HK40112977A - Method for determining aav genomes - Google Patents

Description

Methods for determining the AAV genome

This invention belongs to the field of gene therapy. More specifically, this paper reports a method for determining the viral genome copy number in a process and purified sample using ddPCR, wherein the sample is incubated with proteinase K prior to PCR in the presence of a detergent.

Background Technology

Adeno-associated virus (AAV) particles are commonly used as gene transfer vectors in research and clinical approaches due to their favorable safety profile, high therapeutic efficacy, and the potential for target-specific engineering. For recombinant production, accurate and robust analytical methods are required for the characterization of viral vectors. Typically, vector genome titration is performed using droplet digital PCR (ddPCR), a method for absolute quantification of nucleic acids.

Several pretreatment methods for AAV vector genome titration have been reported in the literature for both real-time and droplet digital PCR. Before absolute quantification of the target sequence within the capsid, free transient plasmids and other unpackaged 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 to obtain better accessibility to the vector genome. Thermal inactivation of the AAV particles and the resulting denaturation of their capsid proteins are sufficient to achieve this (Wang, Y. et al. 2019). However, most publications describe additional protein digestion using proteinase K (Zolotukhin, S. et al. 1999; Fripont, S. et al. 2019; Dobnik, D. et al. 2019; Sanmiguel J. et al. 2019). Furthermore, various suggestions exist for dilution buffers used in subsequent dilution series of 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 antisurfactant Pluronic F-68 (Furuta-Hanawa, B. et al. 2019; Sanmiguel J. et al. 2019) and cleavage of salmon sperm (sss) DNA (Lock, M. et al. 2014; Sanmiguel J. et al. 2019).

Suoranta et al. (Hum. Gen. Ther. 32 (2020) 1270-1279) compared the extraction of AAV1, AAV2, AAV5, AAV6, AAV8, and AAV9 genomes after gradient ultracentrifugation with iodixanol in phosphate-buffered saline solution using kits for qPCR and ddPCR, respectively, by heat denaturation, proteinase K treatment, and extraction using kits. Kit extraction (which contained proteinase K treatment in denaturing buffer in the presence of additional carrier RNA prior to spin column purification) significantly increased the titers obtained for all serotypes in both qPCR and ddPCR. Importantly, Suoranta et al. found that no studies have yet presented conclusive data regarding the availability of genomes in ddPCR.

CN 109957561 discloses a method for extracting nucleic acids from a sample, wherein the method includes the following steps: adding a lysis solution to the sample to be extracted to release nucleic acid molecules; further adding a sodium lauroyl sarcosinate solution; further adding a mixed solution containing sodium iodide, glycogen and isopropanol to form a precipitate containing nucleic acids, and recovering the precipitate by centrifuging the sample.

US 8,652,821 discloses a reagent mixture for purifying RNA-free DNA, comprising a protease, an RNase, and a detergent.

US11,028,372 discloses a scalable purification method for AAV particles of serum rh.10.

WO 03/104413 discloses a dot blot analysis of pseudotyped recombinant AAV virions without further details, which includes proteinase K incubation followed by phenol extraction and ethanol precipitation.

WO 2007/084773 discloses a dot blot analysis of an infectious parvovirus vector produced in insect cells without further details.

Binny, C.J. and Nathwani, A.C. disclosed an agarose gel analysis method for determining whether a genome packaged into a scAAV vector is a short double-stranded hairpin structure, a short ssDNA, or a long unfolded ssDNA as expected (Meth.Mol.Biol.891(2012)109-131).

US2021/0284699 discloses a qPCR method for analyzing rAAV particles purified using a method comprising the following steps: (a) generating a viral particle extract containing a plurality of rAAVs as described herein, wherein the viral particle extract contains a supernatant of lysed production 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 with one or more filters to generate a filtrate; and (f) performing one or more cycles to exchange the filtrate buffer for a final storage buffer.

Summary of the Invention

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

The number of aspects (independent topics) and embodiments (dependent topics) that are not limited are as follows:

1. A method for determining the copy number of viral genomic DNA in a sample, wherein the method comprises the following steps:

- The viral genomic DNA copy number was determined by digital droplet polymerase chain reaction.

The sample contained no DNA that was not encapsulated within the viral particle.

The sample in question has not yet been incubated with nucleases, and

The sample in question has not yet been incubated with the protease.

2. The method according to aspect 1, wherein the nuclease is a restriction enzyme.

3. The method according to any one of aspect 1 or example 2, wherein the nuclease is DNAase I.

4. The method according to aspect 1 or any one of Examples 2 to 3, wherein the protease is proteinase K.

5. A method for determining the copy number of viral genomic DNA in a sample, wherein the method

Includes the following steps:

- Incubate the sample with proteinase K.

- The viral genomic DNA copy number was determined by digital droplet polymerase chain reaction.

6. The method according to aspect 5, wherein the sample is a cell lysate.

7. The method according to any one of aspect 5 or embodiment 6, wherein the sample is a lysed cell sample.

8. The method according to Example 7, wherein the lysed cell sample has been obtained by lysing virus-producing cells with a detergent or by chemical means; optionally, cell debris has been removed from the lysed cell sample.

9. The method according to aspect 5 or any one of embodiments 6 to 8, wherein the sample comprises viral particles and does not contain DNA that is not capped within the viral particles, wherein the viral DNA genome is capped within the viral particles.

10. A method for determining the copy number of viral genomic DNA in a sample, wherein the method comprises the following steps:

- Incubate the sample with proteinase K.

- The viral genomic DNA copy number was determined by digital droplet polymerase chain reaction.

The sample contained no DNA that was not encapsulated within the viral particle.

The incubation with proteinase K was carried out in the presence of a detergent.

11. The method according to aspect 10, wherein the detergent is sodium dodecyl sulfate.

12. The method according to any one of aspect 10 or example 11, wherein the final concentration of the detergent during incubation with proteinase K is from 0.05 (w/v)% to 1.5 (w/v)%.

13. The method according to aspect 10 or any one of examples 11 to 12, wherein the final concentration of the detergent during incubation with proteinase K is about 0.1% (w/v)%.

14. The method according to aspect 10 or any one of examples 11 to 12, wherein the final concentration of the detergent during incubation with proteinase K is about 1 (w/v)%.

15. The method according to any one of aspect 5 or aspect 10 or embodiments 6 to 9 or embodiments 11 to 14, wherein the method comprises the following steps:

- The sample is incubated with a nuclease to obtain a digested sample.

- The digested sample is incubated with proteinase K to obtain a proteinase K-incubated sample.

- The number of viral genomic DNA copies in the proteinase K-incubated sample was determined by digital droplet polymerase chain reaction.

16. The method according to Example 15, wherein the digested sample is diluted up to 2.5 times for the incubation with proteinase K.

17. The method according to any one of Examples 15 to 16, wherein all of the digested sample is incubated with proteinase K.

18. The method according to any one of aspect 1, aspect 5, aspect 10, or examples 2 to 4, or examples 6 to 9, or examples 11 to 17, wherein the digital droplet polymerase chain reaction comprises the following steps:

a) Incubate the sample at approximately 95°C for 10 minutes.

b) Perform the following thermal cycle: incubate the sample at approximately 94°C for 30 seconds, then incubate the sample at approximately 60°C for 1 minute.

c) Repeat step b) 15 to 60 times.

d) Perform the final extension step at 98°C for 10 minutes.

19. The method according to Example 18, wherein the temperature is increased by 2°C in steps a) and d).

20. The method according to any one of aspect 1, aspect 5, aspect 10, or examples 2 to 4, or examples 6 to 9, or examples 11 to 19, wherein the sample is maintained at a temperature of 95°C or lower.

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

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

23. The method according to any one of aspect 5 or aspect 10 or examples 2 to 4 or examples 6 to 9 or examples 11 to 22, wherein the total amount of proteinase K used in the incubation is from 1 mU to 50 mU.

24. The method according to any one of aspect 5 or aspect 10 or examples 2 to 4 or examples 6 to 9 or examples 11 to 23, wherein the total amount of proteinase K used in the incubation is 10 mU to 40 mU.

25. The method according to any one of aspect 5 or aspect 10 or examples 2 to 4 or examples 6 to 9 or examples 11 to 24, wherein the total amount of proteinase K used in the incubation is 15 mU to 35 mU.

26. The method according to any one of aspect 5 or aspect 10 or examples 2 to 4 or examples 6 to 9 or examples 11 to 25, wherein the total amount of proteinase K used in the incubation is 20 mU to 32 mU.

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

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

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

30. The method according to any one of aspect 1, aspect 5, aspect 10, or examples 2 to 4, or examples 6 to 9, or examples 11 to 29, wherein the volume of the sample is about 10 μL.

31. The method according to any one of aspect 5 or aspect 10 or examples 2 to 4 or examples 6 to 9 or examples 11 to 30, wherein the incubation with proteinase K is carried out in a total volume of 100 μL.

32. The method according to any one of Examples 15 to 31, wherein the nuclease is DNAase I.

33. The method according to any one of Examples 15 to 32, wherein the total amount of nuclease used in the incubation is about 5 U.

34. The method according to any one of Examples 15 to 33, wherein the incubation with the nuclease is carried out in a total volume of 50 μL.

35. The method according to any one of Examples 15 to 34, wherein the incubation with the nuclease is carried out at a final concentration of 40 mM Tris*HCl, 10 mM _MgSO4 , and 1 mM _CaCl2 at a pH of about 8.

36. The method according to any one of Examples 15 to 35, wherein the incubation with the nuclease is carried out at 37°C for 30 minutes.

37. The method according to Example 36, wherein the nuclease is inactivated at 95°C for 15 minutes after the incubation.

38. The method according to any one of Examples 15 to 37, wherein the incubation with proteinase K is carried out at a final concentration of 20 mM Tris*HCl, 1 mM EDTA, and 100 mM NaCl at a pH of about 8.

39. The method according to any one of Examples 15 to 37, wherein the incubation with proteinase K is carried out at a final concentration of 20 mM Tris*HCl, 1 mM EDTA, and 100 mM NaCl at a pH of about 8.

40. The method according to any one of Examples 15 to 37, wherein the incubation with proteinase K is carried out at a final concentration of 20 mM Tris*HCl, 1 mM EDTA, 100 mM NaCl, and 1 (w/v)% sodium dodecyl sulfate at a pH of about 8.

41. The method according to any one of Examples 15 to 37, wherein the incubation with proteinase K is carried out at a pH of about 8 with a final concentration of 30 mM Tris*HCl, ₅ mM MgSO4, 0.5 mM _CaCl2 , 0.5 mM EDTA, 50 mM NaCl, and 0.1 (w/v)% sodium dodecyl sulfate.

42. The method according to any one of Examples 15 to 41, wherein the incubation with proteinase K is carried out at 50°C for 60 minutes.

43. The method according to Example 42, wherein the protease is inactivated at 95°C for 15 minutes after the incubation.

44. The method according to any one of aspect 1, aspect 5, aspect 10, or examples 2 to 4, or examples 7 to 9, or examples 11 to 43, wherein the volume of sample is a cell lysate purified by affinity chromatography.

45. The method according to any one of aspect 1, aspect 5, aspect 10, or examples 2 to 4, or examples 6 to 9, or examples 11 to 43, wherein the method is carried out in the absence of a precipitation step and/or glycogen.

Detailed Implementation

This invention is based, at least in part, on the finding that for AAV genome copy number determination, in cases where crude cell lysate samples contain AAV particles and also contain non-viral capsidated DNA, the samples must be incubated with proteinase K prior to PCR. The presence of a detergent can further improve this determination.

This invention is based, at least in part, on the finding that for AAV genome copy number determination, in the case that the purified sample contains AAV particles and is substantially free of non-viral capsidated DNA, the sample must be incubated with proteinase K prior to PCR. Free from this theory, it is assumed that the detergent prevents the formation of aggregates of protein fragments and viral genomic DNA, which in turn prevents subsequent amplification via polymerase chain reaction, and thus leads to an underestimation of the viral genome copy number in the sample.

This invention is based, at least in part, on the finding that for determining the AAV genome copy number after sequential treatment with nucleases and proteases, the protease treatment must be performed in the presence of a detergent. Free from this theory, it is assumed that the detergent prevents the formation of aggregates of protein fragments and viral genomic DNA, which in turn prevents subsequent amplification via polymerase chain reaction, and thus leads to an underestimation of the viral genome copy number in the sample.

This invention is further based, at least in part, on the finding that for purified samples containing AAV particles and substantially free of non-viral capsidated DNA, the determination of the AAV genome copy number must be performed without nuclease and protease pretreatment, or with proteinase K incubation in the presence of detergent prior to polymerase chain reaction. Free from this theory, it is hypothesized that highly purified samples (i.e., containing no substantial amount of non-viral capsidated DNA) incubated with nucleases alone or in combination of nucleases and proteases in the absence of detergent lead to aggregation, thereby artificially reducing the AAV genome copy number.

This invention is further based, at least in part, on the finding that for purified samples containing AAV particles and substantially free of non-viral capsidated DNA, the determination of the AAV genome copy number must be performed at temperatures above 95°C without a heat denaturation step. Free from this theory, it is assumed that heat treatment of highly purified samples (i.e., containing no substantial amount of non-viral capsidated DNA) at temperatures above 95°C causes aggregation, thereby artificially reducing the AAV genome copy number.

This invention is further based, at least in part, on the premise that low amounts of proteinase K in the presence of detergent are sufficient to make substantially all of the AAV genome in a sample available for polymerase chain reaction (PCR) and thus for determination/quantification. Free from this theory, it is assumed that high concentrations of proteinase K interfere with PCR, and that improvements in PCR are achieved by using significantly reduced amounts (i.e., low amounts) of proteinase K through reduced PCR inhibition. In particular, incubation with proteinase K in the presence of detergent can reduce or even eliminate the inhibition of PCR by proteinaceous substances in cell lysates from AAV-producing cells.

The present invention is further based, at least in part, on the following finding: for the sequential incubation of crude cell lysate samples with nucleases and proteases, the samples should not be diluted after nuclease incubation, and the total volume of the incubation mixture should be used for the protease incubation step.

Generally, the more preprocessing steps required before determining the AAV genome copy number in ddPCR, the higher the likelihood of contamination.

definition

Methods and techniques applicable to carrying out the present invention are described in, for example, Ausubel, F.M. (ed.), Current Protocols in Molecular Biology, Volumes I through III (1997); Glover, N.D. and Hames, B.D. (eds.), DNA Cloning: A Practical Approach, Volumes I and II (1985), Oxford University Press; Freshney, R.I. (ed.), Animal Cell Culture – a practical approach, IRL Press Limited (1997). 986); Watson, J.D. et al., Recombinant DNA, 2nd edition, CHSL Press (1992); Winnacker, E.L., From Genes to Clones; N.Y., VCH Publishers (1987); Celis, J. (ed.), Cell Biology, 2nd edition, Academic Press (1998); Freshney, R.I., Culture of Animal Cells: A Manual of Basic Technique, 2nd edition, Alan R. Liss, Inc., N.Y. (1987).

It is important to note that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly specifies otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells and their equivalents known to those skilled in the art, and so on. Similarly, the terms “a,” “one or more,” and “at least one” are used interchangeably herein. It should also be noted that the terms “comprising,” “including,” and “having” are used interchangeably.

The term "AAV helper function" refers to an AAV-derived coding sequence (protein) that can be expressed to provide AAV gene products and AAV particles, which in turn function trans-AAV for productive AAV replication and packaging. Therefore, 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 numerous functions, including: recognizing, binding to, and cleaving AAV origins of DNA replication; DNA helicase activity; and regulating transcription from AAV (or other heterologous) promoters. The cap gene expression product (capsid) provides the necessary packaging function. AAV helper functions complement the trans-AAV function missing in the AAV vector genome.

