Compositions and methods for in VIVO nuclease-mediated treatment of phenylketonuria (PKU)

Abstract

A dual vector system for treating phenylketonuria is provided. The system includes (a) a gene editing AAV comprising a first AAV rh79 capsid and a first vector genome comprising a 5' ITR, a sequence encoding a meganuclease that targets PCSK9 under control of regulatory sequences that direct expression of the meganuclease in a target cell comprising a PCSK9 gene, and a 3' ITR; and (b) a donor AAV vector comprising a second AAV capsid and a second AAV rh79 vector genome comprising: a 5'ITR, a 5' homology directed recombination (HDR) arm, a transgene encoding phenylalanine hydroxylase (PAH) and regulatory sequences that direct expression of the transgene in the target cell, a 3' HDR arm, and a 3' ITR.

Description

[0001] COMPOSITIONS AND METHODS FOR IN VIVO NUCLEASE-MEDIATED TREATMENT OF PHENYLKETONURIA (PKU)

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit under 35 USC 119(e) of US Provisional Patent Application No. 63 / 631,282, filed April 8, 2024, which is incorporated herein by reference.

[0004] INCORPORA TION-BY-REFERENCE OF MATERIAL SUBMITTED IN ELECTRONIC FORM

[0005] The Contents of the electronic sequence listing (24-10723. PCT Seq-Listing.xml; Size: 36.0kb: and Date of Creation: April 08, 2025) is herein incorporated by reference in its entirety.

[0006] Background of the Invention

[0007] As one of the most common inborn errors of metabolism. Phenylketonuria (PKU) occurs in 1 in 10,000 to 15,000 newborns in the United States. The current treatment approaches require the affected individual to adhere consistently to an unpalatable and expensive dietary restriction and / or take enzy me substitution with phenylalanine ammonia lyase from birth for their whole life.

[0008] The most common cause of PKU is deficiency of phenylalanine hydroxylase (PAH) due to a recessively inherited mutation in the PAH gene. PAH is expressed primarily in the liver that catalyzes the irreversible hydroxylation of phenylalanine to tyrosine. Thus, deficiency in PAH affects the catabolic pathway of phenylalanine, resulting in accumulation of phenylalanine. High plasma phenylalanine levels result in build-up of phenylalanine in the brain and can affect brain development and function, resulting in intellectual disability and seizures. Furthermore, reduction of plasma phenylalanine via dietary restriction and enzy me substitution is expensive, inconvenient and has been linked with various adverse complications, such as persistent mild cognitive deficits.

[0009] An alternative approach to achieve sustained therapeutic levels of PAH is through continuous in vivo production of the native enzyme in the hepatocytes using gene transfer mediated by a cell-directed adeno-associated virus (AAV) or other viral or non- viral vector. Several attempts of vector-mediated PAH expression have been tested preliminary on mouse studies. See, e.g.. Harding et al, Complete correction of hyperphenylalaninemia following liver-directed, recombinant AAV2 / 8 vector mediated gene therapy in murine phenylketonuria Gene Ther. 2006 Mar; 13(5):457-6 and Viecelli et al, Treatment of Phenylketonuria Using Minicircle-Based Naked-DNA Gene Transfer to Murine Liver Hepatology. 2014 Sep; 60(3): 1035-1043, which are incorporated herein by reference. However, the evaluations of delivery efficiency, immune stimulation, longterm expression stability and safety are either lacking or not optimal. Thus, more efficient AAV.hPAH vectors are needed for PKU treatment.

[0010] Meganucleases generate double strand breaks (DSBs) in the chromosome, leading to DNA repair. In the presence of donor DNA, homolog}’ directed repair (HDR) occurs and replaces genetic information in the chromosome with new information from the donor gene.

[0011] Safe harbor sites (SHS) are genomic loci where genes or other genetic elements can be safely inserted and expressed. These SHS are critical for effective human disease gene therapies; for investigating gene structure, function and regulation; and for cell marking and tracking.

[0012] Nuclease-mediated, site-specific integration of a transgene cassette in a safe harbor in the genome would provide long-term therapeutic benefits to patients with PKU.

[0013] What are needed are improved compositions and methods for treatment of PKU.

[0014] Summary of the Invention

[0015] Provided herein are compositions, methods, systems, and kits for treatment of PKU in a subject in need thereof, which allow knockdown or ablation of the native PCSK9 gene and insertion and / or expression of an exogenous PKU transgene in the PCSK9 gene locus.

[0016] In one aspect, a dual vector system for treating phenylketonuria is provided. The system includes (a) a gene editing AAV comprising a first AAV capsid and a first vector genome comprising a 5’ ITR, a sequence encoding a meganuclease that targets PCSK9 under control of regulatory sequences that direct expression of the meganuclease in a target cell comprising a PCSK9 gene, and a 3’ ITR; and (b) a donor AAV vector comprising a second AAV capsid and a second vector genome comprising: a 5’ITR. a 5’ homology directed recombination (HDR) arm, a transgene encoding phenylalanine hydroxylase (PAH) and regulatory sequences that direct expression of the transgene in the target cell, a 3’ HDR arm, and a 3’ ITR. In certain embodiments, the meganuclease is the ARCUS meganuclease having the sequence of SEQ ID NO: 3. In certain embodiments, the sequence encoding a meganuclease comprises nucleotides (nt) 1089- 2183 of SEQ ID NO: 2, or a sequence at least 90% identical to nucleotides (nt) 1089- 2183 of SEQ ID NO: 2. In certain embodiments, the transgene encoding PAH comprises SEQ ID NO: 4, or a sequence at least 90% identical to SEQ ID NO: 4. In certain embodiments, the first and second AAV capsid are AAVrh79 capsids of SEQ ID NO: 16.

[0017] In another aspect, a method of treating PKU in a subject in need thereof is provided. The method includes co-administering to the subject having PKU (a) a gene editing AAV comprising a first AAV capsid and a first vector genome comprising a 5’ ITR. a sequence encoding a meganuclease that targets PCSK9 under control of regulatory sequences that direct expression of the meganuclease in a target cell comprising a PCSK9 gene, and a 3’ ITR; and (b) a donor AAV vector comprising a second AAV capsid and a second vector genome comprising: a 5’ITR, a 5’ homology directed recombination (HDR) arm. a transgene encoding phenylalanine hydroxylase (PAH) and regulatory sequences that direct expression of the transgene in the target cell, a 3’ HDR arm, and a 3’ ITR. In certain embodiments, i) the first vector genome comprises nt 211 to 2964 of SEQ ID NO: 2, or a sequence sharing at least 90% identity with nt 211 to 2964 of SEQ ID NO: 2; and ii) the second vector genome comprises nt 178 to 3281 of SEQ ID NO: 6 or a sequence sharing at least 90% identity with nt 178 to 3281 of SEQ ID NO: 6.

[0018] Other aspects and advantages of the invention will be apparent from the following detailed description of the invention. Brief Description of the Drawings

[0019] FIG. 1: Nuclease vector map.

[0020] FIG. 2: Donor vector map.

[0021] FIG. 3A-3B: Rhesus Macaques study showing efficacy of dual vector approach. FIG. 3A: Table showing dosing and biopsy schedule for each vector dose. FIG. 3B: Bimonthly analysis of liver enzymes alanine transaminase (ASL), aspartate transaminase (AST), and bilirubin.

[0022] FIG. 4: Vector GCs in liver by quantitative PCR analysis and RNA expression analysis of Donor vector, and AAVrh79 vector at Low Dose 1 :3, Mid Dose 1 : 1, and High Dose 1:3.

[0023] FIG. 5: PCSK9 ELISA analysis and insertion-deletion (indel%) analysis of Donor vector, and AAVrh79 vector at Low Dose 1 :3, Mid Dose 1: 1, and High Dose 1 :3

[0024] FIG. 6: Biopsy results of each subject 100 days after vector administration (D100) and 365 days after vector administration (D365).

[0025] FIG. 7: Magnified biopsy results of subjects 22-071 (Donor Only) and 22- 123(1: 1 Mid Dose) at D100 and D365.

[0026] FIG. 8: Quantification summary of Rhesus Macaques study.

[0027] FIG. 9: Project Data summary of Rhesus Macaques study.

[0028] FIG. 10: Full Data summary' of Rhesus Macaques study.

[0029] FIG. 11 : Codon optimized donor vector map.

[0030] FIG. 12: Evaluation of Homology Arm Size on Gene Targeting Efficacy and Integration Characteristics

[0031] Detailed Description of the Invention

[0032] Provided herein are compositions, kits, and methods which provide stable, long term therapeutic effects to patients with certain genetic disorders, including liver metabolic disorders. The compositions, kits, and methods utilize a nuclease that targets the PCSK9 locus of the target cell, and a donor vector provides a template which includes an exogenous product for integration into, and expression from, the PCSK9 locus, wherein the inserted nucleic acid sequence does not encode PCSK9, and the expression of the endogenous PCSK9 is disrupted and expression levels are reduced.

[0033] In one embodiment, the test article described herein is comprised of 2 vectors both using a clade E capsid. AAVrh79. The first vector is the nuclease, ARCUS, and the second is the hPAH donor gene cassette flanked by 500bp arms of homology for PCSK9 exon 7.

[0034] In certain embodiments, the test article is comprised of two non-replicating recombinant adeno associated virus (AAV) rh79 vectors: AAVrh79.TBG.M2PCSK9.WPRE.bGH (nuclease vector) and AAVrh79.hHDR.TBG.hPAHco.bGH (donor vector), which are mixed at a ratio determined by genome copies (GC) just before dosing. As per nonclinical studies, the ratio may be a 1:3 ratio of nuclease vector and donor vector. The test article is, in certain embodiments, administered as a single dose given as intravenous (IV) infusion and dose administered is based on GC / kg of the subject's body weight.

