Compositions and methods for reducing nuclease expression and off-target activity using promoters with low transcriptional activity

Abstract

[0003] Provided are gene-editing nuclease expression cassettes, the gene-editing nuclease expression cassettes comprising a nucleic acid sequence comprising a nuclease coding sequence operably linked to a regulatory sequence that directs expression of the nuclease after delivery to a host cell, the regulatory sequence comprising a weak promoter. Provided are vectors comprising the gene-editing nuclease expression cassettes. Also provided are compositions containing the same and methods of use.

Description

[Background technology]

[0001] The use of engineered nucleases has been described to edit dysfunctional genes. AAV-mediated delivery of such nucleases has also been described. However, although AAV-mediated delivery of nucleases avoids the need for repeated re-administration, the resulting nucleases are continuously expressed in target tissues after vector transduction, which can induce immune responses and cytotoxicity.

[0002] Furthermore, both in vitro and in vivo studies have shown that nucleases, regardless of delivery vehicle, generate indels (insertions and deletions) in other regions of the genome, suggesting off-target activity. This off-target activity is undesirable, and reducing or eliminating this off-target activity while retaining high on-target efficacy is essential, especially for clinical studies.

[0003] What is needed are improved compositions and methods for gene editing. Summary of the Invention

[0004] In one aspect, a gene-targeting nuclease expression cassette is provided. In one embodiment, the expression cassette comprises a nucleic acid comprising a nuclease coding sequence operably linked to a regulatory sequence that directs expression of the nuclease after delivery to a host cell having a sequence targeted by the nuclease, the regulatory sequence comprising a promoter with low transcriptional activity. In one embodiment, the promoter is a liver-specific promoter. In another embodiment, the promoter is a TBG-S1 promoter variant. In yet another embodiment, the promoter is TBG-S1-F64. In another embodiment, the promoter is TBG-S1-F113. In another embodiment, the promoter is TBG-S1-F140. In another embodiment, the promoter is a CCL16 promoter. In another embodiment, the promoter is an SCLC22A9 promoter. In another embodiment, the promoter is a CYP26A1 promoter. In yet another embodiment, the nuclease is a meganuclease, a CRISPR / Cas nuclease, a zinc finger nuclease, or a TALEN.

[0005] In another aspect, a recombinant AAV useful for gene editing is provided. The rAAV comprises an AAV capsid and a vector genome packaged in the AAV capsid, the vector genome comprising an expression cassette described herein and the AAV inverted terminal repeats necessary for packaging the expression cassette into the capsid.

[0006] In another aspect, a method for editing a targeted gene is provided, the method comprising delivering to a subject a nuclease expression cassette, composition, or viral vector described herein.

[0007] In another aspect, a method is provided for reducing off-target activity of a gene-targeting nuclease, the method comprising delivering to a subject a nuclease expression cassette, composition, or viral vector described herein.

[0008] In another aspect, novel "weak promoters" are provided. In another embodiment, the promoter is TBG-S1-F64. In another embodiment, the promoter is TBG-S1-F113. In another embodiment, the promoter is TBG-S1-F140. In another embodiment, the promoter has the sequence of SEQ ID NO: 6. In another embodiment, the promoter has the sequence of SEQ ID NO: 7. In another embodiment, the promoter has the sequence of SEQ ID NO: 8.

[0009] In another aspect, there is provided a pharmaceutical composition comprising a nuclease expression cassette, composition, or viral vector described herein, wherein the composition comprises one or more of a carrier, a suspending agent, and / or an excipient.

[0010] Other aspects and advantages of the present invention will become apparent from the following detailed description of the invention. [Brief explanation of the drawings]

[0011] [Figure 1]

[0023] Figure 1 is a schematic diagram of an AAV construct containing a "weak" promoter for the vector used in Example 1 (data shown in Figures 2-5). Promoter: derived from a truncated version of the human thyroxine-binding globulin (TBG) gene or the promoter sequence of the following liver-enriched genes: CCL16, CYP26A1, or SLC22A9 (identified using the Human Protein Atlas database). M2PCSK9: engineered I-CreI meganuclease (sometimes referred to herein as ARCUS meganuclease) targeting a 22-bp sequence in the human PCSK9 gene. PolyA: bovine growth hormone polyadenylation signal. [Figure 2A] Shown are the levels of indels in the region corresponding to the target sequence of ARCUS nuclease at 7 weeks post-AAV, as quantified by next-generation sequencing assay. Linear scale. [Figure 2B] Logarithmic scale, showing the same levels as in Figure 2A. [Figure 2C]Mean serum recombinant PCSK9 levels at week 9 as determined by ELISA assay are shown for each treatment group. [Figure 3] Figure 1 shows the number of off-target loci in genomic DNA as a result of nuclease activity, as determined using an NGS-based method called ITR-Seq. [Figure 4] Figure 1 shows indels in the set of genomic locations corresponding to identified off-targets. The indel level for each off-target is shown relative to the indel level in the TBG control group (arbitrary value 1). [Figure 5] 1 shows hPCSK9 levels (as a percentage of baseline) at 7 weeks post-treatment for the vectors tested in Example 1. [Figure 6-10] Data from the NHP pilot study described in Example 2 are shown. NHPs were injected with 6 x 10 GS / kg of the indicated vector. Liver biopsies were performed on days 18 and 128, and DNA / RNA analysis was performed to detect on-target and off-target genome editing by next-generation sequencing. A summary of some of the data presented in Figures 7-10 is shown in Figure 6. The % indels (Figure 7) and number of off-targets (Figure 8) were determined in DNA from liver biopsies 18 days post-AAV. PCSK9 levels (as a percentage of baseline) are shown in Figure 9 (7 weeks post-AAV). LDL levels (as a percentage of baseline) are shown in Figure 10. [Figure 11] FIG. 1 is a schematic diagram of the NHP pharmacology / toxicology study design described in Example 3. [Figure 12] 1 is an alignment of the sequences of the TBG-S1 promoter and the F64, F113, and F140 promoters described herein. DETAILED DESCRIPTION OF THE INVENTION

[0012] The compositions and methods provided herein are designed to result in less expression of persistently expressed enzymes (e.g., after delivery of an expression cassette), minimize their off-target activity, and / or adjust the activity of the expressed enzyme. The use of these compositions and methods with non-secreted enzymes that may accumulate in cells and / or enzymes that accumulate at higher than desired levels prior to secretion is particularly desirable. The compositions and methods of the present invention are useful for gene editing enzymes, It is particularly well suited for use with meganucleases, however other applications will be apparent to those skilled in the art.

[0013] Low-transcription promoters ("weak" promoters) In one aspect, novel promoters with low transcriptional activity (i.e., weak promoters) are provided. As used herein, the term "promoter with low transcriptional activity" or "weak promoter" refers to an expression control sequence that results in low levels of expression of a coding sequence. In one embodiment, the term "low transcriptional activity" refers to a level of transcription that is less than the level induced by a reference "strong promoter." In one embodiment, the reference strong promoter is the thyroxine-binding globulin (TBG) promoter or the TBG-S1 promoter. Other reference "strong" promoters are known in the art.

[0014] In one embodiment, the promoter is a weakened version of the liver-specific thyroxine-binding globulin (TBG) promoter. In one embodiment, the weak promoter is truncated at the 5' or 3' end of the native promoter or TBG-S1 sequence. In one embodiment, the promoter retains only the 3'-most 64 nt from the TBG-S1 promoter and is designated F64 (also referred to as TBG-S1-F64) (SEQ ID NO: 6). In another embodiment, the promoter retains only the 3'-most 113 nt from the TBG-S1 promoter and is designated F113 (also referred to as TBG-S1-F113) (SEQ ID NO: 7). In one embodiment, the promoter retains only the 3'-most 140 nt from the TBG-S1 promoter and is designated F140 (also referred to as TBG-S1-F140) (SEQ ID NO: 8). An alignment of the TBG-S1, F64, F113, and F140 sequences is provided in Figure 12. In one embodiment, the promoter shares at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity with SEQ ID NO: 6. In one embodiment, the promoter shares at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity with SEQ ID NO: 7. In one embodiment, the promoter shares at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity with SEQ ID NO: 8.

[0015] In other embodiments, weak promoters useful herein include known promoters. In one embodiment, the weak promoter is the CCL16 promoter (SEQ ID NO: 3). In another embodiment, the weak promoter is the SLC22A9 promoter (SEQ ID NO: 4). In yet another embodiment, the weak promoter is the CYP26A1 promoter (SEQ ID NO: 5).

[0016] Expression cassettes and vectors In another aspect, an expression cassette is provided. In one embodiment, the expression cassette comprises a weak promoter operably linked to a coding sequence, as described herein. In one embodiment, the expression cassette comprises a coding sequence for a nuclease under the control of a regulatory sequence comprising a promoter with low transcriptional activity, as described herein. In another aspect, a vector comprising the expression cassette (and promoter) is provided.

[0017] The examples herein demonstrate the use of AAV vectors containing a promoter with low transcriptional activity (weak promoter) in the vector genome. However, the use of weak promoters is not limited to AAV constructs and can be used in other vectors. In certain embodiments, the vector genome can be packaged in a different vector (e.g., a recombinant bocavirus). In certain embodiments, the expression cassette can be packaged in a different viral vector, a non-viral vector, and / or a different delivery system. Preferably, the coding sequence of the transgene is engineered within the expression cassette and operably linked to regulatory elements including a weak promoter in the cell that contains a target site for the enzyme.

[0018] As used herein, "expression cassette" refers to a nucleic acid molecule comprising a coding sequence (or transgene), a promoter, and optionally other regulatory sequences therefor, which may be engineered into a genetic element and / or packaged into a viral vector capsid (e.g., a viral particle). Typically, such expression cassettes for generating viral vectors contain the sequences described herein adjacent to the packaging signal of the viral genome and other expression control sequences, such as those described herein. A transgene is a nucleic acid sequence heterologous to the vector sequences adjacent to the transgene, encoding a polypeptide, protein, or other product of interest. The nucleic acid coding sequence is operably linked to regulatory components in a manner that allows transcription, translation, and / or expression of the transgene in a target cell. The heterologous nucleic acid sequence (transgene) may be derived from any organism. An AAV may contain one or more transgenes. The use of the weak promoters described herein in combination with a gene-editing nuclease (specifically, a meganuclease) is exemplified herein. However, weak promoters can be incorporated into any expression cassette where lower expression and / or a shorter promoter sequence is desired.

[0019] In one embodiment, the coding sequence encodes a nuclease selected from meganucleases, zinc finger nucleases, transcription activator-like (TAL) effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR) / endonucleases (Cas9, Cpf1, etc.). Examples of suitable meganucleases are described, for example, in U.S. Patent Nos. 8,445,251, 9,340,777, 9,434,931, 9,683,257, and WO2018 / 195449. Other suitable enzymes include the nuclease-inactive S. pyogenes CRISPR / Cas9, which can bind to RNA in a nucleic acid programmable manner (Nelles et al., Programmable RNA Tracking in Live Cells with CRISPR / Cas9, Cell, 165(2):P488-96 (April 2016)), and base editors (e.g., Levy et al. Cytosine and adenine base editing of the Brain, liver, retina, heart and skeletal muscle of mice via adeno-associated viruses, Nature Biomedical Engineering, 4, 97-110 (Jan 2020)). In certain embodiments, the nuclease is not a zinc finger nuclease. In certain embodiments, the nuclease is not a CRISPR-associated nuclease. In certain embodiments, the nuclease is not a TALEN. In one embodiment, the nuclease is not a meganuclease.

[0020] In certain embodiments, the nuclease is a member of the LAGLIDADG (SEQ ID NO: 1) family of homing endonucleases. In certain embodiments, the nuclease is a member of the I-CreI family of homing endonucleases, which recognizes and cleaves the 22-base pair recognition sequence SEQ ID NO: 2-CAAAACGTCGTGAGACAGTTTG. See, e.g., WO 2009 / 059195. A method for rationally designing mono-LAGLIDADG homing endonucleases has been described (WO 2007 / 047859), which comprehensively redesigns ICreI and other homing endonucleases to target a wide variety of DNA sites, including sites in mammalian, yeast, plant, bacterial, and viral genomes. In one embodiment, the nuclease is not the nuclease encoded by the sequence set forth at nt 1089-2183 of SEQ ID NO: 15. In one embodiment, the nuclease is not the protein sequence set forth in SEQ ID NO: 16.

[0021] One of the objectives of the present invention is to provide a method for the production of nucleases that can be used in a variety of applications without compromising the potent on-target activity of the nucleases. The aim is to reduce its off-target activity. We hypothesized that high expression of nuclease in transduced cells is not necessary to achieve editing of the target DNA sequence, and that off-target activity results from increased accumulation of nuclease in cells. To reduce nuclease expression, the high-expression promoter was replaced with a promoter with lower transcriptional activity. Therefore, the expression cassette contains a promoter sequence as part of the expression control sequence or regulatory sequence. As described herein, the promoter is a promoter with lower transcriptional activity, i.e., a "weak promoter."

[0022] In one embodiment, the weak promoter is the CCL16 promoter (SEQ ID NO: 3). In another embodiment, the weak promoter is the SLC22A9 promoter (SEQ ID NO: 4). In yet another embodiment, the weak promoter is the CYP26A1 promoter (SEQ ID NO: 5).

[0023] Additionally, in another embodiment, the promoter is a weakened version of a tissue-specific promoter. In one example, the tissue-specific promoter is the liver-specific thyroxine-binding globulin (TBG) promoter. In one embodiment, the weak promoter is truncated at the 5' or 3' end of the native promoter or TBG-S1 sequence. In one embodiment, the promoter retains only the 3'-most 64 nt from the TBG-S1 promoter and is designated F64 (SEQ ID NO: 6). In one embodiment, the promoter retains only the 3'-most 113 nt from the TBG-S1 promoter and is designated F113 (SEQ ID NO: 7). In one embodiment, the promoter retains only the 3'-most 140 nt from the TBG-S1 promoter and is designated F140 (SEQ ID NO: 8).

