Improved recombinant adeno-associated virus production method

Abstract

Applications of adeno-associated virus (AAV) vectors in large animal studies and clinical trials generally require high titer and high potency vectors fabricated in scales that can support large doses and / or large populations. Many currently used methods for producing vectors for transient transfection or assisted viral infection based on cell lines have the advantages and limitations thereof. Herein, we report novel methods and compositions for high titer AAV production, which have several key improvements and advantages: (1) a therapeutic AAV vector cassette is cloned in one step into a serotype specific packaging plasmid; and (2) carrying out single plasmid transfection and selection on the stable AAV vector production cell line.

Description

[0001] Related Applications

[0002] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 353,793, filed on June 20, 2022, the disclosure of which is hereby incorporated by reference in its entirety. Background Art

[0003] The delivery of therapeutic nucleic acids to cells is an important biomedical technology, both as a tool for basic research and as a means of cell and gene therapy. Recombinant adeno-associated virus (rAAV) is an engineered form of adeno-associated virus (AAV) that has been used for gene delivery in vitro and in vivo due to its high transduction efficiency, safety, and long-term stable gene expression. This series of favorable characteristics has led to the approval of rAAV-based gene therapies; Glybera for lipoprotein lipase deficiency, Luxturna for Leber congenital amaurosis, and Zolgensma for spinal muscular atrophy type 1.

[0004] All native AAVs and rAAVs have in common a small, non-enveloped icosahedral capsid with a diameter of approximately Contains a linear single-stranded DNA genome. The genome size of natural AAV is about 4.7 kb. Both ends of the natural AAV genome contain inverted terminal repeats (ITRs); these are 145 nucleotides (nt) that form a T-shaped hairpin secondary structure that is important for genome replication and packaging. Between these ITRs, two open reading frames (ORFs) encode a series of replication proteins (Rep) and capsid proteins (Cap), as well as other proteins (e.g., assembly activation protein (AAP)).

[0005] The ITRs contain all the cis-acting elements involved in AAV genome replication and packaging, so rAAV vector design can rely on the removal of viral genes. In this design, rAAV particles are produced by introducing into cells a plasmid containing a cloned rAAV genome (AAV cis-plasmid), which consists of foreign DNA flanked by 145 nucleotide-long ITRs and a separate construct expressed in trans AAV viral genes (rep, cap, AAP, etc.). Auxiliary factors are provided by adenovirus infection of cells or by the introduction of a third plasmid that provides these adenovirus auxiliary factors.

[0006] It can be speculated that successfully delivering three plasmids to one cell is a relatively inefficient process. For large-scale production, transient delivery of plasmids requires excess DNA, thereby increasing the overall cost of production and purification. In addition, transient delivery of rep / cap genes in the presence of auxiliary genes may also lead to product heterogeneity, including rAAV capsids lacking genomes. These "empty capsids" account for a large proportion of the viruses produced during transient transfection. Therefore, there is still an urgent need in the art for improved methods, systems and compositions that allow efficient production of rAAV used as vectors for somatic cell gene therapy. Summary of the invention

[0007] rAAV is often used as a powerful tool for in vivo gene transfer. rAAV has been successfully used to establish efficient and long-term gene transfer in a variety of target tissues as well as throughout the body. Although the application of rAAV offers great potential for curing many genetic diseases, current rAAV production methods still have room for improvement to meet clinical needs, especially those that require large doses of high-quality vectors and disease indications that affect a large number of people. Therefore, this has prompted us to develop unique methods and compositions to meet these needs.

[0008] Disclosed herein is a system for producing a recombinant adeno-associated virus (AAV), comprising a stably transfected mammalian cell line and an auxiliary nucleic acid. The stably transfected cell line has an exogenous DNA sequence consisting of an AAV rep gene and an AAV cap gene, a gene of interest, and an RNAi sequence targeting the AAV rep gene. The auxiliary nucleic acid has a sequence that increases the expression of the AAV rep gene. In some embodiments, the exogenous DNA sequence also comprises a second RNAi targeting the auxiliary nucleic acid.

[0009] In some embodiments, an isolated nucleic acid is disclosed herein, comprising an adeno-associated virus (AAV) rep gene, an AAV cap gene, an RNAi sequence (the RNAi sequence targets an AAV gene transcript), a selectable marker capable of selecting both prokaryotic cells and eukaryotic cells, and a gene of interest, such as a therapeutic nucleic acid (which may also be a marker gene) flanked by AAV inverted terminal repeats (ITRs) at each end. In other embodiments, the isolated nucleic acid further comprises a second RNAi sequence, which targets an auxiliary nucleic acid.

[0010] Further disclosed herein is a method for producing a stably transfected cell line, the method comprising transfecting a host cell line with an isolated nucleic acid comprising an AAV rep gene, an AAV cap gene, an interfering RNA (RNAi) sequence (the RNAi sequence targets an AAV gene transcript), a selectable marker capable of selecting both prokaryotic cells and eukaryotic cells, and a gene of interest flanked by an AAV inverted terminal repeat sequence at each end. In some embodiments, the method further comprises a second RNAi sequence, the second RNAi sequence targeting an auxiliary nucleic acid.

[0011] Also disclosed herein is a method for producing a rAAV drug substance, the method comprising introducing a helper nucleic acid into a stably transfected mammalian cell line, thereby producing the rAAV drug substance. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 An exemplary plasmid map for establishing a cell line for producing a rAAV drug substance. Starting at approximately the 12 o'clock position on the plasmid map and continuing in a clockwise direction, the plasmid includes promoters driving transcription of AAV rep and cap, a promoter driving transcription of shRNA directed against the rep transcript, an ITR sequence, a promoter driving transcription of a gene of interest (GOI) (in this case, the promoter is from cytomegalovirus (CMV)), another ITR, resulting in the GOI being flanked by ITR sequences, a next promoter driving transcription of shRNA directed against an adenoviral transcript, another promoter driving transcription of a selectable marker (in this case, a promoter capable of initiating transcription in both prokaryotes and eukaryotes), and a marker capable of selecting prokaryotes and eukaryotes carrying the plasmid.

[0013] Figure 2.Another exemplary plasmid map for establishing a cell line for producing rAAV drug substances. Starting at approximately the 12 o'clock position on the plasmid map and continuing in a clockwise direction, the plasmid includes an insulator sequence, a promoter for driving transcription of the AAV rep and cap (in this case, the rep and cap sequences are derived from AAV2, but they can be derived from any AAV serotype or even a mix, such as AAV2 rep and AAV8 cap), an insulator sequence, a promoter for driving transcription of a shRNA directed against an adenoviral transcript (in this case, the tripartite leader (TPL)) and an insulator sequence, an ITR sequence, a promoter for driving transcription of the GOI (in this case, the promoter is from cytomegalovirus (CMV) and the GOI is a fusion transcript of human β-globin and emerald green fluorescent protein (emGFP)), another ITR, resulting in the GOI being flanked by ITR sequences, an insulator sequence, a promoter for driving transcription of the shRNA directed against the rep transcript, an insulator, and a selectable marker that enables selection of prokaryotic and eukaryotic cells carrying the plasmid, in this case, the selectable marker is Zeocin TM Choose an antibiotic.

