CN117043347A

CN117043347A - Production and use of recombinant AAV vectors

Info

Publication number: CN117043347A
Application number: CN202280024491.6A
Authority: CN
Inventors: 马提亚·查尔斯·杰罗姆·赫本; 廖晶; 卡门·吴; 威尔哈德·汉斯·路特; 托马斯·马修·爱德华兹
Original assignee: Logic Biotherapy Co
Current assignee: Logic Biotherapy Co
Priority date: 2021-02-26
Filing date: 2022-02-25
Publication date: 2023-11-10

Abstract

Provided herein are techniques and methods for improving AAV vector production.

Description

Production and use of recombinant AAV vectors

Cross reference to related applications

The present application claims priority from U.S. provisional application Ser. No. 63/154,474, filed on month 26 of 2021, ser. No. 63/234,610, filed on month 8 of 2021, and Ser. No. 63/257,036, filed on month 10 of 2021, each of which is incorporated herein by reference in its entirety.

Background

Genetic diseases caused by dysfunctional genes account for a significant portion of the world's diseases. Gene therapy is becoming a promising form of treatment aimed at alleviating the effects of genetic diseases.

Disclosure of Invention

The present disclosure provides methods and techniques for improving the design and/or production of viral vectors, including AAV vectors. According to various embodiments, the present disclosure provides the following insights: certain design elements and/or transfection conditions of an expression construct (e.g., a plasmid) may significantly affect one or more characteristics and/or features of viral (e.g., AAV) production (including, for example, one or more of viral vector yield, packaging efficiency, and/or replication competent AAV levels).

The present disclosure demonstrates, among other things, that a two-plasmid transfection system with a specific combination of sequence elements (e.g., rep genes or gene variants, cap genes or gene variants, one or more helper virus genes or gene variants, and/or one or more genes of interest) can effectively enhance downstream production, particularly viral vectors for gene therapy. For example, in some embodiments, the present disclosure provides the following insight: a two-plasmid transfection system with a specific combination of wild-type sequence elements (e.g., rep genes or gene variants, one or more helper virus genes or gene variants, one or more viral promoters) may be effective in enhancing viral vector production.

In some embodiments, the present disclosure demonstrates that a dual plasmid transfection system with a combination of specific sequence elements can be combined with a variety of transfection reagents (e.g., chemical transfection reagents, including lipids, polymers, and cationic molecules [ e.g., one or more cationic lipids ]) can be effective in enhancing viral vector production.

In some embodiments, the present disclosure provides the following insight: optimizing the plasmid ratio in a two-plasmid system may further improve production of one or more aspects of a viral vector, e.g., an AAV vector (including, e.g., one or more of viral vector yield, packaging efficiency, and/or replication competent AAV levels). Without wishing to be bound by any particular theory, the present disclosure demonstrates that transfection with a two plasmid system comprising a first plasmid having a viral accessory gene (e.g., an adenovirus gene or a herpes virus gene) and an AAV rep gene or an AAV cap gene, and a second plasmid having a payload and an AAV rep gene or an AAV cap gene, can produce increased viral vector yield relative to a reference. In some embodiments, the disclosure demonstrates that specific transfection ratios comprising a large number of first plasmids with helper viral genes can result in increased viral vector yield and packaging efficiency relative to a reference as compared to a second plasmid with a payload.

In some embodiments, the disclosure provides plasmids comprising at least one of a polynucleotide sequence encoding an AAV cap gene, a polynucleotide sequence encoding an AAV rep gene, a polynucleotide sequence encoding a payload and flanking ITRs, and/or a polynucleotide sequence encoding one or more viral accessory genes. In some embodiments, the provided plasmids also include polynucleotide sequences encoding a promoter, such as a native p5 promoter, a native p40 promoter, a CMV promoter, and/or one or more wild-type promoters. In some embodiments, the provided plasmid further comprises a polyA sequence. In some embodiments, the provided plasmid further comprises an intron, such as an intron between the promoter and the AAV rep gene. In some embodiments, the provided plasmid further comprises a polynucleotide sequence encoding a wild-type viral accessory gene. In some embodiments, the provided plasmids further comprise a transgene, such as one or more of propionyl-coa carboxylase, ATP7B, factor IX, methylmalonyl-coa Mutase (MUT), alpha 1-antitrypsin (A1 AT), UGT1A1, or variants thereof. In some embodiments, the plasmids provided do not include a polynucleotide sequence encoding a nuclease.

In some embodiments, the first and second provided plasmids are present in a composition, wherein each plasmid comprises a different sequence element (e.g., a polynucleotide sequence encoding an AAV cap gene, a polynucleotide sequence encoding an AAV rep gene, a polynucleotide sequence encoding a payload and flanking ITRs, and/or a polynucleotide sequence encoding one or more viral accessory genes). In some embodiments, provided compositions include a first plasmid comprising a polynucleotide sequence encoding an AAV cap gene and a second plasmid comprising a polynucleotide sequence encoding an AAV rep gene. In some embodiments, provided compositions include a first plasmid comprising a polynucleotide sequence encoding a payload and flanking ITRs and a second plasmid comprising a polynucleotide sequence encoding one or more viral accessory genes. In some embodiments, provided compositions include a first plasmid comprising a polynucleotide sequence encoding one or more viral accessory genes and a second plasmid comprising a polynucleotide sequence encoding a payload and flanking ITRs. In some embodiments, the provided compositions are formulated for co-delivery of the first and second plasmids to the cells. In some embodiments, compositions are provided that include specific ratios of first and second plasmids to achieve specific ratios between the two plasmids. In some embodiments, the provided compositions include a greater amount of the first plasmid relative to the second plasmid. In some embodiments, compositions are provided comprising a first and a second plasmid, wherein the ratio of the first plasmid to the second plasmid is greater than or equal to 1.5:1 up to 10:1. In some embodiments, provided compositions include a first plasmid comprising a polynucleotide sequence encoding one or more viral accessory genes and a second plasmid comprising a polynucleotide sequence encoding a payload and flanking ITRs. In some embodiments, provided compositions include a first plasmid comprising polynucleotide sequences encoding one or more viral helper genes and rep genes and a second plasmid comprising polynucleotide sequences encoding a payload and flanking ITR and cap genes. In some embodiments, provided compositions include a first plasmid comprising polynucleotide sequences encoding one or more viral accessory genes and cap genes and a second plasmid comprising polynucleotide sequences encoding a payload and flanking ITR and rep genes.

In some embodiments, provided compositions include one or more of the following: polynucleotide sequences encoding one or more enhancer sequences, polynucleotide sequences encoding one or more promoter sequences, polynucleotide sequences encoding one or more intron sequences, polynucleotide sequences encoding genes, and polynucleotide sequences comprising polyA sequences. In some embodiments, provided are polynucleotide sequences encoding a payload comprising a polynucleotide sequence comprising a first nucleic acid sequence and a second nucleic acid sequence, wherein the first nucleic acid sequence comprises at least one gene and the second nucleic acid sequence is located 5 'or 3' to the first nucleic acid sequence and facilitates production of two independent gene products upon integration to a target integration site, a third nucleic acid sequence located 5 'to the polynucleotide and comprising a sequence homologous to a genomic sequence located 5' to the target integration site, and a fourth nucleic acid sequence located 3 'to the polynucleotide and comprising a sequence homologous to a genomic sequence located 3' to the target integration site. In some embodiments, the provided integration site of interest comprises the 3' end of the endogenous gene. In some embodiments, the third nucleic acid sequence is provided homologous to a DNA sequence upstream of a stop codon in the endogenous gene. In some embodiments, the fourth nucleic acid sequence is provided homologous to DNA downstream of the stop codon in the endogenous gene. In some embodiments, the provided integration site of interest is located in the genome of the cell. In some embodiments, the provided integration site of interest is located in the genome of a hepatocyte, a myocyte, or a CNS cell.

In some embodiments, provided compositions include compositions for packaging AAV vectors. In some embodiments, the compositions provided are used in methods of making packaged AAV vectors. In some embodiments, the provided compositions are delivered to cells, including mammalian cells, hepatocytes, myocytes, CNS cells, or cells isolated from a subject. In some embodiments, the provided compositions are delivered to the cells by chemical transfection reagents (including cationic molecules and/or cationic lipids). In some embodiments, provided compositions include packaged AAV vector compositions. In some embodiments, provided compositions may be administered to a subject in need thereof in a method of treatment, including a subject suffering from or suspected of suffering from one or more of the following: propioni, wilson's Disease, hemophilia, krigler-Najjar syndrome (MMA), alpha-1 antitrypsin deficiency (A1 ATD), glycogen Storage Disease (GSD), duchenne's muscular dystrophy, limb-area muscular dystrophy, X-linked myotube myopathy, parkinson's Disease, mucopolysaccharidosis, hemophilia a, hemophilia B, or Hereditary Angioedema (HAE). In some embodiments, the provided compositions do not comprise a nuclease.

Drawings

FIG. 1 compares two-plasmid and three-plasmid systems for cell transfection. (A) The viral vector yields (vg/mL) produced by different double plasmid ratios compared to the three plasmid system are depicted. (B) The relative fold change in viral vector yield relative to the three plasmid system is depicted. The cap gene encodes AAV-DJ, and the gene of interest (GOI) is human factor IX flanked by murine albumin homology arms (mHA-hFIX).

FIG. 2 compares two-plasmid and three-plasmid systems for cell transfection. (A) The viral vector yields (vg/mL) produced by different double plasmid ratios compared to the three plasmid system are depicted. (B) The relative fold change in viral vector yield relative to the three plasmid system is depicted. The cap gene encodes AAV-DJ, and the gene of interest (GOI) is human factor IX flanked by murine albumin homology arms (mHA-hFIX).

FIG. 3 compares two-plasmid and three-plasmid systems for cell transfection. (A) The viral vector yields (vg/mL) produced by different double plasmid ratios compared to the three plasmid system are depicted. (B) The relative fold change in viral vector yield relative to the three plasmid system is depicted. The cap gene encodes AAV-DJ, and the gene of interest (GOI) is human factor IX flanked by murine albumin homology arms (mHA-hFIX).

FIG. 4 compares two-plasmid and three-plasmid systems for cell transfection. (A) The viral vector yields (vg/mL) produced by different double plasmid ratios compared to the three plasmid system are depicted. (B) The relative fold change in viral vector yield relative to the three plasmid system is depicted. The cap gene encodes multiple chimeric AAV serotypes (DJ, LK03, AAVC11.04, AAVC11.11, AAVC 11.12), and the gene of interest (GOI) is human factor IX flanked by murine albumin homology arms (mHA-hFIX).

FIG. 5 compares two-plasmid and three-plasmid systems for cell transfection. (A) The viral vector yields (vg/mL) produced by different double plasmid ratios compared to the three plasmid system are depicted. (B) The relative fold change in viral vector yield relative to the three plasmid system is depicted. The cap gene encodes multiple chimeric AAV serotypes (DJ, AAVC11.01, AAVC11.04, AAVC11.06, AAVC11.09, AAVC11.11, AAVC11.12, AAVC11.13, AAVC11.15, LK 03), and the gene of interest (GOI) is human factor IX under the control of a liver-specific promoter (LSP-hFIX).

FIG. 6 depicts viral vector yields (vg/mL) of two-plasmid (2P) and three-plasmid (3P) systems with different transfection reagents (PEIMAX and FectoVIR AAV) in different culture vessels (shake flask and bioreactor). (A) Viral vector yields of the two-plasmid and three-plasmid systems in shake flasks with human UGT1A1 or human factor IX (hFIX) payloads are depicted. (B) Viral vector production in AmBr250 bioreactor using peimx reagent compared to the two plasmid system using FectoVir-AAV reagent.

FIG. 7 depicts viral vector yield (vg/mL) of a two plasmid system of different plasmid ratios compared to a three plasmid system (3P) in adherent 293T cells grown in 12 well plates and transfected with a lipid transfection agent (Fugene HD). The cap gene encodes AAV-DJ, and the gene of interest (GOI) is human factor IX flanked by murine albumin homology arms (mHA-hFIX).

FIG. 8 depicts viral vector yield (vg/mL) of a two plasmid system at different plasmid ratios compared to a three plasmid system for a larger cell culture volume (> 1L). The cap gene encodes an AAV-DJ serotype, and the gene of interest (GOI) is human factor IX flanked by murine albumin homology arms (mHA-hFIX).

Figure 9 depicts in vivo efficacy of AAV vectors in wild type mice. AAV vectors were made using either a three-plasmid system (3P) or a two-plasmid system (2P) in different media (Expi 293 and F17). The cap gene encodes AAV-DJ, and the gene of interest (GOI) is human factor IX flanked by murine albumin homology arms (mHA-hFIX). Viral vectors were inoculated intravenously at a dose of 1E13 vg/kg. Seven weeks after dosing, in vivo expression levels of payload (FIX) and integration marker (ALB-2A) were quantified in mouse plasma. Protein expression levels are related to the percentage of FIX gene integrated into the albumin locus (DNA INT) and the level of fusion RNA consisting of albumin mRNA followed by FIX mRNA.

FIG. 10 depicts viral vector yields (vg/mL) for different combinations of genetic elements in a two-plasmid system as compared to a three-plasmid system. Different combinations include plasmids containing helper genes and Cap (helper/Cap), as compared to plasmids containing helper genes and Rep (helper/Rep). In addition, different combinations include plasmids containing a payload and a Rep (payload/Rep) or plasmids with a payload and a Rep followed by a polyA (payload/Rep-polyA), as compared to plasmids containing a payload and a Cap (payload/Cap). In this example, the cap gene was of LK03 serotype, and two different payloads (FIX and MMUT) were tested. Different ratios of helper plasmid to payload plasmid are presented.

FIG. 11 depicts viral vector yield (vg/mL) for a two-plasmid system containing additional introns between AAV p5 promoter and rep gene at different plasmid ratios compared to a two-plasmid system and a three-plasmid system without introns. (A) One 1.4kb intron and one 133bp intron were tested, the cap gene was LK03, and the payload was hFIX. (B) One 1.4kb intron and one 3.3kb intron were tested, the cap gene was LK03, and the payload was MMUT.

FIG. 12 depicts a schematic of helper plasmids for AAV production using the 3 plasmid system. Helper plasmids may contain several adenovirus genes, such as the E2A DNA Binding Protein (DBP) gene, E4 Open Reading Frame (ORF) 2, ORF3, ORF4 and ORF6/7. Plasmids may also contain elements necessary for bacterial culture, such as the colE1 origin of replication (ori) and an antibiotic resistance gene (e.g., kanamycin).

FIG. 13 depicts a schematic of payload plasmids for AAV production using a 3 plasmid system. The payload plasmid may contain AAV Inverted Terminal Repeats (ITRs) flanking the payload. As shown in the schematic, the payload may contain the human factor IX gene (human FIX) as the gene of interest, and the mouse albumin gene sequence (mouse HA) as the homology arm, located at the 5 'and 3' positions of the gene of interest. Peptide 2A is located between the 5' homology arm and the gene of interest to allow independent translation of the gene of interest. Plasmids may also contain elements necessary for bacterial culture, such as the colE1 origin of replication (ori) and an antibiotic resistance gene (e.g., kanamycin).

FIG. 14 depicts a schematic of rep-cap plasmids for AAV production using a 3 plasmid system. The helper plasmid may contain the rep gene and the cap gene in its native genomic tissue, e.g., the p5 promoter is located upstream of the rep gene and the p40 promoter is located upstream of the cap gene and in the coding sequence of the rep gene. The cap gene may encode a variety of AAV serotypes and synthetic variants. Plasmids may also contain elements necessary for bacterial culture, such as the colE1 origin of replication (ori) and an antibiotic resistance gene (e.g., kanamycin).

FIG. 15 depicts a schematic of payload-Cap plasmids for AAV production using the 2 plasmid system. The plasmid may contain AAV Inverted Terminal Repeats (ITRs) flanking the payload. The payload may contain the human factor IX gene (human FIX) as the gene of interest, and the mouse albumin gene sequence (mouse HA) as a homology arm, located at the 5 'and 3' positions of the gene of interest. Peptide 2A is located between the 5' homology arm and the gene of interest to allow independent translation of the gene of interest. In addition, the plasmid may contain an AAV cap gene downstream of the p40 promoter and upstream of the polyA. The cap gene may encode a variety of AAV serotypes and synthetic variants. Plasmids may also contain elements necessary for bacterial culture, such as the colE1 origin of replication (ori) and an antibiotic resistance gene (e.g., kanamycin).

FIG. 16 depicts a schematic of helper-Rep plasmids for AAV production using the 2 plasmid system. Plasmids may contain several adenovirus genes, such as the E2A DNA Binding Protein (DBP) gene, E4 Open Reading Frame (ORF) 2, ORF3, ORF4 and ORF6/7. In addition, the plasmid may contain an AAV rep gene downstream of the p5 promoter. Plasmids may also contain elements necessary for bacterial culture, such as the colE1 origin of replication (ori) and an antibiotic resistance gene (e.g., kanamycin).

FIG. 17 depicts a schematic diagram of helper-Rep-intron plasmids for AAV production using a 2 plasmid system. Plasmids may contain several adenovirus genes, such as the E2A DNA Binding Protein (DBP) gene, E4 Open Reading Frame (ORF) 2, ORF3, ORF4 and ORF6/7. In addition, the plasmid may contain an AAV rep gene downstream of the p5 promoter and intron. Introns may be selected from a variety of sizes, for example 1.4kb in the schematic. Plasmids may also contain elements necessary for bacterial culture, such as the colE1 origin of replication (ori) and an antibiotic resistance gene (e.g., kanamycin).

FIG. 18 depicts a schematic diagram of helper-Cap plasmids for AAV production using the 2 plasmid system. Plasmids may contain several adenovirus genes, such as the E2A DNA Binding Protein (DBP) gene, E4 Open Reading Frame (ORF) 2, ORF3, ORF4 and ORF6/7. In addition, the plasmid may contain an AAV cap gene downstream of the p40 promoter and upstream of the polyA. The cap gene may encode a variety of AAV serotypes and synthetic variants. Plasmids may also contain elements necessary for bacterial culture, such as the colE1 origin of replication (ori) and an antibiotic resistance gene (e.g., kanamycin).

FIG. 19 depicts a schematic of payload-Rep plasmid production of AAV using a 2 plasmid system. The plasmid may contain AAV Inverted Terminal Repeats (ITRs) flanking the payload. The payload may contain the human factor IX gene (human FIX) as the gene of interest, downstream of liver-specific promoters (LSPs) and introns, and upstream of polyA. In addition, the plasmid may contain an AAV rep gene downstream of the p5 promoter. Plasmids may also contain elements necessary for bacterial culture, such as the colE1 origin of replication (ori) and an antibiotic resistance gene (e.g., kanamycin).

FIG. 20 depicts SDS-PAGE gels measuring purity of AAV LK03 capsid Viral Proteins (VP) produced via a three plasmid system ("standard plasmid PEIMAX") and a two plasmid system ("novel plasmid FectoVIR-AAV").

FIG. 21 depicts viral titer levels (vg/mL) in crude lysates produced by HEK293F cells transfected with a three plasmid system and PEIMAX (3 P|PEIMAX), a three plasmid system and Fectovir-AAV (3 P|Fectovir-AAV), and a two plasmid system and Fectovir-AAV (LOGC|Fectovir-AAV). The residual level of plasmid DNA (rKan) was measured under each transfection condition. Cells were cultured in ambr250 bioreactor or 50L bioreactor apparatus.

Figure 22 depicts a first round of screening DOE analysis of different transfection conditions to determine which combination can produce the maximum viral titer at the lowest cost. The conditions tested were plasmid DNA amount, fectovir-AAV amount and HEK293F cell density. (A) measurement of viral titers under the indicated conditions using qPCR. (B) analysis of viral titres predictive model as DOE results. (C) Comparison of viral titers and cost predictions under different conditions. (D) specifying a graphical representation of the condition optimization.

Figure 23 depicts a quadratic optimized DOE analysis of different transfection conditions to determine which combination can produce the maximum viral titer at the lowest cost. The conditions tested were the amount of plasmid DNA and the amount of Fectovir-AAV. (A) measurement of viral titers under the indicated conditions using qPCR. (B) viral titer analysis as a predictive model for the second DOE. (C) comparison of predictions of viral titers under different conditions. (D) specifying a graphical representation of the condition optimization.

FIG. 24 depicts viral titer levels (vg/mL) in crude lysates produced by HEK293F cells transfected with a three-plasmid system and PEIMAX (3P+PEIMAX), a two-plasmid system and PEIMAX (2P+PEIMAX), a three-plasmid system and Fectovir-AAV (3P+Fectovir-AAV), and a two-plasmid system and Fectovir-AAV (2P+Fectovir-AAV). (B) The relative fold change in viral vector yield relative to the three plasmid system and PEIMAX (3p+peimax) is depicted. The cap gene encodes a variety of natural and chimeric AAV serotypes (AAV 2, AAV5, AAV6, AAV8, AAV9, DJ, LK03 and sL 65), and the gene of interest (GOI) is human factor IX under the control of a liver-specific promoter (LSP-hFIX).

FIG. 25 depicts viral vector yields (vg/mL) for three plasmid (3P) and two plasmid (2P) systems for cell transfection with different plasmid designs and adenovirus gene numbers.

FIG. 26 depicts a schematic diagram of helper (pXX 6) -Rep plasmids for AAV production using the 2 plasmid system. Plasmids may contain several adenovirus genes, such as the E2A DNA Binding Protein (DBP) gene, E4 Open Reading Frame (ORF) 2, ORF3, ORF4 and ORF6/7. In addition, the plasmid may contain an AAV rep gene downstream of the p5 promoter. Plasmids may also contain elements necessary for bacterial culture, such as the colE1 origin of replication (ori) and an antibiotic resistance gene (e.g., kanamycin).

FIG. 27 depicts a schematic of helper (pXX 6) -Rep-intron plasmids for AAV production using the 2 plasmid system. Plasmids may contain several adenovirus genes, such as the E2A DNA Binding Protein (DBP) gene, E4 Open Reading Frame (ORF) 2, ORF3, ORF4 and ORF6/7. In addition, the plasmid may contain an AAV rep gene downstream of the p5 promoter and intron. Introns may be selected from a variety of sizes, for example 1.4kb in the schematic. Plasmids may also contain elements necessary for bacterial culture, such as the colE1 origin of replication (ori) and an antibiotic resistance gene (e.g., kanamycin).

FIG. 28 depicts an exemplary set of steps for producing a viral vector, including upstream processes (e.g., using various expression systems), capture steps (e.g., affinity chromatography, IEX), refining steps (e.g., 1EX, ultracentrifugation), formulation steps (e.g., tangential flow filtration), and/or filling and finishing steps (e.g., sterile filtration and sterile filling). In some embodiments, the compositions and methods disclosed herein aim to improve one or more characteristics and/or features of viral (e.g., AAV) production (including, for example, one or more of viral vector yield, packaging efficiency, and/or replication competent AAV levels) via modification of one or more upstream processing steps.

FIG. 29 provides an exemplary expression construct including certain sequence features and combinations thereof. Exemplary viral vector products that can be produced with such expression construct combinations are also provided.

Definition of the definition

For easier understanding of the present invention, certain terms are first defined below. Additional definitions of the following terms and other terms are set forth throughout the specification. Publications and other references cited herein to describe the background of the invention and provide additional details concerning its practice are hereby incorporated by reference.

The article "a/an" is used herein to refer to one or more than one (i.e., at least one) of the grammatical object of the article. For example, "an element" refers to one element or more than one element.

About: the term "about" or "approximately," when used herein in reference to a value, refers to a value that is similar in context to the reference value. In general, those skilled in the art who are familiar with the context will understand the relative degree of variation that is covered by "about" in that context. For example, in some embodiments, the term "about" or "approximately" may encompass a range of values within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of a reference value.

The password is optimized: as used herein, the term "codon optimization" refers to the following process: the codons of a given gene are altered in a manner such that the polypeptide sequence encoded by the gene remains the same, while the altered codons improve the expression of the polypeptide sequence. For example, if a polypeptide belongs to a human protein sequence and is expressed in E.coli, expression will generally be improved if the DNA sequence is codon optimized to change the human codon to one that is more efficiently expressed in E.coli.

Combination therapy: as used herein, the term "combination therapy" refers to a clinical intervention in which a subject is simultaneously exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents). In some embodiments, two or more treatment regimens may be administered simultaneously. In some embodiments, two or more treatment regimens may be administered sequentially (e.g., the first regimen is administered prior to administering any dose of the second regimen). In some embodiments, the two or more treatment regimens are administered in an overlapping dosing regimen. In some embodiments, administration of the combination therapy may include administration of one or more therapeutic agents or forms to a subject receiving other agents or forms. In some embodiments, combination therapies do not necessarily require that the individual agents be administered together (or even must be administered simultaneously) in a single composition. In some embodiments, two or more therapeutic agents or forms of the combination therapy are administered separately to the subject, e.g., in separate compositions, via separate routes of administration (e.g., one agent is administered orally and the other agent is administered intravenously), and/or at different points in time. In some embodiments, two or more therapeutic agents may be administered together in a combination composition, or even in a combination compound (e.g., as part of a single chemical complex or covalent entity), via the same route of administration, and/or simultaneously.

The comparison is that: as used herein, the term "comparable" refers to two or more agents, entities, conditions, sets of conditions, etc., that may differ from one another, but are similar enough to allow comparison therebetween, so that one of ordinary skill in the art will understand that a conclusion may be reasonably drawn based on observed differences or similarities. In some embodiments, a comparable set of conditions, environment, individual, or population is characterized by a plurality of substantially identical features and one or a small number of different features. Those of ordinary skill in the art will understand what degree of identity is required in any given instance to render two or more such agents, entities, situations, sets of conditions, etc. to be considered comparable. For example, one of ordinary skill in the art will understand that when characterized by a sufficient number and type of substantially identical features, the environments, individuals, or groups of individuals are comparable to one another to ensure a reasonable conclusion as follows: differences in the results or observed phenomena obtained from or through different environments, individuals or populations are caused by or indicative of those changes characterized by the changes.

Comprising: a composition or method described herein as "comprising" one or more specified elements or steps is open ended, meaning that the specified elements or steps are necessary, but that other elements or steps may be added within the scope of the composition or method. To avoid redundancy, it is also to be understood that any composition or method described as "comprising" one or more specified elements or steps also describes a corresponding, more limited composition or method "consisting essentially of (consisting essentially of) the same specified elements or steps (or" consisting essentially of (consists essentially of) ") meaning that the composition or method includes the specified essential elements or steps and may also include additional elements or steps that do not materially affect the basic and novel characteristics of the composition or method. It will be further understood that any composition or method described herein as "comprising" or "consisting essentially of" one or more specified elements or steps also describes a corresponding, more limited, and closed composition or method "consisting of" (or "consisting of") the specified elements or steps, excluding any other unspecified elements or steps. Known or disclosed equivalents of any named essential elements or steps may be substituted for those elements or steps in any of the compositions or methods disclosed herein.

