Data pipeline systems, devices, and methods

Abstract

A data pipeline for string extraction, clustering, and comparison is disclosed. The method may include extracting sequence regions from each plasmid genome sequence sequenced from the plasmid genome based on the presence of fixed adjacent sequence markers (FFSMs) in the plasmid genome sequence. Each sequence region is located within an FFSM and contains a candidate inverted end repeat (ITR) sequence. An exemplary method further includes clustering two or more sequence regions of the sequence regions based on complete sequence identity to generate clusters, merging two or more clusters of the clusters based on alignment between their corresponding sequence regions, identifying the genotype of the candidate ITR sequence of the single cluster based on local alignment if a single cluster remains, and producing multiple AAV vectors using plasmids having ITR sequences having the genotype of the candidate ITR sequence, based on the genotype of the candidate ITR sequence.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 63 / 507,918, filed on 13 June 2023, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Adeno-associated viruses (AAVs) are small, non-enveloped viruses belonging to the parvoviridae family. AAVs are known for their ability to infect both dividing and non-dividing cells and are currently not associated with any disease. These characteristics make AAVs an attractive candidate for use as vectors in gene therapy applications.

[0003] Gene therapy is a technique that treats or prevents disease by inserting genes into a patient's cells. Gene therapy includes, for example, replacing disease-causing mutated genes with healthy copies of those genes, inactivating or "knocking out" improperly functioning mutated genes, and introducing new genes that help fight disease.

[0004] In the context of gene therapy, a vector is a vehicle for delivering a therapeutic gene to a patient's cells. The use of AAV as a vector in gene therapy has been widely studied due to its ability to deliver genes to a wide range of tissues and its low immunogenicity. AAV vectors are constructed by removing viral genes and replacing them with the therapeutic gene of interest. The resulting recombinant AAV (rAAV) vector is then used to deliver the therapeutic gene to the patient's cells.

[0005] The AAV genome consists of single-stranded DNA molecules flanked by inverted end repeats (ITRs). ITRs are DNA sequences that are identical when read from 5' to 3' on one strand and from 3' to 5' on the complementary strand. These sequences play a role in the replication, packaging, and integration of the AAV genome. ITRs are the only cis-acting elements retained in the rAAV vector genome and function as the origin of viral replication and packaging signal. ITR genotypes can affect the efficiency of vectors in delivering therapeutic genes to patient cells. Therefore, identifying ITR genotypes is an essential part of the production of AAV vectors for gene therapy applications. [Overview of the project]

[0006] A method and system are disclosed, comprising: sequencing the genomes of multiple plasmids to obtain multiple plasmid genome sequences; receiving specifications for fixed adjacent sequence markers; extracting from each plasmid genome sequence multiple sequence regions, each sequence region being within a fixed adjacent sequence marker and containing a candidate inverted end repeat (ITR) sequence, based on the presence of fixed adjacent sequence markers in the plasmid genome sequence; clustering two or more of the sequence regions based on complete sequence identity to generate multiple clusters; merging two or more of the clusters based on alignment between their corresponding sequence regions; identifying the genotype of a candidate ITR sequence of the single cluster based on local alignment if a single cluster remains; and producing multiple AAV vectors using plasmids containing ITR sequences having the genotype of the candidate ITR sequence, based on the genotype of the candidate ITR sequence.

[0007] A method and system are disclosed, comprising: sequencing the genomes of multiple plasmids to obtain multiple plasmid genome sequences; receiving specifications for fixed adjacent sequence markers; extracting from each plasmid genome sequence multiple sequence regions, each of which is located within a fixed adjacent sequence marker and contains a candidate inverted end repeat (ITR) sequence, based on the presence of fixed adjacent sequence markers in that plasmid genome sequence; clustering two or more of the sequence regions based on sequence identity to generate multiple clusters; merging two or more of the clusters based on the alignment between their corresponding sequence regions; and determining that multiple plasmids are unsuitable for the production of recombinant vector genomes based on the two or more clusters remaining after merging.

[0008] A method and system are disclosed, comprising: sequencing the genomes of multiple plasmids to obtain multiple plasmid genome sequences; receiving specifications for fixed adjacent sequence markers; extracting from each plasmid genome sequence multiple sequence regions, each of which is located within a fixed adjacent sequence marker and contains a candidate inverted end repeat (ITR) sequence, based on the presence of a fixed adjacent sequence marker in that plasmid genome sequence; clustering two or more of the sequence regions based on sequence identity to generate multiple clusters; merging two or more of the clusters based on the alignment between their corresponding sequence regions; and, if a single cluster remains after the merge, identifying the genotype of a representative sequence region of the single cluster based on local alignment.

[0009] A method and system are disclosed, comprising: sequencing AAV vector genomes from multiple adeno-associated virus (AAV) vectors to obtain multiple AAV vector genome sequences; receiving specifications for fixed adjacent sequence markers; extracting a sequence region from each of the multiple AAV vector genome sequences that contains a candidate ITR sequence, based on the presence of a fixed adjacent sequence marker in that AAV vector genome sequence, that is immediately to the left or right of the fixed adjacent sequence marker; clustering two or more sequence regions from the multiple sequence regions based on complete sequence identity to generate multiple clusters; merging two or more of the multiple clusters based on the alignment between their corresponding sequence regions; and determining that multiple AAV vector genomes are unsuitable based on the two or more clusters remaining after merging.

[0010] A method and system are disclosed, comprising: sequencing multiple adeno-associated virus (AAV) vector genomes to obtain multiple AAV vector genome sequences; receiving specifications for fixed adjacent sequence markers; extracting a sequence region from each of the multiple AAV vector genome sequences that contains a candidate ITR sequence, based on the presence of a fixed adjacent sequence marker in that AAV vector genome sequence, that is immediately to the left or right of the fixed adjacent sequence marker; clustering two or more sequence regions from the multiple sequence regions based on complete sequence identity to generate multiple clusters; merging two or more of the multiple clusters based on the alignment between their corresponding sequence regions to generate multiple modified clusters; and, if a single cluster remains after the merge, identifying the genotype of a representative sequence region of the single cluster based on local alignment.

[0011] A method for treating a subject in need of treatment is disclosed, comprising administering to the subject a therapeutically effective amount of an AAV vector comprising a vector genome encapsulated by an adeno-associated virus (AAV) capsid, wherein the AAV genome comprises at least two AAV inverted terminal repeats (ITRs), nucleic acid sequences encoding a therapeutic agent, and the genotypes of at least two AAV ITRs are identical to a reference AAV ITR determined based on the method disclosed herein.

[0012] Further advantages of the disclosed methods and compositions are partially described in the following description, partially understood from the description, or can be acquired through the practice of the disclosed methods and compositions. The advantages of the disclosed methods and compositions will be realized and achieved by the elements and combinations specifically indicated in the appended claims. It should be understood that both the above summary and the modes for carrying out the invention below are merely illustrative and descriptive and do not limit the claimed invention.

[0013] The accompanying drawings incorporated herein and constituting part thereof illustrate several embodiments of the disclosed methods and compositions and, together with the description, serve to illustrate the principles of the disclosed methods and systems. [Brief explanation of the drawing]

[0014] [Figure 1] An example diagram of an AAV vector is shown. [Figure 2A] This shows the secondary structure of the ITR of the AAV virus genome. [Figure 2B] The flipped L-ITR chain and the flipped R-ITR chain are shown. [Figure 3-1] This document provides an overview of sample preparation before next-generation sequencing. [Figure 3-2] Same as above. [Figure 4] An exemplary method for determining the ITR genotype is shown. [Figure 5A] An exemplary fixed-adjacent sequence marker method is shown. [Figure 5B]An exemplary fixed-adjacent sequence marker method is shown. [Figure 5C] An exemplary fixed-adjacent sequence marker method is shown. [Figure 5D] An exemplary fixed-adjacent sequence marker method is shown. [Figure 6] This example demonstrates a method for clustering sequence regions to compare candidate sequence regions with reference sequences. [Figure 7] This example demonstrates a method for clustering and merging sequence regions to compare candidate sequence regions with a reference sequence. [Figure 8] An exemplary method for determining the ITR genotype is shown. [Figure 9] This shows the Integrated Genomics Viewer (IGV). [Figure 10-1] This shows the visualization of pINT alignment to a reference sequence using PacBio CCS (Circular Consensus Sequence) data. [Figure 10-2] Same as above. [Figure 11] This shows regions of IGV where some deletions are associated with triple SNP mutations, but others are not. [Figure 12-1] This shows another visualization of pINT alignment relative to a reference sequence using Illumina 150bp data. [Figure 12-2] Same as above. [Figure 13-1] This shows another visualization of pINT alignment relative to a reference sequence using an Oxford Nanopore external data source (Plasmidsaurus). [Figure 13-2] Same as above. [Figure 13-3] Same as above. [Figure 14-1] This shows how the L-ITR CC' deletion induces a linear isoform of the FIX plasmid. [Figure 14-2] Same as above. [Figure 15-1] This shows L-ITR BB' deletion. [Figure 15-2] Same as above. [Figure 16]This shows how a payload deletion generates the same 100bp motif as an L-ITR CC' deletion. [Figure 17] This shows a large homology region (125 bp) between the L-ITR flop sequence and the R-ITR flip sequence. [Figure 18] An exemplary operating environment is shown. [Figure 19] This document describes an exemplary method for evaluating ITR in plasmids. [Figure 20] This document describes an exemplary method for evaluating ITR in plasmids. [Figure 21] Another exemplary method for evaluating ITR in plasmids is shown. [Figure 22] This document presents an exemplary method for evaluating ITR in AAV vectors. [Figure 23] Another exemplary method for evaluating ITR in AAV vectors is presented. [Modes for carrying out the invention]

[0015] The methods and compositions disclosed may be more readily understood by referring to the following detailed descriptions of specific embodiments and the examples contained herein, as well as the drawings and their preceding and succeeding descriptions.

[0016] A.Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the invention pertains. Methods and materials similar to or equivalent to those described herein may be used in carrying out or testing the invention, but preferred methods and materials are described herein. It should be understood that the disclosed methods and compositions are not limited to the specific methodologies, protocols, and reagents described herein, as they may vary. It should also be understood that the terms used herein are solely for the purpose of describing specific embodiments and are not intended to limit the scope of the invention, which would be limited only by the appended claims.

[0017] As used herein, “adapter” or “sequencing adapter” typically refers to a short nucleic acid (e.g., less than about 500 nucleotides, less than about 100 nucleotides, less than about 50 nucleotides, 20-30 nucleotides in length) that is at least partially double-stranded and used to ligate to one or both ends of a given sample nucleic acid molecule. An adapter may include sequencing primer binding sites at both ends, which include nucleic acid primer binding sites that enable amplification of the nucleic acid molecule adjacent to the adapter, and / or primer binding sites for sequencing applications (e.g., various next-generation sequencing (NGS) applications). An adapter may also include binding sites for capture probes, such as oligonucleotides bound to a flow cell support. An adapter may also include nucleic acid tags as described herein. Nucleic acid tags are typically positioned relative to amplification primer and sequencing primer binding sites so that the nucleic acid tag is included in the amplicon and sequencing reads of a given nucleic acid molecule. Adapters with the same or different sequences may be ligated to each end of a nucleic acid molecule. In certain embodiments, the same adapter is ligated to each end of a nucleic acid molecule, except that the nucleic acid tag differs in its sequence. In some embodiments, the adapter is a Y-shaped adapter, with one end being blunt-ended or tail-added for linking to a nucleic acid molecule, as described herein (the nucleic acid molecule is also blunt-ended or tail-added with one or more complementary nucleotides). In yet other exemplary embodiments, the adapter is a bell-shaped adapter, with one end being blunt-ended or tail-added for linking to the nucleic acid molecule to be analyzed. Other exemplary adapters include T-tailed adapters and C-tailed adapters.

[0018] As used herein, “administer” or “dosage” a therapeutic agent (e.g., an immunological therapeutic agent, a DNA damage response (DDR) inhibitor (e.g., a poly(ADP-ribose) polymerase (PARP) inhibitor (PARPi)), an adeno-associated virus (AAV), a liquid nanoparticle (LNP), etc.) to a subject means giving, applying, or bringing the composition into contact with the subject. Administration can be achieved by any of several routes, including, for example, topical, oral, subcutaneous, intramuscular, intraperitoneal, intravenous, intrathecal, and / or intradermal.

[0019] As used herein, “align,” “alignment,” “the act of aligning,” “mapping,” and “the act of mapping” in the context of nucleic acids refer to arranging DNA or RNA sequences to identify regions of similarity. Similarity may relate to nucleotide sequences, structural, functional, and / or evolutionary relationships between sequences. Alignment of DNA or RNA sequences involves the alignment of one sequence's DNA or RNA to at least one other sequence's DNA or RNA. Such alignments may exclude non-genomic DNA or non-transcribed RNA, such as molecular barcodes or padding bases. For example, the DNA of a sequence read may be aligned with the genomic DNA of a reference DNA sequence, excluding any molecular tags or adapter sequences that may bind to the sequence read. Alignment may be performed using any number of tools, including, but not limited to, BLAST, BLASR, BWA-MEM, DAMAPPER, NGMLR, GraphMap, and Minimap, among others.

[0020] Throughout this specification and the claims, the word “comprise,” and variations thereof such as “comprising” and “comprises,” mean “including, but not limited to,” and are not intended to exclude, for example, other additives, components, elements, or steps. In particular, in methods described to include one or more steps or operations, each step is specifically intended to include the enumerated items (unless the step contains a limiting term such as “consisting of”), and each step is not intended to exclude, for example, other additives, components, elements, or steps not enumerated in the step.

[0021] As used herein, the enumeration of nucleotides "corresponding" to a sequence refers to nucleotides identified during alignment with a sequence to maximize identity using any alignment method, such as Smith-Waterman or any related method.

[0022] Gene therapy involves modifying the genetic material of host cells (e.g., addition, substitution, or removal) with the aim of treating or curing a disease. One approach to correcting defective gene expression is to replace a non-functional or defective disease-causing gene by inserting a normal gene (transgene) into a specific location in the genome. Gene therapy can also be used as a platform for delivering therapeutic proteins or RNA to treat various diseases, so that the therapeutic product is expressed for an extended period, eliminating the need for repeated medication. To deliver the transgene to the patient's target cells, a carrier molecule called a vector must be used, and the most common vector is a virus that has been genetically modified to carry a normal human gene. Viruses have evolved ways of encapsulating their genes and delivering them to human cells in a pathogenic manner. Therefore, viral genomes can be manipulated to insert therapeutic genes. Stable transgene expression can be achieved after in vivo delivery of adenovirus or adeno-associated virus (AAV)-based vectors to non-dividing cells, and also by transplantation of stem cells transduced ex vivo with embedded and non-embedded vectors, such as retrovirus and lentivirus-based vectors.

[0023] The term "DNA (deoxyribonucleic acid)" refers to a chain of nucleotides containing deoxyribonucleosides, each containing one of four nucleic acid bases: adenine (A), thymine (T), cytosine (C), and guanine (G). The term "RNA (ribonucleic acid)" refers to a chain of nucleotides containing four types of ribonucleosides, each containing one of four nucleic acid bases: A, uracil (U), G, and C. Specific pairs of nucleotides bind to each other specifically in a complementary manner (called complementary base pairing). In DNA, adenine (A) pairs with thymine (T), and cytosine (C) pairs with guanine (G). In RNA, adenine (A) pairs with uracil (U), and cytosine (C) pairs with guanine (G). When a first nucleic acid strand binds to a second nucleic acid strand consisting of nucleotides complementary to the nucleotides in the first strand, the two strands join to form a double heddle. As used herein, “nucleic acid sequencing data,” “nucleic acid sequencing information,” “nucleic acid sequence,” “nucleotide sequence,” “genome sequence,” “gene sequence,” or “fragment sequence,” or “nucleic acid sequencing read” refers to any information or data indicating the order of nucleotide bases (e.g., adenine, guanine, cytosine, and thymine, or uracil) in a nucleic acid molecule such as DNA or RNA (e.g., whole genome, whole transcriptome, exome, oligonucleotide, polynucleotide, or fragment). It should be understood that this instruction intends sequence information to be obtained using all types of available techniques, platforms, or technologies, including but not limited to capillary electrophoresis, microarrays, ligation-based systems, polymerase-based systems, hybridization-based systems, direct or indirect nucleotide identification systems, pyrosequencing, ion or pH-based detection systems, and digital signature-based systems.

[0024] "Exemplary" means "an example of ~" and is not intended to suggest a preferred or ideal configuration. "For example (such as)" is used for explanatory purposes, not in a restrictive sense.

[0025] Nucleic acid sequences may have eukaryotic origins. In this respect, eukaryotic genes are composed of “exons” and “introns.” As used herein, the term “exon” refers to a nucleic acid sequence present in a gene that is represented in the mature form of the RNA molecule after the excision of introns during transcription. Exons are translated into proteins. As used herein, the term “intron” refers to a nucleic acid sequence present in a given gene that is not translated into proteins and is generally found between exons. During transcription, introns are removed from precursor messenger RNA (premRNA), and exons are ligated via RNA splicing. Therefore, in one embodiment, a nucleic acid sequence may contain one or more exons and introns. As used herein, the term “transcription” refers to the process of creating an equivalent RNA copy of a DNA sequence, involving steps of initiation, extension, termination, and RNA processing (including splicing) (see, for example, Griffiths et al., eds., Modern Genetic Analysis: Integrating Genes and Genomes, 2nd ed., WH Freeman and Co., New York (2002)).

[0026] A vector sequence containing an AAV vector may contain one or more “expression regulatory elements.” Typically, an expression regulatory element is a nucleic acid sequence(s) that affects the expression of an operablely linked polynucleotide. Regulatory elements, including expression regulatory elements described herein such as promoters and enhancers present in the vector, are included to facilitate proper transcription and, as appropriate, translation of heterologous polynucleotides (e.g., promoters, enhancers, intron splicing signals, maintenance of the correct reading frame of a gene to enable in-frame translation of mRNA, and stop codons). Such elements typically act in the cis (referred to as “cis-acting” elements) but can also act in the trans (referred to as “trans-acting” elements). Expression regulation can occur at levels such as transcription, translation, splicing, and message stability. Typically, expression regulatory elements that modulate transcription are juxtaposed near the 5' end of the transcribed polynucleotide (i.e., “upstream”). Expression regulatory elements can also be located at the 3' end of the transcription sequence (i.e., “downstream”) or within the transcript (e.g., within an intron). Expression regulatory elements can be located adjacent to the transcription sequence, away from it (e.g., 1–10, 10–25, 25–50, 50–100, 100–500 nucleotides, or more, from the polynucleotide), or even at considerable distances. Nevertheless, due to the polynucleotide length limitations of certain vectors, such as AAV vectors, such expression regulatory elements are typically within 1–1000 nucleotides of the transcribed polynucleotide. Functionally, the expression of operably linked heterologous polynucleotides is at least partially regulated by elements (e.g., promoters), so that the elements regulate the transcription of the polynucleotide and, where appropriate, the translation of the transcript. A specific example of an expression regulatory element is a promoter, usually located at the 5' end of the transcription sequence. Another example of an expression regulatory element is an enhancer, which can be located at the 5' end, 3' end of the transcription sequence, or within the transcription sequence itself.As used herein, “promoter” may refer to a nucleic acid (e.g., DNA) sequence located adjacent to a polynucleotide sequence encoding a recombinant product. Promoters are typically operably ligated to adjacent sequences (e.g., heterologous polynucleotides). Promoters typically increase the amount expressed from heterologous polynucleotides compared to the amount expressed in the absence of the promoter. As used herein, “enhancer” may refer to a sequence located adjacent to heterologous polynucleotides. Enhancer elements are typically located upstream of promoter elements but are also functional and can be located downstream or within the DNA sequence (e.g., heterologous polynucleotide). Thus, enhancer elements can be located 100, 200, or 300 base pairs, or more, upstream or downstream of heterologous polynucleotides. Enhancer elements typically increase the expression of heterologous polynucleotides beyond the increase in expression brought about by promoter elements. Expression regulatory elements (e.g., promoters and / or enhancers) include those that are active in specific tissues or cell types, which are referred herein to as “tissue-specific expression regulatory elements / promoters.” Tissue-specific regulatory elements are typically active in specific cells or tissues (e.g., liver, brain, central nervous system, spinal cord, eye, retina, bone, muscle, lung, pancreas, heart, and kidney cells). Regulatory elements are typically active in specific cells, tissues, or organs because they are recognized by transcription-activating proteins or other transcriptional regulators specific to those cells, tissues, or organ types. Examples of promoters active in skeletal muscle include promoters derived from genes encoding α-actin, myosin light chain 2A, dystrophin, and muscle creatine kinase, as well as synthetic muscle promoters with higher activity than naturally occurring promoters (see Li, et al., Nat. Biotech. 17:241-245 (1999)).Examples of liver-tissue-specific promoters include the human alpha-1 antitrypsin (hAAT) promoter, albumin (Miyatake, et al. J. Virol., 71:5124-32 (1997)), hepatitis B virus core promoter (Sandig, et al., Gene Ther. 3:1002-9 (1996)), and alpha-fetoprotein (AFP) (Arbuthnot, et al., Hum. Gene. Ther., 7:1503-14 (1996)1). Examples of bone-tissue-specific promoters include osteocalcin (Stein, et al., Mol. Biol. Rep., 24:185-96 (1997)) and bone sialoprotein (Chen, et al., J. Bone Examples of promoters that are tissue-specific to lymphocytes include CD2 (Hansal, et al., J.Immunol., 161:1063-8 (1998)), immunoglobulin heavy chain, and T cell receptor α chain, while examples of promoters that are tissue-specific to neurons include the neuron-specific enolase (NSE) promoter (Andersen, et al., Cell.Mol.Neurobiol., 13:503-15 (1993)), neurofilament light chain gene (Piccioli, et al., Proc.Natl.Acad.Sci.USA, 88:5611-5 (1991)), and neuron-specific vgf gene (Piccioli, et al., Neuron, 15:373-84 (1995)). Examples of enhancers active in the liver include HCR-1 and HCR-2 of apolipoprotein E (apoE) (Allan et al., J. Biol. Chem., 272:29113-19 (1997)). Expression regulatory elements also include ubiquitous or promiscuous promoters / enhancers that can drive polynucleotide expression in many different cell types.Such elements include, but are not limited to, the cytomegalovirus (CMV) earliest promoter / enhancer sequence, the Roussarcoma virus (RSV) promoter / enhancer sequence, and other viral promoters / enhancers active in various mammalian cell types, or synthetic elements not found in nature (see, e.g., Boshart et al, Cell, 41:521-530 (1985)), the SV40 promoter, the dihydrofolate reductase promoter, the cytoplasmic β-actin promoter, and the phosphoglycerol kinase (PGK) promoter. Expression regulatory elements can also confuse expression in a moduloable manner, i.e., a signal or stimulus increases or decreases the expression of operably linked heterologous polynucleotides. Moduloable elements that increase the expression of operably linked polynucleotides in response to a signal or stimulus are also called “inducible elements” (i.e., signal-induced). Specific examples include, but are not limited to, hormone (e.g., steroid) inducible promoters. A moduloable element that operably reduces the expression of a polynucleotide in response to a signal or stimulus is called a “suppressive element” (i.e., the signal reduces expression, and therefore, if the signal is removed or absent, expression increases). Typically, the amount of increase or decrease conferred by such an element is proportional to the amount of signal or stimulus present, with greater amounts of signal or stimulus resulting in a greater increase or decrease in expression.Specific non-limiting examples include the zinc-inducible sheep metallothionein (MT) promoter, the steroid hormone-inducible mouse mammary cancer virus (MMTV) promoter, the T7 polymerase promoter system (WO98 / 10088), the tetracycline-inhibiting system (Gossen, et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)), the tetracycline-inducible system (Gossen, et al., Science. 268:1766-1769 (1995), and see also Harvey, et al., Curr. Opin. Chem. Biol. 2:512-518 (1998)), and the RU486-inducible system (Wang, et al., Nat. Biotech. 15:239-243 (1997) and Wang, et al., Gene Examples include Ther. 4:432-441 (1997)1), as well as the rapamycin-inducible system (Magari, et al., J. Clin. Invest. 100:2865-2872 (1997), Rivera, et al., Nat. Medicine. 2:1028-1032 (1996)). Other modulotable regulatory elements that may be useful in this context are those regulated by specific physiological conditions (e.g., temperature, acute phase, differentiation). Expression regulatory elements also include native elements of heterologous polynucleotides. Native regulatory elements (e.g., promoters) may be used when it is desired that the expression of heterologous polynucleotides should mimic native expression. Native elements may be used when the expression of heterologous polynucleotides is regulated transiently, differentiationally, in a tissue-specific manner, or in response to specific transcriptional stimuli. Other native expression regulatory elements, such as introns, polyadenylation sites, or Kozak consensus sequences, may also be used.

[0027] As used herein, “gene” refers to any segment of DNA. Therefore, a gene includes a coding sequence and, optionally, regulatory sequences necessary for their expression. A gene also optionally includes non-expressed DNA segments that form, for example, recognition sequences for other proteins.

[0028] In recombinant plasmids, the vector "genome" refers to the portion of the recombinant plasmid sequence that is ultimately packaged or capsidized to form a viral (e.g., AAV) particle. When a recombinant plasmid is used to construct or manufacture a recombinant vector, the vector genome does not include any portion of the "plasmid" that does not correspond to the vector genome sequence of the recombinant plasmid. This non-vector genome portion of the recombinant plasmid is called the "plasmid backbone" and is crucial for plasmid cloning and amplification, and is a necessary process for proliferation and recombinant virus production, but is not packaged or capsidized into a viral (e.g., AAV) particle itself. Therefore, the vector "genome" refers to the portion of the vector plasmid that is packaged or capsidized by the virus (e.g., AAV) and contains a heterologous polynucleotide sequence. The non-vector genome portion of the recombinant plasmid is the "plasmid backbone" that is crucial for plasmid cloning and amplification, and has a selection marker such as kanamycin, but is not packaged or capsidized by the virus (e.g., AAV) itself. The amount of rAAV in the capsidated / packaged vector genome can be determined, for example, by quantitative PCR. This assay measures the physical number of vector genomes packaged by real-time quantitative polymerase chain reaction and can be performed at various stages of the manufacturing / purification process (e.g., bulk AAV vector and final product).

[0029] As used herein, “global alignment” is an alignment that aligns two sequences from beginning to end, aligning each base of each sequence only once. The alignment is generated regardless of whether there is similarity or identity between the sequences. For example, 50% sequence identity based on “global alignment” means that 50% of the bases are the same in the alignment of the entire sequences of two compared sequences, each 100 nucleotides long. It is understood that global alignment can be used to determine sequence identity even when the aligned sequences are not the same length. Unless “no penalty for terminal gaps” is selected, differences at the ends of the sequences will be taken into consideration when determining sequence identity. Generally, global alignment is used for sequences that share significant similarity over most of their length. An exemplary method for performing global alignment is the Needleman-Wunsch method (Needleman et al. J.Mol.Biol.48:443(1970)). Exemplary programs for performing global alignment include the global sequence alignment tool, which is publicly available and can be found on the National Center for Biotechnology Information (NCBI) website (ncbi.nlm.nih.gov / ), and the program available at blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=GlobalAln.

[0030] A “heterogeneic” polynucleotide refers to a polynucleotide inserted into a vector (e.g., AAV) for the purpose of vector-mediated introduction / delivery of the polynucleotide into a cell. Heterogeneic polynucleotides are typically different from the vector (e.g., AAV) nucleic acid, i.e., they are non-natural with respect to the viral (e.g., AAV) nucleic acid. Once introduced / delivered into a cell, the heterogeneic polynucleotide contained within the vector can be expressed (e.g., transcribed and, if appropriate, translated). Alternatively, the heterogeneic polynucleotide contained within the vector, to be introduced / delivered into a cell, does not need to be expressed. The term “heterogeneic” is not always used herein with respect to polynucleotides, but even without the modifier “heterogeneic,” references to polynucleotides, whether omitted or not, are intended to include heterogeneic polynucleotides. In some embodiments, a heterogeneic polynucleotide can be a transgene. For example, a transgene may be a therapeutic agent delivered to a cell using a recombinant AAV vector, and the transgene is heterogeneous with respect to the AAV vector.

[0031] The term “homology” or “homology” means that two or more referenced entities share at least partial identity across a given region or portion. Homologous or identical “areas, regions, or domains” means that parts of two or more referenced entities share homology or are identical. Therefore, if two sequences are identical across one or more sequence regions, they share identity in those regions. “Substantial homology” means that a molecule has, or is expected to have, at least partial structure or function of a reference molecule that is structurally or functionally conserved and therefore shares homology, or of one or more of the structure or function of a relevant / corresponding region or portion of the reference molecule (e.g., biological function or activity). The degree of identity (homology) between two sequences can be determined using computer programs and / or mathematical algorithms. Such algorithms for calculating percent sequence identity (homology) generally take into account gaps and mismatches in sequences across the comparison region or area. For example, the BLAST (e.g., BLAST 2.0) search algorithm (see, e.g., Altschul et al., J.Mol.Biol.215:403 (1990), publicly available through NCBI) has the following exemplary search parameters: mismatch -2, gap open 5, gap extension 2. For polypeptide sequence comparison, the BLASTP algorithm is typically used in combination with scoring matrices such as PAM100, PAM250, BLOSUM62, or BLOSUM50. FASTA (e.g., FASTA2 and FASTA3) and SSEARCH sequence comparison programs are also used to quantify the degree of identity (Pearson et al., Proc. Natl. Acad. Sci. USA 85:2444 (1988), Pearson, Methods Mol Biol. 132:185 (2000), and Smith et al., J. Mol. Biol. 147:195 (1981)).A program has also been developed to quantify the structural similarity of proteins using Delaunay-based topological mapping (Bostick et al., Biochem Biophys Res Commun. 304:320 (2003)).

[0032] The term “identical,” and its grammatical variations thereof, means that two or more referenced entities (e.g., polypeptide or polynucleotide sequences) are the same if they are “aligned” sequences. An “aligned” sequence refers to a series of polynucleotide or protein (amino acid) sequences that, compared to a reference sequence, often include corrections for missing or added bases or amino acids. Thus, for example, if two polypeptide sequences are identical, they have the same amino acid sequence, at least within a reference region or portion. If two polynucleotide sequences are identical, they have the same polynucleotide sequence, at least within a reference region or portion. Identity can extend across a defined area (region or domain) of a sequence. An “area” or “domain” of identity refers to a portion of two or more identical referenced entities (e.g., polypeptide or polynucleotide). Thus, if two protein or nucleic acid sequences are identical across one or more sequence regions or domains, they share identity within that region. Identity can extend across the entire length or portion of a sequence. In certain embodiments, the length of a sequence sharing percent identity is 2, 3, 4, 5, or more consecutive polynucleotides or amino acids, e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive polynucleotides or amino acids. In additional specific embodiments, the length of a sequence sharing identity is 21 or more consecutive polynucleotides or amino acids, e.g., 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 consecutive polynucleotides or amino acids. In further specific embodiments, the length of a sequence sharing identity is 41 or more consecutive polynucleotides or amino acids, e.g., 42, 43, 44, 45, 45, 47, 48, 49, 50 consecutive polynucleotides or amino acids.In a more specific embodiment, the length of the sequences sharing identity is 50 or more consecutive polynucleotides or amino acids, for example, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, 95-100, 100-110 consecutive polynucleotides or amino acids.

