Optimization of HEK293 suspension platform for improved rAAV titers

Abstract

The present invention provides a method and system for the production of high-titer rAAV. High-titer rAAV can be used in pharmaceutical compositions, gene therapy and research. In one embodiment, a method for producing high-titer rAAV is provided, comprising the steps of providing HEK293-derived cells, co-transfecting HEK293-derived cells with a plasmid containing an ITR expression vector, an AAV helper construct, Rep and Cap, incubating the transfected cells, and harvesting rAAV.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority pursuant to 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 329,856, filed April 11, 2022, and U.S. Provisional Patent Application No. 63 / 331,438, filed April 15, 2022, each of which is incorporated by reference in its entirety.

[0002] Description of sequence listing An official copy of the Sequence Listing has been submitted contemporaneously herewith as an XML file in WIPO standard ST.26 format with file name "17234-037WO1_SequenceListing.xml", created on April 6, 2023, and size in bytes. This Sequence Listing, as submitted through the USPTO Patent Center, is a part of the present specification and is hereby incorporated by reference in its entirety.

[0003] FIELD OF THEINVENTION The present invention relates to the field of recombinant AAV production. [Background technology]

[0004] background AAV is a 4.7 kb single-stranded DNA virus. Because wild-type AAV is non-pathogenic and has no etiological association with any known disease, rAAV virions are associated with excellent clinical safety. Recent clinical and non-clinical successes using recombinant adeno-associated virus (rAAV) vectors as in vivo gene delivery vehicles have stimulated interest in producing highly pure and effective viral vectors for various natural and engineered serotypes. AAV requires the expression of helper genes for efficient replication. Current rAAV production in human embryonic kidney (HEK) 293 cell lines includes a helper plasmid that provides helper genes instead of a helper virus, and is therefore referred to as a helper-free system.

[0005] AAV vectors used for administering therapeutic nucleic acids typically have approximately 96% of the parent genome deleted, leaving only the terminal repeats (ITRs) that contain the recognition signals for DNA replication and packaging. This reduces immunological or toxic side effects due to the expression of viral genes. In addition, delivery of specific AAV proteins to producer cells allows the integration of AAV vectors containing AAV ITRs into specific regions of the cell genome, if desired. See, for example, U.S. Patent Nos. 6,342,390 and 6,821,511. Host cells containing integrated AAV genomes show little or no change in cell growth or morphology (see, for example, U.S. Patent No. 4,797,368).

[0006] The AAV ITRs are flanked by unique coding nucleotide sequences for the nonstructural replication (Rep) proteins and the structural capsid (Cap) proteins (also known as virion proteins (VPs)). The terminal 145 nucleotides are self-complementary and organized in such a way that energetically stable intramolecular duplexes forming T-shaped hairpins can form. These hairpin structures act as primers for cellular DNA polymerase complexes, thereby serving as origins of viral DNA replication.

[0007] Several AAV serotypes and over 100 AAV variants have been isolated from adenovirus stocks or from human or non-human primate tissues. In general, AAV serotypes have genome sequences with significant homology at the nucleic acid and amino acid sequence levels, so that different serotypes have the same set of gene functions, produce physically and functionally equivalent virions, and are replicated and assembled by nearly identical mechanisms. AAV serotype 2 (AAV2) is widely used for gene therapy applications. AAV Rep and ITR sequences are particularly conserved across many AAV serotypes. Moreover, Rep sequences and ITRs of many AAV serotypes are known to efficiently cross-complement (i.e., functionally replace) corresponding sequences from other serotypes during production of AAV particles in mammalian cells. In general, Cap proteins and related Cap protein coding sequences, which determine the cellular tropism of AAV particles, are significantly less conserved across different AAV serotypes than Rep genes. Given the ability of Rep and ITR sequences to cross-complement corresponding sequences of other serotypes, AAV vectors can include mixtures of serotypes, and thus "chimeric" or "pseudotyped" AAV vectors. Chimeric AAV vectors contain AAV capsid proteins from two or more (e.g., 2, 3, 4, etc.) different AAV serotypes. In contrast, pseudotyped AAV vectors contain one or more ITRs of one AAV serotype packaged into the capsid of another AAV serotype. Chimeras and pseudotypes are further described in, e.g., U.S. Pat. No. 6,723,551; Flotte (2006) Mol Ther 13(1):1-2; Gao et al (2004) J. Virol 6381-6388; Gao et al (2002) Proc Natl Acad Sci USA 99:11854-11859; De et al (2006) Mol Ther 13:67-76, and Gao et al (2006) Mol Ther 13:77-87. The most commonly used method to produce recombinant adeno-associated virus (rAAV) in the laboratory is by transient triple transfection of 293 cells with an AAV cis plasmid and an AAV trans plasmid along with an adenovirus helper plasmid. The triple transfection HEK293 platform is routinely used for rAAV production and utilizes an AAV cis plasmid encoding the gene of interest, an AAV trans plasmid containing the capsid and replicase genes, and a helper plasmid. However, most of the currently used HEK293 production platforms have low titers and are not scalable. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] U.S. Patent No. 6,342,390 [Patent Document 2] U.S. Patent No. 6,821,511 [Patent Document 3] U.S. Pat. No. 4,797,368 [Patent Document 4] U.S. Patent No. 6,723,551 [Non-patent literature]

[0009] [Non-Patent Document 1] Flotte(2006) Mol Ther 13(1):1-2 [Non-Patent Document 2] Gao et al (2004) J. Virol 6381-6388 [Non-Patent Document 3] Gao et al (2002) Proc Natl Acad Sci USA 99:11854-11859 [Non-Patent Document 4] De et al (2006) Mol Ther 13:67-76 [Non-Patent Document 5] Gao et al (2006) Mol Ther 13:77-87 Summary of the Invention

[0010] overview Provided herein are methods and systems for producing high titer rAAV, which can be used in pharmaceutical compositions, gene therapy compositions, for research purposes, and other applications.

[0011] In one embodiment, a method for producing high titer rAAV is provided, comprising providing HEK293 derived cells, co-transfecting the HEK293 derived cells with a plasmid comprising an ITR expression vector, an AAV helper construct, Rep and Cap, incubating the transfected cells, and harvesting the rAAV. In one aspect of this method, Rep and Cap are on separate plasmids. In one aspect, Rep and Cap are on the same plasmid. In an aspect of this method, the transfection reagent used in co-transfecting the cells is selected from the group comprising AAV-Max and PEI-Pro. In various aspects, the HEK239 derived cells are selected from the group comprising VPC2.0 and Oxford cells. In one aspect, the HEK293 derived cells are selected from the group comprising VPC2.0 and Oxford cells, and the transfection reagent used in co-transfecting the cells is selected from the group comprising AAV-Max and PEI-Pro. In an embodiment of the method, the HEK293-derived cells are VPC2.0 cells and the transfection reagent is AAV-Max. In one embodiment, the titer of the harvested rAAV is at least 5×10 10vg / ml. In various embodiments, the transfection reagent used to co-transfect the cells is selected from the group including AAV-Max and PEI-Pro, and Rep and Cap are on separate plasmids. In various embodiments, the transfection reagent used to co-transfect the cells is selected from the group including AAV-Max and PEI-Pro, and Rep and Cap are on the same plasmid. In one embodiment, the transfection reagent used to co-transfect the cells is AAV-Max, and Rep and Cap are on separate plasmids. In one embodiment, the transfection reagent used to co-transfect the cells is TRX, Rep and Cap are on separate plasmids, and the HEK293-derived cells are VPC2.0 cells. In various embodiments, the HEK293-derived cells are selected from the group including VPC2.0 cells and Oxford cells, and the transfection reagent used to co-transfect the cells is selected from the group including AAV-Max and PEI-Pro, and Rep and Cap are on separate plasmids. In some embodiments, the HEK293-derived cells are VPC2.0 cells, the transfection reagent is AAV-Max, and Rep and Cap are on separate plasmids. In various embodiments, the titer of the harvested rAAV is at least 1×10 11 vg / ml.

[0012] A four-plasmid system for producing high-titer rAAV is provided. The four-plasmid system for producing high-titer rAAV includes the steps of providing a set of plasmids including a first plasmid comprising a nucleotide sequence encoding an AAV Rep polypeptide operably linked to a first promoter, a second plasmid comprising a nucleotide sequence encoding an AAV Cap polypeptide operably linked to a second promoter, a third plasmid comprising an ITR expression vector comprising a gene of interest, and a fourth plasmid comprising an AAV helper construct, transfecting the set of plasmids into HEK293-derived cells, incubating the transfected cells, lysing the transfected cells, and harvesting the rAAV. In an embodiment of the system, the second promoter is a constitutively active promoter. In a particular embodiment, the constitutively active promoter is a CMV promoter. In an embodiment of the system, the second promoter is a p40 promoter. In an embodiment of the system, the first promoter is an AAV promoter. In a particular embodiment, the AAV promoter is selected from the group comprising a p5 promoter and a p19 promoter. In an embodiment of this system, the HEK293 derived cells are selected from the group including HEK293 cells, VPC2.0 cells, and Oxford cells. In an embodiment of this system, the molar ratio of the first plasmid to the third plasmid and the molar ratio of the second plasmid to the third plasmid are comparable when normalized to plasmid size. In various embodiments, the four plasmids of the set of plasmids are co-transfected at a molar ratio of approximately 1:1:1:1 when normalized to plasmid size. In various embodiments, the molar ratio of the first plasmid to either the third plasmid or the fourth plasmid is greater than 1:1 when normalized to plasmid size. In various embodiments, the molar ratio of the second plasmid to either the third plasmid or the fourth plasmid is greater than 1:1 when normalized to plasmid size.In some embodiments, the molar ratios of the first plasmid to the fourth plasmid and the molar ratios of the second plasmid to the fourth plasmid are comparable when normalized for plasmid size, hi certain embodiments, the molar ratios of the Rep plasmid to the Cap plasmid are comparable when normalized for plasmid size.