Corresponding to: as used herein, the term "corresponding to" may be used to designate the location/identity of a structural element in a compound or composition via comparison with an appropriate reference compound or composition. For example, in some embodiments, monomer residues in a polymer (e.g., amino acid residues in a polypeptide or nucleic acid residues in a polynucleotide) can be identified as "corresponding to" residues in an appropriate reference polymer. For example, it will be appreciated by one of ordinary skill in the art that for simplicity, residues in a polypeptide are typically specified using a canonical numbering system based on the reference related polypeptide, so for example, an amino acid "corresponding to" a residue at position 190 need not actually be the 190 th amino acid in a particular amino acid chain, but rather corresponds to a residue found at 190 in the reference polypeptide; one of ordinary skill in the art will readily understand how to identify "corresponding" amino acids. For example, one of skill in the art will recognize various sequence alignment strategies, including software programs such as BLAST, CS-BLAST, CUSASW++, DIAMOND, FASTA, GGSEARCH/GLSEARCH, genoogle, HMMER, HHpred/HHsearch, IDF, infernal, KLAST, USEARCH, parasail, PSI-BLAST, PSI-Search, scalaBLAST, sequilab, SAM, SSEARCH, SWAPHI, SWAPHI-LS, SWIMM, or SWIPE, which may be used, for example, to identify "corresponding" residues in polypeptides and/or nucleic acids according to the present disclosure.

Derivatives: as used herein, the term "derivative" refers to a structural analogue of a reference substance. That is, a "derivative" is a substance that has significant structural similarity to a reference substance, such as sharing a core or common structure, but also differs in some discrete manner. In some embodiments, the derivative is a substance that can be generated from a reference substance by chemical manipulation. In some embodiments, a derivative is a substance that can be produced via a synthetic process that performs substantially similar (e.g., shares multiple steps with) a process that produces a reference substance.

Engineering: in general, the term "engineering" refers to aspects manipulated by a human hand. For example, a polynucleotide is considered "engineered" when two or more sequences that are not joined together in the order described in nature are manually manipulated to join each other directly in the engineered polynucleotide. For example, in some embodiments of the invention, the engineered polynucleotide comprises a regulatory sequence that is found in nature to be operably associated with the first coding sequence but not the second coding sequence, by artificial ligation, and thus is operably associated with the second coding sequence. Similarly, a cell or organism is considered "engineered" if it is manipulated to alter its genetic information (e.g., introduce new genetic material that was not previously present, such as by transformation, mating, somatic hybridization, transfection, transduction, or other mechanisms, or the pre-existing genetic material is altered or removed, such as by substitution or deletion mutations, or by mating protocols). As is conventional and understood by those skilled in the art, the progeny of an engineered polynucleotide or cell are still generally referred to as "engineered" even if the actual manipulation was performed on a prior entity.

Excipient: as used herein, refers to non-therapeutic agents that may be included in a pharmaceutical composition, for example, to provide or aid in a desired consistency or stabilization. In some embodiments, suitable pharmaceutical excipients may include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.

Expression: as used herein, "expression" of a nucleic acid sequence refers to one or more of the following events: (1) Generating an RNA template from the DNA sequence (e.g., by transcription); (2) Processing of the RNA transcript (e.g., by splicing, editing, 5 'cap formation, and/or 3' end formation); (3) translating the RNA into a polypeptide or protein; and/or (4) post-translational modification of the polypeptide or protein.

Gene: as used herein, the term "gene" refers to a DNA sequence in a chromosome that encodes a gene product (e.g., an RNA product and/or a polypeptide product). In some embodiments, a gene comprises a coding sequence (e.g., a sequence encoding a particular gene product); in some embodiments, the gene comprises a non-coding sequence. In some particular embodiments, a gene may include coding (e.g., exons) and non-coding (e.g., introns) sequences. In some embodiments, a gene may include one or more regulatory elements (e.g., promoters, enhancers, silencers, termination signals) that, for example, can control or affect one or more aspects of gene expression (e.g., cell type specific expression, inducible expression).

Gene product or expression product: as used herein, the term "gene product" or "expression product" generally refers to RNA transcribed from a gene (pre-and/or post-processing) or a polypeptide encoded by RNA transcribed from a gene (pre-and/or post-modification).

Homology; as used herein, the term "homology" refers to the overall relatedness between polymeric molecules, e.g., between polypeptide molecules. In some embodiments, polymeric molecules, such as antibodies, are considered "homologous" to each other if they are at least 80%, 85%, 90%, 95%, or 99% identical in sequence. In some embodiments, polymer molecules are considered "homologous" to each other if their sequences are at least 80%, 85%, 90%, 95%, or 99% similar.

Identity: as used herein, the term "identity" refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. In some embodiments, polymeric molecules are considered "substantially identical" to each other if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. Calculating the percent identity of two nucleic acid or polypeptide sequences may be performed, for example, by aligning the two sequences for optimal comparison purposes (e.g., gaps may be introduced in one or both of the first and second sequences for optimal alignment, and non-identical sequences may be ignored for comparison purposes). In certain embodiments, the length of the sequences aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of the reference sequence. The nucleotides at the corresponding positions are then compared. When a position in a first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in a second sequence, then the molecules are identical at that position. Taking into account the number of gaps and the length of each gap, the percent identity between two sequences is a function of the number of identical positions shared by the sequences, which length needs to be introduced to achieve optimal alignment of the two sequences. Sequence comparison and determination of percent identity between two sequences can be accomplished using mathematical algorithms. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17), which has been incorporated into the ALIGN program (version 2.0). In some exemplary embodiments, the nucleic acid sequence comparison using the ALIGN program uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. The percent identity between two nucleotide sequences may alternatively be determined using the GAP program in the GCG software package using the nwsgapdna.

"increased", "increased" or "decreased"; as used herein, these terms or grammatically comparable comparison terms refer to values relative to a comparable reference measurement. For example, in some embodiments, an evaluation value obtained using an agent of interest (e.g., a therapeutic agent) may be "improved" relative to an evaluation value obtained using a comparable reference agent. Alternatively or additionally, in some embodiments, the evaluation value obtained in a subject or system of interest may be "increased" relative to an evaluation value obtained in the same subject or system under different conditions (e.g., before or after an event such as administration of an agent of interest), or in a different, comparable subject (e.g., in a comparable subject or system that differs from the subject or system of interest in the presence of one or more indicators of a particular disease, disorder, or condition of interest, or prior exposure to a condition or agent, etc.). In some embodiments, the comparative term refers to a statistically relevant difference (e.g., a difference having a prevalence and/or magnitude sufficient to achieve a statistical correlation). Those skilled in the art will know or will be readily able to determine the degree of difference and/or prevalence needed or sufficient to achieve such statistical significance in a given context.

In vitro: as used herein, the term "in vitro" refers to an event that occurs in an artificial environment, such as in a test tube or reaction vessel, in a cell culture, etc., rather than in a multicellular organism.

In vivo: as used herein, refers to events that occur within multicellular organisms, such as humans and non-human animals. In the context of a cell-based system, the term may be used to refer to events that occur within living cells (e.g., as opposed to an in vitro system).

The marker is as follows: as used herein, a marker refers to an entity or portion whose presence or level is characteristic of a particular state or event. In some embodiments, the presence or level of a particular marker may be characteristic of the presence or stage of a disease, disorder, or condition. By way of example only, in some embodiments, the term refers to a gene expression product that is characteristic of a particular tumor, tumor subclass, tumor stage, and the like. Alternatively or additionally, in some embodiments, the presence or level of a particular marker is correlated with the activity (or activity level) of a particular signaling pathway, e.g., may be characteristic of a particular class of tumor. The statistical significance of the presence or absence of a marker may vary with the particular marker. In some embodiments, detection of the marker is highly specific in that it reflects the high probability that the tumor belongs to a particular subclass. Such specificity may come at the cost of sensitivity (i.e., negative results may occur even if the tumor is one for which a marker is expected to be expressed). In contrast, markers with high sensitivity may not be as specific as less sensitive markers. According to the present invention, useful markers do not need to distinguish between specific subclasses of tumors with 100% accuracy.

Nucleic acid: as used herein, in its broadest sense, refers to any compound and/or substance that is or can be incorporated into an oligonucleotide chain. In some embodiments, the nucleic acid is a compound and/or substance that is or can be incorporated into the oligonucleotide chain via a phosphodiester linkage. As will be apparent from the context, in some embodiments, "nucleic acid" refers to a single nucleic acid residue (e.g., nucleotide and/or nucleoside); in some embodiments, "nucleic acid" refers to an oligonucleotide strand comprising a single nucleic acid residue. In some embodiments, a "nucleic acid" is or comprises RNA; in some embodiments, a "nucleic acid" is or comprises DNA. In some embodiments, the nucleic acid is, comprises, or consists of one or more natural nucleic acid residues. In some embodiments, the nucleic acid is, comprises, or consists of one or more nucleic acid analogs. In some embodiments, the nucleic acid analog differs from the nucleic acid in that it does not utilize a phosphodiester backbone. For example, in some embodiments, the nucleic acid is, comprises, or consists of one or more "peptide nucleic acids" that are known in the art and have peptide bonds rather than phosphodiester bonds in the backbone, are considered to be within the scope of the present invention. Alternatively or additionally, in some embodiments, the nucleic acid has one or more phosphorothioate and/or 5' -N-phosphoramidite linkages instead of phosphodiester linkages. In some embodiments, the nucleic acid is, comprises, or consists of one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine). In some embodiments, the nucleic acid is, comprises, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0 (6) -methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, the nucleic acid comprises one or more modified sugars (e.g., 2 '-fluororibose, ribose, 2' -deoxyribose, arabinose, and hexose) as compared to the sugars in the natural nucleic acid. In some embodiments, the nucleic acid has a nucleotide sequence encoding a functional gene product, such as RNA or a protein. In some embodiments, the nucleic acid comprises one or more introns. In some embodiments, the nucleic acid is prepared by one or more of the following: isolated from natural sources, complementary template-based polymerase synthesis (in vivo or in vitro), propagation in recombinant cells or systems, and chemical synthesis. In some embodiments, the nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, or more residues in length. In some embodiments, the nucleic acid is partially or fully single stranded; in some embodiments, the nucleic acid is partially or fully double stranded. In some embodiments, the nucleic acid has a nucleotide sequence comprising at least one element that encodes a polypeptide, or is a complement of a sequence encoding a polypeptide. In some embodiments, the nucleic acid has enzymatic activity.

Peptide: as used herein, the term "peptide" refers to a polypeptide that is generally relatively short, e.g., less than about 100 amino acids, less than about 50 amino acids, less than about 40 amino acids, less than about 30 amino acids, less than about 25 amino acids, less than about 20 amino acids, less than about 15 amino acids, or less than 10 amino acids in length.

A pharmaceutically acceptable carrier; as used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient or solvent encapsulating material, that participates in carrying or transporting the subject compound from one organ or portion of the body to another organ or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the subject. Some examples of materials that may be used as pharmaceutically acceptable carriers include: sugars such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; diols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; non-thermal raw water; isotonic saline; ringer's solution; ethanol; a pH buffer solution; polyesters, polycarbonates and/or polyanhydrides; and other non-toxic compatible substances for pharmaceutical formulations.

Pharmaceutical composition: as used herein, the term "pharmaceutical composition" refers to an active agent formulated with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose suitable for administration in a treatment regimen that, when administered to a relevant population, exhibits a statistically significant probability of achieving a predetermined therapeutic effect. In some embodiments, the pharmaceutical compositions may be specifically formulated for administration in solid or liquid form, including those suitable for: oral administration, e.g., infusion (aqueous or non-aqueous solutions or suspensions), e.g., tablets, boluses, powders, granules, pastes for application to the tongue that target buccal, sublingual, and systemic absorption; parenteral administration, for example by subcutaneous, intramuscular, intravenous or epidural injection, such as sterile solutions or suspensions, or sustained release formulations; topical application, for example as a cream, ointment or controlled release patch or spray, to the skin, lungs or oral cavity; intravaginal or intrarectal, for example as pessaries, creams or foams; sublingual; ocular menstruation; transdermal; or nasally, pulmonary and applied to other mucosal surfaces.

Polypeptide: as used herein, the term "polypeptide" generally has its art-recognized meaning, i.e., a polymer of at least three amino acids. It will be understood by those of ordinary skill in the art that the term "polypeptide" is intended to be comprehensive enough to encompass not only polypeptides having the complete sequences described herein, but also polypeptides that represent functional fragments (i.e., fragments that retain at least one activity) of such complete polypeptides. Furthermore, one of ordinary skill in the art will appreciate that protein sequences typically tolerate some substitution without disrupting activity. Thus, the relevant term "polypeptide" as used herein encompasses any of the following polypeptides: retains activity and shares at least about 30-40%, typically greater than about 50%, 60%, 70% or 80% overall sequence identity with another polypeptide of the same class, and typically also includes at least one region of much higher identity, typically greater than 90% or even 95%, 96%, 97%, 98% or 99%, typically comprising at least 3-4 and typically up to 20 or more amino acids, in one or more highly conserved regions. The polypeptide may contain L-amino acids, D-amino acids, or both, and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, and the like. In some embodiments, the protein may include natural amino acids, unnatural amino acids, synthetic amino acids, and combinations thereof. The term "peptide" is generally used to refer to polypeptides that are less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids in length. In some embodiments, the protein is an antibody, an antibody fragment, a biologically active portion thereof, and/or a characteristic portion thereof.

Prevention (pre) or prophylaxis (pre): as used herein, when used in connection with the occurrence of a disease, disorder, and/or condition, refers to reducing the risk of developing the disease, disorder, and/or condition and/or delaying the onset of one or more features, signs, or symptoms of the disease, disorder, and/or condition. Prevention may be considered complete when the onset of the disease, disorder or condition is delayed for a predetermined period of time.

Risk: as should be understood from the context, the "risk" of a disease, disorder and/or condition refers to the likelihood that a particular individual will suffer from the disease, disorder and/or condition. In some embodiments, risk is expressed as a percentage. In some embodiments, the risk is 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% up to 100%. In some embodiments, the risk is expressed as a risk relative to a risk associated with a reference sample or a reference sample set. In some embodiments, the reference sample or group of reference samples has a known risk of a disease, disorder, condition, and/or event. In some embodiments, the reference sample or group of reference samples is from an individual similar to the particular individual. In some embodiments, the relative risk is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or higher.

The subject: as used herein, the term "subject" refers to an organism, typically a mammal (e.g., a human, including in some embodiments prenatal human forms). In some embodiments, the subject has a related disease, disorder, or condition. In some embodiments, the subject is susceptible to a disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms or features of a disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or features of the disease, disorder, or condition. In some embodiments, the subject is a human having one or more characteristics of a disease, disorder, or susceptibility or risk of a disorder. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual who is administered and/or has been administered a diagnosis and/or treatment.

Basically: as used herein, the term "substantially" refers to a qualitative condition that exhibits all or nearly all of the range or degree of a feature or characteristic of interest. Those of ordinary skill in the art of biology will appreciate that biological and chemical phenomena rarely, if ever, complete and/or continue to complete or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of integrity inherent in many biological and chemical phenomena.

Is easy to suffer from: an individual who is "susceptible to" a disease, disorder and/or condition is an individual who has a higher risk of developing the disease, disorder and/or condition than a member of the general public. In some embodiments, an individual susceptible to a disease, disorder, and/or condition may not have been diagnosed with the disease, disorder, and/or condition. In some embodiments, an individual susceptible to a disease, disorder, and/or condition may exhibit symptoms of the disease, disorder, and/or condition. In some embodiments, an individual susceptible to a disease, disorder, and/or condition may not exhibit symptoms of the disease, disorder, and/or condition. In some embodiments, an individual susceptible to a disease, disorder, and/or condition will suffer from the disease, disorder, and/or condition. In some embodiments, an individual susceptible to a disease, disorder, and/or condition does not suffer from the disease, disorder, and/or condition.

Therapeutic agent: as used herein, the phrase "therapeutic agent" refers to an agent that has a therapeutic effect and/or elicits a desired biological and/or pharmacological effect when administered to a subject. In some embodiments, a therapeutic agent is any substance that can be used to alleviate, ameliorate, alleviate, inhibit, prevent, delay the onset of, reduce the severity of, and/or reduce the incidence of one or more symptoms or features of a disease, disorder, and/or condition.

Treatment: as used herein, the term "treatment" (and "treatment") or "treatment") refers to the administration of a therapy that partially or completely alleviates, ameliorates, alleviates, inhibits, delays the onset of, reduces the severity of, and/or reduces the incidence of one or more signs, symptoms, features, and/or etiologies of a particular disease, disorder, and/or condition. In some embodiments, such treatment may be directed to subjects that do not exhibit signs of the associated disease, disorder, and/or condition and/or subjects that exhibit only early signs of the disease, disorder, and/or condition. Alternatively or additionally, such treatment may be directed to a subject exhibiting one or more established signs of the associated disease, disorder, and/or condition. In some embodiments, the treatment may be directed to a subject that has been diagnosed with a related disease, disorder, and/or condition. In some embodiments, the treatment may be directed to a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of developing the associated disease, disorder, and/or condition. Thus, in some embodiments, the treatment may be prophylactic; in some embodiments, the treatment may be therapeutic.

Variants: as used herein, the term "variant" refers to an entity that exhibits significant structural identity to a reference entity, but is structurally different from the reference entity in the presence or absence or amount of one or more chemical moieties as compared to the reference entity. In some embodiments, the variant is also functionally different from its reference entity. In general, whether a particular entity is properly considered a "variant" of a reference entity depends on the degree of structural identity with the reference entity. As will be appreciated by those skilled in the art, any biological or chemical reference entity has certain characteristic structural elements. Variants, by definition, are unique chemical entities that share one or more such characteristic structural elements. Small molecules may have a characteristic core structural element (e.g., a macrocyclic core) and/or one or more characteristic overhangs, such that variants of the small molecule are variants that share the core structural element and the characteristic overhangs, but other overhangs and/or variants that differ in the type of bond present within the core (single pair double, E pair Z, etc.), the polypeptide may have a characteristic sequence element composed of multiple amino acids that have a specified position relative to one another in linear or three-dimensional space and/or contribute to a particular biological function, and the nucleic acid may have a characteristic sequence element composed of multiple nucleotide residues that have a specified position relative to one another in linear or three-dimensional space. In some embodiments, a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid by one or more differences in amino acid or nucleotide sequence and/or one or more differences in chemical moieties (e.g., carbohydrates, lipids, phosphate groups) that are covalent components of the polypeptide or nucleic acid (e.g., attached to the polypeptide or nucleic acid backbone). In some embodiments, the variant polypeptide or nucleic acid exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% overall sequence identity with a reference polypeptide or nucleic acid. In some embodiments, the variant polypeptide or nucleic acid exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% overall sequence identity to a portion of a reference polypeptide or nucleic acid. Alternatively or additionally, in some embodiments, the variant polypeptide or nucleic acid does not share at least one characteristic sequence element with a reference polypeptide or nucleic acid. In some embodiments, the reference polypeptide or nucleic acid has one or more biological activities. In some embodiments, the variant polypeptide or nucleic acid shares one or more biological activities of a reference polypeptide or nucleic acid. For example, in some embodiments, the variant polypeptide or nucleic acid shares one or more biological activities of a reference polypeptide or nucleic acid and also comprises one or more sequence variations (e.g., deletions, insertions, truncations, codon optimization, etc.). In some embodiments, the variant polypeptide or nucleic acid lacks one or more biological activities of the reference polypeptide or nucleic acid. In some embodiments, the variant polypeptide or nucleic acid exhibits a reduced level of one or more biological activities as compared to a reference polypeptide or nucleic acid. In some embodiments, a polypeptide or nucleic acid of interest is considered to be a "variant" of a parent or reference polypeptide or nucleic acid if the polypeptide or nucleic acid of interest has the same amino acid or nucleotide sequence as the reference but has a small sequence change at a particular position. Typically, less than about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, or about 2% of the residues in the variant are substituted, inserted, or deleted as compared to the reference. In some embodiments, a variant polypeptide or nucleic acid comprises about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 substituted residues as compared to a reference. Typically, a variant polypeptide or nucleic acid contains a very small number (e.g., less than about 5, about 4, about 3, about 2, or about 1) of functional residues that are substituted, inserted, or deleted (i.e., residues that are involved in a particular biological activity) relative to a reference. In some embodiments, the variant polypeptide or nucleic acid comprises no more than about 5, about 4, about 3, about 2, or about 1 additions or deletions as compared to the reference, and in some embodiments, no additions or deletions. In some embodiments, a variant polypeptide or nucleic acid comprises less than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and typically less than about 5, about 4, about 3, or about 2 additions or deletions as compared to a reference. In some embodiments, the reference polypeptide or nucleic acid is found in nature. In some embodiments, the reference polypeptide or nucleic acid is a human polypeptide or nucleic acid.

Detailed Description

Gene therapy

It has been reported that genetic diseases caused by dysfunctional genes account for nearly 80% of approximately 7,136 diseases reported by 2019 (see Genetic and rare Diseases Information Center and Global Genes). Over 3.3 million people worldwide are affected by genetic disease and nearly half of these cases are estimated to be children. However, it is estimated that only about 500 human diseases can be treated with existing drugs, indicating that new therapies and treatment options are necessary to address a significant portion of these genetic disorders. Gene therapy is an emerging form of treatment aimed at modulating the effects of genetic disorders via delivery of genetic material to a subject. In some embodiments, gene therapy may include transcription and/or translation of the transferred genetic material, and/or integration of the transferred genetic material into the host genome via administration of nucleic acids, viruses, or genetically engineered microorganisms (see FDA Guidelines). Gene therapy may allow for the delivery of therapeutic genetic material to any particular cell, tissue and/or organ of a subject being treated. In some embodiments, gene therapy involves the transfer of a therapeutic gene or transgene to a host cell.

Viral gene therapy

Viruses have become an attractive vehicle for gene therapy because of their ability to express high levels of payload (e.g., transgene), and in some embodiments, their ability to stably express the payload (e.g., transgene) in the host genome. Recombinant AAV is an epidemic viral vector for gene therapy because it generally produces high viral yields, a mild immune response, and is capable of infecting different cell types.

In conventional AAV gene therapies, rAAV can be engineered to deliver a therapeutic payload (e.g., transgene) to a target cell without integration into chromosomal DNA. One or more payloads (e.g., transgenes) may be expressed from non-integrated genetic elements known as episomes that are present in the nucleus. While conventional gene therapy may be effective for initially transduced cells, episomal expression is transient and gradually decreases over time, particularly with cell renewal. Episomal expression may be effective for cells that are longer in life (e.g., cells that are present most of the subject's lifetime). However, conventional gene therapy may have drawbacks when applied to a subject early in life (e.g., during childhood) because rapid tissue growth during development can result in dilution of the payload (e.g., transgene) and eventual loss of therapeutic benefit.

Second AAV Gene therapy GENERIDE ^TM Homologous Recombination (HR) is utilized, a naturally occurring DNA repair process that maintains fidelity of the cellular genome. GENERIDE ^TM HR is used to insert one or more payloads (e.g., transgenes) into a particular target locus within a genomic sequence. In some embodiments, GENERIDE ^TM Endogenous promoters at one or more target loci are utilized to drive high levels of tissue-specific expression. GENERIDE ^TM No exogenous nucleases or promoters need to be used, thereby reducing the deleterious effects normally associated with these elements. Furthermore, GENERIDE ^TM Platform technology is possible to overcome conventional gene therapy and conventional gene editing methodsSo that genetic diseases can be treated well, especially in pediatric subjects. GENERIDE ^TM AAV vectors are used to deliver genes into the nucleus. It then uses HR to stably integrate the correction gene into the subject's genome at a location regulated by an endogenous promoter, allowing for lifelong protein production, which is not feasible in conventional AAV gene therapies, even if the body grows and changes over time.

Previous work on non-destructive gene targeting is described in WO 2013/158309, which is incorporated herein by reference. Previous work on genome editing without nucleases is described in WO 2015/143177, incorporated herein by reference. Previous work on non-destructive gene therapy for the treatment of MMA is described in WO 2020/032986, which is incorporated herein by reference. Previous work on monitoring gene therapy is described in WO/2020/214582, incorporated herein by reference.

Viral structure and function

Viral vectors

Viral vectors comprise viral or viral chromosomal material into which heterologous nucleic acid sequences may be inserted for transfer into a target sequence of interest (e.g., into genomic DNA within a cell). Various viruses may be used as viral vectors, including, for example, single stranded DNA (ssDNA) viruses, double stranded DNA (dsDNA) viruses, and/or RNA viruses having a DNA stage in their life cycle. In some embodiments, the viral vector is or comprises an adeno-associated virus (AAV) or AAV variant.

In some embodiments, the vector particle is a single viral unit comprising a capsid (e.g., a wild-type viral genome or a recombinant viral vector) encapsulating a viral-based polynucleotide. In some embodiments, the vector particle is or comprises an AAV vector particle. In some embodiments, an AAV vector particle refers to a vector particle consisting of at least one AAV capsid protein and a packaged AAV vector. In some embodiments, the vector particle (also referred to as a viral vector) comprises at least one AAV capsid protein and a packaged AAV vector, wherein the vector further comprises one or more heterologous polynucleotide sequences.

Capsid proteins

In some embodiments, the expression construct comprises a polynucleotide sequence encoding a capsid protein from one or more AAV subtypes, including naturally occurring AAV and recombinant AAV. In some embodiments, the expression construct comprises a polynucleotide sequence encoding a capsid protein from: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVC11.01, AAVC11.02, AAVC11.03, AAVC11.04, AAVC11.05, AAVC11.06, AAVC11.07, AAVC11.08, AAVC11.09, AAVC11.10, AAVC11.11 (interchangeably referred to herein as sL 65), AAVC11.12, AAVC11.13, AAVC11.14, AAVC11.15, AAVC11.16, AAVC11.17, AAVC11.18, AAVC11.19, AAV-DJ, AAV-LK03, AAV-LK19, aavrh.74, aavrh.10, aavhu.37, aavrh.k, aavrh.39, AAV12, AAV13, aah.8, avian, bovine, canine, equine, AAV, or a primate, AAV, and AAV (or AAV) may comprise one or more than one or more variant or more of their AAV capsid(s) or capsid sequences derived from an AAV capsid or a variant of the two or more of the AAV capsid types.

In some embodiments, the viral vector is packaged in a capsid protein (e.g., a capsid protein from one or more AAV subtypes). In some embodiments, the capsid protein provides for increased or enhanced transduction of cells (e.g., human or murine cells) relative to a reference capsid protein. In some embodiments, the capsid protein provides for increased or enhanced transduction of certain cell or tissue types (e.g., hepatic tropism, muscle tropism, CNS tropism) relative to a reference capsid protein. In some embodiments, the capsid protein increases or enhances transduction of a cell or tissue (e.g., liver, muscle, and/or CNS) by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more relative to a reference capsid protein. In some embodiments, the capsid protein increases or enhances cellular or tissue (e.g., liver, muscle, and/or CNS) transduction by at least about 1.2x, 1.5x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 11x, 12x, 13x, 14x, 15x, 16x, 17x, 18x, 19x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, or more relative to a reference capsid protein.

