WO2023081807A1

WO2023081807A1 - Compositions and methods for reducing pcsk9 levels in a subject

Info

Publication number: WO2023081807A1
Application number: PCT/US2022/079283
Authority: WO
Inventors: Camilo BRETON; James M. Wilson
Original assignee: The Trustees of the University of Pennsylvania Penn Center for Innovation
Priority date: 2021-11-04
Filing date: 2022-11-04
Publication date: 2023-05-11

Abstract

A gene editing nuclease expression cassette is provided which comprises a nucleic acid sequence comprising a meganuclease coding sequence which is operably linked to regulatory sequences which direct expression of the meganuclease following delivery to a host cell, wherein the regulatory sequences comprise a weak promoter. A vector is provided comprising the gene editing nuclease expression cassette. Also provided are compositions containing same and methods of use.

Description

COMPOSITIONS AND METHODS FOR REDUCING PCSK9 LEVELS IN A SUBJECT Background of the Invention Patients with severe, refractory familial hypercholesterolemia (FH) have severely elevated LDL-C levels, which puts them at high risk for atherosclerotic disease progression and major adverse cardiovascular events. As per 2018 Guideline on the Management of Blood Cholesterol from the American College of Cardiology/American Heart Association (ACC/AHA), the 2016 Canadian Cardiovascular Society (CCS) Guidelines on the Management of Dyslipidemia, and the 2013 European Atherosclerosis Society/European Society of Cardiology (EAS/ESC) Guidelines for Homozygous Familial Hypercholesterolaemia, FH patients should be treated with high-intensity statin therapy to achieve a goal LDL-C level decrease >50% and/or LDL-C <100 mg/dL (LDL-C <70 mg/dL in adults with cardiovascular disease) (Cuchel et al., 2014; Anderson et al., 2016; Grundy et al., 2019). Many FH patients are unable to achieve these target LDL-C levels even while on a maximally tolerated dose of a lipid lowering therapy. Further, both in vitro and in vivo studies have shown that nucleases, regardless of delivery vehicle, generate indels (insertions and deletions) in other regions of the genome, suggesting off-target activity. This off-target activity is undesirable and, especially for clinical studies, it is imperative to reduce or eliminate this off-target activity, while retaining the high on-target efficacy. What are needed are improved compositions and methods for dislipidemia. Summary of the Invention In another aspect, a recombinant AAV useful for gene editing is provided. The rAAV includes an AAVrh79 capsid and a vector genome packaged in the AAV capsid, wherein the vector genome includes an expression cassette and AAV inverted terminal repeats (ITRs). The expression cassette includes a nucleotide sequence that encodes a PCSK9 meganuclease having the amino acid sequence of SEQ ID NO: 16, and regulatory sequences that direct expression of the PCKS9 meganuclease, wherein the expression cassette comprises nt 206 to 1649 of SEQ ID NO: 13, or a sequence sharing at least 70% identity therewith. In another aspect, a pharmaceutical composition is provided. The pharmaceutical composition includes a recombinant AAV useful for gene editing is provided. The rAAV includes an AAVrh79 capsid and a vector genome packaged in the AAV capsid, wherein the vector genome includes an expression cassette and AAV inverted terminal repeats (ITRs). The expression cassette includes a nucleotide sequence that encodes a PCSK9 meganuclease having the amino acid sequence of SEQ ID NO: 16, and regulatory sequences that direct expression of the PCKS9 meganuclease, wherein the expression cassette comprises nt 206 to 1649 of SEQ ID NO: 13, or a sequence sharing at least 70% identity therewith. The composition includes one or more of a carrier, suspending agent, and/or excipient. In another aspect, a method for reducing total serum cholesterol levels in a subject having familial hypercholesterolemia is provided. The method includes delivering a recombinant adeno-associated virus (rAAV) or pharmaceutical composition as described herein. Other aspects and advantages of the invention will be apparent from the following detailed description of the invention. Brief Description of the Drawings FIG.1A shows a timeline for in vivo mouse experiments. Human PCSK9 (hPCSK9)-expressing AAV was intravenously (IV) injected in RAG KO mice. Two weeks later, a single dose of the AAV suicide vectors or corresponding control (AAV8.M2PCSK9) were IV injected into treated mice. FIG.1B is a schematic representation of AAV constructs containing “weak” promoters for vectors used in Example 1 (data shown in FIGs.2-5). Promoter: Shortened versions of human Thyroxine-binding Globulin (TBG) gene or derived from the promoter sequence of liver-enriched genes: CCL16, CYP26A1, or SLC22A9 (identified using Human Protein Atlas database). M2PCSK9: Engineered I-CreI meganuclease targeting a 22bp sequence in the human PCSK9 gene. PolyA: Bovine growth hormone polyadenylation signal. FIG.2A shows the levels at 7 weeks post-AAV of indels in the region corresponding to the target sequence of the ARCUS nuclease, quantified by a next- generation sequencing assay. Linear scale. FIG.2B shows the same levels as FIG.2A, logarithmic scale. FIG.2C shows average levels at week 9 of recombinant PCSK9 in serum, determined by an ELISA assay, per treated group. FIG.3 shows the number of off-target loci in the genomic DNA as a result of the nuclease activity as determined using an NGS-based method called ITR-Seq. FIG.4 shows the indels in a set of genomic locations corresponding to the identified off-targets. Indels levels for each off-target are shown relative to the indels levels in TBG control group (arbitrary value of 1). FIG.5 shows the hPCSK9 levels at 7 weeks after treatment (as a percentage of baseline) for vectors tested in Example 1. FIG.6A and FIG.6B show an in vivo test of self-targeting and short-promoter AAV. (FIG.6A) Schematic representation of the AAV genome of the vectors used in the mouse study. (FIG.6B) Rag1 knockout mice were intravenously injected with AAV9.hPCSK9. Two weeks later, mice received an additional dose of the indicated AAV. Circulating hPCSK9 at the indicated time points were quantified and plotted as a percentage of baseline. (Data presented as mean + SEM, n=5) FIG.7A – FIG.7D show M2PCSK9 editing in vivo expressed by AAV vectors. Rag1 knockout mice treated with AAV9.hPCSK9 and AAV expressing M2PCSK9 were euthanized at either four or nine weeks post-AAV9.hPCSK9. We then isolated liver DNA from the euthanized mice. (FIG.7A) Indel% in the target region present in AAV9.hPCSK9. (FIG.7B) Indel%, at nine weeks post-AAV, in the target region. (FIG. 7C) Number of M2PCSK9 off-target loci identified by ITR-Seq. (FIG.7D) Indel% in selected top-ranking off-targets at nine weeks post-AAV. We have indicated the genomic location for each off-target. NT, indicates that no target sequences were presented in that vector group. Data is shown as mean ± SEM (n=5). * indicates groups that are statistically different from the AAV8.M2PCSK9 group (p<0.05, Wilcoxon rank-sum test). FIG.8 shows M2PCSK9 on-target editing in mice treated with shortened- promoter AAV vectors. Rag1 knockout mice were treated with AAV9.hPCSK9 and shortened-promoter AAV vectors expressing M2PCSK9. At nine weeks post AAV9.hPCSK9, livers were collected and Indel% in the target region present in AAV9.hPCSK9 was calculated. FIG.9 shows liver transduction and transgene RNA expression in NHP. AAV genome copies per diploid cell and M2PCSK9 RNA per microgram of total RNA calculated by qPCR from day 18 or day 128 liver DNA of AAV-treated NHP. FIG.10 shows PCSK9 and LDL serum levels at different time points post-AAV. Here we show values for PCSK9 and LDL (top and bottom rows, respectively) as a percentage of baseline. AAV vector and NHP identification number for each group are displayed on top. * indicates statistically significant averages (day 56 and until last time point) with respect to average levels pre-AAV dosage (p<0.05, one-sided one-sample t- test). FIG.11A and FIG.11B show on and off-target activity of M2PCSK9 in NHP. Rhesus macaques received AAV at the indicated doses. We performed liver biopsies at 18 and 128 days (d18 and d128) post-injection. (FIG.11A) Indel% in M2PCSK9 target region in the rhesus PCSK9 gene calculated by AMP-Seq. (FIG.11B) Number of ITR- Seq-identified off-targets. Results for day 18 (gray bars) and day 128 (black bars) liver biopsies are indicated for each NHP. FIG.12 is a table showing Indel% in a subset of M2PCSK9 off-targets at day 18 post-AAV injection. Rhesus macaques were treated with the selected AAV vectors at the indicated dose. For each NHP (NHP ID shown below the dose) and for each off-target location (first column), the indel% in PBMC before AAV treatment (Pre) and in liver DNA at 18 days post-AAV treatment (d18) was calculated. For each off-target, bold indicates d18 values that are statistically higher than values from control cells (Pre) for the corresponding NHP (p<0.05, Fisher's Exact test). FIG.13A – FIG.13H show T-cell responses to AAV8- and M2PCSK9-derived peptide pools. Number of spot-forming unit (SFU) per million lymphocytes as quantified in the IFN-γ ELISpot using three different pools for AAV8 capsid (AAV8-A, AAV8-B, and AAV8-C) or peptide pools derived from M2PCSK9 sequence (Pool A, Pool B, and Pool C). NHP were treated with AAV8.M2PCSK9 (FIG.13A and FIG. 13B), AAV8.MutTarget.M2PCSK9+PEST (FIG.13C and FIG.13D), AAV8.Target.M2PCSK9 (FIG.13E and FIG.13F) or AAV8.TBG-S1-F113.M2PCSK9 (FIG.13G and FIG.13H). For the AAV8.MutTarget.M2PCSK9+PEST group, we replaced the Pool C with a peptide pool derived from the PEST amino acid sequence. TNTC, too numerous to count. * indicates a positive T cell activation, defined as >55 SFU per million cells and threefold higher than the negative (medium only) control (P). NA indicates that samples are not available as the study was ongoing. NHP identification number and AAV dose (in GC/kg) are displayed below the timepoints. FIG.14 shows liver transaminases levels in treated NHP. We quantified ALT and AST (top and bottom rows, respectively) in serum samples collected at different times post-AAV. Values are shown as units per liter (U/L). AAV and NHP identification number for each group are displayed on top. FIG.15 is a schematic of the NHP Pharmaceutical/Toxicity Study design described in Example 3. FIG.16 is an alignment of the sequences of TBG-S1 promoter and F64, F113, and F140 promoters described herein. FIG.17 is a schematic of the proposed Phase 1/2 FIH clinical trial design. This FIH clinical trial will enroll up to 16 subjects. Cohorts 1 and 2 will encompass the dose escalation portion of the study. The dose escalation cohorts will follow a 3+3 design: initially, three subjects will be enrolled in Cohorts 1 and 2. Dosing will be at least 6 weeks between each subject to allow for monitoring for safety and pharmacodynamics. If the safety data from the three subjects in Cohort 1 are determined to be acceptable by the Sponsor and DSMB, Cohort 2 may be enrolled. These subjects will also be staggered by at least 6 weeks or until a DSMB review is completed, whichever is longer. Abbreviations: DSMB, Data Safety Monitoring Board; FIH, first-in-human; LTFU, long- term follow-up; MTD, maximum tolerated dose. FIG.18A-18D shows the results of a preclinical evaluation of AAVrh79.TBG- S1-F113.M2PCSK9 and AAVhu37.TBG-S1-F113.M2PCSK9 in Rag1-KO mice (episomal model). FIG.18A shows the study design. FIG.18B shows the results of in situ hybridization for the rh79 vector (top) and the hu37 vector (bottom) at 2E13 GC/kg (left panels) 4E12 GC/kg (middle panels), and 8E11 GC/kg (right panels). FIG.18C shows hPCSK9 protein levels as a percentage of day 13 for the rh79 vector (left panels) and the hu37 vector (right panels) at 2E13 GC/kg (left group) 4E12 GC/kg (middle group), and 8E11 GC/kg (right group). FIG.18D shows % indels and number of off target loci measured for each dosage group for the rh79 vector (left panels) and the hu37 vector (right panels). Indels were measured as well as off-target loci using amplicon-seq and ITR-seq respectively. FIG.19 shows a schematic of detection of nuclease off-targets (in vitro) using GUIDE-seq. FIG.20 shows a schematic of identification and quantification of indel- containing alleles using Amplicon-seq. FIG.21 shows a schematic of identification and quantification of indel- containing alleles using AMP-seq. FIG.22 shows a schematic of detection of nuclease off-targets (in vivo) using ITR-seq. FIG.23A-23D shows the results of an experiment where two adult rhesus macaques received a single IV infusion of AAV8.F113.M2PCSK9 vector (6.0 x 10¹² GC/kg). FIG.23A shows a time course of serum PCSK9 protein levels. FIG.23B shows a time course of serum LDL-C levels. FIG.23C shows serum PCSK9 protein levels and FIG.23D shows serum LDL-C levels segregated as pre-dosing data (pre) and all data points post-dosing starting from Day 18 and including all data up until to the most recent time point). Individual data points and the mean ± SEM are shown. All data are shown as the percentage of mean pre-dosing levels (Pre). Pre- and post-dosing levels were analyzed by a one-sided one-sample t-test using R Statistical Software (version R.4.0.0). Abbreviations: AAV8, adeno-associated virus serotype 8; GC, genome copies; IV, intravascular; LDL-C, low density lipoprotein cholesterol; SEM, standard error of the mean. **, p <0.01; ***, p <0.001. FIG.24A-24B shows the results of an experiment where two adult rhesus macaques received a single IV infusion of AAV8.F113.M2PCSK9 (6.0 x 1012 GC/kg). Serum samples collected from each animal before and after AAV vector treatment were assayed for total cholesterol, HDL-C, LDL-C, and triglyceride levels. The left panel (FIG.24A) shows data for animal 181368 and the right panel (FIG.24B) shows data for animal 181373. Abbreviations: AAV8, adeno-associated virus serotype 8; GC, genome copies; HDL-C, high-density lipoprotein cholesterol; IV, intravascular; LDL-C, low density lipoprotein cholesterol; SEM, standard error of the mean; TRIG, triglycerides. FIG.25A-25B shows the results of an experiment where two adult rhesus macaques received a single IV infusion of AAV8.F113.M2PCSK9 (6.0 x 10¹² GC/kg). LFTs, including ALT, AST, ALP, and GGTP tests, were performed on serum samples collected from each animal before and after an intravenous administration of AAV8.F113.M2PCSK9. ALT levels in samples from 181373 on days 21, 28, and 131 were below the detection limit (3 IU/L) and were graphed at 2 IU/L for graphing purpose. The left panel(FIG.25A) shows data for animal 181368 and the right panel(FIG.25A) shows data for animal 181373. Abbreviations: AAV8, adeno-associated virus serotype 8; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; GC, genome copies; GGTP, gamma-glutamytranspeptidase; IV, intravascular; LFTs, liver function tests. FIG.26A-26B shows the results of an experiment where two adult rhesus macaques received a single IV infusion of AAV8.F113.M2PCSK9 (6.0 x 10¹² GC/kg). ELISpot was performed on PBMCs isolated before vector dosing (Pre), and on Day 56, Day 112, and Day 168 post dosing to measure T cell responses to the AAV8 capsid and M2PCSK9 transgene. The left panel (FIG.26A) shows data for animal 181368 and the right panel (FIG.26B) shows data for animal 181373. Abbreviations: AAV8, adeno- associated virus serotype 8; ELISpot, enzyme-linked immune absorbent spot; GC, genome copies; IV, intravascular; PBMC, peripheral blood mononuclear cell. FIG.27A and FIG.27B show the results of an experiment where two adult rhesus macaques received a single IV infusion of AAV8.F113.M2PCSK9 (6.0 x 10¹² GC/kg). Liver samples were taken in Day 18 and Day 127 and analyzed by qPCR to determine vector GCs in the liver (FIG.27A), by reverse transcription qPCR to measure expression of M2PCSK9, presented as transgene-specific mRNA/1 μg total RNA (FIG.27B). FIG.28A-28D shows ISH using transgene-specific probes (M2PCSK9) for liver biopsy samples (ISH signal: red; nuclear DAPI stain: blue) for the experiment performed for FIG.27A and B. Abbreviations: AAV8, adeno-associated virus serotype 8; DAPI, 4’,6-diamidino-2-phenylindole; GC, genome copies; ISH, in situ hybridization; IV, intravascular; qPCR, quantitative polymerase chain reaction. FIG.29A and FIG.29B provide results of an experiment wherein two adult rhesus macaques received a single IV infusion of AAV8.F113.M2PCSK9 (6.0 x 10¹² GC/kg). Liver samples were taken on Day 18 and Day 127 and on-target editing analyzed by amplicon-seq (FIG.29A) and AMP-seq (FIG.29B). Abbreviations: AAV8, adeno-associated virus serotype 8; DAPI, 4’,6-diamidino-2-phenylindole; GC, genome copies; ISH, in situ hybridization; IV, intravascular; qPCR, quantitative polymerase chain reaction. FIG.30 shows the results of an experiment where two adult rhesus macaques received a single IV infusion of AAV8.F113.M2PCSK9 (6.0 x 10¹² GC/kg). Liver samples were taken on Day 18 and Day 127 and off-target editing was evaluated by ITR- seq. Each dot represents a unique potential off-target site detected by ITR-seq and its relative frequency with respect to on-target editing. The number above each data set indicates the number of unique off-target sites detected. Abbreviations: AAV8, adeno- associated virus serotype 8; DAPI, 4’,6-diamidino-2-phenylindole; GC, genome copies; ISH, in situ hybridization; IV, intravascular; OT, off-target; qPCR, quantitative polymerase chain reaction. FIG.31A-31D shows the results of an experiment where two adult rhesus macaques received a single IV infusion of AAV8.F113.M2PCSK9 (6.0 x 10¹² GC/kg). Liver samples were taken on Day 18 and Day 127 and analyzed by IHC using hematoxylin and eosin staining. Representative images are shown. Abbreviations: AAV8, adeno-associated virus serotype 8; GC, genome copies; IHC, immunohistochemistry; IV, intravascular. FIG.32 is a chart showing indel% recorded at the pre-specified off-target sites as discussed in Example 3. Detailed Description of the Invention The compositions and methods provided herein are designed to produce lower expression of, or minimize off-target activity of, a persistently expressed enzyme (e.g., following delivery of an expression cassette) and/or modulating the activity of the expressed enzyme. Use of these compositions and methods with non-secreting enzymes which may accumulate in a cell and/or enzymes which accumulate at higher than desired levels prior to secretion is particularly desirable. The compositions and methods of the invention are well suited for use with gene editing enzymes, particularly meganucleases. However, other applications will be apparent to one of skill in the art. Low-transcription promoters (“Weak” promoters) As used herein, the term “promoter having low-transcriptional activity” or “weak promoter” refers to an expression control sequence which produces a low level of expression of the coding sequence. In one embodiment, the term “low-transcriptional activity” refers to a level of transcription less than the level induced by a reference “strong promoter”. In one embodiment, the reference strong promoter is the thyroxin binding globulin (TBG) promoter or TBG-S1 promoter. Other reference “strong” promoters are known in the art. In one embodiment, the promoter is a weakened version of the liver-specific thyroxin binding globulin (TBG) promoter. In one embodiment, the weak promoter is truncated at the 5’ or 3’ end of the native promoter, or TBG-S1 sequence. In one embodiment, the promoter retains only the 3’ terminal 113 nt from the TBG-S1 promoter and is termed F113 (also called TBG-S1-F113) (SEQ ID NO: 7). An alignment of the TBG-S1, F64, F113 and F140 sequences is shown in FIG.16. Expression cassettes and Vectors In another aspect, an expression cassette is provided. In one embodiment, the expression cassette includes a weak promoter, as described herein, operably linked to a coding sequence. In one embodiment, the expression cassette includes the coding sequence for a nuclease under the control of regulatory sequences which comprise a promoter having low-transcriptional activity, as described herein. In another aspect, vectors comprising the expression cassette (and promoter) are provided. As used herein, an “expression cassette” refers to a nucleic acid molecule which comprises a coding sequence (or transgene), promoter, and may include other regulatory sequences therefor, which cassette may be engineered into a genetic element and/or packaged into the capsid of a viral vector (e.g., a viral particle). Typically, such an expression cassette for generating a viral vector contains the sequences described herein flanked by packaging signals of the viral genome and other expression control sequences such as those described herein. The transgene is a nucleic acid sequence, heterologous to the vector sequences flanking the transgene, which encodes a polypeptide, protein, or other product, of interest. The nucleic acid coding sequence is operatively linked to regulatory components in a manner which permits transgene transcription, translation, and/or expression in a target cell. The heterologous nucleic acid sequence (transgene) can be derived from any organism. The AAV may comprise one or more transgenes. Exemplified herein is the use of the weak promoters described herein in conjunction with a gene editing nuclease (specifically, a meganuclease). In one embodiment, the coding sequence encodes a meganuclease. Examples of suitable meganucleases are described, e.g., in US Patent 8,445,251; US 9,340,777; US 9,434,931; US 9,683,257, and WO 2018/195449. In certain embodiments, the nuclease is a member of the LAGLIDADG (SEQ ID NO: 1) family of homing endonucleases. In certain embodiments, the nuclease is a member of the I-CreI family of homing endonucleases which recognizes and cuts a 22 base pair recognition sequence SEQ ID NO: 2 - CAAAACGTCGTGAGACAGTTTG. See, e.g., WO 2009/059195. Methods for rationally-designing mono-LAGLIDADG homing endonucleases were described which are capable of comprehensively redesigning ICreI and other homing endonucleases to target widely-divergent DNA sites, including sites in mammalian, yeast, plant, bacterial, and viral genomes (WO 2007/047859). In one embodiment, the nuclease is encoded by the sequence shown in nt 1089 to 2183 of SEQ ID NO: 15, or a sequence sharing at least 95%, 96%, 97%, 98%, 99%, or 99.9% identity thereto. In one embodiment, the nuclease protein sequence is the sequence shown in SEQ ID NO: 16, or a sequence sharing at least 95%, 96%, 97%, 98%, 99%, or 99.9% identity thereto. One of the aims of the invention is to reduce the off-target activity of a nuclease without compromising its strong on-target activity. It was hypothesized that high expression of the nuclease in transduced cells is not needed to achieve editing of the target DNA sequence, and that the off-target results from an elevated accumulation of the nuclease in the cell. To reduce nuclease expression, high-expressing promoters were replaced by promoters with lower transcriptional activity. Thus, the expression cassette contains a promoter sequence as part of the expression control sequences or the regulatory sequences. As described herein, the promoter is a promoter having lower transcriptional activity, or “weak promoter”. In addition, in one embodiment, the promoter is a weakened version of a tissue- specific promoter. In one example, the tissue-specific promoter is the liver-specific thyroxin binding globulin (TBG) promoter. In one embodiment, the weak promoter is truncated at the 5’ or 3’ end of the native promoter, or TBG-S1 sequence. In one embodiment, the promoter retains only the 3’ terminal 113 nt from the TBG-S1 promoter and is termed F113 (SEQ ID NO: 7). In addition to a promoter, the expression cassette and/or a vector may contain one or more appropriate “regulatory elements” or “regulatory sequences”, which comprise but are not limited to an enhancer; transcription factor; transcription terminator; efficient RNA processing signals such as splicing and polyadenylation signals (polyA); sequences that stabilize cytoplasmic mRNA, for example Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE); sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product. Examples of suitable polyA sequences include, e.g., SV40, bovine growth hormone (bGH), and TK polyA. Examples of suitable enhancers include, e.g., the alpha fetoprotein enhancer, the TTR minimal promoter/enhancer, LSP (TH-binding globulin promoter/alpha1- microglobulin/bikunin enhancer), amongst others. These control sequences or the regulatory sequences are operably linked to the nuclease coding sequences. In certain embodiments the polyA is the bGH polyA shown in nt 1435 to 1649 of SEQ ID NO: 13. In certain embodiments, the weak promoters, constructs containing same and methods described herein, are useful in targeting liver-directed therapies, such as proprotein convertase subtilisin/kexin type 9 (PCSK9) (cholesterol related disorders). In one embodiment, a nucleic acid molecule is provided which encodes a PCSK9 meganuclease operably linked to a weak promoter. In another embodiment, the weak promoter is F113. In certain embodiments, a meganuclease may be selected from those described in WO 2018/195449A1. In one embodiment, the nucleic acid molecule comprises the F113 promoter operably linked to the PCSK9 meganuclease coding sequence of nt 1089 to 2183 of SEQ ID NO: 15, or a sequence sharing at least 95%, 96%, 97%, 98%, 99%, or 99.9% identity thereto. In one embodiment, the nucleic acid molecule comprises the F113 promoter operably linked to the sequence encoding the PCSK9 meganuclease of SEQ ID NO: 16, or a sequence sharing at least 95%, 96%, 97%, 98%, 99%, or 99.9% identity thereto. In certain embodiments, the nucleic acid molecule comprises the F113 promoter operably linked to the PCSK9 meganuclease coding sequence of nt 1089 to 2183 of SEQ ID NO: 15. In another embodiment, the transgene comprises more than one transgene. This may be accomplished using a single vector carrying two or more heterologous sequences, or using two or more AAV each carrying one or more heterologous sequences. In one embodiment, the AAV is used for gene suppression (or knockdown) and gene augmentation co-therapy. In knockdown/augmentation co-therapy, the defective copy of the gene of interest is silenced and a non-mutated copy is supplied. In one embodiment, this is accomplished using two or more co-administered vectors. See, Millington-Ward et al, Molecular Therapy, April 2011, 19(4):642–649 which is incorporated herein by reference. The transgenes may be readily selected by one of skill in the art based on the desired result. Viral and Non-Viral Vectors The expression cassette described herein, containing a weak promoter and heterologous coding sequence, may be engineered into any suitable genetic element for delivery to a target cell, such as a vector. A “vector” as used herein is a biological or chemical moiety comprising a nucleic acid sequence which can be introduced into an appropriate host cell for replication or expression of said nucleic acid sequence. Common vectors include non-viral vectors and viral vectors. As used herein, a non-viral system might be selected from nanoparticles, electroporation systems and novel biomaterials, naked DNA, phage, transposon, 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). “Plasmid” or “plasmid vector” generally is designated herein by a lower case p preceded and/or followed by a vector name. Plasmids, other cloning and expression vectors, properties thereof, and constructing/manipulating methods thereof that can be used in accordance with the present invention are readily apparent to those of skill in the art. In one embodiment, the nucleic acid sequence as described herein or the expression cassette as described herein are engineered into a suitable genetic element (a vector) useful for generating viral vectors and/or for delivery to a host cell, e.g., naked DNA, phage, transposon, cosmid, episome, etc., which transfers the nuclease sequences carried thereon. The selected vector may be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high velocity DNA-coated pellets, viral infection and protoplast fusion. The methods used to make such constructs are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY. In certain embodiments, the expression cassette is located in a vector genome for packaging into a viral capsid. For example, for an AAV vector genome, the components of the expression cassette are flanked at the extreme 5’ end and the extreme 3’ end by AAV inverted terminal repeat sequences. For example, a 5’ AAV ITR, expression cassette, 3’ AAV ITR. In certain embodiments, the expression cassette comprises or consists of nt 206 to 1649 of SEQ ID NO: 13 or a sequence sharing at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least at least 99.9% identity therewith. In other embodiments, a self-complementary AAV may be selected. In other embodiments, retroviral system, lentivirus vector system, or an adenoviral system may be used. In one embodiment, the vector genome is that shown in any of SEQ ID NO: 9- 14. In one embodiment, the vector genome is that shown in SEQ ID NO: 9 or a sequence sharing at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least at least 99.9% identity therewith. In one embodiment, the vector genome is that shown in SEQ ID NO: 10 or a sequence sharing at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least at least 99.9% identity therewith. In one embodiment, the vector genome is that shown in SEQ ID NO: 11 or a sequence sharing at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least at least 99.9% identity therewith. In one embodiment, the vector genome is that shown in SEQ ID NO: 12 or a sequence sharing at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least at least 99.9% identity therewith. In one embodiment, the vector genome is that shown in SEQ ID NO: 13 or a sequence sharing at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least at least 99.9% identity therewith. In one embodiment, the vector genome is that shown in SEQ ID NO: 14 or a sequence sharing at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least at least 99.9% identity therewith. SEQ ID NO: 13: Features