[0024] In addition to a promoter, the expression cassette and / or vector may contain one or more appropriate "regulatory elements" or "regulatory sequences," including, but not limited to, enhancers; transcription factors; transcription terminators; efficient RNA processing signals, such as splicing and polyadenylation signals (polyA); sequences that stabilize cytoplasmic mRNA, such as the woodchuck hepatitis virus (WHP) posttranscriptional regulatory element (WPRE); sequences that improve translation efficiency (i.e., Kozak consensus sequences); sequences that improve protein stability; and, if desired, sequences that improve secretion of the encoded product. Examples of suitable polyA sequences include, for example, SV40, bovine growth hormone (bGH), and TK polyA. Examples of suitable enhancers include, for example, the alpha-fetoprotein enhancer, TTR minimal promoter / enhancer, and LSP (TH-binding globulin promoter / alpha1-microglobulin / bikunin enhancer), among others. These control or regulatory sequences are operably linked to the nuclease-coding sequence.

[0025] In certain embodiments, the weak promoters, constructs containing the same, and methods described herein are useful for targeting liver-directed therapies. Thus, liver-expressed genes, as well as nucleases targeting these genes or their adjacent sequences, are useful herein. Liver-expressed genes include, but are not limited to, proprotein convertase subtilisin / kexin type 9 (PCSK9) (cholesterol-related disorders), transthyretin (TTR) (transthyretin amyloidosis), HAO, apolipoprotein C-III (APOC3), factor VIII, factor IX, low-density lipoprotein receptor (LDLr), lipoprotein lipase (LPL) (lipoprotein lipase deficiency), lecithin-cholesterol acyltransferase (LTP), and lecithin-cholesterol acyltransferase (LTP). LCAT, ornithine transcarbamylase (OTC), carnosinase (CN1), sphingomyelin phosphodiesterase (SMPD1) (Niemann-Pick disease), hypoxanthine-guanine phosphoribosyltransferase (HGPRT), branched-chain alpha-ketoacid dehydrogenase complex (BCKDC) (maple syrup urine disease), erythropoietin (EPO), carbamoyl phosphate synthetase (CPS1), N-acetylglutamate synthetase These include argininosuccinate synthetase (NAGS), argininosuccinate synthetase (citrullinemia), argininosuccinate lyase (ASL) (argininosuccinic aciduria), and arginase (AG).

[0026] In certain embodiments, rAAV may be used in gene editing systems, which may involve the co-administration of one rAAV or multiple rAAV stocks. For example, rAAV may be engineered to deliver SpCas9, SaCas9, ARCUS, Cpfl, and other suitable gene editing constructs.

[0027] In one embodiment, a nucleic acid molecule encoding a PCSK9 meganuclease is provided that is operably linked to a weak promoter. In one embodiment, the weak promoter is F64. In another embodiment, the weak promoter is F113. In yet another embodiment, the weak promoter is F140. In another embodiment, the weak promoter is a CCL16 promoter. In another embodiment, the weak promoter is a SLC22A9 promoter. In yet another embodiment, the weak promoter is a CYP26A1 promoter. In certain embodiments, the meganuclease may be selected from those described in WO2018 / 195449A1. In one embodiment, the nucleic acid molecule comprises the F113 promoter operably linked to a PCSK9 meganuclease coding sequence from nt 1089 to 2183 of SEQ ID NO: 15, or a sequence sharing at least 95%, 96%, 97%, 98%, 99%, or 99.9% identity thereto. In one embodiment, the nucleic acid molecule comprises an F113 promoter operably linked to a sequence encoding the PCSK9 meganuclease of SEQ ID NO: 16, or a sequence sharing at least 95%, 96%, 97%, 98%, 99%, or 99.9% identity thereto.

[0028] In another embodiment, the nucleic acid molecule comprises an F64 promoter operably linked to the PCSK9 meganuclease coding sequence of nt 1089 to 2183 of SEQ ID NO: 15, or a sequence sharing at least 95%, 96%, 97%, 98%, 99%, or 99.9% identity thereto. In one embodiment, the nucleic acid molecule comprises an F64 promoter operably linked to the PCSK9 meganuclease coding sequence of SEQ ID NO: 16, or a sequence sharing at least 95%, 96%, 97%, 98%, 99%, or 99.9% identity thereto.

[0029] In another embodiment, the nucleic acid molecule comprises an F140 promoter operably linked to the PCSK9 meganuclease coding sequence from nt 1089 to 2183 of SEQ ID NO: 15, or a sequence sharing at least 95%, 96%, 97%, 98%, 99%, or 99.9% identity thereto. In one embodiment, the nucleic acid molecule comprises an F140 promoter operably linked to the PCSK9 meganuclease coding sequence of SEQ ID NO: 16, or a sequence sharing at least 95%, 96%, 97%, 98%, 99%, or 99.9% identity thereto.

[0030] In another embodiment, the nucleic acid molecule comprises an SLC22A9 promoter operably linked to a PCSK9 meganuclease coding sequence from nt 1089 to 2183 of SEQ ID NO: 15, or a sequence sharing at least 95%, 96%, 97%, 98%, 99%, or 99.9% identity thereto. In one embodiment, the nucleic acid molecule comprises an SLC22A9 promoter operably linked to a PCSK9 meganuclease encoding sequence of SEQ ID NO: 16, or a sequence sharing at least 95%, 96%, 97%, 98%, 99%, or 99.9% identity thereto.

[0031] In another embodiment, the nucleic acid molecule comprises a CCL16 promoter operably linked to a PCSK9 meganuclease coding sequence from nt 1089 to 2183 of SEQ ID NO: 15, or In one embodiment, the nucleic acid molecule comprises a CCL16 promoter operably linked to a sequence encoding a PCSK9 meganuclease of SEQ ID NO: 16, or a sequence sharing at least 95%, 96%, 97%, 98%, 99%, or 99.9% identity thereto.

[0032] In another embodiment, the nucleic acid molecule comprises a CYP26A1 promoter operably linked to a PCSK9 meganuclease coding sequence from nt 1089 to 2183 of SEQ ID NO: 15, or a sequence sharing at least 95% to 99.9% identity thereto. In one embodiment, the nucleic acid molecule comprises a CYP26A1 promoter operably linked to a PCSK9 meganuclease-encoding sequence from SEQ ID NO: 16, or a sequence sharing at least 95%, 96%, 97%, 98%, 99%, or 99.9% identity thereto.

[0033] In one embodiment, a nucleic acid molecule encoding a TTR meganuclease is provided that is operably linked to a weak promoter. In one embodiment, the weak promoter is F64. In another embodiment, the weak promoter is F113. In yet another embodiment, the weak promoter is F140. In another embodiment, the weak promoter is a CCL16 promoter. In another embodiment, the weak promoter is a SLC22A9 promoter. In yet another embodiment, the weak promoter is a CYP26A1 promoter.

[0034] In one embodiment, a nucleic acid molecule encoding an HAO meganuclease is provided that is operably linked to a weak promoter. In one embodiment, the weak promoter is F64. In another embodiment, the weak promoter is F113. In yet another embodiment, the weak promoter is F140. In another embodiment, the weak promoter is a CCL16 promoter. In another embodiment, the weak promoter is a SLC22A9 promoter. In yet another embodiment, the weak promoter is a CYP26A1 promoter.

[0035] In one embodiment, a nucleic acid molecule encoding a BCKDC meganuclease is provided that is operably linked to a weak promoter. In one embodiment, the weak promoter is F64. In another embodiment, the weak promoter is F113. In yet another embodiment, the weak promoter is F140. In another embodiment, the weak promoter is a CCL16 promoter. In another embodiment, the weak promoter is a SLC22A9 promoter. In yet another embodiment, the weak promoter is a CYP26A1 promoter.

[0036] In one embodiment, a nucleic acid molecule encoding an APOC3 meganuclease is provided that is operably linked to a weak promoter. In one embodiment, the weak promoter is F64. In another embodiment, the weak promoter is F113. In yet another embodiment, the weak promoter is F140. In another embodiment, the weak promoter is a CCL16 promoter. In another embodiment, the weak promoter is a SLC22A9 promoter. In yet another embodiment, the weak promoter is a CYP26A1 promoter.

[0037] In one embodiment, a nucleic acid molecule encoding a CRISPR / Cas9 nuclease is provided that is operably linked to a weak promoter. In one embodiment, the weak promoter is F64. In another embodiment, the weak promoter is F113. In yet another embodiment, the weak promoter is F140. In another embodiment, the weak promoter is a CCL16 promoter. In another embodiment, the weak promoter is SLC2 In yet another embodiment, the weak promoter is a CYP26A1 promoter. In one embodiment, the promoters, cassettes, and rAAVs described herein are useful in the CRISPR-Cas dual vector system described in WO2016 / 176191, which is incorporated herein by reference.

[0038] In another embodiment, transgene is selected for use in gene correction therapy.This can be achieved, for example, by using zinc finger nuclease (ZFN)-induced DNA double-strand break in combination with exogenous DNA donor substrate.For example, see Ellis et al., Gene Therapy (epub January 2012) 20:35-42, which is incorporated herein by reference.Transgene can be easily selected by those skilled in the art based on desired results.

[0039] In one embodiment, a nucleic acid molecule encoding a zinc finger nuclease is provided that is operably linked to a weak promoter. In one embodiment, the weak promoter is F64. In another embodiment, the weak promoter is F113. In yet another embodiment, the weak promoter is F140. In another embodiment, the weak promoter is a CCL16 promoter. In another embodiment, the weak promoter is a SLC22A9 promoter. In yet another embodiment, the weak promoter is a CYP26A1 promoter.

[0040] In one embodiment, a nucleic acid molecule is provided that encodes a transcription activator-like effector nuclease (TALEN) operably linked to a weak promoter. In one embodiment, the weak promoter is F64. In another embodiment, the weak promoter is F113. In yet another embodiment, the weak promoter is F140. In another embodiment, the weak promoter is a CCL16 promoter. In another embodiment, the weak promoter is a SLC22A9 promoter. In yet another embodiment, the weak promoter is a CYP26A1 promoter.

[0041] Useful products encoded by transgenes include various gene products that replace missing or defective genes, inactivate or "knock out," or "knock down," or reduce expression of genes that are expressed at undesirably high levels, or deliver gene products with a desired therapeutic effect. In some embodiments, the therapy is "somatic cell gene therapy," i.e., the introduction of genes into cells of the body that do not produce sperm or eggs. In certain embodiments, the transgene-expressed protein has a sequence of a native human sequence. However, in other embodiments, synthetic proteins are expressed. Such proteins may be intended for human treatment, or in other embodiments, may be designed for the treatment of animals, including companion animals such as dog or cat populations, or for the treatment of livestock or other animals that come into contact with the human population.

[0042] Examples of suitable gene products may include those associated with familial hypercholesterolemia, muscular dystrophy, cystic fibrosis, and rare or orphan diseases, such as spinal muscular atrophy (SMA), Huntington's disease, Rett syndrome (e.g., methyl-CpG binding protein 2 (MeCP2), UniProtKB-P51608), amyotrophic lateral sclerosis (ALS), Duchenne muscular dystrophy, Friedreich's ataxia (e.g., frataxin), ATXN2 associated with spinocerebellar ataxia type 2 (SCA2) / ALS, TDP-43 associated with ALS, and progranulin (PRGN) (associated with non-Alzheimer's brain degeneration, including frontotemporal dementia (FTD), progressive non-fluent aphasia (PNFA), and semantic dementia), among others. For example, www.orpha.net / consor / cgi-bin / Disease_Search_List.php;rarediseases.info.nih.gov / diseases Please refer to.

[0043] Examples of suitable genes include, but are not limited to, insulin, glucagon, glucagon-like peptide-1 (GLP1), growth hormone (GH), parathyroid hormone (PTH), growth hormone-releasing factor (GRF), follicle-stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiopoietin, angiostatin, granulocyte colony-stimulating factor (GCSF), erythropoietin (EPO) (including, for example, human, canine, or feline EPO), connective tissue growth factor (CTGF), e.g., basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin growth factor I and II (IGF-I and IGF-II), and the like. Neurotrophic factors including I), any one of the transforming growth factor α superfamily including TGFα, activin, and inhibin, or any one of the bone morphogenetic proteins (BMP) BMP1 to 15, the heregluin / neuregulin / ARIA / neu differentiation factor (NDF) family of growth factors, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophins NT-3 and NT-4 / 5, ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), neurturin, any one of agrin, semaphorin / collapsin, netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrin, noggin, sonic hedgehog, and any one of the tyrosine hydroxylase families.

[0044] Other useful transgene products include proteins that regulate the immune system, including cytokines and lymphokines such as thrombopoietin (TPO), interleukins (IL), IL-1 through IL-36 (e.g., human interleukins IL-1, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-6, IL-8, IL-12, IL-11, IL-12, IL-13, IL-18, IL-31, and IL-35), monocyte chemotactic protein, leukemia inhibitory factor, granulocyte-macrophage colony-stimulating factor, Fas ligand, tumor necrosis factors α and β, interferons α, β, and γ, stem cell factor, and flk-2 / flt3 ligand. Gene products produced by the immune system are also useful in the present invention. These include, but are not limited to, immunoglobulins IgG, IgM, IgA, IgD, and IgE, chimeric immunoglobulins, humanized antibodies, single-chain antibodies, T cell receptors, chimeric T cell receptors, single-chain T cell receptors, class I and class II MHC molecules, and engineered immunoglobulins and MHC molecules. For example, in certain embodiments, rAAV antibodies can be designed to deliver canine or feline antibodies, such as anti-IgE, anti-IL31, anti-IL33, anti-CD20, anti-NGF, and anti-GnRH. Useful gene products also include complement regulatory proteins, such as complement regulatory proteins, membrane cofactor protein (MCP), decay-accelerating factor (DAF), CR1, CF2, CD59, and C1 esterase inhibitor (C1-INH).

