Rhabdovirus-free SF9 insect cell line for large-scale AAV production for cardiovascular gene therapies

Abstract

Provided herein are invertebrate cell lines for production of AAV viral proteins (such as capsid proteins) and AAV viral particles that are rhabdovirus-negative or have rhabdovirus genomes below the limit of detection, and methods of deriving such cell lines. Certain cell lines described herein, and methods of deriving such cell lines, achieve significantly greater AAV production over the cell lines and methods known in the art.

Description

RHABDO VIRUS-FREE SF9 INSECT CELL LINE FOR LARGE-SCALE AAV PRODUCTION FOR CARDIOVASCULAR GENE THERAPIESCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 636,981 filed April 22, 2024, the entire disclosure of which is incorporated herein by reference in its entirety.FIELD

[0001] The present disclosure relates generally to populations of insect host cells devoid of detectable rhabdovirus genomes, methods of producing such populations, and methods of use thereof.BACKGROUND

[0002] For successful clinical use of virus-based gene therapy, large-scale, reproducibly high yield production of AAV proteins, in particular AAV capsid proteins, and AAV viral particles needs to be achieved. There is a need to improve viral production in cells to achieve improved viral titer than that achieved using known production methods.

[0003] Existing invertebrate cell lines used for baculovirus expression vector (BEV) systems, including the Sf9 cell line, comprise a rhabdovirus (SfRV). Populations of insect host cells devoid of detectable rhabdovirus genomes, methods of producing such populations, and methods of use thereof, are needed for robust, reliable expression of AAV proteins and virions.SUMMARY

[0004] The present disclosure relates generally to populations of insect host cells devoid of detectable rhabdovirus genomes, methods of producing such populations, and methods of use thereof.In some embodiments, the present disclosure provides a population of insect host cells devoid of detectable rhabdovirus genomes. In some embodiments, the population comprises less than 1, less than 2, or less than 10 rhabdovirus genome copies per milliliter. In some embodiments, the number of rhabdovirus genomes in the population is measured by a Loop- Mediated Isothermal Amplification (LAMP) assay, a RT-qPCR assay, or an RNA-Seq assay.

[0005] In some embodiments, the insect host cells are Spodoptera frugiperda (Sf) cells. In some embodiments, the population is derived from a Sf9 or Sf21 cell line. In some embodiments, the population is derived from a qualified producer cell line for baculovirusand / or AAV production at normal culturing temperature, wherein the normal culturing temperature is between about 25°C and about 30°C. In some embodiments, the normal culturing temperature is between about 28°C.

[0006] In some embodiments, the population provides enhanced production of at least one serotype of AAV virions, compared to a population of insect host cells that contains detectable rhabdovirus genomes. In some embodiments, the serotype of AAV is AAV9. In some embodiments, the serotype of AAV is AAV5.

[0007] In some embodiments, the population produces AAV with comparable transduction of a target cell compared to a population of insect host cells that contains detectable rhabdovirus genomes. In some embodiments, the population produces AAV with a comparable full capsid ratio compared to a population of insect host cells that contains detectable rhabdovirus genomes. In some embodiments, the population produces AAV with a comparable capsid composition compared to a population of insect host cells that contains detectable rhabdovirus genomes.

[0008] In some embodiments, the population demonstrates faster thaw recovery compared to a population of insect host cells that contains detectable rhabdovirus genomes. In some embodiments, the population has comparable or improved viability during thaw recovery compared to a population of insect host cells that contains detectable rhabdovirus genomes. In some embodiments, the population has comparable or improved viable cell density during thaw recovery compared to a population of insect host cells that contains detectable rhabdovirus genomes.

[0009] In some embodiments, the population retains undetectable levels of rhabdovirus following culture at about 28°C to about 30°C for greater than 250 days.In some embodiments, the population is derived using a method comprising adapting a population of insect host cells that contains detectable rhabdovirus genomes to a non-typical culturing temperature, optionally wherein the non-typical culturing temperature is between about 31°C and about 37°C. In some embodiments, the non-typical culturing temperature is about 33°C. In some embodiments, the population is derived using a method comprising adapting the population that contains detectable rhabdovirus genomes to a non-typical culturing temperature by culturing at the non-typical culturing temperature for 0 to 100 days after thaw recovery. In some embodiments, the population is derived using a method comprising adapting the population that contains detectable rhabdovirus genomes to a non- typical culturing temperature by culturing at the non-typical culturing temperature for 10 days to 30 days after thaw recovery. In some embodiments, the population is derived using amethod comprising adapting the population that contains detectable rhabdovirus genomes to a non-typical culturing temperature by culturing at the non-typical culturing temperature for about 18 days after thaw recovery.

[0010] In some embodiments, the population is derived using a method comprising limited dilution adherent plating to generate clones or limited pools of cell lines.

[0011] In some embodiments, the population is derived using a method that does not comprise use of an antiviral compound. In some embodiments, the population is derived using a method comprising culturing the cells in rhabdovirus-free conditioned medium comprising between 0 and 10% FBS.

[0012] In some embodiments, the present disclosure provides a method of producing a population of insect host cells devoid of detectable rhabdovirus, wherein the method comprises adapting a population of insect host cells that contains detectable rhabdovirus genomes to a non-typical culturing temperature.

[0013] In some embodiments, the method comprises culturing at the non-typical culturing temperature for 0 to 100 days after thaw recovery. In some embodiments, the method comprises culturing at the non-typical culturing temperature for 20 to 50 days after thaw recovery. In some embodiments, the method comprises culturing at the non-typical culturing temperature about 42 days after thaw recovery.

[0014] In some embodiments, the method comprises performing a limited dilution adherent plating step to generate at least one clone or limited pool of producer cell lines. In some embodiments, the limited dilution adherent plating step generates at least one clonal or limited pool population of insect cells. In some embodiments, the method comprises culturing the at least one clone or limited pool of producer cell lines in rhabdovirus-free conditioned medium comprising between 0 and 10% FBS for 0 to 200 days after limited dilution plating.

[0015] In some embodiments, the present disclosure provides a method of producing a population of insect host cells devoid of detectable rhabdovirus comprising: i) recovering a population of insect host cells that contains detectable rhabdovirus genomes at normal culturing temperatures for 0-100 days after thawing, wherein the normal culturing temperature is between about 25°C and about 30°C; ii) adapting the population of insect host cells to a non-typical culturing temperature; and iii) performing a limited dilution adherent plating of the population of insect host cells to generate clones or limited pools of producer cell lines.

[0016] In some embodiments, the non-typical culturing temperature is between about 31 °C and about 37°C. In some embodiments, the non-typical culturing temperature is about 33°C.

[0017] In some embodiments, the method does not comprise use of an antiviral compound. In some embodiments, the insect host cells are Spodoptera frugiperda (Sf) cells. In some embodiments, the population of insect host cells that contains detectable rhabdovirus genomes is, or is derived from, a Sf9 or Sf21 cell line.

[0018] In some embodiments, the population of insect host cells that contains detectable rhabdovirus genomes is a qualified producer cell line for baculovirus and / or AAV production at normal culturing temperature, wherein the normal culturing temperature is between about 25°C and about 30°C. In some embodiments, the normal culturing temperature is about 28°C.

[0019] In some embodiments, the present disclosure provides a method of producing AAV particles using a population of insect host cells devoid of detectable rhabdovirus genomes described herein, wherein the population of insect host cells are contacted with one, two, or three baculovirus expression vectors. In some embodiments, the AAV particles are AAV9 particles. In some embodiments, the AAV particles are AAV5 particles.

[0020] In some embodiments, the method produces AAV with comparable transduction of a target cell compared to a method using a population of insect host cells that contains detectable rhabdovirus genomes. In some embodiments, the method produces AAV with a comparable full capsid ratio compared to a method using a population of insect host cells that contains detectable rhabdovirus genomes. In some embodiments, the method produces AAV with a comparable capsid composition compared to a method using a population of insect host cells that contains detectable rhabdovirus genomes.

[0021] In some embodiments, the present disclosure provides a method of producing a population of insect host cells devoid of detectable rhabdovirus comprising: i) recovering a population of insect host cells that contains detectable rhabdovirus genomes at normal culturing temperatures for 18 days after thawing, wherein the normal culturing temperature is about 28°C; ii) adapting the population of insect host cells to a non-typical culturing temperature by culturing the population of insect cells at a non-typical culturing temperature for about 42 days, wherein the non-typical culturing temperature is about 33°C; and iii) performing a limited dilution adherent plating of the population of insect host cells to generate clones or limited pools of producer cell lines.

[0022] In some embodiments, the present disclosure provides a cell stock comprising a population of insect host cells described herein, wherein the cell stock comprises at least 1% DMSO by volume. In some embodiments, the present disclosure provides a cell stock comprising a population of insect host cells described herein, wherein the cell stock comprisesat least 1% DMSO by volume and the population of insect host cells is derived by a method described herein.BRIEF DESCRIPTION OF DRAWINGS

[0023] FIG. 1 provides a schematic of the method described herein, including the steps of the method and the temperature and duration of each step.

[0024] FIG. 2 provides qRT-PCR data quantifying Sf9 rhabdovirus (SfRV) genomes per cell in the parental cell line used for derivation of rhabdovirus-free cell lines. Intracellular SfRV genomes were quantified in samples collected from the parental cell culture at various passages, culture densities, and cell pellet sizes after thaw recovery. TCD = total cell density.

[0025] FIG. 3 shows screening of AAV9 productivity by cell lines that were derived from selected clones and pools following adaptation to non-typical culturing temperature and cloning. AAV9 productivity was measured in shake flasks at non-typical culturing temperatures.