AAV Vectors In certain embodiments, a recombinant AAV is provided. A “recombinant AAV” or “rAAV” is a DNAse-resistant viral particle containing two elements, an AAV capsid and a vector genome containing at least non-AAV coding sequence packaged within the AAV capsid. Unless otherwise specified, this term may be used interchangeably with the phrase “rAAV vector”. The rAAV is a “replication-defective virus” or “viral vector”, as it lacks any functional AAV rep gene or functional AAV cap gene and cannot generate progeny. In certain embodiments, the only AAV sequences are the AAV inverted terminal repeat sequences (ITRs), typically located at the extreme 5’ and 3’ ends of the vector genome in order to allow the gene and regulatory sequences located between the ITRs to be packaged within the AAV capsid. The source of the AAV capsid may be one of any of the dozens of naturally occurring and available adeno-associated viruses, as well as engineered AAVs. An adeno-associated virus (AAV) viral vector is an AAV DNase-resistant particle having an AAV protein capsid into which is packaged nucleic acid sequences for delivery to target cells. An AAV capsid is composed of 60 capsid (cap) protein subunits, VP1, VP2, and VP3, that are arranged in an icosahedral symmetry in a ratio of approximately 1:1:10 to 1:1:20, depending upon the selected AAV. Various AAVs may be selected as sources for capsids of AAV viral vectors as identified above. See, e.g., US Published Patent Application No.2007-0036760-A1; US Published Patent Application No.2009- 0197338-A1; EP 1310571. See also, WO 2003/042397 (AAV7 and other simian AAV), US Patent 7790449 and US Patent 7282199 (AAV8), WO 2005/033321 and US 7,906,111 (AAV9), and WO 2006/110689, WO 2003/042397 (rh.10) and WO 2018/160582 (AAVhu68). These documents also describe other AAV which may be selected for generating AAV and are incorporated by reference. In certain embodiments, the 5’ ITR is nt 1 to 130 of SEQ ID NO: 13. In certain embodiments, the 3’ ITR is nt 1737 to 1866 of SEQ ID NO: 13. Unless otherwise specified, the AAV capsid, ITRs, and other selected AAV components described herein, may be readily selected from among any AAV, including, without limitation, the AAVs commonly identified as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV8bp, AAV7M8, AAVAnc80, AAVrh10, and AAVPHP.B and variants of any of the known or mentioned AAVs or AAVs yet to be discovered or variants or mixtures thereof. See, e.g., WO 2005/033321, which is incorporated herein by reference. In one embodiment, the AAV capsid is an AAV1 capsid or variant thereof, AAV8 capsid or variant thereof, an AAV9 capsid or variant thereof, an AAVrh.10 capsid or variant thereof, an AAVrh64R1 capsid or variant thereof, an AAVhu.37 capsid or variant thereof, or an AAV3B or variant thereof. In one aspect, the capsid is an AAVhu.37 capsid. See, also WO 2019/168961 and WO 2019/168961, which are incorporated by reference herein in their entirety. In other embodiments, the AAV capsid is an AAVrh.79 capsid or variant thereof. In other embodiments, the AAV capsid is an AAVrh.90 or variant thereof. In other embodiments, the AAV capsid is an AAVrh.91 or variant thereof. In other embodiments, the AAV capsid is an AAVhu.68 or variant thereof. In certain embodiments, the rAAV comprises an AAVhu37 capsid. An AAVhu37 capsid comprises: a heterogeneous population of vp1 proteins which are the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 45, a heterogeneous population of vp2 proteins which are the product of a nucleic acid sequence encoding the amino acid sequence of at least about amino acids 138 to 738 of SEQ ID NO: 45, and a heterogeneous population of vp3 proteins which are the product of a nucleic acid sequence encoding at least amino acids 204 to 738 of SEQ ID NO: 45 wherein: the vp1, vp2 and vp3 proteins contain subpopulations with amino acid modifications comprising at least two highly deamidated asparagines (N) in asparagine - glycine pairs in SEQ ID NO: 45 and optionally further comprising subpopulations comprising other deamidated amino acids, wherein the deamidation results in an amino acid change. AAVhu37 is characterized by having highly deamidated residues, e.g., at positions N57, N263, N385, and/or N514 based on the numbering of the AAVhu37 VP1 (SEQ ID NO: 45). Deamidation has been observed in other residues, as shown in the table below, and in, e.g., WO 2019/168961, published September 6, 2019, which is incorporated herein by reference. In certain embodiments, an AAVhu37 capsid is modified in one or more of the following positions, in the ranges provided below, as determined using mass spectrometry with a trypsin enzyme. In certain embodiments, one or more of the following positions, or the glycine following the N is modified as described herein. For example, in certain embodiments, a G may be modified to an S or an A, e.g., at position 58, 264, 386, or 515. In one embodiment, the AAVhu37 capsid is modified at position N57/G58 to N57Q or G58A to afford 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, an increase in deamidation is observed when NG is altered to NS or NA. In certain embodiments, an N of an NG pair is modified to a Q while retaining the G. In certain embodiments, both amino acids of an NG pair are modified. In certain embodiments, N385Q results in significant reduction of deamidation in that location. In certain embodiments, N499Q results in significant increase of deamidation in that location. In certain embodiments, AAVhu37 may have these or other residues deamidated, e.g., typically at less than 10% and/or may have other modifications, including methylations (e.g, ~R487) (typically less than 5%, more typically less than 1% at a given residue), isomerization (e.g., at D97) (typically less than 5%, more typically less than 1% at a given residue, phosphorylation (e.g., where present, in the range of about 10 to about 60%, or about 10 to about 30%, or about 20 to about 60%) (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 the W may oxidize to kynurenine.

Still other positions may have such these or other modifications (e.g., acetylation or further deamidations). In certain embodiments, the nucleic acid sequence encoding the AAVhu37 vp1 capsid protein is provided in SEQ ID NO: 44. In other embodiments, a nucleic acid sequence of 70% to 99.9% identity to SEQ ID NO: 44 may be selected to express the AAVhu37 capsid proteins. 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: 44. However, other nucleic acid sequences which encode the amino acid sequence of SEQ ID NO: 45 may be selected for use in producing rAAVhu37 capsids. In certain embodiments, the nucleic acid sequence has the nucleic acid sequence of SEQ ID NO: 44 or a sequence at least 70% to at least 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 SEQ ID NO: 44 which encodes SEQ ID NO: 45. In certain embodiments, the nucleic acid sequence has the nucleic acid sequence of SEQ ID NO: 44 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 about nt 412 to about nt 2214 of SEQ ID NO: 44 which encodes the vp2 capsid protein (about aa 138 to 738) of SEQ ID NO: 45. In certain embodiments, the nucleic acid sequence has the nucleic acid sequence of 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 nt SEQ ID NO: 44 which encodes the vp3 capsid protein (about aa 204 to 738) of SEQ ID NO: 45. See, EP 2345731 B1 and SEQ ID NO: 88 therein, which are incorporated by reference. Provided herein is an AAVhu.37.F113.PCS7-8L vector. In certain embodiments, the rAAV comprises an AAV8 capsid. An AAV8 capsid comprises: a heterogeneous population of VP isoforms which are deamidated as defined in the following table, based on the total amount of VP proteins in the capsid, as determined using mass spectrometry. Suitable modifications include those described in the paragraph above labelled modulation of deamidation, which is incorporated herein. In certain embodiments, the AAV capsid is modified at one or more of the following position, in the ranges provided below, as determined using mass spectrometry. In certain embodiments, one or more of the following positions, or the glycine following the N is modified as described herein. In certain embodiments, an artificial NG is introduced into a different position than one of the positions identified below. In certain embodiments, one or more of the following positions, or the glycine following the N is modified as described herein. For example, in certain embodiments, a G may be modified to an S or an A, e.g., at position 58, 67, 95, 216, 264, 386, 411, 460, 500, 515, or 541 of SEQ ID NO: 43. Significant reduction in deamidation is observed when NG57/58 is altered to NS 57/58 or NA57/58. However, in certain embodiments, an increase in deamidation is observed when NG is altered to NS or NA. In certain embodiments, an N of an NG pair is modified to a Q while retaining the G. In certain embodiments, both amino acids of an NG pair are modified. In certain embodiments, N385Q results in significant reduction of deamidation in that location. In certain embodiments, N499Q results in significant increase of deamidation in that location. In certain embodiments, an NG mutation is made at the pair located at N263 (e.g., to N263A). In certain embodiments, an NG mutation is made at the pair located at N514 (e.g., to N514A). In certain embodiments, an NG mutation is made at the pair located at N540 (e.g., N540A). In certain embodiments, AAV mutants containing multiple mutations and at least one of the mutations at these positions are engineered. 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 mutation is made at position Q467. In certain embodiments, no mutation is made at position N479. In certain embodiments, no mutation is made at position N653. In certain embodiments, the capsid is modified to reduce “N” or “Q” at positions other than then “NG” pairs. Residue numbers are based on the published AAV8 sequence, reproduced in SEQ ID NO: 43. A representative DNA coding sequence of AAV8 is provided in SEQ ID NO: 42. Provided herein is an AAV8.F113.PCS7-8L vector.

In certain embodiments, the rAAV comprises a AAVrh79 capsid, as described in WO 2019/169004, published September 6, 2019, which is incorporated herein by reference. In one embodiment, an AAVrh79 capsid comprises a heterogeneous population of AAVrh79 vp1 proteins, AAVrh79 vp2 proteins, and AAVrh79 vp3 proteins. In one embodiment, the AAVrh79 capsid is produced by expression from a nucleic acid sequence which encodes the predicted amino acid sequence of 1 to 738 of SEQ ID NO: 41. Optionally, sequences co-expressing the vp3 protein from a nucleic acid sequence excluding the vp1-unique region (about aa 1 to 137) or the vp2-unique region (about aa 1 to 203), vp1 proteins produced from SEQ ID NO: 40, or vp1 proteins produced from a nucleic acid sequence at least 70% identical to SEQ ID NO: 40 which encodes the predicted amino acid sequence of 1 to 738 of SEQ ID NO: 41. In other embodiments, the AAVrh79 vp2 proteins produced by expression from a nucleic acid sequence which encodes the predicted amino acid sequence of at least about amino acids 138 to 738 of SEQ ID NO: 41, vp2 proteins produced from a sequence comprising at least nucleotides 412 to 2214 of SEQ ID NO: 40, or vp2 proteins produced from a nucleic acid sequence at least 70% identical to at least nucleotides 412 to 2214 of SEQ ID NO: 40 which encodes the predicted amino acid sequence of at least about amino acids 138 to 738 of SEQ ID NO: 41, AAVrh79 vp3 proteins produced by expression from a nucleic acid sequence which encodes the predicted amino acid sequence of at least about amino acids 204 to 738 of SEQ ID NO: 41, vp3 proteins produced from a sequence comprising at least nucleotides 610 to 2214 of SEQ ID NO: 40, or vp3 proteins produced from a nucleic acid sequence at least 70% identical to at least nucleotides 610 to 2214 of SEQ ID NO: 40 which encodes the predicted amino acid sequence of at least about amino acids 204 to 738 of SEQ ID NO: 41. In certain embodiments, an AAVrh79 capsid comprises: a heterogeneous population of vp1 proteins which are the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 41, a heterogeneous population of vp2 proteins which are the product of a nucleic acid sequence encoding the amino acid sequence of at least about amino acids 138 to 738 of SEQ ID NO: 41, and a heterogeneous population of vp3 proteins which are the product of a nucleic acid sequence encoding at least amino acids 204 to 738 of SEQ ID NO: 41. The AAVrh79 vp1, vp2 and vp3 proteins contain subpopulations with amino acid modifications comprising at least two highly deamidated asparagines (N) in asparagine - glycine pairs in SEQ ID NO: 41 and optionally further comprising subpopulations comprising other deamidated amino acids, wherein the deamidation results in an amino acid change. High levels of deamidation at N-G pairs N57, N263, N385 and/or N514 are observed, relative to the number of SEQ ID NO: 41. Deamidation has been observed in other residues, as shown in the table below and in the examples. In certain embodiments, AAVrh79 may have other residues deamidated, e.g., typically at less than 10% and/or may have other modifications, including methylations (e.g, ~R487) (typically less than 5%, more typically less than 1% at a given residue), isomerization (e.g., at D97) (typically less than 5%, more typically less than 1% at a given residue, phosphorylation (e.g., where present, in the range of about 10 to about 60%, or about 10 to about 30%, or about 20 to about 60%) (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 the W may oxidize to kynurenine. Table C – AAVrh79 Deamidation