[0045] Still other useful gene products include any one of receptors for hormones, growth factors, cytokines, lymphokines, regulatory proteins, and immune system proteins. The present invention encompasses receptors for cholesterol regulation and / or lipid modulation, including low-density lipoprotein (LDL) receptors, high-density lipoprotein (HDL) receptors, very-low-density lipoprotein (VLDL) receptors, and scavenger receptors. The present invention also encompasses gene products such as members of the steroid hormone receptor superfamily, including glucocorticoid receptors, estrogen receptors, vitamin D receptors, and other nuclear receptors. In addition, useful gene products include transcription factors, e.g., jun, fos, max, mad, serum response factor (SRF), AP-1, AP2, myb, MyoD, and myogenin, ETS-box-containing proteins, TFE3, E2F, ATF1, ATF2, ATF3, ATF4, ZF5, NFAT, CREB, HNF-4, C / EBP, SP1, and CCAA. These include T-box binding proteins, interferon regulatory factor (IRF-1), Wilms tumor protein, ETS binding proteins, STATs, GATA-box binding proteins such as GATA-3, and the forkhead family of winged helix proteins.

[0046] Other useful gene products include hydroxymethylbilane synthase (HMBS), carbamoyl synthetase I, ornithine transcarbamylase (OTC), argininosuccinate synthetase, argininosuccinate lyase (ASL) for the treatment of argininosuccinate lyase deficiency, arginase, fumaryl acetate hydrolase, phenylalanine hydroxylase, alpha-1 antitrypsin, rhesus alpha-fetoprotein (AFP), rhesus chorionic gonadotropin (CG), glucose-6-phosphatase, porphobilinogen deaminase, and cis- These include thionine beta-synthase, branched-chain keto acid decarboxylase, albumin, isovaleryl-CoA dehydrogenase, propionyl-CoA carboxylase, methylmalonyl-CoA mutase, glutaryl-CoA dehydrogenase, insulin, beta-glucosidase, pyruvate carboxylate, hepatic phosphorylase, phosphorylase kinase, glycine decarboxylase, H-protein, T-protein, cystic fibrosis transmembrane conductance regulator (CFTR) sequence, and dystrophin gene products (e.g., mini-dystrophin or micro-dystrophin). Still other useful gene products include enzymes that may be useful in enzyme replacement therapy, which is useful in various conditions resulting from insufficient enzyme activity. For example, enzymes containing mannose-6-phosphate can be used to treat lysosomal storage diseases (e.g., suitable genes include those encoding β-glucuronidase (GUSB)). In another example, the gene product is ubiquitin protein ligase E3A (UBE3A). Further useful gene products include UDP glucuronosyltransferase family 1 member A1 (UGT1A1).

[0047] Still other useful gene products include those used to treat hemophilia, including hemophilia B (including factor IX) and hemophilia A (including factor VIII and its variants, e.g., heterodimers and B-deleted domain light and heavy chains; U.S. Pat. Nos. 6,200,560 and 6,221,349). In some embodiments, the minigene contains the first 57 base pairs of the factor VIII heavy chain encoding a 10-amino acid signal sequence and the human growth hormone (hGH) polyadenylation sequence. In alternative embodiments, the minigene further contains the A1 and A2 domains, and 5 amino acids from the N-terminus of the B domain and / or the C-terminal 85 amino acids of the B domain, as well as the A3, C1, and C2 domains. In yet other embodiments, nucleic acids encoding the factor VIII heavy and light chains are provided in a single minigene separated by 42 nucleic acids encoding the 14 amino acids of the B domain [U.S. Pat. No. 6,200,560].

[0048] Additional exemplary genes that can be delivered via rAAV include, but are not limited to, glucose-6-phosphatase, which is associated with glycogen storage disease or type 1A deficiency (GSD1), phosphoenolpyruvate-carboxykinase (PEPCK), which is associated with PEPCK deficiency, cyclin-dependent kinase-like 5 (CDKL5), also known as serine / threonine kinase 9 (STK9), which is associated with seizures and severe neurodevelopmental disorders, galactose-1-phosphate uridyltransferase, which is associated with galactosemia, and phenylketone kinase. Gene products associated with primary hyperoxaluria type 1, including phenylalanine hydroxylase (PAH), hydroxyacid oxidase 1 (GO / HAO1), and AGXT, are associated with primary hyperoxaluria type 1 (PKU). Branched-chain alpha-ketoacid dehydrogenases (including BCKDH, BCKDH-E2, BAKDH-E1a, and BAKDH-E1b) are associated with maple syrup urine disease. Fumarylacetoacetate hydrolase is associated with tyrosinemia type 1. Methylmalonyl-CoA mutase is associated with methylmalonic acidemia. Medium-chain acetyl-CoA deficiency is also associated with Chain acyl-CoA dehydrogenase, ornithine transcarbamylase (OTC) associated with ornithine transcarbamylase deficiency, argininosuccinate synthetase (ASS1) associated with citrullinemia, lecithin-cholesterol acyltransferase (LCAT) deficiency, methylmalonic acidemia (MMA), NPC1 associated with Niemann-Pick disease (type C1), propionic acidemia (PA), transthyretin (TTR) associated with TTR-related hereditary amyloidosis, and low-density lipoprotein (LDL) associated with familial hypercholesterolemia (FH). Protein receptor (LDLR) proteins, LDLR variants such as those described in WO2015 / 164778, PCSK9, ApoE and ApoC proteins associated with dementia, UDP-glucourosyltransferase associated with Crigler-Najjar disease, adenosine deaminase associated with severe combined immunodeficiency disease, hypoxanthine guanine phosphoribosyltransferase associated with gout and Lesch-Naird syndrome, biotin associated with biotin deficiency, alpha-galactosidase A (a-Ga) associated with Fabry disease lA), beta-galactosidase (GLB1) associated with GM1 gangliosidosis, ATP7B associated with Wilson's disease, beta-glucocerebrosidase associated with Gaucher disease types 2 and 3, peroxisomal membrane protein 70 kDa associated with Zellweger syndrome, arylsulfatase A (ARSA) associated with metachromatic leukodystrophy, galactocerebrosidase (GALC) enzyme associated with Krabbe disease, alpha-glucosidase (GAA) associated with Pompe disease, sphingomyelinase associated with Niemann-Pick disease type A (SMPD1) gene, argininosuccinate synthase associated with adult-onset type II citrullinemia (CTLN2), carbamoyl phosphate synthase 1 (CPS1) associated with urea cycle disorders, survival motor neuron (SMN) protein associated with spinal muscular atrophy, ceramidase associated with Farber lipogranulomatosis, b-hexosaminidase associated with GM2 gangliosidosis and Tay-Sachs and Sandhoff disease, aspartylglucosaminidase associated with aspartyl-glucosaminuria, α-fucosidase associated with fucosidosis,α-mannosidase associated with α-mannosidosis, porphobilinogen deaminase associated with acute intermittent porphyria (AIP), α-1 antitrypsin for the treatment of α-1 antitrypsin deficiency (emphysema), erythropoietin for the treatment of anemia due to thalassemia or renal failure, vascular endothelial growth factor, angiopoietin-1, and fibroblast growth factor for the treatment of ischemic disease, inhibitors of the thrombomodulin and tissue factor pathways for the treatment of blocked blood vessels, such as those found in atherosclerosis, thrombosis, or embolism, aromatic amino acid decarboxylase (AADC) for the treatment of Parkinson's disease. and tyrosine hydroxylase (TH), beta-adrenergic receptors for the treatment of congestive heart failure, antisense to phospholamban or its variants, sarco(endo)plasmic reticulum adenosine triphosphatase-2 (SERCA2), and cardiac adenylate cyclase, tumor suppressor genes such as p53 for the treatment of various cancers, cytokines such as one of the various interleukins for the treatment of inflammatory and immune disorders and cancer, dystrophin or mini-dystrophin and utrophin or mini-utrophin for the treatment of muscular dystrophies, and insulin or GLP-1 for the treatment of diabetes.

[0049] In another embodiment, the transgene comprises more than one transgene. This can be achieved using a single vector with two or more heterologous sequences, or using two or more AAVs, each with one or more heterologous sequences. In one embodiment, AAVs are used in gene suppression (or knockdown) combination therapy and gene enhancement combination therapy. In knockdown / enhancement combination therapy, a defective copy of the gene of interest is silenced and a non-mutated copy is provided. In one embodiment, this is achieved using two or more co-administered vectors. See Millington-Ward et al., Molecular Therapy, April 2011, 19(4):642-649, which is incorporated herein by reference. The transgene can be easily selected by those skilled in the art based on the desired outcome.

[0050] Viral and non-viral vectors The expression cassettes described herein contain a weak promoter and heterologous coding sequence and can be engineered into any suitable genetic element, such as a vector, for delivery to a target cell. As used herein, a "vector" refers to a biological or chemical entity containing a nucleic acid sequence that can be introduced into an appropriate host cell for replication or expression of the nucleic acid sequence. Common vectors include non-viral and viral vectors. As used herein, non-viral systems can be selected from nanoparticles, electroporation systems, and novel biomaterials, naked DNA, phages, transposons, plasmids, cosmids (Phillip McClean, www.ndsu.edu / pubweb / ~mcclean / -plsc731 / cloning / cloning4.htm), and artificial chromosomes (Gong, Shiaoching, et al., "A gene expression atlas of the central nervous system based on bacterial artificial chromosomes," Nature 425.6961 (2003):917-925).

[0051] A "plasmid" or "plasmid vector" is generally designated herein by a lowercase p before and / or after the name of the vector. Plasmids, other cloning and expression vectors that can be used in accordance with the present invention, their properties, and methods for their construction / manipulation will be readily apparent to those of skill in the art. In one embodiment, the nucleic acid sequences described herein or the expression cassettes described herein are engineered within suitable genetic elements (vectors) that transcribe the nuclease sequences carried thereon, useful for generating viral vectors and / or for delivery into host cells, e.g., naked DNA, phage, transposons, cosmids, episomes, etc. The selected vector can be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high-speed DNA-coated pellets, viral infection, and protoplast fusion. Methods used to generate such constructs are known to those skilled in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY.

[0052] In certain embodiments, the expression cassette is positioned within the vector genome for packaging into a viral capsid. For example, for an AAV vector genome, the components of the expression cassette are flanked at the extreme 5' and 3' ends by AAV inverted terminal repeat sequences. For example, 5' AAV ITR, expression cassette, 3' AAV ITR. In other embodiments, a self-complementary AAV may be selected. In other embodiments, a retroviral, lentiviral, or adenoviral system may be used. In one embodiment, the vector genome is set forth in any of SEQ ID NOS: 9-14. In one embodiment, the vector genome is set forth in SEQ ID NOS: 9. In one embodiment, the vector genome is set forth in SEQ ID NOS: 10. In one embodiment, the vector genome is set forth in SEQ ID NOS: 11. In one embodiment, the vector genome is set forth in SEQ ID NOS: 12. In one embodiment, the vector genome is set forth in SEQ ID NOS: 13. In one embodiment, the vector genome is set forth in SEQ ID NOS: 14.

[0053] AAV vectors In certain embodiments, recombinant AAV is provided. A "recombinant AAV" or "rAAV" is a DNAse-resistant viral particle that contains two elements: an AAV capsid and a vector genome that includes at least a non-AAV coding sequence packaged within the AAV capsid. Unless otherwise specified, this term is used interchangeably with the phrase "rAAV vector." rAAVs can be used in a variety of applications. Because rAAVs lack any functional AAV rep or cap genes and are unable to generate progeny, they are "replication-deficient viruses" or "viral vectors." In certain embodiments, the only AAV sequences are AAV inverted terminal repeats (ITRs), which are typically located at the extreme 5' and 3' ends of the vector genome to enable packaging of genes and regulatory sequences located between the ITRs into the AAV capsid.

[0054] The source of the AAV capsid can be any one of several dozen naturally occurring and available adeno-associated viruses and engineered AAVs. Adeno-associated virus (AAV) viral vectors are AAV DNase-resistant particles containing an AAV protein capsid, within which a nucleic acid sequence for delivery to a target cell is packaged. The AAV capsid is composed of 60 capsid protein subunits, VP1, VP2, and VP3, arranged in an icosahedral symmetry in a ratio of approximately 1:1:10 to 1:1:20, depending on the AAV selected. Various AAVs may be selected as the source of the capsid for the above-identified AAV viral vectors. See, for example, U.S. Patent Application Publication No. 2007-0036760-A1, U.S. Patent Application Publication No. 2009-0197338-A1, and EP 1310571. See also WO2003 / 042397 (AAV7 and other simian AAVs), U.S. Patent No. 7,790,449 and U.S. Patent No. 7,282,199 (AAV8), WO2005 / 033321 and U.S. Patent No. 7,906,111 (AAV9), and WO2006 / 110689, WO2003 / 042397 (rh.10), and WO2018 / 160582 (AAVhu68), which also describe other AAVs that can be selected to generate AAVs and are incorporated by reference. Unless otherwise specified, the AAV capsids, ITRs, and other selected AAV components described herein can be readily selected from among any AAV, including, but not limited to, the AAVs commonly identified as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV8bp, AAV7M8, AAVAnc80, AAVrhlO, and AAVPHP.B, as well as variants of any of the known or referenced AAVs, or as yet undiscovered AAVs or variants thereof, or mixtures thereof. See, e.g., WO2005 / 033321, incorporated herein by reference.In one embodiment, the AAV capsid is an AAV1 capsid or a variant thereof, an AAV8 capsid or a variant thereof, an AAV9 capsid or a variant thereof, an AAVrh.10 capsid or a variant thereof, an AAVrh64R1 capsid or a variant thereof, an AAVhu.37 capsid or a variant thereof, or an AAV3B or a variant thereof. In one aspect, the capsid is an AAVhu.37 capsid. See also WO2019 / 168961 and WO2019 / 168961, which are incorporated herein by reference in their entireties.

[0055] In one embodiment, the AAV capsid is an AAVrh.79 capsid or a variant thereof. In another embodiment, the AAV capsid is an AAVrh.90 capsid or a variant thereof.