[0026] FIG. 4 shows a second round of AAV9 productivity testing in cell lines derived by adapting the non-typical culturing temperature-adapted cell lines to normal culturing temperature. AAV9 productivity was measured in shake flasks at normal culturing temperatures.

[0027] FIG. 5 shows AAV9 production in the top five normal culturing temperature-adapted cell lines (derived from 4 clones & 1 pool) and the parental cell line, at normal temperature, in triplicate.

[0028] FIG. 6A shows AAV9 production in the top three normal culturing temperature- adapted cell lines (1-2, 2-10, and 1-12) and the parental cell line, at normal temperature. AAV9 productivity was measured in 3L bioreactors (BRX) and satellite cultures (Sat).

[0029] FIG. 6B shows the efficiency of iPSC-cardiomyocyte transduction by AAV9:GFP produced by each of the top three normal culturing temperature-adapted cell lines (1-2, 2-10, and 1-12), measured by GFP fluorescence.

[0030] FIG. 6C shows the capsid ratios of AAV9:GFP produced by each of the top three normal culturing temperature-adapted cell lines (1-2, 2-10, and 1-12), measured by capsid ELISA.

[0031] FIG. 6D the capsid composition of AAV9:GFP produced by each of the top three normal culturing temperature-adapted cell lines (1-2, 2-10, and 1-12), measured by western blot.

[0032] FIG. 7 shows production of AAV9:4.8Kb (AAV9 packaging a 4.8Kb cassette) by each of the three top normal culturing temperature-adapted cell lines (1-2, 2-10, and 1-12), compared to parental MCB infected with SfRVz-produced baculovirus. Historic AAV9 productivity of the MCB cell line is also shown for reference (Historic, Baseline).

[0033] FIG. 8 shows production of AAV5:4.8kB (AAV5 packaging a 4.8Kb cassette) in normal culturing temperature-adapted cell line 2-10.

[0034] FIG. 9 shows cell growth, population doubling levels and viability of the three top normal culturing temperature-adapted cell lines (1-2, 2-10, and 1-12) from banked vial thaw through 135hrs post-thaw, compared to the parental MCB cell line.

[0035] FIG. 10 shows reduction of rhabdovirus levels by PCR, in seed trains over time, during non-typical culturing temperature adaption.DETAILED DESCRIPTION

[0036] In some aspects, provided herein are populations of insect host cells devoid of detectable rhabdovirus genomes. In some aspects, provided herein are methods of producing such populations, and methods of use thereof.Terminology

[0037] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the disclosure also contemplates that in some embodiments, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

[0038] Also as used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0039] The term “a” or “an” refers to one or more of that entity, i.e. can refer to plural referents. As such, the terms “a,” “an,” “one or more,” and “at least one” are used interchangeably herein. In addition, reference to “an element” by the indefinite article “a” or “an” does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there is one and only one of the elements.

[0040] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device or the method being employed to determine thevalue, or the variation that exists among the samples being measured. Unless otherwise stated or otherwise evident from the context, the term “about” means within 10% above or below the reported numerical value (except where such number would exceed 100% of a possible value or go below 0%). When used in conjunction with a range or series of values, the term “about” applies to the endpoints of the range or each of the values enumerated in the series, unless otherwise indicated. As used in this application, the terms “about” and “approximately” are used as equivalents.

[0041] “AAV” is an abbreviation for adeno-associated virus. The term covers all subtypes of AAV, except where a subtype is indicated, and to both naturally occurring and recombinant forms. The abbreviation “rAAV” refers to recombinant adeno-associated virus. “AAV” includes AAV or any subtype. “AAV5” refers to AAV subtype 5. “AAV9” refers to AAV subtype 9. The genomic sequences of various serotypes of AAV, as well as the sequences of the native inverted terminal repeats (ITRs), Rep proteins, and capsid subunits may be found in the literature or in public databases such as GenBank. See, e.g., GenBank Accession Numbers NC_002077 (AAV1), AF063497 (AAV1), NC_001401 (AAV2), AF043303 (AAV2), NC_001729 (AAV3), NC_001829 (AAV4), U89790 (AAV4), NC_006152 (AAV5), AF513851 (AAV7), AF513852 (AAV8), NC_006261 (AAV8), and AY530579 (AAV9). Publications describing AAV include Srivistava et al. (1983) J. Virol. 45:555; Chiorini et al. (1998) J. Virol. 71 :6823; Chiorini et al. (1999) J. Virol. 73:1309; Bantel-Schaal et al. (1999) J. Virol. 73:939; Xiao et al. (1999) J. Virol. 73:3994; Muramatsu et al. (1996) Virol. 221 :208; Shade et al. (1986) J. Virol. 58:921; Gao et al. (2002) Proc. Nat. Acad. Sci. USA 99: 11854; Moris et al. (2004) Virology 33:375-383; Int’l Pat. Publ Nos. WO2018 / 222503 Al, WO2012 / 145601A2, W02000 / 028061A2, WO 1999 / 61601A2, and WO1998 / 11244A2; U.S. Pat. Appl. Nos. 15 / 782,980 and 15 / 433,322; and U.S. Pat. Nos. 10,036,016, 9,790,472, 9,737,618, 9,434,928, 9,233,131, 8,906,675, 7,790,449, 7,906,111, 7,718,424, 7,259,151, 7,198,951, 7,105,345, 6,962,815, 6,984,517, and 6,156,303.

[0042] An “AAV particle” refers to an extracellular viral particle including at least one viral capsid protein (e.g. VP1) and an encapsidated AAV vector (or fragment thereof), including the capsid proteins.

[0043] For brevity and clarity, the disclosure refers to “capsid protein” or “capsid proteins” of AAV. Those skilled in the art understand that such references refer to VP1, VP2, or VP3, or combinations of VP1, VP2, and VP3. As in wild-type AAV and most recombinant expression systems VP1, VP2, and VP3 are expressed from the same open reading frame, engineering of the sequence that encodes VP3 inevitably alters the sequences of the C-terminal domain ofVP1 and VP2. One may also express the capsid proteins from different open reading frames, in which case the capsid of the resulting rAAV virion could contain a mixture of wild-type and engineered capsid proteins, and mixtures of different engineered capsid proteins.

[0044] A viral particle or virion referenced herein is an infectious viral particle that comprises a competently assembled viral capsid and is capable of delivering a polynucleotide component into a cell for which the viral particle is tropic. In some embodiments, the viral particles described herein are replication-competent (i.e., capable of being replicated in an infected cell).

[0045] The term “culture” or “cell culture” means the maintenance of cells in an artificial, in vitro environment. A “cell culture system” is used herein to refer to culture conditions in which a population of cells may be grown as monolayers or in suspension. “Culture medium” is used herein to refer to a nutrient solution for the culturing, growth, or proliferation of cells. Culture medium may be characterized by functional properties such as, but not limited to, the ability to maintain cells in a particular state (e.g., a pluripotent state, a quiescent state, etc.), to mature cells - in some instances, specifically, to promote the differentiation of progenitor cells into cells of a particular lineage (e.g., a cardiomyocyte).

[0046] As used herein, the term “rhabdovirus-free conditioned medium” refers to a culture medium comprising a portion of fresh medium and a portion of conditioned medium. As used herein, “conditioned” refers to previous incubation of the medium in the presence of rhabdovirus-free cells. In some embodiments, the rhabdovirus-free cells are the parental pre- MCB cells incubated continuously at non-typical culturing temperature, and passaged as needed. In some embodiments, the portion of conditioned medium is between about 10% and about 90% by volume. In some embodiments, the portion of conditioned medium is between about 30% and about 70% by volume. In some embodiments, the portion of conditioned medium is between about 40% and about 60% by volume. In some embodiments, the portion of conditioned medium about 50% by volume.

[0047] The term “expression cassette” refers to a polynucleotide cassette comprising a coding sequence which encodes a gene product of interest used to effect the expression of the gene product in target cells, which is operably linked to a promoter. Unless otherwise specified, the expression cassette of an AAV vector includes only the polynucleotides between (and not including) the ITRs.

[0048] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rdedition; Ausubel et al. eds. (2007)Current Protocols in Molecular Biology; Methods in Enzymology (Academic Press, Inc., N. Y.); MacPherson et al. (1991) PCR 1 : A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5thedition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Pat. No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984) Transcription and Translation; IRL Press (1986) Immobilized Cells and Enzymes; Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Herzenberg et al. eds (1996) Weir’s Handbook of Experimental Immunology; Manipulating the Mouse Embryo: A Laboratory Manual, 3rdedition (2002) Cold Spring Harbor Laboratory Press; Sohail (2004) Gene Silencing by RNA Interference: Technology and Application (CRC Press); and Sell (2013) Stem Cells Handbook.Populations of Insect Host Cells Provided Herein

[0049] The present disclosure provides populations of insect host cells devoid of detectable rhabdovirus genomes. In some embodiments, the insect cells are selected from the group consisting of the following cells or cell lines: Spodoptera frugiper da-derived insect cell or cell line, Trichoplusia / / / -derived or Tni insect cell or cell line, and Drosophila melanogaster- derived or S2 insect cell or cell line. In some embodiments, the insect cells are Spodoptera frugiper da-derived insect cells. In some embodiments, the insect cells are derived from Sf9 insect cells. In some embodiments, the insect cells are derived from Sf21 insect cells.