The term "about" indicates a range of +/- 20% of the value that follows it. In some embodiments, the term "about" indicates a range of +/- 10% of the value that follows it. In some embodiments, the term "about" indicates a range of +/- 5% of the value that follows it.

The term "including" also covers the term "contains...".

The terms "empty capsid" and "empty particle" refer to AAV particles, which possess an AAV protein shell but lack all or part of the nucleic acid encoding the protein or the nucleic acid transcribed into a target transcript flanked by an AAV ITR (i.e., a vector). Therefore, an empty capsid cannot function as a means of transferring the nucleic acid encoding the protein or transcribed into the target transcript into the host cell.

The term "endogenous" means that something is naturally present in the cell; produced naturally by the cell; similarly, "endogenous locus/cellular endogenous locus" is a locus that is naturally present in the cell.

As used herein, the term "exogenous" refers to a nucleotide sequence that is not derived from a specific cell but is introduced into the cell via DNA delivery methods (e.g., transfection, electroporation, or transduction by a viral vector). Therefore, an exogenous nucleotide sequence is an artificial sequence, where artificiality can originate from, for example, a combination of subsequences from different sources (e.g., a combination of a recombinase recognition sequence having an SV40 promoter and a coding sequence for a green fluorescent protein is an artificial nucleic acid) or from the deletion of portions of a sequence (e.g., a sequence encoding only the extracellular domain of a membrane-binding receptor or cDNA), or nucleobase mutations. The term "endogenous" refers to a nucleotide sequence derived from a cell. An "exogenous" nucleotide sequence may have an "endogenous" counterpart with the same base composition, but wherein the sequence becomes an "exogenous" sequence, for example, through introduction into the cell via recombinant DNA technology.

"Separated" compositions are compositions that have been separated from one or more components of their natural environment. In some embodiments, the composition is purified to a purity greater than 95% or 99%, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis, CE-SDS) or chromatography (e.g., size exclusion chromatography, ion exchange, or reversed-phase HPLC). For a review of methods used to assess, for example, antibody purity, see Flatman, S. et al., J. Chrom. B 848 (2007) 79-87.

"Isolated" nucleic acids refer to nucleic acid molecules that have been separated from one or more components of their natural environment. Isolated nucleic acids include nucleic acid molecules that are contained in cells that normally contain nucleic acid molecules, but which are located outside the chromosome or at a chromosomal location different from their natural chromosomal location.

"Separated" polypeptides or antibodies refer to polypeptide or antibody molecules that have been separated from one or more components of their natural environment.

The term "mammalian cell containing a foreign nucleotide sequence" encompasses cells in which one or more foreign nucleic acids have been introduced, including progeny of such cells. These can serve as a starting point for further genetic modifications. Therefore, the term "mammalian cell containing a foreign nucleotide sequence" encompasses a cell containing a foreign nucleotide sequence at a single site within a locus integrated into the genome of the mammalian cell, wherein the foreign nucleotide sequence contains at least a first recombination recognition site and a second recombination recognition site flanked by at least one first selection marker (these recombination recognition sites are distinct). In some embodiments, a mammalian cell containing a foreign nucleotide sequence is a cell containing a foreign nucleotide sequence at a single site within a locus integrated into the cell's genome, wherein the foreign nucleotide sequence contains a first recombination recognition sequence flanked by at least one first selection marker and a second recombination recognition sequence, and a third recombination recognition sequence located between the first and second recombination recognition sequences, and all recombination recognition sequences are distinct.

"Mammalian cells containing exogenous nucleotide sequences" and "recombinant cells" are both terms for "transformed cells." This term includes primary transformed cells and their offspring, regardless of passage number. Offspring may not, for example, have completely identical nucleic acid contents to the parent cells, but may contain mutations. It also encompasses mutant offspring with the same function or biological activity as in the originally transformed cells.

"Nucleic acid encoding AAV packaging proteins" generally refers to one or more nucleic acid molecules that include nucleotide sequences that provide AAV function and have been deleted from an AAV vector. This nucleic acid molecule is used to produce transducible recombinant AAV particles. Nucleic acids encoding AAV packaging proteins are typically used to provide expression of the AAV rep and/or cap genes to supplement the missing AAV function required for AAV replication; however, the nucleic acid construct lacks the AAV ITR and cannot self-replicate or self-package. Nucleic acids encoding AAV packaging proteins can be in the form of plasmids, phages, transposons, granules, viruses, or particles. Many nucleic acid constructs have been described, such as the commonly used plasmids pAAV/Ad and pIM29+45, which encode both the rep gene expression product and the cap gene expression product. See, for example, Samulski et al., J. Virol. 63 (1989) 3822-3828; and McCarty et al., J. Virol. 65 (1991) 2936-2945. Numerous plasmids encoding rep and/or cap gene expression products have been described (e.g., US 5,139,941 and US 6,376,237). Any of these nucleic acids encoding AAV packaging proteins may contain DNA elements or nucleic acids according to the invention.

The term "nucleic acid encoding accessory proteins" generally refers to one or more nucleic acid molecules that include nucleotide sequences encoding proteins and/or RNA molecules that provide accessory functions for adenoviruses. Plasmids containing nucleic acids encoding accessory proteins can be transfected into suitable cells, whereby the plasmid is then able to support the production of AAV particles in said cells. Any of these nucleic acids encoding accessory proteins may contain DNA elements or nucleic acids according to the invention. The term explicitly excludes infectious viral particles, as they are present in nature, such as adenovirus, herpesvirus, or vaccinia virus particles.

As used herein, the term "operably linked" refers to the juxtaposition of two or more components in a manner in which the relationship between these components allows them to function in a desired manner. For example, if a promoter and/or enhancer functions to regulate transcription of a coding sequence/open reading frame/gene, then the promoter and/or enhancer is operably linked to the coding sequence/open reading frame/gene. In some embodiments, the DNA sequences "operably linked" are adjacent. In some embodiments, for example, when two protein-coding regions (such as a secretory leader region and a polypeptide) must be joined, these sequences are adjacent and within the same reading frame. In some embodiments, the operably linked promoter is located upstream of the coding sequence/open reading frame/gene and may be adjacent to it. In some embodiments, for example, with respect to an enhancer sequence regulating coding sequence/open reading frame/gene expression, the two components may be operably linked but not adjacent. If the enhancer increases transcription of the coding sequence/open reading frame/gene, then the enhancer is operably linked to the coding sequence/open reading frame/gene. Operable enhancers can be located upstream, inside, or downstream of the coding sequence/open reading frame/gene, and can be located at a considerable distance from the promoter of the coding sequence/open reading frame/gene.

The term "packaging protein" refers to non-AAV-derived viral and/or cellular functions upon which AAV replication depends. Therefore, the term encompasses the proteins and RNA required for AAV replication, including those involved in AAV gene transcriptional activation, stage-specific AAV mRNA splicing, AAV DNA replication, Cap expression product synthesis, and AAV capsid assembly. Viral helper functions can originate from any of the known helper viruses, such as adenoviruses, herpesviruses (except type I herpes simplex virus), and vaccinia virus.

As used herein, “AAV packaging protein” refers to an AAV-derived sequence whose trans-AAV function is required for productive AAV replication. Therefore, AAV packaging proteins are encoded by the major AAV open reading frame (ORF), rep, and cap. The rep protein has been shown to have numerous functions, including: recognizing, binding to, and cleaving the AAV origin of DNA replication; DNA helicase activity; and regulating transcription from AAV (or other heterologous) promoters. The cap protein provides the necessary packaging function. In this paper, AAV packaging proteins are used to supplement the trans-AAV function missing in AAV vectors.

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

As used herein, the term "recombinant cell" refers to a cell that has undergone final genetic modification, such as expressing a target polypeptide or producing target rAAV particles, and can be used to produce said target polypeptide or target rAAV particles on any scale. For example, a "mammalian cell containing a foreign nucleotide sequence" that has undergone recombinase-mediated cassette exchange (RMCE), whereby the coding sequence of the target polypeptide has been introduced into the host cell genome, is a "recombinant cell." Although the cell may still be capable of further RMCE reactions, it is not desirable to do so.

"Recombinant AAV vectors" are derived from the wild-type genome of a virus (such as AAV) by using molecular methods to remove the wild-type genome from the virus (e.g., AAV) and replace it with non-natural nucleic acids (such as nucleic acids transcribed into transcripts or encoding proteins). For AAV, one or both inverted terminal repeat (ITR) sequences of the wild-type AAV genome are typically retained in recombinant AAV vectors. "Recombinant" AAV vectors are distinguished from wild-type viral AAV genomes because all or part of the viral genome has been replaced with non-natural (i.e., heterologous) sequences relative to the viral genome nucleic acids. Therefore, the incorporation of non-natural sequences defines a viral vector (e.g., AAV) as a "recombinant" vector, and in the case of AAV, this vector may be called an "rAAV vector."

Recombinant vector (e.g., AAV) sequences can be packaged—referred to herein as “particles”—for subsequent cell infection (transduction) in vitro, in vitro, or in vivo. When a recombinant vector sequence is encapsulated or packaged into an AAV particle, the particle may also be referred to as “rAAV”. Such particles include proteins that encapsulate or package the vector genome. Specific examples include viral envelope proteins, and in the case of AAV, capsid proteins such as AAV VP1, VP2, and VP3.

As used herein, the term "selection marker" refers to a gene that allows for the specific selection or exclusion of cells carrying that gene in the presence of a suitable selective agent. For example, but not as a limitation, a selection marker may allow for the positive selection of host cells transformed with the selection marker gene in the presence of a suitable selective agent (selective culture conditions); untransformed host cells will not be able to grow or survive under those selective culture conditions. Selection markers can be positive, negative, or bifunctional. Positive selection markers allow for the selection of cells carrying the marker, while negative selection markers allow for the selective elimination of cells carrying the marker. Selection markers can confer resistance to drugs or compensate for metabolic or catabolistic defects in host cells. In prokaryotic cells, genes conferring resistance to ampicillin, tetracycline, kanamycin, or chloramphenicol, as well as other genes, may be used. Resistance genes that can be used as selection markers in eukaryotic cells include, but are not limited to, genes targeting aminoglycoside phosphotransferases (APHs) (e.g., hygromycin phosphotransferase (HYG), neomycin, and G418APH), dihydrofolate reductase (DHFR), thymidine kinase (TK), glutamine synthase (GS), asparagine synthase, tryptophan synthase (indole), histamine dehydrogenase (histamine D), and genes encoding resistance to puromycin, blastomycin, bleomycin, fulvicin, chloramphenicol, zeocin, and mycophenolic acid. Additional marker genes are described in WO 92/08796 and WO 94/28143.

In addition to aiding selection in the presence of appropriate selective reagents, selection markers can alternatively be molecules 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. Cells expressing this molecule can be distinguished from cells lacking the gene, for example, by detecting the fluorescence emitted by the encoded polypeptide or by the absence of such fluorescence.

As used herein, the term "serotype" is based on the serological differentiation of the AAV capsid. Serological differentiation is determined by the lack of cross-reactivity between antibodies against one AAV and antibodies against another. Such differences in cross-reactivity are typically due to differences in capsid protein sequence/antigenic determinants (e.g., due to differences in the VP1, VP2, and/or VP3 sequences of AAV serotypes). Although AAV variants, including capsid variants, may be serologically indistinguishable from a reference AAV or other AAV serotypes, they differ from the reference AAV serotype or other AAV serotypes by at least one nucleotide or amino acid residue.

Under the traditional definition, a serotype refers to a serum that has been tested against a target virus, which is specific for neutralizing activity against all existing and characterized serotypes, and for which no antibodies neutralizing the target virus have been found. As more naturally occurring viral isolates are discovered and/or capsid mutants are generated, serological differences with any of the currently existing serotypes may or may not exist. Therefore, in cases where a new virus (e.g., AAV) does not exhibit serological differences, that new virus (e.g., AAV) will be a subgroup or variant of the corresponding serotype. In many cases, serological tests for neutralizing activity have not been performed on mutant viruses with capsid sequence modifications to determine whether they belong to another serotype according to the traditional definition of serotype. Therefore, for convenience and to avoid duplication, the term "serotype" broadly refers to both serologically distinct viruses (e.g., AAV) and serologically indistinguishable viruses (e.g., AAV), which may fall within a subgroup or variant of a given serotype.

The terms “transduction” and “transfection” refer to the introduction of molecules such as nucleic acids (viral vectors, plasmids) into cells. Cells are “transduced” or “transfected” when a foreign nucleic acid has been introduced into the cell membrane. Therefore, a “transduced cell” is a cell in which a “nucleic acid” or “polynucleotide” has been introduced, or its progeny in which a foreign nucleic acid has been introduced. In certain embodiments, “transduced” cells (e.g., in mammals, such as cell or tissue or organ cells) undergo genetic changes after incorporation of a foreign molecule such as a nucleic acid (e.g., a transgene). “Transduced” cells can proliferate and transcribe the introduced nucleic acid and/or express proteins.

In transduced or transfected cells, nucleic acids (viral vectors, plasmids) may or may not integrate into the genomic nucleic acid. If the introduced nucleic acid integrates into the nucleic acid (genomic DNA) of the recipient cell or organism, it can be stably maintained in that cell or organism and further transferred to or inherited by the daughter cells or organisms of the recipient cell or organism. Finally, the introduced nucleic acid may exist extrachromosomally in the recipient cell or host organism, or may exist only transiently. Many techniques are known, for example see 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. Such techniques can be used to introduce one or more portions of foreign DNA into a suitable host cell.

The term "transgenic" is used herein for convenience to refer to nucleic acids that are intended to be or have been introduced into cells or organisms. Transgenic includes any nucleic acid, such as genes that are transcribed into transcripts or encode polypeptides or proteins.

A "vector" refers to a portion of a recombinant plasmid sequence that is ultimately packaged or encapsulated, either directly or as a single strand or RNA, to form a viral (e.g., AAV) particle. When a recombinant plasmid is used to construct or manufacture recombinant viral particles, the viral particle does not include a "plasmid" portion 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 essential for plasmid cloning and amplification—processes required for propagation and recombinant viral production—but it is not itself packaged or encapsulated into a viral (e.g., AAV) particle. Therefore, a "vector" refers to nucleic acid packaged or encapsulated by a viral particle (e.g., AAV).

Recombinant cells

Generally, in order to efficiently and on a large scale produce target protein compounds, such as rAAV particles or therapeutic peptides, cells that express and, if possible, also secrete said protein compounds are necessary. Such cells are called “recombinant cells” or “recombinant production cells.”

To generate "recombinant production cells," suitable mammalian cells are transfected with the desired nucleic acid sequence encoding the target protein compound. Additional helper peptides may also be required.

To generate stable recombinant production cells, the next step is to select single cells that stably express the target protein compound. This can be accomplished, for example, based on the co-expression of a selection marker that has been co-transfected with the nucleic acid sequence encoding the target protein compound, or through the expression of the protein compound itself.

For expression of the coding sequence, i.e., the open reading frame (OPF), additional regulatory elements are required, such as a promoter and a polyadenylation signal (sequence). Therefore, the ORF is operatively linked to these additional regulatory elements for transcription. This can be achieved by integrating it into a so-called expression cassette. The minimum regulatory elements required for the expression cassette to function in mammalian cells are the promoter, which functions in the mammalian cell, located upstream of the ORF (5'), and the polyadenylation signal (sequence), which functions in the mammalian cell, located downstream of the ORF (3'). Additionally, a terminator sequence may be present at the 3' of the polyadenylation signal (sequence). For expression, the promoter, ORF/coding region, and polyadenylation signal sequence must be arranged in an operatively linked manner.