[0056] In certain embodiments, the rAAV comprises an AAVhu37 capsid. The AAVhu37 capsid comprises a heterogeneous population of vp1 proteins that are the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:22, a heterogeneous population of vp2 proteins that are the product of a nucleic acid sequence encoding the amino acid sequence of at least about amino acids 138-738 of SEQ ID NO:22, and a heterogeneous population of vp3 proteins that are the product of a nucleic acid sequence encoding at least amino acids 204-738 of SEQ ID NO:22, wherein the vp1, vp2, and vp3 proteins contain subpopulations with amino acid modifications comprising at least two highly deamidated asparagines (N) in the asparagine-glycine pairings of SEQ ID NO:22, and optionally further subpopulations comprising other deamidated amino acids, wherein the deamidation results in an amino acid change. AAVhu37 is a heterogeneous population of AAVhu37 capsids. 7 are characterized by having highly deamidated residues, for example, at positions N57, N263, N385, and / or N514, based on the numbering of VP1 (SEQ ID NO: 22).

[0057] Deamidation has been observed at other residues, as shown in the table below and, for example, in WO2019 / 168961, published September 6, 2019, which is incorporated herein by reference. In certain embodiments, an AAVhu37 capsid is modified at one or more of the following positions, in the ranges provided below, as determined using mass spectrometry with the enzyme trypsin. In certain embodiments, one or more of the following positions, or the glycine following an N, is modified as described herein. For example, in certain embodiments, a G can be modified to an S or an A, e.g., at positions 58, 264, 386, or 515. In one embodiment, an AAVhu37 capsid is modified at positions N57 / G58 to N57Q or G58A, resulting in a capsid with reduced deamidation at this position. In another embodiment, N57 / G58 is altered to NS57 / 58 or NA57 / 58. However, in certain embodiments, when NG is modified to NS or NA, increased deamidation is observed. In certain embodiments, the N of the NG pair is modified to Q while retaining G. In certain embodiments, both amino acids of the NG pair are modified. In certain embodiments, N385Q results in a significant decrease in deamidation at that position. In certain embodiments, N499Q results in a significant increase in deamidation at that position.

[0058] In certain embodiments, AAVhu37 may have these or other residues deaminated (e.g., typically less than 10%) and / or methylated (e.g., at ∼R487) (typically less than 5% at a given residue, more typically less than 1%), isomerized (e.g., at D97) (typically less than 5% at a given residue, more typically less than 1%), phosphorylated (e.g., if present, about 10 to about 60%, or about 10 to about 30%, or or in the range of about 20 to about 60%) (e.g., at one or more of S149, S153, S474, S570, S665), or other modifications, including oxidation (e.g., at one or more of W248, W307, W307, M405, M437, M473, W480, W480, W505, M526, M544, M561, W621, M637, and / or W697). Optionally, W can be oxidized to kynurenine. [Table 1]

[0059] Still other positions may have these or other modifications (e.g., acetylation or further deamidation). In certain embodiments, the nucleic acid sequence encoding the AAVhu37 vp1 capsid protein is provided in SEQ ID NO:21. In other embodiments, nucleic acid sequences between 70% and 99.9% identical to SEQ ID NO:21 may be selected to express the AAVhu37 capsid protein. In certain other embodiments, the nucleic acid sequence is at least about 75% identical, at least 80% identical, at least 85%, at least 90%, at least 95%, at least 97% identical, or at least 99% identical to SEQ ID NO:21. However, other nucleic acid sequences encoding the amino acid sequence of SEQ ID NO:22 may be selected for use in producing rAAVhu37 capsids. In certain embodiments, the nucleic acid sequence has the nucleic acid sequence of SEQ ID NO:21, or has a sequence at least 70% to at least 99% identical, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO:21, and encodes SEQ ID NO:22. In certain embodiments, the nucleic acid sequence has the nucleic acid sequence of SEQ ID NO:21, or has a sequence at least 70% to 99%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to about nt 412 to about nt 2214 of SEQ ID NO:21, and encodes the vp2 capsid protein (about aa 138-738) of SEQ ID NO:22. The nucleic acid sequence has the nucleic acid sequence from about nt 610 to about nt 2214 of SEQ ID NO: 21, or has a sequence at least 70% to 99%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to nt of SEQ ID NO: 21, and encodes the vp3 capsid protein (about aa 204 to 738) of SEQ ID NO: 22. See EP 2 345 731 B1 and SEQ ID NO: 88 therein, which are incorporated by reference.

[0060] In certain embodiments, the rAAV comprises an AAV8 capsid. The AAV8 capsid comprises a heterogeneous population of VP isoforms that are deamidated as defined in the table below, based on the total amount of VP protein in the capsid, as determined using mass spectrometry. Suitable modifications include the labeled deamidation modifications described in the section above and are incorporated herein. In certain embodiments, the AAV capsid is modified at one or more of the following positions, within the ranges provided below, as determined using mass spectrometry. In certain embodiments, one or more of the following positions, or a glycine following an N, are modified as described herein. In certain embodiments, an artificial NG is introduced at a position different from one of the positions identified below. In certain embodiments, one or more of the following positions, or a glycine following an N, are modified as described herein. For example, in certain embodiments, G can be modified to S or A at, for example, positions 58, 67, 95, 216, 264, 386, 411, 460, 500, 515, or 541. When NG57 / 58 is modified to NS57 / 58 or NA57 / 58, a significant reduction in deamidation is observed. However, in certain embodiments, when NG is modified to NS or NA, an increase in deamidation is observed. In certain embodiments, the N of the NG pair is modified to Q while retaining the G. In certain embodiments, both amino acids of the NG pair are modified. In certain embodiments, N385Q results in a significant reduction in deamidation at that position. In certain embodiments, N499Q results in a significant increase in deamidation at that position. In certain embodiments, the NG mutation is made in the pair located at N263 (e.g., N263A). In certain embodiments, an NG mutation is made in the pair located at N514 (e.g., N514A). In certain embodiments, an NG mutation is made in the pair located at N540 (e.g., N540A). In certain embodiments, an AAV variant is engineered that includes multiple mutations, and at least one of the mutations at these positions. In certain embodiments, no mutation is made at position N57. In certain embodiments, no mutation is made at position N94. In certain embodiments, no mutation is made at position N305.In certain embodiments, no mutation is made at position G386. In certain embodiments, no mutation is made at position Q467. In certain embodiments, no mutation is made at position N479. In certain embodiments, no mutation is made at position N653. In certain embodiments, the capsid is modified to reduce an "N" or a "Q" at a position other than an "NG" pair. Residue numbers are based on the published AAV8 sequence reproduced in SEQ ID NO:20. [Table 2-1] [Table 2-2]

[0061] In certain embodiments, the rAAV comprises an AAVrh79 capsid, as described in WO2019 / 169004, published September 6, 2019, which is incorporated herein by reference. In one embodiment, the AAVrh79 capsid comprises a heterogeneous population of AAVrh79 vp1, vp2, and vp3 proteins. In one embodiment, the AAVrh79 capsid is produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of 1-738 of SEQ ID NO: 18. Optionally, the sequence co-expresses a vp3 protein from a nucleic acid sequence excluding the vp1 unique region (about aa 1-137) or the vp2 unique region (about aa 1-203), a vp1 protein produced from SEQ ID NO: 17, or a vp1 protein produced from a nucleic acid sequence at least 70% identical to SEQ ID NO: 17 that encodes the predicted amino acid sequence of 1-738 of SEQ ID NO: 18. In other embodiments, AAVrh79 vp2 protein is produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of at least about amino acids 138-738 of SEQ ID NO:18; vp2 protein is produced from a sequence comprising at least nucleotides 412-2214 of SEQ ID NO:17; vp2 protein is produced from a nucleic acid sequence that encodes the predicted amino acid sequence of at least about amino acids 138-738 of SEQ ID NO:18 and that is at least 70% identical to at least nucleotides 412-2214 of SEQ ID NO:18; AAVrh79 vp3 protein is produced by expression from a nucleic acid sequence that encodes the predicted amino acid sequence of at least about amino acids 204-738 of SEQ ID NO:18; vp3 protein, or a vp3 protein produced from a nucleic acid sequence at least 70% identical to at least nucleotides 610 to 2214 of SEQ ID NO:17, which encodes the predicted amino acid sequence of at least about amino acids 204 to 738 of SEQ ID NO:18.

[0062] In certain embodiments, the AAVrh79 capsid comprises a heterogeneous population of vp1 proteins that are the product of nucleic acid sequences encoding the amino acid sequence of SEQ ID NO:18, a heterogeneous population of vp2 proteins that are the product of nucleic acid sequences encoding the amino acid sequence of at least about amino acids 138-738 of SEQ ID NO:18, and a heterogeneous population of vp3 proteins that are the product of nucleic acid sequences encoding at least amino acids 204-738 of SEQ ID NO:18.

[0063] The AAVrh79 vp1, vp2, and vp3 proteins include subpopulations with amino acid modifications that include at least two highly deamidated asparagines (N) in the asparagine-glycine pair in SEQ ID NO: 18, and optionally further include subpopulations that include other deamidated amino acids, where deamidation results in an amino acid change. High levels of deamidation are observed at N57, N263, N385, and / or N514 of the N-G pair relative to the numbering in SEQ ID NO: 18. Deamidation has been observed at other residues, as shown in the table and examples below. In certain embodiments, AAVrh79 may have other residues that are deaminated (e.g., typically less than 10%) and / or methylated (e.g., at up to R487) (typically less than 5% at a given residue, more typically less than 1%), isomerized (e.g., at D97) (typically less than 5% at a given residue, more typically less than 1%), phosphorylated (e.g., if present, about 10 to about 60%, or about 10 to about 30%, or about 20 to about 60%) (e.g., at one or more of S149, S153, S474, S570, S665), or other modifications, including oxidation (e.g., at one or more of W248, W307, W307, M405, M437, M473, W480, W480, W505, M526, M544, M561, W621, M637, and / or W697). Optionally, W can be oxidized to kynurenine. [Table 3]

[0064] In certain embodiments, the AAVrh79 capsid is modified at one or more of the positions identified in the table above, to the ranges shown below, as determined using mass spectrometry with the enzyme trypsin. In certain embodiments, one or more of the following positions, or the glycine following an N, is modified as described herein. Residue numbers are based on the AAVrh79 sequence provided herein. See SEQ ID NO: 18.

[0065] In certain embodiments, the nucleic acid sequence encoding the AAVrh79 vp1 capsid protein is provided in SEQ ID NO: 17. In other embodiments, a nucleic acid sequence 70% to 99.9% identical to SEQ ID NO: 17 may be selected to express the AAVrh79 capsid protein. In certain other embodiments, the nucleic acid sequence is at least about 75% identical, at least 80% identical, at least 85%, at least 90%, at least 95%, at least 97% identical, at least 99%, or at least 99.9% identical to SEQ ID NO: 17. However, other nucleic acid sequences that encode the amino acid sequence of SEQ ID NO: 18 may be selected for use in producing rAAV capsids. In certain embodiments, the nucleic acid sequence has the nucleic acid sequence of SEQ ID NO: 17 or has a sequence at least 70% to at least 99% identical, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or at least 99.9% identical to SEQ ID NO: 17 and encodes SEQ ID NO: 18. In certain embodiments, the nucleic acid sequence is SEQ ID NO: 17 or has a nucleic acid sequence from about nt 412 to about nt 2214 of SEQ ID NO: 17 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%, at least 99%, or at least 99.9% identical to about nt 412 to about nt 2214 of SEQ ID NO: 17, and encodes the vp2 capsid protein (about aa 138 to 738) of SEQ ID NO: 18. In certain embodiments, the nucleic acid sequence has a nucleic acid sequence from about nt 610 to about nt 2214 of SEQ ID NO: 17, or has a sequence 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%, at least 99%, or at least 99.9% identical to nt 412 to about nt 2214 of SEQ ID NO: 17, and encodes the vp3 capsid protein (about aa 204 to 738) of SEQ ID NO: 18.

[0066] The present invention also encompasses nucleic acid sequences encoding mutant AAVrh79 in which one or more residues have been altered to reduce deamidation or other modifications identified herein, and such nucleic acid sequences can be used in the production of mutant rAAVrh79 capsids.

[0067] In certain embodiments, the rAAV comprises an AAVrh.90 capsid, as described in WO2020 / 223232, published November 5, 2020, which is incorporated herein by reference. In a further aspect, a recombinant adeno-associated virus (rAAV) is provided, comprising: (A) an AAVrh.90 capsid; (1) a heterogeneous population of AAVrh.90 vp1 proteins selected from a vp1 protein produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of 1-738 of SEQ ID NO:24, a vp1 protein produced from SEQ ID NO:23, or a vp1 protein produced from a nucleic acid sequence that encodes the predicted amino acid sequence of 1-738 of SEQ ID NO:24 and that is at least 70% identical to SEQ ID NO:23; a vp2 protein produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of at least about amino acids 138-738 of SEQ ID NO:24, a vp2 protein produced from a sequence comprising at least nucleotides 412-2214 of SEQ ID NO:23, or a vp2 protein produced from a nucleic acid sequence that encodes the predicted amino acid sequence of at least about amino acids 138-738 of SEQ ID NO:24 that is at least 70% identical to at least nucleotides 412-2214 of SEQ ID NO:23 that encodes the predicted amino acid sequence of at least about amino acids 138-738 of SEQ ID NO:24; AAVrh selected from a heterologous population of vp2 proteins, vp3 proteins produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of at least about amino acids 204-738 of SEQ ID NO:24, vp3 proteins produced from a sequence comprising at least nucleotides 610-2214 of SEQ ID NO:23, or vp3 proteins produced from a nucleic acid sequence that encodes the predicted amino acid sequence of at least about amino acids 204-738 of SEQ ID NO:24 and that is at least 70% identical to at least nucleotides 610-2214 of SEQ ID NO:23.and / or (2) a heterogeneous population of vp1 proteins that are the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:24, a heterogeneous population of vp2 proteins that are the product of a nucleic acid sequence encoding the amino acid sequence of at least about amino acids 138-738 of SEQ ID NO:24, and a heterogeneous population of vp3 proteins that are the product of a nucleic acid sequence encoding at least amino acids 204-738 of SEQ ID NO:24, wherein the vp1, vp2, and vp3 proteins contain at least two highly deamidated asparagine-glycine pairs in the asparagine-glycine pairs of SEQ ID NO:24. Provided is a recombinant adeno-associated virus (rAAV), comprising: (A) an AAVrh.90 capsid containing a subpopulation having an amino acid modification that includes paragine (N), and optionally further including a subpopulation that includes other deamidated amino acids, wherein the deamidation results in one or more of the amino acid changes; and (B) a vector genome in the AAVrh.90 capsid, the vector genome comprising a nucleic acid molecule that includes an AAV inverted terminal repeat and a non-AAV nucleic acid sequence encoding a product operably linked to a sequence that directs expression of the product in a host cell.