AAV structure and function

Adeno-associated virus (AAV) is a parvovirus consisting of an icosahedral protein capsid and a single-stranded DNA genome. AAV viral capsids comprise three subunits VP1, VP2 and VP3 and two Inverted Terminal Repeat (ITR) regions, which are located at the ends of the genomic sequence. ITRs act as origins of replication and play a role in viral packaging. The viral genome also contains rep and cap genes, which are associated with replication and capsid packaging, respectively. In most wild-type AAV, the rep gene encodes four proteins required for viral replication: rep 78, rep68, rep52, and Rep40. The cap gene encodes a capsid subunit and an Assembly Activating Protein (AAP), which facilitates viral particle assembly. AAV is often replication-defective, requiring the presence of helper or helper functions, such as Herpes Simplex Virus (HSV) and/or adenovirus (AdV), in order to replicate within infected cells. For example, in some embodiments, AAV requires adenovirus E1A, E2A, E and a VA RNA gene in order to replicate within a host cell.

Recombinant AAV

In general, recombinant AAV (rAAV) vectors can comprise many of the same elements found in wild-type AAV, including similar capsid sequences and structures, as well as polynucleotide sequences that are not AAV-derived (e.g., polynucleotides heterologous to AAV). In some embodiments, the rAAV will replace the native wild-type AAV sequence with a polynucleotide sequence encoding the payload. For example, in some embodiments, a rAAV will comprise a polynucleotide sequence encoding one or more genes intended for therapeutic purposes (e.g., for gene therapy). The rAAV may be modified to remove one or more wild-type viral coding sequences. For example, the rAAV may be engineered to contain only one ITR, and/or one or more fewer genes required for packaging (e.g., rep and cap genes) than wild-type AAV. Gene expression of rAAV is typically limited to one or more genes that total 5kb or less, because larger sequences cannot be efficiently packaged within the viral capsid. In some embodiments, two or more rAAV may be used to provide portions of a larger payload, e.g., to provide complete coding sequences for genes that are typically too large to be accommodated in a single AAV.

The present disclosure provides, inter alia, viral vectors comprising one or more polypeptides described herein. In some embodiments, the rAAV may comprise one or more capsid proteins (e.g., one or more capsid proteins from: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVC11.01, AAVC11.02, AAVC11.03, AAVC11.04, AAVC11.05, AAVC11.06, AAVC11.07, AAVC11.08, AAVC11.09, AAVC11.10, AAVC11.11 (interchangeably referred to herein as sL 65), AAVC11.12, AAVC11.13, AAVC11.14, AAVC11.15, AAVC11.16, AAVC11.17, AAVC11.18, AAVC11.19, AAV-DJ, AAV-LK03, AAV-LK19, aavrh.74, aavrh.10, aavhu.37, aavrh.k, aavrh.39, AAV12, AAV13, aah.8, avian, bovine, canine, equine, AAV, non-primate, AAV, and AAV comprising one or more than one or more AAV capsid(s) or AAV (e.g., a capsid) of the AAV and a capsid, in some embodiments, a rAAV may comprise one or more polynucleotide sequences encoding a gene or nucleic acid of interest (e.g., a gene and/or inhibitory nucleic acid sequence for treating a genetic disease/disorder).

AAV vectors may be capable of replication in an infected host cell (replication competent) or incapable of replication in an infected host cell (replication deficient). Replication competent AAV (rcAAV) requires the presence of one or more functional AAV packaging genes. Recombinant AAV vectors are typically designed to have insufficient replication capacity in mammalian cells in order to reduce the likelihood of producing rcAAV via recombination with sequences encoding AAV packaging genes. In some embodiments, a rAAV vector formulation as described herein is designed to include a small amount (if any) of an rcAAV vector. In some embodimentsEvery 10 rAAV vector formulations ² Each rAAV vector comprises less than about 1 rcAAV. In some embodiments, each 10 of the rAAV vector formulations ⁴ Each rAAV vector comprises less than about 1 rcAAV. In some embodiments, each 10 of the rAAV vector formulations ⁸ Each rAAV vector comprises less than about 1 rcAAV. In some embodiments, each 10 of the rAAV vector formulations ¹² Each rAAV vector comprises less than about 1 rcAAV. In some embodiments, the rAAV vector formulation does not comprise an rcAAV vector.

Heterologous nucleic acid

Payload

In some embodiments, one or more vectors or constructs described herein may comprise a polynucleotide sequence encoding one or more payloads. According to various aspects, any of a variety of payloads (e.g., those having diagnostic and/or therapeutic purposes) may be used alone or in combination. In some embodiments, the payload may be or comprise a polynucleotide sequence encoding a peptide or polypeptide. In some embodiments, the payload is a peptide having an intrinsic or extrinsic activity that facilitates a biological process for treating a medical condition. In some embodiments, the payload may be or comprise a transgene (also referred to herein as a gene of interest (GOI)). In some embodiments, the payload may be or comprise one or more Inverted Terminal Repeat (ITR) sequences (e.g., one or more AAV ITRs). In some embodiments, the payload may be or comprise one or more transgenes having flanking ITR sequences. In some embodiments, the payload may be or comprise one or more transgenes having flanking homology arm sequences. In some embodiments, the payload may be or comprise one or more transgenes having flanking homology arm sequences and flanking ITRs. In some embodiments, the payload may be or comprise one or more heterologous nucleic acid sequences encoding a reporter gene (e.g., a fluorescent or luminescent reporter gene). In some embodiments, the payload may be or comprise one or more biomarkers (e.g., an indicator of payload expression). In some embodiments, the expression construct comprises one or more transcription termination sequences (e.g., polyA sequences). In some embodiments, the expression construct comprises one or more promoter sequences. In some embodiments, the expression construct comprises one or more enhancer sequences. In some embodiments, the expression construct comprises one or more intron sequences. In some embodiments, the payload may comprise sequences for polycistronic expression (including, for example, 2A peptide or intron sequences, internal ribosome entry sites). In some embodiments, the 2A peptide is a small (e.g., about 18-22 amino acids) peptide sequence, enabling co-expression of two or more discrete protein products within a single coding sequence. In some embodiments, the 2A peptide allows for co-expression of two or more discrete protein products regardless of the arrangement of the protein coding sequences. In some embodiments, the 2A peptide is or comprises a common motif (e.g., DVEXNPGP). In some embodiments, the 2A peptide facilitates protein cleavage. In some embodiments, the 2A peptide is or comprises a viral sequence (e.g., foot-and-mouth disease virus (F2A), equine type a rhinitis virus, porcine teschovirus-1 (P2A), or thorn vein agrimony (Thosea asigna) virus (T2A)).

In some embodiments, the biomarker is or comprises a 2A peptide (e.g., P2A, T2A, E2A and/or F2A). In some embodiments, the biomarker is or comprises a furin cleavage motif (see Tian et al, furinDB: A Database of 20-Residue Furin Cleavage Site Motifs, substrates and Their Associated Drugs, (2011), int.J.mol.Sci., volume 12: 1060-1065). In some embodiments, the biomarker is or comprises a tag (e.g., an immune tag). In some embodiments, the payload may comprise one or more functional nucleic acids (e.g., one or more sirnas or mirnas). In some embodiments, the payload may comprise one or more inhibitory nucleic acids (including, e.g., ribozymes, mirnas, sirnas, shrnas, or the like). In some embodiments, the payload can comprise one or more nucleases (e.g., cas protein, endonuclease, TALEN, ZFN).

Transgenic plants

In some embodiments, the transgene is a correction gene selected for ameliorating one or more signs and/or symptoms of a disease, disorder, or condition. In some embodiments, the transgene may be integrated into the host cell genome via use of a vector encompassed by the present disclosure. In some embodiments, the transgene is a functional form of a disease-associated gene (i.e., a gene isoform associated with the manifestation or exacerbation of a disease, disorder or condition) found in the host cell. In some embodiments, the transgene is an optimized form of a disease-associated gene found in the host cell (e.g., a codon-optimized or expression-optimized variant). In some embodiments, the transgene is a variant (e.g., a functional gene fragment or variant thereof) of a disease-associated gene found in the host cell. In some embodiments, the transgene is a gene that causes expression of a peptide that is normally expressed in one or more healthy tissues. In some embodiments, the transgene is a gene that causes expression of a peptide that is normally expressed in hepatocytes. In some embodiments, the transgene is a gene that causes expression of a peptide that is normally expressed in a muscle cell. In some embodiments, the transgene is a gene that causes expression of a peptide that is normally expressed in cells of the central nervous system.

In some embodiments, the transgene may be or comprise a gene that causes expression of a peptide that is not normally expressed in one or more healthy tissues (e.g., an ectopically expressed peptide). In some embodiments, a transgene is a gene that causes expression of a peptide that is ectopically expressed in one or more healthy tissues, such as the liver, muscle, central Nervous System (CNS). In some embodiments, a transgene is a gene that causes expression of a peptide that is ectopically expressed in one or more healthy tissues and normally expressed in one or more healthy tissues (e.g., liver, muscle, central Nervous System (CNS)).

In some embodiments, the transgene may be or comprise a gene encoding a functional nucleic acid. In some embodiments, the therapeutic agent is or comprises an agent having a therapeutic effect on a host cell or subject (including, for example, ribozymes, guide RNAs (grnas), antisense oligonucleotides (ASOs), mirnas, sirnas, and/or shrnas). For example, in some embodiments, the therapeutic agent promotes a biological process to treat a medical condition, such as at least one symptom of a disease, disorder, or condition.

In some embodiments, transgene expression in the subject results substantially from integration at the target locus. In some embodiments, 75% or more (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 99% or more, 99.5% or more) of the total transgene expression in the subject results from transgene integration at the target locus. In some embodiments, 25% or less (e.g., 20% or less, 15% or less, 10% or less, 5% or less, 1% or less, 0.5% or less, 0.1% or less) of the total transgene expression in the subject is from a source other than transgene integration at the target locus (e.g., episomal expression, integration at a non-target locus).

In some embodiments, the transgene is transiently expressed in the subject (e.g., episomal expression from a plasmid, small loop DNA, virus, etc.). In some embodiments, 75% or more (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 99% or more, 99.5% or more) of the total transgene expression in the subject is from transient expression. In some embodiments, 25% or less (e.g., 20% or less, 15% or less, 10% or less, 5% or less, 1% or less, 0.5% or less, 0.1% or less) of the total transgene expression in the subject is from a source other than transient expression (e.g., integration at a non-target locus). In some embodiments, the transgene is transiently expressed in the subject (e.g., episomal expression from a plasmid, small circular DNA, virus, etc.) within one or more weeks after treatment. In some embodiments, the transgene is transiently expressed in the subject (e.g., episomal expression from a plasmid, small loop DNA, virus, etc.) for one or more months after treatment.

In some embodiments, the transgene is transiently expressed in the subject (e.g., episomal expression from a plasmid, small loop DNA, virus, etc.) one or more weeks after treatment in an amount comparable to that observed during one or more days after treatment. In some embodiments, the transgene is transiently expressed in the subject (e.g., episomal expression from a plasmid, small loop DNA, virus, etc.) one or more months after treatment in an amount comparable to that observed during one or more days after treatment.

In some embodiments, the transgene is transiently expressed in the subject (e.g., episomal expression from a plasmid, small loop DNA, virus, etc.) one or more weeks after treatment in an amount that is reduced relative to the amount observed during one or more days after treatment. In some embodiments, the transgene is transiently expressed in the subject (e.g., episomal expression from a plasmid, small loop DNA, virus, etc.) at one or more months after treatment, in an amount that is reduced relative to the amount observed during one or more days after treatment.

In some embodiments, the transgene is transiently expressed in the subject (e.g., episomal expression from a plasmid, small circular DNA, virus, etc.) within no more than one month after treatment. In some embodiments, the transgene is transiently expressed in the subject (e.g., episomal expression from a plasmid, small circular DNA, virus, etc.) no more than two months after treatment. In some embodiments, the transgene is transiently expressed in the subject (e.g., episomal expression from a plasmid, small circular DNA, virus, etc.) no more than three months after treatment. In some embodiments, the transgene is transiently expressed in the subject (e.g., episomal expression from a plasmid, small circular DNA, virus, etc.) for no more than four months after treatment. In some embodiments, the transgene is transiently expressed in the subject (e.g., episomal expression from a plasmid, small loop DNA, virus, etc.) for no more than five months after treatment. In some embodiments, the transgene is transiently expressed in the subject (e.g., episomal expression from a plasmid, small circular DNA, virus, etc.) for no more than six months after treatment.

In some embodiments, the selected transgene may be or comprise a polynucleotide sequence encoding: c1NH, fumaric Acetoacetate Hydrolase (FAH), ATP7B, UGT A1, G6PC, G6PT1, SLC17A3, SLCA4, GAA, DDC, factor IX, factor VIII, COL7A1, COL17A1, MMP1, KRT5, LAMA3, LAMB3, LAMC2, ITGB4, CBS, CPS1, ARG1, ASL, OTC, IDUA, SGSH, NAGLU, HGSNAT, GNS, GALNS, GLB1, ARSB, GUSB, HYAL1, OCTN2, CPT1, CACT, CPT2, HADHA, HADHB, LCHAD, ACADM, ACADVL, BCKDH complex (E1 a, E1b and E2 subunits), methylmalonyl-CoA Mutase (MUT), propionyl-CoA carboxylase, LAMC2, ITGB4, CBS, CPS1, ARG1, ASL, OTC, IDUA, SGSH, NAGLU, HGSNAT, GNS, GALNS, GLB, ARSB, GUSB, HYAL1, OCTN2, CPT1, CPT2, HADHA, HADHB, LCHAD, ACADM, ACADVL, BCKDH complex (E1 a, E1b and E2 subunits) isovaleryl coa dehydrogenase, argininosuccinate lyase (ASL), caps 3, ANO5, DYSF, SGCG, SGCA, SGCB, calpain 3, neutrophin-3, SCN1a, SCN8a, SCN1b, SCN2a, NPC1, NPC2, LMNA, SYNE1, SYNE2, FHL1, TTR, factor XII, SERPINA1, AGL, mini-dystrophin (microdystrophin), mini-dystrophin (ministrataphin), AADC, αsarc, γsarc, β SARC, FKRP, MTM1, SMN2, or variants thereof.

Homology arm

In some embodiments, the viral vectors described herein comprise one or more flanking polynucleotide sequences (e.g., homology arms) having substantial sequence homology to the target locus. In some embodiments, homology arms flank a polynucleotide sequence encoding a payload (e.g., a transgene). In some embodiments, the homology arms flank the polynucleotide sequence encoding the transgene. In some embodiments, the homology arms direct site-specific integration of the payload (e.g., transgene). In some embodiments, the payload may comprise a homology arm and a transgene, wherein the homology arm directs site-specific integration of the transgene.

In some embodiments, the homology arms have the same length (also referred to herein as balanced homology arms or balanced homology arms). In some embodiments, viral vectors comprising homology arms of the same length, wherein the homology arms are at least a certain length, provide improved effects (e.g., improved target integration rate). In some embodiments, the homology arms are between 50nt and 500nt in length. In some embodiments, the homology arms are between 50nt and 100nt in length. In some embodiments, the homology arm is between 100nt and 1000nt in length. In some embodiments, the homology arm is between 200nt and 1000nt in length. In some embodiments, the homology arm is between 500nt and 1500nt in length. In some embodiments, the homology arm is between 1000nt and 2000nt in length. In some embodiments, the homology arm is greater than 2000nt in length. In some embodiments, each homology arm is at least 750nt in length. In some embodiments, each homology arm is at least 1000nt in length. In some embodiments, each homology arm is at least 1250nt in length. In some embodiments, the homology arm is less than 1000nt in length.

In some embodiments, the homology arms have different lengths (also referred to herein as unbalanced homology arms or unbalanced homology arms). In some embodiments, viral vectors comprising unbalanced homology arms of different lengths provide improved effects (e.g., increased target site integration rate) compared to a reference sequence. In some embodiments, a viral vector comprising homology arms of different lengths (wherein each homology arm is at least a certain length) provides an improved effect (e.g., increased target site integration rate) compared to a reference sequence (e.g., a viral vector comprising homology arms of the same length or a viral vector comprising one or more homology arms less than 1000nt in length).

In some embodiments, each homology arm is greater than 50nt in length. In some embodiments, each homology arm is greater than 100nt in length. In some embodiments, each homology arm is greater than 200nt in length. In some embodiments, each homology arm is greater than 500nt in length. In some embodiments, each homology arm is at least 750nt in length. In some embodiments, each homology arm is at least 1000nt in length. In some embodiments, one homology arm is at least 750nt in length and the other homology arm is at least 1000nt in length. In some embodiments, one homology arm is at least 750nt in length and the other homology arm is at least 1100nt in length. In some embodiments, one homology arm is at least 750nt in length and the other homology arm is at least 1200nt in length. In some embodiments, one homology arm is at least 750nt in length and the other homology arm is at least 1300nt in length. In some embodiments, one homology arm is at least 750nt in length and the other homology arm is at least 1400nt in length. In some embodiments, one homology arm is at least 750nt in length and the other homology arm is at least 1500nt in length. In some embodiments, one homology arm is at least 750nt in length and the other homology arm is at least 1600nt in length. In some embodiments, one homology arm is at least 750nt in length and the other homology arm is at least 1700nt in length. In some embodiments, one homology arm is at least 750nt in length and the other homology arm is at least 1800nt in length. In some embodiments, one homology arm is at least 750nt in length and the other homology arm is at least 1900nt in length. In some embodiments, one homology arm is at least 750nt in length and the other homology arm is at least 2000nt in length. In some embodiments, one homology arm is at least 1000nt in length and the other homology arm is at least 1100nt in length. In some embodiments, one homology arm is at least 1000nt in length and the other homology arm is at least 1200nt in length. In some embodiments, one homology arm is at least 1000nt in length and the other homology arm is at least 1300nt in length. In some embodiments, one homology arm is at least 1000nt in length and the other homology arm is at least 1400nt in length. In some embodiments, one homology arm is at least 1000nt in length and the other homology arm is at least 1500nt in length. In some embodiments, one homology arm is at least 1000nt in length and the other homology arm is at least 1600nt in length. In some embodiments, one homology arm is at least 1000nt in length and the other homology arm is at least 1700nt in length. In some embodiments, one homology arm is at least 1000nt in length and the other homology arm is at least 1800nt in length. In some embodiments, one homology arm is at least 1000nt in length and the other homology arm is at least 1900nt in length. In some embodiments, one homology arm is at least 1000nt in length and the other homology arm is at least 2000nt in length. In some embodiments, one homology arm is at least 1300nt in length and the other homology arm is at least 1400nt in length. In some embodiments, the 5 'homology arm is longer than the 3' homology arm. In some embodiments, the 3 'homology arm is longer than the 5' homology arm.

In some embodiments, the homology arm has at least 70% homology to the target locus. In some embodiments, the homology arm has at least 80% homology to the target locus. In some embodiments, the homology arm has at least 90% homology to the target locus. In some embodiments, the homology arm has at least 95% homology to the target locus. In some embodiments, the homology arm has at least 99% homology to the target locus. In some embodiments, the homology arm has 100% homology to the target locus.

In some embodiments, a viral vector comprising a homology arm provides increased integration of a target site compared to a reference sequence (e.g., a viral vector lacking a homology arm). In some embodiments, a viral vector comprising homology arms provides a target site integration rate of 0.01% or more (e.g., 0.05% or more, 0.1% or more, 0.2% or more, 0.3% or more, 0.4% or more, 0.5% or more, 0.6% or more, 0.7% or more, 0.8% or more, 0.9% or more, 1% or more, 1.5% or more, 2% or more, 5% or more, 10% or more, 20% or more, 30% or more). In some embodiments, a viral vector comprising a homology arm provides an increased rate of integration of the target site over time. In some embodiments, the rate of target site integration increases over time relative to an initial measurement of target site integration. In some embodiments, the target site integration rate over time is at least 1.5X (e.g., 1.5X, 2X, 3X, 4X, 5X, 10X, 20X, 30X, 40X, 50X, 60X, 70X, 80X, 90X, 100X, 200X) higher than the initial measurement of target site integration. In some embodiments, the target site integration rate is measured after one or more days. In some embodiments, the target site integration rate is measured after one or more weeks. In some embodiments, the target site integration rate is measured after one or more months. In some embodiments, the target site integration rate is measured after one or more years. In some embodiments, the target site integration rate is measured via evaluation of one or more biomarkers (e.g., biomarkers comprising 2A peptide). In some embodiments, the target site integration rate is measured via evaluation of one or more isolated nucleic acids (e.g., mRNA, gDNA). In some embodiments, the target site integration rate is measured via assessment of gene expression (e.g., via immunohistochemical staining).

Table 1: exemplary methods for assessing target site integration

In some embodiments, viral vectors comprising homology arms of different lengths may provide improved gene editing in a species or model system of a species (e.g., mouse, human, or model thereof). In some embodiments, the viral vectors may comprise different combinations of homology arm lengths when optimized for expression in a particular species or model system of a particular species (e.g., mouse, human, or model thereof). In some embodiments, a viral vector comprising a particular combination of homology arm lengths may provide improved gene editing in a species or model system of a species (e.g., human, humanized mouse model) as compared to a second species or model system of a second species (e.g., mouse, pure mouse model). In some embodiments, a viral vector comprising a particular combination of homology arm lengths may be optimized for high level gene editing in a species or model of a species (e.g., human, humanized mouse model) as compared to a model system of a second species or second species (e.g., mouse, pure mouse model).

In some embodiments, the homology arms direct integration of the transgene immediately following the highly expressed endogenous gene. In some embodiments, the homology arms direct transgene integration without disrupting endogenous gene expression (non-destructive integration).

Therapeutic method

The compositions and constructs disclosed herein can be used for any in vitro or in vivo application in which a payload (e.g., transgene) from a particular target locus is expressed in a cell while maintaining expression of an endogenous gene at and around the target locus. For example, the compositions and constructs disclosed herein can be used to treat a disorder, disease, or medical condition in a subject (e.g., via gene therapy).

In some embodiments, the treatment comprises achieving or maintaining a desired pharmacological and/or physiological effect. In some embodiments, the desired pharmacological and/or physiological effect may include complete or partial prevention of a disease (e.g., prevention of disease symptoms). In some embodiments, the desired pharmacological and/or physiological effect may include complete or partial cure of the disease (e.g., cure of side effects associated with the disease). In some embodiments, the desired pharmacological and/or physiological effect may include preventing disease recurrence. In some embodiments, the desired pharmacological and/or physiological effect may include slowing the progression of the disease. In some embodiments, the desired pharmacological and/or physiological effect may include alleviation of symptoms of a disease. In some embodiments, the desired pharmacological and/or physiological effect may include preventing disease progression. In some embodiments, the desired pharmacological and/or physiological effect may include stabilization and/or alleviation of symptoms associated with the disease.

In some embodiments, the treatment comprises administering the composition before, during, or after the onset of the disease (e.g., before, during, or after the occurrence of symptoms associated with the disease). In some embodiments, the treatment comprises a combination therapy (e.g., using one or more therapies, including different types of therapies).

Diseases of interest

In some embodiments, the compositions and constructs disclosed herein can be used to treat any disease of interest including genetic defects or abnormalities as part of the disease.

As a specific example, in some embodiments, compositions and constructs such as those disclosed herein may be used to treat branched chain organic aciduria (e.g., maple diabetes (MSUD), methylmalonic acidemia (MMA), propionic Acidemia (PA), isovaleric acidemia (IVA)). In some embodiments, the treatment comprises introducing polynucleotide sequences encoding one or more transgenes of interest (e.g., BCKDH complex (E1 a, E1b, and E2 subunits), methylmalonyl-coa mutase, propionyl-coa carboxylase (α and β subunits), isovaleryl-coa dehydrogenase, and/or variants thereof). In some embodiments, the treatment comprises reducing abnormal proteins (e.g., nonfunctional proteins) associated with branched-chain organic aciduria. In some embodiments, the treatment comprises alleviating symptoms and/or signs associated with branched chain organic aciduria (e.g., hypotonic, bradykinesia, seizures, optic atrophy, acute encephalopathy, hyperventilation, respiratory distress, unstable body temperature, repeated vomiting, ketoacidosis, pancreatitis, constipation, neutropenia, whole blood cytopenia, secondary hemophagocytosis, cardiac arrhythmias, cardiomyopathy, chronic renal failure, dermatitis, hearing loss).

In some embodiments, the compositions and constructs disclosed herein are useful for treating fatty acid oxidation disorders (e.g., trifunctional protein deficiency, long chain L-3 hydroxyacyl-coa dehydrogenase (LCAD) deficiency, medium chain acyl-coa dehydrogenase (MCHAD) deficiency, very long chain acyl-coa dehydrogenase (VLCHAD) deficiency). In some embodiments, the treatment comprises introducing a polynucleotide sequence encoding one or more transgenes of interest (e.g., HADHA, HADHB, LCHAD, ACADM, ACADVL and/or variants thereof). In some embodiments, the treatment comprises reducing an abnormal protein (e.g., a nonfunctional protein) associated with a fatty acid oxidation disorder. In some embodiments, the treatment comprises alleviating the signs and/or symptoms associated with fatty acid oxidation disorders (e.g., hepatomegaly, mental and physical retardation, cardiac muscle weakness, cardiac arrhythmias, nerve damage, liver dysfunction, rhabdomyolysis, myoglobin urine, hypoglycemia, metabolic acidosis, respiratory distress, hepatomegaly, hypotonic, cardiomyopathy).

In some embodiments, the compositions and constructs disclosed herein can be used to treat glycogen storage disease (e.g., type 1 glycogen storage disease (GSD 1), type 2 glycogen storage disease (Pompe disease), GSD2, type 3 glycogen storage disease (GSD 3)). In some embodiments, the treatment comprises introducing a polynucleotide sequence encoding one or more transgenes of interest (e.g., G6PC (GSD 1 a), G6PT1 (GSD 1 b), SLC17A3, SLC37A4 (GSD 1 c), AGL, acid alpha-glucosidase, and/or variants thereof). In some embodiments, the treatment comprises reducing abnormal proteins (e.g., nonfunctional proteins) associated with a glycogen storage disease. In some embodiments, the treatment comprises alleviating the signs and/or symptoms associated with glycogen storage disease (e.g., liver enlargement, hypoglycemia, muscle weakness, muscle spasm, fatigue, developmental delay, obesity, hemorrhagic condition, liver dysfunction, kidney dysfunction, respiratory dysfunction, cardiac dysfunction, canker sore, gout, liver cirrhosis, fibrosis, liver tumor).