In certain embodiments, an AAVrh79 capsid is modified in one or more of the positions identified in the preceding table, in the ranges provided below, as determined using mass spectrometry with a trypsin enzyme. In certain embodiments, one or more of the following positions, or the glycine following the N is modified as described herein. Residue numbers are based on the AAVrh79 sequence provided herein. See, SEQ ID NO: 41. In certain embodiments, the nucleic acid sequence encoding the AAVrh79 vp1 capsid protein is provided in SEQ ID NO: 40. In other embodiments, a nucleic acid sequence of 70% to 99.9% identity to SEQ ID NO: 40 may be selected to express the AAVrh79 capsid proteins. 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, at least 99%, or at least 99.9% identical to SEQ ID NO: 40. However, other nucleic acid sequences which encode the amino acid sequence of SEQ ID NO: 41 may be selected for use in producing rAAV capsids. In certain embodiments, the nucleic acid sequence has the nucleic acid sequence of SEQ ID NO: 40 or 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 SEQ ID NO: 40 which encodes SEQ ID NO: 41. In certain embodiments, the nucleic acid sequence has the nucleic acid sequence of SEQ ID NO: 40 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 about nt 412 to about nt 2214 of SEQ ID NO: 40 which encodes the vp2 capsid protein (about aa 138 to 738) of SEQ ID NO: 41. In certain embodiments, the nucleic acid sequence has the nucleic acid sequence of about nt 610 to about nt 2214 of SEQ ID NO: 40 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 nt SEQ ID NO: 40 which encodes the vp3 capsid protein (about aa 204 to 738) of SEQ ID NO: 41. Provided herein is an AAVrh79.F113.PCS7-8L vector. In certain embodiment, an rAAV79 vector has an AAVrh79 capsid containing a vector genome comprising a nucleic acid molecule comprising AAV inverted terminal repeat sequences and a non-AAV nucleic acid sequence encoding a product operably linked to sequences which direct expression of the product. In certain embodiments, the AAVrh79 capsid is characterized by comprising a heterogeneous population of AAVrh79 vp1 proteins, AAVrh79 vp2 proteins, and AAVrh79 vp3 proteins selected from: vp1 proteins produced by expression from a nucleic acid sequence which encodes the predicted amino acid sequence of 1 to 738 of SEQ ID NO: 41, vp1 proteins produced from SEQ ID NO: 40, or vp1 proteins produced from a nucleic acid sequence at least 70% to 100% identical to SEQ ID NO: 40 which encodes the predicted amino acid sequence of 1 to 738 of SEQ ID NO: 41, a heterogeneous population of AAVrh79 vp2 proteins selected from: vp2 proteins produced by expression from a nucleic acid sequence which encodes the predicted amino acid sequence of at least about amino acids 138 to 738 of SEQ ID NO: 41, vp2 proteins produced from a sequence comprising at least nucleotides 412 to 2214 of SEQ ID NO: 40, or vp2 proteins produced from a nucleic acid sequence at least 70% to 100% identical to at least nucleotides 412 to 2214 of SEQ ID NO: 40 which encodes the predicted amino acid sequence of at least about amino acids 138 to 738 of SEQ ID NO: 41, and a heterogeneous population of AAVrh79 vp3 proteins selected from: vp3 proteins produced by expression from a nucleic acid sequence which encodes the predicted amino acid sequence of at least about amino acids 204 to 738 of SEQ ID NO: 41, vp3 proteins produced from a sequence comprising at least nucleotides 610 to 2214 of SEQ ID NO: 40, or vp3 proteins produced from a nucleic acid sequence at least 70% identical to at least nucleotides 610 to 2214 of SEQ ID NO: 40 which encodes the predicted amino acid sequence of at least about amino acids 204 to 738 of SEQ ID NO: 41. In certain embodiments, the rAAVrh79 capsid is characterized by a heterogeneous population of AAVrh79 vp1 proteins, AAVrh79 vp2 and AAVrh79 vp3 proteins which are the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 41, a heterogeneous population of vp2 proteins which are the product of a nucleic acid sequence encoding the amino acid sequence of at least about amino acids 138 to 738 of SEQ ID NO: 41, and a heterogeneous population of vp3 proteins which are the product of a nucleic acid sequence encoding at least amino acids 204 to 738 of SEQ ID NO: 41. In certain embodiments, the AAVrh79 capsid is characterized by AAVrh79 vp1proteins, vp2 proteins and vp3 proteins which comprise heterogenous populations relative to amino acids 1 to 738 (vp1), 138 to 738 (vp2), and 204 to 738 (vp3), respectively, of SEQ ID NO: 41, wherein: the heterogenous population of AAVrh79 vp1, AAVrh79 vp2 and AAVrh79 vp3 proteins contain subpopulations with amino acid modifications comprising at least 50% to 100% two highly deamidated asparagines (N) in asparagine - glycine pairs in at least two positions relative to SEQ ID NO: 41 and optionally further comprising subpopulations comprising other deamidated amino acids, wherein the deamidation results in an amino acid change. In certain embodiment, the highly deamidated positions are N57, N263, N385, and N514, based on SEQ ID NO: 41, and as measured using mass spectrometry. In certain embodiment, the AAVrh79 capsid proteins are each individually deamidated at 60% to about 100% at position N57, at 60% to about 100% at position N263, at 60% to about 100% at position N385, and at 60% to about 100% at position N514, based on SEQ ID NO: 41, and as measured using mass spectrometry. Other suitable techniques for measuring deamidation or other post- translational modifications may be selected. In certain embodiments, the rAAV79 capsid comprises AAVrh79 VP1 proteins having about 80 to 85% deamidation at position N57 of SEQ ID NO: 41; about 82% to about 88% deamidation at position N263 of SEQ ID NO: 41; about 90% to about 96% deamidation at position N385 of SEQ ID NO: 41; and/or about 85% to about 90% deamidation at position N514 of SEQ ID NO: 41, optionally with further post- translational modifications at other positions, as determined using mass spectrometry. Optionally, there is deamidation at position N94, N254, N305, N410, N479, Q601, N653; generally deamidation in found in these positions at less than 10% of the population of VP1, VP2 and VP3 proteins of AAVrh79, less than 5%, less than 3%, or less than 2%. Optionally, phosphorylation is observed at position S149, based on the residues of SEQ ID NO: 41; in certain embodiments, no more than 0% of the capsid proteins have phosphorylation at this position. In certain embodiments, oxidation is observed at positions W248, W307, M437, M473, M480, W505, M637, and/or W697; in certain embodiments, less than 10% of the capsid proteins are oxidated at any one of these positions. Post-translational modifications may be determined using mass spectrometry or another suitable technique. The invention also encompasses nucleic acid sequences encoding mutant AAVrh79, in which one or more residues has been altered in order to decrease deamidation, or other modifications which are identified herein. Such nucleic acid sequences can be used in production of mutant rAAVrh79 capsids. In certain embodiments, the rAAV comprises an AAV8 capsid. An AAV8 capsid comprises: a heterogeneous population of VP isoforms which are deamidated as defined in the following table, based on the total amount of VP proteins in the capsid, as determined using mass spectrometry. Suitable modifications include those described in the paragraph above labelled modulation of deamidation, which is incorporated herein. In certain embodiments, the AAV capsid is modified at one or more of the following position, in the ranges provided below, as determined using mass spectrometry. In certain embodiments, one or more of the following positions, or the glycine following the N is modified as described herein. In certain embodiments, an artificial NG is introduced into a different position than one of the positions identified below. In certain embodiments, one or more of the following positions, or the glycine following the N is modified as described herein. For example, in certain embodiments, a G may be modified to an S or an A, e.g., at position 58, 67, 95, 216, 264, 386, 411, 460, 500, 515, or 541 of SEQ ID NO: 43. Significant reduction in deamidation is observed when NG57/58 is altered to NS 57/58 or NA57/58. However, in certain embodiments, an increase in deamidation is observed when NG is altered to NS or NA. In certain embodiments, an N of an NG pair is modified to a Q while retaining the G. In certain embodiments, both amino acids of an NG pair are modified. In certain embodiments, N385Q results in significant reduction of deamidation in that location. In certain embodiments, N499Q results in significant increase of deamidation in that location. In certain embodiments, an NG mutation is made at the pair located at N263 (e.g., to N263A). In certain embodiments, an NG mutation is made at the pair located at N514 (e.g., to N514A). In certain embodiments, an NG mutation is made at the pair located at N540 (e.g., N540A). In certain embodiments, AAV mutants containing multiple mutations and at least one of the mutations at these positions are engineered. 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 mutation is made at position Q467. In certain embodiments, no mutation is made at position N479. In certain embodiments, no mutation is made at position N653. In certain embodiments, the capsid is modified to reduce “N” or “Q” at positions other than then “NG” pairs. Residue numbers are based on the published AAV8 sequence, reproduced in SEQ ID NO: 43. A representative DNA coding sequence of AAV8 is provided in SEQ ID NO: 42. Provided herein is an AAV8.F113.PCS7-8L vector.

As used herein, a “vector genome” refers to the nucleic acid sequence packaged inside the rAAV capsid which forms a viral particle. Such a nucleic acid sequence contains AAV inverted terminal repeat sequences (ITRs). In the examples herein, a vector genome contains, at a minimum, from 5’ to 3’, an AAV 5’ ITR, expression cassette containing the transgene or coding sequence(s) operably linked to regulatory sequences directing expression thereof, and an AAV 3’ ITR. The ITRs are the genetic elements responsible for the replication and packaging of the genome during vector production and are the only viral cis elements required to generate rAAV. In one embodiment, the ITRs are from an AAV different than that supplying a capsid. In a preferred embodiment, the ITR sequences from AAV2, or the deleted version thereof (ΔITR), which may be used for convenience. However, ITRs from other AAV sources may be selected. Where the source of the ITRs is from AAV2 and the AAV capsid is from another AAV source, the resulting vector may be termed pseudotyped. Typically, AAV vector genome comprises an AAV 5’ ITR, the nucleic acid sequences encoding the gene product(s) and 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 provided. A shortened version of the 5’ ITR, termed ΔITR, has been described in which the D-sequence and terminal resolution site (trs) are deleted. In certain embodiments, the vector genome includes a shortened AAV2 ITR of 130 base pairs, wherein the external “a” element is deleted. The shortened ITR is reverted back to the wild-type length of 145 base pairs during vector DNA amplification using the internal A element as a template. In other embodiments, the full-length AAV 5’ and 3’ ITRs are used. In other embodiments, a full-length or engineered ITR may be selected. Further, the vector genome contains regulatory sequences that modulate expression of the gene products (e.g, directly or indirectly by modulating transcription and/or translation). Suitable components of a vector genome are discussed in more detail herein. For use in producing an AAV viral vector (e.g., a recombinant (r) AAV), the expression cassettes can be carried on any suitable vector, e.g., a plasmid, which is delivered to a packaging host cell. The plasmids useful in this invention may be engineered such that they are suitable for replication and packaging in vitro in prokaryotic cells, insect cells, mammalian cells, among others. Suitable transfection techniques and packaging host cells are known and/or can be readily designed by one of skill in the art. In one embodiment, the vector genome shown in SEQ ID NO: 13 is packaged into an AAVhu.37 capsid. In another embodiment, the vector genome shown in SEQ ID NO: 13 is packaged into an AAVrh.90 capsid. In one embodiment, the vector genome shown in SEQ ID NO: 13 is packaged into an AAVrh.79 capsid. In one embodiment, the vector genome shown in SEQ ID NO: 13 is packaged into an AAV8 capsid. In one embodiment, the vector genome shown in SEQ ID NO: 13 is packaged into an AAV3B capsid. Methods for generating and isolating AAVs suitable for use as vectors are known in the art. See generally, e.g., Grieger & Samulski, 2005, “Adeno-associated virus as a gene therapy vector: Vector development, production and clinical applications,” Adv. Biochem. Engin/Biotechnol.99: 119-145; Buning et al., 2008, “Recent developments in adeno-associated virus vector technology,” J. Gene Med.10:717-733; and the references cited below, each of which is incorporated herein by reference in its entirety. For packaging a transgene into virions, the ITRs are the only AAV components required in cis in the same construct as the nucleic acid molecule containing the expression cassettes. The cap and rep genes can be supplied in trans. The term “AAV intermediate” or “AAV vector intermediate” refers to an assembled rAAV capsid which lacks the desired genomic sequences packaged therein. These may also be termed an “empty” capsid. Such a capsid may contain no detectable genomic sequences of an expression cassette, or only partially packaged genomic sequences which are insufficient to achieve expression of the gene product. These empty capsids are non-functional to transfer the gene of interest to a host cell. The recombinant adeno-associated virus (AAV) described herein may be generated using techniques which are known. See, e.g., WO 2003/042397; WO 2005/033321, WO 2006/110689; US 7588772 B2. Such a method involves culturing a host cell which contains a nucleic acid sequence encoding an AAV capsid protein; a functional rep gene; an expression cassette composed of, at a minimum, AAV inverted terminal repeats (ITRs) and a transgene; and sufficient helper functions to permit packaging of the expression cassette into the AAV capsid protein. Methods of generating the capsid, coding sequences therefor, and methods for production of rAAV viral vectors have been described. See, e.g., Gao, et al, Proc. Natl. Acad. Sci. U.S.A.100 (10), 6081- 6086 (2003) and US 2013/0045186A1. In one embodiment, a production cell culture useful for producing a recombinant AAV is provided. Such a cell culture contains a nucleic acid which expresses the AAV capsid protein in the host cell; a nucleic acid molecule suitable for packaging into the AAV capsid, e.g., a vector genome which contains AAV ITRs and a non-AAV nucleic acid sequence encoding a gene product operably linked to sequences which direct expression of the product in a host cell; and sufficient AAV rep functions and adenovirus helper functions to permit packaging of the nucleic acid molecule into the 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). Optionally the rep functions are provided by an AAV other than the AAV providing the capsid. For example the rep may be, but is not limited to, AAV1 rep protein, AAV2 rep protein, AAV3 rep protein, AAV4 rep protein, AAV5 rep protein, AAV6 rep protein, AAV7 rep protein, AAV8 rep protein; or rep 78, rep 68, rep 52, rep 40, rep68/78 and rep40/52; or a fragment thereof; or another source. Optionally, the rep and cap sequences are on the same genetic element in the cell culture. There may be a spacer between the rep sequence and cap gene. Any of these AAV or mutant AAV capsid sequences may be under the control of exogenous regulatory control sequences which direct expression thereof in a host cell. In one embodiment, cells are manufactured in a suitable cell culture (e.g., HEK 293) cells. Methods for manufacturing the gene therapy vectors described herein include methods well known in the art such as generation of plasmid DNA used for production of the gene therapy vectors, generation of the vectors, and purification of the vectors. In some embodiments, the gene therapy vector is an AAV vector and the plasmids generated are an AAV cis-plasmid encoding the AAV genome and the gene of interest, an AAV trans-plasmid containing AAV rep and cap genes, and an adenovirus helper plasmid. The vector generation process can include method steps such as initiation of cell culture, passage of cells, seeding of cells, transfection of cells with the plasmid DNA, post-transfection medium exchange to serum free medium, and the harvest of vector-containing cells and culture media. The harvested vector-containing cells and culture media are referred to herein as crude cell harvest. In yet another system, the gene therapy vectors are introduced into insect cells by infection with baculovirus-based vectors. For reviews on these production systems, see generally, e.g., Zhang et al., 2009, “Adenovirus-adeno-associated virus hybrid for large-scale recombinant adeno-associated virus production,” Human Gene Therapy 20:922-929, the contents of each of which is incorporated herein by reference in its entirety. 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 is incorporated herein by reference in its entirety: 5,139,941; 5,741,683; 6,057,152; 6,204,059; 6,268,213; 6,491,907; 6,660,514; 6,951,753; 7,094,604; 7,172,893; 7,201,898; 7,229,823; and 7,439,065. The crude cell harvest may thereafter be subject method 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 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. A two-step affinity chromatography purification at high salt concentration followed anion exchange resin chromatography are used to purify the vector drug product and to remove empty capsids. These methods are described in more detail in International Patent Publication No. WO 2017/160360, which is incorporated by reference herein. Purification methods for AAV8, International Patent Publication No. WO 2017/100676, and rh10, International Patent Publication No. WO 2017/100704, and for AAV1, International Patent Publication No. WO 2017/100674 are all incorporated by reference herein. To calculate empty and full particle content, VP3 band volumes for a selected sample (e.g., in examples herein an iodixanol gradient-purified preparation where # of GC = # of particles) are plotted against GC particles loaded. The resulting linear equation (y = mx+c) is used to calculate the number of particles in the band volumes of the test article peaks. The number of particles (pt) per 20 µL loaded is then multiplied by 50 to give particles (pt) /mL. Pt/mL divided by GC/mL gives the ratio of particles to genome copies (pt/GC). Pt/mL–GC/mL gives empty pt/mL. Empty pt/mL divided by pt/mL and x 100 gives the percentage of empty particles. Generally, methods for assaying for empty capsids and AAV vector particles with packaged genomes have been known in the art. See, e.g., Grimm et al., Gene Therapy (1999) 6:1322-1330; Sommer et al., Molec. Ther. (2003) 7:122-128. To test for denatured capsid, the methods include subjecting the treated 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 the buffer, then running the gel until sample material is separated, and blotting the gel onto nylon or nitrocellulose membranes, preferably nylon. Anti-AAV capsid antibodies are then used as the primary antibodies that bind to denatured capsid proteins, preferably an anti-AAV capsid monoclonal antibody, most preferably the B1 anti-AAV-2 monoclonal antibody (Wobus et al., J. Virol. (2000) 74:9281-9293). A secondary antibody is then used, one that binds to the primary antibody and contains a means for detecting binding with the primary antibody, more preferably an anti-IgG antibody containing a detection molecule covalently bound to it, most preferably a sheep anti-mouse IgG antibody covalently linked to horseradish peroxidase. A method for detecting binding is used to semi- quantitatively determine binding between the primary and secondary antibodies, preferably a detection method capable of detecting radioactive isotope emissions, 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 reducing agent (e.g., DTT), and capsid proteins were resolved on pre-cast 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 method, i.e., SYPRO ruby or coomassie stains. In one embodiment, the concentration of AAV vector genomes (vg) in column fractions can be measured by quantitative real time PCR (Q-PCR). Samples are diluted and digested with DNase I (or another suitable nuclease) to remove exogenous DNA. After inactivation of the nuclease, the samples are further diluted and amplified using primers and a TaqMan™ fluorogenic probe specific for 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 identical sequences to that contained in the AAV vector is employed to generate a standard curve in the Q-PCR reaction. The cycle threshold (Ct) values obtained from the samples are used to determine vector genome titer by normalizing it to the Ct value of the plasmid standard curve. End-point assays based on the digital PCR can also be used. In one aspect, an optimized q-PCR method is used which utilizes a broad spectrum serine protease, e.g., proteinase K (such as is commercially available from Qiagen). More particularly, the optimized qPCR genome titer assay is similar to a standard assay, except that after the DNase I digestion, samples are diluted with proteinase K buffer and treated with proteinase K followed by heat inactivation. Suitably samples are diluted with proteinase K buffer in an amount equal to the sample size. The proteinase K buffer may be concentrated to 2-fold or higher. Typically, proteinase K treatment is about 0.2 mg/mL, but may be varied from 0.1 mg/mL to about 1 mg/mL. The treatment step is generally conducted at about 55 °C for about 15 minutes, but may be performed at a lower temperature (e.g., about 37 °C to about 50 °C) over a longer time period (e.g., about 20 minutes to about 30 minutes), or a higher temperature (e.g., up to about 60 °C) for a shorter time period (e.g., about 5 to 10 minutes). Similarly, heat inactivation is generally at about 95 °C for about 15 minutes, but the temperature may be lowered (e.g., about 70 to about 90 °C) and the time extended (e.g., about 20 minutes to about 30 minutes). Samples are then diluted (e.g., 1000 fold) and subjected to TaqMan analysis as described in the standard assay. Additionally, or alternatively, droplet digital PCR (ddPCR) may be used. For example, methods for determining single-stranded and self-complementary AAV vector genome titers by ddPCR have been described. See, e.g., 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. In brief, the method for separating rAAV particles having packaged genomic sequences from genome-deficient AAV intermediates involves subjecting a suspension comprising recombinant AAV viral particles and AAV capsid intermediates to fast performance liquid chromatography, wherein the AAV viral particles and AAV intermediates are bound to a strong anion exchange resin equilibrated at a high pH, and subjected to a salt gradient while monitoring eluate for ultraviolet absorbance at about 260 and about 280. The pH may be adjusted depending upon the AAV selected. See, e.g., WO2017/160360 (AAV9), WO2017/100704 (AAVrh10), WO 2017/100676 (e.g., AAV8), and WO 2017/100674 (AAV1)] which are incorporated by reference herein. In this method, the AAV full capsids are collected from a fraction which is eluted when the ratio of A260/A280 reaches an inflection point. In one example, for the Affinity Chromatography step, the diafiltered product may be applied to a Capture Select^TM Poros- AAV2/9 affinity resin (Life Technologies) that efficiently captures the AAV2 serotype. Under these ionic conditions, a significant percentage of residual cellular DNA and proteins flow through the column, while AAV particles are efficiently captured. In a specific embodiment, an rAAV having an AAVrh79 capsid is provided, which encapsulates a vector genome. The vector genome includes a 5’ ITR, TBG-S1- F113 promoter (F113), PSCK9-targeting meganuclease (sometimes referred to as the ARCUS meganuclease), polyA signal, and 3’ ITR. In one embodiment, the vector genome is SEQ ID NO: 13:

