AAV particle processing using affinity chromatography with gradient elution

Abstract

Herein is reported a method for separating full AAV particles from empty AAV particles, the method comprising the steps of applying a solution comprising full, empty and partially filled AAV particles to an AAV affinity column comprising an AAV affinity material and thereby binding the AAV particles to the AAV affinity material, wherein the solution has a pH in the range of 7.4 to pH 4,5 eluting full AAV particles from the AAV affinity column by applying a pH gradient from the pH of the solution of the first step to a pH in the range of 4 to 2.5, wherein the pH of the applying step and the pH of the eluting step differ in a least 0.5 pH units, and recovering full AAV particles from the eluate, and thereby separating full AAV particles from empty and partially filled AAV particles.

Description

[0001] P39840-WQ-1 (GH)

[0002] AAV PARTICLE PROCESSING USING AFFINITY CHROMATOGRAPHY WITH GRADIENT ELUTION

[0003] The current invention is in the field of adeno-associated virus particle processing, especially the current invention relates to methods for the purification or / and separation or / and enrichment of adeno- associated virus particles of interest from product- or / and process-related impurities. In more detail, herein is reported a method comprising the elution of adeno-associated virus particles of interest from the adeno-associated virus affinity column by applying a pH or salt concentration gradient, whereby at least a partial separation or enrichment of the adeno-associated virus particles from product- or / and process-related impurities.

[0004] BACKGROUND

[0005] Gene therapy is opening unprecedented opportunities for novel therapeutic approaches. Based on the concept of rescuing function mutations by co-expressing the correct gene, to allow biological functions to be restored, it requires the use of specific delivery to ensure the proper delivery of therapeutic genes to the intended site of action. In this context, recombinant adeno-associated virus particles (rAAVp) are most widely used for therapeutic gene delivery.

[0006] Bioprocessing of viruses is complex and requires systematic and coordinated steps both in upstream processing as well as downstream processing. However, the use of traditional cultivation processes for the production of therapeutic virus particles does not support effective commercial manufacturing strategies. This is even more pronounced by the large size of therapeutic viral particles as compared to therapeutic biomolecules, such as, e.g., antibodies. Additionally, viral particles are much more complex.

[0007] The manufacturing process of therapeutic recombinant adeno-associated virus particles requires the insertion of the therapeutic transgene into the recombinant AAV capsid shell (full viral particles, i.e. recombinant viral particles comprising an encapsidated nucleic acid).

[0008] However, a certain percentage of the recombinantly produced viral particles that does not contain the desired transgene might also be produced, as well as partly filled recombinant viral particles.

[0009] SUMMARY OF INVENTION The present invention overcomes problems associated with the separation or purification of full AVV particles from product- or / and process-related impurities, such as empty AAV particles.

[0010] The current invention is based, at least in part, on the finding that elution of adeno-associated virus particles of interest from the adeno-associated virus affinity column by applying a pH or / and salt concentration gradient, leads to at least a partial separation or enrichment of the adeno-associated virus particles from product- or / and process-related impurities.

[0011] One exemplary aspect of the current invention is directed to a method for separating full AAV particles from empty (and partially filled) AAV particles, the method comprising a) Applying a solution comprising full, empty and partially filled AAV particles to an AAV affinity column (comprising an AAV affinity material) and thereby binding the AAV particles to the AAV affinity column (material), wherein the solution has a pH in the range of 7.4 to 4, b) Eluting full AAV particles from the AAV affinity column by applying a pH gradient from the pH of the solution of step a) to a pH in the range of 4 to 2.5, wherein the pH of step a) (applying step) and the pH of step b) (eluting step) differ in a least 0.5 pH units, c) Recovering full AAV particles from the eluate of step b), and thereby separating full AAV particles from empty (and partially filled) AAV particles.

[0012] One exemplary aspect of the current invention is directed to a method for separating full AAV particles from empty (and partially filled) AAV particles, the method comprising a) Applying a solution comprising full, empty and partially filled AAV particles to an AAV affinity column (comprising an AAV affinity material) and thereby binding the AAV particles to the AAV affinity column (material), wherein the solution has a pH in the range of pH 6 to pH 3, b) Eluting full AAV particles from the column applying a salt gradient from the salt concentration of the solution of step a) to a salt concentration in the range of 0 mM to 1000 mM, c) Recovering full AAV particles from the eluate of step b), and thereby separating full AAV particles from empty (and partially filled) AAV particles.

