WO2026020116A1 - Methods and compositions for enriching the production of dna cargo loaded recombinant adeno-associated virus capsids - Google Patents

Abstract

Improved methods for producing recombinant adeno-associated viral capsids (rAAVs) that results in an increase in the percent of filled AAVs are disclosed, and compositions of cells that contain a higher percentage of filled rAAVs. The method uses at least four constructions to provide gene required for rAAV packaging, and transformation of rAAV producing host cells is performed in two temporally separated stages. The filled rAAVs produced by the disclosed methods contain both the viral DNA and the desired transgene. The disclosed methods also minimize the percentage of empty AAV capsids. The four-vector transfection rAAV production method includes a two stage transfection of host cells, temporally separated by at least 7 hrs. Stage 1 involves transfecting host cells with: (1) a Helper construct (2) a Rep construct; and (3) a third construct containing the gene of interest. Stage 2 involves transfecting the cells from Stage 1 with: (4) a fourth construct, containing Rep and Cap genes.

Description

METHODS AND COMPOSITIONS FOR ENRICHING THE PRODUCTION OF DNA CARGO LOADED RECOMBINANT ADENO-ASSOCIATED VIRUS CAPSIDS CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of, and priority to U.S. Serial No. 63/673,015, filed July 18, 2024, the contents of which is hereby incorporated by references in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant Number FD006584 awarded by The Food and Drug Administration, Grant Number 75F40121C00131 awarded by The Food and Drug Administration, Grant Number FD007226 awarded by The Food and Drug Administration, Grant Number FD007458 awarded by The Food and Drug Administration and Grant number 75F40124C00130 EOM, awarded by The Food and Drug Administration. The government has certain rights in the invention.

REFERENCE TO SEQUENCE LISTING PARAGRAPH

The Sequence Listing XML submitted as a file named “MIT_25585_PCT_ST26.xml,” created on July 18, 2025, and having a size of 49,131 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.834(c)(1).

FIELD OF THE INVENTION

The disclosed invention is generally in the field of viral vector production and specifically in the area of recombinant adeno-associated viruses.

BACKGROUND OF THE INVENTION

Adeno-associated viruses (AAVs) are widely used vectors for gene therapy due to their ability to deliver genetic material to a variety of cell types with minimal immunogenicity. AAV is a commonly preferred vehicle for transferring therapeutic genes in patients with genetic disorders. Typically, ~10¹⁵ intact AAV particles carrying DNA cargo of therapeutic gene of interest are required for a single dose. These particles are produced using human or insect cell fines along with defective particles.

One significant challenge in the production of recombinant AAVs (rAAVs) is the low yield of virus particles that are filled with the desired genetic material. High proportions of empty or partially filled capsids can reduce the efficacy of gene therapy and complicate downstream purification processes. Current AAV cell culture production processes typically yield < 30% of the particles produced per cell intact. Separating intact AAV particles from defective empty particles is an expensive and time-consuming process. Several rounds of standard and non-standard purification steps are the only means to achieve > 99% product purity. This often leads to significant loss of the valuable product which increases the overall cost of the drug. Further, increased production of intact functional AAVs in cells is likely to go awry due to the barriers in the cellular environment to produce viral components such as the temporal mismatch between rAAV capsid production and viral DNA replication. Therefore, there is a need for improved methods of minimizing the percentage of empty or partially filled AAV capsids, thereby producing cells containing a higher percentage of filled rAAVs, i.e., AAV capsids containing viral DNA and the desired genetic material.

It is an object of the invention to provide methods of making rAAVs with a higher percentage of capsids filled with the desired recombinant viral RNA.

It is also an object of the invention to provide transformed cells containing a higher percentage of filled rAAVs.

BRIEF SUMMARY OF THE INVENTION

Disclosed are improved methods for producing a higher percentage of filled recombinant adeno-associated viral capsids (rAAVs) and compositions of cells that contain a higher percentage of filled rAAVs. The method involves a transfection scheme which uses four constructs which provide the genes required for rAAV packaging, and these constructs are introduced into the rAAV producing host cells in two temporally separated stages. This transformation strategy results in an improvement in the % of filled AAV capsids (i.e., containing both the viral DNA and the desired transgene).

The four- vector transfection rAAV production method includes the following steps:

(A) Stage 1 Transfection: transiently transfecting host cells with three constructs: (i) a first construct containing necessary genes of helper virus required for AAV production such as adenoviral E2, E40rf6, and VA genes (H) (Helper construct); (ii) a second construct containing nucleic acid encoding rep proteins and which expresses Rep isoforms only (R) (Rep construct); and (iii) a third construct a heterologous gene of interest (GOI (gene of interest) (G) construct) i.e., H, R, G;

(B) Stage 2 Transfection: transiently transfecting the cells from Stage 1 with: (4) a fourth construct, containing Rep and Cap genes (RC), which provides the necessary genes for AAV replication and capsid formation (RepCap construct).

Thus after the Stage I and Stage II transfections, the host cells contain (H, R, G), and (RC) In some forms, Stage 2transfection includes transfecting cells with RepCap and Helper constructs after Stage 1 transfection. Thus, in these forms, Thus after the Stage I and Stage II transfections, the host cells contain (H, R, G), and (RC, H)

(C) maintaining the transformed host cells from the combination of step (A) and (B) in cell culture for an effective amount of time for rAAV packaging; and

(D) optionally, purifying rAAV from the host cells.

This two-stage transfection process facilitates coordinated kinetics between viral DNA replication and capsid protein expression, resulting in a higher percentage of filled rAAV capsids. The constructs are provided using suitable vectors, such as a plasmid, viral vector, etc.

In some forms, the time interval between Stage 1 Transfection, and Stage 2 Transfection is at least 7 hrs., and up to about 24 hours, preferably between about 18 to about 24 hrs.

In some forms the second construct contains the rep gene sequence under the control of endogenous AAV promoters, for example, p5 and p!9. In preferred embodiments, the second construct does not employ an inducible promoter such as Tet-on.

In some forms, inducible promoters can be used to control the kinetics of Cap gene expression.

In some forms, the fourth construct contains the cap gene under the control of endogenous AAV promoters, e.g., p40 and p81.

The disclosed methods are generally applicable to a wide range of AAV serotypes and can be used in various host cells, including human embryonic kidney cells (HEK 293) and insect cells. The improved methods are suitable for producing rAAVs for a variety of therapeutic applications, e.g., gene delivery.

Also disclosed are transformed cells produced by the disclosed four-vector transfection method.

In some forms the transformed cells include (i) a first construct containing necessary genes of helper virus required for AAV production such as adenoviral E2, E40rf6, and VA genes (Helper construct); (ii) a second construct containing an AAV rep coding region which expresses Rep isoforms only (Rep construct); and (iii) a third construct containing AAV inverted terminal repeats (ITR) flanking a heterologous gene of interest (GOI (gene of interest) construct). Following the second stage of transformation, the transformed cells additionally include, (iv) a fourth construct, containing Rep and Cap genes. In some forms, the host cell is a human cell, such as human embryonic kidney cells (HEK 293). In some forms, the host cell is an insect cell. Also disclosed are transformed cells contain a high percentage of filled recombinant adeno-associated viruses (rAAVs) with both viral DNA and the desired transgene, produced according to the methods disclosed herein. Cells transformed according to the improved methods herein accumulate up to 90 % and in some forms, up to 100% filled rAAVs, following transfection and cell culture.

Additional advantages of the disclosed method and compositions will be set forth in part in the description which follows, and in part will be understood from the description, or can be learned by practice of the disclosed method and compositions. The advantages of the disclosed method and compositions will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings illustrate several embodiments of the disclosed method and compositions and together with the description serve to explain the principles of the disclosed method and compositions.

FIG. 1 is a schematic of the process for transient transfection for triple plasmid approach. G, RC and H refer to pAAV-GFP, pAAV-RC5 and pHelper plasmids, respectively. A cationic transfection reagent, polyethyleneimine (PEI) was used to prepare the transfection mix. I pg of plasmids per million cells at 1:2 DNA:PEI ratio was added to 30ml suspension HEK293 cell culture at ~1.5 million cells/mL. The culture was proceeded until 72 hours (h) post transfection. At 48h and 72h time points -1 million cells were drawn to estimate the capsid and Vector genome (vg) titers. Remaining cells at 72h post transfection were used for analytical characterization experiments.

FIG. 2 is a schematic of the process for transient transfection for four-plasmid approach. G, R, RC and H refer to pAAV-GFP, pRep, pAAV-RC5 and pHelper plasmids respectively. A cationic transfection reagent, polyethyleneimine (PEI), was used to prepare the transfection mix. 1 fig of plasmids per million cells at 1:2 DNA:PEI mass ratio was added to 30 ml suspension HEK293 cell culture at -1.5 million cells/mL. The cell culture proceeded until 96h post first transfection. At 72h and 96h time points -1 million cells were drawn to estimate the capsid and vg titers. Remaining cells at 96h post transfection were for analytical characterization experiments. FIGs. 3A and 3B are bar graphs showing the comparison of the capsid and vg titers of samples from four-plasmid and triple plasmid transfection schemes. Capsid and vg titers of the samples drawn at indicated time points were estimated using AAV5 Capsid ELISA and ddPCR assays, respectively. Error bars denote standard error of mean of n=3 replicates. FIG. 3A shows that both capsid and vg titer values are almost the same for four-plasmid transfection. Out of a total of -6.0x10⁹ capsids/mL of lysate, ~5.8xl0⁹ capsids/mL (-97%) were filled with vDNA at 72h post second transfection. FIG 3B shows that the vg titer for triple-plasmid transfection is orders of magnitude lesser than the capsid titer. Out of a total of -3.2x10” capsids/mL of lysate, only ~1.7xlO¹⁰ capsids/mL (-5.3%) were filled with vDNA at 72h post transfection.

FIG. 4 shows UV absorbance spectra for biophysical characterization of filled and empty capsid samples. UV absorbance spectra of purified filled and empty capsids purchased from Virovek were obtained using a Nanodrop spectrophotometer. Absorbance ratio, AR = Abs260/Abs280 values can be direct readouts to assess the quality of AAV samples. Purified samples enriched with filled and empty capsids have AR- 1.3 and -0.62 respectively.

FIG. 5 shows biophysical characterization of AAVs produced via four-plasmid transient transfection. (Top) Size exclusion chromatography (SEC) separation of molecules indicated by UV absorbance (260nm and 280nm) traces within a chosen retention time interval. Sample eluant that corresponds to the major peak at 10.3 min has AR -1.3 suggesting approximately -100% enrichment with filled capsids. (Bottom) Dynamically synchronized Multi-angle light scattering (MALS) measurement indicates scattering intensity of single particle and aggregated AAVs were higher than that of other molecules in the sample. Scattering intensity depends both on number and size of the molecules and is very sensitive to the size (scales sixth power of a size). Hence, the number of aggregated AAV particles in the sample is less compared to single particle AAVs.

FIG. 6 shows the absorbance ratio as a function of percentage of filled capsids. Theoretically evaluated absorbance ratio, AR using the extinction coefficients of filled and empty capsids published in the literature (J. M. Sommer et al., Molecular Therapy, 7, 122-128, 2003) as a function of fill fraction. For a sample containing a binary mixture of filled and empty capsids, the absorbance ratio follows a non-linear trend as the percentage of filled capsids increases from 0 to 100%.

FIGs. 7A and 7B show the effect of the timing of the second dose on the percentage of filled capsids. FIG. 7A shows the percentage of filled capsids produced increase as the timing of the second dose is delayed and it reaches >90% after 24h post first transfection. FIG. 7B shows the increase in the percentage of filled capsids produced via four-plasmid transient transfection was due to the reduction in the population of empty capsids. The population of empty capsids produced was very high when all four plasmids were delivered at the same time. However, when pAAV-RC5 was delivered later by a few hours, the population of empty capsids dropped drastically. In contrast, the population of filled capsids produced was comparable to that produced with Oh time interval.

FIG.S 8A and 8B are negatively stained transmission electron micrographs of capsid population. FIG. 8A shows standard samples are a heterogenous mixture of empty (dark contrast in the middle), filled (white contrast in the middle) and partially filled (dark and white contrast shades) capsids. FIG. 8B is a representative image of a filled capsid population from a sample purified after four-plasmid transient transfection. A total of 5 different test samples were analyzed. At least 12 different fields of view were imaged for each sample. Greater than 95% of the capsids in the samples were filled with vDNA from all the fields of view analyzed.

FIG. 9A-9C are dot plots showing media replacement after low-density transfection did not increase the population of filled capsids. Four-plasmid transient transfection experiments were performed dosing the cells to the transfection mix briefly for 6hrs, followed by replacement with the fresh media. FIG. 9A shows that filled capsids percentage reaches -100% at 48h and 72h but eventually drops faster due to removal of the transfection mix. FIG. 9B shows that vector genome titer is comparable to that achieved without media replacement. FIG. 9C shows cell density (filled dot) and percentage viability (empty dot) of HEK293 suspension cell culture. The first dose was delivered at Oh and the second dose at 24h.

FIG. 10A is a map of pAAV-GFP purchased from Cell Biolabs (Catalog # VPK-405). FIG. 10B is a map of pAAV-RC5 purchased from Cell Biolabs (Catalog # VPK-405). FIG. 10C is a map of pHelper purchased from Cell Biolabs (Catalog # VPK-405). FIG. 10D is a map of an exemplary engineered pRep plasmid.

FIG. 11 shows AAV helper-free system reproduced from Cell Biolabs product manual for Cat number VPK-405.

FIG. 12. Scheme 5: pMock plasmid created using pAAV-GFP as a template via Gibson assembly reaction.

FIG. 13 is a bar graph showing the effect of pHelper plasmid on capsid production in single and sequential transient transfection. G, R, H, RC and M refers to pAAV-GFP, pRep, pHelper, pAAV-RC5 and pMock plasmids respectively. ‘+’ and symbols refer to inclusion and exclusion of a particular plasmid, respectively. Data shown are the mean and standard deviation of three capsid ELISA estimates for a single dose (in green background) and sequential dose (in blue background) transient transfections.

FIG. 14. Baculovirus mediated AAV production using insect sf9 cells. The two-Bac platform includes recombinant baculovirus Rep and Cap genes under the control of very late promoters, polyhedrin and plO, respectively. The second type of recombinant baculovirus encodes the transgene (Gene of Interest) flanked by the inverted terminal repeats.

FIG. 15. Schematics of the modified BEVS, comprising three baculoviruses that express Rep, Cap, and GOI genes individually. The recombinant baculovirus that expresses Rep contains two Rep cassettes under the control of immediate early and late promoters. The immediate early promoters can either be Hr5-IE1 or OPIE2 promoters. The recombinant Cap baculovirus under the control of the PIO promoter can either be introduced along with the Rep and GOI baculovirus at t=0 or introduced at t=4 hours after the inoculation of Rep and Cap baculovirus.

FIG. 16. Baculovirus dosing schemes to achieve high percentage of filled capsids via temporal modulation of Rep genes under the control of different promoters. Six conditions were performed to improve the percentage of filled capsids. All groups, except group 5, received a single inoculation of the baculovirus. Group 5 received the second dose 4 hours after the initial infection. Baculovirus titer quantified from ddPCR was used to infect sf9 cells. High: 100- 300 rBV/cell, Medium: 30-60 rBV/cell, Low: 2-10 rBV/cell. rAAVs packaged in Sf9 cells were quantified using mass photometry

Figure 17A-17G: Mass histograms for conditions 1-6 and commercial empty AAV5 capsids. The light and dark blue rectangles gate the empty and filled regions. FIG. 17A) condition 1 FIG. 17B) condition2 FIG. 17C) conditions FIG. 17D) condition4 FIG. 17E) conditions FIG. 17F) condition6 and FIG. 17G) Empty capsids.

DETAILED DESCRIPTION OF THE INVENTION

The disclosed methods and compositions are based at least on the identification of an improved cell transformation scheme and culture approach for producing intact or filled recombinant adeno-associated viruses (rAAVs) in cells i.e., higher quantities of rAAV capsids containing the recombinant viral DNA. The improved cell culture approach includes a two-stage transfection strategy that employs transforming cells with four plasmids. Studies were conducted to compare the effects of the conventional triple plasmid transfection approach and the improved four- vector transfection strategy on the yield of intact rAAVs in cells. The studies aimed to eliminate temporal mismatch between the synthesis of viral genomes and the assembly of capsid proteins, which can lead to a high proportion of empty or partially filled capsids (which are suboptimal for therapeutic applications). The disclosed transfection method addresses this issue by coordinating the timing of gene expression of functional genes needed for viral replication and structural genes necessary for viral assembly. Data from the Examples demonstrate that this four- vector transfection method (exemplified therein using plasmids as the vector for transfection) is capable of producing 97 % filled rAAV capsids compared to methods that do not use the four plasmid transfection method disclosed herein.

The non-limiting Examples demonstrate that transfecting cells in a first stage with three plasmids for a period of time e.g., 24 hours, and then transfecting the cells with a fourth plasmid for a period of time e.g., 96 hours, results in a drastically higher percentage of filled rAAVs compared to conventional triple-plasmid transfection approaches. The data shows that the four- vector transfection strategy can be used as a scalable and inexpensive method for producing a higher percentage, e.g., more than 55%, of filled rAAVs, while reducing the percentage of empty AAV capsids in transformed cells.

I. DEFINITIONS

The singular forms “a” and “an” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

The term “about,” as used herein when referring to a measurable value such as an amount of the length of a polynucleotide or polypeptide sequence, dose, time, temperature, and the like, is meant to encompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount.

As used herein, the term “construct” refers to a recombinant genetic molecule having one or more isolated polynucleotide sequences. Genetic constructs used for transgene expression in a host organism, also referred to “expression constructs”, include in the 5 ’-3’ direction, a promoter sequence; a sequence encoding a gene of interest; and a termination sequence. The construct may also include selectable marker gene(s) and other regulatory elements for expression.

As used herein, the term “operably linked to” refers to the functional relationship of a nucleic acid with another nucleic acid sequence. Promoters, enhancers, transcriptional and translational stop sites, and other signal sequences are examples of nucleic acid sequences operably linked to other sequences. For example, operable linkage of gene to a transcriptional control element refers to the physical and functional relationship between the gene and promoter such that the transcription of the gene is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to and transcribes the DNA.

As used herein, term “expression control sequence” refers to a DNA sequence that controls and regulates the transcription and/or translation of another DNA sequence. Control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, a ribosome binding site, and the like. Eukaryotic cells are known to utilize promoters, poly adenylation signals, and enhancers.

As used herein, the term “promoter” refers to a regulatory nucleic acid sequence, typically located upstream (5’) of a gene or protein coding sequence that, in conjunction with various elements, is responsible for regulating the expression of the gene or protein coding sequence. These include constitutive promoters, inducible promoters, tissue- and cell-specific promoters, and developmentally regulated promoters.

As used herein, the terms “transformed,” “transgenic,” “transfected” and “recombinant” refer to a host organism into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome of the host or the nucleic acid molecule can also be present as an extrachromosomal molecule. Such an extrachromosomal molecule can be auto-replicating. Transformed cells, tissues, or plants are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof. A “non-transformed,” “non-transgenic,” or “non-recombinant” host refers to a wildtype organism, e.g., a bacterium or plant, which does not contain the heterologous nucleic acid molecule.

As used herein, the term “adeno-associated virus” (AAV), includes but is not limited to, AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, AAV type 12, AAV type 13, snake AAV, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, goat AAV, shrimp AAV, engineered serotypes (such as AAV-DJ, AAV-DJ/8) and any other AAV now known or later discovered. See, e.g., FIELDS et al. VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers). Other AAV serotypes and clades have been identified (See, e.g., Gao et al., J. Virol. 78:6381 (2004); Moris et al., Virol. 33:375 (2004)).

As used herein, the term “endogenous” with regard to a nucleic acid refers to nucleic acids normally present in the host.

As used here, the term “heterologous” refers to elements occurring where they are not normally found. For example, a promoter may be linked to a heterologous nucleic acid sequence, e.g., a sequence that is not normally found operably linked to the promoter. When used herein to describe a promoter element, heterologous means a promoter element that differs from that normally found in the native promoter, either in sequence, species, or number. For example, a heterologous control element in a promoter sequence may be a control/ regulatory element of a different promoter added to enhance promoter control, or an additional control element of the same promoter. The term “heterologous” thus can also encompass “exogenous” and “non-native” elements. The terms “heterologous nucleotide sequence” and “heterologous nucleic acid” are used interchangeably herein and refer to a sequence that is not naturally occurring in the virus. Generally, the heterologous nucleic acid comprises an open reading frame that encodes a polypeptide or non-translated RNA of interest (e.g., for delivery to a cell or subject).