or a sequence sharing at least 80%, at least 85%, at least 86%, at least 87%, at

least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least at least 99.9% identity therewith. In another embodiment, the vector genome of SEQ ID NO: 13 encapsulated by a AAV8 capsid. In another embodiment, the vector genome of SEQ ID NO: 13 encapsulated by a AAVrh90 capsid. In another embodiment, the vector genome of SEQ ID NO: 13 is encapsulated by a AAVrh79 capsid. In another embodiment, the vector genome of SEQ ID NO: 13 is encapsulated by a AAVhu37 capsid. In a specific embodiment, an rAAV having an AAV8 capsid is provided, which encapsulates a vector genome. The vector genome includes a 5’ ITR, TBG-S1-F113 promoter (F113), PSCK9-targeting meganuclease (sometimes referred to as the ARCUS meganuclease), polyA signal, and 3’ ITR. In a specific embodiment, an rAAV having an AAVrh.90 capsid is provided, which encapsulates a vector genome. The vector genome includes a 5’ ITR, TBG-S1- F113 promoter (F113), PSCK9-targeting meganuclease (sometimes referred to as the ARCUS meganuclease), polyA signal, and 3’ ITR. In a specific embodiment, an rAAV having an AAVrh.79 capsid is provided, which encapsulates a vector genome. The vector genome includes a 5’ ITR, TBG-S1- F113 promoter (F113), PSCK9-targeting meganuclease (sometimes referred to as the ARCUS meganuclease), polyA signal, and 3’ ITR. In a specific embodiment, an rAAV having an AAVhu37 capsid is provided, which encapsulates a vector genome. The vector genome includes a 5’ ITR, TBG-S1- F113 promoter (F113), PSCK9-targeting meganuclease (sometimes referred to as the ARCUS meganuclease), polyA signal, and 3’ ITR. In a specific embodiment, an rAAV having an AAVrh.91 capsid is provided, which encapsulates a vector genome. The vector genome includes a 5’ ITR, TBG-S1- F113 promoter (F113), PSCK9-targeting meganuclease (sometimes referred to as the ARCUS meganuclease), polyA signal, and 3’ ITR. In a specific embodiment, an rAAV having an AAV3B capsid is provided, which encapsulates a vector genome. The vector genome includes a 5’ ITR, TBG-S1-F113 promoter (F113), PSCK9-targeting meganuclease (sometimes referred to as the ARCUS meganuclease), polyA signal, and 3’ ITR. Pharmaceutical Compositions A pharmaceutical composition comprises one or more of an expression cassette, vector containing same (viral or non-viral) or another system containing the expression cassette and one or more of a carrier, suspending agent, and/or excipient. In certain embodiments, compositions containing at least one rAAV stock (e.g., an rAAV stock) and an optional carrier, excipient and/or preservative. An rAAV stock refers to a plurality of rAAV vectors which are the same, e.g., such as in the amounts described below in the discussion of concentrations and dosage units. As used herein, “carrier” includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Supplementary active ingredients can also be incorporated into the compositions. The phrase “pharmaceutically-acceptable” refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a host. Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, may be used for the introduction of the compositions of the present invention into suitable host cells. In particular, the rAAV vector delivered vector genomes may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like. In certain embodiments, an expression cassette is delivered via a lipid nanoparticle. The term “lipid nanoparticle” refers to a lipid composition having a typically spherical structure with an average diameter of 10 to 1000 nanometers, e.g.75 nm to 750 nm, or 100 nm and 350 nm, or between 250 nm to about 500 nm. In some formulations, lipid nanoparticles can comprise at least one cationic lipid, at least one noncationic lipid, and at least one conjugated lipid. Lipid nanoparticles known in the art that are suitable for encapsulating nucleic acids, such as mRNA, may be used. “Average diameter” is the average size of the population of nanoparticles comprising the lipophilic phase and the hydrophilic phase. The mean size of these systems can be measured by standard methods known by the person skilled in the art. Examples of suitable lipid nanoparticles for gene therapy is described, e.g., L. Battaglia and E. Ugazio, J Nanomaterials, Vol 2019, Article ID 283441, pp.1-22; US2012/0183589A1; and WO 2012/170930 which are incorporated herein by reference in their entirety. In one embodiment, a composition includes a final formulation suitable for delivery to a subject, e.g., is 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 may be transported as a concentrate which is diluted for administration to a subject. In other embodiments, the composition may be lyophilized and reconstituted at the time of administration. 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, for example, contain excipients, carriers, stabilizers, or diluents such as sterile water, saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes, preservatives (such as 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 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 counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). The active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). A suitable surfactant, or combination of surfactants, may be selected from among non-ionic surfactants that are nontoxic. In one embodiment, a difunctional block copolymer surfactant terminating in primary hydroxyl groups is selected, e.g., such as Pluronic® F68 [BASF], also known as Poloxamer 188, which has a neutral pH, has an average molecular weight of 8400. Other surfactants and other Poloxamers may be selected, i.e., nonionic triblock copolymers composed of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)), SOLUTOL HS 15 (Macrogol-15 Hydroxystearate), LABRASOL (Polyoxy capryllic glyceride), polyoxy 10 oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid esters), ethanol and polyethylene glycol. In one embodiment, the formulation contains a poloxamer. These copolymers are commonly named with the letter “P” (for poloxamer) followed by three digits: the first two digits x 100 give the approximate molecular mass of the polyoxypropylene core, and the last digit x 10 gives the percentage polyoxyethylene content. In one embodiment Poloxamer 188 is selected. The surfactant may be present in an amount up to about 0.0005 % to about 0.001% of the suspension. In one embodiment, the composition is formulated in PBS containing about 0.001% Poloxamer 188. The vectors are administered in sufficient amounts to transfect the cells and to provide sufficient levels of gene transfer and expression to provide a therapeutic benefit without undue adverse effects, or with medically acceptable physiological effects, which can be determined by those skilled in the medical arts. Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to a desired organ (e.g., the liver (optionally via the hepatic artery), lung, heart, eye, kidney,), oral, inhalation, intranasal, intrathecal, intratracheal, intraarterial, intraocular, intravenous, intramuscular, subcutaneous, intradermal, and other parental routes of administration. Routes of administration may be combined, if desired. Dosages of the viral vector depend primarily on factors such as the condition being treated, the age, weight and health of the patient, and may thus vary among patients. For example, a therapeutically effective human dosage of the viral vector is generally in the range of from about 25 to about 1000 microliters to about 100 mL of solution containing concentrations of from about 1 x 10⁹ to 1 x 10¹⁶ genomes virus vector. The dosage is adjusted to balance the therapeutic benefit against any side effects and such dosages may vary depending upon the therapeutic application for which the recombinant vector is employed. The levels of expression of the transgene product can be monitored to determine the frequency of dosage resulting in viral vectors, preferably AAV vectors containing the minigene. Optionally, dosage regimens similar to those described for therapeutic purposes may be utilized for immunization using the compositions of the invention. The replication-defective virus compositions can be formulated in dosage units to contain an amount of replication-defective virus that is in the range of about 1.0 x 10⁹ GC to about 1.0 x 10¹⁶ GC (to treat an average subject of 70 kg in body weight) including all integers or fractional amounts within the range, and preferably 1.0 x 10¹² GC to 1.0 x 10¹⁴ GC for a human patient. In one embodiment, the compositions are formulated to contain at least 1x10⁹, 2x10⁹, 3x10⁹, 4x10⁹, 5x10⁹, 6x10⁹, 7x10⁹, 8x10⁹, or 9x10⁹ GC per dose including all integers or fractional amounts within the range. In another embodiment, the compositions are formulated to contain at least 1x10¹⁰, 2x10¹⁰, 3x10¹⁰, 4x10¹⁰, 5x10¹⁰, 6x10¹⁰, 7x10¹⁰, 8x10¹⁰, or 9x10¹⁰ GC per dose including all integers or fractional amounts within the range. In another embodiment, the compositions are formulated to contain at least 1x10¹¹, 2x10¹¹, 3x10¹¹, 4x10¹¹, 5x10¹¹, 6x10¹¹, 7x10¹¹, 8x10¹¹, or 9x10¹¹ GC per dose including all integers or fractional amounts within the range. In another embodiment, the compositions are formulated to contain at least 1x10¹², 2x10¹², 3x10¹², 4x10¹², 5x10¹², 6x10¹², 7x10¹², 8x10¹², or 9x10¹² GC per dose including all integers or fractional amounts within the range. In another embodiment, the compositions are formulated to contain at least 1x10¹³, 2x10¹³, 3x10¹³, 4x10¹³, 5x10¹³, 6x10¹³, 7x10¹³, 8x10¹³, or 9x10¹³ GC per dose including all integers or fractional amounts within the range. In another embodiment, the compositions are formulated to contain at least 1x10¹⁴, 2x10¹⁴, 3x10¹⁴, 4x10¹⁴, 5x10¹⁴, 6x10¹⁴, 7x10¹⁴, 8x10¹⁴, or 9x10¹⁴ GC per dose including all integers or fractional amounts within the range. In another embodiment, the compositions are formulated to contain at least 1x10¹⁵, 2x10¹⁵, 3x10¹⁵, 4x10¹⁵, 5x10¹⁵, 6x10¹⁵, 7x10¹⁵, 8x10¹⁵, or 9x10¹⁵ GC per dose including all integers or fractional amounts within the range. In one embodiment, for human application the dose can range from 1x10¹⁰ to about 1x10¹² GC per dose including all integers or fractional amounts within the range. These above doses may be administered in a variety of volumes of carrier, excipient or buffer formulation, ranging from about 25 to about 1000 microliters, or higher volumes, including all numbers within the 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. Any suitable route of administration may be selected. Accordingly, pharmaceutical compositions may be formulated for any appropriate route of administration, for example, in the form of liquid solutions or suspensions (as, for example, for intravenous administration, for oral administration, etc.). Alternatively, pharmaceutical compositions may be in solid form (e.g., in the form of tablets or capsules, for example for oral administration). In some embodiments, pharmaceutical compositions may be in the form of powders, drops, aerosols, etc. Methods The compositions provided herein are useful for reducing off-target activity of enzymes delivered in vivo. In certain embodiments, the compositions are useful in reducing off-target activity of an enzyme expressed following non-viral mediated delivery of an expression cassette comprising the enzyme coding sequence under the control of a weak promoter, as described herein. In certain embodiments, the compositions are useful in reducing off-target activity of an enzyme expressed following AAV-mediated delivery of a vector genome. In one embodiment, a method for editing a targeted gene is provided. The method includes delivering a nuclease expression cassette comprising a nucleic acid comprising a nuclease coding sequence which is operably linked to regulatory sequences which direct expression of the nuclease following delivery to a host cell having a sequence to which the nuclease is targeted, wherein the regulatory sequences comprise a promoter which has low transcriptional activity. Such promoters are described herein. In another embodiment, the method includes delivering a composition, viral vector or rAAV comprising the expression cassette, as described herein. In another embodiment, a method for reducing off-target activity of a gene targeting nuclease is provided. The method includes delivering a nuclease expression cassette comprising a nucleic acid comprising a nuclease coding sequence which is operably linked to regulatory sequences which direct expression of the nuclease following delivery to a host cell having a sequence to which the nuclease is targeted, wherein the regulatory sequences comprise a promoter which has low transcriptional activity. Such promoters are described herein. In another embodiment, the method includes delivering a composition, viral vector or rAAV comprising the expression cassette, as described herein. In one embodiment, the rAAV is an AAV8 capsid having a vector genome that includes a 5’ ITR, TBG-S1-F113 promoter (F113), PSCK9-targeting meganuclease (sometimes referred to as the ARCUS meganuclease), polyA signal, and 3’ ITR. In another embodiment, the rAAV is an AAVrh.90 capsid having a vector genome that includes a 5’ ITR, TBG-S1-F113 promoter (F113), PSCK9-targeting meganuclease (sometimes referred to as the ARCUS meganuclease), polyA signal, and 3’ ITR. In another embodiment, the rAAV is an AAVrh.79 capsid having a vector genome that includes a 5’ ITR, TBG-S1-F113 promoter (F113), PSCK9-targeting meganuclease (sometimes referred to as the ARCUS meganuclease), polyA signal, and 3’ ITR.. In another embodiment, the rAAV is an AAVhu37 capsid having a vector genome that includes a 5’ ITR, TBG-S1-F113 promoter (F113), PSCK9-targeting meganuclease (sometimes referred to as the ARCUS meganuclease), polyA signal, and 3’ ITR. In one embodiment, the rAAV is an AAVrh.91 capsid having a vector genome that includes a 5’ ITR, TBG-S1-F113 promoter (F113), PSCK9-targeting meganuclease (sometimes referred to as the ARCUS meganuclease), polyA signal, and 3’ ITR. In one embodiment, the rAAV is an AAV3B capsid having a vector genome that includes a 5’ ITR, TBG-S1-F113 promoter (F113), PSCK9-targeting meganuclease (sometimes referred to as the ARCUS meganuclease), polyA signal, and 3’ ITR. In certain embodiments, the effectiveness of a weak promoter may be assessed in vitro. For example, the half-life of a nuclease may be assessed in vitro (in cultured cells) by treating the cells to stop translation of the protein (e.g., with cycloheximide (CHX)) and then performing a western blot at different times post-treatment. Other suitable methods for assessing off-targeting activity of a nuclease may be readily determined by one of skill in the art. Meganuclease-based editing results in both on-target and off-target editing. On- target editing is quantified as the percentage of alleles containing indels in the target sequence (i.e., the PCSK9 gene), and is also referred to as indel percent (indel%). In off- target editing sites, indel% of the meganuclease is defined as the percentage of alleles containing indels in a region other than the intended target-sequence (i.e., the PCSK9 gene) and represents the number of genomic locations in addition to the on-target sequence in which the meganuclease has editing activity. Given the importance of characterizing on-target editing and off-target editing in genome editing studies, different methods have been developed to measure both types of editing (Bennett et al., 2020). There are advantages and disadvantages for each of the methods and none of them can both identify and quantify indels for evaluation of on- target and off-target editing. Table 1. Methods for Identifying and Quantifying Indel-Containing Alleles