[0035] In certain embodiments, the test article described herein is comprised of two non- replecating recombinant adeno associated virus (AAV) vectors:

[0036] AAVrh79.TBG.M2PCSK9.WPRE.bGH (nuclease vector) and

[0037] AAVrh79.hHDR.TBG.hPAHco5fix.bGH (donor vector), which are mixed at a ratio determined by genome copies (GC) just before dosing. In certain embodiments, the ratio may be a 1:3 ratio of nuclease vector and donor vector. The test article is, in certain embodiments, administered as a single dose given as intravenous (IV) infusion and dose administered is based on GC / kg of the subject's body weight.

[0038] PKU

[0039] Phenylketonuria is an inherited error of metabolism caused predominantly by mutations in the phenylalanine hydroxy lase (PAH) gene. Mutations in the PAH gene result in decreased catalytic activity- affecting the catabolic pathway of phenylalanine (Phe). PAH is a hepatic enzyme that requires the cofactor tetrahydrobiopterin (BH4) to convert Phe to tyrosine (Tyr). A deficiency in PAH or its cofactor BH4 results in the accumulation of excess phenylalanine, whose toxic effects can cause severe and irreversible intellectual disability and other disorders, if untreated. See, Havid and Cristodoulou, Transl Pediatr, 2015 Oct, 4(4):304-17, which is incorporated herein by reference.

[0040] Over 550 mutations of the PAH gene have been described, the majority of which result in deficient enzyme activity. See. Phenylalanine Hydroxylase Locus Knowledgebase, accessed at http: / / www.pahdb.mcgill.ca / , which is incorporated herein by reference. Due to the large number of known PKU mutations, and the autosomal recessive nature of the disease, a wide range of disease severity is seen. The severity of the disease is generally classified by blood phenylalanine levels, which are sometimes classified as classic PKU, moderate or variant PKU, mild PKU, or hyperphenylalaninemia. Based on blood Phe levels at diagnosis, there are 4 levels of PKU severity’.

[0041] • Hyperphenylalaninemia, with Phe levels that are slightly above normal range: 120-600 pmol / L (2-10 mg / dL)

[0042] • Mild, with the lowest blood Phe levels: 600-900 pmol / L (10-15 mg / dL)

[0043] • Moderate or variant, with blood Phe levels somewhere in the middle: 900- 1200 pmol / L (15-20 mg / dL)

[0044] • Severe or “classic’’ PKU, with extremely high blood Phe levels: >1200 pmol / L (20 mg / dL)

[0045] The goal of therapies described herein would provide functional PAH enzyme resulting in Phe levels in the 120-600 pmol / L range, e.g., a 25% or greater reduction in plasma Phe levels. In another embodiment, the vector dose is intended to deliver PAH to result in a reduction of plasma phenylalanine levels by 25% or greater. In another embodiment, the vector dose is intended to deliver PAH to result in a reduction of plasma phenylalanine levels by 30% or greater. In another embodiment, the vector dose is intended to deliver PAH to result in a reduction of plasma phenylalanine levels by 35% or greater. In another embodiment, tire vector dose is intended to deliver PAH to result in a reduction of plasma phenylalanine levels by 40% or greater. In another embodiment, the vector dose is intended to deliver PAH to result in a reduction of plasma phenylalanine levels by 45% or greater. In another embodiment, the vector dose is intended to deliver PAH to result in a reduction of plasma phenylalanine levels by 50% or greater. In another embodiment, the vector dose is intended to deliver PAH to result in a reduction of plasma phenylalanine levels by 60% or greater. In another embodiment, the vector dose is intended to deliver PAH to result in a reduction of plasma phenylalanine levels by 70% or greater. In another embodiment, the vector dose is intended to deliver PAH to result in a reduction of plasma phenylalanine levels by 75% or greater.

[0046] In one embodiment, tire “subject” or “patient” is a mammalian subject having PKU as described above. It is intended that a patient having PKU of any severity is the intended subject.

[0047] In one embodiment, the hPAH gene encodes the hPAH protein shown in SEQ ID NO: 6. Thus, in one embodiment, the hPAH transgene can include, but is not limited to, the sequence provided by SEQ ID NO: 4 or SEQ ID NO: 5 which are provided in tire attached Sequence Listing, which is incorporated by reference herein. SEQ ID NO: 5 provides the cDNA for native human PAH. SEQ ID NO: 4 provides an engineered cDNA for human PAH, which has been, inter alia, codon optimized for expression in humans (sometimes referred to herein as hPAHco). It is to be understood that reference to hPAH herein may, in some embodiments, refer to the hPAH native or codon optimized sequence. Alternatively, or additionally, web-based or commercially available computer programs, as well as service based companies may be used to back translate the amino acid sequences to nucleic acid coding sequences, including both RNA and / or cDNA. See, e.g., backtranseq by EMBOSS, http: / / www.ebi.ac.uk / Tools / st / ; Gene Infinity (http: / / www.geneinfmity.org / sms- / sms backtranslation.html); ExPasy (http: / / www.expasy.org / tools / ). It is intended that all nucleic acids encoding the described hPAH polypeptide sequences are encompassed, including nucleic acid sequences which have been optimized for expression in the desired target subject (e.g., by codon optimization).

[0048] In one embodiment, the nucleic acid sequence encoding hPAH shares at least 95% identity with the native hPAH coding sequence of SEQ ID NO: 5. In another embodiment, the nucleic acid sequence encoding hPAH shares at least 90, 85, 80, 75, 70, or 65% identity with die native hPAH coding sequence of SEQ ID NO: 5. In one embodiment, the nucleic acid sequence encoding hPAH shares about 78% identity with the native hPAH coding sequence of SEQ ID NO: 5.

[0049] In one embodiment, the nucleic acid sequence encoding hPAH is SEQ ID NO: 4. In one embodiment, tire nucleic acid sequence encoding hPAH shares at least 95% identity with the hPAH coding sequence of SEQ ID NO: 4. In another embodiment, the nucleic acid sequence encoding hPAH shares at least 90, 85, 80, 75, 70, or 65% identity with the hPAH coding sequence of SEQ ID NO: 4.

[0050] In one embodiment, the nucleic acid sequence encoding hPAH is SEQ ID NO: 11. In one embodiment, the nucleic acid sequence encoding hPAH shares at least 95% identity with the hPAH coding sequence of SEQ ID NO: 11. In another embodiment, the nucleic acid sequence encoding hPAH shares at least 90, 85, 80. 75. 70, or 65% identity with the hPAH coding sequence of SEQ ID NO: 11.

[0051] In one embodiment, the PAH coding sequence is optimized for expression in the target subject. Codon-optimized coding regions can be designed by various different methods. This optimization may be performed using methods which are available on-line (e.g., GeneArt,), published methods, or a company which provides codon optimizing services, e.g., as DNA2.0 (Menlo Park, CA). One codon optimizing approach is described, e.g., in International Patent Publication No. WO 2015 / 012924, which is incorporated by reference herein. See also, e.g.. US Patent Publication No. 2014 / 0032186 and US Patent Publication No. 2006 / 0136184. Suitably, the entire length of the open reading frame (ORF) for the product is modified. However, in some embodiments, only a fragment of the ORF may be altered. By using one of these methods, one can apply the frequencies to any given polypeptide sequence, and produce a nucleic acid fragment of a codon-optimized coding region which encodes tire polypeptide.

[0052] A number of options are available for performing the actual changes to the codons or for synthesizing the codon-optimized coding regions designed as described herein. Such modifications or synthesis can be performed using standard and routine molecular biological manipulations well known to those of ordinary skill in the art. In one approach, a series of complementary oligonucleotide pairs of 80-90 nucleotides each in length and spanning the length of the desired sequence are synthesized by standard methods. These oligonucleotide pairs are synthesized such that upon annealing, they form double stranded fragments of 80- 90 base pairs, containing cohesive ends, e.g., each oligonucleotide in the pair is synthesized to extend 3, 4, 5, 6. 7, 8, 9, 10, or more bases beyond the region that is complementary to the other oligonucleotide in the pair. The single-stranded ends of each pair of oligonucleotides are designed to anneal with the single-stranded end of another pair of oligonucleotides. The oligonucleotide pairs are allowed to anneal, and approximately five to six of these double- stranded fragments are then allowed to anneal together via the cohesive single stranded ends, and then they ligated together and cloned into a standard bacterial cloning vector, for example, a TOPO® vector available from Thermo Fisher Scientific Inc. The construct is then sequenced by standard methods. Several of these constructs consisting of 5 to 6 fragments of 80 to 90 base pair fragments ligated together, i.e., fragments of about 500 base pairs, are prepared, such that the entire desired sequence is represented in a series of plasmid constructs. The inserts of these plasmids are then cut with appropriate restriction enzymes and ligated together to form the final construct. The final construct is then cloned into a standard bacterial cloning vector, and sequenced. Additional methods would be immediately apparent to the skilled artisan. In addition, gene synthesis is readily available commercially.

[0053] PCSK9

[0054] Proprotein convertase subtilisin kexin 9 (PCSK.9) is a serine protease that reduces both hepatic and extrahepatic low-density lipoprotein (LDL) receptor (LDLR; 606945) levels and increases plasma LDL cholesterol. PCSK9 is critical in the regulation of plasma cholesterol homeostasis. PCSK9 binds to the low-density lipid receptor family members low density lipoprotein receptor (LDLR). very low-density lipoprotein receptor (VLDLR), apolipoprotein E receptor (LRP1 / APOER) and apolipoprotein receptor 2 (LRP8 / APOER2), and promotes their degradation in intracellular acidic compartments.

