Method for determining an AAV genome

Abstract

Reported herein is a method for determining viral genomic DNA copy number in a sample, comprising incubating the sample with proteinase K and determining the viral genomic DNA copy number by digital droplet polymerase chain reaction, wherein the sample does not contain DNA that is not encapsidated within viral particles, and wherein the incubation with proteinase K is in the presence of 0.05 (w / v)% to 1.5 (w / v)% sodium dodecyl sulfate.

Description

[Technical field]

[0001] The present invention belongs to the field of gene therapy.More specifically, this paper reports a method for determining the viral genome copy number in processing and purified samples by ddPCR, in which the sample is incubated with proteinase K in the presence of detergent before PCR. [Background technology]

[0002] 2. Background of the Invention Adeno-associated virus (AAV) particles are commonly used as gene transfer vehicles for research and clinical approaches due to their good safety profile, high therapeutic efficacy and the possibility of target-specific manipulation. Accurate and robust analytical methods for viral vector characterization are required for recombinant production. In general, vector genome titration is performed by droplet digital PCR (ddPCR), an absolute quantification method for nucleic acids.

[0003] In the literature, several pretreatment methods for AAV vector genome titration have been reported for both real-time PCR and droplet digital PCR. Prior to absolute quantification of the target sequence within the capsid, free transient plasmids and other non-packaged nucleic acids such as host cell DNA need to be degraded with nucleases such as DNase I (Zolotukhin, S., et al. 1999; Furuta-Hanawa, B., et al. 2019; Fripont, S., et al. 2019; Dobnik, D., et al. 2019; Sanmiguel J., et al. 2019). Furthermore, the capsid needs to be degraded for better vector genome accessibility. Heat inactivation of AAV particles and the resulting denaturation of their capsid proteins are sufficient for that purpose (Wang, Y., et al. 2019). However, most publications describe additional protein digestion with proteinase K (Zolotukhin, S., et al. 1999; Fripont, S., et al. 2019; Dobnik, D., et al. 2019; Sanmiguel J., et al. 2019). Furthermore, there are various recommendations on dilution buffers for the subsequent dilution series of the treated samples. In addition to nuclease-free water (Zolotukhin, S., et al. 1999), TE buffer (Furuta-Hanawa, B., et al. 2019; Wang, Y., et al. 2019) or PCR buffer (Sanmiguel J., et al. 2019), additives such as the anti-surfactant Pluronic F-68 (Furuta-Hanawa, B., et al. 2019; Sanmiguel J., et al. 2019) and sheared salmon sperm (sss) DNA (Lock, M., et al. 2014; Sanmiguel J., et al. 2019) have been described in the literature.

[0004] Suoranta, T., et al. (Hum. Gen. Ther. 32 (2020) 1270-1279) compared the extraction of AAV1, AAV2, AAV5, AAV6, AAV8, and AAV9 genomes after heat denaturation, proteinase K treatment, and iodixanol gradient ultracentrifugation in phosphate-buffered saline by kit extraction with qPCR and ddPCR. The kit extraction included proteinase K treatment in the presence of additional carrier RNA in denaturing buffer prior to spin column purification, significantly increasing the titers obtained for all serotypes in both qPCR and ddPCR. Importantly, Suoranta et al. found that no studies presented conclusive data on genome availability in ddPCR.

[0005] China Patent No. 109957561 discloses a method for extracting nucleic acids from a sample, which includes the steps of adding a lysis solution to the sample to be extracted to release nucleic acid molecules, further adding a lauroyl sarcosine sodium salt solution, and further adding a mixed solution containing sodium iodide, glycogen and isopropanol to form a precipitate containing nucleic acids, which is collected by centrifuging the sample.

[0006] US Pat. No. 8,652,821 discloses a reagent mixture for purifying DNA free of RNA, comprising a protease, an RNase, and a detergent.

[0007] US Pat. No. 11,028,372 discloses a scalable purification method for AAV particles of serotype rh.10.

[0008] WO 03 / 104413 discloses a non-specific dot blot analysis of pseudotyped recombinant AAV virions involving proteinase K incubation followed by phenol extraction and ethanol precipitation.

[0009] WO 2007 / 084773 discloses a non-specific dot blot analysis of infectious parvovirus vectors produced in insect cells.

[0010] Binny, CJ and Nathwani, AC disclose an agarose gel analysis method to determine whether the genome packaged in the scAAV vector is a short double-stranded hairpin structure or a short ssDNA or a long unfolded ssDNA, as intended (Meth. Mol. Biol. 891 (2012) 109-131).

[0011] U.S. Patent Application Publication No. 2021 / 0284699 discloses a qPCR method for analyzing rAAV particles purified using a method comprising: (a) generating a viral particle extract comprising a plurality of rAAVs provided herein, wherein the viral particle extract comprises a supernatant of lysed producer cells or a derivative thereof; (b) contacting the viral particle extract with an ionic detergent to generate a first mixture; (c) contacting the first mixture with an acid to generate a second mixture; (d) centrifuging the second mixture to generate a supernatant; (e) filtering the supernatant through one or more filters to generate a filtrate; and (f) performing one or more cycles of buffer exchange of the filtrate into a final storage buffer. Summary of the Invention

[0012] Reported herein is a method for determining viral genomic DNA copy number in a sample, comprising incubating the sample with proteinase K and determining the viral genomic DNA copy number by digital droplet polymerase chain reaction, wherein the sample does not contain DNA that is not encapsidated within viral particles, and wherein the incubation with proteinase K is in the presence of 0.05 (w / v)% to 1.5 (w / v)% sodium dodecyl sulfate.

[0013] A non-limiting number of aspects (independent subject matter) and embodiments (dependent subject matter) are as follows.

[0014] 1. A method for determining the viral genomic DNA copy number in a sample, said method comprising: determining the viral genomic DNA copy number by digital droplet polymerase chain reaction; Including, the sample does not contain DNA that is not encapsidated within viral particles; The sample was not incubated with nuclease, The sample has not been incubated with a protease. The method.

[0015] 2. The method of embodiment 1, wherein the nuclease is a restriction enzyme.

[0016] 3. The method of any one of aspect 1 or embodiment 2, wherein the nuclease is DNase I.

[0017] 4. The method of aspect 1 or embodiment 2 or 3, wherein the protease is proteinase K.

[0018] 5. A method for determining the viral genomic DNA copy number in a sample, comprising: - incubating the sample with proteinase K; determining the viral genomic DNA copy number by digital droplet polymerase chain reaction; The method comprising:

[0019] 6. The method of embodiment 5, wherein the sample is a cell lysate.

[0020] 7. The method of any one of aspect 5 or embodiment 6, wherein said sample is a lysed cell sample.

[0021] 8. The method of embodiment 7, wherein the lysed cell sample is obtained by lysing the virus-producing cells with a detergent or by chemical means, and optionally, cellular debris is removed from the lysed cell sample.

[0022] 9. The method of aspect 5 or any one of embodiments 6-8, wherein the sample comprises viral particles in which the viral DNA genome is encapsidated and free DNA that is not encapsidated within viral particles.

[0023] 10. A method for determining the number of viral genomic DNA copies in a sample, the method comprising: - incubating the sample with proteinase K; determining the viral genomic DNA copy number by digital droplet polymerase chain reaction; Including, the sample does not contain DNA that is not encapsidated within viral particles; The incubation with proteinase K is in the presence of a detergent. The method.

[0024] 11. The method of claim 10, wherein the detergent is sodium dodecyl sulfate.

[0025] 12. The method according to any one of aspects 10 or embodiment 11, wherein the final concentration of the detergent during incubation with Proteinase K is between 0.05% (w / v) and 1.5% (w / v).

[0026] 13. The method according to any one of aspects 10 or embodiments 11 to 12, wherein the final concentration of the detergent during incubation with Proteinase K is about 0.1% (w / v).

[0027] 14. The method according to any one of aspects 10 or embodiments 11-12, wherein the final concentration of the detergent during incubation with Proteinase K is about 1% (w / v).

[0028] 15. A step of incubating the sample with a nuclease to obtain a digested sample; - A step of incubating the digested sample with proteinase K to obtain a proteinase K-incubated sample; - A step of determining the number of copies of the viral genomic DNA in the proteinase K-incubated sample by digital droplet polymerase chain reaction The method according to any one of embodiment 5 or embodiment 10 or embodiments 6-9 or embodiments 11-14, comprising:

[0029] 16. The method according to embodiment 15, wherein the digested sample is diluted up to 2.5-fold for incubation with the proteinase K.

[0030] 17. The method according to any one of embodiments 15-16, wherein the completely digested sample is incubated with proteinase K.

[0031] 18. The digital droplet polymerase chain reaction is a) A step of incubating the sample at about 95°C for 10 minutes; b) A step of performing a thermal cycle of incubating the sample at about 94°C for 30 seconds and then at about 60°C for 1 minute; c) A step of repeating step b) 15 to 60 times; d) A step of performing a final extension step at 98°C for 10 minutes The method according to any one of embodiment 1 or embodiment 5 or embodiment 10 or embodiments 2-4 or embodiments 6-9 or embodiments 11-17, comprising:

[0032] 19. The method according to embodiment 18, wherein steps a) and d) are carried out with a temperature gradient of 2°C.

[0033] 20. The method of any one of aspect 1, or aspect 5, or aspect 10, or embodiment 2-4, or embodiment 6-9, or embodiment 11-19, wherein the sample is maintained at a temperature of 95° C. or less.

[0034] 21. The method of any one of aspect 1, or aspect 5, or aspect 10, or embodiment 2-4, or embodiment 6-9, or embodiment 11-19, wherein the sample is not exposed to a temperature above 95° C.

[0035] 22. The method according to any one of aspect 1, or aspect 5, or aspect 10, or embodiment 2-4, or embodiment 6-9, or embodiment 11-19, wherein, except for the final extension step of the digital droplet polymerase chain reaction, the method is performed at a temperature of up to 95°C.

[0036] 23. The method according to any one of aspect 5 or aspect 10 or embodiment 2-4 or embodiment 6-9 or embodiment 11-22, wherein the total amount of Proteinase K used in the incubation is between 1 mU and 50 mU.

[0037] 24. The method according to any one of aspect 5 or aspect 10 or embodiment 2-4 or embodiment 6-9 or embodiment 11-23, wherein the total amount of Proteinase K used in the incubation is between 10 mU and 40 mU.

[0038] 25. The method according to any one of aspect 5 or aspect 10 or embodiment 2-4 or embodiment 6-9 or embodiment 11-24, wherein the total amount of Proteinase K used in the incubation is between 15 mU and 35 mU.

[0039] 26. The method according to any one of aspect 5 or aspect 10 or embodiment 2-4 or embodiment 6-9 or embodiment 11-25, wherein the total amount of Proteinase K used in the incubation is between 20 mU and 32 mU.

[0040] 27. The method according to any one of aspect 5 or aspect 10 or embodiment 2-4 or embodiment 6-9 or embodiment 11-26, wherein the total amount of Proteinase K used in the incubation is about 20 mU.

[0041] 28. The method according to any one of aspect 5 or aspect 10 or embodiment 2-4 or embodiment 6-9 or embodiment 11-26, wherein the total amount of Proteinase K used in the incubation is about 32 mU.

[0042] 29. The method according to any one of aspect 1 or aspect 5 or aspect 10 or embodiment 2-4 or embodiment 6-9 or embodiment 11-28, wherein the determination of the viral genomic DNA copy number is a quantification of the viral genomic DNA copy number.

[0043] 30. The method of any one of aspect 1, or aspect 5, or aspect 10, or embodiment 2-4, or embodiment 6-9, or embodiment 11-29, wherein the volume of the sample is about 10 μL.

[0044] 31. The method according to any one of aspect 5, or aspect 10, or embodiment 2-4, or embodiment 6-9, or embodiment 11-30, wherein the incubating with proteinase K is in a total volume of 100 μL.

[0045] 32. The method of any one of embodiments 15 to 31, wherein the nuclease is DNase I.

[0046] 33. The method of any one of embodiments 15 to 32, wherein the total amount of nuclease used in the incubation is about 5 U.

[0047] 34. The method of any one of embodiments 15-33, wherein the incubating with the nuclease is in a total volume of 50 μL.

[0048] 35. Incubating with the nuclease is performed in 40 mM Tris*HCl, 10 mM MgSO at a pH value of about 8. 4 , 1 mM CaCl 2 The method according to any one of embodiments 15 to 34, wherein the final concentration is

[0049] 36. The method of any one of embodiments 15-35, wherein incubating with the nuclease is at 37° C. for 30 minutes.

[0050] 37. The method of embodiment 36, wherein said incubating is followed by inactivation of said nuclease at 95°C for 15 minutes.

[0051] 38. The method of any one of embodiments 15 to 37, wherein the incubation with Proteinase K is in a final concentration of 20 mM Tris*HCl, 1 mM EDTA, 100 mM NaCl at a pH value of about 8.

[0052] 39. The method of any one of embodiments 15 to 37, wherein the incubation with Proteinase K is in a final concentration of 20 mM Tris*HCl, 1 mM EDTA, 100 mM NaCl at a pH value of about 8.

[0053] 40. The method of any one of embodiments 15 to 37, wherein the incubation with Proteinase K is in a final concentration of 20 mM Tris*HCl, 1 mM EDTA, 100 mM NaCl, 1 (w / v)% sodium dodecyl sulfate at a pH value of about 8.

[0054] 41. The incubation with proteinase K is carried out in 30 mM Tris*HCl, 5 mM MgSO at a pH value of about 8. 4 , 0.5 mM CaCl 2 38. The method of any one of embodiments 15 to 37, wherein the final concentrations are 0.5 mM EDTA, 50 mM NaCl, 0.1 (w / v)% sodium dodecyl sulfate.

[0055] 42. The method of any one of embodiments 15 to 41, wherein the incubation with proteinase K is at 50° C. for 60 minutes.

[0056] 43. The method of embodiment 42, wherein the incubating is followed by inactivation of the proteinase at 95°C for 15 minutes.

[0057] 44. The method according to any one of aspect 1, or aspect 5, or aspect 10, or embodiment 2-4, or embodiment 7-9, or embodiment 11-43, wherein said sample volume is an affinity chromatography purified cell lysate.

[0058] 45. The method according to any one of aspect 1 or aspect 5 or aspect 10 or embodiment 2-4 or embodiment 6-9 or embodiment 11-43, wherein the method is carried out in the absence of a precipitation step or / and glycogen. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0059] Detailed Description of the Invention The present invention is based, at least in part, on the discovery that for AAV genome copy number determination, for crude cell lysate samples that contain AAV particles and also contain non-virally encapsidated DNA, it is necessary to incubate the sample with proteinase K prior to PCR. The presence of a detergent can further improve the determination.

[0060] The present invention is based, at least in part, on the discovery that for AAV genome copy number determination, for purified samples that contain AAV particles and are essentially free of non-virally encapsidated DNA, the sample must be incubated with proteinase K in the presence of a detergent prior to PCR. Without being bound by this theory, it is believed that the detergent prevents the formation of aggregates of protein fragments and viral genomic DNA, which would prevent subsequent amplification by polymerase chain reaction, thereby resulting in an underestimation of the viral genome copy number in the sample.

[0061] The present invention is based at least in part on the discovery that for AAV genome copy number determination after sequential treatment with nuclease and protease, protease treatment must be performed in the presence of detergent.Without being bound by this theory, it is believed that detergent prevents the formation of protein fragments and aggregates of viral genome DNA, which prevents subsequent amplification by polymerase chain reaction, thereby underestimating the viral genome copy number in the sample.

[0062] The present invention is further based, at least in part, on the discovery that for purified samples that contain AAV particles and are essentially free of non-virally encapsidated DNA, AAV genome copy number determinations must be performed without nuclease and proteinase pretreatment or with proteinase K incubation in the presence of detergent prior to polymerase chain reaction. Without being bound by this theory, it is believed that incubation of highly purified samples, i.e., samples that do not contain a requisite amount of non-virally encapsidated DNA, with nuclease alone or in combination with protease in the absence of detergent leads to aggregation, thereby artificially lowering the AAV genome copy number.

[0063] The present invention is further based, at least in part, on the discovery that for purified samples that contain AAV particles and that are essentially free of non-virally encapsidated DNA, determination of AAV genome copy number must be performed without a heat denaturation step at temperatures greater than 95° C. Without being bound by this theory, it is believed that heat treatment of highly purified samples, i.e., samples that do not contain a requisite amount of non-virally encapsidated DNA, at temperatures greater than 95° C. leads to aggregation, thereby artificially lowering the AAV genome copy number.

[0064] The invention is further based, at least in part, on the fact that a small amount of proteinase K in the presence of a detergent is sufficient to render substantially all AAV genomes in a sample accessible for polymerase chain reaction and thus determination / quantification. Without being bound by this theory, it is believed that high concentrations of proteinase K interfere with polymerase chain reaction, and that using substantially reduced, i.e. low amounts of proteinase K improves polymerase chain reaction by reducing PCR inhibition. In particular, inhibition of polymerase chain reaction by proteinaceous material in cell lysates of AAV producing cells can be reduced or even eliminated by incubation of proteinase K in the presence of a detergent.

[0065] The present invention is further based, at least in part, on the discovery that for sequential incubation of a crude cell lysate sample with a nuclease and a protease, the sample should not be diluted after the nuclease incubation, and the total volume of the incubation mixture is that used in the protease incubation step.

[0066] In general, the more preparative steps required before determining AAV genome copy number by ddPCR, the greater the chance of contamination.

[0067] definition Useful methods and techniques for carrying out the invention are described, for example, in Ausubel, FM (ed.), Current Protocols in Molecular Biology, Volumes I to III (1997); Glover, ND, and Hames, BD, ed., DNA Cloning: A Practical Approach, Volumes I and II (1985), Oxford University Press; Freshney, RI (ed.), Animal Cell Culture-a practical approach, IRL Press Limited (1986); Watson, JD, et al., Recombinant DNA, Second Edition, CHSL Press (1992); Winnacker, EL, From Genes to Clones; NY, VCH Publishers (1987); Celis, J., ed., Cell Biology, Second Edition, Academic Press (1998); Freshney, RI, Culture of Animal Cells: A Manual of Basic Technique, second edition, Alan R. Liss, Inc., NY (1987).