In some embodiments, the compositions and constructs disclosed herein are useful for treating a carnitine circulatory disorder. In some embodiments, the treatment comprises introducing a polynucleotide sequence encoding one or more transgenes of interest (e.g., OCTN2, CPT1, CACT, CPT2, and/or variants thereof). In some embodiments, the treatment comprises reducing an abnormal protein (e.g., a nonfunctional protein) associated with a carnitine circulatory disorder. In some embodiments, the treatment comprises alleviating the signs and/or symptoms associated with a carnitine circulatory disorder (e.g., ketotic hypoglycemia, cardiomyopathy, muscle weakness, fatigue, bradykinesia, edema).

In some embodiments, the compositions and constructs disclosed herein are useful for treating urea cycle disorders. In some embodiments, the treatment comprises introducing polynucleotide sequences encoding one or more transgenes of interest (e.g., CPS1, ARG1, ASL, OTC, and/or variants thereof). In some embodiments, the treatment comprises reducing abnormal proteins (e.g., nonfunctional proteins) associated with a urea cycle disorder. In some embodiments, the treatment comprises alleviating the signs and/or symptoms associated with a urea cycle disorder (e.g., vomiting, nausea, behavioral abnormalities, fatigue, coma, psychosis, somnolence, periodic vomiting, myopia, hyperammonemia, elevated ornithine levels).

In some embodiments, the compositions and constructs disclosed herein are useful for treating Homocystinuria (HCU). In some embodiments, the treatment comprises introducing a polynucleotide sequence encoding one or more transgenes of interest, such as Cystathionine Beta Synthase (CBS) and/or variants thereof. In some embodiments, the treatment comprises reducing abnormal proteins (e.g., nonfunctional proteins) associated with the HCU. In some embodiments, the treatment comprises alleviating signs and/or symptoms associated with HCU (e.g., lens dislocation, myopia, iris reduction, cataract, optic atrophy, glaucoma, retinal detachment, retinal damage, delayed development milestone, mental retardation, depression, anxiety, obsessive compulsive disorder, elongated fingers, gonyeas, high arches, scoliosis, chicken breast, funnel breast, osteoporosis, increased thrombosis, thromboembolism, pulmonary embolism, skin fragility, hypopigmentation, cheekbone flushing, inguinal hernia, pancreatitis, kyphosis, spontaneous pneumothorax).

In some embodiments, the compositions and constructs disclosed herein are useful for treating kriging-naljeer syndrome. In some embodiments, the treatment comprises introducing a polynucleotide sequence encoding one or more transgenes of interest (e.g., UGT1A1 and/or variants thereof). In some embodiments, the treatment comprises reducing abnormal proteins (e.g., nonfunctional proteins) associated with kriging-naltrexone syndrome. In some embodiments, the treatment comprises alleviating the signs and/or symptoms associated with kriging-naltrexone syndrome (e.g., jaundice, nuclear jaundice, somnolence, vomiting, fever, abnormal reflexes, muscle spasms, starburst, spasms, hypotonia, athetosis, elevated bilirubin levels, diarrhea, aphtha, confusion, dysphagia, seizures).

In some embodiments, the compositions and constructs disclosed herein are useful for treating hereditary tyrosinemia. In some embodiments, the treatment comprises introducing polynucleotide sequences encoding one or more transgenes of interest, such as Fumaroyl Acetoacetate Hydrolase (FAH) and/or variants thereof. In some embodiments, the treatment comprises reducing abnormal proteins (e.g., nonfunctional proteins) associated with hereditary tyrosinemia. In some embodiments, the treatment comprises alleviating the signs and/or symptoms associated with hereditary tyrosinemia (e.g., hepatomegaly, jaundice, liver disease, liver cirrhosis, liver cancer, fever, diarrhea, jettisonia, vomiting, splenomegaly, edema, coagulation disorders, renal dysfunction, rickets, weakness, hypertension, ileus, tachycardia, hypertension, nervous system crisis, respiratory failure, cardiomyopathy).

In some embodiments, the compositions and constructs disclosed herein are useful for treating epidermolysis bullosa. In some embodiments, the treatment comprises introducing polynucleotide sequences encoding one or more transgenes of interest (e.g., COL7A1, COL17A1, MMP1, KRT5, LAMA3, LAMB3, LAMC2, ITGB4, and/or variants thereof). In some embodiments, the treatment comprises reducing abnormal proteins (e.g., nonfunctional proteins) associated with epidermolysis bullosa. In some embodiments, the treatment comprises alleviating the signs and/or symptoms associated with vesicular epidermolysis (e.g., skin fragility, abnormal nail growth, blisters, skin roughness, cicatricial alopecia, atrophic scars, papules, dental problems, dysphagia, skin itching, and pain).

In some embodiments, the compositions and constructs disclosed herein are useful for treating alpha-1 antitrypsin deficiency (A1 ATD). In some embodiments, the treatment comprises introducing a polynucleotide sequence encoding one or more transgenes of interest, such as alpha-1 antitrypsin (A1 AT) and/or variants thereof. In some embodiments, the treatment comprises reducing abnormal proteins (e.g., nonfunctional proteins) associated with alpha-1 antitrypsin. In some embodiments, the treatment comprises alleviating the signs and/or symptoms associated with A1ATD (e.g., emphysema, chronic cough, excessive phlegm, wheezing, chronic respiratory tract infection, jaundice, hepatomegaly, hemorrhage, abnormal dropsy, elevated liver enzymes, liver dysfunction, portal hypertension, fatigue, edema, chronic active hepatitis, cirrhosis, liver cancer, lipid membrane inflammation).

In some embodiments, the compositions and constructs disclosed herein are useful for treating wilson's disease. In some embodiments, the treatment comprises introducing a polynucleotide sequence encoding one or more transgenes of interest (e.g., ATP7B and/or variants thereof). In some embodiments, the treatment comprises reducing abnormal proteins (e.g., nonfunctional proteins) associated with wilson's disease. In some embodiments, the treatment comprises alleviating symptoms and/or symptoms associated with wilson's disease (e.g., fatigue, anorexia, abdominal pain, jaundice, kayser-Fleischer ring), edema, speech problems, swallowing problems, loss of physical coordination, loss of motion, muscle stiffness, liver disease, anemia, depression, schizophrenia, amenorrhea, infertility, kidney stones, tubular injury, arthritis, osteoporosis, osteophytes.

In some embodiments, the compositions and constructs disclosed herein are useful for treating hematological disorders (e.g., hemophilia a, hemophilia B). In some embodiments, the treatment comprises introducing polynucleotide sequences encoding one or more transgenes of interest (e.g., factor IX (FIX), factor VIII (FVIII), and/or variants thereof). In some embodiments, the treatment comprises reducing abnormal proteins (e.g., nonfunctional proteins) associated with the hematological disorder. In some embodiments, the treatment comprises alleviating signs and/or symptoms associated with hematological disorders (e.g., excessive bleeding, abnormal bruising, joint pain and swelling, hematuria, bloody stool, abnormal nasal bleeding, headache, somnolence, vomiting, double vision, weakness, tics, seizures).

In some embodiments, the compositions and constructs disclosed herein are useful for treating hereditary angioedema. In some embodiments, the treatment comprises introducing a polynucleotide sequence encoding one or more transgenes of interest, such as a C1 esterase inhibitor (C1-inh). In some embodiments, the treatment comprises reducing abnormal proteins (e.g., nonfunctional proteins) associated with hereditary angioedema. In some embodiments, the treatment comprises alleviating the signs and/or symptoms associated with hereditary angioedema (e.g., edema, itching, urticaria, nausea, vomiting, acute abdominal pain, dysphagia, dysphonia, wheezing).

In some embodiments, the compositions and constructs disclosed herein are useful for treating Parkinson's disease. In some embodiments, the treatment comprises introducing a polynucleotide sequence encoding one or more transgenes of interest, such as Dopamine Decarboxylase (DDC). In some embodiments, the treatment comprises reducing abnormal proteins (e.g., nonfunctional proteins) associated with parkinson's disease. In some embodiments, the treatment comprises alleviating signs and/or symptoms associated with parkinson's disease (e.g., tremors, bradykinesia, muscle stiffness, impaired posture and balance, automatic movement loss, speech changes, writing changes).

In some embodiments, the compositions and constructs disclosed herein are useful for treating muscle disorders. In some embodiments, the treatment comprises introducing a polynucleotide sequence encoding one or more transgenes of interest (e.g., muscular dystrophy, duchenne Muscular Dystrophy (DMD), limb muscular dystrophy, X-linked myotube myopathy). In some embodiments, the treatment comprises reducing abnormal proteins (e.g., nonfunctional proteins) associated with the muscle disorder. In some embodiments, the treatment comprises alleviating the signs and/or symptoms associated with muscle disorders (e.g., dyskinesia, calf muscle enlargement, muscle pain and stiffness, bradykinesia, learning disorders, gait abnormalities, scoliosis, respiratory problems, dysphagia, arrhythmia, cardiomyopathy, joint dysfunction, hypotonia, respiratory distress, loss of reflex).

In some embodiments, the compositions and constructs disclosed herein are useful for treating Mucopolysaccharidoses (MPS) (e.g., MPS IH/S, MPS IS, MPS II, MPS IIIA, MPS IIIB, MPS IIIC, MPS IIID, MPS IVA, MPS IVB, MPS V, MPS vi, MPS VII, MPS IX). In some embodiments, the treatment comprises introducing a polynucleotide sequence encoding one or more transgenes of interest (e.g., IDUA, IDS, SGSH, NAGLU, HGSNAT, GNS, GALNS, GLB1, ARSB, GUSB, HYAL 1). In some embodiments, the treatment comprises reducing abnormal proteins (e.g., nonfunctional proteins) associated with mucopolysaccharidosis. In some embodiments, treatment comprises alleviating the signs and/or symptoms associated with MPS (e.g., heart abnormalities, breathing irregularities, liver enlargement, spleen enlargement, nerve abnormalities, developmental delay, recurrent infections, persistent runny nose, respiratory upsets, corneal clouding, tongue enlargement, spinal deformity, joint stiffness, carpal tunnel, aortic valve insufficiency, progressive hearing loss, seizures, gait instability, heparan sulfate accumulation, enzyme deficiency, skeletal and muscle tissue abnormalities, heart disease, cysts, soft tissue mass).

In some embodiments, the compositions and constructs disclosed herein are useful for treating aromatic 1-Amino Acid Decarboxylase (AADC) deficiency. In some embodiments, the treatment comprises introducing a polynucleotide sequence encoding one or more transgenes of interest (e.g., DDC, AADC). In some embodiments, the treatment comprises reducing abnormal proteins (e.g., nonfunctional proteins) associated with AADC deficiency. In some embodiments, the treatment comprises alleviating symptoms and/or signs associated with AADC deficiency (e.g., hypotonic, ocular crisis, hypokinesia, hypertone, hypomuscle tone, athetosis, chorea, tremor, hyperhidrosis, hypersalivation, ptosis, nasal obstruction, body temperature instability, hypotension, behavioral problems, insomnia, somnolence, hyporeflexia, hyperreflexia, gastrointestinal problems).

In some embodiments, the compositions and constructs disclosed herein are useful for treating Duchenne Muscular Dystrophy (DMD). In some embodiments, the treatment comprises introducing a polynucleotide sequence encoding one or more transgenes of interest (e.g., a dystrophin, a microdystrophin). In some embodiments, the treatment comprises reducing aberrant proteins (e.g., nonfunctional proteins) associated with DMD. In some embodiments, the treatment comprises alleviating signs and/or symptoms associated with DMD (e.g., bradykinesia, pseudohypertrophy, muscle weakness, gait changes, gao Ershi action (gold's maneuver), cardiomyopathy, respiratory problems, scoliosis, contractures, cognitive disorders).

In some embodiments, the compositions and constructs disclosed herein are useful for treating X-linked myopathy (xltm). In some embodiments, the treatment comprises introducing a polynucleotide sequence encoding one or more transgenes of interest (e.g., MTM 1). In some embodiments, the treatment comprises reducing abnormal proteins (e.g., nonfunctional proteins) associated with xltm. In some embodiments, the treatment comprises alleviating the symptoms and/or symptoms associated with xltm (e.g., muscle weakness, hypotonia, respiratory distress, muscular dysplasia, mid-face dysplasia, long head malformation, malocclusion, ocular muscle paralysis, myopia, large head malformation, reflex disappearance, cryptorch, contracture, scoliosis, hip dysplasia, premature adrenal glands, pyloric stenosis, gall stones, kidney stones, soul (anima), globoid red blood cell disorders, bleeding abnormalities, liver dysfunction).

In some embodiments, the compositions and constructs disclosed herein are useful for treating one or more limb muscular dystrophies (LGMD). In some embodiments, the treatment comprises introducing a polynucleotide sequence encoding one or more transgenes of interest (e.g., a myoglycan gene, alpha myoglycan (SGCA), beta myoglycan (SGCB), gamma myoglycan (SGCG), dysferlin, calpain 3, anoctamin 5, fukutin-related protein (FKRP), etc.). In some embodiments, the treatment comprises reducing abnormal proteins (e.g., nonfunctional proteins) associated with one or more LGMDs. In some embodiments, the treatment comprises alleviating the signs and/or symptoms associated with one or more LGMD (e.g., muscle weakness, atrophy, scoliosis, lordosis, contracture, hypertrophy, cardiomyopathy, fatigue, heart block, arrhythmia, heart failure, dysphagia, dysarthria).

In some embodiments, the compositions and constructs disclosed herein are useful for treating Spinal Muscular Atrophy (SMA). In some embodiments, the treatment comprises introducing a polynucleotide sequence encoding one or more transgenes of interest (e.g., SMN 1). In some embodiments, the treatment comprises reducing abnormal proteins (e.g., nonfunctional proteins) associated with SMA. In some embodiments, the treatment comprises alleviating symptoms and/or signs associated with SMA (e.g., muscle weakness, atrophy, hypotonia, hyporeflexia, loss of reflexes, muscle beam tremor, congenital heart defect, dysphagia, tremor, scoliosis, heart problems).

In some embodiments, the compositions and constructs disclosed herein are useful for treating Parkinson's Disease (PD). In some embodiments, the treatment comprises introducing polynucleotide sequences encoding one or more transgenes of interest (e.g., PRKN, SNCA, PARK, UCHL1, LRRK2, GIGYF2, HTRA2, EIF4G1, TMEM230, CHCHD2, RIC3, VPS35, etc.). In some embodiments, the treatment comprises reducing abnormal proteins (e.g., nonfunctional proteins) associated with parkinson's disease. In some embodiments, the treatment comprises alleviating signs and/or symptoms associated with parkinson's disease (e.g., tremors, rigidity, bradykinesia, akinesia, postural instability, gait disorders, posture disorders, speech and swallowing disorders, cognitive abnormalities).

In some embodiments, the compositions and constructs disclosed herein are useful for treating diseases associated with genetic defects. In some embodiments, the treatment comprises introducing a polynucleotide sequence encoding one or more transgenes of interest disclosed herein. In some embodiments, the treatment comprises reducing abnormal proteins (e.g., nonfunctional proteins) associated with the disease. In some embodiments, the treatment comprises alleviating signs and/or symptoms associated with the disease.

Targeted integration

In some embodiments, the compositions and constructs provided herein direct the integration of a payload (e.g., a transgene and/or a functional nucleic acid) at a target locus (e.g., an endogenous gene). In some embodiments, the compositions and constructs provided herein direct the integration of a payload at a target locus (e.g., a tissue-specific locus) in a particular cell type. In some embodiments, the integration of the payload occurs in a specific tissue (e.g., liver, central Nervous System (CNS), muscle, kidney, blood vessel). In some embodiments, the integration of the payload occurs in multiple tissues (e.g., liver, central Nervous System (CNS), muscle, kidney, blood vessel).

In some embodiments, the compositions and constructs provided herein direct integration at a target locus (e.g., albumin, apolipoprotein A2 (ApoA 2), heme binding) that is effectively supported as a safe harbor site. In some embodiments, the target locus may be selected from any genomic locus suitable for use with the methods and compositions provided herein. In some embodiments, the target locus encodes a polypeptide. In some embodiments, the target locus encodes a polypeptide that is highly expressed in a subject (e.g., a subject that does not have a disease, disorder, or condition, or a subject that has a disease, disorder, or condition). In some embodiments, the integration of the payload occurs at the 5 'or 3' end of one or more endogenous genes (e.g., genes encoding polypeptides). In some embodiments, the integration of the payload occurs between the 5 'or 3' ends of one or more endogenous genes (e.g., genes encoding polypeptides).

In some embodiments, the compositions and constructs provided herein direct integration that is effectively supported at a target locus with little or no off-target integration (e.g., integration at a non-target locus). In some embodiments, the compositions and constructs provided herein direct the integration of a payload at a target locus with reduced off-target integration compared to a reference composition or construct (e.g., relative to a composition or construct that does not have flanking homologous sequences).

In some embodiments, integration of the transgene at the target locus allows expression of the payload without disrupting endogenous gene expression. In some embodiments, integration of the transgene at the target locus allows expression of the payload from the endogenous promoter. In some embodiments, integration of the transgene at the target locus disrupts endogenous gene expression. In some embodiments, integration of the transgene at the target locus disrupts endogenous gene expression without adversely affecting the target cell and/or subject (e.g., by targeting a safe harbor site). In some embodiments, integration of the transgene at the target locus does not require the use of nucleases (e.g., cas protein, endonuclease, TALEN, ZFN). In some embodiments, integration of the transgene at the target locus is facilitated by the use of nucleases (e.g., cas protein, endonuclease, TALEN, ZFN).

In some embodiments, integration of the transgene at the target locus confers a selective advantage (e.g., increased survival in multiple cells relative to other cells in the tissue). In some embodiments, the selective advantage can result in an increased percentage of cells in one or more tissues expressing the transgene.

Composition and method for producing the same

In some embodimentsIn cases, the methods and constructs (e.g., viral vectors) provided herein can be used to produce compositions. In some embodiments, the composition comprises a liquid, solid, and gaseous composition. In some embodiments, the composition comprises additional ingredients (e.g., diluents, stabilizers, excipients, adjuvants). In some embodiments, the additional ingredients may include buffers (e.g., phosphate, citrate, organic acid buffers), antioxidants (e.g., ascorbic acid), low molecular weight polypeptides (e.g., less than 10 residues), various proteins (e.g., serum albumin, gelatin, immunoglobulins), hydrophilic polymers (e.g., polyvinylpyrrolidone), amino acids (e.g., glycine, glutamine, asparagine, arginine, lysine), carbohydrates (e.g., monosaccharides, disaccharides, glucose, mannose, dextrins), chelators (e.g., EDTA), sugar alcohols (e.g., mannitol, sorbitol), salt-forming counter ions (e.g., sodium, potassium), and/or nonionic surfactants (e.g., tween ^TM 、Pluronics ^TM Polyethylene glycol (PEG)), and the like. In some embodiments, the aqueous carrier is an aqueous pH buffered solution.

In some embodiments, the compositions provided herein may be provided in a range of dosages. In some embodiments, the compositions provided herein may be provided in a single dose. In some embodiments, the compositions provided herein may be provided in multiple doses. In some embodiments, the composition is provided over a period of time. In some embodiments, the composition is provided at specific time intervals (e.g., varying time intervals, set time intervals). In some embodiments, the dosage may vary depending on the dosage form and route of administration. In some embodiments, the compositions provided herein may be provided at a dose of between 1e11 and 1e14 vg/kg. In some embodiments, the compositions provided herein may be provided at a dose of between 1e12 and 1e13 vg/kg. In some embodiments, the compositions provided herein may be provided at a dose of between 1e12 and 1e14 vg/kg. In some embodiments, the compositions provided herein may be provided at a dose of between 1e14 and 1e15 vg/kg. In some embodiments, the compositions provided herein may be provided at a dose of no more than 1e14 vg/kg. In some embodiments, the compositions provided herein may be provided at a dose of no more than 1e15 vg/kg.

Route of administration

In some embodiments, the compositions provided herein can be administered to a subject (e.g., parenterally, subcutaneously, intravenously, intracranially, intraspinal, intraocular, intramuscular, intravaginally, intraperitoneally, epidermially, intradermal, rectally, pulmonary, intraosseously, orally, buccally, portal intravenously, intraarterially, intratracheally, or nasally) via any one (or more) of a variety of routes known in the art. In some embodiments, the compositions provided herein can be introduced into cells, which are then introduced into a subject (e.g., liver, muscle, central Nervous System (CNS), blood cells). In some embodiments, the compositions provided herein can be introduced via delivery methods known in the art (e.g., injection, catheter).

Method for producing viral vectors

Production of viral vectors

Prior to the present disclosure, production of viral vectors has typically involved the use of three separate expression constructs (e.g., plasmids), one comprising a viral rep gene or gene variant (e.g., an AAV rep gene) and a viral cap gene or gene variant (e.g., an AAV cap gene), one comprising one or more viral helper genes or gene variants (e.g., an adenovirus helper gene), and one comprising a payload (e.g., a transgene with flanking ITRs). As used herein, an upstream production process refers to steps involved in the production of a viral vector, while a downstream production process refers to steps involved in subsequent processing after the viral vector is produced (i.e., after the desired payload and other components have been integrated into the vector). The present disclosure recognizes, among other things, the limitations of the three plasmid systems previously used to produce viral vectors. In some embodiments, the constructs and methods described in the present disclosure are designed to overcome limitations in previous three plasmid systems for the production of viral vectors via the use of the two plasmid systems described herein.

In some embodiments, production of a viral vector (e.g., an AAV viral vector) may include an upstream step of producing the viral vector (e.g., cell-based culture) and a downstream step of processing the viral vector (e.g., purification, formulation, etc.). In some embodiments, the upstream step may include one or more of the following: cell expansion, cell culture, cell transfection, cell lysis, viral vector production and/or viral vector harvesting.

In some embodiments, the downstream steps may include one or more of the following: separation, filtration, concentration, clarification, purification, chromatography (e.g., affinity, ion exchange, hydrophobic, mixed mode), centrifugation (e.g., ultracentrifugation), and/or formulation.

In some embodiments, the constructs and methods described herein are designed to increase viral vector yield (e.g., AAV vector yield), reduce the level of replication competent viral vectors (e.g., replication competent AAV (rcAAV)), increase viral vector packaging efficiency (e.g., AAV vector capsid packaging), and/or any combination thereof relative to reference constructs or methods, such as those in Xiao et al, 1998, and Grieger et al, 2015, each of which is incorporated herein by reference in its entirety.

Cell lines and transfection reagents

In some embodiments, viral vector production includes the use of cells (e.g., cell cultures). In some embodiments, viral vector production includes cell culture using one or more cell lines (e.g., mammalian cell lines). In some embodiments, viral vector production includes the use of HEK293 cell lines or variants thereof (e.g., HEK293T, HEK293F cell lines). In some embodiments, the cells are capable of growing in suspension. In some embodiments, the cells are comprised of adherent cells. In some embodiments, the cells are capable of growing in a medium that does not contain animal components (e.g., animal serum). In some embodiments, the cells are capable of growing in serum-free medium (e.g., F17 medium, expi293 medium). In some embodiments, viral vector production includes transfecting cells with an expression construct (e.g., a plasmid). In some embodiments, the cells are selected for high expression of a viral vector (e.g., an AAV vector). In some embodiments, the cells are selected for high packaging efficiency of the viral vector (e.g., capsid packaging of AAV vectors). In some embodiments, the cells are selected to increase transfection efficiency (e.g., using chemical transfection reagents, including cationic molecules). In some embodiments, the cells are engineered for high expression of a viral vector (e.g., an AAV vector). In some embodiments, the cells are engineered for high packaging efficiency of the viral vector (e.g., capsid packaging of AAV vectors). In some embodiments, the cells are engineered to increase transfection efficiency (e.g., using chemical transfection reagents, including cationic molecules). In some embodiments, cells may be engineered or selected for two or more of the above attributes. In some embodiments, the cells are contacted with one or more expression constructs (e.g., plasmids). In some embodiments, the cells are contacted with one or more transfection reagents (e.g., chemical transfection reagents, including lipids, polymers, and cationic molecules) and one or more expression constructs. In some embodiments, the cells are contacted with one or more cationic molecules (e.g., cationic lipids, PEI agents) and one or more expression constructs. In some embodiments, the cells are contacted with a peimx agent and one or more expression constructs. In some embodiments, the cell is contacted with a Fectovir-AAV agent and one or more expression constructs. In some embodiments, the cells are contacted with one or more transfection reagents and one or more expression constructs in a particular ratio. In some embodiments, the ratio of transfection reagent to expression construct increases viral vector production (e.g., increased vector yield, increased packaging efficiency, and/or increased transfection efficiency).

Expression constructs

In some embodiments, the expression construct is or comprises one or more polynucleotide sequences (e.g., a plasmid). In some embodiments, the expression construct comprises a specific polynucleotide sequence element (e.g., a payload, a promoter, a viral gene, etc.). In some embodiments, the expression construct comprises a polynucleotide sequence encoding a viral gene (e.g., a rep or cap gene or gene variant, one or more helper viral genes or gene variants). In some embodiments, a particular type of expression construct comprises a particular combination of polynucleotide sequence elements. In some embodiments, a particular type of expression construct does not comprise a particular combination of polynucleotide sequence elements. In some embodiments, the specific expression construct does not comprise polynucleotide sequence elements encoding the rep and cap genes and/or gene variants.

In some embodiments, the expression construct comprises a polynucleotide sequence encoding a wild-type viral gene (e.g., a wild-type rep gene, cap gene, viral accessory gene, or a combination thereof). In some embodiments, the expression construct comprises a polynucleotide sequence encoding a viral accessory gene or gene variant (e.g., a herpes virus gene or gene variant, an adenovirus gene or gene variant). In some embodiments, the expression construct comprises a polynucleotide sequence encoding one or more gene copies that express one or more wild-type Rep proteins (e.g., 1 copy, 2 copies, 3 copies, 4 copies, 5 copies, etc.). In some embodiments, the expression construct comprises a polynucleotide sequence encoding a single gene copy that expresses one or more wild-type Rep proteins (e.g., rep68, rep40, rep52, rep78, or a combination thereof). In some embodiments, the expression construct comprises a polynucleotide sequence encoding one or more wild-type Rep proteins (e.g., rep68, rep40, rep52, rep78, or a combination thereof). In some embodiments, the expression construct comprises a polynucleotide sequence encoding at least four wild-type Rep proteins (e.g., rep68, rep40, rep52, rep 78). In some embodiments, the expression construct comprises a polynucleotide sequence encoding each of Rep68, rep40, rep52, and Rep 78. In some embodiments, the expression construct comprises a polynucleotide sequence encoding one or more wild-type adenovirus helper proteins (e.g., E2 and E4).

