescued by delivering enzyme function to less than 100% activity levels, and optionally may 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 "normal" healthy subjects. In yet other embodiments, where reduction of gene expression is desired, a 20%-50% reduction or up to about 100% reduction may provide the desired benefit. As described herein, the therapies described herein may be used in conjunction with other therapies (i.e., standard of care for the subject's (patient's) diagnosis).

[0145] 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.

[0146] 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 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 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. One or more of these drugs may be continued at the same dose or at an adjusted dose after gene therapy administration. Such therapy may be for about 1 week (7 days), 2 weeks, 3 weeks, about 60 days, or more, as needed. In certain embodiments, a regimen that does not include tacrolimus is selected.

[0147] In another embodiment, the method includes co-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).

[0148] 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).

[0149] In one aspect, a method is provided for treating a patient with ornithine transcarbamylase (OTC) deficiency using a nuclease expression cassette comprising a meganuclease coding sequence that recognizes a site in the human PCSK9 gene under the control of a promoter as described herein. The method further comprises administration of an expression cassette carrying an OTC transgene of SEQ ID NO: 17 or a sequence sharing at least 90% identity therewith, as described herein. Such an expression cassette may be delivered via a viral or non-viral vector. In certain embodiments, the expression cassette may be delivered using LNP. The native human OTC coding sequence is shown in SEQ ID NO: 30. SEQ ID NO: 17 and SEQ ID NO: 30 are , sharing approximately 75.89% identity.

[0150] In another aspect, a method is provided for treating a patient with ornithine transcarbamylase (OTC) deficiency using a nuclease expression cassette comprising a sgRNA and a Cas9 coding sequence that recognizes a site in the human PCSK9 gene. The method further comprises administering an expression cassette carrying an OTC transgene of SEQ ID NO: 17 or a sequence that shares at least 90% identity therewith, as described herein. Such an expression cassette can be delivered via a viral or non-viral vector. In certain embodiments, the expression cassette can be delivered using LNP.

[0151] 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 about 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 about 20 micromoles / mmol creatine, indicates OTCD, as does elevated urinary orotic acid following an allopurinol challenge test. 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 Disease s 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.

[0152] In certain embodiments, any of the nuclease expression cassettes described herein, non-viral vectors, viral vectors (e.g., rAAV), or in pharmaceutical compositions thereof can be administered for gene editing in a patient. In certain embodiments, the method is useful for non-embryonic gene editing. In certain embodiments, the patient is an infant (e.g., birth to about 9 months). In certain embodiments, the patient is a toddler or older, e.g., 12 months or older.

[0153] 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.

[0154] 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.

[0155] The abbreviation "sc" refers to self-complementary. "Self-complementary AAV" refers to constructs in which the coding region carried by the recombinant AAV nucleic acid sequence is designed to form an intramolecular double-stranded DNA template. Upon infection, rather than waiting for cell-mediated synthesis of the second strand, the two complementary halves of the scAAV will associate to form one double-stranded DNA (dsDNA) unit ready for immediate replication and transcription. See, for example, DM McCarty et al, "Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis", Gene Therapy, (August 2001), Vol 8, Number 16, Pages 1248-1254. Self-complementary AAVs are described, for example, in U.S. Patent Nos. 6,596,535, 7,125,717, and 7,456,683, each of which is incorporated by reference in its entirety.

[0156] 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.

[0157] 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).

[0158] 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.

[0159] 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.

[0160] 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.

[0161] "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.

[0162] 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.

[0163] The terms "substantial homology" or "substantial similarity," when referring to a nucleic acid, or a fragment thereof, indicates that when optimally aligned with another nucleic acid (or its complementary strand) with appropriate amino acid insertions or deletions, the nucleic acid has at least about 95-99% amino acid sequence identity with the aligned sequence. Preferably, the homology extends over the full length sequence, or a protein thereof, such as the cap protein, rep protein, or a fragment thereof that is at least 8 amino acids in length, or more preferably, at least 15 amino acids in length. Examples of suitable fragments are described herein.

[0164] The term "highly conserved" means at least 80% identity, preferably at least 90% identity, more preferably more 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.

[0165] 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 the examples, the 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 may also be 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 listed above. As another example, polynucleotide sequences 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 and search sequences. For example, percent sequence identity between nucleic acid sequences can be determined using Fasta™ using its default parameters (word size 6 and NOPAM factor for scoring matrix) provided in GCG Version 6.1, which is incorporated herein by reference. Several sequence alignment programs for amino acid sequences are also available, such as, for example, "Clustal Omega", "Clustal X", "MAP", "PIMA", "MSA", "BLOCKMAKER", "MEME", and "Match-Box" programs. Generally, any of these programs are used with default settings, but if necessary, those skilled in the art can change these settings.Alternatively, one of skill in the art can utilize another algorithm or computer program that provides at least the same level of identity or alignment as that provided by the referenced algorithms and programs. See, e.g., JD Thomson et al, Nucl. Acids. Res., "A comprehensive comparison of multiple sequence alignments", 27(13):2682-2690 (1999).

[0166] 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 the 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" refers to ranges that include both integers and fractions. Includes all values ​​in.

[0167] Throughout this application, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to specifically disclose all possible subranges as well as individual values ​​within that range. For example, the description of a range such as 1-6 should be considered to specifically disclose subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, and individual numbers within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0168] 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 excludes other components, elements, integers, steps, etc.