[0013] In various embodiments, the method of lysing the transfected cells comprises at least one step selected from the group comprising a freeze-thaw step, a sonication step, a thermochemical step, and an alternative chemical method step.

[0014] In one embodiment, the titer of the harvested rAAV is at least 5×10 10 In one embodiment, the titer of the harvested rAAV is at least 1×10 11 In other embodiments, the titer of the harvested rAAV is at least 3×10 11 vg / ml.

[0015] In various aspects of this system, the step of transfecting the set of plasmids into the HEK293-derived cells is a co-transfection of the set of plasmids, while in other aspects, the steps of transfecting the set of plasmids into the HEK293-derived cells are performed sequentially.

[0016] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. [Brief description of the drawings]

[0017] [Figure 1]A chart summarizing the titers of recombinant virus (rAAV2.7m8-Fluc) in viral genomes per ml (vg / ml) produced from the indicated HEK293-derived cells after co-transfection with the indicated transfection reagents. Co-transfection of AAV ITR expression vector, pAAV Rep-Cap gene, and pHelper was performed in the presence of either AAV-Max (TRX) transfection reagent or PEIPro (PEIPRO) transfection reagent. Plasmids were transfected into either VPC2.0 cells (VPC2.0) or Oxford cells (OX). Data in columns labeled OX-TRX-molar, OX-PEIPro-molar, VPC2.0-TRX-molar, and VPC2.0-PEIPro-molar were obtained from experiments in which cells were cotransfected with pAAV ITR expression vector, pAAV Rep-Cap gene, and pHelper at a molar ratio of 1:2:0.5, respectively, normalized to plasmid size. Recombinant AAV titers obtained with VPC2.0-PEIPro-molar conditions in this set of experiments were greater than 5×1010 vg / ml. Recombinant AAV titers obtained with VPC2.0-TRX-molar conditions in this set of experiments were at least 1×1011 vg / ml. Data in columns labeled OX-mass were obtained from experiments using equimolar concentrations of Rep-Cap plasmid, helper plasmid, and gene of interest (GOI) plasmid (1:1:1).

[0018] [Diagram 2] A chart summarizing rAAV titers (y-axis, vg / ml) obtained from Oxford cells co-transfected with pAAV ITR expression vector, pAAV Rep-Cap gene, and pHelper in the presence of PEIPro transfection reagent after lysis by the methods indicated on the x-axis. Results are shown from cells lysed by freeze-thaw (FT), sonication (SC), thermochemical (Thermo), or alternative chemical (ALT) methods.

[0019] [Diagram 3] A chart summarizing the rAAV titers obtained from VPC2.0 cells co-transfected with pAAV ITR expression vector, pAAV Rep-Cap gene, and pHelper at the indicated molar ratios normalized to plasmid size is shown.

[0020] [Figure 4] A chart summarizing rAAV titers obtained from the indicated cell types (Oxford (OX) or VPC2.0 (VPC2.0)) co-transfected with the indicated transfection reagents (PEIpro or TRX) is shown. The same gene of interest or transgene was inserted into the same backbone, but the transgene was linked to either the 886 signal peptide (886, SEQ ID NO:1) or the 1176 signal peptide (1176, SEQ ID NO:2). The 1176 construct with the different signal peptide resulted in higher titers.

[0021] [Diagram 5] A schematic diagram of the three-plasmid system for rAAV production in two different types of HEK-derived cells (Oxford or VPC2.0) is shown.

[0022] [Figure 6] A chart summarizing rAAV titers produced in VPC2.0 cells after co-transfection with either a three-plasmid system (3 plasmid) or a four-plasmid system (4 plasmid). In the traditional three-plasmid system, cells were co-transfected with a pAAV ITR expression vector, pAAV Rep-Cap gene, and pHelper. In the three-plasmid system, the Rep and Cap genes are on the same plasmid. In the four-plasmid system, cells were co-transfected with a pAAV ITR expression vector, pAAV Rep gene, pAAV-CMV-Cap gene, and pHelper. The Rep and Cap genes were on separate plasmids. The increase in titer obtained from the four-plasmid system is substantial.

[0023] [Figure 7] Schematic diagrams of three-plasmid and four-plasmid systems for rAAV production in HEK-derived cells are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] Provided herein are methods and systems for producing high titer rAAV, which allow for the production of rAAV at substantially higher titers than typically achieved with standard production processes.

[0025] In the following passages, different aspects of the invention are described in more detail. Each aspect, embodiment or feature of the invention may be combined with any other aspect, embodiment or feature of the invention, unless expressly indicated to the contrary.

[0026] A.Definition Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

[0027] The terms used herein are for the purpose of describing particular examples only and are not intended to be limiting. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms unless the context clearly indicates otherwise. Furthermore, to the extent that "including," "includes," "having," "has," "including," or variations thereof are used anywhere in the detailed description and / or claims, such terms are intended to be inclusive in a similar manner to the term "comprising." The term "comprising," as used herein, is synonymous with "including" or "containing," and is inclusive or open-ended. Any reference to "or" herein is intended to include "and / or" unless otherwise specified.

[0028] As used herein, the term "about" following a number refers to that number plus or minus 10% of that number. The term "about" following a range refers to that range from minus 10% of the minimum value of that range to plus 10% of the maximum value of that range. Reference to "about" following a value or parameter herein includes (and describes) embodiments that are directed to the value or parameter itself.

[0029] "High titer" contemplates a particle or virion concentration equal to or greater than a predefined threshold before dilution; a virus preparation with a lower titer, either by dilution or by using a larger volume when harvesting particles or virions, is encompassed by the term "high titer" if the predefined threshold is obtained when harvested. Titer types can be selected from, but are not limited to, total capsid titer, genomic titer, physical titer, infectious titer of a virus preparation, and full capsid to empty capsid ratio. It is recognized that different titer types are assessed using different methods and that the value of each titer type for a particular aliquot of virus particles may differ. It is understood that a "high titer" preparation requires a high particle concentration only for at least one titer type. The terms "genomic titer" and "physical titer" are used interchangeably herein. Genomic titer is understood to be the concentration of viral genome per ml (vg / ml). Methods for assessing genome titer are known in the art and include, but are not limited to, quantitative real-time PCR (qPCR) and digital droplet PCR (ddPCR). Total capsid titer represents the amount of all intact (fully assembled) viral capsids in a sample, including both full and empty capsids. Methods for assessing capsid titer are known in the art and include, but are not limited to, antibody-based methods, ELISA, and dot blots. Methods for determining the ratio of full to empty particles are known in the art and include, but are not limited to, cryo-electron microscopy, negative staining, and combinations of methods for determining genome titer and capsid titer. See, e.g., Gimpel et al (2021) Mol Therapy: Methods & Clin Dev 20:740-754. The predefined threshold for "high titer" rAAV is 5×10 10 vg / ml, 5.5×10 10 vg / ml, 6 × 10 10 vg / ml, 6.5 × 10 10 vg / ml, 7 × 10 10 vg / ml, 7.5 × 1010 ing / ml、8×10 10 ing / ml、8.5×10 10 in g / ml, 9×10 10 ing / ml、9.5×10 10 ing / ml、1×10 11 ing / ml、1.5×10 11 in g / ml, 2×10 11 ing / ml、2.5×10 11 in g / ml, 3×10 11 ing / ml、3.5×10 11 in g / ml, 4×10 11 in g / ml, 4.5×10 11 ing / ml、5×10 11 ing / ml、5.5×10 11 in g / ml, 6×10 11 ing / ml、6.5×10 11 in g / ml, 7×10 11 ing / ml、7.5×10 11 ing / ml、8×10 11 ing / ml、8.5×10 11 in g / ml, 9×10 11 ing / ml、9.5×10 11 ing / ml、1×10 12 vg / ml、&5×10 12 vg / ml is included, but it is limited to 5×10 10 ing / ml、5.5×10 10 in g / ml, 6×10 10 ing / ml、6.5×10 10 in g / ml, 7×10 10 ing / ml、7.5×10 10 ing / ml、8×10 10 ing / ml、8.5×10 10 in g / ml, 9×10 10 ing / ml、9.5×10 10 ing / ml、1×10 11 ing / ml、1.5×10 11 in g / ml, 2×10 11 ing / ml、2.5×10 11 in g / ml, 3×10 11 ing / ml、3.5×1011 vg / ml, 4 × 10 11 vg / ml, 4.5 × 10 11 vg / ml, 5 × 10 11 vg / ml, 5.5×10 11 vg / ml, 6 × 10 11 vg / ml, 6.5 × 10 11 vg / ml, 7 × 10 11 vg / ml, 7.5 × 10 11 vg / ml, 8 × 10 11 vg / ml, 8.5 × 10 11 vg / ml, 9 × 10 11 vg / ml, 9.5×10 11 vg / ml, 1×10 12 vg / ml, and 5 × 10 12 It may have a concentration in the range of vg / ml.

[0030] In some embodiments, the methods and systems of the present application can be used to generate rAAV virions for use in a subject. In various aspects, a subject refers to a mammal. Mammals include, but are not limited to, livestock (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents. In certain embodiments, a "subject," "patient," or "individual" refers to a primate, for example a human and a non-human primate, such as an African green monkey and a rhesus monkey. In some embodiments, a subject is a human.