[0033] As used herein, “local alignment” is an alignment that aligns two sequences, but only those portions of the sequences that share similarity or identity. Thus, local alignment determines whether a subsegment of one sequence exists in another sequence. If there is no similarity, no alignment is returned. Exemplary local alignment methods include, but are not limited to, the BLAST method or the Smith-Waterman method (Adv.Appl.Math.2:482(1981)). For example, 50% sequence identity based on “local alignment” means that in the alignment of the entire sequences of two sequences being compared of any length, a 100-nucleotide-long region of similarity or identity means that 50% of the bases in the similarity or identity region are the same.

[0034] Polynucleotides, polypeptides, and their subsequences include modified and variant forms. As used herein, the terms “modified” or “variant,” and their grammatical variations, mean that a polynucleotide, polypeptide, or its subsequence deviates from a reference sequence. Thus, modified and variant sequences may have substantially the same, higher, or lower activity or function as the reference sequence, but retain at least some partial activity or function of the reference sequence. In certain embodiments, a variant ITR has one or more deletions, additions, or substitutions compared to a wild-type AAV ITR. An example of an amino acid substitution is a conserved amino acid substitution. Another example of an amino acid substitution is an arginine substitution of a lysine residue (e.g., one or more arginine substitutions of lysine as described in any of 4-1, 15-1, 15-2, 15-3 / 15-5, 15-4, and / or 15-6). Further modifications include additions (e.g., insertions or deletions of 1-3, 3-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-40, 40-50, 50-100, or more nucleotides or residues) and deletions (e.g., subsequences or fragments) of the reference sequence. In certain embodiments, the modified or variant sequence retains at least a portion of the function or activity of the unmodified sequence. Such modified forms and variants may have the same, less, or greater function or activity as the reference sequence (but at least a portion thereof), as described herein, for example.

[0035] As described herein, variants may have one or more non-conservative or conservative amino acid sequence differences or modifications, or both. A “conservative substitution” is a substitution of one amino acid by a biologically, chemically, or structurally similar residue. Biological similarity means that the substitution does not disrupt biological activity. Structural similarity means that the amino acids have side chains of similar length or similar size, such as alanine, glycine, and serine. Chemical similarity means that the residues have the same charge or are both hydrophilic or hydrophobic. Specific examples include substitutions of one hydrophobic residue with another, such as isoleucine, valine, leucine, or methionine, or substitutions of one polar residue with another, such as arginine with lysine, glutamic acid with aspartic acid, glutamine with asparagine, or serine with threonine. Specific examples of conserved substitutions include substitutions of hydrophobic residues such as isoleucine, valine, leucine, or methionine with another, or substitutions of polar residues such as arginine with lysine, glutamic acid with aspartic acid, or glutamine with asparagine. For example, conserved amino acid substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. "Conservative substitution" also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid.

[0036] As used herein, “nucleic acid sequencing data,” “nucleic acid sequencing information,” “nucleic acid sequence,” “nucleotide sequence,” “genome sequence,” “gene sequence,” “fragment sequence,” “nucleic acid sequencing read,” and “sequence data” refer to any information or data that shows the order of nucleotide bases (e.g., adenine, guanine, cytosine, and thymine or uracil) in a nucleic acid molecule such as DNA or RNA (e.g., whole genome, whole transcriptome, exome, oligonucleotide, polynucleotide, or fragment). It should be understood that this instruction intends sequence information to be obtained using all types of techniques, platforms, or technologies available, including but not limited to: NGS, capillary electrophoresis, microarrays, ligation-based systems, polymerase-based systems, hybridization-based systems, direct or indirect nucleotide identification systems, pyrosequencing, ion or pH-based detection systems, and digital signature-based systems.

[0037] As used herein, the terms “operable linkage” or “operable linkage” refer to the physical or functional juxtaposition of components described in such a way that the components function in their intended manner. In the example of nucleic acids and operable linkages of regulatory expression elements, the relationship is such that the regulatory element modulates the expression of the nucleic acid. More specifically, for example, two operable linkages of DNA sequences mean that at least one of the DNA sequences is arranged (cis or trans) in such a relationship that it can exert a physiological effect on the other sequence. Thus, modified nucleic acid sequences encoding human proteins, as well as vectors and plasmids, including viral vectors such as AAV vectors, and their compositions, may contain additional nucleic acid elements. These elements may include, but are not limited to, one or more copies of an AAV ITR sequence, regulatory expression elements (e.g., promoter / enhancer), transcription termination signals or stop codons, 5' or 3' untranslated regions adjacent to polynucleotide sequences (e.g., polyadenylated (poly-A) sequences), or introns. The nucleic acid element further includes, for example, filler or stuffer polynucleotide sequences to improve packaging and reduce the presence of contaminating nucleic acids (e.g., to reduce the packaging of the plasmid backbone). As disclosed herein, AAV vectors typically accept DNA inserts having a defined size range, generally about 4 kb to about 5.2 kb, or slightly larger. Therefore, for shorter sequences, stuffers or fillers are included in the insert fragment to adjust its length to be close to, or to, the normal size of a viral genome sequence acceptable for packaging the AAV vector into a viral particle. In various embodiments, the filler / stuffer nucleic acid sequence is an untranslated (non-protein-coding) segment of the nucleic acid.In certain embodiments of the AAV vector, heterologous polynucleotide sequences have a length of less than 4.7 kb, and filler or stuffer polynucleotide sequences, when combined with heterologous polynucleotide sequences (e.g., inserted into the vector), have a total length of approximately 3.0–5.5 kb, or approximately 4.0–5.0 kb, or approximately 4.3–4.8 kb. Introns can also function as filler or stuffer polynucleotide sequences to achieve the length necessary to package the AAV vector into viral particles. Introns and intron fragments functioning as filler or stuffer polynucleotide sequences can also enhance expression. Inclusion of intron elements may enhance expression compared to expression in the absence of intron elements (Kurachi et al., 1995, above). The lengths of expression regulatory elements, ITRs, poly(A) sequences, filler or stuffer polynucleotide sequences can vary. In certain embodiments, the expression regulatory element, ITR, polyA, or filler or stuffer polynucleotide sequence is a sequence of approximately 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-75, 75-100, 100-150, 150-200, 200-250, 250-300, 300-400, 400-500, 500-750, 750-1,000, 1,000-1,500, 1,500-2,000, or 2,000-2,500 nucleotides in length.

[0038] "Optional" or "optionally" means that the events, situations, or substances described later may or may not occur or exist, and the descriptions include cases where the events, situations, or substances occur or exist, and cases where they do not occur or do not exist.

[0039] The terms “polynucleotide” and “nucleic acid” are used interchangeably herein to refer to all forms of nucleic acids, oligonucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Polynucleotides include genomic DNA, cDNA, and antisense DNA, as well as splicing or unsplicing mRNA, rRNA, tRNA, and repressive DNA or RNA (RNAi, e.g., small or short hairpin (sh)RNA, microRNA (miRNA), small or short interfering (si)RNA, trans-splicing RNA, or antisense RNA). Polynucleotides include naturally occurring polynucleotides, synthetic polynucleotides, and intentionally modified or altered polynucleotides (e.g., those having reduced CpG dinucleotides). Polynucleotides can be single-stranded, double-stranded, or triple-stranded, linear or circular, and of any length. In discussing polynucleotides, the sequence or structure of a particular polynucleotide may be described herein in accordance with the convention of providing the sequence in the 5' to 3' direction. Polynucleotides include additions and insertions (e.g., one or more heterogeneous domains). Additions (e.g., heterogeneous domains) can be covalent or non-covalent bonding of any type of molecule to the composition.

[0040] Typically, additions and insertions (e.g., heterogeneous domains) confer complementary or distinct functions or activities. Additions and insertions include chimeric and fusion sequences. These are polynucleotide or protein sequences that have one or more molecules covalently bonded to a reference native (wild-type) sequence that are not normally present in the original sequence. The terms "fusion" or "chimera" and their grammatical variations, when used in relation to molecules, mean that a portion or part of a molecule contains a different (heterogeneous) entity from the molecule, since they do not typically exist together in nature. That is, for example, a portion of a fusion or chimera contains or consists of structurally different parts that do not inherently exist together.

[0041] Polynucleotides, proteins, and peptides encoded by a "polynucleotide" or "nucleic acid" sequence include, like naturally occurring proteins, full-length native sequences as well as functional subsequences, modified forms, or sequence variants, insofar as the subsequences, modified forms, or variants retain some degree of functionality of the native full-length protein. In the methods and uses of the present invention, such polypeptides, proteins, and peptides encoded by a polynucleotide sequence may, but are not required, be identical to endogenous proteins that are defective, underexpressed, or absent in the treated mammal.

[0042] A "promoter" or "promoter sequence" is a DNA regulatory region in a cell that binds to RNA polymerase and initiates the transcription of any type of RNA, such as messenger RNA, ribosomal RNA, micronucleus of nucleolar RNA, or RNA of any class I, II, or III.

[0043] As used herein, the term “recombinant” means that a composition (e.g., AAV or sequence) has been manipulated (i.e., modified) in a manner not generally found in nature, such as a modifier of a viral vector, such as a recombinant AAV vector, and a modifier of a sequence, such as recombinant polynucleotides and polypeptides. A specific example of a recombinant vector, such as an AAV vector, is when a polynucleotide that is not normally present in the wild-type viral (e.g., AAV) genome is inserted into the viral genome. For example, an example of a recombinant polynucleotide is when a heterologous polynucleotide (e.g., a gene) that codes for a protein is cloned into a vector, with or without the 5' region, 3' region, and / or intron region, where the gene normally associates in the viral (e.g., AAV) genome. The term “recombinant” is not always used herein with respect to vectors, such as viruses and AAV vectors, and sequences, such as polynucleotides and polypeptides, but recombinant forms of viruses, AAV, and sequences containing polynucleotides and polypeptides are expressly included despite such omissions.

[0044] Recombinant viral “vectors” or “AAV vectors” are derived from the wild-type genome of a virus, such as AAV, by using molecular methods to remove the wild-type genome from the virus (e.g., AAV) and replace it with non-natural nucleic acids, such as heterologous polynucleotide sequences. Typically, in the case of AAV, one or both inverted end repeat (ITR) sequences of the AAV genome are retained in the AAV vector. A “recombinant” viral vector (e.g., AAV) is distinguished from the viral (e.g., AAV) genome because all or part of the viral genome is replaced with non-natural sequences with respect to the viral (e.g., AAV) genome nucleic acids, such as heterologous polynucleotide sequences. Thus, the incorporation of non-natural sequences defines the viral vector (e.g., AAV) as a “recombinant” vector, and in the case of AAV, it may be referred to as an “rAAV vector.” The recombinant vector (e.g., rAAV) sequence may, as herein it may be referred to, “particles” for ex vivo, in vitro, or subsequent in vivo infection (transduction) of cells. When a recombinant vector sequence is encapsulated or packaged in an AAV particle, the particle may also be referred to as “rAAV”. Such a particle contains a protein that encapsulates or packages the vector genome. Specific examples include viral envelope proteins, and in the case of AAV, a capsid protein. Recombinant vector sequences are manipulated by polynucleotide insertions or incorporations. As disclosed herein, a vector plasmid generally comprises at least one origin of replication for intracellular proliferation and one or more expression regulatory elements.

[0045] As used herein, “sequence identity,” “sequence homology,” or “identity” refers to the number of identical or similar nucleotide bases in an alignment between two or more polynucleotide sequences. In one non-limiting example, “at least 90% identical to” refers to 90–100% percent identity with respect to a reference polynucleotide. Identity at a level of 90% or higher means, for illustrative purposes, that assuming a 100-nucleotide length test polynucleotide and a reference polynucleotide are compared, that no more than 10% of the nucleotides in the test polynucleotide (i.e., 10 out of 100) differ from the nucleotides in the reference polynucleotide. Such differences can be represented as point mutations randomly distributed over the entire length of the nucleotide sequence, or they can be clustered at one or more positions of varying lengths up to a maximum acceptable value, e.g., 10 nucleotide differences out of 100 (approximately 90% identity). Differences are defined as substitutions, insertions, or deletions of nucleic acids. Sequence identity can be determined by sequence alignment of nucleic acid sequences to identify regions of similarity or identity. For the purposes of this specification, sequence identity is generally determined by alignment to identify identical bases. Alignment can be local or global. Matches, mismatches, and gaps can be identified between the compared sequences. A gap is a null nucleotide inserted between bases of an aligned sequence so that identical or similar letters are aligned. Generally, internal gaps and terminal gaps may exist. Sequence identity can be determined by considering gaps as number of identical bases / shortest sequence length × 100. When a gap penalty is used, sequence identity can be determined without penalty for terminal gaps (e.g., terminal gaps are not penalized). Alternatively, sequence identity can be determined without considering gaps as number of identical positions / total aligned sequence length × 100.

[0046] As used herein, the terms “sequencing” or “sequencer” refer to any of several techniques used to determine the sequence of a biomolecule (for example, nucleic acids such as DNA or RNA). Examples of sequencing methods, though not limited to these, include targeted sequencing, single-molecule real-time sequencing, exon sequencing, electron microscope-based sequencing, panel sequencing, transistor-mediated sequencing, direct sequencing, random shotgun sequencing, Sanger dideoxy termination sequencing, whole-genome sequencing, hybridization sequencing, pyrosequencing, double-strand sequencing, cycle sequencing, single-nucleotide extension sequencing, solid-phase sequencing, high-throughput sequencing, large-scale parallel signature sequencing, emulsion PCR, cold-temperature co-amplification PCR (COLD-PCR), multiplex PCR, reversible dye-terminator sequencing, paired-end sequencing, near-term sequencing, exonuclease sequencing, ligation sequencing, short-read sequencing, single-molecule sequencing, synthesis sequencing, real-time sequencing, reversible terminator sequencing, nanopore sequencing, 454 sequencing, and Solexa Genome sequencing. Examples include Analyzer sequencing, SOLiD® sequencing, MS-PET sequencing, and combinations thereof. In some embodiments, sequencing can be performed by a gene analyzer (e.g., gene analyzers commercially available from Illumina, Applied Biosystems, Oxford Nanopore Technologies, and Pacific Biosciences).

[0047] As used herein, “subject” or “patient” means an animal such as a mammalian species (e.g., human) or a bird species (e.g., bird), or another living organism such as a plant. More specifically, a subject may be a vertebrate, such as a mouse, primate, monkey, or mammal such as a human. Animals include livestock (e.g., beef cattle, dairy cows, poultry, horses, pigs, etc.), sports animals, and companion animals (e.g., pets or support animals). A subject may be a healthy individual, an individual with or suspected of having a disease or being predisposed to a disease, or an individual in need of treatment or suspected of needing treatment. The terms “individual” or “patient” are intended to be interchangeable with “subject.” In some embodiments, a subject is a human being with or suspected of having a disease. The disease may be a genetic disease (e.g., a monogenic disease). The disease may be cancer. For example, a subject may be an individual diagnosed with cancer, an individual seeking cancer treatment, and / or an individual who has received at least one cancer therapy. A subject may be in remission of cancer. As another example, a subject may be an individual diagnosed with an autoimmune disease. As yet another example, a subject may be a female individual who is pregnant or planning to become pregnant and has been diagnosed with or is suspected of having a disease (e.g., cancer, autoimmune disease). A “reference subject” refers to a subject known to have or lack a specific characteristic (e.g., a known cancer or disease condition, a known nucleic acid variant, a known cellular origin, a known tumor fraction, known coverage, and / or similar).

[0048] In one embodiment, a “therapeutic agent” is a peptide or protein capable of alleviating or reducing symptoms resulting from the absence or deficiency of a protein in a cell or subject. Alternatively, a “therapeutic” peptide or protein encoded by a transgene is, for example, one that provides a benefit to a subject by correcting a gene deficiency or correcting a gene (expression or function) deficiency. All mammalian and non-mammalian forms of polynucleotides encoding gene products, including the non-limiting genes and proteins disclosed herein, whether known or unknown, are expressly included. Accordingly, the present invention includes genes and proteins of non-mammalian, non-human mammalian, and human origin, and these genes and proteins function in a manner substantially similar to the human genes and proteins described herein.

[0049] The terms “transform” and “transfect” refer to the introduction of molecules such as polynucleotides into cells or host organisms. Cells into which a transgene has been introduced are called “transformed cells.” Therefore, “transformed” cells (e.g., cells, tissues, or organ cells in mammals) signify the genetic changes in cells after the incorporation of exogenous molecules, such as polynucleotides or proteins (e.g., transgenes), into the cells. Thus, “transformed” cells are, for example, cells into which exogenous molecules have been introduced or their offspring. Cells can grow, express the introduced protein, or transcribe nucleic acids. Transformed cells may be within the scope of the use and methods of gene therapy. The introduced polynucleotide may or may not be incorporated into the nucleic acid of the recipient cell or organism. If the introduced polynucleotide is incorporated into the nucleic acid (genomic DNA) of the recipient cell or organism, that polynucleotide may be stably maintained in that cell or organism and may be further inherited by or passed on to the offspring cells or organisms of the recipient cell or organism. Finally, introduced nucleic acids may only be present transiently in recipient cells or host organisms. Cells that can be transduced include cells of any tissue or organ type of any origin (e.g., mesoderm, ectoderm, or endoderm). Non-limiting examples of cells include liver (e.g., hepatocytes, sinusoidal endothelial cells), pancreas (e.g., beta-islet cells), lungs, central or peripheral nervous system (e.g., brain (e.g., nerves, glial cells, or ependymal cells) or spine), kidneys, eyes (e.g., retina, cellular components), spleen, skin, thymus, testes, lungs, diaphragm, heart (myocardium), muscle or psoas muscle, or intestines (e.g., endocrine), adipose tissue (white, brown, or beige), muscle (e.g., fibroblasts), synovial cells, chondrocytes, osteoclasts, epithelial cells, endothelial cells, salivary gland cells, inner ear nerve cells, or hematopoietic cells (e.g., blood or lymph).Additional examples include stem cells such as pluripotent or multipotent progenitor cells that develop or differentiate into liver (e.g., hepatocytes, sinusoidal endothelial cells), pancreas (e.g., beta islet cells), lungs, central or peripheral nervous system (e.g., brain (e.g., nerves, glial cells, or ependymal cells) or spine), kidneys, eyes (e.g., retina, cellular components), spleen, skin, thymus, testes, lungs, diaphragm, heart (myocardium), muscle or psoas muscle, or intestines (e.g., endocrine), adipose tissue (white, brown, or beige), muscle (e.g., fibroblasts), synovial cells, chondrocytes, osteoclasts, epithelial cells, endothelial cells, salivary gland cells, inner ear nerve cells, or hematopoietic cells (e.g., blood or lymph).

[0050] Nucleic acid sequences can be introduced into cells by “transfection,” “transformation,” or “transduction.” “Transfection,” “transformation,” or “transduction,” as used herein, refer to the introduction of one or more exogenous polynucleotides into host cells by physical or chemical methods, for example, by using expression vectors. Many transfection techniques are known in the art and include, for example, calcium phosphate DNA coprecipitation (e.g., Murray EJ (ed.), Methods in Molecular Biology, Vol. 7, Gene Transfer and Expression Protocols, Humana Press (1991)), DEAE-dextran, electroporation, cationic liposome-mediated transfection, tungsten particle-enhanced microparticle guns (Johnston, Nature, 346:776-777 (1990)), and strontium phosphate DNA coprecipitation (see Brash et al., Mol. Cell. Biol., 7:2031-2034 (1987)). Phage or viral vectors can be introduced into host cells after the proliferation of infectious particles within suitable packaging cells, and these are commercially available.

[0051] As used herein, “transgene” refers to a polynucleotide that can be expressed in a non-natural environment or in a heterologous cell under appropriate conditions via recombinant technology. The coding region of the transgene may be inserted into a viral vector. In one embodiment, the viral vector is an adeno-associated virus vector. The transgene may originate from the same type of cell in which it is expressed, but may be introduced from an exogenous source, modified compared to the corresponding native form, and / or expressed from a non-natural site, or may originate from a heterologous cell. “Transgene” is synonymous with “exogenous gene,” “foreign gene,” “heterologous coding sequence,” and “heterologous gene.” In the context of vectors, “heterologous polynucleotide,” “heterologous gene,” or “transgene” is any polynucleotide or gene that is not present in the corresponding wild-type vector or virus. The transgene coding sequence may be a naturally occurring sequence encoding a particular protein. Alternatively, the transgene coding sequence may be a non-natural coding sequence. For example, those skilled in the art can easily recode the coding sequence and use a codon usage table to optimize the codons for expression in a particular species. In one embodiment, the recoded sequence still encodes the same amino acid sequence as the original coding sequence of the transgene. The transgene may be a therapeutic gene. The transgene does not necessarily encode a protein.

[0052] The terms “vector” or “expression vector,” as used herein, refer to a molecule (typically a nucleic acid molecule) containing the sequence necessary to enable the transcription and / or translation of a gene cloned within it. A “vector” can be a plasmid, phage, cosmid, virus or viral construct (e.g., an AAV vector), or other vehicle that can be manipulated by the insertion or incorporation of a polynucleotide. Such a vector can be used for genetic engineering (i.e., a “cloning vector”) to introduce / transfer a polynucleotide into a cell and cause the inserted polynucleotide to be transcribed or translated in the cell. A vector nucleic acid sequence generally includes at least an origin of replication for growth in a cell, and optionally additional elements, such as heterologous polynucleotide sequences, expression regulatory elements (e.g., promoters, enhancers), introns, ITRs (or plurals), selection markers (e.g., antibiotic resistance), and polyadenine (also called polyadenylation) sequences. A viral vector is derived from or based on one or more nucleic acid elements, including a viral genome. Certain viral vectors include adeno-associated virus (AAV) vectors. As described, polynucleotides can be stably or transiently introduced / delivered into cells and their progeny using AAV vectors containing AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74, or AAV-2i8, as well as related AAV variants such as AAV-Rh74 variants (e.g., capsid variants such as 4-1, 15-1, 15-2, 15-3 / 15-5, 15-4, and 15-6).

[0053] In some embodiments, the vector has a circular genome. In some embodiments, the vector has a linear genome. In some embodiments, the vector or expression vector is a plasmid or virus designed to carry the gene of interest and to express the gene of interest in a cell. The expression vector may be inserted into a specific (e.g., targeted) locus. The expression vector may be an “episome.” In some embodiments, an “episome” is a vector that can replicate in a host cell and can persist as an extrachromosomal segment of DNA in the host cell in the presence of appropriate selective pressure (see, for example, Conese et al., Gene Therapy, 11:1735-1742 (2004)). Representative commercially available episome expression vectors include, but are not limited to, episome plasmids utilizing Epstein-Barr nuclear antigen 1 (EBNA1) and Epstein-Barr virus (EBV) origin of replication (oriP). Vectors pREP4, pCEP4, pREP7, and pcDNA3.1 from Invitrogen (Carlsbad, Calif.), and pBK-CMV from Stratagene (La Jolla, Calif.), represent non-limiting examples of episome vectors that use the T antigen and SV40 origin of replication instead of EBNA1 and oriP. In some embodiments, the episomes may be AAV episomes that do not replicate in the host cell. In some embodiments, the AAV episomes are not integrated into the host genome and therefore may be lost in dividing cells as cell division is repeated. Other suitable vectors include incorporating expression vectors, which may be randomly integrated into the host cell's DNA or may contain recombination sites that allow specific recombination between the expression vector and the host cell's chromosomes. Such integrated expression vectors may utilize endogenous expression regulatory sequences on the host cell's chromosomes to result in the expression of the desired protein.Examples of vectors that integrate in a site-specific manner include, for example, the flp-in system components from Invitrogen (Carlsbad, Calif.) (e.g., pcDNA® 5 / FRT), or the cre-lox system found in the pExchange-6 Core vector from Stratagene (La Jolla, Calif.). Examples of vectors that are randomly integrated into host cell chromosomes include, for example, pcDNA3.1 from Invitrogen (Carlsbad, Calif.) (when introduced in the absence of a T-antigen), and pCI or pFN10A(ACT)FLEXI® from Promega (Madison, Wis.). Other suitable vectors may include non-viral vectors or liquid nanoparticle (LNP) vectors. Expression vectors can be viral vectors. Typical viral expression vectors include, but are not limited to, adeno-associated virus (AAV) vectors, adenoviruses, hybrid adenovirus systems, parts of any of these, fragments of any of these, or any combination thereof.

[0054] As used herein, the singular forms "a," "and," and "the" include multiple referents unless the context otherwise clearly indicates otherwise. Thus, for example, a reference to "nucleic acid" includes multiple such nucleic acids, a reference to "vector" includes multiple such vectors, and a reference to "virus" or "particle" includes multiple such virions / particles. Similarly, for example, a reference to "a sequence" includes multiple sequences, and a reference to "the sequence" is a reference to one or more sequences and their equivalents known to those skilled in the art.

[0055] Where used herein, all numbers or ranges include values ​​and integer fractions within such ranges, as well as integer fractions within such ranges, unless the context otherwise clearly indicates. For example, references to identity of 80% or more include 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, etc., as well as 81.1%, 81.2%, 81.3%, 81.4%, 81.5%, etc., 82.1%, 82.2%, 82.3%, 82.4%, 82.5%, etc. References to numerical ranges such as 1 to 10 include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc. Therefore, references to the range 1-50 include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc., up to a maximum of 50, as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc., and 2.1, 2.2, 2.3, 2.4, 2.5, etc. References to a series of ranges include ranges that combine boundary values ​​of different ranges within the series. Therefore, to give an example, a series of ranges, for example, 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-75, 75-100, 100-150, 150-200, 200-250, 250-300, 300-400, 400-500, 500-750, 750-1,000, 1,000-1,500, 1,500-2,000, 2,000-2,500 References to 2,500-3,000, 3,000-3,500, 3,500-4,000, 4,000-4,500, 4,500-5,000, 5,500-6,000, 6,000-7,000, 7,000-8,000, or 8,000-9,000 include ranges such as 10-50, 50-100, 100-1,000, 1,000-3,000, 2,000-4,000, etc. Also, ranges may be expressed herein as “about” one particular value and / or “about” another particular value. Where such ranges are expressed, unless the context indicates otherwise, ranges from one particular value and / or other particular values ​​are also considered to be specifically intended and disclosed.Similarly, when a value is expressed as an approximation, the use of the antecedent "approximately" will be understood to mean that a particular value forms another specifically intended embodiment which should be considered disclosed unless the context indicates otherwise. Furthermore, unless the context indicates otherwise, it will be understood that each endpoint of a range is important both in relation to and independently of the other endpoints. Finally, it should be understood that all individual values ​​and sub-ranges of values ​​contained within an explicitly disclosed range should also be considered specifically intended and disclosed unless the context specifically indicates otherwise. The above applies in any particular case whether some or all of these embodiments are explicitly disclosed.

[0056] The present invention is generally disclosed herein using affirmative language to describe a number of embodiments and aspects. The present invention also includes embodiments in which certain subject matter, such as substances or materials, steps and conditions of a method, protocols, or procedures, is excluded in whole or in part. For example, in certain embodiments or aspects of the present invention, materials and / or steps of a method are excluded. Thus, the present invention is not generally expressed in terms of what is excluded, but aspects that are not expressly excluded in the present invention are nevertheless disclosed herein.

[0057] All features disclosed herein can be combined in any combination. Each feature disclosed herein may be replaced by an alternative feature that serves the same, equivalent, or similar purpose. Thus, unless otherwise specified, the disclosed features (e.g., modified nucleic acids, vectors, plasmids, recombinant vector (e.g., rAAV) sequences, vector genomes, or recombinant viral particles) are examples of a genus of equivalent or similar features.

[0058] All applications, publications, patents, and other references cited herein, as well as GenBank and ATCC citations, are incorporated by reference in their entirety. In case of any conflict, this specification, including definitions, shall prevail.

[0059] B. Adeno-associated virus (AAV) vector Figure 1 shows an exemplary diagram of AAV vector 100 and DNA 101 packaged within AAV vector 100. In some embodiments, the AAV vector is referred to as a recombinant AAV vector. Recombinant AAV vectors contain non-natural sequences, such as xenotransgenes. Two inverted end repeat (ITR) sequences 102 and 105 derived from the AAV genome, one at the 5' end (102) and the other at the 3' end (105), are required for DNA 101 to be packaged in AAV vector 100. Foreign DNA (including the target transgene / gene) can be introduced between ITR 102 and ITR 105. The foreign DNA may include, but is not limited to, the target transgene / gene 103 and / or poly(A) 104 shown in Figure 1. Optionally, promoters may be included in the foreign DNA (with or without enhancer elements).

[0060] The transgene sequence 103 encodes the gene product of interest, which may be an RNA molecule (e.g., mRNA, tRNA, and / or shRNA) or a polypeptide (also referred to herein as a “protein”). In some cases, the gene product of interest may be factor 9 (“F9”). Examples of suitable proteins include, for example, surface proteins, intracellular proteins, membrane proteins, and secreted proteins of any unmodified or synthetic source. The gene product of interest may be an antibody heavy chain or a portion thereof, an antibody light chain or a portion thereof, an enzyme, a receptor, a structural protein, a cofactor, a polypeptide, a peptide, an intrabody, a selection marker, a toxin, a growth factor, or a peptide hormone.

[0061] The target gene product can be any suitable enzyme, including enzymes related to microbial fermentation, metabolic pathway engineering, protein production, bioremediation, and plant growth and development (see, for example, Olsen et al., Methods Mol. Biol., 230:329-349 (2003), Turner, Trends Biotechnol., 21(11):474-478 (2003), Zhao et al., Curr. Opin. Biotechnol., 13(2):104-110 (2002), and Mastrobattista et al., Chem. Biol., 12(12):1291-300 (2005)).

[0062] The gene product of interest may be an antigen. An "antigen" is any molecule that induces an immune response in mammals. An "immune response" may involve, for example, antibody production and / or activation of immune effector cells (e.g., T cells). An antigen may include any subunit, fragment, or epitope of any proteinaceous or non-proteinaceous (e.g., carbohydrate or lipid) molecule that elicits an immune response in mammals. An "epitope" means a sequence on an antigen that is recognized by an antibody or antigen receptor. An epitope is also referred to in the art as an "antigenic determinant."