The term “tropism” as used herein refers to entry of the virus into the cell, optionally and preferably followed by expression (e.g., transcription and, optionally, translation) of sequences carried by the viral genome in the cell, e.g., for a recombinant virus, expression of the heterologous nucleotide sequences(s). In the case of AAV, gene expression from the viral genome may be from a stably integrated provirus, from a non-integrated episome, as well as any other form in which the virus may take within the cell.

The terms “5' portion” and “3’ portion” are relative terms to define a spatial relationship between two or more elements. Thus, for example, a “3' portion” of a polynucleotide indicates a segment of the polynucleotide that is downstream of another segment. The term “3' portion” is not intended to indicate that the segment is necessarily at the 3' end of the polynucleotide, or even that it is necessarily in the 3' half of the polynucleotide, although it may be. Likewise, a “5' portion” of a polynucleotide indicates a segment of the polynucleotide that is upstream of another segment. The term “5' portion” is not intended to indicate that the segment is necessarily at the 5' end of the polynucleotide, or even that it is necessarily in the 5' half of the polynucleotide, although it may be.

As used herein, the term “polypeptide” encompasses both peptides and proteins, unless indicated otherwise.

A “polynucleotide” is a sequence of nucleotide bases, and may be RNA, DNA or DNA- RNA hybrid sequences (including both naturally occurring and non-naturally occurring nucleotide), and can be either single or double stranded DNA sequences.

As used herein, the terms “virus vector,” “vector” or “gene delivery vector” refer to a virus (e.g., AAV) particle that functions as a nucleic acid delivery vehicle, and which comprises the vector genome (e.g., viral DNA) packaged within a virion. Alternatively, in some contexts, the term “vector” may be used to refer to the vector genome/viral DNA alone.

The term “terminal repeat” or “TR” includes any viral terminal repeat or synthetic sequence that forms a hairpin structure and functions as an inverted terminal repeat (i.e., mediates the desired functions such as replication, virus packaging, integration and/or provirus rescue, and the like). The TTR can be an AAV TTR or a non-AAV TTR. For example, a non-AAV ITR sequence such as those of other parvoviruses (e.g., canine parvovirus, bovine parvovirus, mouse parvovirus, porcine parvovirus, human parvovirus B-19) or the SV40 hairpin that serves as the origin of SV40 replication can be used as an ITR, which can further be modified by truncation, substitution, deletion, insertion and/or addition. Further, the ITR can be partially or completely synthetic, such as the “double-D sequence” as described in U.S. Pat. No. 5,478,745 to Samulski et al.

As used herein, AAV “Rep coding sequences” indicate the nucleic acid sequences that encode the AAV non- structural proteins that mediate viral replication and the production of new virus particles.

The “Rep coding sequences” need not encode all of the AAV Rep proteins. For example, with respect to AAV, the Rep coding sequences do not need to encode all four AAV Rep proteins (Rep78, Rep68, Rep52 and Rep40), in fact, it is believed that AAV5 only expresses the spliced Rep68 and Rep40 proteins. In representative embodiments, the Rep coding sequences encode at least those replication proteins that are necessary for viral genome replication and packaging into new virions. The Rep coding sequences will generally encode at least one large Rep protein (i.e., Rep78/68) and one small Rep protein (i.e., Rep52/40). In particular embodiments, the Rep coding sequences encode the AAV Rep78 protein and the AAV Rep52 and/or Rep40 proteins. In other embodiments, the Rep coding sequences encode the Rep68 and the Rep52 and/or Rep40 proteins. In a still further embodiment, the Rep coding sequences encode the Rep68 and Rep52 proteins, Rep68 and Rep40 proteins, Rep78 and Rep52 proteins, or Rep78 and Rep40 proteins.

As used herein, the term “large Rep protein” refers to Rep68 and/or Rep78. Large Rep proteins may be either wild-type or synthetic. A wild-type large Rep protein may be from any AAV, including but not limited to serotypes 1, 2, 3a, 3b, 4, 5, 6, 7, 8, 9, 10, 11, or 13, or any other AAV now known or later discovered (see, e.g., Table 1). As used herein, the AAV “cap coding sequences” encode the structural proteins that form a functional AAV capsid (i.e., can package DNA and infect target cells). Typically, the cap coding sequences will encode all of the AAV capsid subunits, but less than all of the capsid subunits may be encoded as long as a functional capsid is produced. Typically, but not necessarily, the cap coding sequences will be present on a single nucleic acid molecule. The capsid structure of AAV is described in more detail in BERNARD N. FIELDS et al., Virology, volume 2, chapters 69 & 70 (4th ed., Lippincott-Raven Publishers) on, deletion, truncation and/or missense mutations.

As used herein “% filled rAAV” refers to % capsids produced that are filled with viral DNA cargo i.e., containing both the viral DNA elements and the desired transgene.

Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.

Use of the term “about” is intended to describe values either above or below the stated value in a range of approx. +/- 10%; in other embodiments the values may range in value either above or below the stated value in a range of approx. +/- 5%; in other embodiments the values may range in value either above or below the stated value in a range of approx. +/- 2%; in other embodiments the values may range in value either above or below the stated value in a range of approx. +/- 1%. The preceding ranges are intended to be made clear by context, and no further limitation is implied. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any nonclaimed element as essential to the practice of the invention.

Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed method and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a ligand is disclosed and discussed and a number of modifications that can be made to a number of molecules including the ligand are discussed, each and every combination and permutation of ligand and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited, each is individually and collectively contemplated. Thus, in this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Further, each of the materials, compositions, components, etc. contemplated and disclosed as above can also be specifically and independently included or excluded from any group, subgroup, list, set, etc. of such materials.

These concepts apply to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed.

Unless otherwise indicated, the disclosure encompasses conventional techniques of molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. Unless otherwise noted, technical terms are used according to conventional usage, and in the art, such as in the references cited herein, each of which is specifically incorporated by reference herein in its entirety.

II. IMPROVED METHODS OF PRODUCING FILLED rAAV CAPSIDS

Improved methods of producing a higher percentage of filled recombinant adeno- associated viral capsids (rAAVs) and cells containing a higher percentage of filled rAAVs are disclosed. The filled rAAVs produced by the disclosed methods contain the viral DNA and the desired transgene. Further, the disclosed methods also minimize the percentage of empty AAV capsids.

The disclosed methods in some forms require transfecting host cells with four plasmids (as opposed to three plasmids used in conventional cell culture approaches). The data in the Examples demonstrate that the disclosed four-vector transfection method is capable of producing 97% filled rAAV capsids, is effective in reducing the number of downstream purification steps and consequently, increasing the overall yield of filled rAAVs. A. The Four-Vector Transfection Method

The disclosed four- vector transfection method effectively coordinates the timing of gene expression of functional genes needed for viral replication and structural genes necessary for viral assembly, by temporally separating the introduction of necessary genes into a host cell and providing combinations of genes effective to achieve a higher proportion of filled rAAV capsids, significantly enhancing the overall yield and quality of the viral vectors. This improvement not only reduces the need for extensive downstream purification but also, increases the efficiency and efficacy of gene therapy applications.

As used herein, the term “vector” refers to a polynucleotide capable of transporting into a cell another polynucleotide to which the vector sequence has been linked. The term “expression vector” includes any vector, (e.g., a plasmid, cosmid, minimalistic, immunologically defined gene expression (MIDGE) vector, or phage chromosome) containing a gene construct in a form suitable for expression by a cell (e.g., linked to a transcriptional control element). “Plasmid” is a commonly used vector. MIDGE is described for example in Schakowski, et al., Molecular Therapy, Volume 3, Issue 5, 2001, Pages 793-800.

An understanding of AAV replication is important to appreciating the significance of the four-vector transfection method. AAV is a non-pathogenic DNA virus that can replicate only in the presence of helper viruses (such as Adenovirus, Herpes Simplex virus, etc.). AAV is a ~24 nm nucleocapsid with icosahedral structure shielding a single- stranded viral DNA (vDNA) cargo [1]. The wild-type AAV (wtAAV) genome has evolved as a compactly arranged genetic architecture that is self-contained and replication competent when assisted by helper viruses (e.g., Adenovirus) [1, 2]. The wtAAV genome contains several genes - rep (replicase), cap (capsid), AAP (assembly-activating protein) and MAAP (membrane-associated accessory protein) [3]. Of these, rep and cap sequences are arranged in tandem between two inverted terminal repeat (ITR) sequences that form a hairpin loop at the 3’ and 5’ end of the viral DNA (vDNA). cap codes for three viral proteins namely, VP1, VP2, VP3 that assemble into an icosahedral capsid structure at 1:1: 10 stoichiometric ratio. VP1 and VP2 can assemble into threefold and five-fold symmetric tunnel on the capsid surface formed by VP3 [4]. rep codes for four different Rep protein isoforms namely Rep78, Rep68, Rep52 and Rep40. Rep52 is encoded within the Rep78 sequence but at an alternate start site. Rep68 and Rep40 are the splice variants of Rep78 and Rep52 respectively. Rep78/68 assist in vDNA replication, targeting and packaging while Rep52/40 are required for packaging vDNA into pre-formed assembled capsids. The fivefold symmetry tunnel on the capsid surface facilitates vDNA insertion into preformed capsid using Rep78/Rep52 complex (King, et al., EMBO J., 20:3282-3291, 2001). AAP and MAAP sequences are +1 frame shifted with respect to the reading frame of cap (Maurer, et al., Human Gene Therapy, 31:499-507, 2020). AAP is involved in assembling viral proteins (Maurer, et al., Cell Reports, 23: 1817-18380, 2018) and MAAP limits AAV production (Ogden, el al., Science. 366, 1139-1143, 2019), and egress from host cells (Elmore, et al., Nature Communications, 12:6239, 2021). For gene therapy vector production, it is critical to render AAV particles replication-incompetent. Hence, the tandem rep and cap sequences between ITRs are usually replaced with the gene of interest (GOI) and the rep can genes are provided separately. The tandemly arranged rep and cap sequence is approximately ~4.7kbp that represents the maximum packaging capacity of an AAV capsid. Most therapeutic genes are < 4.7kbp and therefore olfer opportunities to use AAVs as a carrier for in vivo gene delivery.

The disclosed four- vector transfection method is designed to be performed in two stages, a Stage 1 Transfection and a Stage 2 Transfection. The time interval between the Stage 1 transfection and Stage 2 transfection can be between about 7 hours to about 28 hours, for about 10 hours to about 28 hours, for about 12 hours to about 28 hours, for about 18 hours to about 28 hours, or for about 18 to about 24 hours. In preferred forms, the time interval between Stage 1 transfection and Stage 2 transfection is about 24 hours.

1. Stage 1 Transfection

Stage 1 Transfection requires first transfecting host cells with three constructs: (i) a first construct containing adenoviral E2, E40rf6, and VA genes (H), operably linked to an origin of replication element and one or more other regulatory sequences; (ii) a second construct containing an AAV rep coding region (R), operably linked to one or more regulatory sequences; and (iii) a third construct containing AAV inverted terminal repeats flanking a heterologous gene of interest (G), operably linked to one or more regulatory sequences. Optionally, the first construct, the second construct, and/or the third construct are provided in plasmids.

In an exemplary form, Stage 1 Transfection can be performed by mixing a transfection mix having equimolar 1: 1: 1 ratio of pAAV-GFP:pRep:pHelper for each transfection reaction. In some forms, the amount of total plasmids dosed per million cells is aboutl pg at PELDNA plasmid mass ratio of about 2:1. i. First Construct (Helper construct)

The first construct can be a helper AAV plasmid, referred to as the "helper plasmid". The helper plasmid provides the necessary helper functions that support the replication and packaging of the AAV vector. In some forms, the helper construct e.g., the helper plasmid is derived from adenovirus or herpes simplex virus (HS V), which are natural helpers for AAV replication. Helper factors are provided by helper viruses from the herpesvirus family (e.g., HSV-1 and human cytomegalovirus, HCMV), adenoviruses (e.g., AdV5), and papillomaviruses (e.g., human papillomavirus type 16, HPV-16), as well as other viruses such as baculovirus and human bocavirus (doi: 10.1126/science.149.3685.754; doi: 10.1016/0042-6822(70)90248-5; doi: 10.1016/0042-6822(84)90272-1 ; doi: 10.1 128/JVI.80.4.1874-1885.2006). Helper genes from HSV-1 that support AAV replication include UL8,UL52, ICP8, ICP0 etc.

In some forms, the helper construct contains genes from an adenovirus, that are essential for AAV replication e.g., El A. E1B, E2A, E4orf6, and/or VA RNA. The genes that are essential for AAV replication are referred to herein as helper genes (H). Therefore, H is used to represent one or more helper genes. These genes do not encode for the structural proteins of adenovirus but rather, provide the functions needed to replicate the AAV genome and package it into capsids. Expression of Adenovirus early region 1A (E1A) initiates viral DNA replication and produces a variety of E1A proteins. The proteins encoded by E1A tend to localize in the nucleus and affect genetic regulation by the host cell. After viral infection, these proteins stimulate expression of other viral genes and can either enhance or repress expression of cellular genes depending on cellular context and coordination with other viral genes. Adenovirus E1B protein usually refers to one of two proteins transcribed from the E1B gene of the adenovirus: a 55kDa protein and a 19kDa protein. These two proteins are needed to block apoptosis in adenovirus- infected cells. E1B proteins work to prevent apoptosis that is induced by the small adenovirus E1A protein, which stabilizes p53, a tumor suppressor. E4orf6 is a nuclear protein and contains both a putative nuclear localization signal (NLS) and an amphipathic arginine-rich a-helical nuclear retention signal (NRS) that are believed to be responsible for the targeting of E4orf6 and E4orf6-E1B55K complexes to the nucleus. In addition, E4orf6 contains a nuclear export signal (NES) believed to play a role in nuclear-cytoplasmic shuttling of the E1B55K-E4orf6 complex and to be essential for the ability of E4orf6 to support viral replication. Adenovirus E2a encodes a single-stranded DNA-binding protein that stimulates viral DNA replication and gene transcription. Finally, VA RNA (Virus-Associated RNA) inhibits the cellular antiviral response mediated by PKR (Protein Kinase R), thereby enhancing the translation of viral mRNAs.

In one exemplary form, the helper plasmid contains the Adenovirus E2A, Adenovirus E4 (E4orf6), and VA genes.

In some forms, the helper plasmid contains a selectable marker to facilitate the identification and selection of successfully transfected cells. Exemplary selectable marker genes include antibiotic resistance genes e.g., Ampicillin Resistance (AmpR), Kanamycin Resistance (KanR); Neomycin Resistance (NeoR), Hygromycin Resistance (HygR), Puromycin Resistance (PuroR), and Zeocin Resistance (ZeoR); fluorescent protein genes e.g., green fluorescent protein (GFP), red fluorescent protein (RFP), and yellow fluorescent protein (YFP); luciferase genes e.g., firefly luciferase (Luc) and Renilla Luciferase (Rluc).

AAV Helper plasmids are commercially available, and can be obtained from Addgene (Catalog # 112867), and GENEMEDI (Catalog # P-HP01). An exemplary AAV Helper plasmid that can be used is commercially available from Cell Biolabs (Catalog # VPK-405), has the nucleic acid sequence represented by SEQ ID NO:7 and is depicted in the AAV map of Figure IOC.

Cell Biolabs’ AAV Helper- Free System allows the production of infectious recombinant human adeno-associated virus (rAAV) virions without the use of a helper virus. In the AAV Helper-Free System, most of the adenovirus gene products required for the production of infective AAV particles are supplied on the plasmid pHelper (i.e. E2A, E4, and VA RNA genes) that is co-transfected into cells with human AAV vector DNA. The remaining adenoviral gene product is supplied by the host cell, such as 293 host cells, which stably express the adenovirus El gene. By eliminating the requirement for live helper virus, the AAV Helper-Free System provides a safer and more convenient gene delivery system. In the AAV Helper-Free System, the rep and cap genes have been removed from the viral vector that contains A AV-2 ITRs and are supplied in trans on the plasmid pAAV-RC. The removal of the AAV rep and cap genes allows for insertion of a gene of interest in the viral genome.

An exemplary AAV helper plasmid represented by SEQ ID NO:7 contains an Adenovirus E2A gene (represented by nucleotides 1-5336); an Adenovirus E4 gene (represented by nucleotides 5337-8537); an Adenovirus VA gene (represented by nucleotides 8538-9280), and an ampicillin resistance (bla) gene (complement) (represented by nucleotides 10182-11042). GGTACCCAACTCCATGCTTAACAGTCCCCAGGTACAGCCCACCCTGCGTCGCAACCAGGAACAGCTCT ACAGCTTCCTGGAGCGCCACTCGCCCTACTTCCGCAGCCACAGTGCGCAGATTAGGAGCGCCACTTCT TTTTGTCACTTGAAAAACATGTAAAAATAATGTACTAGGAGACACTTTCAATAAAGGCAAATGTTTTT ATTTGTACACTCTCGGGTGATTATTTACCCCCCACCCTTGCCGTCTGCGCCGTTTAAAAATCAAAGGGG TTCTGCCGCGCATCGCTATGCGCCACTGGCAGGGACACGTTGCGATACTGGTGTTTAGTGCTCCACTTA AACTCAGGCACAACCATCCGCGGCAGCTCGGTGAAGTTTTCACTCCACAGGCTGCGCACCATCACCAA CGCGTTTAGCAGGTCGGGCGCCGATATCTTGAAGTCGCAGTTGGGGCCTCCGCCCTGCGCGCGCGAGT TGCGATACACAGGGTTGCAGCACTGGAACACTATCAGCGCCGGGTGGTGCACGCTGGCCAGCACGCTC TTGTCGGAGATCAGATCCGCGTCCAGGTCCTCCGCGTTGCTCAGGGCGAACGGAGTCAACTTTGGTAG CTGCCTTCCCAAAAAGGGTGCATGCCCAGGCTTTGAGTTGCACTCGCACCGTAGTGGCATCAGAAGGT GACCGTGCCCGGTCTGGGCGTTAGGATACAGCGCCTGCATGAAAGCCTTGATCTGCTTAAAAGCCACC