[0050] Rhabdovirus has a negative-stranded RNA linear genome. Rhabdovirus genomes A.g, SfR viral genomes) can be detected by any method known in the art for detecting nucleic acids. Non-limiting examples of methods for rhabdovirus detection include q-RT-PCR, ddPCR, nextgeneration (NGS) sequencing (e.g., Illumina NGS), cryo-EM and antibody affinity methods. In some embodiments, the method for detecting SfR viral genomes is a loop-mediated isothermal amplification (LAMP) assay. In some embodiments, the limit of detection of the LAMP assay is about 2 SfR viral genome copies per ml. In some embodiments, the method for detecting SfR viral genomes is a RT-qPCR assay. In some embodiments, the limit of detection of the RT-qPCR assay is about 10 SfR viral genome copies per ml. In some embodiments, the method for detecting SfR viral genomes is a RNA-Seq assay (e.g., Illumina). In someembodiments, the limit of detection of the RNA-Seq assay is 1 SfR viral genome copy, where in the 1 SfR viral genome copy is an absolute copy number per sample. Populations of insect host cells can be used to produce recombinant baculovirus (rBV) and / or AAV. In some embodiments, the rBV is any suitable strain known in the art. In some embodiments, the AAV is any AAV known in the art. In some embodiments, an AAV is an AAV selected from the group consisting of serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, or a chimeric AAV derived therefrom. In some embodiments, AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12, or a variant thereof. In some embodiments, the AAV capsid protein described herein is a wild type AAV capsid protein from AAV serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or a variant thereof. In some embodiments, the AAV Rep protein is a wild type AAV Rep protein from AAV serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or a variant thereof. Exemplary AAV vectors and capsids are provided in U.S. Pat. Pub. Nos. US10011640B2; US7892809B2, US8632764B2, US8889641B2, US9475845B2, US10889833B2, US10480011B2, and US10894949B2, the contents of which are herein incorporated by reference; and Int’l Pat. Pub. Nos. WO2020198737A1, W02019028306A2, WO2016054554A1, WO2018152333A1, WO2017106236A1, WO2008124724A1, W02017212019A1, WO2020117898A1, WO2017192750A1, W02020191300A1, and W02017100671 Al, the contents of which are herein incorporated by reference.

[0051] In some embodiments, the AAV virions are recombinant (rAAV). In some embodiments, the rAAV virions comprise an engineered capsid protein. Engineered capsid proteins can be derived from a parental, e.g. wild type, capsid and include, for example, variant polypeptide sequence with respect to a parental capsid sequence at one or more sites. For example, variant sites of the parental capsid can occur at the VR-IV site, VR-V site, VR-VII site and / or VR- VIII site (see, e.g. Biining and Srivastava. Mol Ther Methods Clin Dev. 12:248- 265 (2019)).

[0052] In some embodiments, the capsid protein is an AAV5 / AAV9 chimeric capsid protein. In some embodiments, the chimeric capsid protein comprises at least 1, 2, 3, 4, 5 or more polypeptide segments that are derived from AAV5 capsid protein. In some embodiments, the chimeric capsid protein comprises at least 1, 2, 3, 4, 5 or more polypeptide segments that are derived from AAV9 capsid protein. In some embodiments, at least one polypeptide segment is derived from the AAV5 capsid protein and at least one polypeptide segment is derived from the AAV9 capsid protein.

[0053] In some embodiments, the capsid protein is a combinatory capsid protein. As used herein, “combinatory capsid protein” refers to a AAV5 / AAV9 chimeric capsid protein, which further comprises amino acid variations with respect to the chimeric parental sequence at one or more sites. In some embodiments, the one or more sites of the chimeric parental sequence are selected from those equivalent to the VR-IV site, the VR-V site, the VR-VII site and the VR-VIII site of the AAV9 capsid protein.

[0054] In some embodiments, the rAAV virions described herein comprise any capsid protein or variant capsid protein, e.g., any AAV9 variant capsid protein (e.g., comprising one or more substitutions or insertions), described in WO2021 / 163357A2 and / or U.S. Patent No. 11,129,908, both of which are incorporated by reference herein in their entirety.

[0055] In some embodiments, the rAAV virions described herein comprise any capsid protein or variant capsid protein, e.g., any AAV9 variant capsid protein (e.g., comprising one or more substitutions or insertions), described in WO 2021 / 216456, which is incorporated by reference herein in its entirety.

[0056] Populations of insect host cells can be qualified according to defined criteria as producer cell lines for baculovirus and / or AAV manufacturing (e.g., a research cell bank [RCB] RCB or master cell bank [MCB]). In some embodiments, the population of insect cells described herein is derived from a qualified producer cell line (e.g., an RCB or MCB cell line) for baculovirus and / or AAV production at normal culturing temperature, wherein the normal culturing temperature is between about 25°C and about 30°C.

[0057] During production of baculovirus and / or AAV, populations of insect host cells may be cultured under a range of conditions. Culture conditions for populations of insect host cells are known in the art. Non-limiting examples of culture conditions include culture temperature, culture medium, and culture format. In some embodiments, populations of insect host cells may be cultured under one or more sets of culture conditions (e.g., a set temperature, culture medium, and culture format).

[0058] In some embodiments, populations of insect host cells are cultured at a temperature between about 25°C and about 30°C. In some embodiments, populations of insect host cells are cultured in a culture medium comprising between 0% and about 10% fetal bovine serum (FBS). In some embodiments, populations of insect host cells are cultured in a serum-free culture medium. In some embodiments, populations of insect host cells are cultured in a chemically defined culture medium. In some embodiments, populations of insect host cells are cultured in adherent culture. In some embodiments, populations of insect host cells are cultured as an adherent culture in 96 well culturing plates through Cell Factory culturing and productionformats. In some embodiments, populations of insect host cells are cultured in suspension culture. Non-limiting examples of suspension culture formats include shake flask, benchtop bioreactor (e.g., 3L), and pilot-scale bioreactor (e.g., 200L). In some embodiments, populations of insect host cells are cultured in suspension at volumes of 0.01L to greater than lOOOL.

[0059] In some embodiments, a population of insect host cells devoid of detectable rhabdovirus genomes, as described herein, may provide enhanced production of at least one serotype of AAV compared to a population of insect host cells that contains detectable rhabdovirus genomes. The serotype of AAV may be any described herein. In some embodiments, the AAV is AAV9 or a variant thereof. In some embodiments, the AAV is AAV5 or a variant thereof.

[0060] In some embodiments, a population of insect host cells devoid of detectable rhabdovirus genomes, as described herein, provides enhanced production of AAV particles. In some embodiments, enhanced production is in comparison to production of AAV particles by a population of insect host cells that contains detectable rhabdovirus genomes. In some embodiments, enhanced production is in comparison to a qualified producer cell line (e.g., an RCB or pre-MCB).

[0061] In some embodiments, a population of insect host cells devoid of detectable rhabdovirus genomes, as described herein, is capable of producing AAV particles with volumetric yields greater than greater than 1E15 vg / L. In some embodiments, a population of insect host cells devoid of detectable rhabdovirus genomes described herein is capable of producing AAV9 viral particles with a volumetric yield greater than 1.5E15 vg / L. In some embodiments, a population of insect host cells devoid of detectable rhabdovirus genomes described herein is capable of producing AAV5 viral particles with a volumetric yield greater than 2.5E15 vg / L.

[0062] The quality of AAV particles produced by an insect host cell line can be quantified by methods known in the art, including transduction of target cells, full capsid ratio, and capsid composition. Methods of determining transduction efficiency levels, full capsid ratio, and capsid composition are known in the art.

[0063] In some embodiments, a population of insect host cells devoid of detectable rhabdovirus genomes, as described herein, is capable of producing AAV with comparable transduction of a target cell or target cells, such as cardiac cells (e.g., cardiomyocytes), compared to a population of insect host cells that contains detectable rhabdovirus genomes. Methods of determining transduction efficiency levels are known in the art. In some embodiments, transduction efficiency is measured by %GFP signal to control following transduction of GFP- expressing AAV.

[0064] In some embodiments, a population of insect host cells devoid of detectable rhabdovirus genomes, as described herein, is capable of producing AAV with a comparable ratio or percentage (%) of full capsids (full capsid ratio or %) compared to a population of insect host cells that contains detectable rhabdovirus genomes. In some embodiments, the AAV has a comparable full capsid ratio or % compared to a method using a population of insect host cells that contains detectable rhabdovirus genomes. Methods of measuring the full capsid ratio or % are known in the art. In some embodiments, the full capsid ratio or % is measured by capsid ELISA.

[0065] In some embodiments, a population of insect host cells devoid of detectable rhabdovirus genomes, as described herein, is capable of producing AAV with a comparable capsid composition compared to a population of insect host cells that contains detectable rhabdovirus genomes. In some embodiments, capsid composition is measured by quantifying expression of VP1, VP2 and VP3 proteins in a population of insect host cells. In some embodiments, the VP1:VP2:VP3 ratio is comparable to that achieved with a method using a method using a population of insect host cells that contains detectable rhabdovirus genomes. Methods of measuring the capsid composition are known in the art. In some embodiments, the capsid composition is measured by capsid western blot.

[0066] Populations of insect host cells, if cryopreserved, may be thawed prior to culture. In some embodiments, thawed populations of insect host cells may undergo a cell thaw recovery period (i.e., “post-thaw recovery” or “thaw recovery”). In some embodiments, a population of insect host cells devoid of detectable rhabdovirus genomes described herein has faster thaw recovery compared to a population of insect host cells that contains detectable rhabdovirus genomes. In some embodiments, a population of insect host cells devoid of detectable rhabdovirus genomes described herein has comparable or improved viability during thaw recovery compared to a population of insect host cells that contains detectable rhabdovirus genomes. Methods for measuring viability are known in the art. In some embodiments, a population of insect host cells devoid of detectable rhabdovirus genomes described herein is capable of between about 85% and about 99% viability at 135 hours after thaw.