[0169] 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 published documents which provide general guidance to those of ordinary skill in the art for many of the terms used herein. EXAMPLES

[0170] Ornithine transcarbamylase (OTC) deficiency 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 will only provide short-term therapeutic benefit because unintegrated genomes are lost during hepatocyte proliferation. Nuclease-mediated site-specific integration of an OTC minigene cassette in a genomic safe harbor will provide long-term therapeutic benefit to patients with OTC deficiency. One of the safe harbors for gene targeting is the PCSK9 gene (e.g., exon 7 region). The nuclease can be an engineered meganuclease (ARCUS2) targeting PCSK9 or CRISPR / Cas9 with a specific sgRNA targeting PCSK9. The donor vector contains a minigene containing a liver-specific promoter (e.g., TBG promoter), a codon-optimized hOTC coding sequence, and a polyA sequence. Both the nuclease and the donor template can be delivered by AAV vectors (dual AAV vector system). We demonstrated sustained transgene expression and efficient gene targeting in 12% of hepatocytes 12 weeks after a single intravenous injection of dual AAV vectors in neonatal non-human primates (NHPs). The minigene in the donor vector is flanked by homologous recombination (HDR) arms.

[0171] This is the first demonstration in NHPs of in vivo nuclease-mediated gene targeting of the PCSK9 locus to express a therapeutic protein after a single injection of dual AAV vectors as newborns or infants. The composition of a donor vector containing an OTC minigene for gene targeting of the PCSK9 locus in humans / NHPs has not been clinically tested for the treatment of OTC deficiency. The hOTC donor vector will be tested in neonatal NHPs for gene targeting efficiency and in neonatal transgenic OTC-deficient mice for efficacy.

[0172] Many metabolic diseases require early intervention and therapy, but AAV-mediated neonatal gene therapy is a promising approach to treat these conditions by preventing rapid liver proliferation during the neonatal period. and is unstable due to the non-integrative nature of AAV vectors. Targeted integration of therapeutic minigene cassettes into the safe harbor will result in persistent expression of therapeutic genes at the genomic level, maintaining therapeutic effects through cell division. For many metabolic diseases, such as OTC deficiency, sufficient transduction efficiency in the liver needs to be achieved for clinical benefit.

[0173] 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 propose to 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 studies of genome editing for OTCD were performed in OTC-deficient mice that were rendered sensitive to PCSK9 ARCUS nuclease via germline modification of exon 7 of the endogenous PCSK9 gene. 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 trials, 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 influence of the following parameters on editing efficiency and toxicity: transgenes (human factor IX and human OTC), promoters driving ARCUS, clade E capsid, donor length flanking the transgene, and age of the macaques at the time of dosing. Here we report preliminary data from 16 of 24 animals with at least 3-month biopsy results.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 the primate model 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 believed to provide 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 vector genome knock-ins were 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.

[0174] Example 1 - Materials and Methods Materials and Methods AAV vectors were constructed according to previously established procedures and manufacturer's instructions. AAVhu37 capsids were used for the experiments described herein where indicated.

[0175] All animal procedures were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania.

[0176] Example 2 - Pilot Study: Knock-in of hFIX Minigene at the PCSK9 Locus with ARCUS2 or SaCas9 in Neonatal NHPs In this study, we evaluated the efficiency of on-target (PSK9) SaCas9 or ARCUS-mediated gene editing and hFIX or OTC minigene knock-in in neonatal non-human primates (NHPs). Figure 1 shows a schematic diagram of the rhPCSK9 locus, showing the donor splice site in exon 7 and the HDR donor vector containing the donor template of interest (e.g., hFIX, hOTC). Additionally, Figures 3A-3C show schematic diagrams of a dual AAV vector system for SaCas9 or ARCUS-mediated gene correction. Figure 3A shows a schematic diagram of a dual AAVhu37 vector system for ARCUS2-mediated gene correction, where the AAVhu37 donor vector contains the hOTC donor template sequence. Figure 3B shows a schematic diagram of a dual AAVhu37 vector system for Sa-Cas9-mediated gene correction (trans; AAVhu37-SaCas9), where the AAV.hu37.shRNA donor vector contains the hOTC donor template sequence. FIG. 3C shows a schematic diagram of the dual AAVhu37 vector system (cis; AAVhu37.PCSK9-sgRN.SaCas9) for Sa-Cas9-mediated gene correction, in which the AAV.hu37 donor vector contains the hOTC donor template sequence.

[0177] The above dual AAVhu37 vectors containing gene editing nucleases and donor templates were used in neonatal NHPs to investigate knock-in of the hFIX minigene at the PCSK9 locus mediated by either SaCas9 or ARCUS2. Gene editing AAVhu37 vectors were injected at 1×10 13 The donor template AAVhu37 vector was delivered at a dose of 3 × 10 13GC / kg. Overall, there were three treatment NHP groups: 1) AAVhu37.EGFP and AAVhu37.Donor-HDR-hFIX.U6.sgR, 2) AAVhu37.ARCUS2 and AAVhu37.Donor-HDR-hFIX, and 3) AAVhu37.SaCas9 and AAVhu37.Donor-HDR-hFIX.U6.sgR. Figure 2 shows the timeline of a pilot study involving hFIX minigene knock-in at the PCSK9 locus with ARCUS2 or SaCas9 in neonatal NHPs. In this study, NHPs were injected on day 0, blood samples were taken every 2-4 weeks (to examine serum chemistry, hFIX expression in plasma, PCSK9 levels in serum, LDL levels, and neutralizing antibody (NAb) levels), and the first liver biopsy was performed on day 84 (to examine vector genome levels, gene expression levels, off-target editing, and histology).

