Compositions and methods for increasing AAV productivity

Abstract

Provided are vectors and vector systems for the production of recombinant adeno-associated virus vectors, as well as methods for optimizing productivity using the above vectors and vector systems. This disclosure relates to vectors comprising one or more modified adenovirus nucleic acids for the production of recombinant adeno-associated virus (rAAV). In some embodiments, the vector comprises nucleic acid E4, nucleic acid E2a, and virus-associated (VA)RNA from the adenovirus genome, including deletions.

Description

Background Art

[0001] Background Adeno-associated virus (AAV) vectors are commonly used for nucleic acid delivery into cells. The success of AAV-mediated gene replacement, gene silencing, and gene editing has made AAV a desirable therapeutic vector, and AAV-based therapeutic agents have received regulatory approval in Europe and the United States. However, existing production systems can be difficult to optimize and often produce only insufficient amounts of vector genomes for gene therapy applications. Thus, an improved AAV vector production system is needed.

Summary of the Invention

Means for Solving the Problems

[0002] Summary of the Invention Some of the main aspects of the present invention are outlined below. Further aspects are described in the detailed description, examples, and claims sections of the present disclosure. The description in each section of the present disclosure is intended to be read in conjunction with the other sections. Furthermore, the various embodiments described in each section of the present disclosure can be combined in various ways, and all such combinations are intended to fall within the scope of the present invention.

[0003] The present disclosure relates to a vector comprising one or more modified adenovirus nucleic acids for the production of recombinant adeno-associated virus (rAAV). In some embodiments, the vector comprises nucleic acid E4, nucleic acid E2a, and virus-associated (VA) RNA, which includes deletions, from the adenovirus genome. In some embodiments, the nucleic acid E2a is a modified nucleic acid E2 (e.g., nucleic acid E2 including deletions).

[0004] In some embodiments, the vector comprises nucleic acid E4, nucleic acid E2a, and virus-associated (VA) RNA of the adenovirus genome, wherein the nucleic acid E4 includes deletions of open reading frames 1-3 (orf 1-3).

[0005] In some embodiments, the nucleic acid E4 deletion is a deletion of at least 1000 base pairs (for example, a deletion of at least 1000 base pairs in E4orf1 to E4orf3, or a deletion of at least 1400 base pairs in E4orf1 to E4orf4). In some embodiments, the nucleic acid E4 includes the nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 4; or a nucleic acid sequence that is at last 85% identical to SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 4.

[0006] In some embodiments, nucleic acid E4 includes an open reading frame 6 (orf6), i.e., nucleic acid E4 containing deletions of orf 1-4 and 7. In some embodiments, nucleic acid E4 includes the nucleic acid sequence of SEQ ID NO: 3 or a nucleic acid sequence having at last 85% identity with SEQ ID NO: 3.

[0007] In some embodiments, the nucleic acid E4 includes E4orf6 and E4orf7, i.e., nucleic acid E4 containing deletions of orf 1 to 5. In some embodiments, the vector includes the nucleic acid sequence of SEQ ID NO: 4 or a nucleic acid sequence having at last 85% identity with SEQ ID NO: 4.

[0008] In some embodiments, the vector further comprises an E2a deletion. In some embodiments, the E2 deletion comprises a deletion of more than 400 base pairs in the 5' untranslated region of the E2a gene. In some embodiments, the vector comprises the nucleic acid sequence of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12, or a nucleic acid sequence having at least 85% identity with SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12.

[0009] In some embodiments, the vector includes the nucleic acid sequence of SEQ ID NOs. 13, 14, 15, 16, 17, or 18, or a nucleic acid sequence having at least 85% identity with SEQ ID NOs. 13, 14, 15, 16, 17, or 18.

[0010] Furthermore, a vector system comprising at least two vectors is provided, wherein the first vector comprises a helper plasmid, wherein the helper plasmid is a plasmid comprising modified E4 nucleic acid, E2a nucleic acid, and virus-associated (VA) RNA from the adenovirus genome as described herein. In some embodiments, the second vector is a plasmid comprising a transgene.

[0011] Furthermore, a vector system is provided comprising a portion of nucleic acid E4 of the adenovirus genome, at least a portion of nucleic acid E2a, and virus-associated (VA)RNA, wherein at least a portion of nucleic acid E4 includes a deletion configured to increase the production of AAV products, including the AAV capsid, compared to production using a vector system lacking the above-mentioned nucleic acid E4 deletion. In some embodiments, at least a portion of the nucleic acid E2a includes a deletion.

[0012] A vector system comprising at least two vectors is further provided, wherein the first vector comprises one or more of the following from the adenovirus genome: a portion of nucleic acid E4, at least a portion of nucleic acid E2a, and virus-associated (VA)RNA, wherein the portion of nucleic acid E4 comprises a deletion configured to increase the production of AAV products, including the AAV capsid, compared to production using a vector system lacking the nucleic acid E4 deletion. In some embodiments, at least a portion of nucleic acid E2a comprises the deletion. Optionally, the first vector comprises the portion of nucleic acid E4, at least a portion of nucleic acid E2a, and virus-associated (VA)RNA from the adenovirus genome.

[0013] In some embodiments, the AAV product has a higher level of vector genome per liter, a greater number of capsids per liter, or both, compared to a control AAV product produced by an AAV vector system lacking a deletion of nucleic acid E4 or lacking deletions of both nucleic acid E4 and nucleic acid E2a. In some embodiments, the second vector includes a transgene.

[0014] Furthermore, a method for producing an AAV product containing an AAV capsid is provided. The method comprises introducing one of the vector systems described herein into a host cell, incubating the host cell under culture conditions that promote AAV product production, and purifying the AAV product.

[0015] Another aspect of the present invention is a method for delivering a transgene to cells, the method comprising introducing the AAV product produced by the method provided above into the cells. In some embodiments, the method is an in vitro method. In some embodiments, the transgene is expressed by the cells. Some embodiments provide the use of the AAV product of the present disclosure in a method for delivering a transgene to cells, where the transgene is expressed by the cells as necessary. [Brief explanation of the drawing]

[0016] This application includes the following drawings. The drawings are intended to illustrate certain embodiments and / or features of the above compositions and methods, and to supplement any(s) of the above compositions and methods. The drawings do not limit the scope of the above compositions and methods unless expressly indicated that the same applies to the written description.

[0017] [Figure 1]Figure 1 is a schematic diagram illustrating a vector construct containing helper genes E2a, E4, and VA RNA. The E2a gene is approximately 5.3 kilobases (kb) and is regulated by the E2E promoter to produce a gene product. The E2a and L4 genes are duplicated on opposite strands, resulting in the L4 region being included in the helper plasmid. The E4 gene contains six open reading frames (orfs) and is approximately 3.2 kb. The VA (virus-associated) RNA gene is approximately 0.74 kb.

[0018] [Figure 2] Figure 2 shows, from top to bottom: (a) VA RNA, nucleic acid E4, nucleic acid E2a, and an adenovirus skeleton without deletions (referred to as control); (b) VA RNA, nucleic acid E4 including E4 deletion 1 (i.e., nucleic acid E4 including deletions E4orf1~E4orf3), nucleic acid E2a, and an adenovirus skeleton with deletions (referred to as del-1); (c) VA RNA, E4 nucleic acid including E4 deletion 2 (i.e., nucleic acid E4 including deletions E4orf1~E4orf4 and having only E4orf6~7), and an adenovirus skeleton with deletions (referred to as del-2); (d) VA RNA, E4 nucleic acid, E2a nucleic acid with deletions, and an adenovirus skeleton with deletions (referred to as del-3); (e) VA The following are exemplary helper vector constructs comprising RNA, nucleic acid E4 (above), E2a nucleic acid with deletion (approximately 900 base pairs), and an adenovirus skeleton (referred to as del-4) with deletion; and (f)VA RNA, E4 nucleic acid (above), E2a nucleic acid with deletion, and an adenovirus skeleton (referred to as del-5) with deletion. The above skeleton deletions consist of deletions of nucleic acid residues or regions not required for functional plasmid formation.

[0019] [Figure 3]Figure 3 provides graphs showing the effects of various helper plasmids described in Figure 2 on viral genome (VG) concentration (left) and capsid concentration (right), using AAV9 serotypes and GFP / luciferase genomes. All plasmids with E4 deletions increased production, as evidenced by higher concentrations of VG and capsid per liter, compared to production by control plasmids or plasmids lacking the E4 deletion (e.g., del-3 with E2a deletion and skeletal deletion). VG titer was measured from crude lysates by ddPCR, and capsid titer was measured by capsid ELISA.

[0020] [Figure 4] Figure 4 shows graphs illustrating the effects of a control plasmid, as well as a helper plasmid (del-5) containing E2a nucleic acid deletion, E4 nucleic acid with deletion 2, VA RNA, and adenovirus scaffold deletion, on VG concentration (left) and AAV capsid concentration (right) for multiple AAV serotypes. For each serotype, the increase in viral genome and capsid production was observed using the plasmid referred to as del-5 compared to the control plasmid. VG titer was measured from crude lysates by ddPCR, and capsid titer was measured by capsid ELISA. [Modes for carrying out the invention]

[0021] Detailed explanation The following description outlines various aspects and embodiments of the compositions and methods of the present invention. No particular embodiment is intended to define the scope of the compositions and methods described above. Rather, the embodiment merely provides non-limiting examples of various compositions and methods that fall within the scope of the compositions and methods of this disclosure. The description should be read from the perspective of those skilled in the art; therefore, information that is well known to those skilled in the art is not necessarily included.

[0022] Any headings provided in this specification are not limitations of the various aspects or embodiments of the invention that may be obtained by referring to the specification as a whole. Thus, the terms defined immediately below are more fully defined by referring to the specification as a whole in its entirety.

[0023] All references cited in this disclosure are hereby incorporated by reference in their entirety into this specification. Further, any manufacturer's instructions or catalogs regarding any products cited or referred to in this specification are incorporated by reference. Any document incorporated by reference into this text, or any teachings therein, may be used in the practice of the invention. The documents incorporated by reference into this text are not admitted to be prior art.

[0024] Definitions The nomenclature or terminology used in this disclosure is for descriptive purposes only and not for limiting purposes, and as a result, the nomenclature or terminology of this specification should be interpreted by one of ordinary skill in the art in light of the above teachings and guidance.