TGAGCCTTTGCGCCTTCAGAGAAGAACATGCCGCAAGACTTGCCGGAAAACTGATTGGCCGGACAGG

CCGCGTCATGCACGCAGCACCTTGCGTCGGTGTTGGAGATCTGCACCACATTTCGGCCCCACCGGTTCT

TCACGATCTTGGCCTTGCTAGACTGCTCCTTCAGCGCGCGCTGCCCGTTTTCGCTCGTCACATCCATTTC

AATCACGTGCTCCTTATTTATCATAATGCTCCCGTGTAGACACTTAAGCTCGCCTTCGATCTCAGCGCA

GCGGTGCAGCCACAACGCGCAGCCCGTGGGCTCGTGGTGCTTGTAGGTTACCTCTGCAAACGACTGCA

GGTACGCCTGCAGGAATCGCCCCATCATCGTCACAAAGGTCTTGTTGCTGGTGAAGGTCAGCTGCAAC

CCGCGGTGCTCCTCGTTTAGCCAGGTCTTGCATACGGCCGCCAGAGCTTCCACTTGGTCAGGCAGTAG

CTTGAAGTTTGCCTTTAGATCGTTATCCACGTGGTACTTGTCCATCAACGCGCGCGCAGCCTCCATGCC

CTTCTCCCACGCAGACACGATCGGCAGGCTCAGCGGGTTTATCACCGTGCTTTCACTTTCCGCTTCACT

GGACTCTTCCTTTTCCTCTTGCGTCCGCATACCCCGCGCCACTGGGTCGTCTTCATTCAGCCGCCGCAC

CGTGCGCTTACCTCCCTTGCCGTGCTTGATTAGCACCGGTGGGTTGCTGAAACCCACCATTTGTAGCGC

CACATCTTCTCTTTCTTCCTCGCTGTCCACGATCACCTCTGGGGATGGCGGGCGCTCGGGCTTGGGAGA

GGGGCGCTTCTTTTTCTTTTTGGACGCAATGGCCAAATCCGCCGTCGAGGTCGATGGCCGCGGGCTGG

GTGTGCGCGGCACCAGCGCATCTTGTGACGAGTCTTCTTCGTCCTCGGACTCGAGACGCCGCCTCAGC

CGCTTTTTTGGGGGCGCGCGGGGAGGCGGCGGCGACGGCGACGGGGACGACACGTCCTCCATGGTTG

GTGGACGTCGCGCCGCACCGCGTCCGCGCTCGGGGGTGGTTTCGCGCTGCTCCTCTTCCCGACTGGCC

ATTTCCTTCTCCTATAGGCAGAAAAAGATCATGGAGTCAGTCGAGAAGGAGGACAGCCTAACCGCCCC

CTTTGAGTTCGCCACCACCGCCTCCACCGATGCCGCCAACGCGCCTACCACCTTCCCCGTCGAGGCAC

CCCCGCTTGAGGAGGAGGAAGTGATTATCGAGCAGGACCCAGGTTTTGTAAGCGAAGACGACGAGGA

TCGCTCAGTACCAACAGAGGATAAAAAGCAAGACCAGGACGACGCAGAGGCAAACGAGGAACAAGT

CGGGCGGGGGGACCAAAGGCATGGCGACTACCTAGATGTGGGAGACGACGTGCTGTTGAAGCATCTG

CAGCGCCAGTGCGCCATTATCTGCGACGCGTTGCAAGAGCGCAGCGATGTGCCCCTCGCCATAGCGGA

TGTCAGCCTTGCCTACGAACGCCACCTGTTCTCACCGCGCGTACCCCCCAAACGCCAAGAAAACGGCA

CATGCGAGCCCAACCCGCGCCTCAACTTCTACCCCGTATTTGCCGTGCCAGAGGTGCTTGCCACCTATC

ACATCTTTTTCCAAAACTGCAAGATACCCCTATCCTGCCGTGCCAACCGCAGCCGAGCGGACAAGCAG

CTGGCCTTGCGGCAGGGCGCTGTCATACCTGATATCGCCTCGCTCGACGAAGTGCCAAAAATCTTTGA

GGGTCTTGGACGCGACGAGAAACGCGCGGCAAACGCTCTGCAACAAGAAAACAGCGAAAATGAAAG

TCACTGTGGAGTGCTGGTGGAACTTGAGGGTGACAACGCGCGCCTAGCCGTGCTGAAACGCAGCATCG

AGGTCACCCACTTTGCCTACCCGGCACTTAACCTACCCCCCAAGGTTATGAGCACAGTCATGAGCGAG

CTGATCGTGCGCCGTGCACGACCCCTGGAGAGGGATGCAAACTTGCAAGAACAAACCGAGGAGGGCC

TACCCGCAGTTGGCGATGAGCAGCTGGCGCGCTGGCTTGAGACGCGCGAGCCTGCCGACTTGGAGGA

GCGACGCAAGCTAATGATGGCCGCAGTGCTTGTTACCGTGGAGCTTGAGTGCATGCAGCGGTTCTTTG

CTGACCCGGAGATGCAGCGCAAGCTAGAGGAAACGTTGCACTACACCTTTCGCCAGGGCTACGTGCGC

CAGGCCTGCAAAATTTCCAACGTGGAGCTCTGCAACCTGGTCTCCTACCTTGGAATTTTGCACGAAAA

CCGCCTCGGGCAAAACGTGCTTCATTCCACGCTCAAGGGCGAGGCGCGCCGCGACTACGTCCGCGACT

GCGTTTACTTATTTCTGTGCTACACCTGGCAAACGGCCATGGGCGTGTGGCAGCAATGCCTGGAGGAG

CGCAACCTAAAGGAGCTGCAGAAGCTGCTAAAGCAAAACTTGAAGGACCTATGGACGGCCTTCAACG

AGCGCTCCGTGGCCGCGCACCTGGCGGACATTATCTTCCCCGAACGCCTGCTTAAAACCCTGCAACAG

GGTCTGCCAGACTTCACCAGTCAAAGCATGTTGCAAAACTTTAGGAACTTTATCCTAGAGCGTTCAGG AATTCTGCCCGCCACCTGCTGTGCGCTTCCTAGCGACTTTGTGCCCATTAAGTACCGTGAATGCCCTCC

GCCGCTTTGGGGTCACTGCTACCTTCTGCAGCTAGCCAACTACCTTGCCTACCACTCCGACATCATGGA

AGACGTGAGCGGTGACGGCCTACTGGAGTGTCACTGTCGCTGCAACCTATGCACCCCGCACCGCTCCC

TGGTCTGCAATTCGCAACTGCTTAGCGAAAGTCAAATTATCGGTACCTTTGAGCTGCAGGGTCCCTCGC

CTGACGAAAAGTCCGCGGCTCCGGGGTTGAAACTCACTCCGGGGCTGTGGACGTCGGCTTACCTTCGC

AAATTTGTACCTGAGGACTACCACGCCCACGAGATTAGGTTCTACGAAGACCAATCCCGCCCGCCAAA

TGCGGAGCTTACCGCCTGCGTCATTACCCAGGGCCACATCCTTGGCCAATTGCAAGCCATCAACAAAG

CCCGCCAAGAGTTTCTGCTACGAAAGGGACGGGGGGTTTACCTGGACCCCCAGTCCGGCGAGGAGCTC

AACCCAATCCCCCCGCCGCCGCAGCCCTATCAGCAGCCGCGGGCCCTTGCTTCCCAGGATGGCACCCA

AAAAGAAGCTGCAGCTGCCGCCGCCGCCACCCACGGACGAGGAGGAATACTGGGACAGTCAGGCAG

AGGAGGTTTTGGACGAGGAGGAGGAGATGATGGAAGACTGGGACAGCCTAGACGAAGCTTCCGAGGC

CGAAGAGGTGTCAGACGAAACACCGTCACCCTCGGTCGCATTCCCCTCGCCGGCGCCCCAGAAATTGG

CAACCGTTCCCAGCATCGCTACAACCTCCGCTCCTCAGGCGCCGCCGGCACTGCCTGTTCGCCGACCC

AACCGTAGATGGGACACCACTGGAACCAGGGCCGGTAAGTCTAAGCAGCCGCCGCCGTTAGCCCAAG

AGCAACAACAGCGCCAAGGCTACCGCTCGTGGCGCGGGCACAAGAACGCCATAGTTGCTTGCTTGCA

AGACTGTGGGGGCAACATCTCCTTCGCCCGCCGCTTTCTTCTCTACCATCACGGCGTGGCCTTCCCCCG

TAACATCCTGCATTACTACCGTCATCTCTACAGCCCCTACTGCACCGGCGGCAGCGGCAGCGGCAGCA

ACAGCAGCGGTCACACAGAAGCAAAGGCGACCGGATAGCAAGACTCTGACAAAGCCCAAGAAATCC

ACAGCGGCGGCAGCAGCAGGAGGAGGAGCGCTGCGTCTGGCGCCCAACGAACCCGTATCGACCCGCG

AGCTTAGAAATAGGATTTTTCCCACTCTGTATGCTATATTTCAACAAAGCAGGGGCCAAGAACAAGAG

CTGAAAATAAAAAACAGGTCTCTGCGCTCCCTCACCCGCAGCTGCCTGTATCACAAAAGCGAAGATCA

GCTTCGGCGCACGCTGGAAGACGCGGAGGCTCTCTTCAGCAAATACTGCGCGCTGACTCTTAAGGACT

AGTTTCGCGCCCTTTCTCAAATTTAAGCGCGAAAACTACGTCATCTCCAGCGGCCACACCCGGCGCCA

GCACCTGTCGTCAGCGCCATTATGAGCAAGGAAATTCCCACGCCCTACATGTGGAGTTACCAGCCACA

AATGGGACTTGCGGCTGGAGCTGCCCAAGACTACTCAACCCGAATAAACTACATGAGCGCGGGACCC

CACATGATATCCCGGGTCAACGGAATCCGCGCCCACCGAAACCGAATTCTCCTCGAACAGGCGGCTAT

TACCACCACACCTCGTAATAACCTTAATCCCCGTAGTTGGCCCGCTGCCCTGGTGTACCAGGAAAGTC

CCGCTCCCACCACTGTGGTACTTCCCAGAGACGCCCAGGCCGAAGTTCAGATGACTAACTCAGGGGCG

CAGCTTGCGGGCGGCTTTCGTCACAGGGTGCGGTCGCCCGGGCGTTTTAGGGCGGAGTAACTTGCATG

TATTGGGAATTGTAGTTTTTTTAAAATGGGAAGTGACGTATCGTGGGAAAACGGAAGTGAAGATTTGA

GGAAGTTGTGGGTTTTTTGGCTTTCGTTTCTGGGCGTAGGTTCGCGTGCGGTTTTCTGGGTGTTTTTTGT

GGACTTTAACCGTTACGTCATTTTTTAGTCCTATATATACTCGCTCTGTACTTGGCCCTTTTTACACTGT

GACTGATTGAGCTGGTGCCGTGTCGAGTGGTGTTTTTTAATAGGTTTTTTTACTGGTAAGGCTGACTGT

TATGGCTGCCGCTGTGGAAGCGCTGTATGTTGTTCTGGAGCGGGAGGGTGCTATTTTGCCTAGGCAGG

AGGGTTTTTCAGGTGTTTATGTGTTTTTCTCTCCTATTAATTTTGTTATACCTCCTATGGGGGCTGTAAT

GTTGTCTCTACGCCTGCGGGTATGTATTCCCCCGGGCTATTTCGGTCGCTTTTTAGCACTGACCGATGTT

AACCAACCTGATGTGTTTACCGAGTCTTACATTATGACTCCGGACATGACCGAGGAACTGTCGGTGGT

GCTTTTTAATCACGGTGACCAGTTTTTTTACGGTCACGCCGGCATGGCCGTAGTCCGTCTTATGCTTAT

AAGGGTTGTTTTTCCTGTTGTAAGACAGGCTTCTAATGTTTAAATGTTTTTTTTTTTGTTATTTTATTTTG

TGTTTAATGCAGGAACCCGCAGACATGTTTGAGAGAAAAATGGTGTCTTTTTCTGTGGTGGTTCCGGA ACTTACCTGCCTTTATCTGCATGAGCATGACTACGATGTGCTTGCTTTTTTGCGCGAGGCTTTGCCTGAT

TTTTTGAGCAGCACCTTGCATTTTATATCGCCGCCCATGCAACAAGCTTACATAGGGGCTACGCTGGTT

AGCATAGCTCCGAGTATGCGTGTCATAATCAGTGTGGGTTCTTTTGTCATGGTTCCTGGCGGGGAAGTG

GCCGCGCTGGTCCGTGCAGACCTGCACGATTATGTTCAGCTGGCCCTGCGAAGGGACCTACGGGATCG

CGGTATTTTTGTTAATGTTCCGCTTTTGAATCTTATACAGGTCTGTGAGGAACCTGAATTTTTGCAATCA

TGATTCGCTGCTTGAGGCTGAAGGTGGAGGGCGCTCTGGAGCAGATTTTTACAATGGCCGGACTTAAT

ATTCGGGATTTGCTTAGAGACATATTGATAAGGTGGCGAGATGAAAATTATTTGGGCATGGTTGAAGG

TGCTGGAATGTTTATAGAGGAGATTCACCCTGAAGGGTTTAGCCTTTACGTCCACTTGGACGTGAGGG

CAGTTTGCCTTTTGGAAGCCATTGTGCAACATCTTACAAATGCCATTATCTGTTCTTTGGCTGTAGAGT

TTGACCACGCCACCGGAGGGGAGCGCGTTCACTTAATAGATCTTCATTTTGAGGTTTTGGATAATCTTT

TGGAATAAAAAAAAAAAAACATGGTTCTTCCAGCTCTTCCCGCTCCTCCCGTGTGTGACTCGCAGAAC

GAATGTGTAGGTTGGCTGGGTGTGGCTTATTCTGCGGTGGTGGATGTTATCAGGGCAGCGGCGCATGA

AGGAGTTTACATAGAACCCGAAGCCAGGGGGCGCCTGGATGCTTTGAGAGAGTGGATATACTACAAC

TACTACACAGAGCGAGCTAAGCGACGAGACCGGAGACGCAGATCTGTTTGTCACGCCCGCACCTGGTT

TTGCTTCAGGAAATATGACTACGTCCGGCGTTCCATTTGGCATGACACTACGACCAACACGATCTCGG

TTGTCTCGGCGCACTCCGTACAGTAGGGATCGCCTACCTCCTTTTGAGACAGAGACCCGCGCTACCAT

ACTGGAGGATCATCCGCTGCTGCCCGAATGTAACACTTTGACAATGCACAACGTGAGTTACGTGCGAG

GTCTTCCCTGCAGTGTGGGATTTACGCTGATTCAGGAATGGGTTGTTCCCTGGGATATGGTTCTGACGC

GGGAGGAGCTTGTAATCCTGAGGAAGTGTATGCACGTGTGCCTGTGTTGTGCCAACATTGATATCATG

ACGAGCATGATGATCCATGGTTACGAGTCCTGGGCTCTCCACTGTCATTGTTCCAGTCCCGGTTCCCTG

CAGTGCATAGCCGGCGGGCAGGTTTTGGCCAGCTGGTTTAGGATGGTGGTGGATGGCGCCATGTTTAA

TCAGAGGTTTATATGGTACCGGGAGGTGGTGAATTACAACATGCCAAAAGAGGTAATGTTTATGTCCA

GCGTGTTTATGAGGGGTCGCCACTTAATCTACCTGCGCTTGTGGTATGATGGCCACGTGGGTTCTGTGG

TCCCCGCCATGAGCTTTGGATACAGCGCCTTGCACTGTGGGATTTTGAACAATATTGTGGTGCTGTGCT

GCAGTTACTGTGCTGATTTAAGTGAGATCAGGGTGCGCTGCTGTGCCCGGAGGACAAGGCGTCTCATG

CTGCGGGCGGTGCGAATCATCGCTGAGGAGACCACTGCCATGTTGTATTCCTGCAGGACGGAGCGGCG

GCGGCAGCAGTTTATTCGCGCGCTGCTGCAGCACCACCGCCCTATCCTGATGCACGATTATGACTCTAC

CCCCATGTAGGCGTGGACTTCCCCTTCGCCGCCCGTTGAGCAACCGCAAGTTGGACAGCAGCCTGTGG

CTCAGCAGCTGGACAGCGACATGAACTTAAGCGAGCTGCCCGGGGAGTTTATTAATATCACTGATGAG

CGTTTGGCTCGACAGGAAACCGTGTGGAATATAACACCTAAGAATATGTCTGTTACCCATGATATGAT

GCTTTTTAAGGCCAGCCGGGGAGAAAGGACTGTGTACTCTGTGTGTTGGGAGGGAGGTGGCAGGTTGA

ATACTAGGGTTCTGTGAGTTTGATTAAGGTACGGTGATCAATATAAGCTATGTGGTGGTGGGGCTATA

CTACTGAATGAAAAATGACTTGAAATTTTCTGCAATTGAAAAATAAACACGTTGAAACATAACATGCA

ACAGGTTCACGATTCTTTATTCCTGGGCAATGTAGGAGAAGGTGTAAGAGTTGGTAGCAAAAGTTTCA

GTGGTGTATTTTCCACTTTCCCAGGACCATGTAAAAGACATAGAGTAAGTGCTTACCTCGCTAGTTTCT

GTGGATTCACTAGAATCGATGTAGGATGTTGCCCCTCCTGACGCGGTAGGAGAAGGGGAGGGTGCCCT

GCATGTCTGCCGCTGCTCTTGCTCTTGCCGCTGCTGAGGAGGGGGGCGCATCTGCCGCAGCACCGGAT

GCATCTGGGAAAAGCAAAAAAGGGGCTCGTCCCTGTTTCCGGAGGAATTTGCAAGCGGGGTCTTGCAT

GACGGGGAGGCAAACCCCCGTTCGCCGCAGTCCGGCCGGCCCGAGACTCGAACCGGGGGTCCTGCGA

CTCAACCCTTGGAAAATAACCCTCCGGCTACAGGGAGCGAGCCACTTAATGCTTTCGCTTTCCAGCCT AACCGCTTACGCCGCGCGCGGCCAGTGGCCAAAAAAGCTAGCGCAGCAGCCGCCGCGCCTGGAAGGA

AGCCAAAAGGAGCGCTCCCCCGTTGTCTGACGTCGCACACCTGGGTTCGACACGCGGGCGGTAACCGC

ATGGATCACGGCGGACGGCCGGATCCGGGGTTCGAACCCCGGTCGTCCGCCATGATACCCTTGCGAAT

TTATCCACCAGACCACGGAAGAGTGCCCGCTTACAGGCTCTCCTTTTGCACGGTCTAGAGCGTCAACG

ACTGCGCACGCCTCACCGGCCAGAGCGTCCCGACCATGGAGCACTTTTTGCCGCTGCGCAACATCTGG

AACCGCGTCCGCGACTTTCCGCGCGCCTCCACCACCGCCGCCGGCATCACCTGGATGTCCAGGTACAT

CTACGGATTACGTCGACGTTTAAACCATATGATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACA

GAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAA

AAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTC

AAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTC

GTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTG

GCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGT

GTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCC

GGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTA

GGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTAT

CTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCA

CCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAA

GATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTC

ATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTA

AAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGA

TCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCT

TACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCA

ATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTC

TATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCAT

TGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATC

AAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGT

CAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCAT

GCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCG

GCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAG

TGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTT

CGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAG

CAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCA

TACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGA

ATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTAAATTGT

AAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGC

CGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTT

GGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGG

CGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAA

ATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAA GGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGT AACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGATGGATCC (SEQ ID NO: 7). ii. Second Construct (rep construct)

The second construct can be an engineered rep plasmid, which encodes only the replicase gene, responsible for producing four non- structural proteins: Rep78, Rep68, Rep52, and Rep40. These proteins are generated via alternative splicing and alternative promoter usage within the rep gene. Rep78 and Rep68 are involved in initiating and regulating AAV DNA replication. They bind to the AAV inverted terminal repeats (ITRs) at the origins of replication and introduce site-specific nicks to initiate the replication process. Rep52 and Rep40 are primarily involved in the packaging of the AAV genome into capsids. They help in the assembly of the viral particles by interacting with the AAV capsid proteins and the viral DNA.

In some forms, the engineered rep plasmid contains the rep sequence under the control of endogenous AAV promotors e.g., p5 and pl9. In one preferred form, the rep sequence is under the control of both AAV promoters p5 and pl 9. The p5 promoter is responsible for the expression of Rep78 and Rep68, while the pl9 promoter drives the expression of Rep52 and Rep40. In preferred forms, the AAV promoters p5 and pl9 are derived from AAV Serotype 2 (AAV2). However, the AAV promoters p5 and pl9 can also be derived from other AAV serotypes including but not limited to AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9. In preferred forms, the rep plasmid does not include an inducible promoter, for example, when expressed in mammalian cells.

In some forms, for example insect cells, Rep can be expressed using constitutive promoters or baculovirus promoters such as polyhedrin, plO, etc. For example, Rep can be expressed with constitutive promoters in Sf9 cells. In some forms, the second construct does not employ an inducible promoter such as Tet-on or the cumate gene-switch system/ expression of rep genes is not initiated by adding cumate the cells containing the rep plasmid (Mullick, et al., BMC Biotechnol. 2006 3:6:43. doi: 10.1186/1472-6750-6-4).

In some forms, inducible promoters such as Tet-on or the cumate gene-switch system can be used to control the kinetics of Cap gene expression. GeneSwitch (ThermoFisher Scientific) is another induction promoter system that offers the possibility to reduce the background leaky expression but this has not been used in AAV production so far (Kallunki, et al., Cells. 2019 lul 30;8(8):796. doi: 10.3390/cells8080796). For gene therapy vectors, promoters of genes of interest are typically tissue specific to reduce off targets and to evoke high expression ( Walther, et al.. J Mol Med 74, 379-392 (1996); https://doi.org/10.1007/BF00210632)). Regulation of transgene expression was earlier reported via chemical (Barrett, et al. Cancer Gene Ther 25, 106-116 (2018). and radiation (Xiong, et al., Cancer, 118(2):536-548 (2012)) induction.

