Packaging cells with tunable and leakproof gene expression to optimize production of AAV vectors

Abstract

This disclosure provides a system for improving productivity and quality of adeno-associated virus (AAV) vectors obtained from hybrid packaging cells. Different groups of genetic elements required to produce AAV vectors (rep, cap, and helper genes) are separated into different modules, which are each placed under control of a different inducible regulatory element. The regulatory elements are designed to be leakproof, to prevent overexpression of genes that would reduce yield. The packaging cells also contain a landing pad to integrate a payload gene. To manufacture the intended AAV vector or particle, the packaging cells are contacted with (1) a pay load vector encoding a therapeutic cargo, and (2) inducer molecules for each of the regulatory elements. Viral production and ratio of full capsids can be optimized by tuning the inducer compounds for each of the modules.

Description

Packaging cells with tunable and leakproof gene expression to optimize production of AAV vectors REFERNCE TO EARLIER APPLICATIONS

[0001] This application claims the priority benefit of U.S. patent applications 63 / 662,870, filed June 21, 2024; and 18 / 806,560, filed August 15, 2024. This application also claims the priority benefit of U.S. patent application 63 / 795,149, filed April 25, 2025. For all purposes in the U.S., the aforelisted priority applications are hereby incorporated herein by reference in their entireties. TECHNICAL FIELD

[0002] The technology of this disclosure relates generally to the fields of virology, gene therapy, and production of medicaments that contain viral components. It provides a technology for the modification, selection, and genetic alteration of host cells for high levels of production of viral vectors and particles with improved biological and pharmacological characteristics. BACKGROUND

[0003] The past decade has seen viral vector based therapies become a bona fide option in clinical medicine.. A dozen therapies using viral vectors have been approved by the FDA. Adenovirus vectors have been approved as immunogenic compositions for treatment of infections diseases such as COVID 19. With about 25 viral vector therapeutics currently in late-stage development and another 120 in Phase II trials, the number of viral vectors approved for commercial production will increase rapidly (E. Capra et al., McKinsey and Company, 2022).

[0004] The first gene therapy vectors were typically developed for treatment of rare diseases. The emerging interest in treating more common conditions requires higher yields and a lower cost of goods. Over the last few years, large contract development and manufacturing organizations (CDMOs) have invested billions of dollars in production facilities for viral vectors. This burgeoning interest is promising, but the rapid influx of money and development of new technology have not solved the bottlenecks and challenges of viral vector manufacturing.

[0005] Currently, lack of standardization and low yields are part of the challenge. Physical characteristics and functional requirements vary considerably between different vectors. A high degree of process optimization is still needed for each product. Low recovery from chromatography steps means that yields are typically below 50 percent (M. May, Biotech. Eng. News, August 2, 2021). By way of comparison, the manufacture of therapeutic antibodies like Humira® and Rituxan® and biosimilars is done using standardized platforms, and typically achieves yields higher than 90 percent.PCT patent application Tunable AAV packaging cells

[0006] The owners of the technology described below previously developed the use of cell hybrids as producer cells, increasing yield of monoclonal antibodies by over four-fold (U.S. Patent Nos. 10,329,594 and 11,649,449), and increasing production of viral vectors by over six-fold (U.S. Patent No.12,252,713).

[0007] This disclosure provides further advancements in the technology for configuring vector packaging cells for high levels of production of viral vectors and particles with improved biological and pharmacological characteristics. SUMMARY OF THE INVENTION

[0008] This disclosure provides a system for improving productivity and quality of adeno- associated virus (AAV) vectors obtained from hybrid packaging cells. Different groups of genetic elements required to produce AAV vectors (rep, cap, and helper genes) are separated into different modules, which are each placed under control of a different inducible regulatory element. The regulatory elements are designed to be leakproof, to prevent overexpression of genes that would reduce yield. The packaging cells also contain a landing pad to integrate a payload gene. To manufacture the intended AAV vector or particle, the packaging cells are contacted with (1) a payload vector encoding a therapeutic cargo, and (2) inducer molecules for each of the regulatory elements. Viral production and ratio of full capsids can be optimized by tuning the inducer compounds for each of the modules. Definitions and technical background

[0009] For purposes of this disclosure, the terms “packaging cell line” and “producer cell line” interchangeably refer to cell lines that are transiently or permanently genetically altered to produce AAV viral vectors and / or particles. The packaging or producer cell line may also be transiently or permanently genetically altered to include a payload. A “viral vector” comprises a gene that constitutes or encodes a product of interest, contained in an AAV capsid. A “viral particle” comprises a protein or other gene product, contained in an AAV capsid. Depending on context, the technology of this disclosure may be used to produce either vectors or particles or both, mutatis mutandis.

[0010] Each of the “modules” is a transgene (or portion thereof) that includes at least one encoding region under transcriptional control of a regulatory element that is “tunable”. This means that the level of expression of encoding regions in the module can be increased or otherwise adjusted by the presence in the culture medium of a particular small molecule (<3,000 Da, typically < 1,000 kDa, or 200 to 800 kDa), or by other condition of the culture, such as light, magnetism, or temperature. The inducing compound or condition binds to or otherwise influences an inducible regulatory element, thereby increasing or decreasing the expression of genes controlled by the respective inducible regulatory element. Each module may contain more than one encoding and one or more regulatoryPCT patent application Tunable AAV packaging cells regions. Genes within each module may interact to invert or otherwise control gene expression or function in other modules.

[0011] Tunability of each module may or may not be reversible by reducing the concentration of the small molecule drug or reverting to previous culture conditions. For example, an inducible promoter that responds to the concentration of a small molecule drug can be retuned as needed. Other inducible regulatory elements which (for example) cause gene inversion or removal of a stop codon may not be reversible after the gene has been altered. The system may be retuned by taking a fresh batch of packaging cells and culturing them with the new or adjusted concentration of inducer compounds from the outset.

[0012] Some tunable constructs presented in this disclosure are “leakproof” which means that they are designed or constructed to minimize or eliminate constitutive basal levels of expression when inducer compounds or conditions are not present. A single inducible promoter by definition is not leakproof. Examples of leakproof systems are: (1) systems in which there is at least one inducible gene alteration event, such as excision, inversion, or translocation, optionally in combination with another inducible element, such as an inducible promoter; (2) systems in which there are at least two superimposed or inter-reliant events that are both needed for expression to occur: for example, an inducible promoter, in combination with removal of a gene repressor. A “leakproof regulatory element” is the combination of one or more inducible or tunable elements that when contacted with an inducing agent, results in expression of a gene or gene cluster.

[0013] An “attenuated promoter” is a promoter characterized as promoting gene expression at a rate that is 1 / 3 or less of the rate of a CMV (cytomegalovirus) promoter. J.Y. Qin et al., PLoS One. 2010 May 12;5(5):e10611. The attenuated promoter may be constitutive or inducible. Exemplary is the PGK (phosphoglycerate kinase) promoter.

[0014] Different genes required for production of an AAV vector or particle include (1) AAV replication (Rep) genes; (2) AAV capsid (Cap) genes; (3) AAV assembly (aav) genes; (4) helper genes required for AAV production, and (5) a gene encoding the intended payload. Depending on context, the helper function may be supplied, for example, by one or more genes from adenovirus (Ad), specifically E1 and / or E4; by one or more genes from herpes simplex virus (HSV), specifically ICP27, ICP5, and UL5; or one or more genes from bovine papilloma virus (BVP), specifically E2A and E2B.

[0015] In the tunable technology of this disclosure, a plurality of such gene categories or individual genes thereof are each placed in a different module under control of a different inducible element such that the expression of such genes may be individually adjusted and optimized. The plurality may be 2, 3, 4, or more than 4. Each of the modules may be but is not necessarily on a different chromosome or transgene.

[0016] Typically, the suite of transgenes is stably integrated into the genome of the packaging cell line, for example, using an integrating vector based on lentivirus, a transposase such as Sleeping BeautyPCT patent application Tunable AAV packaging cells transposase, or a gene editing tool such as CRISPR. Also contemplated is a system in which a suite of three modules is transiently transfected into the cell together or separately, for example, using a DNA or RNA vectors. In this case, the packaging cell line may be optimized for virus production beforehand by other means. Typically the modularized components don’t include the payload, which is on a separate vector to be transfected into the cell transiently or permanently. Exemplary implementations

[0017] The technology of this disclosure is illustrated by a packaging cell line comprising three separately inducible modules on three separate transgenes integrated into the genome of the cells. The first module comprises at least one AAV cap gene under control of a first inducible regulatory element; the second module comprises one or a plurality of AAV helper genes under control of a second inducible regulatory element; the third module comprises at least one AAV rep gene under control of a third inducible regulatory element. One, two, or all three of these modules may be leakproof. Separate and not included in the packaging cell in this example is payload module that contains an expressible gene encoding a vector payload for the AAV vector or particle to be produced by the packaging cell. The modules may be “inheritably integrated,” which means that they will continue to be expressible in progeny of the transfected cells for at least five doublings. Usually, they will be permanently transfected into the genome of the packaging cell.

[0018] Another aspect of the technology provided in this disclosure is to provide the packaging cell with an apoptosis inhibitor such as Bcl-2, to counter pro-apoptotic effects on human host cells due to expression of one or more AAV components, such as Rep52, Rep78 or other Rep variant.

[0019] The tunable vector systems of this disclosure can be implemented together with other technologies that increase vector production and / or the proportion of filled vector capsids. For example, the packaging cells may be progeny of hybrid cells, made by fusion of two or more parental cells. Alternatively or in addition, the progeny may have been selected for high mitochondria content, or a high or low level of reactive oxygen species per cell.

[0020] The AAV vectors and particles of this disclosure usually carry a payload of nucleic acid or protein that constitutes or encodes a protein or nucleic acid of interest. To help develop and characterize the packaging cells, the vector payload can be a marker protein such as enhanced green fluorescent protein (eGFP). This can be replaced subsequently with a vector payload that is an immunogenic peptide or a nucleic acid encoding said peptide for eliciting a specific immune response in a subject in need thereof; or a nucleic acid encoding a gene product that is deficient in a subject in need of thereof.

[0021] Once the packaging cells are built comprising a plurality of modules under separate inducible control, they can be used to optimize culture conditions to produce more vector capsids per cell and / or to increase the proportion of “full” capsids that include the desired payload. The tunablePCT patent application Tunable AAV packaging cells packaging cell line is contacted, transfected, or genetically altered with a payload vector comprising a gene that encodes a vector payload between two AAV inverted terminal repeat sequences (ITRs). The transfected cells or their progeny are cultured in the presence of different amounts and / or ratios of inducer compounds for each of said three inducible regulatory elements, thereby producing said AAV vector.

[0022] The amounts and / or the ratios of the inducer compounds in the culture medium can then be tuned or adjusted to obtain a desired titer of said AAV vector and / or proportion of viral capsids bearing said payload. In cases where the inducible regulatory element controls expression of its respective module be gene inversion or other alteration, then the consequence of the amount of inducing compound initially selected may not be reversible. Instead of tuning the packaging cells by readjusting the level of the various inducer compounds in the original culture, a fresh lot of the packaging cells is transfected with the payload vector and cultured by the new readjusted level of each of the inducer compounds at the outset.

[0023] Once optimal conditions are identified, then an industrial scale of the AAV vector or particle can be manufactured by culturing an expanded population of cells from the tunable packaging cell line in a medium that contains the predetermined amount of each inducer compound for each of the multiple inducible regulatory elements; and harvesting the AAV vector from the culture. Productivity of at least 1011, 1012, and 1013or more viral particles (Vp) / mL, and from 1010or 1011up to 1013or 1014Vp / mL are contemplated. Productivity of at least 5,000, 10,000, and 20,000 VP / cell are contemplated. Ratio of full to empty capsids of at least 1, 2, and 3 are contemplated. Percent of capsids that contain payload of at least 30% 40%, 50%, and 60% are contemplated.

[0024] The selection of particular inducible regulatory elements and payloads and the construction and use of the tunable modules are detailed in the sections that follow. These and other aspects, embodiments, features, and characteristics of the invention are described below, and in the accompanying drawings and the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] FIG.1 is a set of three gene maps constituting an example of how the technology provided in this disclosure can be used to construct a tunable packaging cell line for an adeno-associated (AAV) viral vector or virus-like particle. Three gene constructs or “modules” are present in the cells, interacting to form AAV vectors or particles in a tunable fashion.

[0026] FIG.2A is a gene map of a vector comprising a payload for inclusion in the packaged AAV vector, positioned between two inverted terminal repeats (ITRs). FIG.2B show the gene and protein components assembled by the packaging cell line containing Modules A, B, and C. The payload may be a marker protein or a pharmaceutical agent.PCT patent application Tunable AAV packaging cells

[0027] FIG.3 is a flow diagram showing how the genetic elements mapped in FIG.1 interact to form AAV vectors or particles in a tunable fashion. In Module B, the CreERT protein is inducible by the small molecule 4-OHT (4-hydroxytamoxifen). This causes expression of Flp5 which inverts the expression cassettes on Modules A and B. Inversion of the expression cassette on Module B results in expression of the adenovirus helper genes E4orf6 and DBP. In Module A, the EcR protein is inducible by the small molecule tebutemazide, which activates the expression cassette once inverted by Flp5. This causes expression of the AAV genes Rep52 and Cap, and also causes expression of Flp. In Module C, the Flp causes removal of the stop codon, thereby causing expression of Bcl-2 to inhibit apoptosis. Module C also comprises the AAV Rep68 gene under control of a TRE3G promoter, which is inducible by doxycycline.

[0028] In this example, Module A comprises a gene resistant to the antibiotic blasticidin (Bsd), Module B comprises a gene that is resistant to the antibiotic puromycin (Puro), and Module C comprises a gene that is resistant to the antibiotic hygromycin (Hygro). Culturing transfected cells in the presence of all three antibiotics will select for cells that have integrated all three modules.

[0029] FIG.4A is a workflow for obtaining hybrid packaging cell lines that have both high virus production capacity and tunable virus components. Cells are fused together to produce an engineered cell population. The population is then cloned or separated into separate aliquots, optionally taking into account desirable cell phenotypes (such as intracellular organelle content). The separate clones or aliquots are stably transfected to express viral elements (Module A, B and C) \and high producer cells are identified. The chosen cells are expanded to establish a bank of packaging cell lines.

[0030] FIG.4B is a workflow for equipping a packaging cell line obtained according to FIG.4A for production of a target AAV vector with a selected payload. The packaging cells are transfected transiently or permanently with a payload vector and tuned using small molecule inducers for each of the modules — thereafter producing a viral vector or particle encapsulating a therapeutic payload for gene therapy or immunization.