Similarly, nucleic acids transcribed into RNA that encodes non-proteins are called "RNA genes." The expression of RNA genes also requires additional regulatory elements, such as promoters and transcription termination signals or polyadenylation signals (sequences). The nature and location of these elements depend on the RNA polymerase designed to drive RNA gene expression. Therefore, RNA genes are often also integrated into expression cassettes.

If the target protein compound is an AAV particle composed of different (monomeric) capsid polypeptides and single-stranded DNA molecules, and additional adenoviral helper functions are required for production and encapsulation, then multiple expression cassettes are needed, differing only in the open reading frames/coding sequences they contain. In this case, at least one expression cassette is required for each of the following: the different polypeptides for transgenic AAV vector capsid formation, the required helper functions, and the VA RNA. Therefore, separate expression cassettes are needed for each of the helper genes E1A, E1B, E2A, E4orf6, VA RNA, rep, and cap.

As outlined in the preceding paragraphs, the more complex the target protein compound or the higher the number of additional accessory peptides and/or RNA required, the greater the number of different expression cassettes needed. The total size of the nucleic acid is also inherently related to the number of expression cassettes. However, there is a practical limit to the size of transferable nucleic acids, ranging from approximately 15 kbps (kilobase pairs). Beyond this limit, processing and handling efficiency drops significantly. This problem can be addressed by using two or more separate plasmids. Thus, different expression cassettes are assigned to different plasmids, with each plasmid containing only a subset of the expression cassettes.

In all aspects and embodiments, each of the expression cassettes comprises a promoter, an open reading frame/coding sequence or RNA gene, and a polyadenylation signal sequence, and/or a terminator sequence along the 5' to 3' direction. In some 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 some embodiments, the expression cassette comprises an RNA gene, 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 some embodiments, the expression cassette comprises an RNA gene, a type 2 Pol III promoter, and a polyU terminator sequence.

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

In all aspects and in certain embodiments, the promoter is the human CMV promoter with intron A, the polyadenylation signal sequence is the bGH polyadenylation signal sequence and the terminator is hGT, except for expression cassettes of RNA genes and expression cassettes of selection markers, wherein for selection markers, the promoter is the SV40 promoter and the polyadenylation signal sequence is the SV40 polyadenylation signal sequence and there is no terminator, and wherein for RNA genes, the promoter is the wild-type type 2 polymerase III promoter and the terminator is a polymerase II or III terminator.

Adeno-associated virus (AAV)

For a general review of the auxiliary functions of AAV and adenovirus or herpesvirus, 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. Design considerations for constructing recombinant AAV vectors are described in US 4,797,368 (see also WO 93/24641). Other references describing AAV vectors include West et al., Virol. 160 (1987) 38-47; Kotin, Hum. Gene Ther. 5 (1994) 793-801; and Muzyczka J. Clin. Invest. 94 (1994) 1351. The construction of recombinant AAV vectors is described in US 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.

Adeno-associated virus (AAV) is a replication-defective parvovirus. It can only replicate in cells, where some viral functions are provided by co-infected helper viruses, such as adenoviruses, herpesviruses, and in some cases poxviruses, such as cowpox. Nevertheless, AAV can replicate in almost any cell line of human, ape, or rodent origin, provided that appropriate helper viral functions are present.

If the helper viral gene is absent, AAV establishes a latent period in its host cells. Its genome integrates into a specific site on chromosome 19 [(Chr)19(q13.4)], known as adeno-associated virus integration site 1 (AAVS1). For certain serotypes, such as AAV-2, other integration sites have been found, such as on chromosome 5 [(Chr)5(p13.3)], known as AAVS2, and on chromosome 3 [(Chr)3(p24.3)], known as AAVS3.

AAV is classified into different serotypes. These are assigned based on parameters such as hemagglutination, tumorigenicity, and DNA sequence homology. To date, more than 10 different serotypes and more than 100 sequences corresponding to different branches of AAV have been identified.

The type and symmetry of capsid proteins determine the tissue specificity of the corresponding AAVs. 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 lungs.

Pseudo-packaging refers to the process involving cross-packaging of the AAV genome between different serotypes, i.e., the genome is packaged with capsid proteins from different sources.

The wild-type AAV genome is approximately 4.7 kb in size. 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. Viral., 51 (1984) 611-619). The open reading frames encoding the Rep protein provide four proteins of different sizes, referred to as Rep78, Rep68, Rep52, and Rep40. These are involved in AAV replication, rescue, and integration. The open reading frames encoding the Cap protein provide four proteins, referred to as VP1, VP2, VP3, and AAP. VP1, VP2, and VP3 are part of the protein 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, in addition to the Rep and Cap proteins, AAV also requires the products of the adenovirus genes E1A, E1B, E4orf6, E2A and VA, or corresponding factors of another helper virus.

For example, in the case of AAV serotype 2 (AAV-2), each ITR is 145 nucleotides long and flanked by a coding sequence region of approximately 4470 nucleotides. Of the 145 nucleotides in the ITR, 125 nucleotides have palindromic sequences and can form a T-shaped hairpin structure. This structure functions as a primer during viral replication. The remaining 20 unpaired nucleotides are represented as D sequences.

The AAV genome contains three transcription promoters, P5, P19, and P40 (Laughlin et al., Proc. Natl. Acad. Sci. USA 76(1979) 5567-5571) for expressing the rep and cap genes.

ITR sequences must be present in cis configuration within the coding region. ITRs provide a functional origin of replication (ori), the signals required for integration into the target cell genome, and efficient excision and rescue from the host cell chromosome or recombinant plasmid. ITRs further contain origin of replication-like elements such as Rep protein binding sites (RBS) and terminal resolution sites (TRS). ITRs themselves have been found to function as transcription promoters 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).

Separate replication and capsidation of the viral single-stranded DNA genome require trans-organization of the rep and cap gene products.

The rep locus contains two internal promoters, called P5 and P19. It contains open reading frames for four proteins. Promoter P5 is operatively linked to a sequence providing an unspliced 4.2 kb mRNA encoding Rep78 (a chromatin nickase that arrests the cell cycle) and a spliced 3.9 kb mRNA encoding Rep68 (a site-specific endonuclease). Promoter P19 is operatively linked to a sequence providing an unspliced mRNA encoding Rep52 and a spliced 3.3 kb mRNA encoding Rep40 (a DNA helicase used for accumulation and packaging).

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

The larger Rep proteins Rep68 and Rep78 can specifically bind to the hairpin conformation of the AAV ITR. They exhibit defined enzymatic activity required to resolve replication at the AAV terminus. Expression of Rep78 or Rep68 may be sufficient to form infectious particles (Holscher, C. et al. J. Virol. 68 (1994) 7169-7177 and 69 (1995) 6880-6885).

It is believed that all Rep proteins, mainly Rep78 and Rep68, exhibit regulatory activities, such as induction and inhibition of AAV genes and inhibition of cell growth (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).

Recombinant overexpression of Rep78 produces a phenotype of reduced cell growth caused by induced DNA damage. As a result, host cells are arrested in the S phase, thereby promoting latent viral infection (Berthet, C. et al., Proc. Natl. Acad. Sci. USA 102 (2005) 13634-13639).

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 Rep protein expression, it has been reported that expression is only very low in some cell lines after stable integration of AAV (see, for example, Mendelson et al., Virol. 166 (1988) 154-165).

The cap locus contains a promoter called P40. Promoter P40 is operatively linked to a nucleic acid sequence providing 2.6 kb mRNA, which encodes the Cap proteins VP1 (87 kDa, unspliced mRNA transcript), VP2 (72 kDa, spliced mRNA transcript), and VP3 (61 kDa, from an alternative start codon) via alternative splicing and the use of alternative start codons. VP1 through VP3 constitute the components of the viral capsid. The capsid functions to bind to cell surface receptors and allow intracellular transport of the virus. VP3 accounts for approximately 90% of the total protein in the viral particle. However, all three proteins are essential for efficient capsid production.

It has been reported that inactivation of all three capsid proteins, VP1 through VP3, prevents the accumulation of single-stranded progenitor AAV DNA. Mutations at the N-terminus of VP1 (“Lip-negative” or “Inf-negative”) still allow single-stranded DNA to assemble into viral particles, thereby significantly reducing the infectious titer.

The AAP open reading frame encodes the assembly activation protein (AAP). It is approximately 22 kDa in size and transports the native VP protein to the nucleolar region for capsid assembly. This open reading frame is located upstream of the VP3 protein coding sequence.

A single AAV particle contains only one single-stranded DNA molecule. This can be either a "positive" strand or a "negative" strand. AAV viral particles containing DNA molecules are infectious. Inside an infected cell, the parental single-stranded virus is converted to a double-stranded DNA molecule, which is then amplified. This amplification produces a large number of double-stranded DNA molecules, which replace the single strand and are packaged into the capsid.

Adeno-associated virus (AAV) vectors can transduce both dividing and resting cells. It can be assumed that transgenes introduced into target cells using AAV vectors will be expressed long-term. One drawback of using AAV vectors is that the size of the transgene that can be introduced into cells is limited.

Viral vectors, such as parvovirus particles, including AAV serotypes and variants, provide a means of delivering nucleic acids ex vivo, in vitro, and in vivo into cells encoding proteins, enabling the cells to express the encoded proteins. AAVs are viruses that can be used as gene therapy vectors because they can penetrate cells and introduce nucleic acids/genetic material, allowing the nucleic acids/genetic material to be stably maintained within the cell. Furthermore, these viruses can, for example, introduce nucleic acids/genetic material into specific sites. Because AAVs are not associated with pathogenic diseases in humans, AAV vectors can deliver heterologous polynucleotide sequences (e.g., therapeutic proteins and drugs) to human patients without inducing substantial AAV pathogenesis or disease.

The viral vectors that can be used include, but are not limited to, adeno-associated virus (AAV) particles of various serotypes (e.g., AAV-1 to AAV-12, etc.) and hybrid/chimeric AAV particles.

Advantageously, AAV particles can be used as a medium for efficient gene delivery. These particles possess many of the characteristics required for such applications, including tropism towards both dividing and non-dividing cells. Early clinical experience with these vectors has also indicated no persistent toxicity and minimal or undetectable immune responses. AAVs are known to infect multiple cell types in vivo and in vitro via receptor-mediated endocytosis or transcytosis. These vector systems have been tested in humans targeting retinal epithelium, liver, skeletal muscle, airway, brain, joints, and hematopoietic stem cells.

Recombinant AAV particles typically do not include viral genes associated with pathogenesis. Such vectors usually have one or more of the wild-type AAV genes, such as the rep and/or cap genes, which are wholly or partially missing, but retain at least one functional flanking ITR sequence, which is essential for rescuing, replicating, and packaging the recombinant vector into the AAV particle. Alternatively, only the basic parts of the vector may be included, such as ITR and LTR elements, respectively. Therefore, the AAV vector genome will include the cis sequences (e.g., functional ITR sequences) required for replication and packaging.

Recombinant AAV vectors, methods thereof, and uses include any viral strain or serotype. As a non-limiting example, recombinant AAV vectors can be based on any AAV genome, such as, for example, 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 different from each other. As a non-limiting example, a recombinant AAV vector based on a serotype genome can be identical to one or more of the capsid proteins packaging the vector. Additionally, the recombinant AAV vector genome can be based on an AAV (e.g., AAV2) serotype genome that is different from one or more of the AAV capsid proteins packaging the vector. For example, the AAV vector genome can be based on AAV2, and at least one of the three capsid proteins can be AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV rh74, AAV 7m8, or variants thereof. AAV variants include variants and chimeras of the capsids of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV rh74, and AAV 7m8.

In all aspects and in certain embodiments, the adeno-associated virus (AAV) vector includes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV rh74, and AAV7m8 and variants thereof (e.g., capsid variants, such as amino acid insertions, additions, substitutions, and deletions), for example, as shown in WO 2013/158879, WO 2015/013313, and US2013/0059732 (which discloses LK01, LK02, LK03, etc.).

AAV and AAV variant (e.g., capsid variant) serotypes (e.g., VP1, VP2 and/or VP3 sequences) may be different from or may not be different from other AAV serotypes, including, for example, AAV1-AAV12 (e.g., VP1, VP2 and/or VP3 sequences that are different from any of the AAV1 to AAV12 serotypes).

In all aspects and in certain embodiments, the AAV particles associated with the reference serotype have a polynucleotide, polypeptide, or a subsequence thereof that includes or consists of the following: 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 sequences to one or more AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV rh74, or AAV 7m8 (e.g., such as ITR, or VP1, VP2, and/or VP3 sequences).

The compositions, methods, and uses of the present invention include AAV sequences (peptides and nucleotides) and their subsequences that exhibit less than 100% sequence identity with reference AAV serotypes (such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV rh74, or AAV7m8), but are different from 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 genes or proteins). In all aspects and in certain embodiments, the AAV polypeptide or its subsequence includes or consists of the following: a sequence that is at least 75% or more identical (e.g., 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) to any reference AAV sequence or its subsequence (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)). In some embodiments, the AAV variant has 1, 2, 3, 4, 5, 5 to 10, 10 to 15, 15 to 20 or more amino acid substitutions.

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 variants, related, heterozygous, and chimeric sequences, can be constructed using recombinant techniques known to those skilled in the art to include one or more nucleic acid sequences (transgenics) side-joined with one or more functional AAV ITR sequences.

Recombinant particles (e.g., rAAV particles) can be incorporated into pharmaceutical compositions. Such pharmaceutical compositions are particularly suitable for administration and delivery to subjects, either in vivo or ex vivo. In some embodiments, the pharmaceutical composition contains a pharmaceutically acceptable carrier or excipient. Such excipients include any agent that does not induce an immune response harmful to the individual receiving the composition and can be administered without excessive toxicity.

Protocols for generating adenovirus vectors have been described in US 5,998,205; US 6,228,646; US 6,093,699; US 6,100,242; WO 94/17810 and WO 94/23744, the entire contents of which are incorporated herein by reference.

Specific embodiments of the method according to the present invention

To allow for the determination of viral genome copy numbers, the viral genome must be made accessible, i.e., the protective capsid must be opened. Thermal denaturation is a convenient and commonly used method for this purpose. However, it has been found that thermal denaturation artificially reduces the viral genome copy number.

The inventors have demonstrated that thermal denaturation at temperatures above 95°C (such as, for example, 98°C) results in a reduction in the defined viral genome copy number. This has been illustrated using ATCC AAV2 standard VR-1616. The nominal viral genome copy number (vgcn) of the batch used in the experiment was 3.28 × 10⁻¹⁰ vg/mL. The results are shown in Table 1 below.

Table 1:

condition Absolutely vgcn Compared to VGCN No pretreatment 2.735-3.306*10E10/mL 83-101% Heat denaturation, 98℃, 10 minutes 1.012*10E10/mL 31%

Similarly, the inventors have demonstrated that incubation with proteinase K (PK) in aqueous or buffer solutions in the absence of detergent also results in a reduction in the determined viral genome copy number. This has been illustrated using ATCC AAV2 standard VR-1616. The nominal viral genome copy number (vgcn) of the batch used in the experiment was 3.28 × 10⁸ × 10⁸ vg/mL. The results are shown in Table 2 below.

Table 2:

In combination, the inventors have demonstrated that thermal denaturation at temperatures above 95°C (such as, for example, 98°C), even when combined with incubation in a buffer solution with proteinase K (PK) without detergent, results in a reduction in the determined viral genome copy number. This has been illustrated using ATCC AAV2 standard VR-1616. The nominal viral genome copy number (vgcn) of the batch used in the experiments was 3.28 × 10⁻¹⁰ vg/mL. The results are shown in Table 3 below.