[0068] In certain embodiments, the vp1, vp2, and vp3 proteins of AAVrh.90 contain subpopulations with amino acid modifications that include at least two highly deamidated asparagines (N) in the asparagine-glycine pair of SEQ ID NO: 24, and optionally further subpopulations that include other deamidated amino acids, where deamidation results in an amino acid change. High levels of deamidation are observed at N57, ~N263, ~N385, and / or ~N514 of the N-G pair relative to the numbering of SEQ ID NO: 24. Deamidation has been observed at other residues, as shown in the table below. In certain embodiments, AAVrh.90 may have other residues that are deaminated (e.g., at ~N305, ~N499, and / or ~N599, typically less than 20%) and / or have other modifications, including phosphorylation (e.g., if present, in a range of about 2 to about 30%, or about 2 to about 20%, or about 2 to about 10%) (e.g., at S149) or oxidation (e.g., at one or more of ~W23, ~M204, ~M212, W248, W282, M405, M473, W480, W505, M526, ~N544, M561, and / or ~M607). Optionally, W may be oxidized to kynurenine. [Table 4]

[0069] In certain embodiments, the AAVrh.90 capsid is modified at one or more positions identified in the table above, in the ranges provided, as determined using mass spectrometry with trypsin enzyme. In certain embodiments, one or more of the above positions, or the glycine following the N, is modified as described herein. Residue numbers are based on the AAVrh.90 sequence provided herein. See SEQ ID NO:24.

[0070] In certain embodiments, the AAVrh.90 capsid comprises a heterogeneous population of vp1 proteins that are the product of nucleic acid sequences encoding the amino acid sequence of SEQ ID NO:24, a heterogeneous population of vp2 proteins that are the product of nucleic acid sequences encoding the amino acid sequence of at least about amino acids 138-738 of SEQ ID NO:24, and a heterogeneous population of vp3 proteins that are the product of nucleic acid sequences encoding at least amino acids 204-738 of SEQ ID NO:24.

[0071] As used herein, "vector genome" refers to a nucleic acid sequence that is packaged inside the rAAV capsid that forms the viral particle. Such a nucleic acid sequence contains AAV inverted terminal repeats (ITRs). In the example herein, the vector genome contains at least, from 5' to 3', the AAV 5' ITR, an expression cassette containing a transgene or coding sequence operably linked to a regulatory sequence that directs its expression, and the AAV 3' ITR. The ITRs are involved in genome replication and packaging during vector production. The ITRs are genetic elements and are the only viral cis-elements required to generate rAAV. In one embodiment, the ITRs are derived from a different AAV than the one providing the capsid. In a preferred embodiment, ITR sequences from AAV2, or a deleted version thereof (ΔITR), can be used for convenience. However, ITRs from other AAV sources can be selected. When the source of the ITRs is AAV2 and the AAV capsid is derived from another AAV source, the resulting vector can be referred to as pseudotyped. Typically, an AAV vector genome contains the AAV 5' ITR, a nucleic acid sequence encoding a gene product and any regulatory sequences, and the AAV 3' ITR. However, other configurations of these elements may also be suitable. In one embodiment, a self-complementary AAV is used. A shortened version of the 5' ITR, termed ΔITR, in which the D sequence and terminal resolution site (trs) are deleted, has been described. In a specific embodiment, the vector genome contains a 130-base-pair truncated AAV2 ITR, lacking the external "a" element. The shortened ITRs are restored to their wild-type length of 145 base pairs during vector DNA amplification using the internal A element as a template. In other embodiments, full-length AAV 5' and 3' ITRs are used. In other embodiments, full-length or engineered ITRs may be selected. Additionally, the vector genome comprises regulatory sequences that regulate expression of the gene product (e.g., directly or indirectly by regulating transcription and / or translation). Suitable components of the vector genome are discussed in more detail herein.

[0072] For use in producing AAV viral vectors (e.g., recombinant (r)AAV), the expression cassette can be carried on any suitable vector, e.g., a plasmid, that is delivered to a packaging host cell. Plasmids useful in the present invention can be engineered to be suitable for in vitro replication and packaging in prokaryotic, insect, or mammalian cells, among others. Suitable transfection techniques and packaging host cells are known and / or can be readily designed by one of skill in the art. In one embodiment, the vector genome set forth in SEQ ID NO: 13 is packaged into an AAVhu.37 capsid.

[0073] Methods for generating and isolating AAV suitable for use as a vector are known in the art. See generally, for example, Grieger & Samulski, 2005, "Adeno-associated virus as a gene therapy vector: Vector development, production, and characterization." 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). The ITRs are the only AAV components required in cis in the same construct as the nucleic acid molecule containing the expression cassette to package the transgene into virions. The cap and rep genes can be supplied in trans.

[0074] The term "AAV intermediate" or "AAV vector intermediate" refers to an assembled rAAV capsid that lacks the desired genomic sequence packaged therein. These may also be referred to as "empty" capsids. Such capsids may contain no detectable genomic sequence of an expression cassette or may contain only partially packaged genomic sequence that is insufficient to achieve expression of a gene product. These empty capsids are non-functional for introducing a gene of interest into a host cell.

[0075] The recombinant adeno-associated viruses (AAVs) described herein can be produced using known techniques. AAV capsid proteins may also be produced. See, for example, WO 2003 / 042397, WO 2005 / 033321, WO 2006 / 110689, and US 7,588,772 B2. Such methods involve culturing host cells containing an expression cassette consisting of a nucleic acid sequence encoding an AAV capsid protein, a functional rep gene, at least AAV inverted terminal repeats (ITRs), and a transgene, as well as sufficient helper functions to enable packaging of the expression cassette into AAV capsid proteins. Methods for generating capsids, coding sequences therefor, and methods for producing rAAV viral vectors have been described. See, for example, Gao, et al., Proc. Natl. Acad. Sci. USA 100(10), 6081-6086 (2003) and US 2013 / 0045186 A1.

[0076] In one embodiment, a producer cell culture useful for producing recombinant AAV is provided. Such cell culture contains a nucleic acid that expresses AAV capsid proteins in a host cell, a nucleic acid molecule suitable for packaging into an AAV capsid, e.g., a vector genome containing non-AAV nucleic acid sequences encoding a gene product operably linked to AAV ITRs and sequences that direct expression of the product in the host cell, and sufficient AAV rep and adenovirus helper functions to allow packaging of the nucleic acid molecule into a recombinant AAV capsid. In one embodiment, the cell culture is comprised of mammalian cells (e.g., human embryonic kidney 293 cells, among others) or insect cells (e.g., baculovirus).

[0077] Optionally, the rep function is provided by an AAV other than the AAV that provides the capsid. For example, rep can be, but is not limited to, AAV1 rep protein, AAV2 rep protein, AAV3 rep protein, AAV4 rep protein, AAV5 rep protein, AAV6 rep protein, AAV7 rep protein, AAV8 rep protein, or rep 78, rep 68, rep 52, rep 40, rep 68 / 78, and rep 40 / 52, or fragments thereof, or another source. Optionally, the rep sequence and cap sequence are on the same genetic element in cell culture. A spacer can be present between the rep sequence and the cap gene. Any of these AAV or mutant AAV capsid sequences can be under the control of exogenous regulatory control sequences that direct their expression in host cells.

[0078] In one embodiment, the cells are produced in suitable cell culture (e.g., HEK293) cells. Methods for producing gene therapy vectors described herein include methods well known in the art, such as generating plasmid DNA used to produce the gene therapy vector, generating the vector, and purifying the vector. In some embodiments, the gene therapy vector is an AAV vector, and the generated plasmids are 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, passaging the cells, seeding the cells, transfecting the cells with plasmid DNA, changing the medium to serum-free medium after transfection, and harvesting the vector-containing cells and culture medium. The harvested vector-containing cells and culture medium are referred to herein as crude cell harvest. In yet another system, gene therapy vectors are introduced into insect cells by infection with baculovirus-based vectors. For reviews of these production systems, see, generally, for example, Zhang et al. See, e.g., Wang et al., 2009, "Adenovirus-adeno-associated virus hybrid for large-scale recombinant adeno-associated virus production," Human Gene Therapy 20:922-929, the contents of each of which are incorporated herein by reference in their entirety. Methods of making and using these and other AAV production systems are also described in the following U.S. patents, the contents of each of which are incorporated herein by reference: Nos. 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, each of which is incorporated herein by reference in its entirety.

[0079] The crude cell harvest may then be subjected to process steps such as concentration of the vector harvest, diafiltration of the vector harvest, microfluidization of the vector harvest, nuclease digestion of the vector harvest, filtration of the microfluidized intermediate, crude purification by chromatography, crude purification by ultracentrifugation, buffer exchange by tangential flow filtration, and / or formulation and filtration to prepare bulk vector.

[0080] The vector drug product is purified and empty capsids are removed using a two-step affinity chromatography purification at high salt concentrations, followed by anion exchange resin chromatography. These methods are described in further detail in International Patent Publication No. 2017 / 160360, which is incorporated herein by reference. The purification methods of International Patent Publication No. 2017 / 100676 for AAV8, International Patent Publication No. 2017 / 100704 for rh10, and International Patent Publication No. 2017 / 100674 for AAV1 are all incorporated herein by reference.

[0081] To calculate the content of empty and filled particles, the VP3 band volume for a selected sample (e.g., in the present example, a preparation purified on an iodixanol gradient, where the number of GC particles = the number of particles) is plotted against the number of loaded GC particles. The resulting linear equation (y = mx + c) is used to calculate the number of particles in the band volume of the test article peak. The number of particles (pt) per 20 μL loaded is then multiplied by 50 to obtain particles (pt) / mL. Pt / mL is divided by GC / mL to obtain the ratio of particles to genome copies (pt / GC). Pt / mL - GC / mL yields empty pt / mL. Dividing empty pt / mL by pt / mL and multiplying by 100 gives the percentage of empty particles.

[0082] Generally, methods for assaying AAV vector particles, including empty capsids and packaged genomes, are known in the art. See, e.g., Grimm et al. See Wobus et al., Gene Therapy (1999) 6:1322-1330; Sommer et al., Molec. Ther. (2003) 7:122-128. To test for denatured capsids, the method involves subjecting the treated AAV stock to SDS-polyacrylamide gel electrophoresis (e.g., a gradient gel containing 3-8% Tris acetate in buffer) using any gel capable of separating the three capsid proteins, running the gel until the sample material is separated, and blotting the gel onto a nylon or nitrocellulose membrane, preferably nylon. An anti-AAV capsid antibody, preferably an anti-AAV capsid monoclonal antibody, most preferably a B1 anti-AAV-2 monoclonal antibody, is then used as the primary antibody that binds to the denatured capsid proteins (Wobus et al., J. Virol. (2000) 74:9281-9293). Next, a secondary antibody is used that binds to the primary antibody and contains a means for detecting binding to the primary antibody, more preferably an anti-IgG antibody that contains a detection molecule covalently bound to the antibody itself, and most preferably a sheep anti-mouse IgG antibody covalently bound to horseradish peroxidase. To semiquantitatively determine binding between the primary and secondary antibodies, a method for detecting binding is used, preferably a detection method that can detect radioisotope radiation, electromagnetic radiation, or colorimetric changes, most preferably a chemiluminescent detection kit. For example, in SDS-PAGE, samples from column fractions can be taken and heated in SDS-PAGE loading buffer containing a reducing agent (e.g., DTT), and capsid proteins are resolved on a precast gradient polyacrylamide gel (e.g., Novex). SilverXpress (Inv Silver staining may be performed using a chromatograph (Generogen, CA) according to the manufacturer's instructions, or other suitable staining methods, i.e., SYPRO Ruby or Coomassie dye, may be used. In one embodiment, the concentration of AAV vector genome (vg) in column fractions can be measured by quantitative real-time PCR (Q-PCR). Samples are diluted and digested with DNase I (or another suitable nuclease) to remove exogenous DNA. After inactivation of the nuclease, the sample is further diluted and amplified using primers and a TaqMan™ fluorogenic probe specific for the DNA sequence between the primers. The number of cycles required to reach a defined level of fluorescence (threshold cycle, Ct) is measured for each sample on an Applied Biosystems Prism 7700 Sequence Detection System. Plasmid DNA containing the same sequence as that contained in the AAV vector is used to generate a standard curve in the Q-PCR reaction. The cycle threshold (Ct) value obtained from the sample is used to determine the vector genome titer by normalizing it to the Ct value of the plasmid standard curve. An endpoint assay based on digital PCR may also be used.

[0083] In one embodiment, an optimized q-PCR method utilizing a broad-spectrum serum protease, e.g., proteinase K (e.g., commercially available from Qiagen), is used. More specifically, the optimized qPCR genomic titer assay is similar to the standard assay, except that after DNase I digestion, the sample is diluted with proteinase K buffer and treated with proteinase K, followed by heat inactivation. Preferably, the sample is diluted with a volume of proteinase K buffer equal to the sample size. The proteinase K buffer can be concentrated two-fold or more. Typically, proteinase K treatment is about 0.2 mg / mL, but can vary from 0.1 mg / mL to about 1 mg / mL. The treatment step is generally performed at about 55°C for about 15 minutes, but can also be performed at lower temperatures (e.g., about 37°C to about 50°C) for longer periods (e.g., about 20 minutes to about 30 minutes) or at higher temperatures (e.g., up to about 60°C) for shorter periods (e.g., about 5 to 10 minutes). Similarly, heat inactivation is typically at about 95°C for about 15 minutes, although the temperature may be lowered (e.g., about 70 to about 90°C) and the time may be extended (e.g., about 20 to about 30 minutes). The sample is then diluted (e.g., 1000-fold) and subjected to TaqMan analysis as described for standard assays.