[0031] FIGS.5A, 5B, and 5C demonstrate higher productivity of capsids of several serotypes of AAV vectors from fused HEK293 cells. Engineered clone “44” showed 12X increase in AAV1 titer compared with parental cells. Engineered clone “2” showed 14X increase in AAV2 titer compared to parental cells. Engineered clone “121” showed 5X increase in AAV5 titer compared to parental cells. Engineered clone “17” showed 6X increase in AAV9 titer compared to parental cells.

[0032] FIGS.6A, 6B, and 6C show how modules may be tuned to optimize virus production and phenotype. Packaging cell lines were made from HEK293 cells (“parent”), or from progeny of HEK293 cells fused together (“hybrids”). The packaging cell lines were genetically altered to incorporate the three tunable modules shown FIG.1.

[0033] FIG.6A shows the conditions of a trial expression culture. The cells were expanded to a workable sized population, and then put in a medium containing the inducers: a relatively low level ofPCT patent application Tunable AAV packaging cells tebufenozide (inducer A), a moderate level of 4 hydroxytamoxifen (4-OHT, inducer B), and a relatively high level of doxycycline (inducer C). The tuned cells were transfected with the AAV payload (GFP), grown in the adapted culture medium, and virus was harvested. FIG.6B shows the capsid titer obtained. Using separate packaging cells made with cell hybrids, serotypes AAV1, AAV2, and AAV5 were produced at a level that was as much as 4-fold higher or more, compared with packaging cells made from the parental HEK293 line. FIG.6C shows the percentage of capsules that were full (contained the intended payload).

[0034] FIGS.7A, 7B, and 7C show results of another experimental trial in which the concentrations of inducers A and B were higher than in FIGS.6A, 6B, and 6C, whereas the concentration of inducer C was lower. There was a comparable capsid titer (FIG.7B), but a higher percentage of full capsids (FIG.7C).

[0035] FIG.8 depicts integration of an AAV payload gene into a packaging cell line using Sleeping Beauty transposase in protein form. The transposase is electroporated into the target cell along with a transposon plasmid encoding the AAV payload. Positive integrants are isolated by FACS.

[0036] FIGS.9A to 9C are schematic illustrations of an exemplary landing pad technology of site-specific integration of a gene of interest. FIG.9A shows constructs used both to place the landing pad in the cell genome, and use it to dock a desired gene of interest (GOI). The triangles represent recombination sites N-select Mk: negative selection marker. Only the correctly integrated cells survive the negative selection after recombination. FIG.9B illustrates a landing pad construct that contains a puromycin resistance gene fused to a thymidine kinase gene through T2A coding sequence. FIG.9C illustrates a targeting construct that contains a gene of interest (GOI) flanked by two phiC31 attB sites and a thymidine kinase gene in the vector backbone.

[0037] FIG.10A shows schematically the steps of installing the landing pad into a high productivity region of the genome; and then using the landing pad to dock a gene of interest. FIG.10B shows how the landing pad system improves the timeline of producer cell development. FIGS.10C and 10D are schematic depictions of the process used to integrate the landing pad into a targeted region of the genome of the host cell, and then dock it with a gene of interest (GOI). The technology is modeled for the purpose of initial studies using enhanced green fluorescent protein (eGFP) as the GOI.

[0038] FIG.11A is a contour plot of hybrid HEK293 cells that have been installed with a landing pad (labeled with mCherry) and then docked with a model AAV cargo (eGFP). Event densities of cells bearing both markers are depicted with contour lines (gradients). 80 to 85% of the cells bear the intended cargo. FIG.11B shows capsid titer for AAV2 and AAV5 in HEK293 host cells with AAV payload targeted specifically to the H11 locus using landing pad technology. Percent increase was normalized to parental cell line which does not contain stably integrated AAV payload at the H11 locus. FIG.11C shows that the relative mRNA expression of the AAV payload measured by RT-qPCR is stable through 60 doublings of the integration.PCT patent application Tunable AAV packaging cells

[0039] FIG.12 shows the productivity yield (vg / L) from cell hybrids that have an AAV payload gene integrated into a transcription safe harbor using landing pad technology.

[0040] FIG.13A schematically illustrates a two-step chromatographic process for purifying AAV vectors. AAV capsids (both full and empty) are captured by affinity chromatography. Anion exchange chromatography is then used to separate full and empty capsids. FIG.13B shows purification yield of AAV from packaging cells installed with a payload gene by undirected transfection (open bars) or using a landing pad system (striped bars).

[0041] FIG.14A shows the logic circuit for leakproof expression of Rep52 in the three module system shown in FIG.1. FIG.14B shows the logic circuit for leakproof expression of Rep68.

[0042] FIG.15 is a map of a two-module system in which rep and cap genes are on separate modules. Expression of both Rep52 and Rep68 are inducible and leakproof. Helper activity is not on either module: It is either constitutive to the cell, or provided by a separate helper cassette with attenuated expression. DETAILED DESCRIPTION

[0026] This disclosure provides improved virus packaging cell lines for producing viral vectors and virus-like particles for delivering nucleic acid and protein respectively into a target cell. 1. Technology pitch — fast timeline for developing viral vector producing cells for new indications

[0043] Rather than the usual mode of developing new AAV vectors — gene transfection, followed by extensive selection cloning, testing, and optimization — the technology put forth in this disclosure constitutes a mode by which new vectors are ready for preclinical testing in just a few months. The technology starts with a line of cell hybrids, which make at least two-fold more viral particles and a higher percent of full capsids than regular HEK293 cells. The AAV packaging cells are installed with virus encoding genes in a plurality of separate modules that are leakproof and tunable by titrating small molecule inducers. The packaging cells are also installed with a landing pad system for rapid integration of a payload gene at a transcription safe harbor in the cell’s genome.

[0044] To develop producer cells for a new AAV vector, the selected payload gene is exchanged into the landing pad. The cells are cultured to produce viral vectors, tuning each of the separate models to optimize production. Once optimal conditions are established, the virus producing cells are ready to be scaled up for biological testing and industrial production.PCT patent application Tunable AAV packaging cells 2. Overview and selected example of a tunable vector packaging cell

[0045] FIG.1 is a gene map of a non-limiting implementation of this technology. Three modules are introduced into the virus producer or packaging cell line, which interact for separate and tunable expression of AAV Rep and Cap genes, and adenovirus helper function. A map of a suitable payload vector for delivery into the target cell by the vector is shown in FIG.2A. A map of the gene products assembled by the producer cell into an AAV capsid encapsulating the payload is shown in FIG.2B.

[0046] This system can be used to produce viral titer of the level of 1012Vp / mL Features of this selected example are as follows: ^ Replication, cap, and helper components are in 3 independent modules of the host cell; ^ Each module expression is dependent on a single small molecule inducer, which can be used to tune expression; ^ Helper functions are divided between Module A and Module B to allow for finer control of VP proteins; ^ Replication proteins are divided between Module A and Module C for tuning either for higher titer and / or higher percent full capsid; ^ In other systems, overactivation of Rep52 can result in cell death. The effect is minimized in this system by leakproof control of expression, and the presence of the apoptosis-inhibiting gene Bcl-2.

[0047] FIGS.2A is a gene maps showing a prototype payload vector suitable for encapsulating in an AAV vector, and the AAV vector product made therefrom. The payload vector is a bicistronic construct for co-expressing both enhanced green fluorescent protein (eGFP) and infrared fluorescent protein 682 (iFP682). The two fluorescent reporters mimic therapeutic payload size used for gene therapy (~3-4 kb). A strong ubiquitous promoter (CMV) was used to express EGFP and FP682. WPRE was used to enhance transgene expression, while poly A from SV40 (pA) ensures proper transcription termination by adding poly A tail to transcribed mRNA.

[0048] FIG.2B shows the gene products that are produced and assembled from Modules A, B, and C. The AAV capsid produced contains recombinant Rep and Cap (VP1, VP2, and VP3) open reading frames and inverted terminal repeats (ITRs). To ensure correct capsid protein stoichiometry for VP1:VP2:VP3 of 1:1:10, VP1-3 capsid protein production requires both alternative mRNA splicing as well as alternative translational start codons. VP1 is translated from ATG start codon; while VP2 uses a weaker ACG start codon and readthrough translation to the next available ATG codon for the production of the most abundant capsid protein, VP3.PCT patent application Tunable AAV packaging cells

[0049] Abbreviations used in the gene maps are as follows: Module A: SV40 pA = simian virus 40 polyadenylation signal Bsd = blasticidin S deaminase: selectable marker gene conferring resistance to the antibiotic blastocidin CMV = cytomegalovirus promoter: drives high-level gene expression PGK = phosphoglycerate kinase promoter: strong and constitutive gene expression CFDBD = quinic acid fucose dimeric biosensor domain: detects specific molecules or ligands in cellular environments AD = activation domain: interacts with transcriptional machinery to enhance gene expression EcR = enhancer core region: promotes the assembly of transcriptional machinery bgH pA = bovine growth hormone polyadenylation signal miniTK promoter = minimal thymidine kinase promoter: drives transgene expression QUAS = QF-responsive upstream activating sequence for controlling gene expression 5’UTR = 5’ untranslated region of QUAS gene mFRT71 = modified FRT71: modified version of the FRT (flippase recognition target) site, used for site-specific recombination Flp = flippase: enzyme for catalyzing site-specific recombination between FRT sites E2A = sequence from adenovirus 2 that enhances transcription when bound by transcription factors S-Cap = AAV gene (capsid) T2A = Thosea asigna virus 2A: sequence from Thosea asigna virus that mediates ribosome skipping, for expression of multiple proteins from a single mRNA transcript Rep52 = AAV gene (viral DNA replication and regulation of gene expression) T2A = Thosea asigna virus 2A: sequence from Thosea asigna virus that mediates ribosome skipping, for expression of multiple proteins from a single mRNA transcript VP1 / VP2 / VP3 (VP123) = capsid protein complex that self-assembles to form icosahedral capsid : promotes viral entry into host cells by binding to host cell heparan sulfate and is a key target for the host immune response WPRE = woodchuck hepatitis virus posttranscriptional regulatory element: enhances stability and translation efficiency of mRNA transcripts in mammalian cells SV40 pA = simian virus 40 polyadenylation signalPCT patent application Tunable AAV packaging cells Module B: SV40 pA = simian virus polyadenylation signal Puro = Puromycin resistance gene, a selectable marker gene conferring resistance to the antibiotic puromycin T2A = Thosea asigna virus 2A: sequence from Thosea asigna virus that mediates ribosome skipping, for expression of multiple proteins from a single mRNA transcript CreERT = a fusion protein combining the Cre recombinase enzyme with the ligand-binding domain of the estrogen receptor. It allows for inducible and temporally controlled activation of Cre-mediated recombination in response to tamoxifen EF1A = Eukaryotic elongation factor 1 alpha promoter, drives high-level and constitutive gene expression miniCMV = minimal cytomegalovirus promoter: drives transgene expression miniTK = minimal thymidine kinase promoter: drives transgene expression loxP = LoxP site recognized by Cre recombinase lox511 = mutated Lox sites recognized by Cre recombinase DBP = DNA-binding protein T2A = Thosea asigna virus 2A: sequence from Thosea asigna virus that mediates ribosome skipping, for expression of multiple proteins from a single mRNA transcript E4orf6 = early region 4 open reading frame 6, an adenovirus gene that encodes a multifunctional protein that modulates host cell processes to facilitate viral replication WPRE = woodchuck hepatitis virus posttranscriptional regulatory element: enhances stability and translation efficiency of mRNA transcripts in mammalian cells Synt PA = synthetic polyadenylation signal miniTK promoter = minimal thymidine kinase promoter: drives transgene expression Stop = stop codon mFlp5 =modified version of the Flippase (Flp) enzyme for catalyzing site-specific recombination between FRT sites bgH pA = bovine growth hormone polyadenylation signal Module C: Synt PA = synthetic polyadenylation signal Hygro = hygromycin resistance gene, a selectable marker gene conferring resistance to the antibiotic hygromycin T2A = Thosea asigna virus 2A: sequence from Thosea asigna virus that mediates ribosome skipping, for expression of multiple proteins from a single mRNA transcriptPCT patent application Tunable AAV packaging cells rtTA = reverse tetracycline-controlled transactivator: transcriptional activator protein that is regulated by tetracycline for inducible gene expression HSV-TKmini = truncated version of the Herpes simplex virus thymidine kinase promoter for driving transgene expression SV40 pA = simian virus polyadenylation signal WPRE = woodchuck hepatitis virus posttranscriptional regulatory element: enhances stability and translation efficiency of mRNA transcripts in mammalian cells tTS = tetracycline-controlled transcriptional silencer: regulated by tetracycline for inducible gene silencing Cbh = chicken beta actin hybrid promoter for constitutive gene expression TRE3G = tetracycline-responsive element, third generation (3G), responds to tetracycline for inducible gene expression in mammalian cells Rep68 = AAV Replication protein 68: A protein encoded by the Rep gene of AAV, involved in regulation of viral gene expression SV40 pA = simian virus polyadenylation signal miniEF = minimal elongation factor 1 alpha promoter: for driving gene expression FRT = Flippase recognition target for Flp-FRT-mediated gene excision or integration Stop = stop codon Bcl-2 = B-cell lymphoma / leukemia-2 gene: inhibits apoptosis (programmed cell death) bgH pA = bovine growth hormone polyadenylation signal