[0068] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a cell" includes a plurality of such cells and equivalents thereof known to those of skill in the art, and so forth. Similarly, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein. It should also be noted that the terms "comprising," "including," and "having" can be used interchangeably.

[0069] The term "AAV helper functions" refers to AAV-derived coding sequences (proteins) that can be expressed to provide AAV gene products and AAV particles that function in trans for productive AAV replication and packaging. Thus, AAV helper functions include AAV open reading frames (ORFs), including rep and cap, as well as others such as AAP for certain AAV serotypes. The rep gene expression product has been shown to have many functions, including, among others, recognition, binding and nicking of AAV origins of DNA replication; DNA helicase activity; and regulation of transcription from AAV (or other heterologous) promoters. The cap gene expression product (capsid) supplies the necessary packaging functions. AAV helper functions are used to complement AAV functions in trans that are missing from the AAV vector genome.

[0070] The term "about" refers to a range of + / - 20% of the numerical value that follows. In certain embodiments, the term "about" refers to a range of ±10% of the numerical value that follows. In certain embodiments, the term "about" refers to a range of ±5% of the numerical value that follows.

[0071] The term "comprising" also includes the term "consisting of."

[0072] The terms "empty capsid" and "empty particle" refer to AAV particles that have an AAV protein shell but lack all or part of the AAV ITRs, i.e., the nucleic acid that encodes a protein or is transcribed into a transcript of interest adjacent to the vector. Thus, empty capsids do not function to transfer the nucleic acid that encodes a protein or is transcribed into a transcript of interest into a host cell.

[0073] The term "endogenous" refers to something that occurs naturally within a cell; that is naturally produced by a cell; similarly, an endogenous locus / cell-specific locus is a locus that occurs naturally within a cell.

[0074] As used herein, the term "exogenous" indicates that a nucleotide sequence is not native to a particular cell, but is introduced into said cell by a DNA delivery method, such as transfection, electroporation, or transformation with a viral vector. Thus, an exogenous nucleotide sequence is an artificial sequence, which may result, for example, from a combination of subsequences of different origins (e.g., the combination of a recombinase recognition sequence with an SV40 promoter and a coding sequence for green fluorescent protein is an artificial nucleic acid), or from partial deletion or nucleic acid base mutation of a sequence (e.g., a sequence or cDNA coding only for the extracellular domain of a membrane-bound receptor). The term "endogenous" refers to a nucleotide sequence that originates from a cell. An "exogenous" nucleotide sequence may have an "endogenous" counterpart that is identical in base composition, but the sequence has become an "exogenous" sequence by introduction into the cell, for example, via recombinant DNA technology.

[0075] An "isolated" composition is a composition that is separated from one or more components of its natural environment. In some embodiments, the composition is purified to greater than 95% or 99% purity, for example, as measured by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis, CE-SDS) or chromatography (e.g., size exclusion chromatography or ion exchange or reverse phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman, S. et al., J.Chrom.B 848(2007)79-87.

[0076] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from one or more component(s) of its natural environment. Isolated nucleic acid includes a nucleic acid molecule contained in a cell that ordinarily contains the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0077] By "isolated" polypeptide or antibody is meant a polypeptide or antibody molecule that is separated from one or more component(s) of its natural environment.

[0078] The term "mammalian cell comprising an exogenous nucleotide sequence" encompasses cells into which one or more exogenous nucleic acid(s) have been introduced, including the progeny of such cells. These can be the starting point for further genetic modifications. Thus, the term "mammalian cell comprising an exogenous nucleotide sequence" encompasses cells comprising an exogenous nucleotide sequence integrated into a single site within a locus of the genome of the mammalian cell, the exogenous nucleotide sequence comprising at least one first and at least one second recombination recognition site (these recombination recognition sites are different) adjacent to at least one first selection marker. In certain embodiments, a mammalian cell comprising an exogenous nucleotide sequence is a cell comprising an exogenous nucleotide sequence integrated into a single site within a locus of the genome of the cell, the exogenous nucleotide sequence comprising a first recombination recognition sequence and a second recombination recognition sequence adjacent to at least one first selection marker, and a third recombination recognition sequence located between the first recombination recognition sequence and the second recombination recognition sequence, and the recombination recognition sequences are all different.

[0079] Both "mammalian cells containing an exogenous nucleotide sequence" and "recombinant cells" are "transfected cells." The term includes both the primary transfected cell and the progeny derived therefrom, regardless of the number of transfers. The progeny may not be completely identical to the parent cell, for example in nucleic acid content, but may contain mutations. Mutant progeny that have the same function or biological activity as the originally transfected cell are encompassed.

[0080] "Nucleic acid encoding AAV packaging proteins" generally refers to one or more nucleic acid molecules (or molecules) that contain nucleotide sequences that provide AAV functions deleted from an AAV vector used to produce transduction-competent recombinant AAV particles. Nucleic acids encoding AAV packaging proteins are commonly used to provide expression of AAV rep and / or cap genes to complement missing AAV functions required for AAV replication. However, the nucleic acid construct lacks AAV ITRs and cannot replicate or package. Nucleic acids encoding AAV packaging proteins can be in the form of a plasmid, phage, transposon, cosmid, virus or particle. Many nucleic acid constructs have been described, such as the commonly used plasmids pAAV / Ad and pIM29+45, which encode both rep and cap gene expression products. See, for example, Samulski et al., J.Virol. 63 (1989) 3822-3828; and McCarty et al., J.Virol. 65 (1991) 2936-2945. Several plasmids have been described that encode rep and / or cap gene expression products (e.g., U.S. Patent Nos. 5,139,941 and 6,376,237). Any one of these nucleic acids encoding AAV packaging proteins can comprise a DNA element or nucleic acid according to the present invention.

[0081] The term "nucleic acid encoding a helper protein" generally refers to one or more nucleic acid molecule(s) comprising a nucleotide sequence encoding a protein and / or an RNA molecule that provides an adenovirus helper function(s). A plasmid carrying a nucleic acid(s) encoding a helper protein(s) can be transfected into a suitable cell, such that the plasmid can support AAV particle production in the cell. Any one of these nucleic acids encoding a helper protein can include a DNA element or a nucleic acid according to the present invention. Naturally occurring infectious virus particles, such as adenovirus, herpesvirus or vaccinia virus particles, are specifically excluded from this term.

[0082] As used herein, the term "operably linked" refers to the juxtaposition of two or more components, where the components are in a relationship that allows them to function in their intended manner. For example, a promoter and / or enhancer is operably linked to a coding sequence / open reading frame / gene if the promoter and / or enhancer plays a role in regulating the transcription of the coding sequence / open reading frame / gene. In certain embodiments, "operably linked" DNA sequences are contiguous. In certain embodiments, when it is necessary to join the coding regions of two proteins, e.g., a secretory leader and a polypeptide, the sequences are contiguous and in the same reading frame. In certain embodiments, an operably linked promoter can be located upstream of and adjacent to the coding sequence / open reading frame / gene. In certain embodiments, for example, with respect to an enhancer sequence that regulates the expression of a coding sequence / open reading frame / gene, the two components can be operably linked, but not adjacent. An enhancer is operably linked to a coding sequence / open reading frame / gene if it increases the transcription of the coding sequence / open reading frame / gene. An operably linked enhancer can be located upstream, within, or downstream of the coding sequence / open reading frame / gene and can be located at a substantial distance from the promoter of the coding sequence / open reading frame / gene.

[0083] The term "packaging protein" refers to non-AAV derived viral and / or cellular functions that AAV depends on for its replication. Thus, this term captures the proteins and RNAs required for AAV replication, including those involved in AAV gene transcription, stage-specific AAV mRNA splicing, AAV DNA replication, Cap expression product synthesis and AAV capsid assembly activation. Viral-based accessory functions can be derived from any of the known helper viruses, such as adenovirus, herpesvirus (other than type I herpes simplex virus) and vaccinia virus.

[0084] As used herein, "AAV packaging proteins" refer to AAV-derived sequences that function in trans for productive AAV replication. Thus, AAV packaging proteins are encoded by the major AAV open reading frames (ORFs), rep and cap. The rep protein has been shown to have many functions, including, among others, recognition, binding and nicking of the AAV origin of DNA replication; DNA helicase activity; and regulation of transcription from AAV (or other heterologous) promoters. The cap (capsid) protein supplies the necessary packaging functions. AAV packaging proteins are used herein to complement AAV functions in trans that are missing from AAV vectors.

[0085] A "plasmid" is typically a form of nucleic acid or polynucleotide that has additional elements for expression (e.g., transcription, replication, etc.) or propagation (replication) of the plasmid. As used herein, plasmid can also be used to refer to such a nucleic acid or polynucleotide sequence. Thus, in all aspects, the compositions and methods of the present invention are applicable to nucleic acids, polynucleotides, and plasmids, for example, to produce cells that produce viral (e.g., AAV) vectors, to produce viral (e.g., AAV) particles, to produce cell culture media containing viral (e.g., AAV) particles, and the like.

[0086] As used herein, the term "recombinant cell" refers to a cell after final genetic modification, e.g., a cell that expresses a polypeptide of interest or produces a rAAV particle of interest, and can be used for the production of said polypeptide of interest or rAAV particle of interest on any scale. For example, a "mammalian cell containing an exogenous nucleotide sequence" that has been subjected to recombinase-mediated cassette exchange (RMCE), whereby a coding sequence for a polypeptide of interest has been introduced into the genome of the host cell, is a "recombinant cell". The cell is still capable of performing further RMCE reactions, but it is not intended to do so.

[0087] A "recombinant AAV vector" is derived from the wild-type genome of a virus, such as AAV, by using molecular biology methods to remove the wild-type genome from the virus (e.g., AAV) and replace it with a non-natural nucleic acid, such as a nucleic acid transcribed into a transcript or a nucleic acid encoding a protein. Typically, for AAV, one or both inverted terminal repeat (ITR) sequences of the wild-type AAV genome are retained in the recombinant AAV vector. A "recombinant" AAV vector is distinguished from the wild-type viral AAV genome because all or part of the viral genome has been replaced with a non-natural (i.e., heterologous) sequence with respect to the viral genome nucleic acid. Thus, the incorporation of a non-natural sequence defines a viral vector (e.g., AAV) as a "recombinant" vector, which in the case of AAV can be called a "rAAV vector."

[0088] Recombinant vectors (e.g., AAV) can be packaged and referred to herein as "particles" for subsequent infection (transduction) of cells ex vivo, in vitro or in vivo. When recombinant vector sequences are enclosed or packaged in AAV particles, the particles can also be referred to as "rAAV". Such particles contain proteins that encapsulate or package the vector genome. Particular examples include viral envelope proteins, and in the case of AAV, capsid proteins, such as AAV VP1, VP2 and VP3.

[0089] As used herein, the term "selection marker" refers to a gene that allows cells carrying the gene to be specifically selected, positively or negatively, in the presence of a corresponding selection agent. For example, but not limited to, a selection marker can allow host cells transformed with the selection marker gene to be positively selected in the presence of the respective selection agent (selective culture conditions), while untransformed host cells cannot grow or survive under the selective culture conditions. Selection markers can be positive, negative, or bifunctional. Positive selection markers can allow for the selection of cells carrying the marker, whereas negative selection markers can allow for the selective elimination of cells carrying the marker. Selection markers can confer resistance to drugs in host cells or complement metabolic or catabolic defects. In prokaryotic cells, genes that confer resistance to ampicillin, tetracycline, kanamycin, or chloramphenicol, among others, can be used. Resistance genes useful as selectable markers in eukaryotic cells include, but are not limited to, genes for aminoglycoside phosphotransferase (APH) (e.g., hygromycin phosphotransferase (HYG), neomycin, and G418 APH), dihydrofolate reductase (DHFR), thymidine kinase (TK), glutamine synthetase (GS), asparagine synthetase, tryptophan synthase (indole), histidinol dehydrogenase (histidinol D)), as well as genes encoding resistance to puromycin, blasticidin, bleomycin, phleomycin, chloramphenicol, zeocin, and mycophenolic acid. Additional marker genes are described in WO 92 / 08796 and WO 94 / 28143.

[0090] Beyond facilitating selection in the presence of a corresponding selection agent, a selection marker may alternatively be a molecule not normally present in cells, such as green fluorescent protein (GFP), enhanced GFP (eGFP), synthetic GFP, yellow fluorescent protein (YFP), enhanced YFP (eYFP), cyan fluorescent protein (CFP), mPlum, mCherry, tdTomato, mStrawberry, J-red, DsRed monomer, mOrange, mKO, mCitrine, Venus, YPet, Emerald, CyPet, mCFPm, Cerulean, and T-Sapphire. For example, cells expressing such molecules can be distinguished from cells that do not harbor this gene based on the detection or absence, respectively, of fluorescence emitted by the encoded polypeptide.

[0091] As used herein, the term "serotype" is a distinction based on serologically different AAV capsids. Serological specificity is determined based on the lack of cross-reactivity between antibodies to one AAV compared to other AAVs. Such cross-reactivity differences are usually due to differences in capsid protein sequences / antigenic determinants (e.g., due to differences in VP1, VP2 and / or VP3 sequences of AAV serotypes). AAV variants, including capsid variants, differ in at least one nucleotide or amino acid residue compared to the reference or other AAV serotypes, even though they may be serologically indistinguishable from the reference or other AAV serotypes.

[0092] Under the traditional definition, serotype means that the virus of interest has been tested against all existing and characterized serotype-specific sera for neutralizing activity, and no antibodies have been found that neutralize the virus of interest. As more naturally occurring virus isolates are discovered and / or capsid mutants are generated, they may or may not be serologically distinct from any of the currently existing serotypes. Thus, if a new virus (e.g., AAV) does not have serological differences, this new virus (e.g., AAV) is a subgroup or variant of the corresponding serotype. In many cases, serological testing for neutralizing activity has not yet been performed on mutant viruses with capsid sequence modifications to determine whether they are other serotypes according to the traditional definition of serotype. Therefore, for convenience and to avoid repetition, the term "serotype" refers broadly to both serologically distinct viruses (e.g., AAV) as well as serologically non-distinguishable viruses (e.g., AAV) that may be within a subgroup or variant of a given serotype.

[0093] The terms "transduction" and "transfect" refer to the introduction of a molecule, such as a nucleic acid (viral vector, plasmid), into a cell. A cell is "transduced" or "transfected" when an exogenous nucleic acid is introduced inside the cell membrane. Thus, a "transduced cell" is a cell into which a "nucleic acid" or "polynucleotide" has been introduced, or its progeny into which an exogenous nucleic acid has been introduced. In certain embodiments, a "transduced" cell (e.g., in a mammal, e.g., a cell or tissue or organ cell) has a genetic change following incorporation of an exogenous molecule, e.g., a nucleic acid (e.g., a transgene). The "transduced" cell(s) can be propagated and the introduced nucleic acid can be transcribed and / or a protein can be expressed.

[0094] In a "transduced" or "transfected" cell, the nucleic acid (viral vector, plasmid) may or may not be integrated into the genomic nucleic acid. When the introduced nucleic acid is integrated into the nucleic acid (genomic DNA) of the recipient cell or organism, it can be stably maintained in the cell or organism and can be passed on to or inherited by the descendant cells or organisms of the recipient cell or organism. Finally, the introduced nucleic acid can be present extrachromosomally or only transiently in the recipient cell or host organism. Several techniques are known, see for example Graham et al., Virology 52 (1973) 456; Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York; Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier; and Chu et al., Gene 13 (1981) 197. Using such techniques, one or more exogenous DNA moieties can be introduced into a suitable host cell.

[0095] The term "transgene" is used herein to conveniently refer to a nucleic acid that is intended or introduced into a cell or organism. A transgene includes any nucleic acid, e.g., a gene that is transcribed into a transcript or that encodes a polypeptide or protein.

[0096] "Vector" refers to the portion of the recombinant plasmid sequence that is ultimately packaged or enclosed, either directly or in single-stranded or RNA form, to form a viral (e.g., AAV) particle. When a recombinant plasmid is used to construct or produce a recombinant viral particle, the viral particle does not contain the portion of the "plasmid" that does not correspond to the vector sequence of the recombinant plasmid. This non-vector portion of the recombinant plasmid is called the "plasmid backbone", which is important for the cloning and amplification of the plasmid, a process necessary for propagation and recombinant viral production, but is not itself packaged or enclosed in the viral (e.g., AAV) particle. Thus, "vector" refers to the nucleic acid that is packaged or enclosed by the viral particle (e.g., AAV).

[0097] Recombinant cells Generally, for efficient and large-scale production of a proteinaceous compound of interest, such as a rAAV particle or a therapeutic polypeptide, cells are required that express and possibly secrete the proteinaceous compound. Such cells are called "recombinant cells" or "recombinant production cells."

[0098] For the generation of a "recombinant production cell", suitable mammalian cells are transfected with the necessary nucleic acid sequence encoding the proteinaceous compound of interest. Transfection of additional helper polypeptides may be required.

[0099] To generate a stable recombinant production cell, a second step follows, in which single cells stably expressing the proteinaceous compound of interest are selected. This can be done, for example, on the basis of the co-expression of a selectable marker co-transfected with the nucleic acid sequence encoding the proteinaceous compound of interest, or it can be the expression of the proteinaceous compound itself.

[0100] The expression of the coding sequence, i.e., the open reading frame, requires additional regulatory elements such as a promoter and a polyadenylation signal (sequence). The open reading frame is therefore operably linked to said additional regulatory elements for transcription. This can be achieved by incorporating it into a so-called expression cassette. The minimum control elements required for an expression cassette to be functional in a mammalian cell are a promoter functional in said mammalian cell, located upstream, i.e., 5', of the open reading frame, and a polyadenylation signal (sequence) functional in said mammalian cell, located downstream, i.e., 3', of the open reading frame. Furthermore, a terminator sequence may be present 3' of the polyadenylation signal (sequence). For expression, the promoter, the open reading frame / coding region and the polyadenylation signal sequence must be arranged in an operably linked form.