Table 3:

The inventors have now discovered that the reduction in the determined viral genome copy number can be overcome by incubating with proteinase K in the presence of sodium dodecyl sulfate (SDS). This was demonstrated in this example using ATCC AAV2 standard VR-1616. The nominal viral genome copy number (vgcn) of the batch used in the experiment was 3.28 × 10⁻¹⁰ vg/mL. The results are shown in Table 4 below.

Table 4:

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

The inventors have further demonstrated that treatment with DNase I prior to determination of the viral genome number results in a reduction of the determined viral copy number. This has been illustrated using ATCC AAV2 standard VR-1616. The nominal viral genome copy number (vgcn) of the batch used in the experiments was 3.28 × 10⁻¹⁰ vg/mL. The results are shown in Table 5 below.

Table 5:

The reduction induced by DNase I treatment was independent of further sample processing, i.e., unrelated to heat denaturation or proteinase K incubation. Table 6 summarizes the different test conditions, all of which showed a definitive reduction in viral copy number. ATCC AAV2 standard VR-1616 was used. The nominal viral genome copy number (vgcn) of the batch used in the experiments was 3.28 × 10⁸ × 10⁸ vg/mL.

Table 6:

The inventors' findings were confirmed using lysates from HEK293 cell cultures producing AAV2. The viral genome copy number (vgcn) determined in the lysates was 1.746 × 10⁻¹⁰ vg/mL (lysate 18). The results are shown in Table 7 below.

As can be seen, incubation with proteinase K allowed for a viral genome recovery rate of 96%. The recovery rate could be further increased to 100% by incubating the sample with proteinase K in the presence of SDS.

Table 7:

It must be pointed out that the lysate incubated with proteinase K was not further purified; that is, for example, there was no column-based purification or extraction purification.

The determined viral genome copy number after treatment with additional DNase I was only in the range of 4% to 16%, as shown in Table 8 below.

Table 8:

The inventors have discovered that, for sequential incubation with DNase I and proteinase K, it is advantageous to use the entire incubation mixture incubated with DNase I for proteinase K incubation. This allows the determined viral genome copy number to be increased to 80% of the intended amount. The intended viral genome copy number (vgcn) in the lysates was 1.746 × 10⁻¹⁰ vg/mL (lysate 18) and 2.340 × 10⁻⁹ vg/mL (lysate 31). This effect may not be observed in purified samples, such as ATCC standards (nominal viral genome copy number (vgcn) of 3.28 × 10⁻¹⁰ vg/mL). The results are shown in Table 9.

Table 9:

The inventors have discovered that incubation using a combination of DNase I and proteinase K can achieve viral genome recovery rates exceeding 80% without dilution of cell lysates purified by affinity chromatography. The predetermined viral genome copy numbers (vgcn) in the affinity-purified lysates were 5.110 × 10⁻¹⁰ vg/mL (affinity-purified lysate 31) and 7.320 × 10⁻¹⁰ vg/mL (affinity-purified lysate 33). The results are shown in Table 10.

Table 10:

Viral genome quantification

For proper viral genome quantification to be possible, the sample must be free of plasmid DNA and unpackaged vector genome, both of which must contain at least a portion of the viral genome. Furthermore, the packaged AAV genome must be available from the first PCR cycle.

To remove unpackaged DNA that may interfere with the ddPCR process, DNase I digestion is typically used.

To enable ddPCR of encapsulated DNA, the capsid needs to be opened. This can be accomplished through high-temperature incubation or digestion with proteinase K. The need for proteinase K digestion has been extensively discussed in the art.

Microdroplet digital polymerase chain reaction

Droplet digital PCR (ddPCR) allows for absolute quantification of viral genomes without the need to generate a standard curve.

More specifically, droplet digital polymerase chain reaction (ddPCR) achieves absolute quantification of nucleic acids by randomly distributing the PCR reaction mixture into discrete partitions, some of which lack the target nucleic acid sequence and others contain one or more copies of the template (Hindson, B. et al., Anal. Chem. 83 (2011) 8604-8610). Due to the partitioning, thousands of independent PCR reactions are performed during thermal cycling. At the endpoint, the fraction of target-positive partitions is read out and used to calculate the initial template DNA concentration (Pinheiro, L. et al., Anal. Chem. 84 (2011) 1003-1011).

First, up to 20,000 droplets of approximately 1 nL each are formed in an oil-water emulsion. The PCR reaction mixture is then partitioned, consisting of a nucleic acid template, forward (fwd) and reverse (rev) primers, a TaqMan probe, and a ddPCR ultramix 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). During thermal cycling, individual PCR reactions are performed in each droplet, depending on the presence or absence of the DNA target.

In a droplet 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. As the probe degrades into smaller fragments, the 5' fluorophore is no longer close to its 3' quencher. This eliminates signal quenching and generates a fluorescent signal. Parts lacking the template sequence do not show amplification and therefore do not exhibit TaqMan probe hydrolysis or fluorescence generation, as the fluorescence of the 5' fluorophore remains quenched (see, for example, Holland, P. et al., Proc. Natl. Acad. Sci. USA 88(1991) 7276-7580). Because probes with different fluorophores and different excitation and emission wavelengths can be used, ddPCR reactions can be performed as multiplex reactions within a single droplet. The 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, for example, Furuta-Hanawa, B. et al. Hum. Gen. Therap. Meth. 30 (2019) 127-136).

As an endpoint analysis, the fluorescence signal of each droplet after thermal cycling is read out. The copy number (λ) of the target sequence can be calculated using Poisson statistics according to Equation 1 from the ratio (p) of positive readings to total readings (see, for example, Hindson, B. et al. (2011)).

λ = - ln (1 – p) (1)

Because ddPCR relies on endpoint measurements, target sequence quantification is to some extent independent of PCR reaction efficiency. This contrasts with real-time PCR (qPCR), which is typically used for viral genome titration (see, for example, Taylor, S et al. (2017)). Furthermore, the use of standards or calibration samples is not required (see, for example, Dorange, F., Bec, C., Cell Gen. Therap. Ins. 4 (2018) 119-129).

Recombinant AAV particles

Various methods for generating rAAV particles are known in the art. For example, transfection using an AAV plasmid and an AAV helper sequence, co-infection with an AAV helper virus (e.g., adenovirus, herpesvirus, or vaccinia virus), or transfection using a recombinant AAV plasmid, an AAV helper plasmid, and a helper functional plasmid. Non-limiting methods for generating rAAV particles are described, for example, in US 6,001,650, US 6,004,797, WO 2017/096039, and WO 2018/226887. After recombinant rAAV particle production (i.e., particle generation in a cell culture system), rAAV particles can be obtained from host cells and cell culture supernatant and purified.

For the generation of recombinant AAV particles, Rep and Cap proteins, accessory proteins E1A, E1B, E2A, and E4orf6, and adenovirus VA RNA need to be expressed in a single mammalian cell. Accessory proteins E1A, E1B, E2A, and E4orf6 can be expressed using any promoter, as shown by Matsushita et al. (Gene Ther. 5 (1998) 938-945), especially the CMV IE promoter. Therefore, any promoter can be used.

Generally, to produce recombinant AAV particles, different complementary plasmids are co-transfected into host cells. One of these plasmids contains a transgene sandwiched between two cis-acting AAV ITRs. The deleted AAV elements (i.e., open reading frames targeting Rep and Cap proteins) required for progeny recombinant genome replication and subsequent packaging are contained in trans form on the second plasmid. Overexpression of the Rep protein causes inhibition of cell growth (Li, J. et al., J. Virol. 71 (1997) 5236-5243). In addition, AAV replication requires a third plasmid containing genes from helper viruses (i.e., E1, E4orf6, E2A, and VA from adenovirus).

To reduce the number of plasmids required, Rep, Cap, and adenovirus helper genes can be combined on a single plasmid.

Alternatively, the host cells may have already stably expressed the E1 gene product. Such cells are HEK293 cells. The human embryonic kidney clone, designated 293, was first generated in 1977 by integrating adenovirus DNA into human embryonic kidney cells (HEK cells) (Graham, F.L. et al., J. Gen. Virol. 36 (1977) 59-74). The HEK293 cell line contains base pairs 1 through 4344 of the adenovirus serotype 5 genome. This covers the E1A and E1B genes as well as adenovirus packaging signals (Louis, N. et al., Virology 233 (1997) 423-429).

When using HEK293 cells, the missing E2A, E4orf6, and VA genes can be introduced by co-infection with adenovirus or by co-transfection with plasmids expressing E2A, E4orf6, and VA (see, for example, Samulski, R.J. et al., J.Virol. 63 (1989) 3822-3828; Allen, J.M. et al., J.Virol. 71 (1997) 6816-6822; Tamayose, K. et al., Hum. Gene Ther. 7 (1996) 507-513; Flotte, T.R. et al., Gene Ther. 2 (1995) 29-37; Conway, J.E. et al., J.Virol. 71 (1995) 29-37). 7) 8780-8789; Chiorini, J.A. et al., Hum. Gene Ther. 6 (1995) 1531-1541; Ferrari, F.K. 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 may be used (see, for example, Conway, J.E. et al., J.Virol. 71 (1997) 8780-8789; Johnston, K.M. et al., Hum. Gene Ther. 8 (1997) 359-370; Thrasher, A.J. et al., Gene Ther. 2 (1995) 481-485; Fisher, J.K. et al., Hum. Gene Ther. 7 (1996) 2079-2087; Johnston, K.M. et al., Hum. Gene Ther. 8 (1997) 359-370).

Therefore, cell lines that integrate and express the rep gene tend to grow slowly or express the Rep protein at very low levels.

To restrict transgene activity to a specific tissue, i.e., to a restricted integration site, the transgene can be operatively linked to an inducible or tissue-specific promoter (see, for example, Yang, Y. et al. Hum. Gene. Ther. 6 (1995) 1203-1213).

One challenge in rAAV particle production is the inefficient packaging of the rAAV carrier, resulting in low titers. Packaging has been difficult for several reasons, including:

- Preferred capsiding of the wild-type AAV genome in its presence;

-Due to the repressive effects associated with the rep gene product, it is difficult to generate sufficient complementary functions, such as those provided by the wild-type rep and cap genes;

- The efficiency of co-transfection of plasmid constructs is limited.

All of this is based on the biological properties of Rep proteins. In particular, the inhibitory (cell-suppressive and cytotoxic) properties of Rep proteins and their ability to reverse the immortalization phenotype of cultured cells are problematic. In addition, Rep proteins downregulate their own expression when the widely used AAV P5 promoter is employed (see, for example, Tratschin et al., Mol. Cell. Biol. 6 (1986) 2884-2894).

In all aspects and in some embodiments, the rAAV particles are derived from AAVs selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, Rh.10, Rh74, and 7m8.

In all aspects and in certain embodiments, the rAAV particle comprises a capsid sequence having 70% or higher sequence identity with the capsid sequence of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, Rh.10, Rh74, or 7m8.

In all aspects and in certain embodiments, the rAAV particle comprises an ITR sequence having 70% or higher sequence identity with an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10 ITR sequence.

E1A, E1B, E2 and E4

The coding sequences (open reading frames) for E1A and E1B can be derived from human adenoviruses, such as, for example, human adenovirus serotype 2 or serotype 5, in particular. Exemplary sequences of human Ad5 (adenovirus serotype 5) are found in GenBank entries X02996, AC_000008, and exemplary sequences of human Ad2 are found in GenBank entry AC_000007. Nucleotides 505 to 3522 contain the nucleic acid sequences encoding E1A and E1B of human adenovirus serotype 5. Plasmid pSTK146 reported in EP 1 230 354 and plasmids pGS119 and pGS122 reported in WO2007/056994 can also be used as sources of the open reading frames for E1A and E1B.

E1A is the first viral accessory gene expressed after adenovirus DNA enters the cell nucleus. The E1A gene encodes the 12S and 13S proteins, which are selectively spliced from the same E1A mRNA. Expression of the 12S and 13S proteins activates other viral functions, E1B, E2, E3, and E4. Furthermore, expression of the 12S and 13S proteins forces the cell into the S phase of the cell cycle. If only E1A-derived proteins are expressed, the cell will die (apoptosis).

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

The E2 gene encodes various proteins. The E2A transcript encodes a single-stranded binding protein (SSBP), which is crucial for AAV replication.

The E4 gene also encodes a variety of proteins. The 34kDa protein derived from the E4 gene (E4orf6), along with the 55kDa E1B protein, prevents the accumulation of cellular mRNA in the cytoplasm, but also promotes the transport of viral RNA from the nucleus to the cytoplasm.

Adenovirus VA RNA gene

Virus-associated RNA (VA RNA) is the 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 from a type 2 polymerase III promoter by RNA polymerase III (see, for example, Machitani, M. et al., J. Contr. rel. 154 (2011) 285-289). For recombinant production, the adenovirus VA RNA gene can be driven by any promoter.

Ma, Y. and Mathews, M.B. (J. Virol. 70 (1996) 5083-5099) used phylogenetic methods to study the structure, function, and evolution of adenovirus-associated RNAs. They provided alignments based on 47 known human adenovirus serotypes and shared VA RNA sequences. The disclosure described herein is incorporated herein by reference in its entirety.

VA RNA, VAI, and VAII consist of 157 to 160 nucleotides (nt).

Depending on the serotype, adenoviruses contain one or two VA RNA genes. VA RNAI is thought to play a major proviral role, while VA RNAII can partially compensate for the absence of VA RNAI (Vachon, V.K. and Conn, G.L., VirusRes. 212 (2016) 39-52).

VA RNA is not essential, but it plays a crucial role in effective viral growth by overcoming cellular antiviral mechanisms. That is, although VA RNA is not essential for viral growth, adenoviruses lacking VA RNA cannot grow during the initial steps of vector generation, with only a few copies of the viral genome present in each cell, possibly because viral genes that block cellular antiviral mechanisms other than VA RNA may not be adequately expressed (see Maekawa, A. et al., Nature Sci. Rep. 3 (2013) 1136).

Maekawa, A. et al. (Nature Sci. Rep. 3 (2013) 1136) reported the efficient production of adenovirus vectors lacking genes for virus-associated RNA that disrupt cellular RNAi mechanisms, in which HEK293 cells composed of and highly expressing flippant recombinase were infected to obtain VA RNA-deficient adenovirus via FLP recombinase-mediated excision of the VA RNA locus.

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

Method for producing rAAV particles

Carter et al. have shown that the entire rep and cap open reading frames in the wild-type AAV genome can be deleted and replaced with transgenes (Carter, B.J., "Handbook of Parvoviruses", edited by P. Tijssen, CRC Press, pp. 155-168 (1990)). Furthermore, it has been reported that ITRs must be maintained to preserve their function of replicating, rescuing, packaging, and integrating transgenes into the target cell genome.

When cells containing the corresponding viral helper gene are transduced by an AAV vector, or vice versa, when cells containing integrated AAV provirus are transduced by a suitable helper virus, the AAV provirus is activated and re-enters the lysis infection cycle (Clark, K.R. et al., Hum. Gene Ther. 6 (1995) 1329-1341; Samulski, R.J., Curr. Opin. Genet. Dev. 3 (1993) 74-80).

An aspect of the invention is a method for transducing cells with nucleic acids (e.g., plasmids) containing all the necessary elements for producing recombinant AAV particles, wherein the viral genome copy number is determined using the method according to the invention. Thus, when the plasmid encodes viral packaging proteins and/or accessory proteins, the cells can produce recombinant viral particles comprising nucleic acids encoding a target protein or containing a sequence transcribed into a target transcript.