[0050] The function of these components in the expression system is shown in Table 1. TABLE 1: Function of viral elements in Modules A, B, and C Full Name Function SV40 pA Poly A tail for simian Stabilize mRNA and proper exportation from nucleus to virus 40 cytoplasm for protein translation bGH pA Stabilize mRNA and proper exportation from nucleus tocytoplasm for protein translation Synt pA Synthetic poly A Stabilize mRNA and proper exportation from nucleus to cytoplasm for protein translation Woodchuck Hepatitis WPRE Virus Posttranscriptional Enhance transgene expression Regulatory Element CMV enhancer and Cbh chicken β-actin hybrid Ubiquitous, strong promoter driving transgene promoter expressionPCT patent application Tunable AAV packaging cells TABLE 1: Function of viral elements in Modules A, B, and C Full Name Function TRE3G Tetracycline regulatory Inducible promoter that is activated upon binding of rtTA element, third generation and addition of doxycycline activating Rep68 CMV Cytomegalovirus Ubiquitous, strong promoter driving transgene promoter expression PGK 3-phosphoglycerate Ubiquitous, weak promoter driving transgene expressionEF1A Human elongation factor- Ubiquitous, strong promoter driving transgene 1 alpha expression miniCMV Minimal cytomegalovirus promoter Minimal weak promoter driving transgene expression miniTK Minimal thymidine kinase promoter Minimal weak promoter driving transgene expression Minimal herpes simplex HSV-TKmini virus (HSV) thymidine Minimal weak promoter driving transgene expression kinase promoter Minimal human miniEF elongation factor-1 alpha Minimal weak promoter driving transgene expression promoter Bsd Blasticidin Antibiotic selection marker for Module A Hygro Hygromycin Antibiotic selection marker for Module B Puro Puromycin Antibiotic selection marker for Module C Fusion protein containing: 1) DNA binding domain of QF QAT or qa-1F transcription factor; and 2) VP16 activation QFDBD-EcRAD fusion domain fused to modified Ecdysone receptor that is protein activated upon addition of tebufenozide ligand (Teb). Upon addition of Teb, QAT is activated and binds to QUAS to activate any transgene downstream of QUAS. QUAS QF upstream activation Upon binding of QAT to QUAS, any downstream gene is sequence activated 5’UTR 5’ untranslated region Enhance translation efficiency of messenger RNA Recombinase that catalyzes homologous recombination Flp Flippase at two Flippase recognition targets (FRT) activating Bcl-2 gene to inhibit cell death Mutant Flippase recombinase that catalyzes mFlp5 mutant Flippase 5 homologous recombination at Flippase recognition target sites, mFRT71, activating E4orf6 and DBP FRT Flippase recognition targets Homologous recombination sites FRT catalyzed by Flp mFRT71 Mutant Flippase Homologous recombination sites mFRT71 catalyzed by recognition targets 71 mFlp5PCT patent application Tunable AAV packaging cells TABLE 1: Function of viral elements in Modules A, B, and C Full Name Function Cre recombinase (Cre) CreERT fused to a mutant Fused protein that is activated upon addition of estrogen ligand-binding 4-hydroxytamoxifen (4-OHT) domain (ERT2) loxP Locus of X-over P1 34 base pair DNA sequence recognized by Cre recombinase to induce homologous recombination Mutant loxP site recognized by Cre recombinase that lox511 Locus of X-over 511 can result homologous recombination with another lox511 site Reve Transcription activator that requires the addition of rtTA rsible tetracycline transcriptional activator doxycycline. rtTA binds to TRE site to activation transgene downstream of TRE tTS Tetracycline controlled Reduces the leakage of gene activation in the absence transcriptional silencer of doxycycline Stop Stop cassette Terminate gene transcription E2A 2A peptide from equine Allows for polycistronic gene expression: transcription of rhinitis A virus multiple genes from a single open reading frame P2A 2A peptide from porcine Allows for polycistronic gene expression: transcription of teschovirus-1 multiple genes from a single open reading frame T2A 2A peptide from thosea Allows for polycistronic gene expression: transcription of asigna virus multiple genes from a single open reading frame S-Cap Serotype-specific capsid genes Capsid genes essential for forming proper AAV capsid Rep52 Rep52 Required for proper formation of AAV capsid Rep68 Rep68 Required for proper formation of AAV capsid VP123 VP1, VP2, and VP3 Structural protein for AAV E4orf6 E4orf6 Required for transportation of viral RNA DBP D-Box Binding PAR BZIP Transcription Factor Enhance posttranslational expression of AAV Bcl-2 B-cell lymphoma 2 Cell death inhibitor

[0051] FIG.3 is a flow chart showing functional interaction and flow of the vector system mapped in FIG.1. Starting with Module B, the CreERT protein is a gene inverting enzyme, comprising Cre recombinase and estrogen receptor ligand binding domain. It responds to the small molecule inducer 4-hydroxytamoxifen (4-OHT) by activating recombinase activity. This acts on Module B to invert and activate the expression cassette bounded by two loxP sites to put the cassette in anPCT patent application Tunable AAV packaging cells expressible configuration. CreERT also excises the stop codon between the two lox511 sites on Module B to activate expression of mFlp5.

[0052] Module A is under control of QAT fusion protein. The QAT fusion protein contains an activation domain of VP16 fused to modified but truncated Ecdysone receptor (EcR), which responds to the small molecule inducer tebufenozide. Activation of QAT allows binding QFDBD domain to QUAS, causing gene expression from the expression cassette that was inverted by Module B — which, in turn, causes expression of the Cap and Rep52 proteins for assembly into the viral vector. QAT also activates the other recombinase Flp, which removes the stop codon on Module C.

[0053] Module C has two components. One is under control of the small molecule inducer doxycycline. It activates the inducible promoter TRE3G, which causes expression of Rep68, the other component needed for viral production. Also residing on Module C is the human apoptosis inhibitor gene Bcl-2. This is included in the system to counter the overexpression of Rep52, which can cause death, resulting in reduced viral production in the packaging cell.

[0054] Put another way, Module A is a packaging module that contains some components of Rep and Cap. S-Cap is the serotype specific capsid used to package each AAV serotype, under the control of tebufenozide. Upon expression of mFlp5 (a variant of Flp recombinase that only induces homologous recombination at mFRT71 sites) from Module B and 4-OHT. This results in expression of VP1 / VP2 / VP3, Rep52, and serotype-specific Cap protein (S-Cap). Tebufenozide binds QAT, activating QF transcriptional activator (QFDBD), which then binds to QUAS enhancer sites. This in turn promotes inversion of cassette containing VP1 / VP2 / VP3, Rep52, S-Cap, and Flp.

[0055] Module B contains components E4orf6 and DBP to allow for sufficient helper functions. Helper function is under the control of 4-OHT. Expression of mFlp5 is normally OFF and activated upon induction of 4-OHT, resulting in Cre expression, excising the stop codon upstream of mFlp5, resulting in its expression.

[0056] Module C contains the gene encoding Rep68, which confers a viral replication function under the control of doxycycline. Module C also contains the human Bcl-2 gene under control of the Flp enzyme on Module A. In some previous systems, Rep52 overactivation resulted in apoptosis (cell death) of the packaging cell. In this example, Rep52 is linked to Flp, which induces homologous recombination at FRT sites in Module C, which leads to activation of Bcl-2, inhibiting apoptosis. 3. Procedure for building packaging cell lines

[0057] Selecting parental cell line for producing packaging cells according to this technology is influenced by viral type, and whether the cell line has elements that may be missing in a replication deficient virus. Suitable cell lines for adeno-associated virus (AAV): HEK293, HeLa, A549, BHK-21, Vero; Adenovirus: HEK293, HeLa, A549, COS-7, Vero; Herpes simplex virus (HSV): Vero, HeLa, BHK-21, CHO-K1, NIH / 3T3.PCT patent application Tunable AAV packaging cells

[0058] Potential parental cells are as follows: ^ Vero cells, derived from African green monkey kidney tissue, ATCC # CCL-81; ^ HEK293 cells, originating from human embryonic kidney tissue, ATCC # CRL-1573; ^ HeLa cells, derived from cervical cancer cells, ATCC # CCL-2; ^ A549 cells, derived from human lung carcinoma tissue, ATCC # CCL-185; ^ BHK-21 cells, derived from baby hamster kidney tissue, ATCC # CCL-10; ^ COS-7 cells, originating from African green monkey kidney cells transformed with SV40, ATCC # CRL-1651; ^ NIH / 3T3 cells, derived from mouse embryonic fibroblasts, ATCC # CRL-1658.

[0059] By way of illustration, a packaging system using the modules shown in FIG.1 can be produced as follows: 1. Linearize plasmid DNA containing Module A, B, and C by restriction enzyme digest and purify; 2. Grow HEK293 cells in shake flask so that they are in log phase; 3. Triple transfect cells by electroporation with linearized DNA containing Module A, B, and C; 4. Grow cells in selection medium containing blasticidin, hygromycin, puromycin for 3 weeks in shaking culture flasks to select for cells that have integrated all three modules. 5. Prepare frozen aliquots of stable cell pool expressing Module A, Module B, Module C. 4. Procedure to use packaging cell lines and to characterize vectors and particles obtained thereby

[0060] Step 1. Transfection and small molecule induction: 1. Thaw vial of frozen HEK293 cells expressing Module A, Module B, and Module C; 2. Grow HEK293 cells to VCD of 3 x 106cells / mL in growth media without antibiotics; 3. Transfect HEK293 cells by chemical-based methods with transfer vector containing payload flanked by AAV inverted terminal repeats; 4. Culture cells for 4 h at 37 C, 8% CO2, and 125 rpm; 5. Add tebufenozide (4-OHT) and doxycycline to transfected cell g075 6. Culture cells for additional 2 h at 37 C, 8% CO2, and 125 rpm;

[0061] Step 2. Lyse and harvest cells: 7. After 72 hours, add benzonase and lysis buffer to transfected cells. Culture cells for 2 h at 37 C, 8% CO2, and 125 rpm; 8. Harvest lysed cells by centrifugation at 4000 rpm at 4ºC for 30 min; 9. Transfer supernatant containing AAV particles to new centrifuge tube to assay for AAV production.PCT patent application Tunable AAV packaging cells

[0062] Step 3: Assay for viral genome titer 10. Measure AAV genomic copy number by real-time quantitative PCR. Treat sample with DNase I to remove non-viral host genomic DNA; 11. Real-time quantitative PCR by fluorescent detection performed using DNA primers that bind to coding regions of fluorescent reporter within transfected transfer vector; 12. Assay for viral genome titer.

[0063] Step 4: Measure concentration of AAV serotype-specific capsids: 13. Bio-layer interferometry (BLI) is an optical biosensing technology that analyzes biomolecular interactions in real-time without the need for fluorescent labeling. Interference patterns of white light or phase shift caused by analyte sample binding to immobilized ligand on biosensor probe was used to quantify the amount of AAV virus in an unknown sample. (The apparatus comprises a small biosensor that binds specifically to AAV capsid protein for multiple serotypes (AAV1, AAV2, and AAV5). For each serotype (AAV1, AAV2, and AAV5), a standard curve of commercial AAV reference standard with known capsid concentration measured by other validated methods are used to back- calculate the concentration of AAV serotypes in unknown sample.)

[0064] Step 5: Measure the ratio of full to empty capsids: 14. Ratio is measured in unknown samples by biolayer interferometry; 15. Determine AAV serotype-specific capsid concentration of AAV as described above; 16. For each serotype being measured, AAV capsid at a normalized concentration are first captured and immobilized on biosensor; 17. Following immobilization, AAV particles are lysed to release packaged single stranded DNA (ssDNA) and ssDNA is captured and measured using biosensor probe that is conjugated to SSB protein wherein SSB protein binds specifically to only ssDNA; 18. Perform binding kinetics assay of unknown samples and commercial AAV reference standard, with known full-to-empty capsids ratio measured by other validated methods; 19. Back-calculate the ratio of AAV serotypes in unknown sample using commercial AAV reference standard, with known full-to-empty capsids ratio.

[0065] Step 6: Measure functional titer: 20. HEK293 cells are infected with undiluted samples of AAV1, AAV2, and AAV5 at a range of dilutions and added to a fixed population of HEK293 cells; 21. Functional titer is measured in transduction units (TU) per mL. Infectivity was determined by quantifying percentage of fluorescent-positive cells by flow cytometry at different multiplicity of infection (MOI).PCT patent application Tunable AAV packaging cells 5. Procedure for tuning the packaging cell to maximize viral capsid production and payload incorporation

[0066] The user may employ the packaging cells of this invention to optimize production of AAV vectors or virus like particles, for example, in terms of viral particles per cell or per mL culture fluid, or in terms of the percent of viral capsids that contain a payload. Different modules or cassettes in the cell may be dialed up or down by adjusting the amount of the inducer element. By way of illustration, the system mapped in FIG.1 can be dialed as shown in Table 2. TABLE 2: Dialable components of the modules Module Controlled by inducer Dial Activates Function A tebufenozide Teb Dial containing Rep52, S-Cap, VP1, Packaging: Produces QAT cassette VP2, VP3, and Flp packaging genes and Flp B 4-OHT OHT Dial containing E4orf6, DBP, and Helper: Produces helper CreERT2 mFlp5 proteins and mFlp5 Replication: inhibits apoptosis C doxycycline Dox Dial containing when Rep52 is too high and TRE3G Rep68 and Bcl-2 increase viral capsid production

[0067] Module A is a packaging module that contains some components of Rep and Cap. S-Cap is the serotype specific capsid used to package each AAV serotype. It is under the control of tebufenozide. Upon expression of mFlp5 (a variant of Flp recombinase that only induces homologous recombination at mFRT71 sites) from transfer vector and tebufenozide. Expression of VP1 / VP2 / VP3, Rep52, and serotype-specific Cap protein (abbreviated as S-Cap). Tebufenozide binds the EcRAD fusion protein (truncated ecdysone receptor fused to activation domain of VP16), activating QAT fusion protein. QAT then binds to QUAS enhancer sites, promoting the inversion of cassette containing VP1 / VP2 / VP3, Rep52, serotype-specific Cap protein (abbreviated as S-Cap), and Flp. As Rep52 overactivation can result in cell death; Rep52 is indirectly linked to Flp, which induces homologous recombination at FRT sites in Module C, activating a cell death inhibitor, Bcl-2.

[0068] Module B contains components E4orf6 and DBP to allow for sufficient helper functions. Helper function Is under the control of 4-OHT. Expression of mFlp5 is normally OFF and activated upon induction of 4-OHT, resulting in Cre expression, excising the stop codon upstream of mFlp5, resulting in its expression.PCT patent application Tunable AAV packaging cells

[0069] Module C: contains components additional Rep protein, Rep68 (function only viral replication machinery), Flp recombinase protein, and hBCL2 (under the control of Flp expression in Module A). The level of Rep68 expression is under the control of doxycycline. Tuning Modules A, B, and C for high viral titer, high percent filled ratio, and / or high functional titer

[0070] Once the packaging cell has been constructed, it is used to produce AAV vectors as follows. First, the cells are transfected with a transfer vector containing a payload. The transfected cells are tuned with a measured concentration of the respective small molecule inducer: 1. Thaw vial of frozen HEK293 cells expressing Module A, Module B, and Module C. 2. Grow HEK293 cells to VCD of 3 x 106cells / mL in growth media without antibiotics. 3. Transfect HEK293 cells by chemical-based methods with transfer vector containing payload flanked by AAV inverted terminal repeats. 4. Culture cells for 4 h at 37 C, 8% CO2, and 125 rpm. 5. Add tebufenozide at concentration range of 1 µM-10 µM to transfected cells. 6. Add 4-OHT at concentration range of 1-10 µM to transfected cells. 7. Add doxycycline at concentration range 100-500 ug / mL to transfected cells. Subsequent steps include lysis and harvesting of the cells, assaying for viral genome titer, measuring AAV serotype specific capsids, measuring ratio of full to empty capsids, and measuring functional titer, as described above. Depending on the results, a fresh lot of packaging cells can be retuned and remeasured.