[0014] Figure 3 Table and BLAST search describing unique short hairpin RNA (shRNA) targets within adenovirus tripartite leader (TPL) and AAV rep nucleic acid sequences demonstrating the absence of off-target homology in the human genome, human adenovirus type 5 off-target sequences, and AAV serotype 2 off-target sequences, indicated by an "X". Target homology sequences are indicated by a √ symbol. The targeted TPL sequences and rep sequences lack homology to each other.

[0015] Figure 4 .Evaluate various RNA pol III promoters, U6, H1, H1-Hy and 7SK, to drive shRNA-mediated gfp mRNA level knockdown in transfected A549 cells. To test these promoters, A549 cells were transfected with one of four plasmids capable of transcribing green fluorescent protein (gfp) and shRNA targeting gfp, wherein the transcription of shRNA was driven by one of the four promoters. Then reverse transcriptase polymerase chain reaction (RT-PCR) was used to measure GFP transcription levels. The figure shows that each tested promoter, U6, H1, H1-Hy and 7SK, drives sufficient transcription of shRNA targeting gfp, reducing gfpmRNA levels relative to the control "disordered". X-axis, compares the drive of each promoter to shRNA targeting gfp. Y-axis, relative mRNA levels of gfp.

[0016] Figure 5Adenoviral vector genome titers in the presence of control-expressing A549 cells and various shRNAs targeting the adenoviral tripartite leader sequence (TPL). The ability of different shRNA sequences targeting the adenoviral TPL sequence to reduce adenoviral genome titers was tested at various multiplicities of infection (MOI) in transfected A549 cells. At each MOI, shRNAs targeting TPL reduced the concentration of adenoviral genomes detected. X-axis, multiplicity of infection. Y-axis, adenoviral genomes per ml of culture medium. Negative control – “scrambled”, a randomly generated shRNA sequence with no identity to TPL. TPL#1–4 represent shRNA sequences targeting TPL. IVa2 represents shRNA sequence targeting the adenoviral mid-stage gene IVa2.

[0017] Figure 6 .Rep mRNA levels in HEK293 cells in the presence of a control shRNA, "scrambled" (sequence lacking identity to rep) or five shRNAs targeting rep (REP 1-5). The ability of various shRNA sequences to reduce rep mRNA levels was evaluated. In each condition, rep transcript levels were normalized using endogenous 18S rRNA levels. X-axis, test shRNA. Y-axis, relative mRNA levels.

[0018] Figure 7 Comparison of linearized and circular plasmids in transfected cell lines. Various cell lines, BHK21, HeLa, HeLa S, and A549, were transfected with either a linearized plasmid or a circular plasmid carrying the zeocin antibiotic resistance cassette. The number of viable colonies for each cell line transfected with either the linearized plasmid or the circular plasmid at two different zeocin concentrations was compared. X-axis, test conditions, including cell line and zeocin concentration. Y-axis, number of colonies (counts). DETAILED DESCRIPTION

[0019] "Recombinant adeno-associated virus" (rAAV) refers to viral particles that are used as nucleic acid delivery vehicles and carry therapeutic nucleic acids packaged in AAV capsids. Gene therapy based on rAAV vectors has been adapted for use in more than 100 clinical trials. This is due to its excellent safety profile, ability to target a wide range of tissues, stable transgene expression, and significant clinical benefits. However, the main challenge is to mass-produce high-titer, high-efficiency vectors to achieve better therapeutic effects.

[0020] Commonly used rAAV production methods rely on transient transfection of mammalian cells, or infection of mammalian or insect cells. These strategies require the successful introduction of multiple independent nucleic acid molecules into the same cell. In transient transfection, two or even three plasmids need to be introduced into the same cell within a narrow time window. Similarly, infection-based strategies require the co-infection of the same cell with two or three viruses. The need to introduce multiple nucleic acid molecules into the same cell in a short period of time makes these methods labor-intensive, difficult to scale and expensive.

[0021] Stably transfected production cell lines are alternative methods to overcome the various limitations of transient transfection and infection-based methods. In this method, the cell line produced is a copy of the AAV viral gene and the therapeutic nucleic acid of the side joint ITR is incorporated into the genome of the production cell, and the copy of the AAV viral gene and the therapeutic nucleic acid of the side joint ITR can be passed to the offspring of the production cell. In this method, the production of rAAV is induced by infecting the stable cell line with a helper virus (e.g., adenovirus). In order to engineer this stable production cell line, exogenous nucleic acid, such as a nucleic acid vector, is utilized.

[0022] Nucleic acid vector

[0023] Nucleic acid vector refers to the medium that carries exogenous DNA sequence into target cell.Nucleic acid vector can be engineered to have one or more selection genes, so that the cell carrying exogenous DNA sequence is selected or identified, one or more stabilizing elements (for example, cer sequence) and / or one or more integration elements (for example, LTR virus sequence, transposon and recombination site) that are used to maintain nucleic acid vector in cell, these integration elements promote nucleic acid vector to be integrated into the genome of host cell.The example of nucleic acid vector includes bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), artificial chromosome (PAC) derived from P1, cosmid and recombinant DNA plasmid.In some embodiments, nucleic acid vector is recombinant plasmid.

[0024] In addition to the selection gene, stabilizing element or integration element, the nucleic acid vector also carries other exogenous genetic material into the target cell. In some embodiments, the exogenous DNA sequence carried in part by the nucleic acid vector includes one or more sequences encoding factors that inhibit the translation of AAV Rep proteins, and one or more sequences encoding factors that inhibit the translation of auxiliary viral proteins; for example, the translation of adenovirus late proteins is inhibited by targeting the adenovirus tripartite leader sequence (TPL).

[0025] "AAV Rep protein" refers to one or more nonstructural proteins that mediate AAV replication. The four AAV Rep proteins, Rep78, Rep68, Rep52, and Rep40, are produced by translation of alternatively spliced ​​transcripts initiated by two promoters, p5 and p19. The p5 promoter produces transcripts encoding Rep78 and Rep68, while p19 produces transcripts encoding Rep52 and Rep40. In some embodiments, the nucleic acid vector encodes a factor that represses the translation of Rep78 and Rep68. In other embodiments, the nucleic acid vector encodes a factor that represses the translation of Rep52 and Rep40. In yet other embodiments, the nucleic acid vector encodes a factor that represses the translation of Rep78, Rep68, Rep52, and Rep40.