In some embodiments, the expression construct comprises a wild-type polynucleotide sequence encoding a wild-type viral gene (e.g., rep gene, cap gene, helper gene). In some embodiments, the expression construct comprises a modified polynucleotide sequence (e.g., codon optimization) encoding a wild-type viral gene (e.g., rep gene, cap gene, helper gene). In some embodiments, the expression construct comprises a modified polynucleotide sequence encoding a modified viral gene (e.g., rep gene, cap gene, helper gene). In some embodiments, the modified viral genes are designed and/or engineered for certain improvements (e.g., improved transduction, tissue specificity, reduced size, reduced immune response, improved packaging, reduced rcAAV levels, etc.).

According to various embodiments, the expression constructs disclosed herein may provide increased flexibility and modularity compared to previous techniques. In some embodiments, the expression constructs disclosed herein may allow for the exchange of various polynucleotide sequences (e.g., different rep genes, cap genes, payloads, helper genes, promoters, etc.) while providing certain improvements (e.g., increased viral vector yield, increased packaging, reduced rcAAV levels, etc.). In some embodiments, the expression constructs disclosed herein are compatible with various upstream production processes (e.g., different cell culture conditions, different transfection reagents, etc.), while providing certain improvements (e.g., increased viral vector yield, increased packaging, reduced rcAAV levels, etc.).

In some embodiments, different types of expression constructs comprise different combinations of polynucleotide sequences. In some embodiments, one type of expression construct comprises one or more polynucleotide sequence elements (e.g., payload, promoter, viral gene, etc.) that are not present in a different type of expression construct. In some embodiments, one type of expression construct comprises a polynucleotide sequence element encoding a viral gene (e.g., rep or cap gene or gene variant) and a polynucleotide sequence element encoding a payload (e.g., transgene and/or functional nucleic acid). In some embodiments, one type of expression construct comprises polynucleotide sequence elements encoding one or more viral genes (e.g., rep or cap genes or gene variants and/or one or more helper viral genes). In some embodiments, one type of expression construct comprises polynucleotide sequence elements encoding one or more viral genes, wherein the viral genes are from one or more viral types (e.g., genes or gene variants from AAV and adenovirus). In some embodiments, the viral gene from adenovirus is a gene and/or a gene variant. In some embodiments, the viral gene from adenovirus is one or more of the following: in some embodiments, the expression constructs are used to produce viral vectors (e.g., via cell culture) & in some embodiments, the expression constructs are contacted with cells that are contacted with one or more transfection reagents (e.g., chemical transfection reagents) & in some embodiments, the expression constructs are contacted with cells that are contacted with one or more transfection reagents at a particular ratio, the cells are contacted with one or more transfection reagents in some embodiments, different types of expression constructs are contacted with cells at a particular ratio (e.g., weight ratio), the cells are contacted with one or more transfection reagents, in some embodiments, a first expression construct comprising one or more viral accessory genes and a second expression construct comprising one or more payloads are contacted with a cell in a ratio (e.g., weight ratio) of about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1.5:1, 1:1.5, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10, in about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1.5:1, 1:5, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, about 10:1, 9:1, 8:1, 7:1, 6:1, 5, 5:1, A ratio of 1:8, 1:9, or 1:10 (e.g., weight ratio) is contacted with the cells. In some embodiments, a first expression construct comprising one or more payloads and a second expression construct comprising one or more viral accessory genes are contacted with the cell at a ratio (e.g., weight ratio) of about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1.5:1, 1:1, 1:1.5, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10 of the first expression construct to the second expression construct. In some embodiments, the cell is contacted (e.g., sequentially or substantially simultaneously) with two or more expression constructs.

In some embodiments, a first expression construct comprising one or more viral accessory genes and a second expression construct comprising one or more payloads are expressed in a sequence greater than or equal to 1:1 up to a 3:1 ratio, wherein the viral titer yield is at least 1.5X greater than that obtained via administration of a reference system (e.g., three plasmids comprising separate plasmids, each plasmid encoding one of 1) AAV rep and AAV cap sequences, 2) related sequences from helper virus, and 3) payload). In some embodiments, a first expression construct comprising one or more viral accessory genes and a second expression construct comprising one or more payloads are expressed in a sequence greater than or equal to 1:1 up to a 5:1 ratio, wherein the viral titer yield is at least 1.5X greater than that obtained via administration of a reference system (e.g., three plasmids comprising separate plasmids, each plasmid encoding one of 1) AAV rep and AAV cap sequences, 2) related sequences from helper virus, and 3) payload). In some embodiments, a first expression construct comprising one or more viral accessory genes and a second expression construct comprising one or more payloads are present in a ratio of greater than or equal to 1:1 up to 6:1, wherein the viral titer yield is at least 1.5X greater than the yield obtained via administration of a reference system (e.g., three plasmids comprising separate plasmids, each plasmid encoding one of 1) AAV rep and AAV cap sequences, 2) related sequences from helper virus, and 3) payload). In some embodiments, a first expression construct comprising one or more viral accessory genes and a second expression construct comprising one or more payloads are contacted with the cell at a ratio of greater than or equal to 1:1 up to 8:1, wherein the viral titer yield is at least 1.5X greater than that obtained via administration of a reference system (e.g., three plasmids comprising separate plasmids, each plasmid encoding one of 1) AAV rep and AAV cap sequences, 2) related sequences from an accessory virus, and 3) payloads). In some embodiments, a first expression construct comprising one or more viral accessory genes and a second expression construct comprising one or more payloads are contacted with the cell at a ratio of greater than or equal to 1:1 up to 10:1, wherein the viral titer yield is at least 1.5X greater than that obtained via administration of a reference system (e.g., three plasmids comprising separate plasmids, each plasmid encoding one of 1) AAV rep and AAV cap sequences, 2) related sequences from an accessory virus, and 3) payloads).

In some embodiments, a first expression construct comprising one or more viral accessory genes and a second expression construct comprising one or more payloads are contacted with a cell at a ratio between 10:1 and 1:1. In some embodiments, a first expression construct comprising one or more viral accessory genes and a second expression construct comprising one or more payloads are contacted with the cell in a ratio between 9:1 and 1:1. In some embodiments, a first expression construct comprising one or more viral accessory genes and a second expression construct comprising one or more payloads are contacted with a cell at a ratio between 8:1 and 1:1. In some embodiments, a first expression construct comprising one or more viral accessory genes and a second expression construct comprising one or more payloads are contacted with the cell in a ratio between 7:1 and 1:1. In some embodiments, a first expression construct comprising one or more viral accessory genes and a second expression construct comprising one or more payloads are contacted with a cell in a ratio between 6:1 and 1:1. In some embodiments, a first expression construct comprising one or more viral accessory genes and a second expression construct comprising one or more payloads are contacted with a cell in a ratio between 5:1 and 1:1. In some embodiments, a first expression construct comprising one or more viral accessory genes and a second expression construct comprising one or more payloads are contacted with the cell in a ratio between 4:1 and 1:1. In some embodiments, a first expression construct comprising one or more viral accessory genes and a second expression construct comprising one or more payloads are contacted with the cell in a ratio between 3:1 and 1:1. In some embodiments, a first expression construct comprising one or more viral accessory genes and a second expression construct comprising one or more payloads are contacted with the cell in a ratio between 2:1 and 1:1.

In some embodiments, a first expression construct comprising one or more viral accessory genes and a second expression construct comprising one or more payloads are contacted with the cell in a ratio between 1:1 and 2:1. In some embodiments, a first expression construct comprising one or more viral accessory genes and a second expression construct comprising one or more payloads are contacted with the cell in a ratio between 1:1 and 3:1. In some embodiments, a first expression construct comprising one or more viral accessory genes and a second expression construct comprising one or more payloads are contacted with the cell in a ratio between 1:1 and 4:1. In some embodiments, a first expression construct comprising one or more viral accessory genes and a second expression construct comprising one or more payloads are contacted with a cell in a ratio between 1:1 and 5:1. In some embodiments, a first expression construct comprising one or more viral accessory genes and a second expression construct comprising one or more payloads are contacted with a cell in a ratio between 1:1 and 6:1. In some embodiments, a first expression construct comprising one or more viral accessory genes and a second expression construct comprising one or more payloads are contacted with the cell in a ratio between 1:1 and 7:1. In some embodiments, a first expression construct comprising one or more viral accessory genes and a second expression construct comprising one or more payloads are contacted with a cell in a ratio between 1:1 and 8:1. In some embodiments, a first expression construct comprising one or more viral accessory genes and a second expression construct comprising one or more payloads are contacted with the cell in a ratio between 1:1 and 9:1. In some embodiments, a first expression construct comprising one or more viral accessory genes and a second expression construct comprising one or more payloads are contacted with a cell at a ratio between 1:1 and 10:1. In some embodiments, a first expression construct comprising one or more viral accessory genes and a second expression construct comprising one or more payloads are contacted with a cell at a ratio of 1.5:1.

In some embodiments, the expression construct comprises one or more polynucleotide sequences encoding elements (e.g., selectable markers, origins of replication) necessary for cell culture (e.g., bacterial cell culture, mammalian cell culture). In some embodiments, the expression construct comprises one or more polynucleotide sequences encoding an antibiotic resistance gene (e.g., kanamycin resistance gene, ampicillin resistance gene). In some embodiments, the expression construct comprises one or more polynucleotide sequences encoding a bacterial origin of replication (e.g., colE1 origin of replication).

In some embodiments, the expression construct comprises one or more transcription termination sequences (e.g., polyA sequences). In some embodiments, the expression construct comprises one or more of BGH polyA, FIX polyA, SV40 polyA, synthetic polyA, or a combination thereof. In some embodiments, the expression construct comprises one or more transcription termination sequences downstream of a particular sequence element (e.g., rep or cap gene or gene variant). In some embodiments, the expression construct comprises one or more transcription termination sequences upstream of a particular sequence element (e.g., rep or cap gene or gene variant).

In some embodiments, the expression construct comprises one or more intron sequences. In some embodiments, the expression construct comprises one or more introns of different origin (e.g., known genes), including but not limited to FIX introns, albumin introns, or combinations thereof. In some embodiments, the expression construct comprises one or more introns of different lengths (e.g., 133bp to 4 kb). In some embodiments, the expression construct comprises one or more intron sequences upstream of a specific sequence element (e.g., rep or cap gene or gene variant). In some embodiments, the expression construct comprises one or more intron sequences within a specific sequence element (e.g., rep or cap gene or gene variant). In some embodiments, the expression construct comprises one or more intron sequences downstream of a specific sequence element (e.g., rep or cap gene or gene variant). In some embodiments, the expression construct comprises one or more intron sequences following the promoter (e.g., p5 promoter). In some embodiments, the expression construct comprises one or more intron sequences prior to the rep gene or gene variant. In some embodiments, the expression construct comprises one or more intron sequences between the promoter and the rep gene or gene variant. In some embodiments, the compositions provided herein comprise an expression construct. In some embodiments, the composition comprises: (i) A first expression construct comprising a polynucleotide sequence encoding one or more rep genes and a polynucleotide sequence encoding one or more wild-type adenovirus helper proteins; and (ii) a second expression construct comprising a polynucleotide sequence encoding one or more cap genes and one or more payloads.

In some embodiments, the composition comprises a first expression construct comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to a sequence in table 1C below, or a variant thereof. In some embodiments, the composition comprises a first expression construct comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to a portion of a sequence in table 1C below, or a variant thereof. In some embodiments, the composition comprises a first expression construct consisting of the sequences in table 1C below. In some embodiments, the composition comprises a first expression construct consisting of the sequences in table 1C below. In some embodiments, the composition comprises a first expression construct consisting of a portion of the sequence in table 1C below.

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In some embodiments, the compositions provided herein comprise an expression construct. In some embodiments, the composition comprises: (i) A first expression construct comprising a polynucleotide sequence encoding one or more rep genes and a polynucleotide sequence encoding one or more wild-type adenovirus helper proteins; and (ii) a second expression construct comprising a polynucleotide sequence encoding one or more cap genes and one or more payloads.

In some embodiments, the composition comprises a second expression construct comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to a sequence in table 1D below, or a variant thereof. In some embodiments, the composition comprises a second expression construct comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to a portion of the sequence in table 1D below, or a variant thereof. In some embodiments, the composition comprises a second expression construct consisting of the sequences in table 1D below. In some embodiments, the composition comprises a second expression construct consisting of a portion of the sequence in table 1D below.

In some embodiments, the composition comprises a second expression construct comprising a sequence that hybridizes to SEQ ID NO:11 has a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity. In some embodiments, the composition comprises a second expression construct consisting of: (i) SEQ ID NO:11; (ii) a polynucleotide sequence encoding a cap gene; and (iii) a polynucleotide sequence encoding a payload (e.g., a transgene, ITR, 2A peptide, homology arm, or combination thereof). In some embodiments, the composition comprises a second expression construct comprising the amino acid sequence of SEQ ID NO:11, wherein a polynucleotide sequence comprising a sequence encoding a cap gene is inserted before position 2025 and a polynucleotide sequence encoding a payload comprising a polynucleotide sequence encoding a transgene is inserted after position 2663. In some embodiments, the composition comprises a second expression construct consisting of SEQ ID NO:11, wherein a polynucleotide sequence encoding a cap gene is inserted before position 2025 and a polynucleotide sequence encoding a payload comprising a polynucleotide sequence encoding a transgene is inserted after position 2663.

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In some embodiments, the composition comprises: (i) A first expression construct comprising a polynucleotide sequence encoding one or more rep genes and a polynucleotide sequence encoding one or more wild-type adenovirus helper proteins; and (ii) a second expression construct comprising a polynucleotide sequence encoding a capsid protein and a polynucleotide sequence encoding a payload comprising a polynucleotide sequence encoding a gene (or variant thereof). In some embodiments, the composition comprises: (i) A first expression construct comprising the sequence outlined in figure 29; and (ii) a second expression construct comprising a polynucleotide sequence encoding the capsid outlined in figure 29 and a polynucleotide sequence encoding a payload comprising a polynucleotide sequence encoding the gene (or variant thereof) outlined in figure 29. In some embodiments, the composition comprises: (i) A first expression construct comprising the sequence outlined in figure 29; and (ii) a second expression construct comprising a polynucleotide sequence encoding the capsid outlined in figure 29 and a polynucleotide sequence encoding a payload comprising a polynucleotide sequence encoding the gene (or variant thereof) outlined in figure 29, wherein the first and second expression constructs are present in combination, as outlined in a single line of figure 29. In some embodiments, the composition comprises: (i) A first expression construct comprising the sequence outlined in figure 29; and (ii) a second expression construct comprising a polynucleotide sequence encoding the capsid outlined in figure 29 and a polynucleotide sequence encoding a payload comprising a polynucleotide sequence encoding the gene (or variant thereof) outlined in figure 29, wherein the first and second expression constructs are present in combination, as outlined in a single line of figure 29, and wherein a composition comprising such a combination of the first expression construct and the second expression construct is administrable to one or more cells to produce an exemplary viral vector product, as outlined in figure 29.

In some embodiments, the composition comprises: (i) A first expression construct consisting of the sequence outlined in figure 29; and (ii) a polypeptide consisting of SEQ ID NO:11, wherein the sequence in SEQ ID NO:11, and a polynucleotide sequence encoding a payload comprising a polynucleotide sequence encoding the gene (or variant thereof) outlined in fig. 29, and in SEQ ID NO:11 into the polynucleotide sequence encoding the capsid outlined in figure 29. In some embodiments, the composition comprises: (i) a first expression construct consisting of the sequence in figure 29; and (ii) a polypeptide consisting of SEQ ID NO:11, wherein the sequence in SEQ ID NO:11, and a polynucleotide sequence encoding a payload comprising a polynucleotide sequence encoding the gene (or variant thereof) outlined in fig. 29, and in SEQ ID NO:11 into the capsid as outlined in figure 29, wherein the first and second expression constructs are present in combination, as outlined in a single line of figure 29. In some embodiments, the composition comprises: (i) a first expression construct consisting of the sequence in figure 29; and (ii) a polypeptide consisting of SEQ ID NO:11, wherein the sequence in SEQ ID NO:11, comprising a polynucleotide sequence encoding a payload comprising a polynucleotide sequence encoding a gene (or variant thereof) as outlined in figure 29, and in SEQ ID NO:11, wherein the first and second expression constructs are present in combination, as outlined in a single line of fig. 29, and wherein a composition comprising such a combination of the first expression construct and the second expression construct can be administered to one or more cells to produce an exemplary viral vector product, as outlined in fig. 29.

In some embodiments, the composition comprises: (i) A first expression construct consisting of the sequence outlined in figure 29; and (ii) a polypeptide consisting of SEQ ID NO:12, wherein the sequence in SEQ ID NO:12, a polynucleotide sequence encoding a payload comprising a polynucleotide sequence encoding the gene (or variant thereof) outlined in fig. 29, and having the sequence set forth in SEQ ID NO:12, between positions 2446-2453, a polynucleotide sequence encoding the capsid outlined in figure 29 is inserted. In some embodiments, the composition comprises: (i) a first expression construct consisting of the sequence in figure 29; and (ii) a polypeptide consisting of SEQ ID NO:12, wherein the sequence in SEQ ID NO:12, a polynucleotide sequence encoding a payload comprising a polynucleotide sequence encoding the gene (or variant thereof) outlined in fig. 29, and having the sequence set forth in SEQ ID NO:12, wherein the first and second expression constructs are present in combination, as outlined in the single line of fig. 29. In some embodiments, the composition comprises: (i) a first expression construct consisting of the sequence in figure 29; and (ii) a polypeptide consisting of SEQ ID NO:12, wherein the sequence in SEQ ID NO:12, a polynucleotide sequence encoding a payload comprising a polynucleotide sequence encoding the gene (or variant thereof) outlined in fig. 29, and having the sequence set forth in SEQ ID NO:12, wherein the first and second expression constructs are present in combination, as outlined in a single line of fig. 29, and wherein a composition comprising such a combination of the first expression construct and the second expression construct can be administered to one or more cells to produce an exemplary viral vector product, as outlined in fig. 29. In some embodiments, the polynucleotide sequence is inserted into SEQ ID NO:12 (e.g., an insertion between positions 2011-2026 results in a deletion of a previous nucleotide at positions 2012-2025 and an insertion of a polynucleotide sequence).

In some embodiments, the composition comprises a first expression construct (e.g., a plasmid) comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to a sequence in table 1C or a variant thereof, and a second expression construct (e.g., a plasmid) comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to a sequence in table 1D or a variant thereof. In some embodiments, the composition comprises a first plasmid (e.g., rep/helper plasmid) comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to a sequence in table 1C or a variant thereof, and a second plasmid (e.g., payload/Cap plasmid) comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to a sequence in table 1D or a variant thereof.

Methods of characterizing AAV viral vectors

According to various embodiments, the viral vectors may be characterized via evaluation of various characteristics and/or features. In some embodiments, viral vector evaluation may be performed at different points in the production process. In some embodiments, viral vector evaluation may be performed after the upstream production step is completed. In some embodiments, viral vector evaluation may be performed after the downstream production steps are completed.

Viral yield

In some embodiments, characterization of the viral vector includes assessing viral yield (e.g., viral titer). In some embodiments, characterization of the viral vector includes assessing viral yield prior to purification and/or filtration. In some embodiments, characterization of the viral vector includes assessing viral yield after purification and/or filtration. In some embodiments, characterization of the viral vector includes assessing whether the viral yield is greater than or equal to 1e10 vg/mL.

In some embodiments, characterization of the viral vector includes assessing whether the viral yield in the crude cell lysate is greater than or equal to 1e11 vg/mL. In some embodiments, characterization of the viral vector includes assessing whether the viral yield in the crude cell lysate is greater than or equal to 5e11vg/mL. In some embodiments, characterization of the viral vector includes assessing whether the viral yield in the crude cell lysate is greater than or equal to 1e12 vg/mL. In some embodiments, characterization of the viral vector includes assessing whether the viral yield in the crude lysate is between 5e9 vg/mL and 5e11vg/mL. In some embodiments, characterization of the viral vector includes assessing whether the viral yield in the crude lysate is between 5e9 vg/mL and 1e10 vg/mL. In some embodiments, characterization of the viral vector includes assessing whether the viral yield in the crude lysate is between 1e10 vg/mL and 1e11 vg/mL. In some embodiments, characterization of the viral vector includes assessing whether the viral yield in the crude lysate is between 1e11 vg/mL and 1e12 vg/mL. In some embodiments, characterization of the viral vector includes assessing whether the viral yield in the crude lysate is between 1e12vg/mL and 1e13 vg/mL.

In some embodiments, characterization of the viral vector includes assessing whether the viral yield in the purified drug is greater than or equal to 1e11 vg/mL. In some embodiments, characterization of the viral vector includes assessing whether the viral yield in the purified drug is greater than or equal to 1e12 vg/mL. In some embodiments, characterization of the viral vector includes assessing whether the viral yield in the purified drug is between 1e10 vg/mL and 1e15 vg/mL. In some embodiments, characterization of the viral vector includes assessing whether the viral yield in the purified drug is between 1e11 vg/mL and 1e15 vg/mL. In some embodiments, characterization of the viral vector includes assessing whether the viral yield in the purified drug is between 1e12 vg/mL and 1e14 vg/mL. In some embodiments, characterization of the viral vector includes assessing whether the viral yield in the crude lysate is between 1e13 vg/mL and 1e14 vg/mL.

In some embodiments, the methods and compositions provided herein can provide similar or increased viral vector production compared to previous methods known in the art. For example, in some embodiments, provided methods for producing and/or manufacturing viral vectors include the use of a two-plasmid transfection system that provides similar or increased viral vector yield as compared to a three-plasmid system. In some embodiments, provided methods for producing and/or manufacturing viral vectors include the use of a two plasmid transfection system with a combination of specific sequence elements that provides similar or increased viral vector yield as compared to a two plasmid system with a combination of different sequence elements. In some embodiments, provided methods for producing and/or manufacturing viral vectors include the use of a two-plasmid transfection system with a specific plasmid ratio that provides similar or increased viral vector yield as compared to a two-plasmid system with a different plasmid ratio. In some embodiments, provided methods for producing and/or manufacturing viral vectors include the use of a dual plasmid transfection system with specific plasmid ratios that provide similar or increased viral vector yield under specific culture conditions as compared to a reference (e.g., dual plasmid system, three plasmid system with different plasmid ratios). In some embodiments, provided methods for producing and/or making viral vectors include the use of a dual plasmid transfection system with specific plasmid ratios that provide similar or increased viral vector yield under large scale culture conditions (e.g., greater than 100mL, greater than 250mL, greater than 1L, greater than 10L, greater than 20L, greater than 30L, greater than 40L, greater than 50L, etc.) as compared to a reference (e.g., dual plasmid system, three plasmid system with different plasmid ratios).

Virus package

In some embodiments, characterization of the viral vector includes assessing viral packaging efficiency (e.g., percentage of intact capsids relative to empty capsids). In some embodiments, characterization of the viral vector includes assessing viral packaging efficiency prior to purification and/or whole capsid enrichment (e.g., cesium chloride-based density gradient, iodixanol-based density gradient, or ion exchange chromatography). In some embodiments, characterization of the viral vector includes assessing whether the viral packaging efficiency is greater than or equal to 20% (e.g., 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%) prior to purification and/or filtration. In some embodiments, characterization of the viral vector includes assessing viral packaging efficiency after purification and/or whole capsid enrichment. In some embodiments, characterization of the viral vector includes assessing whether the viral packaging efficiency is greater than or equal to 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%) after purification and/or filtration.

In some embodiments, the methods and compositions provided herein can provide similar or increased packaging efficiency as compared to previous methods known in the art. For example, in some embodiments, provided methods for producing and/or manufacturing viral vectors include the use of a two-plasmid transfection system that provides similar or increased packaging efficiency as compared to a three-plasmid system. In some embodiments, provided methods for producing and/or manufacturing viral vectors include the use of a two plasmid transfection system with a combination of specific sequence elements that provides similar or increased packaging efficiency as compared to a two plasmid system with a different combination of sequence elements. In some embodiments, provided methods for producing and/or manufacturing viral vectors include the use of a two-plasmid transfection system with a specific plasmid ratio that provides similar or increased packaging efficiency as compared to a two-plasmid system with a different plasmid ratio.

Replication competent vector levels

In some embodiments, characterization of the viral vector includes assessing replication competent vector levels. In some embodiments, characterization of the viral vector includes assessing replication competent vector levels prior to purification and/or filtration. In some embodiments, characterization of the viral vector includes assessing replication competent vector levels after purification and/or filtration. In some embodiments, characterization of the viral vector includes assessing whether replication competent vector levels are less than or equal to 1rcAAV/1E10vg.

In some embodiments, the methods and compositions provided herein can provide similar or reduced replication competent vector levels compared to previous methods known in the art. For example, in some embodiments, provided methods for producing viral vectors include the use of a dual plasmid transfection system that provides similar or reduced replication competent vector levels compared to a three plasmid system. In some embodiments, provided methods for producing viral vectors include the use of a two plasmid transfection system with a combination of specific sequence elements that provides similar or reduced replication competent vector levels as compared to a two plasmid system with a combination of different sequence elements. In some embodiments, provided methods for producing viral vectors include the use of a two plasmid transfection system with one or more intron sequences inserted into the rep gene, which methods provide similar or reduced replication competent vector levels compared to a two plasmid system without the intron sequences.

Example

Example 1: the dual plasmid system can increase volume yield

This example demonstrates, among other things, that a two-plasmid system can produce increased viral yields compared to a three-plasmid system at a particular plasmid ratio.

HEK293F cells were expanded for vector production. In 100mL of Expi293 medium in a 500mL flask, the cells divide into 2e6 cells/mL. Plasmid mixtures for the various transfection conditions outlined in tables 1 and 1A below were prepared and filtered through a 0.22 μm filter unit. The transfection reagent mixture (e.g., PEI) was prepared according to the manufacturer's protocol. The plasmid and transfection reagent mixture are combined to produce a single transfection mixture. 20mL of the transfection mixture was added to 100mL of HEK293F cells in a 500mL flask and incubated at 37℃for 72 hours.

In some embodiments, plasmids tested in the two-plasmid system comprise AAV Rep sequences and related sequences from helper virus ("Rep/helper plasmid") or AAV Cap sequences and payloads ("payload/Cap plasmid"). In some embodiments, the plasmids tested in the three plasmid system comprise separate plasmids, each encoding one of the following: 1) AAV rep and AAV cap sequences, 2) related sequences from helper viruses, and 3) payloads. In the experiments outlined below, the human factor IX gene sequence ("mHA-FIX") with a flanking mouse albumin homology arm was tested as the payload and AAV-DJ was tested as the viral capsid.

Table 1: transfection conditions for the two plasmid system. The relative amounts are shown, normalized so that the sum of the Rep/assist and payload/Cap amounts is 1.

Capsid shell	Payload	Rep/helper plasmid	payload/Cap plasmid
				AAV-DJ	mHA-hFIX	0.75	0.25
AAV-DJ	mHA-hFIX	0.667	0.333
				AAV-DJ	mHA-hFIX	0.6	0.4
AAV-DJ	mHA-hFIX	0.556	0.444
				AAV-DJ	mHA-hFIX	0.5	0.5
AAV-DJ	mHA-hFIX	0.444	0.556
				AAV-DJ	mHA-hFIX	0.4	0.6
AAV-DJ	mHA-hFIX	0.333	0.667
				AAV-DJ	mHA-hFIX	0.25	0.75

Table 1A: transfection conditions for the three plasmid system.