[0067] In some embodiments, a population of insect host cells devoid of detectable rhabdovirus genomes described herein has comparable or improved viable cell density during thaw recovery compared to a population of insect host cells that contains detectable rhabdovirus genomes. Methods for measuring viable cell density are known in the art. In some embodiments, a population of insect host cells devoid of detectable rhabdovirus genomesdescribed herein is capable of a viable cell density of about 0.5E6 to about 6.5E6 cells / ml over the 135 hours after thaw.

[0068] In some embodiments, a population of insect host cells devoid of detectable rhabdovirus genomes described herein has comparable or improved post-recovery population doubling levels (PDL) compared to a population of insect host cells that contains detectable rhabdovirus genomes during the cell thaw recovery period. Methods for measuring PDL are known in the art. In some embodiments, a population of insect host cells devoid of detectable rhabdovirus genomes described herein is capable of PDL of between 19.5 and 27.4 in average hours (seed train). In some embodiments, a population of insect host cells devoid of detectable rhabdovirus genomes described herein is capable of PDL of between 21 and 35 in average hours during the first 120 hours of the cell thaw recovery period.

[0069] Stability of rhabdovirus clearance from populations of insect host cells may enhance reproducibility of AAV production yield and quality (e.g., transduction efficiency, full capsid ratio, and capsid composition of produced AAV). In some embodiments, a population of insect host cells devoid of detectable rhabdovirus genomes described herein retains undetectable levels of rhabdovirus is adapted following culture at normal culture temperature for greater than 250 days.

[0070] In some embodiments, the population of insect host cells devoid of detectable rhabdovirus genomes described herein is derived by a method comprising one or more defined culture conditions during one or more steps. In some embodiments, the population of insect host cells devoid of detectable rhabdovirus genomes is derived using a method comprising adapting a population of insect host cells that contains detectable rhabdovirus genomes to a non-typical culturing temperature. In some embodiments, the non-typical culturing temperature is between about 31°C and about 45°C. In some embodiments, the non-typical culturing temperature is between about 31°C and about 37°C. In some embodiments, the non- typical culturing temperature is between about 31 °C and about 34°C. In some embodiments, the non-typical culturing temperature is about 30.5°C, 31°C, 31.5°C, 32°C, 32.5°C, 33°C, 33.5°C, or 34°C. In some embodiments, the non-typical culturing temperature is about 33°C.

[0071] In some embodiments, the population of insect cells devoid of detectable rhabdovirus genomes is cultured at the non-typical culturing temperature for 0 to 100 days. In some embodiments, the non-typical culturing temperature period lasts for a duration of about 4 days to about 100 days. In some embodiments, the non-typical culturing temperature period lasts for a duration of about 10 days to about 100 days. In some embodiments, the non-typical culturing temperature period lasts for a duration of 10 to 100 days. In some embodiments, the non-typicalculturing temperature period lasts for a duration of 20 to 50 days. In some embodiments, the non-typical culturing temperature period lasts for a duration of 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 44 days, 45 days, 46 days, 47 days, 48 days, 49 days, or 50 days. In some embodiments, the non-typical culturing temperature period lasts for a duration of about 41 days. In some embodiments, the non-typical culturing temperature period lasts for a duration of about 42 days.

[0072] In some embodiments, rhabdovirus genomes are reduced by greater than l-log, greater than 2-logs, greater than 3-logs, greater than 4-logs, greater than 5-logs, greater than 6-logs, greater than 7-1 ogs, greater than 8-1 ogs, greater than 9-logs, or greater than 10-logs, in the population of insect host cells following the non-typical culturing temperature step, compared to the population of insect cells prior to the non-typical culturing temperature step. In some embodiments, rhabdovirus genomes are reduced by greater than greater than 3-logs, greater than 4-logs, greater than 5-logs, greater than 6-logs, or greater than 7-logs, in the population of insect host cells following the non-typical culturing temperature step, compared to the population of insect cells prior to the non-typical culturing temperature step. In some embodiments, rhabdovirus genomes are reduced by greater than greater than 4-logs, greater than 5-logs, or greater than 6-logs, in the population of insect host cells following the non- typical culturing temperature step, compared to the population of insect cells prior to the non- typical culturing temperature step. In some embodiments, rhabdovirus genomes are reduced by greater than 5-logs in the population of insect host cells following the non-typical culturing temperature step, compared to the population of insect cells prior to the non-typical culturing temperature step.

[0073] In some embodiments, the population is derived using a method comprising a cell thaw recovery period at normal culturing temperatures. In some embodiments, the population is derived using a method comprising limited dilution adherent plating to generate clones or limited pools of cell lines.

[0074] In some embodiments, the population is derived using a method that does not comprise use of an antiviral compound.

[0075] In some embodiments, the population is derived using a method comprising culturing the cells in rhabdovirus-free conditioned medium comprising between 0 and 10% FBS.Methods of Rhabdovirus-Free Cell Line Production Provided Herein

[0076] The present disclosure also provides methods for producing one or more rhabdovirus- free cell lines. In some embodiments, the method comprises culturing a population of insect host cells under one or more sets of culture conditions (e.g., a set temperature, culture medium, and culture format) at one or more steps or periods of the method. The methods disclosed herein may comprise one or more of the following steps or periods: cell thaw recovery period, a nontypical culturing temperature step, a limited dilution adherent plating step, and a clone or limited pool suspension adaptation step. FIG. 1 provides a schematic showing the steps of the methods described herein. For each step, the range of temperatures at which the step may be performed, and the duration of the step, is provided.

[0077] The population of insect cells may initially be frozen. In some embodiments, a population of insect cells is thawed from a frozen state. In some embodiments, the population of insect cells is thawed at about 37°C. In some embodiments, the population of insect cells is centrifuged and resuspended in fresh medium to a target seeding density prior to culture at normal culturing temperature. In some embodiments, the target seeding density is between about 0.1E6 and about 0.9E6 viable cells per ml (VCD). In some embodiments, the target seeding density is about 0.1E6 VCD, 0.2E6 VCD, 0.3E6 VCD, 0.4E6 VCD, 0.5E6 VCD, 0.6E6 VCD, 0.7E6 VCD, 0.8E6 VCD, 0.9E6 VCD In some embodiments, the target seeding density is about 0.5E6 VCD.

[0078] In some embodiments, the method comprises a cell thaw recovery period (i.e., a thaw recovery step) wherein the population of insect host cells that contains detectable rhabdovirus genomes is cultured at a normal culturing temperature. In some embodiments, the normal culturing temperature is between about 25°C and about 30°C. In some embodiments, the normal culturing temperature is about 25°C, 25.5°C, 26°C, 26.5°C, 27°C, 27.5°C, 28°C, 28.5°C, 29°C, 29.5°C, or 30°C. In some embodiments, the normal culturing temperature is between about 27°C and about 28°C. In some embodiments, the normal culturing temperature is about 28°C. In some embodiments, the cell thaw recovery period is between about 10 days and about 30 days. In some embodiments, the cell thaw recovery period is about 18 days.

[0079] In some embodiments, the population of insect cells is passaged one or more times during the cell thaw recovery period. In some embodiments, the population of insect cells is passaged 2 to 10 times. In some embodiments, the population of insect cells is passaged 8 times. In some embodiments, the population of insect cells is passaged to generate a first target passage density. In some embodiments, the first target passage density is between about 0.1E6and about 0.9E6 viable cells per ml (VCD). In some embodiments, first target passage density is about 0.5E6 VCD.

[0080] In some embodiments, the method comprises a non-typical culturing temperature period after the cell thaw recovery period. In some embodiments, the non-typical culturing temperature period comprises adapting a population of insect host cells that contains detectable rhabdovirus genomes to a non-typical culturing temperature. In some embodiments, the non- typical culturing temperature is between about 31 °C and about 45°C. In some embodiments, the non-typical culturing temperature is between about 31°C and about 37°C. In some embodiments, the non-typical culturing temperature is between about 31°C and about 33°C. In some embodiments, the non-typical culturing temperature is about 30.5°C, 31°C, 31.5°C, 32°C, 32.5°C, 33°C, 33.5°C, or 34°C. In some embodiments, the non-typical culturing temperature is about 33°C. In some embodiments, the non-typical culturing temperature period lasts for a duration of 0 days to about 100 days. In some embodiments, the non-typical culturing temperature period lasts for a duration of about 4 days to about 100 days. In some embodiments, the non-typical culturing temperature period lasts for a duration of about 10 days to about 100 days. In some embodiments, the non-typical culturing temperature period lasts for a duration of 10 to 100 days. In some embodiments, the non-typical culturing temperature period lasts for a duration of 20 to 50 days. In some embodiments, the non-typical culturing temperature period lasts for a duration of about 40 days, about 41 days, about 42 days, about 43 days, or about 45 days. In some embodiments, the non-typical culturing temperature period lasts for a duration of about 42 days.

[0081] In some embodiments, the population of insect cells is passaged one or more times during the non-typical culturing temperature period. In some embodiments, the population of insect cells is passaged 2 to 30 times. In some embodiments, the population of insect cells is passaged 8-14 times. In some embodiments, the population of insect cells is passaged on a 1- 3-3 schedule as follows, beginning on the first day of the non-typical culturing temperature period: (i) after 3 days, the population of insect cells is diluted 1 : 1 in fresh culture media and culture at non-typical temperature is continued; (ii) 1 day later, the culture is diluted to a second target passage density; (iii) 3 days later, the culture is again diluted to a second target passage density; and (iv) steps (i) through (iii) are repeated until the end of the non-typical culturing temperature period. In some embodiments, the second target passage density is between about 0.1E6 and about 0.9E6 viable cells per ml (VCD). In some embodiments, the second target passage density is about 0.5E6 VCD. In some embodiments, the population of insect cells ispassaged 2 to 10 times. In some embodiments, the population of insect cells is passaged 8 times.