[0055] While the PCSK9 gene has been targeted for treatment of cholesterol related diseases, it is demonstrated herein that the PSCK.9 gene locus is a safe harbor for gene targeting for insertion of other, non-PCSK9 transgenes. Thus, the compositions, kits, and methods provided herein utilize nucleases which target the PCSK9 gene locus, and insert a therapeutic transgene into the target PCSK9 locus, using a donor template.

[0056] The compositions, kits, and methods provided herein include a gene editing vector, and a donor vector which provides the therapeutic PKU transgene to be expressed in the host cell.

[0057] GENE EDITING COMPONENT

[0058] The compositions, kits, and methods provided herein include a gene editing component that comprises a nuclease (or the coding sequence therefore) and sequences which direct the nuclease to specifically target the native PCSK9 gene locus on chromosome 1. As used herein, the “target PCSK9 locus” or “PCSK9 gene locus” is in Exon 7 of the PCSK9 coding sequence.

[0059] Described herein are compositions, particularly nucleases, which are useful targeting a gene for the insertion of a transgene, for example, nucleases that are specific for PCSK9. In certain embodiments, the nuclease is a meganuclease that targets PCSK9. Meganucleases are endodeoxyribonucleases characterized by a large recognition site (double-stranded DNA sequences of 12 to 40 base pairs), for example, I-Scel. When combined with a nuclease, DNA can be cut at a specific location. The restriction enzy mes can be introduced into cells, for use in gene editing or for genome editing in situ. In certain embodiments, the nuclease is a member of the I-Crel family of homing endonucleases which recognizes and cuts a 22 base pair recognition sequence SEQ ID NO: 1 - CAAAACGTCGTGAGACAGTTTG. See, e.g., WO 2009 / 059195. In one embodiment, the nuclease is encoded by the sequence shown in SEQ ID NO: 2, nt 1089 to 2183, or a sequence sharing at least 95%, 98%, or 99% identity thereto. In one embodiment, the nuclease protein sequence is the sequence shown in SEQ ID NO: 3. Such nuclease is sometimes referred to herein as the ARCUS nuclease. 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.

[0060] In certain embodiments, the compositions, kits, and methods the nuclease coding sequence is comprised in a gene editing vector. The gene editing vector includes an expression cassette comprising a nucleic acid sequence encoding a nuclease and regulatory7sequences that direct expression of the nuclease in a target cell comprising a PCSK9 gene. 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. The vector comprising the nuclease coding sequence is an Adeno- Associated Virus (AAV) vector.

[0061] As used herein, an “expression cassette” refers to a nucleic acid molecule which comprises a biologically useful nucleic acid sequence (e.g., a gene cDNA encoding a protein, enzyme or other useful gene product, mRNA, etc) and regulatory sequences operably linked thereto which direct or modulate transcription, translation, and / or expression of the nucleic acid sequence and its gene product. As used herein, “operably linked’7sequences include both regulatory sequences that are contiguous with the nucleic acid sequence and regulatory sequences that act in trans or at a distance to control the sequence. Such regulatory' sequences ty pically include, e.g., one or more of a promoter, an enhancer, an intron, a Kozak sequence, a poly adenylation sequence, and a TATA signal. The expression cassette may contain regulatory sequences upstream (5’ to) of the gene sequence, e.g., one or more of a promoter, an enhancer, an intron, etc, and one or more of an enhancer, or regulatory sequences downstream (3’ to) a gene sequence, e.g., 3’ untranslated region comprising a polyadenylation site, among other elements. In other embodiments, the term “transgene” refers to one or more DNA sequences from an exogenous source which are inserted into a target cell. Typically, such an expression cassette for generating a viral vector contains the coding sequence for the gene product described herein flanked by packaging signals of the viral genome and other expression control sequences such as those described herein.

[0062] In addition to the coding sequence for the nuclease, the gene editing vector includes regulatory sequences which direct expression of the nuclease in a host cell. The regulatory elements include a promoter, e.g., the liver-specific promoter thyroxin binding globulin (TBG) promoter. In certain embodiments, the TBG promoter has the sequence of nucleotides 211 to 907 of SEQ ID NO: 2, which includes enhancer sequences.

[0063] In addition to a promoter, the gene editing cassette, expression cassette and / or 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 (poly A); sequences that stabilize cytoplasmic mRNA. for example Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE); sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product. In certain embodiments, the vector includes a bovine growth hormone (bGH) poly A, e.g., such as that shown in nucleotides 2750 to 2964 of SEQ ID NO: 2. A suitable enhancer includes the alphal -microglobulin / bikunin enhancer. A suitable WPRE includes that shown in nucleotides 2202 to 2743 of SEQ ID NO: 2. These control sequences or the regulatory sequences are operably linked to the nuclease coding sequence or transgene coding sequence. In certain embodiments, a SV40 intron is included, such as that shown in nucleotides 939 to 1071 of SEQ ID NO: 2.

[0064] In certain embodiments, the nuclease vector genome includes the following components. Inverted Terminal Repeat (ITR): The ITRs are identical, reverse complementary sequences derived from AAV2 (145 base pairs [bp], GenBank: NC_001401) that flank all components of the vector genome. The ITRs function as both the origin of vector DNA replication and the packaging signal for the vector genome when AAV and adenovirus helper functions are provided in trans. As such, the ITR sequences represent the only cis sequences required for vector genome replication and packaging. Human Thyroxine-Binding Globulin (TBG) Promoter: This regulatory element confers tissue specific transgene expression in liver (410 bp, GenBank: L13470.1). Coding Sequence: The transgene is an engineered meganuclease (ARCUS; 1095 bp. 365 amino acids). It is derived from a variant of a homing endonuclease. I-Crel, isolated from Chlamydomonas reinhcirdtii, that recognizes and edits PCSK9 gene with high efficiency and specificity. WPRE (Woodchuck Hepatitis Virus Post-Transcriptional Regulator^' Element): A c / .s-acting RNA element derived from the Woodchuck Hepatitis Virus (WHV) (GenBank: MT612432.1) has been inserted in the 3' untranslated region of the coding sequence upstream of the PolyA signal. The WPRE is a hepadnavirus-derived sequence and has been previously used as a / .s-actmg regulator}' module in viral gene vectors to achieve sufficient levels of transgene product expression and to improve the viral titers during manufacturing. The WPRE is believed to increase transgene product expression by improving transcript termination and enhancing 3’ end transcript processing, thereby increasing the amount of poly adenylated transcripts and the size of the PolyA tail and resulting in more transgene mRNA available for translation. The WPRE included in the vector is a mutated version containing 5 point mutations in the putative promoter region of the woodchuck hepatitis virus X protein (WHX) protein open reading frame (ORF), along with an additional point mutation in the start codon of the WHX protein ORF (ATG mutated to TTG). This mutant WPRE (termed mut6) is considered sufficient to eliminate expression of truncated WHX protein based on sensitive flow cytometry analyses of various human cell lines transduced with lentivirus containing a WPRE mut6-GFP fusion construct (Zanta-Boussif et al.. 2009).

[0065] Bovine Growth Hormone Poly A (bGH Poly A): The bGH PolyA signal (208 bp, GenBank: MT267334) facilitates efficient poly adenylation of the transgene mRNA in cis. This element functions as a signal for transcriptional termination, a specific cleavage event at the 3’ end of the nascent transcript and the addition of a long polyadenyl tail.

[0066] In certain embodiments, the gene editing vector further includes one or more nuclear localization signal (NLSs). See, e.g., Lu et al. Types of nuclear localization signals and mechanisms of protein import into the nucleus, Cell Commun Signal (May 2021) 19:60. In one embodiment, i e vector contains the NLS shown in nt 1095 to 1115 of SEQ ID NO: 2.

[0067] DONOR VECTOR

[0068] The compositions, kits, and methods include a donor vector, which provides the coding sequence for the PKU therapeutic transgene. The donor vector contains an expression cassette comprising a nucleic acid sequence encoding a transgene, and regulatory sequences that direct expression of the transgene in the target cell. The compositions, kits, and methods for nuclease-mediated, site-specific integration of an PKU transgene cassette in a PCSK9 safe harbor in the genome that provides long-term therapeutic benefits to patients with PKU. An engineered, coding sequence for PKU, and shown in SEQ ID NO: 4 is provided. Nucleic acids having the sequence of SEQ ID NO: 4 or sequences sharing at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.9% identity with SEQ ID NO: 4 are provided. Nucleic acids having the sequence of SEQ ID NO: 11 or sequences sharing at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%. at least 98%, at least 99%, or at least 99.9% identity with SEQ ID NO: 11 are provided. In one embodiment, the nucleic acid shares less than 80%, less than 79%, less than 78%, less than 77%, less than 76%, less than 75%, less than 74%, less than 73%, less than 72%, less than 71%, or less than 70% identity with the native PKU coding sequence that is shown in SEQ ID NO: 5.

[0069] In addition to a promoter, the transgene cassette, expression cassette and / or vector (editing or donor) 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 (poly A); sequences that stabilize cytoplasmic mRNA, for example Woodchuck Hepatitis Virus (WHP) Posttranscri phonal 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 / alphal-microglobulin / bikunin enhancer), amongst others. These control sequences or the regulatory- sequences are operably linked to the nuclease coding sequences or transgene coding sequence.