Capsid shell	Payload	Helper plasmid	Rep/Cap plasmid	Payload plasmid
					AAV-DJ	mHA-hFIX	0.43	0.35	0.22

Benzonase was added to 10 Xlysis buffer (10% v/v Tween 20, 500mM Trix-HCl pH8.0, 20mM MgCl) ₂ pH8.0, milli-Q water), 100U benzonase per mL of lysis buffer. 22mL of the lysate and benzonase mixture was added to each cell culture flask and placed in an incubator and shaken at 120 or 130rpm for 90 minutes at 37 ℃. Next, 24.2mL of sterile filtered 5M NaCl was added to each flask (to reach a target concentration of 0.5M naci) and incubated at 37 ℃ for 30 minutes while shaking at 130 rpm. The whole lysed culture or a 40mL aliquot was used for the next step. The lysed culture was then spun at 5000rpm for 10 minutes at 4 ℃. The supernatant was retained for analysis of vector titer by ddPCR and pellet was discarded.

The present disclosure demonstrates, among other things, that a two plasmid transfection system with specific sequence features can increase volumetric yield. In some embodiments, as shown in fig. 1 and 2, transfecting a two plasmid system with certain relative plasmid ratios can further increase yield, e.g., as compared to a three plasmid "triple transfection" system.

Example 2: the dual plasmid system can increase volume yield at a certain plasmid ratio

HEK293F cells were expanded for vector production. In 200mL of Expi293 medium in a 500mL flask, the cells split into 2e6 cells/mL. Plasmid mixtures for the various transfection conditions outlined in tables 2 and 2A below were prepared and filtered through a 0.22 μm filter unit. The transfection reagent mixture (e.g., PEI) was prepared according to the manufacturer's protocol. The plasmid and transfection reagent mixture are combined to produce a single transfection mixture. 20mL of the transfection mixture was added to 100mL of HEK293F cells in a 500mL flask and incubated at 37℃for 72 hours.

Table 2: transfection conditions for the two plasmid system. The relative amounts are shown, normalized so that the sum of the Rep/assist and payload/Cap amounts is 1.

Capsid shell	Payload	Rep/helper plasmid	payload/Cap plasmid
				AAV-DJ	mHA-hFIX	0.909	0.091
AAV-DJ	mHA-hFIX	0.888	0.111
				AAV-DJ	mHA-hFIX	0.857	0.143
AAV-DJ	mHA-hFIX	0.8	0.2
				AAV-DJ	mHA-hFIX	0.667	0.333
AAV-DJ	mHA-hFIX	0.5	0.5
				AAV-DJ	mHA-hFIX	0.333	0.667
AAV-DJ	mHA-hFIX	0.2	0.8
				AAV-DJ	mHA-hFIX	0.143	0.857
AAV-DJ	mHA-hFIX	0.111	0.888
				AAV-DJ	mHA-hFIX	0.091	0.909

Table 2A: transfection conditions for the three plasmid system.

Benzonase was added to 10 Xlysis buffer (10% v/v Tween 20, 500mM Trix-HCl pH8.0, 20mM MgCl) ₂ pH8.0, milli-Q water), 100U benzonase per mL of lysis buffer. 22mL of the lysate and benzonase mixture was added to each cell culture flask and placed in an incubator and shaken at 120rpm for 90 minutes at 37 ℃. Next, 24.2mL of sterile filtered 5M NaCl was added to each flask (to reach the target concentration of 0.5M NaCl) and incubated at 37 ℃ for 30 minutes upon shaking at 120 rpm. A40 mL aliquot was taken for the next step. The lysed culture was then spun at 5000rpm for 10 minutes at 4 ℃. The supernatant was retained for analysis of vector titer by ddPCR and pellet was discarded.

The present disclosure demonstrates, among other things, that certain transfection conditions of a two-plasmid transfection system can yield surprising and unexpected volumetric yield improvements (e.g., as compared to a three-plasmid "triple transfection" system). As shown in fig. 3, a relatively small change in the ratio between the two plasmids can produce a significant change in viral yield.

Example 3: the dual plasmid system can increase the volumetric yield of multiple AAV capsids

This example demonstrates that various AAV capsids are particularly useful in a two plasmid system to produce high viral yields.

HEK293F cells were expanded for vector production. In 200mL of Expi293 medium in a 500mL flask, the cells split into 2e6 cells/mL. Plasmid mixtures for the various transfection conditions outlined in tables 3 and 3A below were prepared and filtered through a 0.22 μm filter unit. The transfection reagent mixture (e.g., PEI) was prepared according to the manufacturer's protocol. The plasmid and transfection reagent mixture are combined to produce a single transfection mixture. 20mL of the transfection mixture was added to 100mL of HEK293F cells in a 500mL flask and incubated at 37℃for 72 hours.

In some embodiments, plasmids tested in the two-plasmid system comprise AAV Rep sequences and related sequences from helper virus ("Rep/helper plasmid") or AAV Cap sequences and payloads ("payload/Cap plasmid"). In some embodiments, the plasmids tested in the three plasmid system comprise separate plasmids, each encoding one of the following: 1) AAV rep and AAV cap sequences, 2) related sequences from helper viruses, and 3) payloads. In the experiments outlined below, a human factor IX gene sequence ("mHA-FIX") compatible with the GeneRide system with a flanking mouse albumin homology arm was tested as a payload. Using different plasmid ratios as described in table 3, multiple AAV Cap genes encoding different chimeric capsids were evaluated in the payload/Cap plasmid.

Table 3: transfection conditions for two plasmid systems with various AAV capsids. The plasmid ratio (w/w) is shown as Rep/helper plasmid: payload/Cap plasmid.

Table 3A: transfection conditions for the three plasmid system.

5mL of sample was collected per 500mL flask. Benzonase was mixed with Expi293 medium using 2uL Benzonase (about 250U/uL) and 50uL medium. Preparation for 30 samples (60 uL benzonase and 1500uL Medium)And (5) mother mixed liquor. 50uL of the master mix was added to each sample, 100U benzonase per 1mL culture volume. The samples were incubated at 37℃for 15 minutes with shaking. Preparation of 10 Xlysis buffer (10% v/v Tween 20, 500mM Trix-HCl pH8.0, 20mM MgCl) ₂ pH8.0, milli-Q water) and 500uL (10% culture volume) was added to each sample, followed by shaking incubation at 37℃for 90 minutes. Next, 500uL of sterile filtered 5M NaCl was added to each flask (to achieve the target concentration of 0.5M NaCl) and incubated at 37 ℃ for 30 minutes upon shaking. The lysed culture was then spun at 3900rpm for 10 minutes. The supernatant was retained for analysis of vector titer by ddPCR and pellet was discarded.

The present disclosure demonstrates, among other things, that a dual plasmid transfection system can produce surprising and unexpected improvements in volumetric yield for a variety of different capsids (e.g., as compared to a three plasmid "triple transfection" system). As shown in FIG. 4, AAV-DJ, LK03, AAVC11.04, AAVC11.11, and AAVC11.12 all appear to produce similar viral yield trends for the three different plasmid ratios tested. 1.5: ratio 1 Rep/helper plasmid: the payload/Cap plasmid always produced the highest yield for each capsid. These data indicate that in some embodiments, a two plasmid system with a specific ratio of Rep/helper plasmid to payload/Cap plasmid can be widely applied to different capsids of interest to increase volumetric yield.

Example 4: dual plasmid systems can use alternative payloads to increase volumetric yields of multiple AAV capsids

This example demonstrates that a variety of AAV capsids and payloads are particularly useful in a two plasmid system to produce high viral yields.

HEK293F cells were expanded for vector production. In 200mLExpi293 medium in a 500mL flask, the cells divide into 2e6 cells/mL. Plasmid mixtures for the various transfection conditions outlined in tables 4 and 4A below were prepared and filtered through a 0.22 μm filter unit. The transfection reagent mixture (e.g., PEI) was prepared according to the manufacturer's protocol. The plasmid and transfection reagent mixture are combined to produce a single transfection mixture. 20mL of the transfection mixture was added to 100mL of HEK293F cells in a 500mL flask and incubated at 37℃for 72 hours.

In some embodiments, plasmids tested in the two-plasmid system comprise AAV Rep sequences and related sequences from helper virus ("Rep/helper plasmid") or AAV CAP sequences and payload ("payload/CAP plasmid"). In some embodiments, the plasmids tested in the three plasmid system comprise separate plasmids, each encoding one of the following: 1) AAV rep and AAV cap sequences, 2) related sequences from helper viruses, and 3) payloads. In the experiments outlined below, human factor IX gene sequences under the control of Liver Specific Promoters (LSPs) were tested as payloads. Using different plasmid ratios as described in table 4, multiple AAV Cap genes encoding different chimeric capsids were evaluated in the payload/Cap plasmid.

Table 4: transfection conditions for two plasmid systems with various AAV capsids. The plasmid ratio (w/w) is shown as Rep/helper plasmid: payload/Cap plasmid.

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Table 4A: transfection conditions for the three plasmid system.

Capsid shell	Payload	Helper plasmid	Rep/Cap plasmid	Payload plasmid
					AAV-DJ	LSP-hFIX	0.43	0.35	0.22
LK03	LSP-hFIX	0.43	0.35	0.22
					AAVC11.01	LSP-hFIX	0.43	0.35	0.22
AAVC11.04	LSP-hFIX	0.43	0.35	0.22
					AAVC11.06	LSP-hFIX	0.43	0.35	0.22
AAVC11.09	LSP-hFIX	0.43	0.35	0.22
					AAVC11.11	LSP-hFIX	0.43	0.35	0.22
AAVC11.12	LSP-hFIX	0.43	0.35	0.22
					AAVC11.13	LSP-hFIX	0.43	0.35	0.22
AAVC11.15	LSP-hFIX	0.43	0.35	0.22

5mL of sample was collected per 500mL flask. Benzonase was mixed with Expi293 medium using 2uL Benzonase (about 250U/uL) and 50uL medium. A master mix was prepared for 30 samples (60 uL benzonase and 1500uL medium). 50uL of the master mix was added to each sample, 100U benzonase per 1mL culture volume. The samples were incubated at 37℃for 15 minutes with shaking. Preparation of 10 Xlysis buffer (10% v/v Tween 20, 500mM Trix-HCl pH8.0, 20mM MgCl) ₂ pH8.0, milli-Q water) and 500uL (10% culture volume) was added to each sample, followed by shaking incubation at 37℃for 90 minutes. Next, 500uL of sterile filtered 5M NaCl was added toIn each flask (to achieve the target concentration of 0.5M NaCl) and at 37 ℃ under shaking for 30 minutes. The lysed culture was then spun at 3900rpm for 10 minutes at room temperature. The supernatant was retained for analysis of vector titer by ddPCR and pellet was discarded.

The present disclosure demonstrates, among other things, that dual plasmid transfection systems can yield surprising and unexpected volumetric yield improvements (e.g., compared to a three plasmid "triple transfection" system) for different capsids with payloads suitable for use in conventional gene therapy (e.g., human factor IX). As shown in FIG. 5, AAV-DJ, LK03, AAVC11.01, AAVC11.04, AAVC11.06, AAVC11.09, AAVC11.11, AAVC11.12, AAVC11.13, and AAVC11.15 all appear to produce similar viral yield trends for the three different plasmid ratios tested. The 1.5:1 ratio of Rep/helper plasmid to payload/Cap plasmid consistently produced the highest yields for each capsid, similar to that observed in mHA-hFIX payload, except for AAVC13, where the 1.5:1 ratio produced yields similar to those seen in the three plasmid system. These data indicate that in some embodiments, a two plasmid system with a specific ratio between Rep/helper plasmid and payload/Cap plasmid can be successfully used for different capsids and different genes of interest (e.g., for both conventional gene therapy and GeneRide methods) to increase volumetric yield.

Example 5: the dual plasmid system can be combined with various transfection reagents, various media and different culture containers (shake flask and stirred tank bioreactor)

This example shows that the two plasmid system can be combined with various transfection reagents (PEIMAX and FectoVIR-AAV), various media (Expi 293 and F17) and different culture systems (shake flask and stirred tank bioreactor, amBr250 system) to further increase viral genome yield.

HEK293F cells were expanded in 500-mL shake flasks for vector production. Cell counts were first recorded on a ViCell XR cell counter to ensure VCD was between 2.0e6-2.6e6 cells/mL and survival was higher than 95% at transfection. The transfection mixture was then prepared by first pre-weighing the Expi293 medium in two separate containers "DNA medium" and "transfection reagent medium", each containing equal volume requirements from the transfection mixture calculation. The transfection reagent was then added to the flask labeled "transfection reagent medium" and set aside. For the 3 plasmid transfection system, the mass fractions of p-helper, pRep/Cap and pGOI were 0.43, 0.35 and 0.22, respectively. For the 2 plasmid transfection system, the mass fractions of Rep/helper plasmid and payload/Cap plasmid were 0.60 and 0.40, respectively (1.5:1 plasmid ratio). The plasmid was sterile filtered through a Corning 0.22um PES bottle top filter by first wetting the membrane with medium in the bottle labeled "DNA medium", adding the appropriate amount of pDNA to the bottle top, opening the vacuum of the filter, and finally rinsing the remaining DNA on the filter with the remaining medium in the "DNA medium" bottle. After preparing the transfection reagent/medium and DNA/medium solutions at a 1:1 volume ratio, the two mixtures were combined into separate containers and inverted 10 times to begin the compounding process. The transfection mixture was then transferred to an incubator at 37℃and shaken at 95RPM for 15 minutes when PEIMAX was used and 30 minutes when FectoVIR-AAV was used. Unless otherwise indicated, after the lapse of time, the transfection mixture was added to the medium at a 10% culture volume fraction (e.g., 20mL of transfection mixture was added to 200mL of culture) and grown at 37 ℃ for 72 hours.

Cells were harvested 72 hours after transfection of the culture. 5mL of the culture was transferred to a 15mL centrifuge tube and 50uL of an Expi293 medium solution containing 10 units/uL of benzonase was added to the tube and shaken horizontally in an incubator at 37℃and 145RPM for 15 minutes. 500uL of lysis buffer (500 mM Tris pH 8, 20mM MgCl2, 10% polysorbate-20) was then added to the tube and incubated under the same conditions for 90 minutes. Finally, 500ul 5m NaCl was added to the tube and incubated for 30 minutes under the same conditions. After NaCl incubation, the cell lysate was centrifuged at 3200g in a centrifuge to clarify the harvested medium. 1mL of supernatant containing AAV particles was collected in a 1.5mL microcentrifuge tube and stored at-80℃until sample analysis was ready. The results of volume titer production are presented in figure 6.

Table 5: the conditions of the transfection system and the transfection reagent were evaluated.

Conditions (conditions)	Description of the Carrier	Transfection system	Transfection reagent
				l	LK03/hHA-hUGT1A1	3 plasmid	PEIMAX
2	LK03/hHA-hUGT1A1	3 plasmid	FectoVIR-AAV
				3	LK03/LSP-hFIX	3 plasmid	PEIMAX
4	LK03/LSP-hFIX	3 plasmid	FectoVIR-AAV
				5	LK03/LSP-hFIX	2 plasmid	PEIMAX
6	LK03/LSP-hFIX	2 plasmid	FectoVIR-AAV

Table 5A: transfection parameters of different transfection reagents.

Parameters (parameters)	PEIMAX	FectoVIR-AAV
			Total DNA (ug) of 6 cells per 1e	0.75	0.75
TR: DNAw/w ratio	1.5	1.5
			Percentage of transfection mix of culture volume (%)	10	10
Time of compounding (minutes)	15	30

The same transfection conditions were then tested in an AmBr250 bioreactor to determine if similar trends in viral yield could be obtained in a laboratory scale stirred tank bioreactor that simulates the conditions of larger scale manufacturing.

Table 5B: the bioreactor verifies the conditions studied.

Conditions (conditions)	Description of the Carrier	Transfection system	Transfection reagent
				1	LK03/LSP-hFIX	3 plasmid	PEIMAX
2	LK03/LSP-hFIX	2 plasmid	PEIMAX
				3	LK03/LSP-hFIX	2 plasmid	FectoVIR-AAV

Table 5C: the bioreactor studies titer and fold changes between conditions.

Conditions (conditions)	Titer (vg/mL)	Fold increase
			3 plasmid PEIMAX	2.31e10	1
2 plasmid PEIMAX	1.10e11	4.8
			2 plasmid FectoVIR	6.18e11	26.8

In another experiment, the 2 plasmid system was tested in HEK293F, HEK293F was amplified in different media: expi293 and F17. In 200mL of Expi293 medium or F17 medium in a 500mL flask, the cells split into 2e6 cells/mL. Plasmid mixtures for the various transfection conditions outlined in table 5D below were prepared and filtered through a 0.22 μm filter unit. The transfection reagent mixture (e.g., PEI) was prepared according to the manufacturer's protocol. The plasmid and transfection reagent mixture are combined to produce a single transfection mixture. 20mL of the transfection mixture was added to 100mL HEK293 cells in a 500mL flask and incubated at 37℃for 72 hours.

In some embodiments, plasmids tested in the two-plasmid system comprise AAV Rep sequences and related sequences from helper virus ("Rep/helper plasmid") or AAV Cap sequences and payloads ("payload/Cap plasmid"). In some embodiments, the plasmids tested in the three plasmid system comprise separate plasmids, each encoding one of the following: 1) AAV rep and AAV cap sequences, 2) related sequences from helper viruses, and 3) payloads. In the experiments outlined below, the human factor IX gene (hFIX) flanked by mouse albumin homology arm sequences (mHA) was tested as a payload. The plasmid ratio of the 2 plasmid system was Rep/helper to payload/cap=1.5:1, and the plasmid ratio of the 3 plasmid system was helper to Repcap to payload=0.43:0.35:0.22.

The results in table 5D show similar trends in different media, with the 2 plasmid system providing higher titers than the 3 plasmid system.

Table 5D: transfection parameters of different media.

Description of the Carrier	System and method for controlling a system	Culture medium	Crude volume yield (transfected vg/mL)
				DJ-mHA-hFIX	3 plasmid	Expi293	2.30E+10
DJ-mHA-hFIX	2 plasmid	Expi293	4.61E+10
				DJ-mHA-hFIX	3 plasmid	F17	2.90E+10
DJ-mHA-hFIX	2 plasmid	F17	4.13E+10

The present disclosure demonstrates, among other things, that a dual plasmid system can be combined with various transfection reagents to produce high viral yields in both small scale and manufacturing settings. As shown in FIG. 6, the Fectovir-AAV transfection system increased the yield of both plasmid systems, with an approximately 4-fold increase in vector genome titres compared to PEIMAX. Fectovir-AAV increased vector genome titres by more than 16-fold under small scale shake flask conditions when combined with a two plasmid system at an optimized plasmid ratio (1.5:1 Rep/helper: payload/Cap), and increased vector genome titres by approximately 27-fold under laboratory scale bioreactor conditions (Table 5C). Furthermore, as shown in table 5D above, the increase in virus yield was consistent between different types of cell culture media. The present disclosure demonstrates that optimizing transfection conditions via a combination of a two-plasmid system (e.g., at a specific plasmid ratio) and a specific transfection reagent (e.g., fectovir-AAV) can provide a substantial increase in viral vector yield for mammalian cells, which can be consistent between different cell culture conditions (e.g., different media and different culture containers).

Example 6: the dual plasmid system can increase the volumetric yield of different cell lines grown in adherent culture.

Previous examples 1 to 5 show the production of AAV vectors using a two plasmid system in suspension HEK 293F. This example shows that the dual plasmid system also increases AAV production in adherent 293T cells.

Experiments were performed on 293T cells in 12-well plates. Cells were plated in dmem+10% fcs at 8E5 cells/Kong Tupu. After one day, cells were transfected with a mixture of plasmids in OptiMEM combined with a lipid-based transfection reagent (Fugene HD). To quantify AAV vectors, benzonase was added to each culture at 100U/mL. After 15 min at 37℃the cells were lysed by adding 200. Mu.L of lysis buffer (10% Tween20, 500mM Tris, 20mM MgCl2, pH 8) and incubated for 90 min at 37 ℃. Then, naCl was added to a final concentration of 0.5M, and the sample was centrifuged at 3900rpm for 10 minutes at room temperature. The supernatant was collected for vector genome titration using ddPCR.

In this example, a two plasmid system was tested that contained a plasmid containing AAV Rep sequences and related sequences from helper virus ("Rep/helper plasmid") and a plasmid containing AAV Cap sequences and payload ("payload/Cap plasmid"). For comparison, a three plasmid system comprising separate plasmids, each encoding one of the following: 1) AAV rep and AAV cap sequences, 2) related sequences from helper viruses, and 3) payloads. In the experiments outlined below, the human factor IX gene sequence ("mHA-FIX") with a flanking mouse albumin homology arm was tested as a payload.

Table 6: transfection conditions for the two plasmid system. The relative amounts of Rep/helper and payload/Cap plasmids are shown.

Capsid shell	Payload	Rep/helper plasmid	payload/Cap plasmid
				AAV-DJ	mHA-hFIX	10	1
AAV-DJ	mHA-hFIX	6	1
				AAV-DJ	mHA-hFIX	1	1
AAV-DJ	mHA-hFIX	1	6
				AAV-DJ	mHA-hFIX	1	10

Table 6A: transfection conditions for the three plasmid system.

The present disclosure demonstrates, among other things, that a two plasmid system at different ratios can produce AAV vector yields similar to or higher than a three plasmid system in 293T cells grown under adherent culture conditions. As shown in fig. 7, when rep/assist: when the payload/cap plasmid ratio is greater than or equal to 1, the two plasmid system can produce up to 4 times more vector than the three plasmid system (3P). The present disclosure also demonstrates that the dual plasmid system can use lipid-based transfection agents (e.g., fugene HD) to generate high AAV vector yields.

Example 7: the dual plasmid system can increase volumetric yield and alter packaging efficiency of AAV vectors

This example demonstrates that the dual plasmid system can be used for larger scale cell culture conditions (above 1L culture) to provide increased volumetric yields, similar to the trends observed under smaller scale conditions. Furthermore, this example demonstrates that specific plasmid ratios affect capsid packaging efficiency.

HEK293F cells were tested in 2.8L flasks. HEK293F cells were expanded and split into 2e6 cells/mL in 1.4L expi293 medium in a 2.8L flask. Plasmid mixtures for the various transfection conditions outlined in table 6 below were prepared and filtered through a 0.22 μm filter unit. The transfection reagent mixture (e.g., PEI) was prepared according to the manufacturer's protocol. The plasmid and transfection reagent mixture are combined to produce a single transfection mixture. 140mL of the transfection mixture was added to 1.4L HEK293F cells in a 2.8L flask and incubated at 37℃for 72 hours. To harvest the vector, the cell culture was dispensed into 1L flasks and centrifuged at 3500rpm for 5 minutes. The supernatant was discarded and each cell pellet was lysed by adding 130mL of lysis buffer (PBS, 1mM MgCl2,0.5%Triton-X100) and 7800U benzonase. The lysate was then subjected to 3 freeze-thaw cycles (-80 ℃ and 37 ℃). After removal of cell debris by centrifugation at 3900rpm for 5 minutes, lysates were assayed via ddPCR to determine volumetric yield of viral vectors. A portion of the lysate was purified by affinity chromatography on POROS AAVX resin. After elution at pH 2.5, the purified vector was dialyzed against PBS using an Amicon cartridge. The dialyzed vector was then tested for capsid packaging efficiency (percentage of packaged (intact) relative to unpacked (empty) capsids) via SDS-PAGE and sedimentation velocity analysis ultracentrifugation (SV-AUC).

In some embodiments, plasmids tested in the two-plasmid system comprise AAV Rep sequences and related sequences from helper virus ("Rep/helper plasmid") or AAV Cap sequences and payloads ("payload/Cap plasmid"). In some embodiments, the plasmids tested in the three plasmid system comprise separate plasmids, each encoding one of the following: 1) AAV rep and AAV cap sequences, 2) related sequences from helper viruses, and 3) payloads. In the experiments outlined below, the human factor IX gene sequence ("mHA-FIX") with a flanking mouse albumin homology arm was tested as a payload.

Table 7: the conditions of the transfection system and the transfection reagent were evaluated.

Capsid shell	Payload	Rep/helper plasmid: payload/Cap plasmid ratio
			AAV-DJ	mHA-hFIX	10∶1
AAV-DJ	mHA-hFIX	8∶1
			AAV-DJ	mHA-hFIX	6∶1
AAV-DJ	mHA-hFIX	4∶1
			AAV-DJ	mHA-hFIX	3∶1
AAV-DJ	mHA-hFIX	2∶1
			AAV-DJ	mHA-hFIX	1.5∶1
AAV-DJ	mHA-hFIX	1.25∶1
			AAV-DJ	mHA-hFIX	1∶1
AAV-DJ	mHA-hFIX	1∶1.25
			AAV-DJ	mHA-hFIX	1∶1.5
AAV-DJ	mHA-hFIX	1∶2
			AAV-DJ	mHA-hFIX	1∶3
AAV-DJ	mHA-hFIX	1∶4
			AAV-DJ	mHA-hFIX	1∶6
AAV-DJ	mHA-hFIX	1∶8
			AAV-DJ	mHA-hFIX	1∶10

Table 7A: packaging efficiency of the two plasmid system at the selected ratio compared to the three plasmid system.

The present disclosure demonstrates, among other things, that dual plasmid transfection systems can produce increased volumetric vector yields as compared to three plasmid transfection systems. As shown in fig. 8, similar volumetric yield trends were observed in the two plasmid systems of different plasmid ratios under larger scale culture conditions.

Furthermore, as shown in table 7A, the dual plasmid transfection system can also produce different packaging efficiencies depending on the ratio between Rep/helper and payload/Cap plasmids. Some plasmid ratios produced a higher percentage of intact capsids than the three plasmid system, while other plasmid ratios showed similar or lower percentages.

Example 8: viral vectors produced using a two plasmid system are functional in vivo

This example demonstrates that viral vectors generated using a two plasmid system are functional in vivo.

The vector produced in table 5D was purified by affinity chromatography using POROS AAVX and dialyzed against PBS. Mice (FVB/NJ) were injected with a composition comprising packaged viral vectors produced in two types of media using two-plasmid and three-plasmid transfection conditions at a dose of 1e13 vg/kg (Table 5D). The payload contains a murine homology arm (mHA) and 2A peptide sequence that allows recombination into the albumin locus, followed by human factor IX (hFIX). In vivo vector efficacy was demonstrated by measuring 2 expression products produced by inserted hFIX in mouse plasma: albumin (ALB-2A) with a 2A peptide at the C-terminus and human factor IX (hFIX). In addition, liver samples were extracted to measure the copy number of hFIX gene integrated into albumin loci and to measure mRNA of albumin-hFIX fusion. As shown in fig. 9, vectors produced via the two-plasmid and three-plasmid systems exhibited similar factor IX and ALB-2A expression and similar DNA integration and mRNA expression in the liver.