[0101] Similarly, a nucleic acid that is transcribed into a non-protein-coding RNA is called an "RNA gene". For the expression of an RNA gene, additional regulatory elements such as a promoter and a transcription termination signal or a polyadenylation signal (sequence) are also required. The nature and localization of such elements depend on the RNA polymerase intended to drive the expression of the RNA gene. Therefore, the RNA gene is usually also incorporated into an expression cassette.

[0102] If the proteinaceous compound of interest is an AAV particle, which is composed of different (monomeric) capsid polypeptides and single-stranded DNA molecules, and which also requires other adenovirus helper functions for production and encapsidation, multiple expression cassettes containing different open reading frames / coding sequences are required. In this case, at least one expression cassette is required for each of the transgenes, the different polypeptides that form the capsid of the AAV vector, and the VA RNA for the required helper functions. Thus, individual expression cassettes for each of the helper E1A, E1B, E2A, E4orf6, VA RNA, rep and cap genes are required.

[0103] As outlined in the previous paragraph, the more complex the proteinaceous compound of interest or the greater the number of additional needed helper polypeptides and / or RNAs, respectively, the greater the number of different expression cassettes required. Essentially, along with the number of expression cassettes, also the total size of the nucleic acid. However, there is a practical upper limit to the size of the transferable nucleic acid, which is in the range of about 15 kbp (kilobase pairs). Beyond this limit, handling and processing efficiency is greatly reduced. This problem can be addressed by using two or more separate plasmids. This allows different expression cassettes to be assigned to different plasmids, with each plasmid containing only a part of the expression cassette.

[0104] In certain embodiments of all aspects and embodiments, each expression cassette comprises, from 5' to 3', a promoter, an open reading frame / coding sequence or an RNA gene, and a polyadenylation signal sequence and / or a terminator sequence. In certain embodiments, the open reading frame encodes a polypeptide and the expression cassette comprises a polyadenylation signal sequence with or without an additional terminator sequence. In certain embodiments, the expression cassette comprises an RNA gene, the promoter is a type 2 Pol III promoter, and a polyadenylation signal sequence or a polyU terminator is present. See, for example, Song et al. Biochemical and Biophysical Research Communications 323 (2004) 573-578. In certain embodiments, the expression cassette comprises an RNA gene, the promoter is a type 2 Pol III promoter and a polyU terminator sequence.

[0105] In certain embodiments of all aspects and embodiments, the open reading frame encodes a polypeptide, the promoter is the human CMV promoter with or without intron A, the polyadenylation signal sequence is the bGH (bovine growth hormone) polyA signal sequence, and the terminator is hGT (human gastrin terminator).

[0106] In particular embodiments of all aspects and embodiments, the promoter is a human CMV promoter with intron A, the polyadenylation signal sequence is a bGH polyadenylation signal sequence, and the terminator is hGT; except for the expression cassette of the RNA gene and the expression cassette of the selection marker, for the selection marker, the promoter is an SV40 promoter, the polyadenylation signal sequence is an SV40 polyadenylation signal sequence, and the terminator is absent; and for the RNA gene, the promoter is a wild-type type 2 polymerase III promoter and the terminator is a polymerase II or III terminator.

[0107] Adeno-associated virus (AAV) For a general review of AAV and adenovirus or herpesvirus helper functions, see Berns and Bohensky, Advances in Virus Research, Academic Press., 32 (1987) 243-306. The genome of AAV is described in Srivastava et al., J. Virol., 45 (1983) 555-564. U.S. Patent No. 4,797,368 describes design considerations for constructing recombinant AAV vectors (see also WO 93 / 24641). Further references describing AAV vectors are West et al., Virol. 160 (1987) 38-47; Kotin, Hum. Gene Ther. 5 (1994) 793-801; and Muzyczka J. Clin. Invest. 94 (1994) 1351. Construction of recombinant AAV vectors, as described in U.S. Patent No. 5,173,414; Lebkowski et al., Mol. Cell. Biol. 8 (1988) 3988-3996; Tratschin et al., Mol. Cell. Biol. 5 (1985) 3251-3260; Tratschin et al., Mol. Cell. Biol., 4 (1994) 2072-2081; Hermonat and Muzyczka Proc. Natl. Acad. Sci. USA 81 (1984) 6466-6470; Samulski et al. J. Virol. 63 (1989) 3822-3828.

[0108] Adeno-associated virus (AAV) is a replication-deficient parvovirus. It can only replicate in cells where certain viral functions are provided by co-infecting helper viruses such as adenovirus, herpesvirus, and sometimes poxviruses such as vaccinia. Nevertheless, AAV can replicate in virtually any cell line of human, monkey or rodent origin, provided that the appropriate helper virus functions are present.

[0109] In the absence of helper virus genes, AAV establishes latency in its host cells. Its genome integrates into a specific site on chromosome 19 [(Chr)19(q13.4)], called adeno-associated virus integration site 1 (AAVS1). For certain serotypes, such as AAV-2, other integration sites have been found, e.g., on chromosome 5 [(Chr)5(p13.3)], called AAVS2, and on chromosome 3 [(Chr)3(p24.3)], called AAVS3.

[0110] AAVs are classified into different serotypes, which are assigned based on parameters such as hemagglutination, oncogenicity and DNA sequence homology. To date, more than 10 different serotypes and more than 100 sequences corresponding to different clades of AAVs have been identified.

[0111] The type and symmetry of the capsid protein determine the tissue tropism of each AAV. For example, AAV-2, AAV-4 and AAV-5 are specific to the retina, AAV-2, AAV-5, AAV-8, AAV-9 and AAVrh-10 are specific to the brain, AAV-1, AAV-2, AAV-6, AAV-8 and AAV-9 are specific to heart tissue, AAV-1, AAV-2, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9 and AAV-10 are specific to the liver, and AAV-1, AAV-2, AAV-5 and AAV-9 are specific to the lung.

[0112] Pseudotyping refers to a process involving cross-packaging of the AAV genome between various serotypes, i.e. the genome is packaged with capsid proteins of different origins.

[0113] The wild-type AAV genome has a size of about 4.7 kb. The AAV genome further contains two overlapping genes called rep and cap, which contain multiple open reading frames (see, for example, Srivastava et al., J. Viral., 45 (1983) 555-564; Hermonat et al., J. Viral. 51 (1984) 329-339; Tratschin et al., J. Virol., 51 (1984) 611-619). The open reading frame coding Rep protein provides four proteins of different sizes called Rep78, Rep68, Rep52 and Rep40. These are involved in AAV replication, rescue and integration. The open reading frame coding Cap protein provides four proteins called VP1, VP2, VP3 and AAP. VP1, VP2 and VP3 are part of the proteinaceous capsid of the AAV particle. The combined rep and cap open reading frames are flanked at their 5' and 3' ends by so-called inverted terminal repeats (ITRs). For replication, AAV requires, in addition to the Rep and Cap proteins, the products of the adenoviral genes E1A, E1B, E4orf6, E2A and VA or the corresponding factors of another helper virus.

[0114] For example, in the case of AAV serotype 2 (AAV-2), each ITR has a length of 145 nucleotides and flanks a coding sequence region of about 4470 nucleotides. Of the 145 nucleotides of the ITR, 125 nucleotides have a palindrome and can form a T-shaped hairpin structure. This structure has the function of a primer during viral replication. The remaining 20 unpaired nucleotides are shown as D sequences.

[0115] The AAV genome has three transcriptional promoters, P5, P19, and P40, for expression of the rep and cap genes (Laughlin et al., Proc. Natl. Acad. Sci. USA 76 (1979) 5567-5571).

[0116] ITR sequence must be in cis with respect to the coding region. ITR provides a functional origin of replication (ori), a signal required for integration into the genome of target cells, and efficient excision and rescue from host cell chromosomes or recombinant plasmids. ITR also contains origin of replication-like elements such as Rep protein binding site (RBS) and terminal release site (TRS). It has been found that ITR itself can have the function of a transcription promoter in AAV vectors (Flotte et al., J. Biol. Chem. 268 (1993) 3781-3790; Flotte et al., Proc. Natl. Acad. Sci. USA 93 (1993) 10163-10167).

[0117] Replication and encapsidation of the viral single-stranded DNA genome require trans -assembly of the rep and cap gene products, respectively.

[0118] The rep locus contains two internal promoters, designated P5 and P19. It contains open reading frames for four proteins. Promoter P5 is operably linked to a nucleic acid sequence that provides an unspliced ​​4.2 kb mRNA encoding the Rep protein Rep78 (a chromatin nickase for arresting the cell cycle) and a spliced ​​3.9 kb mRNA encoding the Rep protein Rep68 (a site-specific endonuclease). Promoter P19 is operably linked to a nucleic acid sequence that provides an unspliced ​​mRNA encoding the Rep protein Rep52 and a spliced ​​3.3 kb mRNA encoding the Rep protein Rep40 (a DNA helicase for accumulation and packaging).

[0119] The two larger Rep proteins, Rep78 and Rep68, are essential for AAV double-stranded DNA replication, whereas the smaller Rep proteins, Rep52 and Rep40, appear to be essential for progeny single-stranded DNA accumulation (Chejanovsky & Carter, Virology 173 (1989) 120-128).

[0120] The larger Rep proteins, Rep68 and Rep78, can specifically bind to the hairpin conformation of the AAV ITR. They exhibit certain enzymatic activities required to resolve replication at the AAV termini. Expression of Rep78 or Rep68 can be sufficient for infectious particle formation (Holscher, C., et al. J. Virol. 68 (1994) 7169-7177 and 69 (1995) 6880-6885).

[0121] All Rep proteins, mainly Rep78 and Rep68, are thought to exhibit regulatory activities such as induction and repression of AAV genes and inhibitory effects on cell proliferation (Tratschin et al., Mol. Cell. Biol. 6 (1986) 2884-2894; Labow et al., Mol. Cell. Biol., 7 (1987) 1320-1325; Khleif et al., Virology, 181 (1991) 738-741).

[0122] Recombinant overexpression of Rep78 results in a phenotype involving reduced cell proliferation due to the induction of DNA damage, which arrests host cells in S phase, thereby promoting latent infection by the virus (Berthet, C., et al., Proc. Natl. Acad. Sci. USA 102 (2005) 13634-13639).

[0123] Tratschin et al. reported that the P5 promoter is negatively autoregulated by Rep78 or Rep68 (Tratschin et al., Mol. Cell. Biol. 6 (1986) 2884-2894). Due to the toxic effects of expression of Rep proteins, only very low expression has been reported for certain cell lines after stable integration of AAV (see, for example, Mendelson et al., Virol. 166 (1988) 154-165).

[0124] The cap locus contains one promoter, called P40. Promoter P40 is operably linked to a nucleic acid sequence that provides a 2.6 kb mRNA encoding the Cap proteins VP1 (87 kDa, unspliced ​​mRNA transcript), VP2 (72 kDa from spliced ​​mRNA transcript) and VP3 (61 kDa from alternative start codons) by alternative splicing and the use of alternative start codons. VP1 to VP3 constitute the components of the viral capsid. The capsid has the function of binding to cell surface receptors and allowing intracellular transport of the virus. VP3 accounts for approximately 90% of the total virus particle protein. Nevertheless, all three proteins are essential for efficient capsid production.

[0125] It has been reported that inactivation of all three capsid proteins, VP1-VP3, prevents the accumulation of single-stranded progeny AAV DNA. Mutations in the VP1 amino terminus ("lipid negative" or "Inf negative") still allow assembly of single-stranded DNA into viral particles, thereby greatly reducing infectious titers.

[0126] The AAP open reading frame encodes the assembly activating protein (AAP), which is approximately 22 kDa in size and transports native VP proteins to the nucleolar region for capsid assembly. This open reading frame is located upstream of the VP3 protein coding sequence.

[0127] Each AAV particle contains only one single-stranded DNA molecule. This can be either the "plus" or "minus" strand. AAV viral particles containing a DNA molecule are infectious. Inside the infected cell, the parental infectious single strand is converted to a double strand and then amplified. Amplification results in a large pool of double-stranded DNA molecules from which the single strand is displaced and packaged into a capsid.

[0128] Adeno-associated virus (AAV) vectors can transduce dividing cells as well as quiescent cells. The transgene introduced into target cells using AAV vectors is expected to be expressed for a long period of time. One drawback of using AAV vectors is the size restriction of the transgene that can be introduced into cells.

[0129] Viral vectors, such as parvovirus particles, including AAV serotypes and variants thereof, provide a means of delivery of nucleic acids to cells ex vivo, in vitro and in vivo, encoding proteins so that cells express the encoded proteins. AAV is a useful virus as a gene therapy vector, since it can penetrate cells and introduce nucleic acid / genetic material so that the nucleic acid / genetic material can be stably maintained in the cells. Furthermore, these viruses can, for example, introduce nucleic acid / genetic material to specific sites. Because AAV is not associated with pathogenic diseases in humans, AAV vectors can deliver heterologous polynucleotide sequences (e.g., therapeutic proteins and drugs) to human patients without causing substantial AAV pathogenesis or disease.

[0130] Viral vectors that can be used include, but are not limited to, adeno-associated virus (AAV) particles of multiple serotypes (eg, AAV-1 through AAV-12, etc.) and hybrid / chimeric AAV particles.

[0131] AAV particles can be advantageously used as vehicles for effective gene delivery. Such particles have several desirable characteristics for such applications, including tropism for dividing and non-dividing cells. Early clinical experience with these vectors has also shown no persistent toxicity and minimal or undetectable immune responses. AAV is known to infect a wide variety of cell types in vivo and in vitro by receptor-mediated endocytosis or transcytosis. These vector systems have been tested in humans targeting retinal epithelium, liver, skeletal muscle, airways, brain, joints and hematopoietic stem cells.

[0132] Recombinant AAV particles typically do not contain viral genes related to pathogenesis.Such vectors typically have one or more of the wild-type AAV genes deleted in whole or in part, such as the rep and / or cap genes, but retain at least one functional adjacent ITR sequence as necessary for rescue, replication and packaging of the recombinant vector into AAV particles.For example, only the essential parts of the vector, such as the ITR and LTR elements, respectively, are included.Thus, the AAV vector genome will include sequences (e.g., functional ITR sequences) required in cis for replication and packaging.

[0133] Recombinant AAV vectors and their methods and uses include any virus strain or serotype.As a non-limiting example, recombinant AAV vectors can be based on any AAV genome, such as AAV-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, 2i8, AAV rh74 or AAV 7m8. Such vectors can be based on the same strain or serotype (or subgroup or variant), or can be different from each other.As a non-limiting example, recombinant AAV vectors based on one serotype genome can be identical to one or more of the capsid proteins that package the vector.Furthermore, recombinant AAV vector genomes can be based on AAV (e.g., AAV2) serotype genomes that are different from one or more of the AAV capsid proteins that package the vector. For example, the AAV vector genome can be based on AAV2, but at least one of the three capsid proteins can be, for example, AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV rh74, AAV 7m8 or a variant thereof. AAV variants include variants and chimeras of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV rh74 and AAV 7m8 capsids.

[0134] In certain embodiments of all aspects and embodiments, adeno-associated virus (AAV) vectors include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV rh74, and AAV 7m8, and variants thereof (e.g., capsid variants, e.g., amino acid insertions, additions, substitutions and deletions), as described, for example, in WO 2013 / 158879, WO 2015 / 013313, and U.S. Patent Application Publication No. 2013 / 0059732 (disclosing LK01, LK02, LK03, etc.).

[0135] AAV and AAV variant (e.g., capsid variant) serotypes (e.g., VP1, VP2 and / or VP3 sequences) may or may not be distinct from other AAV serotypes, including, for example, AAV1-AAV12 (e.g., distinct from the VP1, VP2 and / or VP3 sequences of any of the AAV1-AAV12 serotypes).

[0136] In certain embodiments of all aspects and embodiments, AAV particles related to a reference serotype have a polynucleotide, polypeptide or subsequence thereof that comprises or consists of a sequence at least 80% or more (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc.) identical to one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV rh74 or AAV 7m8 (e.g., ITR sequences, or VP1, VP2 and / or VP3 sequences, etc.).

[0137] The compositions, methods and uses of the invention include AAV sequences (polypeptide and nucleotide) and subsequences thereof that show less than 100% sequence identity to a reference AAV serotype, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV rh74, or AAV 7m8, but are different and not identical to known AAV genes or proteins, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV rh74, or AAV 7m8. In certain embodiments of all aspects and embodiments, the AAV polypeptide or subsequence thereof comprises or consists of a sequence that is at least 75% identical or more identical to any reference AAV sequence or subsequence thereof, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV rh74, or AAV 7m8 (e.g., VP1, VP2 and / or VP3 capsid or ITR), such as 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identical. In certain embodiments, the AAV variant has 1, 2, 3, 4, 5, 5-10, 10-15, 15-20 or more amino acid substitutions.

[0138] Recombinant AAV particles, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV rh74, or AAV 7m8, as well as variant, related, hybrid and chimeric sequences, can be constructed using recombinant techniques known to those of skill in the art to contain one or more nucleic acid sequences (transgenes) flanked by one or more functional AAV ITR sequences.

[0139] Recombinant particles (e.g., rAAV particles) can be incorporated into pharmaceutical compositions. Such pharmaceutical compositions are useful, inter alia, for administration and delivery to subjects in vivo or ex vivo. In certain embodiments, the pharmaceutical composition contains a pharma- ceutically acceptable carrier or excipient. Such excipients include any pharmaceutical agent that does not itself induce a harmful immune response in the individual receiving the composition and that can be administered without undue toxicity.

[0140] Protocols for the production of adenoviral vectors are described in U.S. Pat. Nos. 5,998,205; 6,228,646; 6,093,699; 6,100,242; WO 94 / 17810 and WO 94 / 23744, which are incorporated by reference in their entireties.

[0141] Specific embodiments of the method according to the invention To allow the determination of viral genome copies, the viral genome must be made accessible, i.e., the shielding capsid must be opened. For this purpose, heat denaturation is convenient and commonly used. However, it has been found that heat denaturation artificially reduces viral genome copy number.

