HK40112402A - Methods for treatment of ornithine transcarbamylase (otc) deficiency - Google Patents

Description

Methods for treating ornithine carbamoyltransferase (OTC) deficiency

Background Technology

Ornithine carbamoyltransferase (OTC) deficiency (OTCD) is an X-linked urea cycle disorder associated with high mortality. Although adeno-associated virus (AAV) neonatal gene therapy is a promising treatment for late-onset OTC deficiency, it only provides short-term therapeutic effects due to non-integrated genome loss during hepatocyte proliferation.

Extensive nucleases induce double-strand breaks (DSBs) in chromosomes, leading to DNA repair. In the presence of donor DNA, homology-directed repair (HDR) occurs, replacing the genetic information in the chromosome with new information from the donor gene.

Safe harbor sites (SHS) are genomic loci where genes or other genetic elements can be safely inserted and expressed. These SHS are crucial for effective gene therapy for human diseases; for studying gene structure, function, and regulation; and for cell labeling and tracking.

Nuclease-mediated site-specific integration of transgenic boxes into the safe harbor of the genome will provide long-term therapeutic benefits for patients with OTC deficiency.

What is needed are improved compositions and methods for treating OTC.

Summary of the Invention

This article provides compositions, methods, systems, and kits for treating subjects in need of OTC, which allow knockdown or ablation of the native PCSK9 gene and insertion and/or expression of exogenous OTC transgenes in the PCSK9 locus.

On one hand, a dual-vector system for treating ornithine carbamoyltransferase deficiency is provided. The system comprises: (a) a gene-editing AAV comprising a first AAV capsid and a first vector genome, the first vector genome comprising a 5' ITR, a sequence encoding a macronuclease, and a 3' ITR, the macronuclease targeting PCSK9 under the control of a regulatory sequence, the regulatory sequence guiding the expression of the macronuclease in target cells including the PCSK9 gene; and (b) a donor AAV vector comprising a second AAV capsid and a second vector genome, the second vector genome comprising: a 5' ITR, a 5' homologous directed recombination (HDR) arm, a transgene encoding ornithine carbamoyltransferase (OTC), and a regulatory sequence guiding the expression of the transgene in the target cells, a 3' HDR arm, and a 3' ITR. In some embodiments, the macronuclease is an ARCUS macronuclease having the sequence SEQ ID NO:3. In some embodiments, the sequence encoding a wide range of nucleases includes nucleotides (nt) 1089 to 2183 of SEQ ID NO:2 or a sequence that is at least 90% identical to nucleotides (nt) 1089 to 2183 of SEQ ID NO:2. In some embodiments, the transgene encoding an OTC includes SEQ ID NO:5 or a sequence that is at least 90% identical to SEQ ID NO:5. In some embodiments, the first AAV capsid and the second AAV capsid are the AAVrh79 capsid of SEQ ID NO:16.

On the other hand, a method for treating OTC deficiency in a subject in need is provided. The method comprises co-administering to the subject suffering from OTC: (a) a gene-edited AAV comprising a first AAV capsid and a first vector genome, the first vector genome comprising a 5' ITR, a sequence encoding a macronuclease, and a 3' ITR, the macronuclease targeting PCSK9 under the control of a regulatory sequence, the regulatory sequence guiding the expression of the macronuclease in target cells including the PCSK9 gene; and (b) a donor AAV vector comprising a second AAV capsid and a second vector genome, the second vector genome comprising a 5' ITR, a 5' homologous directed recombination (HDR) arm, a transgene encoding ornithine carbamoyltransferase (OTC), and a regulatory sequence guiding the expression of the transgene in the target cells, a 3' HDR arm, and a 3' ITR. In some embodiments, i) the first vector genome includes nt 211 to 2964 of SEQ ID NO:2 or a sequence that shares at least 90% identity with nt 211 to 2964 of SEQ ID NO:2; and ii) the second vector genome includes nt 178 to 3281 of SEQ ID NO:6 or a sequence that shares at least 90% identity with nt 178 to 3281 of SEQ ID NO:6.

Other aspects and advantages of the invention will become apparent from the following detailed description.

Attached Figure Description

Figure 1A shows the timeline of the study including the hOTC mini gene knock-in of ARCUS2 at the PCSK9 locus. Figure 1B shows the study design for groups 1–7 (G). Before administration, animals 21-111, 21-122, and 21-113 were positive for AAV-binding antibodies (BAb).

Figure 2 shows a schematic representation of a dual AAV vector system for ARCUS2-mediated gene correction, where the AAV donor vector includes an hOTC donor template sequence, as used in the studies shown in Figures 1A-1B. Different HDR arms were used, as illustrated.

Figure 3 is a graph showing the experimental results of the experiment described in Figure 1A-2. Details are provided in Figures 4A-4K.

Figures 4A-4K illustrate the experimental results described in Figures 1A-2. Figure 4A shows the PCSK9 levels on day 0 for each group, expressed as a percentage (%). Figure 4B shows the ALT levels for each group, expressed as U/L. Liver biopsies were performed at specified time points, and double in situ hybridization (ISH) was performed using specific probes to detect hOTC and ARCUS. Figure 4C shows the transduction efficiency of the OTC transgene as quantified by ISH, plotted as the percentage of transduced hepatocytes. Figure 4D shows the transduction efficiency of the OTC transgene as quantified by IF. Figure 4E shows the body weight of NHPs. Figure 4F shows the analysis of vector GC in the liver by quantitative PCR. Figure 4G shows the expression of hOTC and nucleases in rhesus liver, measured by quantitative PCR followed by reverse transcription of total RNA isolated from liver biopsy samples, and expressed as relative expression levels normalized relative to GAPDH levels. Figure 4H shows the insertion/deletion analysis performed on rhPCSK9-targeted loci by amplicon sequencing at specified time points. Figure 4I shows the in-target insertions/deletions in specified tissues. The only non-hepatic tissue with insertions and deletions was the pancreas. Figure 4J shows a comparison of LFT (IU/mL) between neonatal and juvenile NHP treated with 4 x ^10¹³ GC/kg and adult NHP treated with AAV. Arcus. Figure 4K shows staining for OTC enzyme activity at 1-year necropsy in some animals from groups 2 and 3. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GC, genomic copy; hOTC, human ornithine carbamoyltransferase; OT, off-target; PCR, polymerase chain reaction; rhPCSK9, proprotein convertase subtilisin/kexin type 9 (rhesus monkey gene); RNA, ribonucleic acid.

Figures 5A and 5B illustrate schematic representations of the PCSK9-hE7-KI mouse model. Figure 5A shows a schematic representation of mouse PCSK9 exon 7 replaced with human PCSK9 exon 7 (hE7 containing the ARCUS targeting sequence). Figure 5B shows a schematic representation of the PCSK9-hE7-KI mouse model crossed with other disease mouse models (such as OTC spf ^ash and KI-spf ^ash models). The PCSK9-hE7-KI knock-in mouse model is first generated by replacing the region containing exon 7 of the mouse Pcsk9 gene with the region containing exon 7 of the human PCSK9 gene. The PCSK9-hE7-KI mice are then crossed with sparse-haired ash (spf ^ash ) mice, which exhibit a 20-fold reduction in OTC expression due to a G-to-A point mutation at the splice donor site at the end of exon 4 of the Otc ^gene . Mice from this cross are designated PCSK9-hE7-KI.spf ^ash mice and are utilized as described herein. Abbreviations: bp, base pair; E6, exon 6; E7, exon 7; E8, exon 8; HDR, homology-dependent recombination; PCSK9, proprotein convertase subtilisin/kexin type 9 (gene, human); Pcsk9, proprotein convertase subtilisin/kexin type 9 (gene, mouse). Figure 5C shows the sequence of the human exon 7 region exchanged in the mouse Pcsk9 locus (SEQ ID NO:17) and a portion of the adjacent intron sequence.

Figure 6 shows the sequence alignment of the human PCSK9 sequence, mouse PCSK9 (mPCSK9), and rhesus monkey PCSK9 (rhPCSK9) representing the pcsk9-hE7 knock-in allele.

Figure 7 shows a schematic representation of the donor construct of a dual AAV vector system for ARCUS2-mediated gene correction, wherein the AAV donor vector includes an hOTC donor template sequence. Homology of the HDR arm in the construct with the knock-in mouse model (Figure 5), NHP, and human target region is shown.

Figure 8A shows a timeline of studies including hOTC mini gene knock-in performed via ARCUS2 at the PCSK9 locus in PCSK9-hE7-KI.spf-ash pups (partial OTC deficiency model).

Figure 8B shows the carriers and doses that each group will receive for the study in Figure 8A.

Figures 9A-9F show the results of the study on the mice shown in Figures 8A-8B, which were treated with or untreated with the vector shown in Figure 7 (KI WT) and fed a high-protein (HP) diet for 10 days. Figure 9A shows the survival probability. Figure 9B shows the weight before HP diet introduction as a percentage by weight. Figure 9C shows the plasma _NH3 level on day 10 of the HP diet. Figure 9D shows the mPCSK9 protein level on day 48. Figure 9E shows the insertion/deletion percentage as measured by amplicon sequencing on day 59. Figure 9F shows the vector transduction level in liver biopsy samples measured on day 59, plotted as AAV genome copies (GC) per diploid cell.

Figure 10 is a schematic diagram of a dual-vector approach for treating OTC deficiency. Both vectors utilize the E-clade AAVrh79 and the liver-specific TBG promoter. The first vector is the nuclease ARCUS, and the second vector is an hOTC donor gene cassette with a 500bp homologous arm laterally attached to exon 7 of PCSK9.

Figure 11 is a plasmid diagram of the donor construct for the dual-vector method described in Figure 10. The plasmid sequences from ITR to ITR are shown in SEQ ID NO:6.

Figure 12 is a plasmid diagram of the nuclease construct using the dual-vector method described in Figure 10. The sequence of the plasmid from ITR to ITR is shown in SEQ ID NO:2.

Figure 13 is a table illustrating exemplary HDR sequences used in the donor constructs provided herein.

Figures 14A-14B show the results of amplicon sequencing validation of the off-target edits for the experiments described in Figures 4A-4K. Figure 14A provides a list of off-target sites, along with their chromosomal locations and best matches with common off-target sequences. Figure 14B is a graph showing the percentage of insertions and deletions for OT1-OT10. Edits to OT1, OT4, and OT5 were significantly higher in ARCUS+ donor animals than in the non-nuclease controls.

Figure 15A shows a timeline of MED studies including hOTC mini gene knock-in performed via ARCUS2 at the PCSK9 locus in PCSK9-hE7-KI.spf-ash pups, as discussed in Example 7.

Figure 15B shows the research design of the study shown in Figure 15A.

Figure 16 shows a partial study design of a 1-year toxicity study performed in NHP, as discussed in Example 9.

Detailed Implementation

This article provides compositions, kits, and methods for providing stable, long-term therapeutic effects for patients with certain inherited diseases, including liver metabolic disorders. The compositions, kits, and methods utilize a nuclease targeting the PCSK9 locus in target cells, and the donor vector provides a template containing 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 endogenous PCSK9 is disrupted and its expression level is reduced.

In one embodiment, the test article described herein comprises two vectors, both using the clade E capsid AAVrh79 and the liver-specific TBG promoter. The first vector is the nuclease ARCUS, and the second vector is an hOTC donor gene cassette with a 500 bp homologous arm laterally attached to exon 7 of PCSK9.

In Pcsk9-hE7-KI.spf-ash mice, the test item reduced mPCSK9 levels and improved weight loss and survival after high-protein diet challenge. hOTC was well transduced and induced good insertion/deletion rates compared to untreated and GFP-treated control mice.

In newborn nonhuman primates, the test item induced 18.6% and 11.9% hOTC transduction, respectively, as assessed in liver biopsies on day 84, both exceeding the threshold for substantial benefit to patients, which is approximately 5% of cells expressing OTC.

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

In some embodiments, the test article comprises two non-replicating recombinant adeno-associated virus (AAV) rh79 vectors: AAVrh79.TBG.M2PCSK9.WPRE.bGH (nuclease vector) and AAVrh79.hHDR.TBG.hOTCco.bGH (donor vector), which are mixed prior to administration at a ratio determined by genome copy number (GC). According to non-clinical studies, this ratio may be a 1:3 ratio of nuclease vector to donor vector. In some embodiments, the test article is administered as a single dose via intravenous (IV) infusion, and the dose administered is based on GC/kg of the subject's body weight.

PCSK9

Proprotein convertase subtilisin 9 (PCSK9) is a serine protease that reduces hepatic and extrahepatic low-density lipoprotein (LDL) receptor (LDLR; 606945) levels and increases plasma LDL cholesterol. PCSK9 is crucial in the regulation of plasma cholesterol homeostasis. PCSK9 binds to members of the LDL receptor family, including LDL receptor (LDLR), very low-density lipoprotein receptor (VLDLR), apolipoprotein E receptor (LRP1/APOER), and apolipoprotein receptor 2 (LRP8/APOER2), and promotes their degradation in the acidic compartment of the cell.

While the PCSK9 gene has been targeted for the treatment of cholesterol-related diseases, this paper demonstrates that the PCSK9 locus is a safe harbor for targeted insertion of other non-PCSK9 transgenes. Therefore, the compositions, kits, and methods presented herein utilize nucleases targeting the PCSK9 locus and employ donor templates to insert therapeutic transgenes into the target PCSK9 locus.

The compositions, kits, and methods provided herein comprise gene editing vectors and donor vectors, the donor vectors providing therapeutic OTC transgenes to be expressed in host cells.

Gene editing components

The compositions, kits, and methods provided herein comprise gene-editing components comprising a nuclease (or its coding sequence) and a sequence guiding the nuclease to specifically target the native PCSK9 locus on chromosome 1. As used herein, the “target PCSK9 locus” or “PCSK9 locus” is located in exon 7 of the PCSK9 coding sequence. Figure 6 provides a comparison of PCSK9 exon 7 splicing sites in humans (h), rhesus monkeys (rh), and mice (m), which are illustrated herein using SaCas9 and a broad-spectrum nuclease targeting PCSK9 (referred to as ARCUS).