[0026] Rep protein can be from any AAV, including but not limited to natural serotype 1, 2, 3a, 3b, 4, 5, 6, 7, 8, 9, 10, 11 or 13, and any other AAV serotype or variant known at the time of application. Codon optimization / codon changes can be applied to the rep sequence. Such changes can change the translation of rep in a cell-specific manner, increase or decrease the translation in the cell. In some embodiments, the nucleic acid vector encodes a factor that suppresses the translation of naturally occurring rep. In other embodiments, the nucleic acid vector encodes a factor that suppresses the translation of non-natural rep. In other embodiments, the nucleic acid vector encodes a factor that suppresses the translation of non-natural rep, wherein the rep is a codon-optimized or otherwise modified rep.

[0027] "AVV cap" refers to a protein encoded by the cap gene present in the AAV genome. The cap gene encodes three capsid proteins VP1, VP2 and VP3, all of which form the capsid. Many naturally occurring capsid proteins and engineered capsid proteins were known at the time of the present application. Some variants are called serotypes, named 1, 2, 3 (A and B), 4-11. In some embodiments, the exogenous DNA sequence provides the AAV cap gene encoding the AAV cap.

[0028] "Gene of interest" or "GOI" refers to a polynucleotide sequence, such as a gene, open reading frame, or protein coding sequence. The encoded protein is used to alleviate or treat a disease or indication. The gene of interest can be packaged within an AAV cap when flanked by AAV inverted terminal repeats (ITRs).

[0029] In addition to one or more sequences encoding factors that repress translation of the AAV Rep protein, in some cases the exogenous DNA sequence also encodes one or more factors that repress translation of an auxiliary nucleic acid."Auxiliary nucleic acid" is a polynucleotide that increases translation of rep.

[0030] "Helper virus" refers to a virus used to induce rAAV production. Helper viruses infect cells carrying exogenous DNA sequences (such as nucleic acid vectors) and provide the necessary factors required for rAAV replication and production. Helper viruses for the production of rAAV include adenovirus, herpes virus, Epstein-Barr virus, cytomegalovirus, poxvirus, bocavirus and vaccinia virus. In some embodiments, the exogenous DNA sequence encodes a factor that suppresses the translation of one or more adenovirus, herpes virus, Epstein-Barr virus, cytomegalovirus, poxvirus, bocavirus or vaccinia virus proteins. In some embodiments, the exogenous DNA sequence encodes a factor that suppresses the translation of one or more adenovirus proteins. In other embodiments, the exogenous DNA sequence encodes a factor that suppresses the translation of one or more adenovirus proteins but does not suppress the translation of adenovirus early region 1 (E1). In some embodiments, the exogenous DNA sequence encodes a factor that suppresses the translation of adenovirus mid-transcription units. Adenovirus mid-transcription units include IX, IVa2, and L4 intermediates. In other embodiments, the exogenous DNA sequence encodes a factor that suppresses the protein translated from the major late transcription unit (MLTU). MLTU encodes multiple proteins from L1 to L5 five regions by differential splicing and polyadenylation. In other embodiments, the exogenous DNA sequence encodes a factor that individually represses the protein translated from the major late transcription unit. In other embodiments, the exogenous DNA sequence encodes a factor that represses L1, L2, L3, L4 or L5. In some embodiments, the exogenous DNA sequence encodes a factor that represses L1 or L4. In some embodiments, the exogenous DNA sequence encodes a factor that represses L4. In each case, the repression percentage relative to the control without repression can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100%. In some embodiments, the factor is RNAi (see below). In other embodiments, the factor is shRNA (see below).

[0031] A variety of mRNAs are produced from MLTU, all of which contain a triplet leader sequence (TPL). In some embodiments, the exogenous DNA sequence encoding suppresses the factor of translation of the protein encoded by the mRNA containing the TPL sequence. In other embodiments, the exogenous DNA sequence encoding suppresses the factor of translation of the protein encoded by the mRNA containing the TPL sequence alone. In each case, the suppression percentage relative to the control without suppression can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100%.

[0032] In some embodiments, a system for producing a recombinant adeno-associated virus is provided, the system comprising a stably transfected mammalian cell line and a helper virus, wherein the stably transfected mammalian cell line has an exogenous DNA sequence, the exogenous DNA sequence has an AAV rep gene, an AAV cap gene, a gene of interest, and an RNAi sequence. In other embodiments, the system includes a second RNAi sequence.

[0033] RNAi

[0034] RNAi molecules can regulate the expression of one or more genes in a production cell line. Typically, RNAi is used to reduce the translation of one or more proteins.

[0035] RNAi molecules can be designed to antagonize translation by targeting corresponding RNA sequences based on sequence homology. Such RNAi will usually be small interfering RNA (siRNA), small hairpin RNA (shRNA), double-stranded RNA (dsRNA) or micro RNA (miRNA). The sequence of such RNAi will contain a portion corresponding to a sequence of a portion of an mRNA encoding one or more proteins whose translation will be inhibited. This portion will usually be 100% complementary to the target portion within the mRNA transcribed from one or more genes, but may also have only a lower level of complementarity (e.g., 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more). Typically, the complementarity percentage (%) is determined over a continuous stretch of nucleic acid residues. For example, the RNAi molecule can have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% complementarity to a target portion within an mRNA transcribed from one or more genes, wherein complementarity is measured over at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or more nucleic acid residues of the mRNA transcribed from one or more genes of interest, including a gene of interest of rep, MLTU and / or TPL, up to the length of the mRNA. In some cases, for example, a dsRNA molecule can have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% complementarity to a target portion within an mRNA transcribed from one or more genes of interest, wherein complementarity is measured over at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or more nucleic acid residues of the mRNA transcribed from one or more genes of interest (including genes of interest of rep, MLTU and / or TPL, but excluding genes of interest of adenovirus E1) up to the length of the mRNA.

[0036] In some embodiments, RNAi is shRNA. The shRNA can be delivered to the host cell line by any appropriate means. Suitable techniques are known in the art, including the use of plasmids, viruses and bacterial vectors to deliver shRNA to the production cell line. In some embodiments, a plasmid delivery system is used to deliver shRNA.

[0037] Generally speaking, once shRNA has been delivered to a cell, it is transcribed in the nucleus and subsequently processed. The resulting pre-shRNA is exported from the nucleus and loaded into the RNA-induced silencing complex (RISC) after being processed by Dicer. The sense (passenger) strand is degraded. The antisense (guide) strand directs RISC to an mRNA with a complementary sequence. In the case of complete complementarity, RISC cuts the mRNA. In the case of incomplete complementarity, RISC represses the translation of the mRNA. In both cases, shRNA causes target gene silencing.