[0025] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. The terms "a" (or "an") and the terms "one or more" and "at least one" may be used interchangeably.

[0026] Furthermore, "and / or" should be understood as a specific disclosure of each of the two identified features or components, in the presence or absence of another feature or component. Thus, when used in a phrase such as "A and / or B," the term "and / or" is intended to include A and B, A or B, A (alone), and B (alone). Similarly, when used in a phrase such as "A, B, and / or C," the term "and / or" is intended to include A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A (alone); B (alone); and C (alone).

[0027] Whenever an embodiment is described with the words “comprising” or “having,” it encompasses other similar embodiments described with respect to “consisting of” and / or “essentially consisting of.”

[0028] Units, prefixes, and symbols are presented in the form recognized by their International System of Units (SI). A numeric range encompasses the numeric values ​​that define that range, and any individual values ​​provided herein may act as endpoints to a range that includes other individual values ​​provided herein. For example, a group of values ​​such as 1, 2, 3, 8, 9, and 10 is also a disclosure of numeric ranges such as 1–10, 1–8, and 3–9. Similarly, a disclosed range is a disclosure of each individual value encompassed by that range. For example, the indicated value 5–10 is also a disclosure of 5, 6, 7, 8, 9, and 10. If “approximately” precedes a numeric term, that term includes the indicated numeric value and values ​​within ±10% of that numeric value.

[0029] vector One or more modified vectors comprising adenovirus nucleic acids are provided herein. In some embodiments, the vector comprises nucleic acid E4, nucleic acid E2a, and virus-associated (VA)RNA from the adenovirus genome, wherein nucleic acid E4 includes deletions.

[0030] When used in its entirety, "vector" refers to a nucleic acid construct. Any nucleic acid construct containing one or more elements required for AAV production may be used as one of the vectors described above or as a component of the vector system described herein. Suitable vectors include plasmids, minimal vectors (e.g., minicircles, nanoplasmids). TM Examples of suitable DNA minima vectors include, but are not limited to, doggybone, MIDGE vectors, viruses, cosmids, artificial chromosomes, linear DNA, and mRNA. Suitable DNA minima vectors include linear covalently closed DNA (e.g., ministring DNA), linear covalently closed dumbbell-shaped DNA (e.g., doggybone DNA, dumbbell DNA), minicircles, and nanoplasmids. TMExamples include, but are not limited to, minimalistic immunologically defined gene expression (MIDGE) vectors and others known to those skilled in the art. Minimal DNA vectors and methods for producing them are described, for example, in U.S. Patent Publications 20100233814, 20120282283, 20130216562, 20150218565, 20150218586, 20160008488, 20160215296, 20160355827, 20190185924, 20200277624, and 20210010021 (all of which are incorporated herein by reference in their entirety). In some embodiments, the vector is a circular single-stranded or double-stranded nucleic acid sequence construct, such as a double-stranded DNA plasmid. In some cases, the vector is an extrachromosomal circular DNA containing one or more origins of replication that have the ability to autonomously replicate in a given cell (e.g., a eukaryotic cell or a bacterial cell). In some embodiments, the vector does not contain one or more sequences necessary for autonomous replication in a bacterial cell, such as a bacterial origin of replication. In some embodiments, the vector contains one or more elements required for AAV replication. In some embodiments, the vector system includes a vector or combination of vectors containing the elements required for AAV replication. In some embodiments, the vector system includes a vector containing the elements required for AAV replication and a vector containing a gene of interest.

[0031] As used herein, the terms nucleic acid or nucleotide refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof, in either single-stranded or double-stranded form. Unless specifically limited, the above terms encompass nucleic acids, including known analogs of natural nucleotides having properties similar to the reference nucleic acid. Nucleic acid sequences may include combinations of deoxyribonucleic acid and ribonucleic acid. Such deoxyribonucleic acid and ribonucleic acid include both naturally occurring molecules and synthetic analogs. Nucleic acids also include all forms of sequences, including, but not limited to, single-stranded, double-stranded, hairpin, and stem-and-loop structures.

[0032] In some embodiments, the nucleic acids in the vectors described herein are optimized, for example, by codon / RNA optimization, substitution with heterologous signal sequences, and / or elimination of mRNA instability elements. Methods for generating polynucleotides optimized for recombinant expression by introducing codon changes and / or eliminating inhibitory regions in mRNA may, depending on the context, be adapted to, for example, U.S. Patents 5,965,726; 6,174,666; 6,291,664; 6,414,132; and 6,794,498 (all of which are incorporated herein by reference in their entirety). For example, potential splice sites and instability elements in RNA (e.g., A / T or A / U rich elements) may be mutated without altering the amino acids encoded by the nucleic acid sequence in order to increase the stability of the RNA for recombinant expression. The above changes may, for example, utilize the degeneracy of the genetic code by using alternative codons for the same amino acids. In certain embodiments, it may be desirable to alter one or more codons to encode a conservative mutation (e.g., a similar amino acid having a similar chemical structure and properties and / or function to the original amino acid).

[0033] When used as a whole, nucleic acid E4 may contain coding and / or non-coding sequences from the E4 gene of the adenovirus genome. The gene may contain exon regions, intron regions, and / or untranslated regions from the genome sequence. In some cases, the gene (e.g., E4) may have one or more coding sequences due to alternative splicing or alternative translation initiation, etc. The coding sequences may be wild-type or naturally occurring coding sequences (e.g., codon-optimized E4 coding sequences).

[0034] In some cases, nucleic acid E4 encodes one or more E4 gene products, e.g., one or more E4 proteins or fragments thereof. The one or more E4 gene products or fragments thereof may be encoded by one or more open reading frames (orfs) (i.e., orf1-7) of the adenovirus E4 genome sequence. The one or more E4 gene products or fragments thereof may modulate transcription, the cell cycle, cell signaling, and / or DNA repair. For example, E4orf1 encodes a protein that interacts with cellular factors containing a PDZ domain binding motif. E4orf3 encodes an 11kDa protein that is functionally redundant with the 34kD protein encoded by E4orf6. The E4orf3 protein may be involved in viral DNA replication, late viral protein synthesis, blockade of host protein synthesis, and / or virogenesis. E4orf4 encodes a protein that can regulate protein phosphorylation during infection through its ability to bind to protein phosphatase 2A (PP2A), one of the major serine / threonine-specific phosphatases in cells. E4orf6 encodes a 34kDa protein that forms a multifunctional complex involved in viral DNA replication, RNA processing, nucleocytoplasmic transport of late viral mRNA, and blockade of host protein synthesis. The gene products of E4orf6 and E4orf7 are fusions of the E4orf6 and E4orf7 proteins. For example, in AdV type 5, the above protein consists of 58 residues from the amino terminus of E4orf6 fused to 92 amino acids of E4orf7. The above E4orf6 / 7 proteins modulate transcriptional activity through their interaction with the cellular E2F / DP family of cellular transcription factors.

[0035] When used as a whole, nucleic acid E2a contains coding and / or non-coding sequences from the E2a gene of the adenovirus genome. Nucleic acid E2a may contain exon regions, intron regions, and / or untranslated regions from the E2a sequence of the genome. In some cases, nucleic acid E2a encodes the E2a protein or a fragment thereof. E2A is a single-strand DNA-binding protein that stimulates viral DNA replication and gene transcription.

[0036] As used herein, VA RNA is an RNA that functions by inhibiting double-stranded RNA-activated kinase (PKR), a cellular innate immune protein, which ensures efficient viral protein synthesis. VA RNA has also been shown to promote the synthesis and assembly of AAV structural proteins. It will be readily apparent to those skilled in the art that the above VA RNA nucleic acid sequence is the untranslated nucleic acid sequence that gives rise to the above VA RNA.

[0037] The vectors described herein can function as helper plasmids in plasmid systems for generating recombinant AAV capsids. Typically, adenovirus (AdV) helper factors E1A, E1B, E2A, E4, and VA RNA are required for viral replication. Any of the helper viral factors described herein may be derived from the adenovirus (AdV) genome, for example, from the AdV type 2 genome or the AdV type 5 genome. If a nucleic acid sequence for an AdV type 2 helper viral factor, such as E4, E2a, or VA, is provided herein, it is understood that the corresponding AdV type 5 nucleic acid sequence will also be provided. The sequences provided herein correspond to AdV type 2, but comparable sequences may be obtained for other types of AdV. The nucleic acids encoding these AdV helper factors or parts thereof (i.e., E1A, E1B, E2A, E4, and VA RNA) are transfected into host cells on a single plasmid. Alternatively, one or more plasmids containing these helper functions may be transfected into host cells. The above helper factors may also be expressed in host cells by transfecting them with one or more plasmids encoding helper factors that are not endogenously expressed by these host cells. In some cases, certain host cells (such as HEK293T cells) endogenously provide some (but not all) of the required helper factors, and the remaining helper factors may be provided exogenously via plasmid transfection.For example, HEK293T cells endogenously express the AdV E1A and E1B genes and can be transfected with any of the vectors described herein (e.g., helper plasmids) to provide the host cells with at least the minimum helper elements required for AAV replication (e.g., one or more AdV E4 gene products or fragments thereof, E2a gene products or fragments thereof, and virus-associated (VA) RNA) to generate the necessary helper elements (e.g., E1A, E1B, E2A, one or more E4 gene products, and VA RNA) for AAV production in the host cells.

[0038] In any of the helper plasmids described herein, nucleic acid sequences encoding one or more of the above-mentioned helper factors or functional portions thereof for AAv replication (e.g., E4 gene product, E2a gene product, and / or VA RNA) can be operably ligated to a transcriptional regulatory element that controls the expression of the above-mentioned helper factors. In certain embodiments, the transcriptional regulatory element comprises a promoter selected from the group consisting of constitutive promoters, inducible promoters, or native promoters. Suitable promoters are known to those skilled in the art and include, but are not limited to, the RSV LTR promoter, the CMV early promoter, the SV40 promoter, the dihydrofolate reductase promoter, the cytoplasmic β-actin promoter, the phosphoglycerate kinase (PGK) promoter, the metallothionein (MT) promoter, the mouse mammary tumor virus (MMTV) promoter, the T7 promoter, the insect ecdysone promoter, the tetracycline repressive promoter, the tetracycline inducible promoter, the RU486 inducible promoter, and the rapamycin inducible promoter.