[0070] In certain embodiments, the donor vector genome includes the following: Inverted Terminal Repeat (ITR): The ITRs are identical, reverse complementary sequences derived from AAV2 (145 bp, GenBank: NC_001401) that flank all components of the vector genome. The ITRs function as both the origin of vector DNA replication and the packaging signal for the vector genome when AAV and adenovirus helper functions are provided in trans. As such, the ITR sequences represent the only cis sequences required for vector genome replication and packaging. 5' and 3' Homology Arms: Homology-dependent recombination arms (also referred to as hHDR) consisting of sequences flanking the cleavage site in exon 7 of the endoenous human PCSK9 gene locus. The homology arms comprise the sequence 500 bp upstream (5' homology arm) and 500 bp downstream (3' homology arm) of the ARCUS meganuclease cleavage site in exon 7 of the PCSK9 gene. Alpha mic / bic Enhancer: 2 copies of this element that provides increased expression of the transgene (100 bp). Human thyroxine binding globulin (TBG) promoter: This regulatory- element confers tissue-specific transgene expression in liver (434 bp, GenBank: L13470. 1). Coding Sequence: The transgene is a codon-optimized version of the human PAH gene (1356 bp, 452 amino acids). Bovine growth hormone poly A (BGH Poly A): The bGH Poly A signal (215 bp, GenBank: MT267334) facilitates efficient polyadenylation of the transgene mRNA in cis. This element functions as a signal for transcriptional termination, a specific cleavage event at the 3' end of the nascent transcript and the addition of a long poly adenyl tail.

[0071] In certain embodiments, the donor vector genome includes the following: Inverted Terminal Repeat (ITR): The ITRs are identical, reverse complementary sequences derived from AAV2 (168 bp, GenBank: NC_001401) that flank all components of the vector genome. The ITRs function as both the origin of vector DNA replication and the packaging signal for the vector genome when AAV and adenovirus helper functions are provided in trans. As such, the ITR sequences represent the only cis sequences required for vector genome replication and packaging. 5' and 3' Homology Arms: Homology-dependent recombination arms (also referred to as hHDR) consisting of sequences flanking the cleavage site in exon 7 of the endoenous human PCSK9 gene locus. The homology arms comprise the sequence -500 bp upstream (5' homolog}' arm) and -500 bp downstream (3' homology' arm) of the ARCUS meganuclease cleavage site in exon 7 of the PCSK9 gene. Human thyroxine binding globulin (TBG) promoter: This regulatory element confers tissue-specific transgene expression in liver (477 bp. GenBank: L13470.1). Coding Sequence: The transgene is a codon-optimized version of the human PAH gene (1356 bp, 452 amino acids). Bovine grow th hormone poly A (BGH Poly A): The bGH PolyA signal (208 bp, GenBank: MT267334) facilitates efficient poly adenylation of the transgene mRNA in cis. This element functions as a signal for transcriptional termination, a specific cleavage event at the 3' end of the nascent transcript and the addition of a long polyadenyl tail.

[0072] In addition to the transgene cassette, the donor vector also includes homology- directed recombination (HDR) arms 5’ and 3’ to the transgene cassette, to facilitate homology directed recombination of the transgene into the endogenous genome. The homology arms are directed to the target PCSK.9 locus and can be of varying length. In another embodiment, the HDR arms are about 500bp. The HDR arms ideally share a high level of complementarity with the target PCSK9 locus, although it need not be 100% complementarity. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16. 17. 18. 19, 20, or more mismatches are permitted in each HDR arm. In one embodiment, the HDR arm sequences are those shown in SEQ ID NO: 7, nt 178-677 and nt 3070-3569. In another embodiment, the HDR arm sequences are those shown in SEQ ID NO: 10, nt 178-678 and nt 3017-3517.

[0073] In certain embodiments, the donor vector has a vector genome as shown in SEQ ID NO: 7 or 10. In other embodiments, the vector genome has a sequence sharing at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.9% identity with SEQ ID NO: 7 or 10. In one embodiment, the nucleic acid shares less than 80%. less than 79%, less than 78%, less than 77%, less than 76%, less than 75%. less than 74%, less than 73%, less than 72%, less than 71%, or less than 70% identity with SEQ ID NO: 7 or 10. AAV Viral Vectors

[0074] The gene editing vector and the donor vector are provided as a recombinant AAV. 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” or “AAV vecor”. 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.

[0075] 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.

[0076] 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.

[0077] The source of the AAV capsid for both the donor and nuclease vectors is AAVrh79, 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 vpl 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: 9. Optionally, sequences co-expressing the vp3 protein from a nucleic acid sequence excluding the vpl-unique region (about aa 1 to 137) or the vp2-unique region (about aa 1 to 203), vpl proteins produced from SEQ ID NO: 8, or vpl proteins produced from a nucleic acid sequence at least 70% identical to SEQ ID NO: 8 which encodes the predicted amino acid sequence of 1 to 738 of SEQ ID NO: 9. 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: 9, vp2 proteins produced from a sequence comprising at least nucleotides 412 to 2214 of SEQ ID NO: 8, or vp2 proteins produced from a nucleic acid sequence at least 70% identical to at least nucleotides 412 to 2214 of SEQ ID NO: 8 which encodes the predicted amino acid sequence of at least about amino acids 138 to 738 of SEQ ID NO: 9, 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: 9, vp3 proteins produced from a sequence comprising at least nucleotides 610 to 2214 of SEQ ID NO: 8, or vp3 proteins produced from a nucleic acid sequence at least 70% identical to at least nucleotides 610 to 2214 of SEQ ID NO: 8 which encodes the predicted amino acid sequence of at least about amino acids 204 to 738 of SEQ ID NO: 9.

[0078] In certain embodiments, an AAVrh79 capsid comprises: a heterogeneous population of vpl proteins which are the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 9, 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: 9, 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: 9.

[0079] The AAVrh79 vpl, 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: 9 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: 9. 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, ~S 153, -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 1 - AAVrh79 Deamidation

[0080] 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.

[0081] Residue numbers are based on the AAVrh79 sequence provided herein. See, SEQ ID NO: 9.

[0082] In certain embodiments, the nucleic acid sequence encoding the AAVrh79 vpl capsid protein is provided in SEQ ID NO: 8. In other embodiments, a nucleic acid sequence of 70% to 99.9% identity to SEQ ID NO: 8 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: 8. However, other nucleic acid sequences which encode the amino acid sequence of SEQ ID NO: 9 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: 8 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: 8 which encodes SEQ ID NO: 9. In certain embodiments, the nucleic acid sequence has the nucleic acid sequence of SEQ ID NO: 8 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: 8 which encodes the vp2 capsid protein (about aa 138 to 738) of SEQ ID NO: 9. In certain embodiments, the nucleic acid sequence has the nucleic acid sequence of about nt 610 to about nt 2214 of SEQ ID NO: 8 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: 8 which encodes the vp3 capsid protein (about aa 204 to 738) of SEQ ID NO: 9.

[0083] 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 vpl proteins, AAVrh79 vp2 proteins, and AAVrh79 vp3 proteins selected from: vpl proteins produced by expression from a nucleic acid sequence which encodes the predicted amino acid sequence of 1 to 738 of SEQ ID NO: 9. vpl proteins produced from SEQ ID NO: 8, or vpl proteins produced from a nucleic acid sequence at least 70% to 100% identical to SEQ ID NO: 8 which encodes the predicted amino acid sequence of 1 to 738 of SEQ ID NO: 9, 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: 9, vp2 proteins produced from a sequence comprising at least nucleotides 412 to 2214 of SEQ ID NO: 8, 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: 8 which encodes the predicted amino acid sequence of at least about amino acids 138 to 738 of SEQ ID NO: 9, 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: 9, vp3 proteins produced from a sequence comprising at least nucleotides 610 to 2214 of SEQ ID NO: 8, or vp3 proteins produced from a nucleic acid sequence at least 70% identical to at least nucleotides 610 to 2214 of SEQ ID NO: 8 which encodes the predicted amino acid sequence of at least about amino acids 204 to 738 of SEQ ID NO: 9. In certain embodiments, the rAAVrh79 capsid is characterized by a heterogeneous population of AAVrh79 vpl 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: 9. 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: 9, 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: 9. In certain embodiments, the AAVrh79 capsid is characterized by AAVrh79 vplproteins, vp2 proteins and vp3 proteins which comprise heterogenous populations relative to amino acids 1 to 738 (vpl). 138 to 738 (vp2). and 204 to 738 (vp3), respectively, of SEQ ID NO: 9, wherein: the heterogenous population of AAVrh79 vpl, 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: 9 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: 9, 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: 9, and as measured using mass spectrometry. Other suitable techniques for measuring deamidation or other post- translational modifications may be selected.

[0084] In certain embodiments, the rAAV79 capsid comprises AAVrh79 VP1 proteins having about 80 to 85% deamidation at position N57 of SEQ ID NO: 9; about 82% to about 88% deamidation at position N263 of SEQ ID NO: 9; about 90% to about 96% deamidation at position N385 of SEQ ID NO: 9; and / or about 85% to about 90% deamidation at position N514 of SEQ ID NO: 9, 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: 9; 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.

[0085] 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.

[0086] As used herein, a "vector genome7’ 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 a preferred embodiment, the ITR sequences from AAV2. or the deleted version thereof (AITR), which may be used for convenience. In certain embodiments, the ITRs are those shown in nucleotides 1 to 130 and 3052 to 3181 of SEQ ID NO: 2, and nucleotides 1 to 130 and 3633 to 3762 of SEQ ID NO: 7. In certain embodiments, the ITRs are those shown in nucleotides 1 to 168 and 3527 to 3694 of SEQ ID NO: 10. A shortened version of the 5’ ITR, termed AITR, has been described in which the D-sequence and terminal resolution site (trs) are deleted. In 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. ITRs from AAV2, a different source AAV than the capsid, or other than full-length ITRs may be selected. The ITRs are from the same AAV source as the AAV which provides the rep function during production or a transcomplementing AAV.

[0087] 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.

[0088] 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,’' A dv. 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.

[0089] 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.

[0090] 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.

[0091] 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).

[0092] 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.

[0093] 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.