[0142] The inventors have shown that heat denaturation at temperatures above 95°C, such as 98°C, results in a reduction in the determined viral copy genome number. This is exemplified using ATCC AAV2 standard VR-1616. The lot used in the experiment has a nominal viral genome copy number (vgcn) of 3.28 x 10E10 vg / mL. The results are shown in Table 1 below.

[0143] (Table 1) TIFF2025516117000001.tif20170

[0144] Similarly, the inventors have shown that incubation with proteinase K (PK) in aqueous or buffered solutions in the absence of detergent also results in a reduction in the determined viral copy genome number. This is exemplified using ATCC AAV2 standard VR-1616. The lot used in the experiment has a nominal viral genome copy number (vgcn) of 3.28 x 10E10 vg / mL. The results are shown in Table 2 below.

[0145] (Table 2) TIFF2025516117000002.tif38170

[0146] In combination, we have shown that heat denaturation at temperatures above 95°C, e.g. 98°C, even in combination with incubation with proteinase K (PK) in buffer in the absence of detergent, results in a reduction in the determined viral copy genome number. This is exemplified using ATCC AAV2 standard VR-1616. The lot used in the experiment has a nominal viral genome copy number (vgcn) of 3.28 x 10E10 vg / mL. The results are shown in Table 3 below.

[0147] (Table 3) TIFF2025516117000003.tif32170

[0148] We have now found that the reduction in the determined viral genome copy number can be overcome by incubation with proteinase K in the presence of sodium dodecyl sulfate (SDS). This is shown in this example using ATCC AAV2 standard VR-1616. The lot used in the experiment has a nominal viral genome copy number (vgcn) of 3.28 x 10E10 vg / mL. The results are shown in Table 4 below.

[0149] (Table 4) TIFF2025516117000004.tif32170

[0150] The recovery values ​​obtained under the conditions in Table 4 meet the acceptance criteria for validation of the assay procedure according to the EMA and FDA guidelines, i.e., within + / - 15% of the nominal value.

[0151] The present inventors further demonstrated that treatment with DNase I prior to viral genome number determination reduces the determined viral copy genome number. This is exemplified using ATCC AAV2 standard VR-1616. The lot used in the experiment has a nominal viral genome copy number (vgcn) of 3.28×10E10vg / mL. The results are shown in Table 5 below.

[0152] (Table 5) TIFF2025516117000005.tif36170

[0153] The reduction by DNase I treatment is independent of further processing of the samples, i.e. heat denaturation or proteinase K incubation. The different test conditions, all showing a reduction in the determined viral copy number, are summarized in Table 6. ATCC AAV2 standard VR-1616 was used. The lot used in the experiments has a nominal viral genome copy number (vgcn) of 3.28 x 10E10 vg / mL.

[0154] (Table 6) TIFF2025516117000006.tif117170

[0155] Our findings were confirmed using lysates of AAV2-producing HEK293 cell cultures. The established viral genome copy number (vgcn) in the lysates was 1.746×10E10vg / mL (lysate 18). The results are shown in Table 7 below.

[0156] It is found that incubation with proteinase K allows the recovery of 96% of the viral genome, which can be further increased by incubating the sample with proteinase K in the presence of SDS to 100%.

[0157] (Table 7) TIFF2025516117000007.tif74170

[0158] It should be pointed out that no further purification of the proteinase K incubated lysates was performed, ie, no column-based or extractive purification, for example.

[0159] The determined viral genome copy numbers with additional DNase I treatment only ranged from 4% to 16%, as shown in Table 8 below.

[0160] (Table 8) TIFF2025516117000008.tif92170

[0161] We found that for sequential incubation with DNase I and proteinase K, it was advantageous to use the entire incubation mixture of the DNase I incubation for the proteinase K incubation. It allowed us to increase the determined viral genome copy number to 80% of the established number. The established viral genome copy number (vgcn) in the lysates was 1.746×10E10 vg / mL (lysate 18) and 2.340×10E9 vg / mL (lysate 31). Such an effect cannot be seen in purified samples such as the ATCC standard (nominal viral genome copy number (vgcn) of 3.28×10E10 vg / mL). The results are shown in Table 9.

[0162] (Table 9) TIFF2025516117000009.tif49170

[0163] We found that greater than 80% viral genome recovery could be achieved using a combination of DNase I and proteinase K incubation without dilution using affinity chromatography purified cell lysates. The established viral genome copy numbers (vgcn) in the affinity purified lysates were 5.110×10E10 vg / mL (affinity purified lysate 31) and 7.320×10E10 vg / mL (affinity purified lysate 33). The results are shown in Table 10.

[0164] (Table 10) TIFF2025516117000010.tif36170

[0165] Viral genome quantification To allow accurate viral genome quantification, samples must be free of plasmid DNA as well as unpackaged vector genomes, and must contain at least a portion of the viral genome. Furthermore, the packaged AAV genome must be available from the first PCR cycle.

[0166] DNase I digestion is commonly used to remove unpackaged DNA that may interfere with the ddPCR process.

[0167] To make the encapsulated DNA accessible for ddPCR, capsid opening is required, which can be accomplished either by incubation at high temperature or by proteinase K digestion. The necessity of proteinase K digestion has been much debated in the art.

[0168] Droplet Digital Polymerase Chain Reaction Droplet digital PCR (ddPCR) allows absolute quantification of viral genomes without the need for the creation of a standard curve.

[0169] More specifically, droplet digital polymerase chain reaction (ddPCR) allows absolute quantification of nucleic acids by randomly distributing the PCR reaction mixture into separate compartments, some of which do not contain the nucleic acid target sequence and others of which one or more template copies are present (Hindson, B., et al., Anal. Chem. 83 (2011) 8604-8610). By distributing, thousands of independent PCR reactions are performed during thermal cycling. At the end point, the percentage of target-positive distributions is read out and used to calculate the initial template DNA concentration (Pinheiro, L., et al., Anal. Chem. 84 (2011) 1003-1011).

[0170] First, up to 20,000 droplets with a volume of about 1 nL are formed in a water-oil emulsion. A PCR reaction mixture is dispensed therein, which includes a nucleic acid template, forward (fwd) and reverse (rev) primers, TaqMan probe, and a ddPCR supermix containing Thermus aquaticus (Taq) DNA polymerase, dNTPs, and PCR buffer (see, for example, Hindson, B., et al. 2011; Taylor, S, et al., Sci. Rep. 7 (2017) 2409). In each droplet, an individual PCR reaction is carried out during thermal cycling depending on the presence or absence of a DNA target.

[0171] In droplets containing template DNA, the target sequence is amplified. During amplification, the 5' to 3' exonuclease activity of Taq polymerase hydrolyzes the TaqMan probe bound to the template strand. Due to the degradation of the probe into smaller fragments, the 5'-located fluorophore is no longer in close proximity to its 3'-located quencher. This eliminates signal quenching and generates a fluorescent signal. Partitions lacking the template sequence show no amplification of the TaqMan probe, and thus no hydrolysis and no fluorescence generation, respectively, since the fluorescence of the 5'-located fluorophore remains quenched (see, for example, Holland, P., et al., Proc. Natl. Acad. Sci. USA 88 (1991) 7276-7580). Probes with different fluorophores are available, which have different excitation and emission wavelengths, allowing ddPCR reactions to be performed as multiplex reactions within one droplet. Commonly used fluorophores for two-dimensional ddPCR are 6-carboxyfluorescein (FAM) and hexachloro-6-carboxyfluorescein (HEX), both of which are quenched by black hole quencher 1 (BHQ1) (see, e.g., Furuta-Hanawa, B., et al. Hum. Gen. Therap. Meth. 30 (2019) 127-136).

[0172] As an end-point analysis, the fluorescent signal of each droplet after thermal cycling is read out. Using Poisson statistics, the copy number (λ) of the target sequence can be calculated from the ratio of positive reads to total reads (p) according to Equation 1 (see, for example, Hindson, B., et al. (2011)). λ=-ln(1-p) (1)

[0173] Because ddPCR relies on end-point measurements, quantification of target sequences is somewhat independent of PCR reaction efficiency. This is in contrast to real-time PCR (qPCR), which is commonly used for viral genome titration (see, for example, Taylor, S, et al. (2017)). Furthermore, there is no need to use standards or calibration samples (see, for example, Dorange, F., Bec, C., Cell Gen. Therap. Ins. 4 (2018) 119-129).

[0174] Recombinant AAV particles Various methods are known in the art for producing rAAV particles. For example, transfection using AAV plasmid and AAV helper sequence in conjunction with co-infection with one AAV helper virus (e.g., adenovirus, herpesvirus, or vaccinia virus), or transfection with recombinant AAV plasmid, AAV helper plasmid, and helper function plasmid. Non-limiting methods for producing rAAV particles are described, for example, in U.S. Patent No. 6,001,650, U.S. Patent No. 6,004,797, International Publication No. WO 2017 / 096039 and International Publication No. WO 2018 / 226887. After recombinant rAAV particle production (i.e., particle production in cell culture system), rAAV particles can be obtained and purified from host cells and cell culture supernatant.

[0175] The production of recombinant AAV particles requires the expression of Rep and Cap proteins, helper proteins E1A, E1B, E2A and E4orf6, and adenovirus VA RNA in a single mammalian cell.Helper proteins E1A, E1B, E2A and E4orf6 can be expressed using any promoter, particularly the CMV IE promoter, as shown by Matsushita et al. (Gene Ther.5 (1998) 938-945).Therefore, any promoter can be used.

[0176] In general, different complementary plasmids are co-transfected into host cells to produce recombinant AAV particles. One of the plasmids contains the transgene sandwiched between two cis-acting AAV ITRs. The defective AAV elements required for replication and subsequent packaging of the progeny recombinant genome, namely the open reading frames of Rep and Cap proteins, are contained in trans on the second plasmid. Overexpression of Rep proteins leads to an inhibitory effect on cell growth (Li, J., et al., J. Virol. 71 (1997) 5236-5243). In addition, a third plasmid containing genes of the helper virus, namely E1, E4orf6, E2A and VA from adenovirus, is required for AAV replication.

[0177] To reduce the number of plasmids required, the Rep, Cap and adenoviral helper genes may be combined on a single plasmid.

[0178] Alternatively, the host cell may already stably express the E1 gene product. Such a cell is the HEK293 cell. The human embryonic kidney clone designated 293 was generated in 1977 by integrating adenovirus DNA into human embryonic kidney cells (HEK cells) (Graham, FL, et al., J. Gen. Virol. 36 (1977) 59-74). The HEK293 cell line contains base pairs 1 to 4344 of the adenovirus serotype 5 genome. It encompasses the E1A and E1B genes as well as the adenovirus packaging signal (Louis, N., et al., Virology 233 (1997) 423-429).

[0179] When using HEK293 cells, the missing E2A, E4orf6 and VA genes can be introduced by coinfection with adenovirus or by cotransfection with E2A, E4orf6 and VA expression plasmids (e.g., Samulski, RJ, et al., J. Virol. 63 (1989) 3822-3828; Allen, JM, et al., J. Virol. 71 (1997) 6816-6822; Tamayose, K., et al., Hum. Gene Ther. 7 (1996) 507-513; Flotte, TR, et al., Gene Ther. 2 (1995) 29-37; Conway, JE, et al., J. Virol. 71 (1997) 8780-8789; Chiorini, JA, et al., Hum. Gene Ther. 2 (1995) 29-37). Ther.6(1995)1531-1541;Ferrari,FK,et al.,J.Virol.70(1996)3227-3234;Salvetti,A.,et al.,Hum.Gene Ther.9(1998)695-706;Xiao,X.,et al., J. Virol. 72 (1998) 2224-2232; Grimm, D., et al., Hum. Gene Ther. 9 (1998) 2745-2760; Zhang, X., et al., Hum. Gene Ther. 10 (1999) 2527-2537). Alternatively, adenovirus / AAV or herpes simplex virus / AAV hybrid vectors can be used (see, e.g., Conway, JE, et al., J. Virol. 71 (1997) 8780-8789; Johnston, KM, et al., Hum. Gene Ther. 8 (1997) 359-370; Thrasher, AJ, et al., Gene Ther. 2 (1995) 481-485; Fisher, JK, et al., Hum. Gene Ther. 7 (1996) 2079-2087; Johnston, KM, et al., Hum. Gene Ther. 8 (1997) 359-370).

[0180] Thus, cell lines in which the rep gene is integrated and expressed tend to grow slowly or express very low levels of the Rep protein.

[0181] To restrict transgene activity to specific tissues, i.e., to limit the site of integration, the transgene can be operably linked to an inducible or tissue-specific promoter (see, e.g., Yang, Y., et al. Hum. Gene. Ther. 6 (1995) 1203-1213).

[0182] The main difficulty in producing rAAV particles is the inefficient packaging of the rAAV vector, resulting in low titers. Packaging has been difficult for several reasons, including the following: - preferred encapsidation of wild-type AAV genomes, if present; - the difficulty of generating sufficient complementation functions such as those provided by wild-type rep and cap genes due to the inhibitory effects associated with the rep gene product; -Limited efficiency of co-transfection of plasmid constructs.

[0183] All this is based on the biological properties of Rep proteins. In particular, the inhibitory (cytostatic and cytotoxic) properties of Rep proteins and their ability to reverse the immortalized phenotype of cultured cells are problematic. Furthermore, Rep proteins down-regulate their own expression when the widely used AAV P5 promoter is used (see, for example, Tratschin et al., Mol. Cell. Biol. 6 (1986) 2884-2894).

[0184] In certain embodiments of all aspects and embodiments, the rAAV particles are derived from an AAV selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, Rh.10, Rh74 and 7m8.

[0185] In certain embodiments of all aspects and embodiments, the rAAV particles comprise a capsid sequence having 70% or greater sequence identity to the capsid sequence of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, Rh.10, Rh74, or 7m8.

[0186] In certain embodiments of all aspects and embodiments, the rAAV particles comprise ITR sequences having 70% or greater sequence identity to an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10 ITR sequence.

[0187] E1A, E1B, E2, and E4 The coding sequences for E1A and E1B (open reading frames) can be derived from human adenovirus, for example, human adenovirus serotype 2 or serotype 5, among others. An exemplary sequence for human Ad5 (adenovirus serotype 5) can be found in GenBank entry X02996, AC_000008, and an exemplary sequence for human Ad2 can be found in GenBank entry AC_000007. Nucleotides 505-3522 contain the nucleic acid sequence encoding E1A and E1B of human adenovirus serotype 5. The plasmid pSTK146 reported in EP 1230354, and the plasmids pGS119 and pGS122 reported in WO 2007 / 056994 can also be used as sources of E1A and E1B open reading frames.

[0188] E1A is the first viral helper gene expressed after adenoviral DNA enters the cell nucleus. The E1A gene encodes the 12S and 13S proteins, which are based on the same E1A mRNA by alternative splicing. Expression of the 12S and 13S proteins leads to the activation of other viral functions E1B, E2, E3 and E4. Furthermore, expression of the 12S and 13S proteins pushes the cell into the S phase of the cell cycle. If only E1A-derived proteins are expressed, the cell will die (apoptosis).

[0189] E1B is the second viral helper gene expressed. It is activated by E1A-derived proteins 12S and 13S. E1B gene-derived mRNA can be spliced ​​in two different ways, resulting in a first 55 kDa transcript and a second 19 kDa transcript. The E1B 55 kDa protein is involved in regulating the cell cycle, preventing the transport of cellular mRNA in the late stages of infection, and preventing E1A-induced apoptosis. The E1B 19 kDa protein is involved in preventing E1A-induced apoptosis of cells.

[0190] The E2 genes encode different proteins: The E2A transcript encodes a single-stranded binding protein (SSBP) that is essential for AAV replication.

[0191] The E4 gene also encodes several proteins: the 34 kDa protein from the E4 gene (E4orf6), which, together with the E1B 55 kDa protein, prevents the accumulation of cellular mRNA in the cytoplasm but also promotes the transport of viral RNA from the cell nucleus to the cytoplasm.

[0192] Adenovirus VA RNA gene Virus-associated RNA (VA RNA) is a non-coding RNA of adenovirus (Ad) that regulates translation. The adenovirus genome contains two independent copies: VAI (VA RNAI) and VAII (VA RNAII). Both are transcribed by RNA polymerase III (see, for example, Machitani, M., et al., J. Contr. Rel. 154 (2011) 285-289) from a type 2 polymerase III promoter. For recombinant production, the adenovirus VA RNA gene can be driven by any promoter.

[0193] The structure, function and evolution of adenovirus-associated RNAs using a phylogenetic approach have been investigated by Ma, Y. and Mathews, MB (J. Virol. 70 (1996) 5083-5099), who provided an alignment and consensus VA RNA sequence based on 47 known human adenovirus serotypes, the disclosure of which is incorporated herein by reference in its entirety.

[0194] VA RNAs, VAI and VAII, consist of 157 to 160 nucleotides (nt).

[0195] Depending on the serotype, adenoviruses contain one or two VA RNA genes. VA RNAI is thought to play a dominant proviral role, whereas VA RNAII can partially compensate for the absence of VA RNAI (Vachon, VK and Conn, GL, Virus Res. 212 (2016) 39-52).

[0196] Although VA RNA is not essential, it plays an important role in efficient viral propagation by overcoming the cellular antiviral mechanism. That is, although VA RNA is not essential for viral propagation, VA RNA-deficient adenoviruses cannot propagate during the early stages of vector production when only a few copies of the viral genome are present per cell, possibly because viral genes other than VA RNA may not be sufficiently expressed, which blocks the cellular antiviral mechanism (see Maekawa, A., et al. Nature Sci. Rep. 3 (2013) 1136).

[0197] Maekawa, A., et al. (Nature Sci. Rep. 3 (2013) 1136) reported efficient production of adenovirus vectors lacking genes for virus-associated RNAs that disrupt the cellular RNAi machinery, in which they infected HEK293 cells that constitutively and highly express flippase recombinase to obtain VA RNA-deleted adenoviruses by FLP recombinase-mediated excision of the VA RNA locus.