[0082] In some embodiments, rhabdovirus genomes were reduced by greater than 5-logs in the population of insect host cells following the non-typical culturing temperature period, compared to the population of insect cells prior to the non-typical culturing temperature period. In some embodiments, the non-typical culturing temperature period results in a population of non-typical culturing temperature-adapted insect host cells. In some embodiments, the population of non-typical culturing temperature-adapted insect host cells does not comprise rhabdovirus above the limit of detection of assay detection. The assay may be any known the art that is suitable for detecting rhabdovirus.

[0083] In some embodiments, the non-typical culturing temperature period comprises adapting a population of insect host cells that contains detectable rhabdovirus genomes to a non-typical culturing temperature in a single adapting step or in multiple adapting steps. In some embodiments, the single adapting step comprises culturing the population of insect host cells at a single intermediate culturing temperature. In some embodiments, the multiple adapting steps comprise culturing the population of insect host cells at multiple intermediate culturing temperatures, wherein each adapting step comprises a single intermediate culturing temperature. In some embodiments, the multiple adapting steps comprise at most two, at most three, or at most four adapting steps. In some embodiments, the intermediate culturing temperatures are between about 30°C and about 34°C. In some embodiments, the non-typical culturing temperature is about 30.5°C, 31°C, 31.5°C, 32°C, 32.5°C, 33°C, 33.5°C, or 34°C. In some embodiments, the intermediate culturing temperatures are between about 31°C and about 33°C.

[0084] In some embodiments, the method comprises a limited dilution adherent plating step after the non-typical culturing temperature period. In some embodiments, the limited dilution adherent plating step comprises plating the population of non-typical culturing temperature- adapted insect host cells at a concentration suitable for isolating cell clones or limited cell pools. In some embodiments, the limited dilution adherent plating step comprises adherent culture in 96 well culturing plates through Cell Factory culturing and / or production formats. In some embodiments, the limited dilution adherent plating step comprises culturing the population of non-typical culturing temperature-adapted insect host cells in rhabdovirus-free conditioned medium. In some embodiments, the rhabdovirus-free conditioned medium comprises between 0 and 10% FBS.

[0085] In some embodiments, the population of insect cells is plated at a dilution target density. In some embodiments, the dilution target density is between about 0.1 and 0.9 viable cells per well. In some embodiments, dilution target density is between about 0.5 viable cells per well. In some embodiments, the limited dilution adherent plating step comprises culturing the population of non-typical culturing temperature-adapted insect host cells at a non-typical culturing temperature as described herein.

[0086] In some embodiments, the method comprises monitoring of the wells to identify wells with clones (single cell per well) and wells with limited pools (multiple cells per well). In some embodiments, the clones and / or limited pools are monitored visually for colony formation and expansion to confluency. In some embodiments, the clones and / or limited pools are passaged one or more times during the limited dilution adherent plating step. In some embodiments, the clones and / or limited pools are passaged 2 to 10 times. In some embodiments, the clones and / or limited pools are passaged 8 times. In some embodiments, the clones and / or limited pools are passaged by tip scraping. In some embodiments, the clones and / or limited pools are passaged by use of a scraping tool followed by pipetting. In some embodiments, the clones and / or limited pools are passaged to a new culture container without the use of dissociation reagent. In some embodiments, at least one passage comprises transferring the clones and / or limited pools to a culture container of greater volume. In some embodiments, the clones and / or limited pools are sequentially passaged into a series of about 1 to about 5 culture containers, wherein at least 2, at least 3, at least 4, or 5 of the culture containers are each of greater volume than the previous culture container.

[0087] In some embodiments, the limited dilution adherent plating step lasts for a duration of about 7 days to about 100 days. In some embodiments, the limited dilution adherent plating step lasts for a duration of about 30 days to about 100 days. In some embodiments, the limited dilution adherent plating step lasts for a duration of about 40 to about 60 days. In some embodiments, the limited dilution adherent plating step lasts for a duration of about 50 days, about 51 days, or about 52 days. In some embodiments, the limited dilution adherent plating step generates at least one clone or limited pool of producer cell lines. In some embodiments, the limited dilution adherent plating step generates at least one clonal or limited pool population of insect cells.

[0088] In some embodiments, the method comprises a clone or limited pool suspension adaptation step (e.g., a clone or limited pool culturing step) after the limited dilution adherent plating step. In some embodiments, the clone or limited pool suspension adaptation step comprises adapting at least one clonal or limited pool population of insect cells (henceforth“clonal and / or pool population”) to suspension culture. In some embodiments, the clonal and / or pool populations are scraped and passaged to shake flasks.

[0089] In some embodiments, the clonal and / or pool populations are allowed to recover by culturing for 2 or 3 days, then counted. In some embodiments, cultures above a threshold density are passaged at a third target passage density. In some embodiments, the threshold density is between about 3.0E6 and about 5.0E6 viable cells per ml (VCD). In some embodiments, threshold density is between about 4.0E6. In some embodiments, the third target passage density is between about 0.2E6 and about 2.0E6 viable cells per ml (VCD). In some embodiments, the second target passage density is about 1.0E6 VCD.

[0090] In some embodiments, the clonal and / or pool populations are passaged when they exceed the threshold density, until each clonal and / or pool population achieves a consistent doubling time. In some embodiments, the clonal and / or pool populations are then passaged on a 1-3-3 schedule as described herein. In some embodiments, the clone or limited pool suspension adaptation step comprises culturing the clonal and / or pool populations in rhabdovirus-free conditioned medium. In some embodiments, the rhabdovirus-free conditioned medium does not comprise FBS. In some embodiments, the clone or limited pool suspension adaptation step comprises culturing the at least one clone or limited pool at a non-typical culturing temperature as described herein. In some embodiments, the clone or limited pool suspension adaptation step lasts for a duration of about 3 to about 10 days. In some embodiments, the clone or limited pool suspension adaptation step lasts for a duration of about 5 to about 7 days.

[0091] In some embodiments, the clone or limited pool suspension adaptation step generates at least one suspension-adapted population of insect cells derived from a clone or limited pool (henceforth “suspension-adapted population”). In some embodiments, the at least one suspension-adapted population is then cultured at a normal culturing temperature and / or a non- typical culturing temperature, as described herein. In some embodiments, the clonal and / or pool populations are assessed for AAV and / or baculovirus productivity. Assessment for AAV and / or baculovirus productivity may be performed using any suitable method known in the art or described herein.In some embodiments, the at least one suspension-adapted population is banked (e.g., frozen and stored) as a research cell bank (RCB). In some embodiments, the at least one suspension- adapted population is centrifuged and resuspended in the presence of between about 5% and about 20% DMSO. In some embodiments, the at least one suspension-adapted population is centrifuged and resuspended in the presence of between about 10% DMSO. In someembodiments, the at least one suspension-adapted population is banked at a target RCB density. In some embodiments, the target RCB density is between about 5.0E6 and about 1.0E8 viable cells per ml (VCD). In some embodiments, the target RCB density is between about 0.8E6 and about 3.0E7 VCD. In some embodiments, the target RCB density is about 1.5E7 VCD. In some embodiments, the at least one suspension-adapted population is aliquoted into at least one vial. In some embodiments, the at least one vial containing a suspension-adapted population is frozen. In some embodiments, freezing comprises freezing the vial in a cool cell to about -80°C, then transferring to liquid nitrogen for long-term storage.

[0092] In some embodiments, the method comprises: i) a cell thaw recovery period; ii) a non-typical culturing temperature period; and iii) a limited dilution adherent plating step.

[0093] In some embodiments, the method comprises producing a population of insect host cells devoid of detectable rhabdovirus comprising: i) recovering a population of insect host cells that contains detectable rhabdovirus genomes at normal culturing temperatures for 0-100 days after thawing, wherein the normal culturing temperature is between about 25°C and about 30°C; ii) adapting the population of insect host cells to a non-typical culturing temperature; and iii) performing a limited dilution adherent plating of the population of insect host cells to generate clones or limited pools of producer cell lines.

[0094] In some embodiments, the method comprises: i) a cell thaw recovery period, wherein a population of insect host cells that contains detectable rhabdovirus genomes is cultured at normal culturing temperatures after thawing; ii) a non-typical culturing temperature period, wherein the population of insect host cells is adapted to a non-typical culturing temperature, wherein the non-typical culturing temperature period is of a duration of 0 to about 100 days and wherein the non-typical culturing temperature period results in a non-typical culturing temperature-adapted population of insect host cells; and iii) a limited dilution adherent plating step, wherein the non-typical culturing temperature- adapted population of insect host cells are diluted using limited dilution adherent plating, such as to generate clones or limited pools.

[0095] In some embodiments, the method does not comprise use of an antiviral compound.

[0096] The method may be performed with any insect host cell line that contains detectable rhabdovirus genomes and is suitable for production of AAV, as described herein or known in the art. In some embodiments, the insect host cells are Spodoptera frugiperda (Sf) cells. In some embodiments, the population of insect host cells that contains detectable rhabdovirus genomes is, or is derived from, a Sf9 cell line. In some embodiments, the population of insect host cells that contains detectable rhabdovirus genomes is, or is derived from, a Sf21 cell line.