The present invention provides a virus (e.g., AAV) particle production platform that includes features that distinguish it from current 'industry standard' virus (e.g., AAV) particle production processes by using a method according to the present invention.

More generally, cells transfected or transduced with DNA for the recombinant production of AAV particles can be called “recombinant cells.” These cells can be, for example, yeast cells, insect cells, or mammalian cells, and have been used as recipients for nucleic acids (plasmids) encoding packaging proteins (such as AAV packaging proteins), nucleic acids (plasmids) encoding accessory proteins, and nucleic acids (plasmids) encoding proteins or transcribed into the target transcript (i.e., transgenes placed between two AAV ITRs). The term includes the offspring of the original cell that has been transduced or transfected. It should be understood that the offspring of a single parent cell may not necessarily be identical to the original parent in morphology or genome or total nucleic acid complementarity due to natural, accidental, or intentional mutations.

Many 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 those used to maintain the viability of mammalian (e.g., human) cells or to provide 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 may 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.

Helper protein particles can take the form of plasmids, bacteriophages, transposons, or granules. In particular, it has been shown that helper functions do not require complete complementation of adenoviral genes. For example, adenoviral mutants that are unable to replicate DNA and synthesize late-stage genes have been shown to allow AAV replication. (Ito et al., J. Gen. Virol. 9 (1970) 243; Ishibashi et al., Virology 45 (1971) 317.)

Mutants within the E2B and E3 regions have been shown to support AAV replication, indicating that the E2B and E3 regions may not be involved in providing accessory functions. (Carter et al., Virology 126 (1983) 505). However, adenoviruses with defects in the E1 region or lacking the E4 region cannot support AAV replication. Therefore, for adenoviral accessory proteins, the E1A and E4 regions may be directly or indirectly necessary for AAV replication (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 characteristic adenovirus mutants include: E1B (Laughlin et al. (1982), ibid.; Jank et al. (1981), ibid.; 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. U. SA 78 (1981) 2927; Myers et al., J. Biol. Chem. 256 (1981) 567); E2B (Carter, Adeno-Associated Virus Helper Functions, I CRC Handbook of Parvoviruses (edited by P. Tijssen, 1990)); E3 (Carter et al. (1983), ibid.); and E4 (Carter et al. (1983), ibid.; Carter (1995)).

Studies of accessory proteins provided by E1B mutant adenoviruses have been reported; the E1B 55kDa protein is essential for AAV particle production, while the E1B 19kDa protein is not. Furthermore, WO 97/17458 and Matshushita et al. (Gene Therapy 5 (1998) 938-945) describe accessory functional plasmids encoding various adenovirus genes. Examples of accessory plasmids include adenovirus VA RNA coding regions, adenovirus E4orf6 coding regions, adenovirus E2A 72kDa coding regions, adenovirus E1A coding regions, and adenovirus E1B regions lacking the complete E1B 55kDa coding region (see, for example, WO 01/83797).

Therefore, this document provides a method for producing a recombinant AAV vector or AAV particles containing said recombinant AAV vector using the method for determining viral genome number according to the present invention, wherein the recombinant AAV vector contains nucleic acids encoding proteins or transcribed into target transcripts.

One aspect of the present invention is a method for producing a recombinant AAV vector or AAV particles containing said recombinant AAV vector, the recombinant AAV vector containing nucleic acid encoding a protein or transcribed into a target transcript, the method comprising the following steps:

(i) Provide one or more plasmids containing nucleic acids encoding AAV packaging proteins and/or nucleic acids encoding accessory proteins;

(ii) Provide a plasmid containing nucleic acid encoding the target protein or transcribed into the target transcript;

(iii) Contact one or more mammalian or insect cells with a provided plasmid;

(iv) Further add transfection reagent and optionally incubate the plasmid/transfection reagent/cell mixture;

Alternatively, physical means, such as electric current, can be used to introduce nucleic acids into cells;

(v) Culture the transfected cells;

(vi) Harvesting cultured cells and/or culture media derived from cultured cells to produce cell and/or culture media harvests; and

(vii) Lyse the cells and optionally isolate the recombinant AAV vector or AAV particles from the cell and/or culture medium harvest lysate;

(viii) Determine the viral genome copy number in or after step (vi) and/or step (vii) using the method according to the invention;

This allows for the production of recombinant AAV vectors or AAV particles containing nucleic acids encoding the target protein or transcribed into the target transcript.

One aspect of the present invention is a method for producing a recombinant AAV vector or AAV particles containing said recombinant AAV vector, the recombinant AAV vector containing nucleic acid encoding a protein or transcribed into a target transcript, the method comprising the following steps:

(i) Provide one or more plasmids containing nucleic acids encoding AAV packaging proteins and/or nucleic acids encoding accessory proteins;

(ii) Provide a plasmid containing nucleic acid encoding the target protein or transcribed into the target transcript;

(iii)

(a) Generating stably transfected cells by the following methods

- To bring one or more mammalian or insect cells into contact with the provided plasmid of (i);

- Further add transfection reagent and optionally incubate the plasmid/transfection reagent/cell mixture; or provide physical means, such as electric current, to introduce nucleic acids into the cells;

- Select the first stable transfected cells;

- Contact the selected first stable transfected cells with the provided plasmid from (ii); and

- Further add transfection reagent and optionally incubate the plasmid/transfection reagent/cell mixture; or provide physical means, such as electric current, to introduce nucleic acids into the cells;

(b) or generate transiently transfected cells by the following methods

- To contact one or more mammalian or insect cells with the provided plasmids (i) and (ii); and

- Further add transfection reagent and optionally incubate the plasmid/transfection reagent/cell mixture; or provide physical means, such as electric current, to introduce nucleic acids into the cells;

(iv) Culture of (iii) transfected cells;

(v) Harvesting cultured cells and/or culture media derived from cultured cells to produce cell and/or culture media harvests;

(vi) Lyse the cells and optionally isolate the recombinant AAV vector or AAV particles from the cell and/or culture medium harvest lysate;

(vii) Determine the viral genome copy number in or after step (v) and/or step (vi) using the method according to the invention;

This allows for the production of recombinant AAV vectors or AAV particles containing nucleic acids encoding the target protein or transcribed into the target transcript.

One aspect of the present invention is a method for producing a recombinant AAV vector or AAV particles containing said recombinant AAV vector, the recombinant AAV vector containing nucleic acid encoding a protein or transcribed into a target transcript, the method comprising the following steps:

(i) Providing mammalian or insect cells containing nucleic acids encoding AAV packaging proteins and/or nucleic acids encoding accessory proteins;

(ii) Provide a plasmid containing nucleic acid encoding the target protein or transcribed into the target transcript;

(iii)

(a) Generating stably transfected cells by the following methods

- To bring one or more mammalian or insect cells into contact with the provided plasmid of (i);

- Further add transfection reagent and optionally incubate the plasmid/transfection reagent/cell mixture; or provide physical means, such as electric current, to introduce nucleic acids into the cells;

- Select the first stable transfected cells;

- Contact the selected first stable transfected cells with the provided plasmid from (ii); and

- Further add transfection reagent and optionally incubate the plasmid/transfection reagent/cell mixture; or provide physical means, such as electric current, to introduce nucleic acids into the cells;

(b) or generate transiently transfected cells by the following methods

- To contact one or more mammalian or insect cells with the provided plasmids (i) and (ii); and

- Further add transfection reagent and optionally incubate the plasmid/transfection reagent/cell mixture; or provide physical means, such as electric current, to introduce nucleic acids into the cells;

(iv) Culture of (iii) transfected cells;

(v) Harvesting cultured cells and/or culture media derived from cultured cells to produce cell and/or culture media harvests;

(vi) Lysing cells and optionally isolating and/or purifying the recombinant AAV vector or AAV particles from the cell and/or culture medium harvest; and

(vii) Determine the viral genome copy number in or after step (v) and/or step (vi) using the method according to the invention;

This allows for the production of recombinant AAV vectors or AAV particles containing nucleic acids encoding the target protein or transcribed into the target transcript.

Nucleic acid (plasmid) can be introduced into cells in a variety of ways.

Various methods for transferring DNA into mammalian cells have been reported in the art. These can all be used in the methods according to the present invention. In some embodiments of all aspects and examples, electroporation, nuclear transfection, or microinjection for nucleic acid transfer/transfection are used. In some embodiments of all aspects and examples, inorganic substances (such as, for example, calcium phosphate/DNA coprecipitation), cationic polymers (such as, for example, polyethyleneimine, DEAE-glucan), or cationic lipids (lipotransfection) for nucleic acid transfer/transfection are used. Calcium phosphate and polyethyleneimine are the most commonly used reagents for transfection in large-scale nucleic acid transfer (see, for example, Baldi et al., Biotechnol. Lett. 29 (2007) 677-684), with polyethyleneimine being preferred.

In some embodiments across all aspects and examples, the nucleic acid (plasmid) is provided in the form of a composition in combination with polyethyleneimine (PEI), optionally in combination with cells. In some embodiments, the composition comprises a plasmid/PEI mixture having several components: (a) one or more plasmids containing nucleic acids encoding AAV packaging proteins and/or nucleic acids encoding accessory proteins; (b) plasmids containing nucleic acids encoding proteins or transcribed into target transcripts; and (c) a polyethyleneimine (PEI) solution. In some embodiments, the molar ratio of plasmids ranges from about 1:0.01 to about 1:100, or from about 100:1 to about 1:0.01, and the mixture of components (a), (b), and (c) has optionally been incubated for a period of about 10 seconds to about 4 hours.

In all aspects and in certain embodiments, the composition further comprises cells. In some embodiments, the cells are contacted with the plasmid/PEI mixture of components (a), (b), and/or (c).

In all aspects and in certain embodiments, the composition optionally combined with cells further comprises free PEI. In some embodiments, the cells are contacted with free PEI.

In all aspects and in certain embodiments, the cells have been contacted with a 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 have been contacted with a mixture of components (a), (b), and/or (c) and optionally free PEI for at least about 4 hours.

The composition may further comprise plasmids and/or cells. Such plasmids and cells may be contacted with free PEI. In some embodiments, the plasmids and/or cells have been contacted with free PEI for at least about 4 hours, or about 4 hours to about 140 hours, or about 4 hours to about 96 hours.

The present invention also provides a method for producing transfected cells. The method includes providing one or more plasmids; providing a solution containing polyethyleneimine (PEI); and mixing the plasmid with the PEI solution to produce a plasmid/PEI mixture. In some embodiments, such a mixture is incubated for a period ranging from about 10 seconds to about 4 hours. In such methods, cells are then contacted with the plasmid/PEI mixture to produce a plasmid/PEI cell culture; then free PEI is added to the produced plasmid/PEI cell culture to produce a free PEI/plasmid/PEI cell culture; and then the produced free PEI/plasmid/PEI cell culture is incubated for at least about 4 hours to produce transfected cells. In some embodiments, the plasmid contains one or more or all of the following: a rep open reading frame, a cap open reading frame, E1A, E1B, E2, and E4orf6 open reading frames, and nucleic acids encoding proteins or transcribed into a target transcript.

Further, a method for producing transfected cells that produce recombinant AAV vectors or AAV particles is provided. This method includes providing one or more plasmids containing nucleic acids encoding AAV packaging proteins and/or nucleic acids encoding accessory proteins; providing plasmids containing nucleic acids encoding proteins or transcribed into target transcripts; providing a solution containing polyethyleneimine (PEI); and mixing the plasmids with the PEI solution, wherein the molar ratio of the plasmids ranges from about 1:0.01 to about 1:100, or from about 100:1 to about 1:0.01, to produce a plasmid/PEI mixture (and any...). The process involves incubating the plasmid/PEI mixture for a period ranging from about 10 seconds to about 4 hours; contacting the cells 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 to produce transfected cells that produce a recombinant AAV vector or particle containing nucleic acid encoding a protein or transcribed into a target transcript, wherein the viral genome copy number is determined using the method according to the invention.

Additionally, a method is provided for producing a recombinant AAV vector or AAV particle containing nucleic acid encoding a protein or transcribed into a target transcript, the method comprising: providing one or more plasmids containing nucleic acids encoding AAV packaging proteins and/or nucleic acids encoding accessory proteins; providing plasmids containing nucleic acids encoding a target protein or transcribed into a target transcript; providing a solution containing polyethyleneimine (PEI); mixing the plasmids with the PEI solution, wherein the molar ratio of the plasmids ranges from about 1:0.01 to about 1:100, or from about 100:1 to about 1:0.01, to produce a plasmid/PEI mixture (and optionally incubating the plasmid/PEI mixture for a period of time ranging from about 10 seconds to about 4 hours); and mixing cells with the plasmid/PEI produced as described above. The process involves contacting the cells with a medium to produce a plasmid/PEI cell culture; adding free PEI to the plasmid/PEI cell culture produced as described above 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 the 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 particles from the cell and/or culture medium harvest lysate, thereby determining the viral genome copy number in the lysate or isolated AAV particles using the method according to the invention; and thus producing a recombinant AAV vector or particles containing nucleic acids encoding proteins or transcribed into target transcripts.

A method for producing recombinant AAV vectors or AAV particles using the method according to the invention may 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 cell and/or culture medium harvests. Additional exemplary steps or features include, but are not limited to, lysing the harvested cells and optionally isolating the recombinant AAV vectors or AAV particles from the cell and/or culture medium harvest lysate; wherein the viral genome copy number is determined using the method according to the invention; thereby producing recombinant AAV vectors or AAV particles containing nucleic acids encoding proteins or transcribed into target transcripts.

In all aspects and in some embodiments, PEI is added to the plasmid and/or cells at different time points. In some embodiments, free PEI is added to the cells before, simultaneously with, or after contacting the plasmid/PEI mixture with the cells.

In certain embodiments across all aspects and examples, cells are at a specific 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 cell density is in the range of about 1 x 10E5 cells/mL to about 1 x 10E8 cells/mL when contacted with the plasmid/PEI mixture and/or with free PEI. In certain embodiments, cell viability is about 60% or greater than 60% when contacted with the plasmid/PEI mixture or with free PEI, or wherein cells are in the logarithmic growth phase when contacted with the plasmid/PEI mixture, or wherein cell viability is about 90% or greater when contacted with the plasmid/PEI mixture or with free PEI, or wherein cells are in the logarithmic growth phase when contacted with the plasmid/PEI mixture or with free PEI.

In some embodiments of all aspects and examples, the encoded AAV packaging proteins include AAV rep and/or AAV cap. In some embodiments of all aspects and examples, such AAV packaging proteins include AAVrep and/or AAV cap proteins of any AAV serotype.

In all aspects and in certain embodiments, the encoded accessory proteins include adenovirus E1A and E1B, adenovirus E2 and/or E4, VA RNA and/or non-AAV accessory proteins.

In all aspects and certain embodiments, nucleic acids (plasmids) are used in specific amounts or ratios. In some embodiments, the total amount of plasmids containing nucleic acids encoding proteins or transcribed into target transcripts and one or more plasmids containing nucleic acids encoding AAV packaging proteins and/or nucleic acids encoding accessory proteins is in the range of about 0.1 μg to about 15 μg per mL of cells. In some embodiments, the molar ratio of plasmids containing nucleic acids encoding proteins or transcribed into target transcripts to one or more plasmids containing nucleic acids encoding AAV packaging proteins and/or nucleic acids encoding accessory proteins is in the range of about 1:5 to about 1:1, or in the range of about 1:1 to about 5:1.

In all aspects and in some embodiments, the first plasmid contains nucleic acid encoding an AAV packaging protein and the second plasmid contains nucleic acid encoding an accessory protein.