[0198] Human adenovirus 2 VA RNAI corresponds to nucleotides 10586 to 10810 of the sequence of GenBank entry AC_000007. Human adenovirus 5 VA RNAI corresponds to nucleotides 10579 to 10820 of the sequence of GenBank entry AC_000008.

[0199] Methods for Producing rAAV Particles Carter et al. showed that the entire rep and cap open reading frames in the wild-type AAV genome can be deleted and replaced with a transgene (Carter, BJ, in "Handbook of Parvoviruses", ed. by P. Tijssen, CRC Press, pp. 155-168 (1990)). Furthermore, it has been reported that the ITRs must be maintained to retain the functions of replication, rescue, packaging, and integration of the transgene into the genome of the target cell.

[0200] When cells containing the respective viral helper genes are transduced by an AAV vector, or vice versa, when cells containing an integrated AAV provirus are transduced by the appropriate helper virus, the AAV provirus becomes activated and again enters the lytic infection cycle (Clark, KR, et al., Hum. Gene Ther. 6 (1995) 1329-1341; Samulski, RJ, Curr. Opin. Genet. Dev. 3 (1993) 74-80).

[0201] An embodiment of the invention is a method of transducing a cell with a nucleic acid (e.g., a plasmid) that contains all the elements necessary for the production of a recombinant AAV particle, and the viral genome copy number is determined by the method according to the invention. Thus, because the plasmid encodes viral packaging proteins and / or helper proteins, the cell is capable of producing recombinant viral particles that contain a nucleic acid encoding a protein of interest or that contain a sequence that is transcribed into a transcript of interest.

[0202] The present invention provides a viral (e.g., AAV) particle production platform that includes features that distinguish it from current "industry standard" viral (e.g., AAV) particle production processes by using methods according to the present invention.

[0203] More generally, a cell that has been transfected or transduced with DNA for recombinant production of AAV particles can be referred to as a "recombinant cell". Such cells can be, for example, yeast cells, insect cells or mammalian cells, and have been used as recipients of nucleic acids (plasmids) encoding packaging proteins, such as AAV packaging proteins, nucleic acids (plasmids) encoding helper proteins, and nucleic acids (plasmids) encoding proteins or transcribed into a transcription product of interest, i.e., a transgene, placed between two AAV ITRs. This term includes the progeny of the original cell that has been transduced or transfected. It is understood that the progeny of a single parent cell may not necessarily be completely identical in morphology or genome or total nucleic acid complement to the original parent due to natural, accidental or deliberate mutations.

[0204] A number of cell growth media suitable for maintaining cell viability or providing cell growth and / or proliferation are commercially available. Examples of such media include serum-free eukaryotic growth media, such as media for maintaining viability or media for providing mammalian (e.g., human) cell growth. Non-limiting examples include Ham's F12 or F12K medium (Sigma-Aldrich), FreeStyle (FS) F17 medium (Thermo-Fisher Scientific), MEM, DMEM, RPMI-1640 (Thermo-Fisher Scientific) and mixtures thereof. Such media can be supplemented with vitamins and / or trace minerals and / or salts and / or amino acids, such as essential amino acids for mammalian (e.g., human) cells.

[0205] Helper protein plasmids can be in the form of plasmids, phages, transposons or cosmids. In particular, it has been demonstrated that a perfect complement of the adenovirus gene is not required for helper function. For example, adenovirus mutants incapable of DNA replication and late gene synthesis have been shown to permit AAV replication. Ito et al., J. Gen. Virol. 9 (1970) 243; Ishibashi et al, Virology 45 (1971) 317.

[0206] Mutations in the E2B and E3 regions have been shown to support AAV replication, indicating that the E2B and E3 regions are probably not involved in providing helper functions. Carter et al., Virology 126 (1983) 505. However, adenoviruses with defective E1 regions or deleted E4 regions cannot support AAV replication. Thus, in the case of adenovirus helper proteins, the E1A and E4 regions are likely required for AAV replication, either directly or indirectly (see, for example, Laughlin et al., J. Virol. 41 (1982) 868; Janik et al., Proc. Natl. Acad. Sci. USA 78 (1981) 1925; Carter et al., Virology 126 (1983) 505). Other characterized adenovirus variants include E1B (Laughlin et al. (1982), supra; Janik et al. (1981), supra; Ostrove et al., Virology 104 (1980) 502); E2A (Handa et al., J. Gen. Virol. 29 (1975) 239; Strauss et al., J. Virol. 17 (1976) 140; Myers et al., J. Virol. 35 (1980) 665; Jay et al., Proc. Natl. Acad. Sci. USA 78 (1981) 2927; Myers et al., J. Biol. Chem. 256 (1981) 567); E2B (Carter, Adeno-Associated Virus Helper Functions, in I CRC Handbook of Parvoviruses (P. Tijssen ed., 1990)); E3 (Carter et al. (1983), supra); and E4 (Carter et al. (1983), supra; Carter (1995)).

[0207] Studies of helper proteins provided by adenoviruses with mutations in E1B have reported that the E1B 55kDa protein is required for AAV particle production, but the E1B 19kDa protein is not. Furthermore, WO 97 / 17458 and Matshushita et al. (Gene Therapy 5 (1998) 938-945) described helper function plasmids encoding various adenovirus genes. Examples of helper plasmids include the adenovirus VA RNA coding region, the adenovirus E4orf6 coding region, the adenovirus E2A 72kDa coding region, the adenovirus E1A coding region, and the adenovirus E1B region lacking an intact E1B 55kDa coding region (see, for example, WO 01 / 83797).

[0208] Thus, provided herein is a method for producing a recombinant AAV vector or AAV particle comprising said recombinant AAV vector comprising a nucleic acid encoding a protein or transcribed into a transcription product of interest using the method according to the invention for determining viral genome number.

[0209] One aspect of the invention is a method for producing a recombinant AAV vector comprising a nucleic acid encoding a protein or a nucleic acid that is transcribed into a transcript of interest, or an AAV particle comprising said recombinant AAV vector, comprising the steps of: (i) providing one or more plasmids comprising nucleic acids encoding AAV packaging proteins and / or nucleic acids encoding helper proteins; (ii) providing a plasmid that contains a nucleic acid encoding a protein of interest or that is to be transcribed into a transcription product of interest; (iii) contacting one or more mammalian or insect cells with the provided plasmid; (iv) further adding a transfection reagent and, optionally, incubating the plasmid / transfection reagent / cell mixture; or providing physical means, such as an electric current, to introduce the nucleic acid into the cells; (v) culturing the transfected cells; (vi) harvesting the cultured cells and / or culture medium from the cultured cells to produce a harvest of cells and / or culture medium; and (vii) lysing the cells and, optionally, isolating the recombinant AAV vector or AAV particles from the cell and / or medium harvested lysate; (viii) determining the viral genome copy number during or after step (vi) or / and step (vii) using a method according to the invention, thereby producing a recombinant AAV vector or AAV particle that contains a nucleic acid encoding a protein of interest or that is transcribed into a transcription product of interest. Includes.

[0210] One aspect of the invention is a method for producing a recombinant AAV vector comprising a nucleic acid encoding a protein or a nucleic acid that is transcribed into a transcript of interest, or an AAV particle comprising said recombinant AAV vector, comprising the steps of: (i) providing one or more plasmids comprising nucleic acids encoding AAV packaging proteins and / or nucleic acids encoding helper proteins; (ii) providing a plasmid that contains a nucleic acid encoding a protein of interest or that is to be transcribed into a transcription product of interest; (iii) (a) Stably transfected cells, - contacting one or more mammalian or insect cells with the plasmid provided in (i); - either further adding a transfection reagent and optionally incubating the plasmid / transfection reagent / cell mixture; or applying physical means such as an electric current to introduce the nucleic acid into the cells; - selecting a first stably transfected cell; - contacting the selected first stably transfected cell with the provided plasmid of (ii); and - either further adding a transfection reagent and optionally incubating the plasmid / transfection reagent / cell mixture; or applying physical means such as an electric current to introduce the nucleic acid into the cells; a step of producing the compound by any one of the following methods; (b) or transiently transfecting cells, - contacting one or more mammalian or insect cells with the provided plasmids of (i) and (ii); and - generating the nucleic acid by either further adding a transfection reagent and, optionally, incubating the plasmid / transfection reagent / cell mixture; or by applying physical means, such as an electric current, to introduce the nucleic acid into the cells; (iv) culturing the transfected cells of (iii); (v) harvesting the cultured cells and / or culture medium from the cultured cells to produce a harvest of cells and / or culture medium; (vi) lysing the cells and, optionally, isolating the recombinant AAV vector or AAV particles from the cell and / or medium harvested lysate; (vii) determining the viral genome copy number during or after step (v) or / and step (vi) using a method according to the invention, thereby producing a recombinant AAV vector or AAV particle that contains a nucleic acid encoding a protein of interest or that is transcribed into a transcription product of interest. Includes.

[0211] One aspect of the invention is a method for producing a recombinant AAV vector comprising a nucleic acid encoding a protein or a nucleic acid that is transcribed into a transcript of interest, or an AAV particle comprising said recombinant AAV vector, comprising the steps of: (i) providing a mammalian or insect cell containing nucleic acid encoding an AAV packaging protein and / or nucleic acid encoding a helper protein; (ii) providing a plasmid that contains a nucleic acid encoding a protein of interest or that is to be transcribed into a transcription product of interest; (iii) (a) Stably transfected cells, - contacting one or more mammalian or insect cells with the plasmid provided in (i); - either further adding a transfection reagent and optionally incubating the plasmid / transfection reagent / cell mixture; or applying physical means such as an electric current to introduce the nucleic acid into the cells; - selecting a first stably transfected cell; - contacting the selected first stably transfected cell with the provided plasmid of (ii); and - either further adding a transfection reagent and optionally incubating the plasmid / transfection reagent / cell mixture; or applying physical means such as an electric current to introduce the nucleic acid into the cells; a step of producing the compound by any one of the following methods; (b) or transiently transfecting cells, - contacting one or more mammalian or insect cells with the provided plasmids of (i) and (ii); and - generating the nucleic acid by either further adding a transfection reagent and, optionally, incubating the plasmid / transfection reagent / cell mixture; or by applying physical means, such as an electric current, to introduce the nucleic acid into the cells; (iv) culturing the transfected cells of (iii); (v) harvesting the cultured cells and / or culture medium from the cultured cells to produce a harvest of cells and / or culture medium; (vi) lysing the cells and, optionally, isolating and / or purifying the recombinant AAV vectors or AAV particles from the cells and / or medium harvest; and (vii) determining the viral genome copy number during or after step (v) or / and step (vi) using a method according to the invention, thereby producing a recombinant AAV vector or AAV particle that contains a nucleic acid encoding a protein of interest or that is transcribed into a transcription product of interest. Includes.

[0212] Introduction of nucleic acids (plasmids) into cells can be accomplished in several ways.

[0213] A variety of methods for DNA transfer into mammalian cells have been reported in the art. All of these are useful in the method according to the invention. In certain embodiments of all aspects and embodiments, electroporation, nucleofection or microinjection for nucleic acid transfer / transfection are used. In certain embodiments of all aspects and embodiments, inorganic substances (e.g. calcium phosphate / DNA co-precipitation, etc.), cationic polymers (e.g. polyethyleneimine, DEAE-dextran, etc.) or cationic lipids (lipofection) are used for nucleic acid transfer / transfection. Calcium phosphate and polyethyleneimine are the most commonly used reagents for transfection for nucleic acid transfer on a larger scale (see, for example, Baldi et al., Biotechnol. Lett. 29 (2007) 677-684), with polyethyleneimine being preferred.

[0214] In certain embodiments of all aspects and embodiments, the nucleic acid (plasmid) is provided as a composition in combination with polyethylenimine (PEI), optionally in combination with cells. In certain embodiments, the composition comprises a plasmid / PEI mixture having the following components: (a) one or more plasmids containing nucleic acids encoding AAV packaging proteins and / or nucleic acids encoding helper proteins; (b) a plasmid containing a nucleic acid encoding a protein or to be transcribed into a transcript of interest; (c) a polyethylenimine (PEI) solution. In certain embodiments, the plasmids are in a molar ratio range of about 1:0.01 to about 1:100, or in a molar ratio range of about 100:1 to about 1:0.01, and the mixture of components (a), (b) and (c) is optionally incubated for a period of about 10 seconds to about 4 hours.

[0215] In certain embodiments of all aspects and embodiments, the composition further comprises a cell, hi certain embodiments, the cell is in contact with the plasmid / PEI mixture of components (a), (b) and / or (c).

[0216] In certain embodiments of all aspects and embodiments, the composition further comprises free PEI, optionally in combination with the cells. In certain embodiments, the cells are in contact with free PEI.

[0217] In certain embodiments of all aspects and embodiments, the cells are contacted with the mixture of components (a), (b) and / or (c) for at least about 4 hours, or from about 4 hours to about 140 hours, or from about 4 hours to about 96 hours. In a preferred embodiment, the cells are contacted with the mixture of components (a), (b) and / or (c), and optionally free PEI, for at least about 4 hours.

[0218] The composition may include additional plasmids and / or cells. Such plasmids and cells may be in contact with free PEI. In certain embodiments, the plasmids and / or cells are in contact with free PEI for at least about 4 hours, or from about 4 hours to about 140 hours, or from about 4 hours to about 96 hours.

[0219] The present invention also provides a method for producing a transfected cell. The method includes providing one or more plasmids, preparing a solution containing polyethyleneimine (PEI), and mixing the plasmid(s) with the PEI solution to produce a plasmid / PEI mixture. In certain embodiments, such a mixture is incubated for a period ranging from about 10 seconds to about 4 hours. In such a method, cells are then contacted with the plasmid / PEI mixture to produce a plasmid / PEI cell culture, free PEI is then added to the produced plasmid / PEI cell culture to produce a free PEI / plasmid / PEI cell culture, and the produced free PEI / plasmid / PEI cell culture is then incubated for at least about 4 hours, thereby producing a transfected cell. In certain embodiments, the plasmid includes one or more or all of the rep open reading frame, cap open reading frame, E1A, E1B, E2 and E4orf6 open reading frames, and nucleic acids that encode proteins or are transcribed into transcripts of interest.

[0220] A method of producing a transfected cell that produces a recombinant AAV vector or AAV particles, comprising providing one or more plasmids comprising nucleic acids encoding AAV packaging proteins and / or nucleic acids encoding helper proteins; providing a plasmid comprising a nucleic acid that encodes a protein or is to be transcribed into a transcript of interest; providing a solution comprising polyethylenimine (PEI); and mixing the plasmid with the PEI solution to produce a plasmid / PEI mixture, wherein the plasmids are in a molar ratio range of about 1:0.01 to about 1:100, or in a molar ratio range of about 100:1 to about 1:0.01. contacting a cell with the plasmid / PEI mixture to produce a plasmid / PEI cell culture; adding free PEI to the produced plasmid / PEI cell culture to produce a free PEI / plasmid / PEI cell culture; and incubating the free PEI / plasmid / PEI cell culture for at least about 4 hours, thereby producing transfected cells that produce recombinant AAV vectors or particles that include a nucleic acid encoding a protein or that is transcribed into a transcript of interest, thereby determining viral genome copy number by a method according to the invention.

[0221] Further provided is a method of producing a recombinant AAV vector or AAV particle comprising a nucleic acid encoding a protein or to be transcribed into a transcript of interest, comprising providing one or more plasmids comprising a nucleic acid encoding an AAV packaging protein and / or a nucleic acid encoding a helper protein; providing a plasmid comprising a nucleic acid encoding a protein of interest or to be transcribed into a transcript of interest; providing a solution comprising polyethylenimine (PEI); mixing said plasmid with a PEI solution to produce a plasmid / PEI mixture, wherein the plasmids are in a molar ratio range of about 1:0.01 to about 1:100, or in a molar ratio range of about 100:1 to about 1:0.01 (and optionally incubating the plasmid / PEI mixture for a period of time ranging from about 10 seconds to about 4 hours); and incubating cells with the plasmid / PEI mixture produced as described. adding free PEI to the plasmid / PEI cell culture produced as described to produce a free PEI / plasmid / PEI cell culture; incubating the produced plasmid / PEI cell culture or the free PEI / plasmid / PEI cell culture for at least about 4 hours to produce transfected cells; harvesting the produced transfected cells and / or culture medium from the produced transfected cells to produce a cell and / or culture medium harvest; lysing the cells and optionally isolating the recombinant AAV vector or particle from the cell and / or medium harvest lysate, thereby determining the viral genome copy number in the lysate or isolated AAV particle by a method according to the invention; and thereby producing a recombinant AAV vector or particle comprising a nucleic acid encoding a protein or transcribed into a transcript of interest.

[0222] The method of producing a recombinant AAV vector or AAV particle using the method according to the invention can include one or more additional steps or features. Exemplary steps or features include, but are not limited to, harvesting the produced cultured cells and / or harvesting the culture medium from the produced cultured cells to produce a cell and / or culture medium harvest. Further exemplary steps or features include, but are not limited to, lysing the harvested cells and optionally isolating the recombinant AAV vector or AAV particle from the cell and / or culture medium harvest lysate, whereby the viral genome copy number is determined using the method according to the invention, thereby producing a recombinant AAV vector or AAV particle that includes a nucleic acid that encodes a protein or is transcribed into a transcript of interest.

[0223] In certain embodiments of all aspects and embodiments, PEI is added to the plasmid and / or cells at various times, hi certain embodiments, free PEI is added to the cells before, at the same time as, or after contacting the plasmid / PEI mixture with the cells.

[0224] In certain embodiments of all aspects and embodiments, the cells are at a particular density and / or cell growth phase and / or viability when contacted with the plasmid / PEI mixture and / or with free PEI. In a preferred embodiment, the cells are at a density ranging from about 1×10E5 cells / mL to about 1×10E8 cells / mL when contacted with the plasmid / PEI mixture and / or with free PEI. In certain embodiments, the viability of the cells when contacted with the plasmid / PEI mixture or free PEI is about 60% or more than 60%, or the cells are in logarithmic growth phase when contacted with the plasmid / PEI mixture, or the viability of the cells when contacted with the plasmid / PEI mixture or free PEI is about 90% or more than 90%, or the cells are in logarithmic growth phase when contacted with the plasmid / PEI mixture or free PEI.