[0094] 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 rhlO, International Patent Publication No. WO 2017 / 100704, and for AAV1, International Patent Publication No. WO 2017 / 100674 are all incorporated by reference herein.

[0095] 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 pL 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.

[0096] 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 Bl 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.

[0097] 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 I 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.

[0098] Additionally, or alternatively, droplet digital PCR (ddPCR) may be used. For example, methods for determining single-stranded and sei f-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): l 15-25. doi: 10.1089 / hgtb.2013. 131. Epub 2014 Feb 14. The ddPCR method directly measures the concentration of encapsidated vector genomes. The sample is treated with DNase I to digest any non-encapsi dated DNA present in the sample followed by treatment with Proteinase K to disrupt the capsid. The sample is then diluted to fit the assay range. The sample is mixed with ddPCR Supermix, and detection is accomplished using sequencespecific primers targeting the Meganuclease specific to the PCSK9 gene (ARCUS) in combination with a fluorescently -tagged probe hybridizing to this same region. Twenty microliters of ddPCR reaction mixture is processed in the Bio-Rad droplet generator, and the ddPCR reaction mixture is partitioned into >10.000 droplets. After droplet generation, the ddPCR reaction mixture undergoes PCR amplification, and the amplified ddPCR reaction mixture is read using a Bio-Rad Droplet Reader.

[0099] The infectious unit (IU) assay may be used to determine the productive uptake and replication of rAAV vector in RC32 cells (rep2 expressing HeLa cells). A 96-well endpoint format has been employed similar to that previously published. Briefly, RC32 cells will be co-infected by serial dilutions of rAAV BDS and a uniform dilution of Ad5 with 12 replicates at each dilution of rAAV. Seventy -two hours after infection, the cells will be lysed, and qPCR will be performed to detect rAAV vector amplification over input. An endpoint dilution 50% tissue culture infectious dose (TCIDso) calculation (Spearman-Karber) will be performed to determine a infectious titer expressed as lU / mL. Since “infectivity” values are dependent on each particle’s contact with cells, receptor binding, internalization, transport to the nucleus, and genome replication, they are influenced by assay geometry and the presence of appropriate receptors and post-binding pathways in the cell line used. Receptors and post-binding pathways are not usually maintained in immortalized cell lines, and thus infectivity7assay titers are not an absolute measure of the number of “infectious'’ particles present. However, the ratio of encapsidated GC to “infectious units” (described as GC / IU ratio) can be used as a measure of product consistency from lot to lot.

[0100] 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.. W02017 / 160360 (AAV9). W02017 / 100704 (AAVrhlO). 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™ 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.

[0101] DUAL VECTOR SYSTEM

[0102] In another aspect, a dual vector system for treating a genetic disorder is provided. The system includes (a) a gene editing component that includes a nucleic acid sequence encoding a meganuclease that targets PCSK9 and regulatory sequences that direct expression of the nuclease in a target cell comprising a PCSK9 gene; and (b) a donor vector comprising a nucleic acid sequence encoding PKU for expression from the PCSK9 locus, and wherein the system further comprises sequences that direct the nuclease to specifically targets the native PCSK9 gene locus. The components of the dual vector are as those described herein. In one embodiment, the expression cassette of the gene editing vector includes the sequence of nucleotides 211 to 2964 of SEQ ID NO: 2 or a sequence sharing at least 80%, at least 85%, at least 90%, at least 95%. at least 96%. at least 97%. at least 98%, at least 99%. or at least 99.9% identity to the sequence of nucleotides 211 to 2964 of SEQ ID NO: 2

[0103] In one embodiment, the expression cassette of the donor vector includes the sequence of nucleotides 178 to 3569 of SEQ ID NO: 7 or a sequence sharing at least 80%, at least 85%, at least 90%, at least 95%. at least 96%. at least 97%. at least 98%. at least 99%, or at least 99.9% identity to the sequence of nucleotides 178 to 3569 of SEQ ID NO: 7.

[0104] In one embodiment, the expression cassette of the donor vector includes the sequence of nucleotides 178 to 3517 of SEQ ID NO: 10 or a sequence sharing at least 80%, at least 85%, at least 90%. at least 95%. at least 96%. at least 97%. at least 98%. at least 99%, or at least 99.9% identity to the sequence of nucleotides 178 to 3517 of SEQ ID NO: 10.

[0105] While the system can be effective if the ratio of gene editing vector to template vector is about 1 to about 1. it is desirable for the donor template vector to be present in excess of the gene editing vector. In one embodiment, the ratio of editing vector (a) to donor vector (b) is about 1:3 to about 1 :100, or about 1: 10. In certain embodiments, the ratio of editing vector (a) to donor vector (b) is about 1:3. In certain embodiments, the ratio of editing vector (a) to donor vector (b) is about 1:2. In certain embodiments, the ratio of editing vector (a) to donor vector (b) is about 1 :2.5. In certain embodiments, the ratio of editing vector (a) to donor vector (b) is about 1 :3.5. In certain embodiments, the ratio of editing vector (a) to donor vector (b) is about 1:4. In certain embodiments, the ratio of editing vector (a) to donor vector (b) is about 1:4.5. In certain embodiments, the ratio of editing vector (a) to donor vector (b) is about 1:5.

[0106] In one embodiment, the dual vector system includes a gene editing AAV comprising an AAV capsid and a first vector genome comprising a 5’ ITR, a sequence encoding a meganuclease that targets PCSK9 under control of regulator ' sequences that direct expression of the meganuclease in a target cell comprising a PCSK9 gene, and a 3’ ITR; and (b) a donor AAV vector comprising an AAV capsid and a second vector genome comprising: a 5’ITR, a 5‘ homology directed recombination (HDR) arm, an PKU transgene and regulatory sequences that direct expression of the transgene in the target cell, a 3‘ HDR arm. and a 3’ ITR.

[0107] Pharmaceutical Compositions

[0108] In another aspect, a pharmaceutical composition is provided which contains a first rAAV stock comprising rAAV gene editing vectors comprising an expression cassette comprising a nucleic acid sequence encoding a meganuclease that targets PCSK9 (e.g., the protein sequence of SEQ ID NO: 3) and regulatory sequences that direct expression of the nuclease in a target cell comprising a PCSK9 gene; and a second rAAV stock comprising rAAV donor vectors comprising a transgene cassette comprising a nucleic acid sequence encoding an PKU transgene (e.g., coding sequence of SEQ ID NO: 4) and regulatory sequences that direct expression of the transgene in the target cell. The pharmaceutical composition contains an optional carrier, excipient, and / or preservative. In some embodiments, the donor vector further includes homology-directed recombination (HDR) arms 5’ and 3’ to the transgene cassette. In one embodiment, the AAV capsid for the donor vector, gene editing vector, or both, is an AAVrh79 capsid.

[0109] 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 one embodiment, a composition includes a final formulation suitable for delivery7to 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.

[0110] 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).

[0111] 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 (polypropylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)), SOLUTOL HS 15 (Macrogol-15 Hydroxy stearate), LABRASOL (Poly oxy capryllic glyceride), poly oxy 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 poly oxypropylene 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.

[0112] 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. In certain embodiments, the route of administration is IV. Routes of administration may be combined, if desired.

[0113] Dosages of the viral vector depend primarily on factors such as the condition being treated, the age, w eight 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 109to 1 x 1016genomes 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.

[0114] 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 109GC to about 1.0 x 1016GC (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 1012GC to 1.0 x 1014GC for a human patient. In one embodiment, the compositions are formulated to contain at least IxlO9, 2xl09, 3xl09, 4xl09, 5xl09, 6xl09, 7xl09, 8xl09, or 9x109GC per dose including all integers or fractional amounts within the range. In another embodiment, the compositions are formulated to contain at least IxlO10, 2xlO10. 3xl010, 4xlO10, 5xl010, 6xlO10, 7xlO10, 8xlO10, or 9xlO10GC per dose including all integers or fractional amounts within the range. In another embodiment, the compositions are formulated to contain at least IxlO11, 2xlOn, 3xl0n, 4xlOn, 5xl0n, 6xlOn, 7xlOn, 8xl0n, or 9xlOnGC per dose including all integers or fractional amounts within the range. In another embodiment, the compositions are formulated to contain at least IxlO12, 2xl012, 3xl012, 4xl012, 5xl012, 6xl012, 7xl012, 8xl012, or 9xl012GC per dose including all integers or fractional amounts within the range. In another embodiment, the compositions are formulated to contain at least IxlO13, 2xl013, 3xl013, 4x1013, 5x1013, 6x1013, 7x1013, 8x1013, or 9x1013GC per dose including all integers or fractional amounts within the range. In another embodiment, the compositions are formulated to contain at least IxlO14, 2xl014, 3xl014, 4xl014, 5xl014, 6xl014, 7xl014, 8xl014, or 9xl014GC per dose including all integers or fractional amounts within the range. In another embodiment, the compositions are formulated to contain at least IxlO15, 2xl015, 3xl015, 4xl015, 5xl015, 6xl015, 7xlO15, 8xl015. or 9xl015GC per dose including all integers or fractional amounts within the range. In one embodiment, for human application the dose can range from lxl010to about IxlO12GC per dose including all integers or fractional amounts within the range.

[0115] These above doses may be administered in a variety of volumes of earner, 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.

[0116] 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.

[0117] In one aspect, provided herein is a pharmaceutical composition comprising at least parvovirus vector comprising at least one gene editing vector and at least one donor vector as described herein in a formulation buffer. In certain embodiments, the pharmaceutical composition comprises a combination of different vector populations. In one embodiment, provided is a pharmaceutical composition comprising a single rAAV population described herein in a formulation buffer. The methods provided herein provide for co-administration of two separate vector-containing suspensions.