In some forms, the replicase gene incorporated into the Rep plasmid is isolated from AAV2. Generally, nearly all recombinant AAV production processes use the Rep gene from AAV2, regardless of the capsid serotype chosen. However, the Rep gene can also be isolated from other AAV serotypes e.g., AAV1 , AAV5, AAV6, AAV8, and AAV9.

In some forms, the engineered rep plasmid contains a selectable marker to facilitate the identification and selection of successfully transfected cells. Exemplary selectable marker genes that can be included in the engineered rep plasmid include antibiotic resistance genes e.g., Ampicillin Resistance (AmpR), Kanamycin Resistance (KanR); Neomycin Resistance (NeoR), Hygromycin Resistance (HygR), Puromycin Resistance (PuroR), and Zeocin Resistance (ZeoR); fluorescent protein genes e.g., green fluorescent protein (GFP), red fluorescent protein (RFP), and yellow fluorescent protein (YFP); luciferase genes e.g., firefly luciferase (Luc) and Renilla Luciferase (Rluc).

An exemplary rep plasmid is represented by the nucleic acid sequence - SEQ ID NO:9 and is depicted in Figure 10D. ATCGTTAACGCCCCGCGCCGGCCGCTCTAGAACTAGTGGATCCCCCGGAAGATCAGAAGTTCCTATTC CGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCTGATCTGCGCAGCCGCCATGCCGGGGTTTTACG AGATTGTGATTAAGGTCCCCAGCGACCTTGACGAGCATCTGCCCGGCATTTCTGACAGCTTTGTGAACT GGGTGGCCGAGAAGGAATGGGAGTTGCCGCCAGATTCTGACATGGATCTGAATCTGATTGAGCAGGC ACCCCTGACCGTGGCCGAGAAGCTGCAGCGCGACTTTCTGACGGAATGGCGCCGTGTGAGTAAGGCCC CGGAGGCCCTTTTCTTTGTGCAATTTGAGAAGGGAGAGAGCTACTTCCACATGCACGTGCTCGTGGAA ACCACCGGGGTGAAATCCATGGTTTTGGGACGTTTCCTGAGTCAGATTCGCGAAAAACTGATTCAGAG AATTTACCGCGGGATCGAGCCGACTTTGCCAAACTGGTTCGCGGTCACAAAGACCAGAAATGGCGCCG GAGGCGGGAACAAGGTGGTGGATGAGTGCTACATCCCCAATTACTTGCTCCCCAAAACCCAGCCTGAG CTCCAGTGGGCGTGGACTAATATGGAACAGTATTTAAGCGCCTGTTTGAATCTCACGGAGCGTAAACG GTTGGTGGCGCAGCATCTGACGCACGTGTCGCAGACGCAGGAGCAGAACAAAGAGAATCAGAATCCC AATTCTGATGCGCCGGTGATCAGATCAAAAACTTCAGCCAGGTACATGGAGCTGGTCGGGTGGCTCGT GGACAAGGGGATTACCTCGGAGAAGCAGTGGATCCAGGAGGACCAGGCCTCATACATCTCCTTCAAT GCGGCCTCCAACTCGCGGTCCCAAATCAAGGCTGCCTTGGACAATGCGGGAAAGATTATGAGCCTGAC TAAAACCGCCCCCGACTACCTGGTGGGCCAGCAGCCCGTGGAGGACATTTCCAGCAATCGGATTTATA AAATTTTGGAACTAAACGGGTACGATCCCCAATATGCGGCTTCCGTCTTTCTGGGATGGGCCACGAAA AAGTTCGGCAAGAGGAACACCATCTGGCTGTTTGGGCCTGCAACTACCGGGAAGACCAACATCGCGG AGGCCATAGCCCACACTGTGCCCTTCTACGGGTGCGTAAACTGGACCAATGAGAACTTTCCCTTCAAC GACTGTGTCGACAAGATGGTGATCTGGTGGGAGGAGGGGAAGATGACCGCCAAGGTCGTGGAGTCGG CCAAAGCCATTCTCGGAGGAAGCAAGGTGCGCGTGGACCAGAAATGCAAGTCCTCGGCCCAGATAGA CCCGACTCCCGTGATCGTCACCTCCAACACCAACATGTGCGCCGTGATTGACGGGAACTCAACGACCT

TCGAACACCAGCAGCCGTTGCAAGACCGGATGTTCAAATTTGAACTCACCCGCCGTCTGGATCATGAC

TTTGGGAAGGTCACCAAGCAGGAAGTCAAAGACTTTTTCCGGTGGGCAAAGGATCACGTGGTTGAGGT

GGAGCATGAATTCTACGTCAAAAAGGGTGGAGCCAAGAAAAGACCCGCCCCCAGTGACGCAGATATA

AGTGAGCCCAAACGGGTGCGCGAGTCAGTTGCGCAGCCATCGACGTCAGACGCGGAAGCTTCGATCA

ACTACGCAGACAGGTACCAAAACAAATGTTCTCGTCACGTGGGCATGAATCTGATGCTGTTTCCCTGC

AGACAATGCGAGAGAATGAATCAGAATTCAAATATCTGCTTCACTCACGGACAGAAAGACTGTTTAG

AGTGCTTTCCCGTGTCAGAATCTCAACCCGTTTCTGTCGTCAAAAAGGCGTATCAGAAACTGTGCTACA

TTCATCATATCATGGGAAAGGTGCCAGACGCTTGCACTGCCTGCGATCTGGTCAATGTGGATTTGGAT

GACTGCATCTTTGAACAATAACCCATTCATGTCGCATACCCTCAATAAACCGGTTAATTCGTGTCAGTT

GAACTTTGGTCTCATGTCGTTATTATCTTATCTGGTCACCAGATCCCCGTAGATAAGTAGCATGGCGGG

TTAATCATTAACTACAGCCCGGGCGTTTAAACAGCGGGCGGAGGGGTGGAGTCGTGACGTGAATTACG

TCATAGGGTTAGGGAGGTCCTGTATTAGAGGTCACGTGAGTGTTTTGCGACATTTTGCGACACCATGT

GGTCTCGCTGGGGGGGGGGGCCCGAGTGAGCACGCAGGGTCTCCATTTTGAAGCGGGAGGTTTGAAC

GAGCGCTGGCGCGCTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAA

CTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCG

CCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGAAATTGTAAGCGTTAATATTTTGTTAAAATTC

GCGTTAAATTTTTGTTAAATCAGCTCATTTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAA

ATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAGAA

CGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCA

CCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCG

ATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGC

GGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAAT

GCGCCGCTACAGGGCGCGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATT

TTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTG

AAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCT

TCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAG

TGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTT

CCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGA

GCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGC

ATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCG

GCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGA

TCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACA

CCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCT

TCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCT

TCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAG

CACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATG

GATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCA

AGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGAT

CCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGT AGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAA AACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACT GGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAA GAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGA TAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAA CGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCG TGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGG GTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCG GGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAA AACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTG CGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCC GAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTC TCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGT GAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCG GCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTA CGCCAAGCGCGCCGAT (SEQ ID N0:9).

The rep construct e.g., the rep plasmid can be generated using molecular cloning approaches known in the art such as described by Russell et al., Chem Biol 8(10) :941-50, 2001). An exemplary method for engineering a rep plasmid includes one or more of the following steps:

(a) amplifying rep and origin of replication PCR products from a pAAV-RC5 template using DNA Polymerase master mix (e.g., Q5 High-Fidelity DNA polymerase master mix II from New England Biolabs). Exemplary sequences of the primer sets that can be used for this reaction are: poriF: 5’-GTGAGCGAGGAAGCGGAAGAG-3’ (SEQ ID NO:1) pRepR: 5’-TTATTGTTCAAAGATGCAGTCATCCAAATC-3’ (SEQ ID NO:2) pRepF: 5’-ACTGCATCTTTGAACAATAACCCATTCATGTCGCATACCCTCAA TAAAC-3’(SEQ ID NO:3) poriR: 5’-CTCTTCCGCTTCCTCGCTCAC-3’ (SEQ ID NO:4);

(b) separating the PCR amplified products using agarose gel electrophoresis (1% agarose gel) and extracting the PCR amplified products using a DNA recovery kit (e.g., Zymoclean gel DNA recovery kit from Zymo Research);

(c) assembling the extracted products using an assembly mix (e.g., HiFi 2X Assembly mix from New England Biolabs);

(d) transforming the products of the assembly reaction into the E. coli Stbl3 competent cells (Invitrogen); and (e) screening for positive clones for colonies e.g., ampicillin resistant colonies, formed after overnight incubation of plates at 37 °C. iii. Third Construct (GOI construct)

The third construct can be a transfer AAV plasmid, referred to as the "transfer plasmid”. The transfer plasmid contains the gene of interest such as a therapeutic gene and several elements necessary for the successful replication, packaging, and expression of the rA AV vector e.g., inverted terminal repeat sequences.

The transfer plasmid contains a transgene expression cassette containing a promoter, a transgene of interest, a polyadenylation signal e.g., SV40 polyA signal, and optionally, one or more enhancer elements. In some forms, the choice of promoter depends on the target tissue and desired expression levels. In some forms, the promoter is a ubiquitous promoter. Exemplary ubiquitous promoters include the cytomegalovirus (CMV) promoter, the chicken P-actin promoter (CBA), elongation factor-1 alpha (EF-la), nuclear factor-Kb (NF-kB), proximal sequence element 7 (PSE-7), murine phosphoglycerate kinase promoter (mPGK), murine small nuclear RNA promoter (U a), and RNA polymerase III U6 promoter (U6). In one form, the ubiquitous promoter is CMB which provides strong, ubiquitous expression. In some forms, the promoter is a synthetic promoter e.g., a CAG promoter. CAG promoters are composite promoters that combine elements from different sources to achieve robust and widespread expression. CAG promoters include a CMV early enhancer element, a chicken P-actin promoter, and rabbit P-globin intron.

In some forms, the transfer plasmid contains the transgene of interest under the control of a tissue-specific promoter. Exemplary tissue specific promoter include liver- specific promoters e.g., hybrid human liver promoter (HLP), human A-l antitrypsin promoter (hAAT), liver promoter (LP1), thyroxine-binding globulin (TBG), and transthyretin promoter (TTR); neuronspecific promoters e.g., proximal region of the Synapsin 1 promoter (SYN1), and rat neuronspecific enolase (NSE); muscle-specific promoters e.g., Triple tandem copies of Mouse Muscle Creatine Kinase enhancer ligated to its basal promoter (tMCK), Mouse Creatine Kinase promoter/enhancer element (CK8), Murine Muscle Creatine Kinase (CK) and a-myosin heavychain genes (MHCK7), survival motor neuron promoter (SMN), and human desmin enhancer/ promo ter (DES); retina- specific promoters e.g., human rhodopsin kinase (RK), 776 bp- long rho promoter fragment carrying the zf6-cis deletion (RHO), Human Rhodopsin Kinase promoter (hGRKl), Human Cone Arrestin promoter (hCAR), Human retinal pigmented epithelium (hRPE65p); truncated mecp2-promoter (P546), and 1.7 kb L-opsin promoter; and cardiac-specific promoters e.g., a-myosin heavy chain promoter (a-MHC). Other examples include Adiponectin Promoter, Albumin Promoter, Alpha-Fetoprotein (AFP) Promoter, Cardiac Myosin Light Chain-2 (MLC2) Promoter, CD68 Promoter, Desmin Promoter, Erythropoietin (EPO) Promoter, Insulin Promoter, Keratin 14 (KI 4) Promoter, Muscle Creatine Kinase (MCK) Promoter, Neuron-Specific Enolase (NSE) Promoter, Pdxl Promoter, Surfactant Protein C (SP- C) Promoter, Synapsin-1 Promoter, a-Myosin Heavy Chain (a-MHC) Promoter. Also human thyroxine binding globulin (TBG) and human alpha- 1- antitrypsin (hAAT) promoters were used for targeting liver tissues.

The transgene of interest is the therapeutic gene that is intended for delivery to the target cells. This could be a gene that corrects a genetic defect, expresses a therapeutic protein, or provides another beneficial function. Generally, the maximum size of the transgene of interest that can be incorporated into the transfer plasmid is about 4.7 kbp (including includes the ITRs, enhancer elements, introns, and transgene sequences). In some forms, the size of the transgene of interest that can be incorporated into the transfer plasmid is less than 4.7 kbp. For example, in some forms, the transgene of interest can have a size ranging from about 900 bases to about 4.5 kbp, from about 1 kbp to about 4.5 kbp, from about 1.5 kbp to about 4.5 kbp, from about 2.0 kbp to about 4.5 kbp, from about 2.5 kbp to about 4.5 kbp, from about 3.0 to about 4.5 kbp, or from about 3.5 kbp to about 4.5 kbp.

Preferably, the transgene encodes a therapeutic (poly)peptide or therapeutic protein. Therapeutic (poly)peptides and proteins for use in the context of the present invention include, but are not limited to, (soluble) cluster of differentiation 39 (CD39) protein, (soluble) cluster of differentiation 73 (CD73) protein, Recombinant Anti-Inflammation fusioN protein (RAIN) (CD73-39 fusion), interleukin- 1 inhibitor, tumor necrosis factor-a inhibitor, interleukin- 12 inhibitor, interleukin- 1 receptor antagonist, interleukin- 18 binding protein, soluble tumor necrosis factor-a receptor p55 or soluble tumor necrosis factor-a protein 75, dominant negative IKB kinase- P, inter leukin-4, interleukin- 10, interleukin- 13, interferon- , vasoactive intestinal polypeptide, cystic fibrosis transmembrane regulator protein (CFTR), dystrophin, utrophin, blood coagulation (clotting) factor (e.g., Factor XIII, Factor IX, Factor X, Factor VIII, Factor Vila, protein C, Factor VII, B domain-deleted Factor VIII, or a high-activity or longer half-life variant of coagulation factor, or an active or inactive form of a coagulation factor), a monoclonal antibody (e.g., against tumor necrosis factor-a or interleukin- 12), retinal pigment epithelium-specific 65 kDa protein (RPE65), erythropoietin, LDL receptor, lipoprotein lipase, ornithine transcarbamylase, P- globin, a-globin, spectrin, a-antitrypsin, adenosine deaminase (ADA), a metal transporter (ATP7A or ATP7), sulfamidase, an enzyme involved in lysosomal storage disease (ARSA), hypoxanthine guanine phosphoribosyl transferase, P-25 glucocerebrosidase, sphingomyelinase, lysosomal hexosaminidase, branched chain keto acid dehydrogenase, a hormone, a growth factor, insulinlike growth factor 1 or 2, platelet derived growth factor, epidermal growth factor, nerve growth factor, neurotrophic factor-3 and -4, brain-derived neurotrophic factor, glial derived growth factor, transforming growth factor a and , a cytokine, interferon-a, interferon-y, inter leukin-2, interleukin- 12, granulocyte-macrophage colony stimulating factor, lymphotoxin, a suicide gene product, herpes simplex virus thymidine kinase, cytosine deaminase, diphtheria toxin, cytochrome P450, deoxycytidine kinase, tumor necrosis factor, a drug resistance protein, a tumor suppressor protein (e.g., p53, Rb, Wt-1, NF1 , Von Hippel-Lindau (VHL), SERCA2a, adenomatous polyposis coli (APC)), VEGF, microdystrophin, lysosomal acid lipase, arylsulfatase A and B, ATP7A and B, a peptide with immunomodulatory properties, a tolerogenic or immunogenic peptide or protein Tregitope or hCDRl , insulin, glucokinase, guanylate cyclase 2D (LCA- GUCY2D), Rab escort protein 1 (Choroideremia), LCA 5 (LCA-Lebercilin), ornithine ketoacid aminotransferase (Gyrate Atrophy), Retinoschisin 1 (X-linked Retinoschisis), USH1C (Usher's Syndrome IC), X-linked retinitis pigmentosa GTPase (XLRP), MERTK (AR forms of RP: retinitis pigmentosa), DFNB1 (Connexin 26 deafness), ACHM 2, 3 and 4 (Achromatopsia), PKD-1 or PKD-2 (Polycystic kidney disease), TPP1 , CLN2, a gene product implicated in lysosomal storage diseases (e.g., sulfatases, N-acetylglucosamine-1- phosphate transferase, cathepsin A, GM2-AP, NPC1 , VPC2, a sphingo lipid activator protein), or one or more zinc finger nucleases, transcription activation- like effector nucleases (TALENs), or CRISPER-Cas9 protein for genome editing, or donor sequences used as repair templates for genome editing, and any other peptide or protein that has a therapeutic effect in an individual in need thereof. Preferably, the therapeutic protein is a therapeutic antiinflammatory protein, preferably selected from the group consisting of (soluble) cluster of differentiation 39 (CD39) protein, (soluble) cluster of differentiation 73 (CD73) protein, interleukin- 1 inhibitor, tumor necrosis factor-a inhibitor, interleukin- 1 receptor antagonist, interleukin- 18 binding protein, soluble tumor necrosis factor-a receptor p55 or soluble tumor necrosis factor-a protein 75, dominant negative IKB kinase- f>, inter leukin-4, interleukin- 10, interleukin- 13, interferon-P and vasoactive intestinal polypeptide. Further exemplary therapeutic peptides or proteins encoded by transgenes include those that may be used in the treatment of a disease or disorder including, but not limited to, rheumatoid arthritis (RA), juvenile rheumatoid arthritis, osteoarthritis (OA), gout, spondlyarthritis (SpA), psoriasis, psoriatic arthritis, ankylosing spondylitis, inflammatory bowel disease including Crohn’s disease or ulcerative colitis, hepatitis, sepsis, alcoholic liver disease, and non-alcoholic steatosis, cystic fibrosis (and other diseases of the lung), hemophilia A, hemophilia B, thalassemia, anemia and other blood disorders, AIDS, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, epilepsy, and other neurological disorders, cancer, diabetes mellitus, muscular dystrophies (e.g., Duchenne, Becker), Gaucher's disease, Hurler's disease, adenosine deaminase deficiency, glycogen storage diseases and other metabolic defects, retinal degenerative diseases (and other diseases of the eye), and diseases of solid organs (e.g., brain, liver, kidney, heart).

As set forth herein, the transgene of the invention may be an inhibitory and/or antisense nucleic acid sequence. Inhibitory, antisense, siRNA, miRNA, shRNA, RNAi and antisense oligonucleotides can modulate expression of a target gene. Such molecules include those able to inhibit expression of a target gene involved in mediation of a disease process, thereby reducing, inhibiting or alleviating one or more symptoms of a disease.

The transfer plasmid generally contains two inverted terminal repeat (ITR) sequences flanking the transgene expression cassette. The ITRs form T-shaped hairpin structures that are recognized by the Rep proteins and serve as the origins of replication and packaging signals, facilitating initiation of DNA replication and packaging of the viral genome into AAV capsids. Typically, the ITRs incorporated into the transfer plasmid is isolated from AAV2, regardless of the capsid serotype chosen. However, the ITRs can also be isolated from other AAV serotypes e.g., AAV1, AAV5, AAV6, AAV8, and AAV9.

The transfer plasmid generally contains two inverted terminal repeat (ITR) sequences flanking the transgene expression cassette. The ITRs form T-shaped hairpin structures that are recognized by the Rep proteins and serve as the origins of replication and packaging signals, facilitating initiation of DNA replication and packaging of the viral genome into AAV capsids. Typically, the ITRs incorporated into the transfer plasmid is isolated from AAV2, regardless of the capsid serotype chosen. However, the ITRs can also be isolated from other AAV serotypes e.g., AAV1, AAV5, AAV6, AAV8, and AAV9.

An exemplary left ITR has the nucleic sequence

CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGGGCGACCTTTGGTCGCCC GGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT (SEQ ID NO:11). An exemplary right ITR has the nucleic acid sequence represented by nucleotides 2638-2778 of SEQ ID NO:8. Other ITR sequences include AAV1,2,3,4, 6 and 7 (Human Gene Therapy, 31(3-4), 151-162, 2020). In some forms, the helper plasmid contains a selectable marker to facilitate the identification and selection of successfully transfected cells. Exemplary selectable marker genes that can be included in the engineered rep plasmid include antibiotic resistance genes e.g., Ampicillin Resistance (AmpR), Kanamycin Resistance (KanR); Neomycin Resistance (NeoR), Hygromycin Resistance (HygR), Puromycin Resistance (PuroR), and Zeocin Resistance (ZeoR); fluorescent protein genes e.g., green fluorescent protein (GFP), red fluorescent protein (RFP), and yellow fluorescent protein (YFP); luciferase genes e.g., firefly luciferase (Luc) and Renilla Luciferase (Rluc).