[0038] Selecting genes

[0039] Nucleic acid vectors can include one or more selection genes to select or identify transfected cells, such as by complementation of cell auxotrophy or by antibiotic resistance. Certain antibiotics can only be used to select microbial cells, while other antibiotics can be used to select both prokaryotic cells and eukaryotic cells with antibiotic resistance genes. Blasticidin, hygromycin B, mycophenolic acid, puromycin and Zeocin are examples of antibiotics that can be used to select prokaryotic cells and eukaryotic cells containing appropriate antibiotic resistance genes.

[0040] Therefore, in some embodiments, the nucleic acid vector is a plasmid, which comprises a selection gene. In some embodiments, the selection gene supplements a cell auxotrophic defect. In other embodiments, the selection gene confers antibiotic resistance. In other embodiments, the selection gene confers antibiotic resistance in both prokaryotic and eukaryotic cells.

[0041] Nucleic acid vectors can exist as extrachromosomal nucleic acids capable of autonomous replication. BACs, YACs, PACs, cosmids and plasmids have non-mammalian origins of replication. Non-mammalian origins of replication are nucleic acid sequences that enable nucleic acid vectors to be stably replicated and separated together with endogenous chromosomes in suitable host cells (microbial cells, such as bacterial cells or yeast cells). Examples of non-mammalian origins of replication include bacterial origins of replication, such as oriC, oriV, oriS or yeast origins of replication, also referred to as autonomous replication sequences (ARS elements).

[0042] Nucleic acid vector can be introduced into host cell as linear nucleic acid or as closed loop (such as plasmid).In some embodiments, nucleic acid vector is introduced into host cell as linear nucleic acid.In other embodiments, nucleic acid vector is introduced into host cell as circular nucleic acid.

[0043] In some embodiments, the nucleic acid vector is less than 25 kb, 20 kb, 19 kb, 18 kb, 17 kb, 16 kb, 15 kb, 14 kb, 13 kb, 12 kb, 11 kb, 10 kb, 9 kb, 8 kb, 7 kb, 6 kb, or 5 kb in length.

[0044] Nucleic acid vectors can be integrated into the host cell genome, thus becoming unable to replicate autonomously. The integration of nucleic acid vectors into the host cell genome can occur randomly or be targeted. Integration can occur at one or more positions within the host cell genome. In some embodiments, nucleic acid vectors are integrated into the host cell genome at non-targeted random positions. In other embodiments, nucleic acid vectors are integrated into the host cell genome at predetermined targeting positions. In some embodiments, nucleic acid vectors are integrated into the host cell genome at predetermined targeting positions, which are on the long arm of chromosome 19, referred to as AAV1 sites. In some embodiments, nucleic acid vectors are integrated into the host cell genome at predetermined targeting positions, which are engineered within the genome of the host cell, such as safe harbor sites.

[0045] Integration into the safe harbor locus

[0046] Nucleic acid vectors can be inserted into specific safe harbor locations in the genome. Several safe harbor loci have been described, including AAVS1, CCR5, and Rosa26. Other safe harbor loci have been identified, for example, as described in WO / 2019 / 169232 entitled "Identifying and characterizing genomic safe harbors (gsh) in humans and murine genomes, and viral and non-viral vector compositions for targeted integration at an identified gsh loci". In alternative embodiments, the disclosure herein also relates to a nucleic acid vector composition comprising a -safe harbor (SH)-5' homology arm, and a 3'SH homology arm flanking a nucleic acid containing a restriction cloning site, wherein the vector can be used to integrate the flanked nucleic acid into the genome at SH by homologous recombination. In all aspects as disclosed herein, the nucleic acid vector composition can be a plasmid, a cosmid, or an artificial chromosome (e.g., BAC), a small circular nucleic acid, a dog bone DNA, or a recombinant viral vector (e.g., rAd, AAV, rHSV, BEV, or a variant thereof).

[0047] Thus, one aspect of the technology described herein relates to a nucleic acid vector composition comprising: (a) a genomic safe harbor (GSH) 5' homology arm (also referred to herein as a "5'GSH-specific homology arm" or "5'GSH-HA"), (b) a nucleic acid sequence comprising a restriction cloning site, and (c) a GSH 3' homology arm (also referred to herein as a "3'GSH-specific homology arm" or "3'GSH-HA"), wherein the 5' homology arm and the 3' homology arm bind to a target site located in a genomic safe harbor locus, and wherein the 5' homology arm and the 3' homology arm allow for insertion of a nucleic acid located between the two homology arms into a locus located within the genomic safe harbor by homologous recombination. In some embodiments, a nucleic acid vector composition for integrating a nucleic acid of interest into a GSH locus comprises a nucleic acid of interest and / or an expressible transgene cassette (e.g., a sequence encoding a gene, a gene editing molecule described herein, or a reporter protein), and may include one or more gene editing molecules.

[0048] In some embodiments, the nucleic acid vector composition for integrating a nucleic acid of interest into a GSH locus as described herein comprises, in order: a) a 5'GSH-specific homology arm, c) a restriction cloning site, and d) a 3'GSH-specific homology arm. In some embodiments, the 3' homology arm and the 5' homology arm are complementary base paired with a region of GSH. In some embodiments, the 3' homology arm and the 5' homology arm are flanked by a target integration site, for example, a target insertion locus in GSH. In some embodiments, the 3' homology arm is complementary base paired with a nucleic acid region 3' (i.e., upstream) of a target integration site or a target insertion locus of GSH, and the 5' homology arm is complementary base paired with a nucleic acid region 5' (i.e., downstream) of a target integration site or a target insertion locus of GSH. In some embodiments, the 5' homology arm and the 3' homology arm are complementary to a portion of GSH, e.g., at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% complementary.

[0049] In order to increase the possibility of integration at a precise position, the 5' homology arm and the 3' homology arm can include enough nucleic acids, such as 50 to 5,000 base pairs, or 100 to 5,000 base pairs, or 500 to 5,000 base pairs, which have a high degree of sequence identity or homology with the corresponding target sequence to increase the probability of homologous recombination. The 5' homology arm and the 3' homology arm can be any sequence homologous to the GSH target sequence in the host cell genome. That is, the 5' homology arm and the 3' homology arm are partially complementary to the GSH target sequence identified herein. In addition, the 5' homology arm and the 3' homology arm can be non-coding nucleotide sequences or coding nucleotide sequences. In some embodiments, the homology between the 5' homology arm and the corresponding sequence on the chromosome is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%. In embodiments, the homology between the 3' homology arm and the corresponding sequence on the chromosome is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%. In embodiments, the 5' homology arm and / or 3' homology arm can be homologous to the sequence upstream and / or downstream of the integration site or DNA cleavage site next to the chromosome. Alternatively, the 5' homology arm and / or 3' homology arm can be homologous to the sequence that is away from the integration site or DNA cleavage site (such as leaving the integration site or DNA cleavage site at least 1bp, 2bp, 5bp, 10bp, 15bp, 20bp, 25bp, 30bp, 50bp, 100bp, 200bp, 300bp, 400bp or 500bp) or partially or completely overlapped with the DNA cleavage site. In embodiments, the 3' homology arm of the nucleotide sequence is adjacent to ITR.