[0071] The drawings provide two examples in which Modules A, B, and C were variously tuned to increase capsid titer or the percentage of full capsids made by HEK293 producer cells.

[0072] FIGS.6A to 6C is a first example in which modules were tuned to increase virus production. In FIG.6A, HEK293 producer cells with an eGFP payload were expanded to a workable population; then passaged to a medium comprising each of the inducer compounds at a chosen concentration. Module A, B, and C were turned to levels of low, low-medium, and high using the small molecule inducers Teb, 4-OHT, and Dox, respectively. FIGS.6B and 6C compare capsid titer and the percentage of full capsids measured in supernatants for cells adapted to package three different serotypes of AAV: specifically, AAV1, AAV2, and AAV5. Tuning the modules in this fashion increased capsid titer up to 4-fold while maintaining similar percentage of full capsids.

[0073] FIG.7A to 7C show a second example in which Modules A, B, and C were tuned to produce a higher percentage of full capsids. In FIG.7A, packaging cells containing the three modules were further transfected with a payload vector. Modules A, B, and C were turned to levels of medium, medium-high, and medium using the small molecule inducers Teb, 4-OHT, and Dox dials, respectively. FIGS.7B and 7C compares capsid titer and percent full capsids harvested from packaging cells for thePCT patent application Tunable AAV packaging cells three AAV serotypes. Tuning the modules in this manner increased the percentage of full capsids produced from packaging cells made with hybridized parental cells. 6. Inducible regulatory elements for use in a tunable system

[0074] A hallmark of the tunable packaging cells in this disclosure is the use of multiple genetic elements that each responds to small molecule inducers (or other effects that can be imposed upon culturing of the cells).

[0075] In FIG.1, the inducible element for Module A is the hybrid bipartite QF and ecdysone receptor (ECR) system: two un-related truncated proteins fused together for controlling gene expression in a regulated manner. QFDBD serves as a transcription factor that binds to Q-response elements (QREs) but requires activation of Teb ligand which binds to truncated Ecdysone receptor (EcR). Other singular and bi-partite regulatory elements, which offer inducible gene expression, include: ^ Tetracycline-Inducible Systems: These systems are based on the tetracycline repressor (TetR) protein and its interaction with tetracycline or its derivatives (e.g., doxycycline). Examples include Tet-On and Tet-Off systems. ^ Lac Operon / Isopropyl β-D-1-thiogalactopyranoside (IPTG)-Inducible Systems: These systems utilize the Lac repressor (LacI) protein and its interaction with the inducer IPTG to control gene expression. ^ Cre-LoxP Recombination System: This system involves the Cre recombinase enzyme and its ability to catalyze site-specific recombination between DNA sequences known as LoxP sites. Inducible versions of this system can be controlled by promoters that regulate Cre expression. ^ Flp-FRT Recombination System: Similar to the Cre-LoxP system, this system involves the Flp recombinase enzyme and its ability to catalyze recombination between FRT sites. ^ GeneSwitch® System: This system utilizes a fusion protein comprising a mutant progesterone receptor ligand-binding domain and a transcriptional activator domain. Gene expression is induced by the synthetic ligand, mifepristone (RU486). ^ Chemically-Inducible Systems: These systems use small molecules or chemical compounds as inducers to regulate gene expression. Examples include the rapamycin-inducible system (based on the FK506 binding protein (FKBP) and rapamycin) and the Shield-1-inducible system (based on the FKBP protein and Shield-1 ligand). ^ Heat Shock-Inducible Promoters: These promoters drive gene expression in response to elevated temperatures, typically in the range of 37-42°C.

[0076] The inducible element in Module B is CreERT. The CreERT protein is a fusion protein that combines the Cre recombinase enzyme with the ligand-binding domain of the estrogen receptorPCT patent application Tunable AAV packaging cells (ER). This fusion protein allows for inducible and temporally controlled activation of Cre-mediated recombination in response to the estrogen receptor agonist, typically tamoxifen or its active metabolite 4-hydroxytamoxifen (4-*). When tamoxifen or 4-OHT binds to the estrogen receptor domain of CreERT, it induces a conformational change in the protein, activating its recombinase activity. This activated CreERT can then catalyze site-specific recombination between loxP sites, leading to the deletion, inversion, or translocation of DNA sequences flanked by loxP sites.

[0077] The inducible element in Module C is the Tetracycline-Responsive Element 3G (TRE3G) promoter: a synthetic DNA sequence engineered to regulate gene expression in response to the presence or absence of tetracycline or its derivative, doxycycline.

[0078] More generally, inducible elements that can be used to control virus gene expression include the following: ^ Promoters: Promoters are DNA sequences that initiate the transcription of a gene. In viral vectors, viral promoters (e.g., CMV promoter, SV40 promoter) or tissue-specific promoters (e.g., promoters specific to liver, muscle, or neuronal cells) are often used to control where and when the transgene is expressed. ^ Enhancers: Enhancers are DNA sequences that can increase the transcriptional activity of a promoter. They can be viral enhancers or cellular enhancers, and they contribute to fine-tuning the expression levels of the transgene. ^ Polyadenylation (polyA) signal: The polyA signal is a sequence of RNA that signals the termination of mRNA synthesis and the addition of a polyadenine tail to the mRNA. It enhances mRNA stability and translation efficiency. ^ Transcriptional terminators: Transcriptional terminators are sequences that signal the end of transcription. They help prevent read-through transcription from adjacent sequences and ensure proper processing of mRNA. ^ Regulatory elements for viral replication and packaging: These elements are necessary for viral vector replication and packaging. They include sequences essential for viral genome replication, packaging signals, and sequences required for viral particle assembly. ^ Response elements: Response elements are sequences that respond to specific cellular or environmental signals, allowing for inducible or regulated expression of the transgene. Examples include tetracycline-responsive elements (TRE), doxycycline-inducible promoters, or heat shock-responsive elements. ^ Insulators: Insulators are DNA sequences that can block the effects of nearby enhancers or silencers, preventing inappropriate activation or repression of neighboring genes.

[0079] The inducible elements in the modules shown in FIG.1 are as follows:

[0080] QAT fusion protein consisting of QF DNA-binding domain combined with truncated EcR protein fused to VP16 activation domain. QF serves a transcription factor while truncated EcR enablesPCT patent application Tunable AAV packaging cells the ligand-receptor activation by Teb. QF is under the category of transcription factors or regulatory proteins. It should be noted the QF can be activated by quinic acid but this response to quinic acid have been abrogated in QAT fusion protein in Module A.

[0081] CreERT protein is a fusion protein that consists of two main parts: Cre recombinase and a modified estrogen receptor ligand-binding domain (ERT). The Cre recombinase recognizes specific DNA sequences known as loxP sites and induces recombination between them, leading to DNA rearrangement. The ERT domain allows the activity of Cre recombinase to be controlled by the presence or absence of tamoxifen or its derivatives. Thus, the CreERT is a type of inducible regulatory protein.

[0082] TRE3G promoter contains tetracycline operator (TetO) sequences that bind to the tetracycline-controlled transcriptional activator (tTA) or reverse tetracycline-controlled transactivator (rtTA) protein in the absence or presence of tetracycline or its derivatives. This system allows for tight control of gene expression in response to the presence or absence of the inducer molecule. Thus, TRE3G promoter is an inducible regulatory DNA element.

[0083] Other inducible or regulatory elements that can be used in the context of this technology include the following: Transcription Factors: ^ Gal4: Utilized in the Gal4-UAS system for inducible gene expression. ^ LexA: Another transcription factor used in inducible gene expression systems. ^ TetR: The tetracycline repressor protein, used in tetracycline-inducible systems. Recombinases: ^ FlpERT: Similar to CreERT, FlpERT is a fusion protein comprising Flp recombinase and a modified estrogen receptor ligand-binding domain, enabling conditional gene manipulation. ^ Split-Cre Systems: Divides Cre recombinase into two inactive fragments, reassembling and becoming active in the presence of specific inducers. ^ Optogenetic Recombinases: Light-inducible recombinases activated by specific wavelengths of light, offering precise spatiotemporal control over genetic manipulation. Promoters: ^ Tet-On / Tet-Off Systems: Utilize tetracycline-controlled transcriptional activators (tTA / rtTA) along with tetracycline-responsive promoters for inducible gene expression. ^ RU486-Inducible Promoters: Responsive to the synthetic steroid mifepristone (RU486) for controlled gene expression. ^ Gal4-UAS System: Relies on the yeast transcription factor Gal4 and its upstream activating sequence (UAS) for inducible gene expression.PCT patent application Tunable AAV packaging cells Chemically-Inducible Systems: ^ RU486-Inducible Systems: These systems use the synthetic steroid mifepristone (RU486) to control gene expression through engineered ligand-responsive promoters. ^ Ecdysone-Inducible Systems: Based on the insect steroid hormone ecdysone and its receptor, these systems enable regulated gene expression upon addition of ecdysone or its analogs. Light inducible and heat shock inducible promoters Galactose inducible and nutrient inducible promoters Oxygen-Sensitive Systems: Synthetic Biology Approaches: ^ Riboswitches: RNA elements that modulate gene expression in response to specific small molecules. ^ RNA-Based Systems: Utilize RNA interference (RNAi) or RNA-guided gene regulation for inducible gene expression or silencing.

[0084] Depending on the inducible system used, expression may depend on small molecules such as tetracycline, tebufenozide, 4-OHT (4-hydroxytamoxifen), doxycycline, arabinose, estradiol (or 17β-estradiol), rapamycin (or sirolimus), RU486 (mifepristone), IPTG (isopropyl β-D-1-thiogalactopyranoside), X-Gal (5-bromo-4-chloro-3-indolyl β-D-galactopyranoside), IPTG (isopropyl β-D-1-thiogalactopyranoside), AHT (anhydro-tetracycline), AAL (ara-ab-lactose), cumate, and lactose. Depending on the inducible system used, expression may depend on physical parameters such as temperature (heat), light level, and the presence of oxygen or galactose in the medium.

[0085] Table 3 provides a list of inducible elements that may be adapted and incorporated into the technology described herein. TABLE 3: Inducible elements to control expression of tunable AAV vector modules Inducible Element Inducer Molecule CreERT2 4-OHT, Tamoxifen EcR Teb Cumate operator Cumate TRE3G, Tet-ON Tetracycline, doxycycline QF2 Quinic acid FKBP12-ZFHD1-FRAP-p65 RapamycinPCT patent application Tunable AAV packaging cells TABLE 3: Inducible elements to control expression of tunable AAV vector modules Inducible Element Inducer Molecule FKBP12-Gal4DBD, Cyclophilin-VP16AD, and FKCsA UAS cassettes PYL1 / ABI1 (ABA activator cassette) ABA Cry2 / CIB1, other photoactivatable (PA) switches Blue light Riboswitch Aptazymes: tetracycline RNA aptamer, engineered ribozymes Geneswitch Mifepristone 7. Obviating leaky promoters using components that cause gene alteration

[0086] Some types of tunable regulators (such as inducible promoters) can be titrated — the amount of expression can be adjusted precisely by titrating the amount of the inducing small molecule or condition. This titratable expression is often reversible, meaning that a high level of expression induced by a high concentration of the inducing agent can be reversed by decreasing the concentration of the inducing agent. These types of regulatory elements can be “leaky”: even in the absence of the inducing element, they may support a low but not insubstantial level of expression. This may occur, for example, if the regulatory element has a floor level of constitutive expression, or if there are other components in the reaction mixture that cross-react and cause a low level of expression in the absence of the usual inducer.

[0087] Leakiness can be prevented by using an inducible element that results in gene alteration: for example, by rearrangement, inversion, or excision of part of the gene. Each inducer can initiate one, two three, or several gene alterations. Gene rearrangements can cascade and cross over to other modules.

[0088] For example, in FIG.1, the inducer 4~OHT initiates several gene alterations. It causes excision of a STOP codon on Module B, causing expression of mFlp5. This in turn inverts a gene cluster on Module A, which includes sequences encoding Rep52 and S~Cap, which can be induced to express with the inducer tebufenozide. Concurrently, 4~OHT controls expression a cluster of helper genes on Module B.

[0089] Components that regulate expression via gene rearrangement include the following: (A) Recombinase recognition sites ^ loxP – Cre recombinase target; allows excision or inversion; ^ FRT – Flp recombinase target; allows excision or inversion; ^ rox – Dre recombinase target; orthogonal to Cre / Flp;PCT patent application Tunable AAV packaging cells ^ attB / attP / attL / attR – Sites for integrases (e.g., PhiC31, Bxb1); used for site-specific integration / excision. (B) Blocking or conditional expression elements ^ STOP cassette (loxP-STOP-loxP) – Transcriptional block removed by Cre; ^ PolyA terminator cassettes – Cause early termination; excised to allow expression; ^ Transcriptional pause signals – Removed or bypassed by recombination. (C) Inversion-based control elements ^ Inverted gene cassettes – Gene placed in antisense orientation; flipped ON by recombinase; ^ Inverted promoter elements – Promoter flipped into correct orientation to drive expression; ^ Double loxP / FRT switches – Enable inversion depending on recombinase directionality. (D) Structural boundary elements (when used with rearrangement) ^ Insulators such as cHS4 – Block enhancer-promoter interaction; repositioned by recombination; ^ Enhancer-blocking sequences – Prevent activation until excised or moved. (E) Other rearrangement-responsive components ^ Splice acceptor sites – Enable gene trap expression post-recombination; ^ Landing pads (e.g., attP / loxP cassette) – Allow targeted gene insertion via recombinase; ^ Recombinase-activated transcription units – Genes or reporters turned on only after structural rearrangement.

[0090] A gene rearrangement system is typically a binary switch. The inducing element has no effect at low concentrations. At higher concentrations, it causes the rearrangement to occur, switching expression on or off. Such gene rearrangements are typically not reversible. After the rearrangement occurs, there may be no need to maintain the same concentration of inducing agent in the mixture.