Abbreviations: AAV, adeno-associated virus; AMP-seq, anchored multiplex PCR- sequencing; DNA, deoxyribonucleic acid; DSBs, double-stranded breaks; GUIDE-seq, genome-wide, unbiased identification of DSBs enabled by sequencing; indels, insertions and deletions; ITR-seq, inverted terminal repeat sequencing; PCR, polymerase chain reaction; UPenn-GTP, University of Pennsylvania Gene Therapy Program. For on-target editing, two next-generation sequencing (NGS)-based methods are routinely used to accurately identify and quantify indel-containing alleles: amplicon-seq and AMP-seq. In Amplicon-Seq, DNA is isolated from the tissue of interest in treated animals. The locus containing the region of interest is amplified using primers encompassing the nuclease target region (FIG.20) using commercially available PCR and NGS kits. NGS- compatible libraries are generated from the amplicons and sequenced on a MiSeq instrument (Illumina). The resulting reads contain a mixture of edited and non-edited alleles. Using custom computer scripts, the number of reads containing indels in the nuclease target region are quantified. Briefly, all the reads are mapped to the reference genome using NovoAlign (developed by Novocraft). The alignment information that results from this mapping step are stored as a Sequence Alignment Map (SAM) file (Li et al., 2009). Indels are then identified directly from the Compact Idiosyncratic Gapped Alignment Report (CIGAR) string that is available for each mapped read present in the SAM file. Reads containing indels within the region of interest are quantified and expressed as a percentage of the total reads mapped to the region of interest (indel%). In some embodiments, the on-target activity is assessed by amplicon-seq. While amplicon-seq can be used in both in vitro- and in vivo-edited samples to quantify editing efficiency at pre-identified on- and off-target sites, this method is unable to capture large insertions or identify editing sites de novo, and only quantifies editing efficiency in a pre-defined locus. One of the limitations of the amplicon-seq analysis is that it will not detect large insertions in the region of interest because smaller DNA fragments are preferentially amplified by PCR and large insertions (i.e., mostly vector integration) might not be representatively amplified in the PCR reaction (Wang et al., 2018; Hanlon et al., 2019; Breton et al., 2021). Additionally, the NGS for amplicon-seq only captures short sequence reads (<500 bp). To circumvent these issues, a technique called anchored multiplex PCR-sequencing (AMP-seq) was used (FIG.21) (Zheng et al., 2014). This method quantifies insertions and deletions, including vector integration and translocations, at a pre-defined locus. In this method, DNA is isolated from the tissue of interest in treated animals and is then randomly sheared by sonication. DNA is end- repaired and A-tailed. Y-adapters are ligated to these DNA fragments. Following two rounds of PCR using adapter- and gene-specific primers, NGS libraries are produced. Because only two gene-specific primer binding sites are required and DNA is fragmented prior to the amplification (eliminating possible fragment-length bias), it is possible to quantify alleles containing large insertions and deletions, as well as less common gene rearrangements,as long as those deletions do not remove the primer binding sites. Thus, the AMP-seq method results in a more complete analysis of the target region edits. This method quantifies both small and large indels at pre-identified loci in both in vitro and in vivo edited samples, but is unable to identify editing sites de novo. In some embodiments, the on-target activity is assessed by AMP-seq. A reduction in off-target nuclease activity can be determined using a variety of approaches which have been described in the literature. Such methods for determining nuclease specificity, include cell-free methods such as Site-Seq [Cameron, P., et al, (2017) Mapping the genomic landscape of CRISPR-Cas9 cleavage. Nat Methods, 14, 600-606], Digenome-seq [Kim, D., et al, (2015) Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells. Nat Methods, 12, 237-243, 231 p following 243], and Circle-Seq [Tsai, S.Q., et al, (2017) CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR-Cas9 nuclease off-targets. Nat Methods, 14, 607-614] and in vitro-based methods such as, e.g., GUIDE-Seq [Tsai (2017) Nat Methods, 14, 607-614] and Integrative-Deficient Lentiviral Vectors Capture (IDLV) [Gabriel, R., et al. (2011) An unbiased genome-wide analysis of zinc-finger nuclease specificity. Nat Biotechnol, 29, 816-823; Wang, X., et al., (2015) Unbiased detection of off-target cleavage by CRISPR-Cas9 and TALENs using integrase-defective lentiviral vectors. Nat Biotechnol, 33, 175-178], CAST-seq [Turchiano et al, Quantitative evaluation of chromosomal rearrangements in gene-edited human stem cells by CAST- Seq, Cell Stem Cell, 2021 Jun 3;28(6):1136-1147.e5. doi: 10.1016/j.stem.2021.02.002. Epub 2021 Feb 23], BLISS [Ballarino et al, Genome-Wide CRISPR Off-Target DNA Break Detection by the BLISS Method, Methods Mol Biol, 2021;2162:261-281. doi: 10.1007/978-1-0716-0687-2_15], BLESS [Mirzazadeh et al, Genome-Wide Profiling of DNA Double-Strand Breaks by the BLESS and BLISS Methods, Methods Mol Biol, 2018;1672:167-194. doi: 10.1007/978-1-4939-7306-4_14], CHIP-seq [Robertson et al, Genome-wide profiles of STAT1 DNA association using chromatin immunoprecipitation and massively parallel sequencing, Nature Methods, 4(8):651-7 (August 2007)], and LAM-HGTGTS [Hu J et al, Detecting DNA double-stranded breaks in mammalian genomes by linear amplification-mediated high-throughput genome-wide translocation sequencing. Nat Protoc.2016 May;11(5):853-71. doi: 10.1038/nprot.2016.043. Epub 2016 Mar 31]. In some embodiments, the off-target activity is assessed by ITR-seq. See, e.g., the publication Breton et al, ITR-Seq, a next-generation sequencing assay, identifies genome-wide DNA editing sites in vivo following adeno-associated viral vector- mediated genome editing, BMC Genomics, (2020):21:239 which is incorporated herein by reference in its entirety. Briefly, the DNA is isolated from the tissue of interest. DNA is sheared and ligated to adapters. An NGS-compatible library is generated by PCR, using adapter- and AAV-ITR-specific primers. After sequencing, hybrid reads (containing AAV-ITR and cellular genome sequences) are retained. The location of the AAV-ITR integration is determined and a list of all the sites of the nuclease off-target editing is generated as well as their frequency in the analyzed DNA samples. In contrast with GUIDE-seq, ITR-seq can be used in vivo without additional reagents. Moreover, ITR-seq has been shown to be more sensitive in detecting ITR integration than other next-generation sequencing- (NGS)-based methods, such as the combined approach of GUIDE-seq and subsequent amplicon sequencing. ITR-seq has already been utilized in published studies to investigate the specificity of different genome editing nucleases in liver samples collected from animals following administration of AAV nuclease vectors (Breton et al., 2021; Wang et al., 2021). For example, in the liver DNA harvested 7 weeks after IV administration of a single dose of AAV3B.TBG.M2PCSK9.WPRE.BGH (5.0 x10¹³ GC/kg) in immune-deficient, humanized FRG mice, ITR-seq detected 93 to 233 potential off-target sites in the human genome through analysis of 8 different samples from two mice, with 29 off-target sites commonly found in both mice (Wang et al., 2021). In the Day 127 liver biopsy samples collected from the two NHPs treated with the clinical candidate construct packaged with AAV8 (AAV8.TBG-S1-F113.M2PCSK9.BGH), ITR-seq showed the overall ratio of on- to off-target editing at 1:116 and 1: 146, respectively. ITR-seq has been validated through its application to successfully detect off- target sites that were previously identified by GUIDE-seq (Breton et al., 2020). In the study discussed above, ITR-seq detected 32–54 off-target sites, varying between the eight samples from the two FRG mice, that were also detected by GUIDE-seq in the iPSC-derived human hepatocytes, although the ranking orders of the off-target sites were not consistent between the in vivo ITR-seq and in vitro GUIDE-seq data. The reproducibility of ITR-seq in detecting potential off-target sites has been investigated by repeating the assay on the same sample by two independent experiments (i.e., two ITR-seq assays on the same DNA sample to evaluate reproducibility). Liver samples collected from NHPs 18 days after being administered with AAV8-M2PCSK9 showed that about half of the off-target sites were commonly detected by both experiments (Wang et al., 2021). Those off-target sites that were only detected by one of the ITR-seq experiments usually had very low number of reads by NGS, indicating they may not be true off-target sites. A few off-target sites that are repeatedly detected in biopsy samples from the same animal at different time points, or from different animals treated with the same vector, or by both ITR-seq and GUIDE-seq warrant further investigation. In addition, amplicon-seq on the off-target sites of interest can be performed to validate the off-target editing efficiency. ITR-Seq can be used for both in vivo- and in vitro-edited samples, is unbiased, allows for a genome-wide analysis, and is sensitive. However, the method is limited to AAV-delivered genome editing. Cumulatively, the qualities and capabilities of ITR-seq make this a highly valuable and appropriate method for measuring the off-target editing of M2PCSK9 in vivo. In some embodiments, the off-target activity is assessed by GUIDE-seq. GUIDE- seq (genome-wide, unbiased identification of DSBs enabled by sequencing) allows the detection of DSBs induced by genome editing nucleases (FIG.19) (Tsai et al., 2015). In this method, DSBs are tagged by integration of a short, double-stranded oligodeoxynucleotide (dsODN), delivered to the cells by transfection or electroporation. The integration of the dsODN occurs during the DSB repair events and thus they are captured in an unbiased way. As the dsODN integration occurs at the DSB, identification of the genomic location of the dsODN allows for the identification of the on- and off- target editing by the meganuclease. DNA from treated cells is purified and sheared by sonication into small fragments. The generated fragments are end-repaired and A-tailed. Y-adapters are ligated to all the generated fragments. Adapter- and dsODN-specific primers are used to amplify only the genomic DNA containing an integrated dsODN. NGS-compatible libraries are generated by PCR and sequenced. Identification of the sites of dsODN integration, and therefore on- and off-target sites, is achieved by bioinformatics methods using the corresponding open-source software (Tsai et al., 2016). Since the sequence of the on-target sites is known, GUIDE-seq can also be used to confirm on-target editing. This method is unbiased, allows for a genome-wide analysis (compared to computational methods) and is sensitive. However, it can only be used in vitro because it requires delivering dsODN directly to cells using a viral vector or electroporation and is influenced by transfection efficiency of the cell line. As previously mentioned, none of the methods discussed above can both identify and quantify indels for evaluation of on-target and off-target editing, thus a composite approach where different methods are used is necessary. Based on the non-clinical data from the completed or ongoing non-clinical studies and from AAV6.2-transduced human iPSCs (Wang et al., 2018), a common set of 41 potential off-target editing sites following M2PCSK9 nuclease administration have been identified using a composite of 3 methods (GUIDE-seq, amplicon-seq and ITR-seq). These sites and others identified by amplicon- seq and/or ITR-seq will be assessed in the proposed FIH clinical trial (Off-Target Editing Safety Monitoring Section). Off-target sites were chosen based on non-clinical studies including GUIDE-seq on AAV6.2-transduced human iPSCs (Wang et al., 2018) and were confirmed to have above-background editing at these locations in FRG mice by amplicon-seq (Wang et al., 2021). Off-target sites in white cells were identified by ITR-seq in all 8 samples from both AAV3B.M2PCSK9-treated humanized FRG mice (Wang et al., 2021) and will be verified by amplicon-seq before commencing the FIH clinical trial; the list of off-target sites may be modified based on these pending amplicon-seq data. Abbreviations: N.D., not detected; TBC, to be conducted. In one aspect, a method for editing a targeted gene is provided which comprises delivering a nuclease expression cassette under control of a weak promoter as described herein. In one embodiment, the dosage of an rAAV is about 1 x 10⁹ GC to about 1 x 10¹⁵ genome copies (GC) per dose (to treat an average subject of 70 kg in body weight), and preferably 1.0 x 10¹² GC to 2.0 x 10¹⁵ GC for a human patient. In another embodiment, the dose is less than about 1 x 10¹⁴ GC/kg body weight of the subject. In certain embodiments, the dose administered to a patient is at least about 1.0 x 10⁹ GC/kg, about 1.5 x 10⁹ GC/kg, about 2.0 x 10⁹ GC/g, about 2.5 x 10⁹ GC/kg, about 3.0 x 10⁹ GC/kg, about 3.5 x 10⁹ GC/kg, about 4.0 x 10⁹ GC/kg, about 4.5 x 10⁹ GC/kg, about 5.0 x 10⁹ GC/kg, about 5.5 x 10⁹ GC/kg, about 6.0 x 10⁹ GC/kg, about 6.5 x 10⁹ GC/kg, about 7.0 x 10⁹ GC/kg, about 7.5 x 10⁹ GC/kg, about 8.0 x 10⁹ GC/kg, about 8.5 x 10⁹ GC/kg, about 9.0 x 10⁹ GC/kg, about 9.5 x 10⁹ GC/kg, about 1.0 x 10¹⁰ GC/kg, about 1.5 x 10¹⁰ GC/kg, about 2.0 x 10¹⁰ GC/kg, about 2.5 x 10¹⁰ GC/kg, about 3.0 x 10¹⁰ GC/kg, about 3.5 x 10¹⁰ GC/kg, about 4.0 x 10¹⁰ GC/kg, about 4.5 x 10¹⁰ GC/kg, about 5.0 x 10¹⁰ GC/kg, about 5.5 x 10¹⁰ GC/kg, about 6.0 x 10¹⁰ GC/kg, about 6.5 x 10¹⁰ GC/kg, about 7.0 x 10¹⁰ GC/kg, about 7.5 x 10¹⁰ GC/kg, about 8.0 x 10¹⁰ GC/kg, about 8.5 x 10¹⁰ GC/kg, about 9.0 x 10¹⁰ GC/kg, about 9.5 x 10¹⁰ GC/kg, about 1.0 x 10¹¹ GC/kg, about 1.5 x 10¹¹ GC/kg, about 2.0 x 10¹¹ GC/kg, about 2.5 x 10¹¹ GC/kg, about 3.0 x 10¹¹ GC/kg, about 3.5 x 10¹¹ GC/kg, about 4.0 x 10¹¹ GC/kg, about 4.5 x 10¹¹ GC/kg, about 5.0 x 10¹¹ GC/kg, about 5.5 x 10¹¹ GC/kg, about 6.0 x 10¹¹ GC/kg, about 6.5 x 10¹¹ GC/kg, about 7.0 x 10¹¹ GC/kg, about 7.5 x 10¹¹ GC/kg, about 8.0 x 10¹¹ GC/kg, about 8.5 x 10¹¹ GC/kg, about 9.0 x 10¹¹ GC/kg, about 9.5 x 10¹¹ GC/kg, about 1.0 x 10¹² GC/kg, about 1.5 x 10¹² GC/kg, about 2.0 x 10¹² GC/kg, about 2.5 x 10¹² GC/kg, about 3.0 x 10¹² GC/kg, about 3.5 x 10¹² GC/kg, about 4.0 x 10¹² GC/kg, about 4.5 x 10¹² GC/kg, about 5.0 x 10¹² GC/kg, about 5.5 x 10¹² GC/kg, about 6.0 x 10¹² GC/kg, about 6.5 x 10¹² GC/kg, about 7.0 x 10¹² GC/kg, about 7.5 x 10¹² GC/kg, about 8.0 x 10¹² GC/kg, about 8.5 x 10¹² GC/kg, about 9.0 x 10¹² GC/kg, about 9.5 x 10¹² GC/kg, about 1.0 x 10¹³ GC/kg, about 1.5 x 10¹³ GC/kg, about 2.0 x 10¹³ GC/kg, about 2.5 x 10¹³ GC/kg, about 3.0 x 10¹³ GC/kg, about 3.5 x 10¹³ GC/kg, about 4.0 x 10¹³ GC/kg, about 4.5 x 10¹³ GC/kg, about 5.0 x 10¹³ GC/kg, about 5.5 x 10¹³ GC/kg, about 6.0 x 10¹³ GC/kg, about 6.5 x 10¹³ GC/kg, about 7.0 x 10¹³ GC/kg, about 7.5 x 10¹³ GC/kg, about 8.0 x 10¹³ GC/kg, about 8.5 x 10¹³ GC/kg, about 9.0 x 10¹³ GC/kg, about 9.5 x 10¹³ GC/kg, or about 1.0 x 10¹⁴ GC/kg body weight of the subject. In another embodiment, the method further comprises administering a co-therapy to the subject. Such co-therapy may be a known treatment for high cholesterol, including high low-density lipoprotein cholesterol ((LDL-C) >200 mg/dL), and/or subjects with familial hypercholesterolemia (FH). Such therapies include statins, ezetimibe, lomitapide, inclisiran, PCSK9 monoclonal antibodies (evolocumab, alirocumab), or LDL-C apheresis. Such co-therapies may be administered prior to delivery of an rAAV or a composition as disclosed. In certain embodiments, such co-therapies are administered after delivery of an rAAV or a composition as disclosed, for additional lipid-lowering effects. In one embodiment, the method further comprises administering an immunosuppressive co-therapy to the subject. Such immunosuppressive co-therapy may be started prior to delivery of an rAAV or a composition as disclosed, e.g., if undesirably high neutralizing antibody levels to the AAV capsid are detected. In certain embodiments, co-therapy may also be started prior to delivery of the rAAV as a precautionary measure. In certain embodiments, immunosuppressive co-therapy is started following delivery of the rAAV, e.g., if an undesirable immune response is observed following treatment. Immunosuppressants for such co-therapy include, but are not limited to, a glucocorticoid, steroids, antimetabolites, T-cell inhibitors, a macrolide (e.g., a rapamycin or rapalog), and cytostatic agents including an alkylating agent, an anti-metabolite, a cytotoxic antibiotic, an antibody, or an agent active on immunophilin. The immune suppressant may include prednisolone, a nitrogen mustard, nitrosourea, platinum compound, methotrexate, azathioprine, mercaptopurine, fluorouracil, dactinomycin, an anthracycline, mitomycin C, bleomycin, mithramycin, IL-2 receptor- (CD25-) or CD3- directed antibodies, anti-IL-2 antibodies, ciclosporin, tacrolimus, sirolimus, IFN-β, IFN- γ, an opioid, or TNF-α (tumor necrosis factor-alpha) binding agent. In certain embodiments, the immunosuppressive therapy may be started 0, 1, 2, 7, or more days prior to the rAAV administration, or 0, 1, 2, 3, 7, or more days post the rAAV administration. Such therapy may involve a single drug (e.g., prednisolone) or co- administration of two or more drugs, the (e.g., prednisolone, micophenolate mofetil (MMF) and/or sirolimus (i.e., rapamycin)) on the same day. One or more of these drugs may be continued after gene therapy administration, at the same dose or an adjusted dose. Such therapy may be for about 1 week (7 days), two weeks, three weeks, about 60 days, or longer, as needed. In certain embodiments, a tacrolimus-free regimen is selected. In one aspect, a method for editing a targeted gene is provided which comprises delivering a composition as described herein. In one aspect, a method for editing a targeted gene is provided which comprises delivering a viral or non-viral vector as described herein. In one aspect, a method for editing a targeted gene is provided which comprises delivering an rAAV as described herein. In one aspect, a method for treating a patient having a cholesterol-related disorder(s), such as hypercholesterolemia, using a nuclease expression cassette comprising a meganuclease which recognizes a site within the human PCSK9 gene, under the control of a weak promoter as described herein. In one embodiment, the weak promoter is F113. Such expression cassettes may be delivered via a viral or non-viral vector. In certain embodiments, the expression cassettes may be delivered using an LNP. In one embodiment, the method includes administering F113.Arcus. In one aspect, a method for editing a gene using a meganuclease is provided, using an expression cassette comprising a coding sequence for a meganuclease which recognizes a site within the desired gene, under the control of a weak promoter as described herein. In one embodiment, the weak promoter is F113. Such expression cassettes may be delivered via a viral or non-viral vector. In certain embodiments, the expression cassettes may be delivered using an LNP. In certain embodiments, nucleases other than meganucleases targeting any of the above-described genes are contemplated. In certain embodiments, a nuclease expression cassette, non-viral vector, viral vector (e.g., rAAV), or any of the same in a pharmaceutical composition, as described herein is administrable 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 older than an infant, e.g, 12 months or older. In certain embodiments, the subject is an adult subject at least 35 years of age. In some embodiments, the subject is at least 35 years of age and less than 76 years of age. In certain embodiments, the subject has familial hypercholesteremia (FH). In some embodiments, the subject has severe, refractory FH with an unmet need for treatment due to uncontrollably high LDL-C levels even in the presence of stable background lipid- lowering therapy, or intolerance or lack of access to other anti-PCSK9 therapies. That is, in certain embodiments, the subject has been previously treated for high LDL-C levels. In certain embodiments, the subject has a clinical diagnosis of severe, refractory FH, defined as an LDL-C level in blood of >200 mg/dL. In certain embodiments, the subject has at least one mutation causative for FH; namely, at least one loss-of-function mutation in the LDLR gene or APOB gene, at least one gain-of-function mutation in the PCSK9 gene, or a loss-of-function mutation in both alleles of the LDLRAP1 gene. In certain embodiments, the subject has clinical evidence of symptomatic or asymptomatic atherosclerotic disease as determined by a physician. As used herein, “a,” “an,” or “the” can mean one or more than one. For example, “a” cell can mean a single cell or a multiplicity of cells. In certain embodiments, the term “meganuclease” refers to an endonuclease that binds double-stranded DNA at a recognition sequence that is greater than 12 base pairs. Preferably, the recognition sequence for a meganuclease of the invention is 22 base pairs. A meganuclease can be an endonuclease that is derived from I-CreI, and can refer to an engineered variant of I-CreI that has been modified relative to natural I-CreI with respect to, for example, DNA-binding specificity, DNA cleavage activity, DNA-binding affinity, or dimerization properties. Methods for producing such modified variants of I-CreI are known in the art. See, e.g., WO 2007/047859). A meganuclease as used herein binds to double-stranded DNA as a heterodimer. A meganuclease may also be a “single-chain meganuclease” in which a pair of DNA-binding domains are joined into a single polypeptide using a peptide linker. The term “homing endonuclease” is synonymous with the term “meganuclease.” See, WO 2018/195449, describing certain PCSK9 meganucleases, which is incorporated herein in its entirety. As used herein, the term “specificity” means the ability of a meganuclease to recognize and cleave double-stranded DNA molecules only at a particular sequence of base pairs referred to as the recognition sequence, or only at a particular set of recognition sequences. The set of recognition sequences will share certain conserved positions or sequence motifs, but may be degenerate at one or more positions. A highly- specific meganuclease is capable of cleaving only one or a very few recognition sequences. Specificity can be determined by any method known in the art. The abbreviation “sc” refers to self-complementary. “Self-complementary AAV” refers a construct in which a coding region carried by a recombinant AAV nucleic acid sequence has been designed to form an intra-molecular double-stranded DNA template. Upon infection, rather than waiting for cell mediated synthesis of the second strand, the two complementary halves of scAAV will associate to form one double stranded DNA (dsDNA) unit that is ready for immediate replication and transcription. See, e.g., D M 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 in, e.g., U.S. Patent Nos.6,596,535; 7,125,717; and 7,456,683, each of which is incorporated herein by reference in its entirety. As used herein, the term “operably linked” refers to both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. The term “exogenous” as used to describe a nucleic acid sequence or protein means that the nucleic acid or protein does not naturally occur in the position in which it exists in a chromosome, or host cell. An exogenous nucleic acid sequence also refers to a sequence derived from and inserted into the same expression cassette or host cell, but which is present in a non-natural state, e.g., a different copy number, or under the control of different regulatory elements. The term “heterologous” when used with reference to a protein or a nucleic acid indicates that the protein or the nucleic acid comprises two or more sequences or subsequences which are not found in the same relationship to each other in nature. For instance, the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid. For example, in one embodiment, the nucleic acid has a promoter from one gene arranged to direct the expression of a coding sequence from a different gene. As used herein, the term “host cell” may refer to the packaging cell line in which a vector (e.g., a recombinant AAV) is produced from a production plasmid. In the alternative, the term “host cell” may refer to any target cell in which expression of the transgene is desired. Thus, a “host cell,” refers to a prokaryotic or eukaryotic cell that contains a exogenous or heterologous nucleic acid sequence that has been introduced into the cell by any means, e.g., electroporation, calcium phosphate precipitation, microinjection, transformation, viral infection, transfection, liposome delivery, membrane fusion techniques, high velocity DNA-coated pellets, viral infection and protoplast fusion. In certain embodiments herein, the term “host cell” refers to cultures of cells of various mammalian species for in vitro assessment of the compositions described herein. In other embodiments herein, the term “host cell” refers to the cells employed to generate and package the viral vector or recombinant virus. Still in other embodiment, the term “host cell” is intended to reference the target cells of the subject being treated in vivo for the diseases or conditions as described herein. In certain embodiments, the term “host cell” is a liver cell or hepatocyte. A “replication-defective virus” or “viral vector” refers to a synthetic or artificial viral particle in which an expression cassette containing a gene of interest is packaged in a viral capsid or envelope, where any viral genomic sequences also packaged within the viral capsid or envelope are replication-deficient; i.e., they cannot generate progeny virions but retain the ability to infect target cells. In one embodiment, the genome of the viral vector does not include genes encoding the enzymes required to replicate (the genome can be engineered to be “gutless” - containing only the gene of interest flanked by the signals required for amplification and packaging of the artificial genome), but these genes may be supplied during production. Therefore, it is deemed safe for use in gene therapy since replication and infection by progeny virions cannot occur except in the presence of the viral enzyme required for replication. The terms “sequence identity” “percent sequence identity” or “percent identical” in the context of nucleic acid sequences refers to the residues in the two sequences which are the same when aligned for maximum correspondence. The length of sequence identity comparison may be over the full-length of the genome, the full-length of a gene coding sequence, or a fragment of at least about 500 to 5000 nucleotides, is desired. However, identity among smaller fragments, e.g., of at least about nine nucleotides, usually at least about 20 to 24 nucleotides, at least about 28 to 32 nucleotides, at least about 36 or more nucleotides, may also be desired. Similarly, “percent sequence identity” may be readily determined for amino acid sequences, over the full-length of a protein, or a fragment thereof. Suitably, a fragment is at least about 8 amino acids in length and may be up to about 700 amino acids. Examples of suitable fragments are described herein. The term “substantial homology” or “substantial similarity,” when referring to amino acids or fragments thereof, indicates that, when optimally aligned with appropriate amino acid insertions or deletions with another amino acid (or its complementary strand), there is amino acid sequence identity in at least about 95 to 99% of the aligned sequences. Preferably, the homology is over full-length sequence, or a protein thereof, e.g., a cap protein, a rep protein, or a fragment thereof which is at least 8 amino acids, or more desirably, at least 15 amino acids in length. Examples of suitable fragments are described herein. By the term “highly conserved” is meant at least 80% identity, preferably at least 90% identity, and more preferably, over 97% identity. Identity is readily determined by one of skill in the art by resort to algorithms and computer programs known by those of skill in the art. Generally, when referring to “identity”, “homology”, or “similarity” between two different adeno-associated viruses, “identity”, “homology” or “similarity” is determined in reference to “aligned” sequences. “Aligned” sequences or “alignments” refer to multiple nucleic acid sequences or protein (amino acids) sequences, often containing corrections for missing or additional bases or amino acids as compared to a reference sequence. In the examples, AAV alignments are performed using the published AAV9 sequences as a reference point. Alignments are 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 for such programs are known to those of skill in the art. Alternatively, Vector NTI utilities are also used. There are also a number of algorithms known in the art that can be used to measure nucleotide sequence identity, including those contained in the programs described above. As another example, polynucleotide sequences can be compared using Fasta™, a program in GCG Version 6.1. Fasta™ provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences. For instance, percent sequence identity between nucleic acid sequences can be determined using Fasta™ with its default parameters (a word size of 6 and the NOPAM factor for the scoring matrix) as provided in GCG Version 6.1, herein incorporated by reference. Multiple sequence alignment programs are also available for amino acid sequences, e.g., the “Clustal Omega”, “Clustal X”, “MAP”, “PIMA”, “MSA”, “BLOCKMAKER”, “MEME”, and “Match-Box” programs. Generally, any of these programs are used at default settings, although one of skill in the art can alter these settings as needed. Alternatively, one of skill in the art can utilize another algorithm or computer program which provides at least the level of identity or alignment as that provided by the referenced algorithms and programs. See, e.g., J. D. Thomson et al, Nucl. Acids. Res., “A comprehensive comparison of multiple sequence alignments”, 27(13):2682-2690 (1999). As used herein, the term “about” refers to a variant of ±10% from the reference integer and values therebetween. For example, “about” 40 base pairs, includes ±4 (i.e., 36 – 44, which includes the integers 36, 37, 38, 39, 40, 41, 42, 43, 44). For other values, particularly when reference is to a percentage (e.g., 90% identity, about 10% variance, or about 36% mismatches), the term “about” is inclusive of all values within the range including both the integer and fractions. As used throughout this specification and the claims, the terms “comprising”, “containing”, “including”, and its variants are inclusive of other components, elements, integers, steps and the like. Conversely, the term “consisting” and its variants are exclusive of other components, elements, integers, steps and the like. Unless defined otherwise in this specification, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art and by reference to published texts, which provide one skilled in the art with a general guide to many of the terms used in the present application. EXAMPLES Example 1 – Materials and Methods Materials and Methods AAV plasmids and vectors The procedure to clone the vectors used in AAV production is shown below (primers sequences are shown in the table below- SEQ ID NOs: 29-39, from top to bottom). Table. Primer sequences used to generate the plasmids for AAV production. Sequences corresponding to the target or mutant target sequences are in bold.