In some forms, the transfer plasmid includes one or more intron sequences and/or insulator sequences. For example, introns can be included in the transfer plasmid to enhance gene expression through RNA processing mechanisms. Exemplary introns include the human 0- globin intron, simian virus 40 (SV40) intron, CMV intron A, human growth hormone (hGH) intron, and the human intron 1 from the EFla gene. Additional examples include human TPI introns, intron 2 of the human al -antitrypsin (hAAT) minigene, 0-globin/IgG chimeric intron. In a second example, insulator sequences can be included in the transfer plasmid to protect the expression cassette from positional effects and enhance consistent expression levels.

In some forms, the transfer plasmid is multi cistronic, i.e., carries more than one gene. Unlike promoters which will create unique mRNA transcripts for each gene that is expressed, multicistronic plasmids simultaneously express two or more separate proteins from the same mRNA. In such cases, the multiple genes are separated by an element that allows for separate translation for each gene (e.g., internal ribosomal entry sites (IRES) or 2A peptides). RES and Viral 2A peptides (such as P2A, T2A and E2A) can be employed to express multiple genes using four plasmid approach (Liu, Sci Rep 7, 2193 (2017)).In some forms, the transgene may contain a nucleotide sequence that is either homologous or heterologous to a particular nucleotide sequence in the mammal's endogenous genetic material or is a hybrid sequence (i.e. one or more portions of the transgene are homologous, and one or more portions are heterologous to the mammal's genetic material).

Any heterologous nucleic acid sequence(s) of interest may be delivered in the virus vectors produced by the present invention. Nucleic acids of interest include nucleic acids encoding polypeptides or RNAs, including reporter, therapeutic (e.g., for medical or veterinary uses), immunogenic (e.g., for vaccines), or diagnostic polypeptides or RNAs. The heterologous nucleic acid can encode any polypeptide or RNA that is desirably produced in a cell in vitro, ex vivo, or in vivo. For example, the virus vectors may be introduced into cultured cells and the expressed gene product isolated therefrom. In some forms, the heterologous nucleic acid sequence can be a heterologous gene of interest encoding one or more peptide, polypeptide, or protein. In some forms, the heterologous nucleic acid sequence can encode a peptide, polypeptide, or protein that binds to a specific target of interest, which can be useful for the treatment or prevention of disease in a subject. Examples of heterologous nucleic acid sequences and associated peptides, polypeptides, or proteins include, but are not limited to, a gene encoding antibodies, T-cell receptors, B-cell receptors, aptamers, receptor-binding ligands, or targeting peptides.

It will be understood by those skilled in the art that the heterologous nucleic acid(s) of interest can be operably associated with appropriate control sequences. For example, the heterologous nucleic acid can be operably associated with expression control elements, such as transcription/translation control signals, origins of replication, polyadenylation signals, internal ribosome entry sites (IRES), promoters, and/or enhancers, and the like.

A variety of promoter/enhancer elements can be used depending on the level and tissuespecific expression desired. The promoter/enhancer can be constitutive or inducible, depending on the pattern of expression desired. The promoter/enhancer can be native or foreign and can be a natural or a synthetic sequence. By foreign, it is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced.

In particular embodiments, the promoter/enhancer elements can be native to the target cell or subject to be treated. In representative embodiments, the promoters/enhancer element can be native to the heterologous nucleic acid sequence. The promoter/enhancer element is generally chosen so that it functions in the target cell(s) of interest. Further, in particular embodiments the promoter/enhancer element is a mammalian promoter/enhancer element. The promoter/enhancer element may be constitutive or inducible.

Transfer AAV plasmids are commercially available e.g., from Vector Biolabs and Charles River. Also, transfer AAV plasmids for selected therapeutic genes can be customized and ordered from commercial providers e.g., Vector Biolabs and Addgene. An exemplary transfer plasmid is commercially available from Cell Biolabs (Catalog # VPK-405) and has the nucleic acid sequence represented by SEQ ID NO:8 (see Figure 10A). The exemplary transfer plasmid includes a left inverted terminal repeat sequence (nucleotides 1-130); a CMV promoter (represented by nucleotides 139-798); a human P-globin intron (represented by nucleotides 806- 1298); a green fluorescent protein (GFP) transgene (represented by nucleotides 1321-2061); a polyadenylation (Poly A) signal (represented by nucleotides 2120-2598); a right inverted terminal repeat sequence (represented by nucleotides 2638-2778) and an ampicillin resistance gene (represented by nucleotides 3695-4555).

CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGGGCGACCTTTGGTCGCCC

GGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGC CGCACGCGTCTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCC

GCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCA

ATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTT

ACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCA ATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGT

ACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGAT

AGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACC

AAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGT

GTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCC ACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGATTCGAATCCCGGCCGG GAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATAGAGTCTATAG GCCCACAAAAAATGCTTTCTTCTTTTAATATACTTTTTTGTTTATCTTATTTCTAATACTTTCCCTAATCT CTTTCTTTCAGGGCAATAATGATACAATGTATCATGCCTCTTTGCACCATTCTAAAGAATAACAGTGAT AATTTCTGGGTTAAGGCAATAGCAATATTTCTGCATATAAATATTTCTGCATATAAATTGTAACTGATG

TAAGAGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTACCATTCTGCTTTTATTTTATGGTTGGGA

TAAGGCTGGATTATTCTGAGTCCAAGCTAGGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTCCTCC CACAGCTCCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAGAATTGGGATTCGAA

CATCGATTGAATTCTGAATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTC

GAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCT

ACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTG

ACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTT CAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACA

AGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGA

CTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTAT ATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACG

GCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCC

GACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGG

TCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTACTCAGAT

CTCGAGCTCAAGTAGGGATCCTCTAGAGTCGACCTGCAGAAGCTTGCCTCGAGCAGCGCTGCTCGAGA

GATCTACGGGTGGCATCCCTGTGACCCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCACTCCAGT GCCCACCAGCCTTGTCCTAATAAAATTAAGTTGCATCATTTTGTCTGACTAGGTGTCCTTCTATAATAT TATGGGGTGGAGGGGGGTGGTATGGAGCAAGGGGCAAGTTGGGAAGACAACCTGTAGGGCCTGCGGG GTCTATTGGGAACCAAGCTGGAGTGCAGTGGCACAATCTTGGCTCACTGCAATCTCCGCCTCCTGGGT TCAAGCGATTCTCCTGCCTCAGCCTCCCGAGTTGTTGGGATTCCAGGCATGCATGACCAGGCTCAGCTA _{ATTTTTGTTTTTTTGGTAGAGACGGGGTTTCACCATATTGGCCAGGCTGGTCTCCAACTCCTAATCTCA} GGTGATCTACCCACCTTGGCCTCCCAAATTGCTGGGATTACAGGCGTGAACCACTGCTCCCTTCCCTGT

CCTTCTGATTTTGTAGGTAACCACGTGCGGACCGAGCGGCCGCAGGAACCCCTAGTGATGGAGTTGGC

CACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCT

TTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCCTGATGCGGTATTTT

CTCCTTACGCATCTGTGCGGTATTTCACACCGCATACGTCAAAGCAACCATAGTACGCGCCCTGTAGC

GGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGC

GCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAAT

CGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTTGGGT

GATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTC

TTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGGCTATTCTTTTGATTTA

TAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAA

TTTTAACAAAATATTAACGTTTACAATTTTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATA

GTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCAT

CCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCG

AAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGT

TTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAAT

ACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGA

AGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTT

TGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTAC

ATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGAT

GAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCG

GTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACG

GATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTT

ACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAA

CTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGAT

GCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGC

AACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCT

GGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGG

GCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAAC

GAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTAC

TCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTG

ATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAG

ATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCG

CTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGC

AGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGT

AGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTG

TCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGT

TCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATG

AGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAAC

AGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCC ACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGC AACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGT (SEQ ID N0:8)

2. Stage 2 of Transfection

Stage 2 Transfection requires transfecting the cells from Stage 1 Transfection with at least fourth construct i.e., the Rep/Cap construct, preferably, a plasmid. The Rep/Cap construct provides the necessary genes for AAV replication and capsid formation. Thus, the cells are transfected in Stage 1 so they contain and express H, R, G and in Stage 2, they are transfected so they contain and express RC.

In some forms, Stage 2 Transfection additionally includes transfecting the transformed cells from Stage 1 with a helper construct. In these forms, the cells are transfected in Stage 1 so they contain and express H, R, G and in Stage 2, they are transfected so they contain and express RC and H. i. Cap Gene

The cap (capsid) gene encodes the capsid proteins, VP1 (Viral Protein 1), VP2 (Viral Protein 2), and VP3 (Viral Protein 3), which form the viral capsid. The cap gene also determines the serotype of the AAV vector. Different serotypes have different capsid proteins, which influence tissue tropism, and transduction efficiency. ii. Rep gene

The rep gene is responsible for producing four non-structural proteins: Rep78, Rep68, Rep52, and Rep40. These proteins are generated via alternative splicing and alternative promoter usage within the rep gene. Rep78 and Rep68 are involved in initiating and regulating AAV DNA replication. They bind to the AAV inverted terminal repeats (ITRs) at the origins of replication and introduce site-specific nicks to initiate the replication process. Rep52 and Rep40 are primarily involved in the packaging of the AAV genome into capsids. They help in the assembly of the viral particles by interacting with the AAV capsid proteins and the viral DNA.

Significant reduction in the capsid titer was observed when Rep coding sequence was removed even if the Cap promoter region was kept intact. While not being bound by theory, the upstream Rep coding sequence may transactivate Cap genes which would lead to reduced Cap protein expression when Rep coding sequence was removed.

In some forms, the Rep/Cap plasmid contains the rep sequence under the control of endogenous AAV promotors e.g., p5 and pl9. In one preferred form, the rep sequence in the Rep/Cap plasmid is under the control of both AAV promoters p5 and pl9. In preferred forms, the AAV promoters p5 and p 19 are derived from AAV Serotype 2 (AAV2). However, the AAV promoters p5 and p!9 can also be derived from other AAV serotypes including but not limited to AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9.

In some forms, the Rep/Cap plasmid contains the cap sequence under the control of endogenous AAV promoters e.g., p40 or p81. In one preferred form, the cap sequence is under the control of the AAV promoter p40. In preferred forms, the AAV promoter p40 is derived from AAV Serotype 2 (AAV2). However, the AAV promoter p40 can also be derived from other AAV serotypes including but not limited to AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9.

In some forms, Stage 2 Transfection is performed by dosing about 1 pg of pAAV-RC5 at a PELDNA (polyethyleneimine: DNA) plasmid mass ratio of about 2:1.

In some forms, the Stage 1 transfected cells are incubated with the fourth construct at Staege 2, for about 40 hours to about 76 hours. The stagel transfected cells are typically incubated for 24-120 hrs post second transfection. The percentage of filled capsids are preferably obtained between 20 and 60h post second transfection, i.e. Stage 2.

Rep/Cap plasmids are available for purchase and are commercially available. Cell Biolabs’ AAV Helper-Free System allows the production of infectious recombinant human adeno associated virus (rAAV) virions without the use of a helper virus. In the AAV Helper-Free System, most of the adenovirus gene products required for the production of infective AAV particles are supplied on the plasmid pHelper (i.e. E2A, E4, and VA RNA genes) that is co-transfected into cells with human AAV vector DNA. The remaining adenoviral gene product is supplied by the 293 host cells, which stably express the adenovirus El gene. By eliminating the requirement for live helper virus the AAV Helper-Free System provides a safer and more convenient gene delivery system. In the AAV Helper-Free System, the rep and cap genes have been removed from the viral vector that contains A AV-2 ITRs and are supplied in trans on the plasmid pAAV- RC. The removal of the AAV rep and cap genes allows for insertion of a gene of interest in the viral genome.

Rep/Cap plasmids are available for purchase from commercial providers such as Cell Biolabs (Cell Biolabs (Cat. # VPK-405), Addgene, and Vector Biolabs.

Exemplary Rep-Cap Plasmid: ATCGTTAACGCCCCGCGCCGGCCGCTCTAGAACTAGTGGATCCCCCGGAAGATCAGAAGTTCCTATTC CGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCTGATCTGCGCAGCCGCCATGCCGGGGTTTTACG AGATTGTGATTAAGGTCCCCAGCGACCTTGACGAGCATCTGCCCGGCATTTCTGACAGCTTTGTGAACT GGGTGGCCGAGAAGGAATGGGAGTTGCCGCCAGATTCTGACATGGATCTGAATCTGATTGAGCAGGC ACCCCTGACCGTGGCCGAGAAGCTGCAGCGCGACTTTCTGACGGAATGGCGCCGTGTGAGTAAGGCCC

CGGAGGCCCTTTTCTTTGTGCAATTTGAGAAGGGAGAGAGCTACTTCCACATGCACGTGCTCGTGGAA

ACCACCGGGGTGAAATCCATGGTTTTGGGACGTTTCCTGAGTCAGATTCGCGAAAAACTGATTCAGAG

AATTTACCGCGGGATCGAGCCGACTTTGCCAAACTGGTTCGCGGTCACAAAGACCAGAAATGGCGCCG

GAGGCGGGAACAAGGTGGTGGATGAGTGCTACATCCCCAATTACTTGCTCCCCAAAACCCAGCCTGAG

CTCCAGTGGGCGTGGACTAATATGGAACAGTATTTAAGCGCCTGTTTGAATCTCACGGAGCGTAAACG

GTTGGTGGCGCAGCATCTGACGCACGTGTCGCAGACGCAGGAGCAGAACAAAGAGAATCAGAATCCC

AATTCTGATGCGCCGGTGATCAGATCAAAAACTTCAGCCAGGTACATGGAGCTGGTCGGGTGGCTCGT

GGACAAGGGGATTACCTCGGAGAAGCAGTGGATCCAGGAGGACCAGGCCTCATACATCTCCTTCAAT

GCGGCCTCCAACTCGCGGTCCCAAATCAAGGCTGCCTTGGACAATGCGGGAAAGATTATGAGCCTGAC

TAAAACCGCCCCCGACTACCTGGTGGGCCAGCAGCCCGTGGAGGACATTTCCAGCAATCGGATTTATA

AAATTTTGGAACTAAACGGGTACGATCCCCAATATGCGGCTTCCGTCTTTCTGGGATGGGCCACGAAA

AAGTTCGGCAAGAGGAACACCATCTGGCTGTTTGGGCCTGCAACTACCGGGAAGACCAACATCGCGG

AGGCCATAGCCCACACTGTGCCCTTCTACGGGTGCGTAAACTGGACCAATGAGAACTTTCCCTTCAAC

GACTGTGTCGACAAGATGGTGATCTGGTGGGAGGAGGGGAAGATGACCGCCAAGGTCGTGGAGTCGG

CCAAAGCCATTCTCGGAGGAAGCAAGGTGCGCGTGGACCAGAAATGCAAGTCCTCGGCCCAGATAGA

CCCGACTCCCGTGATCGTCACCTCCAACACCAACATGTGCGCCGTGATTGACGGGAACTCAACGACCT

TCGAACACCAGCAGCCGTTGCAAGACCGGATGTTCAAATTTGAACTCACCCGCCGTCTGGATCATGAC

TTTGGGAAGGTCACCAAGCAGGAAGTCAAAGACTTTTTCCGGTGGGCAAAGGATCACGTGGTTGAGGT

GGAGCATGAATTCTACGTCAAAAAGGGTGGAGCCAAGAAAAGACCCGCCCCCAGTGACGCAGATATA

AGTGAGCCCAAACGGGTGCGCGAGTCAGTTGCGCAGCCATCGACGTCAGACGCGGAAGCTTCGATCA

ACTACGCAGACAGGTACCAAAACAAATGTTCTCGTCACGTGGGCATGAATCTGATGCTGTTTCCCTGC

AGACAATGCGAGAGAATGAATCAGAATTCAAATATCTGCTTCACTCACGGACAGAAAGACTGTTTAG

AGTGCTTTCCCGTGTCAGAATCTCAACCCGTTTCTGTCGTCAAAAAGGCGTATCAGAAACTGTGCTACA

TTCATCATATCATGGGAAAGGTGCCAGACGCTTGCACTGCCTGCGATCTGGTCAATGTGGATTTGGAT

GACTGCATCTTTGAACAATAAATGATTTGTAAATAAATTTAGTAGTCATGTCTTTTGTTGATCACCCTC

CAGATTGGTTGGAAGAAGTTGGTGAAGGTCTTCGCGAGTTTTTGGGCCTTGAAGCGGGCCCACCGAAA

CCAAAACCCAATCAGCAGCATCAAGATCAAGCCCGTGGTCTTGTGCTGCCTGGTTATAACTATCTCGG

ACCCGGAAACGGTCTCGATCGAGGAGAGCCTGTCAACAGGGCAGACGAGGTCGCGCGAGAGCACGAC

ATCTCGTACAACGAGCAGCTTGAGGCGGGAGACAACCCCTACCTCAAGTACAACCACGCGGACGCCG

AGTTTCAGGAGAAGCTCGCCGACGACACATCCTTCGGGGGAAACCTCGGAAAGGCAGTCTTTCAGGCC

AAGAAAAGGGTTCTCGAACCTTTTGGCCTGGTTGAAGAGGGTGCTAAGACGGCCCCTACCGGAAAGC

GGATAGACGACCACTTTCCAAAAAGAAAGAAGGCTCGGACCGAAGAGGACTCCAAGCCTTCCACCTC

GTCAGACGCCGAAGCTGGACCCAGCGGATCCCAGCAGCTGCAAATCCCAGCCCAACCAGCCTCAAGT

TTGGGAGCTGATACAATGTCTGCGGGAGGTGGCGGCCCATTGGGCGACAATAACCAAGGTGCCGATG

GAGTGGGCAATGCCTCGGGAGATTGGCATTGCGATTCCACGTGGATGGGGGACAGAGTCGTCACCAA

GTCCACCCGAACCTGGGTGCTGCCCAGCTACAACAACCACCAGTACCGAGAGATCAAAAGCGGCTCC

GTCGACGGAAGCAACGCCAACGCCTACTTTGGATACAGCACCCCCTGGGGGTACTTTGACTTTAACCG

CTTCCACAGCCACTGGAGCCCCCGAGACTGGCAAAGACTCATCAACAACTACTGGGGCTTCAGACCCC

GGTCCCTCAGAGTCAAAATCTTCAACATTCAAGTCAAAGAGGTCACGGTGCAGGACTCCACCACCACC ATCGCCAACAACCTCACCTCCACCGTCCAAGTGTTTACGGACGACGACTACCAGCTGCCCTACGTCGT

CGGCAACGGGACCGAGGGATGCCTGCCGGCCTTCCCTCCGCAGGTCTTTACGCTGCCGCAGTACGGTT

ACGCGACGCTGAACCGCGACAACACAGAAAATCCCACCGAGAGGAGCAGCTTCTTCTGCCTAGAGTA

CTTTCCCAGCAAGATGCTGAGAACGGGCAACAACTTTGAGTTTACCTACAACTTTGAGGAGGTGCCCT

TCCACTCCAGCTTCGCTCCCAGTCAGAACCTGTTCAAGCTGGCCAACCCGCTGGTGGACCAGTACTTGT

ACCGCTTCGTGAGCACAAATAACACTGGCGGAGTCCAGTTCAACAAGAACCTGGCCGGGAGATACGC

CAACACCTACAAAAACTGGTTCCCGGGGCCCATGGGCCGAACCCAGGGCTGGAACCTGGGCTCCGGG

GTCAACCGCGCCAGTGTCAGCGCCTTCGCCACGACCAATAGGATGGAGCTCGAGGGCGCGAGTTACC

AGGTGCCCCCGCAGCCGAACGGCATGACCAACAACCTCCAGGGCAGCAACACCTATGCCCTGGAGAA

CACTATGATCTTCAACAGCCAGCCGGCGAACCCGGGCACCACCGCCACGTACCTCGAGGGCAACATG

CTCATCACCAGCGAGAGCGAGACGCAGCCGGTGAACCGCGTGGCGTACAACGTCGGCGGGCAGATGG

CCACCAACAACCAGAGCTCCACCACTGCCCCCGCGACCGGCACGTACAACCTCCAGGAAATCGTGCCC

GGCAGCGTGTGGATGGAGAGGGACGTGTACCTCCAAGGACCCATCTGGGCCAAGATCCCAGAGACGG

GGGCGCACTTTCACCCCTCTCCGGCCATGGGCGGATTCGGACTCAAACACCCACCGCCCATGATGCTC

ATCAAGAACACGCCTGTGCCCGGAAATATCACCAGCTTCTCGGACGTGCCCGTCAGCAGCTTCATCAC

CCAGTACAGCACCGGGCAGGTCACCGTGGAGATGGAGTGGGAGCTCAAGAAGGAAAACTCCAAGAG

GTGGAACCCAGAGATCCAGTACACAAACAACTACAACGACCCCCAGTTTGTGGACTTTGCCCCGGACA

GCACCGGGGAATACAGAACCACCAGACCTATCGGAACCCGATACCTTACCCGACCCCTTTAACCCATT

CATGTCGCATACCCTCAATAAACCGGTTAATTCGTGTCAGTTGAACTTTGGTCTCATGTCGTTATTATC

TTATCTGGTCACCAGATCCCCGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACAGCCCGGGCGT

TTAAACAGCGGGCGGAGGGGTGGAGTCGTGACGTGAATTACGTCATAGGGTTAGGGAGGTCCTGTATT

AGAGGTCACGTGAGTGTTTTGCGACATTTTGCGACACCATGTGGTCTCGCTGGGGGGGGGGGCCCGAG

TGAGCACGCAGGGTCTCCATTTTGAAGCGGGAGGTTTGAACGAGCGCTGGCGCGCTCACTGGCCGTCG

TTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTT

TCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAAT

GGCGAATGGAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCAT

_{TTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTT}

GAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAGAACGTGGACTCCAACGTCAAAGGGCGAAA

AACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGT

GCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGC

GAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGC

GGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCAGGTGGCA

CTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGC

TCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACAT

TTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGG

TGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAG

CGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCT

ATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTC

AGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGA

ATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAG GACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAA CCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAA CGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATG GAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAA ATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCC GTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGA GATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGA TTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAAT CCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAG ATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTT TGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAAT ACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTC GCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCA AGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCT TGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCC CGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGA GCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCG _{ATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGT} TCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCG TATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGA GCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAG CTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGC GGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCGCGCCGAT (SEQ ID NO: 10).