[0063] In one embodiment, the gene product of interest is an antibody or a portion thereof. For example, the gene product of interest may be an antibody heavy chain or a portion thereof, or an antibody light chain or a portion thereof. The nucleic acid sequence may encode an antibody or a fragment thereof directed against any suitable antigen. All naturally occurring germline, affinity-mature, synthetic, or semi-synthetic antibodies, as well as nucleic acid sequences encoding fragments thereof, can be used. The gene product may be any suitable antibody fragment, such as F(ab')2, Fab', Fab, Fv, scFv, dsFv, dAb, or single-chain linked polypeptide. The antibody or fragment thereof is preferably a mammalian antibody (e.g., a human antibody or a non-human antibody). The antibody may also be a human antibody. Human antibodies, non-human antibodies, or chimeric antibodies can be obtained by any means, including in vitro sources (e.g., hybridomas or recombinant antibody-producing cell lines) and in vivo sources (e.g., rodents). Methods for generating antibodies are known in the art and are described, for example, in Kohler and Milstein, Eur. J. Immunol., 5:511-519 (1976), Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988), and CA Janeway et al. (eds.), Immunobiology, 5th Ed., Garland Publishing, New York, NY (2001).

[0064] In certain embodiments, human antibodies or chimeric antibodies can be generated using transgenic animals (e.g., mice) in which one or more endogenous immunoglobulin genes are replaced by one or more human immunoglobulin genes. Examples of transgenic mice in which endogenous antibody genes are effectively replaced by human antibody genes include, but are not limited to, HUMAB-MOUSE®, Kirin TC MOUSE®, and KM-MOUSE® (see, for example, Lonberg N., Nat. Biotechnol., 23(9):1117-25 (2005) and Lonberg N., Handb. Exp. Pharmacol., 181:69-97 (2008)).

[0065] In some embodiments, the gene product of interest may be a fusion protein (also referred to in the art as a “chimeric protein”). A fusion protein is produced by transcriptionally linking two or more nucleic acid sequences encoding distinct proteins. Translation of the linked genes produces a single polypeptide having functional properties derived from each of the individual proteins. Fusion proteins may exist naturally (e.g., antibody proteins or bcr-abl fusion proteins), or they may be produced synthetically using recombinant DNA techniques known in the art. For example, a nucleic acid sequence encoding a peptide tag can be ligated to a second nucleic acid sequence encoding the gene product of interest to facilitate protein purification and / or identification. Suitable peptide tags include, for example, glutathione-S-transferase (GST) protein, FLAG peptide, or polyhistidine (HIS) tags. Fc fusion proteins are another type of synthetic fusion protein that can be used. Fc fusion proteins contain a soluble antibody constant fragment (Fc). Soluble Fc fusion proteins can be used as reagents for several in vitro and in vivo applications, including but not limited to immunotherapy, flow cytometry, immunohistochemistry, and in vitro activity assays. Fc fusion proteins are described, for example, in Flanagan et al., “Soluble Fc Fusion Proteins for Biomedical Research,” M. Albitar, ed., Monoclonal Antibodies: Methods and Protocols (Methods in Molecular Biology), Human Press, Inc., pp. 33-52 (2008). Fusion proteins can be used for therapeutic or diagnostic purposes.For example, a therapeutic fusion protein can be generated, in which one portion of the fusion protein is capable of directing the fusion protein to a specific cell or tissue, while the other portion of the fusion protein is a biologically active protein or peptide (also referred to in the art as the "payload"), such as an antibody or cytotoxic protein.

[0066] As shown in Figure 2A, the ITRs of the AAV virus genome (e.g., ITR102 and / or 105) can be characterized by secondary structures (201, 202). These secondary structures (201, 202) may be referred to as hairpin structures (201, 202). This is a cross-shaped (e.g., "T") configuration that can be formed when a single strand of DNA folds back into itself and pairs with a complementary sequence within the same strand. These hairpin structures (201, 202) are defining features of the ITR and play a role in the replication, packaging, and integration of the AAV genome. The hairpin structures (201, 202) of the ITR may include a stem region (203, 204) consisting of fully or nearly fully base-paired sequences, and a loop region (205, 206) where the DNA strand folds back and pairs with itself. The stability and sequence of these hairpin structures (201, 202) may affect the efficiency of AAV vectors in gene delivery, as they are recognized by cellular and viral proteins involved in replication initiation and mediating replication and packaging. In some cases, the hairpin structures (201, 202) may also contain palindromic sequences. This can further enhance hairpin formation and facilitate folding and base pairing by providing complementary bases. Identifying the genotypes of these ITR sequences, including any variations in their hairpin structures (201, 202), may be essential for optimizing AAV vector production and therapeutic efficacy.

[0067] In some embodiments, the ITR hairpin structure (201, 202) may exist in two variant forms known as “flip” (207) and “flop” (208). These variants can be distinguished by the orientation of the palindromic sequences within the loop region (205, 206) of the hairpin structure (201, 202). In the “flip” variant (207), the palindromic sequences are oriented in a way that facilitates the formation of hairpins (201) with a specific polarity. Conversely, in the “flop” variant (202), the orientation of the palindromic sequences is reversed, leading to the formation of hairpins (202) with the opposite polarity. The presence of these flip (207) and flop (208) variants may have implications for AAV genome replication and packaging, as the orientation of the hairpins (201, 202) may affect their interaction with replication and packaging mechanisms. The produced viruses will contain flip (207) and flop (208) variants in proportion to each ITR (four different combinations). As shown in Figure 2B, the flip L-ITR at (+) strand 209 has the same sequence as the flip R-ITR at (-) strand 210. In some cases, the flip-flop configuration may also affect the site-specific integration of the AAV genome into the host genome. Therefore, identifying the flip (207) or flop (208) variant morphology of the ITR hairpin structure (201, 202) in candidate ITR sequences can provide valuable information for the design and optimization of AAV vectors for gene therapy applications.

[0068] This specification provides nucleic acid sequences, expression vectors, and plasmids used to generate AAV vectors. In some embodiments, the AAV vector includes a nucleic acid sequence encoding a therapeutic agent. The nucleic acid encoding the desired therapeutic agent can be included in the nucleic acid sequence, expression vector, and plasmid used to generate the AAV vector. As a vector for nucleic acid sequence delivery, the AAV vector drives the expression of a transgene in cells. The transgene, i.e., a polynucleotide encoding a protein such as the nucleic acid encoding the therapeutic agent, can be optionally expressed at therapeutic levels after administration. In some embodiments, the recombinant AAV vector includes a viral genome containing the transgene, the viral genome containing two inverted end repeat (ITR) sequences derived from the AAV genome, one at the 3' end and the other at the 5' end, which are required for the DNA packaging the AAV gene therapy vector. For example, each ITR may be 145 bp long (the left ITR and the right ITR are identical). The DNA between the two ITRs may include, but are not limited to, a promoter (with or without an enhancer element), the transgene (e.g., the gene of interest, the therapeutic agent), poly(A), etc.

[0069] Therefore, recombinant AAV vectors are provided that include a vector genome (capsidized, packaged) containing nucleic acids encoding one or more genetherapy drugs. In certain embodiments, recombinant AAV particles capsidize or package the vector genome. Such recombinant AAV particles include a viral vector genome containing heterologous polynucleotide sequences (e.g., nucleic acids encoding therapeutic drugs). In one embodiment, the vector genome containing nucleic acids encoding a desired therapeutic drug is capsidized or packaged by an AAV capsid or an AAV capsid variant.

[0070] In recombinant AAV vectors, heterologous polynucleotide sequences can be transcribed and subsequently translated into proteins. In various embodiments, the heterologous polynucleotide sequence encodes a therapeutic protein. In more specific embodiments, the vector contains nucleic acids encoding a therapeutic protein. In some embodiments, the therapeutic protein can be any protein encoded by any known gene therapy drug.

[0071] AAV variants, such as AAV and capsid variants, can deliver polynucleotides and / or proteins that provide desirable or therapeutic benefits, thereby enabling the treatment of a variety of diseases. For example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74, or AAV-2i8, as well as their variants and AAV capsid variants (e.g., 4-1), are useful vectors for delivering therapeutic genes to cells, tissues, and organs.

[0072] Recombinant viral and AAV vectors containing a vector genome (virus or AAV) (capsidized, packaged) include additional elements that function in cis or trans. In certain embodiments, a recombinant viral (e.g., AAV) vector containing a vector genome (capsidized, packaged) also includes one or more inverted end repeat (ITR) sequences adjacent to the 5' or 3' end of a heterologous polynucleotide sequence (e.g., a nucleic acid encoding a therapeutic agent), expression regulatory elements (e.g., constitutive or regulated regulatory elements, or tissue-specific expression regulatory elements) that drive the transcription of the heterologous polynucleotide sequence (e.g., a nucleic acid encoding a therapeutic protein) (e.g., promoters or enhancers), intronic sequences, stuffer or filler polynucleotide sequences, and / or polyadenylated sequences located on the 3' side of the heterologous polynucleotide sequence.

[0073] Therefore, the vector may further include introns, expression regulatory elements (e.g., constitutive or modulotable regulatory elements, or tissue-specific expression (e.g., for liver expression) regulatory elements or promoters, e.g., human α1-antitrypsin (hAAT) promoter and / or apolipoprotein E (ApoE) HCR-1 and / or HCR-2 enhancers), one or more adeno-associated virus (AAV) inverted terminal repeats (ITRs) (e.g., ITR sequences of any of the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74, or AAV-2i8 AAV serotypes), and / or filler polynucleotide sequences. The positions of such additional elements may vary. In certain embodiments, an intron is located within a sequence encoding a therapeutic protein, and / or an expression regulatory element is operably ligated to a sequence encoding a therapeutic protein, and / or an AAV ITR(or ITR) is adjacent to the 5' or 3' end of a sequence encoding a therapeutic protein, and / or a filler polynucleotide sequence is adjacent to the 5' or 3' end of a sequence encoding a therapeutic protein.

[0074] The remarkable AAV vectors include any of the following AAV capsid sequences: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74, or AAV-2i8, or capsid variants of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74, or AAV-2i8. The disclosed recombinant AAV particles also include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74, or AAV-2i8, and their variants.

[0075] Methods and uses for administration or delivery include any form compatible with the subject. In certain embodiments, lentivirus or parvovirus (e.g., AAV) vectors (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74, and AAV-2i8, as well as variants) or multiple such viral particles are administered or delivered parenterally, for example, intravenously, intra-arterially, intramuscularly, subcutaneously, or via catheter.

[0076] The subjects include mammals such as humans and non-humans (e.g., primates). In certain embodiments, the subjects may benefit from or require the expression of heterologous polynucleotide sequences.

[0077] Methods for producing variants including recombinant AAV vectors, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74, and AAV-2i8, as well as (capsidate, package) vector genomes, are well known in the art. In one embodiment, the method comprises introducing a recombinant vector (e.g., AAV) plasmid into packaging cells to induce a productive viral infection, and culturing the packaging cells under conditions for producing recombinant viral particles. In another embodiment, a method for producing recombinant viral particles or AAV particles in which the amount of recombinant viral particles containing contaminating nucleic acids in a recombinant viral vector is reduced comprises introducing a recombinant vector (e.g., AAV) plasmid into packaging cells and culturing the packaging cells under conditions for producing recombinant viral particles, wherein the number of viral particles having a vector genome containing contaminating nucleic acids is reduced compared to the number of viral particles containing contaminating nucleic acids produced under conditions in which the recombinant viral vector does not contain filler or stuffer polynucleotide sequences. In certain embodiments, the contaminating nucleic acid is bacterial nucleic acid or a sequence other than a heterologous polynucleotide sequence (i.e., an ITR, promoter, enhancer, origin of replication, polyA sequence, or selection marker).

[0078] Packaging cells include mammalian cells. In certain embodiments, packaging cells include helper (e.g., AAV) functions that package (heterogeneous polynucleotide) sequences (e.g., modified nucleic acids encoding therapeutic agents), expression vectors (e.g., vector genomes), into viral particles (e.g., AAV particles). In certain embodiments, packaging cells provide AAV Rep and / or Cap proteins (e.g., Rep78 and / or Rep68 proteins), and packaging cells are stably or transiently transfected with polynucleotides encoding Rep and / or Cap protein sequences(s), and / or packaging cells are stably or transiently transfected with polynucleotides encoding Rep78 and / or Rep68 protein polynucleotide sequences(s).

[0079] Recombinant AAV vectors and their associated cis-elements (e.g., expression regulatory elements, ITRs, poly(A)) or trans-elements (e.g., packaging functions such as capsid proteins and Rep / Cap proteins) can be based on any organism, species, strain, or serotype. Recombinant AAV virus particles may be based on AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74, and AAV-2i8, as well as variants, but also include hybrids or chimeras of different serotypes. Representative AAV serotypes include, but are not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74, and AAV-2i8 serotypes. Therefore, recombinant virus (e.g., AAV) particles containing a vector genome may contain capsid proteins derived from different serotypes, mixtures of serotypes, or hybrids or chimeric forms of different serotypes, such as VP1, VP2, or VP3 capsid proteins of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74, or AAV-2i8 serotypes. Furthermore, recombinant lentivirus or parvovirus (e.g., AAV) vectors, sequences, plasmids, and vector genomes such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74, and AAV-2i8 may contain elements derived from any one serotype, mixtures of serotypes, or hybrids or chimeric forms of different serotypes. In various embodiments, the recombinant AAV vector contains ITRs, Capsules, Repules, and / or sequences derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74, and / or AAV-2i8 serotypes, or mixtures, hybrids, or chimeric sequences of any of the aforementioned AAV serotypes.

[0080] As described herein, viral vectors such as AAV vectors provide a means for delivering polynucleotide sequences to cells ex vivo, in vitro, and in vivo, thereby encoding proteins so that the cells can express the encoded proteins. For example, recombinant AAV vectors may contain heterologous polynucleotides encoding a desired protein or peptide. Thus, delivery or administration of the vector to a subject (e.g., a mammal) provides the encoded protein and peptide to the subject. Therefore, viral vectors such as AAV vectors can be used to introduce / deliver heterologous polynucleotides for expression and, optionally, to treat a variety of diseases.

[0081] In certain embodiments, the recombinant vector (e.g., AAV) is a parvovirus vector. Parvoviruses are small viruses with a single-stranded DNA genome. Adeno-associated viruses (AAVs) belong to the parvovirus family.

[0082] Parvoviruses, including AAV, are useful as gene therapy vectors because they can enter cells and introduce nucleic acids / genetic material, thereby allowing the nucleic acids / genetic material to be stably maintained within the cell. In addition, these viruses can introduce nucleic acids / genetic material to specific sites (e.g., specific sites on chromosome 19). Since AAV is not associated with pathogenic disease in humans, AAV vectors can deliver heterologous polynucleotide sequences (e.g., therapeutic proteins and drugs) to human patients without causing substantial AAV pathogenesis or disease.

[0083] AAV and AAV variants (e.g., capsid variants such as 4-1) serotypes (e.g., VP1, VP2, and / or VP3 sequences) may or may not be different from other AAV serotypes (e.g., including AAV1-AAV11, Rh74, or Rh10) (e.g., different from any of the VP1, VP2, and / or VP3 sequences of any of the AAV1-AAV11, Rh74, or Rh10 serotypes).

[0084] The disclosed methods and uses provide means for delivering (transduction) heterologous polynucleotides (transgenes) to host cells, including dividing cells and / or non-dividing cells. The disclosed recombinant vectors (e.g., rAAV) sequences, vector genomes, recombinant viral particles, methods, uses, and pharmaceutical formulations are further useful in methods for delivering, administering, or providing nucleic acids or proteins to subjects in need as therapeutic methods. In this way, nucleic acids are transcribed and proteins can be produced in vivo in subjects. Subjects may have a deficiency in nucleic acids or proteins, or may benefit from or require nucleic acids or proteins because the production of nucleic acids or proteins in the subject may confer some degree of therapeutic effect as a therapeutic method or otherwise.

[0085] In general, recombinant AAV vector sequences, vector genomes, recombinant viral particles, methods, and uses may be used to deliver any heterologous polynucleotide (transgene) that has a biological effect of treating or improving one or more symptoms associated with any disorder related to insufficient or undesirable gene expression. Recombinant AAV vector sequences, plasmids, vector genomes, recombinant viral particles, methods, and uses may be used to provide therapies for various disease conditions.

[0086] Nucleic acids, vectors, recombinant vectors (e.g., rAAV), vector genomes, and recombinant viral particles, methods, and uses that enable the treatment of hereditary diseases are disclosed herein. Generally, disease states are classified into two classes: deficiency states (usually enzyme deficiencies, generally inherited in a recessive manner) and imbalance states (involving at least sometimes regulatory or structural proteins, inherited in a dominant manner). For deficiency states, gene transfer can be used to bring normal genes into affected tissue for replacement therapy, and antisense mutations can be used to create animal models of the disease. For imbalance states, gene transfer can be used to create the disease state in a model system, which can then be used in efforts to combat the disease state. Site-directed incorporation of nucleic acid sequences to correct defects is also possible.

[0087] The present invention provides therapeutic methods and uses comprising nucleic acids, vectors, recombinant vectors (e.g., rAAV), vector genomes, and recombinant viral particles. The methods and uses of the present invention are broadly applicable to providing, increasing, or stimulating gene expression or function (e.g., gene addition or substitution).

[0088] C. Method for producing viral vectors Methods for producing viral vectors are disclosed herein. For example, methods for producing viral vectors may include plasmid preparation, transformation, selection, amplification, and extraction of plasmid DNA, transfection into producing cells, production of viral vectors, recovery and purification, and quality control.

[0089] The first step involves creating plasmids, which are small circular fragments of DNA that can replicate independently of chromosomal DNA in bacteria. The plasmid DNA is then engineered to carry the desired transgene along with other necessary elements. These may include a strong promoter for transgene expression, an antibiotic resistance gene for selecting successfully transformed bacterial cells, and sequences necessary for the replication and packaging of the viral genome.

[0090] Once a plasmid carrying the transgene is constructed, it is introduced into Escherichia coli (E. coli) using a process known as transformation. This can be achieved by various methods, such as heat shock or electroporation, which make the bacterial cell wall permeable to the plasmid DNA.

[0091] After transformation, E. coli cells are cultured in a growth medium containing a specific antibiotic. Only cells that have incorporated the plasmid (and therefore the antibiotic resistance gene) can survive and grow in this environment. This process effectively selects transformed cells.

[0092] Selected bacterial cells are grown in larger quantities to amplify plasmid DNA. The cells are then lysed (disrupted), and the plasmid DNA is purified from the cell debris using various extraction and purification procedures.

[0093] Next, the purified plasmid DNA is transfected into a set of producing cells (e.g., mammalian cell lines) in which viral vector production occurs. This can be achieved using chemical, physical, or viral methods. For example, in the case of chemical methods, calcium phosphate precipitation or lipofection can be used; in the case of physical methods, electroporation can be used; and in the case of viral methods, lentiviruses or adenoviruses can be used.

[0094] Within the producing cell, the viral sequence contained in the plasmid DNA is transcribed and translated, leading to the production of viral proteins. These proteins then self-assemble to form viral particles, and in the process, the transgene is packaged into the genome.

[0095] The viral vector carrying the transgene is recovered from the producing cells and then purified using methods such as ultracentrifugation or column chromatography.

[0096] Finally, one or more quality control procedures may be performed. For example, viral vectors may be tested for transduction efficiency (the ability to deliver the transgene to target cells), safety (the absence of reproducible viruses and residual producing cells), and purity.

[0097] Quality control processes are crucial because the hairpin structure of ITRs and their high GC content (e.g., 70%) contribute to the instability of ITR-containing plasmids. One or more quality control assays may be performed, including but not limited to Xmal digestion, agarose gel electrophoresis, capillary electrophoresis, Sanger sequencing, and / or AAV-ITR sequencing. Xmal digestion (CCCGGG) is traditionally performed to confirm the integrity of ITRs. Agarose gel electrophoresis provides qualitative size separation and indicates the presence of ITR mutations, but does not indicate the nature of the ITR mutations. Capillary electrophoresis (CE) provides size separation and quantification (peak area calculation), but does not reveal the nature of any ITR mutations. Sanger sequencing cannot reliably sequence through ITR structures. AAV-ITR sequencing requires that the variant be at a certain threshold for reliable detection and cannot quantify the relative amount of variants present in the sample.

[0098] A quality control process utilizing next-generation sequencing (NGS) technology is provided herein. While not limited to any particular theory, the provided NGS quality control technology offers greater depth than Sanger sequencing, enabling the quantification of variants present in the plasmid pool and the identification of major and minor variants.

[0099] NGS quality control techniques may be performed to identify ITR sequence variants in AAV-ITR packaging plasmids and / or recombinant AAV samples. Figure 3 provides an overview of sample preparation before next-generation sequencing. Briefly, sample preparation is carried out as described below.

[0100] For samples containing an AAV-ITR packaging plasmid (not shown), the plasmid is first linearized by digesting it with a restriction endonuclease that does not recognize cleavage sites within the ITR-gene payload-ITR region of the packaging plasmid. The linearized double-stranded packaging plasmid DNA sample is then purified using a commercially available method such as column purification. In some embodiments, 1 microgram of an AAV-ITR packaging plasmid is linearized as described above.

[0101] For recombinant AAV samples, in step 301, the AAV sample is first treated with DNase to remove uncapsidized DNA contamination. Next, in step 302, the AAV sample is treated with proteinase K to release the viral genome sequence. Then, the viral genome sequence, including both the + and - strands, is purified using a commercially available method such as column purification. Next, in step 303, the + and - strands of the purified viral genome are annealed to produce a sample of double-stranded DNA molecules containing the ITR-gene payload-ITR region of the recombinant AAV genome. In some embodiments, approximately 1 × 10⁻⁶ 12 An AAV sample containing a single viral genome serves as the starting material for the above steps.

[0102] Next, using a double-stranded DNA sample prepared from an AAV-ITR packaging plasmid or recombinant AAV sample (as described above), a PacBio® sequencing library is prepared using an SMRTbell® adapter according to the manufacturer's instructions. In some embodiments, in step 304, 800 nanograms of the double-stranded DNA sample serves as the starting material for generating the sequencing library, but other amounts are also intended. Briefly, in preparing the ligation with the SMRTbell® adapter in step 306, the double-stranded DNA sample is first subjected to end repair in step 305 (e.g., using the PacBio® template preparation kit) and purified before ligation (e.g., using AMPure® PB bead purification). Then, the blunt-end SMRTbell® adapter is attached to the repaired ends of the double-stranded DNA sample using blunt-end ligation according to the manufacturer's instructions. Next, the single-strand exonuclease treatment in step 307 is performed to remove failed ligation products and purify the sample (e.g., using AMPure® PB bead purification). The sample is then pooled and concentrated in step 308, followed by next-generation PacBio® sequencing in step 309. In some embodiments, 100 nanograms of the pooled concentrated sample is used for next-generation PacBio® sequencing.

[0103] How to identify D.ITR In some embodiments, accurately genotyping ITR regions using existing genotyping techniques can be difficult for several reasons, including high GC content, secondary structures (hairpins), and reverse complementary sequences of left and right ITRs (L-ITR and R-ITR, respectively). High GC content DNA can be an obstacle to library preparation workflows requiring PCR reactions, as it is difficult to "unzip" for PCR amplification. High GC content DNA is also an obstacle for some sequencing approaches. In some embodiments, secondary structures (e.g., hairpins) can be problematic because synthetic sequencing may not allow polymerase to sequence via the ITR sequence. Finally, the reverse complementary nature of L-ITR and R-ITR can be problematic during alignment of both WT reads and variant-carrying reads. Short-read sequencing cannot distinguish between L-ITR and R-ITR because alignment software is designed for double-stranded DNA and expects both left and right ITR sequences to map to the same locus. In addition, some different variants may result in the same DNA motif that short-read sequencing could not correctly map to the left and right loci.

[0104] Some problems with genotyping techniques include the fact that they are not locus-specific; for example, long-read sequencing generally generates noisy reads, which can lead to inaccurate variant calling. Even when reads are long and accurate, conventional analytical tools are primarily designed for noisy reads, which can be problematic.

[0105] In some aspects, not only sequencing but also bioinformatics challenges can cause problems in determining ITR genotypes. Even with long, accurate reads, conventional variant calling tools cannot accurately call ITR variants. Two types of variants are particularly problematic: ITR insertions at the ends of the reference genome and overlapping deletions (by reference coordinate) in different molecules. In the case of ITR insertions, conventional alignment software "soft-clips" excessive ITR insertions, which downstream variant calling software ignores. In the case of overlapping deletions, while the alignment may appear accurate, the variant caller homogenizes the signal rather than calling multiple deletions. Consequently, many variants are missed.

[0106] Due to these challenges, technical improvements are needed in ITR genotyping. The novel methods disclosed herein include solutions to overcome the previous sequencing and bioinformatics problems discussed above. Conventional alignment-and-variant calling approaches have been avoided, at least by directly extracting relevant sequences from raw sequencer data using fixed adjacent sequence markers. Conventional alignment-and-variant calling approaches have been avoided, at least by selecting the obtained sequences by abundance and thus clustering them by identity. Conventional alignment-and-variant calling approaches have been avoided, at least by using local alignment to characterize the obtained sequences. In some embodiments, the methods disclosed herein differ from and improve upon previously known approaches because there is no risk of homogenization of signals from multiple molecules, they all reside in separate “clusters,” and variants are called independently in each cluster, as described in more detail herein.

[0107] In some embodiments, systems, devices, and methods for identifying ITRs in plasmids used to produce AAV or adenovirus vectors, and methods for identifying ITRs in the viral genome of the produced AAV or adenovirus vectors are disclosed. In some embodiments, the disclosed methods may be performed on sequencing data associated with any viral vector containing ITRs in its genome, including but not limited to AAV vectors and adenovirus vectors. While the methods described herein target AAV vectors and their ITRs, the same methods can be used to identify ITRs in adenovirus vectors (or plasmids used to produce adenovirus vectors).

[0108] 1. ITR in plasmids Aspects of the methods disclosed herein can be at least partially carried out by the system 1800 shown in Figure 18.

[0109] A method is disclosed that includes sequencing the genomes of multiple plasmids (for example, by sequencer 1830 and / or other sequencing devices(may be) communicably coupled to device 1801 and / or server 1802) to obtain multiple plasmid genome sequences; receiving specifications for fixed adjacent sequence markers (for example, by processor 1808); extracting from each plasmid genome sequence (for example, by processor 1808) multiple sequence regions, each sequence region being within a fixed adjacent sequence marker and containing a candidate ITR sequence, based on the presence of fixed adjacent sequence markers in that plasmid genome sequence; clustering two or more of the sequence regions (for example, by processor 1808) based on complete sequence identity to generate multiple clusters; merging two or more of the clusters (for example, by processor 1808) based on the alignment between their corresponding sequence regions; and considering the multiple plasmids unsuitable for the production of recombinant vector genomes if two or more clusters remain after merging.

[0110] A method is disclosed that includes sequencing the genomes of multiple plasmids (for example, by sequencer 1830 and / or other sequencing devices(may be) communicably coupled to device 1801 and / or server 1802) to obtain multiple plasmid genome sequences; receiving specifications for fixed adjacent sequence markers (for example, by processor 1808); extracting from each plasmid genome sequence (for example, by processor 1808) multiple sequence regions, each sequence region being within a fixed adjacent sequence marker and containing a candidate ITR sequence, based on the presence of fixed adjacent sequence markers in the plasmid genome sequence; clustering two or more of the sequence regions (for example, by processor 1808) based on complete sequence identity to generate multiple clusters; merging two or more of the clusters (for example, by processor 1808) based on alignment between their corresponding sequence regions; and, if a single cluster remains after the merge, identifying the genotype of a representative candidate ITR sequence based on local alignment.

[0111] In some embodiments, multiple plasmids from different batches of plasmids can be tested to determine the genotype / identity of the ITR in the plasmid genome. Finally, for each batch, a decision can be made to approve or reject the continued use of AAV vector production.

[0112] Sequence the genomes of multiple plasmids from two or more batches of plasmids (for example, by sequencer 1830 and / or other sequencing devices(or more) communicably coupled to device 1801 and / or server 1802) to obtain multiple plasmid genome sequences from each batch of plasmids; receive specifications for fixed adjacent sequence markers (for example, by processor 1808); extract multiple sequence regions from each plasmid genome sequence, where each sequence region is within a fixed adjacent sequence marker and contains a candidate ITR sequence (for example, by processor 1808), based on the presence of fixed adjacent sequence markers in the plasmid sequence; and (for example, by processor A method is disclosed that includes clustering two or more sequence regions from a plurality of sequence regions based on complete sequence identity (by processor 1808) to generate multiple clusters, and merging two or more of the clusters based on alignment between their corresponding sequence regions (e.g., by processor 1808), wherein if, after merging, two or more clusters remain for at least one batch of plasmid, further use of that batch of plasmid in the production of an AAV vector is rejected; and if, if a single cluster remains for at least one batch of plasmid, the genotype of a representative candidate ITR sequence is identified based on local alignment, and then the use of that batch of plasmid for the production of an AAV vector is approved if the candidate ITR is identical (or, in some embodiments, substantially identical) to a reference AAV ITR (e.g., wild-type AAV ITR and / or manipulated AAV ITR).

[0113] As shown in Figure 4, methods, devices (e.g., device 1801 in Figure 18), and systems (e.g., system 1800 in Figure 18) are provided for identifying the genotype of inverted terminal repeats (ITRs) in plasmids used to produce AAV vectors.

[0114] Step 410 discloses a method 400 which includes determining plasmid genome sequence data. Plasmid genome sequence data may include multiple plasmid genome sequences. Multiple plasmid genome sequences may be sequences from multiple plasmids. Determining plasmid genome sequence data may include sequencing the genomes of multiple plasmids (for example, by sequencer 1830 and / or other sequencing instruments(s) communicably coupled to device 1801 and / or server 1802) to obtain multiple plasmid genome sequences. Determining plasmid genome sequence data may include receiving plasmid genome sequence data.

[0115] In some embodiments, sequencing includes sequencing the genomes of multiple plasmids to obtain multiple plasmid genome sequences. In some embodiments, sequencing includes sequencing the AAV vector genomes from multiple AAV vectors to obtain multiple AAV vector genome sequences.

[0116] Methods for sequencing plasmid genomes and AAV vector genomes are disclosed. Plasmid genomes and / or AAV vector genomes can be sequenced and analyzed as part of the AAV vector production quality control process.