[0091] Titratable systems are advantageous for precise control and adjustment of multiple cassettes. Non-leaky systems are advantageous for reproducing a desired effect under pre-established conditions. 8. Genes to include that inhibit cell death caused by expression of viral proteins

[0092] Rep protein expression (Rep52 or Rep78) can induce apoptosis of an AAV infected cell line or inhibit expression of other viral genes in the absence of adenovirus. M. Schmidt et al., J Virol. 2000 Oct; 74(20): 9441–9450. To prevent this effect from decreasing production of AAV, the tunable packaging system shown in FIG.1 comprises the human gene for (Bcl-2) B-cell lymphoma 2) protein. This acts as an anti-apoptotic regulator by inhibiting the release of cytochrome c from mitochondria, thereby preventing the activation of caspases and apoptosis.PCT patent application Tunable AAV packaging cells

[0093] Other genes that perform a similar function and are potential substitutes for Bcl-2 in this context are Bcl-xL (B-cell lymphoma-extra large), Mcl-1 (Myeloid cell leukemia 1), Bcl-w (Bcl-2-like protein 2), Bcl-B (Bcl-2-like protein 10), Bax (Bcl-2-associated X protein), Bak (Bcl-2 homologous antagonist / killer), Bad (Bcl-2-associated death promoter), Bim (Bcl-2-interacting mediator of cell death), Bid (BH3 interacting-domain death agonist), and the BHRF1 apoptosis regulator of Epstein Barr virus.

[0094] Referring to FIG.3, activation of CreERT protein in Module B results in expression of AAV Cap, and AAV Rep52 on Module A. It also results in expression of Bcl-2 on Module C. Thus, expression of Rep 52 which may cause apoptosis, and Bcl-2 which inhibits apoptosis, are under control of the same inducible elements on both Modules A and B. More generally, any Rep gene or AAV gene that promotes apoptosis can be placed under control of any apoptosis inhibiting protein to prevent overexpression of the AAV gene from limiting AAV packaging. 9. Using cell hybrids as the host packaging cell

[0095] Previous disclosures by CHO Plus provide improved cell lines for manufacture of pharmaceutical agents containing viral elements, considerably reducing the cost of commercial production. See, for example, US Patent No.12,252,713, entitled Cell hybrids as host cells for high efficiency production of gene therapy vectors and viral vaccines.

[0096] FIG.4 is a general scheme that outlines a suitable workflow by which cell hybrids may be generated, selected, and expanded to yield AAV packaging cell lines.

[0097] Cells from a chosen source (for example, an established cell line such as HEK293) are fused together in multiple cycles to generate a population of hybrids that are heterogeneous in their ability to synthesize viral vectors or particles. To obtain cells from the population that are high producers, the population is partitioned into a plurality of separate aliquots or clones. As part of the partitioning, the cells may be screened or separated according to particular phenotypic features that are known or suspected of being beneficial to high levels of viral capsid production or filling. A sample from each of the separate aliquots or clones are individually tested for their ability to produce high quantities or high titers of vectors or particles. Aliquots of cells that are now proven to be high producers are expanded, and used to establish one or more packaging cell lines.

[0098] The banked packaging cells can then be sourced for industrial-scale production of one or a variety of therapeutic viral vectors or particles, especially using viral components of the same species and serotype that were used for screening. The cells selected for industrial production can be transfected in the same manner used for screening, with the exception that the reporter gene is substituted with a therapeutic payload. The final transfection can be transient, or the viral elements can be integrated into the genome of the producer cell with an inducible or ubiquitous promoter — whereafter different payloads can be encapsulated into the same viral system by transient transfection.PCT patent application Tunable AAV packaging cells

[0099] Cell hybrids are made by obtaining a cell mixture of cells to be fused: a plurality of cells from one cell line, or more than one cell line, or a mixture of at least one cell line and at least one primary cell population. The cell mixture is then subjected to an appropriate fusion protocol: for example, by culturing under culture conditions that promote the formation of hybrids, by conducting an electrofusion, by combining with a fusogenic virus such as Sendai virus, by placing cells into contact (for example, by gentle centrifugation), by treating with a fusogenic agent such as polyethylene glycol (PEG) or using any effective combination thereof.

[0100] Cells may be fused into hybrids using any suitable technique. For example, cells may be cultured in the presence of a fusogenic agent and / or under culture conditions that promote the formation of hybrids, or may be forced into contact, for example, by gentle centrifugation, optionally in combination with a fusogenic agent such as polyethylene glycol (PEG). Typically, a fused cell is obtained by fusing two cells together, although fusion of three or more cells is possible. It is recognized that fusion of two different cell populations will result in mixed cell products (isotopic, allotypic, or xenotypic hybrids, depending on the parental cell lines), and autotypic hybrids. Autotypic or isotopic hybrids can be separated from allotypic or xenotypic hybrids, if desired, using fluorescently labeled or surface bound antibody specific for a ligand expressed on one of the cell lines in the mixture, but not another. 10. Detailed protocol for making cell hybrids

[0101] By way of illustration (and without implying any limitation on the claimed invention and equivalents thereof), packaging cell lines for AAV vectors have been obtained according to the following protocol:

[0102] Step 1: Production of hybrids. A starting cell population of HEK293 cells was used to make cell hybrids by using polyethylene glycol as fusogenic agent combined with gentle centrifugation to promote cell contact. Hybrids were cloned. Each clone was separated into aliquots, and sampled for transfection testing.

[0103] Step 2: Transfection. Sampled hybrid cell clones in suspension were transfected with chemical-based methods using a lipid polymer that complexes with negatively charged DNA to form lipopolyplexes via electrostatic interactions. Three plasmid vectors were used for transfection: 1) transfer vector expressing a fluorescent protein under the control of ubiquitous CMV promoter cassette flanked by AAV inverted terminal repeats; 2) helper vector cassette expressing adenovirus E4 gene for AAV DNA replication, adenovirus E2a gene and adenovirus VA RNA (virus-associated RNA) genes to enhance AAV mRNA stability and promote AAV capsid transcripts; and 3) packaging vector expressing Rep and Cap proteins specific serotype being assayed (AAV1, AAV2, and AAV5). Cells were harvested 72 hours post-transfection, lysed and assayed for AAV production.PCT patent application Tunable AAV packaging cells

[0104] Step 3: Determining production capability of cloned hybrids. AAV genomic copy number was measured by real-time quantitative PCR. Cell lysates were treated with DNase I to remove non- viral host genomic DNA. Real-time quantitative PCR by fluorescent detection was performed to determine viral genomic copy number. DNA primers bind to coding regions of fluorescent reporter within the transfected transfer vector in the assembled AAV and copy number was detected using fluorescence (methods used by previous figures).

[0105] Step 4: Determining AAV serotype-specific capsids. Bio-layer interferometry (BLI) is an optical biosensing technology that analyzes biomolecular interactions in real-time without the need for fluorescent labeling. Interference patterns of white light or phase shift caused by analyte sample binding to immobilized ligand on biosensor probe was used to quantify the amount of AAV virus in an unknown sample. A small biosensor that binds specifically to AAV capsid protein for multiple serotypes (AAV1, AAV2, AAV5). For each serotype (AAV1, AAV2, and AAV5), a standard curve of a commercial AAV reference standard, with known concentration measured by other validated methods are used to back-calculate the concentration of AAV serotypes in unknown sample.

[0106] Step 5: Measuring the ratio of full to empty capsids. Ratio of full to empty capsids can be measured in unknown samples by biolayer interferometry. First, concentrations of AAV serotypes of unknown samples are measured as described above. For each serotype being measured, AAV capsids at a normalized concentration are first captured and immobilized on the biosensor. Following immobilization, AAV particles are lysed to release the packaged ssDNA and ssDNA is captured and measured using a biosensor probe that is conjugated to SSB protein wherein SSB protein binds specifically to ssDNA. For each serotype (AAV1, AAV2, and AAV5), a standard curve of a commercial AAV reference standard, with known full-to-empty capsids ratio measured by other validated methods are used to back-calculate the ratio of AAV serotypes in unknown sample.

[0107] Step 6: Measuring functional titer. For measurements of functional titer, undiluted samples of AAV1, AAV2 and AAV5 produced using the cloned hybrids were infected at a range of dilutions and added to fixed population of un-infected HEK293 cells. Functional titers were measured in transduction units (TU) per mL. Infectivity was determined by quantifying percent fluorescent- positive cells by flow cytometry.

[0108] Step 7: Expand high producer packaging clones. The original aliquots corresponding to samples that showed high levels of capsid production and functional titer were expanded to establish producer cell lines for transduction and expression of other types of viral vectors and particles. 11. Selecting high producer packaging cell lines and preferred phenotypic features

[0109] This disclosure provides a variety of means for identifying and selecting cell hybrids that have the capacity of generating high producer packaging cell lines. Cells can be transfected with a reporter gene (for example, genes that encode fluorogenic products such as green florescent protein),PCT patent application Tunable AAV packaging cells along with genes that encode a viral capsid for testing purposes. High producers can be selected on the basis of viral capsids produced and / or encapsulated promoter gene products.

[0110] FIG.4B is a workflow for obtaining producer cell line for a particular AAV vector from a packaging cell line already optimized for high virus production capacity. A packaging cell line obtained according to FIG.4A is transfected with transfer vector containing payload and a secondary fluorescent marker (such as shown in FIG.2A). Cells are then sorted by flow cytometry for expression of secondary fluorescent reporter. Positive cells are cloned or separated into separate aliquots. Small molecule inducers that activate Modules A, B, and C are then added to separate clones or aliquots at varying concentrations and high producer cells are identified. The identified cells are expanded to establish a bank of producer cell lines. The producer cells can be used to produce AAV vectors for gene therapy or immunization without any additional transfection.

[0111] FIGS.5A, 5B, and 5C demonstrate higher productivity from cells fused, cloned, and sampled as described. Host cells were transiently transfected with (1) transfer plasmid expressing fluorescent reporter; (2) packaging plasmid expressing Rep and Cap proteins specific for AAV1, AAV2, or AAV5; and (3) helper plasmid. Capsid concentration or titer were measured by biolayer interferometry (BLI) using a biosensor that binds to AAV1, AAV2, or AAV5 capsids. Cumulative capsids productivity (FIG.5A), cell specific productivity (VP / cell) (FIG.5B), and percent full capsids (FIG.5C) of AAV1, AAV2, AAV5, AAV9 of HEK293 parent, engineered pool (7A) and clones (44, 2, 121, and 17). Engineered clones (44, 2, 121, and 17) showed 12-fold, 14-fold, 5-fold, and 6-fold increase compared with parent host, respectively. 12. Selecting for mitochondria phenotype

[0112] Virus producing cells (hybrids or standard diploid cells) can be selected for characteristic phenotypes that correlate generally with high levels of protein and / or virus product. Previous disclosures from CHO Plus demonstrate that fused cells sorted for higher amounts of mitochondria per cell and higher levels of reactive oxygen species (ROS) can be used to make producer cells that generate viral capsids that are as much as two-fold higher in the proportion of capsids that contain an intended pharmaceutical payload, such as a polynucleotide for purposes of gene therapy or vaccination.

[0113] Virus biosynthesis and assembly depend in part on mitochondria function. The cellular content of mitochondria and mitochondria properties are used as basis for sorting or selection without damaging the cell using vital dyes. Such dyes can be obtained commercially, for example from the companies: Invitrogen and Sigma Aldrich. Example of vital dyes for the mitochondria include: MitoTracker Green FM; MitoTracker Orange CMTMRos; MitoTracker Red CMXRos; MitoTracker Red FM; MitoTracker Deep Red FM; BioTracker 488 Green Mitochondria dye; BioTracker 633 Red Mitochondria dye; BioTracker 405; and Blue Mitochondria.PCT patent application Tunable AAV packaging cells

[0114] Functional dyes to measure the membrane or redox potential of the mitochondria can also be used to sort or select for cells with enhanced mitochondria function. Mitochondria membrane potential is generated by Complexes I, III and IV, and serves as a reliable read-out to assess mitochondria function. Membrane depolarization shifts fluorescence signal from one wavelength to another. These membrane potential dyes are available from companies: Invitrogen and Sigma Aldrich: JC-1 Dye (Invitrogen T3168; Sigma CS0390); JC-9 Dye (Invitrogen D-22421); and C10 Dye (Sigma MAK160, MAK159).

[0115] Additional characteristics to sort for enhanced mitochondria includes vital dyes to measure mitochondria calcium, superoxide production, and dyes selective to the mitochondria. These include: Rhod-2 AM Reagent (Invitrogen R1245MP); and MitoSOX Red (Invitrogen M36008).

[0116] Alternatively or in addition, the user can test expression-based labeling systems that would introduce a fluorescent protein targeted to the mitochondria. They are fusion proteins comprising a portion that expresses an optical label, fused with a protein sequence that targets or is processed by the organelle to be labeled. Examples include the following. From Invitrogen: CellLight™ Mitochondria- GFP (C10600); and CellLight™ Mitochondria-RFP (C10505, C10601). From Evrogen: pTagCFP- mito (FP117); pTagYFP-mito (FP137); pTagRFP-mito (FP147); pmKate-mito (FP187); pTagGFP2- mito (FP197); pTurboRFP-mito (FP237); pTurboGFP-mito (FP517); pPhi-Yellow-mito (FP607); and pTurboFP602-mito (FP717). From Takara Bio: pAcGFP1-Mito Vector (632432); pDsRed2-Mito Vector (632421); pHcRed1-Mito Vector (632434); and pPAmCherry-Mito Vector (632591).

[0117] After staining with any of these dyes, cells may be selected (for example, by flow cytometry and sorting) that have on average a level of staining that is at least 1.2, 1.5, 2, or more than 2- fold higher than the parental cell line or lines, in terms of staining, for example, for mitochondria or an optically labeled gene product.

[0118] By way of illustration, to screen for different phenotypes of mitochondria and reactive oxygen species (ROS), hybrids were stained with CellROX® Deep Red Reagent, a fluorogenic probe for measuring cellular oxidative stress in cells; TMRM (tetramethyl rhodamine methyl ester), which measures the membrane potential of mitochondria in living cells; and Biotracker 405 Blue Mitochondria, which stains the mitochondria membrane. LIVE / DEAD Fixable NIR was used in this experiment to stain live cells.

[0119] Fused cells were cultured in complete, animal origin free (AOF), chemically defined cell culture medium: CDM4 PerMAb + 6 mM L-Glutamine and detached using StemPro™ Accutase™ Cell Dissociation Reagent. Samples of cells were combined with a calculated volume of each dye to final concentrations in ~200 mL of cell suspension containing 1 x 108cells in a 500 mL shake flask. Samples were incubated @ 120 rpm in shaker overnight at 37ºC with 8% CO2.