[0178] In vivo studies of nuclease-mediated gene targeting were performed in newborn and infant NHPs. As shown in Figures 4A, 4B, and 5G, animals were dosed with 1 x 10 13 GC / kg AAVhu37.ARCUS2.WPRE and 3 x 10 13 GC / kg of AAVhu37.hFIXco-HDR or 1 × 10 13 GC / kg AAVhu37.SaCas9.WPRE and 3 x 10 13 GC / kg AAVhu37.hFIXco-HDR.U6.sgR or 1 × 10 13 GC / kg AAVhu37.GFP.WPRE and 3 x 10 13 GC / kg of AAVhu37.hFIXco-HDR.U6.sgR. Figure 4C shows hFIX levels at the indicated time points, 0 days to 13 months after treatment (plotted as ng / mL). Figure 4D shows PCSK9 levels at the indicated time points, 0 days to 12 months after treatment (plotted as percentage of baseline on day 0). Figure 4E shows ALT (alanine aminotransferase) levels at the indicated time points, 0 days to 196 days after treatment (plotted as U / L). Figure 4F shows hFIX levels at the indicated time points, 0 days to 13 months after treatment (plotted as percentage of baseline on day 0). FIG. 4G shows anti-FIX IgG levels at the indicated time points from days 0 to 196 (plotted as dilution factor, 1 / dilution). FIG. 4G shows PCSK9 levels at the indicated time points from days 0 to 196 after treatment (plotted as ng / mL). FIG. 4H shows body weights measured at the indicated time points from days 0 to 196 after treatment (plotted as g). FIG. 5A shows hFIX levels at the indicated time points (plotted as ng / mL) in infant NHPs. FIG. 5B shows PCSK9 levels at the indicated time points (plotted as percentage of baseline on day 0) in infant NHPs. FIG. 5C shows ALT (alanine aminotransferase) levels at the indicated time points (plotted as U / L) in infant NHPs. FIG. 5D shows anti-FIX IgG levels at the indicated time points (plotted as dilution factor, 1 / dilution) in infant NHPs. Figure 5E shows PCSK9 levels (plotted as ng / mL) at the indicated time points in infant NHPs. Figure 5F shows body weights measured at the indicated time points (plotted as g) in infant NHPs. Figure 5G is a summary table showing data from the experiments described in Figures 4A-5G. Figure 5H shows a comparison of various data between neonatal and infant NHPs tested.

[0179] Figures 6A-6E show vector transduction (GC) and transgene expression in liver biopsy samples taken on the indicated days after treatment in NHPs. Figure 6A shows the level of vector transduction in liver biopsy samples plotted as AAV genome copies (GC) per diploid cell. Figure 6B shows the relative expression of transgene RNA in liver biopsy samples. Figure 6C shows dual in situ hybridization (ISH) using specific probes to detect FIX and ARCUS in liver biopsies. Figure 6D shows digitized ISH images used to quantify transduction. Figure 6E shows transduction efficiency of the FIX transgene quantified by ISH and plotted as percent transduction. Figure 6F shows genome copies in the liver. At both time points, higher GC of FIXco was observed in infants (3-fold) than in neonates. In the second biopsy, there was a decrease in GC of FIXco than in the first biopsy (1.5-2-fold). Figure 6G shows transgene mRNA in liver biopsies. FIXco mRNA was stable between 3 months and 1 year in infants, whereas FIXco mRNA decreased 3-fold between 3 months and 1 year in neonatally treated animals. Figure 6H shows the results of molecular analysis on liver samples. Indels measured by amplicon-seq and off-targets measured by ITR-seq are shown. In addition, nanopore long-read sequencing results for animal 20-196 at day 366 are shown. 0.6% of reads showed HDR incorporation on both sides. Figure 6I is a summary table of the data presented in Figures 6A-H.

[0180] 7A-7L show dual in situ hybridization (ISH) using specific probes to detect FIX and ARCUS in liver biopsies taken 84 days after treatment in NHPs (NHPs treated with AAVhu37.ARCUS2 and AAVhu37.Donor-HDR-hFIX) shown in various magnification fields. FIG. 7A shows ISH-detected ARCUS in liver biopsies viewed at 4x magnification. FIG. 7B shows ISH-detected hFIX in liver biopsies viewed at 4x magnification. FIG. 7C shows overlaid images of ISH-detected ARCUS and hFIX viewed at 4x magnification. FIG. 7D shows ISH-detected ARCUS and hFIX viewed at 4x magnification as overlaid with DAPI (nuclear stain). FIG. 7E shows ISH-detected ARCUS in liver biopsies viewed at 10x magnification. Figure 7F shows ISH-detected hFIX in liver biopsies viewed at 10x magnification. Figure 7G shows an overlay image of ISH-detected ARCUS and hFIX viewed at 10x magnification. Figure 7H shows ISH-detected ARCUS and hFIX viewed at 10x magnification overlaid with DAPI (nuclear stain). Figure 7I shows ISH-detected ARCUS expression in liver biopsies viewed at 20x magnification. Figure 7J shows ISH-detected hFIX in liver biopsies viewed at 20x magnification. Figure 7K shows an overlay image of ISH-detected ARCUS and hFIX viewed at 20x magnification. Figure 7L shows ISH-detected ARCUS and hFIX viewed at 20x magnification as an overlay image with DAPI (nuclear stain). A summary of vector transduction (GC / diploid genome) is shown in Table 1 below. [Table 5]