[0039] In some embodiments, the vector comprises the nucleic acid E4, nucleic acid E2a, and virus-associated (VA)RNA of the adenovirus genome, where the nucleic acid E4 includes a deletion of one or more E4 open reading frames (orfs). When used as a whole, an orf deletion means the deletion or removal of at least a portion of the E4orf such that reduced expression and / or activity of one or more gene products encoded by the E4orf occurs. As used herein, the reduction may be a reduction of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the expression and / or activity. In some examples, the E4 deletion results in reduced expression and / or activity of one or more E4 gene products selected from the group consisting of E4orf1, E4orf2, E4orf3, E4orf4, and E4orf6 / 7.

[0040] In some embodiments, the nucleic acid E4 deletion is a deletion of open reading frames 1-3 (E4orf1-3). That is, the nucleic acid E4 deletion includes E4orf4, Eorf6, and Eorf7. In some embodiments, the nucleic acid E4 deletion is a deletion of at least 1000 base pairs (e.g., at least about 1000, 1025, 1050, or 1075 base pairs) of the nucleic acid sequence (SEQ ID NO: 2) containing E4orf1-E4orf3. In some embodiments, the nucleic acid E4 containing the deletions of E4orfs1-3 includes SEQ ID NO: 1, or a nucleic acid sequence having at least 60%, 65%, 70%, 75%, 80%, 90%, 95%, or 99% identity with SEQ ID NO: 1.

[0041] In some embodiments, the vector comprises nucleic acid E4 orf6 of the adenovirus genome; nucleic acid E2a or a portion thereof; and VA RNA, where nucleic acid E4 includes a deletion.

[0042] In some embodiments, nucleic acid E4, including E4orf6 and / or E4orf7, includes one or more orf deletions selected from the group consisting of E4orf1, E4orf2, and E4orf4 (e.g., deletions of E4orfs1-4). In some embodiments, the nucleic acid E4 deletion is a deletion of at least 1000 base pairs (e.g., at least about 1000, 1025, 1050, 1075, 1100, 1125, 1150, 1175, 1200, 1225, 1250, 1275, 1300, 1325, 1350, or 1400 base pairs) of nucleic acid sequences containing the deletions of E4orf1-E4orf4 (SEQ ID NO: 5). Exemplary nucleotide sequences containing nucleic acids E4orf6, nucleic acids E4orf7, and nucleic acid E4 deletions E4orfs1-5 are shown herein as Sequence ID No. 4. In some embodiments, nucleic acid E4 containing nucleic acids E4orf6, nucleic acids E4orf7, and nucleic acid E4 deletions E4orfs1-5 have at least 60%, 65%, 70%, 75%, 80%, 90%, 95%, or 99% identity with Sequence ID No. 4.

[0043] In some embodiments, the vector comprises nucleic acid E4 containing a deletion (as described above) and nucleic acid E2a without a deletion (i.e., wild-type adenovirus nucleic acid E2a). Exemplary wild-type nucleic acid E4 and wild-type nucleic acid E2a sequences are shown herein as Sequence ID No. 6 and 7, respectively.

[0044] In some embodiments, the vector comprises nucleic acid E4 (as described above) containing a deletion, and nucleic acid E2a containing a deletion. In some embodiments, the E2a deletion comprises a deletion of at least about 475, 500, 525, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, or 1000 base pairs in the untranslated genomic region (e.g., the 5' untranslated region (UTR)) of nucleic acid E2a. Exemplary nucleic acid sequences comprising nucleic acid E2a containing a deletion of at least 500 base pairs in the 5' UTR are shown herein as SEQ ID NOs: 8-12. SEQ ID NO: 8 is nucleic acid E2a containing a deletion of about 2 kb in the 5' UTR. SEQ ID NO: 9 is nucleic acid E2a containing a deletion of about 1 kb in the 5' UTR. Sequence ID 10 is nucleic acid E2a containing a deletion of approximately 1.4 kb in the 5' UTR. Sequence ID 11 is nucleic acid E2a containing a deletion of approximately 500 base pairs in the 5' UTR. Sequence ID 12 is nucleic acid E2a containing a deletion of approximately 900 base pairs in the 5' UTR.

[0045] Any of the vectors described herein may further include a nucleotide sequence encoding a VA RNA, for example, a nucleotide sequence containing SEQ ID NO: 13, or a nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 90%, 95%, or 99% identity with SEQ ID NO: 13.

[0046] In some embodiments, the vector includes nucleic acid E4 containing a deletion (e.g., sequence number 1, sequence number 3 or sequence number 4, or a sequence having at least 60%, 65%, 70%, 75%, 80%, 90%, 95%, or 99% identity with sequence number 1, sequence number 3 or sequence number 4), nucleic acid E2a (e.g., sequence number 7, or a nucleic acid sequence having at least 60%, 65%, 70%, 75%, 80%, 90%, 95%, or 99% identity with sequence number 7), and VA RNA (e.g., sequence number 13, or a nucleic acid sequence having at least 60%, 65%, 70%, 75%, 80%, 90%, 95%, or 99% identity with sequence number 13).

[0047] In some embodiments, the vector includes a deletion-containing nucleic acid E4 (e.g., sequence number 1, sequence number 3, or sequence number 4, or a sequence having at least 60%, 65%, 70%, 75%, 80%, 90%, 95%, or 99% identity with sequence number 1, sequence number 3, or sequence number 4), a deletion-containing nucleic acid E2a (e.g., sequence number 8, 9, 10, 11, or 12; or a nucleic acid sequence having at least 60%, 65%, 70%, 75%, 80%, 90%, 95%, or 99% identity with sequence number 8, 9, 10, 11, or 12), and VA RNA (e.g., sequence number 13, or a nucleic acid sequence having at least 60%, 65%, 70%, 75%, 80%, 90%, 95%, or 99% identity with sequence number 13).

[0048] An exemplary vector sequence (plasmid pOX-02010) containing SEQ ID NOs: 1, 7, and 13 is shown herein as SEQ ID NO: 14. An exemplary vector sequence (plasmid pOX-02011) containing SEQ ID NOs: 4, 7, and 13 is shown herein as SEQ ID NO: 15.

[0049] In some embodiments, the vector comprises a deletion-containing nucleic acid E4 (e.g., SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 4), a deletion-containing nucleic acid E2a (SEQ ID NO: 8, 9, 10, 11, or 12), and VA RNA (SEQ ID NO: 13). An exemplary vector sequence (plasmid pOX-02013) containing SEQ ID NO: 1, SEQ ID NO: 12, and SEQ ID NO: 13 is shown herein as SEQ ID NO: 16. An exemplary vector sequence (plasmid pOX-02014) containing SEQ ID NO: 4, SEQ ID NO: 12, and SEQ ID NO: 13 is shown herein as SEQ ID NO: 17.

[0050] In some embodiments, the vector comprises nucleic acid E4 without deletion (e.g., SEQ ID NO: 6), nucleic acid E2a with deletion (SEQ ID NO: 8, 9, 10, 11, or 12), and VA RNA (SEQ ID NO: 13). An exemplary vector sequence (plasmid pOX-02012) containing SEQ ID NO: 6, SEQ ID NO: 12, and SEQ ID NO: 13 is shown herein as SEQ ID NO: 18.

[0051] Any of the vectors described herein, for example, a helper plasmid, may contain a skeletal nucleic acid sequence comprising one or more regulatory elements (e.g., promoters, enhancers, sequences, transcription termination factors, polyadenylation sites, etc.), one or more origins of replication, and / or a selection marker gene). In some embodiments, the helper plasmid vector comprises a skeletal nucleic acid sequence without deletions or a skeletal nucleic acid sequence containing deletions.

[0052] Any of the vectors provided herein may contain nucleic acid sequences having at least 60%, 65%, 70%, 80%, 90%, 91%, 93%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with the sequences described herein (e.g., SEQ ID NOs. 1-18). The term, identity, when used in the context of polynucleotide or polypeptide sequences described herein, refers to a sequence having at least 60% sequence identity with a reference sequence (e.g., any one of SEQ ID NOs. 1-18). Alternatively, percent identity can be any integer between 60% and 100%. Such identity with respect to a reference sequence can be determined using BLAST with the program described herein; preferably with standard parameters (as described below). Those skilled in the art will recognize that these values ​​can be appropriately adjusted to determine the corresponding identity of proteins encoded by two nucleotide sequences, taking into account codon degeneracy, amino acid similarity, reading frame positioning, etc.

[0053] For sequence comparison, typically one sequence acts as a reference sequence (to which the test sequence is compared). When using a sequence comparison algorithm, the test sequence and reference sequence are input into the computer, and if necessary, subsequence coordinates and sequence algorithm program parameters are specified. Default program parameters may be used, or alternative parameters may be specified. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence to the reference sequence based on the program parameters.

[0054] The comparison window includes references to one segment of a number of consecutive positions selected from a group consisting of 20 to 600, typically about 50 to 200, and more typically about 100 to 150, where one sequence can be compared to the same number of consecutive reference positions after the two sequences, the sequence itself and the reference sequence, have been optimally aligned. Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed by the local homology algorithm of Smith and Waterman Add. APL. Math. 2:482 (1981), the homology alignment algorithm of Needleman and Wunsch J. Mol. Biol. 48:443 (1970), the similarity search method of Pearson and Lipman Proc. Natl. Acad. Sci. (USA) 85: 2444 (1988), by computerized implementations of these algorithms (e.g., BLAST), or by manual alignment and visual inspection.

[0055] Suitable algorithms for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (NCBI) website. The algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short word lengths W in the query sequence (which, when aligned with words of the same length in the database sequence, either match or satisfy a certain positive threshold score T). T is called the adjacent word score threshold (Altschul et al.). These initial adjacent word hits act as seeds to initiate a search to find longer HSPs that contain them. Next, the word hits are extended in both directions along each sequence as long as the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score is calculated using parameters M (reward score for matching residue pairs; always > 0) and N (penalty score for mismatched residues; always < 0). For amino acid sequences, the scoring matrix is ​​used to calculate the cumulative score. The extension of word hits in each direction is stopped when the cumulative alignment score decreases by an amount X from its maximum achieved value; when the cumulative score becomes zero or less due to the accumulation of 1 or more negative scoring residue alignments; or when it reaches the end of either sequence. The BLAST algorithm parameters W, T, and X above determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses, by default, a word size (W) of 28, an expected value (E) of 10, M=1, N=-2, and a comparison of both strands.For amino acid sequences, the BLASTP program uses a word size (W) of 3, an expected value (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) as default.