[0225] In certain embodiments of all aspects and embodiments, the encoded AAV packaging proteins include AAV rep and / or AAV cap, In certain embodiments of all aspects and embodiments, such AAV packaging proteins include AAV rep and / or AAV cap proteins of any AAV serotype.

[0226] The encoded helper proteins include, in certain embodiments of all aspects and embodiments, adenovirus E1A and E1B, adenovirus E2 and / or E4, VA RNA, and / or non-AAV helper proteins.

[0227] In certain embodiments of all aspects and embodiments, the nucleic acids (plasmids) are used in certain amounts or ratios. In certain embodiments, the total amount of the plasmid containing the nucleic acid encoding the protein or to be transcribed into the transcript of interest and the one or more plasmids containing the nucleic acid encoding the AAV packaging protein and / or the nucleic acid encoding the helper protein ranges from about 0.1 μg to about 15 μg per mL of cells. In certain embodiments, the molar ratio of the plasmid containing the nucleic acid encoding the protein or to be transcribed into the transcript of interest and the one or more plasmids containing the nucleic acid encoding the AAV packaging protein and / or the nucleic acid encoding the helper protein ranges from about 1:5 to about 1:1, or from about 1:1 to about 5:1.

[0228] In certain embodiments of all aspects and embodiments, a first plasmid comprises nucleic acid encoding an AAV packaging protein and a second plasmid comprises nucleic acid encoding a helper protein.

[0229] In certain embodiments of all aspects and embodiments, the molar ratio of a plasmid containing a nucleic acid encoding a protein or to be transcribed into a transcript of interest, a first plasmid containing a nucleic acid encoding an AAV packaging protein, and a second plasmid containing a nucleic acid encoding a helper protein ranges from about 1-5:1:1, or 1:1-5:1, or 1:1:1-5 in a co-transfection.

[0230] In certain embodiments of all aspects and embodiments, the cell is a eukaryotic cell. In certain embodiments, the eukaryotic cell is a mammalian cell. In a preferred embodiment, the cell is a HEK293 cell or a CHO cell.

[0231] The culture was carried out in a temperature range of approximately 37°C, 95% humidity and 8% CO, which is commonly used for culturing eukaryotic cells. 2 The culture can be carried out using conditions of. Cultivation can be carried out in serum-containing or serum-free medium, in adherent or suspension culture. Suspension culture can be carried out in any fermentation vessel, for example stirred tank reactors, wave reactors, rocking bioreactors, shaker or spinner vessels, or so-called roller bottles. Transfection can be carried out in, for example, 96 or 384 well formats, in high throughput formats and screening, respectively.

[0232] The methods according to the invention include AAV particles of any serotype or variants thereof. In certain embodiments of all aspects and embodiments, the recombinant AAV particles comprise any of AAV serotypes 1-12, AAV VP1, VP2 and / or VP3 capsid proteins, or modified or variant AAV VP1, VP2 and / or VP3 capsid proteins, or wild-type AAV VP1, VP2 and / or VP3 capsid proteins. In certain embodiments of all aspects and embodiments, the AAV particles comprise an AAV serotype or AAV pseudotype, and the AAV pseudotype comprises an AAV capsid serotype that is different from the ITR serotype.

[0233] The method according to the present invention, which provides or includes an AAV vector or particle, can also include other elements. Examples of such elements include, but are not limited to, introns, expression control elements, one or more adeno-associated virus (AAV) inverted terminal repeats (ITRs) and / or filler / stuffer polynucleotide sequences. Such elements can be present within or adjacent to the nucleic acid that encodes a protein or is transcribed into a transcript of interest, or the expression control elements can be operably linked to the nucleic acid that encodes a protein or is transcribed into a transcript of interest, or the AAV ITR(s) can be adjacent to the 5' or 3' end of the nucleic acid that encodes a protein or is transcribed into a transcript of interest, or the filler polynucleotide sequence can be adjacent to the 5' or 3' end of the nucleic acid that encodes a protein or is transcribed into a transcript of interest.

[0234] Expression control elements include tissue-specific expression control elements or constitutive or regulatable control elements, such as promoters.

[0235] The ITRs can be any of the AAV2 or AAV6 or AAV8 or AAV9 serotypes, or combinations thereof. The AAV particles can comprise any VP1, VP2 and / or VP3 capsid protein having 75% or greater sequence identity to any of the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV10, AAV11, AAV-2i8, AAV rh74 or AAV 7m8 VP1, VP2 and / or VP3 capsid proteins, or comprise modified or variant VP1, VP2 and / or VP3 capsid proteins selected from any of the following: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV10, AAV11, AAV-2i8, AAV rh74 and AAV 7m8 AAV serotypes.

[0236] Following production of recombinant viral (e.g., AAV) particles as described herein, if desired, the viral (e.g., rAAV) particles can be purified and / or isolated from the host cells using a variety of conventional methods, including column chromatography, CsCl gradients, iodixanol gradients, and the like.

[0237] For example, multiple column purification steps can be used, such as purification by anion exchange columns, affinity columns, and / or cation exchange columns. (See, for example, WO 02 / 12455 and US 2003 / 0207439). Alternatively or additionally, iodixanol or CsCl gradient steps can be used (see, for example, US 2012 / 0135515 and US 2013 / 0072548). Furthermore, when the use of infectious viruses is used to express packaging and / or helper proteins, various methods can be used to inactivate residual virus. For example, adenovirus can be inactivated by heating to a temperature of approximately 60° C. for, for example, 20 minutes or more. This treatment effectively inactivates the helper virus, since AAV is heat stable, but the helper adenovirus is heat labile.

[0238] The objective of the rAAV vector production and purification system is to implement a strategy to minimize / control the generation of production-related impurities such as protein, nucleic acid and vector-related impurities, including wild-type / pseudo-wild-type AAV species (wtAAV) and AAV-encapsulated residual DNA impurities.

[0239] Considering that rAAV particles represent a very small fraction of the biomass, they need to be purified to a level of purity that allows them to be used as a clinical human gene therapy product (e.g., Smith PH, et al., Mo. Therapy 7 (2003) 8348; Chadeuf G., et al, Mo. Therapy 12 (2005) 744; reports from the CHMP Gene Therapy Expert Group Meeting, European Medicines Agency EMEA / CHMP 2005, 183989 / 2004).

[0240] As a first step, typically, the cultured cells that produce rAAV particles are harvested, optionally in combination with the harvested cell culture supernatant (medium) in which the cells that produce rAAV particles (suspension or adherence) were cultivated. The harvested cells and optionally the cell culture supernatant can be used as is, dissolved, or concentrated as required. Furthermore, if infection is used to express helper functions, residual helper virus can be inactivated. For example, adenovirus can be inactivated by heating to a temperature of approximately 60°C for, for example, 20 minutes or more, which inactivates only the helper virus, since AAV is heat stable while the helper adenovirus is heat unstable.

[0241] The cells and / or harvest supernatant are lysed by disrupting the cells, e.g., by chemical or physical means such as detergents, microfluidization and / or homogenization, to release the rAAV particles. During or after cell lysis, a nuclease, e.g., benzonase, is added to degrade contaminating DNA. Typically, the resulting lysate is clarified to remove cellular debris, e.g., by filtration or centrifugation, to give a clarified cell lysate. In certain instances, the lysate is filtered through a micron diameter pore size filter (e.g., a filter with a pore size of 0.1-10.0 μm, e.g., a filter with a pore size of 0.45 μm and / or 0.2 μm) to produce a clarified lysate.

[0242] The lysate (optionally clarified) contains AAV particles (including rAAV vectors and empty capsids) and production / process-related impurities, such as soluble cellular components from host cells, which may include cellular proteins, lipids and / or nucleic acids, among others, as well as cell culture medium components.The clarified lysate is then optionally subjected to a purification step to purify the AAV particles (including rAAV vectors) from impurities using chromatography.The clarified lysate can be diluted or concentrated with an appropriate buffer before the first chromatography step.

[0243] Following cell lysis, optional clarification, and optional dilution or concentration, multiple subsequent sequential chromatographic steps can be used to purify the rAAV particles.

[0244] The first chromatography step can be cation exchange chromatography or anion exchange chromatography. If the first chromatography step is cation exchange chromatography, the second chromatography step can be anion exchange chromatography or size exclusion chromatography (SEC). Thus, in certain embodiments of all aspects and embodiments, rAAV particle purification is by cation exchange chromatography, followed by purification by anion exchange chromatography.

[0245] Alternatively, if the first chromatography step is cation exchange chromatography, the second chromatography step can be size exclusion chromatography (SEC). Thus, in certain embodiments of all aspects and embodiments, rAAV particle purification is by cation exchange chromatography, followed by purification by size exclusion chromatography (SEC).

[0246] Further alternatively, the first chromatography step may be affinity chromatography. If the first chromatography step is affinity chromatography, the second chromatography step may be anion exchange chromatography. Thus, in certain embodiments of all aspects and embodiments, rAAV particle purification is by affinity chromatography, followed by purification by anion exchange chromatography.

[0247] Optionally, a third chromatography step can be added to the above chromatography steps. Typically, the optional third chromatography step follows a cation exchange, anion exchange, size exclusion or affinity chromatography.

[0248] Thus, in certain embodiments of all aspects and embodiments, rAAV particle purification is by cation exchange chromatography, followed by purification by anion exchange chromatography, followed by purification by size exclusion chromatography (SEC).

[0249] Moreover, in certain embodiments of all aspects and embodiments, further rAAV particle purification is by cation exchange chromatography, followed by purification by size exclusion chromatography (SEC), followed by purification by anion exchange chromatography.

[0250] In further embodiments of all aspects and embodiments, rAAV particle purification is by affinity chromatography, followed by purification by anion exchange chromatography, followed by purification by size exclusion chromatography (SEC).

[0251] In further embodiments of all aspects and embodiments, rAAV particle purification is by affinity chromatography, followed by purification by size exclusion chromatography (SEC), followed by purification by anion exchange chromatography.

[0252] Cation exchange chromatography serves to separate AAV particles from cellular and other components present in the lysate and / or column eluate clarified from affinity or size exclusion chromatography. Examples of strong cation exchange resins capable of binding rAAV particles over a wide pH range include any sulfonic acid-based resin as indicated by the presence of sulfonate functional groups, including, but not limited to, aryl and alkyl substituted sulfonates such as sulfopropyl or sulfoethyl resins. Representative matrices include, but are not limited to, POROS HS, POROS HS 50, POROS XS, POROS SP, and POROS S (strong cation exchangers available from Thermo Fisher Scientific, Inc., Waltham, MA, USA). Further examples include Capto S, Capto S ImpAct, Capto S ImpRes (strong cation exchangers available from GE Healthcare, Marlborough, MA, USA), and the commercially available DOWEX®, AMBERLITE®, and AMBERLYST® resin families available from Aldrich Chemical Company, Milliwaukee, WI, USA. Weak cation exchange resins include, but are not limited to, any carboxylic acid-based resin. Exemplary cation exchange resins include carboxymethyl (CM), phospho (based on phosphate functional groups), methyl sulfonate (S) and sulfopropyl (SP) resins.

[0253] Anion exchange chromatography serves to separate AAV particles from proteins, cellular components and other components present in the lysate and / or column eluate clarified from affinity or cation exchange or size exclusion chromatography. Anion exchange chromatography can also be used to reduce and thereby control the amount of empty capsids in the eluate. For example, an anion exchange column with bound rAAV particles can be washed with a solution containing a moderate concentration (e.g., about 100-125 mM, e.g., 110-115 mM) of NaCl, and a portion of the empty capsids can be eluted in the flow-through without substantially eluting the rAAV particles. The rAAV particles bound to the anion exchange column can then be eluted with a solution containing a higher concentration (e.g., about 130-300 mM NaCl) of NaCl to produce a column eluate with reduced or depleted amounts of empty capsids and a proportionally increased amount of rAAV vector-containing rAAV particles.

[0254] Exemplary anion exchange resins include, but are not limited to, those based on polyamine resins and other resins. Examples of strong anion exchange resins include those that are generally based on quaternized nitrogen atoms, including, but are not limited to, quaternary ammonium salt resins such as trialkylbenzylammonium resins. Suitable exchange chromatography materials include, but are not limited to, MACRO PREP Q (strong anion exchanger available from BioRad, Hercules, CA, USA); UNOSPHERE Q (strong anion exchanger available from BioRad, Hercules, CA, USA); POROS 50HQ (strong anion exchanger available from Applied Biosystems, Foster City, CA, USA); POROS XQ (strong anion exchanger available from Applied Biosystems, Foster City, CA, USA); POROS SOD (weak anion exchanger available from Applied Biosystems, Foster City, CA, USA); POROS 50PI (weak anion exchanger available from Applied Biosystems, Foster City, CA, USA); Capto Q, Capto XQ, Capto Q ImpRes, and SOURCE 30Q (GE Examples of anion exchange resins include: DEAE Sepharose (a strong anion exchanger available from Amersham Healthcare, Marlborough, MA, USA); DEAE Sepharose (a weak anion exchanger available from Amersham Biosciences, Piscataway, NJ, USA); Q Sepharose (a strong anion exchanger available from Amersham Biosciences, Piscataway, NJ, USA). Further exemplary anion exchange resins include aminoethyl (AE), diethylaminoethyl (DEAE), diethylaminopropyl (DEPE) and quaternary aminoethyl (QAE).

[0255] A manufacturing process for purifying recombinant AAV particles intended as a product for treating human disease should achieve the following objectives: 1) consistent particle purity, potency and safety; 2) scalability of the manufacturing process; and 3) acceptable production costs.

[0256] An exemplary process for recombinant AAV particle purification is reported in WO 2019 / 006390.

[0257] The recombinant adeno-associated viral particle (rAAV particle) purification and production methods outlined below are scalable to large scale, e.g., up to 5, 10, 10-20, 20-50, 50-100, 100-200 or more liter volumes of suspension cultures. The recombinant adeno-associated viral particle purification and production methods are applicable to a wide variety of AAV serotypes / capsid variants.

[0258] In certain embodiments of all aspects and embodiments, purification of rAAV particles comprises: (a) harvesting the cell culture supernatant containing the cells and / or rAAV particles to produce a harvest; (b) optionally concentrating the harvest produced in step (a) to produce a concentrated harvest; (c) lysing the harvest produced in step (a) or the concentrated harvest produced in step (b) to produce a lysate; (d) treating the lysate produced in step (c) to reduce contaminating nucleic acids in the lysate, thereby producing a nucleic acid-reduced lysate; (e) optionally filtering the nucleic acid reduced lysate produced in step (d) to produce a clarified lysate, and optionally diluting the clarified lysate to produce a diluted clarified lysate; (f) subjecting the nucleic acid reduced lysate of step (d), the clarified lysate of step (e), or the diluted clarified lysate produced in step (e) to cation exchange column chromatography to produce a column eluate comprising rAAV particles, thereby separating the rAAV particles from protein impurities or other production / process related impurities, and optionally diluting the column eluate to produce a diluted column eluate; (g) subjecting the column eluate or diluted column eluate produced in step (f) to anion exchange chromatography to produce a second column eluate comprising the rAAV particles, thereby separating the rAAV particles from protein impurities or production / process related impurities, and optionally concentrating the second column eluate to produce a concentrated second column eluate; (h) subjecting the second column eluate or the concentrated second column eluate produced in step (g) to size exclusion column chromatography (SEC) to produce a third column eluate comprising rAAV particles, thereby separating the rAAV particles from protein impurities or production / process related impurities, and optionally concentrating the third column eluate to produce a concentrated third column eluate; and (i) filtering the third column eluate or the concentrated third column eluate produced in step (h), thereby producing purified rAAV particles. Including, The viral genome copy number is determined using a method according to the invention at or after one or more of steps (a) to (i).

[0259] In certain embodiments, steps (a)-(f) are maintained and include the steps of: (g) subjecting the column eluate or the concentrated column eluate produced in step (f) to size exclusion column chromatography (SEC) to produce a second column eluate comprising the rAAV particles, thereby separating the rAAV particles from protein impurities or other production / process related impurities, and optionally diluting the second column eluate to produce a concentrated second column eluate; (h) subjecting the second column eluate produced in step (g) or the diluted second column eluate to anion exchange chromatography to produce a third column eluate comprising rAAV particles, thereby separating the rAAV particles from production / process-related protein impurities, and optionally diluting the third column eluate to produce a diluted third column eluate; and (i) filtering the third column eluate or the concentrated third column eluate produced in step (h), thereby producing purified rAAV particles. Combined with The viral genome copy number is determined using a method according to the invention at or after one or more of steps (a) to (i).

[0260] In certain embodiments, steps (a)-(g) are maintained and include the steps of: (h) filtering the second column eluate or the concentrated second column eluate produced in step (g), thereby producing purified rAAV particles. Combined with The viral genome copy number is determined using a method according to the invention at or after one or more of steps (a) to (h).

[0261] In an embodiment, steps (a) to (e) are maintained and the following steps are performed: (f) subjecting the nucleic acid reduced lysate of step (d), or the clarified lysate or diluted clarified lysate produced in step (e), to AAV affinity chromatography to produce a column eluate comprising rAAV particles, thereby separating the rAAV particles from protein impurities or other production / process related impurities, and optionally concentrating the column eluate to produce a concentrated column eluate; (g) subjecting the column eluate or concentrated column eluate produced in step (f) to size exclusion column chromatography (SEC) to produce a second column eluate comprising the rAAV particles, thereby separating the rAAV particles from protein impurities or other production / process related impurities, and optionally diluting the second column eluate to produce a diluted second column eluate; (h) optionally, subjecting the second column eluate produced in step (g) or the diluted second column eluate to anion exchange chromatography to produce a third column eluate comprising rAAV particles, thereby separating the rAAV particles from protein impurities or other production / process related impurities, and optionally diluting the third column eluate to produce a diluted third column eluate; and (i) filtering the second column eluate or the diluted second column eluate produced in step (g), or filtering the third column eluate or the concentrated third column eluate produced in step (h), thereby producing purified rAAV particles. Combined with The viral genome copy number is determined using a method according to the invention at or after one or more of steps (a) to (i).