[0118] Methods

[0119] The compositions provided herein are useful for treatment of phenylketonuria. Provided herein is a method of treating a disorder in a human by co-administering the dual vector system as described herein. In certain embodiments, provided herein is a composition comprising non-replicating recombinant adeno-associated virus serotype rh79 vectors: AAVrh79.TBG.M2PCSK9.WPRE.bGH and AAVrh79.hHDR.TBG.hPAHco.bGH for treatment of phenylketonuria. In one embodiment, a method of treating PKU in a subject is provided. The method includes co-administering to the subject having PKU a gene editing AAV vector comprising a sequence encoding a nuclease that targets PCSK9 and regulatory sequences that direct expression of the nuclease in a target cell comprising a PCSK9 gene; and a donor AAV vector comprising a transgene and regulatory sequences that direct expression of the PAH transgene in the target cell. In one embodiment, the subject is a neonate.

[0120] In certain embodiments, the gene editing AAV vector and the donor vector of are delivered essentially simultaneously via the same route. In other embodiments, the gene editing vector is delivered first. In other embodiments, the donor vector is delivered first.

[0121] In one embodiment, the dosage of an rAAV is about 1 x 109GC to about 1 x 1015genome copies (GC) per dose (to treat an average subject of 70 kg in body weight), and preferably 1.0 x 1012GC to 2.0 x 1015GC for a human patient. In another embodiment, the dose is less than about 1 x 1014GC / kg body weight of the subject. In certain embodiments, the dose administered to a patient is at least about 1.0 x 109GC / kg, about 1.5 x 109GC / kg, about 2.0 x 109GC / g, about 2.5 x 109GC / kg. about 3.0 x 109GC / kg, about 3.5 x 109GC / kg, about 4.0 x 109GC / kg, about 4.5 x 109GC / kg. about 5.0 x 109GC / kg, about 5.5 x 109GC / kg, about 6.0 x 109GC / kg, about 6.5 x 109GC / kg, about 7.0 x 109GC / kg , about 7.5 x 109GC / kg , about 8.0 x 109GC / kg , about 8.5 x 109GC / kg , about 9.0 x 109GC / kg , about 9.5 x 109GC / kg , about 1.0 x 1010GC / kg , about 1.5 x 1010GC / kg . about 2.0 x 1010GC / kg . about 2.5 x 1010GC / kg . about 3.0 x 1010GC / kg . about 3.5 x 1010GC / kg , about 4.0 x IO10GC / kg , about 4.5 x IO10GC / kg , about 5.0 x 1010GC / kg , about 5.5 x 1010GC / kg , about 6.0 x 1010GC / kg , about 6.5 x 1010GC / kg , about 7.0 x 1010GC / kg , about 7.5 x 1010GC / kg , about 8.0 x 1010GC / kg , about 8.5 x

[0122] 1010GC / kg . about 9.0 x 1010GC / kg . about 9.5 x 1010GC / kg . about 1.0 x 1011GC / kg . about 1.5 x 10" GC / kg , about 2.0 x 10" GC / kg , about 2.5 x 10" GC / kg , about 3.0 x

[0123] 1011GC / kg , about 3.5 x 1011GC / kg , about 4.0 x 1011GC / kg , about 4.5 x 1011GC / kg , about 5.0 x 1011GC / kg , about 5.5 x 1011GC / kg , about 6.0 x 1011GC / kg , about 6.5 x 10" GC / kg , about 7.0 x 10" GC / kg . about 7.5 x 10" GC / kg . about 8.0 x 10" GC / kg , about 8.5 x 10" GC / kg , about 9.0 x 10" GC / kg , about 9.5 x 10" GC / kg , about 1.0 x 1012GC / kg , about 1.5 x 1012GC / kg , about 2.0 x 1012GC / kg , about 2.5 x 1012GC / kg , about 3.0 x 1012GC / kg , about 3.5 x 1012GC / kg , about 4.0 x 1012GC / kg , about 4.5 x 1012GC / kg , about 5.0 x 1012GC / kg . about 5.5 x 1012GC / kg . about 6.0 x 1012GC / kg . about 6.5 x 1012GC / kg , about 7.0 x 1012GC / kg , about 7.5 x 1012GC / kg , about 8.0 x

[0124] 1012GC / kg , about 8.5 x 1012GC / kg , about 9.0 x 1012GC / kg , about 9.5 x 1012GC / kg , about 1.0 x 1013GC / kg , about 1.5 x 1013GC / kg , about 2.0 x 1013GC / kg , about 2.5 x

[0125] 1013GC / kg , about 3.0 x 1013GC / kg . about 3.5 x 1013GC / kg . about 4.0 x 1013GC / kg . about 4.5 x 1013GC / kg , about 5.0 x 1013GC / kg , about 5.5 x 1013GC / kg , about 6.0 x 1013GC / kg , about 6.5 x 1013GC / kg , about 7.0 x 1013GC / kg , about 7.5 x 1013GC / kg , about 8.0 x 1013GC / kg , about 8.5 x 1013GC / kg , about 9.0 x 1013GC / kg , about 9.5 x 1013GC / kg . or about 1.0 x 1014GC / kg body weight or the subject.

[0126] 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. In certain embodiments, IV administration is used.

[0127] The system described herein may be therapeutically useful if a sufficient amount of functional enzyme or protein is generated to improve the patient’s condition. In certain embodiments, gene expression levels as low as 5% of the level of a healthy patients will provide sufficient therapeutic effect for the patient. In other embodiments, gene expression levels are at least about 5%, 6%, 7%, 8%, 9%. 10%. 11%. 12%. 13%. 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%,

[0128] 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%,

[0129] 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,

[0130] 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,

[0131] 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,

[0132] 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the normal range (levels) observed in humans (or other vetennary subject). For example, by ‘‘functional enzyme'’, is meant a gene which encodes the wild-type enzyme (e.g., PAH) which provides at least about 5%. 6%, 7%, 8%. 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%,

[0133] 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%,

[0134] 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,

[0135] 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,

[0136] 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,

[0137] 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about the same, or greater than 100% of the biological activity level of the wild-type enzyme, or a natural variant or polymorph thereof which is not associated with disease. Similarly, patients having no detectable amounts of enzyme may be rescued by delivering enzyme function to less than 100% activity levels, and may optionally be subj ect to further treatment subsequently. In certain embodiments, where gene function is being delivered by the donor template, patients may express higher levels than found in “normal", healthy subjects. As described herein, the therapy described herein may be used in conjunction with other treatments, z.e., the standard of care for the subject’s (patient’s) diagnosis.

[0138] In one embodiment, the method further comprises administering an immunosuppressive co-therapy to the subject. Such immunosuppressive co-therapy may be started prior to delivers’ 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.

[0139] 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- , IFN- y, an opioid, or TNF-a (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 coadministration of two or more drugs, the (e.g., prednisolone, mi cophenolate 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.

[0140] In another embodiment, the method includes co-treatment with a standard PKU therapy. In some embodiments, rAAV.hPAH is administered in combination with one or more therapies for the treatment of PKU, such as a low Phe diet or administration of sapropterin dihydrochloride.

[0141] In one aspect, a method is provided for treating a patient having phenylketonuria (PKU), using a nuclease expression cassette comprising a meganuclease which recognizes a site within the human PCSK9 gene, under the control of a TBG promoter as described herein. The method further includes administration of an expression cassette carrying the PKU transgene of SEQ ID NO: 4, or a sequence sharing at least 90% identity therewith, as described herein. The method further includes administration of an expression cassette carrying the PKU transgene of SEQ ID NO: 11, or a sequence sharing at least 90% identity therewith, as described herein.

[0142] As discussed above, a subject having PKU of any severity is the intended recipient of the compositions and methods described herein. In certain embodiments, the subject has, or is at risk of developing, PKU. In certain embodiments, the subject has a documented genetic confirmation of an PKUD mutation. In certain embodiments, tire subject has, or has had previously, Hyperammonemic Crisis (HAC). In certain embodiments, the subject is currently being treated for PKU, e.g., with at least one nitrogen scavenger therapy and / or a protein restricted diet.

[0143] In certain embodiments, the subject is male. In certain embodiments, the subject is female. In certain embodiments, it is desirable to treat the subject within hours of birth, e.g., at least 12 hours of age, 24 hours of age, 36 hours of age, or 48 hours of age. In other embodiments, it is desirable to treat the subject within days of birth, e.g., at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days from birth. In other embodiments, it is desirable to treat the subject within weeks of birth, e.g., at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks from birth. In other embodiments, it is desirable to treat the subject within months of birth, e.g., at 1. 2, 3, 4, 5. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15. 16, 17, 18, 19, 20, 21, 22. 23 or 24 months from birth. In certain embodiments, the subject is treated at 24 hours to 4 months of age. In other embodiments, the subject is treated at less than 1 year of age, less than 2 years of age, less than 3 years of age, less than 4 years of age, less than 5 years of age, less than 6 years of age, less than 7 years of age, less than 8 years of age, less than 9 years of age, less than 10, 15, 20, 25, 30, 35, 40, 45, or 50 years of age. In certain embodiments, the subject is older than 50 years of age.

[0144] Subjects may be permitted to continue their standard of care treatment(s) (e.g.. diet low in Phe; treatment with sapropterin dihydrochloride) prior to and concurrently with the gene therapy treatment at the discretion of their caring physician. In the alternative, the physician may prefer to stop standard of care therapies prior to administering the gene therapy treatment and, optionally, resume standard of care treatments as a co-therapy after administration of the gene therapy.