[0056] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which represents the probability that a match between two nucleotide or amino acid sequences occurs by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in the comparison of the test nucleic acid to the reference nucleic acid is less than about 0.01, more preferably less than about 10 to 5, and most preferably less than about 10 to 20.

[0057] Any of the vectors disclosed herein can be introduced into cells (using any technique known in the art) for the proliferation of the vector and / or for the expression of the protein(s) encoded by the vector. Recombinant cells and populations of cells containing the vectors disclosed herein are provided herein. Furthermore, any of the vectors described herein may be used in any method described herein or later developed to produce rAAV, the method which includes, for example, culturing the recombinant cells under conditions that produce rAAV capsids and, optionally, the expression of the transgene (i.e., the gene of interest).

[0058] Various host cells and expression systems may be used to grow any of the vectors described herein. These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli and B. subtilis), yeast (e.g., Saccharomyces Pichia); plant cell systems; insect cells, or mammalian cell systems (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK293, NS0, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa, and NIH 3T3, HEK293T, HEK293F, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, and BMT10 cells).

[0059] Vector System A vector system comprising at least two vectors is also provided, where the first vector comprises one or more helper sequences (e.g., a helper plasmid comprising modified E4 nucleic acid, E2a nucleic acid, and virus-associated (VA)RNA from an adenovirus genome). In some embodiments, the second vector comprises a transgene.

[0060] The above vector system optionally comprises at least a portion of nucleic acid E4, at least a portion of nucleic acid E2a, and / or virus-associated (VA)RNA of the adenovirus genome, wherein at least a portion of nucleic acid E4 includes a deletion configured to increase the production of AAV products, including the AAV capsid, compared to production using a vector system lacking the nucleic acid E4 deletion. In some embodiments, at least a portion of nucleic acid E2a includes a deletion.

[0061] The first vector, as specified, comprises one or more of the following: a portion of nucleic acid E4 of the adenovirus genome, at least a portion of nucleic acid E2a, and virus-associated (VA)RNA, wherein at least a portion of nucleic acid E4 comprises a deletion configured to increase the production of AAV products, including the AAV capsid, compared to production using a vector system lacking the nucleic acid E4 deletion. In some embodiments, at least a portion of nucleic acid E2a comprises a deletion. The first vector, as specified, comprises a portion of nucleic acid E4 of the adenovirus genome, at least a portion of nucleic acid E2a, and virus-associated (VA)RNA.

[0062] The vector systems described herein may be used as packaging systems for the preparation of rAAV. In some embodiments, the packaging system comprises or consists of (1) a first vector containing a helper virus gene (e.g., a helper plasmid described herein); and (2) a second vector containing a first nucleotide sequence encoding the AAV Rep protein, a second nucleotide sequence containing the rAAV genome including the transgene, and a third nucleotide sequence encoding the AAV capsid protein. In some embodiments, the vector system comprises or consists of (1) a first vector containing one or more helper virus genes (e.g., a helper plasmid described herein); (2) a second vector containing the rAAV genome including the transgene; and (3) a third vector containing a nucleotide sequence encoding the AAV Rep protein and a nucleotide sequence encoding the AAV capsid protein.

[0063] In some embodiments, two or more vectors of the above packaging system may together provide all the components required for the production of rAAV. In a particular embodiment, certain components required for the production of rAAV are provided by the host cell on which the rAAV is produced. In such embodiments, two or more vectors of the above packaging system, together with the host cell, may provide all the components required for the production of rAAV. The packaging systems described herein function within a cell to form the rAAV by enclosing the AAV genome within a capsid.

[0064] In some vector systems, the first vector comprises at least a portion of nucleic acid E4 of the adenovirus genome, at least a portion of nucleic acid E2a, and virus-associated (VA)RNA, wherein the portion of nucleic acid E4 includes a deletion configured to increase the production of AAV products, including the AAV capsid, compared to production using a vector lacking the nucleic acid E4 deletion.

[0065] In some embodiments, the nucleic acid E4 deletion is a deletion of orf 1-3, resulting in nucleic acid E4 containing E4orf4, Eorf6, and Eorf7. In some embodiments, the nucleic acid E4 deletion is a deletion of at least 1000 base pairs (e.g., at least about 1000, 1025, 1050, or 1075 base pairs) of the nucleic acid sequence (SEQ ID NO: 2) containing E4orf1-E4orf3. In some embodiments, nucleic acid E4 containing the deletion of E4orfs1-3 contains the nucleic acid sequence containing SEQ ID NO: 1, or a nucleic acid sequence having at least 60%, 65%, 70%, 75%, 80%, 90%, 95%, or 99% identity with SEQ ID NO: 1.

[0066] In some embodiments, the vector comprises the adenovirus genome, nucleic acid E4 orf6-7; nucleic acid E2a or a portion thereof; and VA RNA, where nucleic acid E4 includes a deletion. In some embodiments, nucleic acid E4 including orf6-7 also includes a deletion of one or more orfs selected from the group consisting of E4orf1, E4orf2, and E4orf4. In some embodiments, the nucleic acid E4 deletion is a deletion of at least 1000 base pairs (e.g., at least about 1000, 1025, 1050, 1075, 1100, 1125, 1150, 1175, 1200, 1225, 1250, 1275, 1300, 1325, 1350, or 1400 base pairs) of the nucleic acid sequence including E4orf1-E4orf4 (SEQ ID NO: 5). Exemplary nucleotide sequences containing nucleic acids E4orf6, nucleic acids E4orf7, and nucleic acid E4 deletions E4orfs1-4 are shown herein as Sequence ID No. 4. In some embodiments, the nucleic acids E4 containing E4orf6, nucleic acids E4orf7, and nucleic acid E4 deletions E4orfs1-4 have at least 60%, 65%, 70%, 75%, 80%, 90%, 95%, or 99% identity with Sequence ID No. 4.

[0067] In some embodiments, at least a portion of the nucleic acid E2a contains a deletion in the untranslated genomic region of the nucleic acid E2a (e.g., the 5' untranslated region (UTR)) (e.g., a deletion of at least about 475, 500, 525, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, or 1000 base pairs). Exemplary nucleic acid sequences containing nucleic acid E2a with at least 500 base pairs of deletion in the 5' UTR are shown herein as SEQ ID NOs: 8-12. SEQ ID NO: 8 is nucleic acid E2a with about 2kb of deletion in the 5' UTR. SEQ ID NO: 9 is nucleic acid E2a with about 1kb of deletion in the 5' UTR. Sequence ID 10 is nucleic acid E2a containing a deletion of approximately 1.4 kb in the 5' UTR. Sequence ID 11 is nucleic acid E2a containing a deletion of approximately 500 base pairs in the 5' UTR. Sequence ID 12 is nucleic acid E2a containing a deletion of approximately 900 base pairs in the 5' UTR.

[0068] In some embodiments, the AAV product has a higher level of vector genome per liter, a greater number of capsids per liter, or both, compared to a control AAV product produced by an AAV vector system lacking a deletion of nucleic acid E4 or lacking deletions of both nucleic acid E4 and nucleic acid E2a. In some embodiments, the second vector contains a transgene. When used whole, the increase may be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, or greater. The increase may also be a multiplicative increase, e.g., at least 2 to 4 times. For example, if the control VG / L value is about 2 × 10¹⁴, the AAV product produced using the vector system described herein may be about 3 to 8 × 10¹⁴, e.g., 3 to 7 × 10¹⁴ VG / L. Similarly, if the control capsid / L is 0.75 × 10¹⁴, the AAV product produced using the vector system described herein may be about 1 to 5 × 10¹⁵, for example, 1 to 3 × 10¹⁵ capsid / L.

[0069] Transgene In some embodiments, the transgene comprises one or more sequences encoding an RNA molecule. Suitable RNA molecules include, but are not limited to, miRNA, shRNA, siRNA, antisense RNA, gRNA, antagomyl, miRNA sponge, RNA aptazyme, RNA aptamer, mRNA, lncRNA, ribozyme, and synthetic RNA known in the art.

[0070] In some embodiments, the transgene encodes one or more polypeptides or fragments thereof. Such a transgene may consist only of the complete coding sequence of a polypeptide or only fragments of the coding sequence of a polypeptide. In certain embodiments, the transgene encodes a polypeptide useful for treating a disease or disorder in a subject. Suitable polypeptides include, but are not limited to, β-globin, hemoglobin, tissue plasminogen activator, and coagulation factors; colony-stimulating factor (CSF); interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, etc.); growth factors (e.g., keratinocyte growth factor (KGF), stem cell factor (SCF), fibroblast growth factor (FGF, e.g., basic FGF and acidic FGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), bone morphogenetic protein (BMP), epidermal growth factor ( EGF), growth differentiation factor-9 (GDF-9), hepatocellular carcinoma-derived growth factor (HDGF), myostatin (GDF-8), nerve growth factor (NGF), neurotrophic factor, platelet-derived growth factor (PDGF), thrombopoietin (TPO), transforming growth factor α (TGF-α), transforming growth factor β (TGF-β), etc.; soluble receptors (e.g., soluble TNF-α receptor, soluble interleukin receptors (e.g., soluble IL-1 receptor and soluble type II IL-1 receptor), soluble γ / Δ T cell receptors, ligand-binding fragments of soluble receptors, etc.; enzymes (e.g., α-glucosidase, imiglucerase, β-glucocerebrosidase, and alglucerase); enzyme activators (e.g., tissue plasminogen activator); chemokines (e.g., IP-10, interferon-γ-induced monokine (Mig), Groα / IL-8, RANTES, MIP-1a, MIP-1β, MCP-1, PF-4, etc.); angiogenic factors (e.g., vascular endothelial growth factor (VEGF, e.g., VEGF121, VEGF165, VEGF-C, VEGF-2), glioma-derived growth factor, angiogenin, angiogenin-2; etc.);Anti-angiogenic factors (e.g., soluble VEGF receptor); protein vaccines; neuroactive peptides (e.g., nerve growth factor (NGF), bradykinin, cholecystokinin, gastrin, secretin, oxytocin, gonadotropin-releasing hormone, β-endorphin, enkephalin, substance P, somatostatin, prolactin, galanin, growth hormone-releasing hormone, bombesin, dynorphin, warfarin, neurotensin, motilin, thyrotropin, neuropeptide Y, luteinizing hormone) (e.g., calcitonin, insulin, glucagon, vasopressin, angiotensin II, thyrotropin-releasing hormone, vasoactive intestinal peptides, sleep peptides, etc.); thrombolytic agents; atrial natriuretic peptide; relaxin; glial fibrillary acidic protein; follicle-stimulating hormone (FSH); human α-1 antitrypsin; leukemia inhibitor (LIF); tissue factor; macrophage activator; tumor necrosis factor (TNF); neutrophil chemotactic factor (NCF); metalloproteinase tissue inhibitors; vasoactive intestinal peptides, etc.) Butide; angiogenin; angiotropin; fibrin; hirudin; IL-1 receptor antagonist; ciliary neurotrophic factor (CNTF); brain-derived neurotrophic factor (BDNF); neurotrophic factors 3 and 4 / 5 (NT-3 and -4 / 5); glial cell-derived neurotrophic factor (GDNF); aromatic amino acid decarboxylase (AADC); factor VIII, factor IX, factor X; dystrophin or minidystrophin; lysosomal acid lipase; phenylalanine hydroxylase (PAH); Glycogen storage disease-related enzymes (e.g., glucose-6-phosphatase, acid maltase, glycogen debranchase, muscle glycogen phosphorylase, hepatic glycogen phosphorylase, muscle phosphofructokinase, phosphorylase kinase, glucose transporter, aldolase A, β-enolase, glycogen synthase); Lysosomal enzymes (e.g., iduronate-2-sulfatase (I2S), and arylsulfatase A); and mitochondrial proteins (e.g., frataxin).