[0031] The terms "treat", "treating", "treatment", "ameliorate" or "ameliorating", and other grammatical equivalents, refer to alleviating, reducing or improving a disease or disorder or a symptom of a disease or disorder, preventing additional symptoms of a disease or disorder, improving or preventing the underlying metabolic cause of a symptom, inhibiting a disease or condition, e.g., arresting the onset of a disease or condition, relieving a disease or condition, causing regression of a disease or condition, or arresting a symptom of a disease or condition, and are intended to include prevention. The term further includes achieving a therapeutic benefit and / or a prophylactic benefit. The term "therapeutic benefit" refers to the eradication or amelioration of the disease or disorder being treated. Therapeutic benefit is also achieved by the eradication or amelioration of one or more of the physiological symptoms associated with a disease or disorder, such that in some embodiments, an improvement is observed in the subject, even though the subject is still afflicted with the disease or disorder. With regard to prophylactic benefit, the pharmaceutical compositions are administered to subjects at risk of developing a disease or disorder, or who report one or more physiological symptoms of a disease or disorder, even if the disease or disorder has not been diagnosed.

[0032] As used herein, the terms "administer," "administering," "administration," and the like can refer to methods used to enable delivery of a therapeutic agent or pharmaceutical composition to a desired site of biological action.

[0033] As used herein, the terms "effective amount," "therapeutically effective amount," or "pharmaceutical effective amount" can refer to a sufficient quantity of at least one pharmaceutical composition or compound being administered that relieves to some extent one or more of the symptoms of the ocular disease or disorder being treated. An "effective amount," "therapeutically effective amount," or "pharmaceutical effective amount" of a pharmaceutical composition can be administered to a subject in need thereof as a unit dose (described in more detail elsewhere herein).

[0034] The term "pharmaceutical acceptable" as used herein can refer to a substance, such as a carrier or diluent, that does not abrogate the biological activity or properties of the compounds disclosed herein and that is relatively non-toxic (i.e., it does not cause undesirable biological effects when administered to an individual or interact in a deleterious manner with any of the components of the composition in which it is contained).

[0035] The term "pharmaceutical composition" or simply "composition" as used herein can refer to a biologically active compound optionally mixed with at least one pharma- ceutically acceptable chemical component, such as, but not limited to, a carrier, stabilizer, diluent, dispersant, suspending agent, thickener, excipient, and the like.

[0036] As used herein, "AAV vector" or "rAAV vector" refers to an adeno-associated virus (AAV) vector or recombinant AAV (rAAV) vector that contains a polynucleotide sequence that is not of AAV origin (e.g., a polynucleotide heterologous to AAV, such as a nucleic acid sequence encoding a therapeutic transgene, e.g., aflibercept) for transduction into a target cell or tissue. Generally, the heterologous polynucleotide is flanked by at least one, and generally two, AAV inverted terminal repeats (ITRs). The term rAAV vector encompasses both rAAV vector particles and rAAV vector plasmids. rAAV vectors can be either single-stranded (ssAAV) or self-complementary (scAAV).

[0037] "AAV virus" or "AAV virus particle" or "rAAV vector particle" or "rAAV particle" or "rAAV virion" or "rAAV virus" refers to a virus particle that includes at least one AAV capsid protein and a polynucleotide rAAV vector. In some cases, the at least one AAV capsid protein is derived from wild-type AAV or is a variant AAV capsid protein (e.g., an AAV capsid protein that includes an insertion, e.g., an insertion of the 7m8 amino acid sequence shown below). When a particle includes a heterologous polynucleotide (e.g., a polynucleotide other than the wild-type AAV genome, such as a transgene that is delivered to a target cell or tissue), it is referred to as a "rAAV particle," "rAAV vector particle," or "rAAV vector." Thus, the production of rAAV particles necessarily includes the production of rAAV vectors, such as the vectors contained within the rAAV particles.

[0038] As used herein, the term "packaging" can refer to a series of intracellular events that lead to assembly and encapsidation of rAAV particles.

[0039] AAV "rep" and "cap" genes refer to polynucleotide sequences encoding the replication and encapsidation proteins of adeno-associated virus and its variants. The Rep genes encode the Rep proteins Rep78, Rep68, Rep52, and Rep40. Rep78 and Rep68 are transcribed from the p5 promoter, while Rep52 and Rep40 are transcribed from the p19 promoter. The Rep79 and Rep68 proteins are multifunctional DNA-binding proteins that perform helicase and nickase functions during productive replication and allow dissociation of AAV ends (see, e.g., Im et al 1990 Cell 61:447-57). These proteins also control transcription from endogenous AAV promoters and promoters in helper viruses (see, e.g., Periera et al (1997) J. Virol 71:1079-1088). Other Rep proteins modify the function of Rep78 and Rep68. In the four-plasmid system of the present application, Rep can be operably linked to an AAV promoter, including, but not limited to, p19 and p5. "Rep" encompasses variant Rep, chimeric Rep, and modified Rep.

[0040] The Cap gene encodes the capsid proteins VP1, VP2, and VP3. The Cap gene is transcribed from the p40 promoter. In the four-plasmid system of the present application, the Cap is operably linked to a constitutively active promoter. Cap includes variant Cap, chimeric Cap, pseudotyped Cap, and modified Cap. A person skilled in the art would know which Rep and Cap forms are preferred for use together and would make the appropriate selection.

[0041] The term "polypeptide" can encompass both naturally occurring and non-naturally occurring proteins (e.g., fusion proteins), peptides, fragments, variants, derivatives, and analogs thereof. A polypeptide may be monomeric, dimeric, trimeric, or polymeric. Furthermore, a polypeptide may comprise multiple distinct domains, each of which has one or more distinct activities. For the avoidance of doubt, a "polypeptide" may be of any length greater than two amino acids in length.

[0042] As used herein, "polypeptide variant" or simply "variant" refers to a polypeptide whose sequence contains an amino acid modification. In some embodiments, the modification is an insertion, duplication, deletion, rearrangement or substitution of one or more amino acids compared to the amino acid sequence of a reference protein or polypeptide, such as a natural or wild-type protein. A variant may have one or more amino acid point substitutions, in which a single amino acid at a position is changed to another amino acid, one or more insertions and / or deletions, in which one or more amino acids are inserted or deleted, respectively, in the sequence of the reference protein, and / or a truncation of the amino acid sequence at either or both the amino or carboxy terminus. A variant may have the same or different biological activity compared to the reference protein or the unmodified protein.

[0043] In some embodiments, the variants may have, for example, at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overall sequence homology to the corresponding reference protein. In some embodiments, the variants may have at least about 90% overall sequence homology to the wild-type protein. In some embodiments, the variants exhibit at least about 95%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9% overall sequence identity.

[0044] As used herein, "recombinant" can refer to a biological molecule, e.g., a gene or protein, that is (1) removed from its naturally occurring environment, (2) not associated with all or a portion of a polynucleotide with which it is found in nature, (3) operatively linked to a polynucleotide with which it is not naturally linked, or (4) not naturally occurring. The term "recombinant" can be used in reference to cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs biologically synthesized by heterologous systems, as well as proteins and / or mRNAs encoded by such nucleic acids. Thus, for example, a protein synthesized by a microorganism is recombinant when the protein is synthesized from an mRNA synthesized from a recombinant gene present in a cell.

[0045] "Operably linked" or "operably linked" or "coupled" refers to the juxtaposition of genetic elements, where these elements are in a relationship permitting them to function in an expected manner. For example, a promoter can be operably linked to a coding region if the promoter helps to initiate transcription of the coding sequence. There can be intervening residues between the promoter and the coding region so long as this functional relationship is maintained.

[0046] The term "expression vector" or "expression construct" or "cassette" or simply "vector" can include any type of genetic construct, including AAV or rAAV vectors, that contains a nucleic acid or polynucleotide encoding a gene product in which some or all of the nucleic acid coding sequence is transcribable and is adapted for gene therapy. The transcript can be translated into a protein. In some embodiments, the transcript is partially translated or not translated. In certain aspects, expression includes both transcription of the gene and translation of the mRNA into a gene product. In other aspects, expression includes only transcription of the gene of interest encoding nucleic acid. The expression vector can also include control elements operably linked to the coding region to promote expression of the protein in the target cell. The combination of control elements and the gene or genes to which they are operably linked for expression may be referred to as an "expression cassette," many of which are known and available in the art or can be readily constructed from components available in the art.

[0047] A "gene product" is a molecule resulting from the expression of a particular gene. Gene products include, for example, polypeptides, aptamers, interfering RNA, mRNA (messenger RNA), and the like. In certain embodiments, a "gene product" is a polypeptide, peptide, protein, or interfering RNA, including short interfering RNA (siRNA), miRNA (microRNA), or small hairpin RNA (shRNA). In certain embodiments, the gene product is a therapeutic gene product, e.g., a therapeutic protein. In some aspects, the gene product provides knockdown of gene function. In some aspects, the gene product provides increase in gene function.

[0048] In some embodiments, the gene of interest may encode a reporter gene product, such as, but not limited to, GFP and RFP. In some embodiments, the gene of interest may encode a therapeutic gene product, such as a therapeutic protein. Therapeutic gene products are known in the art and include polypeptide hormones, cytokines or growth factors (e.g., insulin or erythropoietin), interferons, blood clotting factors, vaccines, anti-angiogenic polypeptides, vascular endothelial growth factor (VEGF) binding proteins, anti-VEGF agents, anti-VEGF proteins, opsin proteins, anti-C3 antibodies, anti-C5 antibodies, hormone receptors (such as, but not limited to, mineralocorticosteroid, glucocorticoid, and thyroid hormone receptors), intramembrane proteins (such as, but not limited to, TM-1 and TM-7), intracellular receptors (such as, but not limited to, orphan, retinoid, vitamin D3, and vitamin A receptors), signaling molecules (such as, but not limited to, kinases, transcription factors and signal transducers and cytokine superfamilies). Activators of transcriptional receptors, such as erythropoietin, growth hormone, interferons, interleukins, and colony stimulating factors; G protein-coupled receptors, such as, but not limited to, hormones, calcitonin, epinephrine, gastrin, paracrine or autocrine mediators, such as somatostatin or prostaglandins; neurotransmitter receptors, such as norepinephrine, dopamine, serotonin, or acetylcholine; ligands for tyrosine kinase receptors, such as insulin growth factor and nerve growth factor; and anti-dry AMD gene products. Anti-VEGF agents are known in the art and include, but are not limited to, bevacizumab, brolucizumab, ranibizumab, faricimab, abicipar pegol (abicipar pegol), and the like. pegol), conbercept, OPT-302, KSI-301, sunitinib maleate for injection (GB-102), PAN-90806 (PanOptica), and / or aflibercept.