[0084] Additionally or alternatively, droplet digital PCR (ddPCR) may be used. For example, a method for determining single-stranded and self-complementary AAV vector genome titers by ddPCR has been described. See, for example, M. Lock et al., Hu Gene Therapy Methods, Hum Gene Ther Methods. 2014 Apr;25(2):115-25. doi:10.1089 / hgtb.2013.131. Epub 2014 Feb 14.

[0085] Briefly, a method for separating rAAV particles containing packaged genome sequences from genome-defective AAV intermediates involves subjecting a suspension containing recombinant AAV viral particles and AAV capsid intermediates to high-performance liquid chromatography, in which the AAV viral particles and AAV intermediates are bound to a strong anion exchange resin equilibrated at a high pH and subjected to a salt gradient while monitoring the eluate for ultraviolet absorbance at about 260 and about 280. The pH can be adjusted depending on the AAV selected. See, e.g., WO2017 / 160360 (AAV9), WO2017 / 100704 (AAVrhlO), WO2017 / 100676 (e.g., AAV8), and WO2017 / 100674 (AAVl), which are incorporated herein by reference. In this method, AAV complete capsids are recovered from the fraction eluted when the A260 / A280 ratio reaches an inflection point. In one example, for the affinity chromatography step, the diafiltered product is purified using Capture Select™, which efficiently captures AAV2 serotypes. ) Poros-AAV2 / 9 affinity resin (Life Technologies). Under these ionic conditions, AAV particles are efficiently captured while a significant proportion of residual cellular DNA and proteins flow through the column.

[0086] Pharmaceutical Compositions Pharmaceutical compositions comprise one or more of the expression cassette, a vector (viral or non-viral) containing it, or another system containing the expression cassette and one or more of a carrier, suspending agent, and / or excipient.

[0087] In certain embodiments, a composition contains at least one rAAV stock (e.g., an rAAV stock), and optionally a carrier, excipient, and / or preservative. An rAAV stock refers to multiple rAAV vectors that are the same (e.g., in amounts such as those described below in the discussion of concentrations and dosage units).

[0088] As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Supplementary active ingredients can also be incorporated into the composition. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce allergic or similar adverse reactions when administered to a host. Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like can be used to introduce the compositions of the present invention into suitable host cells. In particular, the rAAV vector delivery vector genome can be formulated for delivery encapsulated in either lipid particles, liposomes, vesicles, nanospheres, nanoparticles, or the like.

[0089] In certain embodiments, the expression cassette is delivered via lipid nanoparticles. The term "lipid nanoparticle" refers to a lipid composition having a typical spherical structure with an average diameter of 10 to 1000 nanometers, e.g., 75 to 750 nm, or 100 to 350 nm, or 250 to about 500 nm. In some formulations, the lipid nanoparticles can contain at least one cationic lipid, at least one non-cationic lipid, and at least one conjugated lipid. Lipid nanoparticles known in the art that are suitable for encapsulating nucleic acids, such as mRNA, can be used. The "average diameter" refers to the average size of a population of nanoparticles containing a lipophilic phase and a hydrophilic phase. The average size of these systems can be measured by standard methods known to those skilled in the art. Examples of lipid nanoparticles suitable for gene therapy are described, for example, in L. Battaglia and E. Ugazio, J Nanomaterials, Vol. 2019, Article ID 283441, pp. 1-22, US2012 / 0183589A1, and WO2012 / 170930, which are incorporated herein by reference in their entireties.

[0090] In one embodiment, the composition comprises a final formulation suitable for delivery to a subject, for example, an aqueous liquid suspension buffered to a physiologically compatible pH and salt concentration. Optionally, one or more surfactants are present in the formulation. In another embodiment, the composition can be delivered as a concentrate that is diluted for administration to a subject. In other embodiments, the composition can be lyophilized and reconstituted at the time of administration.

[0091] Methods and agents well known in the art for making formulations are described, for example, in "Remington's Pharmaceutical Sciences," Mack Publishing Company, Easton, Pa. Formulations may contain, for example, an excipient, carrier, stabilizer, or diluent, for example, sterile water, saline, polyalkylene glycols such as polyethylene glycol, vegetable oils, or hydrogenated naphthalenes, preservatives, or the like. Preservatives (e.g., octadecyldimethylbenzyl, ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight polypeptides, proteins such as serum albumin, gelatin, or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as , glycine, glutamine, asparagine, histidine, arginine, and lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, and dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0092] The active ingredient may also be encapsulated in microcapsules, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or macroemulsions, respectively, prepared, for example, by coacervation techniques or by interfacial polymerization. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0093] A suitable surfactant or surfactant combination may be selected from non-toxic non-ionic surfactants. In one embodiment, a difunctional block copolymer surfactant terminated with a primary hydroxyl group, such as Pluronic® F68 (BASF), which has a neutral pH and an average molecular weight of 8400 and is also known as Poloxamer 188, is selected. Other surfactants and poloxamers may be selected, i.e., non-ionic triblock copolymers composed of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)), such as SOLUTOL HS 15 (macrogol-15 hydroxystearate), LABRASOL (polyoxycaprylic acid glyceride), polyoxy 10 oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid ester), ethanol, and polyethylene glycol. In one embodiment, the formulation contains a poloxamer. These copolymers are generally named using the letter "P" (for poloxamer) followed by three digits, where the first two digits x 100 give the approximate molecular mass of the polyoxypropylene core and the last digit x 10 gives the percentage polyoxyethylene content. In one embodiment, poloxamer 188 is selected. The surfactant may be present in an amount up to about 0.0005% to about 0.001% of the suspension.

[0094] The vector is administered in an amount sufficient to transfect cells and provide sufficient levels of gene transfer and expression to provide a therapeutic effect without undue adverse effects or with a medically acceptable physiological effect, which can be determined by one of ordinary skill in the art. Conventional 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, intraarterial, intraocular, intravenous, intramuscular, subcutaneous, intradermal, and other parenteral routes of administration. Routes of administration may be combined, if desired.

[0095] The dosage of a viral vector depends primarily on factors such as the condition being treated, the age, weight, and health of the patient, and may therefore vary between patients. For example, a therapeutically effective human dosage of a viral vector is generally about 1×10 9 ~1×10 16 Genomic viral vector concentration The dosage ranges from about 25 to about 1000 microliters to about 100 mL, containing the composition. The dosage is adjusted to balance the therapeutic benefit against any side effects, and such dosage may vary depending on the therapeutic application for which the recombinant vector is utilized. The level of expression of the transgene product can be monitored to determine the frequency of dosage resulting in a viral vector, preferably an AAV vector containing a minigene. Optionally, a dosing regimen similar to that described for therapeutic purposes can be utilized for immunization using the compositions of the present invention.

[0096] The replication-deficient virus composition may be administered to a human patient in an amount of approximately 1.0 x 10 (to treat an average subject weighing 70 kg), including all integers or fractions within the range. 9 GC~approx. 1.0×10 16 GC range, preferably 1.0 x 10 12 GC~1.0×10 14 The composition can be formulated into dosage units containing an amount of replication-deficient virus in the range of GC. In one embodiment, the composition contains at least 1 x 10 per dose, including all integers or fractions within the range. 9, 2 × 10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , or 9 x 10 9 In another embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, including all integers or fractions within the range. 10 , 2 × 10 10 , 3×10 10 , 4×10 10 , 5×10 10 , 6×10 10 , 7×10 10 , 8×10 10 , or 9 x 10 10 In another embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, including all integers or fractions within the range. 11 , 2 × 10 11 , 3×10 11 , 4×10 11 , 5×10 11 , 6×10 11 , 7×10 11 , 8×10 11 , or 9 x 10 11 In another embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, including all integers or fractions within the range. 12 , 2 × 10 12 , 3×10 12 , 4×10 12 , 5×10 12 , 6×10 12 , 7×10 12 , 8×10 12 , or 9 x 10 12 In another embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, including all integers or fractions within the range. 13 , 2 × 10 13 , 3×10 13 , 4×10 13 , 5×10 13 , 6×10 13 , 7×10 13 , 8×1013 , or 9 x 10 13 In another embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, including all integers or fractions within the range. 14 , 2 × 10 14 , 3×10 14 , 4×10 14 , 5×10 14 , 6×10 14 , 7×10 14 , 8×10 14 , or 9 x 10 14 In another embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, including all integers or fractions within the range. 15 , 2 × 10 15 , 3×10 15 , 4×10 15 , 5×10 15 , 6×10 15 , 7×10 15 , 8×10 15 , or 9 x 10 15 In one embodiment, for human applications, the dose is 1 x 10 per dose, including all integers or fractions within the range. 10 ~Approx. 1×10 12 It can be in the range of GC.

[0097] These above-mentioned doses may be administered in various volumes of carrier, excipient, or buffer formulation, such as in the range of about 25 to about 1000 microliters, or even larger volumes, including all integers or fractions 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.

[0098] Any suitable route of administration may be selected. Thus, the pharmaceutical composition may be formulated for any appropriate route of administration, for example, in the form of a liquid solution or suspension (e.g., for intravenous administration, oral administration, etc.). Alternatively, the pharmaceutical composition may be in solid form (e.g., for oral administration, for example, in the form of a tablet or capsule). In some embodiments, the pharmaceutical composition may be in the form of a powder, a drip infusion, an aerosol, etc.

[0099] method The compositions provided herein are useful for reducing off-target activity of enzymes delivered in vivo. In certain embodiments, the compositions are administered as described herein. In certain embodiments, the compositions are useful for reducing off-target activity of enzymes expressed after non-viral-mediated delivery of an expression cassette containing an enzyme-coding sequence under the control of a weak promoter, such as those described herein. In certain embodiments, the compositions are useful for reducing off-target activity of enzymes expressed after AAV-mediated delivery of a vector genome.

[0100] In one embodiment, a method for editing a targeted gene is provided. The method includes delivering a nuclease expression cassette comprising a nucleic acid comprising a nuclease coding sequence operably linked to a regulatory sequence that directs expression of the nuclease after delivery to a host cell having a sequence targeted by the nuclease, the regulatory sequence comprising a promoter with low transcriptional activity. Such promoters are described herein. In another embodiment, the method includes delivering a composition, viral vector, or rAAV comprising the expression cassette, as described herein.

[0101] In another embodiment, a method for reducing off-target activity of a gene-targeting nuclease is provided. The method comprises delivering a nuclease expression cassette comprising a nucleic acid comprising a nuclease-encoding sequence operably linked to a regulatory sequence that directs expression of the nuclease after delivery to a host cell having a sequence targeted by the nuclease, the regulatory sequence comprising a promoter with low transcriptional activity. Such promoters are described herein. In another embodiment, the method comprises delivering a composition, viral vector, or rAAV comprising the expression cassette, as described herein.

[0102] In certain embodiments, the effectiveness of weak promoter can be evaluated in vitro.For example, the half-life of nuclease can be evaluated in vitro (in cultured cells) by treating cells to stop protein translation (for example, with cycloheximide (CHX)), and then performing Western blot at different times after treatment.Other suitable methods for evaluating the off-target activity of nuclease can be easily determined by those skilled in the art.

[0103] The reduction of off-target nuclease activity can be determined using various approaches described in the literature. Such methods for determining nuclease specificity include cell-free methods, such as Site-Seq [Cameron, P., et al., (2017) Mapping the genomic landscape of CRISPR-Cas9 cleavage. Nat Methods, 14, 600-606], Digenome-seq [Kim, D., et al., (2015) Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells. Nat Methods, 12, 237-243, 231 p following 243], and Circle-Seq [Tsai, SQ, et al, (2017) CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR-Cas9 nuclease off-targets. Nat Methods, 14, 607-614], and in vitro-based methods, such as GUIDE-Seq [Tsai (2017) Nat Methods, 14, 607-614] and integration-deficient lentiviral vector capture (IDLV) [Gabriel, R., et al. (2011) An unbiased genome-wide analysis of zinc-finger nuclease specificity. Nat Biotechnol, 29, 816-823, Wang, X., et al. (2015) Unbiased detection of off-target cleavage by CRISPR-Cas9 and TALENs using integrase-defective lentiviral vectors. Nat Biotechnol, 33, 175-178]. It can be obtained.

[0104] In some embodiments, off-target activity is assessed by ITR-seq. See, e.g., Breton et al., "ITR-Seq, a next-generation sequencing assay, identifies genome-wide DNA editing sites in vivo following adeno-associated viral vector-mediated genome editing," BMC Genomics, (2020):21:239, a publication incorporated herein by reference in its entirety.

[0105] In one aspect, a method is provided for editing a targeted gene, comprising delivering a nuclease expression cassette under the control of a weak promoter as described herein.

[0106] In one aspect, a method is provided for editing a targeted gene comprising delivering a composition described herein.

[0107] In one aspect, a method is provided for editing a targeted gene, comprising delivering a viral or non-viral vector described herein.

[0108] In one aspect, a method is provided for editing a targeted gene, comprising delivering an rAAV as described herein.

[0109] In one aspect, a method for treating a patient with a cholesterol-related disorder, such as hypercholesterolemia, is used to treat a patient with a cholesterol-related disorder, such as hypercholesterolemia, using a nuclease expression cassette comprising a meganuclease that recognizes a site in the human PCSK9 gene under the control of a weak promoter described herein. In one embodiment, the weak promoter is F64. In another embodiment, the weak promoter is F113. In another embodiment, the weak promoter is F140. In yet another embodiment, the weak promoter is the CCL16 promoter. In yet another embodiment, the weak promoter is the SCLC22A9 promoter. In yet another embodiment, the weak promoter is the CYP26A1 promoter. Such expression cassettes can be delivered via viral or non-viral vectors. In certain embodiments, the expression cassette can be delivered using LNP.