This document describes compositions, particularly nucleases, that can be used to target genes for transgenic insertion, such as nucleases specific to PCSK9. In some embodiments, the nuclease is a broad-spectrum nuclease targeting PCSK9. Broad-spectrum nucleases are deoxyribonucleases characterized by large recognition sites (double-stranded DNA sequences with 12 to 40 base pairs), such as I-SceI. When combined with a nuclease, DNA can be cleaved at a specific location. Restriction enzymes can be introduced into cells for gene editing or in situ genome editing. In some embodiments, the nuclease is a member of the homing nuclease I-CreI family that recognizes and cleaves the 22-base-pair recognition sequence SEQ ID NO:1-CAAAACGTCGTGAGACAGTTTG. See, for example, 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 with it. In one embodiment, the nuclease protein sequence is the sequence shown in SEQ ID NO:3. Such nucleases are sometimes referred to herein as ARCUS nucleases. The term "homing endonuclease" is synonymous with the term "widespread nuclease." See WO 2018/195449, which describes certain PCSK9 widespread nucleases, the entire document of which is incorporated herein by reference.

In some embodiments, the compositions, kits, and methods include nuclease-encoding sequences contained in gene-editing vectors. The gene-editing vectors contain expression cassettes that include a nucleic acid sequence encoding a nuclease and a regulatory sequence guiding the expression of the nuclease in target cells including the PCSK9 gene. As used herein, a “vector” is a biological or chemical portion comprising a nucleic acid sequence that can be introduced into a suitable host cell to replicate or express the nucleic acid sequence. A vector comprising a nuclease-encoding sequence is an adeno-associated virus (AAV) vector.

As used herein, an "expression cassette" refers to a nucleic acid molecule comprising a biologically useful nucleic acid sequence (e.g., gene cDNA, mRNA, etc. encoding a protein, enzyme, or other useful gene product) and a regulatory sequence operatively linked thereto, said regulatory sequence directing or regulating the transcription, translation, and/or expression of the nucleic acid sequence and its gene product. As used herein, an "operatively linked" sequence comprises a regulatory sequence adjacent to the nucleic acid sequence and a regulatory sequence acting trans- or at a distance to control the sequence. Such regulatory sequences typically include one or more of, for example, promoters, enhancers, introns, Kozak sequences, polyadenylation sequences, and TATA signals. An expression cassette may contain a regulatory sequence upstream (5') of the gene sequence, such as one or more promoters, enhancers, introns, etc., and one or more enhancers, or a regulatory sequence downstream (3') of the gene sequence, such as a 3' untranslated region including a polyadenylation site, and other elements. In other embodiments, the term "transgenic" refers to one or more DNA sequences derived from an exogenous source and inserted into a target cell. Typically, such expression cassettes used to generate viral vectors contain the coding sequence of the gene product described herein, which is adjacent to the packaging signal of the viral genome and other expression control sequences, such as those described herein.

In addition to the coding sequence of the nuclease, the gene editing vector also contains a regulatory sequence that guides the expression of the nuclease in the host cell. The regulatory element includes a promoter, such as a liver-specific promoter or a thyroxine-binding globulin (TBG) promoter. In some embodiments, the TBG promoter has the sequence of nucleotides 211 to 907 of SEQ ID NO:2, which includes an enhancer sequence.

In addition to promoters, gene editing cassettes, expression cassettes, and/or vectors may contain one or more suitable "regulatory elements" or "regulatory sequences," which include, but are not limited to: enhancers; transcription factors; transcription terminators; effective RNA processing signals, such as splicing and polyadenylation signals (polyA); sequences that stabilize cytoplasmic mRNA, such as the marmot hepatitis virus (WHP) posttranscriptional regulatory element (WPRE); sequences that enhance translation efficiency (i.e., Kozak concordant sequences); sequences that enhance protein stability; and sequences that, when desired, enhance the secretion of the encoded product. In some embodiments, the vector contains bovine growth hormone (bGH) polyA, such as the bGH polyA shown in nucleotides 2750 to 2964 of SEQ ID NO:2. Suitable enhancers include α1 microglobulin/bisKunitz inhibitor enhancers. Suitable WPREs include the WPREs shown in nucleotides 2202 to 2743 of SEQ ID NO:2. These control or regulatory sequences are operatively linked to nuclease-coding sequences or transgene-coding sequences. In some embodiments, the SV40 intron is included, such as the SV40 intron shown in nucleotides 939 to 1071 of SEQ ID NO:2.

In some embodiments, the nuclease vector genome comprises the following components: Inverted Terminal Repeat (ITR): The ITR is the same inverted complementary sequence to all components of the vector genome flanked by AAV2 (145 base pairs [bp], library: NC_001401). When AAV and adenovirus helper functions are provided in a trans configuration, the ITR functions as both the initiation point for vector DNA replication and the packaging signal for the vector genome. Thus, the ITR sequence represents the unique cis sequence required for vector genome replication and packaging. Human thyroxine-binding globulin (TBG) promoter: This regulatory element confers tissue-specific transgene expression in the liver (410 bp, library: L13470.1). Coding sequence: The transgene is an engineered, wide-range nuclease (ARCUS; 1095 bp, 365 amino acids). The transgene is derived from a variant of the homing endonuclease I-CreI isolated from *Chlamydomonas reinhardtii* to efficiently and specifically recognize and edit the PCSK9 gene. WPRE (WHV Posttranscriptional Regulatory Element): A cis-acting RNA element derived from marmot hepatitis virus (WHV) (gene bank: MT612432.1) has been inserted into the 3' untranslated region of the coding sequence upstream of the PolyA signal. WPRE is a hepatotropic DNA virus-derived sequence and has previously been used as a cis-acting regulatory module in viral gene vectors to achieve sufficient levels of transgenic product expression and increase viral titers during manufacturing. WPRE is believed to increase transgenic product expression by improving transcript termination and enhancing 3' transcript processing, thereby increasing the amount of polyadenylated transcript and the size of the PolyA tail, and producing more transgenic mRNA available for translation. The WPRE included in the vector is a mutant version containing five point mutations in the putative promoter region of the WHV X protein (ORF) and additional point mutations (ATG to TTG) in the start codon of the WHX protein ORF. Based on sensitive flow cytometry analysis of various human cell lines transduced with lentiviruses containing the WPRE mut6-GFP fusion construct, this mutant WPRE (called mut6) is considered sufficient to eliminate the expression of the truncated WHX protein ( Zanta-Boussif et al., 2009 ).

Bovine growth hormone PolyA (bGH PolyA): The bGH PolyA signal (208 bp, gene bank: MT267334) promotes efficient polyadenylation of cis-transgenic mRNA. This element acts as a signal for transcription termination, a signal for specific cleavage events at the 3' end of nascent transcripts, and a signal for the addition of a long polyadenylated tail.

In some embodiments, the gene-editing vector further comprises one or more nuclear localization signals (NLS). (See, for example, Lu et al., Types of nuclear localization signals and mechanisms of protein import into the nucleus, Cell Communications (May 2021) 19:60. In one embodiment, the vector contains the NLS shown in nt 1095 to 1115 of SEQ ID NO:2.)

donor carrier

The compositions, kits, and methods comprise a donor vector that provides a coding sequence for an OTC therapeutic transgene. The donor vector contains an expression cassette comprising a nucleic acid sequence encoding the transgene and a regulatory sequence guiding the expression of the transgene in target cells.

Compositions, kits, and methods for nuclease-mediated site-specific integration of OTC transgenic cassettes into the PCSK9 safe harbor of the genome provide long-term therapeutic benefits for patients with OTC deficiency. An engineered coding sequence of an OTC referred to herein as hOTCco2 and shown in SEQ ID NO:4 is provided. Nucleic acids having the sequence of SEQ ID NO:4 or 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. In one embodiment, the nucleic acid shares less than 80%, less than 79%, less than 78%, less than 77%, less than 76%, less than 75%, less than 74%, less than 73%, less than 72%, less than 71%, or less than 70% identity with the native OTC coding sequence shown in SEQ ID NO:5.

In some embodiments, the transgene cassette contains a TBG promoter, a transgene coding sequence, and a polyA sequence.

In addition to promoters, transgenic cassettes, expression cassettes, and/or vectors (editors or donors) may contain one or more suitable "regulatory elements" or "regulatory sequences," which include, but are not limited to: enhancers; transcription factors; transcription terminators; effective RNA processing signals, such as splicing and polyadenylation signals (polyA); sequences that stabilize cytoplasmic mRNA, such as the marmot hepatitis virus (WHP) posttranscriptional regulatory element (WPRE); sequences that enhance translation efficiency (i.e., Kozak concordant sequences); sequences that enhance protein stability; and sequences that, when desired, enhance the secretion of the encoded product. Examples of suitable polyA sequences include, for example, SV40, bovine growth hormone (bGH) polyA, and TK polyA. Examples of suitable enhancers include, for example, alpha-fetoprotein enhancers, TTR minimal promoters/enhancers, LSPs (TH-binding globulin promoters/alpha-1 microglobulins/bikunitz inhibitor enhancers), etc. These control or regulatory sequences are operatively linked to nuclease-coding sequences or transgenic-coding sequences.

In some embodiments, the donor vector genome contains the following: Inverted terminal repeat (ITR): The ITR is the same inverted complementary sequence of all components of the vector genome flanked by AAV2 (145 bp, gene bank: NC_001401). When AAV and adenovirus helper functions are provided in a trans configuration, the ITR function serves both as the initiation point for vector DNA replication and as a packaging signal for the vector genome. Thus, the ITR sequence represents the only cis sequence required for vector genome replication and packaging. 5' and 3' homologous arms: Homologous-dependent recombination arms (also known as hHDRs) consisting of sequences flanking cleavage sites in exon 7 of the endogenous human PCSK9 locus. The homologous arms include sequences 500 bp upstream (5' homologous arm) and 500 bp downstream (3' homologous arm) of the ARCUS extensive nuclease cleavage site in exon 7 of the PCSK9 gene. Human thyroxine-binding globulin (TBG) promoter: This regulatory element confers tissue-specific transgene expression in the liver (434 bp, gene bank: L13470.1). Coding sequence: The transgene is a codon-optimized version of the human ornithine carbamoyltransferase (OTC) gene (1068 bp, 356 amino acids). Bovine growth hormone polyA (BGH PolyA): The bGH PolyA signal (215 bp, gene bank: MT267334) promotes efficient polyadenylation of the cis-transgene mRNA. This element acts as a signal for transcription termination, a signal for specific cleavage events at the 3' end of nascent transcripts, and a signal for the addition of a long polyadenylated tail.

In addition to the transgenic cassette, the donor vector also includes homologous directed recombination (HDR) arms 5' and 3' of the transgenic cassette to facilitate homologous directed recombination of the transgene into the endogenous genome. The homologous arms target the PCSK9 locus and can have different lengths. In another embodiment, the HDR arms are approximately 500 bp. The HDR arms ideally share high complementarity with the target PCSK9 locus, but 100% complementarity is not required. 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. Suitable HDR arm sequences for targeting PCSK9 exon 7 are shown in Figure 14 and SEQ ID NO: 7-12. In one embodiment, the HDR arm sequences are those shown in SEQ ID NO: 13 and 14.

AAV viral vector

Gene editing vectors and donor vectors are provided as recombinant AAV. "Recombinant AAV" or "rAAV" is a DNase-resistant viral particle containing two elements: an AAV capsid and a vector genome containing at least a non-AAV coding sequence packaged within the AAV capsid. Unless otherwise stated, this term may be used interchangeably with the phrase "rAAV vector" or "AAV vector." rAAV is a "replication-defective virus" or "viral vector" because it lacks any functional AAV rep gene or functional AAV cap gene and cannot produce progeny. In some embodiments, only the AAV sequence is an AAV inverted terminal repeat (ITR), typically located at the 5' and 3' ends of the vector genome to allow genes and regulatory sequences located between the ITRs to be packaged within the AAV capsid.

Adeno-associated virus (AAV) viral vectors are AAV DNase-resistant particles with an AAV protein capsid containing a nucleic acid sequence for delivery to target cells. The AAV capsid consists of 60 capsid protein subunits, VP1, VP2, and VP3, arranged in an icosahedral symmetry at a ratio of approximately 1:1:10 to 1:1:20, depending on the AAV selected.

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

The AAV capsids of both the donor vector and the nuclease vector are derived from AAVrh79, as described in WO 2019/169004, published on September 6, 2019, which is incorporated herein by reference. In one embodiment, the AAVrh79 capsid comprises a heterogeneous population of AAVrh79 vp1, AAVrh79 vp2, and AAVrh79 vp3 proteins. In one embodiment, the AAVrh79 capsid is generated by expression of a nucleic acid sequence encoding the predicted amino acid sequence of SEQ ID NO: 16 from 1 to 738. Optionally, the sequence co-expresses a vp3 protein from a nucleic acid sequence that does not contain a vp1 unique region (about aa1 to 137) or a vp2 unique region (about aa1 to 203), a vp1 protein generated from SEQ ID NO:15, or a vp1 protein generated from a nucleic acid sequence encoding a predicted amino acid sequence of SEQ ID NO:16 that is at least 70% identical to SEQ ID NO:15. In other embodiments, the expression of an AAVrh79 vp2 protein generated from a nucleic acid sequence encoding a predicted amino acid sequence of at least about 138 to 738 amino acids of SEQ ID NO:16, a vp2 protein generated from a sequence comprising at least nucleotides 412 to 2214 of SEQ ID NO:15, or a vp3 protein generated from a nucleic acid sequence encoding a predicted amino acid sequence of at least about 138 to 738 amino acids of SEQ ID NO:16 that is at least 70% identical to at least nucleotides 412 to 2214 of SEQ ID NO:15, is also possible. 2. AAVrh79 vp3 protein generated by expression of a nucleic acid sequence encoding at least about 204 to 738 amino acids of SEQ ID NO:16, vp3 protein generated from a sequence including at least 610 to 2214 nucleotides of SEQ ID NO:15, or vp3 protein generated from a nucleic acid sequence encoding at least about 204 to 738 amino acids of SEQ ID NO:16 that is at least 70% identical to at least 610 to 2214 nucleotides of SEQ ID NO:15.

In some embodiments, the AAVrh79 capsid comprises: a heterogeneous population of vp1 protein, said vp1 protein being a product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:16; a heterogeneous population of vp2 protein, said vp2 protein being a product of a nucleic acid sequence encoding at least about 138 to 738 amino acids of SEQ ID NO:16; and a heterogeneous population of vp3 protein, said vp3 protein being a product of a nucleic acid sequence encoding at least 204 to 738 amino acids of SEQ ID NO:16.