[0050] In some embodiments, 5' homology arm and / or 3' homology arm can be any length, for example, between 30bp and 2000bp. In some embodiments, the length of 5' homology arm and / or 3' homology arm is between 200bp and 350bp. Details about the length of homology arm and recombination frequency are reported by Zhang et al., "Efficient precise knockin with adouble cut HDR donor after CRISPR / Cas9-mediated double-stranded DNA cleavage." Genome biology 18.1 (2017): 35, which is incorporated herein by reference in its entirety.

[0051] In some embodiments, the GSH 5' homology arm and the GSH 3' homology arm bind to target sites that are spatially distinct nucleic acid sequences in a genomic safe harbor identified according to the methods disclosed herein. In some embodiments, the nucleic acid vector composition for integrating a nucleic acid of interest into a GSH locus as described herein comprises a 5'GSH-specific homology arm and a 3'GSH-specific homology arm, both of which are at least 65% complementary to a target sequence in a genomic safe harbor locus identified according to the methods disclosed herein. In some embodiments, the nucleic acid vector composition for integrating a nucleic acid of interest into a disclosed GSH locus as described herein comprises a 5'GSH-specific homology arm and a 3'GSH-specific homology arm that bind to a target site located in a PAX5 genomic safe harbor sequence. In one embodiment, the nucleic acid vector composition for integrating a nucleic acid of interest into a GSH locus as described herein does not comprise any prokaryotic DNA sequence elements, such as minicircular DNA (mcDNA), but it is expected that some prokaryotic DNA (such as antibiotic resistance genes) can be inserted as exogenous sequences. In some embodiments, the nucleic acid vector composition for integrating a nucleic acid of interest into a GSH locus as described herein is a plasmid or double-stranded DNA. In one aspect, the nucleic acid vector composition for integrating a nucleic acid of interest into a GSH locus as described herein comprises the following components in sequence, or is obtained by a plasmid encoding the following components in sequence: a nucleotide sequence of interest (e.g., an expression cassette of exogenous DNA, a gene editing sequence, or a donor sequence) positioned between the 5' homology arm and the 3' homology arm.

[0052] Transcription control sequence

[0053] Gene expression is controlled at the transcription initiation level by proteins that bind to specific regulatory sequences. These regulatory sequences include promoters. A "promoter" is a DNA sequence that is recognized by the transcription machinery to initiate transcription of a downstream polynucleotide sequence. The promoter controls the binding of RNA polymerase to DNA to initiate transcription. There are three types of RNA polymerases that transcribe different genes. RNA polymerase I transcribes genes that encode ribosomal RNA.

[0054] RNA polymerase II synthesizes most of the small nuclear RNA. "RNA polymerase II promoter" or "RNA pol II promoter" or "polymerase II promoter" or "pol II promoter" means any invertebrate, vertebrate or mammalian promoter, such as human, mouse, porcine, bovine, primate, ape, etc., which associates or interacts with RNA polymerase II in the natural state of the cell to transcribe its operably linked gene, or any variant of the gene (whether natural or engineered), which will interact with RNA polymerase II in the selected host cell to transcribe the operably linked nucleic acid sequence. RNA pol II promoters include promoters of any higher eukaryote (including any vertebrate or mammal) containing any sequence variation or alteration (whether natural or generated in the laboratory) that maintains or enhances but does not eliminate the binding of RNA polymerase II to the promoter and is capable of transcribing a gene or nucleotide sequence (whether natural or engineered) that is operably linked to the promoter sequence. Examples of RNA polymerase II promoters include CMV, SV40, bacteriophage T7, and SP6.

[0055] "RNA polymerase III promoter" or "RNA pol III promoter" or "polymerase III promoter" or "polIII promoter" means any invertebrate, vertebrate or mammalian promoter, such as human, mouse, porcine, bovine, primate, monkey, etc., which associates or interacts with RNA polymerase III in the natural state of the cell to transcribe its operably linked gene, or any variant of the gene (whether natural or engineered) that will interact with RNA polymerase III in the selected host cell to transcribe the operably linked nucleic acid sequence. The so-called U6 promoter (e.g., human U6, mouse U6), H1 promoter or 7SK promoter means any invertebrate, vertebrate or mammalian promoter, or polymorphic variants or mutants found in nature, which interact with RNA polymerase III to transcribe its cognate RNA product, i.e., U6 RNA, H1 RNA or 7SK RNA, respectively. Preferred in some applications are type III RNA pol III promoters present in the 5' flanking region, including U6, H1 and 7SK, which include a TATA box but lack an internal promoter sequence. For pol III 5S rRNA, tRNA or VA RNA genes, an internal promoter is present. The 7SK RNA pol III gene contains a weak internal promoter, as well as sequences in the 5' flanking region of the gene necessary for transcription. RNA pol III promoters include promoters of any higher eukaryote (including any vertebrate or mammal) containing any sequence variation or alteration (whether natural or generated in the laboratory) that maintains or enhances but does not eliminate RNA polymerase III binding to the promoter and is capable of transcribing a gene or nucleotide sequence (whether natural or engineered) that is operably linked to the promoter sequence.

[0056] In some embodiments, the RNAi promoter is an RNA pol II promoter. In other embodiments, the RNAi promoter is an RNA pol III promoter. In still other embodiments, the second RNAi promoter is an RNA pol II promoter or an RNA pol III promoter.

[0057] RNA transcripts typically include the coding sequence of a gene and a poly(A) tail. "Poly(A) tail" refers to a chain of adenine nucleotides added to the end of an RNA transcript or encoded by a DNA template. The length of the poly(A) tail is typically 5 to 300 nucleotides.

[0058] In some embodiments, an exogenous nucleic acid is provided that provides an AAV rep gene, an AAV cap gene, an RNAi sequence targeting expression of the AAV rep gene, a selectable marker, and a gene of interest flanked by two AAV inverted terminal repeats, with one ITR at each end of the gene of interest. In other embodiments, the exogenous nucleic acid comprises a second RNAi sequence that targets a helper viral nucleic acid or a helper nucleic acid.

[0059] Insulator sequence

[0060] Eukaryotic genomes are organized into domains containing individual genes and gene clusters that have different expression patterns during development and in differentiated cells. These genomes contain regulatory elements, such as enhancers that can cis-activate target genes over considerable distances. "Insulators" are names given to a class of DNA sequence elements that have the common ability to protect genes from inappropriate signal effects from their surroundings (e.g., enhancers). In some cases, nucleic acid vectors include one or more insulator sequences.