[0117] In some embodiments, plasmids and / or AAV vectors can be purified or isolated first before sequencing the genome. In some embodiments, the AAV capsid can be purified to remove nucleotides outside the AAV capsid, leaving only nucleotides inside the AAV capsid. The AAV capsid can be denatured to obtain nucleotides inside the AAV capsid. The plasmid genome or AAV vector genome can be subjected to one or more DNA sequencing techniques. Sequencing methods that can be performed by sequencer 1830 include, for example, long-read sequencing and / or circular consensus sequencing (CCS). Other sequencing methods or commercially available formats that may be used at will include, for example, Sanger sequencing, high-throughput sequencing, bisulfite sequencing, pyrosequencing, synthesis sequencing, single-molecule sequencing, nanopore-based sequencing, semiconductor sequencing, ligation sequencing, hybridization sequencing, RNA-Seq (Illumina), digital gene expression (Helicos), next-generation sequencing (NGS), synthesis single-molecule sequencing (SMSS) (Helicos), large-scale parallel sequencing, cloned single-molecule arrays (Solexa), shotgun sequencing, Ion Torrent, Oxford Nanopore, Roche Genia, Maxim-Gilbert sequencing, primer walking, sequencing using PacBio, SOLiD, Ion Torrent, or nanopore platforms. Sequencing reactions can be performed in a variety of sample processing units, which may include other means of processing multiple lanes, multiple channels, multiple wells, or multiple sample sets substantially simultaneously. The sample processing unit may also include multiple sample chambers, allowing for the simultaneous processing of multiple runs.

[0118] In some embodiments, sequencing multiple AAV genomes involves sequencing via long-read sequencing. In some embodiments, long-read sequencing generates sequences of 1kb, 2kb, 3kb, 4kb, 5kb, 6kb, 7kb, 8kb, 9kb, 10kb, or longer.

[0119] In some embodiments, the method may further include a step of receiving specifications for fixed adjacent sequence markers (e.g., by processor 1808) after the sequencing step.

[0120] Method 400 may include, in step 420, extracting one or more sequence regions from plasmid genome sequence data. Extracting one or more sequence regions from plasmid genome sequence data may include extracting multiple sequence regions from each plasmid genome sequence in the plasmid genome sequence data. The sequence regions may include candidate ITR sequences. Extracting one or more sequence regions (or multiple sequence regions) from plasmid genome sequence data may be based on fixed adjacent sequence markers. Extracting one or more sequence regions from plasmid genome sequence data may include receiving specifications for fixed adjacent sequence markers. Extracting one or more sequence regions from plasmid genome sequence data may include extracting multiple sequence regions from each plasmid genome sequence based on the presence of fixed adjacent sequence markers in that plasmid sequence, each sequence region being within a fixed adjacent sequence marker and including candidate ITR sequences.

[0121] As shown in Figure 5A, extraction can be achieved, for example, by analyzing and / or localizing the positions of fixed neighboring sequence markers in plasmid genome sequence data and extracting some or all of the positions from the fixed neighboring sequence markers to the ends of the plasmid genome sequence. As shown in Figures 5B and 5C, extraction can be achieved, for example, by analyzing the positions of fixed neighboring sequence markers in plasmid genome sequence data and extracting some or all of the positions between the fixed neighboring sequence markers. The results of the extraction may be sequence regions that can be stored as strings or other data structures.

[0122] In some embodiments, the plasmid genome sequence includes fixed facultative sequence markers located at both ends of the candidate ITR sequence. In some embodiments, the fixed facultative sequence markers may include known plasmid genome sequences adjacent to the candidate ITR sequence. In some embodiments, multiple regions having fixed facultative sequence markers may indicate that the sequence region containing the fixed facultative sequence has a mutation (e.g., concatenation), and therefore different fixed facultative sequence markers can be used, or the sequence region can be discarded from subsequent analysis. The fixed facultative sequence markers may include sequences of at least 10 nucleotides.

[0123] Because plasmid genomes are circular, any sequence region within a plasmid genome containing a candidate ITR is flanked by sequences on both sides. Therefore, in some embodiments, extracting a sequence region (e.g., a candidate ITR sequence) from a plasmid involves extracting a sequence region between two fixed flanking sequence markers.

[0124] In some embodiments, the plasmid genome used to produce the AAV vector includes a sequence having a first ITR, a transgene (or gene of interest), and a second ITR. In some embodiments, this sequence containing the first ITR, the transgene (or gene of interest), and the second ITR can be called the viral genome sequence region because it is the region that can be packaged as a viral genome during the production of the AAV vector. The remainder of the plasmid genome is called the plasmid backbone and is a known sequence.

[0125] In some embodiments, since the transgene is known, a portion of the transgene sequence can be used as a fixed adjacent sequence marker. For example, the 5' end of the transgene adjacent to the 3' end of the first ITR can be used as a fixed adjacent sequence marker. A portion of the plasmid backbone adjacent to the 5' end of the first ITR can also be used as a fixed adjacent sequence marker. Similarly, for the second ITR, the 3' end of the transgene adjacent to the 5' end of the second ITR can be used as a fixed adjacent sequence marker, and a portion of the plasmid backbone adjacent to the 3' end of the second ITR can also be used as a fixed adjacent sequence marker. Thus, fixed adjacent sequence markers can be present at the 5' and 3' ends of each ITR. In some embodiments, the fixed adjacent sequence markers can be known sequences in the plasmid backbone and / or known sequences in the transgene.

[0126] In some embodiments, the fixed adjacent sequence markers can be predetermined. For example, the plasmid genome can be constructed (or selected) based on specific transgenes present in the plasmid genome that can be used as specific markers in the plasmid backbone or as fixed adjacent sequence markers. Thus, in some embodiments, the fixed adjacent sequence markers to be used for extraction are known even before sequencing and can be provided as input to device 1801.

[0127] In some embodiments, the plasmid genome is circular, so each candidate ITR has fixed adjacent sequence markers at both ends, and sequences between the fixed adjacent sequence markers can be extracted.

[0128] In some embodiments, the extraction allows for the determination of the length of the sequence region. In some embodiments, the method includes discarding a sequence region if its length exceeds a first threshold or falls below a second threshold. In some embodiments, the first threshold is a length of approximately 300 base pairs, and the second threshold is 0, approximately 3 base pairs, approximately 10 base pairs, approximately 50 base pairs, approximately 100 base pairs, approximately 150 base pairs, or more. In some embodiments, the method includes discarding a sequence region if its length is outside the range of approximately 80 bp to approximately 250 bp. In some embodiments, the sequence region can be between 100 bp and 130 bp. For example, in some embodiments, the sequence region can be approximately 101 bp (e.g., a candidate ITR with both hairpin structures of the ITR deleted), and therefore, if the length of the sequence region is not at least 101 bp, it can be discarded. In some embodiments, discarding a sequence region based on length may mean that no further steps need to be performed after the extraction step. In some embodiments, if the length is 300 bp or more, this may indicate the presence of overlapping ITRs and that the sequence region should be discarded. In some embodiments, sequence regions having lengths outside the described range are further analyzed using the claimed method. In some embodiments, sequence regions having lengths outside the range may provide meaningful information, for example, regarding manufacturing process issues. In some embodiments, the step of determining the length of the sequence region may be performed immediately after sequencing and before extraction.

[0129] The extraction steps may differ depending on whether the extraction is from plasmid genome sequencing or AAV vector genome sequencing.

[0130] Returning to Figure 4, one or more extracted sequence regions may be clustered in step 430. Clustering one or more sequence regions may involve aligning the sequence regions containing candidate ITRs. Clustering may be based on sequence identity. In one embodiment, sequence regions may be clustered based on complete sequence identity. In some embodiments, complete identity means 99% or more sequence identity, such as at least 99.5% sequence identity. For example, sequence regions that share 100% sequence identity may be clustered together. One or more clusters may be generated. In some embodiments, a single cluster may be formed, and all extracted sequence regions are identical. In some embodiments, multiple clusters may be formed.

[0131] If all extracted sequence regions have the same identity, only one cluster (or group) is formed. However, there will be as many clusters as there are sequence identities. For example, if there are 100 extracted sequence regions and 70 of them have complete identity, those 70 extracted sequence regions can be clustered together. If another 20 sequence regions have a different sequence identity from those 70 but are identical to each other, those 20 sequence regions can be clustered together. If the remaining 10 sequence regions have a different sequence identity from the 70 and 20 clustered together but are identical to each other, those 10 sequence regions can be clustered together. Therefore, in this example, there are 3 sequence identities, and thus 3 clusters (one for each identity).

[0132] In some embodiments, a cluster may contain 1, 10, 50, 100, 500, 1000, or more extracted sequence regions. In some embodiments, a cluster may contain a single sequence region. For example, if all extracted sequence regions have complete identity with at least one other sequence region, except that one extracted sequence region does not match any of the others, then that sequence region will form a cluster by itself.

[0133] Next, method 400 may perform cluster analysis in step 440. In step 440, the number of clusters may be compared to a threshold. In step 450, if the number of clusters does not meet the threshold, the sequences represented by the clusters may be compared to a reference sequence to determine the genotype of the candidate ITR. For example, the threshold may be 2. Thus, in some embodiments, if only a single cluster is formed (because all sequences are completely identical), the candidate ITR sequence representing the single cluster can be compared to a reference sequence. The reference sequence may be a reference ITR sequence. The reference ITR sequence may be, for example, a wild-type ITR sequence, an engineered ITR sequence, or any other biologically functional ITR sequence. If the candidate ITR sequence and the reference sequence are identical (or substantially identical in some embodiments), the candidate ITR can be used to produce an AAV vector containing a viral genome that matches and therefore includes the reference sequence (e.g., wild-type ITR sequence and / or engineered ITR sequence). In some embodiments, determining the genotype of the candidate ITR may be based on alignment (e.g., local alignment).

[0134] As shown in Figure 6, each of the sequence regions 601, 602, 603, 604, 605, and 606 is determined to have 100% sequence identity with one another and thus form a single cluster 607. Since only a single cluster 607 exists, a representative ITR sequence 608 from the single cluster 607 may be compared (e.g., by alignment, mapping, etc.) to a reference ITR sequence 609. The reference ITR sequence 609 may be, for example, a wild-type ITR sequence, an engineered ITR sequence, or any other biologically functional ITR sequence. If the representative ITR sequence 608 matches the reference ITR sequence 609, the plasmid may be genotyped as the reference ITR sequence 609. In some embodiments, in step 450, candidate ITR genotypes may be identified by aligning (e.g., mapping) the candidate ITR sequences representing the clusters to the reference ITR sequence 609 using one or more sequence alignment methods. Method 400 may utilize a variety of sequence alignment methods. Examples of such methods include BLAST, which quickly identifies regions of local similarity, and Clustal, which is useful for simultaneous alignment of multiple sequences. The Smith-Waterman algorithm provides high accuracy for short sequences through strict local alignment, while the Needleman-Wunsch algorithm is better suited for global sequence alignment and can narrow down regions of perfect sequence identity for clustering. In addition, the MUSCLE (multiple sequence comparison by log-expected value) algorithm may be used. It is known for its speed and effectiveness when handling large datasets and is particularly useful for high-throughput sequencing data.

[0135] Therefore, in some embodiments, the genotype of the candidate ITR sequence is identical (or, in some embodiments, substantially identical) to that of the reference ITR sequence (e.g., the wild-type ITR sequence and / or the manipulated ITR sequence). Method 400 may then approve the plasmid. Approving the plasmid means that several plasmids are deemed suitable for the production of recombinant vector genomes.

[0136] If the cluster analysis in step 440 indicates that the number of clusters satisfies the threshold, method 400 may proceed to the merge step 460. The number of clusters may satisfy the threshold, for example, by being equal to or exceeding the threshold.

[0137] Method 400 may merge two or more clusters in step 460. In some embodiments, merging two or more clusters may be based on sequence alignment. In some embodiments, the two or more clusters have at least one variant between their sequences. In some embodiments, the method includes merging two or more clusters of a plurality based on alignment between their corresponding sequence regions. In some embodiments, merging may produce a modified plurality of clusters or a modified cluster.

[0138] As shown in Figure 7, each of the sequence regions 701, 702, 703, 704, and 705 is determined to have 100% sequence identity with each other and thus forms cluster 708, and each of the sequence regions 706 and 707 is determined to have 100% sequence identity with each other and thus forms cluster 709. Since the two clusters do not have 100% sequence identity with each other, the sequence regions 701, 702, 703, 704, and 705 are not clustered with the sequence regions 706 and 707. Since two clusters exist, the threshold is met and a merge operation is performed at 710 to relax the sequence identity requirement to 99%, so that each of the sequence regions 701, 702, 703, 704, 705, 706, and 707 has at least 99% sequence identity with each other and thus forms a single cluster 711. Since only a single cluster 711 exists, a representative ITR sequence 712 from this single cluster 711 may be compared (e.g., through alignment, mapping) to a reference ITR sequence 713. The reference ITR sequence 713 could be, for example, a wild-type ITR sequence, an engineered ITR sequence, or any other biologically functional ITR sequence. If the representative ITR sequence 712 matches the reference ITR sequence 713, the plasmid can be genotyped using the reference ITR sequence 713.

[0139] In some embodiments, representative sequence regions from each cluster can be aligned with representative sequence regions from each of the other clusters. In some embodiments, representative sequence regions from any given cluster, particularly clusters generated based on less than 100% sequence identity, can generally be selected by any preferred approach, such as ordering the sequence regions within the cluster based on the number and / or position of variants and selecting the central sequence region in that order as the representative sequence region.

[0140] In some embodiments, if only a single variant exists between two aligned clusters, the two clusters can be merged or combined into a single cluster. In some embodiments, a single variant can be a single deletion, addition, or substitution. In some embodiments, one, two, three, four, five, six, seven, eight, nine, or ten variants can exist in the sequence region and clusters can be merged. In some embodiments, five, four, three, or fewer than two variants can exist in the sequence region and clusters can be merged. In some embodiments, clusters can be merged as long as the aligned sequence regions have at least 99% identity.

[0141] In some embodiments, merging may be performed iteratively by processor 1808. A first merge operation may be performed to reduce three or more groups to two or more groups. A second merge operation may be performed to reduce two or more groups to one group. Any number of groups may be merged in a merge operation. The constraints on array identity may change with each merge operation. For example, a first merge operation may be performed on an initial set of clusters requiring 99-100% identity (e.g., 99.8%), a second merge operation may be performed on the remaining clusters requiring 99-100% identity (e.g., 99.5%), and a third operation may be performed on the remaining clusters requiring 99-100% identity (e.g., 99%). The amount of identity change can vary between merge operations; for example, identity may change by 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, etc., between merge operations. Any number of merge operations can be performed to reduce any number of clusters to any reduced number of clusters.

[0142] In some embodiments, merging two or more clusters from a group of clusters may produce modified clusters or modified clusters. In some embodiments, merging two or more clusters from a group of clusters may include iterative merging of two or more clusters from a group of clusters. Each iterative merging of two or more clusters from a group of clusters may be based on a different sequence identity. Iterative merging of two or more clusters from a group of clusters may be performed until a predetermined number of clusters (e.g., one cluster) are produced. In step 470, method 400 may perform another cluster analysis based on the merged clusters. In step 470, the number of merged clusters may be compared to a threshold. In step 450, if the number of merged clusters does not meet the threshold, the sequences represented by the merged clusters may be compared to a reference sequence to identify the genotype of the candidate ITR represented by the merged clusters. For example, the threshold may be 2. Therefore, in some embodiments, if only a single merged cluster is formed (because all sequences have complete identity), the sequence can be compared to a reference sequence, and if the sequences are identical (or substantially identical in some embodiments), the candidate ITR matches the reference sequence and can therefore be used to produce an AAV vector containing a viral genome (e.g., wild-type ITR sequence and / or engineered ITR sequence) that includes the reference sequence. In some embodiments, identification can be based on local alignment. Therefore, in some embodiments, the genotype of the candidate ITR sequence is identical (or substantially identical in some embodiments) to the reference sequence (e.g., wild-type ITR sequence and / or engineered ITR sequence). Method 400 may then approve the plasmid. Approving a plasmid means that multiple plasmids are considered suitable for the production of recombinant vector genomes.

[0143] In step 470, if the cluster analysis indicates that the number of merged clusters meets a threshold, method 400 may proceed to step 480 and reject multiple plasmids. In some embodiments, method 400 includes rejecting multiple plasmids if two or more clusters remain after merging. In some embodiments, rejection means that the multiple plasmids are considered unsuitable for the production of a recombinant vector genome.

[0144] In some embodiments, multiple plasmids from different batches of plasmids can be tested to determine the genotype / identity of the ITR in the plasmid genome. Finally, for each batch, a decision can be made to approve or reject the continued use of AAV vector production.

[0145] In some embodiments, where the plasmid is deemed acceptable according to the approach described herein, the method may further include producing a recombinant AAV vector using a plasmid containing a candidate ITR sequence that is identical (or, in some embodiments, substantially identical) to a reference ITR sequence (e.g., a wild-type ITR sequence and / or a manipulated ITR sequence).

[0146] In some embodiments, AAV vectors can be prepared for further use using only plasmids that have been identified to have a candidate ITR sequence genotype identical (or, in some embodiments, substantially identical) to a reference ITR sequence (e.g., a wild-type ITR sequence and / or a manipulated ITR sequence).

[0147] In some embodiments, if a plasmid is rejected, the method may further include halting the production of the AAV vector produced using the rejected plasmid.

[0148] In some embodiments, if the plasmid is approved, the method may further include the step of administering a therapeutically effective dose of an AAV vector produced from the approved plasmid to the target. Thus, in some embodiments, the AAV vector produced from the approved plasmid may contain a candidate ITR sequence that is identical (or, in some embodiments, substantially identical) to a reference ITR sequence (e.g., a wild-type ITR sequence and / or a manipulated ITR sequence).

[0149] In some embodiments, if the plasmid is approved and used to produce an AAV vector containing a candidate ITR sequence identical (or, in some embodiments, substantially identical) to a reference ITR sequence (e.g., a wild-type ITR sequence and / or a manipulated ITR sequence), the Method may further include packaging the AAV vector for distribution.

[0150] ITR in AAV vectors Aspects of the methods disclosed herein can be at least partially carried out by the system 1800 shown in Figure 18.

[0151] Sequence AAV vector genomes from multiple AAV vectors (for example, by sequencer 1830 and / or other sequencing devices(or more) communicably coupled to computing device 1801 and / or server 1802) to obtain multiple AAV vector genome sequences, receive specifications for fixed adjacent sequence markers (for example, by processor 1808), and (for example, by processor 1808) determine the immediate left of the adjacent sequence marker from each AAV vector genome sequence of the multiple AAV vectors based on the presence of fixed adjacent sequence markers in the AAV vector genome sequences. A method is disclosed which includes extracting a sequence region containing a candidate ITR sequence, clustering two or more sequence regions from among several sequence regions based on complete sequence identity (e.g., by processor 1808) to generate multiple clusters, merging two or more of the multiple clusters based on the alignment between their corresponding sequence regions (e.g., by processor 1808), and considering multiple AAV vectors unsuitable for the production of recombinant vector genomes if two or more clusters remain after merging.

[0152] Sequence AAV vector genomes from multiple AAV vectors (for example, by sequencer 1830 and / or other sequencing devices(or more) communicably coupled to computing device 1801 and / or server 1802) to obtain multiple AAV vector genome sequences, receive specifications for fixed adjacent sequence markers (for example, by processor 1808), and (for example, by processor 1808) determine the presence of fixed adjacent sequence markers in the AAV vector genome sequences from each AAV vector genome sequence of multiple AAV vectors to the immediate left or next of the adjacent sequence markers. A method is disclosed that includes: extracting a sequence region in which the sequence region contains a candidate ITR sequence; clustering two or more sequence regions from among the multiple sequence regions based on complete sequence identity (e.g., by processor 1808) to generate multiple clusters; merging two or more of the multiple clusters based on alignment between their corresponding sequence regions (e.g., by processor 1808); and, if a single cluster remains after merging, identifying the genotype of a representative candidate ITR sequence based on local alignment.

[0153] In some embodiments, multiple AAV vectors from different batches of AAV vectors can be tested to determine the genotype / identity of ITRs in the AAV vector genome. Finally, a decision can be made for each batch to approve or reject continued use.

[0154] Sequence AAV vector genomes from multiple AAV vectors (for example, by sequencer 1830 and / or other sequencing devices(s) communicably coupled to computing device 1801 and / or server 1802) to obtain multiple AAV vector genome sequences from each batch of AAV vectors, wherein the multiple AAV vectors originate from two or more batches of AAV vectors, and receive specifications for fixed adjacent sequence markers (for example, by processor 1808), and (for example, by processor 1808) determine the sequence region immediately to the left or right of the adjacent sequence marker from each AAV vector genome sequence of the multiple AAV vectors, where the sequence region The disclosed method includes extracting sequence regions containing candidate ITR sequences; clustering two or more sequence regions from among multiple sequence regions based on complete sequence identity (e.g., by processor 1808) to generate multiple clusters; and merging two or more clusters from among the multiple clusters based on alignment between their corresponding sequence regions (e.g., by processor 1808), wherein if, after merging, two or more clusters remain for at least one batch of AAV vectors, further use of the AAV vectors in that batch is rejected; and if, if a single cluster remains for at least one batch of AAV vectors, the genotype of a representative candidate ITR sequence is identified based on local alignment, and then the use of the AAV vectors in that batch is approved if the candidate ITR is identical (or, in some embodiments, substantially identical) to a reference AAV ITR sequence (e.g., wild-type AAV ITR and / or manipulated AAV ITR).

[0155] As shown in Figure 8, methods, devices (e.g., device 1801 in Figure 18), and systems (e.g., system 1800 in Figure 18) are provided for identifying the genotype of inverted terminal repeats (ITRs) in AAV vectors.

[0156] Step 810 discloses a method 800 which includes determining AAV vector genome sequence data. The AAV vector genome sequence data may include multiple AAV vector genome sequences. The multiple AAV vector genome sequences may be sequences derived from multiple AAV vectors. Determining AAV vector genome sequence data may include sequencing the genomes of multiple AAV vectors (for example, by sequencer 1830 and / or other sequencing devices(s) communicably coupled to device 1801 and / or server 1802) to obtain multiple AAV vector genome sequences. Determining AAV vector genome sequence data may include receiving AAV vector genome sequence data.

[0157] In some embodiments, sequencing includes sequencing the genomes of multiple AAV vectors to obtain multiple AAV vector genome sequences. In some embodiments, sequencing includes sequencing the AAV vector genomes from multiple AAV vectors to obtain multiple AAV vector genome sequences.

[0158] Methods for sequencing plasmid genomes and AAV vector genomes are disclosed. Plasmid genomes and / or AAV vector genomes can be sequenced and analyzed as part of the AAV vector production quality control process.

[0159] In some embodiments, plasmids and / or AAV vectors can be purified or isolated first before sequencing the genome. In some embodiments, the AAV capsid can be purified to remove nucleotides outside the AAV capsid, leaving only nucleotides inside the AAV capsid. The AAV capsid can be denatured to obtain nucleotides inside the AAV capsid. The plasmid genome or AAV vector genome can be subjected to one or more DNA sequencing techniques. Sequencing methods that can be performed by sequencer 1830 include, for example, long-read sequencing and / or circular consensus sequencing (CCS). Other sequencing methods or commercially available formats that may be used at will include, for example, Sanger sequencing, high-throughput sequencing, bisulfite sequencing, pyrosequencing, synthesis sequencing, single-molecule sequencing, nanopore-based sequencing, semiconductor sequencing, ligation sequencing, hybridization sequencing, RNA-Seq (Illumina), digital gene expression (Helicos), next-generation sequencing (NGS), synthesis single-molecule sequencing (SMSS) (Helicos), large-scale parallel sequencing, cloned single-molecule arrays (Solexa), shotgun sequencing, Ion Torrent, Oxford Nanopore, Roche Genia, Maxim-Gilbert sequencing, primer walking, sequencing using PacBio, SOLiD, Ion Torrent, or nanopore platforms. Sequencing reactions can be performed in a variety of sample processing units, which may include other means of processing multiple lanes, multiple channels, multiple wells, or multiple sample sets substantially simultaneously. The sample processing unit may also include multiple sample chambers, allowing for the simultaneous processing of multiple runs.

[0160] In some embodiments, sequencing multiple AAV genomes involves sequencing via long-read sequencing. In some embodiments, long-read sequencing generates sequences of 1kb, 2kb, 3kb, 4kb, 5kb, 6kb, 7kb, 8kb, 9kb, 10kb, or longer.

[0161] In some embodiments, the method may further include a step of receiving specifications for fixed adjacent sequence markers (e.g., by processor 1808) after the sequencing step.

[0162] Method 800 may include, in step 820, extracting one or more sequence regions from AAV vector genome sequence data. Extracting one or more sequence regions from AAV vector genome sequence data may include extracting one or more sequence regions from each AAV vector genome sequence of multiple AAV vectors in the AAV vector genome sequence data. The sequence regions may include candidate ITR sequences. Extracting one or more sequence regions (or more sequence regions) from AAV vector genome sequence data may be based on fixed adjacent sequence markers. Extracting one or more sequence regions from AAV vector genome sequence data may include receiving specifications for fixed adjacent sequence markers. Extracting one or more sequence regions from AAV vector genome sequence data may include, from each AAV vector genome sequence, extracting a sequence region immediately to the left or immediately to the right of a fixed adjacent sequence marker based on the presence of a fixed adjacent sequence marker in the AAV vector genome sequence, and the sequence regions may include candidate ITR sequences. Extraction can be achieved by analyzing the positions of fixed adjacent sequence markers in the AAV vector genome sequence data (e.g., this can be done by processor 1808), and by extracting some or all of the positions from the fixed adjacent sequence markers to the ends of the AAV vector genome sequence, and / or by extracting some or all of the positions between the fixed adjacent sequence markers. The results of the extraction may be sequence regions that can be stored as strings or other data structures.

[0163] In some embodiments, the AAV vector genome sequence may include a fixed facultative sequence marker present only at one end of the candidate ITR sequence. In some embodiments, the fixed facultative sequence marker may include a known sequence of the AAV vector genome adjacent to one end of the candidate ITR sequence. In some embodiments, multiple regions having fixed facultative sequence markers may indicate that the sequence region containing the fixed facultative sequence has mutations (e.g., concatenation), and therefore different fixed facultative sequence markers may be used, or the sequence region may be discarded from subsequent analysis.

[0164] As shown in Figure 5D, the AAV vector genome is linear and has ITRs at both ends of the genome; therefore, each sequence region containing a candidate ITR is adjacent to a fixed adjacent sequence marker on only one side. Thus, in some embodiments, extracting a sequence region (e.g., a candidate ITR sequence) from the AAV vector genome may involve extracting a sequence region immediately to the left or immediately to the right of a fixed adjacent sequence marker.

[0165] In some embodiments, the AAV vector genome includes a sequence having a first ITR, a transgene (or gene of interest), and a second ITR.

[0166] In some embodiments, since the transgene is known, a portion of the transgene sequence can be designated as a fixed adjacent sequence marker. For example, the 5' end of the transgene adjacent to the 3' end of the first ITR can be used as a fixed adjacent sequence marker. Similarly, for the second ITR, the 3' end of the transgene adjacent to the 5' end of the second ITR can be used as a fixed adjacent sequence marker. Therefore, a single fixed adjacent sequence marker can exist for each ITR. In some embodiments, the fixed adjacent sequence marker can be a known sequence in the transgene.

[0167] In some embodiments, fixed adjacent sequence markers can be predetermined. For example, an AAV vector genome can be constructed based on specific transgenes present in the AAV vector genome that can be used as fixed adjacent sequence markers. Therefore, in some embodiments, the fixed adjacent sequence markers to be used for extraction are known even before sequencing.

[0168] In some embodiments, the AAV vector genome is linear, so each candidate ITR has a fixed adjacent sequence marker on one side, and the sequence from the fixed adjacent sequence marker to the end of the AAV vector genome (e.g., the end of the ITR) can be extracted.

[0169] In some embodiments, the extraction allows for the determination of the length of the sequence region. In some embodiments, the method includes discarding a sequence region if its length exceeds a first threshold or falls below a second threshold. In some embodiments, the first threshold is a length of approximately 300 base pairs, and the second threshold is 0, approximately 3 base pairs, approximately 10 base pairs, approximately 50 base pairs, approximately 100 base pairs, approximately 150 base pairs, or more. In some embodiments, the method includes discarding a sequence region if its length is outside the range of approximately 80 bp to approximately 250 bp. In some embodiments, the sequence region can be between 100 bp and 130 bp. For example, in some embodiments, the sequence region can be approximately 101 bp (e.g., a candidate ITR with both hairpin structures of the ITR deleted), and therefore, if the length of the sequence region is not at least 101 bp, it can be discarded. In some embodiments, discarding a sequence region based on length may mean that no further steps need to be performed after the extraction step. In some embodiments, if the length is 300 bp or more, this may indicate the presence of overlapping ITRs and that the sequence region should be discarded. In some embodiments, sequence regions having lengths outside the described range are further analyzed using the claimed method. In some embodiments, sequence regions having lengths outside the range may provide meaningful information, for example, regarding manufacturing process issues. In some embodiments, the step of determining the length of the sequence region may be performed immediately after sequencing and before extraction.

[0170] Returning to Figure 8, one or more extracted sequence regions may be clustered in step 830. Clustering one or more sequence regions may involve aligning the sequence regions containing candidate ITRs. Clustering may be based on sequence identity. In one embodiment, sequence regions may be clustered based on complete sequence identity. In some embodiments, complete identity means 99% or more sequence identity, such as at least 99.5% sequence identity. For example, sequence regions that share 100% sequence identity may be clustered together. One or more clusters may be generated. In some embodiments, a single cluster may be formed, and all extracted sequence regions are identical. In some embodiments, multiple clusters may be formed.

[0171] If all extracted sequence regions have the same identity, only one cluster (or group) is formed. However, there will be as many clusters as there are sequence identities. For example, if there are 100 extracted sequence regions and 70 of them have complete identity, those 70 extracted sequence regions can be clustered together. If another 20 sequence regions have a different sequence identity from those 70 but are identical to each other, those 20 sequence regions can be clustered together. If the remaining 10 sequence regions have a different sequence identity from the 70 and 20 clustered together but are identical to each other, those 10 sequence regions can be clustered together. Therefore, in this example, there are 3 sequence identities, and thus 3 clusters (one for each identity).

[0172] In some embodiments, a cluster may contain 1, 10, 50, 100, 500, 1000, or more extracted sequence regions. In some embodiments, a cluster may contain a single sequence region. For example, if all extracted sequence regions have complete identity with at least one other sequence region, except that one extracted sequence region does not match any of the others, then that sequence region will form a cluster by itself.