[0181] 8A-8M show dual in situ hybridization (ISH) using specific probes to detect FIX and ARCUS in liver biopsies taken 84 days after treatment in NHPs, shown in various magnification fields (NHPs treated with AAVhu37.EGFP and AAVhu37.Donor-HDR-hFIX.U6.sgR). FIG. 8A shows ISH-detected GFP-WRPE in liver biopsies viewed at 4x magnification. FIG. 8B shows ISH-detected hFIX in liver biopsies viewed at 4x magnification. FIG. 8C shows overlaid images of ISH-detected GFP-WRPE and hFIX viewed at 4x magnification. FIG. 8D shows ISH-detected GFP-WRPE and hFIX viewed at 4x magnification, overlaid with DAPI (nuclear stain). FIG. 8E shows ISH-detected GFP-WRPE in liver biopsies viewed at 10x magnification. FIG. 8F shows ISH-detected hFIX in liver biopsies viewed at 10x magnification. FIG. 8G shows an overlay of ISH-detected GFP-WRPE and hFIX viewed at 10x magnification. FIG. 8H shows ISH-detected GFP-WRPE and hFIX viewed at 10x magnification overlaid with DAPI (nuclear stain). FIG. 8I shows ISH-detected GFP-WRPE expression in liver biopsies viewed at 20x magnification. FIG. 8J shows ISH-detected hFIX in liver biopsies viewed at 20x magnification. FIG. 8K shows an overlay of ISH-detected GFP-WRPE and hFIX viewed at 20x magnification. FIG. 8L shows ISH-detected GFP-WRPE and hFIX viewed at 20x magnification overlaid with DAPI (nuclear stain). Figure 8M shows ISH-detected GFP-WRPE and hFIX in untreated controls at 20x magnification overlaid with DAPI (nuclear stain). A summary of vector transduction (GC / diploid genome) is shown in Table 2 below. [Table 6]

[0182] FIG. 9 shows ARCUS-mediated on-target editing in NHPs treated with AAVhu37.ARCUS2 and AAVhu37.Donor-HDR-hFIX. After 84 days, liver biopsies were taken and the percentage of total indels present in the target regions was calculated. Further, ARCUS-mediated on-target editing in NHPs treated with AAVhu37.ARCUS2 and AAVhu37.Donor-HDR-hFIX. After 84 days of treatment, liver biopsies were taken and the frequency of total indels present in the target regions was calculated and plotted as the frequency of unique UMI OT reads to the target. A summary of indels quantified by amplicon-seq is shown in Table 3 below. [Table 7]

[0183] Example 3 - 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. M2PCSK9 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.

[0184] In this study, neonatal NHPs were administered the ARCUS2 nuclease vector and donor vectors with various lengths of HDR arms (500 bp arms or short HDR arms). A schematic diagram of the vectors is shown in FIG. 11J. FIG. 12A is a summary table showing the data from the experiment. All 14 neonatal macaques tolerated the vector infusion well (i.e., no obvious clinical sequelae) and gained weight over time (FIG. 11I). Liver enzyme levels were within the normal range, except for a transient and mild elevation of ALT levels in some animals on day 14 (FIG. 11C).

[0185] 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 11A). These pre-existing anti-AAVrh79 antibodies would block AAV gene transfer.

[0186] PCSK9 levels were followed over time in all newborn animals, including the donor-only control animals. PCSK9 levels on day 0 differed between newborns (FIG. 11B). Nine animals showed a trend toward decreased PCSK9 levels after vector administration, including one donor-only control animal, while the remaining five animals showed sustained or transient increases in PCSK9 levels after administration (FIG. 11B).

[0187] Liver biopsies were performed by laparotomy on day 84. Transduction efficiency of hOTC in the liver was assessed by dual ISH using hOTC-specific and M2PCSK9-specific probes to detect transgene mRNA, and OTC immunofluorescence to detect human OTC protein, followed by quantification on scanned slides (Figure 11D). Three animals (21-111, 21-113, and 21-122) with pre-existing anti-AAVrh79 binding antibodies at the time of dosing showed OTC-positive hepatocytes by both methods. Two donor-only control animals showed low levels of hOTC transduction (1% or less). The highest transduction efficiency (11.9% and 18.6% by OTC immunofluorescence) was detected in two animals co-administered with AAVrh79.TBG.PI.M2PCSK9.WPRE.bGH and AAVrh79.rhHDR.TBG.hOTCco.bGH donor vectors (G6). Positive hOTC-expressing hepatocytes were also found to be present in the clusters, and these levels are above the threshold (approximately 5% of OTC-expressing cells) that would provide substantial benefit to patients.

[0188] 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 11F). Consistent with the transduction efficiency analysis, two animals in group 6 (21-157 and 21-175) had the highest GC of hOTC vector (Figure 11F), hOTC mRNA (Figure 12I), and on-target indels (%) (Figure 11H). The GC of M2PCSK9 vector in animals was 2- to 7-fold lower than the GC of hOTC vector, while M2PCSK9 mRNA levels were 23- and 765-fold lower than hOTC mRNA levels (Figures 11F and 11G).

[0189] Off-target activity assessed by ITR-seq identified 2-40 potential off-targets in liver biopsy samples at day 84 of this study. Several off-target sites were detected in multiple animals, including hFIX infant and hFIX neonatal animals, in study 2 and study 3, respectively. Off-target editing was further characterized by amplicon-seq of potential off-target sites.

[0190] In summary, we have identified a combination of M2PCSK9 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 threshold for patients with beneficial outcomes (i.e., approximately 5% OTC-expressing hepatocytes). Animals in this study are being followed for long-term efficacy and safety evaluation. We will perform a second liver biopsy at 1 year post-administration to evaluate the stability of hOTC transduction, histopathology in the liver, and on- and off-targeting in the liver.