[0097] In some embodiments, the method uses a population of insect host cells that contains detectable rhabdovirus genomes, wherein the population of insect host cells that contains detectable rhabdovirus genomes is a qualified producer cell line (e.g., an RCB or MCB) for baculovirus and / or AAV production. In some embodiments, the producer line is qualified for baculovirus and / or AAV production at normal culturing temperature, wherein the normal culturing temperature is between about 25°C and about 30°C.Exemplary Methods

[0098] The present disclosure also provides certain exemplary methods for producing a population of insect host cells devoid of detectable rhabdovirus. In some embodiments, the method comprises: i) recovering a population of insect host cells that contains detectable rhabdovirus genomes at normal culturing temperatures for about 10 to about 30 days after thawing, wherein the normal culturing temperature is between about 25°C and about 30°C; ii) adapting the population of insect host cells to a non-typical culturing temperature by culturing the population of insect cells at a non-typical culturing temperature for a duration of about 4 days to about 100 days, wherein the non-typical culturing temperature is about 31°C and about 37°C; and iii) performing a limited dilution adherent plating of the population of insect host cells to generate clones or limited pools of producer cell lines.

[0099] In some embodiments, the present disclosure provides a population of insect host cells produced by any of the methods described herein. In some embodiments, the present disclosure provides a cell stock comprising a population of insect host cells described herein, wherein the cell stock comprises at least 1% DMSO by volume. In some embodiments, the present disclosure provides a cell stock comprising a population of insect host cells described herein, wherein the population is derived by any method described herein. In some embodiments, thepresent disclosure provides a cell stock comprising a population of insect host cells described herein, wherein the cell stock comprises at least 1% DMSO by volume and the population is derived by any method described herein.

[0100] The present disclosure also provides certain exemplary methods for producing a population of insect host cells devoid of detectable rhabdovirus. In some embodiments, the method comprises: i) recovering a population of insect host cells that contains detectable rhabdovirus genomes at normal culturing temperatures for about 12 days and about 24 days after thawing, wherein the normal culturing temperature is between about 27°C and about 28°C; ii) adapting the population of insect host cells to a non-typical culturing temperature by culturing the population of insect cells at a non-typical culturing temperature for a duration of about 20 days to about 50 days, wherein the non-typical culturing temperature is between about 31°C and about 33°C; and iii) performing a limited dilution adherent plating of the population of insect host cells to generate clones or limited pools of producer cell lines.

[0101] In some embodiments, the present disclosure provides a population of insect host cells produced by any of the methods described herein. In some embodiments, the present disclosure provides a cell stock comprising a population of insect host cells described herein, wherein the cell stock comprises at least 1% DMSO by volume. In some embodiments, the present disclosure provides a cell stock comprising a population of insect host cells described herein, wherein the population is derived by any method described herein. In some embodiments, the present disclosure provides a cell stock comprising a population of insect host cells described herein, wherein the cell stock comprises at least 1% DMSO by volume and the population is derived by any method described herein.

[0102] The present disclosure also provides certain exemplary methods for producing a population of insect host cells devoid of detectable rhabdovirus. In some embodiments, the method comprises: i) recovering a population of insect host cells that contains detectable rhabdovirus genomes at normal culturing temperatures for about 16 to about 20 days after thawing, wherein the normal culturing temperature is between about 27°C and about 28°C; ii) adapting the population of insect host cells to a non-typical culturing temperature by culturing the population of insect cells at a non-typical culturing temperature for aduration of about 40 days to about 50 days, wherein the non-typical culturing temperature is between about 31°C and about 33°C; and iii) performing a limited dilution adherent plating of the population of insect host cells to generate clones or limited pools of producer cell lines.

[0103] In some embodiments, the present disclosure provides a population of insect host cells produced by any of the methods described herein. In some embodiments, the present disclosure provides a cell stock comprising a population of insect host cells described herein, wherein the cell stock comprises at least 1% DMSO by volume. In some embodiments, the present disclosure provides a cell stock comprising a population of insect host cells described herein, wherein the population is derived by any method described herein. In some embodiments, the present disclosure provides a cell stock comprising a population of insect host cells described herein, wherein the cell stock comprises at least 1% DMSO by volume and the population is derived by any method described herein.

[0104] The present disclosure also provides certain exemplary methods for producing a population of insect host cells devoid of detectable rhabdovirus. In some embodiments, the method comprises: i) recovering a population of insect host cells that contains detectable rhabdovirus genomes at normal culturing temperatures for about 18 days after thawing, wherein the normal culturing temperature is between about 28°C; ii) adapting the population of insect host cells to a non-typical culturing temperature by culturing the population of insect cells at a non-typical culturing temperature for a duration of about 42 days, wherein the non-typical culturing temperature is about33 °C; and iii) performing a limited dilution adherent plating of the population of insect host cells to generate clones or limited pools of producer cell lines.

[0105] In some embodiments, the present disclosure provides a population of insect host cells produced by any of the methods described herein. In some embodiments, the present disclosure provides a cell stock comprising a population of insect host cells described herein, wherein the cell stock comprises at least 1% DMSO by volume. In some embodiments, the present disclosure provides a cell stock comprising a population of insect host cells described herein, wherein the population is derived by any method described herein. In some embodiments, the present disclosure provides a cell stock comprising a population of insect host cells described herein, wherein the cell stock comprises at least 1% DMSO by volume and the population is derived by any method described herein.Methods of AAV Production Provided Herein

[0106] The present disclosure also provides methods of producing AAV viral particles using an insect host cell or population of insect host cells devoid of detectable rhabdovirus genomes. In some embodiments, the method comprises (i) introducing any one or more expression systems, plasmids or vectors known in the art into the insect host cell or population of host cells, and (ii) culturing the cell under conditions suitable for protein expression and / or suitable for generation of AAV viral particles. In some embodiments, the population of insect host cells are contacted with one, two, or three baculovirus expression vectors as described herein or known in the art.

[0107] In some embodiments, the methods described herein, before step (ii), further comprise introducing into the insect host cell or population of insect host cells devoid of detectable rhabdovirus genomes a plasmid or vector comprising a transgene expression cassette.

[0108] In some embodiments, the methods described herein further comprise introducing into the insect host cell or population of insect host cells one or more vectors or plasmids comprising helper functions, such as a helper plasmid expressing adenovirus helper genes.

[0109] In some embodiments, introducing into a cell is by transfection. In some embodiments, transfection comprises complexing with cationic polyamides, cationic lipids, Calcium phosphate, peptides. In some embodiments, introducing into a cell is by electroporation. In some embodiments, the vectors, promoters, packaging factors, packaging systems, host cells, and / or methods of AAV virion production are any of those known in the art.

[0110] In some embodiments, the methods for production of AAV particles described herein utilize suspension culture (e.g., shake flask or bioreactor).[OHl] In some embodiments, the methods described herein, further comprise collecting the AAV viral particles produced by the cell cultured in step (ii). AAV particles (such as packaged virions) can be collected using any methods described herein or known in the art. In some embodiments, AAV particles are extracted from production cells using cell lysis.

[0112] In some embodiments, the collected AAV viral particles produced by the methods described herein are purified. The purification can be performed using any method known in the art. In some embodiments, the purification is performed using centrifugation and / or filtration. In some embodiments, the purification is performed using an affinity chromatography resin.

[0113] In some embodiments, the AAV particles may be of any AAV serotype described herein. In some embodiments, the AAV particles are AAV9 particles. In some embodiments, the AAV particles are AAV5 particles.

[0114] In some embodiments, the method provides adequate or enhanced production of AAV particles. In some embodiments, the methods described herein achieve viral titers of about lei 1 vg / ml to lei 3 vg / ml. In some embodiments, the methods described herein achieve viral titers of more than el l vg / ml. In some embodiments, the methods described herein achieve viral titers of more than lel2 vg / ml. In some embodiments, the method produces AAV9 viral particles with a volumetric yield greater than 1E12 vg / ml. In some embodiments, the method produces AAV5 viral particles with a volumetric yield greater than 2E12 vg / ml.

[0115] In some embodiments, enhanced production of AAV particles is in comparison to production of AAV particles by a population of insect host cells that contains detectable rhabdovirus genomes. In some embodiments, enhanced production is in comparison to a qualified producer cell line (e.g., an RCB or pre-MCB).

[0116] In some embodiments, the method produces AAV with comparable quality, as measured by one or more attributes including transduction efficiency, full capsid ratio, and capsid composition, to a method using a population of insect host cells that contains detectable rhabdovirus genomes. In some embodiments, the method produces AAV with comparable transduction of a target cell (e.g., a cardiomyocyte) compared to a method using a population of insect host cells that contains detectable rhabdovirus genomes.

[0117] In some embodiments, the method produces AAV with a comparable ratio or percentage (%) of full capsids to empty capsids (full capsid ratio or %) compared to a method using a population of insect host cells that contains detectable rhabdovirus genomes. Methods of measuring the full capsid ratio or % are known in the art. In some embodiments, the full capsid ratio or % is measured by capsid ELISA.

[0118] In some embodiments, the methods described herein produce AAV with a comparable capsid composition compared to a population of insect host cells that contains detectable rhabdovirus genomes. In some embodiments, capsid composition is measured by quantifying expression of VP1, VP2 and VP3 proteins in a population of insect host cells. In some embodiments, the VP1:VP2:VP3 ratio is comparable to that achieved with a method using a method using a population of insect host cells that contains detectable rhabdovirus genomes. Methods of measuring the capsid composition are known in the art. In some embodiments, the capsid composition is measured by capsid western blot.