In all aspects and in some embodiments, the molar ratio of the plasmid containing nucleic acid encoding a protein or transcribed into a target transcript to the first plasmid containing nucleic acid encoding an AAV packaging protein and the second plasmid containing nucleic acid encoding an accessory protein in co-transfection is in the range of about 1-5:1:1 or 1:1-5:1 or 1:1:1-5.

In all aspects and in some embodiments, the cells are eukaryotic cells. In some embodiments, the eukaryotic cells are mammalian cells. In a preferred embodiment, the cells are HEK293 cells or CHO cells.

Culture can be performed using standard conditions for culturing eukaryotic cells, such as approximately 37°C, 95% humidity, and 8% CO2 (by volume ₎ . Cultures can be carried out in serum-containing or serum-free media, adherent cultures, or suspension cultures. Suspension cultures can be performed in any fermentation vessel, such as, for example, stirred tank reactors, oscillating reactors, rocking bioreactors, shaker containers, rotating containers, or so-called roller flasks. Transfection can be performed in high-throughput formats and screening, for example, in 96- or 384-well formats.

The method according to the invention comprises AAV particles of any serotype or variants thereof. In certain embodiments of all aspects and examples, the recombinant AAV particles comprise any of the following: AAV serotypes 1 to 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 examples, the AAV particles comprise an AAV serotype or an AAV pseudotype, wherein the AAV pseudotype comprises an AAV capsid serotype different from the ITR serotype.

The method according to the invention, which provides or includes AAV vectors or particles, may 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 repeat (ITR) sequences and/or filler/filler polynucleotide sequences. Such elements may be located within or flanked by nucleic acids encoding proteins or transcribed into target transcripts, or expression control elements may be operatively linked to nucleic acids encoding proteins or transcribed into target transcripts, or AAV ITRs may be flanked by the 5' or 3' ends of nucleic acids encoding proteins or transcribed into target transcripts, or filler polynucleotide sequences may be flanked by the 5' or 3' ends of nucleic acids encoding proteins or transcribed into target transcripts.

Expression control elements include constitutive or adjustable control elements, such as tissue-specific expression control elements or promoters.

ITR can be any of the AAV2, AAV6, AAV8, or AAV9 serotypes, or a combination thereof. AAV particles may include any VP1, VP2, and/or VP3 capsid protein having 75% or higher sequence identity with 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 contain 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.

After producing recombinant viral (e.g., AAV) particles as described herein, a variety of conventional methods can be used to purify and/or isolate the viral (e.g., rAAV) particles from host cells, if desired. Such methods include column chromatography, CsCl gradients, iodixanol gradients, etc.

For example, multiple column purification steps can be used, such as purification via anion exchange column, affinity column, and/or cation exchange column (see, for example, WO 02/12455 and US 2003/0207439). Alternatively or additionally, iodixanol or CsCl gradient steps can be used (see, for example, US2012/0135515; and US2013/0072548). Furthermore, if an infectious virus is used to express packaging proteins and/or accessory proteins, various methods can be used to inactivate the residual virus. For example, adenovirus can be inactivated by heating to a temperature of approximately 60°C for, for example, 20 minutes or longer. This treatment effectively inactivates accessory viruses because AAV is thermostable, while accessory adenoviruses are thermolabile.

One goal of rAAV vector production and purification systems is to implement strategies to minimize/control the generation of production-related impurities, such as proteins, nucleic acids, and vector-related impurities, including wild-type/pseudo-wild-type AAV species (wtAAV) and residual DNA impurities encapsulated in AAV.

Given that rAAV particles constitute only a small fraction of the biomass, they need to be purified to a purity level suitable for use as clinical human gene therapy products (see, for example, Smith P.H. et al., Mo. Therapy 7 (2003) 8348; Chadeuf G. et al., Mo. Therapy 12 (2005) 744; report from CHMP gene therapy expert group meeting, European Medicines Agency EMEA/CHMP 2005, 183989/2004).

As an initial step, cultured cells producing rAAV particles are typically harvested and optionally combined with the harvested cell culture supernatant (culture medium) in which cells (suspension cells or adherent cells) producing rAAV particles have already been cultured. The harvested cells and optional cell culture supernatant can be used as is, lysed or concentrated as needed. Furthermore, if infection is used to express a helper function, any remaining helper virus can be inactivated. For example, adenovirus can be inactivated by heating to approximately 60°C for, for example, 20 minutes or longer; this only inactivates the helper virus, as AAV is thermostable while helper adenovirus is thermolabile.

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

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

After cell lysis, optional clarification, and optional dilution or concentration, rAAV particles can be purified using multiple subsequent and sequential chromatographic steps.

The first chromatographic step can be cation exchange chromatography or anion exchange chromatography. If the first chromatographic step is cation exchange chromatography, then the second chromatographic step can be anion exchange chromatography or size exclusion chromatography (SEC). Therefore, in all aspects and in some embodiments, rAAV particle purification is performed by cation exchange chromatography followed by anion exchange chromatography.

Alternatively, if the first chromatographic step is cation exchange chromatography, the second chromatographic step may be size exclusion chromatography (SEC). Therefore, in all aspects and in some embodiments, rAAV particle purification is performed via cation exchange chromatography followed by size exclusion chromatography (SEC).

Alternatively, the first chromatographic step can be affinity chromatography. If the first chromatographic step is affinity chromatography, then the second chromatographic step can be anion exchange chromatography. Therefore, in all aspects and in some embodiments, rAAV particle purification is performed via affinity chromatography followed by anion exchange chromatography.

Optionally, a third chromatographic step may be added to the aforementioned chromatographic steps. Typically, the optional third chromatographic step follows cation exchange, anion exchange, size exclusion, or affinity chromatography.

Therefore, in all aspects and in some embodiments, the purification of rAAV particles is performed by cation exchange chromatography, followed by anion exchange chromatography, and then by size exclusion chromatography (SEC).

Furthermore, in all aspects and certain embodiments, further purification of the rAAV particles is performed via cation exchange chromatography, followed by size exclusion chromatography (SEC), and then by anion exchange chromatography.

In a further embodiment of all aspects and embodiments, rAAV particle purification is performed by affinity chromatography, followed by anion exchange chromatography, and then by size exclusion chromatography (SEC).

In a further embodiment of all aspects and embodiments, rAAV particle purification is performed by affinity chromatography, followed by size exclusion chromatography (SEC), and then by anion exchange chromatography.

The purpose of cation exchange chromatography is to separate AAV particles from cells and other components present in clear lysates and/or column eluates from affinity chromatography or size exclusion chromatography. Examples of strong cation exchange resins capable of binding rAAV particles over a wide pH range include, but are not limited to, any sulfonic acid-based resins indicated by the presence of sulfonate functional groups, including 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). Other examples include Capto S, Capto SImpAct, Capto S ImpRes (strong cation exchangers, available from GE Healthcare, Marlborough, MA, USA), and commercial and family-of resins available from Aldrich Chemical Company (Milliwaukee, WI, USA). Weak cation exchange resins include, but are not limited to, any carboxylic acid-based resins. Exemplary cation exchange resins include carboxymethyl (CM) resins, phosphate resins (based on phosphate functional groups), methanesulfonate (S) resins, and sulfopropyl (SP) resins.

Anion exchange chromatography is used to separate AAV particles from proteins, cells, and other components present in clear lysates and/or column eluates from affinity, cation exchange, or size exclusion chromatography. Anion exchange chromatography can also be used to reduce and thus 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 mM to 125 mM, such as 110 mM to 115 mM) of NaCl, and a portion of the empty capsid can be eluted without a large amount of rAAV particles being eluted. Subsequently, a solution containing a higher concentration of NaCl (e.g., about 130 mM to 300 mM NaCl) can be used to elute the rAAV particles bound to the anion exchange column, resulting in a column eluate with a reduced or depleted amount of empty capsids and a proportionally increased amount of rAAV particles containing the rAAV carrier.

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 typically based on quaternary ammonium nitrogen atoms, including, but 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); and POROS SOD (weak anion exchanger, available from Applied Biosystems, Foster City, CA). The following are examples of anion exchange resins 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 (strong anion exchangers, available from GE Healthcare, Marlborough, MA, USA); DEAE SEPHAROSE (weak anion exchanger, available from Amersham Biosciences, Piscataway, NJ, USA); and Q SEPHAROSE (strong anion exchanger, available from Amersham Biosciences, Piscataway, NJ, USA). Other exemplary anion exchange resins include aminoethyl (AE), diethylaminoethyl (DEAE), diethylaminopropyl (DEPE), and quaternary aminoethyl (QAE).

The manufacturing process for purifying recombinant AAV particles intended as products for treating human diseases should achieve the following objectives: 1) consistent particle purity, potency, and safety; 2) scalability of the manufacturing process; and 3) acceptable manufacturing costs.

An exemplary procedure for purifying recombinant AAV particles is reported in WO 2019/006390.

The purification and production methods for recombinant adeno-associated virus (rAAV) particles outlined below are scalable to large-scale operations. For example, volumes to suspension cultures can be 5, 10, 10–20, 20–50, 50–100, 100–200, or more liters. These recombinant adeno-associated virus particle purification and production methods are applicable to a variety of AAV serotypes/capsid variants.

In all aspects and in some embodiments, the purification of rAAV particles includes the following steps:

(a) Harvesting cells and/or cell culture supernatants containing rAAV particles to produce harvest;

(b) Optionally concentrate the harvest produced in step (a) to produce concentrated harvest;

(c) The harvest produced in pyrolysis step (a) or the concentrated harvest produced in step (b) to produce pyrolyte;

(d) Process the lysate produced in step (c) to reduce contaminating nucleic acids in the lysate, thereby producing a lysate with reduced nucleic acids;

(e) Optionally filter the nucleic acid-reduced lysate produced in step (d) to produce a clarified lysate, and optionally dilute the clarified lysate to produce a diluted clarified lysate;

(f) The lysate of the reduced nucleic acid from step (d), the clarified lysate from step (e), or the diluted clarified lysate produced in step (e) is subjected to cation exchange column chromatography to produce a column eluent containing rAAV particles, thereby separating the rAAV particles from protein impurities or other production/process-related impurities, and optionally diluting the column eluent to produce a diluted column eluent.

(g) subject the column eluent or diluted column eluent produced in step (f) to anion exchange chromatography to produce a second column eluent containing rAAV particles, thereby separating the rAAV particles from protein impurities or production/process-related impurities, and optionally concentrate the second column eluent to produce a concentrated second column eluent.

(h) subjecting the second column eluent or concentrated second column eluent produced in step (g) to size exclusion column chromatography (SEC) to produce a third column eluent containing rAAV particles, thereby separating the rAAV particles from protein impurities or production/process-related impurities, and optionally concentrating the third column eluent to produce a concentrated third column eluent; and

(i) The third column eluent produced in step (h) or the concentrated third column eluent is filtered to produce purified rAAV particles;

The viral genome copy number is determined in or after one or more steps (a) to (i) using the method according to the invention.

In some embodiments, steps (a) through (f) are retained and combined with the following steps:

(g) subject the column eluent or concentrated column eluent produced in step (f) to size exclusion column chromatography (SEC) to produce a second column eluent containing rAAV particles, thereby separating the rAAV particles from protein impurities or other production/process-related impurities, and optionally dilute the second column eluent to produce a concentrated second column eluent.

(h) subjecting the second column eluent produced in step (g) or the diluted second column eluent to anion exchange chromatography to produce a third column eluent containing rAAV particles, thereby separating the rAAV particles from impurities related to the production/process of protein impurities, and optionally diluting the third column eluent to produce a diluted third column eluent; and

(i) The third column eluent produced in step (h) or the concentrated third column eluent is filtered to produce purified rAAV particles;

The viral genome copy number is determined in or after one or more steps (a) to (i) using the method according to the invention.

In some embodiments, steps (a) through (g) are retained and combined with the following steps:

(h) Filter the second column eluent produced in step (g) or concentrate the second column eluent to produce purified rAAV particles;

The viral genome copy number is determined in or after one or more steps (a) to (h) using the method according to the invention.

In the embodiment, steps (a) through (e) are retained and combined with the following steps:

(f) The lysate with reduced nucleic acid in step (d) or the clarified lysate or diluted clarified lysate produced in step (e) is subjected to AAV affinity column chromatography to produce a column eluate containing 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) subject the column eluent or concentrated column eluent produced in step (f) to size exclusion column chromatography (SEC) to produce a second column eluent containing rAAV particles, thereby separating the rAAV particles from protein impurities or other production/process-related impurities, and optionally dilute the second column eluent to produce a diluted second column eluent.

(h) Optionally, the second column eluent produced in step (g) or the diluted second column eluent is subjected to anion exchange chromatography to produce a third column eluent containing rAAV particles, thereby separating the rAAV particles from protein impurities or other production/process-related impurities, and optionally the third column eluent is diluted to produce a diluted third column eluent; and

(i) The second column eluent or diluted second column eluent produced in the filtration step (g), or the third column eluent or concentrated third column eluent produced in the filtration step (h), thereby producing purified rAAV particles.

The viral genome copy number is determined in or after one or more steps (a) to (i) using the method according to the invention.

In some embodiments of all aspects and examples, the concentration of steps (b) and/or steps (f) and/or steps (g) and/or steps (h) is performed via ultrafiltration/percolation, for example by tangential flow filtration (TFF).

In all aspects and in some embodiments, the concentration in step (b) reduces the volume of the harvested cells and cell culture supernatant by about 2 to 20 times.

In all aspects and in some embodiments, the concentration of steps (f) and/or steps (g) and/or steps (h) reduces the volume of column eluent by about 5 to 20 times.

In some embodiments across all aspects and examples, the pyrolysis of the harvest produced in step (a) or the concentrated harvest produced in step (b) is carried out 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 nonionic and ionic detergents. Non-limiting examples of nonionic detergents include Triton X-100. Non-limiting examples of detergent concentrations are between about 0.1% (v/v) or (w/v) and 1.0% (v/v) or (w/v), including extreme values.

In all aspects and in some embodiments, step (d) includes treatment with a nuclease to reduce nucleic acid contamination. Non-limiting examples of nucleases include benzonase.

In all aspects and in some embodiments, the filtration of the clarified pyrolyte or diluted clarified pyrolyte in step (e) is performed via a filter. Non-limiting examples of filters are those with pore sizes between about 0.1 micrometers and 10.0 micrometers (inclusive).

In some embodiments across all aspects and examples, the dilution of the clarified lysate in step (e) is performed using an aqueous buffered phosphate, acetate, or Tris solution. Non-limiting examples of solution pH are between about pH 4.0 and pH 7.4, including the extreme values. Non-limiting examples of Tris solution pH are greater than pH 7.5, such as between about pH 8.0 and pH 9.0, including the extreme values.

In some embodiments across all aspects and examples, the dilution of the column eluent from step (f) or the second column eluent from step (g) is performed using an aqueous buffered phosphate, acetate, or Tris solution. Non-limiting examples of solution pH are between about pH 4.0 and pH 7.4, including the extreme values. Non-limiting examples of Tris solution pH are greater than pH 7.5, such as being between about pH 8.0 and pH 9.0, including the extreme values.

In all aspects and in some embodiments, the rAAV particles produced in step (i) are formulated together with a surfactant to produce an rAAV particle formulation.

In all aspects and in certain embodiments, the anion exchange column chromatography of steps (f), (g), and/or (h) includes polyethylene glycol (PEG) regulated column chromatography.

In some embodiments of all aspects and examples, the anion exchange column chromatography in step (g) and/or (h) is washed with PEG solution before eluting rAAV particles from the column.

In all aspects and in some embodiments, the average molecular weight of PEG is in the range of about 1,000 g/mol to 80,000 g/mol (inclusive).