The AAVrh79 vp1, vp2, and vp3 proteins contain subpopulations with amino acid modifications including at least two highly deamidated asparagine (N) residues in the asparagine-glycine pair as shown in SEQ ID NO:16, and optionally further include subpopulations containing other deamidated amino acids, wherein deamidation results in amino acid changes. High levels of deamidation are observed at N57, N263, N385, and/or N514 of the N-G pair relative to the numbering in SEQ ID NO:16. Deamidation is also observed in other residues, as shown in the table and examples below. In some embodiments, AAVrh79 may have other deamidated residues, typically less than 10%, and/or may have other modifications including methylation (e.g., about 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., in the presence, ranging from 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, about S153, about S474, about T570, about S665) or oxidized (e.g., at one or more of W248, W307, W307, M405, M437, M473, W480, W480, W505, M526, M544, M561, W621, M637 and/or W697). Optionally, W may be oxidized to kynurenine.

Table 1 - Deamidation of AAVrh79

In some embodiments, the AAVrh79 capsid is modified at one or more of the positions identified in the table above, within the range determined by mass spectrometry using trypsin as provided below. In some embodiments, one or more of the following positions or the glycine residue following N are modified, as described herein. The number of residues is based on the AAVrh79 sequence provided herein. See SEQ ID NO:16.

In some embodiments, SEQ ID NO:15 provides a nucleic acid sequence encoding the AAVrh79 vp1 capsid protein. In other embodiments, a nucleic acid sequence having 70% to 99.9% identity with SEQ ID NO:15 may be selected to express the AAVrh79 capsid protein. In some other embodiments, the nucleic acid sequence is at least about 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or at least 99.9% identical to SEQ ID NO:15. However, other nucleic acid sequences encoding the amino acid sequence of SEQ ID NO:16 may be selected for generating the rAAV capsid. In some embodiments, the nucleic acid sequence has the nucleic acid sequence of SEQ ID NO:15 or a sequence encoding SEQ ID NO:16 that is at least 70% to 99% identical, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO:15. In some embodiments, the nucleic acid sequence has the nucleic acid sequence of SEQ ID NO:15 or a sequence encoding the vp2 capsid protein (about aa 138 to 738) of SEQ ID NO:15 that is at least 70% to 99.%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to that of SEQ ID NO:15 from about nt 610 to about nt 2214. In some embodiments, the nucleic acid sequence has the nucleic acid sequence of SEQ ID NO:15 from about nt 610 to about nt 2214 or a sequence encoding the vp3 capsid protein (about aa 204 to 738) of SEQ ID NO:15 that is at least 70% to 99.%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to that of nt SEQ ID NO:15.

In some embodiments, the rAAV79 vector has an AAVrh79 capsid containing a vector genome comprising nucleic acid molecules including AAV inverted terminal repeat sequences and non-AAV nucleic acid sequences, the non-AAV nucleic acid sequences encoding a product operatively linked to the sequence, the sequence guiding the expression of the product. In some embodiments, the AAVrh79 capsid is characterized by comprising a heterogeneous population of AAVrh79 vp1, AAVrh79 vp2, and AAVrh79 vp3 proteins selected from: vp1 proteins generated by expression of a nucleic acid sequence encoding the predicted amino acid sequence of SEQ ID NO: 16 (1 to 738), vp1 proteins generated from SEQ ID NO: 15, or vp1 proteins generated from a nucleic acid sequence encoding the predicted amino acid sequence of SEQ ID NO: 16 (at least 70% to 100% identical to SEQ ID NO: 15); and a heterogeneous population of AAVrh79 vp2 proteins selected from: vp2 proteins generated by expression of a nucleic acid sequence encoding the predicted amino acid sequence of at least about 138 to 738 amino acids of SEQ ID NO: 16, or vp2 proteins generated from expression of a nucleic acid sequence including at least nucleotide 41 of SEQ ID NO: 15. The vp2 protein generated from sequences 2 to 2214 or from nucleic acid sequences encoding at least about 138 to 738 amino acids of SEQ ID NO:16 that are at least 70% to 100% identical to at least nucleotides 412 to 2214 of SEQ ID NO:15, and a heterogeneous population of AAVrh79 vp3 proteins selected from: vp3 proteins generated by expression of nucleic acid sequences encoding at least about 204 to 738 amino acids of SEQ ID NO:16, vp3 proteins generated from sequences including at least nucleotides 610 to 2214 of SEQ ID NO:15 or from nucleic acid sequences encoding at least about 204 to 738 amino acids of SEQ ID NO:16 that are at least 70% identical to at least nucleotides 610 to 2214 of SEQ ID NO:15. In some embodiments, the rAAVrh79 capsid is characterized by a heterogeneous population of AAVrh79 vp1, AAVrh79 vp2, and AAVrh79 vp3 proteins, said AAVrh79 vp1, AAVrh79 vp2, and AAVrh79 vp3 proteins as products of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:16; a heterogeneous population of vp2 protein, said vp2 protein as a product of a nucleic acid sequence encoding at least about 138 to 738 amino acids of SEQ ID NO:16; and a heterogeneous population of vp3 protein, said vp3 protein as a product of a nucleic acid sequence encoding at least 204 to 738 amino acids of SEQ ID NO:16. In some embodiments, the AAVrh79 capsid is characterized by AAVrh79 vp1, vp2, and vp3 proteins, which comprise heterogeneous populations of amino acids 1 to 738 (vp1), 138 to 738 (vp2), and 204 to 738 (vp3) respectively relative to SEQ ID NO:16, wherein the heterogeneous populations of AAVrh79 vp1, AAVrh79 vp2, and AAVrh79 vp3 proteins contain subpopulations with amino acid modifications comprising at least 50% to 100% of two highly deamidated asparagine (N) in asparagine-glycine pairs at at least two positions relative to SEQ ID NO:16, and optionally further comprising subpopulations containing other deamidated amino acids, wherein deamidation causes amino acid changes. In some embodiments, based on SEQ ID NO:16 and as measured using mass spectrometry, the highly deamidated sites are N57, N263, N385, and N514. In some embodiments, based on SEQ ID NO:16 and as measured using mass spectrometry, the AAVrh79 capsid protein is individually deamidated at each of the following locations: 60% to about 100% at position N57, 60% to about 100% at position N263, 60% to about 100% at position N385, and 60% to about 100% at position N514. Other suitable techniques may be selected for measuring deamidation or other post-translational modifications.

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

This invention also covers nucleic acid sequences encoding the mutant AAVrh79, wherein one or more residues have been altered to reduce deamidation or other modifications identified herein. Such nucleic acid sequences can be used to generate the mutant rAAVrh79 capsid.

As used herein, “vector genome” refers to the nucleic acid sequence packaged inside the rAAV capsid that forms the viral particle. Such nucleic acid sequences contain AAV inverted terminal repeat (ITR) sequences. In the examples herein, the vector genome contains at least an AAV 5' ITR from 5' to 3', an expression cassette, and an AAV 3' ITR, the expression cassette containing a transgenic or coding sequence operatively linked to a regulatory sequence that guides its expression. The ITR is the genetic element responsible for genome replication and packaging during vector production and is the only viral cis-element required for rAAV production. In a preferred embodiment, an ITR sequence from AAV2 or its deleted version (ΔITR) may be used for convenience. In some embodiments, the ITR is those ITRs shown in nucleotides 1 to 130 and 3052 to 3181 of SEQ ID NO:2 and nucleotides 1 to 130 and 3345 to 3474 of SEQ ID NO:6.

A shortened version of the 5' ITR, referred to as the ΔITR, has been described, in which the D sequence and terminal resolution sites (trs) are missing. In some embodiments, the vector genome contains a shortened AAV2 ITR of 130 base pairs, with the outer "a" element missing. During vector DNA amplification using the inner A element as a template, the shortened ITR is restored to a wild-type length of 145 base pairs. In other embodiments, full-length AAV 5' ITRs and 3' ITRs are used. In other embodiments, full-length or engineered ITRs can be selected. ITRs can be selected from AAV2 (an AAV of a different origin than the capsid) or from sources other than the non-full-length ITR. The ITR is derived from the same AAV source that provides rep functionality during the generation or trans-supplementation of AAV.

For the purpose of generating AAV viral vectors (e.g., recombinant (r)AAV), the expression cassette can be carried on any suitable vector (e.g., plasmid) delivered to the packaging host cell. Plasmids that can be used in this invention can be engineered to be suitable for in vitro replication and packaging in prokaryotic cells, insect cells, mammalian cells, and other cells. Suitable transfection techniques and packaging host cells are known and/or can be readily designed by those skilled in the art.

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

The term "AAV intermediate" or "AAV vector intermediate" refers to an assembled rAAV capsid lacking the desired genomic sequence packaged within it. These can also be referred to as "empty" capsids. Such capsids may not contain a detectable genomic sequence of the expression cassette, or may contain only a partially packaged genomic sequence insufficient to achieve expression of the gene product. These empty capsids are non-functional for transferring the gene of interest into the host cell.

Recombinant adeno-associated virus (AAV) as described herein can be generated using known techniques. See, for example, WO2003/042397, WO 2005/033321, WO 2006/110689; US 7588772 B2. Such methods involve culturing host cells containing a nucleic acid sequence encoding an AAV capsid protein; a functional rep gene; an expression cassette consisting of at least an AAV inverted terminal repeat (ITR) sequence and a transgene; and sufficient auxiliary functions to allow the expression cassette to be packaged into the AAV capsid protein. Methods for generating the capsid, its coding sequence, and methods for generating rAAV viral vectors have been described. See, for example, Gao et al., Proceedings of the National Academy of Sciences of the United States of America (Proc. Natl. Acad. Sci. U.S.A.) 100(10), 6081-6086 (2003) and US2013/0045186A1.

In one embodiment, a generative cell culture for producing recombinant AAV is provided. Such a cell culture contains nucleic acids expressing an AAV capsid protein in a host cell; a nucleic acid molecule suitable for packaging into an AAV capsid, such as a vector genome containing an AAV ITR and a non-AAV nucleic acid sequence encoding a gene product operatively linked to a sequence of a guide product expressed in the host cell; and sufficient AAV rep functionality and adenovirus helper functionality to allow the packaging of the nucleic acid molecule into a recombinant AAV capsid. In one embodiment, the cell culture comprises mammalian cells (e.g., human embryonic kidney 293 cells and other cells) or insect cells (e.g., baculoviruses).

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

In one embodiment, cells are manufactured in a suitable cell culture (e.g., HEK 293). Methods for manufacturing the gene therapy vectors described herein include techniques well-known in the art, such as generating plasmid DNA for producing the gene therapy vector, generating the vector, and purifying the vector. In some embodiments, the gene therapy vector is an AAV vector, and the resulting plasmid is an AAV cis plasmid encoding the AAV genome and the gene of interest, an AAV trans plasmid containing the AAV rep and cap genes, and an adenovirus helper plasmid. The vector production process may include method steps such as initiating cell culture, performing cell passage, seeding cells, transfecting cells with plasmid DNA, exchanging the transfected medium for serum-free medium, and collecting the vector-containing cells and medium. The collected vector-containing cells and medium are referred to herein as crude cell collection. In yet another system, the gene therapy vector is introduced into insect cells by infection with a baculovirus-based vector. For reviews of these production systems, see, for example, 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 these references are incorporated herein by reference in their entirety. Methods for preparing and using these and other AAV generation systems are also described in the following U.S. patents, the contents of each of which are incorporated herein by reference in their entirety: 5,139,941; 5,741,683; 6,057,152; 6,204,059; 6,268,213; 6,491,907; 6,660,514; 6,951,753; 7,094,604; 7,172,893; 7,201,898; 7,229,823 and 7,439,065.

Subsequently, the crude cell collection can be prepared using the methodological steps of this subject, such as concentrating the carrier collection, percolating the carrier collection, microfluidizing the carrier collection, digesting the carrier collection with nucleases, filtering the microfluidized intermediate, crude purification by chromatography, crude purification by ultracentrifugation, buffer exchange and/or formulation and filtration by tangential flow filtration to prepare a large number of carriers.

Purification was performed using a two-step affinity chromatography method at high salt concentrations, followed by anion exchange resin chromatography to purify the carrier drug product and remove empty capsids. These methods are described in more detail in International Patent Publication No. 2017/160360, which is incorporated herein by reference. The purification methods for AAV8 in International Patent Publication No. 2017/100676, rh10 in International Patent Publication No. 2017/100704, and AAV1 in International Patent Publication No. 2017/100674 are also incorporated herein by reference in their entirety.

To calculate the content of empty and intact particles, the VP3 band volume of the selected sample (e.g., in the examples of this paper, a formulation purified by iodixanol gradient purification, where GC# = particle#) was plotted against the loaded GC particles. The resulting linear equation (y = mx + c) was used to calculate the number of particles in the band volume of the test sample peak. The number of particles (pt) per 20 μL loaded was then multiplied by 50 to obtain particles (pt)/mL. Pt/mL was divided by GC/mL to obtain the ratio of particles to genome copies (pt/GC). Pt/mL - GC/mL yielded empty pt/mL. Empty pt/mL was divided by pt/mL and multiplied by 100 to obtain the percentage of empty particles.

Typically, methods for determining empty capsids and AAV vector particles containing packaged genomes are known in the art. See, for example, Grimm et al., Gene Therapy (1999) 6:1322-1330; Sommer et al., Molecular Therapy (2003) 7:122-128. To test for denatured capsids, the method comprises subjecting a treated AAV stock solution to SDS-polyacrylamide gel electrophoresis (consisting of any gel capable of separating the three capsid proteins, such as a gradient gel containing 3-8% triacetate in buffer), then running the gel until sample material is separated, and then imprinting the gel onto a nylon or nitrocellulose membrane (preferably nylon). Then, an anti-AAV capsid antibody is used as the primary antibody to bind to the denatured capsid protein, preferably an anti-AAV capsid monoclonal antibody, most preferably a B1 anti-AAV-2 monoclonal antibody (Wobus et al., *Journal of Virology* (2000) 74:9281-9293). A secondary antibody is then used, which binds to the primary antibody and contains a device for detecting the binding to the primary antibody, more preferably an anti-IgG antibody containing a detection molecule covalently bound thereto, most preferably a sheep anti-mouse IgG antibody covalently linked to horseradish peroxidase. A method for detecting the binding is used to semi-quantitatively determine the binding between the primary and secondary antibodies, preferably a detection method capable of detecting radioisotope emission, electromagnetic radiation, or colorimetric changes, most preferably a chemiluminescent detection kit. For example, for SDS-PAGE, the sample can be extracted from the column fraction and heated in an SDS-PAGE loading buffer containing a reducing agent (e.g., DTT), and the capsid protein can be resolved on a pre-prepared gradient polyacrylamide gel (e.g., Novex). Silver staining can be performed using SilverXpress (Invitrogen, CA) or other suitable staining methods (i.e., SYPRO Ruby or Coomassie staining) according to the manufacturer's instructions. In one embodiment, the concentration of the AAV vector genome (vg) in the column fraction can be measured by quantitative real-time PCR (Q-PCR). The sample is diluted and digested with DNase I (or another suitable nuclease) to remove exogenous DNA. After nuclease inactivation, the sample is further diluted and amplified using primers and TaqMan ^™ fluorescent probes that are specific to the DNA sequence between the primers. The number of cycles (threshold cycles, Ct) required for each sample to reach a defined fluorescence level is measured on an Applied Biosystems Prism 7700 Sequencing System. Plasmid DNA containing the same sequence as that contained in the AAV vector is used to generate a standard curve in the Q-PCR reaction. The value of the cycle threshold (Ct) obtained from the sample is used to determine the vector genome titer by normalizing it relative to the Ct value of the plasmid standard curve. Endpoint assays based on digital PCR can also be used.