3. Host Cells and A A V Serotypes

Any cell that can be used to produce recombinant gene products can be used as an AAV host cell. Therefore, prokaryotic and eukaryotic cells are contemplated to fall within the scope of the instant invention. Accordingly, bacterial cells, yeast cells, insect cells, mammalian cells and so on can serve as the host cell. In some forms, the host cells are human cells e.g., human embryonic kidney cells (HEK 293 cells). Alternate human cell lines that could be used for AAV production include HEK293T, A549, PerC6, and HeLa cells.

The percentage of filled rAAVs in host cells transformed according to the methods disclosed herein ranges from about 50% to 100%, including 50% to 99%, 50% to 95%, 50% to 90%, 50% to 85%, 50% to 75%, and 50% to 65%.

The disclosed methods of producing rAAVs are generally suited for producing rAAVs of a wide range of serotypes such as AAV1, AAV2, AAV3, AAV 3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AVV10, AAV 11, AAV 12, BAAV, AAAV, AAV VR-942, AAV-DJ, or

AAV-DJ/8

Table 1 below gives a summary of the tropism of AAV serotypes, indicating the optimal serotype(s) for transduction of a given organ.

Table 1: Summary of the Tropism of AAV Serotypes

The disclosed methods can include other gene editing elements for targeted gene therapy. Gene editing elements such as CRISPR/Cas can be included in this approach, Also gene editing using Cre-lox could also be included.

When considering insect cell-based systems for Adeno-Associated Virus (AAV) production, several key insect cell lines are frequently utilized, primarily within the baculovirus expression system. Spodoptera frugiperda (Sf9 cells) are perhaps the most common and well- established insect cell line for AAV production. They are derived from the fall armyworm ovary and are widely used for expressing recombinant proteins and producing recombinant baculoviruses, which then carry the AAV genes for replication and packaging within the Sf9 cells. Sf21 are also from Spodoptera frugiperda and are the parent cell line of Sf9.

B. Insect cell Transduction Method

Also disclosed is an in vitro method of producing a recombinant AAV virion in an insect host cells. The method includes contacting insect host cells with: (i) a first nucleic acid sequence comprising a first rep gene (Rl) under the control of an immediate early promoter and a second nucleic acid sequence comprisinga second rep gene (R2) under the control of a late promoter; (ii) a third nucleic acid sequence comprising an AAV cap gene (C) under the control of a late promoter; and (iii) a fourth nucleic acid sequence comprising a heterologous gene of interest (GOI (gene of interest) (G)), to obtain a transformed insect cell containing Rl, R2, C and G. Each of the first, second, third and fourth nucleic acids can be provided in one or more viral expression constructs, for example, one or more baculovirus expression constructs. A baculovirus expression construct is used herein to refer to a modified baculovirus genome used to express foreign genes, ft typically involves replacing a non-essential gene (like polyhedrin) in the baculovirus genome with the gene of interest, allowing the host cells such as insect cells, with which the baculovirus construct is transduced, to produce the desired protein.

Recombinant baculoviruses are generated with a foreign gene of choice placed under control of a native promoter which is then expressed during the infection cycle and proteins are released during cellular lysis. AcMNPV is a highly pathogenic baculovirus that infects lepidopteran insects and is the most commonly used baculovirus for recombinant protein production in insect cells. It consists of a double-stranded DNA genome encoding over 150 genes (Shrestha, et al., J Virol, 2018. 92(23)). The infection process of the baculovirus is temporally regulated and can be divided into immediate early, delayed early, late, and very late phases. Accordingly, the genes necessary for these different phases are under the control of immediate early, delayed early, late, and very late promoters, respectively (reviewed in Grose, et al., Protein Expr Purif. 2021 Oct; 186: 105924. doi: 10.1016/j.pep.2021.105924).

Immediate early and delayed early promoters are activated prior to viral DNA replication. Immediate early promoters are transcribed by host cell RNA polymerase II and also utilize host transcription factors. The structural organization of immediate early and delayed early promoters differ greatly from late and very late baculovirus promoters and instead more closely resembles that of other eukaryotic promoters. The most distinguishing characteristic of baculovirus immediate early promoters is the conserved CAGT motif which functions as a transcription start site and plays an essential role in regulating downstream gene expression. In some instances, the immediate early promoters contain a TATA-like motif and/or an initiator sequence. The IE1 promoter is an immediate early promoter and is the most frequently used constitutive promoter available for baculovirus expression as it is active throughout all temporal phases as well as in uninfected insect cells. As such, it is frequently used in transient expression plasmids; the promoters for IE0/IEI and IE2 are also used in this manner. The 39k promoter is a delayed early promoter which is transactivated by the IE1 protein. The 39k promoter can also be used transiently when co-expressed with the IE1 protein. Both IE1 and 39k promoters have been shown to be particularly useful for secreted protein production. Another early viral promoter is the OPiel promoter.

The gp64 promoter is most commonly used in conjunction with gp64 fusions of heterologous proteins for viral display. More commonly utilized late promoters include the p6.9.

Very late promoters include the polyhydrin promoter and the plO promoter. The p 10 promoter has been described as the 101 nucleotides upstream from the ATG codon of the plO gene and suggested its use for driving foreign protein production. Compared to the polyhedrin promoter, plO becomes active a few hours earlier and drives slightly lower transcription levels. Promoters from other baculovirus species and organisms have also been used to increase very late expression. The orf46 promoter exhibited a high level of late expression in AcMNPV, and when placed downstream of a polyhedrin promoter, expression of eGFP increased 2-fold. Insect promoters that can be used as well as chimeric promoters are known in the art and reviewed in Grose, et al., Protein Expr Purif. 2021 Oct;186:105924. doi: 10.1016/j.pep.2021.105924). Examples include the silkworm Bornbyx mori actin promoter, the Trichoplusia nz'-derived basic juvenile hormone-suppressible protein 2 (pB2) promoter, Drosophila hsp70 promoter, the Sf21- derived GAPDH promoter.

In some forms, the immediate promoter is the immediate early gene (iel) promoter, optionally, fused with the homologous region 5 (Hr5) enhancer (Hr5-IE1) or Orgyia pseudotsugata immediate early 2 promoter (OPIE2).

In some forms, the late promoter is plO or polyhedrin.

In some forms, the insect cells are sf9 cells.

In some forms, the methods include contacting the host cells with the first, second, third and fourth nucleic acids simultaneously, wherein the first, second, third and fourth nucleic acids are in one or more viral expression constructs.

In some forms, the method includes contacting the cells with the first, second, third and fourth nucleic acids in a first transduction step and a second transduction step, wherein the time interval between the first and second transfection steps is at least about 2 to about 6 hours, preferably from about 3 to about 5 hours.

In some forms, the method includes contacting the cells with one or more viral expression constructs containing Rl, R2 and G, in the first transduction step, and contacting the cells with a viral construct comprising C in the second transduction step. Preferably, each viral construct is a baculovirus expression construct. In some forms, the method includes contacting the cells with about 2 to about 1000 baculovirus genome copies containing the cap gene, per cell, for example, from about 10 to about 80 baculovirus genome copies comprising the cap gene, per cell.

Experiments in this application demonstrate high filling by reducing the capsids. This is achieved by reducing the baculovirus titer encoding cap gene.

C. Methods of Harvesting, Verifying, and Storing rAAVs

Also disclosed are methods of harvesting, verifying, and storing the rAAVs from the transfected cells.

The AAV template and AAV rep and cap sequences are provided under conditions such that virus vector comprising the AAV template packaged within the AAV capsid is produced in the cell.

In some forms, the method includes the step of collecting or harvesting the virus vector from the culture. Harvesting the virus vectors from the cells can be done using methods known in the art e.g., via mechanical lysis, chemical lysis, or enzymatic lysis. For example, mechanical lysis requires disrupting the transformed cells using mechanical methods such as freeze-thaw cycles. In some forms, three cycles of freezing at -80°C followed by thawing at 37°C are used to break open the cells and release the rAAV particles. In a second example, cells can be chemically lysed using detergents such as Triton X-100 or sodium deoxy cholate to lyse cells by disrupting the cell membrane. In a third example, the transformed cells can by enzymatically lysed using enzymes such as trypsin or proteases to digest cell membranes and facilitate the release of viral particles.

In some forms, the virus vector can be collected by lysing the cells, e.g., after removing the cells from the culture medium, e.g., by pelleting the cells. In another embodiment, the virus vector can be collected from the medium in which the cells are cultured, e.g., to isolate vectors that are secreted from the cells. Some or all the media can be removed from the culture one time or more than one time, e.g., at regular intervals during the culturing step for collection of rAAV (such as every 12, 18, 24, or 36 hours, or longer extended time that is compatible with cell viability and vector production), e.g., beginning about 48 hours post-transfection.

After removal of the medium, fresh medium, with or without additional nutrient supplements, can be added to the culture. In one form, the cells can be cultured in a perfusion system such that medium constantly flows over the cells and is collected for isolation of secreted rAAV. Collection of rAAV from the medium can continue for as long as the transfected cells remain viable, e.g., 48, 72, 96, or 120 hours or longer post-transfection. In certain embodiments, the collection of secreted rAAV is carried out with serotypes of AAV (such as AAV 8 and AAV9), which do not bind or only loosely bind to the producer cells. In other embodiments, the collection of secreted rAAV is carried out with heparin binding serotypes of AAV (e.g., AAV2) that have been modified so as to not bind to the cells in which they are produced. Examples of suitable modifications, as well as rAAV collection techniques, are disclosed in U.S. Publication No. 2009/0275107, which is incorporated by reference herein in its entirety.

In some forms, one or more purification steps can be performed to remove impurities such as AAV components and host cell components and concentrate the viral vectors. AAV purification methods are known in the art and include but are not limited to density gradient centrifugation, chromatography, and ultrafiltration. For example, one or more purification steps can be performed via density gradient centrifugation using a Cesium Chloride (CsCl) Gradient in which rAAV particles are separated based on their buoyant density by ultracentrifugation in a CsCl gradient or a lodixanol gradient which provide a gentler separation process, preserving the integrity of rAAV particles. In a second example, chromatography methods such as ion exchange chromatography, affinity chromatography, and size exclusion chromatography can also be used to purify the rAAVs. In a third example, ultrafiltration such as by Tangential Flow Filtration (TFF) can be used to concentrate rAAV particles and exchange buffer solutions. TFF is efficient for large-scale purification and is scalable for clinical production.

In some forms, the methods include one or more steps for purifying the quality and purity of the extracted rAAVs. Exemplary quality control tests include:

(a) Quantification of Viral Genome Titer: Quantitative PCR (qPCR) or droplet digital PCR (ddPCR) are used to determine the number of viral genomes in the preparation;

(b) Infectious Titer: Infectious units are quantified using cell-based assays, such as transduction of a suitable cell line followed by flow cytometry or reporter gene expression analysis;

(c) Purity Assessment: SDS-PAGE and silver staining or Western blotting are used to assess the purity and integrity of the viral capsid proteins; and/or

(d) Endotoxin Testing: Limulus Amebocyte Lysate (LAL) assay is used to detect endotoxin contamination, which must be minimized for clinical applications.

In some forms, the methods include one or more steps for storing the extracted rAAVs to maintain their stability and integrity for therapeutic and research applications. Factors affecting the stability of rAAVS during storage include but are not limited to temperature (high temperatures can lead to denaturation of capsid proteins and degradation of viral genomes); pH (extreme pH levels can affect the structural integrity of the virus); freeze-Thaw Cycles (repeated freezing and thawing can cause aggregation and loss of viral particles; and buffer composition (the choice of buffer can impact the stability and solubility of rAAV particles).

Methods for storing rAAVs are known in the art. For example, for long-term storage, rAAVs should be stored at about -80°C. This temperature significantly slows down any biochemical reactions that could lead to degradation. As such, the viral particles can remain stable for several years. Although less ideal than -80°C, storing rAAVs at -20°C is still acceptable for long-term storage. However, stability may be somewhat reduced compared to - 80°C. In another example, for short-term storage such as up to a few weeks, rAAVs can be kept at 4°C. This temperature is suitable for immediate use without the need for repeated freeze-thaw cycles.

In some forms, to avoid repeated freeze-thaw cycles, rAAV preparations can be aliquoted into smaller volumes, e.g., in single-use vials, that can be used for individual applications. This minimizes the number of times each aliquot is thawed and refrozen. In some forms, the rAAVs can be stored in high-quality, sterile cryovials that are designed to withstand ultra-low temperatures.

In some forms, the rAAVs can be stored in a buffer that maintain their stability. Exemplary buffers include Phosphate-Buffered Saline (PBS), HEPES -Buffered Saline, and Tris- Buffered Saline (TBS). In some forms, adding stabilizing agents such as glycerol (up to 10%) or trehalose (1-5%) can help protect rAAV particles from damage during freezing and thawing. III. COMPOSITIONS

A. Compositions of Transformed Cells for rAAV Production

Also disclosed are transformed cells containing a high percentage of filled recombinant adeno-associated viruses (rAAVs), i.e., rAAVs having viral DNA and the desired transgene. The disclosed transformed cells can be one of two populations of intermediate cells produced following transfection of the host cells using the four- vector transfection method described above.

In some forms, the transformed cells are cells produced via Stage I of the transfection strategy. Transformed cells from Stage I of the transfection strategy generally contain three constructs i.e., the helper plasmid, the plasmid containing the transgene of interest, and the rep plasmid (plasmid containing the replicase gene).

In some forms, the transformed cells are cells produced via Stage 2 of the transfection strategy. Transformed cells from Stage 2 of the transfection strategy generally contain four constructs i.e., the helper plasmid, the plasmid containing the transgene of interest, and the rep plasmid (plasmid containing the replicase gene) and the replcap plasmid (i.e., the plasmid containing the replicase and capsid genes).

In some forms, the transformed cells are human cells e.g., human embryonic kidney cells (HEK 293 cells). Exemplary human cell lines that could be used for AAV production include HEK293T, A549, PerC6, and HeLa cells. The transformed cells generally contain more than 50% filled rAAVs. In some forms, the transformed cells contain from about 50% to about 100% filled rAAVs. In some forms, the transformed cells contain from about 50% to about 99%, from about 50% to about 95%, from about 50% to about 90% from about 50% to about 85%, from about 50% to about 75%, or from about 50% to about 65% of filled rAAVs.

B. Kits

Also provided are kits for producing a high percentage of filled recombinant adeno- associated viruses (rAAVs) using the disclosed methods. The kits can include reagents and components for both Stage 1 and Stage 2 transfections. An exemplary kit can include one or more of the first construct (Helper Plasmid), second construct (transfer plasmid), third construct, (Rep plasmid), and fourth construct (Rep/Cap plasmid).

The kits can also include transfection reagents such as polyethyleneimine (PEI) for facilitating the introduction of the constructs into the host cells and one or more buffer solutions for diluting and mixing the plasmids and transfection reagent.

In some forms, the kits can also include one or more vials of host cells e.g., HEK 293 cells suitable for transfection and production of rAAVs.

In some forms, the kits can also include additional materials and reagents such as sterile single-use vials, and pipettes and pipette tips.

In some forms, the kits can also include detailed instructions outlining the steps for Stage 1 and Stage 2 transfections, including incubation times, reagent volumes, and handling procedures.

In some forms, the kits provide a population of transformed cells containing a transgene of interest in a buffer solution and detailed instructions for its use.

IV. METHODS OF USE

Also disclosed are methods of using the recombinant AAVs made by the improved methods described in Section II above. RAAVs produced from the improved methods can be administered to one or more cells or tissue of a subject. For example, the rAAVs can be used to deliver a transgene of interest, such as a heterologous nucleic acid sequence, to cells or tissue of a subject. For example, rAAV can upregulate or downregulate an activity or product of a cell or tissue in the subject.

Inducible expression control elements are typically advantageous in those applications in which it is desirable to provide regulation over expression of the heterologous nucleic acid sequence(s). Inducible promoters/enhancer elements for gene delivery can be tissue-specific or - preferred promoter/enhancer elements, and include muscle specific or preferred (including cardiac, skeletal and/or smooth muscle specific or preferred), neural tissue specific or preferred (including brain-specific or preferred), eye specific or preferred (including retina- specific and cornea-specific), liver specific or preferred, bone marrow specific or preferred, pancreatic specific or preferred, spleen specific or preferred, and lung specific or preferred promoter/enhancer elements. Other inducible promoter/enhancer elements include hormone- inducible and metal-inducible elements. Exemplary inducible promoters/enhancer elements include, but are not limited to, a Tet on/off element, a RU486-inducible promoter, an ecdysone- inducible promoter, a rapamycin-inducible promoter, and a metallothionein promoter.

In embodiments wherein the heterologous nucleic acid sequence(s) is transcribed and then translated in the target cells, specific initiation signals are generally included for efficient translation of inserted protein coding sequences. These exogenous translational control sequences, which may include the ATG initiation codon and adjacent sequences, can be of a variety of origins, both natural and synthetic.

The virus vectors produced according to the present invention provide a means for delivering heterologous nucleic acids into a broad range of cells, including dividing and nondividing cells. The virus vectors can be employed to deliver a nucleic acid of interest to a cell in vitro, e.g., to produce a polypeptide in vitro or for ex vivo gene therapy. The virus vectors are additionally useful in a method of delivering a nucleic acid to a subject in need thereof, e.g., to express an immunogenic or therapeutic polypeptide or a functional RNA. In this manner, the polypeptide or functional RNA can be produced in vivo in the subject. The subject can be in need of the polypeptide because the subject has a deficiency of the polypeptide. Further, the method can be practiced because the production of the polypeptide or functional RNA in the subject may impart some beneficial effect.

The virus vectors can also be used to produce a polypeptide of interest or functional RNA in cultured cells or in a subject (e.g., using the subject as a bioreactor to produce the polypeptide or to observe the effects of the functional RNA on the subject, for example, in connection with screening methods). The present invention can be further understood by means of the following paragraphs and examples.