pAAV.M2PCSK9: This plasmid is similar to pAAV.TBG.PI.PCS 7- 8L.197.WPRE.BGH but without the WPRE sequence¹⁴. It contains the TBG promoter, a synthetic intron, the coding sequence for M2PCSK9 (I-Cre-I engineered Meganuclease, also known as PCS 7-8L.197), and the bovine growth hormone polyadenylation sequence. pAAV.M2PCSK9+PEST: The PEST sequence from mouse ornithine decarboxylase was amplified by PCR using the primers PEST-F/-R. We cloned this fragment in Bsu36I-BglII-digested pAAV.TBG.PI.PCS 7-8L.197.WPRE.BGH¹⁴ using In-Fusion HD kit (Takara, Mountain View, CA) and followed the manufacturer’s instructions. pAAV.Target.M2PCSK9, pAAV.Target.M2PCSK9+PEST, pAAV.MutTarget.M2PCSK9, pAAV.MutTarget.M2PCSK9+PEST: We amplified the intron region in pAAV.M2PCSK9 with primers containing either the M2PCSK9 target (C-Target-F/-R primers) or the mutant target (C-MutTarget-F/-R primers) sequences. Fragments with the target or mutant target (MutTarget) sequences were purified and cloned in the PstI and NotI sites of the plasmid that did (pAAV.M2PCSK9+PEST) or did not (pAAV.M2PCSK9) contain the PEST sequence. pAAV.2xTarget.M2PCSK9+PEST: We amplified the PEST sequence using the primers PEST-Target-F/-R. The reverse primer contains the additional M2PCSK9 target sequence. We cloned this fragment in the HindIII and BglII sites in p0146 plasmid²⁹. We obtained a DNA fragment from this new plasmid by HindIII and XhoI digestion and cloned the fragment in pAAV.Target.M2PCSK9+PEST in the corresponding restriction sites. pAAV.TBG-S1- F113.M2PCSK9 and pAAV.TBG-S1-F140. M2PCSK9: We generated shorter versions of the TBG promoter by PCR using the primer TBG-S1-R and either the primer TBG- S1-F113-F or TBG-S1-F140-F. We cloned PCR products in pAAV.M2PCSK9 in the AflII and NotI restriction sites. Animal experiments All animal procedures were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania. We obtained B6.129S7-Rag1^tm1Mom/J (also known as Rag1 knockout) mice from The Jackson Laboratory (Bar Harbor, ME). We intravenously administered AAV9.hPCSK9¹⁴ to the mice at a dose of 3.5x10¹⁰ GC/mouse. Two weeks later, mice received an intravenous dose of AAV vectors expressing the corresponding M2PCSK9 nuclease at a dose of 10¹⁰ GC/mouse. Two or seven weeks later (four or nine weeks after the initial AAV injection, respectively), mice were euthanized for liver collection. In NHP studies, we intravenously administered AAV vectors at a dose of 6x10¹² or 3x10¹³ GC/kg. We obtained PBMCs and serum samples before and at different times after vector administration. A liver biopsy was collected on day 18 post-vector administration. We performed all the blood tests, including hPCSK9 measurements, as previously described¹⁴. Analyzing on- and off-target activity using NGS We calculated the percentage of total alleles containing insertions or deletions in the region of interest (indel%) using Amplicon-Seq as previously described¹⁴. Briefly, the region of interest was amplified by PCR using the primers indicated in the table below. We generated NGS-compatible libraries from the PCR product and subsequently sequenced them on a MiSeq instrument (Illumina, San Diego, CA). These sequences were then mapped to the corresponding reference genome (Assembly GRCm38.p6 for mouse and Mmul_8.0.1 for rhesus macaque). Using a custom script, we quantified unedited reads and reads containing insertions and deletions¹⁴. In addition to insertions and deletions, we quantified AAV integration and translocations in the region of interest by AMP-Seq^{14, 23}. DNA was purified and sheared using a ME220 focused- ultrasonicator (Covaris, Woburn, MA) and purified with Agencourt AMPure XP beads (Beckman Coulter, Brea, CA). Fragments were end-repaired, A-tailed, and ligated to special adapters. NGS libraries were generated by two rounds of nested PCR using either the negative (Neg_GSP1 and Neg_GSP2) or positive (Pos_GSP1 and Pos_GSP2) primers. Libraries were sequenced on an Illumina MiSeq. Resulting sequences were mapped to the reference genomes in addition to the sequence of the AAV vector used in the study. Edited alleles were characterized and quantified using a custom script as previously reported¹⁴. Table. Primers used to amplify the region of interest for indel% calculation

We carried out an unbiased genome-wide detection of M2PCSK9 off-target sites in the livers of treated mice and NHP using ITR-Seq¹⁵. Liver DNA was purified and sheared using a ME220 focused-ultrasonicator. DNA was end-repaired, A-tailed, and ligated to special adapters as described for AMP-Seq. Using AAV-ITR and adapter- specific primers, we amplified ITR-containing DNA fragments and generated NGS- compatible libraries. We sequenced DNA on a MiSeq and mapped the obtained reads to the reference genome plus the sequence of the AAV vector administered to the animal. We identified off-targets sites from the mapped reads using a custom script as described¹⁵. Statistical analyses AAV9.hPCSK9 editing in mice was analyzed by Wilcoxon rank-sum test comparing each group against AAV8.M2PCSK9. Reduction in rhesus PCSK9 and LDL levels in treated NHP were determined by performing a one-sided one-sample t-test. Indels in selected off-targets were calculated in DNA from liver biopsies taken at day 18 post-AAV and in DNA from PBMCs before treatment. These two values were compared using Fisher's Exact test. For all the analyses, Benjamini–Hochberg procedure was applied to correct for multiple hypothesis testing³⁰. Statistical significance was assessed at the 0.05 level. All the analyses were done using R Statistical Software (version R.4.0.0) Example 2 – The ARCUS nuclease (I-CreI endonuclease, further engineered by Precision BioSciences) recognizes and cuts a 22 bp target sequence in the DNA. Cellular proteins recognize and repair these breaks in the DNA. A consequence of this repair mechanism is the insertions or deletions (indels) of nucleotides in the edited loci, these modifications will affect the expression of the corresponding gene. We, and others, have observed a high percentage of editing in the DNA target region, in both mice and rhesus macaque studies, after adeno-associated viral (AAV) vector-mediated delivery of the ARCUS nuclease. However, sequences similar to the on- target region also were shown to contain indels, indicating off-target activity of the ARCUS nuclease. We hypothesized that a certain level of M2PCSK9 is needed for the on-target editing and that increasing nuclease expression over this threshold results in off-target activity. To reduce the levels of M2PCSK9 expression, we replaced the parental TBG promoter in the AAV constructs with promoters with low transcriptional activity. Our hypothesis is that, in contrast with gene therapy, where high expression of the transgene is desirable, genome editing might require a lower transgene expression, while higher expression will also promote off-target editing. Therefore, the aim of this invention is to reduce the transgene expression by reducing its transcription. This could be achieved by selecting liver-specific promoters with weak transcriptional activity. Selection of candidate promoters was performed by two methods. In the first approach, we identified liver-specific human genes with low RNA expression. We searched the Human Atlas Protein database, using the Consensus transcript expression levels (NX level) as a parameter of the transcriptional activity and we selected genes whose transcription was also enriched on liver. The TBG (thyroid hormone-binding globulin) promoter has been shown to be useful for AAV-mediated delivery of transgenes to the liver. We selected three genes with decreasing NX levels that were also enriched in liver. We obtained the promoter region for these genes from SwitchGear Genomics (Carlsbad, CA).

Table – Weak promoter characteristics

1 Consensus normalized expression (NX) from Human Protein Atlas available from http://www.proteinatlas.org. * Data for parental TBG (without enhancers) For our second approach, we aimed to reduce the transcriptional activity of the TBG-S1 promoter, a smaller (176 bp) version of the TBG promoter, by shortening its sequence. Starting from the upstream region, we remove increasing lengths of this sequence, the resulting promoters TBG-S1-F140 (F140), TBG-S1-F113 (F113), and TBG-S1-F64 (F64), contained 140, 113, or 64 bp of the TBG-S1 promoter respectively. AAV serotype 8 vectors, in which the expression of the ARCUS nuclease, specific for PCSK9, is mediated by one of these six weak promoters, were produced. A schematic representation of the genome of these AAVs is shown in FIG.1B. The following vectors were produced: a) AAV8.CCL16-1k.ARCUS2.bGH b) AAV8.CYP26A1-1k.ARCUS2.bGH c) AAV8.SLC22A9-1k.ARCUS2.bGH d) AAV8.TBG-S1-F64.ARCUS2.bGH e) AAV8.TBG-S1-F113.ARCUS2.bGH f) AAV8.TBG-S1-F140.ARCUS2.bGH An initial test was performed in mice. Briefly, mice were administered with AAV expressing human PCSK9, two weeks later, mice received a second injection of AAV expressing the PCSK9-specific ARCUS nuclease under the different weak promoters. As a positive control we used a construct in which the nuclease expression is mediated by the TBG promoter. At week 2 and 7 post administration of the second vector, mice were euthanized, and liver collected for further analysis (FIG.1A) The levels of indels in the region corresponding to the target sequence of the ARCUS nuclease were quantified by a next-generation sequencing assay (FIG.2A, 2B). The results show that in two of the weak promoters groups (TBG-S1-F113 and TBG-S1- F140) the indel percentage was around 40% at week 7 post-nuclease administration, indicating that the on-target activity is retained. In the rest of the groups the on-target activity was lower than 10%, except for the TBG control group in which the editing was between 60-70% (FIG.2A and FIG.2B - linear and logarithmic scales, respectively). FIG.2C shows average levels of recombinant PCSK9 in serum, determined by an ELISA assay, per treated group. The number of off-target loci in the genomic DNA as a result of the nuclease activity was determined using an NGS-based method called ITR-Seq. The publication Breton et al, ITR-Seq, a next-generation sequencing assay, identifies genome-wide DNA editing sites in vivo following adeno-associated viral vector-mediated genome editing, BMC Genomics, (2020):21:239 is incorporated herein by reference in its entirety. There is a reduction in the number of off-target loci for all the weak promoter groups compared to the TBG control in which the number of off-targets was around 160 (FIG.3). A more quantitative approach to measure the off-target activity of these vectors was to calculate indels in a subset of the identified off-targets. The analysis was only performed in TBG control, TBG-S1-F113 and TBG-S1-F140 groups, as this showed the highest indel%. FIG.4 shows the indels in a set of genomic locations corresponding to the identified off-targets. Indel levels for each off-target are shown relative to the indels levels in TBG control group (arbitrary value of 1). There was an approximately 20-fold reduction in the indels in the analyzed weak promoter groups, indicating that the use of these promoters clearly reduces the nuclease off-target activity. hPCSK9 levels in the injected mice are shown in FIG.5. Overall, these results show that the use of weak liver-specific promoters to mediate the expression of genome editing nucleases is a promising strategy to reduce their off-target activity while retaining on-target activity. Example 3 - Increasing the specificity of AAV-based gene editing through self-targeting and short promoter strategies We previously characterized the use of M2PCSK9—an engineered I-Cre-I meganuclease targeting a conserved DNA sequence (also referred to as ARCUS)—in rhesus and human PCSK9¹⁴. We found that in rhesus macaques, a single intravenous injection of an adeno-associated viral vector (AAV) expressing M2PCSK9 reduced circulating PCSK9 levels in a sustained manner¹⁴. At the molecular level, we observed a dose-dependent formation of indels at the target site. However, a genome-wide analysis of the off-target activity of M2PCSK9 in treated animals revealed that the nuclease also edited other genomic sequences that were homologous to the intended target sequence^14, ¹⁵. It is essential to minimize or eliminate the nuclease off-target activity because off- target editing can disrupt genes that are critical for cell viability or controlling cell growth¹⁶. In this study, we developed a clinically relevant strategy to reduce M2PCSK9 off-target activity and increase its safety profile without impacting its efficacy. Although one can engineer the meganuclease amino acid sequence to enhance its specificity^{14, 15}, this approach is limited. We hypothesized that only a low level of M2PCSK9 nuclease is required to induce editing at the target sequence. Increasing the intracellular nuclease concentration beyond this minimal threshold may increase off- target activity. Thus, our strategy was to reduce cellular nuclease accumulation to reduce off-target activity. We tested two main approaches. The first was a ‘self-targeting’ approach in which the M2PCSK9 target sequence was inserted in the same AAV genome expressing the nuclease, to reduce or prevent further transgene expression as a consequence of the self-induced double strand breaks and/or indels in this target region. An additional ‘self-targeting’ approach consisted of fusing the M2PCSK9 nuclease sequence to a PEST domain [a small peptide rich in proline (P), glutamic acid (E), serine (S), and threonine (T)] which targets the associated protein for degradation^17-19. The second ‘short promoter’ approach entailed reducing M2PCSK9 transcription by replacing the highly active, liver-specific human thyroid hormone-binding globulin (TBG) promoter in the parental AAV¹⁴ with shortened versions of TBG. We evaluated the specificity of M2PCSK9 expressed through AAV containing these regulatory elements in both mice and non-human primates (NHPs). Compared to the parental AAV, we observed reduced M2PCSK9 off-target activity in animals administered with these novel AAV vectors, while the on-target activity was largely conserved. Constructing self-targeting and short promoter AAVs with in vivo editing capability In order to obtain the plasmid pAAV.M2PCSK9, we first modified all subsequent plasmids by removing the WPRE sequence in the parental plasmid pAAV.TBG.M2PCSK9.WPRE.BGH¹⁴ (FIG.6A), as this non-coding sequence can lead to a six- to eight-fold increase in the expression of a transgene^{20, 21}. To generate ‘self- targeting’ AAV vectors, we inserted the M2PCSK9 target sequence (pAAV.Target.M2PCSK9) immediately after the promoter sequence in pAAV.M2PCSK9. To investigate if the self-targeting activity can be modulated, instead of the M2PCSK9 target sequence, we inserted a mutant target sequence containing eight mismatching nucleotides into the vector genome (pAAV.MutTarget.M2PCSK9). To mediate the expression of a M2PCSK9 nuclease with a reduced half-life, we cloned the PEST sequence in-frame with the carboxyl terminal region of the nuclease (pAAV.M2PCSK9+PEST). Additionally, we generated AAV plasmids that contained a combination of target and PEST sequences (pAAV.Target.M2PCSK9+PEST, pAAV.2xTarget.M2PCSK9+PEST, and pAAV.MutTarget.M2PCKS9+PEST) to investigate whether we can obtain an additive or synergistic effect for improving M2PCSK9 specificity. We employed a parallel strategy to create shortened versions of the parental TBG promoter to reduce nuclease expression. We constructed two short-promoter AAV plasmids (pAAV.TBG-S1-F113.M2PCSK9 and pAAV.TBG-S1-F140.M2PCSK9) containing only the last 113 and 140 base pairs (bp) of the 3’ end of the TBG promoter. Using these plasmids, we produced AAV vectors and obtained similar AAV titers (below), indicating these modifications did not negatively affect the vector production process. Table. Titers of the produced AAV vectors