[0071] In some embodiments, the above-mentioned transgene encodes a protein that may be deficient in one or more lysosomal storage disorders. Suitable proteins include α-sialidase, cathepsin A, α-mannosidase, β-mannosidase, glycosyl asparaginase, α-fucosidase, α-N-acetylglucosaminidase, β-galactosidase, β-hexosaminidase α-subunit, β-hexosaminidase β-subunit, GM2 activator protein, glucocerebrosidase, saposin C, arylsulfatase A, saposin B, formyl-glycine-producing enzyme, β-galactosylceramidase, α-galactosidase A, iduronate sulfatase, α-iduronidase, heparan N-sulfatase, and acetaminophen. Examples include, but are not limited to, tyl-CoA transferase, N-acetylglucosaminidase, β-glucuronidase, N-acetylglucosamine 6-sulfatase, N-acetylgalactosamine 4-sulfatase, galactose 6-sulfatase, hyaluronidase, α-glucosidase, acid sphingomyelinase, acid ceramidase, acid lipase, cathepsin K, tripeptidyl peptidase, palmitoyl protein thioesterase, cystinosine, sialine, UDP-N-acetylglucosamine, phosphotransferase γ-subunit, mucolipin-1, LAMP-2, NPC1, CLN3, CLN6, CLN8, LYST, MYOV, RAB27A, melanophilin, and AP3 β-subunit.

[0072] In some embodiments, the transgene encodes an antibody or a fragment thereof (e.g., Fab, scFv, or full-length antibody). Suitable antibodies include, but are not limited to, muromonab-cd3, efalizumab, tocitumomab, daclizumab, nevacumab, catumakisomab, edrecolomab, absiximab, rituximab, basiliximab, palivizumab, infliximab, trastuzumab, adalimumab, ibritumomab tiuxetan, omalizumab, cetuximab, bevacizumab, natalizumab, panitumumab, ranibizumab, eculizumab, certo Lizumab, ustekinumab, canakinumab, golimumab, ofatumumab, tocilizumab, denosumab, belimumab, ipilimumab, brentuximab vedotin, pertuzumab, laxibakumab, obinutuzumab, alemtuzumab, siltuximab, ramucirumab, vedolizumab, blinatumomab, nivolumab, pembrolizumab, idarucizumab, necitumumab, dinutuximab, sek Kinumab, mepolizumab, alirocumab, evolocumab, daratumumab, elotuzumab, ixekizumab, reslizumab, olaratumab, bezlotoxumab, atezolizumab, obiltoxaximab, inotuzumab ozogamicin, brodalumab, guselkumab, dupilumab, sarilumab, avelumab, ocrelizumab, emicizumab, benralizumab, gemtuzumab ozogamicin, Durvalumab, brosumab, erenumab, galcanezumab, lanadelmab, mogamulizumab, tildrakizumab, semiprimab, fremanezumab, ravulizumab, emapalmab, ibalizumab, moxetumomab, caplacizumab, romosozumab, risankizumab, polatuzumab, eptinezumab, leronrimab, sacituzumab, brolucizumab, isatuximab, and teprotumumab.

[0073] In some embodiments, the introduced gene encodes a nuclease. Suitable nucleases include, but are not limited to, zinc finger nucleases (ZFNs) (see, e.g., Porteus, and Baltimore (2003) Science 300: 763; Miller et al. (2007) Nat. Biotechnol. 25:778-785; Sander et al. (2011) Nature Methods 8:67-69; and Wood et al. (2011) Science 333:307 (each of these is incorporated herein by reference in whole)), and transcription activator-like effector nucleases (TALENs) (see, e.g., Wood et al. (2011) Science 333:307; Boch et al. (2009) Science 326:1509-1512; Moscou and Bogdanove (2009) Science See 326:1501; Christian et al. (2010) Genetics 186:757-761; Miller et al. (2011) Nat. Biotechnol. 29:143-148; Zhang et al. (2011) Nat. Biotechnol. 29:149-153; and Reyon et al. (2012) Nat. Biotechnol. 30(5): 460-465 (each of these is incorporated herein by reference in its entirety), homing endonucleases, meganucleases (see, for example, U.S. Patent Publication No. US 2014 / 0121115 (which is incorporated herein by reference in its entirety)), and RNA guide nucleases (see, for example, Makarova et al. (2018) The CRISPR Journal 1(5): See 325-336; and Adli (2018) Nat. Communications 9:1911 (each of these is incorporated herein by reference in whole).

[0074] In some embodiments, the transgene encodes an RNA guide nuclease. Suitable RNA guide nucleases include, but are not limited to, clustered and regularly arranged short palindromic sequence repeat (CRISPR)-associated nucleases of class I and class II. Class I is further divided into types I, III, and IV, and includes, but is not limited to, type I (Cas3), type IA (Cas8a, Cas5), type I (Cas8b), type IC (Cas8c), type ID (Cas10d), type IE (Cse1, Cse2), type IF (Csy1, Csy2, Csy3), type IU (GSU0054), type III (Cas10), type III-A (Csm2), type III-B (Cmr5), type III-C (Csx10 or Csx11), type III-D (Csx10), and type IV (Csf1). Class II nucleases are divided into type II, type V, and type VI, and include, but are not limited to, type II (Cas9), type II-A (Csn2), type II-B (Cas4), type V (Cpf1, C2c1, C2c3), and type VI (Cas13a, Cas13b, Cas13c). RNA guide nucleases also include naturally occurring type II CRISPR nucleases, such as Cas9 (type II) or Cas12a / Cpf1 (type V), and other nucleases derived from or obtained from them. Examples of Cas9 nucleases that may be used in the present invention include, but are not limited to, S. pyogenes Cas9 (SpCas9), S. aureus Cas9 (SaCas9), N. meningitidis Cas9 (NmCas9), C. jejuni Cas9 (CjCas9), and Geobacillus Cas9 (GeoCas9).

[0075] In some embodiments, the transgene encodes one or more reporter sequences, which, upon expression, generate detectable signals. Such reporter sequences include, but are not limited to, DNA sequences encoding β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), red fluorescent protein (RFP), chloramphenicol acetyltransferase (CAT), luciferase, and membrane-bound proteins (e.g., CD2, CD4, CD8, influenza hemagglutinin protein, and, in particular, fusion proteins containing membrane-bound proteins appropriately fused to an antigen-tagged domain derived from hemagglutinin or Myc).

[0076] AAV genome In some embodiments, the rAAV genome includes transcriptional regulatory elements (TREs) operably linked to the transgene to control the expression of the RNA or polypeptide encoded by the transgene. In certain embodiments, the TRE includes a constitutive promoter. In certain embodiments, the TRE may be active in any mammalian cell (e.g., any human cell). In certain embodiments, the TRE is active in a wide range of human cells. Such a TRE may include a constitutive promoter and / or enhancer elements (including any of those described herein). In certain embodiments, the TRE includes an inducible promoter. In certain embodiments, the TRE may be a tissue-specific TRE; that is, it is active in a specific tissue(s) and / or organ(s). A tissue-specific TRE includes one or more tissue-specific promoters and / or enhancer elements, and optionally one or more constitutive promoters and / or enhancer elements. Those skilled in the art will recognize that tissue-specific promoters and / or enhancer elements can be isolated from genes specifically expressed in that tissue by methods well known in the art.

[0077] Suitable promoters include, but are not limited to, the following: cytomegalovirus promoter (CMV) (Stinski et al. (1985) Journal of Virology 55(2): 431-441), CMV early enhancer / chicken β-actin (CBA) promoter / rabbit β-globin intron (CAG) (Miyazaki et al. (1989) Gene 79(2): 269-277), CBSB (Jacobson et al. (2006) Molecular Therapy 13(6): 1074-1084), human elongation factor 1α promoter (EF1α) (Kim et al. (1990) Gene 91(2): 217-223), human phosphoglycerate kinase promoter (PGK) (Singer-Sam et al. (1984) Gene 32(3): 409-417), mitochondrial H chain promoter (Lodeiro et al. (2012) PNAS 109(17): 6513-6518), ubiquitin promoter (Wulff et al. (1990) FEBS Letters 261: 101-105). In certain embodiments, the above TREs are brain-specific (e.g., neuron-specific, glial cell-specific, astrocytocyte-specific, oligodendrocyte-specific, microglia-specific, and / or mesneurin-specific). Exemplary brain-specific TREs may include one or more elements derived from, but not limited to, the following: human glial fibrillary acidic protein (GFAP) promoter, human synapsin 1 (SYN1) promoter, human synapsin 2 (SYN2) promoter, human metallothionein 3 (MT3) promoter, and / or human proteolipide protein 1 (PLP1) promoter. More brain-specific promoter elements are disclosed in WO 2016 / 100575A1 (which is incorporated herein by reference in its entirety).