1. An in vitro method of producing a recombinant AAV virion in a host cell, comprising contacting the cell with two or more expression constructs in two stages, wherein:

(A) Stage T comprises contacting the cell with:

(i) a first expression construct, comprising one or more nucleic acid sequences encoding genes required for AAV production (H) (helper construct), optionally wherein the first construct is a plasmid (pH);

(ii) a second expression construct comprising one or more nucleic acid sequences encoding AAV rep genes (R) (Rep construct) under the control of AAV promoters, optionally wherein the second construct is a plasmid (pR); and

(iii) a third expression construct comprising a heterologous gene of interest (GOI (gene of interest) (G) construct); optionally wherein the third construct is a plasmid; and

(B) Stage II comprises contacting the cell with a fourth expression construct comprising one or more nucleic acid sequence encoding genes required for rAAV replication and AAV capsid assembly (RC) (RepCap construct, and optionally with H; optionally wherein the fourth construct is a plasmid; to obtain transformed cells comprising the H, R, G and RC and optionally, H, wherein the time interval between Stage I and stage II is at least 7 hrs.

2. The method of paragraph 1 wherein the helper construct comprises adenoviral E2, E40rf6, and VAI RNA genes operably linked to an origin of replication element and one or more other regulatory sequences.

3. The method of paragraph 1 or 2, wherein in the Rep construct, wherein the AAV rep coding region is operably linked to one or more regulatory sequences.

4. The method of any of paragraphs 1-3, wherein the GOI construct comprises AAV inverted terminal repats flanking a heterologous gene of interest operably linked to one or more regulatory sequences.

5. The method of any one of paragraphs 1-4, wherein the host cell is a mammalian host cell, wherein the AAV rep genes are under the control of one or more endogenous AAV promoters optionally, wherein the mammalian host cell is selected from the group consisting of HEK293, A549, PerC6, and HeLa cells. 6. The method of any one of paragraphs 1-5 wherein the RepCap construct comprises rep and cap coding sequences regions operably linked to one or more regulatory sequences.

7. The method of any one of paragraphs 1-5, wherein Stage 1 transfection and Stage 2 transfection are separated by a time interval of 12 hour to 24 hours.

8. The method of any of claims 1-7, wherein the heterologous gene of interest in the second construct encodes a therapeutic protein.

9. The method of any one of paragraphs 1-8, wherein the Rep construct comprises one or more nucleic acid sequences encoding Rep78 and Rep68 proteins.

10. The method of any one of paragraphs 1-9, wherein the RepCap construct comprises nucleic acid sequences encoding VP1, VP2, and VP3 proteins.

11. The method of any one of paragraphs 1-10, wherein the regulatory sequences include promoters, enhancers, and polyadenylation signals to optimize expression of the adenoviral, AAV rep, and AAV cap genes.

12. The method of any one of paragraphs 1-9, wherein the first, second, third and fourth constructs each comprise a vector selected from the group consisting of a plasmid, virus and minimalistic, immunologically defined gene expression (MIDGE) vector.

13. The method of any one of paragraphs 1-12, wherein the recombinant AAV virion produced has a capsid derived from AAV serotype 2 (, AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9.).

14. The method of any one of paragraphs 1-13, wherein the Helper construct comprises SEQ ID NO:7.

15. The method of any one of paragraphs 1-14, wherein the GOI construct comprises SEQ ID NO:8.

16. The method of any one of paragraphs 1-15, wherein the Rep construct comprises SEQ ID NO:9.

17. The method of any one of paragraphs 1-16, wherein the RepCap construct comprises SEQ ID NO: 10.

18. The method of any one of claims 1-17, further comprising culturing the host cells for an effective amount of time for rAAV assembly.

19. The method of paragraph 18 comprising culturing the cells for at least 72 hours.

20. The method of any one of paragraphs 1-18, further comprising purifying the recombinant AAV virions from the host cell culture supernatant, cell lyste or cell suspension. 21. A population of transformed cells produced by the method of any of claims 1-18, wherein the transformed cells are capable of producing recombinant AAV virions.

22. The cells of paragraph 21, wherein the transformed cells contain from about 50% to about 100% filled rAAVs.

23. The cells of claim 21, comprising the helper construct, the Rep construct, the GOI construct and the RepCap Construct.

24. The cells of paragraph 22 comprising up to about 90% filled AAV capsids.

25. The cells of paragraph 22, comprising up to about 100% filed AAV capsids.

26. The cells of any one of paragraphs 21-25, wherein the cells are HEK293 cells.

27. An in vitro method of producing a recombinant AAV virion in an insect host cell comprising contacting the cell with:

(i) a first nucleic acid sequence comprising a first rep gene (Rl) under the control of an immediate early promoter and a second nucleic acid sequence comprisinga second rep gene (R2) under the control of a late promoter;

(ii) a third nucleic acid sequence comprising an AAV cap gene (C) under the control of a late promoter; and

(iii) a fourth nucleic acid sequence comprising a heterologous gene of interest (GOI (gene of interest) (G)), to obtain a transformed insect cell comprising Rl, R2, C and G, wherein each of the first, second, third and fourth nucleic acids are in one or more viral expression constructs, optionally, one or more baculovirus expression constructs.

28. The method of claim 28, wherein the immediate promoter is the immediate early gene (iel) promoter, optionally, fused with the homologous region 5 (Hr5) enhancer (Hr5-IE1) or Orgyia pseudotsugata immediate early 2 promoter (OPIE2).

29. The method of paragraph 28 or 29, wherein the late promoter is plO or polyhedrin.

30. The method of any one of paragraphs 27-29, wherein the insect cells are sf9 cells.

31. The method of any one of paragraphs 28-30 comprising contacting the cells with the first, second, third and fourth nucleic acids simultaneously, wherein the first, second, third and fourth nucleic acids are in one or more viral expression constructs.

32. The method of any one of paragraphs 28-30, comprising contacting the cells with the first, second, third and fourth nucleic acids in a first transduction step and a second transduction step, wherein the time interval between the first and second transfection steps is at least about 2 to about 6 hours, preferably from about 3 to about 5 hours.

EXAMPLES

Methods

Materials

FreeStyle™ 293-F cells (Catalog #R79007, Thermofisher Scientific), Ex -Cell CD HEK293 Viral vector medium (Catalog #14385C-1000mL), and Gibco™ Glutamax (lOOx, Catalog # 35050061, Thermofisher Scientific) were used for cell culture experiments. AAV5 Helper free plasmids namely - pAAV-GFP (Part #AAV-400), pAAV-RC5 (Part #VPK-425), and pHelper (Part #340202) were purchased from Cell Biolabs, San Diego, CA. These plasmids produce rAAV2/5 rep and cap genes from AAV2 and AAV5 serotypes respectively) particles with enhanced green fluorescent protein (eGFP) transgene cargo. All three plasmids were amplified via bacterial transformation using One Shot™ Stbl3™ E. coli strain purchased from Thermo Fisher Scientific, Waltham, MA (Catalog #C737303). The plasmids were purified using Plasmid Plus Mega kit (Catalog #12981, Qiagen) and filtered through 0.22 pm polyvinylidene fluoride (PVDF) filter. The concentration of all plasmids was determined using NanoDrop™ ONEc spectrophotometer (Thermo Fisher Scientific, Waltham, MA) and stored at -20°C. Transfection-grade linear polyethyleneimine (Transporter 5 Transfection reagent, Catalog # 26008-5, Polysciences, Warrington, PA) was used for transient transfection reactions. AAV5 titration ELISA kits (Catalog #PRAAV5, Progen) were used for quantifying capsid titer. DNase I (Catalog #M0303S, New England Biolabs) was used for digesting DNA fragments of crude lysate not encapsulated into viral capsids. The ddPCRTM (droplet digital PCR) reagents and consumables namely, QX200 EvaGreen supermix (Catalog #1864035), droplet generation oil for EvaGreen (Catalog #1864112), ddPCRTM droplet reader oil (Catalog #1863004), DG32 automated droplet generator cartridges (Catalog # 1864108), and ddPCR 96-well semi-skirted plates (Catalog #12001925) were all purchased from Bio-Rad Laboratories, Hercules, CA. Custom-made single- stranded DNA oligomers (synthesized using the services of Integrated DNA Technologies, Coralville, IA) dissolved in nuclease-free water as per manufacturer’s instructions were used as primers for amplifying target sequences in ddPCRTM reactions.

Engineering pRep Plasmids pAAV-Rep plasmid were constructed using molecular cloning approaches (PCR amplification, extraction and transformation) previously described (Russell & J., 2001). Q5 High-Fidelity DNA Polymerase 2X master mix (New England Biolabs) was used to amplify PCR products using pAAV-RC5 as the template. The sequences of the primer sets used for this reaction are: poriF: 5’-GTGAGCGAGGAAGCGGAAGAG-3’ (SEQ ID NO:1) pRepR: 5’-TTATTGTTCAAAGATGCAGTCATCCAAATC-3’ (SEQ ID NO:2) pRepF: 5 ’ - ACTGC ATCTTTG A ACAATAACCC ATTC ATGTCGC AT ACCCTCAA TAAAC-3’(SEQ ID NO:3) poriR: 5’-CTCTTCCGCTTCCTCGCTCAC-3’ (SEQ ID N0:4)

The PCR amplified products were separated using agarose gel electrophoresis (1% agarose gel) and extracted using the Zymoclean gel DNA recovery kit (Zymo Research). The extracted products were assembled using HiFi 2X Assembly mix (New England Biolabs) and the products of the assembly reaction were transformed into the E. coli Stbl3 competent cells (Invitrogen). Ampicillin resistant colonies formed after overnight incubation of plates at 37 °C were then screened for a positive clone. Plasmids extracted from the colonies using the Zyppy plasmid miniprep kit (Zymo Research) were fully sequenced to ensure 100% match (Primordium Labs). A large scale pAAV-Rep purification was done using the EndoFree Plasmid Mega Kit (Qiagen).

Cell Culture and Transient Transfection

A vial of frozen cells stored in liquid nitrogen at cryogenic temperature was thawed in a 37 °C water bath and transferred into pre-warmed 30 mL Ex-Cell CD HEK293 viral vector medium supplemented with 300ul of lOOx Glutamax in a 125 mL shake flask (Fisher brand sterile PC flasks, Catalog #PBV125). The culture was maintained at 37 °C inside a humidified incubator (Thermo Scientific HERA cell VIOS 160i) with 5% CO2 on a vibration resistant orbital shaker (ORBI SHAKER™ CO2, Benchmark) at 135 rpm. Cell viability was maintained > 90% before the start of all transfection experiments.

Prior to transfection, the seed cells were maintained in the exponential phase at a cell density between ~1 to 2 million cells/mL. For triple plasmid transfection, a transfection mix was freshly prepared with equimolar 1:1: 1 ratio of pAAV-GFP:pAAV-RC5:pHelper for each transfection reaction. The amount of total plasmids dosed per million cells was ~ 1 pg at PELDNA plasmid mass ratio of 2:1. The transfection mix volume was ~2mL. The transfection mix was prepared by incubating plasmids and PEI in ImL of Ex-cell media in two separate tubes for 1 minute. The contents of the DNA tube were then directly added into the PEI tube drop-by- drop. This step was followed by vortex agitation for 10 seconds and incubation of the mixture at room temperature for 10 minutes. The transfection mix was directly added to the cell culture flask. Cell pellets (~1 million cells) were collected at 72 hours post transfection to estimate the vector genome (vg) and capsid titers.

For four-plasmid transfection, a transfection mix was freshly prepared with equimolar 1:1:1 ratio of pAAV-GFP:pRep:pHelper for each transfection reaction. The amount of total plasmids dosed per million cells was ~1 pg at PELDNA plasmid mass ratio of 2: 1. After 24 hours post first transfection, second transfection reaction was performed dosing -1 pg of pAAV- RC5 at PELDNA (polyethyleneimine: DNA) plasmid mass ratio of 2:1. Cell pellets (-1 million cells) were collected at 48 hours and 72 hours post transfection to estimate the vector genome (vg) and capsid titers. Remaining cells were pelleted in a 50mL tube and stored in -80 °C to purify AAVs for analytical characterization using size-exclusion chromatography (SEC), Multiangle light scattering (MALS) and Transmission electron microscopy (TEM).

Average Capsid Titer Estimation

Capsid titer was estimated from the cell pellet samples prepared previously via AAV5 capsid ELISA (Enzyme-Linked ImmunoSorbent Assay). To quantify the total number of capsids in the cells, one sample tube containing cell pellets was thawed on ice. The pre-chilled RIP A buffer (Catalog #R0278-50ML, Millipore Sigma) was added to the cells. About -100 pL of lysis buffer per 1 million cells were used. The cells were suspended, briefly vortexed, and processed by a total of three cycles of freezing at ethanol bath (mixed by dry ice and 70% ethanol) for 10 minutes and thawing at 37 °C water bath for 10 minutes. The sample was clarified by centrifuging at 12100g, 4 °C for 15 minutes. The supernatant was transferred to clean microtubes and followed by an AAV5 ELISA analysis. Lysed samples were diluted by the diluent buffer provided by the kit. AAV5 capsid standards provided by the ELISA kit were diluted to a range of 1.2OxlO¹⁰ to 1.88xl0⁸ capsids/mL. Two replicates of 100 pL diluted standards or samples were loaded onto the antibody-coated plate and incubated at 37 °C for 1 hour. The plate was then washed by 200 pL lx wash buffer three times, followed by incubation with 100 pL of diluted biotinylated anti-capsid Rep antibody at 37 °C for 1 hour. The plate was washed by 200 pL lx wash buffer three times and followed by further incubation with 100 pL diluted strep enzyme at 37 °C for 1 hour. After the last three washes using 200 pL lx wash buffer, 100 pL substrate solution was added, and the mixture was incubated at room temperature for 15 minutes. The 100 pL stop solution was subsequently added. The absorbance readout of the ELISA reactions was measured using a microplate reader at 450 nm (BioTek Instruments, Winooski, VT). A standard curve was fitted using a four-parameter logistic (4PL) model between the absorbance values and the known capsid concentrations of the standards. Capsid concentration (capsids/mL) of the experimental samples were estimated using the fit equations for the measured absorbance readouts.

Vector Genome (vg) Titer Estimation

To estimate the copy number of AAV vector genome, crude lysate samples previously prepared were used. Firstly, DNA fragments in the lysate that are not encapsulated were digested via DNaseT treatment. A 50 u I . digestion reaction was prepared containing the following components - 2 pL of sample, 5 pL of lOx DNase buffer, 5 pL of DNase I (10 U), and 38 pL DNase-free water. The samples were incubated at 37 °C for 2 hours. A 50 pL volume of 10 mM EDTA was added to the reaction to inhibit the activity of DNase I. The samples were diluted serially so that the readouts of the droplet digital polymerase chain reaction (ddPCR) are less than the saturation value of detector (10⁴ copies/pL). All reactions for estimating genome copies were performed using gfp_forward and gfp_reverse primers whose sequences are provided as below. gfpjbrward: 5’ GCAAAGACCCCAACGAGAAG 3’ (SEQ ID NO:5) gfp_reverse: 5’ TCACGAACTCCAGCAGGACC 3’ (SEQ ID NO:6)

DNase-free water was used as a non-template negative control sample. In each well of a 96-well plate, reaction mixture consisting of the four components were added to 6.6 pL water, namely, 11 pL 2x EvaGreen supermix, 0.22 pL of 10 pM primer solution and 2.2 pL template. Aqueous droplets in oil prepared in QX200™ automated droplet generator using automated droplet generator cartridges was collected in ddPCR 96-well semi-skirted plates. The plates were sealed at the top using Aluminum foil before loading inside a thermal cycler (CFX96 Deep Well™ Real-Time System, Biorad). A thermal cycle program with multiple steps were used to amplify the target sequence: enzyme activation at 95 °C for 5 minutes, 40 cycles comprising two steps (denaturation at 95 °C for 30 seconds followed by primer annealing/extension at 60 °C for 1 minute), and lastly, a signal stabilization step comprising a cool down at 4 °C for 5 minutes followed by heating at 90 °C for 5 minutes. The amplified products were then read using QX200™ droplet reader controlled using QuantaSoft software for data acquisition.

Sample Preparation for SEC-MALS Analysis

Purified AAVs from cell pellets stored in a 50mL tube inside -80°C were prepared using AAVpro purification kit Midi (All serotypes, TaKaRa Bio, Catalog # 6675) following manufacturer’s protocol. Briefly, the frozen pellet was loosened by thawing at 4°C and suspended in lOmL of AAV extraction Solution A plus for 15 seconds by vortex agitation. The suspended solution was incubated at room temperature for 5 minutes and again vortexed for 15 seconds. This was followed by centrifugation at 4000g for 10 minutes at 4°C. The supernatant was collected and transferred to a new 14 ml tube without disturbing the solid debris. About l/10th of volume of AAV Extraction solution B was added to the collected supernatant and mixed gently. To this mixture, l/100th of Cryonase Cold-active nuclease was added and incubated for an hour at 37°C to digest the residual DNA fragments. The digested product was then treated with 1/1 Oth of the volume of Precipitator A, vortex ed for 10 seconds and incubated for 30 minutes at 37°C. The resultant mixture was vortexed for 10 seconds, treated with l/20th of the volume of Precipitator B and vortexed again for 10 seconds followed by a centrifugation at 5000g for 5 minutes at 4°C. The supernatant was collected and filtered through Millex-HV 0.45 mm filter. The filtrate containing AAV particles was added to an Amicon-Ultra-15, lOOkDa filter and spun at 2000g for 5 minutes at 15 °C to ensure the sample volume is < 1.5mL. After discarding the filtrate, the sample collected inside the compartment was mixed with 5ml of suspension buffer and centrifuged for 5 minutes at 15 °C. This step was repeated four times to achieve high degree of purity. The final wash step was concentrated to a volume of 300 l to achieve highly concentrated and pure AAV particles.

Purified and concentrated AAV sample produced via transient transfection was used for biophysical characterization of AAV particles using SEC-MALS. 20pl of purified and concentrated sample was injected into SEC (Agilent 1260 Infinity II) integrated to MALS set up (Wyatt Technologies). The SEC set up consists of an SRT SEC-100 column (Sepax, SN: 3A47249, 5mm, lOOOAo, 4.6 x 300 mm) with lx PBS as a mobile phase at 0.3ml/min. UV absorbance traces at 260nm, and 280nm were recorded at the chosen retention time for all the injected samples. Simultaneously, the light scattering intensity trace characterizing the size of different molecules were obtained at different angular positions (Wyatt Technologies, DAWN) for the chosen retention time synchronized with the SEC trace. The ratio of absorbances at 260nm to 280nm was used to assess the AAV samples (~1.3 for filled capsids and -0.6 for empty capsids).

Sample Preparation for TEM Imaging

AAV samples purified as described above for SEC-MALS contain host cell impurities. These impurities were removed to improve the image quality for the assessment of filled capsids % using TEM microscopy. Affinity purification of the AAV samples (DynabeadsTM CaptureSelect™ AAVX magnetic Beads, ThermoFisher Scientific, Cat. # 2853522001) was used following the manufacturer’s protocol. Briefly, 40 pl of slurry containing beads was placed in 1.5mL Eppendorf tube. The beads were washed with 460 pl of binding/wash buffer via vortex agitation and removal of supernatant after bead sedimentation using magnetic force. 300 pl of AAV sample prepared as described above for SEC-MALS was adjusted to 500pl using binding/wash buffer and the sample was mixed with washed magnetic beads and vortexed briefly for 10 seconds and incubated for 30 minutes at room temperature. The beads were then pulled to the tube wall using magnetic force and the supernatant was removed. The beads bound AAV were washed twice with binding/wash buffer, each time sedimenting the beads to the tube wall using magnetic force. Purified AAV at high concentration was then eluted using 50 pl of elution buffer after incubating with the beads for 10 minutes. The eluted sample was then neutralized by adding 1 pl of neutralization buffer for every 10 pl of eluate. Affinity purified AAV samples were then inactivated using glutaraldehyde (1% final concentration) before Transmission electron microscopy (TEM) evaluation.