[0120] For cell sorting, 200 mL of cell sample was centrifuged at 300 x g for 5 min. The cell pellet was suspended in Accutase cell dissociation reagent, diluted, and strained into a sterile 50 mLPCT patent application Tunable AAV packaging cells centrifuge tubes. Cells were sorted using a Sony SH800S Cell Sorter with the following gates: Gate 1 – cell ID gate; Gate 2 – singlets gate; Gate 3 – live cells gate; Gate 4 – Biotracker 405 Blue Mitochondria (select the top 10%); Gate 5 – TMRE x CellROX Deep Red (select the top 10% quadrant).

[0121] Populations of 500,000 sorted cells were expanded for 4-5 days and used for single-cell cloning in 96-well plates containing 150 µl of medium per well. Once individual wells were 80% confluent, they were expanded stepwise to 125 mL shake flasks, and used to create cell banks. 13. Characterizing high producer cell lines

[0122] Cell hybrid cells that have been optimized for the production of viral vectors and particles can be characterized by one or more criteria in any combination.

[0123] Suitable criteria include cell karyotype. Chromosome patterns can be characteristic of homotypic and heterotypic cell fusions. The following characteristics may be favorable for virus production: ^ duplication of chromosomal segments ^ loss of chromosomal segments (90%, 80%, 70%, 60% or less than 50% of original segment size) ^ differences in heterochromatin distribution and amounts (differences of 10%, 20% or greater than 20% and / or distribution differences of heterochromatin greater than 20%) ^ translocation events (2 or more translocation events on the same or different chromosome segment compared to parental cell line)

[0124] Packaging or producer cells can be characterized on the basis of virus productivity. They can also be characterized on the basis of cell phenotype, such as intracellular content of mitochondria, reactive oxygen species (ROS), endoplasmic reticulum, Golgi apparatus, and so on, using the reagents listed above. 14. Determining production capacity and characteristics of producer cells

[0125] A cell line or mixed cell population that has been selected for amount and / or quality of virus production may be characterized in comparison with the parental or originating cell line by any one or more of several different parameters. For example, the selected cells may have: (1) a genome that is more aneuploid than the starting cells, containing part or all of the genome of two or more parental cell lines (which may or may not be the same), (2) a higher concentration of mitochondria, more reactive oxygen species, a higher mitochondria membrane potential, or other phenotypic feature compared with any one or all of the parental cell lines (for example, between 2 to 5-fold or 4 to 8 fold, or more than 2-, 4-, or 8-fold higher), (3) a capacity to produce a level of virus per cell or per liter ofPCT patent application Tunable AAV packaging cells culture fluid that is substantially higher than the parental cell line (for example, between 2 to 5-fold or 4 to 8 fold, or more than 2-, 4-, or 8-fold higher), (4) a capacity to produce a particular amount of virus per cell (for example, more than 50, 65, 75, 100, 150, 200, 300, 500, 2000, 5000, or 20,000 capsids per cell; (5) a capacity to produce a certain amount of virus per volume of culture fluid (for example, at least 5, 8, 12, 20, or 30 grams, or between 8 and 20 or between 10 and 50 grams of virus per liter of culture fluid; or (6) a capacity to produce viral vectors or particles that have a higher proportion of payload-carrying capsids (50% higher, or 2 or 3-fold).

[0126] For the purpose of making such comparisons, the producer cell line can be compared with a standardized population of the original cell line, either kept on hand, as part of the same system, or obtained from a reference source. For example, CHO derived producer cells may be compared with CRL-12023 cells from the American Type Culture Collection (ATCC®). 15. Genetically altering producer cells to synthesize and produce viral elements

[0127] As explained above, AAV vectors and particles can be produced by transfection of helper, packaging, and transfer vectors into a suitable cell line such as a hybrid cell line. Transfection of rep, cap, and helper modules into host cells can be done, for example, using liposome-based reagents (for example, Lipofectamine™ 3000, Expifectamine 293, FuGENE™ HD, X-Fect nanoparticles polymer, Trans-IT Pro reagents, Trans-IT VirusGen, polyethylenimine), calcium phosphate, electroporation by transfection with an adenovirus, retrovirus or lentivirus-based vector, or using a transposase system such as Sleeping Beauty transposase.

[0128] Following transfection, the cells are tested for production for packaging of the intended virus: for example, by enzyme-linked immunosorbent assay (ELISA), quantitative real-time PCR (qPCR), or biolayer interferometry (BLI). Cells or clones having increased production of the desired virus are selected. The objective can be an increase in virus production that is 1.5, 2, 4, 8, 12, 16, 20, or 100-fold higher than the parental cell line; and / or production at a level of greater than 1012viral genome / ml or capsids / ml for AAV; and / or greater than 108infectious units (IFU) per ml of culture fluid under typical manufacturing conditions. The virus of interest can also be tested for other desired characteristics, such as full to empty capsid ratio and functional titer.

[0129] In principle, the transfection can be done either before, during, or after one or more cycles of fusion of the packaging cell line and selection for other features. For example, the fusion and selection can be done before transfection with the packaging, helper and transfer vector containing gene of interest, thereby establishing a parental cell line suitable for high-level of virus production of the user’s choice. Alternatively, the transfection can be done into the originating parental cell line containing gene(s) of interest and used to track production levels during subsequent fusion and sorting steps, or to provide another basis for such sorting. Alternatively, the transfection can be done as an intermediate step, wherein the cells have already been subject to one or more cycles of fusion andPCT patent application Tunable AAV packaging cells selection for some other feature such as mitochondria and ROS. The resulting hybrid is transfected to express virus of interest, and then subjected to further cycles of fusion and selection for expression of the virus of interest and / or other features referred to earlier in this disclosure.

[0130] Another option is to develop a cell line using a reporter gene as a proxy for the virus payload that ultimately will be manufactured: for example, secreted alkaline phosphatase, secreted luciferase, fluorescent virus payloads such as red fluorescent protein or green fluorescent protein. Again, the transfection can be done before, during, or after multiple cycles of fusion and selection, optionally using the level of expression of the marker as the selection criteria in one or more of the cycles. This creates a parental cell line that is optimized for expression of the marker virus payload, with the expectation that the beneficial characteristics of the cell line will be retained after further genetic alteration to produce a biological product of commercial interest.

[0131] Ultimately, once a cell line has been developed having a desired level of expression of the marker virus payload, the marker is then replaced with the virus payload of interest. Transfection can again be done randomly into the genome, using the techniques listed above, and expression of the reporter gene is curtailed. Alternatively, the gene for the reporter protein can be substituted with a gene that encodes the virus payload of interest using a targeted integration technique. Such techniques comprise, for example, a sequence guided editing tool such as CRISPR / Cas virus payloads, CRISPR / Cas associated transposase (CASTs), recombinase cassette exchange (RMCE), a zinc-finger recombinase (ZFR), serine integrases, or a transcription activator-like effector nuclease (TALEN). That way, the gene of interest is inserted into the genome of the cells from the producer cell line or the mixture at a location that is pre-selected as permitting or supporting a high level of transcription, compared with other locations in the genome. 16. Methods for quantifying levels of viral production from cell hybrids

[0132] Real-time quantitative PCR measures viral transcription, concentration of viral genome (vg / ml). Each viral particle typically contains one viral genome. Viruses are treated with DNase I to remove any of the host genomic DNA. Primers binding to targeted regions in the transfer vector are used and amplicon is detected by either probe-based method or SYBR Green, which binds to the amplicon.

[0133] Indirect ELISA and biolayer interferometry (BLI) are used to measure total capsid AAV particles. These measurements utilize an antibody against an abundant capsid protein present in AAV serotype. Samples are captured by capsid antibody and detected using biotinylated capsid antibody and Streptavidin conjugated to HRP for chemiluminescent detection.

[0134] Functional titer or infectious titer of viruses is the concentration of viral particles that can transduce cells. Functional titer can be measured by cell transduction using a fluorescent or chemiluminescent protein as a reporter. Cell lines are infected or transduced with packaged viruses atPCT patent application Tunable AAV packaging cells specific multiplicity of infections (MOI). % of cells expressing reporter gene are quantified and correlated with the # of virus particles used to transduce cells. 17. Pharmaceutical payloads and therapeutic applications

[0135] The technology of this disclosure is advantageous for delivering a nucleic acid, a protein, or mixture thereof for purposes of inducing a specific immunological response. The packaged nucleic acid encodes one or more epitopes from the intended immune target, and optionally one or more additional proteins that may act as an adjuvant or stimulant to enhance immunogenicity. The target may be an infectious agent, such as a pathogenic virus, bacteria, or protozoan. Alternatively, the target may be a cancer cell, in which case the encoded epitopes are epitopes expressed by the cancer cell that are specific to the cancer or to the tissue type. For example, the technology of this disclosure can be used to prepare a composition to induce a response to the SARS-CoV-2 virus, for the purpose of prevention or treatment of COVID-19. Most current vaccines against SARS-CoV-2 typically include or encode the whole spike protein. Ways to optimize the spike protein were recently discussed by F. Heinz & K. Stiasny, NPJ Vaccines (2021) 6:104.

[0136] The technology of this disclosure can also be used for the purpose of gene therapy: for example, delivery of a nucleic acid encoding a gene product that is missing or defective in the subject being treated, or targeted to pathogenic cells in the subject, particularly cancer cells. Therapeutic purposes include but are not limited to expression of a therapeutic protein encoded in the nucleic acid (such as a cytokine or anti-cancer agent), expression of an essential protein that the subject is unable to produce themselves, or delivery of a gene editing system such as CRISPR / Cas9 or a guide RNA. Other possible therapeutic payloads may include DNA antisense oligonucleotides, DNA aptamers; messenger RNAs, micro RNAs, short interfering RNAs, ribozymes, RNA decoys and circular RNAs that specifically increase or decrease expression of a particular endogenous gene in the subject or an infectious agent. K. Sridharan et al., Br J Clin Pharmacol.2016 Sep; 82(3): 659-672. 18. Integrating a payload gene into an AAV packaging cell

[0137] Packaging cells of this disclosure may be provided as cells in which each of the three modules is inheritably integrated such that progeny of cell division will be able to express the same genes in the same manner. Typically, this is done by stably integrating the modules into the cells’ genome. To make AAV vectors or particles (for purposes of testing or tuning, or for purposes of commercial production), the cells are further transfected with a gene that constitutes or encodes a payload: for example, a marker gene such as enhanced green fluorescent protein (eGFP) or luciferase; or a therapeutic cargo, as illustrated below.

[0138] The payload gene is usually separate and apart from any of the three modules used for making the AAV capsid and replication genes: a fourth module, in which the payload gene isPCT patent application Tunable AAV packaging cells optionally under control of a fourth regulatory element. The fourth module can be introduced by transient transfection: for example, using adenoviral vectors, Sendai virus vectors, vesicular stomatitis virus (VSV) vectors, lipofection, or mRNA transfection.

[0139] Alternatively, the fourth module can be inheritably incorporated into the cell by permanent transfection. Lentiviral vectors and retroviral vectors will integrate the payload gene at an undefined location in the genome. A transposase system can be used for less random insertion: Sleeping Beauty transposase (SB) prefers TA dinucleotide sites; PiggyBac transposase integrates at TTAA sequences; Tol2 transposase has a preference for AT rich regions. U.S. Patent 12,270,055 describes a variant Sleeping Beauty (SB) transposase that has been adapted to be more soluble, delivering a transposon vector into a target cell when used in protein form.

[0140] FIG.8 depicts integration of an AAV payload into a packaging cell line using the Sleeping Beauty transposon system, An SB transposon plasmid encoding the AAV payload between two ITRs, plus the SB transposase in protein form are co-delivered into packaging cell line by electroporation. After transfection, cells are allowed to recover in complete media without selection for 24–48 hours to permit transposition. Antibiotic selection is then applied based on the selection marker present, enriching for cells with successful chromosomal integration of the payload. If the transposon also encodes a fluorescent reporter, fluorescence-activated cell sorting (FACS) can be used to isolate positive integrants. 19. Placing a payload gene into a transcription safe harbor using a landing pad system

[0141] To ensure reliable expression, the payload gene can optionally be placed at or near a particular region of the genome of the host cell. This can be done using; (1) a meganuclease (a homing endonuclease that induces double-strand breaks at specific sites, followed by homologous recombination), (2) a zinc finger nuclease (ZFN) (a recombinant protein that combines zinc finger DNA-binding domains with the FokI nuclease domain); (3) a transcription activator-like effector nuclease (TALEN) a DNA-binding domain from transcription activator-like effectors fused to a FokI nuclease domain; or (4) CRISPR / Cas9 (which uses a guide RNA to direct the Cas9 nuclease to a specific DNA sequence).PCT patent application Tunable AAV packaging cells

[0026] The deliberately targeted chromosomal region is at least a gene expression “safe harbor” in the host cell. Inserting a gene in a safe harbor chromosomal region won’t harm cell viability or productivity; supports stable gene expression; and has a low risk of insertional mutagenesis. Examples are AAVS1, CCR5, and ROSA26 locus. AAVS1 (also known as the PPP1R12C locus) on human chromosome 19 has an open chromatin structure and is transcription-competent.

[0142] The payload gene may also be targeted for integration into a region of increased gene expression (RIDGE). RIDGEs have been identified through transcriptome mapping where clusters of highly expressed genes reside therein (Zhou et al.2003, Can. Res.63:5781-5784; Caron et al., 2001, Science 291:1289-1292). Examples of RIDGEs include Hipp11 (H11) locus, ROSA26 locus, AAVS1 and multiple antibiotic resistance (mar) locus.

[0143] With the objective of optimizing production and minimizing the time needed to develop full virus producing cells, the packaging cells may be provided with a landing pad system that establishes a recombination site at a known location in the genome, ready to receive and integrate the payload gene. For purposes of this disclosure, the term “landing pad” refers to a polynucleotide sequence recombinantly placed in the genome of a cell that serves as a docking station to accept other transgenes such that the transgenes may be reliably placed into the genome at a pre-established location. A landing pad by definition comprises at least two sites that are recognized by recombinase reagents, and optionally one or more selectable markers.

[0144] Landing pad systems and components thereof are provided in U.S. Patent 9,932,607 (Michele Pamela Calos et al., Stanford) describes methods for inserting a polynucleotide sequence of interest into the H11 locus in the genome of a human cell, using a phiC31 integrase and a Bxb1 integrase. U.S. Patent 8,980,579 (V. Mauro et al., Scripps) describes a landing pad system for stable integration of a heterologous polynucleotide in CHO cells at or around the ankyrin 2 gene (Ank2). U.S. Patent 9,233,174 (Z.Y. Chen et al., Stanford) describes a method for introducing a minicircle nucleic acid vector into a target cell that contains a gene of interest and a site-specific recombinase, wherein the minicircle is made in bacteria and devoid of plasmid backbone DNA sequences.