[0078] In some embodiments, a native promoter for the transgene may be used. The native promoter may be preferred when it is desirable that the expression of the transgene mimics native expression. The native promoter may also be used when the expression of the transgene must be regulated temporally, developmentally, in a tissue-specific manner, or in response to a specific transcriptional stimulus. In further embodiments, other native expression regulatory elements (e.g., enhancer elements, polyadenylation sites, or Kozak consensus sequences) may also be used to mimic their native expression.

[0079] In certain embodiments, the rAAV genome includes an editing genome. As used herein, the term editing genome refers to a recombinant AAV genome that can modify a genomic target locus via homologous recombination. The editing genome may be used to edit the genome of a cell by homologous recombination between the editing genome and a genomic region surrounding a target locus in the cell. In certain embodiments, the editing genome is designed to correct a genetic deletion in a gene by homologous recombination. The genome being edited generally includes (i) an element being edited to edit or modify a target locus in a target gene; (ii) a 5' homology arm nucleotide sequence on the 5' side of the element being edited that is homologous to a first genomic region located 5' to the target locus; and (iii) a 3' homology arm nucleotide sequence on the 3' side of the element being edited that is homologous to a second genomic region located 3' to the target locus, wherein the portion of the genome being edited including the 5' homology arm, the element being edited, and the 3' homology arm may be oriented sense or antisense with respect to the target locus.Suitable target genes for editing using the genome being edited include, but are not limited to, phenylalanine hydroxylase (PAH), cystic fibrosis conductance transmembrane regulator (CFTR), β-hemoglobin (HBB), oculocutaneous albinism II (OCA2), Huntington's disease (HTT), myotonic dystrophy-protein kinase (DMPK), low-density lipoprotein receptor (LDLR), apolipoprotein B (APOB), neurofibromin 1 (NF1), polycystic kidney disease 1 (PKD1), polycystic kidney disease 2 (PKD2), coagulation factor VIII (F8), dystrophin (DMD), and X-linked phosphate-regulating endopeptidase homologue. X-linked (PHEX), methyl-CpG binding protein 2 (MECP2), and Y-linked ubiquitin-specific peptidase 9Y (USP9Y).

[0080] In some embodiments, the rAAV genome disclosed herein further comprises a transcription termination factor (e.g., a polyadenylated sequence). In certain embodiments, the transcription termination factor is located at the 3' end of the transgene. The transcription termination factor may be any sequence that effectively terminates transcription, and those skilled in the art will recognize that such a sequence may be isolated from any gene expressed in cells in which transcription of at least a portion of the antibody-coding sequence is desired. In certain embodiments, the transcription termination factor comprises a polyadenylated sequence. In certain embodiments, the polyadenylated sequence is identical or substantially identical to the endogenous polyadenylated sequence of an immunoglobulin gene. In certain embodiments, the polyadenylated sequence is an exogenous polyadenylated sequence. In certain embodiments, the polyadenylated sequence is an SV40 polyadenylated sequence. In certain embodiments, the polyadenylated sequence is a bovine growth hormone (BGH) polyadenylated sequence.

[0081] In a particular embodiment, the rAAV genome further comprises a 5' reverse terminal repeat (5' ITR) nucleotide sequence located at the 5' end of the TRE, and a 3' reverse terminal repeat (3' ITR) nucleotide sequence located at the 3' end of the polyadenylated sequence. Any ITR sequence derived from any AAV serotype or variant thereof may be used in the rAAV genome disclosed herein. The 5' ITR and 3' ITR may be derived from the same serotype of AAV or from different serotypes of AAV.

[0082] In a particular embodiment, the 5' ITR or 3' ITR is derived from AAV2. In a particular embodiment, the 5' ITR or 3' ITR is derived from AAV5. In a particular embodiment, both the 5' ITR and 3' ITR are derived from AAV5 or AAV2.

[0083] In a particular embodiment, the 5' ITR nucleotide sequence and the 3' ITR nucleotide sequence are substantially complementary to each other (for example, complementary except for mismatches at the 1, 2, 3, 4, or 5 nucleotide positions in the 5' ITR or 3' ITR).

[0084] In certain embodiments, the 5' ITR or the 3' ITR is modified to reduce or eliminate degradation by the Rep protein ("non-degradable ITR"). In certain embodiments, the non-degradable ITR includes insertions, deletions, or substitutions in the nucleotide sequence of the terminal degradation site. Such modifications enable the formation of a self-complementary double-stranded DNA genome of the AAV after the rAAV genome has been replicated in infected cells. Exemplary non-degradable ITR sequences are known in the art (see, for example, those provided in U.S. Patents 7,790,154 and 9,783,824, which are incorporated herein by reference in their entirety).

[0085] Rep protein In the vector system described herein, the expression of the AAV Rep gene is controlled through the use of two promoters and alternative splicing, giving rise to four Rep proteins: Rep78, Rep68, Rep52, and Rep40. These Rep proteins are involved in AAV genome replication and packaging of the viral genome. The expression of the Rep proteins is controlled by the p5 promoter and the p19 promoter. The p5 promoter drives the expression of the alternative splice variants Rep78 and Rep68. The p19 promoter drives the expression of the alternative splice variants Rep52 and Rep40. Thus, the vector system provided herein may include a vector containing one or more nucleotide sequences encoding Rep proteins or their functional variants.

[0086] One or more of the above Rep proteins may originate from AAV2. An exemplary AAV2 genome sequence can be found via the NCBI reference sequence NC_001401.2. According to the above NCBI reference sequence, Rep68 is encoded by nucleotides 321-2252; Rep78 is encoded by nucleotides 321-2186; Rep40 is encoded by nucleotides 993-2252; and Rep52 is encoded by nucleotides 993-2186. In certain embodiments, the disclosure provides nucleic acids comprising nucleotide sequences in a different adenovirus serotype, e.g., AAV5, corresponding to the sequences encoding Rep40, Rep68, Rep78, and Rep52 as described with respect to AAV2.

[0087] AAV Capsid The vector systems provided herein include nucleic acid vectors comprising nucleotide sequences encoding AAV capsid protein coding sequences. These nucleotide sequences may encode AAV capsid proteins derived from any AAV capsid known in the art, including natural AAV isolates and their variants.

[0088] The AAV capsid proteins include VP1, VP2, and VP3 capsid proteins. The VP1, VP2, and / or VP3 capsid proteins assemble into a capsid that surrounds the rAAV genome. In certain embodiments, the assembly of the capsid proteins is facilitated by an assembly activating protein (AAP). Capsids of certain AAV serotypes require the role of AAP in transporting the capsid protein to the nucleolus for assembly. For example, AAV1, AAV2, AAV3, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV12 require AAP to form the capsid, while the capsids of AAV4, AAV5, and AAV11 may assemble without AAP. See, for example, Earley et al. (2017) J. Virol. 91(3): e01980-16.

[0089] Different AAV serotypes or their variants contain AAV capsid proteins with different amino acid sequences. Appropriate AAV capsid proteins include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-DJ, AAV-LK03, NP59, VOY101, VOY201, VOY701, VOY801, VOY1101, AAVPHP.N, AAVPHP.A, AAVPHP.B, PHP.B2, PHP.B3, G2A3, G2B4, G2B5, PHP.S, and AAVr Capsid proteins derived from h10, AAVRh32, 33, AAVrh74, AAVHSC1, AAVHSC2, AAVHSC3, AAVHSC4, AAVHSC5, AAVHSC6, AAVHSC7, AAVHSC8, AAVHSC9, AAVHSC10, AAVHSC11, AAVHSC12, AAVHSC13, AAVHSC14, AAVHSC15, AAVHSC16, AAVHSC17, and any variant thereof include, but are not limited to, these. In a particular embodiment, the AAV capsid protein is selected from the group consisting of AAV1, AAV2, AAV5, AAV6, AAV8, AAV9, AAVrh10, and AAVrh74. In a particular embodiment, the AAV capsid protein is selected from the group consisting of AAV1, AAV2, AAV5, AAV6, AAV8, and AAVrh74. Sequences of the various AAV capsid proteins described above are disclosed, for example, in U.S. Patent Publications US20030138772, US20140359799, US20150159173, US20150376607, US20170081680, and US20170360962A1, and PCT Publication WO2020227515 (these disclosures are incorporated herein by reference in their entirety).

[0090] rAAV production A method for producing an AAV product containing an AAV capsid is also provided. The method comprises (a) introducing one of the vector systems described herein into a host cell, (b) incubating the host cell under culture conditions that promote AAV product production, and (c) purifying the AAV product.

[0091] In some embodiments, the host cells are mammalian cells selected from the group consisting of, for example, COS cells, CHO cells, BHK cells, MDCK cells, HEK293 cells, HEK293T cells, HeLa cells, NS0 cells, PER.C6 cells, VERO cells, CRL7O3O cells, HsS78Bst cells, HeLa cells, NIH 3T3 cells, HepG2 cells, SP210 cells, R1.1 cells, BW cells, LM cells, BSC1 cells, BSC40 cells, YB / 20 cells, and BMT10 cells. If necessary, the host cells are cells that can be grown in suspension culture (e.g., HEK293 cells, HEK293T cells, or HEK293F cells).

[0092] In some embodiments, rAAV production generally includes an upstream production process encompassing expanding mammalian cells to an appropriate cell density, introducing one or more vectors into the expanded cells to generate AAV producer cells, culturing the AAV producer cells under conditions that produce rAAV particles (capsids), and harvesting and lysing the AAV producer cells for the subsequent recovery of the rAAV particles. After the recovery of the rAAV particles, a downstream production process ensures that the rAAV particles are sufficiently purified from contaminants.

[0093] As used herein, the term “introducing” (i.e., transfecting or transfecting) refers to the transfer of a nucleic acid sequence (e.g., one or more vectors described herein) from the extracellular space to the intracellular space. In some cases, “introducing” refers to the transfer of the nucleic acid from the extracellular space to the nucleus of the cell. Various methods of such transfer are considered, but are not limited to, electroporation, contact with nanowires or nanotubes, receptor-mediated internalization, transfer via cell-permeable peptides, and liposome-mediated transfer.