[0173] Next, method 800 may perform cluster analysis in step 840. In step 840, the number of clusters may be compared to a threshold. In step 850, if the number of clusters does not meet the threshold, the sequences represented by the clusters may be compared to a reference sequence to identify the genotype of the candidate ITR. For example, the threshold may be 2. Thus, in some embodiments, if only a single cluster is formed (because all sequences are completely identical), the candidate ITR sequence representing the single cluster can be compared to a reference sequence. The reference sequence may be a reference ITR sequence. The reference ITR sequence may be, for example, a wild-type ITR sequence, an engineered ITR sequence, or any other biologically functional ITR sequence. If the sequences are identical (or substantially identical in some embodiments), the candidate ITR matches the reference sequence and can therefore be used to produce an AAV vector containing a viral genome including the reference sequence (e.g., wild-type ITR sequence and / or engineered ITR sequence). In some embodiments, identification may be based on local alignment, for example, local alignment. Examples of clustering and genotyping are shown in Figure 6 and described herein. In some embodiments, identifying the genotype of a candidate ITR can be based on alignment. In some embodiments, in step 850, the candidate ITR genotype can be identified by aligning (e.g., mapping) the candidate ITR sequences representing the cluster to a reference sequence using one or more sequence alignment methods. Method 800 may utilize a variety of sequence alignment methods. Examples of such methods include BLAST, which rapidly identifies regions of local similarity, and Clustal, which is useful for simultaneous multiple sequence alignments. The Smith-Waterman algorithm provides high accuracy for short sequences through strict local alignment, while the Needleman-Wunsch algorithm is suitable for global sequence alignment and can narrow down regions of perfect sequence identity for clustering. In addition, the MUSCLE (multiple sequence comparison by log-expected value) algorithm may be used.This is known for its speed and effectiveness when handling large datasets, and is particularly useful for high-throughput sequencing data.

[0174] Therefore, in some embodiments, the genotype of the candidate ITR sequence is identical (or, in some embodiments, substantially identical) to the reference sequence (e.g., the wild-type ITR sequence and / or the engineered ITR sequence). Method 800 may then approve the AAV vector. Approving the AAV vector means considering multiple AAV vectors suitable for production, distribution, and / or administration.

[0175] If the cluster analysis in step 840 indicates that the number of clusters satisfies the threshold, method 800 may proceed to the merge step 860. The number of clusters may satisfy the threshold, for example, by being equal to or exceeding the threshold.

[0176] Method 800 may merge two or more clusters in step 860. In some embodiments, merging two or more clusters may be based on sequence alignment. In some embodiments, the two or more clusters have at least one variant between their sequences. In some embodiments, the method includes merging two or more clusters of a plurality of clusters based on alignment between their corresponding sequence regions. In some embodiments, the method includes merging two or more clusters of a plurality of clusters based on alignment between their corresponding sequence regions to produce a modified plurality of clusters or modified clusters.

[0177] Examples of clustering, merging, and genotyping are shown in Figure 7 and described herein. In some embodiments, representative sequence regions from each cluster can be aligned with representative sequence regions from each of the other clusters. In some embodiments, representative sequence regions of any given cluster, particularly clusters generated based on less than 100% sequence identity, can generally be selected by any preferred approach, such as ordering the sequence regions within the cluster based on the number and / or position of variants and selecting the central sequence region in that order as the representative sequence region.

[0178] In some embodiments, if only a single variant exists between two aligned clusters, the two clusters can be merged or combined into a single cluster. In some embodiments, a single variant can be a single deletion, addition, or substitution. In some embodiments, one, two, three, four, five, six, seven, eight, nine, or ten variants can exist in the sequence region and clusters can be merged. In some embodiments, five, four, three, or fewer than two variants can exist in the sequence region and clusters can be merged. In some embodiments, clusters can be merged as long as the aligned sequence regions have at least 99% identity.

[0179] In some embodiments, merging may be performed iteratively by processor 1808. A first merge operation may be performed to reduce three or more groups to two or more groups. A second merge operation may be performed to reduce two or more groups to one group. Any number of groups may be merged in a merge operation. The constraints on array identity may change with each merge operation. For example, a first merge operation may be performed on an initial set of clusters requiring 99-100% identity (e.g., 99.8%), a second merge operation may be performed on the remaining clusters requiring 99-100% identity (e.g., 99.5%), and a third operation may be performed on the remaining clusters requiring 99-100% identity (e.g., 99%). The amount of identity change can vary between merge operations; for example, identity may change by 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, etc., between merge operations. Any number of merge operations can be performed to reduce any number of clusters to any reduced number of clusters.

[0180] In some embodiments, merging two or more clusters from a group of clusters may produce modified clusters or modified clusters. In some embodiments, merging two or more clusters from a group of clusters may include iteratively merging two or more clusters from a group of clusters. Each iterative merging of two or more clusters from a group of clusters may be based on a different sequence identity. Iterative merging of two or more clusters from a group of clusters may be performed until a predetermined number of clusters (e.g., one cluster) are produced.

[0181] In step 870, method 800 may perform another cluster analysis based on the merged clusters. In step 870, the number of merged clusters may be compared to a threshold. In step 850, if the number of merged clusters does not meet the threshold, the sequence represented by the merged cluster may be compared to a reference sequence to identify the genotype of the candidate ITR represented by the merged cluster. For example, the threshold may be 2. Thus, in some embodiments, if only a single merged cluster is formed (because all sequences have complete identity), the sequence can be compared to a reference sequence, and if the sequences are identical (or substantially identical in some embodiments), the candidate ITR matches the reference sequence and can therefore be used to produce an AAV vector containing the viral genome (e.g., wild-type ITR sequence and / or engineered ITR sequence) that includes the reference sequence. In some embodiments, identification may be based on local alignment. Thus, in some embodiments, the genotype of the candidate ITR sequence is identical (or substantially identical in some embodiments) to the reference sequence (e.g., wild-type ITR sequence and / or engineered ITR sequence). Method 800 may then approve the AAV vector. Approving the AAV vector means considering multiple AAV vectors suitable for production, distribution, and / or administration.

[0182] In step 870, if the cluster analysis indicates that the number of merged clusters meets a threshold, method 800 may proceed to step 880 and reject multiple AAV vectors. In some embodiments, method 800 includes rejecting multiple AAV vectors if, after merging, two or more clusters remain. In some embodiments, rejection means considering multiple AAV vectors unsuitable for production, distribution, and / or administration.

[0183] In some embodiments, multiple AAV vectors from different batches of AAV vectors can be tested to determine the genotype / identity of ITRs in the AAV vector genome. Finally, for each batch, a decision can be made to approve or reject continued use for production, distribution, and / or administration.

[0184] In some embodiments, where the AAV vector is deemed acceptable according to the approach described herein, the method may further include the continuous production of a recombinant AAV vector using the same plasmid containing a candidate ITR sequence identical (or, in some embodiments, substantially identical) to a reference ITR sequence (e.g., a wild-type AAV ITR sequence and / or a manipulated AAV ITR sequence).

[0185] In some embodiments, AAV vectors can be manufactured for further use using only plasmids associated with AAV vectors that have been identified as having a candidate ITR sequence genotype identical (or, in some embodiments, substantially identical) to a reference ITR sequence (e.g., a wild-type AAV ITR sequence and / or a manipulated AAV ITR sequence).

[0186] In some embodiments, if an AAV vector is rejected, the method may further include ceasing production of the AAV vector produced using the plasmid(s) used to produce the rejected AAV vector.

[0187] In some embodiments, if the AAV vector is approved, the method may further include the step of administering a therapeutically effective dose of the AAV vector to the target. Thus, in some embodiments, the AAV vector may contain a candidate ITR sequence that is identical (or, in some embodiments, substantially identical) to a reference ITR sequence (e.g., a wild-type AAV ITR sequence and / or a manipulated AAV ITR sequence).

[0188] In some embodiments, if the AAV vector is approved, the method may further include packaging the AAV vector for distribution.

[0189] In some embodiments, the method described herein can be used to generate a database of ITR genotypes. For example, in some embodiments, the method may further include generating a database of ITR genotypes based on sequence regions associated with clusters containing two or more sequence regions. Thus, in some embodiments, each generated cluster having two or more sequence regions in the cluster can be placed in a database of known sequence regions (e.g., ITR genotypes).

[0190] In some embodiments, ITRs that match known ITRs in the database can provide information about errors during production. For example, in some embodiments, an ITR in the database can be associated with a buffer that may be produced at an inappropriate temperature or have an inappropriate pH.

[0191] E. Treatment methods Methods for treating subjects with an AAV vector that has been confirmed to have an ITR sequence identical (or, in some embodiments, substantially identical) to a reference ITR sequence (e.g., a wild-type AAV ITR sequence and / or a manipulated AAV ITR sequence) are disclosed herein.

[0192] A method for treating a subject in need of treatment is disclosed, comprising administering to the subject a therapeutically effective amount of an AAV vector, wherein the AAV vector genome comprises at least two AAV ITRs, a nucleic acid sequence encoding a therapeutic agent, and a polyadenylation signal sequence, and the genotypes of at least two AAV ITRs are identical (or, in some embodiments, substantially identical) to a reference ITR (e.g., a wild-type AAV ITR and / or a manipulated AAV ITR) determined based on one or more of the methods disclosed herein.

[0193] In some embodiments, the AAV vector genome can be encapsulated by an AAV capsid.

[0194] In some embodiments, the subject requiring it may have any known disease or disorder to be treated using gene therapy. Thus, in some embodiments, the therapeutic agent may be any gene known to treat the disease or disorder the subject has.

[0195] In some embodiments, when a therapeutically effective dose of AAV vector is administered to a target, the cells within the target are transduced by the AAV vector, and the therapeutic agent is expressed in the cells.

[0196] The methods and uses of the present invention include therapeutic methods that produce any therapeutic or beneficial effect. Various methods and uses of the present invention further include inhibiting, reducing, or mitigating one or more adverse (e.g., physical) symptoms, disorders, illnesses, diseases, or complications caused by or related to a disease.

[0197] Therefore, the therapeutic or beneficial effect of treatment is an objectively or subjectively measurable or detectable improvement or benefit provided to a particular subject. The therapeutic or beneficial effect may be, but does not necessarily have to be, the disappearance of all or any specific adverse symptoms, impairments, diseases, or complications of the disease. Accordingly, a satisfactory clinical endpoint is achieved when there is a gradual improvement or partial reduction of adverse symptoms, impairments, diseases, or complications caused by or related to the disease, or when the worsening or progression of one or more adverse symptoms, impairments, diseases, or complications caused by or related to the disease is inhibited, reduced, mitigated, suppressed, prevented, limited, or controlled over a short or long period (e.g., hours, days, weeks, months).

[0198] Compositions such as nucleic acids, vectors, recombinant vectors (e.g., rAAV), vector genomes, and recombinant viral particles containing vector genomes, as well as the methods and uses of the present invention, can be administered in sufficient or effective amounts to subjects requiring them. "Effective amount" or "sufficient amount" means an amount, in single or multiple doses, alone or in combination with one or more other compositions (such as therapeutic agents), therapeutic agents, protocols, or therapeutic regimens, that provides a detectable response over any period (long-term or short-term), to any measurable or detectable extent, or an expected or desired outcome or benefit to a subject over any period (e.g., minutes, hours, days, months, years, or cure).

[0199] A person skilled in the art can determine whether a single dose of AAV vector is sufficient or whether multiple doses of AAV vector should be administered. For example, if protein levels fall below a predetermined level (e.g., below the minimum level that provides therapeutic benefit), a person skilled in the art can determine, as appropriate, whether it is appropriate to administer additional doses of AAV vector.

[0200] The dose required to achieve a therapeutic effect, for example, the vector genome / kilogram body weight (vg / kg) dose, will vary based on several factors, including but not limited to the route of administration, the expression level of heterologous polynucleotides required to achieve the therapeutic effect, the specific disease being treated, any host immune response to the viral vector, the host immune response to the heterologous polynucleotide or expression product (protein), and the stability of the expressed protein. Those skilled in the art can determine the dose range of AAV vector genomes to treat patients with a specific disease or disorder based on the aforementioned and other factors. Generally, to achieve a therapeutic effect, the dose should be at least 1 × 10¹⁶ vector genomes per kilogram of body weight of the subject. 8 , or more, for example, 1 × 10 9 , 1 x 10 10 , 1 x 10 11 , 1 x 10 12 , 1 x 1013 or 1 × 10 14 (vg / kg), or in a range greater than that.

[0201] In certain embodiments, the therapeutically effective dose of the AAV vector, when administered to a subject, is a dose sufficient to achieve a normal therapeutic activity of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50%, or more. In other embodiments, the therapeutically effective dose is one that achieves a therapeutic activity of 1% or more in a subject otherwise lacking such activity, for example, a dose that achieves a therapeutic activity of 1.5 - 10%, 10 - 15%, 15 - 20%, 20 - 25%, 25 - 30%, or more in the subject.

[0202] In some aspects, the therapeutically effective dose of the AAV vector is such that, to achieve the desired therapeutic effect, the vector genome (vg) per kilogram of the subject's body weight is at least 1 × 10 10 (vg / kg), or about 1 × 10 10 ~1 × 10 11 vg / kg, or about 1 × 10 11 ~1 × 10 12 vg / kg (for example, about 1 × 10 11 ~2 × 10 11 vg / kg, or about 2 × 10 11 ~3 × 10 11 vg / kg, or about 3 × 10 11 ~4 × 10 11 vg / kg, or about 4 × 10 11 ~5 × 10 11 vg / kg, or about 5 × 10 11 ~6 × 10 11 vg / kg, or about 6 × 10 11 ~7 × 10 11vg / kg, or approximately 7 × 10 11 ~8×10 11 vg / kg, or approximately 8 × 10 11 ~9×10 11 vg / kg, or approximately 9 × 10 11 ~1 × 10 12 (vg / kg), or approximately 1 × 10 12 ~1 × 10 13 The dose may be vg / kg. Additional doses may be needed to achieve the desired therapeutic effect, with approximately 5 × 10⁶ vector genomes (vg) per kilogram of body weight of the subject. 10 ~1 × 10 10 The range of the vector genome (vg), or approximately 1 × 10⁻⁶ 10 ~5×10 11 The range is vg / kg, or approximately 5 × 10 11 ~1 × 10 12 In the range of vg / kg, or approximately 1 × 10⁻⁶ 12 ~5×10 13 The range can be in the vg / kg range. In other embodiments, the therapeutically effective dose of the AAV vector is approximately 2.0 × 10⁻⁶. 11 vg / kg, 2.1 × 10 11 vg / kg, 2.2 × 10 11 vg / kg, 2.3 × 10 11 vg / kg, 2.4 × 10 11 vg / kg, 2.5 × 10 11 vg / kg, 2.6 × 10 11 vg / kg, 2.7 × 10 11 vg / kg, 2.8 × 10 11 vg / kg, 2.9 × 10 11 vg / kg, 3.0 × 10 11 vg / kg, 3.1 × 10 11 vg / kg, 3.2 × 10 11 vg / kg, 3.3 × 10 11 vg / kg, 3.4 × 10 11 vg / kg, 3.5 × 10 11 vg / kg, 3.6 × 10 11 vg / kg, 3.7 × 10 11 vg / kg, 3.8 × 10 11 vg / kg, 3.9 × 10 11 vg / kg, 4.0 × 10 11vg / kg、4.1×10 11 vg / kg、4.2×10 11 vg / kg、4.3×10 11 vg / kg、4.4×10 11 vg / kg、4.5×10 11 vg / kg、4.6×10 11 vg / kg、4.7×10 11 vg / kg、4.8×10 11 vg / kg、4.9×10 11 vg / kg、5.0×10 11 vg / kg、5.1×10 11 vg / kg、5.2×10 11 vg / kg、5.3×10 11 vg / kg、5.4×10 11 vg / kg、5.5×10 11 vg / kg、5.6×10 11 vg / kg、5.7×10 11 vg / kg、5.8×10 11 vg / kg、5.9×10 11 vg / kg、6.0×10 11 vg / kg、6.1×10 11 vg / kg、6.2×10 11 vg / kg、6.3×10 11 vg / kg、6.4×10 11 vg / kg、6.5×10 11 vg / kg、6.6×10 11 vg / kg、6.7×10 11 vg / kg、6.8×10 11 vg / kg、6.9×10 11 vg / kg、7.0×10 11 vg / kg、7.1×10 11 vg / kg、7.2×10 11 vg / kg、7.3×10 11 vg / kg、7.4×10 11 vg / kg、7.5×10 11 vg / kg、7.6×10 11 vg / kg、7.7×10 11 vg / kg、7.8×10 11 vg / kg、7.9×10 11 vg / kg、もしくは8.0×1011 This is vg / kg, or some other dosage.

[0203] An “effective dose” or “sufficient dose” for therapeutic purposes (e.g., to improve or provide therapeutic benefit or improvement) is typically effective in providing a measurable response to one, more, or all adverse symptoms, consequences, or complications of a disease, such as one or more adverse symptoms, impairments, illnesses, conditions, or complications caused by or associated with the disease, but it is also a satisfactory outcome to reduce, mitigate, inhibit, suppress, limit, or control the progression or worsening of the disease.

[0204] An effective or sufficient dose may be provided in a single dose, but is not necessarily required, and may require multiple doses; it may be administered alone, but is not necessarily required, or may be administered in combination with another composition (e.g., a drug), treatment, protocol, or treatment regimen. For example, the dose may be increased proportionally, as indicated by the needs of the subject, the type, condition, and severity of the disease being treated, or the side effects of the treatment (if any). In addition, an effective or sufficient dose does not need to be effective or sufficient when administered in a single or multiple doses without a second composition (e.g., another drug or medication), treatment, protocol, or treatment regimen, because an additional dose, amount, or duration, or additional composition (e.g., a drug or medication), treatment, protocol, or treatment regimen exceeding such a dose may be considered effective or sufficient in a given subject. A dose considered effective also includes doses that result in a reduction in the use of another treatment, treatment regimen, or protocol.

[0205] An effective or sufficient dose does not need to be effective in all subjects being treated, or in the majority of subjects being treated in a given group or population. An effective or sufficient dose refers to the effectiveness or sufficiency in a specific subject, not in the group or general population. As is typical with such methods, some subjects may respond more, less, or not at all to a given treatment or use.

[0206] The term "improvement" means a detectable or measurable improvement in the disease or its symptoms, or in the underlying cellular response. Detectable or measurable improvement includes subjective or objective reduction, mitigation, inhibition, suppression, limitation, or control of the onset, frequency, severity, progression, or duration of the disease, or complications caused by or associated with the disease; or improvement of the symptoms, underlying causes, or consequences of the disease; or recovery from the disease.

[0207] Therefore, a successful treatment outcome is a “therapeutic effect” or “benefit” that reduces, alleviates, inhibits, suppresses, limits, controls, or prevents the onset, frequency, severity, progression, or duration of a disease, or one or more adverse symptoms, underlying causes, or consequences of the disease in a subject. Thus, treatment methods and uses that affect one or more underlying causes of a disease or adverse symptoms are considered beneficial. Reducing or alleviating worsening of a disease, such as stabilizing it, or its adverse symptoms, are also considered successful treatment outcomes.

[0208] Therefore, a therapeutic benefit or improvement does not have to be the complete disappearance of the disease, or any one, most, or all adverse symptoms, complications, consequences, or underlying causes associated with the disease. Thus, a satisfactory endpoint is achieved if a gradual improvement in the disease in question, or a partial reduction, mitigation, inhibition, suppression, limitation, control, or prevention of the onset, frequency, severity, progression, or duration of the disease, or inhibition or recovery of the disease (e.g., stabilization of one or more symptoms or complications), is observed over a short or long period (e.g., hours, days, weeks, months). The effectiveness of a method or use, such as a treatment, that provides a potential therapeutic benefit or improvement of the disease can be confirmed by various methods, such as measuring changes in body temperature.

[0209] According to some embodiments, a therapeutically effective dose of AAV vector is sufficient to produce therapeutic activity above a certain level over a period of time when administered to a human subject with a given indication. In some of these embodiments, the effective dose of AAV vector produces at least 1% normal therapeutic activity in a human subject with an indication for at least 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years, 5 years, 5.5 years, 6 years, 6.5 years, 7 years, 7.5 years, 8 years, 8.5 years, 9 years, 9.5 years, 10 years or longer. In other embodiments, an effective dose of the AAV vector yields at least 5% normal therapeutic activity over a period of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 months, or at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10 years, or longer. In other embodiments, an effective dose of the AAV vector yields at least 10% normal therapeutic activity over a period of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 months, or at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10 years, or longer. In other embodiments, an effective dose of the AAV vector yields at least 15% normal therapeutic activity over a period of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 months, or at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10 years, or longer.In other embodiments, an effective dose of the AAV vector yields at least 20% normal therapeutic activity over a period of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 months, or at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10 years, or longer. In other embodiments, an effective dose of the AAV vector yields at least 25% normal therapeutic activity over a period of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 months, or at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10 years, or longer. In other embodiments, an effective dose of the AAV vector yields at least 30% normal therapeutic activity over a period of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 months, or at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10 years, or longer. In other embodiments, an effective dose of the AAV vector yields at least 35% normal therapeutic activity over a period of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 months, or at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10 years, or longer. In other embodiments, an effective dose of the AAV vector yields at least 40% normal therapeutic activity over a period of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 months, or at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10 years, or longer.In other embodiments, an effective dose of the AAV vector yields at least 45% normal therapeutic activity over a period of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 months, or at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10 years, or longer.

[0210] According to other embodiments, a therapeutically effective dose of the AAV vector is sufficient to produce therapeutic activity that, when administered to a human subject with a given indication, is at least 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, or 45% of the normal value over a period of at least six months.

[0211] In some human subjects who have received a therapeutically effective dose of an AAV vector, the therapeutic activity attributable to the vector may decrease over a long period (e.g., several months or several years) to a level that is no longer considered sufficient. In such situations, the subject may be administered again with the same type of AAV vector as the initial treatment. In other embodiments, particularly if the subject has developed an immune response to the initial vector, the patient may be administered an AAV vector having a different serotype or variant serotype capsid that is designed to express a therapeutic agent in target cells but is less immunoreactive compared to the first AAV vector.

[0212] According to certain embodiments, a therapeutically effective dose of an AAV vector, when administered to a human subject with a given indication, is sufficient to reduce or even eliminate the need for recombinant gene replacement therapy in the subject to maintain adequate hemostasis. Thus, in some embodiments, a therapeutically effective dose of an AAV vector can reduce the frequency with which an average human subject with that indication requires gene replacement therapy to maintain adequate hemostasis by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In the relevant embodiments, a therapeutically effective dose of the AAV vector can reduce the dose of recombinant human gene required by the average human subject with the indication to maintain adequate hemostasis by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In any of these embodiments, the AAV vector can be administered to the subject in a pharmaceutically acceptable composition.

[0213] In other embodiments, a therapeutically effective dose of the AAV vector is sufficient to reduce or further eliminate symptoms associated with a given indication when administered to a human subject with that indication. Therefore, in some embodiments, a therapeutically effective dose of the AAV vector can reduce the frequency and / or severity of symptoms by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to an average, untreated human subject with the same indication. In any of these embodiments, the AAV vector can be administered to the subject alone as a pharmaceutically acceptable composition.

[0214] In certain embodiments, the immune response may be an innate immune response, a humoral immune response, or a cellular immune response, or a response of all three types. In some embodiments, the immune response may be a response to a capsid, vector genome, and / or therapeutic protein produced from a transducer.

[0215] According to a particular embodiment, a therapeutically effective dose of an AAV vector provides sufficient therapeutic activity to maintain hemostasis in a subject with a given indication, without generating or minimizing a humoral (i.e., antibody) immune response to the therapeutic protein produced from the capsid, genome, and / or transdextrins. The antibody response to the virus or virus-like particles (e.g., the AAV vector) can be determined by measuring the antibody titer in the subject's serum or plasma using techniques well known to those skilled in the art in the field of immunology. The antibody titer against the capsid protein or any component of the AAV vector (e.g., the gene product encoded by the vector genome and produced in transdextrins) can be measured using such techniques. The antibody titer is typically expressed as a ratio indicating the dilution until the antibody signal is no longer detectable in a particular assay used to detect the presence of the antibody. Different dilution factors, e.g., 2x, 5x, 10x, or several other dilution factors can be used. Any suitable assay for the presence of antibodies can be used, for example, ELISA, FACS, or reporter gene assays as described in WO2015 / 006743, but are not limited to those described. Other assays may also be used according to the knowledge of those skilled in the art. Antibody titers can be measured at different time points after the initial administration of the AAV vector.

[0216] In a particular embodiment, a therapeutically effective dose of the AAV vector is determined to be effective in a subject with a given indication, after a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, 2 years, 3 years, 4 years, 5 years, or longer, if determined to be effective in the capsid, genome, and / or therapeutic proteins produced from transduced cells. In contrast, it produces antibody titers of 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:200, 1:300, 1:400, 1:500 or less, while delivering at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or more therapeutic activity. According to one exemplary, non-limiting embodiment, the AAV vector yields at least 20% therapeutic activity in a subject with a given indication, while inducing antibody titers of 1:2, 1:3, or 1:4 or less against the capsid and / or therapeutic agent produced by transduced cells six months after administration of the AAV vector. In any of these embodiments, the AAV vector can be administered to the subject alone as a pharmaceutically acceptable composition.

[0217] In certain embodiments, a therapeutically effective dose of the AAV vector provides sufficient therapeutic activity to maintain hemostasis in subjects with a given indication, while generating no or minimal cellular immune response to the therapeutic protein produced from the capsid and / or transdextrins. The cellular immune response can be determined at least by assaying T cell activity specific to the capsid protein or therapeutic agent.

[0218] In some embodiments, the cellular immune response is determined by assaying T cell activity specific to the capsid protein and / or therapeutic protein produced by transduced cells. Different assays for T cell responses are known in the art. In one exemplary, non-limiting embodiment, the T cell response is determined by collecting peripheral blood mononuclear cells (PBMCs) from a subject previously treated with an AAV vector for a given indication. The cells are then incubated with a peptide derived from the VP1 capsid protein used in the vector and / or a therapeutic protein produced by transduced hepatocytes. T cells that specifically recognize the capsid protein or therapeutic protein are stimulated to release cytokines such as interferon-gamma or another cytokine, which can then be detected and quantified using the ELISPOT assay or another assay well known to those skilled in the art. (See, for example, Manno, et al., Nat Med 2006;12(3):342-347). T-cell responses can be monitored before and after the subject receives a dose of the AAV vector to treat their indication (e.g., weekly, monthly, or at other intervals). Therefore, according to a particular embodiment, a therapeutically effective dose of AAV vector yields sufficient therapeutic activity (e.g., at least 1%, 5%, 10%, 20%, 30%, or more therapeutic activity) to maintain hemostasis in subjects with the indication while eliciting a T cell response, as measured using ELISPOT, which is 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, or less spot-forming units per million PBMCs assayed, when measured weekly, monthly, or at any other interval after the AAV vector is administered, or at 2 weeks, 1 month, 2 months, 3 months, 6 months, 9 months, 1 year, 2 years, or any different time point after the AAV vector is administered.In some of these embodiments, the ELISPOT assay is designed to detect interferon-gamma (or some other cytokine) production stimulated by the AAV vector's capsid protein or peptides from therapeutic proteins produced by transduced hepatocytes. In any of these embodiments, the AAV vector can be administered to the subject alone as a pharmaceutically acceptable composition.

[0219] As a surrogate for a cellular immune response to transdextrose cells, the presence of elevated cellular enzymes can be assayed using standard methods. While not wishing to be constrained by theory, it is thought that T cells specific to certain AAV vectors, such as those used in previous clinical trials, can attack and kill transdextrose cells, transiently releasing enzymes into the circulating system. Normal levels of these enzymes in the circulating system are typically defined as a range with an upper limit; levels exceeding this are considered elevated and therefore indicate liver damage. The normal range is partly dependent on the standards used by the clinical laboratory performing the assay. In a particular embodiment, a therapeutically effective dose of the AAV vector provides sufficient therapeutic activity (e.g., at least 1%, 5%, 10%, 20%, 30%, or more therapeutic activity) to maintain hemostasis in a subject with the indication, while causing an increase in circulating enzyme (e.g., ALT, AST, or LDH) levels that do not exceed 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 1500%, or 2000% of the upper limit of normal (ULN) value for each range, as the mean or highest level measured in multiple samples taken from the same subject during treatment at different time points after administration of the AAV vector (at weekly or monthly intervals).

[0220] In previous clinical trials using AAV vectors, investigators had to co-administer immunosuppressants, such as steroids, to prevent subjects from developing an immune response that would eliminate transdextrin cells producing therapeutic proteins. However, due to attenuated immune responses observed in subjects receiving experimental treatment with certain AAV vectors, co-administration of immunosuppressants may not be necessary. Therefore, in certain embodiments, a therapeutically effective dose of AAV vector is sufficient to maintain adequate hemostasis in subjects with a given indication without the need for co-administration (before, during, or after) of immunosuppressants (such as steroids or other immunosuppressants). However, since immune responses are not predictable in all subjects, the therapeutic methods herein include AAV vectors administered in combination with immunosuppressants. Co-administration of immunosuppressants can be performed before, during, or after administration of the AAV vector for its indication to the subject. In some embodiments, the immunosuppressant is administered to the subject for several days, weeks, or months after administration of the AAV vector for treating its indication. Examples of immunosuppressants include steroids (e.g., prednisone or prednisolone, but not limited to these) and non-steroidal immunosuppressants (e.g., cyclosporine, rapamycin). The drug dose and treatment duration required to achieve sufficient immunosuppression depend on factors specific to each subject being treated, but determining the dose and treatment duration is within the scope of the art. In some embodiments, immunosuppressants may need to be administered two or more times.

[0221] According to certain embodiments, a therapeutically effective dose of an AAV vector, when administered to a population of human subjects with a given indication, results in a consistent increase in therapeutic activity. Consistency can be determined by calculating the variability of the response in the population of human subjects using statistical methods such as the mean and standard deviation (SD), or other statistical techniques well known to those skilled in the art. In some embodiments, a therapeutically effective dose of an AAV vector, when administered to a population of human subjects with its indication, results in an average therapeutic activity of 1–5% (SD less than 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) or an average therapeutic activity of 2.5–7.5% ( Either yields an SD of 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less than 1, or yields an average therapeutic activity of 5-10% (SD of 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less than 1), or yields an average therapeutic activity of 7.5-12.5% ​​(SD of 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less than 1). It results in full treatment, or an average therapeutic activity of 10-15% (SD of 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less than 1), or an average therapeutic activity of 12.5-17.5% (SD of 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less than 1), or an average therapeutic activity of 15-20% (SD of 15, 14, 13, 12, 11, It yields an average therapeutic activity of 10, 9, 8, 7, 6, 5, 4, 3, 2, or less than 1, or an average therapeutic activity of 17.5-22.5% (SD of 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less than 1), or an average therapeutic activity of 20-25% (SD of 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less than 1), or 22.5-27.It yields an average therapeutic activity of 5% (SD of 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less than 1), or an average therapeutic activity of 25-30% (SD of 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less than 1), or an average therapeutic activity of 27.5-32.5% (SD of 15, 14, 13, 12, 11, 10, 9 It results in 8, 7, 6, 5, 4, 3, 2, or less than 1) or an average therapeutic activity of 30-35% (SD of 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less than 1) or an average therapeutic activity of 32.5-37.5% (SD of 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less than 1) or 35 It yields an average therapeutic activity of ~40% (SD of 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less than 1), or an average therapeutic activity of 37.5-42.5% (SD of 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less than 1), or an average therapeutic activity of 40-45% (SD of 15, 14, 13, 12, 11, 10 It yields either an average therapeutic activity of 42.5–47.5% (SD of 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less than 1), or an average therapeutic activity of 45–50% (SD of 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less than 1). In any of these embodiments, the AAV vector can be administered to the subject alone as a pharmaceutically acceptable composition.