[0049] The term "anti-VEGF agent" includes any therapeutic agent that can reduce, interfere, interrupt, block and / or inhibit the activity or function of endogenous VEGF and / or endogenous VEGF receptor (VEGFR) in vivo, or VEGF-VEGFR interaction or pathway, including proteins, polypeptides, peptides, fusion proteins, multimeric proteins, gene products, antibodies, human monoclonal antibodies, antibody fragments, aptamers, small molecules, kinase inhibitors, receptors or receptor fragments, or nucleic acid molecules. The anti-VEGF agent can be any of the known therapeutic agents that can reduce the growth or formation of new blood vessels and / or edema or swelling when delivered to a cell, tissue or subject in vivo, such as ranibizumab, brolucizumab, or bevacizumab. In some embodiments, the anti-VEGF agent can be naturally occurring, non-naturally occurring, or synthetic. In some embodiments, the anti-VEGF agent can be derived from a naturally occurring molecule that is subsequently modified or mutated to confer anti-VEGF activity. In some embodiments, the anti-VEGF agent is a fusion or chimeric protein. In such proteins, functional domains or polypeptides are artificially fused to moieties or polypeptides to create fusion or chimeric proteins that can sequester VEGF in vivo or function as VEGFR decoys. In some embodiments, the anti-VEGF agent is a fusion or chimeric protein that blocks endogenous VEGFR from interacting with its ligands.

[0050] As used herein, "VEGF" includes VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, or any combination thereof, or any functional fragment or variant, unless otherwise required. Unless otherwise required, "VEGF" can refer to any member of the VEGF family, including members of VEGF-A, placenta growth factor (PGF), VEGF-B, VEGF-C, and VEGF-D, or any combination thereof, functional fragment, or variant. As used herein, "VEGF receptor" or "VEGFR" or "VEGF-R" can be used to refer to any one of the receptors of VEGF, including, but not limited to, VEGFR-1 (or Flt-1), VEGFR-2 (or Flk-1 / KDR), and VEGFR-3 (or Flt-4). VEGFR can be membrane-bound or soluble, or a functional fragment or truncation of the receptor.

[0051] Therapeutic gene products for use in treating ocular diseases or disorders include, but are not limited to, anti-angiogenic polypeptides, VEGF binding proteins, opsin proteins, anti-C3 antibodies, anti-C5 antibodies, anti-dry AMD gene products, aflibercept, sFLT-1, CFI, ranibizumab, and bevacizumab. Anti-dry AMD gene products include, but are not limited to, inhibitors of C3, C5, HtrA1, C1qm, and natural inhibitors of the complement pathway, such as CFI, CFH, and CD59.

[0052] As used herein, a "therapeutic gene" refers to a gene that, when expressed, produces a therapeutic gene product that provides a beneficial effect to a cell or tissue in which it is present, or to the mammal in which it is expressed. Examples of beneficial effects include ameliorating a sign or symptom of a condition or disease, preventing or inhibiting a condition or disease, or imparting a desired characteristic. Therapeutic genes include, but are not limited to, genes that correct a genetic defect in a cell or mammal, and genes that express a therapeutic gene product.

[0053] The term "heterologous" may refer to an entity that is genotypically different from the rest of the entity to which it is being compared. For example, a polynucleotide introduced into a plasmid or vector derived from a different species by genetic engineering techniques may be a heterologous polynucleotide. A promoter that is removed from its native coding sequence and operably linked to a coding sequence with which it is not naturally found linked is a heterologous promoter.

[0054] The methods, systems, and kits described herein may employ conventional techniques and explanations of molecular biology (including recombinant techniques), cell biology, biochemistry, immunochemistry, and virology techniques that are within the skill of the art, unless otherwise specified. Such conventional techniques include cloning and propagation of recombinant viruses, formulation of pharmaceutical compositions, and biochemical purification, and immunochemistry methods. Specific examples of suitable techniques can be obtained by reference to the examples herein. However, equivalent conventional procedures may also be used. Such conventional techniques and explanations may be found in standard laboratory manuals such as Weiner et al., Eds, Genetic Variation: a Laboratory Manual (2007); Dieffenbach, Dveksler (Eds.), PCR Primer: a Laboratory Manual (2003); Sambrook and Russell, Condensed Protocols from Molecular Cloning: a Laboratory Manual (2006), all of which are incorporated herein by reference in their entirety for all purposes.

[0055] Natural and engineered serotypes can be used in the methods and systems provided herein, including, but not limited to, AAV type 1 (AAV1), AAV type 2 (AAV2), AAV type 3 (AAV3), AAV type 4 (AAV4), AAV type 5 (AAV5), AAV type 6 (AAV6), AAV type 7 (AAV7), AAV type 8 (AAV8), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, rhesus AAV, ovine AAV, AAV2.7m8, AAVrhlO, AAVr100, and LSV1. Several AAV serotypes and over 100 AAV variants have been isolated from adenovirus strains or human or non-human primate tissues (reviewed, for example, in Wu et al (2006) Molecular Ther. 14(3):316-327). In general, AAV serotypes have genomic sequences with significant homology at the nucleic acid and amino acid sequence levels, such that different serotypes have identical sets of gene functions, produce essentially physically and functionally equivalent virions, and are replicated and assembled by substantially identical mechanisms. AAV serotypes 1-5 and 7-9 are defined as "true" serotypes in that they do not cross-react efficiently with neutralizing sera specific for all other existing characterized serotypes. In contrast, AAV serotypes 6, 10 (also called Rh10), and 11 are considered "variant" serotypes because they do not conform to the definition of a "true" serotype. AAV serotype 2 (AAV2) is widely used for gene therapy applications due to its lack of pathogenicity, broad infectious range, and ability to establish long-term transgene expression (see Carter, BJ (2005) Hum Gene Ther 16:541-550 and Wu et al).The genomic sequences of various AAV serotypes and comparisons thereof are disclosed, for example, in GenBank accession numbers U89790, J01901, AF043303, and AF085716; Chiorini et al. (1997) J. Virol 71:6823-33, Srivastava et al (1983) J. Virol.45:555-564, Chiorini et al. (1999) J. Virol 73:1309-1319, Rutledge et al. (1998) J. Virol 72:309-319, and Wu et al. (2000) J. Virol 74:8635-8647.

[0056] In some embodiments, the AAV capsid protein is a wild-type AAV capsid protein, including but not limited to AAV type 1 (AAV1), AAV type 2 (AAV2), AAV type 3 (AAV3), AAV type 4 (AAV4), AAV type 5 (AAV5), AAV type 6 (AAV6), AAV type 7 (AAV7), AAV type 8 (AAV8), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, rhesus AAV, or ovine AAV capsid protein. In some embodiments, the AAV capsid protein is a VP1, VP2, or VP3 capsid protein. In some embodiments, the AAV capsid protein is a hybrid of multiple AAV capsid proteins. As used herein, a "variant AAV capsid protein" refers to an AAV capsid protein that contains at least one amino acid difference (e.g., an amino acid substitution, an amino acid insertion, an amino acid deletion) compared to a corresponding parent AAV capsid protein and does not correspond to an amino acid sequence present in a naturally occurring AAV capsid protein. In various embodiments, the variant AAV capsid protein can confer an altered tropism to a recombinant AAV virion compared to the tropism of the recombinant AAV virion containing the parent AAV capsid protein. See US2020 / 0338146, which is incorporated herein by reference in its entirety.

[0057] In some embodiments, the AAV Rep protein is a wild-type AAV Rep protein, including, but not limited to, a Rep protein of AAV type 1 (AAV1), AAV type 2 (AAV2), AAV type 3 (AAV3), AAV type 4 (AAV4), AAV type 5 (AAV5), AAV type 6 (AAV6), AAV type 7 (AAV7), AAV type 8 (AAV8), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, rhesus AAV, or ovine AAV. In some embodiments, the AAV Rep protein is selected from the group including a Rep78 protein, a Rep68 protein, a Rep52 protein, and a Rep40 protein. In some embodiments, the AAV Rep protein is a hybrid of multiple AAV Rep proteins. As used herein, a "variant AAV Rep protein" refers to an AAV Rep protein that contains at least one amino acid difference (e.g., an amino acid substitution, an amino acid insertion, an amino acid deletion) compared to a corresponding parent AAV Rep protein and does not correspond to an amino acid sequence present in a naturally occurring AAV Rep protein.

[0058] As used herein, a "modified sequence" refers to a sequence that contains one or more substitutions, insertions, and / or deletions compared to the corresponding sequence of a parent sequence.

[0059] AAV "helper virus" refers to a virus that allows AAV (e.g., wild-type AAV) to replicate and package in mammalian cells. A variety of such AAV helper viruses are known in the art and include adenoviruses, herpesviruses, and poxviruses such as vaccinia. Adenoviruses encompass several different subgroups, with adenovirus type 5 of subgroup C being the most commonly used. Numerous adenoviruses of human, non-human mammalian, and avian origin are known and available from depositories such as the ATCC. Viruses of the Herpes family include, for example, herpes simplex virus (HSV) and Epstein-Barr virus (EBV), as well as cytomegalovirus (CMV) and pseudorabies virus (PRV).