On the one hand, an optimized q-PCR method was used, which utilizes a broad-spectrum serine protease, such as proteinase K (commercially available from Qiagen). More specifically, the optimized qPCR genomic titer assay is similar to the standard assay, except that after DNase I digestion, the sample is diluted with proteinase K buffer and treated with proteinase K, followed by heat inactivation. Suitablely, the sample is diluted with proteinase K buffer in an amount equal to the sample size. The proteinase K buffer can be concentrated 2-fold or more. Typically, the proteinase K treatment is about 0.2 mg/mL, but it can vary between 0.1 g/mL and about 1 mg/mL. The treatment step is typically performed at about 55°C for about 15 minutes, but it can be performed at lower temperatures (e.g., about 37°C to about 50°C) for a longer period of time (e.g., about 20 minutes to about 30 minutes), or at higher temperatures (e.g., up to about 60°C) for a shorter period of time (e.g., about 5 minutes to 10 minutes). Similarly, thermal inactivation typically occurs at approximately 95°C for approximately 15 minutes, but the temperature can be lowered (e.g., from approximately 70°C to approximately 90°C) and the time extended (e.g., from approximately 20 minutes to approximately 30 minutes). The sample is then diluted (e.g., 1000-fold) and analyzed using TaqMan as described in the standard assay.

Alternatively or alternatively, droplet digital PCR (ddPCR) can be used. For example, methods for determining the genomic titer of single-stranded and self-complementary AAV vectors by ddPCR have been described. See, for example, M. Lock et al., Hu Gene Therapy Methods, Hu Gene Ther Methods. April 2014; 25(2):115-25. doi:10.1089/hgtb.2013.131. e.g. February 14, 2014. The ddPCR method directly measures the concentration of the encapsulated vector genome. The sample is treated with DNase I to digest any unencapsulated DNA present in the sample, followed by treatment with proteinase K to destroy the capsid. The sample is then diluted to fit the assay range. The sample is mixed with ddPCR Supermix and the assay is performed using sequence-specific primers targeting a wide range of nucleases specific to the PCSK9 gene (ARCUS), combined with fluorescently labeled probes that hybridize to the same region. Twenty microliters of ddPCR reaction mixture were processed in a Bio-Rad droplet generator and divided into ≥10,000 droplets. After droplet generation, PCR amplification was performed on the ddPCR reaction mixture, and the amplified ddPCR reaction mixture was read using a Bio-Rad droplet reader.

Infectivity unit (IU) assays were used to determine the production, uptake, and replication of the rAAV vector in RC32 cells (expressing rep2 from HeLa cells). A 96-well endpoint format similar to the previously published 96-well endpoint format was employed. Briefly, RC32 cells were co-infected with serial dilutions of rAAV BDS and homogeneous dilutions of Ad5 (each dilution of rAAV repeated 12 times). Cells were lysed 72 hours post-infection, and qPCR was performed to detect rAAV vector over-infusion amplification. The endpoint dilution 50% tissue culture infection dose ( _TCID50 ) was calculated (Spearman-Karber) to determine the infection titer expressed in IU/mL. Since the “infectivity” value depends on each particle that comes into contact with the cell, receptor binding, internalization, transport to the nucleus, and genome replication, the “infectivity” value is influenced by the assay geometry and the presence of appropriate receptors and post-binding pathways in the cell line used. Receptors and post-binding pathways are typically not maintained in immortalized cell lines, and therefore the infectivity titer is not an absolute measure of the number of “infectious” particles present. However, the ratio of encapsulated GC to "infected units" (described as the GC/IU ratio) can be used to measure product consistency for each batch.

In summary, a method for isolating rAAV particles with packaged genomic sequences from genomically defective AAV intermediates involves subjecting a suspension comprising recombinant AAV viral particles and AAV capsid protein intermediates to high-performance liquid chromatography (HPLC), wherein the AAV viral particles and AAV intermediates are bound to a strong anion exchange resin equilibrated at high pH and subjected to a salt gradient while monitoring the UV absorbance of the eluent at approximately 260 and approximately 280. The pH can be adjusted based on the selected AAV. See, for example, WO2017/160360 (AAV9), WO2017/100704 (AAVrh10), WO 2017/100676 (e.g., AAV8), and WO2017/100674 (AAV1), which are incorporated herein by reference. In this method, the intact AAV capsid is collected from the eluted fraction when the A260/A280 ratio reaches an inflection point. In one example, for the affinity chromatography step, the percolated product can be applied to Capture Select ^™ Poros-AAV2/9 affinity resin (Life Technologies) for efficient capture of AAV2 serotypes. Under these ionic conditions, a significant percentage of residual cellular DNA and proteins flows through the column, while AAV particles are effectively captured.

Dual-carrier system

On the other hand, a dual-vector system for treating hereditary diseases is provided. The system comprises: (a) a gene-editing component containing a nucleic acid sequence encoding a broad range of nucleases targeting PCSK9 and a regulatory sequence guiding the expression of the nuclease in target cells including the PCSK9 gene; and (b) a donor vector containing a nucleic acid sequence encoding an OTC for expression from the PCSK9 locus, and wherein the system further includes a sequence guiding the nuclease to specifically target the native PCSK9 locus. The components of the dual-vector system are as described herein.

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

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

While the system may be effective if the ratio of gene-editing vector to template vector is about 1 to about 1, it is desirable that the donor template vector be present in an amount exceeding that 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 some embodiments, the ratio of editing vector (a) to donor vector (b) is about 1:3. In some embodiments, the ratio of editing vector (a) to donor vector (b) is about 1:2. In some embodiments, the ratio of editing vector (a) to donor vector (b) is about 1:2.5. In some embodiments, the ratio of editing vector (a) to donor vector (b) is about 1:3.5. In some embodiments, the ratio of editing vector (a) to donor vector (b) is about 1:4. In some embodiments, the ratio of editing vector (a) to donor vector (b) is about 1:4.5. In some embodiments, the ratio of the editing carrier (a) to the donor carrier (b) is approximately 1:5.

In one embodiment, the dual-vector system comprises a gene-editing AAV comprising an AAV capsid and a first vector genome comprising a 5' ITR, a sequence encoding a macronuclease, and a 3' ITR, the macronuclease targeting PCSK9 under the control of a regulatory sequence, the regulatory sequence guiding the expression of the macronuclease in target cells including the PCSK9 gene; and (b) a donor AAV vector comprising an AAV capsid and a second vector genome comprising a 5' ITR, a 5' homologous directed recombination (HDR) arm, an OTC transgene, and a regulatory sequence guiding the expression of the transgene in target cells, a 3' HDR arm, and a 3' ITR.

Pharmaceutical Composition

On the other hand, a pharmaceutical composition is provided comprising a first rAAV stock solution comprising an rAAV gene-editing vector comprising an expression cassette comprising a nucleic acid sequence encoding a broad range of nucleases targeting PCSK9 (e.g., the protein sequence of SEQ ID NO: 3) and a regulatory sequence guiding the expression of the nuclease in target cells including the PCSK9 gene; and a second rAAV stock solution comprising an rAAV donor vector comprising a transgenic cassette comprising a nucleic acid sequence encoding an OTC transgene (e.g., the coding sequence of SEQ ID NO: 4) and a regulatory sequence guiding the expression of the transgene in target cells. The pharmaceutical composition contains optional carriers, excipients, and/or preservatives. In some embodiments, the donor vector further comprises homologous directed recombination (HDR) arms 5' and 3' of the transgenic cassette. In one embodiment, the AAV capsid of the donor vector, gene-editing vector, or both is an AAVrh79 capsid.

As used herein, "carrier" includes any and all solvents, dispersion media, mordants, coatings, diluents, antibacterial and antifungal agents, isotonic agents and absorption delay agents, buffers, carrier solutions, suspensions, colloids, etc. The use of such culture media and agents for pharmaceutically active substances is well known in the art. Complementary active ingredients may also be incorporated into the composition. The phrase "pharmaceuticalally acceptable" refers to molecular entities and compositions that do not produce allergic reactions or similar adverse reactions when administered to a host. Delivery media (such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, etc.) can be used to introduce the compositions of the present invention into suitable host cells. Specifically, the vector genome delivered by the rAAV vector can be formulated for delivery or encapsulation in lipid particles, liposomes, vesicles, nanospheres, or nanoparticles, etc.

In one embodiment, the composition comprises a final formulation suitable for delivery to a subject, said composition being, for example, an aqueous liquid suspension buffered to physiologically compatible pH and salt concentrations. Optionally, one or more surfactants are present in the formulation. In another embodiment, the composition may be transported as a concentrate diluted for administration to a subject. In other embodiments, the composition may be lyophilized and reconstituted at the time of administration.

Methods and reagents well-known in the art for preparing formulations are described, for example, in *Remington's Pharmaceutical Sciences*, Mack Publishing Company, Easton, PA. Formulations may contain, for example, excipients; carriers; stabilizers or diluents such as sterile water or saline; polyalkylene glycols such as polyethylene glycol; plant-derived oils or hydrogenated naphthalene; preservatives (such as octadecyl dimethyl benzyl ammonium chloride, hexamethyl ammonium chloride, benzalkonium chloride, benzyl chloride, phenol, butanol or benzyl alcohol, alkyl esters of p-hydroxybenzoate such as methylparaben or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); low molecular weight peptides; 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, or dextran; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn protein complexes); and/or nonionic surfactants such as TWEEN ^™ , PLURONICS ^™ , or polyethylene glycol (PEG).

The active ingredient can also be encapsulated in microcapsules, for example, prepared by coagulation techniques or by interfacial polymerization, such microcapsules being, for example, hydroxymethyl cellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in crude emulsions. Such techniques are disclosed in Remington’s Pharmaceutical Sciences, 16th edition, Osol, A. (1980).

Suitable surfactants or combinations of surfactants can be selected from non-toxic nonionic surfactants. In one embodiment, a bifunctional block copolymer surfactant terminated at a primary hydroxyl group, such as F68 [BASF], also known as poloxamer 188, is selected, having a neutral pH and an average molecular weight of 8400. Other surfactants and other poloxamers, i.e., nonionic triblock copolymers, can be selected, consisting of a central hydrophobic chain of polyoxypropylene (poly(ethylene oxide)) with two hydrophilic chains attached to the sides of polyoxyethylene (poly(ethylene oxide)), SOLUTOL HS15 (polyethylene glycol-15 hydroxystearate), LABRASOL (polyoxyethyl octanoate), polyoxyethylene 10 oleate ether, TWEEN (polyoxyethylene sorbitan fatty acid ester), ethanol, and polyethylene glycol. In one embodiment, the formulation contains poloxamer. These copolymers are generally named with the letter "P" (representing poloxamer) followed by three numbers: the first two numbers multiplied by 100 give the approximate molecular weight of the polyoxypropylene core, and the last number multiplied by 10 gives the percentage of polyoxyethylene content. In one embodiment, poloxamer 188 was selected. The surfactant may be present in an amount of up to about 0.0005% to about 0.001% of the suspension.

Administering the vector in sufficient quantities to transfect cells and provide adequate levels of gene transfer and expression to deliver therapeutic benefit without causing excessive side effects or having medically acceptable physiological effects can be determined by a person skilled in the medical field. Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to the desired organ (e.g., liver (optionally via the hepatic artery), lung, heart, eye, kidney), oral, inhalation, intranasal, intrathecal, intratracheal, intra-arterial, intraocular, intravenous, intramuscular, subcutaneous, intradermal, and other parental routes of administration. In some embodiments, the route of administration is IV. Routes of administration may be combined if desired.

The dosage of the viral vector depends primarily on factors such as the condition being treated, the patient's age, weight, and health status, and therefore may vary between patients. For example, a therapeutically effective human dose of the viral vector typically ranges from about 25 μL to about 1000 μL to about 100 mL of solution containing a genomic viral vector at a concentration of about 1 x ^10⁹ to 1 x ^10¹⁶ . Dosage is adjusted to balance therapeutic benefits with any side effects, and such dosages can vary depending on the therapeutic application employing the recombinant vector. The expression level of the transgenic product can be monitored to determine the dosage frequency of the resulting viral vector, preferably an AAV vector containing a mini-gene. Optionally, a dosage regimen similar to that described for therapeutic purposes can be used for immunization with the compositions of the present invention.