[0222] The methods and uses of the present invention can be combined with any compound, agent, drug, treatment, or other therapeutic regimen or protocol having a desired therapeutic, beneficial, additive, synergistic, or complementary activity or effect. Exemplary combination compositions and treatments include a second activator such as a bioagent (protein), agent, and drug. Such bioagents (proteins), agents, drugs, treatments, and therapies can be administered or performed substantially simultaneously with, or after, any other method or use of the present invention.

[0223] Compounds, agents, drugs, therapies, or other therapeutic regimens or protocols may be administered as a combination composition, or separately, concurrently with, sequentially, or sequentially (before or after) the delivery or administration of nucleic acids, vectors, recombinant vectors (e.g., rAAV), vector genomes, or recombinant viral particles. Accordingly, the present invention provides combinations, and the methods or uses of the present invention can be combined with any compound, agent, drug, therapeutic regimen, therapeutic protocol, process, treatment, or composition described herein or known to those skilled in the art. Compounds, agents, drugs, therapeutic regimens, therapeutic protocols, processes, treatments, or compositions may be administered or performed before, substantially concurrently with, or after the administration of the nucleic acids, vectors, recombinant vectors (e.g., rAAV), vector genomes, or recombinant viral particles of the present invention.

[0224] In certain embodiments, the combination composition comprises one or more immunosuppressants. In certain embodiments, the method comprises administering or delivering one or more immunosuppressants to a mammal. In certain embodiments, the combination composition comprises AAV therapeutic particles and one or more immunosuppressants. In certain embodiments, the method comprises administering or delivering AAV therapeutic particles to a mammal and administering immunosuppressants to a mammal. Those skilled in the art can determine the appropriate need or timing for such a combination composition with one or more immunosuppressants and administer the immunosuppressants to the mammal.

[0225] The methods and uses of the present invention also include, among other things, methods and uses that result in a reduction of the need for or use of another compound, agent, drug, treatment regimen, treatment protocol, process, or treatment. Accordingly, the present invention provides methods and uses that reduce the need for or use of another treatment or therapy.

[0226] The present invention is useful in animals, including for human and veterinary applications. Therefore, preferred subjects include mammals such as humans, as well as non-human mammals. The term "subject" refers to animals, typically mammals, e.g., humans, non-human primates (apes, gibbons, gorillas, chimpanzees, orangutans, macaques), domestic animals (dogs and cats), farm animals (poultry such as chickens and ducks, horses, cattle, goats, sheep, pigs), and laboratory animals (mice, rats, rabbits, guinea pigs). Human subjects include fetuses, neonates, infants, young adults, and adult subjects. Subjects include animal disease models, e.g., mouse and other animal models of blood coagulation disorders, as well as other animal models known to those skilled in the art.

[0227] Suitable subjects for treatment include those who produce or are at risk of producing insufficient amounts of functional gene products (proteins), those who have deficiencies in functional gene products (proteins), or those who produce abnormal, partially functional, or nonfunctional gene products (proteins) that may lead to disease. Suitable subjects for treatment according to the present invention also include those who have or are at risk of producing disease-causing abnormal or deficient (mutated) gene products (proteins) in which a reduction in the amount, expression, or function of the abnormal or deficient (mutated) gene product (protein) would lead to the treatment of the disease, alleviate one or more symptoms, or improve the disease.

[0228] Suitable subjects for treatment according to the present invention include subjects that have been previously treated or are currently being treated with supplemental proteins. Furthermore, suitable subjects for treatment according to the present invention include subjects that do not express a substantial or detectable immune response to the therapeutic protein, or subjects that do not have inhibitory antibodies against the therapeutic protein in amounts that would interfere with or block therapeutic-based gene therapy.

[0229] In other embodiments, human pediatric subjects who have been determined to have a given indication (e.g., by genotyping) but have not yet shown any of the symptoms of that indication can be prophylactically treated with an AAV vector to prevent the initial onset of such symptoms, or, in other embodiments, to prevent the symptoms from becoming more severe compared to untreated subjects. In some embodiments, the human subjects thus prophylactically treated are at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 months of age or older at the time of administration of the AAV vector, and produce and maintain sufficient therapeutic activity to maintain hemostasis, and thus prevent or reduce the severity of one or more symptoms of the indication. In any of these embodiments, the AAV vector can be administered to the subject alone as a pharmaceutically acceptable composition.

[0230] Administration to a subject or in vivo delivery can be performed before the onset of any adverse symptoms, conditions, or complications caused by or associated with the disease. For example, screening (e.g., genetic) can be used to identify such subjects as candidates for the composition, method, and use of the invention. Thus, such subjects include those that have been determined to be positive in screening for insufficient amounts of functional gene products (proteins), or deficiencies of functional gene products (proteins), or for the production of abnormal, partially functional, or non-functional gene products (proteins).

[0231] The methods and uses of the present invention include systemic, regional, or local delivery and administration, or by any route (e.g., by injection or infusion). Such delivery and administration include parenteral delivery, such as intravascular, intravenous, intramuscular, intraperitoneal, intradermal, subcutaneous, or transmucosal delivery. Exemplary routes of administration and delivery include intravenous (IV), intraperitoneal (IP), intraarterial, subcutaneous, intrapleural, intubation, intrapulmonary, intracavitary, iontophoresis, intraorganic, and intralymphatic delivery.

[0232] The dosage may vary depending on the type, onset, progression, severity, frequency, duration, or probability of the disease being treated, the desired clinical endpoint, previous or concurrent treatments, the subject's overall health, age, sex, race, or immunological capacity, and other factors that would be understood by those skilled in the art. The dosage, number, frequency, or duration may increase or decrease proportionally as indicated by any adverse side effects, complications, or other risk factors of the treatment or therapy and the subject's condition. Those skilled in the art will understand the factors that may influence the amount and timing of administration required to provide a sufficient quantity to deliver a therapeutic or preventive benefit.

[0233] The methods and uses of the present invention disclosed herein can be carried out within 1 to 2 hours, 2 to 4 hours, 4 to 12 hours, 12 to 24 hours, or 24 to 72 hours after a subject has been identified as having a disease targeted for treatment, having one or more symptoms of the disease, or even if not having one or more symptoms of the disease, after being screened as described herein and identified as positive. Of course, the methods and uses of the present invention can also be carried out 1 to 7 days, 7 to 14 days, 14 to 21 days, 21 to 48 days, or longer (days, months, or years) after a subject has been identified as having a disease targeted for treatment, having one or more symptoms of the disease, or after being screened as described herein and identified as positive.

[0234] The nucleic acids, vectors, recombinant vectors (e.g., rAAV), vector genomes, and recombinant viral particles of the present invention, as well as other compositions, agents, drugs, and biologics (proteins), can be incorporated into pharmaceutical compositions (e.g., pharmaceutically acceptable carriers or excipients). Such pharmaceutical compositions are particularly useful for administration and delivery to subjects in vivo or ex vivo.

[0235] As used herein, the terms “pharmaceutically acceptable” and “physiologically acceptable” mean a bioacceptable formulation, gas, liquid, or solid, or mixture thereof, that is suitable for one or more routes of administration, in vivo delivery, or contact. A “pharmaceutically acceptable” or “physiologically acceptable” composition is a material that is not biologically or otherwise undesirable, for example, a material that can be administered to a subject without causing substantially undesirable biological effects. Such a pharmaceutically acceptable composition may, for example, be used when administering a viral vector or viral particles to a subject.

[0236] Such compositions include solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (e.g., oil in water or water in oil), suspensions, syrups, elixirs, dispersions and suspension media, coatings, isotonic agents, and absorption enhancers or retarders, all of which are suitable for pharmaceutically effective administration or in vivo contact or delivery. Aqueous and non-aqueous solvents, solutions and suspensions may include suspending agents and thickeners. Such pharmaceutically acceptable carriers include tablets (coated or uncoated), capsules (hard or soft), microbeads, powders, granules, and crystals. Auxiliary active compounds (e.g., preservatives, antimicrobials, antivirals, and antifungals) may also be incorporated into the compositions.

[0237] The pharmaceutical composition can be formulated to be compatible with a particular route of administration or delivery described herein or known to those of skill in the art. Thus, the pharmaceutical composition includes carriers, diluents, or excipients suitable for administration by various routes.

[0238] Compositions suitable for parenteral administration include aqueous and non-aqueous solutions, suspensions or emulsions of the active compound, and these preparations are typically sterilized and can be isotonic with the blood of the intended recipient. Non-limiting exemplary examples include water, saline, dextrose, fructose, ethanol, animal, vegetable, or synthetic oils.

[0239] Cosolvents and adjuvants may be added to the formulation. Non-limiting examples of cosolvents include hydroxyl or other polar groups, alcohols (e.g., isopropyl alcohol), glycols (e.g., propylene glycol, polyethylene glycol, polypropylene glycol, glycol ether), glycerol, polyoxyethylene alcohol, and polyoxyethylene fatty acid esters. Adjuvants include surfactants (e.g., soybean lecithin and oleic acid), sorbitan esters (e.g., sorbitan trioleate), and polyvinylpyrrolidone.

[0240] The compositions, methods, and suitable pharmaceutical compositions and delivery systems of the present invention are known in the art (e.g., Remington: The Science and Practice of Pharmacy (2003) 20th ed., Mack Publishing Co., Easton, Pa.; Remington's Pharmaceutical Sciences (1990) 18th ed., Mack Publishing Co., Easton, Pa.; The Merck Index (1996) 12th ed., Merck Publishing Group, Whitehouse, NJ; Pharmaceutical Principles of Solid Dosage Forms (1993), Technonic Publishing Co., Inc., Lancaster, Pa.; Ansel and Stoklosa, Pharmaceutical Calculations (2001) 11th ed., Lippincott Williams & Wilkins, Baltimore, Md.; and Poznansky et al., Drug Delivery). See Systems (1980), RL Juliano, ed., Oxford, NY, pp. 253–315.

[0241] As used herein, “unit dosage form” refers to a physically distinct unit suitable as a unit dose for a target to be treated, each unit containing a predetermined amount calculated to produce a desired effect (e.g., prophylactic or therapeutic effect) when administered in one or more doses, optionally combined with a pharmaceutical carrier (excipient, diluent, vehicle, or filler). Unit dosage forms may be contained in ampoules and vials, and may include, for example, a liquid composition or a composition in a lyophilized or lyophilized state, and a sterile liquid carrier may be added, for example, before in vivo administration or delivery. Individual unit dosage forms may be included in kits or containers for multiple doses. Recombinant vectors (e.g., rAAV) sequences, vector genomes, recombinant viral particles, and their pharmaceutical compositions may be packaged in single or multiple unit dosage forms for ease of administration and uniformity of dosage.

[0242] F. Manufacturing method A method for producing an AAV vector that has been confirmed to have an ITR sequence identical (or, in some embodiments, substantially identical) to a reference ITR sequence (e.g., a wild-type AAV ITR sequence and / or a manipulated AAV ITR sequence) is disclosed.

[0243] Sequence the genomes of multiple plasmids (for example, by sequencer 1830 and / or other sequencing devices(or more) communicably coupled to computing device 1801 and / or server 1802) to obtain multiple plasmid genome sequences; receive specifications for fixed adjacent sequence markers (for example, by processor 1808); and (for example, by processor 1808) from each plasmid genome sequence, based on the presence of fixed adjacent sequence markers in that plasmid genome sequence, a plurality of sequence regions, each sequence region located within a fixed adjacent sequence marker and candidate reverse sequences. A method for producing AAV vectors is disclosed, comprising: extracting multiple sequence regions containing terminal repeat (ITR) sequences; clustering two or more of the multiple sequence regions based on complete sequence identity (e.g., by processor 1808) to generate multiple clusters; merging two or more of the multiple clusters based on alignment between their corresponding sequence regions (e.g., by processor 1808); identifying the genotype of a representative candidate ITR sequence of a sequence region based on local alignment if a single cluster remains; and producing multiple AAV vectors using multiple plasmids having representative candidate ITR sequences (e.g., ITRs identical or substantially identical to a reference ITR sequence (e.g., a wild-type AAV ITR sequence and / or a manipulated AAV ITR sequence)).

[0244] G. Kitt A kit for identifying the genotype of an ITR, or a kit for producing an AAV vector containing an ITR that is identical (or, in some embodiments, substantially identical) to a reference ITR (e.g., a wild-type ITR and / or a manipulated ITR), is disclosed.

[0245] The present invention provides a kit having a packaging material and one or more components therein. The kit typically includes a label or accompanying document that includes a description of the components therein or instructions for use of the components in vitro, in vivo, or ex vivo. The kit may include a collection of such components, e.g., nucleic acids, recombinant vectors, viral (e.g., AAV) vectors, vector genomes, or viral particles, and optionally a second active ingredient, e.g., another compound, agent, drug, or composition.

[0246] A kit refers to a physical structure that contains one or more components of a kit. Packaging materials can be made from materials that can keep the components sterile and are commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampoules, vials, tubes, etc.).

[0247] The label or package insert may include identification information, dosage, and clinical pharmacology (including mechanism of action, pharmacokinetics, and pharmacodynamics) of one or more of its components. The label or package insert may include information identifying the manufacturer, lot number, place and date of manufacture, and expiration date. The label or package insert may include information identifying the manufacturer, lot number, place and date of manufacture. The label or package insert may include information about diseases in which the components of the kit may be used. The label or package insert may include instructions for clinicians or subjects on how, how, or in a treatment protocol or treatment regimen to use one or more of the components of the kit. The instructions may include instructions on dosage, frequency, or duration, and on how to implement any of the methods, uses, treatment protocols, or prophylactic or therapeutic regimens described herein.

[0248] Labels or package inserts may include information about any benefits that the components may offer, such as preventive or therapeutic benefits. Labels or package inserts may also include information about potential adverse side effects, complications, or reactions, such as warnings to the subject or clinician about situations in which the use of a particular composition would be inappropriate. Adverse side effects or complications may also occur if the subject has taken, plans to take, or is currently taking one or more other medicines that may be incompatible with the composition, or if the subject has taken, plans to take, or is currently taking another treatment protocol or regimen that may be incompatible with the composition, and therefore the instructions may include information about such incompatibility.

[0249] Labels or accompanying documents include “printed materials,” such as paper or cardboard, or those separated or fixed to components, kits or packaging materials (e.g., boxes), or those attached to ampoules, tubes or vials containing kit components. Labels or accompanying documents may further include computer-readable media, such as barcode printed labels, discs, optical discs (CD- or DVD-ROM / RAM, DVD, MP3, magnetic tape, etc.), or electrostorage media (RAM and ROM, etc.), or hybrids thereof (magnetic / optical storage media, flash media, or memory type cards, etc.).

[0250] The following examples illustrate the methods and systems of the present invention. These examples are not intended to be limiting.

[0251] Example 1: Figure 9 shows the Integrated Genomics Viewer (IGV), where each row represents a read value, + indicates a positive lead, and - indicates a negative lead. The bottom row shows the reference sequence.

[0252] Figure 10 shows a visualization of pINT3330 alignment to a reference sequence using PacBio CCS (Circular Consensus Sequence) data. The data exhibits high-quality sequences (0.2% error rate), with a single read covering the entire plasmid. Subsequent high-quality alignments lead to the identification of all distinct variants in the plasmid population. Figure 10 shows the alignment of the pINT3330 plasmid in the left ITR region. Almost all dominant variants of the BB' "deletion," CC' "deletion," and L-ITR flop alleles are shown.

[0253] Figure 11 shows that some deletions are associated with triple SNP mutations, while others are not, which is a problem with alignment visualization. Reads with deletions and SNPs can be described as flop alleles carrying simple deletions. Both events result in the same exact DNA sequence, but the latter may be considered more biologically plausible.

[0254] Figure 12 shows another visualization of pINT3330 alignment to a reference sequence using Illumina 150bp data. Short reads (150bp) with a low error rate (0.3%). The analysis pipeline misses the flip-to-flop transition but quantifies hairpin deletions with some accuracy.

[0255] Figure 13 shows another visualization of pINT3330 alignment to a reference sequence using an Oxford Nanopore external data source (Plasmidsaurus). The data shows noisy long-read data with a high error rate (3.9%). Variant signals are present and it is not suitable for accurate variant calling.

[0256] Figure 14 shows that the L-ITR CC’ deletion causes a linear isoform of the FIX plasmid. Approximately 99.5% of the R-ITR sequence has no mutations. The changes at this locus are detectable by the Xmal assay of the ITR, but this change may be difficult to characterize without sequencing. After deletion, the L-ITR loses the Xmal restriction site, while the R-ITR retains it, resulting in a linear isoform of the plasmid. Pacbio, Illumina, and CE assays report similar abundances of the linear isoform.

Table 1

[0257] Figure 15 shows an L-ITR BB’ deletion that avoids Xma1 restriction enzyme-based assay detection to keep the restriction enzyme site intact. However, PacBio sequencing finds further mutations in the L-ITR. Plasmids with these variants retain the Xmal cleavage site in the L-ITR. Deletion of the BB’ hairpin. This process includes a change in configuration from flip to flop.

Table 2

[0258] Figure 16 shows how the deletion of the payload creates the same 100bp motif as the L-ITR CC’ deletion. This can confuse some short-read pipelines and misquantify any mutations. Illumina overestimates the allele frequency. Other plasmid deletions are miscounted as payload deletion alleles.

Table 3

[0259] Figure 17 shows sequence homology between ITR sequences. Figure 17 shows a large homology region (125 bp) between the L-ITR flop sequence and the R-ITR flip sequence. This can also cause alignment problems with short-read sequences. Flop alleles are detected in Illumina raw data but are missed in the analysis pipeline.

[0260] The method and system may be computer-implemented. Figure 18 shows a block diagram depicting a system / environment 1800, including a non-limiting example of a computing device 1801 and a server 1802 connected via a network 1804. In one embodiment, some or all steps of any method described herein (e.g., receiving, extracting, clustering, merging, and / or similar sequences) may be performed via a computing device(s) described herein. The computing device 1801 may include one or more computers configured to generate and / or store sequencing (e.g., genome) data 1829, software 1822 (e.g., alignment software, mapping software, etc.), etc. The sequencing data 1829 may include any data relating to or containing any of the genomes, genotypes, sequences, sequence data, genomic data, combinations thereof, and / or similar, as described herein. The server 1802 may include one or more computers configured to store the sequencing data 1829. Multiple servers 1802 can communicate with computing devices 1801 via (or through) the network 1804.

[0261] Computing device 1801 and server 1802 may each be computers, in terms of hardware architecture, generally comprising a processor 1808, system memory 1810, input / output (I / O) interface 1812, and network interface 1814. These components (1808, 1810, 1812, and 1814) are communicatively coupled via a local interface 1816. The local interface 1816 may be, but is not limited to, one or more buses or other wired or wireless connections, as is known in the art. The local interface 1816 may have additional elements to enable communication, such as controllers, buffers (caches), drivers, repeaters, and receivers, but these are omitted for simplicity. Furthermore, the local interface may include address, control, and / or data connections to enable proper communication between the aforementioned components.

[0262] The processor 1808 may be a hardware device for executing software, particularly stored in system memory 1810. The processor 1808 may be any custom-made or commercially available processor, a central processing unit (CPU), an auxiliary processor among several processors associated with computing device 1801 and server 1802, a semiconductor-based microprocessor (in the form of a microchip or chipset), or any device in general for executing software instructions. While computing device 1801 and / or server 1802 are operating, the processor 1808 may execute software stored in system memory 1810 to communicate data to and from system memory 1810, and in accordance with the software, generally control the operation of computing device 1801 and server 1802.

[0263] The I / O interface 1812 may be used to receive user input from and / or provide system output to one or more devices or components. User input may be provided, for example, via a keyboard and / or mouse. System output may be provided via a display device and / or printer (not shown). The I / O interface 1812 may include, for example, a serial port, a parallel port, a Small Computer System Interface (SCSI), an infrared (IR) interface, a radio frequency (RF) interface, and / or a Universal Serial Bus (USB) interface.

[0264] The network interface 1814 may be used to send and receive data from computing devices 1801 and / or server 1802 on network 1804. The network interface 1814 may include, for example, a 10BaseT Ethernet adapter, a LAN PHY Ethernet adapter, a Token Ring adapter, a wireless network adapter (e.g., WiFi, cellular, satellite), or any other suitable network interface device. The network interface 1814 may include address, control, and / or data connections to enable proper communication on network 1804.

[0265] The system memory 1810 may include one or a combination of volatile memory elements (e.g., random access memory (RAM such as DRAM, SRAM, SDRAM, etc.)) and non-volatile memory elements (e.g., ROM, hard drives, tapes, CD-ROMs, DVD-ROMs, etc.). Furthermore, the system memory 1810 may incorporate electronic, magnetic, optical, and / or other types of storage media. It should be noted that the system memory 1810 may have a distributed architecture in which various components are located apart from each other but can be accessed by the processor 1808.

[0266] The software in system memory 1810 may include one or more software programs, each of which includes an ordered list of executable instructions for implementing a logical function. In the embodiment of Figure 18, the software in system memory 1810 of computing device 1801 may include sequence determination data 1829, software 1822, and a preferred operating system (O / S) 1818. In the embodiment of Figure 18, the software in system memory 1810 of server 1802 may include sequence determination data 1829 and a preferred operating system (O / S) 1818. The operating system 1818 essentially controls the execution of other computer programs and provides scheduling, input / output control, file and data management, memory management, and communication control, as well as related services.

[0267] The computing device 1801 and the server 1802 can communicate with the sequencer 1830 (for example, via the network 1804) as shown in Figure 18. The sequencer 1830 may be configured to determine the sequence of biomolecules (e.g., nucleic acids such as DNA or RNA). The sequencer 1830 may employ any preferred sequencing method(s). In some embodiments, the sequencer 1830 may include and / or perform the function of a gene analyzer (e.g., a gene analyzer commercially available from Illumina or Applied Biosystems).

[0268] For illustrative purposes, application programs and other executable program components, such as the operating system 1818, are shown herein as separate blocks, but it is recognized that such programs and components may reside at different times in different storage components of the computing device 1801 and / or server 1802. The execution modes of the software 1822 may be stored in or transmitted through any form of computer-readable medium. Any of the disclosed methods may be executed by computer-readable instructions embodied on the computer-readable medium. The computer-readable medium may be any available medium accessible by a computer. For example, and not intended to be limiting, the computer-readable medium may include “computer storage medium” and “communication medium.” “Computer storage medium” may include volatile and non-volatile removable and non-removable media implemented in any method or technique for storing information, such as computer-readable instructions, data structures, program modules, or other data. Exemplary computer storage media may include RAM, ROM, EEPROM, flash memory or other memory technologies, CD-ROM, digital versatile disk (DVD) or other optical storage, magnetic cassette, magnetic tape, magnetic disk storage or other magnetic storage devices, or any other media that may be used to store desired information and may be accessed by a computer.

[0269] In one embodiment, software 1822 may be configured to perform method 1900 as shown in Figure 19. Method 1900 may be performed entirely or partially by a single computing device, multiple electronic devices, and so on. Method 1900 may include receiving sequencing data in 1902. The sequencing data may include multiple plasmid genome sequences. Receiving sequencing data may include determining the sequencing data. Receiving sequencing data may include sequencing the genomes of multiple plasmids to obtain multiple plasmid genome sequences, for example, via sequencer 1830. Sequencing multiple plasmids may include sequencing via long-read sequencing or circular consensus sequencing.

[0270] Method 1900 may include receiving a specification for a fixed adjacent sequence marker in 1904. The specification may include any form of data structure, e.g., a string, a flat file, etc. The fixed adjacent sequence marker may include a sequence of at least 15 nucleotides. The fixed adjacent sequence marker may include at least a portion of the transgene delivered by the AAV vector.

[0271] Method 1900 may include extracting multiple sequence regions from a plasmid genome sequence in 1906. Sequence regions may be extracted from each plasmid genome sequence. Sequence regions may be extracted based on the presence of fixed adjacent sequence markers in the plasmid genome sequence. Each sequence region may be within a fixed adjacent sequence marker. Each sequence region may contain a candidate inverted terminal repeat (ITR) sequence.

[0272] Extracting multiple sequence regions may involve locating fixed adjacent sequence markers in a plasmid genome sequence and extracting sequence regions from the fixed adjacent sequence markers in the 5' to 3' direction relative to the orientation of the fixed adjacent sequence markers.

[0273] Extracting multiple sequence regions involves locating fixed adjacent sequence markers in a plasmid genome sequence and extracting sequence regions from the fixed adjacent sequence markers in the 3' to 5' direction relative to the orientation of the fixed adjacent sequence markers.

[0274] Extracting multiple sequence regions involves locating multiple fixed adjacent sequence markers in a plasmid genome sequence and extracting sequence regions between these fixed adjacent sequence markers.

[0275] Method 1900 may include clustering two or more sequence regions from among several sequence regions in order to generate multiple clusters in 1908. The clustering may be based on sequence identity. The clustering may be based on complete sequence identity. The clustering may be based on at least 99% sequence identity. The clustering may be based on at least 98% sequence identity.

[0276] Method 1900 may further include determining the length of each of a plurality of sequence regions and excluding the sequence region from the clustering step if its length exceeds a first threshold or falls below a second threshold. The first threshold may be a length of approximately 700 base pairs, and the second threshold may be a length of approximately 150 base pairs.

[0277] Method 1900 may include merging two or more clusters from a group of clusters in 1910. Merging two or more clusters from a group of clusters may be based on alignment between their corresponding sequence regions. Merging two or more clusters from a group of clusters may include iteratively merging two or more clusters from a group of clusters into one or more modified clusters. Iteratively merging two or more clusters from a group of clusters may each be based on aligning representative sequence regions from each cluster using a different sequence identity for each iteration. Iteratively merging two or more clusters from a group of clusters is performed until a predetermined number of clusters are generated. The predetermined number of clusters is 1.

[0278] Method 1900 may further include generating an ITR genotype database based on sequence regions associated with clusters containing two or more sequence regions. Method 1900 may further include producing a recombinant AAV vector based on the ITR genotype database.

[0279] Method 1900 may include determining the genotype of a candidate ITR sequence in 1912. Determining the genotype of a candidate ITR sequence may be performed if a single cluster remains. Determining the genotype of a candidate ITR sequence may be based on local alignment. Determining the genotype of a candidate ITR sequence may be based on aligning the candidate ITR sequence to a reference sequence. The reference sequence may be a reference ITR sequence. The reference ITR sequence may be a wild-type ITR sequence. The reference ITR sequence may be an engineered ITR sequence. The genotype of the candidate ITR sequence may be identical to that of the wild-type ITR sequence or the engineered ITR sequence.

[0280] Method 1900 may include, in 1914, the production of multiple AAV vectors. The production of multiple AAV vectors may be based on the genotype of the candidate ITR sequence. The production of multiple AAV vectors may be based on the genotype of the candidate ITR sequence which is identical to the wild-type ITR sequence or the manipulated ITR sequence. The production of multiple AAV vectors may include the production of multiple AAV vectors using plasmids having ITR sequences which have the genotype of the candidate ITR sequence.

[0281] Method 1900 may further include packaging multiple AAV vectors for distribution based on the genotype of a candidate ITR sequence that is identical to a wild-type ITR sequence or an engineered ITR sequence.

[0282] Method 1900 may further include administering a therapeutically effective dose of a manufactured recombinant AAV vector to a human subject.

[0283] In one embodiment, software 1822 may be configured to perform method 2000 as shown in Figure 20. Method 2000 may be performed entirely or partially by a single computing device, multiple electronic devices, and so on. Method 2000 may include receiving sequencing data in 2002. The sequencing data may include multiple plasmid genome sequences. Receiving sequencing data may include determining the sequencing data. Receiving sequencing data may include sequencing the genomes of multiple plasmids to obtain multiple plasmid genome sequences, for example, via sequencer 1830. Sequencing multiple plasmids may include sequencing via long-read sequencing or circular consensus sequencing.

[0284] Method 2000 may include receiving a specification for a fixed adjacent sequence marker in 2004. The specification may include any form of data structure, e.g., a string, a flat file, etc. The fixed adjacent sequence marker may include a sequence of at least 15 nucleotides. The fixed adjacent sequence marker may include at least a portion of the transgene delivered by the AAV vector.

[0285] Method 2000 may include extracting multiple sequence regions from a plasmid genome sequence in 2006. Sequence regions may be extracted from each plasmid genome sequence. Sequence regions may be extracted based on the presence of fixed adjacent sequence markers in the plasmid genome sequence. Each sequence region may be within a fixed adjacent sequence marker. Each sequence region may contain a candidate inverted terminal repeat (ITR) sequence.

[0286] Extracting multiple sequence regions may involve locating fixed adjacent sequence markers in a plasmid genome sequence and extracting sequence regions from the fixed adjacent sequence markers in the 5' to 3' direction relative to the orientation of the fixed adjacent sequence markers.

[0287] Extracting multiple sequence regions involves locating fixed adjacent sequence markers in a plasmid genome sequence and extracting sequence regions from the fixed adjacent sequence markers in the 3' to 5' direction relative to the orientation of the fixed adjacent sequence markers.

[0288] Extracting multiple sequence regions involves locating multiple fixed adjacent sequence markers in a plasmid genome sequence and extracting sequence regions between these fixed adjacent sequence markers.

[0289] Method 2000 may include clustering two or more sequence regions from among several sequence regions in order to generate multiple clusters in 2008. The clustering may be based on sequence identity. The clustering may be based on complete sequence identity. The clustering may be based on at least 99% sequence identity. The clustering may be based on at least 98% sequence identity.

[0290] Method 2000 may further include determining the length of each of a plurality of sequence regions and excluding the sequence region from the clustering step if its length exceeds a first threshold or falls below a second threshold. The first threshold may be a length of approximately 700 base pairs, and the second threshold may be a length of approximately 150 base pairs.

[0291] Method 2000 may include merging two or more clusters from a group of clusters in 2010. Merging two or more clusters from a group of clusters may be based on alignment between their corresponding sequence regions. Merging two or more clusters from a group of clusters may include iteratively merging two or more clusters from a group of clusters into one or more modified clusters. Iteratively merging two or more clusters from a group of clusters may each be based on aligning representative sequence regions from each cluster using a different sequence identity for each iteration. Iteratively merging two or more clusters from a group of clusters is performed until a predetermined number of clusters are generated. The predetermined number of clusters is 1.