[0145] A.H. Rosenstein et al. (bioRxiv, March 4, 2023) described aa flexible transgene integration landing-pad toolkit in human induced pluripotent stem cells. Cell lines were established with a landing pad system facilitating rapid high-efficiency delivery of transgenes to the AAVS1 safe- harbor locus using the Bxb1 large-serine recombinase. By integrating a transgene into the landing pad, a fully selected hiPSC population can be isolated within 1-2 weeks after landing-pad recombinase- mediated cassette exchange. A. Blanch-Asensio et al. (bioRxiv, Oct.17, 2024) described a platform for dual, single-copy integrations of DNA payloads and gene circuits into human induced pluripotent stem cells. InSCREENeX GmbH in Germany commercially distributes landing pad cells with targeted integration for rapid cell line generation and library integration. The company asserts that gene- integrated cell banks can be created in 8-10 weeks for CHO cells, and 10-12 weeks for HEK293 cells.PCT patent application Tunable AAV packaging cells

[0146] An exemplary landing pad system for use in a tunable virus may be adapted from the technology described in U.S. Patent 11,505,792 and PLoS One.2019 Jul 25;14(7):e0219842. The system is distributed by Applied StemCell Inc., Milpitas CA under the tradename TARGATT™.

[0147] Referring to FIGS.9A to 9C, the landing pad is introduced into the genome of a producer cell line using a construct that contains the landing pad itself, along with sequences for recombination and selection markers. The construct includes a first negative selectable marker flanked by a pair of recombinase-recognition sites (RRS1 and RRS2). RRS1 can be the same as RRS2, or they can be different. The construct also contains a recombinase that recognizes at least RRS1 or RRS2.

[0148] By way of illustration, the negative selectable marker may be a small molecule resistance gene: for example, thymidine kinase (TK), an ATP-thymidine 5’-phosphotransferase that converts deoxythymidine into deoxythymidine 5’-monophosphate. Mammalian cell expressing HSV-TK converts GCV into GCV phosphate, which is further phosphorylated and incorporated into the synthesized DNA, leading to the termination of synthesis and apoptosis. Alternatively, the negative selectable marker may be cytosine deaminase, which hydrolyzes cytosine to uracil with release of ammonia. Upon provision of non-toxic prodrug 5-FC, cytosine deaminase converts non-toxic prodrug 5-FC into highly toxic 5-FU (a suicide inhibitor of thymidylate synthetase), leading to apoptosis. Alternatively, the selectable marker can be a gene product that emits light or is fluorescent, whereby selection can occur by fluorescence activated cell sorting (FACS).

[0149] The landing pad construct can be inserted into a particular genomic locus, for example, by homologous recombination. To do this, the landing pad sequence is flanked in the construct by sequences homologous to sequences in the specific genomic locus (homologous arm) is introduced into the cell. This can be catalyzed by introducing a double strand break or a nick in the specific genomic locus created by a site-specific nuclease, such as Cas9, TALEN, or a Zinc finger nuclease. The construct may also contain a positive selectable marker to facilitate the selection of the clones having homologous recombination. The construct may also contain sequences or elements enhance the expression of the selectable marker or the recombinase, such as polyA sequence, T2A coding sequence, or IRES (Internal Ribosome Entry Site).

[0150] Alternatively, the landing pad construct can be introduced into the cell using a transposon: for example, a modified sleeping beauty transposase polypeptide with increased solubility for transfection into cells as an isolated protein, in accordance with U.S. Patent 12,270,055.

[0151] The targeting construct (an “exchange cassette”) for inserting a gene of interest (GOI) encoding a payload into the landing pad contains the GOI flanked by a pair of RRS (RRS3 and RRS4). The vector backbone of the targeting construct contains a second negative selectable marker.

[0152] In the general scheme for a prototype landing pad system shown in FIG.9A, recombination occurs between RRS1 and RRS3 and between RRS2 and RRS4. Typically, RRS1 and RRS3 are recognized by the same site-specific recombinase, and RRS2 and RRS4 are recognized by thePCT patent application Tunable AAV packaging cells same site-specific recombinase. Optionally, RRS1 is the same (or recognized by the same recombinase) as RRS2, and RRS3 is the same (or recognized by the same recombinase) as RRS4. In this case, one site-specific recombinase mediates recombination at both positions. A gene encoding site-specific recombinase can be included in the landing pad construct. Alternatively, it may be introduced into the cell in protein form, or it may be provided as an mRNA or a plasmid that encodes the recombinase.

[0153] Depending on how the landing pad and the targeting construct are configured, the recombinase may have different effects: 1. Two recombination events may occur, one between RRS1 and RRS3 and the other between RRS2 and RRS4. As a result, the gene of interest is integrated to the specific genomic locus and replaces the negative selectable marker. 2. Recombination may occur between RRS1 and RRS4, and also between RRS2 and RRS3, resulting the negative selectable marker replacing the selectable marker in the landing pad. 3. No recombination or insertion occurs, and the landing pad remains unaltered. 4. The targeting construct is inserted randomly into the genome of the cell. In such case, the landing pad sequence will be intact, and the second negative selectable marker may be inserted into the genome. 5. The targeting construct may be inserted into the landing pad through one recombination event.

[0154] The respective outcomes are illustrated in the bottom panel of FIG.9A. If recombination occurs between RRS1 and RRS3, the first negative selectable marker will persist in the landing pad. The cells will contain a negative selectable marker except in the first possibility where the gene of interest replaces the first negative selectable marker. Using the selection agent, all the cells contain the negative selectable marker will be depleted, leaving only the cells in which the GOI is properly docked in the landing pad. A master HEK293 cell line containing the landing pad can then be transfected with a targeting construct as illustrated in FIG.9C. The targeting construct contains a GFP gene (GOI) flanked by a pair of phiC31-recognition site, attB. 20. Demonstration of using landing pads in hybrid packaging cells

[0155] FIG.10A is a workflow diagram, depicting the steps of (1) installing the landing pad into a high productivity region of the genome; and (2) using the landing pad to dock a gene of interest for protein or virus production. First, a genomic landing pad is integrated at a safe harbor locus (H11) using CRISPR / Cas9. A donor plasmid (or placement plasmid) carrying the landing pad cassette (containing a fluorescent reporter) is co-transfected with a Cas9-eGFP gRNA construct targeting the chosen locus. In the second stage, the AAV payload vector is integrated into the pre-installed landing pad by co-transfecting serine integrase and donor vector expressing AAV payload. Seventy two hoursPCT patent application Tunable AAV packaging cells post-transfection by FACS, cells are isolated for co-expression of both primary and secondary markers, enriching for correctly targeted and actively expressing integrants.

[0156] FIG.10B shows how using a landing pad can considerably accelerate the development of producer cells for new vectors: Targeted integration (top line) can generate a stable virus producer cell line within the first month. This may lessen the need for single cell cloning and screening that would be required if the transgene is inserted at a random place in the genome.

[0026] Integration of the landing pad is typically but not necessarily done after the host cell has already been hybridized and selected for desirable cell phenotype and productivity. The integration and docking of the cargo can be done in conjunction with other genetic modifications of the hybrid cells in any suitable order.

[0157] FIGS.10C and 10D are schematic depictions of the process used to integrate the landing pad into a targeted region of the genome of the host cell, and then dock it with a gene of interest (GOI) — in this case, eGFP. Selection of positively transfected and integrated cells can be done by small molecule selection genes (Example 6), or using color genes and cell sorting (Examples 1 and 2). pA = polyadenine encoding region; mCherry = red monomeric fluorescent protein from coral; eGFP = enhanced green fluorescent protein; H11 = Hipp11 (H11) safe harbor locus in host cell genome; AAV ITR = inverted terminal repeats (ITRs) on adeno associated virus (AAV) plasmid; WPRE = woodchuck hepatitis virus posttranscriptional regulatory element to increase gene expression.

[0158] In FIG.10C, the landing pad is installed by chemical-based transfection by transfecting CRISPR / Cas9, gRNA targeting H11 locus of HEK293 cells, and landing pad plasmid DNA containing mCherry reporter gene for stable selection. In FIG.10D, the AAV payload is installed by transfecting donor plasmid containing AAV payload, optionally under control of an inducible promoter, and PhiC31 Integrase. To make cargo-loaded AAV vectors, the cells are contacted with the promoter inducer, and plasmids encoding AAV Rep and Cap genes. Alternatively, the cells may be set up beforehand as packaging cells with integral rep and cap genes. The packaging cell can then be used to make AAV with different cargoes by inserting each cargo into the landing pad.

[0159] Results are shown in FIGS.11A, 11B, and 12. FIG.11A is a contour plot of hybrid HEK293 cells that were installed with a landing pad (marked by mCherry) and then docked with a model AAV cargo (eEFP). Event densities of cells bearing both markers are depicted with contour lines (gradients).80 to 85% of the cells bear the intended cargo.

[0160] FIG.11B shows capsid titer for AAV2 and AAV5 in HEK293 host cells with AAV payload targeted specifically to the H11 locus using landing pad technology. Percent increase was normalized to parental cell line which does not contain stably integrated AAV payload at the H11 locus. FIG.11C shows that the relative mRNA expression of the AAV payload measured by RT-qPCR is stable through 60 doublings of the integration.PCT patent application Tunable AAV packaging cells

[0161] FIG.12 shows genome titer (vg / L) for AAV2 and AAV5 in HEK293 cell hybrids with an AAV payload targeted specifically to the H11 locus using landing pad technology. Viral genome copy number was measured by real-time qPCR. Percent increase was normalized to parental cell line which does not contain stably integrated AAV payload at the H11 locus. 21. Production and Purification of AAV vectors from producer cells

[0162] FIG.13A schematically illustrates a two-step chromatographic process for purifying AAV vectors for clinical and commercial use. First, AAV capsids (both full and empty) are captured by affinity chromatography using a suitable resin, such as AAVX resin, Capto™ AVB resin, or Peptide A10 resin. Second, anion exchange chromatography is used to separate full and empty capsids. Elution of capsids is done using a step or linear gradient of salt concentration, with empty capsids eluting before the more negatively charged full capsids. Drawing adapted from P.R.H. Joshi et al., 2021 Mol Ther Meth Clin Dev 21:341.

[0163] FIG.13B shows purification yield of AAV using this method. HEK293 hybrid cells were transfected to express viral genes for AAV2 or AAV5, plus a model payload. For the open bars, the payload was integrated into the cells at an unknown location. For the striped bars, the payload was installed into a landing pad present in the H11 safe harbor. There was comparable purification yield, showing that yield is not compromised by using the landing pad system. 22. Leakproof regulatory elements and why they are important in AAV vector production

[0164] The genes encoding Rep78, Rep68, Rep52, and Rep40 initiate replication at the AAV inverted terminal repeats (ITRs), regulate AAV transcription, and direct site-specific integration of the AAV genome into host DNA. Overexpression of one or more Rep proteins can cause cytotoxicity and apoptosis of the host cells.

[0165] Single promoter systems are prone to “leaky” or basal levels of expression. For example, the Tet-On / Tet-Off system may exhibit low but detectable transcriptional activity under non-inducing conditions. Similarly, the ecdysone-inducible system can display ligand-independent activation, especially in mammalian cells where endogenous hormone receptors may contribute to off-target activation. Other leaky promoter systems include the cumate-inducible system, based on the CymR repressor and CuO operator, GeneSwitch™,. and LacI / KRAB repressible viral promoters such as CMV and RSV. Basal levels of expression of Rep proteins in AAV packaging cells may result in substantial levels of apoptosis of the packaging cells, leading to lower yields of AAV vectors.

[0166] The leakproof regulator elements of this disclosure require multiple, concurrent signals to activate rep expression. The expression is tightly controlled, protecting packaging cells from rep induced apoptosis and improving yield.PCT patent application Tunable AAV packaging cells

[0167] FIG.14A shows the logic circuit controlling expression of Rep52 in the three module system shown in FIG.1. The “Q” gate requires QAT and Teb, activating transcription downstream of QUAS (Q-ON state). The “C” gate requires two inputs: 4OHT and CreERT2. Turning on Q or C gate alone is insufficient to activate Rep52 expression. When inducers 4-OHT and tebufenozide are both present, 4-OHT causes the stop codon on Module B to be excised, causing expression of mFlp5. This inverts the cassette in Module A that includes the Rep52 gene, placing its expression under control of tebufenozide. In this example, a gene excision and a gene inversion event are needed, followed by titratable gene activation.

[0168] FIG.14B shows the logic circuit controlling expression of Rep68 in the three module system shown in FIG.1. Here, gene activation requires two conditions: the presence of doxycycline, which enables the reverse tetracycline transactivator (rtTA) to bind the TRE promoter, and the absence of tTS-mediated repression, which occurs only when doxycycline is present. In the absence of doxycycline, tTS binds to the TRE promoter and actively represses transcription, even if rtTA is expressed. Doxycycline simultaneously activates rtTA and causes tTS to dissociate from the promoter, lifting repression and allowing gene transcription to proceed. There is no gene excision or rearrangement in this example. Instead, the two inputs are removal of the tTS repressor, followed by activation of the TRE promoter. 23. Leakproof regulation of a two module systems

[0169] FIG.15 shows an example of a two-module system in which rep and cap genes are on separate modules, with leakproof control of expression of both Rep52 and Rep68. Doxycycline on Module B2 activates the TRE3G promoter, which promotes expression of Rep68. Doxycycline also causes expression of mFlp6, which inverts the cassette on Module A2. Expression of Rep52 and S-Cap is under control of QUAS, inducible with tebufenozide. The two module system again includes the apoptosis inhibitor Bcl-2 on Module B2, which is activated when a stop codon is excised upon expression of the cassette on Module A2.

[0170] In this example, helper genes are not on either of Module A2 or Module B2. Instead, helper activity is either constitutive to the host cell, or provided by a separate cassette with constitutive or inducible expression. Overexpression helper genes (such as adenovirus E2A, E4orf6, and VA) can cause cellular stress and cytotoxicity. Accordingly, expression of the helper cassette is attenuated.