The present disclosure demonstrates that viral vectors produced via transfection of cells with a two plasmid system exhibit comparable in vivo performance relative to vectors produced via transfection of cells with a three plasmid system.

Example 9: the sequence elements of the two plasmid system are interchangeable

This example demonstrates that a two plasmid system for cell transfection can provide increased vector yields for several combinations of certain sequence elements.

HEK293F cells were expanded for vector production. In 200mL of Expi293 medium in a 500mL flask, the cells split into 2e6 cells/mL. Plasmid mixtures for the various transfection conditions outlined in table 7 below were prepared and filtered through a 0.22 μm filter unit. The transfection reagent mixture (e.g., PEI) was prepared according to the manufacturer's protocol. The plasmid and transfection reagent mixture are combined to produce a single transfection mixture. 20mL of the transfection mixture was added to 100mL of HEK293F cells in a 500mL flask and incubated at 37℃for 72 hours. In some embodiments, plasmids tested in the two-plasmid system comprise AAV Rep sequences and related sequences from helper virus ("Rep/helper plasmid") or AAV Cap sequences and payloads ("payload/Cap plasmid"). In some embodiments, plasmids tested in the two-plasmid system comprise AAV Cap sequences and related sequences from helper virus ("Cap/helper plasmid") or AAV Rep sequences and payloads ("payload/Rep plasmid"). In some embodiments, the plasmids tested in the three plasmid system comprise separate plasmids, each encoding one of the following: 1) AAV rep and AAV cap sequences, 2) related sequences from helper viruses, and 3) payloads. In the experiments outlined below, human factor IX gene sequences under the control of Liver Specific Promoters (LSPs) were tested as payloads.

The results presented in FIG. 10 demonstrate that a two plasmid system comprising rep/helper plasmid and payload/cap plasmid in a 1.5:1 ratio yields high viral yields. When a 6:1 ratio is used, the exchange of the two plasmid system combination comprising cap/helper and payload/rep plasmids yields similar to or higher than the 3 plasmid system.

The present disclosure demonstrates, among other things, that several combinations of genetic elements in a two-plasmid system can produce similar or higher yields than a 3-plasmid system. Notably, higher AAV yields can be obtained when the ratio between the two plasmids is imbalanced to increase the amount of helper viral sequences relative to the payload (1.5:1 to 6:1 or higher).

Example 10: the combination of a two plasmid system with an intron insertion reduces the level of replication competent AAV (rcAAV)

This example demonstrates that a two plasmid system for cell transfection can reduce the level of replication competent AAV (rcAAV) produced in vivo or in vitro. In particular, the level of rcAAV can be particularly reduced when an intron is inserted between the p5 promoter and the start codon of the rep gene.

In some embodiments, the present examples include expanding HEK293F cells for vector production. In 200mL of Expi293 medium in a 500mL flask, the cells split into 2e6 cells/mL. Plasmid mixtures for the various transfection conditions outlined in table 8 below were prepared and filtered through a 0.22 μm filter unit. The transfection reagent mixture (e.g., PEI) was prepared according to the manufacturer's protocol. The plasmid and transfection reagent mixture are combined to produce a single transfection mixture. 20mL of the transfection mixture was added to 100mL of HEK293F cells in a 500mL flask and incubated at 37℃for 72 hours.

In some embodiments, the plasmids in the two-plasmid system comprise AAV Rep sequences and related sequences from helper virus ("Rep/helper plasmid") or AAV Cap sequences and payload ("payload/Cap plasmid"). In some embodiments, the plasmids in the three plasmid system comprise separate plasmids, each encoding one of the following: 1) AAV rep and AAV cap sequences, 2) related sequences from helper viruses, and 3) payloads. In the experiments outlined below, human factor IX gene sequences under the control of Liver Specific Promoters (LSPs) or human mutases (MMUTs) were tested as payloads. In some embodiments, an intron sequence is inserted between the p5 promoter and the rep gene. In some embodiments, the intron sequences may be present in several lengths (133 bp, 1.43kb, or 3.3 kb)

In this experiment, a two plasmid system comprising various combinations of introns was tested at different ratios of Rep/helper plasmid to payload/Cap plasmid as presented in table 10.

Table 10: transfection conditions for a two plasmid system comprising different introns between the p5 promoter and the rep gene.

Table 10A: transfection conditions for the three plasmid (3P) system.

In the second experiment, a longer intron (3.3 kb) was tested compared to the 1.43kb intron shown in Table 10B

Table 10B: transfection conditions for a two plasmid system containing two different introns between the p5 promoter and the rep gene.

To assess the reduction in rcAAV incidence during AAV manufacture using embodiments of the two plasmid system, the vectors described in table 10B were tested in the rcAAV assay. Similar vectors (LK 03 capsid and MMUT payload) generated using the three plasmid system (no introns inserted in rep gene) were tested side-by-side in the same assay for comparison.

HeLa cells were transduced with 1e6, 1e8 and 1e10 vector genomes (vg) of the test samples in the presence of wild type adenovirus (Ad 5). To demonstrate the limit of detection of the assay, cells were also seeded with test samples (1 e6, 1e8 and 1e10 vg) spiked with 10 wild-type AAV2 (wtAAV 2) infectious particles. After the first amplification cycle, the cells were harvested and samples were collected for qPCR quantification; the remaining cells were frozen. Cell lysates were prepared by three consecutive freeze-thaw cycles, and these samples were used to transduce a second batch of HeLa cells. This procedure was repeated for a total of three rounds of sample amplification.

DNA was extracted from the cell harvest samples using dnasy blood and tissue kit (Qiagen, catalog No. 69506). Real-time qPCR was performed on the isolated DNA samples, amplifying two sequences: AAV Rep2 and human albumin (hAlb). If rcAAV is present in the test sample, AAV Rep2 sequences are amplified, while human albumin serves as a housekeeping gene. The copy number (ranging from 1e2 to 1e8 copies/reaction) of each sequence was determined by comparing Ct values with the values of the measured plasmid standard curve. The relative copy number of Rep2 per cell is determined by calculating the ratio of the copies of Rep2 to the copies of human albumin multiplied by 2. Replication is confirmed if the relative copy number of Rep2 in at least one of the rounds of amplification is ≡10. If the relative copy number of Rep2 is observed to increase with each round of sequential amplification, this indicates the presence of replication competent AAV in the test sample. The results of the rcAAV test are presented in table 10C.

Table 10C: rcAAV detection in AAV vector batches made using either a three plasmid system without introns or a two plasmid system with introns in the rep gene sequence.

Viral vectors made using the two plasmid system were found to be negative for rcAAV replication. In contrast, viral vectors produced using the traditional three plasmid system exhibited rcAAV replication (rcAAV positive) at the highest dose of 1e+10vg.

The present disclosure demonstrates, among other things, that inserting introns between AAV p5 promoter and rep gene in a two plasmid system results in vector yields comparable to or higher than that of the intronless 2 plasmid system and 3 plasmid system (fig. 11). In some embodiments, insertion of an intron between the AAV p5 promoter and the rep gene in a two plasmid system reduces the occurrence of replication competent AAV (rcAAV) compared to a traditional three plasmid system.

Example 11: compared to the three plasmid system, the two plasmid system provides similar capsid Viral Protein (VP) ratio and purity

This example demonstrates that a two plasmid system for cell transfection can provide similar protein purity and observed ratios between VP1, VP2 and VP3 capsid proteins as compared to a three plasmid system.

In this example, HEK293F cells were expanded for vector production. HEK293F cells were expanded for cell growth using an Expi293 basal medium. Cell counts were first recorded to ensure viable cell densities between 2.0e6-2.6e6 cells/mL and survival rates above 95% at transfection. Transfection mixtures were prepared by pre-weighing either the Expi293 medium (two plasmid system) or the optiprusfm medium (three plasmid system) in two separate containers labeled "DNA medium" and "TR medium", each containing equal volume requirements from the calculation of the transfection mixture. PEIMAX was added to plasmid DNA (pDNA) (three plasmid system) and Fectovir-AAV was added to pDNA (two plasmid system). Each mixture was added to a separate bottle labeled "TR medium" and set aside. For the three plasmid transfection system, the mass fractions of helper plasmid, rep/Cap plasmid and payload plasmid were 0.43, 0.35 and 0.22, respectively. For the two plasmid transfection system, the mass fractions of Rep/helper plasmid and payload/Cap plasmid were 0.60 and 0.40, respectively (1.5:1 w/w plasmid ratio). The plasmid was sterile filtered through a Coming 0.22um PES bottle top filter by first wetting the membrane with medium in the bottle labeled "DNA medium", adding the appropriate amount of pDNA to the bottle top, turning on the vacuum of the filter, and finally rinsing the remaining DNA on the filter with the remaining medium in the "DNA medium" bottle. Once the "TR medium" and "DNA medium" solutions were prepared, the two mixtures were combined into separate containers and inverted to begin the compounding process. The transfection mixture was then incubated at room temperature for 20 min when PEIMAX (three plasmid system) was used and at room temperature for 30 min when FectoVIR-AAV (two plasmid system) was used. After time elapses, the transfection mixture is added to the medium at a final culture volume fraction of 10% (e.g., 25mL transfection mixture is added to 225mL culture) and grown at 37 ℃ for 72 hours.

At harvest, benzonase was mixed with Expi293 medium, 100uL Benzonase (about 250U/uL) and 2.5mL medium were used per reactor, 100U Benzonase per 1mL culture volume. The bioreactor culture was incubated at 37℃for 15 minutes. 10 Xlysis buffer (10% v/v Tween 20, 500mM Tris-HCl pH 8.0, 20mM MgCl2, milli-Q water) was prepared and 25mL (10% incubation volume) was added to each bioreactor followed by incubation at 37℃for 90 minutes. Next, 25mL of sterile filtered 5M naci was added to each flask (to reach the target concentration of 0.5M NaCl) and incubated for 30 minutes at 37 ℃. The lysed culture was then spun at 3500 Xg for 10 minutes. The supernatant was filtered through a 0.22um Coming sterile filter and sampled for crude lysate analysis. After sterile filtration, the sample was loaded onto a 5mL POROS GoPure AAVX pre-packed column. The eluate was neutralized to pH 7.0-7.5 using 20% v/v Tris-HCl pH 8.5, followed by sampling and subsequent analysis.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to determine the purity of the three AAV structural proteins (VP 1, VP2, and VP 3) present in the sample. Samples and LK03 capsids manufactured using a three plasmid system were mixed with Lithium Dodecyl Sulfate (LDS) sample buffer and Dithiothreitol (DTT), and then subjected to thermal denaturation. The denatured sample and molecular weight markers are loaded onto Bis-Tris gels, and then an electric field is applied to separate proteinaceous material according to relative size. After electrophoresis, the gel was stained with Imperial Protein Stain, washed and imaged on LI-COR CLx. ImageJ software was used to quantify the protein intensity of each band present in each test sample. The viral protein purity is determined by the percentage of the sum of the peak areas of the VP1, VP2 and VP3 products to the sum of all peak areas. Any peak that is not a product (VP 1, VP2, VP 3) peak is considered an impurity.

The present disclosure demonstrates, among other things, that a two-plasmid system can produce capsid proteins having similar purity and capsid protein ratio as obtained via a three-plasmid system (fig. 20).

Example 12: the dual plasmid system can be used for large-scale production of AAV capsids

This example demonstrates that a two plasmid system for cell transfection can be used in a larger scale system (e.g., a 50L bioreactor) to generate high levels of viral genome titres. This example also illustrates that a two-plasmid system can reduce the amount of plasmid DNA (e.g., comprising a kanamycin resistance gene) that is non-specifically packaged in an AAV capsid during manufacture.

In this example, HEK293F cells were expanded as previously described herein for culture in an ambr250 bioreactor as well as a 50L Sartorius BioSTAT STR bioreactor. For both reactor settings, crude viral titers (vg/mL) and residual levels of transfection-derived plasmid DNA in AAVX purification pools were measured (fig. 21).

HEK293F cells were expanded for vector production. HEK293F cells were expanded for cell growth using an Expi293 basal medium. Cell counts were first recorded to ensure viable cell densities between 2.0e6-2.6e6 cells/mL and survival rates above 95% at transfection. Transfection mixtures were prepared by pre-weighing either the Expi293 medium (two plasmid system) or the OptiPRO SFM medium (three plasmid system) in two separate containers labeled "DNA medium" and "TR medium", each containing equal volume requirements from the calculation of the transfection mixture. PEIMAX was added to plasmid DNA (pDNA) (three plasmid system) and Fectovir-AAV was added to pDNA (two plasmid system). Each mixture was added to a separate bottle labeled "TR medium" and set aside. For the three plasmid transfection system, the mass fractions of helper, rep/Cap and payload plasmids were 0.43, 0.35 and 0.22, respectively (1.5:1 w/w TR: plasmid ratio). For the two plasmid transfection system, the mass fractions of Rep/helper plasmid and payload/Cap plasmid were 0.60 and 0.40, respectively (1:1 w/w TR: plasmid ratio). The plasmid was sterile filtered through a 0.22um filter and finally rinsed with the remaining medium in a "DNA medium" bottle. After the "TR medium" and "DNA medium" solutions were prepared, the two mixtures were combined into separate containers and inverted for 1 minute to begin the compounding process. The transfection mixture was then incubated at room temperature for 20 min when PEIMAX (three plasmid system) was used and at room temperature for 30 min when FectoVIR-AAV (two plasmid system) was used. After time elapses, the transfection mixture is added to the medium at a final culture volume fraction of 10% (e.g., 25mL transfection mixture to 225mL culture for ambr 250; 5L transfection mixture to 45L culture for 50L) and grown at 37 ℃ for 72 hours.

At harvest, benzonase was mixed with Expi293 medium using Benzonase (about 250U/uL) at 10U Benzonase per 1mL culture volume and 1% culture volume medium per reactor. The bioreactor culture was incubated at 37℃for 15 minutes. 10 Xlysis buffer (10% v/v Tween 20, 500mM Tris-HCl pH 8.0, 20mM MgCl2, milli-Q water) was prepared and 10% culture volume was added to each bioreactor followed by incubation at 37℃for 90 minutes. Next, 10% culture volume of sterile filtered 5m NaC1 was added to each flask (to reach the target concentration of 0.5m NaC1) and incubated for 30 minutes at 37 ℃. The lysed culture was then spun at 3500 Xg for 10 minutes. The supernatant was filtered through a 0.22um sterile filter and sampled for crude lysate analysis. After sterile filtration, the samples were loaded via AAVX chromatography resin for additional analysis. The eluate was neutralized to pH 7.0-7.5 using 20% v/v Tris-HCl pH 8.5, followed by sampling and subsequent analysis.

Vector genome titers were quantified by ddPCR in cleaved crude harvest samples. The packaged residual plasmid DNA was quantified from the purified vector using ddPCR and a primer/probe set that targets the kanamycin resistance gene in each plasmid backbone used in the examples. In this assay, the test samples were treated with and without salt-active nucleases to confirm that residual plasmid DNA was packaged in AAV capsids (and thus nuclease resistant). The sample is then treated with proteinase K to extract DNA from the capsid. The sample was diluted and mixed with ddPCR master mix containing primer/probe sets that specifically bind to the kanamycin gene. The Bio-Rad automated droplet generator is used to generate droplets for each sample, which are then thermally cycled to amplify the DNA of interest using standard PCR. Positive and negative droplets were quantified using a Bio Rad QX200Droplet Reader and analyzed using poisson distribution analysis. The copy number of kanamycin amplicon was corrected by sample preparation to yield residual Kan plasmid DNA concentration in copies/mL.

The present disclosure demonstrates, among other things, that two-plasmid systems can produce high levels of viral capsids at a larger scale volume (e.g., 50L or greater) than viral capsids obtained with three-plasmid systems. In some embodiments, the two plasmid system can also provide significantly reduced levels of transfection-derived plasmid DNA in AAVX purification pools as compared to a three plasmid system.

Example 13: various factors may affect viral vector yield in a two plasmid system

This example demonstrates, among other things, that a two plasmid system can produce increased viral yields when specific transfection conditions are optimized. In some embodiments, specific combinations of different levels of transfection reagents (e.g., fectovir), cell density (e.g., HEK293F cells), and/or plasmid DNA (e.g., total plasmid DNA) can increase viral yield while minimizing costs.

In this example, HEK293F cells were expanded for vector production. HEK293F cells were expanded for cell growth using an Expi293 basal medium. Cell counts were first recorded to ensure viable cell densities between 2.0e6-2.6e6 cells/mL and survival rates above 95% at transfection. The transfection mixture was prepared by pre-weighing the Expi293 medium in two separate containers labeled "DNA medium" and "TR medium", each containing equal volume requirements from the calculation of the transfection mixture. FectoVIR-AAV was added to a vessel labeled "TR media" and set aside. For the two plasmid transfection system, the mass fractions of Rep/helper plasmid and payload/Cap plasmid were 0.60 and 0.40, respectively (1.5:1 w/w plasmid ratio). The plasmid was sterile filtered through a Coming 0.22um PES bottle top filter by first wetting the membrane with medium in the bottle labeled "DNA medium", adding the appropriate amount of pDNA to the bottle top, turning on the vacuum of the filter, and finally rinsing the remaining DNA on the filter with the remaining medium in the "DNA medium" bottle. After the "TR medium" and "DNA medium" solutions were prepared, the mixtures were combined into separate containers and inverted to begin the compounding process. When Fectovir-AAV was used, the transfection mixture was then incubated for 30 minutes at room temperature. After time elapses, the transfection mixture is added to the medium at a final culture volume fraction of 10% (e.g., 25mL transfection mixture is added to 225mL culture) and grown at 37 ℃ for 72 hours.

At harvest, benzonase was mixed with Expi293 medium, 100uL Benzonase (about 250U/uL) and 2.5mL medium were used per reactor, 100U Benzonase per 1mL culture volume. The bioreactor culture was incubated at 37℃for 15 minutes. 10 Xlysis buffer (10% v/v Tween 20, 500mM Tris-HCl pH 8.0, 20mM MgCl2, milli-Q water) was prepared and 25mL (10% incubation volume) was added to each bioreactor followed by incubation at 37℃for 90 minutes. Next, 25mL of sterile filtered 5M naci was added to each flask (to reach the target concentration of 0.5M NaCl) and incubated for 30 minutes at 37 ℃. The lysed culture was then spun at 3500 Xg for 10 minutes. The supernatant was filtered through a 0.22um Coming sterile filter and sampled for crude lysate analysis.

Various combinations of transfection conditions (e.g., total plasmid DNA amount, amount of transfection reagent, cell density) were tested to determine which conditions could produce increased viral titer production. The first round of testing was performed by analyzing all three of the following at the levels listed in table 11A: total plasmid DNA amount, fectovir-AAV amount and cell density. Analysis was performed to determine optimal conditions to maximize virus titer while minimizing total cost (fig. 22). Further testing optimizes the combination of total plasmid DNA amounts and Fectovir-AAV amounts, as summarized in Table 11B. The second round of analysis again focused on maximizing virus titer while not exceeding the cost threshold determined in the first round of analysis (fig. 23).

This example demonstrates, among other things, that transfection conditions can be optimized in a two-plasmid system to provide increased viral titer yield compared to a reference (e.g., alternative transfection conditions, three-plasmid system) while minimizing cost.

Table 11A: transfection conditions tested in the first round of analysis (FIG. 22)

Table 11B: transfection conditions tested in the second round of analysis (FIG. 23)

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Example 14: dual plasmid systems with Fectovir-AAV can increase volumetric yields of multiple AAV serotypes

This example demonstrates, among other things, that various AAV serotypes can be used in a two plasmid system with FectoVIR-AAV to produce surprisingly high viral yields.

HEK293F cells were expanded in 500-mL shake flasks for vector production. Cell counts were first recorded on a ViCell XR cell counter to ensure VCD was between 2.0e6-2.6e6 cells/mL and survival was higher than 95% at transfection. The transfection mixture was then prepared by first pre-weighing the Expi293 medium in two separate containers "DNA medium" and "transfection reagent medium", each containing equal volume requirements from the transfection mixture calculation. The transfection reagent was then added to the flask labeled "transfection reagent medium" and set aside. For the 3 plasmid transfection system, the mass fractions of p-helper, pRep/Cap and pGOI were 0.43, 0.35 and 0.22, respectively. For the 2 plasmid transfection system, the mass fractions of Rep/helper plasmid and payload/Cap plasmid were 0.60 and 0.40, respectively (1.5:1 plasmid ratio). The plasmid was sterile filtered through a Corning 0.22um PES bottle top filter by first wetting the membrane with medium in the bottle labeled "DNA medium", adding the appropriate amount of pDNA to the bottle top, opening the vacuum of the filter, and finally rinsing the remaining DNA on the filter with the remaining medium in the "DNA medium" bottle. After preparing the transfection reagent/medium and DNA/medium solutions at a 1:1 volume ratio, the two mixtures were combined into separate containers and inverted 10 times to begin the compounding process. The transfection mixture was then allowed to stand at room temperature for 15 minutes when PEIMAX was used and at room temperature for 30 minutes when Fectovir-AAV was used. Unless otherwise indicated, after the lapse of time, the transfection mixture was added to the medium at a 10% culture volume fraction (e.g., 20mL of transfection mixture was added to 200mL of culture) and grown at 37 ℃ for 72 hours.

Table 12A: the transfection systems of the different serotypes and the conditions of the transfection reagents were evaluated.

Conditions (conditions)	Description of the Carrier	Transfection system	Transfection reagent
				1	plasmid/LSP-hFIX	3 plasmid	PEIMAX
2	plasmid/LSP-hFIX	2 plasmid	PEIMAX
				3	plasmid/LSP-hFIX	3 plasmid	FectoVIR-AAV
4	plasmid/LSP-hFIX	2 plasmid	FectoVIR-AAV

Table 12B: transfection parameters of different transfection reagents.

Parameters (parameters)	PEIMAX	FectoVIR-AAV
			Total DNA (ug) of 6 cells per 1e	0.75	0.75
TR: DNAw/w ratio	1.5	1.0
			Percentage of transfection mix of culture volume (%)	10	10
Time of compounding (minutes)	15	30

Cells were harvested 72 hours after transfection of the culture. 5mL of the culture was transferred to a 15mL centrifuge tube and 50uL of an Expi293 medium solution containing 10 units/uL of benzonase was added to the tube and shaken horizontally in an incubator at 37℃and 145RPM for 15 minutes. 500uL of lysis buffer (500 mM Tris pH 8, 20mM MgCl2, 10% polysorbate-20) was then added to the tube and incubated under the same conditions for 90 minutes. Finally, 500ul 5m NaCl was added to the tube and incubated for 30 minutes under the same conditions. After NaCl incubation, the cell lysate was centrifuged at 3200g in a centrifuge to clarify the harvested medium. 1mL of supernatant containing AAV particles was collected in a 1.5mL microcentrifuge tube and stored at-80℃until sample analysis was ready.

In some embodiments, plasmids tested in the two-plasmid system comprise AAV Rep sequences and related sequences from helper virus ("Rep/helper plasmid") or AAV Cap sequences and payloads ("payload/Cap plasmid"). In some embodiments, the plasmids tested in the 3 plasmid system comprise separate plasmids, each encoding one of the following: 1) AAV rep and AAV cap sequences, 2) related sequences from helper viruses, and 3) payloads. In the experiments outlined herein, the human factor IX gene (hFIX) flanked by albumin homology arm sequences was tested as a payload. The plasmid ratio of the 2 plasmid system was Rep/helper to payload/cap=1.5:1, and the plasmid ratio of the 3 plasmid system was helper to Repcap to payload=0.43:0.35:0.22.

The present disclosure demonstrates, among other things, that certain transfection reagents of a two-plasmid transfection system can yield surprising and unexpected volumetric yield improvements for the native serotype (e.g., as compared to a three-plasmid "triple transfection" system). As shown in fig. 24, the FectoVir-AAV transfection system combined with the two plasmid system provides improved yield (e.g., compared to a three plasmid "triple transfection" system combined with FectoVir).

Example 15: the dual plasmid system can provide high titres independent of adenovirus helper plasmid design

This example demonstrates, among other things, that several combinations of genetic elements in a two-plasmid system can produce similar or higher yields than a 3-plasmid system.

HEK293F cells were expanded in 125-mL shake flasks for vector production. Cell counts were first recorded on a ViCell XR cell counter to ensure VCD was between 2.0e6-2.6e6 cells/mL and survival was higher than 95% at transfection. The transfection mixture was then prepared by first pre-weighing the Expi293 medium in two separate containers "DNA medium" and "transfection reagent medium", each containing equal volume requirements from the transfection mixture calculation. The transfection reagent was then added to the flask labeled "transfection reagent medium" and set aside. For the 3 plasmid transfection system, the mass fractions of p-helper, pRep/Cap and pGOI were 0.43, 0.35 and 0.22, respectively. For the 2 plasmid transfection system, the mass fractions of Rep/helper plasmid and payload/Cap plasmid were 0.60 and 0.40, respectively (1.5:1 plasmid ratio). The plasmid was sterile filtered through a Corning 0.22um PES bottle top filter by first wetting the membrane with medium in the bottle labeled "DNA medium", adding the appropriate amount of pDNA to the bottle top, opening the vacuum of the filter, and finally rinsing the remaining DNA on the filter with the remaining medium in the "DNA medium" bottle. After preparing the transfection reagent/medium and DNA/medium solutions at a 1:1 volume ratio, the two mixtures were combined into separate containers and inverted 10 times to begin the compounding process. The transfection mixture was then allowed to stand at room temperature for 30 minutes using FectoVIR-AAV. Unless otherwise indicated, after the lapse of time, the transfection mixture was added to the medium at a 10% culture volume fraction (e.g., 20mL of transfection mixture was added to 200mL of culture) and grown at 37 ℃ for 72 hours.

Cells were harvested 72 hours after transfection of the culture. 5mL of the culture was transferred to a 15mL centrifuge tube and 50uL of an Expi293 medium solution containing 10 units/uL of benzonase was added to the tube and shaken horizontally in an incubator at 37℃and 145RPM for 15 minutes. 500uL of lysis buffer (500 mM Tris pH 8, 20mM MgCl2, 10% polysorbate-20) was then added to the tube and incubated under the same conditions for 90 minutes. Finally, 500ul 5m NaCl was added to the tube and incubated for 30 minutes under the same conditions. After NaCl incubation, the cell lysate was centrifuged at 3200g in a centrifuge to clarify the harvested medium. 1mL of supernatant containing AAV particles was collected in a 1.5mL microcentrifuge tube and stored at-80℃until sample analysis was ready. Samples were analyzed via ddPCR to determine vector genome titers.

Table 13A: the conditions of the transfection system with different helper genes were evaluated.