[0060] AAV requires the expression of helper genes for efficient replication. "Helper function(s)" refers to the function(s) encoded in the helper virus genome that allows AAV replication and packaging (along with other requirements for replication and packaging). As described herein, "helper functions" can be provided in several ways, including, but not limited to, providing a helper virus or, for example, providing a producer cell in trans with a helper plasmid containing a polynucleotide sequence encoding the required functions. The required functions include, but are not limited to, functions provided by the adeno VA, E4, and E2A genes. For example, a plasmid or other expression vector containing a nucleotide sequence encoding one or more adenoviral helper proteins can be transfected or co-transfected into the producer cell. Cao et al 2000 J. Virol 74:11456-11463 describes a method for producing high titer wild-type free recombinant AAV vectors using helper plasmids.

[0061] By "plasmid" is intended a small, often circular, extrachromosomal DNA molecule that can replicate independently within a cell. A plasmid may contain one or more expression cassettes, open reading frames, polynucleotide cassettes, or expression vectors.

[0062] Polynucleotides and cells Also provided herein are polynucleotides encoding one or more AAV capsid proteins. In certain embodiments, the polynucleotide is an expression vector. In some embodiments, the expression vector comprises a polynucleotide encoding a capsid protein, modified capsid protein, or variant capsid protein operably linked to a promoter sequence that drives expression of the polynucleotide in a cell. In certain embodiments, the cell is a host cell. The host cell can be used to produce virions comprising the capsid protein. By "producer cell" is intended a host cell used to produce rAAV virions. Exemplary host cells include mammalian cells, including but not limited to HEK293 cells, HEK293-derived cells, VPC2.0 cells (HEK293-derived cells), and Oxford cells (HEK293-derived cells). In some embodiments, the polynucleotide comprising a polynucleotide encoding Cap further comprises a polynucleotide encoding Rep. In some embodiments, the polynucleotide comprising a polynucleotide encoding Rep is separate from the polynucleotide comprising a polynucleotide encoding Cap.

[0063] A "promoter" is a DNA region that initiates transcription of a particular gene. Promoters from a wide variety of sources are well known in the art, and any promoter known in the art can be utilized in the methods and systems of the present application. Promoters can be unidirectional (i.e., initiate transcription in one direction) or bidirectional (i.e., initiate transcription in either the 3' or 5' direction). Promoters include, but are not limited to, constitutively active promoters, inducible promoters, and cell type specific promoters. Constitutively active promoters include, but are not limited to, human beta actin, chicken beta actin, cytomegalovirus (CMV), SV40, and CAG promoters. Cell type specific promoters include, but are not limited to, CD19 gene promoter, CaMKII1, and UAS. Inducible promoters include, but are not limited to, the Tet system (U.S. Pat. Nos. 5,464,758 and 5,814,618), the ecdysone-inducible system (No et al., (1996) Proc. Natl Acad Sci 93:3346-3351, the Cre-Lox system, the T-Rex™ system (Invitrogen, Carlsbad Calif.), the Cre-ERT tamoxifen-inducible recombinase system (Indra et al (1999) Nuc Acid Res 27:4324-4327, U.S. Pat. No. 7,112,715, Kramer & Fussenegger 2005 Methods Mol Biol 308:123-144, and the LacSwitch™ system (Stratagene San Diego Calif.).

[0064] In certain embodiments, the promoter operably linked to Cap is a constitutively active promoter.In one aspect, the promoter operably linked to Cap is a CMV promoter.It is recognized that the plasmid comprising the CMV promoter operably linked to Cap can further comprise a promoter operably linked to Rep.In certain embodiments, the promoter operably linked to Cap is a p40 promoter.

[0065] In some embodiments, a polynucleotide cassette is provided that includes a sequence encoding a gene product, such as, but not limited to, a therapeutic gene product. In various aspects, the polynucleotide cassette is flanked by one or more AAV inverted terminal repeats (ITRs). In certain embodiments, the polynucleotide cassette is flanked at its 5' and 3' ends by AAV ITRs. In some embodiments, the one or more ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, rhesus AAV, or ovine AAV ITRs or variants thereof. In some embodiments, one or more ITRs are an ITR of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, rhesus AAV, or ovine AAV. In some embodiments, one or more ITRs are an ITR of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, rhesus AAV, or ovine AAV that comprises one or more insertions, deletions, and / or substitutions of nucleotides. The ITR expression vector comprises one or more of the following nucleic acid elements: a first ITR sequence; a promoter sequence; an intron sequence; a first UTR sequence; a gene of interest encoding a gene product; a second UTR sequence; a polyA sequence; and a second ITR sequence. In some embodiments, a linker sequence(s) is inserted between two or more nucleic acid elements. In some embodiments, the heterologous nucleic acid encodes a therapeutic polypeptide, for example, aflibercept (or a functional fragment or variant thereof).

[0066] The terms "transgene", "gene of interest" and "sequence encoding a gene product" may be used interchangeably. The size of the transgene may affect the titer of the harvested rAAV, as described later in this specification. Without being limited to the mechanism, adjusting or altering the size of the transgene may increase the titer of the harvested rAAV. The size of the transgene may be altered by deletion of one or more portions of the gene product or increased by the addition of one or more filler regions. A filler region is a polynucleotide sequence of a size selected to improve rAAV titer, including, but not limited to, a polynucleotide sequence encoding a signal peptide or a polynucleotide sequence encoding a cleavable sequence.

[0067] rAAV production Numerous methods for the production of recombinant AAV virions are known in the art, including transfection, stable cell line production, and infectious hybrid virus production systems, including adenovirus-AAV hybrids, herpesvirus-AAV hybrids, and baculovirus-AAV hybrids. rAAV production cultures for the production of rAAV viral particles all require: 1) suitable producer cells; 2) suitable helper functions; 3) AAV rep and cap genes; 4) nucleic acid flanked by at least one AAV ITR sequence (such as a sequence encoding a transgene); and 5) suitable media and media components to support rAAV production. In some embodiments, suitable producer cells are primate host cells. In some embodiments, suitable host cells are human-derived cell lines, such as HeLa cells, a549 cells, HEK293 cells, or Perc.6 cells. In preferred embodiments, the host cells are HEK293-derived cells. In particularly preferred embodiments, the host cells are VPC2.0 cells.

[0068] In the current methods and systems, suitable helper functions are provided on a plasmid construct. The AAV helper construct comprises a polynucleotide that includes a suitable helper function coding sequence. Suitable helper function coding sequences include, but are not limited to, adenoVA, E4, and E2A helper coding sequences.

[0069] In some embodiments, the AAV rep gene may be from any AAV serotype or may be a modified AAV rep gene. In some aspects, the AAV rep gene is of the same serotype as the ITRs of the rAAV vector genome. In some aspects, the AAV rep gene is of a different serotype than the ITRs or Cap serotype of the rAAV vector genome. In some aspects, the AAV Rep is a chimeric Rep. In some aspects, the AAV Cap is a chimeric Cap. In three-plasmid systems and methods using three plasmids, Rep and Cap are provided on the same plasmid. In four-plasmid systems and methods using four plasmids, Rep and Cap are provided on different plasmids.

[0070] Suitable media known in the art can be used for the production of rAAV vectors, including, but not limited to, Gibco viral production medium supplemented with 4 mM GlutaMAX, Modified Eagle's Medium (MEM), and media from Hyclone Laboratories and JRH, including Dulbecco's Modified Eagle's Medium (DMEM).

[0071] Suitable rAAV production media may be supplemented with serum or serum-derived recombinant proteins at levels between 0.5% and 20% (v / v or w / v). Alternatively, rAAV virions can be produced in serum-free conditions, which may be referred to as animal-derived product-free media. Commercially available or custom media designed to support the production of rAAV virions may be supplemented with one or more cell culture components known in the art, including, but not limited to, glucose, vitamins, amino acids, and growth factors, to increase the titer of rAAV in the production culture.

[0072] Any transfection reagent known in the art can be utilized in the methods and systems of the present application. Transfection reagents can include, but are not limited to, calcium phosphate, AAV-Max (also known as Trx), and PEI-Pro. The terms "Trx", "TRX", "AAV-MAX", and "AAV-Max" can be used interchangeably.

[0073] By "incubating" is intended to maintain the transfected HEK293-derived cells under conditions, including suitable temperature, so that the cells develop and grow. Methods for incubating HEK293-derived cells are known in the art. The transfected HEK293-derived cells can be incubated in a medium containing an agent, such as an antibiotic to which one or more of the plasmids confer resistance. The transfected HEK293-derived cells can be incubated in a medium containing multiple selective agents.

[0074] rAAV production cultures can be grown under a variety of conditions (wide range of temperatures, various lengths of time, etc.) suitable for the particular host cells utilized. rAAV production cultures include attachment-dependent and attachment-independent cultures. Production cultures can be cultured in attachment-dependent vessels, such as, but not limited to, roller bottles, hollow fiber filters, microcarriers, packed bed bioreactors, fluidized bed bioreactors, and suspension-compatible vessels, such as, but not limited to, disposable systems, including, but not limited to, spinner flasks, stirred tank bioreactors, and wave bag systems.

[0075] Also provided herein are compositions comprising an rAAV produced by the methods or systems of the present application. Pharmaceutical compositions comprising an rAAV produced by the methods or systems of the present application are provided.

[0076] One method of producing rAAV virions is triple transfection method.In brief, the plasmid containing Rep gene and Cap gene, the helper adenovirus plasmid, and the plasmid containing the transgene flanked by AAV ITRs can be co-transfected into HEK293-derived cell line, incubated, and virions can be collected and optionally purified.Transfection and co-transfection methods are known in the art, including but not limited to calcium phosphate transfection.