[0262] In certain embodiments of all aspects and embodiments, the concentration in step (b) and / or step (f) and / or step (g) and / or step (h) is by ultrafiltration / diafiltration, such as tangential flow filtration (TFF).

[0263] In certain embodiments of all aspects and embodiments, the concentration in step (b) reduces the volume of the recovered cells and cell culture supernatant by about 2-20 fold.

[0264] In certain embodiments of all aspects and embodiments, the concentration in step (f) and / or step (g) and / or step (h) reduces the volume of the column eluate by about 5-20 fold.

[0265] In certain embodiments of all aspects and embodiments, lysis of the harvest produced in step (a) or the concentrated harvest produced in step (b) is by physical or chemical means. Non-limiting examples of physical means include microfluidization and homogenization. Non-limiting examples of chemical means include detergents. Detergents include non-ionic detergents and ionic detergents. Non-limiting examples of non-ionic detergents include Triton X-100. Non-limiting examples of detergent concentrations are about 0.1-1.0% (v / v) or (w / v), inclusive.

[0266] In certain embodiments of all aspects and embodiments, step (d) comprises treating with a nuclease, thereby reducing contaminating nucleic acids. Non-limiting examples of nucleases include benzonase.

[0267] In certain embodiments of all aspects and embodiments, the filtration of the clarified lysate or diluted clarified lysate in step (e) is through a filter. Non-limiting examples of filters are those having a pore size of about 0.1 microns to 10.0 microns, inclusive.

[0268] In certain embodiments of all aspects and embodiments, the dilution of the clarified lysate in step (e) is with a buffered phosphate, acetate or Tris aqueous solution. Non-limiting examples of solution pH are from about pH 4.0 to pH 7.4, inclusive. Non-limiting examples of Tris solution pH are above pH 7.5, such as from about pH 8.0 to pH 9.0, inclusive.

[0269] In certain embodiments of all aspects and embodiments, the dilution of the column eluate in step (f) or the second column eluate in step (g) is with a buffered phosphate, acetate or Tris aqueous solution. Non-limiting examples of the solution pH are about pH 4.0 to pH 7.4, inclusive. Non-limiting examples of the Tris solution pH are greater than pH 7.5, such as about pH 8.0 to pH 9.0, inclusive.

[0270] In certain embodiments of all aspects and embodiments, the rAAV particles resulting from step (i) are formulated with a surfactant to produce an rAAV particle formulation.

[0271] In certain embodiments of all aspects and embodiments, the anion exchange column chromatography of steps (f), (g) and / or (h) comprises polyethylene glycol (PEG) conditioned column chromatography.

[0272] In certain embodiments of all aspects and embodiments, the anion exchange column chromatography of steps (g) and / or (h) is washed with a PEG solution prior to elution of the rAAV particles from the column.

[0273] In certain embodiments of all aspects and embodiments, the PEG has an average molecular weight in the range of about 1,000 g / mol to 80,000 g / mol, inclusive.

[0274] In certain embodiments of all aspects and embodiments, the PEG is at a concentration of about 4% to about 10% (w / v), inclusive.

[0275] In certain embodiments of all aspects and embodiments, the anion exchange column in steps (g) and / or (h) is washed with an aqueous detergent solution prior to elution of the rAAV particles from the column.

[0276] In certain embodiments of all aspects and embodiments, the cation exchange column in step (f) is washed with a detergent solution prior to elution of the rAAV particles from the column.

[0277] In certain embodiments of all aspects and embodiments, the PEG solution and / or surfactant solution comprises an aqueous Tris-HCl / NaCl buffer, an aqueous phosphate / NaCl buffer, or an aqueous acetate / NaCl buffer.

[0278] In certain embodiments of all aspects and embodiments, the NaCl concentration in the buffer or solution is within the range of about 20-300 mM NaCl, inclusive, or about 50-250 mM NaCl, inclusive.

[0279] In certain embodiments of all aspects and embodiments, the surfactant comprises a cationic or anionic surfactant.

[0280] In certain embodiments of all aspects and embodiments, the surfactant comprises a 12 carbon chain surfactant.

[0281] In certain embodiments of all aspects and embodiments, the surfactant comprises dodecyltrimethylammonium chloride (DTAC) or sarkosyl.

[0282] In certain embodiments of all aspects and embodiments, the rAAV particles are eluted from the anion exchange column of steps (f), (g) and / or (h) with an aqueous Tris-HCl / NaCl buffer.

[0283] In certain embodiments of all aspects and embodiments, the Tris-HCl / NaCl buffer comprises 100 to 400 mM NaCl, inclusive, optionally at a pH ranging from about pH 7.5 to about pH 9.0, inclusive.

[0284] In certain embodiments of all aspects and embodiments, the anion exchange column of steps (f), (g) and / or (h) is washed with an aqueous Tris-HCl / NaCl buffer.

[0285] In certain embodiments of all aspects and embodiments, the NaCl concentration in the aqueous Tris-HCl / NaCl buffer is within the range of about 75 to 125 mM, inclusive.

[0286] In certain embodiments of all aspects and embodiments, the aqueous Tris-HCl / NaCl buffer has a pH of about pH 7.5 to about pH 9.0, inclusive.

[0287] In certain embodiments of all aspects and embodiments, the anion exchange column of steps (f), (g) and / or (h) is washed one or more times to reduce the amount of empty capsids in the second or third column eluate.

[0288] In certain embodiments of all aspects and embodiments, an anion exchange column wash removes empty capsids from the column prior to and / or in lieu of rAAV particle elution, thereby reducing the amount of empty capsids in the second or third column eluate.

[0289] In certain embodiments of all aspects and embodiments, the anion exchange column wash removes at least about 50% of the total empty capsids from the column prior to and / or in lieu of rAAV particle elution, thereby reducing the amount of empty capsids in the second or third column eluate by about 50%.

[0290] In certain embodiments of all aspects and embodiments, the NaCl concentration in the aqueous Tris-HCl / NaCl buffer is within the range of about 110 to 120 mM, inclusive.

[0291] In certain embodiments of all aspects and embodiments, the ratio and / or amount of eluted rAAV particles and empty capsids is controlled by the wash buffer.

[0292] In certain embodiments of all aspects and embodiments, the rAAV particles are eluted from the cation exchange column in step (f) in an aqueous phosphate / NaCl buffer or an aqueous acetate / NaCl buffer. Non-limiting NaCl concentrations in the buffer range from about 125 to 500 mM NaCl, inclusive. Non-limiting examples of buffer pHs are from about pH 5.5 to about pH 7.5, inclusive.

[0293] In certain embodiments of all aspects and embodiments, the anion exchange column of steps (f), (g) and / or (h) comprises a quaternary ammonium functional group, such as a quaternized polyethyleneimine.

[0294] In certain embodiments of all aspects and embodiments, the size exclusion column (SEC) of steps (g) and / or (h) has a separation / fractionation range (molecular weight) of about 10,000 g / mol to about 600,000 g / mol, inclusive.

[0295] In certain embodiments of all aspects and embodiments, the cation exchange column in step (f) contains a functional group such as sulfonic acid or sulfopropyl.

[0296] In certain embodiments of all aspects and embodiments, the AAV affinity column comprises a protein or ligand that binds to AAV capsid protein. Non-limiting examples of proteins include antibodies that bind to AAV capsid protein. More specific non-limiting examples include single chain llama antibodies (Camelid) that bind to AAV capsid protein.

[0297] In certain embodiments of all aspects and embodiments, the method excludes the step of cesium chloride gradient ultracentrifugation.

[0298] In certain embodiments of all aspects and embodiments, the method recovers approximately 50-90% of all rAAV particles from the harvest produced in step (a) or the concentrated harvest produced in step (b).

[0299] In certain embodiments of all aspects and embodiments, the methods produce rAAV particles having greater purity than rAAV particles produced or purified by a single AAV affinity column purification.

[0300] In certain embodiments of all aspects and embodiments, steps (c) and (d) are performed substantially simultaneously.

[0301] In certain embodiments of all aspects and embodiments, the NaCl concentration is adjusted after step (c) but before step (f) to be in the range of about 100-400 mM NaCl, inclusive, or in the range of about 140-300 mM NaCl, inclusive.

[0302] In certain embodiments of all aspects and embodiments, the cells are suspension grown cells or adherent grown cells.

[0303] In certain embodiments of all aspects and embodiments, the cell is a mammalian cell. Non-limiting examples include HEK cells, such as HEK-293 cells, and CHO cells, such as CHO-K1 cells.

[0304] Methods for determining the infectious titer of rAAV particles containing a transgene are known in the art (see, e.g., Zhen et al., Hum. Gene Ther. 15 (2004) 709). Methods for assaying empty capsids and transgene-packaged rAAV particles are known (see, e.g., Grimm et al., Gene Therapy 6 (1999) 1322-1330; Sommer et al., Malec. Ther. 7 (2003) 122-128).

[0305] To determine the presence or amount of disassembled / denatured capsid, purified rAAV particles are subjected to SDS-polyacrylamide gel electrophoresis consisting of any gel capable of separating the three capsid proteins, such as a gradient gel, and then the gel is run until the sample is separated, and the gel can be blotted onto a nylon or nitrocellulose membrane. An anti-AAV capsid antibody is then used as a primary antibody that binds to the denatured capsid protein (see, for example, Wobus et al., J. Viral. 74 (2000) 9281-9293). A secondary antibody that binds to the primary antibody includes a means to detect the primary antibody. The binding between the primary antibody and the secondary antibody is detected semi-quantitatively to determine the amount of capsid. Another method is analytical HPLC using a SEC column or an analytical ultracentrifuge.

[0306] In addition to the various embodiments depicted and claimed, the subject matter of the present disclosure is also directed to other embodiments having other combinations of the features disclosed and claimed herein. Thus, the specific features presented herein may be combined with each other in other ways within the scope of the subject matter of the present disclosure, such that the subject matter of the present disclosure includes any suitable combination of the features disclosed herein. The foregoing description of specific embodiments of the subject matter of the present disclosure is presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the subject matter of the present disclosure to the disclosed embodiments.

[0307] All references mentioned herein are hereby incorporated by reference.

[0308] The following examples are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit or scope of the invention. EXAMPLES

[0309] General Technology 1) Recombinant DNA Technology Standard methods were used to manipulate DNA as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1989). Molecular biology reagents were used according to the manufacturer's instructions.

[0310] 2) DNA and protein sequence analysis and sequence data management The EMBOSS (European Molecular Biology Open Software Suite) software packages, Vector NTI and Geneious Prime from Invitrogen, are used for creating, mapping, analyzing, annotating and illustrating the sequences.

[0311] 3) Gene and oligonucleotide synthesis The desired gene segments are prepared by chemical synthesis at Geneart GmbH (Regensburg, Germany). The synthesized gene fragments are cloned into E. coli plasmids for propagation / amplification. The DNA sequences of the subcloned gene fragments are confirmed by DNA sequencing. Alternatively, short synthetic DNA fragments are constructed by annealing chemically synthesized oligonucleotides or via PCR. The respective oligonucleotides are prepared by metabion GmbH (Planeg-Martinsried, Germany).

[0312] 4) Reagents Unless otherwise stated, all commercially available chemicals, antibodies, and kits are used as provided and according to manufacturer's protocols.

[0313] 5) Cloning general For plasmids, a restriction enzyme-mediated cloning strategy was used. By choosing the appropriate restriction enzyme, the desired gene of interest can be excised and then inserted into another plasmid by ligation. Therefore, it is preferable to use an enzyme that cuts at a multiple cloning site (MCS), selected in a smart way to ensure that ligation of the fragments in the correct array can be performed. If the plasmid and the fragment have been previously cut with the same restriction enzyme, the sticky ends of the fragment and the plasmid will match perfectly and can then be ligated with DNA ligase. After ligation, competent E. coli cells are transformed with the newly created plasmid.

[0314] Cloning by restriction digestion For digestion of the plasmid with restriction enzymes, pipette the following components together on ice:

[0315] Table 11. Restriction digestion reaction mix TIFF2025516117000011.tif32128

[0316] If more enzymes are used in one digestion, use 1 µL of each enzyme and adjust the volume by adding more or less PCR-grade water. All enzymes are selected with the prerequisite that they are qualified for use in New England Biolabs' CutSmart buffer (100% activity) and at the same incubation temperature (all at 37 °C).

[0317] Incubation is performed using a thermomixer or thermal cycler, allowing the samples to incubate at a constant temperature (37°C). The samples are not stirred during incubation. Set the incubation time to 60 min. The samples are then mixed directly with loading dye and loaded onto an agarose electrophoresis gel or stored at 4°C / on ice for further use.

[0318] A 1% agarose gel is prepared for gel electrophoresis. To do so, 1.5 g of all-purpose agarose is weighed into a 125 Erlenmeyer shake flask and filled with 150 mL of TAE buffer. The mixture is heated in a microwave oven until the agarose is completely dissolved. 0.5 μg / mL of ethidium bromide is added to the agarose solution. The gel is then poured into a mold. After the agarose has solidified, the mold is placed into the electrophoresis chamber and the chamber is filled with TAE buffer. The samples are then loaded. (From the left) In the first pocket, the appropriate DNA molecular weight marker is loaded, followed by the sample. The gel is run for approximately 60 minutes at less than 130 V. After electrophoresis, the gel is removed from the chamber and analyzed with a UV-Imager.

[0319] The target band is excised and transferred to a 1.5 mL Eppendorf tube. For gel purification, use Qiagen's QIAquick Gel Extraction Kit according to the manufacturer's instructions. The DNA fragment is stored at -20°C for further use.

[0320] The fragments for ligation are pipetted together in a molar ratio of 1:2, 1:3, or 1:5 plasmid to insert, depending on the length of the insert and plasmid fragments and their relationship to each other. If the fragment that needs to be inserted into the plasmid is short, use a ratio of 1:5. The longer the insert, the lesser the amount of insert used in relationship to the plasmid. Use a plasmid amount of 50 ng for each ligation and calculate the specific insert amount with the NEBioCalculator. For the ligations, use the T4 DNA Ligation Kit from NEB. An example of a ligation mixture is shown in Table 12 below.

[0321] Table 12. Ligation reaction mix TIFF2025516117000012.tif45151

[0322] Starting with the mixing of DNA and water, then the addition of buffer, and finally the addition of enzyme, pipette all components together on ice. Mix the reaction gently by pipetting up and down, microcentrifuge briefly, then incubate at room temperature for 10 minutes. After incubation, heat inactivate the T4 ligase at 65°C for 10 minutes. Chill the sample on ice. In the final step, transform 10 beta-competent E. coli cells with 2 μL of the ligated plasmid (see below).

[0323] Transform 10 beta-competent E. coli cells For transformation, thaw 10 beta-competent E. coli cells on ice. Then pipette 2 μl of plasmid DNA directly into the cell suspension. Flick the tube and place on ice for 30 min. Then, place the cells in a 42 °C thermal block and heat shock them for exactly 30 s. Immediately after, cool the cells on ice for 2 min. Add 950 μL of NEB10 beta growth medium to the cell suspension. Incubate the cells at 37 °C for 1 h with shaking. Then, pipette 50-100 μL onto pre-warmed (37 °C) LB-Amp agar plates and spread with a disposable spatula. Incubate the plates overnight at 37 °C. Only bacteria that have successfully integrated the plasmid and carry a resistance gene to ampicillin are able to grow on these plates. The next day, pick single colonies and culture them in LB-Amp medium for subsequent plasmid preparation.

[0324] bacterial culture Culture the E. coli in LB medium, short for Luria Bertani, and add 1 mL / L of 100 mg / mL ampicillin to give an ampicillin concentration of 0.1 mg / mL. Inoculate the following volumes with a single bacterial colony for the different plasmid preparation volumes:

[0325] Table 13. Volume of E. coli culture TIFF2025516117000013.tif30164

[0326] For the Mini-Prep, fill a 96-well 2 mL deep-well plate with 1.5 mL of LB-Amp medium per well. Pick up a colony and press a toothpick into the medium. Once all colonies have been picked, close the plate with an adhesive air-porous membrane. Incubate the plate in a 37 °C incubator with a shaking speed of 200 rpm for 23 h.

[0327] For the Mini-Prep, fill a 15 mL tube (with ventilated lid) with 3.6 mL of LB-Amp medium and inoculate evenly with a bacterial colony. Do not remove the toothpick but leave it in the tube during incubation. Incubate the tube at 37 °C and 200 rpm for 23 h, similar to the 96-well plate.

[0328] For Maxi-Prep, fill an autoclaved glass 1 L Erlenmeyer flask with 200 mL of LB-Amp medium and inoculate it with 1 mL of a ~5 h old day culture of bacteria. Close the Erlenmeyer flask with a paper stopper and incubate at 37 °C, 200 rpm for 16 h.

[0329] Plasmid preparation For the Mini-Prep, 50 μL of the bacterial suspension is transferred to a 1 mL deep-well plate. The bacterial cells are then centrifuged in the plate at 3000 rpm for 5 min at 4 °C. The supernatant is removed and the plate with the bacterial pellet is placed in the EpMotion. After approximately 90 min, the run is complete and the eluted plasmid DNA can be removed from the EpMotion for further use.

[0330] For the Mini-Prep, remove the 15 mL tube from the incubator and split 3.6 mL of bacterial culture into two 2 mL Eppendorf tubes. Centrifuge the tubes at 6,800 x g in a tabletop microcentrifuge for 3 min at room temperature. Then perform a Mini-Prep using a Qiagen QIAprep Spin Miniprep kit according to the manufacturer's instructions. Measure the plasmid DNA concentration with a Nanodrop.