[0191] Example 4 - PCSK9-hE7-KI Mouse Model Because the M2PCSK9 targeting sequences in the human and macaque PCSK9 genes are not conserved in the mouse Pcsk9 gene, M2PCSK9 cannot be used for genome editing at the mouse locus. Therefore, we aimed to generate a knock-in mouse model (named PCSK9-hE7-KI mouse) in which a region of the mouse Pcsk9 gene containing exon 7 was replaced with a region of the human PCSK9 gene containing exon 7. We requested The Jackson Laboratory to provide the model (Figures 10A-10C). This model can be used to evaluate in vivo genome editing and gene targeting efficiency. PCSK9-hE7-KI mice were then transfected with sparse fur ash (spf ash ) mice. ashMice 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).

[0192] PCSK9-hE7-KI.spf ash 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.

[0193] Figure 12 shows a sequence alignment of 265 bp sequences representing human PCSK9 sequence, mouse Pcsk9 (mPCSK9) and rhesus Pcsk9 (rhPCSK9) of the PCSK9-hE7 knock-in allele. There are six mismatches between the human and rhesus sequences in this 265 bp region. The rodent and primate sequences diverge beyond this frame due to various LINE and LTR insertions. There are two amino acid differences in exon 7 between human and mouse. hE7-KI mice express normal levels of mPCSK9 by ELISA measured.

[0194] Example 5 - PCSK9-hE7-KI.spf ash In vivo OTC gene targeting to the PCSK9 locus in offspring This ongoing non-GLP pharmacology study is investigating neonatal PCSK9-hE7-KI.spf ashWe aimed to evaluate whether M2PCSK9 meganuclease-mediated knock-in of human OTC gene in mice can achieve therapeutic human OTC expression in target tissues for the treatment of OTC deficiency (liver) after a single co-administration of M2PCSK9 nuclease expression vector and human OTC donor vector via intended clinical ROA (IV). A schematic of the experimental design is shown in Figure 14A, and dosage groups are shown in Figure 14B.

[0195] On day 0, newborn (PND1-2) male PCSK9-hE7-KI.spf ash Mouse, 3.0 x 10 13 in combination with one of three different AAVrh79 hOTCco donor vectors at a dose of GC / kg, 1.0 × 10 13 AAVrh79 vector expressing M2PCSK9 meganuclease (AAVrh79.TBG.PI.M2PCSK9.WPRE.bGH) was co-administered IV at a dose of GC / kg. The M2PCSK9 meganuclease expression vector (AAVrh79.TBG.PI.M2PCSK9.WPRE.bGH) evaluated in this study 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), while the hOTCco donor vectors evaluated in this study contain a mouse-human hybrid HDR sequence (AAVrh79.mhHDR.TBG.hOTCco.bGH), a short version of the human HDR sequence (AAVrh79.shHDR.TBG.hOTCco.bGH), or no HDR sequence (AAVrh79.TBG.hOTCco.bGH). Figure 13 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 In mice, AAVrh that does not express meganuclease The 79 vector (AAVrh79.TBG.PI.EGFP.WPRE.bGH) was administered in combination with AAVrh79.mhHDR.TBG.hOTCco.bGH.

[0196] 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.

[0197] Preliminary results show that mice administered vectors with mhHDR arms showed comparable survival rates to wild-type mice, and shHDR-treated mice achieved 80% survival rates after a 10-day high-protein diet challenge (Figure 14C). All treated mice maintained their body weight better than KI-spf-ash untreated mice (Figure 14D). Plasma ammonia levels in mHDR-treated mice were significantly reduced compared to untreated mice (Figure 14E).

[0198] mPCSK9 levels were measured on day 48 and all treated mice showed a decrease (Figure 14F). The percentage of indels was fairly consistent between HDR types (Figure 14G). hOTC levels were increased in shHDR and mhHDR treated mice (Figure 14H).

[0199] Example 6 - In vivo targeting of the OTC gene to the PCSK9 locus in neonatal rhesus monkeys This ongoing non-GLP pharmacology study aims to evaluate whether M2PCSK9 meganuclease-mediated knock-in of the human OTC gene in neonatal rhesus monkeys can achieve therapeutic human OTC expression in target tissue for treatment of OTC deficiency (liver) following a single co-administration of M2PCSK9 meganuclease expression vector and human OTC donor vector via the intended clinical ROA (IV).

[0200] On day 0, newborn (1–16 days old) rhesus monkeys were administered 3.0 × 10 13 in combination with one of two different AAV hOTCco donor vectors at a dose of GC / kg, 1.0 × 10 13 Two different vectors expressing M2PCSK9 meganuclease were co-administered IV at a dose of 3.0×10 GC / kg. 13 A non-nuclease group receiving only the AAV hOTCco donor vector at a dose of GC / kg was included as a donor-only control.

[0201] For the AAV vector targeting the PCSK9 gene, we compared two AAV vector constructs expressing M2PCSK9 in the liver. AAV.TBG.PI.M2PCSK9.WPRE.bGH contains the full-length TBG promoter and two copies of the enhancer element, and WPRE expresses higher levels of nuclease than AAV.TBG-S1-F113.PI.M2PCSK9.bGH, which contains a short and weak promoter. For the hOTC donor vector, we compared two AAV.hOTCco donor vectors with different lengths of homology arms flanking the hOTCco transgene cassette.

[0202] NHPs are administered the two vectors IV on day 0 and are monitored daily for viability. In-life assessments include measurements of body weight, clinical pathology of blood, and gene editing analysis of plasma. Two laparotomies are planned to isolate liver tissue for analysis of genome editing efficiency, vector genome copies, transgene expression, histopathology, immunostaining, and RNA ISH staining. NHPs will be followed over time and necropsied (date to be determined), at which point tissue will be harvested from the liver and other major organs for evaluation of genome editing efficiency, vector genome copies, transgene expression, histopathology, immunostaining, and RNA ISH staining.