[0077] "Co-transfection" is intended to encompass concurrent transfection, simultaneous transfection, near-synchronous transfection, near-simultaneous transfection, and rapid sequential transfection of multiple plasmids. Co-transfection of a three-plasmid system is difficult. Successful co-transfection of multiple plasmids may occur at a lower rate than successful transfection of a single plasmid. As the number of plasmids to be transfected increases, the challenges of successful co-transfection increase. Without being limited by the mechanism, the challenges of co-transfection may relate to the transfection efficiency of the plasmids, the selection of co-transfected cells, and the maintenance of multiple plasmids in the co-transfected cells. In various embodiments, transfecting the plasmids of the plasmid set is performed sequentially.

[0078] This application provides a four-plasmid system for producing rAAV. Briefly, a first plasmid containing AAV Rep, a second plasmid containing AAV Cap, a third plasmid containing an AAV helper construct, and a fourth plasmid containing a transgene flanked by AAV ITRs can be transfected into HEK293-derived cells, the transfected cells are incubated, the transfected cells are lysed, and the rAAV is harvested and optionally purified. Methods of transfection and co-transfection are known in the art and include, but are not limited to, calcium phosphate transfection.

[0079] It is recognized that the four-plasmid system can be utilized in modified producer cell line methods such as those described in Martin et al. (2013) Human Gene Therapy Methods 24:253-269; US Patent Publication No. 2004 / 0224411 and Liu, et al. (1999) Gene Ther. 6:293-299. Briefly, the Rep and Cap genes are provided on separate plasmids. It is understood that the producer cell line may contain either a Rep plasmid, a Cap plasmid, or both a Rep plasmid and a separate Cap plasmid. In some embodiments, cell lines stably transfected with the plasmid maintain the plasmid over multiple passages. The plasmid may be replicated as the cell replicates, or the plasmid may be integrated into the cell genome.

[0080] In some embodiments, the plasmid(s) may contain a selectable marker (e.g., an antibiotic resistance marker) that allows for the selection of cells that maintain the plasmid. Selectable markers include, but are not limited to, blasticidin, G418, hygromycin B, zeocin, and puromycin. Methods for introducing nucleic acids into cells are known in the art, and include, but are not limited to, viral transduction, cationic transfection, calcium phosphate transfection, microinjection, biolistic bombardment, electroporation, and nanoparticle transfection. See, e.g., Kim & Eberwine (2010) Anal Bioanal Chem 397:3173-3178.

[0081] In systems and methods including both three and four plasmid embodiments, it is recognized that the molar ratio of the plasmids when normalized for size impacts the process yield. In various embodiments, the molar ratio of the plasmids is approximately equivalent. In certain embodiments, the molar ratio of the plasmids when normalized for size can be approximately 1:1:1, 1:2:1, 1:3:1, 1:1:2, 2:1:1, 1:4:1, 1:4:2, 2:1:1, 2:1:2, 3:2:1, or 1:2:3 (helper plasmid:rep cap plasmid:ITR plasmid). In certain embodiments, the molar ratio of the plasmids can be approximately 1:1:1:1 (helper plasmid:rep plasmid:cap plasmid:ITR plasmid). In various embodiments, the molar ratio of Cap Rep plasmid to either the helper plasmid or the plasmid containing an ITR expression vector, when normalized to plasmid size, is in the range of 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1 or more. In various embodiments, the molar ratio of Cap plasmid (second plasmid) to either the helper plasmid (third plasmid) or the plasmid containing an ITR expression vector (fourth plasmid), when normalized to plasmid size, is in the range of 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1 or more. In various embodiments, the molar ratio of Rep plasmid (first plasmid) to either the helper plasmid (third plasmid) or the plasmid containing the ITR expression vector (fourth plasmid), when normalized to plasmid size, is in the range of approximately 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1 or more. In various embodiments, the molar ratio of Rep plasmid (first plasmid) to Cap plasmid (second plasmid), when normalized to plasmid size, is in the range of approximately 0.5:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 1:5, 1:4.5, 1:4, 1:3.5, 1:3, 1:2.5, 1:2, 1:1.5, 1:1, and 1:0.5.

[0082] The terms "harvested" and "collected" can be used interchangeably. Methods of harvesting virions, viral particles, and rAAV are known in the art and can include, but are not limited to, filtration, flow filtration, depth filtration, membrane filtration, centrifugation, and combinations thereof. Any method of harvesting rAAV known in the art can be used in the methods and systems of the present application.

[0083] Eye diseases and disorders are known in the art. Eye diseases and disorders include, but are not limited to, color vision disorders, glaucoma, retinitis pigmentosa, macular degeneration, retinoschisis, Leber's congenital amaurosis, diabetic retinopathy, color vision disorders, diabetic macular edema, choroidal neovascularization, proliferative diabetic retinopathy, retinal vein occlusion, central retinal vein occlusion, branch retinal vein occlusion, diabetic macular edema, diabetic retinal ischemia, ischemic retinopathy, diabetic retinal edema. Macular degeneration can include, but is not limited to, dry macular degeneration, wet macular degeneration, age-related macular degeneration, and acute macular degeneration.

[0084] Age-related macular degeneration (AMD) is a degenerative eye disease that affects the macula, a small light-sensitive area in the center of the retina that is responsible for reading and fine vision. Conditions that affect the macula reduce central vision while sparing peripheral vision. In severe cases, the disease can lead to central blindness. AMD is a significant cause of vision loss in people aged 65 years and older in the U.S. population, with an estimated prevalence of AMD in people over 40 years of age of approximately 6.5% (Klein et al., (2011) Arch Ophthalmol, 129(1):75-80).

[0085] There are two forms of age-related macular degeneration: dry (atrophic) macular degeneration and wet macular degeneration. Dry AMD is more common than wet AMD, but dry AMD can progress to wet AMD. Dry AMD is characterized by thinning of tissue in the macula as cells are lost; dry AMD can affect both eyes. Dry AMD is typically characterized by progressive apoptosis of cells in the retinal pigment epithelium (RPE) layer, the photoreceptor cells above it, and often also the cells below the choriocapillaris layer. Convergent areas of RPE cell death with photoreceptor atrophy above are called geographic atrophy (GA). As dry AMD progresses and GA increases, central vision slowly deteriorates and the ability to see fine details is gradually lost. Dry AMD tends to progress more slowly than wet AMD.

[0086] In some embodiments, the ocular neovascular disease is recurrent and / or persistent wAMD. In some embodiments, the ocular neovascular disease is active subfoveal CNV secondary to AMD. In some embodiments, the active subfoveal CNV secondary to AMD occupies >50% of the total lesion size. In some embodiments, the active subfoveal CNV secondary to AMD occupies >50% of the total lesion size and is associated with evidence of leakage on fluorescein angiography (FA), evidence of fluid on spectral domain optical coherence tomography (SD-OCT), and / or evidence of subretinal hemorrhage on color fundus photography. In some embodiments, active subfoveal CNV secondary to AMD accounts for ≥50% of the total lesion size, is associated with evidence of leakage on fluorescein angiography (FA), evidence of fluid on spectral domain optical coherence tomography (SD-OCT), and / or evidence of subretinal hemorrhage on color fundus photography, and the overall dimensions of the lesion do not exceed 12 macular photocoagulation study disk areas. In some embodiments, prior to administration of a unit dose of rAAV particles of the present disclosure, one eye and / or the contralateral eye of the individual exhibits a best corrected visual acuity (BCVA) based on an ETDRS letter rating of 78 to 25 (e.g., less than any of about 78, about 75, about 70, about 65, about 60, about 55, about 50, about 45, about 40, about 35, about 30, or about 25). In some embodiments, prior to administration of a unit dose of the rAAV particles of the present disclosure, one eye and / or the contralateral eye of the individual exhibited a best corrected visual acuity (BCVA) of greater than any of about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 based on an ETDRS letter score.

[0087] In some embodiments, the individual had polypoidal choroidal vasculopathy (PCV) in one eye and / or the contralateral eye prior to administration of the unit dose of rAAV particles.

[0088] Color vision impairment is a rare autosomal recessive disorder that results in retinal degeneration affecting all three types of cone photoreceptor cells, resulting in reduced vision, photophobia, hemianopsia, and severe loss of color discrimination. Mutations in the CNGB3 gene affect more than 90% of patients and result in complete color vision impairment, meaning that patients have significant impairments in color discrimination and central vision.