[0145] Desirable endpoints of the gene therapy regimen are an increase in PAH activity resulting in Phe levels between 120-360 pmol / L. In another embodiment, the vector dose is intended to deliver PAH to result in a reduction of plasma phenylalanine levels by 25% or greater. In another embodiment, the desirable endpoint is reducing plasma Phe levels to take subject to a “moderate” phenotype from a “severe” phenotype. Methods for measurement of phenylalanine levels are known in the art e.g, as described in Gregory et al, Blood phenylalanine monitoring for dietary compliance among patients with phenylketonuria: comparison of methods. Genetics in Medicine (November 2007) 9, 761-765, which is incorporated herein by reference. In one embodiment, patients achieve desired circulating PAH levels after treatment with rAAV.hPAH, alone and / or combined with the use of adjunctive treatments.

[0146] In certain embodiments, a nuclease expression cassette, donor vector cassette, 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 4 months). In certain embodiments, the patient is older than an infant, e g., 12 months or older.

[0147] 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.

[0148] As used herein, the term “specificity” means the ability of a nuclease 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 nuclease is capable of cleaving only one or a very few recognition sequences. Specificity can be determined by any method know n in the art.

[0149] The (gene editing and donor) expression cassettes described herein, 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.

[0150] “Plasmid” or “plasmid vector” generally is designated herein by a lowercase 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.

[0151] 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.

[0152] 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.

[0153] 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 ty pically 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.

[0154] 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.

[0155] A “subject” is a mammal, e.g., a human, mouse, rat, guinea pig, dog, cat. horse, cow. pig, or non-human primate, such as a monkey, chimpanzee, baboon or gorilla. A patient refers to a human. A veterinary subject refers to a non-human mammal. In certain embodiments, the subject does not have a defect in their PCSK9 gene.

[0156] 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.

[0157] 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.

[0158] 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. Identity7is readily determined by one of skill in the art by resort to algorithms and computer programs known by those of skill in the art.

[0159] Generally, when referring to “identity”, “homology”, or “similarity ” between tw o 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).

[0160] 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.

[0161] 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.

[0162] 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.

[0163] EXAMPLES

[0164] Phenylketonuria (PKU) is an autosomal recessive metabolic disorder caused by missense mutations in the gene encoding phenylalanine hydroxylase (PAH), a hepatic enzyme that catalyzes the conversion of phenylalanine to tyrosine. Although PKU does not directly shorten life expectancy, high phenylalanine concentrations in tissues and blood can result in symptoms ranging from a musty' odor from the skin and urine to intellectual disability', epilepsy, and hyperactivity / behavioral problems. Most PKU cases are detected at birth via newborn screening; overall incidence is 1 / 15,000 in the United States, but this rate vanes around the world. Dietary and pharmacological treatment options exist, but their life-long, inconvenient nature can raise compliance and efficacy issues. Our aim was to establish a long-lasting, single-administration treatment for PKU.

[0165] Here, we developed a dual adeno-associated virus (AAV) vector-delivered, meganuclease-mediated gene insertion approach to insert the human PAH transgene into a previously identified safe harbor site for sustained expression. Our approach comprises two AAVrh79 vectors. The first vector encodes an engineered ARCUS meganuclease, M2PCSK9, that targets the human PCSK9 gene. The second, a “donor” vector, encodes a codon-optimized version of the human PAH transgene flanked by 500 bp homology arms on either side of the meganuclease target site in the human genome (and -95% homologous to rhesus macaques) to aid insertion at the meganuclease-induced doublestrand break. We evaluated our approach in a proof-of-concept study in 6-9-month-old rhesus macaques following intravenous administration. The dual AAVs were administered at a total dose of 4 x 1013GC / kg (n=2; high dose) and 1.33 x 1013GC / kg (n=2; low dose) with a 3: 1 donor-to-meganuclease vector ratio. Another cohort received a 1: 1 ratio for a total dose of 2 x 1013GC / kg (n=2; intermediate dose). Liver biopsies were performed at three months post dosing, and animals were followed with bi-monthly blood tests to monitor liver function. Transient, mild elevations of liver enzymes (alanine transaminase and aspartate transaminase) that resolved without intervention were observed and blood chemistry analyses showed no signs of decreased liver function. As the M2PCSK9 meganuclease specifically disrupts PCSK9 expression, we used total serum PCSK9 protein levels as a surrogate readout of target site editing. We observed a consistent decrease in PCSK9 levels in all three dual treatment cohorts, with the intermediate- and low-dose cohorts achieving a > 50% reduction compared with the donor-only group. We conducted amplicon sequencing to directly assess target site editing at the three-month biopsy timepoint. At the meganuclease target site, we detected an insertion / deletion frequency of 5% in the low-dose, 21.1% in the intermediate-dose, and 11% in the high-dose cohorts compared with 0.08% in the donor-only group. In situ hybridization from liver biopsy tissue analyses of the dual treatment cohorts showed expression of human PAH RNA in 4.5% of cells in the low -dose, 8.5% in the intermediate-dose, and 3.1% in the high-dose animals, and 4.5% in the donor-only group; we confirmed this expression pattern via RNA transcript levels. These results indicate that our dual AAV vector-delivered, meganuclease-mediated gene insertion approach can safely introduce a PAH transgene into the liver of nonhuman primates. Example 1 - mPAH KO Mouse Model

[0166] PAH / _mice were generated by CRISPR / Cas9 technology at Jackson Labs. Wild-type C57BL / 6 mice were injected with Cas9 mRNA and two guide RNAs (sgRNA) targeting the second coding exon of PAH gene directly into mouse zygotes. Mice that developed from these embryos were sequenced to determine the mutation(s) and then bred with C57BL / 6J mice to transmit the allele and confirm germline transmission. Four different mutations were generated. The chosen mouse demonstrated a single base pair insertion in the Mus muscuhis PAH gene [NC 000076.6],

[0167] Example 2 - Natural History Study

[0168] Treatment goal for individuals with PKU is a blood Phe concentration betw een 120-360pmol / L (Untreated >1200pmol / L)

[0169] Data in mice suggest that 10% wild-ty pe PAH levels in the liver is sufficient to maintain normal Phe homeostasis without any treatment, and just 5% yields a significant decrease in serum Phe (<700 pM)

[0170] Example 3 - Study in 6-9mo NHP Rhesus Macaques

[0171] We evaluated our approach in a study in 6-9-month-old rhesus macaques following intravenous administration. The dual AAVs w ere administered at a total dose of 4 x 101?GC / kg (n=2; high dose) and 1.33 x 101?GC / kg (n=2; low dose) with a 3:1 donor-to-meganuclease vector ratio. Another cohort received a 1 : 1 ratio for a total dose of 2 x 1013GC / kg (n=2; intermediate dose). Liver biopsies w ere performed at three months post dosing (FIG. 6 and 7), and animals were followed with bi-monthly blood tests to monitor liver function (FIG. 3B). Although transient, mild elevations of liver enzymes (alanine transaminase and aspartate transaminase) that resolved without intervention were observed and the blood chemistry analyses showed no signs of decreased liver function.

[0172] As the M2PCSK9 meganuclease specifically disrupts PCSK9 expression, we used total serum PCSK9 protein levels as a surrogate readout of target site editing. We observed a consistent decrease in PCSK9 levels in all three dual treatment cohorts, with the intermediate- and low-dose cohorts achieving a > 50% reduction compared with the donor-only group (FIG. 4-5). We conducted amplicon sequencing to directly assess target site editing at the three-month biopsy timepoint.

[0173] At the meganuclease target site we detected an insertion / deletion frequency of 5% in the low-dose. 21.1% in the intermediate-dose, and 11% in the high-dose cohorts compared with 0.08% in the donor-only group (FIG. 5). In situ hybridization from liver biopsy tissue analyses of the dual treatment cohorts showed expression of human PAH RNA in 4.5% of cells in the low-dose, 8.5% in the intermediate-dose, and 3. 1% in the high-dose animals, and 4.5% in the donor-only group; we confirmed this expression pattern via RNA transcript levels (FIG. 5). These results indicate that our dual AAV vector-delivered, meganuclease-mediated gene insertion approach can safely introduce a PAH transgene into the liver of nonhuman primates. Our combination therapy represents a single-administration treatment approach for PKU that is PAH-mutation-agnostic and that may provide a less restrictive, improved quality of lives for those affected by PKU.

[0174] Vector administration was well tolerated in all subjects with minimal, acute and transient increases in ALT consistent with the pattern seen in other NHP editing studies. No apparent dose-effect on ALT elevations were seen within the early phase of the study. Following the early phase, normal serum biochemistry and liver function indices were observed throughout the study.

[0175] Vector DNA in the donor only animal was higher than expected. One of the two low dose 1:3 animals had very low editing and transgene expression. This subject was the only 9mo animal dosed. All other subjects were dosed at ~6mo-6.4mo. Figures 4 and 5 show 1.2% indels and 690 transcripts / lOOng for animal administered the donor transgene alone. For the low dose animal, 8.9% indels and 26,400 transcripts / lOOng were observed. Cells expressing transgene (ISH) and abundance of transgene expression (RT- PCR) and genome copies (PCR) stayed relatively stable from 3mo to lyr regardless of initial expression level.

[0176] Example 4 - Evaluation of Homology Arm Size on Gene Targeting Efficacy and Integration Characteristics Next, we evaluated the effect of arm size on gene targeting efficacy and integration characteristics. A graphical representation of this experiment is shown in FIG. 12. Ten groups of PCSK9 KI were administered a combination of the nuclease vector (FIG. 1) and one of 9 different donor vectors. As a control, one group was administered PBS with the nuclease vector. The donor vectors were identical except for different homology arm lengths. Group 1 had no homology arms, group 2 had two short hHDR (-130 bp) arms, group 3 had two long 500 bp mhHDR arms, group 4 had two icrohomology (~40bp) arms, group 5 had one microhomology arm, group 7 had very short (~5bp) arms, group 8 also had short (~25bp) arms, group 9 had ~50bp arms and group 10 had self complementary ~50bp arms. Group 6 was the control group that received PBS.