[0203] Example 7 - PCSK9-hE7-KI.SPF ash Assessment of efficacy in offspring and determination of vector ratios This planned non-GLP compliant pharmacology study will evaluate the efficacy and safety of neonatal PCSK9-hE7-KI.spf for the treatment of OTC deficiency (liver) following a single co-administration of M2 PCSK9 nuclease expression vector and human OTC donor vector via the intended clinical ROA (IV). ash The aim of this study was to evaluate the ratio of vector components required to achieve the highest efficacy of M2PCSK9 meganuclease-mediated knock-in of the human OTC gene in mice.

[0204] On day 0, newborn (PND1-2) male PCSK9-hE7-KI.spf ashMice are co-administered IV with one of three doses of an AAVrh79 vector expressing M2PCSK9 meganuclease (AAVrh79.TBG.PI.M2PCSK9.WPRE.bGH) in combination with one of three doses of a hOTCco donor vector containing a mouse-human hybrid HDR sequence (AAVrh79.mhHDR.TBG.hOTCco.bGH). The M2PCSK9 meganuclease expressing vector (AAVrh79.TBG.PI.M2PCSK9.WPRE.bH) evaluated in this study was identical to the clinical candidate, while the hOTCco donor vector was identical to the 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), while the hOTCco donor vector evaluated in this study contains a mouse-human hybrid HDR sequence (AAVrh79.mhHDR.TBG.hOTCco.bGH).

[0205] The mouse-human hybrid HDR sequence in the donor vector (AAVrh79.mhHDR.TBG.hOTCco.bGH) was selected for this study, and the donor sequence was PCSK9-hE7-KI.spf ash Direct homology with the murine sequence allowed for evaluation of the pharmacology of this approach.

[0206] In-life evaluations include daily survival monitoring, body weight measurement, and evaluation of plasma NH3 and urinary orotic acid levels after high protein diet challenge. On day 81, mice are challenged with a 10-day high protein diet and necropsied 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 also isolated to evaluate on-target editing (amplicon-seq) and to evaluate vector genome copies.

[0207] Example 8 - PCSK9-hE7-KI.SPF ash Evaluating efficacy in offspring and determining the minimal effective dose This planned GLP-compliant pharmacology study will include neonatal PCSK9-hE7-KI.spf ash To evaluate the efficacy of IV-administered AAV in a mouse model and determine the MED. AAVrh79.hHDR.TBG.hOTCco.bGH) will be the toxicology vector lot manufactured for planned GLP-compliant toxicity. 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 clinical candidate toxicology vector lots.

[0208] For this study, the mouse-human hybrid HDR sequence in the donor vector (AAVrh79.mhHDR.TBG.hOTCco.bGH) (donor sequence 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.

[0209] In this study, N=60 neonatal (PND1-2) PCSK9-hE7-KI.spf ashMice 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). 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. Three dose levels of AAV will be evaluated using IV administration. Dose levels will be selected based on dose ranges evaluated in previous non-clinical studies. Dose levels evaluated encompass the expected clinical doses.

[0210] 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.

[0211] MED is vehicle-treated neonatal PCSK9-hE7-KI.spf ash Compared to control mice, AAV-treated neonatal PCSK9-hE7-KI.spf ash The assay will be based on analysis of survival rates following 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.

[0212] Example 9 - PCSK9-hE7-KI.spf ash Toxicity studies in offspring Neonatal (PND1~2) PCSK9-hE7-KI.spf ash A 6-month GLP-compliant safety study will be conducted in mice to examine the safety, tolerability, pharmacology, and pharmacokinetics of the test article following IV co-administration. 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 GC / kg, N=20 per dose) or vehicle (phosphate-buffered saline [PBS], N After administration of the test article or vehicle, in-life evaluations included 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).

[0213] 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.

[0214] 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.

[0215] For vector biodistribution, qPCR detection specific for the transgenes (M2PCSK9 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 assay to previously performed qualification studies. Tested matrices 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.

[0216] Example 10 - PCSK9-hE7-KI.ldlr - / ldlr - .apobec - / apobec - hLDLR minigene knock-in at the PCSK9 locus by SaCas9 in offspring (hoFH model) This study is a neonatal PCSK9-hE7-KI.ldlr - / ldlr - .apobec - / apobec -The aim of this study is to evaluate whether Cas9-mediated knock-in of the human LDLR gene in mice can achieve therapeutic human LDLR expression in target tissues for the treatment of familial hypercholesterolemia (liver) after a single co-administration of SaCas9 nuclease expression vector and human LDLR donor vector via the intended clinical ROA (IV). A mouse model was generated using the experimental design of Figure 15. In the mouse model, mouse PCSK9 exon 7 is replaced with human PCSK9 exon 7 containing the SaCas9 targeting sequence.

[0217] On day 0, neonatal PCSK9-hE7-KI.ldlr - / ldlr - .apobec - / apobec - Mouse, 3.0 x 10 13 GC / kg, in combination with one of two different AAVrh79 hLDLR donor vectors, and 1.0 × 10 13 AAVrh79 vector expressing Cas9 (AAVrh79.U6.sgR3.PSCK9.APB2.HLP.SaCas9.bGH) was co-administered IV at a dose of GC / kg. A schematic showing the vectors used is shown in FIG. 16. Specifically, one of the donor vectors evaluated in this study contained a mouse-human hybrid HDR sequence (AAVrh79.mhHDR.hLDLR011), while the other contained a shorter version of the human HDR sequence (AAVrh79.shHDR.hLDLR011). As a negative control, an additional age-matched PCSK9-hE7-KI.spf ash Mice, AAVrh79.sh An AAVrh79 vector not expressing saCas9 was administered in combination with HDR.hLDLR011.