[0094] In some embodiments, the vector introduced into the enlarged cells comprises a first vector containing one or more helper virus genes (e.g., helper plasmids described herein) and a second vector containing a first nucleotide sequence encoding the AAV Rep protein, a second nucleotide sequence containing the rAAV genome including the transgene, and a third nucleotide sequence encoding the AAV capsid protein. When the first vector containing one or more helper virus genes and the second vector containing the nucleotide sequences encoding the AAV Rep protein, the transgene, and the AAV capsid protein are introduced into the enlarged cells, this can occur in various molar ratios (e.g., equimolar ratios of 1:1) that are most suitable for the vectors being used. In some embodiments, the vector introduced into the enlarged cells comprises the first vector containing one or more helper virus genes (e.g., helper plasmids described herein); a second vector containing the rAAV genome including the transgene; and a third vector containing the nucleotide sequences encoding the AAV Rep protein and the AAV capsid protein. When the first vector containing one or more helper virus genes, the second vector containing the transgene, and the third vector containing the AAV capsid protein and AAV capsid protein are introduced into the enlarged cells, this can occur in various molar ratios (e.g., 1:2:2 ratio) that are most suitable for the vectors being used. The total amount of nucleic acid introduced into the cells is approximately 0.1 μg DNA / 1 × 10⁶ cells to 4.0 μg DNA / 1 × 10⁶ cells.For example, the total amount of nucleic acids transfected or transduced into the cells, including the first nucleic acid vector, the second nucleic acid vector, and optionally the third nucleic acid vector, is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, or 4 μg DNA / 1 × 10⁶ cells.

[0095] In some embodiments, the mammalian cells are provided in a cell culture. In certain embodiments, the cell culture has a volume of at least 2 to 5000 liters. For example, at least 50 liters, at least 500 liters, or at least 2000 liters. In certain embodiments, the method described herein is carried out in a bioreactor having a volume of at least 2 liters, at least 50 liters, or at least 2000 liters.

[0096] Methods for purifying AAV products are known in the art. As used herein, purification means removing at least a portion of non-AAV products, e.g., host cell proteins, culture medium components, nucleic acids, and / or empty AAV capsids, from a cell culture in order to obtain a purified AAV product. For example, in some methods, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of one or more non-AAV products may be removed or eliminated during purification. In some cases, purification includes (a) a clarification step for the removal of cells and cell debris, for example, using fractional centrifugation, density gradient centrifugation and / or filtration; and / or (b) one or more downstream chromatographic steps for separating the AAV product from various impurities in the clarified cell culture feed. See, for example, International Patent Application No. WO 98 / 22588 (which describes a method for the production and purification of adenovirus vectors). Chromatographic methods for purifying viruses from host cell lysates are also described in U.S. Patents No. 6,008,036, 6,586,226, 5,837,520, 6,261,823, 6,537,793, and International Patent Application Publications WO 00 / 50573, WO 02 / 44348, and WO 03 / 078592 (the entire contents of each of these are incorporated herein by reference). Various chromatographic and non-chromatographic methods may be used (including affinity chromatography, anion exchange chromatography, ultracentrifugation, and other methods in the art).

[0097] How to use A method for delivering a transgene to a cell is provided, which includes introducing an AAV product produced by the method described herein into the cell. The introduction into the cell is typically carried out by infection.

[0098] A method is also provided for treating a subject in need of a protein produced by a transgene, the method comprising administering an effective amount of an AAV product produced by one of the methods for producing AAV products described herein to the subject.

[0099] In some embodiments, the AAV product is an AAV particle or virion. As used herein, a recombinant AAV particle or virion is a viral particle comprising a capsidized recombinant AAV viral genome containing at least one AAV capsid protein and a transgene. If necessary, in a subject, the transgene delivered to the cell may be heterologous to the cell. As used herein, “heterologous” refers to something not typically found in nature. Thus, a heterologous nucleotide sequence may be (a) foreign to the host cell (i.e., exogenous to the cell); (b) naturally found in the host cell (i.e., endogenous) but present in non-natural amounts in the cell (i.e., more or less than naturally found in the host cell); or (c) naturally found in the host cell but located outside its natural locus.

[0100] The term, effective dose, when used in whole, is defined as any amount necessary to produce a desired physiological response (e.g., to reduce or delay one or more effects or symptoms of a disease or disorder). The effective dose and schedule for administering the above AAV product (e.g., recombinant AAV particles) may be determined empirically, and making such determinations is within the scope of the art.

[0101] The dosage range for determination should be large enough to produce the desired effect, affecting (e.g., reducing or delaying) one or more symptoms of the disease or disorder described above. The dosage should not be so high as to cause substantial adverse side effects (e.g., unwanted cross-reactivity, unwanted cell death, etc.). In general, the dosage may vary depending on the subject's species, age, weight, overall health, sex, and diet, as well as the mode and timing of administration and the severity of the particular condition, and may be determined by a person skilled in the art. The dosage may be adjusted by the individual physician in the event of any contraindications. The dosage may vary and may be administered in doses of one or more.

[0102] The effective dose of any of the recombinant AAV virions described herein may vary and be determined by those skilled in the art through experiment and / or clinical trials. For example, the effective dose may be about 10⁶ to about 10¹⁵ recombinant rAAV virions, or any value within this range (e.g., about 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, or 10¹⁵ recombinant AAV particles). Thus, the number of rAAV particles administered to a subject may be in the range of about 10⁶ to 10¹⁵ vector genomes (vg) / ml (e.g., about 10⁶ The effective doses may be approximately 10⁶–10¹⁵ vg / kg, or any value between these amounts (e.g., approximately 10⁶, 10⁸, 10⁹, 10⁹, 10¹⁴, or 10¹⁵ vg / ml). In some embodiments, the number of rAAV particles administered to the subject may be approximately 10⁶–10¹⁵ vg / kg, or any value between these amounts (e.g., approximately 10⁶, 10⁷, 10⁸, 10⁹, 10⁰, 10¹

[0103] The compositions described herein are administered in many ways, depending on whether topical or systemic treatment is desired. These compositions are administered via one of several routes of administration (including intraparenchymal, intravenous, intrathecal, intramuscular, intracisional, intracoronary, intramyocardial, intradermal, endocardial, or combinations thereof). Effective doses for any of the administration methods described herein can be extrapolated from dose-response curves obtained from in vitro or animal model test systems.

[0104] When used as a whole, the patient is used interchangeably with the subject. Subject means an individual. The subject may be an adult subject or a pediatric subject. Pediatric subjects include subjects ranging from the age at birth to 18 years of age. Preferably, the subject is an animal, such as a mammal like a primate, and more preferably a human. Non-human primates are also subjects. The term subject includes domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.) and laboratory animals (e.g., ferrets, chinchillas, mice, rabbits, rats, gerbils, guinea pigs, etc.). Veterinary use and pharmaceutical formulations are thus intended herein.

[0105] When used in general, treat, treating, and treatment refer to a method of reducing or delaying one or more effects or symptoms of a disease or disorder. The subject may be diagnosed with the disease or disorder. Treatment may also refer to a method of reducing the underlying pathology rather than merely the symptoms. The effect of administration to the subject may, but is not limited to, reducing one or more symptoms of the disease, reducing the severity of the disease, completely eliminating the disease, or delaying the onset or exacerbation of one or more symptoms. For example, a disclosed method is considered a treatment if it results in approximately a 10% reduction in one or more symptoms of the disease compared to the subject before treatment, or compared to a control subject or control value. Thus, the reduction may be approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any reduction in between. Embodiment Embodiment 1. A vector comprising nucleic acid E4, nucleic acid E2a, and virus-associated (VA)RNA of an adenovirus genome, wherein nucleic acid E4 includes deletions of open reading frames 1-3. Embodiment 2. The vector according to Embodiment 1, wherein the nucleic acid E4 deletion is a deletion of at least 1000 base pairs. Embodiment 3. The vector according to Embodiment 1 or 2, wherein the nucleic acid E4 deletion is a deletion of at least 1000 base pairs of E4 or f1 to 3. Embodiment 4. The vector according to any one of Embodiments 1 to 3, wherein the nucleic acid E4 deletion is a deletion of at least 1400 base pairs of E4 or f1 to 4. Embodiment 5. The vector according to any one of Embodiments 1 to 4, wherein nucleic acid E4 comprises the nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 4; or a nucleic acid sequence having at last 85% identity with SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 4. Embodiment 6. The nucleic acid E4 is a vector according to any one of Embodiments 1 to 5, comprising an open reading frame 6. Embodiment 7. The vector according to Embodiment 6, wherein the nucleic acid E4 comprises the nucleic acid sequence of SEQ ID NO: 3 or a nucleic acid sequence having at last 85% identity with SEQ ID NO: 3. Embodiment 8. The vector according to Embodiment 6 or 7, wherein nucleic acid E4 further comprises an open reading frame 7. Embodiment 9. The vector according to Embodiment 8, wherein the vector comprises the nucleic acid sequence of SEQ ID NO: 4 or a nucleic acid sequence having at last 85% identity with SEQ ID NO: 4. Embodiment 10. The vector according to any one of Embodiments 1 to 9, further comprising a nucleic acid E2a deletion. Embodiment 11. The vector according to Embodiment 10, wherein the vector comprises the open reading frame 6 (orf6) of nucleic acid E4 of the adenovirus genome, the nucleic acid E2a deletion, and VA RNA. Embodiment 12. The vector according to Embodiment 10, wherein the vector comprises an open reading frame 6 (orf6) of nucleic acid E4 of the adenovirus genome, an open reading frame 7 (orf7) of nucleic acid E4, the nucleic acid E2a deletion; and VA RNA. Embodiment 13. The vector according to any one of Embodiments 10 to 12, wherein the E2 deletion comprises a deletion of more than 800 base pairs in the 5' untranslated region of the E2a gene. Embodiment 14. The vector according to Embodiment 13, wherein the vector comprises the nucleic acid sequence of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12, or a nucleic acid sequence having at least 85% identity with SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. Embodiment 15. The vector according to any one of Embodiments 1 to 14, wherein the vector comprises the nucleic acid sequence of SEQ ID NOs. 13, 14, 15, 16, 17, or 18. Embodiment 16. A vector system comprising at least two vectors, wherein the first vector comprises a helper plasmid, the helper plasmid being selected from the vectors described in any one of Embodiments 1 to 15. Embodiment 17. The vector system according to Embodiment 16, wherein the second vector includes a transgene. Embodiment 18. A vector system comprising at least two vectors, wherein the first vector comprises at least a portion of nucleic acid E4 of an adenovirus genome, at least a portion of nucleic acid E2a, and virus-associated (VA)RNA, wherein at least a portion of nucleic acid E4 comprises a deletion configured to increase the production of AAV products, including AAV capsids, compared to production using a vector lacking the nucleic acid E4 deletion. Embodiment 19. The vector system according to Embodiment 18, wherein at least a portion of nucleic acid E2a is a deletion. Embodiment 20. The vector system according to Embodiment 18 or 19, wherein the AAV product has a higher level of vector genome per liter, a greater number of capsids per liter, or both, compared to a control AAV product produced by an AAV vector system lacking a deletion of nucleic acid E4 or lacking deletions of both nucleic acid E4 and nucleic acid E2a. Embodiment 21. The vector system according to any one of Embodiments 18 to 20, wherein the second vector comprises a transgene. Embodiment 22. A method for producing an AAV product containing an AAV capsid, wherein the method is: (a) Introducing the vector system described in any one of embodiments 18 to 21 into a host cell, (b) Incubating the host cells under conditions that promote AAV product production, and (c) Purify the AAV product, A method of including. Embodiment 23. A method for treating a subject who has a need for a protein produced by a transgene, the method comprising administering to the subject an AAV product produced by the method described in Embodiment 22. Embodiment 24. A vector system comprising one or more vectors containing together nucleic acid E4, nucleic acid E2a, and virus-associated (VA)RNA of an adenovirus genome, wherein nucleic acid E4 contains deletions of open reading frames 1-3. Embodiment 25. The vector system according to Embodiment 24, wherein nucleic acid E4 includes deletions of open reading frames 1-4. Embodiment 26. The vector system according to Embodiments 23-25, wherein the vector further comprises an E2a deletion. [Examples]