The replication-defective viral composition can be formulated in dose units such that the amount of replication-defective virus contained is in the range of about 1.0 x ^10⁹ GC to about 1.0 x ^10¹⁶ GC (for treating subjects with an average weight of 70 kg), including all integer or fractional amounts within the range, and preferably 1.0 x ^10¹² GC to 1.0 x ^10¹⁴ GC for human patients. In one embodiment, the composition is formulated to contain at least 1 x ^10⁹ , 2 x ^10⁹ , 3 x ^10⁹ , 4 x ^10⁹ , 5 x ^10⁹ , 6 x ^10⁹ , 7 x ^10⁹ , 8 x ^10⁹ , or 9 x ^10⁹ GC per dose, including all integer or fractional amounts within the range. In another embodiment, the composition is formulated to contain at least 1 x ^10¹⁰ , 2 x ^10¹⁰ , 3 x ^10¹⁰ , 4 x ^10¹⁰ , 5 x ^10¹⁰ , 6 x ^10¹⁰ , ⁷ x 10¹⁰, 8 x ^10¹⁰ , or 9 x ^10¹⁰ GC per dose, encompassing all integer or fractional quantities within the range. In another embodiment, the composition is formulated to contain at least 1 x ^10¹¹ , 2 x ^10¹¹ , 3 x ^10¹¹ , 4 x ^10¹¹ , 5 x ^10¹¹ , 6 x ^10¹¹ , 7 x ^10¹¹ , ⁸ x 10¹¹, or 9 x ^10¹¹ GC per dose, encompassing all integer or fractional quantities within the range. In another embodiment, the composition is formulated to contain at least 1 x ^10¹² , 2 x ^10¹² , 3 x ^10¹² , 4 x ^10¹² , 5 x ^10¹² , 6 x ^10¹² , 7 x ^10¹² , 8 x ^10¹² , or 9 x ^10¹² GC per dose, encompassing all integer or fractional quantities within the range. In another embodiment, the composition is formulated to contain at least 1 x ^10¹³ , 2 x ^10¹³ , 3 x ^10¹³ , 4 x ^10¹³ , 5 x ^10¹³ , 6 x ^10¹³ , 7 x ^10¹³ , 8 x ^10¹³ , or 9 x ^10¹³ GC per dose, encompassing all integer or fractional quantities within the range. In another embodiment, the composition is formulated to contain at least 1 x ^10¹⁴ , 2 x ^10¹⁴ , 3 x ^10¹⁴ , 4 x ^10¹⁴ , 5 x ^10¹⁴ , 6 x ^10¹⁴ , 7 x ^10¹⁴ , 8 x ^10¹⁴ , or 9 x ^10¹⁴ GC per dose, encompassing all integer or fractional quantities within the range. In another embodiment, the composition is formulated to contain at least 1 x ^10¹⁵ , 2 x ^10¹⁵ , 3 x ^10¹⁵ , 4 x ^10¹⁵ , 5 x ^10¹⁵ , 6 x ^10¹⁵ , 7 x ^10¹⁵ , 8 x ^10¹⁵ , or 9 x ^10¹⁵ GC per dose, encompassing all integer or fractional quantities within the range. In one embodiment, for human applications, the dosage range can be from 1 x 10 ¹⁰ to about 1 x 10 ¹² GC per dose, encompassing all integer or fractional quantities within the range.

These doses can be administered in various volumes of carrier, excipient, or buffer formulation, ranging from about 25 μL to about 1000 μL or more, including all numbers within the range, depending on the size of the area to be treated, the viral titer used, the route of administration, and the desired effect of the method.

Any suitable route of administration can be chosen. Therefore, the pharmaceutical composition can be formulated for any suitable route of administration, for example, in the form of a liquid solution or suspension (e.g., for intravenous administration, for oral administration, etc.). Alternatively, the pharmaceutical composition can be in solid form (e.g., in tablet or capsule form, for example, for oral administration). In some embodiments, the pharmaceutical composition can be in the form of powder, drops, aerosol, etc.

On one hand, this document provides a pharmaceutical composition comprising a formulation buffer containing at least one parvovirus vector, said parvovirus vector including at least one gene-editing vector and at least one donor vector as described herein. In some embodiments, the pharmaceutical composition comprises a combination of different vector populations. In one embodiment, a pharmaceutical composition is provided comprising a formulation buffer containing a single rAAV population as described herein. The methods provided herein provide for the co-administration of two separate vector-containing suspensions.

method

The compositions provided herein can be used to treat ornithine carbamoyltransferase deficiency. This document provides a method for treating the human condition by co-administering a dual-vector system as described herein. In some embodiments, this document provides compositions comprising a non-replicating recombinant adeno-associated virus serotype rh79 vector: AAVrh79.TBG.M2PCSK9.WPRE.bGH and AAVrh79.hHDR.TBG.hOTCco.bGH for the treatment of ornithine carbamoyltransferase deficiency.

In one embodiment, a method for treating OTC deficiency in a subject is provided. The method comprises co-administering to a subject suffering from OTC deficiency a gene-edited AAV vector, the gene-edited AAV vector comprising a sequence encoding a nuclease targeting PCSK9 and a regulatory sequence guiding the expression of the nuclease in target cells including the PCSK9 gene; and a donor AAV vector comprising a transgene and a regulatory sequence guiding the expression of the OTC transgene in target cells. In one embodiment, the subject is a newborn.

In some embodiments, the gene-editing AAV vector and the donor vector are delivered substantially simultaneously via the same route. In other embodiments, the gene-editing vector is delivered first. In still other embodiments, the donor vector is delivered first.

In one embodiment, the dose of rAAV is about 1 x ^10⁹ GC to about 1 x ^10¹⁵ genomic copies (GC) per dose (to treat subjects with an average weight of 70 kg), and preferably 1.0 x ^10¹² GC to 2.0 x ^10¹⁵ GC for human patients. In another embodiment, the dose is less than about 1 x ^10¹⁴ GC/body weight of the subject. In some embodiments, the dose administered to the patient is at least about 1.0 x ^10⁹ GC/kg, about 1.5 x ^10⁹ GC/kg, about 2.0 x ^10⁹ GC/kg, about 2.5 x ^10⁹ GC/kg, about 3.0 x ^10⁹ GC/kg, about 3.5 x ^10⁹ GC/kg, about 4.0 x ^10⁹ GC/kg, about 4.5 x ^10⁹ GC/kg, about 5.0 x 10⁹ GC/kg, about 5.5 x ^10⁹ GC/kg, about 6.0 x ^10⁹ GC/kg, about 6.5 x ^10⁹ GC/kg, about 7.0 x ^10⁹ GC/kg, about 7.5 x ^10⁹ GC/kg, about 8.0 x ^10⁹ GC/kg, and about 8.5 x ^10⁹ GC/kg ^. GC/kg, approximately 9.0 x ^10⁹ GC/kg, approximately 9.5 x ^10⁹ GC/kg, approximately 1.0 x ^10¹⁰ GC/kg, approximately 1.5 x ^10¹⁰ GC/kg, approximately 2.0 x ^10¹⁰ GC/kg, approximately 2.5 x ^10¹⁰ GC/kg, approximately 3.0 x ^10¹⁰ GC/kg, approximately 3.5 x ^10¹⁰ GC/kg, approximately 4.0 x ^10¹⁰ GC/kg, approximately 4.5 x ^10¹⁰ GC/kg, approximately 5.0 x ^10¹⁰ GC/kg, approximately 5.5 x ^10¹⁰ GC/kg, approximately 6.0 x ^10¹⁰ GC/kg, approximately 6.5 x ^10¹⁰ GC/kg, approximately 7.0 x ^10¹⁰ GC/kg, approximately 7.5 x ^10¹⁰ GC/kg, approximately 8.0 x ^10¹⁰ GC/kg, approximately 8.5 x ^10¹⁰ GC/kg, approximately 9.0 x ^10¹⁰ GC/kg, approximately 9.5 x ^10¹⁰ GC/kg, approximately 1.0 x ^10¹¹ GC/kg, approximately 1.5 x ^10¹¹ GC/kg, approximately 2.0 x ^10¹¹ GC/kg, approximately 2.5 x ^10¹¹ GC/kg, approximately 3.0 x ^10¹¹ GC/kg, approximately 3.5 x ^10¹¹ GC/kg, approximately 4.0 x ^10¹¹ GC/kg, approximately 4.5 x ^10¹¹ GC/kg, approximately 5.0 x ^10¹¹ GC/kg, approximately 5.5 x ^10¹¹ GC/kg, approximately 6.0 x ^10¹¹ GC/kg, approximately 6.5 x ^10¹¹ GC/kg, approximately 7.0 x ^10¹¹ GC/kg, approximately 7.5 x ^10¹¹ GC/kg, approximately 8.0 x ^10¹¹ GC/kg, approximately 8.5 x ^10¹¹ GC/kg, approximately 9.0 x ^10¹¹ GC/kg, approximately 9.5 x ^10¹¹ GC/kg, approximately 1.0 x ^10¹² GC/kg, approximately 1.5 x ^10¹² GC/kg, approximately 2.0 x ^10¹² GC/kg, approximately 2.5 x ^10¹² GC/kg, approximately 3.0 x ^10¹² GC/kg, approximately 3.5 x ^10¹² GC/kg, approximately 4.0 x ^10¹² GC/kg, approximately 4.5 x ^10¹² GC/kg, approximately 5.0 x ^10¹² GC/kg, approximately 5.5 x ^10¹² GC/kg, approximately 6.0 x ^10¹² GC/kg, approximately 6.5 x ^10¹² GC/kg, approximately 7.0 x ^10¹² GC/kg, approximately 7.5 x ^10¹² GC/kg, approximately 8.0 x ^10¹² GC/kg, approximately 8.5 x ^10¹² GC/kg, approximately 9.0 x ^10¹² GC/kg, approximately 9.5 x ^10¹² GC/kg, approximately 1.0 x ^10¹³ GC/kg, approximately 1.5 x ^10¹³ GC/kg, approximately 2.0 x ^10¹³ GC/kg, approximately 2.5 x ^10¹³ GC/kg GC/kg, approximately 3.0 x ^10¹³ GC/kg, approximately 3.5 x ^10¹³ GC/kg, approximately 4.0 x ^10¹³ GC/kg, approximately 4.5 x ^10¹³ GC/kg, approximately 5.0 x ^10¹³ GC/kg, approximately 5.5 x ^10¹³ GC/kg, approximately 6.0 x ^10¹³ GC/kg, approximately 6.5 x ^10¹³ GC/kg, approximately 7.0 x ^10¹³ GC/kg, approximately 7.5 x ^10¹³ GC/kg, approximately 8.0 x ^10¹³ GC/kg, approximately 8.5 x ^10¹³ GC/kg, approximately 9.0 x ^10¹³ GC/kg, approximately 9.5 x ^10¹³ GC/kg, or approximately 1.0 x ^10¹⁴ GC/kg body weight of the subject.

Applying the vector in sufficient quantities to transfect cells and provide adequate levels of gene transfer and expression to deliver therapeutic benefit without causing excessive side effects or having medically acceptable physiological effects can be determined by a person skilled in the medical field. In some embodiments, intravenous administration is used.

The system described herein may be therapeutically useful if it produces sufficient amounts of functional enzymes or proteins to improve a patient's condition. In some embodiments, gene expression levels as low as 5% of those in healthy patients will provide sufficient therapeutic efficacy. 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%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47% of the normal range (levels) observed in humans (or other veterinary subjects). 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. For example, "functional enzyme" means a gene encoding a wild-type enzyme (e.g., an OTC enzyme) that 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%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, or 47% of a disease-independent natural variant or polymorph of the wild-type enzyme. 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or approximately the same or greater than 100% of the biological activity level. Similarly, patients lacking detectable amounts of the enzyme can be rescued by delivering the enzyme function to less than 100% of its activity level, and can optionally be subsequently treated further. In some embodiments, the expression level in patients may be higher than in “normal” healthy subjects when the gene function is delivered via a donor template. As described herein, the therapies described herein can be used in conjunction with other treatments (i.e., standards of care for the subject’s (patient's) diagnosis).

In one embodiment, the method further includes administering an immunosuppressive co-therapy to the subject. For example, such immunosuppressive co-therapy may be initiated prior to delivery of rAAV or the disclosed composition if an undesirable high level of neutralizing antibodies against the AAV capsid is detected. In some embodiments, as a precaution, the co-therapy may also be initiated prior to rAAV delivery. In some embodiments, for example, if an undesirable immune response is observed after treatment, the immunosuppressive co-therapy is initiated after rAAV delivery.

Immunosuppressants used in such co-therapy include, but are not limited to, glucocorticoids, steroids, antimetabolites, T-cell inhibitors, macrolides (e.g., rapamycin or rapamycin analogs), and cell growth inhibitors, including alkylating agents, antimetabolites, cytotoxic antibiotics, antibodies, or agents active against immunoaffinity. Immunosuppressants may include prednisolone, nitrogen mustard, nitrosourea, platinum compounds, methotrexate, azathioprine, mercaptopurine, fluorouracil, dactinomycin, anthracycline, mitomycin C, bleomycin, mithramycin, IL-2 receptor (CD25) or CD3 targeted antibodies, anti-IL-2 antibodies, cyclosporin, tacrolimus, sirolimus, IFN-β, IFN-γ, opioids, or TNF-α (tumor necrosis factor-α) binders. In some embodiments, immunosuppressive therapy may be initiated on day 0, day 1, day 2, day 7, or more before rAAV administration, or on day 0, day 1, day 2, day 3, day 7, or more after rAAV administration. Such therapy may involve a single drug (e.g., prednisolone) or co-administration of two or more drugs (e.g., prednisolone, mycophenolate mofetil (MMF), and/or sirolimus (i.e., rapamycin)) on the same day. One or more of these drugs may continue to be administered at the same or adjusted dose after gene therapy administration. Such therapy may continue for approximately one week (7 days), two weeks, three weeks, approximately 60 days, or longer, as needed. In some embodiments, a tacrolimus-free regimen is selected.

In another embodiment, the method includes co-treatment with standard OTC therapy. Treatment of OTC deficiency focuses primarily on dietary management of blood ammonia levels to avoid hyperammonemia or to remove excess ammonia from the blood during an episode of hyperammonemia ( NORD, 2021 ). Individuals with OTC deficiency follow dietary restrictions that limit their protein intake to control blood ammonia levels. Dietary restrictions for infants must be carefully balanced, ensuring adequate protein intake for normal growth while avoiding excessive protein intake to prevent episodes of hyperammonemia ( Berry and Steiner, 2001 ). Therefore, infants are given a high-calorie, low-protein diet supplemented with essential amino acids. During an episode of hyperammonemia, all protein in the patient's 24-hour diet may be removed ( NORD, 2021 ).

Several drugs have been designed to promote the removal of nitrogen from the blood. Sodium phenylbutyrate (Buphenyl) is approved by the U.S. Food and Drug Administration (FDA) for the treatment of chronic hyperammonemia in patients with OTC deficiency. Once metabolized, Buphenyl is converted to phenylacetate, which conjugates with glutamine to form phenylacetylglutamine, which is excreted by the kidneys, providing an alternative pathway for nitrogen excretion. Chlorobutyrate glyceride (Ravicti) is also FDA-approved for the treatment of chronic hyperammonemia in patients with urea cycle disorders. Like Buphenyl, Ravicti is converted to phenylacetate and follows the same nitrogen excretion mechanism ( Lichter-Konecki et al., 1993 ; Gordon, 2003 ; Magellan, 2021 ). Finally, Ammonul (sodium phenylacetate and sodium benzoate) is FDA-approved as adjunctive therapy for the treatment of acute hyperammonemia in patients with urea cycle disorders. The sodium phenylacetate component of ammonur follows the same nitrogen excretion mechanism as the phenylacetic acid metabolites produced by Buphenyl and Revian. The sodium benzoate component of ammonur conjugates with glycine to form hippuric acid, which is excreted by the kidneys and nitrogen is removed through this process. Sodium benzoate is also used as an oral formulation for long-term maintenance of OTC deficiency and is generally superior to Buphenyl and Revian due to its fewer side effects ( Lichter-Konecki et al., 1993 ).