In all aspects and in some embodiments, the concentration of PEG is from about 4% (w/v) to about 10% (w/v), including the end values.

In all aspects and in some embodiments, the anion exchange column of steps (g) and/or (h) is washed with an aqueous surfactant solution before eluting rAAV particles from the column.

In all aspects and in some embodiments, the cation exchange column of step (f) is washed with a surfactant solution before eluting the rAAV particles from the column.

In all aspects and in some 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.

In all aspects and in some embodiments, the NaCl concentration in the buffer or solution is in the range of about 20 mM to 300 mM NaCl (inclusive) or about 50 mM to 250 mM NaCl (inclusive).

In all aspects and in some embodiments, the surfactant comprises a cationic or anionic surfactant.

In all aspects and in some embodiments, the surfactant comprises a twelve-carbon chain surfactant.

In all aspects and in certain embodiments, the surfactant comprises dodecyltrimethylammonium chloride (DTAC) or Sarkosyl.

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

In all aspects and in some embodiments, the Tris-HCl/NaCl buffer contains 100 mM to 400 mM NaCl (inclusive) and optionally has a pH in the range of about pH 7.5 to about pH 9.0 (inclusive).

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

In all aspects and in some embodiments, the NaCl concentration in the aqueous Tris-HCl/NaCl buffer is in the range of about 75 mM to 125 mM (inclusive).

In all aspects and in some embodiments, the aqueous Tris-HCl/NaCl buffer has a pH of about 7.5 to about 9.0 (inclusive).

In all aspects and in some embodiments, the anion exchange column of steps (f), (g), and/or (h) is washed once or more to reduce the amount of empty caps in the eluent from the second or third column.

In all aspects and in some embodiments, anion exchange column washing removes empty caps from the column before and/or instead of eluting rAAV particles, thereby reducing the amount of empty caps in the second or third column eluent.

In all aspects and in some embodiments, before and/or instead of eluting the rAAV particles, anion exchange column washing removes at least about 50% of the total empty capsid from the column, thereby reducing the amount of empty capsid in the second or third column eluent by about 50%.

In all aspects and in some embodiments, the NaCl concentration in the aqueous Tris-HCl/NaCl buffer is in the range of about 110 mM to 120 mM (inclusive).

In all aspects and in certain embodiments, the ratio and/or amount of eluted rAAV particles to empty capsids are controlled by a wash buffer.

In all aspects and certain embodiments, rAAV particles are eluted by the cation exchange column of step (f) in an aqueous phosphate/NaCl buffer or an aqueous acetate/NaCl buffer. The non-limiting NaCl concentration in the buffer is in the range of about 125 mM to 500 mM NaCl (inclusive). Non-limiting examples of buffer pH are between about pH 5.5 and about pH 7.5, inclusive.

In all aspects and in certain embodiments, the anion exchange column of steps (f), (g), and/or (h) contains quaternary ammonium functional groups, such as quaternized polyethyleneimine.

In all aspects and in certain embodiments, the size exclusion column (SEC) of step (g) and/or (h) has a separation/fractionation range (molecular weight) of about 10,000 g/mol to about 600,000 g/mol (including end values).

In all aspects and in some embodiments, the cation exchange column of step (f) contains sulfonic acid or functional groups such as sulfopropyl.

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

In all aspects and in some embodiments, the method does not include the step of cesium chloride gradient ultracentrifugation.

In some embodiments across all aspects and examples, the method recovers approximately 50% to 90% of the total rAAV particles from the harvest produced in step (a) or the concentrated harvest produced in step (b).

In all aspects and in certain embodiments, the rAAV particles produced by this method have a higher purity than those produced or purified by a single AAV affinity column.

In all aspects and in some embodiments, steps (c) and (d) are performed substantially simultaneously.

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

In all aspects and in some embodiments, the cells are suspension-grown or adherent-grown cells.

In all aspects or in some embodiments, the cells are mammalian cells. Non-limiting examples include HEK cells, such as HEK-293 cells, and CHO cells, such as CHO-K1 cells.

Methods for determining the infection titer of transgenic rAAV particles are known in the art (see, for example, Zhen et al., Hum. Gene Ther. 15 (2004) 709). Methods for determining the infection titer of empty capsids and packaged transgenic rAAV particles are known (see, for example, Grimm et al., Gene Therapy 6 (1999) 1322-1330; Sommer et al., Malec. Ther. 7 (2003) 122-128).

To determine the presence or amount of the degraded/denatured capsid, purified rAAV particles can be subjected to SDS-polyacrylamide gel electrophoresis, which consists of any gel capable of separating the three capsid proteins (e.g., a gradient gel). The gel is then run until the sample is separated, and the gel is imprinted onto a nylon or nitrocellulose membrane. An anti-AAV capsid antibody is then used as a primary antibody to bind the denatured capsid protein (see, for example, Wobus et al., J. Viral. 74 (2000) 9281-9293). The secondary antibody binding the primary antibody contains the tools for detecting the primary antibody. Semi-quantitative detection of the binding between the primary and secondary antibodies is used to determine the amount of capsid. Another approach is to use analytical HPLC with an SEC column or an analytical ultracentrifuge.

***

In addition to the various embodiments depicted and claimed, the disclosed subject matter also relates to other embodiments having other combinations of the features disclosed and claimed herein. Thus, the specific features presented herein can be combined with each other in other ways within the scope of the disclosed subject matter, such that the disclosed subject matter includes any suitable combination of the features disclosed herein. The foregoing description of specific embodiments of the disclosed subject matter has been presented for illustrative and descriptive purposes. It is not intended to be exhaustive or to limit the disclosed subject matter to those embodiments.

All references mentioned in this article are included here by way of citation.

***

The following examples are provided to aid in understanding the invention, the true scope of which is set forth in the appended claims. It should be understood that modifications may be made to the described procedures without departing from the spirit of the invention.

Example

General Technology

1) Recombinant DNA technology

Use standard methods to manipulate DNA, as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). Use molecular biology reagents according to the manufacturer's instructions.

2) DNA and protein sequence analysis and sequence data management

The EMBOSS (European Molecular Biology Open Software Suite) software package, Invitrogen's Vector NTI, and Geneious Prime are used for sequence creation, mapping, analysis, annotation, and visualization.

3) Gene and oligonucleotide synthesis

The desired gene segment was prepared by chemical synthesis at Geneart GmbH (Regensburg, Germany). The synthesized gene fragment was cloned into an E. coli plasmid for propagation/amplification. The DNA sequence of the subcloned gene fragment was verified by DNA sequencing. Alternatively, short synthetic DNA fragments were assembled by annealing chemically synthesized oligonucleotides or via PCR. The individual oligonucleotides were prepared by metabion GmbH (Planegg-Martinsried, Germany).

4) Reagents

Unless otherwise specified, all commercial chemicals, antibodies, and kits shall be used in accordance with the manufacturer’s instructions.

5) Cloning

conventional

For plasmids, a cloning strategy via restriction enzymes is used. By selecting a suitable restriction enzyme, the desired target gene can be excised, and then inserted into different plasmids via ligation. Therefore, it is preferable to use and cleverly select enzymes that cut at the multiple cloning site (MCS) so that the fragments can be ligated in the correct array. If the plasmid and fragment were previously cut with the same restriction enzyme, the sticky ends of the fragment and plasmid fit together perfectly and can then be ligated using DNA ligase. After ligation, competent *E. coli* cells are transformed with the newly generated plasmid.

Cloning via restriction digestion

For plasmid digestion with restriction enzymes, transfer the following components together onto ice:

Table 11: Restrictive Digestion Reaction Reactions

If more enzymes are used in a single digestion, use 1 μL of each enzyme and adjust the volume by adding more or less PCR-grade water. All enzymes were chosen only if they were qualified to be used with CutSmart buffer (100% active) from New England Biolabs and had the same incubation temperature (37°C for all).

Incubation is performed using a thermal mixer or thermal cycler, allowing the sample to be incubated at a constant temperature (37°C). The sample should not be stirred during incubation. The incubation time is set to 60 minutes. The sample is then mixed directly with the loading dye and loaded onto an agarose gel or stored at 4°C/ice for further use.

A 1% agarose gel was prepared for gel electrophoresis. 1.5 g of multipurpose agarose was weighed into a 125°C conical shake flask and filled with 150 mL of TAE buffer. The mixture was heated in a microwave oven until the agarose was completely dissolved. 0.5 μg/mL ethidium bromide was added to the agarose solution. The gel was then cast into a mold. After the agarose set, the mold was placed in the electrophoresis chamber and filled with TAE buffer. The samples were then loaded. Appropriate DNA molecular weight markers were loaded into the first pocket (starting from the left), followed by the samples. The gel was run at <130 V for approximately 60 minutes. After electrophoresis, the gel was removed from the chamber and analyzed using a UV-Imager.

Cut the band and transfer it to a 1.5 mL Eppendorf tube. For gel purification, use the Qiagen QIAquick Gel Extraction Kit according to the manufacturer's instructions. Store the DNA fragment at -20°C for further use.

The fragments used for ligation were transferred together at a plasmid/insert molar ratio of 1:2, 1:3, or 1:5, depending on the lengths of the insert and plasmid fragments and their correlation with each other. A 1:5 ratio is used if the fragment to be inserted into the plasmid is short. A smaller amount of plasmid is used if the insert is longer. 50 ng of plasmid is used for each ligation, and the specific amount of insert is calculated using the NEBioCalculator. The T4 DNA Ligation Kit from NEB is used for ligation. Table 12 below depicts examples of ligation mixtures.

Table 12: Connection reaction mixture

Begin by mixing DNA and water, adding buffer, and finally the enzyme, transferring all components together to ice. Gently mix the reaction mixture by pumping up and down, briefly microcentrifuge, and then incubate at room temperature for 10 minutes. After incubation, heat-inactivate the T4 ligase at 65°C for 10 minutes. Cool the sample on ice. In the final step, transform 10-β competent E. coli cells with 2 μL of the ligated plasmid (see below).

Transformation of 10-β competent Escherichia coli cells

For transformation, 10-β competent *E. coli* cells were thawed on ice. Then, 2 μL of plasmid DNA was directly transferred into the cell suspension. The tube was gently tapped and placed on ice for 30 minutes. Afterward, the cells were placed in a 42°C heat block and heat-shocked for exactly 30 seconds. Immediately afterwards, the cells were cooled on ice for 2 minutes. 950 μL of NEB 10-β growth medium was added to the cell suspension. The cells were incubated at 37°C with shaking for one hour. Then, 50 μL to 100 μL were transferred to preheated (37°C) LB-Amp agar plates and spread using a disposable spatula. The plates were incubated overnight at 37°C. Only bacteria successfully incorporating plasmids carrying the ampicillin resistance gene were able to grow on these plates. Single colonies were picked the following day and cultured in LB-Amp medium for subsequent plasmid preparation.

Bacterial culture

Escherichia coli was cultured in LB medium (Luria Bertani abbreviation) with 1 mL/L 100 mg/mL ampicillin added to achieve an ampicillin concentration of 0.1 mg/mL. For different plasmid preparation quantities, the following amounts were inoculated using single bacterial colonies.

Table 13: Culture volume of Escherichia coli

Quantitative plasmid preparation Volume of LB-Amp medium [mL] Incubation time [h] Small-scale fabrication of 96-well plates (EpMotion) 1.5 twenty three Small-scale preparation: 15mL tube 3.6 twenty three Mass production 200 16

For small-scale preparation, fill each well of a 96-well 2mL deep-well plate with 1.5mL of LB-Amp medium. Pick colonies and insert toothpicks into the medium. When all colonies have been picked, seal the plate with a viscous air-porous membrane. Incubate the plate at 200rpm in a 37°C incubator for 23 hours.

For small-scale preparations, 3.6 mL of LB-Amp medium was filled into 15 mL tubes (with vent caps) and bacterial colonies were inoculated equally. Toothpicks were left in the tubes during incubation. The tubes were incubated at 37°C and 200 rpm for 23 hours, as with 96-well plates.

For large-scale preparation, fill 200 mL of LB-Amp medium into a 1 L autoclaved Erlenmeyer glass flask and inoculate with 1 mL of bacterial day culture, approximately 5 hours later. Seal the Erlenmeyer flask with a paper stopper and incubate at 37°C and 200 rpm for 16 hours.

plasmid preparation

For small-scale preparation, transfer 50 μL of bacterial suspension to a 1 mL deep-well plate. Then, centrifuge the bacterial cells in the plate at 3000 rpm, 4 °C for 5 min. Remove the supernatant and place the plate containing the bacterial particles in EpMotion. After approximately 90 minutes, the run is complete, and the eluted plasmid DNA can be removed from EpMotion for further use.

For small-scale preparation, remove a 15 mL tube from the incubator and aliquot 3.6 mL of bacterial culture into two 2 mL Eppendorf tubes. Centrifuge the tubes at 6,800 x g for 3 minutes at room temperature using a benchtop microcentrifuge. Then, perform small-scale preparation using the Qiagen QIAprep Spin Small-Scale Preparation Kit according to the manufacturer's instructions. Measure the plasmid DNA concentration using Nanodrop.

Large-scale preparations were performed using the Macherey-Nagel Xtra MaxiEF kit according to the manufacturer's instructions. DNA concentration was measured using Nanodrop.

Ethanol precipitation

Mix a given volume of DNA solution with 2.5 volumes of 100% ethanol. Incubate the mixture at -20°C for 10 minutes. Then centrifuge the DNA at 14,000 rpm at 4°C for 30 minutes. Carefully remove the supernatant and wash the precipitate with 70% ethanol. Centrifuge the tube again at 14,000 rpm at 4°C for 5 minutes. Carefully remove the supernatant by pipetting and dry the precipitate. While the ethanol evaporates, add an appropriate amount of endotoxin-free water. Allow the DNA time to redissolve in 4°C water overnight. Take a small sample and measure the DNA concentration using a Nanodrop device.

Expression box composition

For open reading frame expression, use transcription units that contain at least the following functional elements:

- Promoter,

- Contains nucleic acids with corresponding open reading frames, which may include signal sequences if necessary.

- Polyadenylation signal sequence.

In addition to the expression unit/cassette containing the desired gene to be expressed, the basic/standard mammalian expression plasmid also contains

- The replication origin from plasmid pUC18, which allows the plasmid to replicate in E. coli, and

The β-lactamase gene confers ampicillin resistance in Escherichia coli.

6) Cell culture technology

Standard cell culture techniques were used, as described in Current Protocols in Cell Biology (2000), Bonifacino, J.S., Dasso, M., Harford, J.B., Lippincott-Schwartz, J., and Yamada, K.M. (eds.), John Wiley & Sons, Inc.

Transient transfection in HEK293 system

Cells producing recombinant AAV particles were generated using the HEK293 system (Invitrogen, now Thermo Scientific) according to the manufacturer's instructions via transient transfection with the appropriate plasmid. In short, HEK293 cells (Invitrogen) grown in suspension in serum-free FreeStyle ^™ 293 expression medium (Invitrogen) in shake flasks or stirred fermentation tubes were transfected with the appropriate plasmid and a mixture of 293fectin ^™ or fectin (Invitrogen). For 2L shake flasks (Corning), HEK293 cells were seeded at a density of 1 × ^10⁶ cells/mL in 600 mL and incubated at 120 rpm and 8% _CO₂ . The following day, cells were transfected at a cell density of approximately 1.5 × ^10⁶ cells/mL with approximately 42 mL of a mixture of the following substances: A) 20 mL of Opti-MEM (Invitrogen) containing 600 μg total plasmid DNA (1 μg/mL) and B) 20 mL of Opti-MEM + 1.2 mL of 293 fectin or fectin (2 μL/mL). Glucose solution was added during fermentation according to glucose consumption.