[0061] Production cell lines

[0062] "Production cell line" refers to a cell line capable of replicating and packaging rAAV vectors. According to the present invention, any suitable production cell line can be modified and used. The production cell line of the present invention is a eukaryotic cell line, typically a mammalian cell line. The AAV vector produced by the present invention is generally intended for human treatment. Therefore, preferably, the production cell line of the present invention is a human cell line. The production cell line of the present invention can be selected from NIH3T3, HT1080, A549, HeLa, BHK21 and HEK293 cell lines. The production cell line of the present invention can be in adherent or suspended form.

[0063] Producer cell lines are used to form viral particles capable of infecting target cells. Viral vectors used in gene therapy are produced by producer cell lines, which package the therapeutic nucleic acid into viral particles. The vectors usually contain the minimum viral sequences required for packaging and subsequent integration into the target, with the other viral sequences replaced by an expression cassette encoding the therapeutic nucleic acid to be expressed. The missing viral functions are provided in trans.

[0064] In a preferred embodiment, the production cell line is the human embryonic kidney (HEK) 293 cell line. The HEK293 cell line expresses the adenoviral early region.

[0065] Other AAV serotypes, including AAV1, AAV3, AAV4, AAV5, AAV6, AAV8, AAV8.2, AAV9, AAV rh10, and pseudotyped AAVs, such as AAV2 / 5, AAV2 / 6, and AAV2 / 8, as well as engineered capsids.

[0066] As used herein, a stably transfected mammalian cell line is a production cell line that has been transfected with nucleic acid to produce cells with one or more genes inserted into the genome. In some cases, this can be achieved by linking the desired gene or genes to a selectable marker.

[0067] Therapeutic applications

[0068] The AAV vectors of the present invention can be used as gene therapy vectors in vivo, or for modifying cells in vitro for cell-based therapy. Gene transfer involves delivering new genetic information into cells. Genetic information can be inserted into cells transiently or stably. The AAV vectors of the present invention are used for gene transfer. Therefore, the AAV vectors of the present invention can be able to integrate into mammalian chromosomes non-site-specifically or site-specifically, with essentially no cytotoxicity, and direct host cell-specific expression of therapeutic polynucleotides. Preferably, the AAV vectors of the present invention are used in mammalian, more preferably human gene therapy.

[0069] The AAV vector of the present invention may comprise any polynucleotide that can be used to treat or prevent a disease or condition. The therapeutic polynucleotide may be DNA or RNA. Typically, the therapeutic polynucleotide is DNA. The therapeutic polynucleotide is preferably a biologically functional nucleic acid that targets (replaces or modifies) a non-functional gene and / or a mutant gene in the individual to be treated. In a preferred embodiment, when the individual to be treated is human, the therapeutic polynucleotide is also human-derived.

[0070] Therapeutic polynucleotides can encode biological functional proteins, i.e., polypeptides or proteins that affect the cellular mechanisms of cells expressing the biological functional proteins. For example, biological functional proteins can be proteins necessary for normal cell growth or maintenance of individual health. Biological functional proteins can also be proteins that improve individual health by supplementing missing proteins, by providing incremental proteins that produce insufficient proteins in individuals, or by providing proteins that inhibit or offset unwanted molecules that may be present in individuals. Biological functional proteins can also be proteins useful for investigations and studies on developing new gene therapies or studying cellular mechanisms.

[0071] Biological functional protein can be a protein necessary for normal growth or repair of the body. Biological functional protein can also be a protein that can be used to fight diseases (such as cancer). Biological functional protein can also be a selective marker for antibiotic resistance, such as a selective marker for neomycin resistance in eukaryotic cells. Other types of selective markers can also be used. The therapeutic polynucleotides encoding these proteins can be provided by any of a variety of methods, such as conventional cloning procedures, excision from a vector containing a gene of interest, or chemical synthesis or enzymatic synthesis based on disclosed sequence information. In many cases, DNA encoding a protein of interest is commercially available.

[0072] Biologically functional proteins can affect cellular mechanisms by providing new or altered functions to cells. For example, a therapeutic polynucleotide can be a multidrug resistance gene (mdr) encoding P-glycoprotein. P-glycoprotein is a cell membrane glycoprotein that affects intracellular drug accumulation and causes multidrug resistance.

[0073] Therapeutic polynucleotides can encode non-biological functional proteins. For example, hybrid genes containing various domains and functions from a variety of sources can be designed and produced by recombinant technology or enzymatic or chemical synthesis.

[0074] Therapeutic polynucleotides can be transcribed into RNA molecules that are fully complementary to the mRNA or DNA of interest (i.e., sense or antisense RNA, shRNA, siRNA, miRNA) to hybridize therewith. Such RNA molecules can be used to prevent or limit the expression of overproduced, defective or other unwanted molecules. The AAV vector of the present invention can include a sequence encoding antisense RNA as a therapeutic polynucleotide, which is fully complementary to the target sequence, thereby binding to the target sequence. For example, the target sequence can be a part of the mRNA encoding a polypeptide, thereby binding to the mRNA encoding the polypeptide and preventing its translation. The target sequence can be a fragment of a gene necessary for transcription, so that the antisense RNA binds to the fragment (e.g., a promoter or coding region) and prevents or limits transcription. Therefore, antisense RNA must have sufficient length and complementarity to prevent its target mRNA from translating or its target DNA from being transcribed. Antisense RNA has sufficient complementarity with the target sequence so that the antisense RNA can bind to the target sequence and thereby inhibit translation or transcription, which can be determined using standard techniques. Therapeutic polynucleotides can be provided, for example, by chemical synthesis or enzymatic synthesis, or provided from commercial sources.

[0075] The function of the AAV vector of the present invention, i.e., the ability to mediate transfer and expression of therapeutic polynucleotides, can be evaluated by monitoring the expression of therapeutic polynucleotides in transduced cells. For example, cells can be transduced with the AAV vector of the present invention, or cells can be infected with different concentrations of virions containing the AAV vector, and then the expression of therapeutic polynucleotides can be evaluated.

[0076] In some embodiments, a method of producing a rAAV vector drug substance is provided, the method comprising introducing a helper virus, a helper viral nucleic acid, or a helper nucleic acid into a stably transfected mammalian cell line.

[0077] Example

[0078] Evaluation of promoters regulating shRNA .

[0079] The plasmid disclosed in the present invention includes two different shRNA species, for example, Figure 2 As depicted, it ultimately provides the realization of a production cell line. Given that there are two shRNA sequences, one targeting rep and the other targeting the adenoviral tripartite leader sequence (TPL), it is necessary to evaluate promoter strength and interactions. To address this issue, four different promoters were tested. This was achieved by inoculating A549 cells to a confluence of 50% to 90%. These cells were transfected with a plasmid encoding green fluorescent protein (GFP) under the expression of the PGK promoter, as well as the gfp shRNA sequence under the control of one of the four promoters. A scrambled shRNA sequence (i.e., a sequence that has no identity with gfp) was used as a negative control. Transfected cells were harvested 48 to 72 hours after transfection and gfp mRNA levels were measured by reverse transcriptase polymerase chain reaction (RT-PCR). The results are shown in Figure 4 As shown in . All tested promoters drove the shRNA sequences sufficiently to produce gfp to reduce gfp levels as determined by RT-PCR.