Table 13B: transfection parameters of the transfection reagent.

Parameters (parameters)	FectoVIR-AAV
		Total DNA (ug) of 6 cells per 1e	0.75
TR: DNAw/w ratio	1.0
		Percentage of transfection mix of culture volume (%)	10
Time of compounding (minutes)	30

The present disclosure demonstrates, inter alia, that AAV titers at harvest of cultures are higher when cells are transfected with a two plasmid system as compared to a 3 plasmid system. As shown in FIG. 25, the increase in titer was independent of the design of the plasmid carrying the adenovirus helper gene and the AAV rep gene.

Example 17: the dual plasmid system allows the production of AAV vectors with various ITRs

This example demonstrates, inter alia, that both single-stranded and double-stranded AAV vectors can be produced at high levels using a two-plasmid system. In some embodiments, various ITR sequences can be used to flank payloads in payload/Cap plasmids in a two plasmid system.

The Inverted Terminal Repeat (ITR) is an AAV sequence element required in cis in the vector genome sequence to allow replication and packaging of the vector genome in the AAV capsid (Samulski et al, 1987; mcLaughlin et al, 1988). As part of the AAV natural replication process, ITR sequences and their reverse complement are alternately associated with the positive and negative strands of the AAV genome, a feature known as the forward and reverse flip direction (flip and flop orientation) (reviewed in wilmot et al, 2019). Wild-type ITR sequences of AAV2 commonly used in AAV vectors are shown in Table 14A in the inverted orientation.

Since ITR sequences are typically GC-rich and exhibit hairpin-like secondary structures, they can be difficult to maintain in plasmids during cloning and production of AAV vectors. ITRs may be unstable and may recombine and/or suffer from partial deletions during plasmid production in E.coli. Thus, several different ITR variants can be experimentally observed. For example, table 14A shows that 22 base pairs are deleted in the B loop, 22 base pairs are deleted in the C loop, 15 base pairs are deleted in the a region, and 40 base pairs are deleted in the D region of AAV2 ITR. The B and C loop deleted and the a region deleted ITR variants can retain full function of replicating and packaging the vector genome within the capsid. However, deletion of ITR variants in the D region results in loss of the packaging signal and terminal resolution sites (trs). The D-region deleted ITR variant has been described as a method of producing self-complementary AAV (scAAV), also known as double-stranded AAV (dsAAV) (Wang et al, 2003).

HEK293F cells were expanded in 125-mL shake flasks. Cell counts were first recorded on a ViCell XR cell counter to ensure VCD was between 2.0e6-2.6e6 cells/mL and survival was higher than 95% at transfection. The transfection mixture was then prepared by first pre-weighing the Expi293 medium in two separate containers "DNA medium" and "transfection reagent medium", each containing equal volume requirements from the transfection mixture calculation. The transfection reagent was added to the flask labeled "transfection reagent medium" and set aside. The mass fractions of Rep/helper plasmid and payload/Cap plasmid were 0.60 and 0.40, respectively (1.5:1 plasmid ratio). The plasmid was sterile filtered through a Corning 0.22um PES bottle top filter by first wetting the membrane with medium in the bottle labeled "DNA medium", adding the appropriate amount of pDNA to the bottle top, opening the vacuum of the filter, and finally rinsing the remaining DNA on the filter with the remaining medium in the "DNA medium" bottle. Transfection reagent/medium and DNA/medium solutions were prepared, and the two mixtures were combined into separate containers in a 1:1 volume ratio and inverted 10 times to begin the compounding process. The transfection mixture was then allowed to stand at room temperature for 30 minutes using FectoVIR-AAV. After time elapses, the transfection mixture is added to the medium at a 10% culture volume fraction (e.g., 20mL of transfection mixture is added to 200mL of culture) and grown at 37 ℃ for 72 hours.

Cells were harvested 72 hours after transfection of the culture. 5mL of the culture was transferred to a 15mL centrifuge tube and 50uL of an Expi293 medium solution containing 10 units/uL of benzonase was added to the tube and shaken horizontally in an incubator at 37℃and 145RPM for 15 minutes. 500uL of lysis buffer (500 mM Tris pH 8, 20mM MgCl2, 10% polysorbate-20) was then added to the tube and incubated under the same conditions for 90 minutes. Finally, 500ul 5m NaCl was added to the tube and incubated for 30 minutes under the same conditions. After incubation with NaC1, the cell lysate was centrifuged at 3200g in a centrifuge to clarify the harvested medium. 1mL of supernatant containing AAV particles was collected in a 1.5mL microcentrifuge tube and stored at-80℃until sample analysis was ready. Samples were analyzed via ddPCR to determine vector genome titers. Vector genomes can be analyzed on alkaline agarose gels to confirm single and double stranded vector genome characteristics.

Example 16: the dual plasmid system provides high titers of native and chimeric AAV serotypes

This example demonstrates, inter alia, that a two-plasmid system can produce increased AAV viral yields compared to a three-plasmid system, independent of AAV serotype.

HEK293F cells were expanded for vector production. In 200mL of Expi293 medium in a 500mL flask, the cells split into 2e6 cells/mL. Plasmid mixtures for various transfection conditions were prepared and filtered through a 0.22 μm filter unit. Transfection reagent mixtures (e.g., PEI or FectoVIR-AAV) were prepared according to the manufacturer's protocol. The plasmid and transfection reagent mixture are combined to produce a single transfection mixture. 20mL of the transfection mixture was added to 100mL of HEK293F cells in a 500mL flask and incubated at 37℃for 72 hours.

In some embodiments, plasmids tested in the two-plasmid system comprise AAV Rep sequences and related sequences from helper virus ("Rep/helper plasmid") or AAV Cap sequences and payloads ("payload/Cap plasmid"). In some embodiments, the plasmids tested in the three plasmid system comprise separate plasmids, each encoding one of the following: 1) AAV rep and AAV cap sequences, 2) related sequences from helper viruses, and 3) payloads. In the experiments, human gene sequences of interest compatible with the GeneRide system with flanking mouse albumin homology arms ("mHA-FIX") were tested as payloads. Multiple AAV Cap genes encoding different AAV capsids were evaluated within the payload/Cap plasmid. In some embodiments, an AAV cap gene can encode an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVC11.01, AAVC11.02, AAVC11.03, AAVC11.04, AAVC11.05, AAVC11.06, AAVC11.07, AAVC11.08, AAVC11.09, AAVC11.10, AAVC11.11, AAVC11.12, AAVC11.13, AAVC11.14, AAVC11.15, AAVC11.16, AAVC11.17, AAVC11.18, AAVC11.19, AAV-DJ, AAV-LK03, AAV-LK19, aavrh.74, aavrh.10, aavhu.37, aavrh.k, aavrh.39, AAV12, AAV13, aah.8, avian, bovine, canine, equine, non-primate, AAV, or AAV of a subhuman, or multiple AAV (e.g., AAV) sequence of one or more than one AAV type or multiple AAV sequences.

The present disclosure demonstrates, among other things, that a dual plasmid transfection system with FectoVIR-AAV can produce volumetric yield improvements (e.g., as compared to a three plasmid "triple transfection" system) for different capsids with payloads suitable for use in conventional gene therapy.

Table 14A: reverse terminal repeat (ITR) variants and sequences thereof (left-terminal ITR in 5 'to 3' direction)

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Table 14B: exemplary ITR combinations in payload/Cap plasmids and expected AAV vector characteristics

Left ITR	Transgenic plants	Right-hand ITR	Capsid shell	AAV vector description
					Wild type	Factor IX	Wild type	AAV8	Single strand
Wild type	Factor IX	B-ring deletion	AAV8	Single strand
					Wild type	Factor IX	C-ring deletion	AAV8	Single strand
Wild type	Factor IX	Region A deletion	AAV8	Single strand
					Region A deletion	Factor IX	Region A deletion	AAV8	Single strand
Wild type	Factor IX	Deletion of D region	AAV8	Double strand

The present disclosure demonstrates, among other things, that a two-plasmid system can produce high levels of double stranded AAV vectors (e.g., self-complementary AAV (scAAV) vectors). In some embodiments, the double stranded AAV vector comprises a D region deletion in at least one ITR flanking the payload. In some embodiments, the two plasmid system can produce high levels of the double stranded vector outlined in table 14B above. In some embodiments, the dual plasmid transfection system can produce similar or higher yields of double stranded AAV vectors (e.g., scAAV) compared to a three plasmid system.

Equivalents (Eq.)

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the invention is not intended to be limited by the foregoing description, but is instead as set forth in the following claims:

exemplary embodiments

Exemplary embodiments as described below are also within the scope of the present disclosure:

1. a plasmid comprising at least one of:

(i) A polynucleotide sequence encoding an AAV cap gene;

(ii) A polynucleotide sequence encoding an AAV rep gene;

(iii) Polynucleotide sequences encoding a payload and flanking ITRs; and/or

(iv) Polynucleotide sequences encoding one or more viral accessory genes.

2. The plasmid of embodiment 1 further comprising a polynucleotide sequence encoding a promoter.

3. The plasmid of embodiment 1 or embodiment 2, wherein the promoter is or comprises a native p5 promoter, a native p40 promoter, or a CMV promoter.

4. The plasmid of embodiment 1 further comprising a polyA sequence.

5. The plasmid of embodiment 1 further comprising an intron.

6. The plasmid of embodiment 5, wherein the intron is located between the promoter and the AAV rep gene.

7. A composition comprising two of the plasmids of any one of embodiments 1-6, a first plasmid and a second plasmid, wherein the first plasmid and the second plasmid each comprise different elements (i) - (iv).

8. The composition of embodiment 7, wherein:

(i) The first plasmid comprises a polynucleotide sequence encoding an AAV cap gene; and is also provided with

(ii) The second plasmid comprises a polynucleotide sequence encoding an AAV rep gene.

9. The composition of embodiment 7, wherein:

(i) The first plasmid comprises a polynucleotide sequence encoding a payload and flanking ITRs; and is also provided with

(ii) The second plasmid comprises a polynucleotide sequence encoding one or more viral accessory genes.

10. The composition of embodiment 8, wherein:

(i) The first plasmid further comprises a polynucleotide sequence encoding a payload and flanking ITRs; and is also provided with

(ii) The second plasmid further comprises a polynucleotide sequence encoding one or more viral accessory genes.

11. The composition of embodiment 8, wherein:

(i) The first plasmid further comprises a polynucleotide sequence encoding one or more viral accessory genes; and is also provided with

(ii) The second plasmid further comprises a polynucleotide sequence encoding a payload and flanking ITRs.

12. The composition of any one of embodiments 7-11, wherein the polynucleotide sequence encoding a payload comprises one or more of the following:

(i) A polynucleotide encoding one or more enhancer sequences;

(ii) A polynucleotide encoding one or more promoter sequences;

(iii) A polynucleotide encoding one or more intron sequences;

(iv) A polynucleotide encoding a gene; and

(v) A polynucleotide comprising a polyA sequence.

13. The composition of any of embodiments 7-11, wherein the polynucleotide sequence encoding a payload comprises:

(i) A polynucleotide comprising a first nucleic acid sequence and a second nucleic acid sequence, wherein the first nucleic acid sequence comprises at least one gene and the second nucleic acid sequence is located 5 'or 3' to the first nucleic acid sequence and facilitates the production of two independent gene products upon integration to a target integration site;

(ii) A third nucleic acid sequence located 5 'to the polynucleotide and comprising a sequence homologous to a genomic sequence located 5' to the integration site of interest; and

(iii) A fourth nucleic acid sequence located 3 'to the polynucleotide and comprising a sequence homologous to a genomic sequence located 3' to the integration site of interest.

14. The composition of embodiment 13, wherein the integration site of interest is in the genome of the cell.

15. The composition of embodiment 13 or 14, wherein:

the target integration site comprises the 3' end of the endogenous gene;

the sequence of the third nucleic acid sequence is homologous to the DNA sequence upstream of the endogenous gene stop codon; and is also provided with

The sequence of the fourth nucleic acid sequence is homologous to the DNA sequence downstream of the endogenous gene stop codon.

16. The composition of any of embodiments 13-15, wherein the cell is a hepatocyte, a muscle cell, or a CNS cell.

17. The composition of any one of embodiments 7-16, for use in packaging an AAV vector.

18. The composition of any one of embodiments 7-16, wherein the composition is formulated for co-delivery of the first plasmid and second plasmid to a cell.

19. The composition of any of embodiments 7-18, wherein the composition comprises specific amounts of the first plasmid and the second plasmid to achieve a specific ratio between the two plasmids.

20. The composition of any one of the preceding embodiments, wherein the composition comprises a greater amount of the first plasmid relative to the second plasmid.

21. The composition of embodiment 19, wherein the plasmid ratio of the first plasmid to the second plasmid is greater than or equal to 1.5:1.

22. the composition of embodiment 20 or 21, wherein the first plasmid comprises a polynucleotide sequence encoding one or more viral accessory genes and the second plasmid comprises a polynucleotide sequence encoding a payload and flanking ITRs.

23. The composition of embodiment 22, wherein the first plasmid further comprises a rep gene and the second plasmid further comprises a cap gene.

24. The composition of embodiment 22, wherein the first plasmid further comprises a cap gene and the second plasmid further comprises a rep gene.

25. The composition of any one of the preceding embodiments, wherein the rep gene is a wild-type gene.

26. The composition of any one of the preceding embodiments, wherein the one or more viral accessory genes are wild-type genes.

27. The composition of any one of the preceding embodiments, wherein the rep and cap genes are under the control of one or more wild-type promoters.

28. A method of making a packaged AAV vector, comprising delivering the composition of any one of embodiments 7-27 to a cell.

29. The method of embodiment 28, wherein the cell is a mammalian cell.

30. The method of embodiment 28, further comprising using a chemical transfection reagent

31. The method of embodiment 30, wherein the chemical transfection reagent is or comprises a cationic molecule.

32. The method of embodiment 30, wherein the chemical transfection reagent is or comprises a cationic lipid.

33. A packaged AAV vector composition prepared by delivering the composition of any one of embodiments 7-27 to a cell.

34. The composition of any one of the preceding embodiments, wherein the payload comprises a transgene that is or comprises one or more of the following: propionyl-coa carboxylase, ATP7B, factor IX, methylmalonyl-coa Mutase (MUT), alpha 1-antitrypsin (A1 AT), UGT1A1 or variants thereof.

35. A method of treatment comprising administering to a subject in need thereof a composition comprising a packaged AAV vector produced by the method of embodiment 28 or 30.

36. The method of embodiment 35, wherein the subject has or is suspected of having a genetic disorder affecting metabolism, liver, skeletal muscle, cardiac muscle, central nervous system and/or blood.

37. The method of embodiment 36, wherein the subject has or is suspected of having one or more of: propioniemia, wilson's disease, hemophilia, krigler-naltrexone syndrome, methylmalonic acid emia (MMA), alpha-1 antitrypsin deficiency (A1 ATD), glycogen Storage Disease (GSD), duchenne muscular dystrophy, limb-area muscular dystrophy, X-linked myotube myopathy, parkinson's disease, mucopolysaccharidosis, hemophilia a, hemophilia B or Hereditary Angioedema (HAE).

38. The method of embodiment 35 or 37, wherein the composition is delivered to a cell.

39. The method of embodiment 38, wherein the cell is a hepatocyte, a muscle cell, or a CNS cell.

40. The method of embodiment 38 or 39, wherein the cells are isolated from a subject.

41. The method of any one of embodiments 35-40, wherein the composition does not comprise a nuclease or nucleic acid encoding a nuclease.

42. A Rep/helper plasmid having a sequence comprising SEQ ID NO:1, which does not comprise a polynucleotide sequence encoding an AAV cap gene.

43. A payload/Cap plasmid comprising a polypeptide comprising SEQ ID NO:11, a polynucleotide sequence encoding an AAV cap gene, and a polynucleotide sequence comprising a payload, wherein the plasmid does not comprise a polynucleotide sequence encoding an AAV rep gene.

44. A method, the method comprising the steps of: in the absence of any plasmid comprising polynucleotide sequences encoding both AAV Rep and AAV Cap, the population of cells used to produce AAV is combined with the Rep/helper plasmid and the payload/Cap plasmid in a transfection reagent mixture for AAV vector production under conditions effective to produce AAV vectors in the transfection reagent mixture.

45. The method of embodiment 44, wherein the Rep/helper plasmid is the Rep/helper plasmid of embodiment 42.

46. The method of any one of embodiments 44 or 45, wherein the payload/Cap plasmid is the payload/Cap plasmid of embodiment 43.

47. The method of any one of embodiments 44-46, wherein the Rep/helper plasmid and the payload/Cap plasmid are combined with the cell population at a relative w/w plasmid ratio between 1:10 and 10:1.

48. The method of any one of embodiments 44-47, wherein the Rep/helper plasmid and the payload/Cap plasmid are combined with the cell population at a relative w/w plasmid ratio between 1:3 and 3:1.

49. The method of any one of embodiments 44-48, wherein the Rep/helper plasmid and the payload/Cap plasmid are combined with the cell population at a relative w/w plasmid ratio of about 1.5:1.

50. A composition comprising two plasmids, wherein:

the first plasmid comprises SEQ ID NO: 1; and is also provided with

The second plasmid comprises SEQ ID NO: 11;

wherein the second plasmid further comprises:

a polynucleotide sequence comprising a sequence encoding a cap gene; and

a polynucleotide sequence encoding a payload;

wherein the second plasmid does not comprise a polynucleotide sequence encoding a rep gene.

51. A composition comprising two plasmids, wherein:

the first plasmid comprises SEQ ID NO:2, a sequence of 2; and is also provided with

The second plasmid comprises SEQ ID NO: 11;

wherein the second plasmid further comprises:

a polynucleotide sequence comprising a sequence encoding a cap gene; and

a polynucleotide sequence encoding a payload;

52. A composition comprising two plasmids, wherein:

the first plasmid consists of SEQ ID NO:1, a sequence composition of 1; and is also provided with

The second plasmid consists of:

SEQ ID NO: 11;

a polynucleotide sequence comprising a sequence encoding a cap gene; and

a polynucleotide sequence encoding a payload;

53. A composition comprising two plasmids, wherein:

the first plasmid consists of SEQ ID NO:2, a sequence composition of 2; and is also provided with

The second plasmid consists of:

SEQ ID NO: 11;

a polynucleotide sequence comprising a sequence encoding a cap gene; and

a polynucleotide sequence encoding a payload;

54. The composition of any one of the preceding embodiments, wherein the cap gene is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVC11.01, AAVC11.02, AAVC11.03, AAVC11.04, AAVC11.05, AAVC11.06, AAVC11.07, AAVC11.08, AAVC11.09, AAVC11.10, AAVC11.11, AAVC11.12, AAVC11.13, AAVC11.14, AAVC11.15, AAVC11.16, AAVC11.17, AAVC11.18, AAVC11.19, AAV-DJ, AAV-LK03, AAV-LK19, aavrh.74, aavrh.10, aavhu.37, aah.k, aah.39, 12, 13, aavrh.8, avian, AAV, a non-primate, AAV, or a non-primate.

55. The composition of any of the preceding embodiments, wherein the polynucleotide sequence comprising a sequence encoding a cap gene is set forth in SEQ ID NO:11 is inserted before position 2025.

56. The composition of any of the preceding embodiments, wherein the polynucleotide sequence encoding a payload comprises a polynucleotide sequence encoding a transgene.

57. The composition of any one of the preceding embodiments, wherein the polynucleotide sequence encoding the payload is set forth in SEQ ID NO:11 is inserted after position 2663.

58. The composition of any of embodiments 56 or 57, wherein the transgene is or comprises a gene listed in figure 29, or a variant thereof.

59. The composition of any one of embodiments 56 or 57, wherein the transgene is or comprises one or more of the following: propionyl-coa carboxylase, ATP7B, factor IX, methylmalonyl-coa Mutase (MUT), alpha 1-antitrypsin (A1 AT), UGT1A1, fumaroyl acetoacetate hydrolase (far), cystathionine Beta Synthase (CBS), or variants thereof.

60. The composition of any of the preceding embodiments, wherein the composition comprises no more than two different plasmids.

61. The composition of any of the preceding embodiments, wherein the composition comprises no less than three different plasmids.

62. A composition comprising two plasmids, wherein:

the first plasmid comprises SEQ ID NO: 1; and is also provided with

The second plasmid comprises SEQ ID NO: 9;

63. A composition comprising two plasmids, wherein:

the first plasmid comprises SEQ ID NO: 1; and is also provided with

The second plasmid comprises SEQ ID NO: 10;

64. A composition comprising two plasmids, wherein:

The second plasmid comprises SEQ ID NO: 9;

65. A composition comprising two plasmids, wherein:

The second plasmid comprises SEQ ID NO: 10;

66. A composition comprising two plasmids, wherein:

The second plasmid consists of SEQ ID NO: 9.

67. A composition comprising two plasmids, wherein:

The second plasmid consists of SEQ ID NO: 10.

68. A composition comprising two plasmids, wherein:

The second plasmid consists of SEQ ID NO: 9.

69. A composition comprising two plasmids, wherein:

The second plasmid consists of SEQ ID NO: 10.

70. The composition of any one of embodiments 62-69, wherein the composition comprises no more than two different plasmids.

71. The composition of any one of embodiments 62-70, wherein the composition comprises no less than three different plasmids.

72. The composition of any one of the preceding embodiments, wherein the plasmid ratio of the first plasmid to the second plasmid is greater than or equal to 1.5:1 up to 10:1.

73. The composition of any one of the preceding embodiments, for use in packaging an AAV vector.

74. The composition of any one of the preceding embodiments, wherein the composition is formulated for co-delivery of the first plasmid and second plasmid to a cell.

75. A method of making a packaged AAV vector, comprising delivering the composition of any of the preceding embodiments to a cell.

76. The method of embodiment 75, wherein the cell is a mammalian cell.

77. The method of any one of embodiments 75 or 76, further comprising using a chemical transfection reagent.

78. The method of embodiment 77, wherein the chemical transfection reagent is or comprises a cationic lipid.

79. The method of embodiment 78, wherein the chemical transfection reagent is or comprises a cationic molecule.

80. A packaged AAV vector composition prepared by delivering the composition of any one of embodiments 50-74 to a cell.

81. A method of treatment comprising administering to a subject in need thereof a composition comprising a packaged AAV vector produced by the method of any one of embodiments 75-79.

82. The method of embodiment 81, wherein the subject has or is suspected of having a genetic disorder affecting metabolism, liver, skeletal muscle, cardiac muscle, central nervous system and/or blood.

83. The method of embodiment 82, wherein the subject has or is suspected of having one or more of: propiolic acid, wilson's disease, hemophilia, krigler-naltrexone syndrome, methylmalonic acid (MMA), alpha-1 antitrypsin deficiency (A1 ATD), glycogen Storage Disease (GSD), duchenne muscular dystrophy, limb-area muscular dystrophy, X-linked myotube myopathy, parkinson's disease, mucopolysaccharidosis, hemophilia a, hemophilia B, homocystinuria, urea cycle disorders, hereditary tyrosinemia (HT 1) or Hereditary Angioedema (HAE).

84. The method of any one of embodiments 82 or 83, wherein the composition is delivered to a cell.

85. The method of any one of embodiment 84, wherein the cell is a hepatocyte, a muscle cell, or a CNS cell.

86. The method of any one of embodiments 84 or 85, wherein the cells are isolated from a subject.

87. The method of any one of embodiments 81-86, wherein the composition does not comprise a nuclease or nucleic acid encoding a nuclease.

Claims

1. A Rep/helper plasmid comprising the polynucleotide sequence of SEQ ID No. 1, wherein the plasmid does not comprise a polynucleotide sequence encoding a cap gene.

2. A Rep/helper plasmid comprising the polynucleotide sequence of SEQ ID No. 2, wherein the plasmid does not comprise a polynucleotide sequence encoding a cap gene.

3. A composition, the composition comprising:

the plasmid according to any one of claims 1 or 2; and

a payload/Cap plasmid comprising the polynucleotide sequence of SEQ ID No. 11;

wherein the payload/Cap plasmid does not comprise a polynucleotide sequence encoding a rep gene.

4. The composition of claim 3, wherein the payload/Cap plasmid comprises:

a polynucleotide sequence comprising a sequence encoding a cap gene; and

a polynucleotide sequence encoding a payload.

5. The composition of claim 4, wherein the cap gene is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVC11.01, AAVC11.02, AAVC11.03, AAVC11.04, AAVC11.05, AAVC11.06, AAVC11.07, AAVC11.08, AAVC11.09, AAVC11.10, AAVC11.11, AAVC11.12, AAVC11.13, AAVC11.14, AAVC11.15, AAVC11.16, AAVC11.17, AAVC11.18, AAVC11.19, AAV-DJ, AAV-LK03, AAV-LK19, aavrh.74, aavrh.10, aavhu.37, aavrh.k, aah.39, AAV12, 13, aah.8, avian, AAV, canine, equine, AAV, non-primate, AAV, or a non-primate.

6. The composition of any one of claims 4 or 5, wherein the polynucleotide sequence comprising a sequence encoding a cap gene is inserted prior to position 2025 of SEQ ID No. 11.

7. The composition of any one of claims 4-6, wherein the polynucleotide sequence encoding a payload comprises a polynucleotide sequence encoding a transgene.

8. The composition of any one of claims 4-7, wherein the polynucleotide sequence encoding a payload is inserted after position 2663 of SEQ ID No. 11.

9. The composition of any one of claims 7 or 8, wherein the transgene is or comprises a gene listed in figure 29, or a variant thereof.

10. The composition of any one of claims 7 or 8, wherein the transgene is or comprises one or more of: propionyl-coa carboxylase, ATP7B, factor IX, methylmalonyl-coa Mutase (MUT), alpha 1-antitrypsin (A1 AT), UGT1A1, fumaroyl acetoacetate hydrolase (far), cystathionine Beta Synthase (CBS), or variants thereof.

11. The composition of any one of claims 7 or 8, wherein the transgene is FAH or a variant thereof.

12. The composition of any one of claims 7 or 8, wherein the transgene is MUT or a variant thereof.

13. The composition of any one of claims 7 or 8, wherein the transgene is CBS or a variant thereof.

14. The composition of any one of claims 7 or 8, wherein the transgene is ATP7B or a variant thereof.

15. The composition of any one of claims 7 or 8, wherein the transgene is factor IX or a variant thereof.

16. The composition of any one of claims 7 or 8, wherein the transgene is UGT1A1 or a variant thereof.

17. The composition of any one of claims 3-16, wherein the composition comprises no more than two different plasmids.

18. The composition of any one of claims 3-17, wherein the plasmid ratio of the Rep/helper plasmid to the payload/Cap plasmid is greater than or equal to 1.5:1 up to 10:1.

19. The composition of any one of the preceding claims, for use in the production of an AAV vector.

20. A method of making a packaged AAV vector, the method comprising delivering the composition of any of claims 3-18 to a cell.

21. The method of claim 20, further comprising using a chemical transfection reagent.