Example 1

No pretreatment

program:

Mix 90 μL of H2O and 10 μL of sample.

-Incubate at 95°C for 15 minutes

Example:

1. Process Sample

2. Preparation of PCR master mix

3. Add the main mixture to the plate.

4. Prepare a 1:10 dilution solution using water.

5. Add the template to the board.

6. Seal the plate and vortex (1 minute, 2,200 rpm) and centrifuge.

7. Use an automated droplet generator (Auto-DG) to form droplets (20 μL final mixture + 70 μL oil) and transfer them to a plate (42 μL).

8. Seal the plate and start the PCR run.

Example 2

thermal denaturation

program:

- Incubate the sample at 98°C for 10 minutes.

Example:

1. Thermal denaturation

2. Preparation of PCR master mix

3. Add the main mixture to the plate.

4. Prepare a 1:10 dilution solution using water.

5. Add the template to the board.

6. Seal the plate and vortex (1 minute, 2,200 rpm) and centrifuge.

7. Use Auto-DG to form droplets (20 μL final mixture + 70 μL oil) and transfer them to a plate (42 μL).

8. Seal the plate and start the PCR run.

Example 3

DNA digestion with enzyme I

Reagents:

1) DNase I buffer (Promega): 400mM Tris-HCl, pH ₈ , 100mM MgSO₄, 10mM _CaCl₂

2) DNase I (Promega): 50 U/mL diluted to 1 U/μL

Procedure (50 μL reaction volume):

Mix 30 μL H2O, 5 μL DNase I buffer, 5 μL DNase I, and 10 μL sample.

-Incubate at 37°C for 30 minutes.

Heat to 95°C for 15 minutes.

Procedure (100 μL reaction volume):

Mix 75 μL H2O, 10 μL DNase I buffer, 5 μL DNase I, and 10 μL sample.

-Incubate at 37°C for 30 minutes.

Heat to 95°C for 15 minutes.

Example:

1. Digestion by DNA enzyme I;

2. Add 50 μL of water to the reaction mixture.

3. Preparation of PCR master mix

4. Add the PCR master mix to the plate (16.5 μL per well).

5. Prepare a 1:10 dilution buffer: 10 μL sample/plasmid/standard + 90 μL H2O

6. Add the template to the plate (5.5 μL per well)

7. Seal the plate and vortex (1 minute, 2,200 rpm) and centrifuge (1 minute, 1000 rcf).

8. Form droplets (20 μL final mixture + 70 μL oil) using Auto-DG and transfer them to a plate (42 μL).

9. Seal the plate and begin the PCR run.

Example 4

proteinase K digestion

Reagents:

1) Proteinase K (Roche; 17.8 mg/mL = ≥50 U/mL): Dilute to 1 U/mL

2) Proteinase K buffer (BioRad): 400mM Tris-HCl, 20mM EDTA, 2000mM NaCl, pH 8

3) Sodium dodecyl sulfate solution (SDS solution): 10%

Program (Water):

Mix 68 μL of water with 10 μL of sample and add 20 μL of proteinase K.

-Incubate at 50°C for 60 minutes

Heat to 95℃ for 15 minutes.

Procedure (SDS-free buffer):

Mix 63 μL of water with 5 μL of proteinase K buffer, then add 10 μL of sample and 20 μL of proteinase K.

-Incubate at 50°C for 60 minutes

Heat to 95℃ for 15 minutes.

Procedure (buffer containing SDS):

Mix 53 μL of water with 5 μL of proteinase K buffer, then add 10 μL of SDS solution, 10 μL of sample, and 20 μL of proteinase K.

-Incubate at 50°C for 60 minutes

Heat to 95℃ for 15 minutes.

Example:

1. Proteinase K digestion

2. Preparation of PCR master mix

3. Add the main mixture to the plate (16.5 μL per well).

4. Prepare a 1:10 dilution: 10 μL sample + 90 μL H2O

5. Add the template to the plate (5.5 μL per well)

6. Seal the plate and vortex (1 minute, 2,200 rpm) and centrifuge (1 minute, 1000 rcf).

7. Use Auto-DG to form droplets (20 μL final mixture + 70 μL oil) and transfer them to a plate (42 μL).

8. Seal the plate and start the PCR run.

Example 5

Heat denaturation, followed by proteinase K digestion.

Reagents:

1) Proteinase K (Roche; 17.8 mg/mL = ≥50 U/mL): 1 U/mL

2) Proteinase K buffer (BioRad): 400mM Tris-HCl, 20mM EDTA, 2000mM NaCl, pH 8

program:

- Thermal 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 minutes; inactivate at 95°C for 10 minutes.

Example:

1. Thermal denaturation

2. Proteinase K digestion

3. Prepare a 1:10 dilution: 10 μL sample + 90 μL H2O

4. Preparation of PCR master mix

5. Add the main mixture to the plate.

6. Add the sample to the plate.

7. Seal the plate and vortex (1 minute, 2,200 rpm) and centrifuge.

8. Form droplets (20 μL final mixture + 70 μL oil) using Auto-DG and transfer them to a plate (42 μL).

9. Seal the plate and begin the PCR run.

Example 6

DNA digestion with enzyme I, followed by proteinase K digestion.

Method 1:

Reagents:

1) DNase I buffer (Promega): 400mM Tris-HCl, pH ₈ , 100mM MgSO₄, 10mM _CaCl₂

2) DNase I (Promega): 1 U/μL

3) Proteinase K (Roche; 17.8 mg/mL = ≥50 U/mL): 1 U/mL

4) Proteinase K buffer (BioRad): 400mM Tris-HCl, 20mM EDTA, 2000mM NaCl, pH 8

5) Sodium dodecyl sulfate solution (SDS solution): 10%

program:

Mix 30 μL H2O, 5 μL DNase I buffer, 5 μL DNase I, and 10 μL sample.

-Incubate at 37°C for 30 minutes.

Heat to 95°C for 15 minutes.

Mix 50 μL of PK- mixture (42 μL H2O + 2 μL proteinase K + 5 μL 20x proteinase K buffer + 1 μL 10% SDS solution) with 50 μL of incubated DNase I- mixture.

-Incubate at 50°C for 60 minutes

Heat to 95°C for 15 minutes.

Example:

1. Digest with DNase I; optionally dilute 1:10

2. Digest with proteinase K; dilute 1:10

3. Preparation of PCR master mix

4. Add the PCR master mix to the plate (16.5 μL per well).

5. Prepare a 1:10 dilution: 10 μL sample + 90 μL H2O

6. Add the template to the plate (5.5 μL per well)

7. Seal the plate and vortex (1 minute, 2,200 rpm) and centrifuge (1 minute, 1000 rcf).

8. Form droplets (20 μL final mixture + 70 μL oil) using Auto-DG and transfer them to a plate (42 μL).

9. Seal the plate and begin the PCR run.

Method 2:

Reagents:

1) Proteinase K (NEB; approximately 20 mg/mL = ≥800 U/mL): Dilute to 16 U/mL

2) Proteinase K buffer (BioRad): 400mM Tris-HCl, 20mM EDTA, 2000mM NaCl

3) Sodium dodecyl sulfate solution (SDS solution): 10%

Procedure (buffer containing SDS):

Mix 42 μL of water with 50 μL of sample (DNase I digestion solution), and add 2 μL of proteinase K, 5 μL of proteinase K buffer, and 1 μL of SDS solution.

-Incubate at 50°C for 60 minutes

Heat to 95℃ for 15 minutes.

Example:

1. Digestion by DNA enzyme I

2. Proteinase K digestion

3. Dilute 1:10

4. Preparation of PCR master mix

5. Add the main mixture to the plate (16.5 μL per well).

6. Prepare a 1:10 dilution: 10 μL sample + 90 μL H2O

7. Add the template to the plate (5.5 μL per well)

8. Seal the plate and vortex (1 minute, 2,200 rpm) and centrifuge (1 minute, 1000 rcf).

9. Use Auto-DG to form droplets (20 μL final mixture + 70 μL oil) and transfer them to a plate (42 μL).

10. Seal the plate and start the PCR run (Example 7)

Condition comparison

The reagents and procedures are as outlined in the previous examples.

The conditions are based on Tables 14 and 15 below.

Table 14:

Table 15:

Example 8

ddPCR

For viral genome titration, dual ddPCR assays were performed. Primers and probes were designed targeting the ITR site and the Amp resistance sequence, which is present on the backbone of all three plasmids used in rAAV production. PCR master mixes were prepared according to Table 16 (Droplet Digital PCR Guide - Bio-Rad).

Table 16: Composition of ddPCR master mix.

Components Volume per well [μL] Final concentration Hypermixture (2x) 11 1x 20μM ITR forward primer 0.99 900nM 20μM ITR reverse primer 0.99 900nM 20 μM FAM-labeled ITR probe 0.275 250nM 20μM Amp forward primer 0.99 900nM 20μM Amp reverse primer 0.99 900nM 20 μM HEX-labeled Amp probe 0.275 250nM template 5.5 1*10E4-1*10E5 copies/mL water 0.99 - total twenty two  

The prepared master mixture was transferred to 96-well plates, 16.5 μL per well. The pretreated sample was then serially diluted: 10 μL of sample was transferred to 90 μL of water in a LoBind tube using LoRentention Tips and mixed thoroughly. Subsequently, 5.5 μL of sample was added to the master mixture solution in the 96-well plates via several dilution steps. The plates were sealed at 180 °C, vortexed at 2,200 rpm for 1 minute, and then centrifuged at 1,000 rpm for 1 minute. Using an automated droplet generator, 20 μL of PCR mixture was removed from each well, producing up to 20,000 droplets per well, and transferred to another 96-well plate. After sealing the droplet plates at 180 °C, a PCR run was performed. The corresponding conditions are shown in Table 17 below.

Table 17: ddPCR thermal cycling program.

Loop count transsexual annealing Final extension Finish 1 95℃, 10 minutes 40 94℃, 30 seconds 60℃, 1min 1 98℃, 10min 12℃, maintain

In the droplet reader, fluorescence signals for each droplet were measured in the FAM and HEX channels. QuantaSoft software processed the reader data and calculated the copy number for the target sequence, ITR site, and Amp per 20 μL well. The initial sample titer could be determined using Equation 1:

Example 9

rAAV production

HEK293-F suspension cells were transfected with three plasmids: pAAV-transgenic (EGFP or EBFP), pAAV-rep/cap, and pAAV-helper. Plasmid DNA (1 μg/1 mL cell culture) and lipid transfection 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). The two solutions were then combined, incubated at room temperature for 15 min, and added to HEK293-F cell suspension (1 × 10⁶ cells/mL) in F17 medium. Cells were incubated at 37 °C, 8% CO₂, and 120 rpm for 48 to 72 h (Grieger, J. et al. (2016)).

Recombinant AAV particles were harvested by adding lysis buffer (100 μL/1 mL cell culture) containing 1% Triton X-100, 500 mM TRIS, and ₂₀ mM _MgCl2 (pH 7.5). Freshly diluted Benzonase (10 μL/1 mL cell culture) was added to a final concentration of 50 U/mL. After 60 minutes, lysis was performed with stirring at 37°C, followed by the addition of MgSO4 (final concentration 37.5 mM), and the cell lysis medium was incubated for another 30 minutes (Chahal, P. et al. (2014)). The lysate was then centrifuged at 4,000 g for 20 minutes, and the supernatant was filtered through a 0.22 μm filter. The resulting product was considered the crude lysate.

Example 10

rAAV purification

YMC glass columns are packed with POROS CaptureSelect AAVx affinity resin, with a column volume of 9.1 mL. These resin beads are coated with antibody fragments that can bind to multiple AAV serotypes with high specificity (POROS CaptureSelect AAV Resin – User Guide 2017).

First, equilibrate the column with phosphate-buffered saline (PBS) to produce the correct binding conditions. Then, load the coarsely filtered lysate at 150 cm/h. After capturing the rAAV capsid, wash the column with 4 column volumes (CV) of PBS, followed by 4 CV of 0.5 M NaCl to remove impurities such as cell debris and DNA residue. Perform another washing step with 4 CV of PBS to prepare the elution conditions (POROS CaptureSelect AAV Resin – User Guide 2017).

The rAAV capsid was then eluted in 100 mM citrate buffer (pH 2.4) (POROS CaptureSelect AAV resin – User Guide 2017). Fractions within the elution peak were combined (UV detection at λ = 280 nm). The pH was increased to 7.5 using 2 M TRIS (pH 9). Finally, the eluent was aseptically filtered using a 0.2 μm syringe filter.

Claims (17)

1. A method for determining the copy number of viral genomic DNA in a sample, wherein the method comprises the following steps: - Incubate the sample with proteinase K. - The viral genomic DNA copy number was determined by digital droplet polymerase chain reaction. The sample contained no DNA that was not encapsulated within the viral particle. The incubation with proteinase K was carried out in the presence of 0.05 (w/v)% to 1.5 (w/v)% sodium dodecyl sulfate. 2. The method according to claim 1, wherein the method comprises the following steps: - The sample is incubated with a nuclease to obtain a digested sample. - The digested sample is incubated with proteinase K to obtain a proteinase K-incubated sample. - The number of viral genomic DNA copies in the proteinase K-incubated sample was determined by digital droplet polymerase chain reaction. 3. The method of claim 2, wherein the digested sample is diluted up to 2.5 times for the incubation with proteinase K. 4. The method according to any one of claims 2 to 3, wherein the entire digested sample is incubated with proteinase K. 5. The method according to any one of claims 1 to 4, wherein the method is carried out at a temperature of up to 95°C, except for the final extension step of the digital droplet polymerase chain reaction. 6. The method according to any one of claims 1 to 5, wherein the total amount of proteinase K used in the incubation is 15 mU to 35 mU. 7. The method according to any one of claims 1 to 6, wherein the volume of the sample is about 10 μL. 8. The method according to any one of claims 1 to 7, wherein the incubation with proteinase K is carried out in a total volume of 100 μL. 9. The method according to any one of claims 2 to 8, wherein the nuclease is DNAase I. 10. The method according to any one of claims 2 to 9, wherein the total amount of nuclease used in the incubation is about 5 U. 11. The method according to any one of claims 2 to 10, wherein the incubation with the nuclease is carried out in a total volume of 50 μL. 12. The method according to any one of claims 2 to 11, wherein the incubation with the nuclease is carried out at a final concentration of 40 mM Tris*HCl, 10 mM _MgSO4 , and 1 mM _CaCl2 at a pH of about 8. 13. The method according to any one of claims 2 to 12, wherein the incubation with the nuclease is carried out at 37°C for 30 minutes, and then the nuclease is inactivated at 95°C for 15 minutes. 14. The method according to any one of claims 1 to 13, wherein the incubation with proteinase K is carried out at a final concentration of 20 mM Tris-HCl, 1 mM EDTA, 100 mM NaCl, and 1 (w/v)% sodium dodecyl sulfate at a pH of about 8. 15. The method according to any one of claims 1 to 13, wherein the incubation with proteinase K is carried out at a pH of about 8 with a final concentration of 30 mM Tris-HCl, ₅ mM MgSO4, 0.5 _mM CaCl2, 0.5 mM EDTA, 50 mM NaCl, and 0.1 (w/v)% sodium dodecyl sulfate. 16. The method according to any one of claims 1 to 15, wherein the incubation with proteinase K is carried out at 50°C for 60 minutes, and then the proteinase is inactivated at 95°C for 15 minutes. 17. The method according to any one of claims 1 to 16, wherein all steps of the method are performed at a temperature up to 95°C, except for the final extension step of the digital droplet polymerase chain reaction.