Many patients with OTC deficiency are treated with arginine or citrulline, both of which are essential to ensure that protein synthesis proceeds at a normal rate. Citrulline is often chosen for chronic treatment compared to arginine because it incorporates aspartic acid into the urea cycle, meaning it contributes an extra nitrogen molecule to this pathway ( Lichter-Konecki et al., 1993 ; Magellan, 2021 ).

In cases of hyperammonemia progressing to vomiting and somnolence, hospitalization may be required, and the individual may need to be treated with an IV infusion containing arginine and amonulol. If these treatments are unsuccessful, hemofiltration or hemodialysis may be necessary to rapidly lower blood ammonia levels ( Lichter-Konecki et al., 1993 ).

Liver transplantation is the most effective strategy for preventing hyperammonemia crises and neurodevelopmental deterioration in patients with severe neonatal onset of OTC deficiency. In patients with mild or well-controlled OTC deficiency, stressors can trigger life-threatening hyperammonemia attacks at any age. In addition to the decreased quality of life due to dietary restrictions and chronic treatment with nitrogen scavengers (which may have significant side effects), the fear of such attacks also motivates many patients to seek liver transplantation, even when their condition is well-controlled ( Lichter-Konecki et al., 1993 ).

On one hand, a method is provided for treating patients with ornithine carbamoyltransferase (OTC) deficiency, the method using a nuclease expression cassette comprising a wide range of nucleases that recognize intragenic sites in the human PCSK9 gene under the control of a TBG promoter as described herein. The method further comprises administering an expression cassette carrying an OTC transgene carrying SEQ ID NO:4 or a sequence sharing at least 90% identity with it, as described herein.

Various assays exist for measuring OTC expression and activity levels in vitro or in vivo. See, for example, X Ye et al., 1996, Prolonged metabolic correction in adult ornithine transcarbamylase-deficient mice with adenoviral vectors. (J Biol Chem 271:3639–3646). For instance, OTC enzyme activity can be measured using liquid chromatography-mass spectrometry with stable isotope dilution to detect the formation of citrulline normalized relative to [1,2,3,4,5-13C5]citrulline (98% 13C). The method described is adapted from a previously developed assay for detecting N-acetylglutamate synthase activity [Morizono H et al., Mammalian N-acetylglutamate synthase. Molecular Genetics and Metabolism (Mol Genet Metab.) 2004; 81(Supplement 1):S4–11.]. Freshly frozen liver sections were weighed and simply homogenized in a buffer containing 10 mM HEPES, 0.5% Triton X-100, 2.0 mM EDTA, and 0.5 mM DTT. The volume of the homogenization buffer was adjusted to obtain 50 mg/ml of tissue. Enzyme activity was measured using 50 mM Tris-acetate, 4 mM ornithine, and 5 mM carbamoyl phosphate at pH 8.3 containing 250 μg of liver tissue. The enzyme activity was initiated by adding freshly prepared 50 mM carbamoyl phosphate dissolved in 50 mM Tris-acetate at pH 8.3, and incubated at 25 °C for 5 minutes. An equal volume of 30% TCA containing 5 mM 13C5-citrulline was then added for quenching. Fragments were separated by microcentrifugation for 5 minutes, and the supernatant was transferred to a vial for mass spectrometry analysis. Ten μL of sample was injected into an Agilent 1100 series LC-MS under isocratic conditions, with a mobile phase of 93% solvent A (1 L water containing 1 mL trifluoroacetic acid): 7% solvent B (1 L 1:9 water/acetonitrile containing 1 mL trifluoroacetic acid). Peaks corresponding to citrulline [176.1 m/z] and 13C5-citrulline (181.1 m/z) were quantified, and their ratios were compared to the ratios of the citrulline standard curve run for each assay. Normalize the sample relative to the total liver tissue or protein concentration measured using the Bio-Rad Protein Assay Kit (Bio-Rad, Hercules, CA). Other assays that do not require liver biopsy may also be used. One such assay is a plasma amino acid assay, in which the ratio of glutamine to citrulline is assessed, and a high glutamine level (>800 μL/L) and a low citrulline level (e.g., single digits) suggests a urea cycle defect. Plasma ammonia levels can be measured, and a concentration of approximately 100 μmol/L indicates OTCD. Blood gases can be assessed if the patient is hyperventilating; respiratory alkalosis is common in OTCD. Urinary orotic acid, such as creatine at a level greater than approximately 20 μmol/mM, indicates OTCD, as does elevated urinary orotic acid after an allopurinol challenge test. The diagnostic criteria for OTCD have been proposed in Tuchman et al., 2008, Urea Cycle Disorders Consortium (UCDC) of the Rare Disease Clinical Research Network (RDCRN). (Tuchman M et al., Consortium of the Rare Diseases Clinical Research Network. Cross-sectional multicenter study of patients with urea cycle disorders in the United States. Molecular Genetics & Metabolism. 2008; 94:397–402, which is incorporated herein by reference.) See also http://www.ncbi.nlm.nih.gov/books/NBK154378/, which discusses the current standards of care for OTCD.

In some embodiments, the nuclease expression cassette, viral vector (e.g., rAAV), or any of the thereof in the pharmaceutical compositions described herein can be administered for gene editing in a patient. In some embodiments, the method can be used for non-embryonic gene editing. In some embodiments, the patient is an infant (e.g., newborn to approximately 4 months of age). In some embodiments, the patient is older than an infant, e.g., 12 months of age or older.

As used herein, “a/an” or “the” can mean one or more. For example, “a” cell can mean a single cell or multiple cells.

As used herein, the term "specificity" refers to the ability of a nuclease to recognize and cleave a double-stranded DNA molecule only at a specific base pair sequence, known as the recognition sequence, or only at a specific set of recognition sequences. This set of recognition sequences will share certain conserved positions or sequence motifs, but may be degenerate at one or more positions. Highly specific nucleases are capable of cleaving only one or a very small number of recognition sequences. Specificity can be determined by any method known in the art.

The (gene editing and donor) expression cassettes described herein can be engineered into any suitable genetic element, such as a vector, for delivery to target cells. As used herein, a "vector" is a biological or chemical portion comprising a nucleic acid sequence that can be introduced into a suitable host cell to replicate or express the nucleic acid sequence.

The term "plasmid" or "plasmid vector" is generally designated herein by a lowercase "p" preceding and/or following the vector name. The plasmids, other cloning and expression vectors that may be used according to the invention, their properties, and their construction/manipulation methods will be apparent to those skilled in the art.

As used herein, the term “operably linked” refers to both the expression control sequence adjacent to the gene of interest and the expression control sequence that acts trans or at a distance to control the expression of the gene of interest.

The term "exogenous" as used to describe nucleic acid sequences or proteins means that the nucleic acid or protein is not naturally present at its location in the chromosome or host cell. Exogenous nucleic acid sequences also refer to sequences that originate from and are inserted into the same expression cassette or host cell, but exist in a non-natural state, for example, with different copy numbers or under the control of different regulatory elements.

The term "heterologity," when used in conjunction with proteins or nucleic acids, indicates that the proteins or nucleic acids comprise two or more sequences or subsequences that are not found to be identical to each other in nature. For example, nucleic acids are often recombinantly produced, having two or more sequences from unrelated genes arranged to produce new functional nucleic acids. For example, in one embodiment, the nucleic acid has a promoter from one gene arranged to guide the expression of the coding sequence from a different gene.

As used herein, the term "host cell" can refer to a packaging cell line in which a vector (e.g., recombinant AAV) is generated from a producing plasmid. Alternatively, the term "host cell" can refer to any target cell in which a transgene is desired to be expressed. Thus, "host cell" refers to a prokaryotic or eukaryotic cell containing an exogenous or heterologous nucleic acid sequence introduced into the cell by any means (e.g., electroporation, calcium phosphate precipitation, microinjection, transformation, viral infection, transfection, liposome delivery, membrane fusion technology, high-speed DNA-coated clumps, viral infection, and protoplast fusion). In some embodiments herein, the term "host cell" refers to a culture of cells from various mammalian species used for in vitro evaluation of the compositions described herein. In other embodiments herein, the term "host cell" refers to a cell used to generate and package a viral vector or recombinant virus. In still other embodiments, the term "host cell" is intended to refer to a target cell in a subject treating a disease or condition as described herein in vivo. In some embodiments, the term "host cell" is a liver cell or hepatocyte.

"Subject" refers to a mammal, such as a human, mouse, rat, guinea pig, dog, cat, horse, cow, or pig, or a non-human primate, such as a monkey, chimpanzee, baboon, or gorilla. "Patient" refers to a human. Veterinary subjects refer to non-human mammals. In some embodiments, the subject's PCSK9 gene is not defective.

In some embodiments, the subject has an OTC deficiency or is at risk of developing an OTC deficiency. In some embodiments, the subject has a documented genetic confirmation of an OTCD mutation. In some embodiments, the subject has or has previously had a hyperammonemia crisis (HAC). In some embodiments, the subject is currently receiving treatment for an OTC deficiency, for example, using at least one nitrogen scavenger therapy and/or a protein-restricted diet.

In some embodiments, the subject is male. In some embodiments, it is desirable to treat the subject within hours of birth, such as at least 12 hours, 24 hours, 36 hours, or 48 hours of age. In other embodiments, it is desirable to treat the subject within days of birth, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after birth. In other embodiments, it is desirable to treat the subject within weeks of birth, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks after birth. In other embodiments, it is desirable to treat the subject within months of birth, such as 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 after birth. In some embodiments, subjects are treated between 24 hours and 4 months of age. In some embodiments, subjects are male infants treated between 24 hours and 4 months of age with documented genetic confirmation of OTCD mutations consistent with neonatal onset of OTC deficiency, currently or previously having or not having hyperammonemia crisis (HAC), and currently receiving at least one nitrogen scavenger therapy and a protein-restricted diet. The proposed study population includes subjects with high unmet needs, and in these subjects, stabilization or improvement in the frequency and severity of recurrent hyperammonemia events may be observed due to reduced morbidity and mortality, and increased survival, while potentially delaying the need for liver transplantation within a 52-week protocol timeframe.

"Replication-deficient virus" or "viral vector" refers to a synthetic or artificial viral particle in which an expression cassette containing the gene of interest is packaged within a viral capsid or envelope, wherein any viral genome sequence also packaged within the viral capsid or envelope is replication-deficient; that is, the synthetic or artificial viral particle cannot generate progeny viruses but retains the ability to infect target cells. In one embodiment, the genome of the viral vector does not contain genes encoding enzymes required for replication (the genome can be engineered to be "gutless"—containing only the gene of interest, flanked by signals required for amplification and packaging of the artificial genome), but these genes can be supplied during production. Therefore, this is considered safe for use in gene therapy because replication and infection via progeny viral particles do not occur unless the viral enzymes required for replication are present.

In the context of nucleic acid sequences, the terms "sequence identity," "sequence identity percentage," or "identity percentage" refer to the fact that residues in two sequences are identical when compared to obtain maximum correspondence. The length of a sequence identity comparison is expected to exceed 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. However, identity between smaller fragments may also be expected, such as at least about nine nucleotides, typically at least about 20 to 24 nucleotides, at least about 28 to 32 nucleotides, or at least about 36 or more nucleotides. Similarly, for amino acid sequences, the "sequence identity percentage" can be readily determined over the full length of a protein or over a fragment thereof. Suitable fragments are at least about 8 amino acids long and can be up to about 700 amino acids long. Examples of suitable fragments are described in this article.

When referring to amino acids or fragments thereof, the terms "fundamental homology" or "fundamental similarity" mean that, when optimally aligned with appropriate amino acid insertions or deletions in 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, homology is found in the full-length sequence or in proteins thereof (e.g., cap proteins, rep proteins, or fragments of at least 8 amino acids, or more preferably at least 15 amino acids). Examples of suitable fragments are described herein.

The term "highly conservative" means at least 80% identity, preferably at least 90% identity, and more preferably more than 97% identity. Identity can be readily determined by those skilled in the art using algorithms and computer programs known to them.

Typically, when referring to “identity,” “homology,” or “similarity” between two different adeno-associated viruses, “identity,” “homology,” or “similarity” is determined by referring to an “aligned” sequence. An “aligned” sequence or “alignment” refers to multiple nucleic acid or protein (amino acid) sequences, typically containing corrections for missing or additional bases or amino acids, compared to a reference sequence. In this example, AAV alignment is performed using a publicly available AAV9 sequence as a reference point. Alignment is performed using any of a variety of publicly available or commercially available multiple sequence alignment programs. Examples of such programs include “ClustalΩ,” “ClustalW,” “CAP Sequence Assembly,” “MAP,” and “MEME,” which are accessible via web servers on the Internet. Other sources of such programs are known to those skilled in the art. Alternatively, the vector NTI utility is also used. Many algorithms known in the art can be used to measure nucleotide sequence identity, including those contained in the above-described programs. As another example, the Fasta ^™ program version 6.1 of GCG can be used to compare polynucleotide sequences. Fasta ^™ provides the alignment of the best overlap region between the query sequence and the search sequence, and the percentage of sequence identity. For example, the percentage of sequence identity between nucleic acid sequences can be determined using Fasta ^™ with its default parameters (font size 6 and NOPAM coefficients of the scoring matrix), as provided in GCG version 6.1, which is incorporated herein by reference. Several sequence alignment programs can also be used for amino acid sequences, such as “ClustalΩ”, “Clustal X”, “MAP”, “PIMA”, “MSA”, “BLOCKMAKER”, “MEME”, and “Match-Box” programs. Typically, any of these programs is used with default settings, but those skilled in the art can change these settings as needed. Alternatively, those skilled in the art can utilize another algorithm or computer program that provides at least the same level of identity or alignment as provided by the reference algorithms and programs. See, for example, JDthomson et al., Nucleic Acid Res., “A comprehensive comparison of multiple sequence alignments”, 27(13):2682-2690 (1999).