[0218] In-life evaluations include daily viability monitoring and evaluation of serum LDL-c levels on days 42, 63, 90, 120, and 150. Partial hepatectomy on day 63 to evaluate the stability of human LDLR transduction, and autopsy on day 150. At autopsy, livers are harvested to evaluate knock-in of the human LDLR gene, including evaluation of human LDLR mRNA expression (in situ hybridization), LDLR protein expression (immunostaining). Liver DNA is isolated to evaluate on-target editing (amplicon-seq, Oxford nanopore long-read sequencing) and to evaluate vector genome copies. The experimental design is shown in Figure 17.

[0219] Preliminary results show that mice administered saCas9 and donor vector had significantly reduced serum LDL levels. There was no change in LDL after 2 / 3 partial hepatectomy indicating stable integration (Figure 18A). Indels were consistent using mhHDR and shHDR donor vectors (Figure 18B). At day 63, shHDR-treated mice showed slightly higher hLDLR levels (Figure 18C), but serum LDL levels were similar for mhHDR and shHDR (saCas9) vectors (Figure 18D).

[0220] FIG. 19 shows immunohistochemical evaluation of hLDLR expression in liver 63 days after partial hepatectomy.

[0221] All documents cited herein are incorporated herein by reference, and the sequences and text of the sequence listing filed herewith are also incorporated by reference. U.S. Provisional Patent Application Nos. 63 / 180,603, filed April 27, 2021, 63 / 242,474, filed September 9, 2021, 63 / 244,205, filed September 14, 2021, 63 / 301,933, filed January 21, 2022, and 63 / 331,385, filed April 15, 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. [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4] [Table 8-5]

Claims

1. 1. A system for treating a genetic disorder, the system comprising: (a) a gene editing vector comprising a nucleic acid sequence encoding a nuclease that targets the PCSK9 gene; (b) a donor vector comprising a transgene cassette comprising a nucleic acid sequence encoding a transgene and a regulatory sequence directing expression of the transgene in the target cell, the donor vector further comprising homologous recombination (HDR) arms on the 5' and 3' sides of the transgene cassette, the transgene being not PCSK9.

2. The system of claim 1, further comprising a regulatory sequence that directs the expression of the nuclease in a target cell that comprises a PCSK9 gene, optionally, the nuclease targets PCSK9 exon 7, optionally, the nuclease is a PCSK9-specific meganuclease, and optionally, the meganuclease is an ARCUS meganuclease.

3. The system according to claim 1 or 2, wherein the gene editing vector comprises a sequence encoding Cas9 adjacent to a nuclear localization signal, and optionally, the gene editing vector further comprises an sgRNA comprising at least 20 nucleotides that specifically binds to a target site in the PCSK9 gene, the target site being 5' to the protospacer adjacent motif (PAM) that is specifically recognized by the Cas9; and optionally, the donor vector further comprises an sgRNA comprising a seed region of at least 20 nucleotides, the sgRNA specifically binds to a target site in the PCSK9 gene, the target site being 5' to the protospacer adjacent motif (PAM) that is specifically recognized by the Cas9.

4. 4. The system of claim 1, further comprising an RNA polymerase promoter, optionally wherein the RNA polymerase promoter is a U6 promoter, and optionally wherein the U6 promoter is located 5' to the sgRNA.

5. 6. The system of claim 3, wherein the seed region is 100% complementary to the target site sequence; optionally, the seed region is less than 100% complementary to the target site sequence; optionally, the transgene is OTC, PKU, CTLN1, or LDLR; optionally, at least one of the donor vector and the gene editing vector is an adeno-associated virus (AAV) vector, wherein the AAV vector comprises an AAV 5' ITR and an AAV 3' ITR; and optionally, the ratio of the gene editing AAV vector of (a) to the donor AAV vector of (b) is such that the donor AAV vector of (b) exceeds the gene editing vector of (a).

6. 1. A system for treating a genetic disorder, the system comprising: (c) 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 comprising the PCSK9 gene, and a 3′ ITR; and (d) a donor AAV vector comprising an AAV capsid and a second vector genome comprising a 5′ ITR, a 5′ homologous recombination (HDR) arm, a transgene and a regulatory sequence directing expression of the transgene in the target cell, a 3′ HDR arm, and a 3′ ITR; Or, (e) a gene-edited AAV comprising an AAV capsid and a first vector genome comprising a 5′ ITR, a 5′ nuclear localization signal (NLS), a sequence encoding Cas9 and a regulatory sequence directing expression of saCas9 in a target cell comprising a PCSK9 gene, a 3′ NLS, and a 3′ ITR; and (f) a donor AAV vector comprising an AAV capsid and a second vector genome comprising a 5' ITR, a 5' homologous recombination (HDR) arm, a transgene and a regulatory sequence directing expression of the transgene in the target cell, a 3' HDR arm, a U6 promoter, an sgRNA comprising at least 20 nucleotides that specifically binds to a target site in the PCSK9 gene, the target site being 5' to a protospacer adjacent motif (PAM) that is specifically recognized by the Cas9, and a 3' ITR; Or, (g) a gene editing AAV vector comprising an AAV capsid; and a first vector genome comprising a 5′ ITR, a U6 promoter, an sgRNA comprising at least 20 nucleotides that specifically binds to a target site in a PCSK9 gene, the target site being 5′ to a protospacer adjacent motif (PAM) that is specifically recognized by Cas9, a 5′ nuclear localization signal (NLS), a sequence encoding Cas9 and a regulatory sequence that directs expression of the Cas9 in a target cell comprising the PCSK9 gene, a 3′ NLS, and a 3′ ITR; and (h) a donor AAV vector comprising an AAV capsid and a second vector genome comprising a 5′ ITR, a 5′ homologous recombination (HDR) arm, a transgene and a regulatory sequence directing expression of the transgene in the target cell, a 3′ HDR arm, and a 3′ ITR; or Or, (i) a gene editing vector, (i) lipid nanoparticles, (ii) an sgRNA comprising at least 20 nucleotides that specifically binds to a target site in a PCSK9 gene, the target site being 5' to a protospacer adjacent motif (PAM) that is specifically recognized by Cas9; (iii) a gene editing vector comprising an mRNA, the mRNA comprising a 5' nuclear localization signal (NLS), a sequence encoding the Cas9, and a 3' NLS; and (j) a donor AAV vector comprising an AAV capsid and a second vector genome comprising a 5′ ITR, a 5′ homologous recombination (HDR) arm, a transgene and a regulatory sequence directing expression of the transgene in the target cell, a 3′ HDR arm, and a 3′ ITR; system.