[0171] By way of illustration, the system shown in FIG.15 can be implemented in HEK293 cells or HEK293 cell hybrids as follows. First, host cells are transfected with plasmid DNA containing a gene cassette stably expressing helper genes under the control of a minimal promoter, and a mScarlet fluorescent reporter flanked by two Sleeping Beauty inverted terminal repeats (ITRs). Cells are sorted for expression of mScarlet (the cells with helper activity) and expanded in culture. The cells are then stably transfected with Module A2 and Module B2, and selected for Bsd and Hygro resistance.PCT patent application Tunable AAV packaging cells 24. Possible cargo for incorporation into viral vectors using a tunable cell line

[0172] The technology of this disclosure can be used to prepare a composition to induce a response to the SARS-CoV-2 virus, for the purpose of prevention or treatment of COVID-19. Representative immunogenic epitopes may be taken from any one or more of the four SARS-CoV-2 structural proteins: namely, membrane glycoprotein (M), envelope protein (E), nucleocapsid protein (N), and the spike protein (S). Most current vaccines against SARS-CoV-2 typically include or encode the whole spike protein. Ways to optimize the spike protein were recently discussed by F. Heinz & K. Stiasny, NPJ Vaccines (2021) 6:104. TABLE 4: Immunogenic payloads for viral vectors and particles Name Disease Encoded antigen Clinical Trials identifier Phase Infections III (EUA mRNA-1273 SARS-CoV-2 Spike NCT04470427 and CMA) III (EUA BNT162b2 SARS-CoV-2 Spike NCT04368728 and CMA) CVnCoV SARS-CoV-2 Spike NCT04652102 III LNP- oVsaRNA SARS-C ISRCTN170726 nC oV-2 Spike 92 I ARCT-021 SARS-CoV-2 Spike NCT04728347 II ARCoV SARS-CoV-2 Receptor-binding ChiCTR2000034 domain 112 I mRNA-1440 Influenza H10N8 Haemagglutinin NCT03076385 I mRNA-1851 Influenza H7N9 Haemagglutinin NCT03345043 I Pre-membrane and mRNA-1893 Zika virus envelope NCT04064905 I glycoproteins Respiratory mRNA-1345 syncytial Fglycoprotein NCT04528719 I virus Metapneumovirus NA-1653 and par MPV and PIV3 F mR ainfluenza virus type 3 NCT03392389 I glycoproteins (MPV / PIV3) Pentameric complex mRNA-1647 Cytomegalovirus and B glycoprotein NCT04232280 II Chikunguny Chikungunya virus mRNA-1388 a virus NCT03325075 I antigensPCT patent application Tunable AAV packaging cells TABLE 4: Immunogenic payloads for viral vectors and particles Name Disease Encoded antigen Clinical Trials identifier Phase CV7202 Rabies virus G glycoprotein NCT03713086 I Cancer I Non-small-cell mRNA-5671 / lung cancer, colorectal cancer, KRAS antigens NCT03948763 V941 pancreatic adenocarcinoma Personalized mRNA-4157 Melanoma NCT03897881 II neoantigens Gastrointestinal Personalized mRNA-4650 NCT03480152 I / II cancer neoantigens FixVac Melanoma NY-ESO-1, tyrosinase, MAGE-A3, TPTE NCT02410733 I Triple-negative Personalized TNBC-MERIT NCT02316457 I breast cancer neoantigens HPV onco -40 HPV proteins E6 HARE -positive cancers NCT03418480 I / II and E7 Personalized RO7198457 Melanoma NCT03815058 II neoantigens MesomiR 1 Non-small cell lung cancer miR-16 NCT02369198 I INT-1B3 Advanced solid tumors miR-193a-3p mimic NCT04675996 I Ovarian cancer W_ova1 Ovarian cancer antigens NCT04163094 I

[0173] The technology of this disclosure can also be used for the purpose of gene therapy: for example, delivery of a nucleic acid encoding a gene product that is missing or defective in the subject being treated, or targeted to pathogenic cells in the subject, particularly cancer ells. Therapeutic purposes include but are not limited to expression of a therapeutic protein encoded in the nucleic acid (such as a cytokine or anti-cancer agent), expression of an essential protein that the subject is unable to produce themselves, or delivery of a gene editing system such as CRISPR / Cas9 or a guide RNA. Other possible therapeutic payloads may include DNA antisense oligonucleotides, DNA aptamers; micro RNAs, short interfering RNAs, ribozymes, RNA decoys and circular RNAs that specifically increase or decrease expression of a particular endogenous gene in the subject or an infectious agent. K. Sridharan et al., Br J Clin Pharmacol.2016 Sep; 82(3): 659-672.PCT patent application Tunable AAV packaging cells

[0174] Illustrative payloads for vaccines or immunogenic products is shown in TABLE 4. Payloads for gene therapy are shown in TABLE 5. In the examples shown, the nucleic acid encodes a therapeutic antibody (for passive immunization), anti-cancer drugs such as cytokines and chemotactic factors (for cancer treatment), and natural human proteins (to promote synthesis of an essential factor that the subject may be lacking, such as in the case of a genetically inherited condition). TABLES 4 and 5 are adapted from X. Hou et al., Nat Rev Materials 2021, 10:1-17. TABLE 5: Nucleic acid sequences for gene therapy Clinical Trials Name Disease Encoded protein identifier Phase Infections Antibody against mRNA-1944 Chikungunya virus NCT03829384 I chikungunya virus Cancer mRNA 2416 Solid tumors OX40L NCT03323398 II mRNA-2752 Solid tumors OX40L, IL-23 and IL-36γ NCT03739931 I MEDI1191 Solid tumors IL-12 NCT03946800 I IL-12sc, IL-15sushi, SAR441000 Solid tumors IFNα or GM-CSF NCT03871348 I Genetic disordersMethylmalonic Methylmalonyl-CoA mRNA-3704 a mut NCT03810690 I / II acidaemi ase Propionic Propionyl-CoA mRNA-3927 acidaemia carboxylase NCT04159103 I / II Ornithine MRT5201 transcarbam Ornithine ylase transcarbamylase NCT03767270 I / II deficiency Cystic fibrosis MRT5005 Cystic fibrosis transmembrane NCT03375047 I / II conductance regulator Transthyretin NTLA-2001 amyloidosis with CRISPR–Cas9 gene editin NCT04601051 I polyneuropathy g systemPCT patent application Tunable AAV packaging cells 25. Medicaments and commercial products

[0175] Preparation and formulation of pharmaceutical agents for use according to this disclosure can incorporate standard technology, as described, for example, in the most recent edition of Remington: The Science and Practice of Pharmacy. The formulation will typically be optimized for administration systemically, either intramuscularly or subcutaneously, or for administration orally or nasally (for example, to stimulate the mucosal immune system).

[0176] Preparations of viral vectors and particles may be provided as one or more unit doses (either combined or separate), each containing an amount of the pharmaceutical payload that is effective in the treatment of a chosen disease, infection, or clinical condition. The commercial product may contain a device such as a syringe for administration of the agent or composition in or around the target tissue of a subject in need thereof. The product may also contain or be accompanied by an informational package insert describing the use and attendant benefits of the vector or particle in treating the condition for which it is indicated and approved. 26. Publications

[0177] M.A. Labant. The next generation of cell factories for viral vector production. Gen Eng News, April 2, 2021 J-H. Wang. Adeno-associated virus as a delivery vector for gene therapy of human diseases. Signal Transduct Target Ther.2024 Apr 3;9(1):78 M. Lu et al. Tuning capsid formation dynamics in recombinant adeno-associated virus producing synthetic cell lines to enhance full particle productivity. Biotechnol J.2024 Mar;19(3):e2400051 D. Catalan-Tatjer et al. Expression of anti-apoptotic genes to enhance rAAV production. In "Advancing Manufacture of Cell and Gene Therapies VII", ECI Symposium Series, Feb 6, 2022. dc.engconfintl.org / cellgenetherapies_vii / 33 L. Jalšić et al. Inducible HEK293 AAV packaging cell lines expressing Rep proteins. Mol Ther Methods Clin Dev.2023 Jul 15:30:259-275. S Sparacio et al. Generation of a flexible cell line with regulatable, high-level expression of HIV Gag / Pol particles capable of packaging HIV-derived vectors. Mol Ther.2001 pr;3(4):602-12. 27. Incorporation by reference

[0178] For all purposes in the United States of America, each and every publication and patent document referred to in this disclosure is incorporated herein by reference in its entirety for all purposes to the same extent as if each such publication or document was specifically and individually indicated to be incorporated herein by reference.PCT patent application Tunable AAV packaging cells 28. Practice of the claimed invention

[0179] The technology provided in this disclosure and its use are described within a hypothetical understanding of general principles of virus and pharmaceutical manufacture. These discussions are provided for the edification and interest of the reader, and are not intended to limit the practice of the claimed invention. All of the products and methods claimed in this application may be used for any suitable purpose without restriction, unless otherwise indicated or required.

[0180] While this disclosure has been described with reference to the specific embodiments, changes can be made and equivalents can be substituted to adapt this disclosure to a particular context or intended use as a matter of routine experimentation, thereby achieving benefits of this disclosure without departing from the scope of what is claimed.

Claims

PCT patent application Tunable AAV packaging cells CLAIMS The invention claimed is:

1. A tunable adeno-associated virus (AAV) vector packaging cell line, comprising inheritably integrated transgenes in at least three modules: a first module, which comprises at least one AAV cap gene under control of a first inducible regulatory element; a second module, which comprises one or a plurality of AAV helper genes under control of a second inducible regulatory element; and a third module, which comprises at least one AAV rep gene under control of a third inducible regulatory element.

2. The tunable packaging cell line of claim 1, wherein activation of one, two, or all three of said inducible regulatory elements is leakproof and / or cause gene excision or rearrangement.

3. A tunable adeno-associated virus (AAV) vector packaging cell line, comprising inheritably integrated transgenes in at least two modules: a first module, which comprises at least one AAV cap gene under control of a first inducible leakproof regulatory element; and a second module, which comprises at least one AAV rep gene under control of a second inducible leakproof regulatory element.

4. The tunable packaging cell line of claim 3, wherein at least one of said inducible leakproof regulatory elements comprises a gene rearrangement event, and at least one of said inducible leakproof regulatory elements comprises removal of a gene repressor.

5. The tunable packaging cell line of claim 3 or claim 4, which further comprises a helper cassette comprising one or a plurality of AAV helper genes, optionally under control of a third inducible regulatory element.

6. The tunable packaging cell line of any preceding claim, further comprising a gene that encodes an apoptosis inhibitor protein under control of an inducible regulatory element.

7. The tunable packaging cell line of claim 6, wherein the apoptosis inhibitor protein is Bcl-2.PCT patent application Tunable AAV packaging cells 8. The tunable packaging cell line of any preceding claim, wherein at least one of said regulatory elements comprises an ecdysone receptor fusion protein.

9. The tunable packaging cell line of any preceding claim, wherein the first inducible regulatory element controls a plurality of AAV cap genes and at least one AAV rep gene.

10. The tunable packaging cell line of claim 6, wherein said cap genes and rep gene are also under control of the second inducible regulatory element.

11. The tunable packaging cell line of any preceding claim, wherein at least one of said regulatory elements comprises a recombinase enzyme with a ligand binding domain (RTtA).

12. The tunable packaging cell line of any preceding claim, wherein the second regulatory element activates expression of the AAV helper genes by inversion.

13. The tunable packaging cell line of claim 12, wherein the second regulatory element activates expression of said cap genes and rep gene by inversion.

14. The tunable packaging cell line of claim 5 or claim 6, wherein the gene that encodes the apoptosis inhibitor protein is under control of both the first and second inducible regulatory elements.

15. The tunable packaging cell line of any preceding claim, wherein at least one of said regulatory elements comprises a reverse tetracycline-controlled transactivator (rtTA).

16. The tunable packaging cell line of any preceding claim, wherein the first regulatory element comprises an ecdysone receptor fused to a DNA binding domain, the second regulatory element comprises a Cre recombinase enzyme with a ligand binding domain, and the third regulatory element comprises a reverse tetracycline-controlled transactivator (rtTA).PCT patent application Tunable AAV packaging cells [Hybrid cells] 17. The tunable packaging cell line of any preceding claim, which is the progeny of hybrid cells made by fusion of cells from a parental cell line.

18. The tunable packaging cell line of claim 17, wherein the parental cell line is a line of human cells, exemplified by HEK293.

19. The tunable packaging cell line of claim 17 or claim 18, wherein the hybrid cells were selected for content of mitochondria and / or reactive oxygen species per cell. [Payload genes] 20. The tunable packaging cell line of any of claims 1 to 19, further comprising a fourth module that contains an expressible payload gene between two AAV inverted terminal repeat sequences (ITRs).

21. The tunable packaging cell line of claim 20, wherein the payload gene is inheritably integrated into the genome of the cell line.

22. The tunable packaging cell line of any of claims 1 to 19, further comprising an inheritably integrated landing pad that comprises at least two recombination sites whereby the landing pad is adapted to receive and express an exchange cassette that comprises a payload gene.

23. The tunable packaging cell line of any of claims 20 to 22, wherein the payload gene is under control of a fourth inducible regulatory element.

24. The tunable packaging cell line of any of claims 20 to 23, wherein the payload gene is a marker protein such as green fluorescent protein (GFP).

25. The tunable packaging cell line of any of claims 20 to 23, wherein the payload gene encodes an immunogenic peptide for eliciting a specific immune response in a subject in need thereof.

26. The tunable packaging cell line of any of claims 20 to 23, wherein the payload gene encodes a gene product that is deficient in a subject.PCT patent application Tunable AAV packaging cells [Methods of use] 27. A method of optimizing production of an AAV vector, comprising: (a) transfecting cells of the tunable packaging cell line of any of claims 1 to 19 to express a payload gene; (b) culturing cells transfected in step (a) or progeny thereof in the presence of different amounts and / or ratios of inducer compounds for each of said three inducible regulatory elements, thereby producing said AAV vector; and (c) adjusting (tuning) the amounts and / or the ratios of the inducer compounds to obtain a desired titer of AAV serotype-specific capsids (viral particles / mL); ratio of full to empty capsids, and / or functional titer (transduction units / mL).

28. A method of preparing cells of the tunable packaging cell line of any of claims 1 to 19 for production of AAV vectors containing a payload, comprising contacting the cells containing the landing pad with a transposon comprising a sequence encoding said payload and a Sleeping Beauty transposase under conditions whereby the payload gene is inheritably integrated into the genome of at least some of the cells.

29. A method of preparing cells of the tunable packaging cell line of claim 21 for production of AAV vectors containing a payload, comprising contacting the cells containing the landing pad with an exchange cassette that contains a gene encoding said payload under conditions whereby the payload gene is inheritably integrated into the genome of at least some of the cells at or around the site of the landing pad.

30. A method of producing AAV vectors, comprising culturing cells of the tunable packaging cell line of any of claims 20 to 26 in a medium that contains a predetermined amount of an inducer compound for each of said inducible regulatory elements; and harvesting the AAV vectors from the culture.

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