As used herein, the term “about” refers to a variation of ±10% of a reference integer and values in between. For example, “about” for 40 base pairs includes ±4 (i.e., 36–44, which includes integers 36, 37, 38, 39, 40, 41, 42, 43, 44). For other values, particularly when referring to percentages (e.g., 90% identity, about 10% difference, or about 36% mismatch), the term “about” includes all values within the range, including both integers and fractions.

As used throughout this specification and claims, the terms "comprising," "containing," "including," and variations thereof encompass other components, elements, integers, steps, etc. Conversely, the terms "consisting of," and variations thereof do not encompass other components, elements, integers, steps, etc.

Unless otherwise defined in this specification, the technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art and by reference to the published text, which provides a general guide for those skilled in the art to the many terms used in this application.

Example

A genome editing approach for treating ornithine carbamoyltransferase deficiency (OTCD), which can lead to fatal onset of hyperammonemia in infancy, is described. The goal of genome editing is to make the therapeutic effect durable and achieve it in all OTCD patients, regardless of mutations in the OTCD. This is achieved by treating surviving newborns with two AAV vectors: one vector delivers a nuclease to create a double-strand break at a safe harbor site, and a second vector delivers an OTC mini-gene to knock into this site. It is hypothesized that hepatocyte division in the neonatal liver will facilitate efficient knock-in of the OTC gene and will eliminate non-integrated vector genomes through dilution. The PCSK9 gene was chosen as the safe harbor site, and a broad-spectrum nuclease called ARCUS was used to target the PCSK9 gene, based on previous work in adult rhesus monkeys that showed a safe, efficient, and stable reduction of PCSK9 after AAV delivery of ARCUS. Preliminary studies of genome editing for OTCD were performed in an OTC-deficient mouse by germline modification of exon 7 of the endogenous PCSK9 gene, making the mouse susceptible to the PCSK9ARCUS nuclease. Both vectors were injected into newborn mice to induce efficient knock-in of the human OTC mini-gene, and the mice were protected from fatal hyperammonemia upon challenge with a high-protein diet. Key safety and efficacy parameters were evaluated in preparation for clinical studies in newborn and juvenile macaques. A total of 24 animals were treated with the AAV vectors, and liver biopsies from 3-month-old and 12-month-old animals were analyzed. In these studies, the effects of the following parameters on editing efficiency and toxicity were evaluated: transgene (human factor IX and human OTC), promoter driving ARCUS, clade E capsid, length of the donor transgene lateralized, and age of the macaque at administration. AAV vector injection was found to be very safe, with no evidence of elevated transaminases or liver histopathology in any ARCUS-treated animals. The key measure of efficacy in primate models was transduction efficiency, measured by in situ hybridization and immunostaining to detect cells expressing human OTC mRNA and protein, respectively. The highest and most consistent results were obtained using a novel clade E capsid driving ARCUS with the TBG promoter in the first vector and a 500 bp homologous arm lateralized on the donor vector. This combination achieved 10.0 ± 6.4% (N = 6) transduction, exceeding the threshold considered to provide substantial benefit to patients, which is approximately 5% of cells expressing OTC. Preliminary data indicate that the editing level is stable over one year and that effective targeted insertion can be achieved when injected into 3-month-old rhesus monkeys. Molecular analysis of the PCSK9 target locus revealed that the vast majority of the vector genome knock-in occurred via non-homologous end joining (NHEJ) rather than homologous directed repair (HDR). In conclusion, the significant unmet need for neonatal OTCD necessitates consideration of experimental therapies, such as the genome editing described in this report.

Example 1 - Materials and Methods

The AAV vector was constructed according to the previously established procedures and the manufacturer's instructions. When indicated, the AAVhu37 or AAVrh79 capsid was used for experiments as described herein.

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

Example 2 - ARCUS2-mediated hOTC gene targeting in neonatal NHP

Newborn (1–16 days old) or juvenile (3–4 months old) rhesus monkeys were used for non-GLP-compliant point-of-care pharmacology studies. ARCUS broadly targets a 22-bp sequence present in the PCSK9 gene in both humans and rhesus monkeys. Therefore, rhesus monkeys can be used to evaluate target editing (pharmacology) and safety/toxicology. Furthermore, newborn and juvenile rhesus monkeys possess anatomical and physiological characteristics similar to human infants, which will allow for the use of the anticipated clinical ROA (IV). The anticipated anatomical and ROA similarity is expected to yield representative vector distribution and transduction profiles, enabling a more accurate assessment of the pharmacology and toxicity of the test article, including targeted and off-target editing, as well as clinicopathology that is impossible to achieve in newborn mice.

Figure 1A shows the timeline of the study presented in this paper. Figure 1B provides information on the dosage groups, while a schematic diagram of the vectors is shown in Figure 2. Neonatal NHPs (days 1–16) were administered the ARCUS2 nuclease vector and the donor vector, which had HDR arms of varying lengths—500 bp arms or short HDR arms. Figure 3 is a summary table showing the experimental data. All 14 neonatal rhesus monkeys tolerated the vector injections well (i.e., no apparent clinical sequelae) and gained weight over time (Figure 4E). Liver enzyme levels were within the normal range, except for a transient and moderate increase in ALT levels in some animals on day 14 (Figure 4B). Liver inflammation was significantly reduced in neonatal and juvenile NHPs compared to adult NHPs administered only AAV. Arcus (Figure 4J). The pancreas was the only non-hepatic tissue in which any insertions or deletions were identified (Figure 4I).

Analysis of day 0 plasma samples collected from newborns prior to drug administration showed that three animals (21-111, 21-113, and 21-122) had high levels (≥400) of AAVrh79-binding antibodies (Figure 3). These pre-existing anti-AAVrh79 antibodies will block AAV gene transfer.

PCSK9 levels were tracked over time in all newborn animals (including donor-only controls). PCSK9 levels varied on day 0 of the newborns (Figure 4A). Nine animals showed a decreasing trend in PCSK9 levels after vector administration, including one donor-only control, while the remaining five animals showed a sustained or transient increase in PCSK9 levels after administration (Figure 4A).

Liver biopsies were performed on day 84 and at 1 year. The transduction efficiency of hOTC in the liver was assessed by dual ISH using hOTC-specific and ARCUS-specific probes to detect transgenic mRNA and by OTC immunofluorescence assay for human OTC protein, followed by quantification on scanned slides (Fig. 4C-ISH; Fig. 4D-4F). Three animals (21-111, 21-113, and 21-122) with pre-existing anti-AAVrh79 binding antibodies showed no OTC-positive hepatocytes by either method. Two donor-only control animals continued to show low levels (≤2%) of hOTC transduction. The highest transduction efficiency was detected in the two animals co-administered with the donor vectors AAVrh79.TBG.PI.ARCUS.WPRE.bGH and AAVrh79.rhHDR.TBG.hOTCco.bGH (GTP-506) (27.8% and 23.6% as detected by ISH). Hepatocytes expressing positive hOTC were also found in the clusters. These levels were above the threshold for substantial benefit to patients, which is approximately 5% of cells expressing OTC. The data indicate that ARCUS driven by the strong TBG promoter and the long homologous arm in the donor vector produced the greatest editing. Furthermore, similarly high levels of editing were observed in the same ARCUS vector and the same homologous arm in the F9 donor vector in the NB study. Mouse OTCD pharmacology studies showed that donors containing different homologous arms exhibited consistently high editing activity, relevant to mice containing the long homologous arm vector in this study. The level of editing in NB NHP with GTP-506 was sufficient to demonstrate a robust therapeutic effect.

On day 84, liver biopsy samples from each animal were subjected to molecular analysis to measure transgene copy number, mRNA expression level, on-target editing, and off-target editing for each diploid genome (Fig. 4H). Consistent with transduction efficiency analysis, two animals in group 6 (21-157 and 21-175) had the highest hOTC vector GC (Fig. 4F), hOTC mRNA (Fig. 4G), and on-target insertion/deletion percentage (Fig. 4H). The ARCUS vector GC in the animals was 1/7 to 1/2 that of the hOTC vector GC, while the ARCUS mRNA level was 1/765 and 1/23 of the hOTC mRNA level, respectively (Fig. 4F and 4G).

In liver biopsy samples taken on day 84 of this study, 2 to 40 potential off-target activities were identified by ITR sequence assessment. Several off-target sites were detected in multiple animals, including those given the human factor IX (hfIX) donor vector. Off-target editing was further characterized by amplicon sequencing at potential off-target sites. Figure 14A provides a list of off-target sites, along with their chromosomal locations and best matches with common off-target sequences. Figure 14B is a graph showing the percentage of insertions and deletions for OT1–OT10. Editing of OT1, OT4, and OT5 was significantly higher in ARCUS+ donor animals than in non-nuclease controls. Off-target editing was less common in neonates/infants than in adult NHPs.

The growing livers of newborn/juvenile primates readily accept site-specific insertion of donor genes. OTC levels consistently exceed the 5% therapeutic threshold, and this effect appears to be highly durable. Furthermore, newborn/juvenile primates exhibit tolerance to the toxicity of systemic AAV. When comparing mouse models of NHP and OTCD, dose- and site-specific integration showed excellent correlation.

In summary, the combination of the ARCUS vector and the hOTCco donor vector, when co-administered in newborn rhesus monkeys, achieved liver transduction efficiencies of 12-18.6% at 3 months post-dose, all exceeding the threshold for substantial patient benefit (approximately 5% of OTC-expressing hepatocytes). Animals in this study are being followed up for long-term efficiency and safety assessments. A second liver biopsy was performed 1 year post-dose to evaluate the stability of hOTC transduction, liver histopathology, and on-target and off-target hepatic activity.

Example 3 - PCSK9-hE7-KI mouse model

Because the ARCUS targeting sequence in the human and macaque PCSK9 gene is not conserved with that in the mouse Pcsk9 gene, ARCUS cannot be used for genome editing at mouse genome loci. Therefore, The Jackson Laboratory was commissioned to develop a knock-in mouse model in which the region containing exon 7 of the mouse Pcsk9 gene is replaced by the region containing exon 7 of the human PCSK9 gene, named the PCSK9-hE7-KI mouse (Figures 5A-5C). This model can be used to evaluate in vivo genome editing and gene targeting efficiency. The PCSK9-hE7-KI mouse was then crossed with spfash mice. ^{The spfash mouse} has a G-to-A point mutation at the splice donor site at the end of exon 4 of the Otc gene, which leads to aberrant splicing of Otc mRNA and a 20-fold reduction in both OTC mRNA and protein expression ( Hodges and Rosenberg, 1989 ). Affected animals have 5%–10% residual OTC activity and can survive on feed, but may develop potentially fatal hyperammonemia when they are fed a high-protein diet ( Yang et al., 2016 ).

The PCSK9-hE7-KI.spf ^ash mouse model can be used to evaluate the in vivo gene-targeting efficacy of human OTCs and demonstrate the correlation between targeting efficiency and efficacy. However, due to the small size of newborn mice, the clinical efficacy of blood clinicopathology and gene targeting can only be evaluated and used as a terminal procedure after the mice are weaned and reach sufficient weight.

Figure 6 shows the sequence alignment of the 265 bp sequences representing the human PCSK9 sequence, mouse PCSK9 (mPCSK9), and rhesus monkey PCSK9 (rhPCSK9) representing the PCSK9-hE7 knock-in allele. Within this 265 bp region, there are 6 mismatches between the human and rhesus monkey sequences. The rodent and primate sequences diverge beyond this window due to the insertion of different LINEs and LTRs. There is a 2-amino acid difference in exon 7 between humans and mice. The expression level of mPCSK9 in hE7-KI mice is normal, as measured by ELISA.

Example 4 - In vivo OTC gene targeting Pcsk9-hE7-KI.spf ^ash Pcsk9 locus in pups

This non-GLP-compliant pharmacology study evaluated whether large-scale nuclease-mediated human OTC gene knock-in in neonatal PCSK9-hE7- ^KI.spfash mice could achieve therapeutic human OTC expression in the target tissue (liver) for the treatment of OTC deficiency, following a single co-administration of an ARCUS nuclease-expressing vector and a human OTC donor vector via a planned clinical ROA (IV). A schematic diagram of the experimental design is shown in Figure 8A.

On day 0, newborn (PND 1–2) male PCSK9-hE7- ^KI.spfash mice were IV co-administered with a combination of 1.0 x ^10¹³ GC/kg of the AAVrh79 vector expressing a wide range of ARCUS nucleases (AAVrh79.TBG.PI.ARCUS.WPRE.bGH) and 3.0 x ^10¹³ GC/kg of one of three different AAVrh79hOTCco donor vectors (Figure 8B). The vector expressing a wide range of ARCUS nucleases (AAVrh79.TBG.PI.ARCUS.WPRE.bGH) evaluated in this study was identical to the lead clinical candidate, and each hOTCco donor vector was identical to the lead clinical candidate except for the HDR arm. Specifically, while the clinical candidates included a long version of the human HDR sequence (AAVrh79.hHDR.TBG.hOTCco.bGH), the hOTCco donor vectors evaluated in this study included a mouse-human hybrid HDR sequence (AAVrh79.mhHDR.TBG.hOTCco.bGH), a shorter version of the human HDR sequence (AAVrh79.shHDR.TBG.hOTCco.bGH), or no HDR sequence (AAVrh79.TBG.hOTCco.bGH). Figure 7 shows a comparison of the HDR arm homology with the human sequence, knock-in mouse sequence, and NHP sequence. As a negative control, another age-matched PCSK9-hE7-KI.spf ^ash mouse was administered a combination of the AAVrh79 vector (AAVrh79.TBG.PI.EGFP.WPRE.bGH) and AAVrh79.mhHDR.TBG.hOTCco.bGH, which does not express a wide range of nucleases.

Vital assessments included daily activity monitoring, weight measurement, evaluation of plasma PCSK9 and plasma _NH3 and urinary orotic acid levels after a high-protein diet challenge, and partial hepatectomy on day 120 to assess the stability of human OTC transduction after two-thirds partial hepatectomy. Subgroups within each group were challenged with a 10-day high-protein diet on days 49 and 170, followed by autopsy at the end of the challenge. At autopsy, livers were collected to assess human OTC gene knock-in, including assessment of human OTC mRNA expression (in situ hybridization), OTC protein expression (immunostaining), and OTC enzyme activity assessed by staining and/or enzyme activity assays. Liver DNA was isolated to assess target editing (amplicon sequencing, long-read sequencing from Oxford Nanopore) and vector genome copy.