[0119] In some embodiments, the methods described herein produce AAV with improved biosafety profiles, reducing the need for extensive rhabdovirus viral clearance studies and validation.EXAMPLESExample 1. Derivation of SfRV-Negative Cell Lines

[0120] Rhabdovirus reduction was performed on a high-productivity enriched population of cells (pre-MCB) generated from an ATCC Sf9 insect cell line that has been shown to be contaminated with an invertebrate rhabdovirus (SfRV). The pre-MCB culture was assessed for intracellular SfR viral genomes by sampling the culture during culture at about 28°C (e.g., normal culturing temperature) at various passages after thaw recovery, as well as at different densities (high total cell density [TCD], early exponential phase, exponential phase). The assessment groups are as follows:

[0121] Cells were pelleted at specific quantities (1E+06 or 4E+06 total cells) and washed prior to RNA isolation, then used to calculate the number of SfR viral genomes per cell (by dividing the number of SfR viral genomes by the number of cells in the pellet). As shown in the fourth and fifth columns from the left (Groups D and E above), 148-688 SfR viral genomes were measured per cell, with an average of 418 SfR viral genomes per cell by qRT-PCR. As shown in FIG. 2, SfR viral genomes) were detected in all samples, regardless of cell culture stage, cell density, and / or pellet size.

[0122] The pre-MCB population of cells, following thaw recovery, were adapted to a nontypical culturing temperature (about 33 °C). Adaptation of the pre-MCB culture to a non-typical culturing temperature reduces the half-life of rhabdovirus over time, resulting in a significantly lower basal level of SfRV genomes and infectious particles. Rhabdovirus reduction was tracked over time at non-typical culturing temperature using LAMP, then tested by RT-ddPCR whenreduction plateaued. During temperature adaptation, rhabdovirus genome levels in the culture supernatant decreased below the limit of assay detection seven days before rhabdovirus genome levels in the cell pellet decreased below the limit of assay detection (FIG. 10).

[0123] As shown in Table 1, SfR viral genome levels were reduced by more than 5 logs during this temperature adaptation, as measured by comparing SfR viral genome levels in the parental cell line (row 1 of Table 1) versus week four of temperature adaptation (row 3 of Table 1).

[0124] Following temperature adaptation, cells were cloned by limiting dilution. Single-cell clones or limited pools were grown in 96-well plates. Cell lines (derived from clones and limited pools) that demonstrated robust growth were scaled up and adapted to suspension culture.

[0125] SfR viral genome levels were measured in single-cell clones, in cell lines at eight weeks after adaptation of selected cell lines to suspension culture at normal temperature, and at the time of banking selected rhabdovirus-free (SfRVz) cell lines. As shown in rows 4-6 of Table 1, SfR viral genome levels in these samples were all below the limit of assay detection (BLOD).

[0126] A total of 21 clones and pools selected were rhabdovirus-negative by PCR (z.e., below the limit of detection of the assay, BLOD) in both normal and non-typical culturing temperatures. For single-cell clones, post-suspension adapted cell lines, and cell lines at time of banking, Table 1 summarizes the data for all measured cell clones / pools / cell lines (all of which had SfRV levels below the limit of detection of the assay).

[0127] SfRV titers were measured by ddPCR or equivalent PCR SfRV (designed to detect RNA and DNA) method at all time points other than the parental cell line measurement. At the time of SfRVz cell line banking, cells were also sequenced using Illumina NGS with a targeted read of >40M to confirm absence of SfR viral genomes. Clones, pools, or cell lines with SfR viral genome levels below the limit of detection of the assay are referred to as “SfRVz” clones, pools, or cell lines.TABLE 1. Reduction of SfR Viral Genome Levels in Cell Line During Adaptation to NonTypical Culturing Temperatures, Cloning, and Adaptation to Suspension.BLOD = below limit of detection; * Represent different time points / samples from the same cultureExample 2. AAV9 Production by SfRV-Negative Cell Lines

[0128] Cell lines derived from all selected clones and limited pools (21 cell lines) were assessed for AAV9 production, in comparison with a standard AAV9 production cell line (MCB). These cell lines are referred to as non-typical culturing condition-adapted cell lines. Baculovirus stock from the parental (MCB) cell line was used for AAV infection in all cell lines. AAV9 production was measured in shake flasks at non-typical culture temperatures (FIG. 3). Cell lines labeled with “1-n” or “2-n”, where n is a number, refer to cell lines derived from clones. Cell lines labeled with a P-n, where n is a number, refer to cell lines derived from a limited cell pool. Three cell lines (1-2, 1-9, and P-11) had comparable productivity compared to the MCB control. Titers were as follows in Table 2. However, overall titer was not optimal, so the cell lines were adapted to normal culturing temperatures as described belowTABLE 2. AAV Production by SfR virus-negative cell lines at non-typical culturing temperature.

[0129] The 21 cell lines were adapted from 33 °C (non-typical culturing temperature) to 28°C (normal culturing temperature). These 21 normal culturing temperature-adapted cell lines were used to assess AAV9 production. Titers were as follows in Table 3. Baculovirus stock derived from each cell line was used for AAV infection of the same cell line. As shown in FIG. 4, five of these normal culturing temperature-adapted cell lines (1-2, 1-3, 1-12, 2-10, and P-7) that produced the highest titers were selected for further characterization. Among these five, several demonstrated a comparable or better titer than the MCB Control.TABLE 3. AAV Production by SfR virus-negative cell lines at normal culturing temperature.

[0130] AAV9 production was measured in triplicate for these five normal culturing temperature-adapted cell lines, of which 4 were derived from clones and one from a pool. Titers were as follows in Table 4. Baculovirus stock derived from each cell line was used for AAV infection of the same cell line. As shown in FIG. 5, AAV9 production in two of these cell lines (normal culturing temperature-adapted 1-2 and 2-10) exceeded that of the MCB Control, while production in a third cell line (normal culturing temperature-adapted 1-12) was comparable to the MCB control or higher.TABLE 4. AAV Production by Selected Cell Lines.

[0131] These three cell lines (normal culturing temperature-adapted 1-2, 2-10, and 1-12) were cultured in 3L bioreactors to confirm AAV9 production and quality. Each cell line and the MCB Control cell line were infected by the same baculovirus stock, which was produced in normal culturing temperature-adapted SfRVz cell line 2-10. All productions utilized the same baculovirus stock in this experiment to limit the number of variables for assessing cell line productivity. As shown in FIG. 5 and Table 5, all five cell lines provided AAV9 production comparable to MCB (infected with SfRV-negative baculovirus (FIG. 6A). Sat = satellite, referring to a production made in parallel by transferring an aliquot of a cell culture to a different production-scale vessel and running the course of infection. BRX = bioreactor.

[0132] Unexpectedly, the MCB cell line provided >1.5-fold higher productivity in this experiment utilizing SfRV-negative baculovirus, compared to historical data for this cell line. Without wishing to be bound by theory, it may be that a high SfR viral load during baculovirus infection reduces AAV9 productivity.TABLE 5. AAV Production by Selected Cell Lines in BioreactorsExample 3. Quality of AAV Produced by SfRV-Negative Cell Lines

[0133] The quality of AAV produced by these three cell lines (normal culturing temperature- adapted 1-2, 2-10, and 1-12) in 3L bioreactors was assessed. Characteristics of quality measured included: transduction of iPSC-cardiomyocytes, full capsid ratios, and capsid composition.

[0134] AAV9-GFP produced by each of the three cell lines and the MCB control cell line was used to transduce iPSC-cardiomyocytes. The efficiency of iPSC-cardiomyocyte transduction by AAV9-GFP produced by each cell line was measured by GFP fluorescence (FIG. 6B). iPSC-cardiomyocyte transduction as measured by GFP was higher in normal culturing temperature-adapted cell lines 1-12 and 2-10 than in normal culturing temperature-adapted cell line 1-2. GFP fluorescence is shown below in Table 6.TABLE 6. GFP Fluorescence in iPSC Cardiomyocytes Transduced by AAV9-GFP Produced by Selected Cell Lines

[0135] The percentage of full capsid ratio (vg / cp) for AAV9-GFP produced by each of the three normal culturing temperature-adapted cell lines and the MCB control cell line was measured by capsid ELISA (FIG. 6C and Table 7), and the capsid composition (VP1 :2:3 ratio) was measured by western blot (FIG. 6D and Table 8). Sat = satellite, referring to a production made in parallel by transferring an aliquot of a cell culture to a different production-scale vessel and running the course of infection. BRX = bioreactor. All SfRVz normal culturingtemperature-adapted cell lines showed comparable % full capsid ratios and capsid composition to the MCB control.TABLE 7. Percentage of Full Capsid RatioTABLE 8. Capsid Composition

[0136] To further confirm the productivity of these three cell lines (normal culturing temperature-adapted 1-2, 2-10, and 1-12), 3L bioreactors were run in parallel for AAV9:4.8Kb (AAV9 packaging a 4.8Kb cassette) for clinical relevance. Each cell line in this experiment was treated as an independent producer cell line, where all baculovirus used in AAV9 production was specifically produced by the respective cell line. Additionally, to confirm the effect of SfRV-negative baculovirus on AAV9 production in the parental MCB cell line, MCB was again infected with baculovirus stock produced by SfRVz normal culturing temperature- adapted cell line 2-10. A baseline, based on historic productivity of the same AAV9:4.8Kb cassette in the MCB parental cell line, is included in the graph for reference (FIG. 7 and Table 9).TABLE 9. AAV9 4.8Kb Productivity in 3L Bioreactors

[0137] Production of A AV9 :4.8Kb by each of the three cell lines (normal culturing temperature-adapted 1-2, 2-10, and 1-12) exceeded that of the parental MCB infected with SfRVz-produced baculovirus, as well as the historic productivity of the MCB cell line (baseline). Normal culturing temperature-adapted cell line 2-10 demonstrated superior productivity, achieving a 3.1 -fold increase in AAV9 titer compared to the historical MCB baseline (FIG. 7).