[0080] Evaluation of shRNAs that modulate adenoviral infection .

[0081] Initially, it was not possible to predict whether an shRNA sequence could be identified that would disrupt adenovirus production or infectivity. To address this issue, a library of shRNA sequences targeting the TPL sequence present on the adenovirus type 5 major late transcription unit was generated ( Figure 3). A549 cells were inoculated and expanded to a confluence of 50% to 90%. These cells were transfected with a plasmid encoding the TPL shRNA sequence under the control of the pol III promoter and the Zeocin resistance box using a transfection reagent. A scrambled shRNA sequence in which the shRNA nucleotide sequence was randomly arranged was used as a control sequence. The transfection medium can be replaced with fresh medium after 24 hours. The cells are subjected to selection in a medium containing zeocin. Surviving colonies are pooled and expanded. The effectiveness of TPL shRNA is evaluated by inoculating cells to a confluence of 50% to 90%. The cells are then infected with wild-type adenovirus type 5 for a duration of 0 to 48 hours, followed by the production of cell lysates 48 to 72 hours after infection. The cells are lysed by three freeze / thaw cycles or using 0.25% to 1% (v / v) Tween 20, and an additional endonuclease treatment step may be included. Cell debris is removed by centrifugation. In the presence of adenovirus, the adenovirus titer of the cell lysate and / or spent culture medium is tested.

[0082] To determine the optimal time point for adenoviral infection, control experiments were performed in which cells were transfected with a plasmid containing the reporter gene gfp with or without a shRNA sequence targeting gfp. Evaluation of GFP protein expression and knockdown efficiency was measured by fluorescence microscopy or fluorescence activated cell sorting (FACS). For FACS analysis, cells were transfected with a plasmid containing the reporter gene gfp with or without a shRNA sequence targeting gfp. TM Express enzyme to detach the cells and resuspend in fresh medium. Other assays used to determine knockdown efficiency include RT-PCR for RNA and Western blotting for protein.

[0083] Evaluation of transcriptional components (promoters and shRNA) regulating rep expression .

[0084] The aim of this work was to identify a promoter and shRNA sequence combination that represses Rep expression in the absence of adenovirus type 5 and fails in the presence of adenovirus type 5. The anti-rep shRNA sequences used in this study (4 to 6 different sequences) were directed against the four mRNAs encoding AAV Rep proteins, namely Rep78, Rep68, Rep52 and Rep40. In this study, suitable shRNA sequences were first identified and then evaluated for their effectiveness in repressing Rep expression under the control of various promoter types (3 to 5 promoters). The ability of variants in promoters to modulate expression control was tested.

[0085] Evaluation of promoters regulating rep shRNA expression .

[0086] To identify suitable promoters for anti-rep shRNA expression, knockdown anti-rep shRNA was placed under the control of three to five different promoters in addition to the U6 promoter. Variations of each promoter were included to further fine-tune expression. Adherent A549 cells and / or adherent HEK293 cells were seeded so that the confluence reached ≥50% the next day. Transfection and infection were performed as described previously. Fluorescence, cell viability, and REP-GFP mRNA levels were measured 48 to 72 hours after infection.

[0087] Evaluation of shRNA targeting the rep gene .

[0088] This study uses a fusion construct of rep and gfp genes, in which GFP is fused to the common C-terminus of four Rep proteins. In order to confirm that the fusion construct is expressed in cells and that expression can be regulated, adherent A549 cells and / or adherent HEK293 cells are inoculated to a confluence of 50% to 90%. These cells are transfected with a plasmid having a fusion construct (rep-gfp) regulated by the PGK promoter. In order to confirm that fusion protein expression can be regulated, the plasmid is designed to include a shRNA sequence targeting gfp (anti-gfp shRNA), wherein the anti-gfp shRNA sequence is under the control of the U6 promoter. Scrambled shRNA sequences are used as control shRNA sequences. Plasmid transfection is performed using a transfection reagent (such as PEI, Lipofectamine, etc.). The transfection medium can be replaced with fresh medium after 24 hours. The fluorescence of the cells after transfection is evaluated as previously described.

[0089] To identify the effectiveness of anti-rep shRNA in reducing rep expression, the rep-gfp fusion construct was under the control of the P5 promoter on a plasmid that also had an anti-rep shRNA sequence under the control of the U6 promoter. HEK293 cells expressed the E1 gene, causing activation of the P5 promoter, thereby driving REP-GFP expression. Cell viability and GFP fluorescence were evaluated using fluorescence microscopy or FACS. Rep knockdown was detected using ELISA or Western blotting for mRNA using RT-PCR.

[0090] In a series of studies to identify the effectiveness of anti-rep shRNA in reducing rep expression, adherent A549 cells and / or adherent HEK293 cells were seeded to 50% to 90% confluence. These cells were transfected with a plasmid having a fusion construct (rep-gfp) under the control of the P5 promoter and an anti-rep shRNA under the control of the U6 promoter. A scrambled shRNA sequence has been used as a control for the anti-rep shRNA. Cells were transfected with the plasmid using polyethyleneimine (PEI), Lipofectamine or another suitable transfection reagent. Evaluation of rep mRNA levels 24 to 72 hours after transfection was determined by reverse transcription polymerase chain reaction (RT-PCR), as Figure 6 As shown. Figure 6 As shown, in the presence of rep shRNA, rep expression levels were reduced relative to the scrambled sequence.

[0091] Evaluation of Antibiotic Selection in Mammalian Cells

[0092] In order to determine the zeocin concentration for stable transfection, mammalian cells were inoculated so that the confluence was >20%. Zeocin kill curves were drawn by subjecting cells to 50 μg / mL to 1000 μg / mL of zeocin, with the culture medium replaced 2 to 4 times per week for a maximum duration of 5 weeks. Cell viability estimates were obtained by observing under an optical microscope. The concentration used to select stable transfection was determined by the minimum concentration that killed cells within the target treatment time.

[0093] To verify the duration that cells can retain transfected plasmids, cells were seeded at 50% to 90% confluence and transfected with a plasmid encoding a gene conferring resistance to zeocin expressed from a promoter identified in eukaryotes and prokaryotes and encoding gfp under the control of the PGK promoter. Cells were placed under selection at the minimum concentration of zeocin determined above. Surviving cells were pooled, cultured, and monitored for fluorescence once or twice a week for 1 to 2 months.