Mice administered the vector with the mhHDR arm showed comparable survival to wild-type mice, with shHDR-treated mice achieving 80% viability after a 10-day high-protein diet challenge (Fig. 9A). All treated mice maintained their weight better than untreated KI-spf-ash mice (Fig. 9B). Mice treated with mHDR showed significantly lower plasma ammonia levels compared to untreated mice (Fig. 9C).

mPCSK9 levels were measured on day 48, and all treated mice showed a decrease (Fig. 9D). The percentage of insertions and deletions was fairly consistent across HDR types (Fig. 9E). In mice treated with shHDR and mhHDR, hOTC levels were increased (Fig. 9F).

Example 5 - Evaluating the efficacy of Pcsk9-hE7-KI.spf ^ash in pups and determining the minimum effective dose

This GLP-compliant pharmacology study aims to evaluate the efficacy of intravenous administration of AAV and determine its medium efficacy (MED) in a neonatal PCSK9-hE7-KI.spf ^ash mouse model. The AAVrh79 vector expressing a wide range of ARCUS nucleases (AAVrh79.TBG.PI.ARCUS.WPRE.bGH) will be part of the toxicology vector batch, which will be manufactured for the planned GLP-compliant toxicology study. This study will utilize the hOTCco donor vector containing the mouse-human hybrid HDR sequence (AAVrh79.mhHDR.TBG.hOTCco.bGH), instead of the test article containing a long version of the human HDR sequence (AAVrh79.hHDR.TBG.hOTCco.bGH). This vector will be manufactured using a method similar to that used for the toxicology vector batches of the clinical candidate.

In this study, the mouse-human hybrid HDR sequence within the donor vector (AAVrh79.mhHDR.TBG.hOTCco.bGH) was selected to enable effective study of the pharmacology of this method, where the donor sequence is directly homologous to the sequence in PCSK9-hE7-KI.spf ^ash mice.

This study will evaluate N = 60 newborn (PND 1–2) PCSK9-hE7-KI.spf ^ash mice, with N = 15 age-matched male PCSK9-hE7-KI.WT (wild-type) mice as controls. The study will include a necropsy timepoint (90 days) (see study design in Figure 15A). To assess efficacy, mice will be challenged with a high-protein diet for 10 days from day 81 to day 90. Survival, body condition, and changes in biomarkers will be evaluated. Mice will receive one of three dose levels of the test item (1.0 x ^10¹² GC/kg nuclease carrier and 3.0 x ^10¹² GC/kg donor carrier, 3.3 x ^10¹² GC/kg nuclease carrier and 1.0 x ^10¹³ GC/kg donor carrier, or 1.0 x ^10¹³ GC/kg nuclease carrier and 3.0 x ^10¹³ GC/kg; N = 20 per dose) or the mediator (phosphate-buffered saline [PBS]; N = 20). Dosages are recorded in Figure 15B.

Vital assessments include daily vitality checks, survival monitoring, weight measurement, and evaluation of serum PCSK9 levels, plasma _NH3 , and urinary orotic acid levels after a high-protein diet challenge. An autopsy will be performed on day 90. At the autopsy, blood will be collected for CBC/differential and serum clinical chemistry analysis. A list of tissues will be collected for histopathological evaluation. A liver will be collected to evaluate human OTC gene knock-in, including assessment of human OTC mRNA expression (in situ hybridization), OTC protein expression (immunostaining), and OTC enzyme activity assessed by staining and/or enzyme activity assays. Liver DNA will also be isolated to evaluate on-target editing (amplicon sequencing) and to assess vector genome copy number.

MED will be determined based on the survival of neonatal PCSK9-hE7-KI.spfash ^mice treated with AAV after a high-protein diet, plasma _NH3 levels at the end of the high-protein diet challenge, human OTC mRNA and protein expression, OTC enzyme activity, and on-target editing analysis compared to mediator-treated neonatal PCSK9-hE7- ^KI.spfash control mice.

Example 8 - Toxicological study of Pcsk9-hE7-KI.spf ^ash pups

A 6-month GLP compliance safety study will be conducted in newborn (PND 1-2) PCSK9-hE7-KI.spf ^ash mice to investigate the safety, tolerability, pharmacology, and pharmacokinetics of the test article after IV co-administration. Interim analyses, including target editing, off-target editing, transgene expression, and histopathological analysis, will be performed at days 60 and 180, as these time points will provide sufficient time for the nuclease-dependent gene insertion to reach a stable plateau level after administration. Neonatal PCSK9-hE7-KI.spf ^ash mice will receive one of three dose levels of test items (1.0 x ^10¹² GC/kg nuclease carrier and 3.0 x ^10¹² GC/kg donor carrier, 3.3 x ^10¹² GC/kg nuclease carrier and 1.0 x ^10¹³ GC/kg donor carrier, or 1.0 x ^10¹³ GC/kg nuclease carrier and 3.0 x ^10¹³ GC/kg; N = 20 per dose) or a mediator (phosphate-buffered saline [PBS]; N = 20). Following administration of the test item or mediator, vital assessments will include daily monitoring for signs of distress and abnormal behavior, weight measurements, and clinical serum chemistry (specifically ALT, AST, and total bilirubin).

On day 60 following administration of the test substance, euthanasia will be performed on groups 1, 3, 5, and 7, and a complete list of tissues will be analyzed for histopathological examination, including but not limited to the brain, spinal cord, heart, liver, spleen, kidneys, lungs, reproductive organs, adrenal glands, and lymph nodes. Organs will be weighed as needed.

Liver samples will be collected for on-target editing analysis using amplicon sequencing and AMP sequencing, off-target editing analysis using ITR sequencing and amplicon sequencing, and vector biodistribution and transgene expression analysis. In liver samples, the following will be performed: biodistribution will be assessed by PCR, and broad-spectrum nuclease RNA expression will be analyzed by RT-PCR. Broad-spectrum nuclease RNA analysis will be performed on highly perfused organs, and on-target editing in tissues with detectable broad-spectrum nuclease RNA expression will be assessed by amplicon sequencing. Tissues with detectable on-target editing will be further evaluated for off-target editing.

For vector biodistribution, a qPCR assay specific to the dual vectors ARCUS and hOTCco will be developed. The efficiency, linearity, precision, reproducibility, and detection limit of the assay will be evaluated using AAV cis plasmids as surrogate target sequences. The limit of quantitation (LLOQ) will be determined before assays are initiated on test tissues or excreta. An identification program will be implemented to link the transgene-specific assay to previous identification studies. The test matrix will contain the intended target, liver for biodistribution. The matrix effect will be further evaluated based on the recovery of spiked target controls from all samples tested during the biodistribution studies and data subtracted from previous identification studies.

Example 9 – Toxicological study of juvenile (6-9 weeks old) NHP

A one-year GLP compliance safety study will be conducted in juvenile (6–9 weeks) rhesus monkeys to investigate the safety, tolerability, pharmacology, and pharmacokinetics of the test article following IV co-administration. Interim analyses, including target editing, off-target editing, transgene expression, and histopathological analysis, will be performed on day 90, as this time point will allow sufficient time for nuclease-dependent gene insertion to reach a stable plateau level post-administration. Juvenile rhesus monkeys will receive either 1.0 x ^10¹³ GC/kg nuclease vector and 3.0 x ^10¹³ GC/kg; N=4, or a mediator (phosphate-buffered saline [PBS]/surfactant; N=2). Vital assessments following administration of the test article or mediator will include daily monitoring for signs of discomfort and abnormal behavior, weight measurement, and blood clinical serological chemistry (specifically ALT, AST, and total bilirubin). The study design is shown in Figure 16.

On day 90 following administration of the test substance, one animal from the control group and group 3 will be euthanized, and a complete list of tissues will be analyzed for histopathological examination, including but not limited to the brain, spinal cord, heart, liver, spleen, kidneys, lungs, reproductive organs, adrenal glands, and lymph nodes. Organs will be weighed as needed. On day 90, the remaining animals will undergo liver biopsy.

Liver samples will be collected for on-target editing analysis using amplicon sequencing and AMP sequencing, off-target editing analysis using ITR sequencing and amplicon sequencing, and vector biodistribution and transgene expression analysis. In liver samples, the following will be performed: biodistribution will be assessed by PCR, and broad-spectrum nuclease RNA expression will be analyzed by RT-PCR. Broad-spectrum nuclease RNA analysis will be performed on highly perfused organs, and on-target editing in tissues with detectable broad-spectrum nuclease RNA expression will be assessed by amplicon sequencing. Tissues with detectable on-target editing will be further evaluated for off-target editing.

For vector biodistribution, a qPCR assay specific to the dual vectors ARCUS and hOTCco will be developed. The efficiency, linearity, precision, reproducibility, and detection limit of the assay will be evaluated using AAV cis plasmids as surrogate target sequences. The limit of quantitation (LLOQ) will be determined before assays are initiated on test tissues or excreta. An identification program will be implemented to link the transgene-specific assay to previous identification studies. The test matrix will contain the intended target, liver for biodistribution. The matrix effect will be further evaluated based on the recovery of spiked target controls from all samples tested during the biodistribution studies and data subtracted from previous identification studies.

All documents referenced in this specification are incorporated herein by reference, as are the sequences and sequence lists submitted with this document. U.S. Provisional Patent Application No. 63/301,917, filed January 21, 2022; U.S. Provisional Patent Application No. 63/331,384, filed April 15, 2022; U.S. Provisional Patent Application No. 63/364,861, filed May 17, 2022; U.S. Provisional Patent Application No. 63/370,049, filed August 1, 2022; and U.S. Provisional Patent Application No. 63/379,067, filed October 11, 2022, are each incorporated herein by reference in their entirety. While the invention has been described with reference to specific embodiments, it should be understood that modifications may be made without departing from the spirit of the invention. Such modifications are intended to fall within the scope of the appended claims.

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Claims (18)

1. A dual-carrier system for treating ornithine carbamoyltransferase deficiency, the system comprising: (a) A gene-edited AAV comprising a first AAV rh79 capsid and a first vector genome, the first vector genome comprising a 5' ITR, a sequence encoding a macronuclease having the sequence of SEQ ID NO:3, and a 3' ITR, the macronuclease targeting PCSK9 under the control of a regulatory sequence, the regulatory sequence guiding the expression of the macronuclease in target cells including the PCSK9 gene; and (b) A donor AAV vector comprising a second AAV rh79 capsid and a second vector genome, the second vector genome comprising: a 5'ITR, a 5' homologous directed recombination (HDR) arm, a transgene encoding ornithine carbamoyltransferase (OTC) and a regulatory sequence guiding the expression of the transgene in the target cells, a 3'HDR arm and a 3'ITR, the transgene comprising the sequence of SEQ ID NO:4 or a sequence at least 90% identical to SEQ ID NO:4. 2. The dual-vector system according to claim 1, wherein the sequence encoding the wide range of nucleases comprises nucleotides (nt) 1089-2183 of SEQ ID NO:2, or a sequence that is at least 90% identical to nucleotides (nt) 1089-2183 of SEQ ID NO:2. 3. The dual-vector system according to claim 1 or claim 2, wherein the transgene encoding OTC includes SEQ ID NO:4. 4. The dual-carrier system according to any one of claims 1 to 3, wherein the first AAV capsid and the second AAV capsid are the AAVrh79 capsid of SEQ ID NO:16. 5. The dual-vector system according to any one of claims 1 to 3, wherein the ratio of the gene-editing AAV vector in (a) to the donor AAV vector in (b) is 1:3. 6. The dual-vector system according to any one of claims 1 to 5, wherein the nuclease is under the control of the TBG promoter. 7. The dual-vector system according to any one of claims 1 to 6, wherein the transgene is under the control of the TBG promoter. 8. The dual-carrier system according to any one of claims 1 to 7, wherein i) The genome of the first vector includes nt 211 to 2964 of SEQ ID NO:2 or a sequence that shares at least 90% identity with nt 211 to 2964 of SEQ ID NO:2; and ii) The second vector genome includes nt 178 to 3281 of SEQ ID NO:6 or a sequence that shares at least 90% identity with nt 178 to 3281 of SEQ ID NO:6. 9. A method for treating human OTC deficiency by co-administering a dual-carrier system according to any one of claims 1 to 8. 10. A method for treating an OTC deficiency in a subject, the method comprising: co-administering to the subject suffering from an OTC deficiency: (a) A gene-edited AAV comprising a first AAV rh79 capsid and a first vector genome, the first vector genome comprising a 5' ITR, a sequence encoding a macronuclease having the sequence of SEQ ID NO:3, and a 3' ITR, the macronuclease targeting PCSK9 under the control of a regulatory sequence, the regulatory sequence guiding the expression of the macronuclease in target cells including the PCSK9 gene; and (b) A donor AAV vector comprising a second AAV rh79 capsid and a second vector genome, the second vector genome comprising: a 5'ITR, a 5' homologous directed recombination (HDR) arm, a transgene encoding ornithine carbamoyltransferase (OTC) and a regulatory sequence guiding the expression of the transgene in the target cells, a 3'HDR arm and a 3'ITR, the transgene comprising the sequence of SEQ ID NO:4 or a sequence at least 90% identical to SEQ ID NO:4. 11. The method of claim 10, wherein i) The genome of the first vector includes nt 211 to 2964 of SEQ ID NO:2 or a sequence that shares at least 90% identity with nt 211 to 2964 of SEQ ID NO:2; and ii) The second vector genome includes nt 178 to 3281 of SEQ ID NO:6 or a sequence that shares at least 90% identity with nt 178 to 3281 of SEQ ID NO:6. 12. The method according to any one of claims 9 to 11, wherein the gene-editing AAV vector of (a) and the donor vector of (b) are delivered substantially simultaneously via IV. 13. The method according to any one of claims 9 to 12, wherein the gene-editing AAV vector of (a) is suspended in a medium at a dose of about 1 x ^10¹³ GC/kg for injection. 14. The method according to any one of claims 9 to 13, wherein the AAV donor carrier of (b) is suspended in a medium at a concentration of about 3 x ^10¹³ GC/kg for injection. 15. The method according to any one of claims 9 to 14, wherein the subject is 1 day old to 4 months old. 16. The method according to any one of claims 9 to 15, wherein the subject is male. 17. Use of a dual-carrier system according to any one of claims 1 to 8 for treating OTC deficiency in a subject in need. 18. Use of a dual-carrier system according to any one of claims 1 to 8 for the preparation of a medicament for treating OTC deficiency in subjects in need.