[0106] Embodiments of this disclosure may be further defined by reference to the following non-limiting examples. It will be apparent to those skilled in the art that many modifications to both materials and methods can be carried out without departing from the scope of this disclosure.

[0107] Producing helper plasmids for AAV production using large-scale, high-density fermentation can be challenging, resulting in low yields and increased production costs. Figure 1 shows a schematic diagram of a conventional helper plasmid construct containing VA, E4, and E2a adenovirus sequences. Therefore, to improve plasmid manufacturability, helper plasmid size was reduced by removing regions of adenovirus genes not essential for adenovirus function. Five helper plasmid (pHelpers) deletion constructs aimed at reducing the size of the E4 and E2a transcription units, as well as shrinking the plasmid backbone, were compared to a control plasmid without the test deletion (control pHelper). See Figure 2. Surprisingly, all five helper plasmids containing the E4 deletion (without or without further E2a deletion) increased vector genome (VG) titers across multiple genomes. See Figure 3. Capsid generation also increased proportionally to VG using helper plasmids with the E4 deletion (without or without E2a deletion). See Figure 3. Removal of the non-coding region of the adenovirus gene described above alters the expression of E2a and E4 proteins, which appears to enable higher capsid expression and, subsequently, increase the number of VGs packaged into available capsids. Productivity was optimized in constructs including deletions of orf1-4 in E4, deletions of E2a as described herein, and skeletal deletions. See Figure 4.

[0108] Example 1: Double plasmid transfection Host cells were expanded for at least one passage, inoculated into a flask containing an appropriate amount of cell culture medium, and incubated at 37°C in 8% CO2 and a shaking incubator at 135 rpm. Cells were transfected when they reached a density of 2E6 cells / mL. For dual plasmid transfection, a total of 0.75 μg DNA / 1 × 10⁶ was transfected into the host cells. The transfection plasmid contained a first vector consisting of the GFP-luciferase transgene, AAV2 Rep gene, and AAV9 Cap gene, and a second vector consisting of either a control or various pHelper plasmid constructs (Del 1-5) (Table 1). The transfection plasmids were combined in a 1:1 equimolar ratio. The transfection mix was prepared by mixing the calculated volume of vector(s) and polyethyleneimine (PEI) at ambient temperature in a molar ratio of 1.5:1 PEI to DNA (Table 1). Next, the transfection mix was added to the flask and incubated in a shaker at 37°C, 8% CO2, and 135 rpm for 72 hours, after which the sample was collected. After 72 hours of incubation, the cells were lysed using a lysis buffer containing 1M Tris (pH 9.5), 10% Triton® X-100, 1M MgCl2, endonuclease (e.g., BENZONASE®, DENARASE®), and 5M NaCl, and the shaking flask was incubated for 60 minutes at 37°C, 8% CO2, and 135 rpm. The crude lysate sample was collected by centrifugation. [Table 1-1] [Table 1-2]

[0109] Example 2: Determination of vector genome and capsid productivity from double plasmid transfection The vector genome titer (vg / L) was determined by standard droplet digital PCR (ddPCR) using a primer / probe set specific to the transgene payload of the vector containing the transgene (i.e., the transgene vector) (Figure 3). The number of capsids per cell was determined by standard enzyme-coupled immunosolvent assay (ELISA) using an immobilized antibody directed to the capsid epitope as encoded by the vector containing the Cap sequence (Figure 3).

[0110] Example 3: Triple plasmid transfection Host cells were expanded for at least one passage, inoculated into flasks containing an appropriate amount of cell culture medium, and incubated at 37°C in 8% CO2 and a shaking incubator at 135 rpm. Cells were transfected when they reached a density of 1 × 10⁶ cells / mL. For triple plasmid transfection, a total of 0.75 μg DNA / 1 × 10⁶ was transfected into the host cells. The transfection plasmids consisted of a first vector comprising the GFP-luciferase transgene, a second vector comprising Rep / Cap proteins across multiple AAV serotype backgrounds including HSC15, AAV9, and AAV2, and a third vector comprising Del 5 (Table 2). The transfection plasmids were combined in an equal ratio of 1:2:2 (GFP-luciferase:Rep / Cap:pHelper). Transfection mixes were prepared by mixing the calculated volume of vector(s) and polyethyleneimine (PEI) at ambient temperature in a ratio of 1.5:1 PEI to DNA (Table 2). The transfection mixes were then added to the flasks and incubated in a shaker at 37°C, 8% CO2, and 135 rpm for 72 hours, after which the samples were collected. After 72 hours of incubation, the cells were lysed using a lysis buffer containing 1M Tris (pH 9.5), 10% Triton® X-100, 1M MgCl2, endonuclease (e.g., BENZONASE®, DENARASE®), and 5M NaCl, and the shaker flasks were incubated for 60 minutes at 37°C, 8% CO2, and 135 rpm. Crude lysate samples were collected by centrifugation. [Table 2]

[0111] Example 4: Determination of vector genome and capsid productivity from triple plasmid transfection The vector genome titer (vg / L) was determined by standard droplet digital PCR (ddPCR) using a primer / probe set specific to the transgene payload of the vector containing the transgene (i.e., the transgene vector) (Figure 4). The number of capsids per cell was determined by standard enzyme-coupled immunosolvent assay (ELISA) using an immobilized antibody directed to the capsid epitope as encoded by the vector containing the Cap sequence (Figure 4).

Claims

1. A vector comprising the adenovirus genome nucleic acid E4, nucleic acid E2a, and virus-associated (VA) RNA, wherein nucleic acid E4 contains deletions of open reading frames 1-3.

2. The vector according to claim 1, wherein the nucleic acid E4 deletion is a deletion of at least 1,000 base pairs.

3. The vector according to claim 2, wherein nucleic acid E4 comprises the nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 4; or a nucleic acid sequence having at last 85% identity with SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO:

4.

4. The vector according to claim 1, wherein nucleic acid E4 includes an open reading frame 6.

5. The vector according to claim 4, wherein the nucleic acid E4 comprises the nucleic acid sequence of sequence number 3 or a nucleic acid sequence having at last 85% identity with sequence number 3.

6. The vector according to claim 4, further comprising nucleic acid E4, open reading frame 7.

7. The vector according to claim 6, wherein the vector comprises the nucleic acid sequence of sequence number 4 or a nucleic acid sequence having at last 85% identity with sequence number 4.

8. The vector according to claim 1, further comprising a nucleic acid E2a deletion.

9. The vector according to claim 8, wherein the E2 deletion includes the deletion of more than 800 base pairs in the 5' untranslated region of the E2a gene.

10. The vector according to claim 9, wherein the vector comprises the nucleic acid sequence of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12, or a nucleic acid sequence having at least 85% identity with SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO:

12.

11. The vector according to claim 1, wherein the vector comprises the nucleic acid sequence of sequence number 13, 14, 15, 16, 17, or 18.

12. A vector system comprising a first vector and a second vector, wherein the first vector is the vector described in claim 1.

13. The vector system according to claim 12, wherein the second vector comprises a transgene.

14. A vector system comprising a first vector and a second vector, wherein the first vector comprises at least a portion of nucleic acid E4 of an adenovirus genome, at least a portion of nucleic acid E2a, and virus-associated (VA) RNA, wherein at least a portion of nucleic acid E4 includes a deletion configured to increase the production of an AAV product, including an AAV capsid, compared to production using a vector lacking the nucleic acid E4 deletion.

15. The vector system according to claim 14, wherein at least a portion of nucleic acid E2a includes a deletion.

16. The vector system according to claim 14, wherein the AAV product has a higher level of vector genome, a greater number of capsids per liter, or both, compared to a control AAV product produced by an AAV vector system lacking a deletion of nucleic acid E4 or lacking deletions of both nucleic acid E4 and nucleic acid E2a.

17. The vector system according to claim 14, wherein the second vector comprises a transgene.

18. A method for producing an AAV product containing an AAV capsid, wherein the method is (a) Introducing the vector system described in claim 14 into a host cell, (b) Incubating the host cells under culture conditions that promote AAV product production, and (c) Purify the AAV product. A method of including.

19. A method for delivering a transgene to a cell, the method comprising introducing an AAV product produced by the method of claim 18 into the cell.

20. The method according to claim 19, which is an in vitro method.

21. The method according to claim 19, wherein the introduced gene is expressed by the cells.

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