[0138] Production of an additional AAV serotype packaging the 4.8Kb cassette (AAV5 :4.8Kb) by the lead cell line (normal culturing temperature-adapted 2-10) was also measured in comparison to that of the parental MCB cell line, utilizing baculovirus produced by each cell line respectively (FIG. 8 and Table 10). Production of AAV5:4.8Kb by the SfRVz normal culturing temperature-adapted cell line 2-10 was higher than that of the parental MCB cell line.TABLE 10. AAV5 4.8Kb Productivity in 3L BioreactorsExample 4. Growth and Viability of SfRV-Negative Cell Lines

[0139] Growth characteristics of these three cell lines (normal culturing temperature-adapted 1-2, 2-10, and 1-12) were assessed and compared to the parental MCB cell line. Characteristics assessed included cell growth, population doubling levels (PDL), and viability from banked vial thaw through 135hrs post-thaw. Each of the three cell lines demonstrated comparable viability and comparable or improved viable cell density (VCD) versus the parental MCB cell line (FIG. 9). Table 11 summarizes the data as average population doubling time in hours. Each of the three normal culturing temperature-adapted cell lines also had a significantly faster recovery period from thaw.TABLE 11. Average Population Doubling Level (PDL) in SfRV-Negative Cell LinesINCORPORATION BY REFERENCE

[0140] Various references such as patents, patent applications, and publications are cited herein, the disclosures of which are hereby incorporated herein by reference in their entireties. Also, all references mentioned herein are specifically incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

Claims

CLAIMS1. A population of insect host cells devoid of detectable rhabdovirus genomes.

2. The population of claim 1, wherein the population comprises less than 1, less than 2, or less than 10 rhabdovirus genome copies per milliliter.

3. The population of claim 1 or claim 2, wherein the number of rhabdovirus genomes in the population is measured by a Loop-Mediated Isothermal Amplification (LAMP) assay, a RT-qPCR assay, or an RNA-Seq assay.

4. The population of any one of claims 1-3, wherein the insect host cells are Spodoptera frugiperda (Sf) cells.

5. The population of any one of claims 1-3, wherein the population is derived from a Sf9 or Sf21 cell line.

6. The population of any one of claims 1-5, wherein the population is derived from a qualified producer cell line for baculovirus and / or AAV production at normal culturing temperature, wherein the normal culturing temperature is between about 25°C and about 30°C.

7. The population of any one of claims 1-6, wherein the normal culturing temperature is between about 28°C.

8. The population of any one of claims 1-7, wherein the population provides enhanced production of at least one serotype of AAV virions, compared to a population of insect host cells that contains detectable rhabdovirus genomes.

9. The population of any one of claims 1-8, wherein the serotype of AAV is AAV9.

10. The population of any one of claims 1-8, wherein the serotype of AAV is AAV5.

11. The population of any one of claims 1-10, wherein the population produces AAV with comparable transduction of a target cell compared to a population of insect host cells that contains detectable rhabdovirus genomes.

12. The population of any one of claims 1-11, wherein the population produces AAV with a comparable full capsid ratio compared to a population of insect host cells that contains detectable rhabdovirus genomes.

13. The population of any one of claims 1-12, wherein the population produces AAV with a comparable capsid composition compared to a population of insect host cells that contains detectable rhabdovirus genomes.

14. The population of any one of claims 1-13, wherein the population demonstrates faster thaw recovery compared to a population of insect host cells that contains detectable rhabdovirus genomes.

15. The population of any one of claims 1-14, wherein the population has comparable or improved viability during thaw recovery compared to a population of insect host cells that contains detectable rhabdovirus genomes.

16. The population of any one of claims 1-15, wherein the population has comparable or improved viable cell density during thaw recovery compared to a population of insect host cells that contains detectable rhabdovirus genomes.

17. The population of any one of claims 1-16, wherein the population retains undetectable levels of rhabdovirus following culture at about 28°C to about 30°C for greater than 250 days.

18. The population of any one of claims 1-17, wherein the population is derived using a method comprising adapting a population of insect host cells that contains detectable rhabdovirus genomes to a non-typical culturing temperature, optionally wherein the nontypical culturing temperature is between about 31°C and about 37°C.

19. The population of claim 18, wherein the non-typical culturing temperature is about33°C.

20. The population of claim 18 or claim 19, wherein the population is derived using a method comprising adapting the population that contains detectable rhabdovirus genomes to a non-typical culturing temperature by culturing at the non-typical culturing temperature for 0 to 100 days after thaw recovery.

21. The population of claim 20, wherein the population is derived using a method comprising adapting the population that contains detectable rhabdovirus genomes to a non- typical culturing temperature by culturing at the non-typical culturing temperature for 10 days to 30 days after thaw recovery.

22. The population of claim 21, wherein the population is derived using a method comprising adapting the population that contains detectable rhabdovirus genomes to a non- typical culturing temperature by culturing at the non-typical culturing temperature for about 18 days after thaw recovery.

23. The population of any one of claims 1-22, wherein the population is derived using a method comprising limited dilution adherent plating to generate clones or limited pools of cell lines.

24. The population of any one of claims 1-23, wherein the population is derived using a method that does not comprise use of an antiviral compound.

25. The population of any one of claims 1-24, wherein the population is derived using a method comprising culturing the cells in rhabdovirus-free conditioned medium comprising between 0 and 10% FBS.

26. A method of producing a population of insect host cells devoid of detectable rhabdovirus, wherein the method comprises adapting a population of insect host cells that contains detectable rhabdovirus genomes to a non-typical culturing temperature.

27. The method of claim 26, wherein the method comprises culturing at the non-typical culturing temperature for 0 to 100 days after thaw recovery.

28. The method of claim 27, wherein the method comprises culturing at the non-typical culturing temperature for 20 to 50 days after thaw recovery.

29. The method of claim 28, wherein the method comprises culturing at the non-typical culturing temperature for about 42 days after thaw recovery.

30. The method of any one of claims 26-29, wherein the method comprises performing a limited dilution adherent plating step to generate at least one clone or limited pool of producer cell lines.

31. The method of claim 30, wherein the method comprises culturing the at least one clone or limited pool of producer cell lines in rhabdovirus-free conditioned medium comprising between 0 and 10% FBS for 0 to 200 days after limited dilution plating.

32. A method of producing a population of insect host cells devoid of detectable rhabdovirus comprising: i) recovering a population of insect host cells that contains detectable rhabdovirus genomes at normal culturing temperatures for 0-100 days after thawing, wherein the normal culturing temperature is between about 25°C and about 30°C; ii) adapting the population of insect host cells to a non-typical culturing temperature; and iii) performing a limited dilution adherent plating of the population of insect host cells to generate clones or limited pools of producer cell lines.

33. The method of any one of claims 26-32, wherein the non-typical culturing temperature is between about 31°C and about 37°C.

34. The method of claim 33, wherein the non-typical culturing temperature is about 33°C.

35. The method of any one of claims 26-34, wherein the method does not comprise use of an antiviral compound.

36. The method of any one of claims 26-35, wherein the insect host cells are Spodoptera frugiperda (Sf) cells.

37. The method of any one of claims 26-36, wherein the population of insect host cells that contains detectable rhabdovirus genomes is, or is derived from, a Sf9 or Sf21 cell line.

38. The method of any one of claims 26-37, wherein the population of insect host cells that contains detectable rhabdovirus genomes is a qualified producer cell line for baculovirus and / or AAV production at normal culturing temperature, wherein the normal culturing temperature is between about 25°C and about 30°C.

39. The method of claim 38, wherein the normal culturing temperature is about 28°C.

40. A method of producing AAV particles using the population of insect host cells devoid of detectable rhabdovirus genomes of any one of claims 1-25, wherein the population of insect host cells are contacted with one, two, or three baculovirus expression vectors.

41. The method of claim 40, wherein the AAV particles are AAV9 particles.

42. The method of claim 40, wherein the AAV particles are AAV5 particles.

43. The method of any one of claims 40-42, wherein the method produces AAV with comparable transduction of a target cell compared to a method using a population of insect host cells that contains detectable rhabdovirus genomes.

44. The method of any one of claims 40-43, wherein the method produces AAV with a comparable full capsid ratio compared to a method using a population of insect host cells that contains detectable rhabdovirus genomes.

45. The method of any one of claims 40-44, wherein the method produces AAV with a comparable capsid composition compared to a method using a population of insect host cells that contains detectable rhabdovirus genomes.

46. A method of producing a population of insect host cells devoid of detectable rhabdovirus comprising: i) recovering a population of insect host cells that contains detectable rhabdovirus genomes at normal culturing temperatures for 18 days after thawing, wherein the normal culturing temperature is about 28°C; ii) adapting the population of insect host cells to a non-typical culturing temperature by culturing the population of insect cells at a non-typical culturing temperature for about 42 days, wherein the non-typical culturing temperature is about 33°C; and iii) performing a limited dilution adherent plating of the population of insect host cells to generate clones or limited pools of producer cell lines.

47. A cell stock comprising a population of insect host cells of any one of claims 1-25, wherein the cell stock comprises at least 1% DMSO by volume.

48. A cell stock comprising a population of insect host cells of any one of claims 1-25, wherein the cell stock comprises at least 1% DMSO by volume and the population of insect host cells is derived by the method of any one of claims 26-36.

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