[0094] To test whether plasmid linearization affects the retention of transfected cells, cells were plated to 50% to 90% confluence and then transfected with linearized or circular plasmids carrying the zeocin resistance cassette and the gfp gene under the control of the PGK promoter. Transfected cells were placed under zeocin selection. Surviving colonies were counted by microscopic observation or crystal violet staining. Results are presented in Figure 7 As shown in . It was found that both the linearized plasmid and the circular plasmid were able to generate stable cell lines. GFP expression was confirmed by fluorescence measurement of pooled colonies by flow cytometry.

[0095] To identify the effectiveness of anti-rep shRNA in reducing rep expression in stable cells, A549 cells were transfected with a plasmid expressing rep-cap under the control of the P5 promoter, an anti-rep shRNA sequence under the control of the U6 promoter, and a zeocin selection marker. Cells were selected in a medium containing zeocin. Surviving colonies were amplified and rep mRNA levels were evaluated by RT-PCR.

[0096] Evaluation of rAAV production in various cells

[0097] To evaluate rAAV production, mammalian cells were seeded at 50% to 90% confluence. Cells were transfected with plasmids encoding gfp between the ITRs, a resistance gene to zeocin, shRNA targeting rep, and the TPL region of adenovirus. The transfection medium could be replaced with fresh medium after 24 hours. Cells were infected with adenovirus 24 to 72 hours after transfection for 48 to 72 hours. Cell lysates were generated. rAAV production was evaluated by vector genome analysis, capsid titer analysis, and infectious titers. The efficiency of the TPL shRNA was verified by measuring the adenovirus infectious titer.

[0098] Generation of AAV producer cell lines

[0099] Cells are transfected with plasmids as described above and subjected to selection using zeocin as described above. Single cell clones are isolated and screened for rAAV production, and these single cell clones can be frozen.

[0100] Clones were thawed and cultured at increasing scale for ≥3 passages, and rAAV production was evaluated by infecting cells with adenovirus. Top producer clones were evaluated for stability of rAAV production for ≥15 cell culture passages. Producer cell clones capable of high-level production of rAAV were master cells stored under GMP for the manufacture of AAV vector drug substances, which were then formulated into rAAV drug products.

Claims

1. A system for producing recombinant adeno-associated virus (rAAV), the system comprising a stably transfected mammalian cell line and a helper virus, the stably transfected mammalian cell line comprising an exogenous DNA sequence, the exogenous DNA sequence comprising an AAV rep gene, an AAV cap gene, a gene of interest, and an RNAi sequence targeting the expression of the AAV rep gene, the helper virus comprising a sequence that increases rAAV vector production after the helper virus is introduced into the stably transfected mammalian cell line.

2. The system of claim 1, wherein the exogenous DNA sequence further comprises a selectable marker.

3. The system of claim 1, wherein the exogenous DNA sequence further comprises a second RNAi sequence, wherein the second RNAi sequence targets a helper virus sequence.

4. The system of claim 1, wherein the RNAi sequence comprises double-stranded RNA (dsRNA), small interfering RNA (siRNA), small hairpin RNA (shRNA), micro RNA, or antisense RNA.

5. The system of claim 4, wherein the RNAi sequence is shRNA.

6. The system of claim 1, wherein the helper virus is an adenovirus, herpes virus, Epstein-Barr virus, cytomegalovirus, papillomavirus, bocavirus, or poxvirus.

7. The system of claim 1, wherein the promoter of the RNAi sequence is an RNA pol II or RNA pol III promoter.

8. The system of claim 7, wherein the promoter of the RNAi sequence is an RNA pol III promoter.

9. The system of claim 3, wherein the promoter of the second RNAi sequence is an RNA pol II or RNA pol III promoter.

10. The system of claim 9, wherein the promoter of the second RNAi sequence is an RNA pol II promoter.

11. The system of claim 9, wherein the promoter of the second RNAi sequence is an RNA pol III promoter.

12. An exogenous nucleic acid comprising an AAV rep gene, an AAV cap gene, an RNAi sequence, a selectable marker, and a gene of interest (GOI) flanked by two AAV inverted terminal repeats (ITRs), wherein the RNAi sequence targets expression of the AAV rep gene.

13. The exogenous nucleic acid according to claim 12, further comprising a second RNAi sequence, wherein the second RNAi sequence targets a helper virus nucleic acid sequence.

14. The exogenous nucleic acid of claim 12, wherein the rep gene and the AAV cap gene are both from the same AAV serotype.

15. The exogenous nucleic acid of claim 12, wherein the rep gene and the AAV cap gene are both from different AAV serotypes.

16. The exogenous nucleic acid of claim 12, wherein the AAV rep gene is selected from AAV serotypes 1, 2, 3, 4-9, or other known or engineered rep genes.

17. The exogenous nucleic acid of claim 12, wherein the AAV cap gene is selected from AAV serotypes 1, 2, 3, 4-9, or other known or engineered cap genes.

18. The exogenous nucleic acid according to claim 12, wherein the RNAi is double-stranded RNA, small interfering RNA, small hairpin RNA, micro RNA or antisense RNA.

19. The exogenous nucleic acid according to claim 18, wherein the RNAi is a small hairpin RNA (shRNA).

20. The exogenous nucleic acid according to claim 13, wherein the second RNAi sequence is double-stranded RNA, small interfering RNA, small hairpin RNA, micro RNA or antisense RNA.

21. The exogenous nucleic acid according to claim 20, wherein the RNAi is a small hairpin RNA (shRNA).

22. A method for producing a stably transfected cell line, the method comprising transfecting a host cell with the exogenous nucleic acid according to claim 12.

23. The method of claim 22, wherein the host cell is a HeLa, HeLa S, BHK, HEK293 or A549 cell.

24. The method of claim 22, wherein the exogenous nucleic acid is targeted for integration into a predetermined site within the host cell genome.

25. A method for producing a recombinant adeno-associated virus (rAAV) vector drug substance, the method comprising introducing a helper virus nucleic acid sequence into the stably transfected mammalian cell line according to claim 22, thereby producing the rAAV drug substance.

26. The method of claim 25, wherein the stably transfected mammalian cell line comprises exogenous nucleic acid, the exogenous DNA sequence comprising an AAV rep gene, an AAV cap gene, a gene of interest flanked by two AAV inverted tandem repeat sequences, and an RNAi sequence targeting expression of the AAV rep gene, wherein the RNAi sequence represses expression of the AAV rep gene.

27. The method of claim 26, wherein expression of the helper viral nucleic acid overcomes the repressive effect of the RNAi sequence that represses AAV rep gene expression.

28. The method of claim 26, wherein the exogenous nucleic acid further comprises a second RNAi targeting a helper viral nucleic acid.

29. The method of claim 28, wherein the second RNAi reduces production of adenovirus.

30. The method of claim 25, wherein the helper virus is an adenovirus.

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