[0331] Maxi-Prep is performed using the Macherey-Nagel NucleoBond® Xtra Maxi EF kit according to the manufacturer's instructions. DNA concentration is measured with Nanodrop.

[0332] Ethanol precipitation Mix the volume of the DNA solution with 2.5 volumes of ethanol 100%. Incubate the mixture at -20°C for 10 minutes. Then centrifuge the DNA at 14,000 rpm for 30 minutes at 4°C. Carefully remove the supernatant and wash the pellet with 70% ethanol. Again, centrifuge the tube at 14,000 rpm for 5 minutes at 4°C. Carefully remove the supernatant by pipetting and dry the pellet. Once the ethanol has evaporated, add an appropriate amount of endotoxin-free water. Allow the DNA time to redissolve in water overnight at 4°C. Take a small aliquot and measure the DNA concentration with a Nanodrop instrument.

[0333] Composition of the expression cassette For expression of the open reading frame, a transcription unit is used which contains at least the following functional elements: -promoter, - a nucleic acid comprising the respective open reading frame, optionally including a signal sequence, -Polyadenylation signal sequence.

[0334] In addition to the expression unit / cassette containing the desired gene to be expressed, a basic / standard mammalian expression plasmid contains: - the origin of replication from the plasmid pUC18, allowing replication of this plasmid in E. coli, and -The beta-lactamase gene that confers ampicillin resistance to E. coli Includes.

[0335] 6)Cell culture technology Standard cell culture techniques are used as described in Current Protocols in Cell Biology (2000), Bonifacino, JS, Dasso, M., Harford, JB, Lippincott-Schwartz, J. and Yamada, KM (eds.), John Wiley & Sons, Inc.

[0336] Transient transfection in HEK293 Cells producing recombinant AAV particles were generated by transient transfection with the respective plasmids using the HEK293 system (Invitrogen, now Thermo Scientific) according to the manufacturer's instructions. Briefly, HEK293 cells (Invitrogen) grown in suspension in serum-free FreeStyle™ 293 Expression Medium (Invitrogen) in either shake flasks or stirred fermenters are transfected with a mix of the respective plasmids and 293fectin™ or fectin (Invitrogen). HEK293 cells were cultured at 1×10 in 600 mL in 2 L shake flasks (Corning). 6 Seed cells at a density of 1000 cells / mL and incubate at 120 rpm and 8% CO 2 Incubate at 20°C for 10 min at 37°C. At a later date, approximately 1.5 x 10 6 Transfect cells at a cell density of 1000 cells / mL with A) 20 mL of Opti-MEM (Invitrogen) containing a total of 600 µg of plasmid DNA (1 µg / mL) as well as B) a mix of 20 mL of Opti-MEM + 1.2 mL of 293fectin or fectin (2 µL / mL) in approximately 42 mL of 293fectin. Add glucose solution during the course of fermentation according to glucose consumption.

[0337] Example 1 No pretreatment procedure: -Mix 10 μL sample with 90 μL H2O Incubate at -95 °C for 15 min. Working Example: 1. Processing the sample 2. Prepare PCR Master Mix 3. Add master mix to plate 4. Prepare a 1:10 dilution in water 5. Add template to plate 6. Seal the plate, vortex (2.200 rpm for 1 minute), and centrifuge. 7. Droplet formation using an automated droplet generator (Auto-DG) (20 µL final mixture + 70 µL oil) and transfer to the plate (42 µL) 8. Seal the plate and start the PCR run

[0338] Example 2 Thermal denaturation procedure: - Incubate the sample at 98°C for 10 minutes Working Example: 1. Heat Denaturation 2. Prepare PCR Master Mix 3. Add master mix to plate 4. Prepare a 1:10 dilution in water 5. Add template to plate 6. Seal the plate, vortex (2.200 rpm for 1 minute), and centrifuge. 7. Droplet formation using Auto-DG (20 µL final mixture + 70 µL oil) and transfer to plate (42 µL) 8. Seal the plate and start the PCR run

[0339] Example 3 DNase I digestion; reagent: 1) DNase I buffer (Promega): 400mM Tris-HCl, pH8, 100mM MgSO 4 , 10 mM CaCl 2 2) DNase I (Promega): 50 U / mL diluted to 1 U / μL Procedure (50 μL reaction volume): - Mix 30 μL HO, 5 μL DNase I buffer, 5 μL DNase I, 10 μL sample Incubate at -37 °C for 30 min. Heat to -95°C for 15 minutes. Procedure (100 μL reaction volume): - Mix 75 μL HO, 10 μL DNase I buffer, 5 μL DNase I, 10 μL sample Incubate at -37 °C for 30 min. Heat to -95°C for 15 minutes. Working Example: 1.DNase I digestion; 2. Add 50 μL of water to the reaction mixture 3. Prepare PCR Master Mix 4. Add PCR master mix to plate (16.5 μL / well) 5. Prepare 1:10 dilutions: 10 µL sample / plasmid / standard + 90 µL HO 6. Add template to plate (5.5 μL / well) 7. Seal the plate, vortex (2,200 rpm for 1 minute), and centrifuge (1000 rcf for 1 minute). 8. Droplet formation using Auto-DG (20 µL final mixture + 70 µL oil) and transfer to plate (42 µL) 9. Seal the plate and start the PCR run

[0340] Example 4 Proteinase K digestion reagent: 1) Proteinase K (Roche; 17.8 mg / mL = ≧50 U / mL): Dilute to 1 U / mL 2) Proteinase K buffer (BioRad): 400 mM Tris-HCl, 20 mM EDTA, 2000 mM NaCl, pH 8 3) Sodium dodecyl sulfate solution (SDS solution): 10% Instructions (water): - Mix 68 μL water with 10 μL sample and add 20 μL proteinase K Incubate at -50 °C for 60 min. Heat to -95°C for 15 minutes Procedure (buffer without SDS): - Mix 63 μL water with 5 μL Proteinase K buffer and add 10 μL sample as well as 20 μL Proteinase K. Incubate at -50 °C for 60 min. Heat to -95°C for 15 minutes Procedure (buffer containing SDS): - Mix 53 μL of water with 5 μL of proteinase K buffer and add 10 μL of SDS solution, 10 μL of sample and 20 μL of proteinase K. Incubate at -50 °C for 60 min. Heat to -95°C for 15 minutes Working Example: 1. Proteinase K Digestion 2. Prepare PCR Master Mix 3. Add master mix to plate (16.5 μL / well) 4. Prepare a 1:10 dilution: 10 μL sample + 90 μL HO 5. Add template to plate (5.5 μL / well) 6. Seal the plate, vortex (2,200 rpm for 1 minute), and centrifuge (1000 rcf for 1 minute) 7. Droplet formation using Auto-DG (20 µL final mixture + 70 µL oil) and transfer to plate (42 µL) 8. Seal the plate and start the PCR run

[0341] Example 5 Heat denaturation followed by proteinase K digestion reagent: 1) Proteinase K (Roche; 17.8 mg / mL = ≧50 U / mL): 1 U / mL 2) Proteinase K buffer (BioRad): 400 mM Tris-HCl, 20 mM EDTA, 2000 mM NaCl, pH 8 procedure: -Heat denaturation: Incubate the sample at 98°C for 10 minutes - Proteinase K digestion: 1 μL Proteinase K per 50 μL sample; incubate at 50° C. for 30 min; inactivate at 95° C. for 10 min. Working Example: 1. Heat Denaturation 2. Proteinase K Digestion 3. Prepare a 1:10 dilution: 10 μL sample + 90 μL HO 4. Prepare PCR Master Mix 5. Add master mix to plates 6. Add samples to plate 7. Seal the plate, vortex (2.200 rpm for 1 minute), and centrifuge. 8. Droplet formation using Auto-DG (20 µL final mixture + 70 µL oil) and transfer to plate (42 µL) 9. Seal the plate and start the PCR run

[0342] Example 6 DNase I digestion followed by proteinase K digestion Method 1: reagent: 1) DNase I buffer (Promega): 400mM Tris-HCl, pH8, 100mM MgSO 4 , 10 mM CaCl 2 2) DNase I (Promega): 1U / μL 3) Proteinase K (Roche; 17.8 mg / mL = ≧50 U / mL): 1 U / mL 4) Proteinase K buffer (BioRad): 400 mM Tris-HCl, 20 mM EDTA, 2000 mM NaCl, pH 8 5) Sodium dodecyl sulfate solution (SDS solution): 10% procedure: - Mix 30 μL HO, 5 μL DNase I buffer, 5 μL DNase I, 10 μL sample Incubate at -37 °C for 30 min. Heat to -95°C for 15 minutes. - Mix 50 μL PK Mix (42 μL H2O + 2 μL Proteinase K + 5 μL 20× Proteinase K Buffer + 1 μL 10% SDS solution) with 50 μL of incubated DNase I-Mix Incubate at -50 °C for 60 min. Heat to -95°C for 15 minutes. Working Example: 1. DNase I digestion; optionally, 1:10 dilution 2. Proteinase K digestion; 1:10 dilution 3. Prepare PCR Master Mix 4. Add PCR master mix to plate (16.5 μL / well) 5. Prepare a 1:10 dilution: 10 μL sample + 90 μL HO 6. Add template to plate (5.5 μL / well) 7. Seal the plate, vortex (2,200 rpm for 1 minute), and centrifuge (1000 rcf for 1 minute). 8. Droplet formation using Auto-DG (20 µL final mixture + 70 µL oil) and transfer to plate (42 µL) 9. Seal the plate and start the PCR run Method 2: reagent: 1) Proteinase K (NEB; approximately 20 mg / mL = ≧800 U / mL): Dilute to 16 U / mL 2) Proteinase K buffer (BioRad): 400 mM Tris-HCl, 20 mM EDTA, 2000 mM NaCl 3) Sodium dodecyl sulfate solution (SDS solution): 10% Procedure (buffer containing SDS): - Mix 42 μL water with 50 μL sample (DNase I digestion solution) and add 2 μL proteinase K, 5 μL proteinase K buffer and 1 μL SDS solution Incubate at -50 °C for 60 min. Heat to -95°C for 15 minutes Working Example: 1.DNase I digestion; 2. Proteinase K Digestion 3. Dilute 1:10 4. Prepare PCR Master Mix 5. Add master mix to plate (16.5 μL / well) 6. Prepare a 1:10 dilution: 10 μL sample + 90 μL HO 7. Add template to plate (5.5 μL / well) 8. Seal the plate, vortex (2,200 rpm for 1 minute), and centrifuge (1000 rcf for 1 minute). 9. Droplet formation using Auto-DG (20 µL final mixture + 70 µL oil) and transfer to plate (42 µL) 10. Seal the plate and start the PCR run

[0343] Example 7 Compare conditions Reagents and procedures were similar to those outlined in the previous examples.

[0344] The conditions were as shown in Tables 14 and 15 below.

[0345] (Table 14) TIFF2025516117000014.tif49170

[0346] (Table 15) TIFF2025516117000015.tif82170

[0347] Example 8 ddPCR For viral genome titration, a duplex ddPCR assay was performed. Primers and probes were designed against the ITR sites and Amp resistance sequences present on the backbone of all three plasmids used for rAAV production. PCR master mixes were prepared according to Table 16 (Droplet Digital PCR Guide - Bio-Rad).

[0348] Table 16. ddPCR master mix composition TIFF2025516117000016.tif74158

[0349] The prepared master mix was pipetted into a 96-well plate at 16.5 μL / well. A dilution series of the pretreated samples was then performed: 10 μL of sample was transferred to 90 μL of water in a LoBind Tube with LoRentention Tips and mixed thoroughly. Then, 5.5 μL of sample was added to the master mix solution in the 96-well plate in several dilution steps. The plate was sealed at 180°C, vortexed at 2,200 rpm for 1 min, and centrifuged at 1,000 rpm for another 1 min. Up to 20,000 droplets per well were produced using an automated droplet generator that removed 20 μL of PCR mixture from each well and transferred to another 96-well plate. After sealing the droplet plate at 180°C, PCR was performed. The respective conditions are shown in Table 17.

[0350] Table 17. ddPCR thermal cycling program TIFF2025516117000017.tif26170

[0351] With a droplet reader, the fluorescent signal was measured for each droplet in the FAM and HEX channels. QuantaSoft software processed the reader data and calculated copy numbers per 20 μL well for both target sequences, ITR sites and Amp. The initial sample titer can be determined using the following Equation 1: TIFF2025516117000018.tif16128

[0352] Example 9 rAAV production HEK293-F suspension cells were transfected with three plasmids, namely pAAV transgene (EGFP or EBFP), pAAV-rep / cap and pAAV-helper. Plasmid DNA (1 μg / 1 mL cell culture) and lipofection reagent PEI pro (2 μL / 1 mL cell culture) were mixed separately with OptiMEM (50 μL / 1 mL cell culture) (see, for example, Grieger, J., et al. 2016). Then, both solutions were combined, incubated for 15 min at room temperature and added to a HEK293-F cell suspension containing 1 × 10E6 cells / mL in F17 medium. Cells were incubated at 37 °C, 8% CO2, 120 rpm for 48–72 h (Grieger, J., et al. (2016)).

[0353] Recombinant AAV particles were cultured in 1% Triton X-100, 500 mM TRIS and 20 mM MgCl 2 The cells were harvested by adding lysis buffer (100 μL / 1 mL cell culture) containing 100 μL of 1 mL of cell culture at pH 7.5. Freshly diluted benzonase was added (10 μL / 1 mL cell culture) to a final concentration of 50 U / mL. After lysis for 60 min at 37 °C with stirring, the cells were incubated with MgSO 4 (final concentration 37.5 mM) was added and the cell lysis broth was incubated for another 30 min (Chahal, P., et al. (2014)). The lysis suspension was then centrifuged at 4,000 g for 20 min and the supernatant was filtered through a 0.22 μm filter. The resulting product was considered as crude lysate.

[0354] Example 10 rAAV purification YMC glass column bodies were packed with POROS CaptureSelect AAVx affinity resin at a column bed volume of 9.1 mL. These resin beads are coated with antibody fragments that bind with high specificity to a wide range of AAV serotypes (POROS CaptureSelect AAV Resins-User Guide 2017).

[0355] First, the column was equilibrated with phosphate buffered saline (PBS) to obtain the correct binding conditions. The crude filtered lysate was then loaded at 150 cm / hr. After capturing the rAAV capsids, the column was washed with 4 CV of 0.5 M NaCl followed by 4 column volumes (CV) of PBS to remove impurities such as cellular debris and DNA residues. Another wash step with 4 CV of PBS was performed to prepare the elution conditions (POROS CaptureSelect AAV Resins-User Guide 2017).

[0356] The rAAV capsids were then eluted in 100 mM citrate buffer (pH 2.4) (POROS CaptureSelect AAV Resins-User Guide 2017). Fractions within the elution peak (UV detection at λ = 280 nm) were pooled. The pH value was raised to pH 7.5 using 2 M TRIS (pH 9). Finally, the eluate was sterile filtered using a syringe filter with a pore size of 0.2 μm.

Claims

**Claim 1** A method for determining the number of viral genomic DNA copies in a sample, said method comprising: - incubating said sample with proteinase K; - determining the number of viral genomic DNA copies by digital droplet polymerase chain reaction wherein said sample does not contain DNA that is not encapsulated within viral particles, said incubation with proteinase K is in the presence of 0.05 (w / v)% to 1.5 (w / v)% sodium dodecyl sulfate, said method. **Claim 2** - incubating said sample with a nuclease to obtain a digested sample; - incubating said digested sample with proteinase K to obtain a proteinase K-incubated sample; - determining the number of viral genomic DNA copies in said proteinase K-incubated sample by digital droplet polymerase chain reaction The method according to claim 1, comprising: **Claim 3** The method according to claim 2, wherein said digested sample is diluted up to 2.5-fold maximum for said incubation with proteinase K. **Claim 4** The method according to claim 2 or 3, wherein the completely digested sample is incubated with proteinase K. **Claim 5** The method according to any one of claims 1 to 4, wherein said method is carried out at a temperature of up to 95 °C, excluding the final extension step of said digital droplet polymerase chain reaction. **Claim 6** The method according to any one of claims 1 to 5, wherein the total amount of proteinase K used in said incubation is 15 mU to 35 mU. **Claim 7** The method according to any one of claims 1 to 6, wherein the volume of said sample is about 10 μL. **Claim 8** The method according to any one of claims 1 to 7, wherein said incubation with proteinase K is carried out in a total volume of 100 μL. **Claim 9** The method according to any one of claims 2 to 8, wherein said nuclease is DNase I. **Claim 10** The method according to any one of claims 2 to 9, wherein the total amount of nuclease used in said incubation is about 5 U. **Claim 11** The method according to any one of claims 2 to 10, wherein said incubation with nuclease is carried out in a total volume of 50 μL. **Claim 12** Incubating with the nuclease is at a final concentration of 40 mM Tris*HCl, 10 mM MgSO ４ , 1 mM CaCl ２ at a pH value of about 8, the method according to any one of claims 2 to 11. **Claim 13** The method according to any one of claims 2 to 12, wherein incubating with the nuclease is carried out at 37 °C for 30 minutes, followed by inactivation of the nuclease at 95 °C for 15 minutes.

14. The method according to any one of claims 1 to 13, wherein incubating with the proteinase K is carried out at a pH value of about 8 in a final concentration of 20 mM Tris*HCl, 1 mM EDTA, 100 mM NaCl, 1 (w / v)% sodium dodecyl sulfate.

15. Incubating with said proteinase K is at a final concentration of 30 mM Tris*HCl, 5 mM MgSO ４ , 0.5 mM CaCl ２ , 0.5 mM EDTA, 50 mM NaCl, 0.1 (w / v)% sodium dodecyl sulfate, the method according to any one of claims 1 to 13.

16. The method according to any one of claims 1 to 15, wherein incubating with the proteinase K is carried out at 50 °C for 60 minutes, followed by inactivation of the proteinase at 95 °C for 15 minutes.

17. The method according to any one of claims 1 to 16, wherein all steps of the method, except for the final extension step of the digital droplet polymerase chain reaction, are carried out at a temperature of up to 95 °C.

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