[0110] In one aspect, a method is provided for treating a patient with a disorder associated with a defect in the alanine dioxylate aminotransferase gene, such as primary hyperoxaluria type 1, using a nuclease expression cassette comprising a meganuclease that recognizes a site in the human HAO gene under the control of a weak promoter described herein. In one embodiment, the weak promoter is F64. In another embodiment, the weak promoter is F113. In another embodiment, the weak promoter is F140. In yet another embodiment, the weak promoter is the CCL16 promoter. In yet another embodiment, the weak promoter is the SCLC22A9 promoter. In yet another embodiment, the weak promoter is the CYP26A1 promoter. Such expression cassettes can be delivered via viral or non-viral vectors. In certain embodiments, the expression cassette can be delivered using LNP. In certain embodiments, the disorder is primary hyperoxaluria (PH1).

[0111] In one aspect, a method is provided for treating a patient having a disorder associated with a defect in the transthyretin (TTR) gene using a nuclease expression cassette comprising a meganuclease that recognizes a site within the human TTR gene under the control of a weak promoter described herein. In one embodiment, the weak promoter is F64. In one embodiment, the weak promoter is F113. In another embodiment, the weak promoter is F140. In yet another embodiment, the weak promoter is the CCL16 promoter. In yet another embodiment, the weak promoter is the SCLC22A9 promoter. In yet another embodiment, the weak promoter is the CYP26A1 promoter. Such expression cassettes can be delivered via viral or non-viral vectors. In certain embodiments, the expression cassette can be delivered using LNP. In certain embodiments, the disorder is TTR-related hereditary amyloidosis.

[0112] In another aspect, a method is provided for treating a patient with a disorder associated with a defect in the apoliprotein C-II (APOC3) gene using a nuclease expression cassette comprising a meganuclease that recognizes a site within the human APOC3 gene under the control of a weak promoter described herein. In one embodiment, the weak promoter is F64. In another embodiment, the weak promoter is F113. In another embodiment, the weak promoter is F140. In yet another embodiment, the weak promoter is the CCL16 promoter. In yet another embodiment, the weak promoter is the SCLC22A9 promoter. In yet another embodiment, the weak promoter is the CYP26A1 promoter. Such expression cassettes can be delivered via viral or non-viral vectors. In certain embodiments, the expression cassette can be delivered using LNP.

[0113] In one aspect, a method is provided for treating a patient with a disorder associated with a defect in the branched-chain α-ketoacid dehydrogenase complex (BCKDC) E1α gene using a nuclease expression cassette comprising a meganuclease that recognizes a site within the human BCKDC E1α gene under the control of a weak promoter described herein. In one embodiment, the weak promoter is F64. In another embodiment, the weak promoter is F113. In another embodiment, the weak promoter is F140. In yet another embodiment, the weak promoter is the CCL16 promoter. In yet another embodiment, the weak promoter is the SCLC22A9 promoter. In yet another embodiment, the weak promoter is the CYP26A1 promoter. Such expression cassettes can be delivered via viral or non-viral vectors. In certain embodiments, the expression cassette can be delivered using LNP. In certain embodiments, the disorder is maple syrup urine disease.

[0114] In one aspect, a method for editing genes using CRISPR / Cas-associated nucleases is provided, using an expression cassette containing a coding sequence for a CRISPR / Cas-associated nuclease that recognizes a site within a desired gene under the control of a weak promoter described herein. In one embodiment, the weak promoter is F64. In another embodiment, the weak promoter is F113. In another embodiment, the weak promoter is F140. In yet another embodiment, the weak promoter is the CCL16 promoter. In yet another embodiment, the weak promoter is the SCLC22A9 promoter. In yet another embodiment, the weak promoter is the CYP26A1 promoter. Such expression cassettes can be delivered via viral or non-viral vectors. In certain embodiments, the expression cassettes can be delivered using LNP.

[0115] In one aspect, a method for editing genes using TALEN is provided, which uses an expression cassette comprising a TALEN coding sequence that recognizes a site in a desired gene under the control of a weak promoter as described herein.In one embodiment, the weak promoter is F64.In another embodiment, the weak promoter is F113.In another embodiment, the weak promoter is F140.In yet another embodiment, the weak promoter is CCL16 promoter.In yet another embodiment, the weak promoter is SCLC22A9 promoter.In yet another embodiment, the weak promoter is CYP26A1 The promoter is a promoter. Such expression cassettes can be delivered via viral or non-viral vectors. In certain embodiments, the expression cassettes can be delivered using LNPs.

[0116] In one aspect, a method for editing genes using zinc finger nucleases is provided, using an expression cassette containing a coding sequence for a zinc finger nuclease that recognizes a site in a desired gene under the control of a weak promoter described herein. In one embodiment, the weak promoter is F64. In another embodiment, the weak promoter is F113. In another embodiment, the weak promoter is F140. In yet another embodiment, the weak promoter is the CCL16 promoter. In yet another embodiment, the weak promoter is the SCLC22A9 promoter. In yet another embodiment, the weak promoter is the CYP26A1 promoter. Such expression cassettes can be delivered via viral or non-viral vectors. In certain embodiments, the expression cassettes can be delivered using LNP.

[0117] In one aspect, a method for editing genes using meganucleases is provided, using an expression cassette containing a coding sequence for a meganuclease that recognizes a site within a desired gene under the control of a weak promoter described herein. In one embodiment, the weak promoter is F64. In another embodiment, the weak promoter is F113. In another embodiment, the weak promoter is F140. In yet another embodiment, the weak promoter is the CCL16 promoter. In yet another embodiment, the weak promoter is the SCLC22A9 promoter. In yet another embodiment, the weak promoter is the CYP26A1 promoter. Such expression cassettes can be delivered via viral or non-viral vectors. In certain embodiments, the expression cassettes can be delivered using LNP.

[0118] In certain embodiments, nucleases other than meganucleases that target any of the above genes are contemplated.

[0119] In certain embodiments, any of the nuclease expression cassettes described herein, non-viral vectors, viral vectors (e.g., rAAV), or in pharmaceutical compositions can be administered for gene editing in a patient. In certain embodiments, the methods are useful for non-embryonic gene editing. In certain embodiments, the patient is an infant (e.g., birth to about 9 months). In certain embodiments, the patient is older than a toddler, e.g., 12 months or older.

[0120] As used herein, "a," "an," or "the" can mean one or more than one. For example, "a" cell can mean a single cell or multiple cells.

[0121] In certain embodiments, the term "meganuclease" refers to an endonuclease that binds to double-stranded DNA with a recognition sequence greater than 12 base pairs. Preferably, the recognition sequence of the meganuclease of the present invention is 22 base pairs. Meganucleases may be endonucleases derived from I-CreI and may refer to engineered variants of I-CreI that have been modified relative to the native I-CreI, for example, with respect to DNA-binding specificity, DNA-cleavage activity, DNA-binding affinity, or dimerization properties. Methods for producing such modified variants of I-CreI are known in the art. See, for example, WO2007 / 047859. Meganucleases as used herein bind to double-stranded DNA as heterodimers. Meganucleases may also be "single-chain meganucleases" in which a pair of DNA-binding domains are connected to a single polypeptide using a peptide linker. The term "homing endonuclease" refers to a "meganuclease" " is synonymous with the term "PCSK9 meganuclease." See WO2018 / 195449, which describes certain PCSK9 meganucleases, and is incorporated herein in its entirety. In one embodiment, the meganuclease is not an ARCUS meganuclease as described herein.

[0122] As used herein, the term "specificity" refers to the ability of a meganuclease to recognize and cleave a double-stranded DNA molecule only at a specific sequence of base pairs, referred to as a recognition sequence, or only at a specific set of recognition sequences. A set of recognition sequences share certain conserved positions or sequence motifs, but may be degenerate at one or more positions. A highly specific meganuclease can cleave only one or a very small number of recognition sequences. Specificity can be determined by any method known in the art.

[0123] The abbreviation "sc" refers to self-complementary. "Self-complementary AAV" refers to a construct in which the coding region carried by the recombinant AAV nucleic acid sequence is designed to form an intramolecular double-stranded DNA template. During infection, rather than waiting for cell-mediated synthesis of the second strand, the two complementary halves of the scAAV will associate to form a single double-stranded DNA (dsDNA) unit ready for immediate replication and transcription. See, for example, D. M. McCarty et al., "Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis," Gene Therapy, (August 2001), Vol. 8, Number 16, Pages 1248-1254. Self-complementary AAVs are described, for example, in U.S. Patent Nos. 6,596,535, 7,125,717, and 7,456,683, each of which is incorporated by reference herein in its entirety.

[0124] As used herein, the term "operably linked" refers both to expression control sequences that are contiguous with a gene of interest and to expression control sequences that act in trans or at a distance to regulate a gene of interest.

[0125] The term "exogenous" when used to describe a nucleic acid sequence or protein means that the nucleic acid or protein does not naturally occur in the chromosome or at the location present in the host cell. An exogenous nucleic acid sequence also refers to a sequence that originates from and is inserted into the same expression cassette or host cell, but exists in a non-native state (e.g., in a different copy number or under the control of different regulatory elements).

[0126] The term "heterologous," when used with reference to a protein or nucleic acid, indicates that the protein or nucleic acid comprises two or more sequences or subsequences that are not found in the same relationship to each other in nature. For example, nucleic acids having two or more sequences from unrelated genes arranged to create a new functional nucleic acid are typically produced recombinantly. For example, in one embodiment, a nucleic acid has a promoter from one gene arranged to direct expression of a coding sequence from a different gene.

[0127] As used herein, the term "host cell" may refer to a packaging cell line in which a vector (e.g., a recombinant AAV) is produced from a production plasmid. Alternatively, the term "host cell" may refer to any target cell in which expression of a transgene is desired. Thus, a "host cell" refers to a prokaryotic or eukaryotic cell that contains an exogenous or heterologous nucleic acid sequence that has been introduced into the cell by any means, such as electroporation, calcium phosphate precipitation, microinjection, transformation, viral infection, transfection, liposome delivery, membrane fusion techniques, high-speed DNA-coated pellets, viral infection, and protoplast fusion. In certain embodiments herein, the term "host cell" refers to a cell that contains an exogenous or heterologous nucleic acid sequence that has been introduced into the cell by any means, such as electroporation, calcium phosphate precipitation, microinjection, transformation, viral infection, transfection, liposome delivery, membrane fusion techniques, high-speed DNA-coated pellets, viral infection, and protoplast fusion. "Host cells" refers to cultures of cells of various mammalian species for in vitro evaluation of the compositions described herein. In other embodiments herein, the term "host cells" refers to cells used to produce and package viral vectors or recombinant viruses. In yet other embodiments, the term "host cells" is intended to refer to target cells of a subject to be treated in vivo for a disease or condition described herein. In certain embodiments, the term "host cells" is a liver cell or hepatocyte.

[0128] A "replication-defective virus" or "viral vector" refers to a synthetic or artificial viral particle in which an expression cassette containing a gene of interest is packaged within a viral capsid or envelope, and any viral genomic sequences packaged within the viral capsid or envelope are also replication-defective, i.e., they are unable to produce progeny virions but retain the ability to infect target cells. In one embodiment, the genome of the viral vector does not contain genes encoding enzymes required for replication (the genome can be engineered to be "gutless," containing only the gene of interest flanked by signals required for amplification and packaging of the artificial genome), although these genes can be supplied during production. Thus, they are considered safe for use in gene therapy because replication and infection by progeny virions cannot occur except in the presence of viral enzymes required for replication.

[0129] The terms "sequence identity," "percent sequence identity," or "percent identical" in the context of nucleic acid sequences refer to the residues in two sequences that are the same when aligned for maximum correspondence. The length of sequence identity comparison can be, and preferably is, over the entire length of a genome, the entire length of a gene coding sequence, or a fragment of at least about 500-5000 nucleotides. However, identity between smaller fragments, e.g., at least about 9 nucleotides, usually at least about 20-24 nucleotides, at least about 28-32 nucleotides, or at least about 36 or more nucleotides, may also be desired. Similarly, "percent sequence identity" can be readily determined for amino acid sequences over the entire length of a protein or a fragment thereof. Preferably, the fragment is at least about 8 amino acids in length and can be up to about 700 amino acids in length. Examples of suitable fragments are described herein.

[0130] The terms "substantial homology" or "substantial similarity," when referring to a nucleic acid, or a fragment thereof, indicate that when optimally aligned with another nucleic acid (or its complementary strand) by appropriate amino acid insertions or deletions, the nucleic acid has at least about 95-99% amino acid sequence identity with the aligned sequence. Preferably, the homology is over the full-length sequence, or a protein thereof, e.g., cap protein, rep protein, or a fragment thereof that is at least 8 amino acids, or more preferably, at least 15 amino acids in length. Examples of suitable fragments are described herein.

[0131] The term "highly conserved" means at least 80% identity, preferably at least 90% identity, and more preferably greater than 97% identity. Identity is readily determined by those skilled in the art by relying on algorithms and computer programs known to those skilled in the art.

[0132] Generally, when referring to "identity," "homology," or "similarity" between two different adeno-associated viruses, the "identity," "homology," or "similarity" is determined with reference to "aligned" sequences. An "aligned" sequence or "alignment" refers to multiple nucleic acid or protein (amino acid) sequences, often including corrections for missing or additional bases or amino acids, compared to a reference sequence. In an example, an AAV alignment is performed using the published AAV9 sequence as a reference point. Alignment is performed using any of a variety of publicly or commercially available multiple sequence alignment programs. Examples of such programs include "Clustal Omega," "Clustal W," "CAP Sequence Assembly," "MAP," and "MEME," all accessible through web servers on the Internet. Other sources of such programs are known to those of skill in the art. Alternatively, the Vector NTI utility can also be used. There are also several algorithms known in the art that can be used to measure nucleotide sequence identity, including those included in the programs described above. As another example, polynucleotide sequences can be compared using Fasta™, a program in GCG Version 6.1. Fasta™ provides alignments and percent sequence identity of the best overlapping regions between the query and search sequences. For example, percent sequence identity between nucleic acid sequences can be determined using Fasta™ using its default parameters (word size 6 and NOPAM factor for the scoring matrix) as provided in GCG Version 6.1, incorporated herein by reference. Multiple sequence alignment programs, such as "Clustal Omega," "Clustal X," "MAP," "PIMA," "MSA," "BLOCKMAKER," "MEME," and "Match-Box" programs, are also available for amino acid sequences. Typically, one of these programs is used with default settings, but one of skill in the art may modify these settings as needed. Alternatively, one of skill in the art may utilize another algorithm or computer program that provides at least the same level of identity or alignment as that provided by the referenced algorithm and program. See, e.g., J.D. Thomson et al., Nucl. Acids. Res., "A comprehensive comparison of multiple sequence alignments," 27(13):2682-2690 (1999).