Given that the M2PCSK9 target sequence is conserved in the rhesus and human genomes but absent from the mouse genome, we tested the in vivo editing efficacy of these novel self-targeting and short-promoter AAVs in a pseudo-murine model of human PCSK9. To generate the pseudo-murine model, we injected immune-deficient Rag1 knockout mice with 3.5x10¹⁰ GC/mouse of AAV expressing human PCSK9 (AAV9.hPCSK9). We then investigated whether M2PCSK9 activity can reduce circulating levels of hPCSK9, which would be indicative of the on-target editing in AAV9.hPCSK9. Two weeks after the AAV9.hPCSK9 injection, mice were treated with 10¹¹ GC/mouse of the different M2PCSK9-expressing AAV. We collected serum samples at different time points after vector administration and quantified hPCSK9 levels by a PCSK9-specific enzyme-linked immunosorbent assay (ELISA). Administering the parental AAV8.M2PCSK9 rapidly reduced hPCSK9 in two weeks (four weeks post- AAV9.hPCSK9); moreover, circulating hPCSK9 levels dropped to less than 30% of baseline (FIG.6B). We observed reduced hPCSK9 in the other groups as well, although the kinetics were slower than AAV8.M2PCSK9. The short promoter AAV (i.e., AAV8.TBG-S1-F113 and AAV8.TBG-S1-F140.M2PCSK9) and the self-targeting AAV8.2xTarget.M2PCSK9+PEST induced the slowest reduction, as they required seven weeks (nine weeks post-AAV9.hPCSK9) to achieve an hPCSK9 reduction to 30% of baseline (FIG.6B) Novel AAV retains on-target activity for M2PCSK9 Next, we investigated whether the different kinetics of hPCSK9 reduction reflect slower editing activity of M2PCSK9 when it was expressed through the novel AAV. DNA was isolated from livers collected at four or nine weeks post-vector administration. We PCR-amplified a region encompassing the nuclease target site in the AAV9.hPCSK9 vector. Using next-generation sequencing and a custom script, we determined the percentage of amplicons containing indels (indel%). The on-target indel% induced by AAV8.M2PCSK9 was, on average, 43% and 67% at four weeks and nine weeks post- AAV9.hPCSK9 administration, respectively (FIG.7A). We observed a similar indel% at four and nine weeks in the rest of the AAV-treated groups. However, the groups treated with AAV8.2xTarget.M2PCSK9+PEST and the short promoter-AAV presented the lowest editing activity (average indel% of 18% and 41% at four and nine weeks post- AAV, respectively). We also investigated if expressing M2PCSK9 through an even shorter TBG promoter than TBG-S1-F113 (i.e., the last 64 bp of the TBG promoter) still mediated on-target editing. At 9 weeks post-AAV, the average indel% in AAV9.hPCSK9 was 2.5%, which is approximately ~16-fold lower than the average on- target indel% obtained in AAV8.TBG-S1-F113 and TBG-S1-F140.M2PCSK9-treated groups (FIG.8). Altogether these data indicate that all the AAV retained on-target activity with varying editing kinetics. To investigate if our self-targeting AAVs—which contain the M2PCSK9 target sequence—were recognized and edited by the nuclease, we calculated the indel% in these regions using the PCR-based method described above (FIG.7B). We observed evidence of editing in the target regions present before (5’ Target) and after (3’ Target) the M2PCSK9 transgene. Whereas indel% was ~60% in the 5’ Target in all the target- containing AAV, editing was only ~13% in the 3’ Target, suggesting a nuclease editing preference. The mutant target sequence showed lower levels of more variable editing, in which the indel% was, at week 9 post-AAV9.hPCSK9, less than 1% in the AAV8.MutTarget.M2PCSK9 group and between 0.39% to 28.7% for the AAV8.MutTarget.M2PCSK9+PEST group (FIG.7B). Evaluating off-target activities of self-targeting and short-promoter AAVs in mice Having characterized the on-target activity, we sought to identify differences in the off-target activity of the expressed M2PCSK9. We performed an unbiased, genome- wide analysis of AAV-treated liver DNA samples using a next-generation sequencing (NGS)-based technique known as ITR-Seq¹⁵. Using this method, we identified an average of 161 different M2PCSK9-edited off-target loci in mice treated with AAV8.M2PCSK9 at nine weeks post-AAV9.hPCSK9 (FIG.7C). In contrast, there was a ~6-fold reduction in off-targets in the remaining mice treated with AAV at four and nine weeks post-AAV9.hPCSK9 administration (FIG.7C). We observed only a minimal reduction in the number of off-targets in the AAV8.MutTarget.M2PCSK9-treated group (130 off-targets at week nine), suggesting that the mutant target sequence by itself is not enough to reduce the nuclease off-target activity. We performed a more quantitative analysis of these off-targets by analyzing a subset of high-rank off-targets from the ITR- Seq results. We used specific primers to amplify the corresponding off-target genomic location and calculated the indel% using an NGS analysis of amplicons (FIG.7D). Compared to AAV8.M2PCSK9, the mice treated with AAV8.MutTarget.M2PCSK9 exhibited a 25% reduction in the average off-target indel% (FIG.7D). There was approximately a nine-fold reduction in the off-target indel% in the AAV self-targeting group and a ~20-fold reduction in off-target editing in mice treated with the short- promoter AAV (i.e., AAV8.TBG-S1-F113. and AAV8.TBG-S1-F140.M2PCSK9, see FIG.2D). These data indicate a marked reduction in nuclease off-target activity in vivo when nuclease expression is mediated by our novel self-targeting and shortened promoter AAV. Retaining M2PCSK9 on-target editing activity in the novel AAV in non-human primates Encouraged by the data in the pseudo-murine model of human PCSK9, we decided to evaluate the genome editing activity of some of these AAVs in NHP. Of importance, at the time of writing, the in vivo study was still ongoing for most of the treated NHP. We selected AAV8.Target.M2PCSK9, AAV8.MutTarget.M2PCSK9+PEST, and AAV8.TBG-S1-F113.M2PCSK9 as they exhibited high on-target and low off-target editing activities; AAV8.M2PCSK9 served as a control. Using previously described methods¹³, we intravenously administered rhesus macaques with a single dose of 6x10¹² GC/kg of each AAV, or a higher dose (3x10¹³ GC/kg) of AAV8.MutTarget.M2PCSK9+PEST. A similar extent of liver transduction was observed in all treated NHPs, as we detected comparable numbers of AAV genome copies per diploid cell in liver biopsies obtained at d18 (FIG.9). M2PCSK9 RNA copies were similar among the groups at d18 and d128; by d128 the M2PCSK9 RNA levels decreased for all groups as shown by two detection methods, qPCR and in situ hybridization. In situ hybridization was performed using specific probes to detect M2PCSK9 RNA along with DAPI nuclei staining in liver biopsies samples taken at the indicated time points. (Data not shown). Blood samples were routinely collected from all animals and liver biopsies were collected on days 18 and 128 post-AAV administration. We first evaluated the editing activity of these AAV by measuring the levels of circulating PCSK9. We compared the average circulating PCSK9 levels for each treated NHP, starting from day 56 up to the latest measurement, to the average PCSK9 levels before AAV dosing. There was a significant reduction in PCSK9 to 40-76% of baseline values in the AAV8.M2PCSK9 and AAV8.Target.M2PCSK9 groups (FIG.10). The higher dose (3x10¹³ GC/kg) of AAV8.MutTarget.M2PCSK9+PEST induced a reduction in PCSK9 (47% of baseline), while the 6x10¹² GC/kg dose did not result in a significant PCSK9 reduction. AAV8.TBG-S1-F113.M2PCSK9 reduced PCSK9 to an average level of 49% of baseline after d56. We investigated if the nuclease-mediated PCSK9 inhibition reduced LDL cholesterol in treated NHP. The AAV8.M2PCSK9-treated group showed a small (average of 89% of baseline) reduction in LDL; two NHP (number 180712 and 181289) exhibited a statistically significant reduction in LDL (84% of baseline. Despite the non- significant PCSK9 reduction in the AAV8.MutTarget.M2PCSK9+PEST group, the 6x10¹² GC/kg dose led to a reduction in LDL to 82% of baseline. Meanwhile, a five-fold higher dose of this AAV reduced LDL to 61% of baseline. Both reductions were statistically significant (p<0.05, one-sided one-sample t-test). In NHP treated with AAV8.TBG-S1-F113.M2PCSK9, at a dose of 6x10¹² GC/kg, LDL reached 64% and 74% of baseline, which was the lowest level compared to the other AAV-treated NHP at the dose of 6x10¹² GC/kg. We performed a detailed, molecular-level analysis of the editing in the M2PCSK9 target region using AMP-Seq, an NGS method capable of detecting small and large insertions and deletions as well as translocations derived from the editing activity of the nuclease^{14, 22}. Analysis of liver biopsies at day 18 showed a similar editing level between 15-43% in all of the NHP treated with a 6x10¹² GC/kg dose. As expected, a higher dose of AAV8.MutTarget.M2PCSK9+PEST resulted in an increased indel percentage (41%) on d18 (FIG.11A). The most common type of editing in the target region in all of the treated NHP was integration of sequences derived from the AAV; this type of editing constituted approximately two-thirds of the total indel% at this time point (FIG.11A). In all of the treated NHP, the percentage of translocation in the on-target region, was less than 0.03%. By d128, we observed a reduction of approximately 50% in the on-target editing levels; this was mostly due to a reduction in insertions of sequences matching the AAV vector (ITR integrations, FIG.11A). M2PCSK9 off-target activity is reduced in animals treated with self-targeting or short- promoter AAVs We used ITR-Seq to test if the reduction in the meganuclease off-target activity observed in mice was also present in NHP (FIG.11B). As expected, the AAV8.M2PCSK9-treated group showed the highest number of off-targets (average=131, n=3) on day 18. In NHP treated with AAV8.MutTarget.M2PCSK9+PEST, AAV8.Target.M2PCSK9, and AAV8.TBG-S1-F113.M2PCSK9, at a dose of 6x10¹² GC/kg, we observed a reduction in the number of off-targets. The greatest reduction in off-targets was in the AAV8.TBG-S1-F113.M2PCSK9 group, where the average number of detected off-targets was six-fold lower than those in the AAV8.M2PCSK9 group at day 18 post-AAV (FIG.11B). However, by d128, most of the off-targets were no longer detectable in liver biopsies from all the treated NHP; we identified a maximum of 14 off- targets in all the tested NHP at d128 (FIG.11B). In addition to characterizing the nuclease off-target activity in vivo, we also quantified the indel% in a subset of off- targets at d18. From the list of identified off-targets in the treated NHP, we selected a subset of M2PCSK9 off-targets previously identified by GUIDE-Seq in vitro^{14, 23}. We calculated the indel% in amplicons generated from the genomic location of this subset of off-targets (FIG.12). The calculated indel in the identified off-target region at d18 was statistically different from untreated cells for some of the selected off-targets (Pre vs d18). While the indel% in the off-target region was on average 27% at d18, the indel% in the analyzed off-targets was lower than 1% in almost all the cases. Immune responses of treated NHP to AAV Given that we detected T cells against M2PCSK9-derived peptides in our previous NHP study¹³, we investigated if there was a similar response in these NHPs as the nuclease expression levels differ between the self-targeting and short-promoter AAV. We used an IFN-γ ELISPOT assay to evaluate peripheral blood mononuclear cells (PBMCs) isolated before or on different days post-AAV using pools of peptides derived from the amino acid sequence of the AAV8 capsid or M2PCSK9. When assayed for peptides derived from the AAV capsid, lymphocytes taken at different time points post- AAV remained mostly negative for T-cell activation (FIG.13A, FIG.13C, FIG.13E, and FIG.13G). In contrast, there was a significant activation of T cells in response to M2PCSK9 in lymphocytes collected at different time points post-AAV administration. In two AAV8.M2PCSK9-treated NHP, this T-cell activation remained positive in all the assayed time points (FIG.13B). Interestingly, in the NHP treated with AAV8.MutTarget.M2PCSK9+PEST at a dose of 6x10¹² GC/kg, there was T-cell activation in response to the meganuclease peptide pool in PBMCs collected at day 56, but not at later time points (FIG.13D). We observed a similar momentary response in one NHP treated with AAV8.TBG-S1-F113.M2PCSK9 (FIG.13H). We also quantified the levels of liver transaminases after AAV administration. One of the NHP treated with AAV8.M2PCSK9 presented a maximum elevation of alanine aminotransferase (ALT) of 1112 U/L while the other two NHP exhibited a maximum ALT elevation of 216 and 162 U/L. On the other hand, AAV8.TBG-S1- F113.M2PCSK9 induced a more modest ALT elevation with a maximum of 39 and 125 U/L on days 98 and 57 post-AAV, respectively (FIG.14). Aspartate aminotransferase (AST) elevation was similar in the treated animals. Only the AAV8.M2PCSK9-treated NHP—with the highest ALT elevation—exhibited AST levels higher than 300 U/L (FIG. 14). We have successfully increased the specificity of M2PCSK9 by mediating its expression through self-targeting and short-promoter AAV vectors. Using a pseudo- murine model of hPCSK9 and NHPs, we showed that all the tested AAV vectors mediated expression of the M2PCSK9 nuclease with similar on-target activity and relatively low off-target activity. Given that our approach is based on the regulatory elements presented in the AAV genome and not in modifying the nuclease-coding sequence, we believe that these strategies could be applied to meganucleases targeting other genes. Other groups have used similar strategies to increase the specificity of different nucleases. For instance, multiple research groups achieved transient Cas9 expression in self-targeting lentivirus²⁴ and AAV^{25, 26} by including additional guide RNA in the vectors to target and disrupt the Cas9 transgene. Similar to our strategy, this self- targeting AAV-Cas9 system presented on-target activity while reducing the off-target activity. While the self-targeting editing decreased Cas9 expression, the number of AAV GC did not decrease. Similarly, in our NHP studies, we did not observe a decrease in the number of AAV GC for AAV8.Target.M2PCSK9, compared to AAV8.M2PCSK9 (FIG. 9). Therefore, the reduction in M2PCSK9 off-target activity in the self-targeting AAV is most likely through a mechanism other than a reduction in M2PCSK9 DNA/RNA levels. M2PCSK9 recognized and edited the target sequence in the AAV, given that we detected indels in this region. However, our PCR-based method only detects small indels, which suggests that we may be missing large insertions/deletions in the vector or in the transcribed RNA that could result in a decrease in translation. In order to elucidate the mechanisms that reduce off-target activity of the nuclease, we need to undertake additional experiments like performing a full-sequencing of M2PCSK9 transcripts and episomal AAV genomes, which can help detect large insertions/deletions. One important part of our self-targeting approach was the insertion of a PEST sequence. This peptide has been fused to reporter genes to increase their turnover¹⁸ and, as such, was the ideal candidate to directly mediate the reduction in intracellular levels of M2PCSK9. The M2PCSK9+PEST fusion protein reduced off-target activity in mice and NHP. There was a low, intermittent T-cell response to the PEST sequence in NHP treated with AAV8.MutTarget.M2PCSK9+PEST at a dose of 6x10¹² and 3x10¹³ GC/kg (Pool C, FIG.13D). While the coding sequence is less than 150 bp, length might be an important factor to consider if a similar approach is used for AAV that encode larger nucleases like SaCas9. In the short-promoter approach, the TBG promoter length was reduced from ~700 bp to only 113 bp (FIG.6A). This reduction in the recombinant genome size is of special interest when AAV is used as an expression vector. While the TBG was shortened to arbitrarily chosen lengths, the minimal promoter size for a functional TBG promoter seems to be close to this length, since an AAV expressing M2PCSK9 through a shortened TBG promoter (64 bp) presented an on-target editing of only 2.5% at nine weeks post-AAV (FIG.8). Nevertheless, compared to the full-length TBG, the transcriptional activity does not seem to be lower for the shortened TBG promoters TBG-S1-F113 and -F140. Indeed, all the AAV-treated NHP in our study presented similar M2PCSK9 RNA levels at day 18 (FIG.9). Interestingly, while the M2PCSK9 RNA levels were similar for all groups, the number of identified off-targets was higher for the AAV8.M2PCSK9 group than the short-promoter group (AAV8.TBG-S1- F113.M2PCSK9, FIG.11B). As with the AAV8.Target.M2PCSK9 vector, the mechanism for the increased specificity of M2PCSK9 expressed through a short promoter could be related to the sequence of the resulting M2PCSK9 RNA. Elucidating the mechanism for this increased specificity requires characterizing the mRNA produced with full-length and shortened TBG promoters as well as quantifying M2PCSK9 protein at different times post-AAV treatment. Additionally, once the in vivo study concludes, the short-promoter tissue specificity will be determined. However, liver-specific disruption of the PCSK9 gene can still be accomplished using AAV serotypes that target the liver, such as AAV8. This report represents a step forward in safely translating AAV/meganuclease therapy into the clinic. We identified AAV8.TBG-S1-F113.M2PCSK9 as the most promising candidate for clinical studies as it showed on-target activity that mediated PCSK9 and reduced LDL cholesterol while minimizing the nuclease off-target activity, all in stark contrast to the parental AAV8.M2PCSK9 vector. The low elevation in liver transaminases in treated NHPs is an additional important benefit, as minimizing any resulting toxicity represents another important goal in clinical studies. In conclusion, we have developed a set of strategies to increase the specificity of a meganuclease’s action in relevant animal models. Future experiments can help determine if this strategy is applicable to other genome-editing nuclease-based therapies. Additional data is provided for the two macaques that received AAV8.TBG‑S1‑F113.M2PCSK9.BGH. Both animals tolerated vector infusions well (i.e., no apparent clinical sequelae). Analysis of interim data (study is ongoing) revealed that both animals showed efficient and persistent reduction of serum PCSK9 protein and LDL-C levels beginning on Day 7 that were sustained through 294 days (10 months) post treatment (the most recent time point evaluated) (FIG.23A and FIG.23B). Further analysis of these results showed that PCSK9 protein levels were reduced to an average of 59% and 48% of pre-dosing levels (FIG.23C). Analysis of LDL-C levels also indicated a reduction in this biomarker to an average of 76% and 64% of pre-dosing levels (FIG. 23D). Analysis of the full serum lipid profiles of each animal thus far in the study has revealed that high-density lipoprotein cholesterol (HDL-C) and triglyceride levels have remained fairly consistent (FIG.24). Total cholesterol levels experienced a reduction near Day 14 that aligned with the reduction observed in LDL-C levels. Both treated animals showed a brief and minor elevation of liver enzyme levels. Animal 181368 had elevated alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels at 3 months post treatment (Day 98) (FIG.25) and Animal 181373 had increased ALT and AST levels were at around 2 months post treatment (Day 56), along with more minor elevations in alkaline phosphatase (ALP) and gamma-glutamyl transpeptidase GGTP at the same time point. For both animals, the elevations in ALT were 5-10 fold above the baseline levels, while the AST levels were increased to a lower extent. All elevations in liver enzymes were transient and resolved without intervention. ELISpot was performed on PBMCs isolated before vector dosing (Pre), and on Days 56, 112, and 168 post-dosing to measure T cell responses to the AAV8 capsid and M2PCSK9 transgene. A transient T cell response specific to the M2PCSK9 transgene was detected in Animal 181368 on Day 56 and in Animal 181373 on Days 112 and 168 (FIG.26). Vector genome copies (GCs) were measured in the liver through quantitative polymerase chain reaction (qPCR) analysis of genomic DNA and revealed that the GCs measured in the Day 18 liver biopsy samples were 24.5 and 22.2 GC/diploid genome (FIG.28A). Analysis of Day 18 liver samples by RT-PCR (FIG.28B) and by ISH (FIG. 28C) showed expression of M2PCSK9. Day 127 liver biopsy samples from both animals showed significant reductions in vector genome and M2PCSK9 expression levels by qPCR, RT-PCR, and ISH (FIG.28). Analyses of on-target editing in liver biopsy samples collected on Day 18 and Day 127 by amplicon-seq showed two-fold higher on-target editing of the PCSK9 gene in Animal 181373 (18.6% and 15.3% on Day 18 and Day 127, respectively) than in Animal 1811368 (7.0% and 6.4% on Day 18 and Day 127, respectively) (FIG.29A). AMP-seq also detected a significant reduction of on-target editing between the Day 18 and Day 127 biopsies in both animals, which was mainly attributed to the reduction of ITR insertion (FIG.29B). Consistent with amplicon-seq, AMP-seq also detected higher on-target editing in Animal 181373 (34.1% and 14.0% on Day 18 and Day 127, respectively) than in Animal 1811368 (17.9% and 9.9% on Day 18 and Day 127, respectively) (FIG.29B)(Wang et al., 2021). ITR-seq to evaluate the off-target editing was also performed. In the Day 127 liver biopsy samples from each animal, only 7 and 2 off-targets, respectively, were detected by ITR-seq (FIG.30). In both animals, the number of off-target sites in the Day 127 liver biopsy samples were significantly lower than those in the Day 18 biopsy samples. In immune-competent NHPs, cells expressing higher levels of meganuclease might be more likely to be targeted by cytotoxic T cells. The asymptomatic and transient immune response to the meganuclease seen in FIG.26 was useful in extinguishing off- target editing and accounts for the reduction in the number of off-target sites between Day 18 and Day 127. In addition to the ITR-seq analysis, amplicon-seq validation of potential off- target sites predicted by GUIDE-seq was used to evaluate on-target PCSK9 gene editing efficiency and off-target editing observed in this study. Reduced off-target editing of M2PCSK9 was demonstrated through the low indel% recorded at the pre-specified off- target sites (FIG.32). Histopathology analysis on the liver biopsy samples taken from each animal on Day 18 and Day 127 was performed and findings were considered within normal limits. Table. Summary of Histology Findings in Liver Biopsy Samples from Macaques Treated with AAV8.F113.M2PCSK9

Two adult rhesus macaques received a single IV infusion of AAV8.F113.M2PCSK9 (6.0 x 10¹² GC/kg). Liver samples were taken on Day 18 and Day 127 and histopathology findings are summarized. Abbreviations: AAV8, adeno-associated virus serotype 8; GC, genome copies; IV, intravascular. In summary, this study demonstrated the sustained in vivo PCSK9 gene editing efficiency and favorable safety profiles of AAV8.F113.M2PCSK9, a vector that only differs from AAVrh79.TBG-S1-F113.M2PCSK9.BGH in its AAV capsid serotype. Moreover, these data indicate that the incorporation of the shortened TBG-S1-F113 promoter reduced off-target editing of M2PCSK9, which reduces risk of adverse events caused by off-target meganuclease activity. Example 4 – GLP toxicity study A one-year GLP-compliant safety study will be conducted in adult rhesus macaques (approximately 3-10 years of age) to investigate the safety, tolerability, pharmacology, and pharmacokinetics of AAVrh79.TBG-S1-F113.M2PCSK9.BGH following IV administration via a Harvard infusion pump at a flow rate ~1 mL/minute over an approximately ten-minute interval. Interim analyses, including on-target editing, off-target editing, transgene expression, and histopathological analyses, will be performed on Day 28 and Day 120 as these time points will allow sufficient time for the nuclease-dependent editing to have reached stable plateau levels following administration. The study design is outlined in FIG.15. Rhesus macaques will receive one of two dose levels of AAVrh79.TBG-S1-F113.M2PCSK9.BGH (1.0 x 10¹³ GC/kg or 3.0 x 10¹³ GC/kg; N=6 per dose) or vehicle (phosphate-buffered saline [PBS]; N=3). Baseline clinical pathology (cell counts with differentials, clinical chemistries, coagulation and lipid panels) and neutralizing antibodies assays will be performed. After AAVrh79.TBG-S1-F113.M2PCSK9.BGH or vehicle administration, the animals will be monitored daily for signs of distress and abnormal behavior. Blood clinical pathology, including a complete lipid panel, anti-AAVrh79 NAbs and cytotoxic T lymphocyte responses to AAVrh79 and the AAVrh79.TBG-S1- F113.M2PCSK9.BGH transgene product will be assessed by interferon gamma (IFN-γ) enzyme-linked immunospot (ELISpot) assay assessments, which will be performed on a Day 28 following AAVrh79.TBG-S1-F113.M2PCSK9.BGH or vehicle administration, followed by every 4 weeks thereafter. Liver biopsies for AAVrh79.TBG-S1- F113.M2PCSK9.BGH or PBS treated animals will be performed on Days 28 and 120. Liver tissue samples will be evaluated for on-target editing, off-target editing, transgene expression, and histopathological analysis. On Day 28 after AAVrh79.TBG-S1-F113.M2PCSK9.BGH or vehicle administration, cohorts 1-A and 2-A of will be euthanized, and a histopathological analysis will be performed on a comprehensive list of tissues, including, but not limited to, brain, spinal cord, DRG, peripheral nerves, heart, liver, spleen, kidney, lungs, reproductive organs, adrenal glands, and lymph nodes. Organs will be weighed as appropriate. Lymphocytes harvested from the circulating compartment (peripheral blood mononuclear cells), liver, spleen, and bone marrow will be evaluated for the presence of T cells reactive to both the capsid and transgene product. Liver biopsy samples will be 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 shedding, and transgene expression. Samples from all other tissues collected at necropsy and serum for vector biodistribution will be harvested and archived for possible future analysis. In liver samples, biodistribution will be evaluated by PCR and meganuclease RNA expression will be analyzed by RT-PCR will be performed. Urine and feces will be collected for vector shedding analysis by qPCR for vector excretion. Tissues with detectable expression of meganuclease RNA will be evaluated for on-target editing. Tissues with detectable on-target editing will be further evaluated for off-target editing. Due to the limitations of the mouse models, dose will be directly translated from NHPs to humans based on body weight. Doses planned for the NHP toxicology study and the equivalent human doses are shown below.