[0292] Method 2000 may further include generating an ITR genotype database based on sequence regions associated with clusters containing two or more sequence regions. Method 2000 may further include producing a recombinant AAV vector based on the ITR genotype database.

[0293] Method 2000 may include, in 2012, considering multiple plasmids unsuitable for the production of recombinant vector genomes. Considering multiple plasmids unsuitable for the production of recombinant vector genomes may be based on two or more clusters remaining after merging. The presence of two or more clusters after merging indicates that the multiple plasmids contain variability within the ITR that exceeds the threshold for AAV vector production. Method 2000 may further include discarding any plasmids associated with the multiple plasmids.

[0294] In one embodiment, software 1822 may be configured to perform method 2100 as shown in Figure 21. Method 2100 may be performed entirely or partially by a single computing device, multiple electronic devices, and so on. Method 2100 may include receiving sequencing data in 2102. Sequencing data may include multiple plasmid genome sequences. Receiving sequencing data may include determining the sequencing data. Receiving sequencing data may include sequencing the genomes of multiple plasmids to obtain multiple plasmid genome sequences, for example, via sequencer 1830. Sequencing multiple plasmids may include sequencing via long-read sequencing or circular consensus sequencing.

[0295] Method 2100 may include receiving a specification for a fixed adjacent sequence marker in 2104. The specification may include any form of data structure, e.g., a string, a flat file, etc. The fixed adjacent sequence marker may include a sequence of at least 15 nucleotides. The fixed adjacent sequence marker may include at least a portion of the transgene delivered by the AAV vector.

[0296] Method 2100 may include extracting multiple sequence regions from a plasmid genome sequence in 2106. Sequence regions may be extracted from each plasmid genome sequence. Sequence regions may be extracted based on the presence of fixed adjacent sequence markers in the plasmid genome sequence. Each sequence region may be within a fixed adjacent sequence marker. Each sequence region may contain a candidate inverted terminal repeat (ITR) sequence.

[0297] Extracting multiple sequence regions may involve locating fixed adjacent sequence markers in a plasmid genome sequence and extracting sequence regions from the fixed adjacent sequence markers in the 5' to 3' direction relative to the orientation of the fixed adjacent sequence markers.

[0298] Extracting multiple sequence regions involves locating fixed adjacent sequence markers in a plasmid genome sequence and extracting sequence regions from the fixed adjacent sequence markers in the 3' to 5' direction relative to the orientation of the fixed adjacent sequence markers.

[0299] Extracting multiple sequence regions involves locating multiple fixed adjacent sequence markers in a plasmid genome sequence and extracting sequence regions between these fixed adjacent sequence markers.

[0300] Method 2100 may include clustering two or more sequence regions from among multiple sequence regions in order to generate multiple clusters in 2108. The clustering may be based on sequence identity. The clustering may be based on complete sequence identity. The clustering may be based on at least 99% sequence identity. The clustering may be based on at least 98% sequence identity.

[0301] Method 2100 may further include determining the length of each of a plurality of sequence regions and excluding the sequence region from the clustering step if its length exceeds a first threshold or falls below a second threshold. The first threshold may be a length of approximately 700 base pairs, and the second threshold may be a length of approximately 150 base pairs.

[0302] Method 2100 may include merging two or more clusters from a group of clusters in 2110. Merging two or more clusters from a group of clusters may be based on alignment between their corresponding sequence regions. Merging two or more clusters from a group of clusters may include iteratively merging two or more clusters from a group of clusters into one or more modified clusters. Iteratively merging two or more clusters from a group of clusters may each be based on aligning representative sequence regions from each cluster using a different sequence identity for each iteration. Iteratively merging two or more clusters from a group of clusters is performed until a predetermined number of clusters are generated. The predetermined number of clusters is 1.

[0303] Method 2100 may further include generating an ITR genotype database based on sequence regions associated with clusters containing two or more sequence regions, and producing a recombinant AAV vector based on the ITR genotype database.

[0304] Method 2100 may include, in 2112, identifying the genotype of a candidate ITR sequence of a representative sequence region of a single remaining cluster. Identifying the genotype of a candidate ITR sequence of a representative sequence region may be performed in 2110 if a single cluster remains after the merge. Identifying the genotype of a candidate ITR sequence may be based on local alignment. Identifying the genotype of a candidate ITR sequence may be based on aligning the candidate ITR sequence to a reference sequence. The reference sequence may be a reference ITR sequence. The reference ITR sequence may be a wild-type ITR sequence. The reference ITR sequence may be an engineered ITR sequence. The genotype of a candidate ITR sequence may be identical to that of a wild-type ITR sequence or an engineered ITR sequence.

[0305] Method 2100 may further include the production of multiple AAV vectors. The production of multiple AAV vectors may be based on the genotype of the candidate ITR sequence. The production of multiple AAV vectors may be based on the genotype of the candidate ITR sequence which is identical to the wild-type ITR sequence or the manipulated ITR sequence. The production of multiple AAV vectors may include the production of multiple AAV vectors using plasmids having ITR sequences which have the genotype of the candidate ITR sequence.

[0306] Method 2100 may further include packaging multiple AAV vectors for distribution based on the genotype of a candidate ITR sequence that is identical to a wild-type ITR sequence or an engineered ITR sequence.

[0307] Method 2100 may further include administering a therapeutically effective dose of a manufactured recombinant AAV vector to a human subject.

[0308] In one embodiment, software 1822 may be configured to perform method 2200 as shown in Figure 22. Method 2200 may be performed entirely or partially by a single computing device, multiple electronic devices, and so on. Method 2200 may include receiving sequencing data in 2202. Sequencing data may include multiple adeno-associated virus (AAV) vector genome sequences. Receiving sequencing data may include determining the sequencing data. Receiving sequencing data may include sequencing the genomes of multiple adeno-associated virus (AAV) vectors to obtain multiple adeno-associated virus (AAV) vector genome sequences, for example, via sequencer 1830. Sequencing multiple adeno-associated virus (AAV) vectors may include sequencing via long-read sequencing or circular consensus sequencing.

[0309] Method 2200 may include receiving a specification for a fixed adjacent sequence marker in 2204. The specification may include any form of data structure, e.g., a string, a flat file, etc. The fixed adjacent sequence marker may include a sequence of at least 15 nucleotides. The fixed adjacent sequence marker may include at least a portion of a transgene delivered by multiple AAV vectors.

[0310] Method 2200 may include extracting sequence regions from each AAV vector genome sequence in 2206. Sequence regions may be extracted based on the presence of fixed adjacent sequence markers in the AAV vector genome sequences. Each sequence region may be located immediately to the left or right of a fixed adjacent sequence marker. Each sequence region may contain candidate inverted terminal repeat (ITR) sequences.

[0311] Extracting a sequence region immediately to the left or right of a fixed adjacent sequence marker from each AAV vector genome sequence, based on the presence of a fixed adjacent sequence marker in that AAV vector genome sequence, involves locating the fixed adjacent sequence marker in the AAV vector genome sequence and extracting a sequence region from the fixed adjacent sequence marker in the 5' to 3' direction relative to the orientation of the fixed adjacent sequence marker.

[0312] Extracting a sequence region immediately to the left or right of a fixed adjacent sequence marker from each AAV vector genome sequence, based on the presence of a fixed adjacent sequence marker in that AAV vector genome sequence, involves locating the fixed adjacent sequence marker in the AAV vector genome sequence and extracting a sequence region from the fixed adjacent sequence marker in the 3' to 5' direction relative to the orientation of the fixed adjacent sequence marker.

[0313] Extracting a sequence region immediately to the left or right of a fixed adjacent sequence marker from the AAV vector genome sequence of multiple AAV vectors, based on the presence of a fixed adjacent sequence marker in that AAV vector genome sequence, involves locating multiple fixed adjacent sequence markers in the AAV vector genome sequence and extracting the sequence region between multiple fixed adjacent sequence markers.

[0314] Method 2200 may include clustering two or more sequence regions from among multiple sequence regions in order to generate multiple clusters in 2208. The clustering may be based on sequence identity. The clustering may be based on complete sequence identity. The clustering may be based on at least 99% sequence identity. The clustering may be based on at least 98% sequence identity.

[0315] Method 2200 may further include determining the length of each of a plurality of sequence regions and excluding the sequence region from the clustering step if its length exceeds a first threshold or falls below a second threshold. The first threshold may be a length of approximately 700 base pairs, and the second threshold may be a length of approximately 150 base pairs.

[0316] Method 2200 may include merging two or more clusters from a group of clusters in 2210. Merging two or more clusters from a group of clusters may be based on alignment between their corresponding sequence regions. Merging two or more clusters from a group of clusters may include iteratively merging two or more clusters from a group of clusters into one or more modified clusters. Iteratively merging two or more clusters from a group of clusters may each be based on aligning representative sequence regions from each cluster using a different sequence identity for each iteration. Iteratively merging two or more clusters from a group of clusters is performed until a predetermined number of clusters are generated. The predetermined number of clusters is 1.

[0317] Method 2200 may further include generating an ITR genotype database based on sequence regions associated with clusters containing two or more sequence regions. Method 2200 may further include producing a recombinant AAV vector based on the ITR genotype database.

[0318] Method 2200 may include, in 2212, deeming the AAV vector genome unsuitable. Deeming the AAV vector genome unsuitable may be based on two or more clusters remaining after merging. The presence of two or more clusters after merging indicates that the AAV vector genome contains variability within the ITR that exceeds the threshold for the AAV vector. Method 2200 may further include discarding any AAV vector associated with multiple AAV vectors.

[0319] In one embodiment, software 1822 may be configured to perform method 2300 as shown in Figure 23. Method 2300 may be performed entirely or partially by a single computing device, multiple electronic devices, and so on. Method 2300 may include receiving sequencing data in 2302. Sequencing data may include multiple adeno-associated virus (AAV) vector genome sequences. Receiving sequencing data may include determining the sequencing data. Receiving sequencing data may include sequencing the genomes of multiple adeno-associated virus (AAV) vectors to obtain multiple adeno-associated virus (AAV) vector genome sequences, for example, via sequencer 1830. Sequencing multiple adeno-associated virus (AAV) vectors may include sequencing via long-read sequencing or circular consensus sequencing.

[0320] Method 2300 may include receiving a specification for a fixed adjacent sequence marker in 2304. The specification may include any form of data structure, e.g., a string, a flat file, etc. The fixed adjacent sequence marker may include a sequence of at least 15 nucleotides. The fixed adjacent sequence marker may include at least a portion of a transgene delivered by multiple AAV vectors.

[0321] Method 2300 may include extracting sequence regions from each AAV vector genome sequence in 2306. Sequence regions may be extracted based on the presence of fixed adjacent sequence markers in the AAV vector genome sequences. Each sequence region may be located immediately to the left or right of a fixed adjacent sequence marker. Each sequence region may contain candidate inverted terminal repeat (ITR) sequences.

[0322] Extracting a sequence region immediately to the left or right of a fixed adjacent sequence marker from each AAV vector genome sequence, based on the presence of a fixed adjacent sequence marker in that AAV vector genome sequence, involves locating the fixed adjacent sequence marker in the AAV vector genome sequence and extracting a sequence region from the fixed adjacent sequence marker in the 5' to 3' direction relative to the orientation of the fixed adjacent sequence marker.

[0323] Extracting a sequence region immediately to the left or right of a fixed adjacent sequence marker from each AAV vector genome sequence, based on the presence of a fixed adjacent sequence marker in that AAV vector genome sequence, involves locating the fixed adjacent sequence marker in the AAV vector genome sequence and extracting a sequence region from the fixed adjacent sequence marker in the 3' to 5' direction relative to the orientation of the fixed adjacent sequence marker.

[0324] Extracting a sequence region immediately to the left or right of a fixed adjacent sequence marker from the AAV vector genome sequence of multiple AAV vectors, based on the presence of a fixed adjacent sequence marker in that AAV vector genome sequence, involves locating multiple fixed adjacent sequence markers in the AAV vector genome sequence and extracting the sequence region between multiple fixed adjacent sequence markers.

[0325] Method 2300 may include clustering two or more sequence regions from among multiple sequence regions in order to generate multiple clusters in 2308. The clustering may be based on sequence identity. The clustering may be based on complete sequence identity. The clustering may be based on at least 99% sequence identity. The clustering may be based on at least 98% sequence identity.

[0326] Method 2300 may further include determining the length of each of a plurality of sequence regions and excluding the sequence region from the clustering step if its length exceeds a first threshold or falls below a second threshold. The first threshold may be a length of approximately 700 base pairs, and the second threshold may be a length of approximately 150 base pairs.

[0327] Method 2300 may include merging two or more clusters from a group of clusters in 2310. Merging two or more clusters from a group of clusters may be based on alignment between their corresponding sequence regions. Merging two or more clusters from a group of clusters may include iteratively merging two or more clusters from a group of clusters into one or more modified clusters. Iteratively merging two or more clusters from a group of clusters may each be based on aligning representative sequence regions from each cluster using a different sequence identity for each iteration. Iteratively merging two or more clusters from a group of clusters is performed until a predetermined number of clusters are generated. The predetermined number of clusters is 1.

[0328] Method 2300 may further include generating an ITR genotype database based on sequence regions associated with clusters containing two or more sequence regions. Method 2300 may further include producing a recombinant AAV vector based on the ITR genotype database.

[0329] Method 2300 may include, in 2312, identifying the genotype of a candidate ITR sequence of a representative sequence region of a single cluster. Identifying the genotype of a candidate ITR sequence of a representative sequence region may be performed in 2310 if a single cluster remains after the merge. Identifying the genotype of a candidate ITR sequence may be based on local alignment. Identifying the genotype of a candidate ITR sequence may be based on aligning the candidate ITR sequence to a reference sequence. The reference sequence may be a reference ITR sequence. The reference ITR sequence may be a wild-type ITR sequence. The reference ITR sequence may be an engineered ITR sequence. The genotype of a candidate ITR sequence may be identical to that of a wild-type ITR sequence or an engineered ITR sequence.

[0330] Method 2300 may further include producing multiple AAV vectors. Producing multiple AAV vectors may be based on the genotype of a candidate ITR sequence. Producing multiple AAV vectors may be based on the genotype of a candidate ITR sequence that is identical to a wild-type ITR sequence or an engineered ITR sequence. Producing multiple AAV vectors may include producing multiple AAV vectors using a plasmid having an ITR sequence having the genotype of the candidate ITR sequence. Method 2300 may further include producing additional multiple AAV vectors using the same plasmid used to produce the multiple AAV vectors. Method 2300 may further include packaging multiple AAV vectors for distribution based on the genotype of a candidate ITR sequence that is identical to a wild-type ITR sequence or an engineered ITR sequence.

[0331] Method 2300 may further include administering a therapeutically effective dose of a manufactured recombinant AAV vector to a human subject.

[0332] With regard to the methods, systems, and apparatus described herein, as well as variations thereof, this specification describes below certain specific embodiments that more specifically describe the invention. However, these particularly enumerated embodiments should not be construed as limiting any different claims containing different or more general teachings described herein, or limiting any “specific” embodiment in any way other than the inherent meaning of the language used therein.

[0333] Embodiment 1 is a method for manufacturing a product, comprising: sequencing the genomes of multiple plasmids to obtain multiple plasmid genome sequences; receiving specifications for fixed adjacent sequence markers; extracting from each plasmid genome sequence multiple sequence regions, each of which is located within a fixed adjacent sequence marker and contains a candidate inverted end repeat (ITR) sequence, based on the presence of fixed adjacent sequence markers in the plasmid genome sequence; clustering two or more of the sequence regions based on complete sequence identity to generate multiple clusters; merging two or more of the clusters based on the alignment between their corresponding sequence regions; identifying the genotype of the candidate ITR sequence of the single cluster based on local alignment if a single cluster remains; and manufacturing multiple AAV vectors using plasmids having ITR sequences having the genotype of the candidate ITR sequence, based on the genotype of the candidate ITR sequence.

[0334] Embodiment 2 is characterized in that the subject of Embodiment 1 includes sequencing of multiple plasmids via long-read sequencing or circular consensus sequencing.

[0335] In Embodiment 3, the subject matter of Embodiments 1 and 2 is characterized in that the fixed adjacent sequence marker includes a sequence of at least 15 nucleotides.

[0336] Embodiment 4 is characterized in that the subject matter of Embodiments 1 to 3 includes at least a portion of the transgene delivered by the AAV vector.

[0337] Embodiment 5 is characterized in that the subject matter of Embodiments 1 to 4 is further characterized in that the extraction of multiple sequence regions includes locating fixed adjacent sequence markers in a plasmid genome sequence and extracting sequence regions from the fixed adjacent sequence markers in the 5' to 3' direction relative to the orientation of the fixed adjacent sequence markers.

[0338] Embodiment 6 is characterized in that the subject matter of Embodiments 1 to 5 is further characterized in that the extraction of multiple sequence regions includes locating fixed adjacent sequence markers in a plasmid genome sequence and extracting sequence regions from the fixed adjacent sequence markers in the 3' to 5' direction relative to the orientation of the fixed adjacent sequence markers.

[0339] Embodiment 7 is characterized in that the subject matter of Embodiments 1 to 6 is further characterized in that the extraction of multiple sequence regions includes locating multiple fixed adjacent sequence markers in a plasmid genome sequence and extracting sequence regions between multiple fixed adjacent sequence markers.

[0340] Embodiment 8 is characterized in that, for each of the multiple array regions, the subject matter of Embodiments 1 to 7 includes determining the length of the array region, and excluding the array region from the clustering step if its length exceeds a first threshold or falls below a second threshold.

[0341] Embodiment 9 is characterized in that the subject of Embodiment 8 has a first threshold of approximately 700 base pairs in length and a second threshold of approximately 150 base pairs in length.

[0342] Embodiment 10 is characterized in that the subject matter of Embodiments 1 to 9 is further developed by generating an ITR genotype database based on sequence regions associated with clusters containing two or more sequence regions, and by producing a recombinant AAV vector based on the ITR genotype database.

[0343] Embodiment 11 is characterized in that the subject matter of Embodiments 1 to 10 is characterized in that merging two or more clusters from a plurality of clusters includes iteratively merging two or more clusters from a plurality of clusters into one or more modified clusters.

[0344] Embodiment 12 is characterized in that the subject of Embodiment 11 is characterized in that the iterative merging of two or more clusters among a plurality of clusters is based on aligning representative sequence regions from each cluster using a different sequence identity for each iteration.

[0345] Embodiment 13 is characterized in that the subject matter of Embodiments 11 to 12 is iteratively merged with two or more clusters from among multiple clusters until a predetermined number of clusters are generated.

[0346] Embodiment 14 is characterized in that the subject of Embodiment 13 has a predetermined number of clusters, which is 1.

[0347] Embodiment 15 is characterized in that the subject matter of Embodiments 1 to 14 is that the genotype of the candidate ITR sequence is identical to that of the wild-type ITR sequence or the manipulated ITR sequence.

[0348] Embodiment 16 is characterized in that the subject matter of Embodiments 1 to 15 is packaged for distribution based on the genotype of a candidate ITR sequence that is identical to a wild-type ITR sequence or an engineered ITR sequence.

[0349] Embodiment 17 is characterized in that the subject matter of Embodiments 1 to 16 is administered to a human subject in a therapeutically effective amount of a manufactured recombinant AAV vector.

[0350] Embodiment 18 is a method comprising: sequencing the genomes of multiple plasmids to obtain multiple plasmid genome sequences; receiving specifications for fixed adjacent sequence markers; extracting from each plasmid genome sequence multiple sequence regions, each of which is located within a fixed adjacent sequence marker and contains a candidate inverted end repeat (ITR) sequence, based on the presence of fixed adjacent sequence markers in the plasmid genome sequence; clustering two or more of the sequence regions based on sequence identity to generate multiple clusters; merging two or more of the clusters based on the alignment between their corresponding sequence regions; and determining that multiple plasmids are unsuitable for the production of recombinant vector genomes based on the two or more clusters remaining after merging.

[0351] Embodiment 19 is characterized in that the subject of Embodiment 18 includes sequencing of multiple plasmids via long-read sequencing or circular consensus sequencing.

[0352] Embodiment 20 is characterized in that the subject matter of Embodiments 18-19 is such that the fixed adjacent sequence marker includes a sequence of at least 15 nucleotides.

[0353] Embodiment 21 is characterized in that the subject matter of Embodiments 18-20 includes at least a portion of the transgene delivered by the AAV vector as a fixed adjacent sequence marker.

[0354] Embodiment 22 is characterized in that the subject matter of Embodiments 18 to 21 is further characterized in that the extraction of multiple sequence regions includes locating fixed adjacent sequence markers in a plasmid genome sequence and extracting sequence regions from the fixed adjacent sequence markers in the 5' to 3' direction relative to the orientation of the fixed adjacent sequence markers.

[0355] Embodiment 23 is characterized in that the subject matter of Embodiments 18 to 22 is further characterized in that the extraction of multiple sequence regions includes locating fixed adjacent sequence markers in a plasmid genome sequence and extracting sequence regions from the fixed adjacent sequence markers in the 3' to 5' direction relative to the orientation of the fixed adjacent sequence markers.

[0356] Embodiment 24 is characterized in that the subject matter of Embodiments 18 to 23 is further characterized in that the extraction of multiple sequence regions includes locating multiple fixed adjacent sequence markers in a plasmid genome sequence and extracting sequence regions between multiple fixed adjacent sequence markers.

[0357] Embodiment 25 is characterized in that, for each of the multiple array regions, the subject matter of Embodiments 18 to 24 includes determining the length of the array region, and excluding the array region from the clustering step if its length exceeds a first threshold or falls below a second threshold.

[0358] Embodiment 26 is characterized in that the subject of Embodiment 25 has a first threshold of approximately 700 base pairs in length and a second threshold of approximately 150 base pairs in length.

[0359] Embodiment 27 is characterized in that the subject matter of Embodiments 18 to 26 is expanded to include generating an ITR genotype database based on sequence regions associated with clusters containing two or more sequence regions, and producing a recombinant AAV vector based on the ITR genotype database.

[0360] In Embodiment 28, the subject matter of Embodiments 18 to 27 is characterized in that the generation of multiple clusters by clustering two or more sequence regions from among multiple sequence regions is based on complete sequence identity.

[0361] In Embodiment 29, the subject matter of Embodiments 18 to 28 is characterized in that the generation of multiple clusters by clustering two or more sequence regions from among multiple sequence regions is based on at least 99% sequence identity.

[0362] In Embodiment 30, the subject matter of Embodiments 18 to 29 is characterized in that the generation of multiple clusters by clustering two or more sequence regions from among multiple sequence regions is based on at least 98% sequence identity.

[0363] Embodiment 31 is characterized in that the subject matter of Embodiments 18 to 30 is characterized in that merging two or more clusters from a plurality of clusters includes iteratively merging two or more clusters from a plurality of clusters into one or more modified clusters.

[0364] Embodiment 32 is characterized in that the subject of Embodiment 31 is characterized in that the iterative merging of two or more clusters among a plurality of clusters is based on aligning representative sequence regions from each cluster using a different sequence identity for each iteration.

[0365] Embodiment 33 is characterized in that the subject matter of Embodiments 31 to 32 is iteratively merged with two or more clusters from among multiple clusters until a predetermined number of clusters are generated.

[0366] Embodiment 34 is characterized in that the subject of Embodiment 33 has a predetermined number of clusters, which is 1.

[0367] Embodiment 35 is characterized in that the subject matter of Embodiments 18 to 34 is the same as that of the wild-type ITR sequence or the manipulated ITR sequence.

[0368] Embodiment 36 is characterized by discarding any plasmids related to the subject matter of Embodiments 18 to 35.

[0369] Embodiment 37 is a method comprising: sequencing the genomes of multiple plasmids to obtain multiple plasmid genome sequences; receiving specifications for fixed adjacent sequence markers; extracting from each plasmid genome sequence, based on the presence of fixed adjacent sequence markers in that plasmid genome sequence, multiple sequence regions, each sequence region being within a fixed adjacent sequence marker and containing candidate inverted end repeat (ITR) sequences; clustering two or more of the sequence regions based on sequence identity to generate multiple clusters; merging two or more of the clusters based on the alignment between their corresponding sequence regions; and, if a single cluster remains after the merge, identifying the genotype of a representative sequence region of the single cluster based on local alignment.

[0370] Embodiment 38 is characterized in that the subject of Embodiment 37 includes sequencing of multiple plasmids via long-read sequencing or circular consensus sequencing.

[0371] Embodiment 39 is characterized in that the subject matter of Embodiments 37-38 is such that the fixed adjacent sequence marker includes a sequence of at least 15 nucleotides.

[0372] Embodiment 40 is characterized in that the subject matter of Embodiments 37-39 is such that the fixed adjacent sequence marker includes at least a portion of the transgene delivered by the AAV vector.

[0373] Embodiment 41 is characterized in that the subject matter of Embodiments 37 to 40 is further characterized in that the extraction of multiple sequence regions includes locating fixed adjacent sequence markers in a plasmid genome sequence and extracting sequence regions from the fixed adjacent sequence markers in the 5' to 3' direction relative to the orientation of the fixed adjacent sequence markers.

[0374] Embodiment 42 is characterized in that the subject matter of Embodiments 37 to 41 is further characterized in that the extraction of multiple sequence regions includes locating fixed adjacent sequence markers in a plasmid genome sequence and extracting sequence regions from the fixed adjacent sequence markers in the 3' to 5' direction relative to the orientation of the fixed adjacent sequence markers.

[0375] Embodiment 43 is characterized in that the subject matter of Embodiments 37 to 42 is further characterized in that the extraction of multiple sequence regions includes locating multiple fixed adjacent sequence markers in a plasmid genome sequence and extracting sequence regions between multiple fixed adjacent sequence markers.

[0376] Embodiment 44 is characterized in that, for each of the multiple array regions, the subject matter of Embodiments 37 to 43 includes determining the length of the array region, and excluding the array region from the clustering step if its length exceeds a first threshold or falls below a second threshold.

[0377] Embodiment 45 is characterized in that the subject of Embodiment 44 has a first threshold of approximately 700 base pairs in length and a second threshold of approximately 150 base pairs in length.

[0378] Embodiment 46 is characterized in that the subject matter of Embodiments 37 to 45 includes generating an ITR genotype database based on sequence regions associated with clusters containing two or more sequence regions, and producing a recombinant AAV vector based on the ITR genotype database.

[0379] In Embodiment 47, the subject matter of Embodiments 37 to 46 is characterized in that the generation of multiple clusters by clustering two or more sequence regions from among multiple sequence regions is based on complete sequence identity.

[0380] In Embodiment 48, the subject matter of Embodiments 37 to 47 is characterized in that the generation of multiple clusters by clustering two or more sequence regions from among multiple sequence regions is based on at least 99% sequence identity.

[0381] In Embodiment 49, the subject matter of Embodiments 37 to 48 is characterized in that the generation of multiple clusters by clustering two or more sequence regions from among multiple sequence regions is based on at least 98% sequence identity.

[0382] Embodiment 50 is characterized in that the subject matter of Embodiments 37 to 49 is such that merging two or more clusters from a plurality of clusters includes iteratively merging two or more clusters from a plurality of clusters into one or more modified clusters.

[0383] Embodiment 51 is characterized in that the subject of Embodiment 50 is characterized in that the iterative merging of two or more clusters among a plurality of clusters is based on aligning representative sequence regions from each cluster using a different sequence identity for each iteration.

[0384] In Embodiment 52, the subject matter of Embodiments 50 to 51 is characterized in that two or more clusters from among a plurality of clusters are merged iteratively until a predetermined number of clusters are generated.

[0385] Embodiment 53 is characterized in that the subject of Embodiment 52 has a predetermined number of clusters, which is 1.

[0386] In Embodiment 54, the subject matter of Embodiments 37 to 53 is characterized in that the genotype of the candidate ITR sequence is identical to that of the wild-type ITR sequence or the manipulated ITR sequence.

[0387] Embodiment 55 is characterized in that, based on the subject matter of Embodiments 37 to 54, a plurality of AAV vectors are produced using a plasmid having an ITR sequence having the genotype of a candidate ITR sequence, based on the genotype of the candidate ITR sequence being identical to the wild-type ITR sequence or the manipulated ITR sequence.

[0388] Embodiment 56 is characterized in that the subject of Embodiment 55 is packaged for distribution based on the genotype of a candidate ITR sequence that is identical to a wild-type ITR sequence or an engineered ITR sequence.

[0389] Embodiment 57 is characterized in that the subject matter of Embodiments 55-56 is administered to a human subject in a therapeutically effective amount of a manufactured recombinant AAV vector.

[0390] Embodiment 58 is a method comprising: sequencing AAV vector genomes from a plurality of adeno-associated virus (AAV) vectors to obtain a plurality of AAV vector genome sequences; receiving specifications for fixed adjacent sequence markers; extracting a sequence region from each of the plurality of AAV vector genome sequences that contains a candidate ITR sequence, based on the presence of a fixed adjacent sequence marker in that AAV vector genome sequence, which is a sequence region immediately to the left or immediately to the right of a fixed adjacent sequence marker; clustering two or more sequence regions from the plurality of sequence regions based on complete sequence identity to generate a plurality of clusters; merging two or more clusters from the plurality of clusters based on the alignment between their corresponding sequence regions; and determining that the plurality of AAV vector genomes are unsuitable based on the two or more clusters remaining after merging.

[0391] Embodiment 59 is characterized in that the subject of Embodiment 58 includes sequencing of multiple AAV genomes via long-read sequencing or circular consensus sequencing.

[0392] In Embodiment 60, the subject matter of Embodiments 58-59 is characterized in that the fixed adjacent sequence marker includes a sequence of at least 15 nucleotides.

[0393] Embodiment 61 is characterized in that the subject matter of Embodiments 58-60 includes at least a portion of the transgene delivered by a plurality of AAV vectors.

[0394] Embodiment 62 is characterized in that, based on the presence of a fixed adjacent sequence marker in the AAV vector genome sequence, extracting a sequence region immediately to the left or right of a fixed adjacent sequence marker from each AAV vector genome sequence of a plurality of AAV vectors is performed, and this includes locating the fixed adjacent sequence marker in the AAV vector genome sequence and extracting a sequence region from the fixed adjacent sequence marker in the 5' to 3' direction relative to the orientation of the fixed adjacent sequence marker.

[0395] Embodiment 63 is characterized in that the subject matter of Embodiments 58 to 62 is further characterized in that, from each AAV vector genome sequence of a plurality of AAV vectors, the extraction of a sequence region immediately to the left or immediately to the right of a fixed adjacent sequence marker, based on the presence of a fixed adjacent sequence marker in that AAV vector genome sequence, includes locating the fixed adjacent sequence marker in the AAV vector genome sequence and extracting a sequence region from the fixed adjacent sequence marker in the 3' to 5' direction relative to the orientation of the fixed adjacent sequence marker.