[0133] As used herein, the term "about" refers to a ±10% variation from a reference integer and values ​​therebetween. For example, "about" 40 base pairs includes ±4 (i.e., 36-44, including the integers 36, 37, 38, 39, 40, 41, 42, 43, and 44). When referring to other values, particularly percentages (e.g., 90% identity, about 10% variance, or about 36% mismatch), the term "about" includes all values ​​within the range, including both integers and fractions.

[0134] As used throughout this specification and claims, the terms "comprising," "containing," "including," and variations thereof are inclusive of other components, elements, integers, steps, etc. Conversely, the term "consisting" and variations thereof exclude other components, elements, integers, steps, etc.

[0135] Unless otherwise defined herein, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art and by reference to the published literature, which provides general guidance to those skilled in the art for many of the terms used herein. [Example]

[0136] ARCUS nuclease (I-CreI endonuclease, further engineered by Precision BioSciences) recognizes and cleaves 22 bp target sequences in DNA. Cellular proteins recognize and repair these breaks in the DNA. The result of this repair mechanism is the insertion or deletion of nucleotides (indels) at the edited locus, and these modifications affect the expression of the corresponding gene.

[0137] Applicants observed high rates of editing in DNA target sequences in both mouse and rhesus monkey studies after adeno-associated virus (AAV) vector-mediated delivery of ARCUS nuclease. However, sequences similar to the on-target region were also shown to contain indels, indicating off-target activity of ARCUS nuclease.

[0138] Applicants hypothesized that a certain level of M2PCSK9 is required for on-target editing, and that increased nuclease expression above this threshold results in off-target activity. To reduce the level of M2PCSK9 expression, the parental TBG promoter in the AAV construct was replaced with a promoter with low transcriptional activity.

[0139] Example 1 - In vivo mouse study The present applicants have observed high percentage of editing in the DNA target sequence in both mouse and rhesus monkey tests after adeno-associated virus (AAV) vector-mediated delivery of ARCUS nuclease.However, sequences similar to the on-target region have also been shown to contain indels, indicating the off-target activity of ARCUS nuclease.This off-target activity is undesirable, and it is essential to reduce or eliminate this off-target activity while maintaining high on-target efficacy, especially for clinical studies.

[0140] The applicant's hypothesis is that, in contrast to gene therapy, in which high transgene expression is desirable, genome editing requires lower transgene expression, while higher expression also promotes off-target editing.Therefore, the objective of the present invention is to reduce transgene expression by reducing its transcription.This can be achieved by selecting a liver-specific promoter with weak transcriptional activity.

[0141] Candidate promoters were selected using two methods. In the first approach, liver-specific human genes with low RNA expression were identified. We searched the Human Atlas Protein database using consensus transcript expression levels (NX levels) as a parameter of transcriptional activity and selected genes whose transcription is also enriched in the liver.

[0142] The TBG (thyroid hormone-binding globulin) promoter has been shown to be useful for AAV-mediated delivery of transgenes to the liver. We selected three genes that reduce NX levels, which are also enriched in the liver. The promoter regions of these genes were obtained from SwitchGear Genomics (Carlsbad, CA) (Table 1). [Table 5] [Table 6]

[0143] 1 Consensus normalized expression (NX) from the Human Protein Atlas, available at http: / / www.proteinatlas.org. *Data for parent TBG (no enhancer)

[0144] For our second approach, we shortened the sequence to TBG We aimed to reduce the transcriptional activity of a smaller (176 bp) version of the promoter, the TBG-S1 promoter. Starting from the upstream sequence, increasing lengths of this sequence were removed, resulting in promoters TBG-S1-F140 (F140), TBG-S1-F113 (F113), and TBG-S1-F64 (F64), which contained 140, 113, or 64 bp of the TBG-S1 promoter, respectively.

[0145] AAV serotype 8 vectors were generated in which expression of the PCSK9-specific ARCUS nuclease was mediated by one of these six weak promoters. A schematic representation of the genome of these AAVs is shown in Figure 1. The following vectors were generated: a) AAV8.CCL16-1k.ARCUS2.bGH b) AAV8.CYP26A1-1k.ARCUS2.bGH c) AAV8.SLC22A9-1k.ARCUS2.bGH d) AAV8.TBG-S1-F64.ARCUS2.bGH e) AAV8.TBG-S1-F113.ARCUS2.bGH f) AAV8.TBG-S1-F140.ARCUS2.bGH

[0146] Initial tests were carried out in mice. Briefly, mice were administered with AAV expressing human PCSK9, and two weeks later, mice were given a second injection of AAV expressing PCSK9-specific ARCUS nuclease under a different weak promoter. As a positive control, a construct was used in which nuclease expression was mediated by the TBG promoter. Seven weeks after the administration of the second vector, mice were euthanized, and their livers were collected for further analysis.

[0147] The levels of indels in the regions corresponding to the ARCUS nuclease target sequence were quantified by next-generation sequencing (Figures 2A and 2B). The results showed that in two of the weak promoter groups (TBG-S1-F113 and TBG-S1-F140), the percentage of indels was approximately 40% at 7 weeks after nuclease administration, indicating that on-target activity was maintained. In the rest of the groups, on-target activity was lower than 10%, except for the TBG control group, where editing was 60-70% (Figures 2A and 2B, linear and logarithmic scales, respectively). Figure 2C shows the mean levels of recombinant PCSK9 in serum, as determined by ELISA assay, for each treatment group.

[0148] The number of off-target loci in genomic DNA as a result of nuclease activity was then determined using an NGS-based method called ITR-Seq. In the publication by Breton et al., ITR-Seq, a next-generation sequencing assay, identifies genome-wide DNA editing sites in vivo following adeno-associated viral vector-mediated genome editing. Edited by BMC Genomics, (2020): 21: 239, which is incorporated herein by reference in its entirety. Compared to the TBG control, which had approximately 160 off-target loci, the number of off-target loci for all weak promoter groups was reduced (Figure 3).

[0149] A more quantitative approach to measuring the off-target activity of these vectors was to calculate indels among a subset of identified off-targets. Analysis was performed only on the TBG control, TBG-S1-F113, and TBG-S1-F140 groups, as these showed the highest indel percentages (Figures 2A and 2B). Figure 4 shows indels among the set of genomic locations corresponding to identified off-targets. The indel level for each off-target is shown relative to the indel level in the TBG control group (arbitrary value 1). A roughly 20-fold reduction in indels was observed among the group of weak promoters analyzed. This indicates that the use of these promoters clearly reduces nuclease off-target activity.

[0150] The hPCSK9 levels in the injected mice are shown in FIG.

[0151] Overall, these results indicate that using a weak, liver-specific promoter to mediate the expression of genome-editing nucleases is a promising strategy to reduce their off-target activity while retaining their on-target activity.

[0152] Example 2 - NHP Pilot Study To observe whether off-target reduction was preserved in NHPs, rhesus monkeys were cultured at 6 x 10 12 Patients were treated with vector at a dose of GC / kg. Biopsy data were collected on day 18 (with a maximum follow-up of 1 year). Weekly AAV bleeds were performed from vector administration until day 28, then every other week until study termination. The vectors tested were AAV8.TBG.M2PCSK9 and AAV8.TBG-S1-F113.M2PCSK9. The following tests were performed: neutralizing antibodies against the AAV8 capsid, CBC / Chem / Coag / lipid panel, serum for PCSK9 expression by ELISA, PBMC isolation every 8 weeks for IFN-g ELISPOT, liver biopsies on days 18 and 128, and DNA / RNA analysis to detect on- and off-target genome editing by next-generation sequencing.

[0153] A summary of some of the data presented in Figures 7-10 is shown in Figure 6. The % indels (Figure 7) and number of off-targets (Figure 8) were determined in DNA from liver biopsies 18 days post-AAV. PCSK9 levels (as a percentage of baseline) are shown in Figure 9 (7 weeks post-AAV). LDL levels (as a percentage of baseline) are shown in Figure 10.

[0154] We observed similar reductions in PCSK9 and LDL levels in treated NHPs. Expressing the ARCUS nuclease through the use of a weak promoter reduces nuclease off-target activity in mice and NHPs while retaining its on-target activity against the PCSK9 gene.

[0155] Example 3 - GLP Toxicity Study A GLP toxicity study will be conducted in NHPs (n=27) with data from day 120 and follow-up up to 1 year (minimum). The study design is shown in Figure 11. IV administration of the AAVhu37.TBG-S1-F113.M2PCSK9 vector containing the vector genome set forth in SEQ ID NO: 13 will be provided at one of three doses: 1.2e12, 6.0e12, or 3.0e13. Weekly bleeds will be administered from vector administration until day 28, then every other week until study termination. The following studies will be performed: Neutralizing antibodies against AAVhu.37 capsid, CBC / Chem / Coag / Lipid panel, serum for PCSK9 expression by ELISA, PBMC isolation every 8 weeks for IFN-g ELISPOT, liver biopsy on day 18 for all NHPs, DNA / RNA analysis to detect on-target and off-target genome editing by next-generation sequencing, necropsy on day 28 of 3 NHPs per group - histopathology and biodistribution for all major organs, necropsy on day 120 of 3 NHPs per group - histopathology and biodistribution for all major organs, and liver biopsies on days 180 and 364 for the final 3 NHPs per group.

[0156] Applicants expect to observe similar reductions in PCSK9 and LDL levels in treated NHPs as in the studies described above. By expressing the ARCUS nuclease using a weak promoter, Applicants believe that the nuclease's off-target activity may be reduced in NHPs while retaining its on-target activity against the PCSK9 gene. I predict that this will happen.

[0157] All documents cited herein, as well as Breton et al., Increasing the Specificity of AAV-Based Gene Editing through Self-Targeting and Short-Promoter Strategies, Mol Ther. 2021 Mar 3;29(3):1047-1056. doi:10.1016 / j.ymthe.2020.12.028. Epub 2020 Dec 25., are hereby incorporated by reference. U.S. Provisional Patent Application No. 63 / 016,541, filed April 27, 2020; U.S. Provisional Patent Application No. 63 / 033,738, filed June 2, 2020; U.S. Provisional Patent Application No. 63 / 089,796, filed October 9, 2020; and U.S. Provisional Patent Application No. 63 / 016,139, filed April 27, 2020, are incorporated by reference in their entirety, along with their sequence listings. The sequence listing filed herewith under the title "20-9267PCT_Seq-Listing_ST25.txt," and the sequences and text therein, are incorporated by reference. While the present invention has been described with reference to specific embodiments, it will be understood that modifications can be made without departing from the spirit of the invention. Such modifications are intended to fall within the scope of the appended claims. [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5]

Claims

1. A nuclease expression cassette comprising a nucleic acid comprising a nuclease coding sequence operably linked to a regulatory sequence that directs expression of the nuclease after delivery to a host cell having a sequence targeted by the nuclease, wherein the regulatory sequence comprises a promoter that is TBG-S1-F64 (SEQ ID NO: 6), TBG-S1-F113 (SEQ ID NO: 7) or TBG-S1-F140 (SEQ ID NO: 8).

2. 2. The nuclease expression cassette of claim 1, wherein the promoter is TBG-S1-F64 (SEQ ID NO: 6).

3. 2. The nuclease expression cassette of claim 1, wherein the promoter is TBG-S1-F113 (SEQ ID NO: 7).

4. 2. The nuclease expression cassette of claim 1, wherein the promoter is TBG-S1-F140 (SEQ ID NO: 8).

5. The nuclease expression cassette of any one of claims 1 to 4, wherein the nuclease is a meganuclease, a CRISPR / Cas nuclease, a zinc finger nuclease, or a TALEN.

6. The nuclease expression cassette of any one of claims 1 to 4, wherein the nuclease is a meganuclease.

7. A pharmaceutical composition comprising the nuclease expression cassette of any one of claims 1 to 6 and one or more of a carrier, suspending agent, and / or excipient.

8. The pharmaceutical composition of claim 7 , wherein the expression is in a non-viral delivery system.

9. 9. The pharmaceutical composition of claim 8, wherein the non-viral delivery system is a lipid nanoparticle.

10. A viral vector comprising the nuclease expression cassette of any one of claims 1 to 6.

11. A recombinant AAV useful for gene editing, comprising an AAV capsid and a vector genome packaged in the AAV capsid, wherein the vector genome comprises: (a) an expression cassette according to any one of claims 1 to 6; and (b) AAV inverted terminal repeats necessary for packaging the expression cassette into the capsid.

12. 12. A composition comprising the viral vector of claim 10 or the recombinant AAV of claim 11 and one or more of a carrier, diluent, and / or excipient.

13. 12. A composition for use in a method for editing a targeted gene, comprising the nuclease expression cassette of any one of claims 1 to 6, the composition of any one of claims 7 to 9, the viral vector of claim 10, or the rAAV of claim 11.

14. A composition for use in a method for reducing off-target activity of a gene-targeting nuclease, the composition comprising a nuclease expression cassette described in any one of claims 1 to 6, a composition described in any one of claims 7 to 9, a viral vector described in claim 10, or an rAAV described in claim 11.

15. A promoter comprising the sequence of SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:

8.

16. An expression cassette comprising the promoter of claim 15.

17. An expression vector comprising the promoter of claim 15.

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