7. The system of claim 6, wherein the gene-edited AAV vector (a) and the donor AAV vector (b), the gene-edited AAV vector (c) and the donor AAV vector (d), or the gene-edited AAV vector (e) and the donor AAV vector (f) have the same AAV capsid, and optionally, the AAV capsid is selected from AAV8, AAV9, rh10, AAV6.2, AAV3B, hu37, rh79, and rh64.

8. The system of any one of claims 6 to 18 or 13 to 7, wherein the Cas9 is selected from Staphylococcus aureus Cas9 or Streptococcus pyogenes Cas9.

9. A system described in any one of claims 2 to 6, wherein the nuclease is under the control of a tissue-specific promoter, optionally wherein the nuclease is under the control of a constitutive promoter, optionally wherein the nuclease is under the control of a liver-specific promoter, optionally wherein the nuclease is under the control of a human thyroxine-binding globulin (TBG) promoter, or a hybrid liver promoter (HLP).

10. A method of treating a disorder in a human by co-administering the system according to any one of claims 1 to 10, optionally comprising administering to said subject having a liver metabolic disorder: (a) a gene-editing AAV vector comprising a sequence encoding a nuclease and a regulatory sequence directing expression of the nuclease in a target cell comprising a PCSK9 gene; and (b) co-administering a donor AAV vector comprising a transgene and a regulatory sequence directing expression of the transgene in the target cell, the donor vector further comprising homologous recombination (HDR) arms 5′ and 3′ to the transgene cassette; Optionally, the gene-edited AAV vector of (a) and the donor vector of (b) are delivered essentially simultaneously via the same route, optionally, the gene-edited AAV vector of (a) is suspended in an injection vehicle at a concentration of about 2×10 11 GC / mL to about 2×10 12 GC / mL, optionally, the AAV targeting vector of (a) is suspended in an injection vehicle at a concentration of about 2×10 12 GC / mL to about 1×10 13 GC / mL, optionally, the liver metabolic disorder is ornithine transcarbamylase, and optionally, the liver metabolic disorder is OTC, FH, citrullinemia type I (CTLN1), or phenylketonuria. method.

11. 1. A system for treating a genetic disorder, the system comprising: (a) a lipid nanoparticle (LNP) comprising an mRNA sequence encoding a nuclease; (b) a donor AAV vector comprising a transgene and a regulatory sequence directing its expression in the target cell, the donor vector further comprising homologous recombination (HDR) arms 5' and 3' to the transgene; Optionally, the nuclease targets the PCSK9 gene, optionally the nuclease targets PCSK9 exon 7, optionally the nuclease is a meganuclease specific for PCSK9, optionally the meganuclease is an ARCUS meganuclease, optionally the nuclease is a Cas9 nuclease, the LNP comprises an sgRNA, optionally the Cas9 nuclease is adjacent to a nuclear localization signal, optionally the sgRNA comprises at least 20 nucleotides that specifically bind to a target site in the PCSK9 gene, and the target site is 5' to a protospacer adjacent motif (PAM) that is specifically recognized by the Cas9. system.

12. The system of claim 11, further comprising an RNA polymerase promoter, optionally the RNA polymerase promoter is a U6 promoter, optionally the U6 promoter is located on the 5' side of the sgRNA, optionally the sgRNA is 100% complementary to the target site sequence, optionally the sgRNA is less than 100% complementary to the target site sequence, optionally the transgene is a gene expressed in the liver, optionally the transgene is selected from OTC, PKU, CTLN1, and FH.

13. 1. A system for treating a genetic disorder, the system comprising: (a) a gene editing vector comprising a nucleic acid sequence encoding a nuclease; (b) a donor vector, comprising a nucleic acid sequence encoding an exogenous product for expression from the PCSK9 locus, wherein the inserted nucleic acid sequence does not encode PCSK9; The system further comprises a sequence that directs the nuclease to specifically target a native PCSK9 locus; Optionally, the native PCSK9 in the target cells is ablated or reduced following administration of the dual vector system.

14. 14. A method of treating a patient using the system of claim 13, wherein the patient's expression level of native PCSK9 is reduced and the patient expresses the exogenous product.

15. An expression cassette comprising an engineered coding sequence of SEQ ID NO:17, or a sequence sharing at least 90% identity thereto, optionally wherein the expression cassette further comprises AAV 5' and 3' ITRs, and optionally wherein the expression cassette is encapsulated in an AAV vector.

Images