[0396] Embodiment 64 is characterized in that the subject matter of Embodiments 58 to 63 is further characterized in that, from each AAV vector genome sequence of a plurality of AAV vectors, the extraction of a sequence region immediately to the left or immediately to the right of a fixed adjacent sequence marker, based on the presence of a fixed adjacent sequence marker in that AAV vector genome sequence, includes locating a plurality of fixed adjacent sequence markers in the AAV vector genome sequence and extracting the sequence region between the plurality of fixed adjacent sequence markers.

[0397] Embodiment 65 is characterized in that, for each of the multiple array regions, the subject matter of Embodiments 58 to 64 includes determining the length of the array region and excluding the array region from the clustering step if its length exceeds a first threshold or falls below a second threshold.

[0398] Embodiment 66 is characterized in that the subject of Embodiment 65 has a first threshold of approximately 700 base pairs in length and a second threshold of approximately 150 base pairs in length.

[0399] Embodiment 67 is characterized in that the subject matter of Embodiments 58 to 66 generates a database of AAV ITR genotypes based on sequence regions associated with clusters containing two or more sequence regions.

[0400] In Embodiment 68, the subject matter of Embodiments 58 to 67 is characterized in that the generation of multiple clusters by clustering two or more sequence regions from among multiple sequence regions is based on complete sequence identity.

[0401] In Embodiment 69, the subject matter of Embodiments 58 to 68 is characterized in that the generation of multiple clusters by clustering two or more sequence regions from among multiple sequence regions is based on at least 99% sequence identity.

[0402] In Embodiment 70, the subject matter of Embodiments 58 to 69 is characterized in that the generation of multiple clusters by clustering two or more sequence regions from among multiple sequence regions is based on at least 98% sequence identity.

[0403] Embodiment 71 is characterized in that the subject matter of Embodiments 58 to 70 is characterized in that merging two or more clusters from a plurality of clusters includes iteratively merging two or more clusters from a plurality of clusters into one or more modified clusters.

[0404] Embodiment 72 is characterized in that the subject of Embodiment 71 is characterized in that iterative merging of two or more clusters among a plurality of clusters is based on aligning representative sequence regions from each cluster using a different sequence identity for each iteration.

[0405] In Embodiment 73, the subject matter of Embodiments 71 to 72 is characterized in that two or more clusters from among a plurality of clusters are merged iteratively until a predetermined number of clusters are generated.

[0406] Embodiment 74 is characterized in that the subject of Embodiment 73 is 1 in a predetermined number of clusters.

[0407] Embodiment 75 is characterized in that the subject matter of Embodiments 58 to 74 is the same as that of the candidate ITR sequence, with the genotype being identical to that of the wild-type AAV ITR sequence or the manipulated AAV ITR sequence.

[0408] Embodiment 76 is characterized in that the subject matter of Embodiments 58 to 75 is discarded any AAV vector associated with a plurality of AAV vectors.

[0409] Embodiment 77 is a method comprising: sequencing multiple adeno-associated virus (AAV) vector genomes to obtain multiple AAV vector genome sequences; receiving specifications for fixed adjacent sequence markers; extracting a sequence region from each of the multiple AAV vector genome sequences that contains a candidate ITR sequence, based on the presence of a fixed adjacent sequence marker in that AAV vector genome sequence, which is a sequence region immediately to the left or immediately to the right of a fixed adjacent sequence marker; clustering two or more sequence regions from the multiple sequence regions based on complete sequence identity to generate multiple clusters; merging two or more of the multiple clusters based on the alignment between their corresponding sequence regions to generate multiple modified clusters; and, if a single cluster remains after the merge, identifying the genotype of a representative sequence region of the single cluster, which is a candidate ITR sequence, based on local alignment.

[0410] Embodiment 78 is characterized in that the subject of Embodiment 77 includes sequencing of multiple AAV vector genomes via long-read sequencing or circular consensus sequencing.

[0411] Embodiment 79 is characterized in that the subject matter of Embodiments 77-78 is maintained, and the fixed adjacent sequence marker includes a sequence of at least 15 nucleotides.

[0412] Embodiment 80 is characterized in that the subject of Embodiment 79 is such that the fixed adjacent sequence marker includes at least a portion of the transgene delivered by a plurality of AAV vectors.

[0413] Embodiment 81 is characterized in that the subject matter of Embodiments 77-80 is further characterized in that, from each AAV vector genome sequence of a plurality of AAV vectors, the extraction of a sequence region immediately to the left or right of a fixed adjacent sequence marker, based on the presence of a fixed adjacent sequence marker in that AAV vector genome sequence, includes locating the fixed adjacent sequence marker in the AAV vector genome sequence and extracting a sequence region from the fixed adjacent sequence marker in the 5' to 3' direction relative to the orientation of the fixed adjacent sequence marker.

[0414] Embodiment 82 is characterized in that, based on the presence of a fixed adjacent sequence marker in the AAV vector genome sequence, extracting a sequence region immediately to the left or right of a fixed adjacent sequence marker from each AAV vector genome sequence of a plurality of AAV vectors is performed, and this includes locating the fixed adjacent sequence marker in the AAV vector genome sequence and extracting a sequence region from the fixed adjacent sequence marker in the 3' to 5' direction relative to the orientation of the fixed adjacent sequence marker.

[0415] Embodiment 83 is characterized in that the subject matter of Embodiments 77 to 82 is further characterized in that, from each AAV vector genome sequence of a plurality of AAV vectors, the extraction of a sequence region immediately to the left or immediately to the right of a fixed adjacent sequence marker, based on the presence of a fixed adjacent sequence marker in that AAV vector genome sequence, includes locating a plurality of fixed adjacent sequence markers in the AAV vector genome sequence and extracting a sequence region between a plurality of fixed adjacent sequence markers.

[0416] Embodiment 84 is characterized in that, for each of the multiple array regions, the subject matter of Embodiments 77 to 83 includes determining the length of the array region, and excluding the array region from the clustering step if its length exceeds a first threshold or falls below a second threshold.

[0417] Embodiment 85 is characterized in that the subject of Embodiment 84 has a first threshold of approximately 700 base pairs in length and a second threshold of approximately 150 base pairs in length.

[0418] Embodiment 86 is characterized in that the subject matter of Embodiments 77 to 85 includes generating an ITR genotype database based on sequence regions associated with clusters containing two or more sequence regions, and producing a recombinant AAV vector based on the ITR genotype database.

[0419] In Embodiment 87, the subject matter of Embodiments 77 to 86 is characterized in that the generation of multiple clusters by clustering two or more sequence regions from among multiple sequence regions is based on complete sequence identity.

[0420] In Embodiment 88, the subject matter of Embodiments 77 to 87 is characterized in that the generation of multiple clusters by clustering two or more sequence regions from among multiple sequence regions is based on at least 99% sequence identity.

[0421] In Embodiment 89, the subject matter of Embodiments 77 to 88 is characterized in that the generation of multiple clusters by clustering two or more sequence regions from among multiple sequence regions is based on at least 98% sequence identity.

[0422] Embodiment 90 is characterized in that the subject matter of Embodiments 77 to 89 is such that merging two or more clusters from a plurality of clusters includes iteratively merging two or more clusters from a plurality of clusters into one or more modified clusters.

[0423] Embodiment 91 is characterized in that the subject of Embodiment 90 is characterized in that iterative merging of two or more clusters among a plurality of clusters is based on aligning representative sequence regions from each cluster using a different sequence identity for each iteration.

[0424] In Embodiment 92, the subject matter of Embodiments 90-91 is characterized in that two or more clusters from among multiple clusters are merged iteratively until a predetermined number of clusters are generated.

[0425] Embodiment 93 is characterized in that the subject of Embodiment 92 has a predetermined number of clusters, which is 1.

[0426] Embodiment 94 is characterized in that the subject matter of Embodiments 77 to 93 is the same as that of the candidate ITR sequence, with the genotype being identical to that of the wild-type AAV ITR sequence or the manipulated AAV ITR sequence.

[0427] Embodiment 95 is characterized in that the subject matter of Embodiments 77-94 is used to produce additional AAV vectors using the same plasmids that were used to produce multiple AAV vectors, based on the genotype of a candidate ITR sequence that is identical to a wild-type AAV ITR sequence or a manipulated AAV ITR sequence.

[0428] Embodiment 96 is characterized in that the subject of Embodiment 95 is packaged for distribution based on the genotype of a candidate ITR sequence that is identical to a wild-type ITR sequence or an engineered ITR sequence.

[0429] Embodiment 97 is characterized in that the subject matter of Embodiments 95-96 is administered to a human subject in a therapeutically effective amount of a manufactured recombinant AAV vector.

[0430] Embodiment 98 is a method for treating a subject in need of treatment, comprising administering to the subject a therapeutically effective amount of an AAV vector comprising a vector genome encapsulated by an adeno-associated virus (AAV) capsid, wherein the AAV genome comprises at least two AAV inverted terminal repeats (ITRs), nucleic acid sequences encoding a therapeutic agent, and the genotypes of at least two AAV ITRs are identical to a reference AAV ITR determined based on the method of Embodiment 77.

[0431] Embodiment 99 is a computer-readable medium having a processor-executable instruction embodiment configured to cause a device to execute any of Embodiments 1 to 98.

[0432] Embodiment 100 is a device configured to implement any of Embodiments 1 to 98.

[0433] Embodiment 101 is a system for implementing any of Embodiments 1 to 98.

[0434] While specific configurations have been described, the configurations described herein are intended to be all possible, not limiting, and are not intended to restrict this scope to the specific configurations described.

[0435] Unless otherwise expressly stated, no method described herein is intended to be construed as requiring its steps to be performed in a specific order. Therefore, if the order in which the steps are performed is not actually listed in the claims of a method, or unless it is otherwise specifically stated in the claims or description that the steps should be limited to a specific order, no order is ever intended to be inferred. This also applies to any possible implicit grounds for interpretation, including logical matters relating to the arrangement of steps or operational flows, obvious meanings arising from grammatical structure or punctuation, and the number or type of configurations described in the specification.

[0436] It will be apparent to those skilled in the art that various modifications and variations can be made without departing from the scope or spirit. Other configurations will be apparent to those skilled in the art from the considerations herein and the practices described herein. The specification and the configurations described herein are for illustrative purposes only, and the true scope and spirit are intended to be indicated by the following claims.

Claims

1. A method of manufacturing, This involves sequencing the genomes of multiple plasmids to obtain multiple plasmid genome sequences, Receiving the specifications for fixed adjacent array markers, From each plasmid genome sequence, based on the presence of the fixed adjacent sequence marker in the plasmid genome sequence, a plurality of sequence regions are extracted, each of which is located within the fixed adjacent sequence marker and contains a candidate inverted end repeat (ITR) sequence. Based on complete sequence identity, clustering two or more sequence regions from the aforementioned plurality of sequence regions generates multiple clusters, Based on the alignment between those corresponding sequence regions, two or more clusters from the aforementioned clusters are merged, If a single cluster remains, the genotype of the candidate ITR sequence of the single cluster is identified based on local alignment, A method comprising: producing a plurality of AAV vectors using a plasmid having an ITR sequence having the genotype of the candidate ITR sequence, based on the genotype of the candidate ITR sequence.

2. The method according to claim 1, wherein sequencing of the plurality of plasmids is performed via long-read sequencing or circular consensus sequencing.

3. The method according to claim 1, wherein the fixed adjacent sequence marker includes a sequence of at least 15 nucleotides.

4. The method according to claim 1, wherein the fixed adjacent sequence marker comprises at least a portion of the transgene delivered by the AAV vector.

5. Extracting the aforementioned multiple sequence regions is In the plasmid genome sequence, the fixed adjacent sequence marker is located, The method according to claim 1, comprising extracting the sequence region from the fixed adjacent sequence marker in the direction from 5' to 3' with respect to the orientation of the fixed adjacent sequence marker.

6. Extracting the aforementioned multiple sequence regions is In the plasmid genome sequence, the fixed adjacent sequence marker is located, The method according to claim 1, comprising extracting the sequence region from the fixed adjacent sequence marker in the direction from 3' to 5' with respect to the orientation of the fixed adjacent sequence marker.

7. Extracting the aforementioned multiple sequence regions is In the plasmid genome sequence, the positioning of multiple fixed adjacent sequence markers is performed, The method according to claim 1, comprising extracting the sequence region between the plurality of fixed adjacent sequence markers.

8. For each of the aforementioned plurality of array regions, Determining the length of that array region, The method according to claim 1, further comprising excluding the sequence region from the clustering step if its length exceeds a first threshold or falls below a second threshold.

9. The method according to claim 8, wherein the first threshold is approximately 700 base pairs long, and the second threshold is approximately 150 base pairs long.

10. To generate an ITR genotype database based on sequence regions associated with clusters containing two or more sequence regions, The method according to claim 1, further comprising producing a recombinant AAV vector based on the ITR genotype database.

11. The method according to claim 1, wherein merging two or more clusters from the plurality of clusters includes iteratively merging two or more clusters from the plurality of clusters into one or more modified clusters.

12. The method according to claim 11, wherein iteratively merging two or more clusters among the plurality of clusters is based on aligning representative sequence regions from each cluster using a different sequence identity for each iteration.

13. The method according to claim 11, wherein two or more clusters from the plurality of clusters are merged iteratively until a predetermined number of clusters are generated.

14. The method according to claim 13, wherein the predetermined number of clusters is 1.

15. The method according to claim 1, wherein the genotype of the candidate ITR sequence is the same as that of a wild-type ITR sequence or a manipulated ITR sequence.

16. The method according to claim 1, further comprising packaging the plurality of AAV vectors for distribution based on the genotype of the candidate ITR sequence which is identical to the wild-type ITR sequence or the manipulated ITR sequence.

17. The method according to claim 1, further comprising administering a therapeutically effective amount of a manufactured recombinant AAV vector to a human subject.

18. It is a method, This involves sequencing the genomes of multiple plasmids to obtain multiple plasmid genome sequences, Receiving the specifications for fixed adjacent array markers, From each plasmid genome sequence, based on the presence of the fixed adjacent sequence marker in that plasmid genome sequence, multiple sequence regions are extracted, each of which is located within the fixed adjacent sequence marker and contains a candidate inverted end repeat (ITR) sequence. Based on sequence identity, clustering two or more sequence regions from the aforementioned plurality of sequence regions generates multiple clusters, Based on the alignment between those corresponding sequence regions, two or more clusters from the aforementioned clusters are merged, A method comprising determining that the plurality of plasmids are unsuitable for the production of recombinant vector genomes based on two or more clusters remaining after the merge.

19. The method according to claim 18, wherein sequencing of the plurality of plasmids includes sequencing via long-read sequencing or circular consensus sequencing.

20. The method according to claim 18, wherein the fixed adjacent sequence marker comprises a sequence of at least 15 nucleotides.

21. The method according to claim 18, wherein the fixed adjacent sequence marker comprises at least a portion of the transgene delivered by the AAV vector.

22. Extracting the aforementioned multiple sequence regions is In the plasmid genome sequence, the fixed adjacent sequence marker is located, The method according to claim 18, comprising extracting the sequence region from the fixed adjacent sequence marker in the direction from 5' to 3' with respect to the orientation of the fixed adjacent sequence marker.

23. Extracting the aforementioned multiple sequence regions is In the plasmid genome sequence, the fixed adjacent sequence marker is located, The method according to claim 18, comprising extracting the sequence region from the fixed adjacent sequence marker in the direction from 3' to 5' with respect to the orientation of the fixed adjacent sequence marker.

24. Extracting the aforementioned multiple sequence regions is In the plasmid genome sequence, the positioning of multiple fixed adjacent sequence markers is performed, The method according to claim 18, comprising extracting the sequence region between the plurality of fixed adjacent sequence markers.

25. For each of the aforementioned plurality of array regions, Determining the length of that array region, The method according to claim 18, further comprising excluding the sequence region from the clustering step if its length exceeds a first threshold or falls below a second threshold.

26. The method according to claim 25, wherein the first threshold is approximately 700 base pairs long, and the second threshold is approximately 150 base pairs long.

27. To generate an ITR genotype database based on sequence regions associated with clusters containing two or more sequence regions, The method according to claim 18, further comprising producing a recombinant AAV vector based on the ITR genotype database.

28. The method according to claim 18, wherein clustering two or more sequence regions from the aforementioned plurality of sequence regions generates a plurality of clusters, wherein the method is based on complete sequence identity.

29. The method according to claim 18, wherein two or more sequence regions from the aforementioned plurality of sequence regions are clustered to generate a plurality of clusters, the method being based on at least 99% sequence identity.

30. The method according to claim 18, wherein two or more sequence regions from the aforementioned plurality of sequence regions are clustered to generate a plurality of clusters, the method being based on at least 98% sequence identity.

31. The method according to claim 18, wherein merging two or more clusters from the plurality of clusters includes iteratively merging two or more clusters from the plurality of clusters into one or more modified clusters.

32. The method according to claim 31, wherein iteratively merging two or more clusters among the plurality of clusters is based on aligning representative sequence regions from each cluster using a different sequence identity for each iteration.

33. The method according to claim 31, wherein two or more clusters from the plurality of clusters are merged iteratively until a predetermined number of clusters are generated.

34. The method according to claim 33, wherein the predetermined number of clusters is 1.

35. The method according to claim 18, wherein the genotype of the candidate ITR sequence is the same as that of a wild-type ITR sequence or a manipulated ITR sequence.

36. The method according to claim 18, further comprising discarding any plasmids associated with the plurality of plasmids.

37. It is a method, This involves sequencing the genomes of multiple plasmids to obtain multiple plasmid genome sequences, Receiving the specifications for fixed adjacent array markers, From each plasmid genome sequence, based on the presence of the fixed adjacent sequence marker in that plasmid genome sequence, multiple sequence regions are extracted, each of which is located within the fixed adjacent sequence marker and contains a candidate inverted end repeat (ITR) sequence. Based on sequence identity, clustering two or more sequence regions from the aforementioned plurality of sequence regions generates multiple clusters, Based on the alignment between those corresponding sequence regions, two or more clusters from the aforementioned clusters are merged, A method comprising, if a single cluster remains after the merge, identifying the genotype of a representative sequence region of the single cluster based on local alignment.

38. The method according to claim 37, wherein sequencing of the plurality of plasmids includes sequencing via long-read sequencing or circular consensus sequencing.

39. The method according to claim 37, wherein the fixed adjacent sequence marker comprises a sequence of at least 15 nucleotides.

40. The method according to claim 37, wherein the fixed adjacent sequence marker comprises at least a portion of the transgene delivered by the AAV vector.

41. Extracting the aforementioned multiple sequence regions is In the plasmid genome sequence, the fixed adjacent sequence marker is located, The method according to claim 37, comprising extracting the sequence region from the fixed adjacent sequence marker in the direction from 5' to 3' with respect to the orientation of the fixed adjacent sequence marker.

42. Extracting the aforementioned multiple sequence regions is In the plasmid genome sequence, the fixed adjacent sequence marker is located, The method according to claim 37, comprising extracting the sequence region from the fixed adjacent sequence marker in the direction from 3' to 5' with respect to the orientation of the fixed adjacent sequence marker.

43. Extracting the aforementioned multiple sequence regions is In the plasmid genome sequence, the positioning of multiple fixed adjacent sequence markers is performed, The method according to claim 37, comprising extracting the sequence region between the plurality of fixed adjacent sequence markers.

44. For each of the aforementioned plurality of array regions, Determining the length of that array region, The method according to claim 37, further comprising excluding the sequence region from the clustering step if its length exceeds a first threshold or falls below a second threshold.

45. The method according to claim 44, wherein the first threshold is approximately 700 base pairs long, and the second threshold is approximately 150 base pairs long.

46. To generate an ITR genotype database based on sequence regions associated with clusters containing two or more sequence regions, The method according to claim 37, further comprising producing a recombinant AAV vector based on the ITR genotype database.

47. The method according to claim 37, wherein clustering two or more sequence regions from the aforementioned plurality of sequence regions generates a plurality of clusters, based on complete sequence identity.

48. The method according to claim 37, wherein two or more sequence regions from the aforementioned plurality of sequence regions are clustered to generate a plurality of clusters, the method being based on at least 99% sequence identity.

49. The method according to claim 37, wherein two or more sequence regions from the aforementioned plurality of sequence regions are clustered to generate a plurality of clusters, the method being based on at least 98% sequence identity.

50. The method according to claim 37, wherein merging two or more clusters from the plurality of clusters includes iteratively merging two or more clusters from the plurality of clusters into one or more modified clusters.

51. The method according to claim 50, wherein iteratively merging two or more clusters among the plurality of clusters is based on aligning representative sequence regions from each cluster using a different sequence identity for each iteration.

52. The method according to claim 50, wherein two or more of the aforementioned clusters are iteratively merged until a predetermined number of clusters are generated.

53. The method according to claim 52, wherein the predetermined number of clusters is 1.

54. The method according to claim 37, wherein the genotype of the candidate ITR sequence is the same as that of a wild-type ITR sequence or a manipulated ITR sequence.

55. The method according to claim 37, further comprising producing a plurality of AAV vectors using a plasmid having an ITR sequence having the genotype of the candidate ITR sequence, based on the genotype of the candidate ITR sequence being identical to a wild-type ITR sequence or an engineered ITR sequence.

56. The method of claim 55, further comprising packaging the plurality of AAV vectors for distribution based on the genotype of the candidate ITR sequence which is identical to the wild-type ITR sequence or the manipulated ITR sequence.

57. The method according to claim 55, further comprising administering a therapeutically effective amount of a manufactured recombinant AAV vector to a human subject.

58. It is a method, This involves sequencing the AAV vector genomes from multiple adeno-associated virus (AAV) vectors to obtain multiple AAV vector genome sequences, and Receiving the specifications for fixed adjacent array markers, From each of the AAV vector genome sequences of the plurality of AAV vectors, based on the presence of the fixed adjacent sequence marker in that AAV vector genome sequence, a sequence region immediately to the left or immediately to the right of the fixed adjacent sequence marker, wherein the sequence region contains a candidate ITR sequence, Based on complete sequence identity, clustering two or more sequence regions from the aforementioned plurality of sequence regions generates multiple clusters, Based on the alignment between those corresponding sequence regions, two or more clusters from the aforementioned clusters are merged, A method comprising considering the plurality of AAV vector genomes unsuitable based on two or more clusters remaining after the merge.

59. The method according to claim 58, wherein sequencing of multiple AAV genomes includes sequencing via long-read sequencing or circular consensus sequencing.

60. The method according to claim 58, wherein the fixed adjacent sequence marker comprises a sequence of at least 15 nucleotides.

61. The method according to claim 58, wherein the fixed adjacent sequence marker comprises at least a portion of the transgene delivered by the plurality of AAV vectors.

62. From each of the AAV vector genome sequences of the plurality of AAV vectors, based on the presence of the fixed adjacent sequence marker in that AAV vector genome sequence, the sequence region immediately to the left or immediately to the right of the fixed adjacent sequence marker is extracted. In the AAV vector genome sequence, the fixed adjacent sequence marker is located, The method according to claim 58, comprising extracting the sequence region from the fixed adjacent sequence marker in the direction from 5' to 3' with respect to the orientation of the fixed adjacent sequence marker.

63. From each of the AAV vector genome sequences of the plurality of AAV vectors, based on the presence of the fixed adjacent sequence marker in that AAV vector genome sequence, the sequence region immediately to the left or immediately to the right of the fixed adjacent sequence marker is extracted. In the AAV vector genome sequence, the fixed adjacent sequence marker is located, The method according to claim 58, comprising extracting the sequence region from the fixed adjacent sequence marker in the direction from 3' to 5' with respect to the orientation of the fixed adjacent sequence marker.

64. From each of the AAV vector genome sequences of the plurality of AAV vectors, based on the presence of the fixed adjacent sequence marker in that AAV vector genome sequence, the sequence region immediately to the left or immediately to the right of the fixed adjacent sequence marker is extracted. In the AAV vector genome sequence, the positioning of multiple fixed adjacent sequence markers is performed, The method according to claim 58, comprising extracting the sequence region between the plurality of fixed adjacent sequence markers.

65. For each of the aforementioned plurality of array regions, Determining the length of that array region, The method according to claim 58, further comprising excluding the sequence region from the clustering step if its length exceeds a first threshold or falls below a second threshold.

66. The method according to claim 65, wherein the first threshold is approximately 700 base pairs long, and the second threshold is approximately 150 base pairs long.

67. The method according to claim 58, further comprising generating a database of AAV ITR genotypes based on sequence regions associated with clusters containing two or more sequence regions.

68. The method according to claim 58, wherein clustering two or more sequence regions from the aforementioned plurality of sequence regions generates a plurality of clusters, wherein the method is based on complete sequence identity.

69. The method according to claim 58, wherein two or more sequence regions from the aforementioned plurality of sequence regions are clustered to generate a plurality of clusters, wherein the clusters are based on at least 99% sequence identity.

70. The method according to claim 58, wherein two or more sequence regions from the aforementioned plurality of sequence regions are clustered to generate a plurality of clusters, the method being based on at least 98% sequence identity.

71. The method according to claim 58, wherein merging two or more clusters from the plurality of clusters includes iteratively merging two or more clusters from the plurality of clusters into one or more modified clusters.

72. The method according to claim 71, wherein iteratively merging two or more clusters among the plurality of clusters is based on aligning representative sequence regions from each cluster using a different sequence identity for each iteration.

73. The method according to claim 71, wherein two or more of the aforementioned clusters are iteratively merged until a predetermined number of clusters are generated.

74. The method according to claim 73, wherein the predetermined number of clusters is 1.

75. The method according to claim 58, wherein the genotype of the candidate ITR sequence is the same as that of a wild-type AAV ITR sequence or a manipulated AAV ITR sequence.

76. The method according to claim 58, further comprising discarding any AAV vectors associated with the plurality of AAV vectors.

77. It is a method, This involves sequencing multiple adeno-associated virus (AAV) vector genomes to obtain multiple AAV vector genome sequences, and Receiving the specifications for fixed adjacent array markers, From each of the AAV vector genome sequences of the plurality of AAV vectors, based on the presence of the fixed adjacent sequence marker in that AAV vector genome sequence, a sequence region immediately to the left or immediately to the right of the fixed adjacent sequence marker, wherein the sequence region contains a candidate ITR sequence, Based on complete sequence identity, clustering two or more sequence regions from the aforementioned plurality of sequence regions generates multiple clusters, Based on the alignment between those corresponding sequence regions, two or more clusters from the aforementioned clusters are merged to generate a modified set of clusters. A method comprising, if a single cluster remains after the merge, identifying the genotype of a representative sequence region of the single cluster based on local alignment.

78. The method according to claim 77, wherein sequencing of the plurality of AAV vector genomes includes sequencing via long-read sequencing or circular consensus sequencing.

79. The method according to claim 77, wherein the fixed adjacent sequence marker comprises a sequence of at least 15 nucleotides.

80. The method according to claim 79, wherein the fixed adjacent sequence marker comprises at least a portion of the transgene delivered by the plurality of AAV vectors.

81. From each of the AAV vector genome sequences of the plurality of AAV vectors, based on the presence of the fixed adjacent sequence marker in that AAV vector genome sequence, the sequence region immediately to the left or immediately to the right of the fixed adjacent sequence marker is extracted. In the AAV vector genome sequence, the fixed adjacent sequence marker is located, The method according to claim 77, comprising extracting the sequence region from the fixed adjacent sequence marker in the direction from 5' to 3' with respect to the orientation of the fixed adjacent sequence marker.

82. From each of the AAV vector genome sequences of the plurality of AAV vectors, based on the presence of the fixed adjacent sequence marker in that AAV vector genome sequence, the sequence region immediately to the left or immediately to the right of the fixed adjacent sequence marker is extracted. In the AAV vector genome sequence, the fixed adjacent sequence marker is located, The method according to claim 77, comprising extracting the sequence region from the fixed adjacent sequence marker in the direction from 3' to 5' with respect to the orientation of the fixed adjacent sequence marker.

83. From each of the AAV vector genome sequences of the plurality of AAV vectors, based on the presence of the fixed adjacent sequence marker in that AAV vector genome sequence, the sequence region immediately to the left or immediately to the right of the fixed adjacent sequence marker is extracted. In the AAV vector genome sequence, the positioning of multiple fixed adjacent sequence markers is performed, The method according to claim 77, comprising extracting the sequence region between the plurality of fixed adjacent sequence markers.

84. For each of the aforementioned plurality of array regions, Determining the length of that array region, The method according to claim 77, further comprising excluding the sequence region from the clustering step if its length exceeds a first threshold or falls below a second threshold.

85. The method according to claim 84, wherein the first threshold is approximately 700 base pairs long, and the second threshold is approximately 150 base pairs long.

86. To generate an ITR genotype database based on sequence regions associated with clusters containing two or more sequence regions, The method according to claim 77, further comprising producing a recombinant AAV vector based on the ITR genotype database.

87. The method according to claim 77, wherein clustering two or more sequence regions from the aforementioned plurality of sequence regions generates a plurality of clusters, wherein the method is based on complete sequence identity.

88. The method according to claim 77, wherein clustering two or more sequence regions from the aforementioned plurality of sequence regions to generate a plurality of clusters is based on at least 99% sequence identity.

89. The method according to claim 77, wherein two or more sequence regions from the aforementioned plurality of sequence regions are clustered to generate a plurality of clusters, the method being based on at least 98% sequence identity.

90. The method according to claim 77, wherein merging two or more clusters from the plurality of clusters includes iteratively merging two or more clusters from the plurality of clusters into one or more modified clusters.

91. The method according to claim 90, wherein iteratively merging two or more clusters among the plurality of clusters is based on aligning representative sequence regions from each cluster using a different sequence identity for each iteration.

92. The method according to claim 90, wherein two or more of the aforementioned clusters are iteratively merged until a predetermined number of clusters are generated.

93. The method according to claim 92, wherein the predetermined number of clusters is 1.

94. The method according to claim 77, wherein the genotype of the candidate ITR sequence is the same as that of a wild-type AAV ITR sequence or a manipulated AAV ITR sequence.

95. The method according to claim 77, further comprising producing additional AAV vectors using the same plasmid used to produce the plurality of AAV vectors, based on the genotype of the candidate ITR sequence which is identical to the wild-type AAV ITR sequence or the engineered AAV ITR sequence.

96. The method of claim 95, further comprising packaging the plurality of AAV vectors for distribution based on the genotype of the candidate ITR sequence which is identical to the wild-type ITR sequence or the manipulated ITR sequence.

97. The method according to claim 95, further comprising administering a therapeutically effective amount of a manufactured recombinant AAV vector to a human subject.

98. A method of treating a subject that requires treatment, The aforementioned subjects are administered a therapeutically effective dose of an adeno-associated virus (AAV) vector containing a vector genome encapsulated by an AAV capsid. The AAV genome comprises at least two AAV inverted terminal repeats (ITRs), nucleic acid sequences encoding therapeutic agents, A method wherein the genotypes of at least two AAV ITRs are identical to a reference AAV ITR determined according to the method of claim 77.

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