Example 5 - PCSK9 Gene Editing First-in-Human Study FIG.17A shows a study design for evaluating AAV vector mediated delivery of a PCSK9 meganuclease, such as AAVrh79.TBG-S1-F113.M2PCSK9. The Phase 1/2 First-in-Human (FIH) clinical trial will be an open-label, proof‑of‑concept, dose-escalation study to evaluate the safety, tolerability, pharmacodynamics, and exploratory efficacy of intravenously (IV) administered AAVrh79.TBG-S1-F113.M2PCSK9.BGH in adult subjects with severe, refractory familial hypercholesterolemia (FH). For the purposes of this protocol, a diagnosis of severe, refractory FH is defined as a patient having low density lipoprotein cholesterol (LDL-C) levels of >200 mg/dL in blood at the time of enrollment despite maximal treatment with oral LDL‑C‑lowering therapies. The dose escalation portion of the study (Cohorts 1 and 2) will assess a single IV administration of either of two dose levels of AAVrh79.TBG-S1-F113.M2PCSK9.BGH followed by a subsequent expansion cohort (Cohort 3) that will further assess the maximum tolerated dose (MTD) of the two dose levels tested. The AAVrh79.TBG-S1-F113.M2PCSK9.BGH dose levels will be determined based on data from the NHP toxicology study Example 4, and will consist of a low dose (administered to Cohort 1) and a high dose (administered Cohort 2). Our proposed approach, based on prior experience, is that a safety margin is applied so that the high dose selected for human subjects is 30–50% of the equivalent MTD in NHPs. The low dose is typically 2–3-fold less than the selected high dose. Both dose levels will have the potential to confer therapeutic benefit, with the understanding that, if tolerated, the higher dose is expected to be advantageous. In addition to safety and tolerability, efficacy of AAVrh79.TBG-S1-F113.M2PCSK9.BGH gene editing of PCSK9 and the corresponding changes from baseline for PCSK9 protein and LDL-C in blood will be assessed to evaluate the potential of AAVrh79.TBG-S1-F113.M2PCSK9.BGH to improve or stabilize the symptoms of severe, refractory FH. Because the proposed clinical trial will be the first to assess meganuclease- targeted gene editing in humans, dosing of subjects with AAVrh79.TBG-S1- F113.M2PCSK9.BGH in Cohorts 1 and 2 will be staggered by at least 6 weeks between each subject to allow for monitoring of liver enzymes and evaluation of adverse events (AEs) indicative of immune reactions, immunogenicity, or other dose-limiting toxicities during the interval following dosing and in which maximal gene expression is expected. The 6 week time point is also hypothesized to be sufficient to achieve an initial reduction in PCSK9 protein and LDL-C levels based on interim data which demonstrated an efficient and persistent reduction in serum PCSK9 protein and LDL-C levels beginning on Day 7 in NHPs administered an AAV vector identical to AAVrh79.TBG-S1- F113.M2PCSK9.BGH except for the capsid at a dose of 6.0 x 10¹² GC/kg. Therefore, the 6 week interval between subjects could also potentially provide an initial read out of efficacy; however, this duration between dosing subjects may be further refined based on emerging nonclinical data to either shorten or prolong the interval between subjects in the final protocol. An independent Data Safety Monitoring Board (DSMB) will conduct a safety review of all accumulated safety data between cohorts and after full enrollment of Cohort 2 to make a recommendation regarding further conduct of the clinical trial. The DSMB also conducts a review any time a Study Treatment Stopping Rule is observed (or two subjects have a safety review trigger [SRT]). The DSMB review conducted at the end of Cohorts 1 and 2 will be performed once 6 weeks of safety data from each subject in the respective cohort have been collected and fully analyzed. This data collection will be expedited by the fact that the planned trial will be open-label, and data can therefore be analyzed in real time. If there is a safety event that triggers a DSMB review outside of the specified checkpoints, this interval may be extended such that no new recruitment occurs until after a decision is made by the DSMB. Provided that the DSMB recommends study continuation after completion of Cohort 2, subjects will be enrolled in an expansion cohort (Cohort 3) that will receive the MTD. Enrolment of these additional subjects in Cohort 3 receiving the MTD will not require a 6 week observation window between subjects (with agreement of DSMB review of available data). The secondary efficacy endpoints for this trial will be percent and absolute change from baseline in levels of PCSK9 protein in plasma and levels of LDL-C, in addition to other lipid parameters, in blood assessed at the 6 month time point. The 6 month time point was chosen from nonclinical data showing a stable reduction in LDL-C levels in NHPs by 6 months post treatment with AAVrh79.TBG-S1- F113.M2PCSK9.BGH. Furthermore, all treated subjects will be followed for 1 year as part of the main study to evaluate the safety profile and characterize the pharmacodynamic and efficacy properties of AAVrh79.TBG-S1-F113.M2PCSK9.BGH. Subjects will be followed for an additional 4 years in a long-term follow-up clinical trial extension (for a total of 5 years post dose), to evaluate long-term safety and pharmacodynamics. Thereafter, subjects will be invited to enroll in a long-term registry for an additional 10 years of continued follow-up of long-term outcomes (for a total of 15 years post treatment). This follow-up duration is in line with the recommendation for gene editing per “FDA Guidance for Industry: Long Term Follow-Up after Administration of Human Gene Therapy Products” (January 2020). The protocol synopsis for the proposed Phase 1/2 FIH clinical trial is provided below.

Example 6: Preclinical Evaluation in Rag1-KO Mouse The objective of this non-GLP compliant dose ranging pharmacology study was to evaluate the editing efficiency of AAVrh79.TBG-S1-F113.M2PCSK9 and AAVhu37.TBG-S1-F113.M2PCSK9 and assess the dose response in the murine model episomally expressing the human PCSK9 gene (AAV9.TBG.hPCSK9-transduced adult Rag1^-/- mice). As summarized in FIG.18A, Rag1^-/- mice first received an IV administration of AAV9.TBG.hPCSK9 at a dose of 4.5 x 10¹⁰ GC (1.8 x 10¹² GC/kg) to provide human PCSK9 cDNA episomally as template for M2PCSK9 editing. The serum expression levels of human PCSK9 protein were measured on Days 7, 13, 21, 28, 42, and 56 of the study. On Day 14, mice were IV administered one of three doses (2.0 x 10¹⁰ GC [8.0 x 10¹¹ GC/kg], 1.0 x 10¹¹ GC [4.0 x 10¹² GC/kg], or 5.0 x 10¹¹ GC [2.0 x 10¹³ GC/kg]) of AAVrh79.TBG-S1-F113.M2PCSK9 or AAVhu37.TBG-S1- F113.M2PCSK9. On Day 56 (42 days after administration of the AAVrh79.TBG-S1- F113.M2PCSK9 or AAVhu37.TBG-S1-F113.M2PCSK9 vector), mice were necropsied and their livers were harvested to assess on-target editing activities by amplicon-seq, off- target editing by ITR-seq, and transduction in the liver by ISH. All mice survived until the 56 day study endpoint. Serum PCSK9 protein levels were detected on Day 7 and increased slightly on Day 13. Following administration of either vector, serum PCSK9 protein levels continuously decreased until the end of the study in a dose-dependent manner (FIG .18C). Less robust and delayed reductions in serum PCSK9 protein levels were observed in mice treated with vector at the low dose. On-target indel analysis conducted using amplicon-seq was performed on liver DNA isolated on Day 56. Dose-related indel frequency ranged from 68.6% (high dose), 43.6%, to 13.3% (low dose; FIG.18D). Off-target editing was evaluated by ITR-seq. The number of off-target loci increased in a dose-related manner, with the mean number of loci in each dose group ranging from 88 (high dose), 16, to 7 (low dose; Fig.18D) for AAVrh79.TBG-S1-F113.M2PCSK9.BGH. AAVhu37.TBG-S1-F113.M2PCSK9.BGH treated mice showed similar levels. Transduction of M2PCSK9 in the liver evaluated by ISH also showed dose-related transduction efficiency and that AAVrh79.TBG-S1- F113.M2PCSK9.BGH -treated mice had slightly higher M2PCSK9 expression levels than mice treated with AAVhu37.TBG-S1-F113.M2PCSK9.BGH at similar doses (Fig. 18B). In summary, this study demonstrated that AAVrh79.TBG-S1- F113.M2PCSK9.BGH can efficiently edit human PCSK9 gene in the murine model episomally expressing the human PCSK9 gene in a dose-related manner. On- and off- target editing correlated with vector dose and M2PCSK9 expression levels.

References 1. Arnould, S, Delenda, C, Grizot, S, Desseaux, C, Paques, F, Silva, GH, et al. (2011). The I-CreI meganuclease and its engineered derivatives: applications from cell modification to gene therapy. Protein Eng Des Sel 24: 27-31. 2. Maeder, ML, and Gersbach, CA (2016). Genome-editing Technologies for Gene and Cell Therapy. Mol Ther 24: 430-446. 3. Hsu, PD, Lander, ES, and Zhang, F (2014). Development and applications of CRISPR-Cas9 for genome engineering. Cell 157: 1262-1278. 4. Li, XF, Zhou, YW, Cai, PF, Fu, WC, Wang, JH, Chen, JY, et al. (2019). CRISPR/Cas9 facilitates genomic editing for large-scale functional studies in pluripotent stem cell cultures. Hum Genet 138: 1217-1225. 5. Tyagi, S, Kumar, R, Das, A, Won, SY, and Shukla, P (2020). CRISPR-Cas9 system: A genome-editing tool with endless possibilities. J Biotechnol 319: 36- 53. 6. Doudna, JA (2020). The promise and challenge of therapeutic genome editing. Nature 578: 229-236. 7. Defesche, JC, Gidding, SS, Harada-Shiba, M, Hegele, RA, Santos, RD, and Wierzbicki, AS (2017). Familial hypercholesterolaemia. Nat Rev Dis Primers 3: 17093. 8. Adhyaru, BB, and Jacobson, TA (2018). Safety and efficacy of statin therapy. Nat Rev Cardiol 15: 757-769. 9. Rosenson, RS, Hegele, RA, Fazio, S, and Cannon, CP (2018). The Evolving Future of PCSK9 Inhibitors. J Am Coll Cardiol 72: 314-329. 10. Ding, Q, Strong, A, Patel, KM, Ng, SL, Gosis, BS, Regan, SN, et al. (2014). Permanent alteration of PCSK9 with in vivo CRISPR-Cas9 genome editing. Circ Res 115: 488-492. 11. Carreras, A, Pane, LS, Nitsch, R, Madeyski-Bengtson, K, Porritt, M, Akcakaya, P, et al. (2019). In vivo genome and base editing of a human PCSK9 knock-in hypercholesterolemic mouse model. BMC Biol 17: 4. 12. Ran, FA, Cong, L, Yan, WX, Scott, DA, Gootenberg, JS, Kriz, AJ, et al. (2015). In vivo genome editing using Staphylococcus aureus Cas9. Nature 520: 186-191. 13. Scully, R, Panday, A, Elango, R, and Willis, NA (2019). DNA double-strand break repair-pathway choice in somatic mammalian cells. Nat Rev Mol Cell Biol 20: 698-714. 14. Wang, L, Smith, J, Breton, C, Clark, P, Zhang, J, Ying, L, et al. (2018). Meganuclease targeting of PCSK9 in macaque liver leads to stable reduction in serum cholesterol. Nat Biotechnol 36: 717-725. 15. Breton, C, Clark, PM, Wang, L, Greig, JA, and Wilson, JM (2020). ITR-Seq, a next-generation sequencing assay, identifies genome-wide DNA editing sites in vivo following adeno-associated viral vector-mediated genome editing. BMC Genomics 21: 239. 16. Dai, WJ, Zhu, LY, Yan, ZY, Xu, Y, Wang, QL, and Lu, XJ (2016). CRISPR- Cas9 for in vivo Gene Therapy: Promise and Hurdles. Mol Ther Nucleic Acids 5: e349. 17. Rogers, S, Wells, R, and Rechsteiner, M (1986). Amino acid sequences common to rapidly degraded proteins: the PEST hypothesis. Science 234: 364-368. 18. Li, X, Zhao, X, Fang, Y, Jiang, X, Duong, T, Fan, C, et al. (1998). Generation of destabilized green fluorescent protein as a transcription reporter. J Biol Chem 273: 34970-34975. 19. Ghoda, L, van Daalen Wetters, T, Macrae, M, Ascherman, D, and Coffino, P (1989). Prevention of rapid intracellular degradation of ODC by a carboxyl- terminal truncation. Science 243: 1493-1495. 20. Donello, JE, Loeb, JE, and Hope, TJ (1998). Woodchuck hepatitis virus contains a tripartite posttranscriptional regulatory element. J Virol 72: 5085-5092. 21. Powell, SK, Rivera-Soto, R, and Gray, SJ (2015). Viral expression cassette elements to enhance transgene target specificity and expression in gene therapy. Discov Med 19: 49-57. 22. Zheng, Z, Liebers, M, Zhelyazkova, B, Cao, Y, Panditi, D, Lynch, KD, et al. (2014). Anchored multiplex PCR for targeted next-generation sequencing. Nat Med 20: 1479-1484. 23. Tsai, SQ, Zheng, Z, Nguyen, NT, Liebers, M, Topkar, VV, Thapar, V, et al. (2015). GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat Biotechnol 33: 187-197. 24. Merienne, N, Vachey, G, de Longprez, L, Meunier, C, Zimmer, V, Perriard, G, et al. (2017). The Self-Inactivating KamiCas9 System for the Editing of CNS Disease Genes. Cell Rep 20: 2980-2991. 25. Li, F, Hung, SSC, Mohd Khalid, MKN, Wang, JH, Chrysostomou, V, Wong, VHY, et al. (2019). Utility of Self-Destructing CRISPR/Cas Constructs for Targeted Gene Editing in the Retina. Hum Gene Ther 30: 1349-1360. 26. Li, A, Lee, CM, Hurley, AE, Jarrett, KE, De Giorgi, M, Lu, W, et al. (2019). A Self-Deleting AAV-CRISPR System for In Vivo Genome Editing. Mol Ther Methods Clin Dev 12: 111-122. 27. Gao, G, Lu, Y, Calcedo, R, Grant, RL, Bell, P, Wang, L, et al. (2006). Biology of AAV serotype vectors in liver-directed gene transfer to nonhuman primates. Mol Ther 13: 77-87. 28. Lock, M, Alvira, M, Vandenberghe, LH, Samanta, A, Toelen, J, Debyser, Z, et al. (2010). Rapid, simple, and versatile manufacturing of recombinant adeno- associated viral vectors at scale. Hum Gene Ther 21: 1259-1271. 29. Wang, L, Bell, P, Lin, J, Calcedo, R, Tarantal, AF, and Wilson, JM (2011). AAV8-mediated hepatic gene transfer in infant rhesus monkeys (Macaca mulatta). Mol Ther 19: 2012-2020. 30. Benjamini, Y, and Hochberg, Y (1995). Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing. Journal of the Royal Statistical Society Series B (Methodological) 57: 289-300. All documents cited in this specification, as well as Breton et al, Increasing the Specificity of AAV-Based Gene Editing through Self-Targeting and Short-Promoter Strategies, Mol Ther.2021 Mar 3;29(3):1047-1056. doi: 10.1016/j.ymthe.2020.12.028. Epub 2020 Dec 25. are incorporated herein by reference. US Provisional Patent Application No.63/275,829, filed November 4, 2021is incorporated by reference in its entirety, together with its sequence listing. The sequence listing filed herewith named “22-9879.PCT_Seq-Listing” and the sequences and text therein are incorporated by reference. While the invention has been described with reference to particular embodiments, it will be appreciated that modifications can be made without departing from the spirit of the invention. Such modifications are intended to fall within the scope of the appended claims.

Claims

WHAT IS CLAIMED IS: 1. A recombinant AAV (rAAV) comprising: (a) an AAVrh79 capsid; and (b) a vector genome packaged in the AAVrh79 capsid, said vector genome comprising AAV inverted terminal repeats (ITRs), and an expression cassette comprising a nucleotide sequence that encodes a PCSK9 meganuclease having the amino acid sequence of SEQ ID NO: 16, and regulatory sequences that direct expression of the PCKS9 meganuclease, wherein the expression cassette comprises nt 206 to 1649 of SEQ ID NO: 13, or a sequence sharing at least 70% identity therewith.

2. The rAAV according to claim 1, wherein the vector genome has a sequence sharing at least 80% identity with SEQ ID NO: 13.

3. A pharmaceutical composition comprising an aqueous liquid suitable for intravenous administration and a recombinant AAV (rAAV), said rAAV comprising: (a) an AAVrh79 capsid; and (b) a vector genome packaged in the AAVrh79 capsid, said vector genome comprising AAV inverted terminal repeats (ITRs), and an expression cassette comprising a nucleotide sequence that encodes a PCSK9 meganuclease having the amino acid sequence of SEQ ID NO: 16, and regulatory sequences that direct expression of the PCKS9 meganuclease, wherein the expression cassette comprises nt 206 to 1649 of SEQ ID NO: 13, or a sequence sharing at least 70% identity therewith.

4. The pharmaceutical composition according to claim 4, wherein the vector genome has a sequence sharing at least 80% identity with SEQ ID NO: 13.

5. A method for reducing total serum cholesterol levels in a subject having familial hypercholesterolemia, comprising delivering the recombinant adeno-associated virus (rAAV) according to claim 1 or 2 or the composition according to claim 2 or 4.

6. The method according to claim 5, wherein the subject is at least 35 years old.

7. The method according to claim 5, wherein the subject is less than 76 years old.

8. The method according to any one of claims 5 to 7, wherein the subject has a diagnosis of severe, refractory FH, as defined as LDL-C levels in blood >200 mg/dL, despite maximal treatment with oral LDL-lowering therapies.

9. The method according to any one of claims 5 to 8, wherein the subject has a loss-of-function mutation in either the LDLR gene or APOB gene, a gain-of-function mutation in the PCSK9 gene, and/or a loss-of-function mutation in both alleles of the LDLRAP1 gene.

10. The method according to any one of claims 5 to 9, wherein the subject shows clinical evidence of symptomatic or asymptomatic atherosclerotic disease.