[0089] In some embodiments, the unit dose of rAAV particles is administered in combination with steroid therapy. In some embodiments, the steroid therapy is corticosteroid therapy. In some embodiments, the steroid therapy is systemic steroid therapy. In some embodiments, the steroid therapy is oral steroid therapy. In some embodiments, the steroid therapy is prednisone therapy. In some embodiments, the steroid therapy is ophthalmic steroid therapy. In some embodiments, the ophthalmic steroid therapy is topical steroid therapy (e.g., eye drops), periocular steroid therapy (e.g., subtenon, subconjunctival), intravitreal steroid therapy, or superchoroidal steroid therapy. In some embodiments, the ophthalmic steroid therapy is a glucocorticoid, including but not limited to anti-inflammatory glucocorticoids. In some embodiments, the topical steroid therapy is a glucocorticoid, including but not limited to anti-inflammatory glucocorticoids. In some embodiments, the topical steroid treatment is difluprednate treatment, medrysone treatment, loteprednol treatment, prednisolone treatment, fluocinolone treatment, triamcinolone treatment, rimexolone treatment, dexamethasone treatment, fluorometholone treatment, fluocinolone treatment, rimexolone treatment, or prednisone treatment. Anti-inflammatory glucocorticoids can include, but are not limited to, difluprednate, dexamethasone, prednisolone, triamcinolone, fluorometholone, rimexolone, fluocinolone, loteprednol, and bioequivalents thereof. In some embodiments, the topical steroid treatment is difluprednate treatment. By "dexamethasone" is intended dexamethasone, dexamethasone biosimilars, dexamethasone bioequivalents, and pharmaceutical compositions comprising dexamethasone, dexamethasone biosimilars, or dexamethasone bioequivalents.Pharmaceutical compositions containing dexamethasone include, but are not limited to, Ozurdex™, Maxidex™, Decadron™, Dexamethasone Intensol™, Ocu-Dex™, Dexycu™, Dextza™, and Zodex™. Ozurdex™ is a pharmaceutical composition containing dexamethasone. By "difluprednate" is intended difluprednate, difluprednate biosimilars, difluprednate bioequivalents, and pharmaceutical compositions containing difluprednate, difluprednate biosimilars, or difluprednate bioequivalents. Pharmaceutical compositions containing difluprednate include, but are not limited to, Durezol™ and difluprednate emulsions. By "triamcinolone" is intended triamcinolone, triamcinolone biosimilars, triamcinolone bioequivalents, and pharmaceutical compositions comprising triamcinolone, triamcinolone biosimilars, or triamcinolone bioequivalents. Pharmaceutical compositions comprising triamcinolone include, but are not limited to, Triesence™, Xpere™, and Trivalis™. In some embodiments, steroid therapy is administered before, during, and / or after administration of a unit dose of rAAV particles. In some embodiments, steroid therapy is administered before administration of a unit dose of rAAV particles. In some embodiments, steroid therapy is administered during administration of a unit dose of rAAV particles. In some embodiments, steroid therapy is administered after administration of a unit dose of rAAV particles. In some embodiments, steroid therapy is administered before and during administration of a unit dose of rAAV particles. In some embodiments, steroid therapy is administered before and after administration of a unit dose of rAAV particles. In some embodiments, steroid therapy is administered during and after administration of a unit dose of rAAV particles, hi some embodiments, steroid therapy is administered before, during, and after administration of a unit dose of rAAV particles.

[0090] In some embodiments, the steroid treatment is an ophthalmic steroid treatment (e.g., difluprednate). In some embodiments, the ophthalmic steroid treatment (e.g., difluprednate) is a daily steroid treatment for up to about 4 weeks, about 6 weeks, or about 8 weeks after administration of a unit dose of rAAV particles. In some embodiments, the ophthalmic steroid treatment comprises about 4 administrations of ophthalmic steroid in about week 1, about 3 administrations of ophthalmic steroid in about week 2, about 2 administrations of ophthalmic steroid in about week 3, and about 1 administration of ophthalmic steroid in about week 4, timing starting with administration of a unit dose of rAAV particles and following administration of a unit dose of rAAV particles. In some embodiments, the ophthalmic steroid is about 0.005% to about 0.5% difluprednate. In some embodiments, the ophthalmic steroid is about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.02%, about 0.03%, about 0.4%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, or about 0.1% difluprednate. In some embodiments, the ophthalmic steroid is 0.05% difluprednate. In some embodiments, the dose of 0.05% difluprednate is one drop of eye drop. In some embodiments, one drop is about 50 μl (e.g., about 25 μl to about 50 μl, about 50 μl to about 100 μl). In some embodiments, the dose of difluprednate comprises about 1 μg to about 5 μg, or about 2 μg to about 3 μg, or about 2.5 μg of difluprednate. In some embodiments, the dose of difluprednate comprises about 2.5 μg of difluprednate.

[0091] In some embodiments, the steroid treatment is an ophthalmic steroid treatment (e.g., difluprednate). In some embodiments, the ophthalmic steroid treatment (e.g., difluprednate) is a daily topical steroid treatment for up to about 4 weeks, about 6 weeks, or about 8 weeks after administration of a unit dose of rAAV particles. In some embodiments, the topical steroid treatment comprises about 4 administrations of topical steroid at about week 1, about 3 administrations of topical steroid at about week 2, about 2 administrations of topical steroid at about week 3, and about 1 administration of topical steroid at about week 4, timing beginning with administration of a unit dose of rAAV particles and occurring after administration of a unit dose of rAAV particles. In some embodiments, the topical steroid treatment comprises, after administration of a unit dose of rAAV particles, about 4 topical steroid doses per day for about 3 weeks (i.e., QID), followed by about 3 topical steroid doses per day for about 1 week (i.e., TID), followed by about 2 topical steroid doses per day for about 1 week (i.e., BID), followed by about 1 topical steroid dose per day for about 1 week (i.e., QD). In some embodiments, the topical steroid comprises 0.05% difluprednate at a dose of about 1 μg to about 3 μg. In some embodiments, the topical steroid comprises 0.05% difluprednate at a dose of about 2.5 μg. In some embodiments, the topical steroid is about 0.005% to about 0.5% difluprednate. In some embodiments, the topical steroid is about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, or about 0.1% difluprednate. In some embodiments, the topical steroid is 0.05% difluprednate. In some embodiments, the dose of 0.05% difluprednate is one drop of eye drop. In some embodiments, one drop is about 50 μl (e.g., about 25 μl to about 50 μl, about 50 μl to about 100 μl). In some embodiments, the dose of difluprednate comprises from about 1 μg to about 5 μg, or from about 2 μg to about 3 μg, or about 2.5 μg of difluprednate.In some embodiments, the dose of difluprednate comprises about 2.5 μg of difluprednate.

[0092] It will be understood that reference to the examples below is for illustrative purposes only and is not intended to limit the scope of the claims. EXAMPLES

[0093] Example 1. AAV production in HEK-derived cells The cells were diluted to approximately 4.5–6.0 × 10 6 Grow to a density of 10000 viable cells / mL. Ensure viability is ≥95% before proceeding with transfection. Dilute cells in medium supplemented with GlutaMax™ supplement (ThermoFisher). Immediately after dilution, add AAV-Max or PEI-Pro to the diluted cell solution. Gently swirl the flask to mix the cells. Incubate the cells in a 37°C incubator with a humidified atmosphere of 8% CO2 on an orbital until the DNA / transfection complexing process is complete. Prepare transfer plasmid, Rep and Cap plasmid (or plasmids), and helper plasmid DNA. The molar ratio of plasmid DNA is optimized based on plasmid size, using approximately 1.5 μg / mL of total plasmid DNA for the culture volume to be transfected.

[0094] Transfection complexes are prepared as described later in this specification. Transfection reagent is gently mixed. Total plasmid DNA is diluted in complexation buffer. The diluted DNA is incubated at room temperature (RT) for 10 minutes. Transfection booster and transfection reagent are gently mixed and incubated together at RT for 10 minutes. Premixed booster and reagent are gently mixed with the diluted plasmid DNA and incubated at room temperature for 20-25 minutes. The combination of plasmid, booster, and reagent is gently added to a cell shaker. Cells are incubated for approximately 72 hours in a 37°C incubator with a humidified atmosphere of 8% CO2 on an orbital shaker. A schematic overview of the general process in the two cell types is provided in Figure 5.

[0095] Example 2. AAV production in VPC2.0 cells On the day of transfection (day 0), the viable cell density and percent survival of Gibco VPC2.0 cells (ThermoFisher catalog number A49784) were determined. 6 Cells with viability ≥ 95% in viable cells / mL were proceeded with transfection. Cells were cultured at 3 x 10 6 The cells were diluted to a final density of 1000 viable cells / mL. Immediately after dilution, AAV-MAX was added. The flask was gently swirled to mix the cells, and the cells were placed in a 37°C incubator with a humidified atmosphere of 8% CO2 on an orbital until the DNA / transfection complexing process was complete.

[0096] For the 3-plasmid system, the transfer plasmid DNA, Rep / Cap plasmid DNA, and helper plasmid DNA were mixed using molar ratio optimization based on plasmid size. Approximately 1.5 μg / mL of total plasmid DNA was prepared for the culture volume to be transfected. Transfection complexes were prepared as described. The AAV-MAX transfection reagent bottle was gently inverted to mix. Total plasmid DNA was diluted with Viral-Plex™ complexing buffer to a final volume of 10% of the culture volume to be transfected. The plasmid DNA and complexing buffer were gently mixed and incubated at room temperature for 10 minutes.

[0097] Undiluted AAV-MAX transfection booster was added to the new tube at 3 μL per mL of culture to be transfected, followed by AAV-MAX transfection reagent at 6 μL per mL of culture to be transfected. The booster and reagent were mixed gently and incubated at RT for 10 min. The mixed AAV-MAX transfection booster and AAV-MAX transfection reagent were added to the diluted plasmid DNA and mixed gently. The plasmid DNA / AAV-MAX booster / AAV-MAX reagent complex was incubated at room temperature for 20-25 min and then gently transferred to the cell solution in the shaker flask. The cells were incubated for approximately 72 h in a 37 °C incubator with a humidified atmosphere of 8% CO2 on an orbital shaker.

[0098] Example 3 Collection AAV particles were harvested 70-72 hours after transfection. AAV MAX lysis buffer was added directly to the culture flask at a 1:10 dilution. The flask was swirled to evenly distribute the lysis buffer. 1M MgCl2 was added to a final concentration of 2-4mM in the culture medium. 90U / mL benzonase was added to the flask. The flask was incubated at 37°C for at least 2 hours on an orbital shaker. The cells were centrifuged at 4200RPM at 4°C for 30 minutes and the supernatant was transferred to an appropriately sized container and stored at -80°C.

[0099] Example 4. Harvesting by freeze / thaw or sonication Cells were counted to determine VCD, TCD, and % viability. Cells were collected in an appropriate size container and centrifuged at 3500RPM, 4°C for 10 minutes. The supernatant was discarded. Cell pellets were resuspended (approximately 1mL / 2.8E+7 cells) in lysis buffer (0.15M NaCl, 50mM Tris-Cl, 0.05% Tween®-20; pH 8). Cells were subjected to 3 rounds of freeze-thaw cycles or sonication (approximately 100ml batches). MgCL2 was added to 2mM. Lysates were treated with benzonase (50ul / ml) for 1 hour. Cells were centrifuged at 4200RPM, 4°C for 30 minutes. Supernatants were transferred to an appropriate size container and stored at 4°C or -80°C.