[0177] After 28 days the mice were sacrificed and analyzed using Amplicon-Seq of Target region. qPCR PAH expression and PAH histology. After analysis, group 3 was chosen for further analysis using human sequences. (FIG. 11).

[0178] All documents cited in this specification are incorporated herein by reference, as are sequences and text of the Sequence Listing filed herewith are incorporated by reference. International Patent Publication No. WO2018 / 126112 is incorporated by reference in its entirety. 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.

[0179] EMBODIMENTS

[0180] 1. A dual vector system for treating phenylketonuria, the system comprising: (a) a gene editing AAV comprising a first AAV rh79 capsid and a first vector genome comprising a 5’ ITR, a sequence encoding a meganuclease having the sequence of SEQ ID NO: 3 that targets PCSK9 under control of regulatory' sequences that direct expression of the meganuclease in a target cell comprising a PCSK9 gene, and a 3’ ITR; and (b) a donor AAV vector comprising a second AAV rh79 capsid and a second vector genome comprising: a 5’ITR, a 5’ homology directed recombination (HDR) arm, a transgene comprising the sequence of SEQ ID NO: 4 or 11, or a sequence at least 90% identical to SEQ ID NO: 4 or 11 encoding phenylalanine hydroxylase (PAH) and regulatory sequences that direct expression of the transgene in the target cell, a 3’ HDR arm, and a 3’ ITR.

[0181] 2. The dual vector system according to embodiment 1, wherein the sequence encoding the meganuclease comprises nucleotides (nt) 1089-2183 of SEQ ID NO: 2, or a sequence at least 90% identical to nucleotides (nt) 1089-2183 of SEQ ID NO: 2.

[0182] 3. The dual vector system according to embodiment 1 or embodiment 2, wherein the transgene encoding PAH comprises SEQ ID NO: 4 or 11.

[0183] 4. The dual vector system according to any one of embodiments 1 to 3, wherein the first and second AAV capsid are AAVrh79 capsids of SEQ ID NO: 9.

[0184] 5. The dual vector system according to any one of embodiments 1 to 3, wherein the ratio of gene editing AAV vector of (a) to donor AAV vector of (b) is 1: 1 to 1:3.

[0185] 6. The dual vector system according to any one of embodiments 1 to 5, wherein the nuclease is under the control of a TBG promoter.

[0186] 7. The dual vector system according to any one of embodiments 1 to 6, wherein the transgene is under the control of a TBG promoter.

[0187] 8. The dual vector system according to any one of embodiments 1 to 7, wherein i) the first vector genome comprises nt 211 to 2964 of SEQ ID NO: 2, or a sequence sharing at least 90% identity with nt 211 to 2964 of SEQ ID NO: 2; and ii) the second vector genome comprises nt 178 to 3569 of SEQ ID NO: 7 or a sequence sharing at least 90% identity with nt 178 to 3569 of SEQ ID NO: 7, or nt 178 to 3517 of SEQ ID NO: 10 or a sequence sharing at least 90% identity with nt 178 to 3517 of SEQ ID NO: 10.

[0188] 9. A method of treating phenylketonuria a human subject by co-administering the dual vector system according to any one of embodiments 1 to 8.

[0189] 10. A method of treating phenylketonuria (PKU) in a subject, the method comprising: co-administering to the subject having PKU:

[0190] (a) a gene editing AAV comprising a first AAV rh79 capsid and a first vector genome comprising a 5' ITR, a sequence encoding a meganuclease having the sequence of SEQ ID NO: 3 that targets PCSK9 under control of regulatory sequences that direct expression of the meganuclease in a target cell comprising a PCSK9 gene, and a 3’ ITR; and

[0191] (b) a donor AAV vector comprising a second AAV rh79 capsid and a second vector genome comprising: a 5’ITR, a 5’ homology directed recombination (HDR) arm. a transgene comprising the sequence of SEQ ID NO: 4 or 11 , or a sequence at least 90% identical to SEQ ID NO: 4 or 11 encoding phenylalanine hydroxylase (PAH) and regulatory sequences that direct expression of the transgene in the target cell, a 3’ HDR arm, and a 3’ ITR.

[0192] 11. The method according to embodiment 10, wherein i) the first vector genome comprises nt 211 to 2964 of SEQ ID NO: 2, or a sequence sharing at least 90% identity with nt 211 to 2964 of SEQ ID NO: 2; and ii) the second vector genome comprises nt 178 to 3569 of SEQ ID NO: 7 or a sequence sharing at least 90% identity with nt 178 to 3569 of SEQ ID NO: 7 , or nt 178 to 3517 of SEQ ID NO: 10 or a sequence sharing at least 90% identity with nt 178 to 12. The method according to any one of embodiments 9 to 11, wherein the gene editing AAV vector of (a) and the donor vector of (b) are delivered essentially simultaneously via IV.

[0193] 13. The method according to any one of embodiments 9 to 12, wherein the gene editing AAV vector of (a) is suspended in a vehicle for injection at a dosage of about 1 x 1013GC / kg.

[0194] 14. The method according to any one of embodiments 9 to 13, wherein the AAV donor vector of (b) is suspended in a vehicle for injection at a concentration of about 3 x 1013GC / kg.

[0195] 15. The method according to any one of embodiments 9 to 14, wherein the AAV donor vector of (b) is suspended in a vehicle for injection at a concentration of about 1 x 1013GC / kg.

[0196] 16. The method according to any one of embodiments 9 to 15, wherein the subject is under 1 year of age.

[0197] 17. Use of the dual vector system according to any one of embodiments 1 to 8. for treatment of phenylketonuria in a subject in need thereof.

[0198] 18. Use of the dual vector system according to any one of embodiments 1 to 8 in the preparation of a medicament for treatment of phenylketonuria in a subj ect in need thereof.

Claims

WHAT IS CLAIMED IS:

1. A dual vector system for treating phenylketonuria, the system comprising:(a) a gene editing AAV comprising a first AAV rh79 capsid and a first vector genome comprising a 5’ ITR, a sequence encoding a meganuclease having the sequence of SEQ ID NO: 3 that targets PCSK.9 under control of regulatory sequences that direct expression of the meganuclease in a target cell comprising a PCSK9 gene, and a 3’ ITR; and(b) a donor AAV vector comprising a second AAV rh79 capsid and a second vector genome comprising: a 5’ITR, a 5’ homology directed recombination (HDR) arm. a transgene comprising the sequence of SEQ ID NO: 4, or a sequence at least 90% identical to SEQ ID NO: 4 encoding phenylalanine hydroxylase (PAH) and regulatory' sequences that direct expression of the transgene in the target cell, a 3‘ HDR arm, and a 3’ ITR.

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

3. The dual vector system according to claim 1, wherein the transgene encoding PAH comprises SEQ ID NO: 4.

4. The dual vector system according to claim 1 . wherein the first and second AAV capsid are AAVrh79 capsids of SEQ ID NO: 9.

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

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

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

8. The dual vector system according to claim 1, wherein i) the first vector genome comprises nt 21 1 to 2964 of SEQ ID NO: 2, or a sequence sharing at least 90% identity with nt 211 to 2964 of SEQ ID NO: 2; and ii) the second vector genome comprises nt 178 to 3569 of SEQ ID NO: 7 or a sequence sharing at least 90% identity with nt 178 to 3569 of SEQ ID NO: 7, or nt 178 to 3517 of SEQ ID NO: 10 or a sequence sharing at least 90% identity with nt 178 to 3517 of SEQ ID NO: 10.

9. A method of treating phenylketonuria a human subject by co-administering the dual vector system according to claim 1.

10. A method of treating phenylketonuria (PKU) in a subject, the method comprising: co-administering to the subject having PKU:(a) a gene editing AAV comprising a first AAV rh79 capsid and a first vector genome comprising a 5’ ITR, a sequence encoding a meganuclease having the sequence of SEQ ID NO: 3 that targets PCSK9 under control of regulatory' sequences that direct expression of the meganuclease in a target cell comprising a PCSK9 gene, and a 3’ ITR; and(b) a donor AAV vector comprising a second AAV rh79 capsid and a second vector genome comprising: a 5’ITR, a 5’ homology directed recombination (HDR) arm, a transgene comprising the sequence of SEQ ID NO: 4, or a sequence at least 90% identical to SEQ ID NO: 4 encoding phenylalanine hydroxylase (PAH) and regulatory sequences that direct expression of the transgene in the target cell, a 3’ HDR arm, and a 3’ ITR.

11. The method according to claim 10, whereini) the first vector genome comprises nt 211 to 2964 of SEQ ID NO: 2, or a sequence sharing at least 90% identity with nt 211 to 2964 of SEQ ID NO: 2; and ii) the second vector genome comprises nt 178 to 3569 of SEQ ID NO: 7 or a sequence sharing at least 90% identity with nt 178 to 3569 of SEQ ID NO: 7 , or nt 178 to 3517 of SEQ ID NO: 10 or a sequence sharing at least 90% identity with nt 178 to 3517 of SEQ ID NO: 10.

12. The method according to claim 10, wherein the gene editing AAV vector of (a) and the donor vector of (b) are delivered essentially simultaneously via IV.

13. The method according to claim 10, wherein the gene editing AAV vector of (a) is suspended in a vehicle for injection at a dosage of about 1 x 1013GC / kg.

14. The method according to claimlO, wherein the AAV donor vector of (b) is suspended in a vehicle for injection at a concentration of about 3 x 1013GC / kg.

15. The method according to claim 10, wherein the AAV donor vector of (b) is suspended in a vehicle for injection at a concentration of about 1 x 1013GC / kg.

16. The method according to claim 10, wherein the subject is under 1 year of age.

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

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

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