Negative stain TEM microscopy was performed at MIT ilab core facility using JEOL 2100 FEG microscope. Briefly, the sample preparation involves applying 10 pl of sample on a 200-mesh copper grid coated with a continuous carbon film which is allowed to settle for about 60 seconds. The excess solution on the grid was removed by gently touching the grid with Kim wipes. This step was followed with applying 10 pl of negative stain uranyl acetate solution. After allowing the staining solution to settle for 30 seconds, the remaining excess staining solution was removed by gently touching the edge of the grid with Kim wipes. The stained sample on the grid was allowed to dry at room temperature before mounting on a JEOL single tilt holder housed in the TEM column. The specimen was cooled down to cryogenic temperature using liquid nitrogen and imaging was done following minimal dose method to avoid sample damage by electron beam. The microscope was operated applying 200 kV electric potential to the electron gun at a magnification factor ranging 10000-60000. Images of particles were acquired in multiple fields of view for each sample using a Gata 2k x 2k UltraScan CCD camera.

Results

Studies based on mechanistic model of AAV production using single cell reaction network also suggested a 20 h time lag for vDNA replication with respect to capsid production [16] attributes for increased production of empty capsids. To achieve coordinated kinetics between vDNA replication and capsid protein expression, transfection strategy using four plasmids was developed. Firstly, a new plasmid, pRep was created from pRC5 using molecular biology approaches. Gibson assembly protocol [17] was used to assemble the PCR amplified DNA fragments of pRC5 which excludes cap gene sequences. pRep plasmid contains rep gene sequence under the control of endogenous AAV promoters (p5 and pl 9) and expresses Rep isoforms only. Since Rep78/68 are necessary for vDNA replication, HEK293 cells was first transfected with triple plasmids containing pGFP, pRep and pHelper. After 24 hours post transfection, the cells were re-transfected with pRC5 plasmid. 24-hour time difference between the first and second transfection was allowed to produce a sufficiently high number of replicated vDNA copies for packaging into empty capsids. Figure 2 shows the population of filled and empty capsids obtained via transient transfection of HEK293 cells using four plasmids with 24- hour time difference between the first and second transfections. It was observed that the average capsid and vector genome titers agree very closely indicating most of the capsids are filled with vDNA cargo. The standard error of the technical repeats of the two measurements overlaps suggesting that within the statistical variability of these measurements, the difference between capsid and vector genome titers if any is very small. This experiment was repeated several times and consistently observed > 90% of the capsids Filed with vDNA in all the biological replicates confirming high per cell enrichment of Filed capsids.

Next, analytical evidence to conFrm per cell enrichment of Filed capsids via the four- plasmid transient transfection scheme was obtained. Samples containing a mixture of Filed and empty capsids in different proportions were characterized using a biophysical metric - the ratio of absorbance at 260nm and 280nm (AR = Abs260nm/Abs280nm). AR is a ratiometric measurement and it is independent of sample concentration. Purihed samples containing only Filed and empty capsids will have an AR of between about ~ 1.3 and ~ 0.61, respectively [18]. Figure 4 shows AR of commercially procured ‘pure’ empty and Filed capsid samples that were treated as standards for comparison. As expected, AR values of Filed and empty capsids are very close to the previously reported values. Figure 6 shows theoretically evaluated AR as a function of fill fraction of GFP transgene sequence using the extinction coefficients of AAV components published in the literature [19]. Interestingly, AR has a non-linear relation with the fill fraction and agrees very closely with similar experimental data reported earlier [18]. AR increases monotonically with increasing number of filled capsids in the sample and almost reaches a plateau when approaching a fill fraction of 1.0 (100% filled).

Next, the AR values of the test samples prepared in-house from cells were compared after a 72 h post second transfection using the four-plasmid scheme. Unlike the commercial standards, test samples prepared in-house were not fully pure and contained other molecules from HEK293 cells. However, the downstream purification of the samples allowed quantification of the population of empty and Filed capsids using sensitive biophysical and biochemical assays. Heterogenous test samples containing AAV particles produced via four- plasmid transfection were analyzed using SEC- MALS (Size exclusion chromatography and Multi-angle light scattering). SEC enables the separation of molecules of different sizes contained in the samples. Test samples using complementary measurements - UV absorbance at 260nm and 280nm and scattering intensity profile were also conducted. Figure 5 shows the recorded traces of the molecules of test samples separated chromatographically based on their size. Large molecules are retained in the column for a shorter duration than smaller molecules and vice-versa. The peaks within the blue-shaded window at 10.3 min where both absorbance and scattering intensity are maximum correspond to single AAV particles. The small shallow peak at 8.5 min likely corresponds to aggregated AAV particles. Also, absorbance and scattering intensity depend on the number and size of the particles. Hence, the lower absorbance and intensity values of eluant at 8.5 min possibly suggests that only a small population of aggregated AAVs is present in the test sample and most of the AAVs are in single particle suspension. Both single particle and aggregated AAV populalon have an AR ~1.3 suggesting that approximately 100% of the particles produced were Filed with vDNA.

Since the scattering intensity scales to the sixth power of a particle size [21], particles as big as the size of AAVs were clearly detected from the scattering profile (Figure 5). However, particles smaller than AAV show a decrease in scattering intensity. Several closely spaced peaks (11.7, 12.1, 13.3, 13.6, 13.9 min) in the absorbance profile were not seen in the MALS trace suggesting the scattering intensity of these molecules are significantly smaller than that of the AAVs. Multiple peaks between 11 and 13 min indicate molecules from HEK293 cells [20] that are known to be eluted during chromatographic separation of AAVs. These signatures were not shown in the MALS trace as expected because their scattering intensities were too small to be detected (Figure 5).

Next, the effect of time difference between the first and second transfection reaction was investigated to understand how this affects the filling of the capsids. Figures 7A and 7B shows the trend of capsid filling as the time difference between the first and second transfection reaction was increased from 0 h to 24 h. The population of Filed capsid production increases with increasing time difference between the transfections. DNA replication in cells is a time- consuming process and vDNA synthesis requires formation of replication compartments inside the nucleus with the assistance of Rep78/68 proteins [15]. Studies showed at least 18 hours are required to create many replicated transgene copies containing forty lacO sites inside the nucleus [15] of live HeLa cells. This was further corroborated by the modeling prediction that indicated a 20-hour time difference to compensate for the mismatch between vDNA replication and capsid production 116]. Also, controlled expression of capsid proteins using Tet-on inducible promoter earlier using four plasmids showed a filled percentage of -50% for a 12 h time difference [22]. However, unlike the four- vector approach reported here, Tet-on inducible promoter is known for leaky gene expression and this can result in the increased production of empty capsids. Our experiment directly confirmed that a sufficiently large pool of vDNA copies become readily available for packaging preformed AAV capsids close to 24 h post first transfection. Furthermore, it was noticed that the increase in capsid Filing appears to gain saturation close to 24 h post first transfection thereby suggesting a majority of the capsids produced are Filed at this time point.

The enrichment of Filed capsid population via four plasmid transfection schemes was also confirmed using Transmission Electron Microscopy (TEM). Figures 8A and 8B show representative TEM micrographs of negatively stained commercial standard and AAV samples produced via four-plasmid scheme (referred as test samples) respectively. Standard samples show heterogeneity in the capsid population - Filled, empty, and partially Filed capsids were clearly discerned (Figures 8A and 8B). In contrast, test samples prepared in-house show population of Filed capsid only. Analysis of TEM micrographs of several test samples from hundreds of Felds of view conFrmed that most of the capsids are Filed with vDNA cargo (Figures 8A and 8B).

Prior studies demonstrate that the highest percentage of Filed capsids produced via transient transfection of HEK293 cells is -40% [13]. There is no AAV production process by which 95% of the capsids produced upstream are Filed with vDNA cargo. The present four- vector approach (exemplified herein using plasmids as the transfection vector), is the Frst to demonstrate such high enrichment of Filed capsid population (approximately 97%) from transient transfection of HEK293 cells in culture.

The results in FIGs. 9A-9C are from experiments set up to test if the cytotoxicity of transient transfection scheme contributes for the reduced vector genome titer in four plasmid transient transfection. In order to verify this, the cells were exposed to transfection mix briefly (for about 6hours) instead of long exposure as done previously. However, the data showed that this does not seem to have any major effect on the achievable vector genome titer although the percentage of filled capsids was close to 100% as observed previously at 48h and 72h post transfection. Role of helper gene in improving the total filled capsids

Previous experiments highlighted the importance of early Rep expression that facilitated replication of the rAAV genome, which is the pivotal mechanism for high filled capsids in mammalian cells. In the scheme shown in Fig. 12, studies further explored the role of helper genes, as shown in Figure 13 (G, R, H, RC and M refers to pAAV-GFP, pRep, pHelper, pAAV- RC5 and pMock plasmids respectively).

Briefly, a small volume (~2mL) suspension HEK293 cell culture (~ 1 Million cells/mL) grown in a 6-well plate at 5% CO2 agitated at 135 rpm were transiently transfected at 1:2 DNA:PEI ratio in equimolar plasmid concentration. Plasmids were mixed with cationic PEI and added either as in a single dose (Dose 1) or in two sequential doses (Dose 1 and Dose 2) separated by 24h. A total of Img/mL of total plasmid was used in all transfections.

Engineering pMock Plasmid

Molecular cloning approaches used to construct pMock plasmid (PCR amplification, extraction and transformation) were previously documented (Russell & J., 2001). Q5 High- Fidelity DNA Polymerase 2X master mix (New England Biolabs) was used to amplify PCR products using pAAV-GFP as the template. The sequences of the primer sets used for this reaction are pvectorF: 5’-CTGGCCTTTTGCTCACATGTGGCGCCTGATGCGGTATTTTC -3’ 9SEQ ID NO: 13);

AmpR: 5’- CATGGTTATGGCAGCACTGC -3’ (SEQ ID NO: 14);

AmpF: 5’- GCAGTGCTGCCATAACCATG -3’ (SEQ ID NO: 15); and pvectorR: 5’- ACATGTGAGCAAAAGGCCAGC -3’(SEQ ID NO: 16). The PCR amplified products were separated using agarose gel electrophoresis (1% agarose gel) and extracted using the Zymoclean gel DNA recovery kit (Zymo Research). The extracted products were assembled using HiFi 2X Assembly mix (New England Biolabs) and the products of the assembly reaction were transformed into E. coli Stbl3 competent cells (Invitrogen). Ampicillin resistant colonies formed after overnight incubation of plates at 37degC were then screened for a positive clone. Plasmids extracted from the colonies using the Zyppy plasmid miniprep kit (Zymo Research) were fully sequenced to ensure 100% match (Primordium Labs). A large scale pAAV-Rep purification was done using the Endotoxin Free Plasmid Mega Kit (Qiagen).

The results show that pHelper plasmid has a major impact on the capsid production in single and sequential transient transfections. The presence of helper genes resulted in an increase the capsid titers, as demonstrated in Condition 1. Hence, pHelper (H) was introduced in the second dose of the sequential transfection as performed in condition 7, which resulted in a higher capsid titer (~ l x l l capsids/mL) compared to Condition 8 without pHelper (H).

Exclusion of H has a major impact on the capsid titer in single dose (Condition 1 vs. Condition 2) and sequential dose (Condition 3 vs. Condition 4) transfections. Also, the act of sequential transfection seems to negatively impact capsid production (Condition 1 vs. Conditions), possibly due to the influence of bacterial gene components. Inclusion of H in the Dose 2 (Conditions 5, 6 and 7) increased the capsid titer by several fold compared to its exclusion (Condition 8) if H is included in Dose 1. Including H in both doses of sequential transfection significantly increased the capsid titer (-1 x 1011 capsids/mL as in Condition 7) tending to approach the capsid production limit of single dose transfection (~ 2 x 1011 capsids/mL as in Condition 1) typically achieved in a traditional triple plasmid scheme. Vector genome estimates using ddPCR revealed that > 90% of the total capsids produced for Conditions 7 and 8 were filled with ssDNA cargo.

Temporal Rep proteins modulation in insect cells using baculovirus expression system (BEVS)

Conventional BEVS system involves use of recombinant baculovirus that express Rep proteins under the control of either an immediate early promoter such as IE1 (immediate early promoter) or a very late promoter (polyhedrin or plO promoters). The low filled capsid from these recombinant baculovirus systems is either due to the delayed onset of vector genome replication, or inadequate Rep proteins. Thus, to improve the package of rAAVs in the insect cells, recombinant baculoviruses were engineered to express Rep under the control of immediate promoters to facilitate the rapid onset of AAV vector replication followed by a strong and delayed Rep expression using polyhedrin promoter as shown in Figure 14. The early onset of Rep expression is expected to increase the replication of the vector genome, as observed in the transient transfection of HEK293 cells.

In the disclosed approach, recombinant BV that encodes Cap gene under the control of a very late promoter, such as plO, was also engineered, to maximize the delay in the capsid production by at least 18 hours post-infection. This approach also allows for the option of a single inoculation of recombinant baculovirus. Specifically, experiments were conducted to study the temporal expression of Rep proteins under the control of both immediate early and very late promoters along with rBV encoding the Cap proteins inoculated at medium/low titer as shown in Figure 16. Interestingly, early onset of Rep expression along with medium titer of rBV encoding the Cap gene resulted in > 90% of filled capsids (condition 1). However, lower cap gene regulation also resulted in higher filling (60-70% filled capsids) (condition 4) compared to rBV_cap inoculation at high titer (condition 6). The requirement for the low copies of cap gene hypothesis is also validated in conditions 2 and 3, with medium and low rBV_cap titer, respectively, resulting in -90% filled capsids. An increase in filled capsids was also observed when the rBV encoding Cap gene was delivered as a second dose to further delay (4 hours) the cap expression, resulting in a 20% increase in filled capsids (condition 2 vs condition 5). These studies identified that 4 hours temporal offset is optimal as the entry of rBV is severely reduced in sf9 cells pre- infected with rBV from the first dose. Figure 17 shows the representative raw data from the mass photometry for conditions 1-6.

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It is understood that the disclosed method and compositions are not limited to the particular methodology, protocols, and reagents described as these can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the method and compositions described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

CLAIMS We claim:

1. An in vitro method of producing a recombinant AAV virion in a host cell, comprising contacting the cell with two or more expression constructs in two stages, wherein:

(A) Stage T comprises contacting the cell with:

(i) a first expression construct, comprising one or more nucleic acid sequences encoding genes required for AAV production (H) (helper construct), optionally wherein the first construct is a plasmid (pH);

(ii) a second expression construct comprising one or more nucleic acid sequences encoding AAV rep genes (R) (Rep construct) under the control of AAV promoters, optionally wherein the second construct is a plasmid (pR); and

(iii) a third expression construct comprising a heterologous gene of interest (GOI (gene of interest) (G) construct); optionally wherein the third construct is a plasmid; and

(B) Stage II comprises contacting the cell with a fourth expression construct comprising one or more nucleic acid sequence encoding genes required for rAAV replication and AAV capsid assembly (RC) (RepCap construct, and optionally with H; optionally wherein the fourth construct is a plasmid; to obtain transformed cells comprising the H, R, G and RC and optionally, H, wherein the time interval between Stage I and stage II is at least 7 hrs.

2. The method of claim 1 wherein the helper construct comprises adenoviral E2, E40rf6, and VAI RNA genes operably linked to an origin of replication element and one or more other regulatory sequences.

3. The method of claim 1 or 2, wherein in the Rep construct, wherein the AAV rep coding region is operably linked to one or more regulatory sequences.

4. The method of any one of claims 1-3, wherein the GOI construct comprises AAV inverted terminal repats flanking a heterologous gene of interest operably linked to one or more regulatory sequences.

5. The method of any one of claims 1-4, wherein the host cell is a mammalian host cell, wherein the AAV rep genes are under the control of one or more endogenous AAV promoters optionally, wherein the mammalian host cell is selected from the group consisting of HEK293, A549, PerC6, and HeLa cells.

6. The method of any one of claims 1-5 wherein the RepCap construct comprises rep and cap coding sequences regions operably linked to one or more regulatory sequences.

7. The method of any one of claims 1-5, wherein Stage 1 transfection and Stage 2 transfection are separated by a time interval of 12 hour to 24 hours.

8. The method of any one of claims 1-7, wherein the heterologous gene of interest in the second construct encodes a therapeutic protein.

9. The method of any one of claims 1-8, wherein the Rep construct comprises one or more nucleic acid sequences encoding Rep78 and Rep68 proteins.

10. The method of any one of claims 1-9, wherein the RepCap construct comprises nucleic acid sequences encoding VP1, VP2, and VP3 proteins.

11. The method of any one of claims 1-10, wherein the regulatory sequences include promoters, enhancers, and polyadenylation signals to optimize expression of the adenoviral, AAV rep, and AAV cap genes.

12. The method of any one of claims 1-11, wherein the first, second, third and fourth constructs each comprise a vector selected from the group consisting of a plasmid, virus and minimalistic, immunologically defined gene expression (MIDGE) vector.

13. The method of any one of claims 1-12, wherein the recombinant AAV virion produced has a capsid derived from AAV serotype 2 (, AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9.).

14. The method of any one of claims 1-13, wherein the Helper construct comprises SEQ ID NO:7.

15. The method of any one of claims 1-14, wherein the GOI construct comprises SEQ ID NO:8.

16. The method of any one of claims 1-15, wherein the Rep construct comprises SEQ ID NO:9.

17. The method of any one of claims 1-16, wherein the RepCap construct comprises SEQ ID NO: 10.

18. The method of any one of claims 1-17, further comprising culturing the host cells for an effective amount of time for rAAV assembly.

19. The method of claim 18 comprising culturing the cells for at least 72 hours.

20. The method of any one of claims 1-18, further comprising purifying the recombinant AAV virions from the host cell culture supernatant, cell lyste or cell suspension.

21. A population of transformed cells produced by the method of any of claims 1-18, wherein the transformed cells are capable of producing recombinant AAV virions.

22. The cells of claim 21, wherein the transformed cells contain from about 50% to about 100% filled rAAVs.

23. The cells of claim 21, comprising the helper construct, the Rep construct, the GOI construct and the RepCap Construct.

24. The cells of claim 22 comprising up to about 90% filled AAV capsids.

25. The cells of claim 21, comprising up to about 100% filed AAV capsids.

26. The cells of any one of claims 21-25, wherein the cells are HEK293 cells.

27. An in vitro method of producing a recombinant AAV virion in an insect host cell comprising contacting the cell with:

(i) a first nucleic acid sequence comprising a first rep gene (Rl) under the control of an immediate early promoter and a second nucleic acid sequence comprisinga second rep gene (R2) under the control of a late promoter;

(ii) a third nucleic acid sequence comprising an AAV cap gene (C) under the control of a late promoter; and

(iii) a fourth nucleic acid sequence comprising a heterologous gene of interest (GOI (gene of interest) (G)), to obtain a transformed insect cell comprising Rl, R2, C and G, wherein each of the first, second, third and fourth nucleic acids are in one or more viral expression constructs, optionally, one or more baculovirus expression constructs.

28. The method of claim 27, wherein the immediate promoter is the immediate early gene (iel) promoter, optionally, fused with the homologous region 5 (Hr5) enhancer (Hr5-IE1) or Orgyia pseudotsugata immediate early 2 promoter (OPIE2).

29. The method of claim 28 or 29, wherein the late promoter is plO or polyhedrin.

30. The method of any one of claims 27-29, wherein the insect cells are sf9 cells.

31. The method of any one of claims 27-30 comprising contacting the cells with the first, second, third and fourth nucleic acids simultaneously, wherein the first, second, third and fourth nucleic acids are in one or more viral expression constructs.

32. The method of any one of claims 27-30, comprising contacting the cells with the first, second, third and fourth nucleic acids in a first transduction step and a second transduction step, wherein the time interval between the first and second transfection steps is at least about 2 to about 6 hours, preferably from about 3 to about 5 hours.

33. The method of claim 32 comprising contacting the cells with one or more viral expression constructs comprising Rl, R2 and G, in the first transduction step, and contacting the cells with a viral construct comprising C in the second transduction step.

34. The method of claim 33, wherein each viral construct is a baculovirus expression construct.

35. The method of any one of claims 27-34, wherein therein cap gene is a baculovirus genome, the method comprising contacting the cells with about 2 to about 1000 baculovirus genome copies comprising the cap gene, per cell.

36. The method of claim 35, comprising contacting the cells with about 10 to about 80 baculovirus genome copies comprising the cap gene, per cell.

37. A population of transformed cells produced by the method of any of claims 27-36, wherein the transformed cells are capable of producing recombinant AAV virions.