[0100] If sonication was used, resuspended cells were chilled on ice before and after sonication using a Branson 250 Digital Sonifier with a flat tip, 50% amplitude, 1-second pulses on and off for 40 seconds.

[0101] Example 5. Evaluation of transfection reagents The pAAV ITR expression vector, pAAV Rep-Cap gene, and pHelper were co-transfected into either Oxford or VPC2.0 cells using either PEIpro® or TRX (AAV-MAX Transfection Reagent) transfection reagent at a molar ratio of 1:2:0.5, respectively, normalized for plasmid size. The results of one such series of experiments are shown in Figure 1.

[0102] Example 6. Titer Determination Titers were determined using ddPCR. The digested and harvested rAAV was diluted 1 / 5000 in dilution buffer containing 0.05% Pluronic® F-68 (Gibco, Invitrogen, Grand Island, NY). Using the ddPCR™ Supermix for Residual DNA Detection Kit (BIO-RAD, Hercules, CA), reaction mixtures were assembled as described in the manufacturer's protocol in a final volume of 25 μL with 0.9 μM primers and 0.1 μM probe. ddPCR was performed as described in the ddPCR Amplification Guide (Bio-Rad 6407). Test samples were then emulsified in QX-100 Droplet Generator Oil using a QX-100 Droplet Generator (BIO-RAD) according to the manufacturer's instructions. PCR amplification of droplets was performed using a conventional thermal cycler with the following parameters: 95°C for 10 min, followed by 39 cycles of 94°C for 30 s and 60°C for 30 s, followed by a final heat treatment at 98°C for 10 min. The PCR plates were then scanned using a QX200 droplet reader (BIO-RAD) and the data were analyzed by QuantaSoft software (BIO-RAD). For 2D ddPCR, two sets of primers and probes, labeled with 5(6)-carboxyfluorescein (FAM) and hexachlorofluorescein (HEX), respectively, were added to each reaction mixture, and ddPCR was performed in the same tube.

[0103] Example 7. Evaluation of dissolution conditions Various lysis conditions were evaluated. rAAV2.7m8-CMV-Fluc capsids were produced in Oxford cells using PEIpro® transfection reagent. Oxford cells containing rAAV were lysed with different lysis conditions: freeze-thaw (FT), sonication (SC), thermochemical method (Thermo), and alternative chemistry (Alt). The buffer for the alternative chemistry is 10 mM EDTA, 20 mM TRIS-Base, pH 9.2, Benzonase 10 u / ml, MgSO4·7H2O 40 mM, and 0.4 M NaCl. The results of one such series of experiments are summarized in Figure 2.

[0104] Example 8. Plasmid molar ratio optimization rAAV2.7m8-Dry AMD was produced in VPC2.0 cells by cotransfection of pAAV ITR expression vector, pAAV Rep-Cap gene, and pHelper at different molar ratios normalized to plasmid size, respectively. The results of one such series of experiments are summarized in Figure 3.

[0105] Example 9. Effects of transgenes rAAVs were generated with the same transgene inserted into the same backbone with two different signal peptides (886 and 1176) and the titers of the harvested rAAVs were determined. p886 has an IgK signal sequence (MDMRVPAQLLGLLLLWLRGARC, SEQ ID NO: 1), while p1176 has a CD33 signal sequence (MPLLLLLPLLWAGALA, SEQ ID NO: 2). rAAV2.7m8-Dry AMD (886 and 1176) were produced in Oxford and VPC2.0 cells using two different transfection reagents (PEIpro® and TRX). The results of such a series of experiments are summarized in Figure 4.

[0106] Example 10. Comparison of four and three plasmid systems rAAV2.7m8-CMV-GFP was produced in VPC2.0 cells using two different transfection systems. The first condition was a conventional three-plasmid system with co-transfection of pAAV ITR expression vector, pAAV Rep-Cap gene, and pHelper. The second condition was a four-plasmid system. The pAAV Rep-Cap gene was split into two separate plasmids (pAAV-Rep gene plasmid and pAAV-CMV-Cap gene plasmid), in which the CMV promoter was used for the Cap gene. The pAAV ITR expression vector, pAAV Rep gene, pAAV-CMV-Cap gene, and pHelper were co-transfected into VPC2.0 cells as described elsewhere herein. The transfected cells were incubated. The rAAV2.7m8-CMV-GFP particles were harvested and titers were determined by ddPCR. The results of one such series of experiments are summarized in Figure 6.

[0107] Surprisingly, the four-plasmid system approach significantly increased rAAV titers: in at least one series of experiments, rAAV2.7m8-CMV-GFP titers increased more than five-fold when the four-plasmid system was used.

Claims

1. A method for producing high-titer recombinant adeno-associated virus (rAAV), (a) A step of providing HEK293-derived cells, (b) A step of cotransfecting HEK293-derived cells with a plasmid comprising an inverted terminal repeat (ITR) expression vector, an AAV helper construct, a nucleotide sequence encoding an AAV Rep polypeptide, and a nucleotide sequence encoding an AAV Cap polypeptide. (c) the step of incubating the transfected cells, and (d) Steps to collect the rAAV, Methods that include...

2. The method according to claim 1, wherein the rAAV is an rAAV with serotype AAV2.7m8.

3. The method according to claim 1 or 2, wherein the nucleotide sequence encoding the AAV Rep polypeptide and the nucleotide sequence encoding the AAV Cap polypeptide are located on separate plasmids.

4. The method according to claim 1 or 2, wherein the nucleotide sequence encoding the AAV Rep polypeptide and the nucleotide sequence encoding the AAV Cap polypeptide are located on the same plasmid.

5. The method according to claim 1 or 2, wherein the ITR expression vector comprises one or more of the following nucleic acid elements: a first ITR sequence; a promoter sequence; an intron sequence; a first UTR sequence; a target gene encoding a gene product; a second UTR sequence; a poly(A) sequence; and a second ITR sequence.

6. The method according to claim 1 or 2, wherein, when normalized to plasmid size, the molar ratio of Cap or Rep plasmid to helper construct or ITR expression vector is in the range of 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, or higher.

7. (i) A transfection reagent is used when co-transfecting the HEK293-derived cells, and the transfection reagent is selected from the group including AAV-MAX and PEI-Pro; and / or (ii) The method according to claim 1 or 2, wherein the HEK293-derived cells are selected from the group including VPC2.0 cells and Oxford cells.

8. The method according to claim 7, wherein the HEK293-derived cells are VPC2.0 cells and the transfection reagent is AAV-Max.

9. The nucleotide sequence encoding the AAV Cap polypeptide is (i) Constitutively active promoter; or (ii) p40 promoter The method according to claim 1 or 2, operably connected to a promoter including the following:

10. The method according to claim 9, wherein the constitutively active promoter is a CMV promoter.

11. The method according to claim 1 or 2, wherein the nucleotide sequence encoding the AAV Rep polypeptide is operably linked to a promoter comprising an AAV promoter.

12. The method according to claim 11, wherein the AAV promoter is a p5 or p19 promoter.

13. The titer of the rAAV collected is at least 5 × 10 １０ The method according to claim 1 or 2, wherein the concentration is vg / ml.

14. The titer of the rAAV collected is at least 1 × 10 １１ The method according to claim 13, wherein the concentration is vg / ml.

15. The titer of the rAAV collected is at least 3 × 10 １１ The method according to claim 13, wherein the concentration is vg / ml.

16. A four-plasmid system for producing high-titer recombinant adeno-associated virus (rAAV) in HEK293-derived cells, comprising: a first plasmid containing a nucleotide sequence encoding an AAV Rep polypeptide operably ligated to a first promoter; a second plasmid containing a nucleotide sequence encoding an AAV Cap polypeptide operably ligated to a second promoter; a third plasmid containing an ITR expression vector containing the gene of interest; and a fourth plasmid containing an AAV helper construct.

17. The method according to claim 16, wherein the rAAV is an rAAV with serotype AAV2.7m8.

18. The aforementioned second promoter, (i) Constitutively active promoter; or (ii) p40 promoter The system according to claim 16 or 17.

19. The method according to claim 18, wherein the constitutively active promoter is a CMV promoter.

20. The system according to claim 16 or 17, wherein the first promoter is an AAV promoter.

21. The system according to claim 20, wherein the AAV promoter is a p19 promoter.

22. The system according to claim 16 or 17, wherein the HEK293-derived cells are selected from the group comprising HEK293 cells, VPC2.0 cells, and Oxford cells.

23. The molar ratio of the first plasmid to the third plasmid and the molar ratio of the second plasmid to the third plasmid are similar when normalized with respect to plasmid size, and / or The molar ratio of the first plasmid to the fourth plasmid and the molar ratio of the second plasmid to the fourth plasmid are approximately the same when normalized with respect to plasmid size. The system according to claim 16 or 17.

24. The system according to claim 23, characterized in that the four plasmids of the set of plasmids are used in such a way that they are cotransfected in a molar ratio of approximately 1:1:1:1 when normalized to plasmid size.

25. The system according to claim 16 or 17, wherein, when normalized with respect to plasmid size, the molar ratio of the first or second plasmid to the third or fourth plasmid is greater than 1:

1.

26. The titer of the rAAV is at least 5 × 10 １０ The system according to claim 16 or 17, wherein the concentration is vg / ml.

27. The titer of the rAAV is at least 1 × 10 １１ The system according to claim 26, wherein the concentration is vg / ml.

28. The titer of the rAAV is at least 3 × 10 １１ The system according to claim 26, wherein the concentration is vg / ml.

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