[0013] Thus, the invention encompasses at least the following embodiments:

[0014] 1. A method for separating full AAV particles from empty (and partially filled) AAV particles, the method comprising a) Applying a solution comprising full, empty and partially filled AAV particles to an AAV affinity column (comprising an AAV affinity material) and thereby binding the AAV particles to the AAV affinity column (material), wherein the solution has a pH in the range of 7.4 to pH 4, b) Eluting full AAV particles from the AAV affinity column by applying a pH gradient from the pH of the solution of step a) to a pH in the range of 4 to 2.5, wherein the pH of step a) (applying step) and the pH of step b) (eluting step) differ in a least 0.5 pH units, c) Recovering full AAV particles from the eluate of step b), and thereby separating full AAV particles from empty (and partially filled) AAV particles.

[0015] 2. The method according to embodiment 1, the method comprising after step a) and before step b) the following step al) Washing the AAV affinity column obtained in step a) with a buffer with a pH in the range of pH 5.5 to pH 4, and step b) is b) Eluting full AAV particles from the AAV affinity column by applying a pH gradient from the pH of the solution of step al) to a pH in the range of 4 to 2.5, wherein the pH of step al) (washing step) and the pH of step b) (eluting step) differ in a least 0.5 pH units.

[0016] 3. The method according to embodiment 1 or 2, the method comprising

[0017] (a) Equilibrating an AAV affinity chromatography material / column (comprising an AAV affinity material) with a solution / buffer with a pH value of about pH 7.4,

[0018] (b) Applying a solution comprising full, empty and partially filled AAV particles in a solution / buffer with a pH value of about pH 7.4 (i.e. about the same as the solution / buffer of step a)) to the column obtained in step a), thereby binding the AAV particles to the AAV affinity column (material),

[0019] (c) Optionally washing the column obtained in step b) with a solution / buffer with a pH value of about 7.4,

[0020] (d) Washing the column of step b) or step c) with a solution / buffer with a pH value in the range of pH 5.5 to pH 4,

[0021] (e) Eluting full AAV particles from the column applying a pH gradient from the pH of the solution / buffer used in step d) to a pH in the range of 4 to 2.5 wherein the pH of step d) (washing step) and the pH of step e) (eluting step) differ in a least 0.5 pH units,

[0022] (f) recovering full AAV particles from the eluate of step e), and thereby separating full AAV particles from empty (and partially filled) AAV particles.

[0023] 4. The method according to any one of embodiments 1 to 3, wherein the pH of step a) or step b) (applying step) is in the range of 7.4 to 5.5.

[0024] 5. The method according to any one of embodiments 1 to 4, wherein the pH of step a) or step b) (applying step) is about 7.4.

[0025] 6. The method according to any one of embodiments 1 to 5, wherein the steps of the method are performed in the presence of a salt.

[0026] 7. The method according to embodiment 6, wherein the salt has a concentration of 0-2000 mM, 0-1200 mM, 0-1000 mM, preferably 100-300 mM, more preferred 100-200 mM, most preferred about 200 mM.

[0027] 8. A method for separating full AAV particles from empty (and partially filled) AAV particles, the method comprising a) Applying a solution comprising full, empty and partially filled AAV particles to an AAV affinity column (comprising an AAV affinity material) and thereby binding the AAV particles to the AAV affinity column (material), wherein the solution has a pH in the range of pH 6 to pH 3, b) Eluting full AAV particles from the column applying a salt gradient from the salt concentration of the solution of step a) to a salt concentration in the range of 0 mM to 1000 mM, c) Recovering full AAV particles from the eluate of step b), and thereby separating full AAV particles from empty (and partially filled) AAV particles.

[0028] 9. The method according to any one of embodiments 1 to 8, the method comprising

[0029] (a) Equilibrating an AAV affinity chromatography material / column (comprising an AAV affinity material) with a solution / buffer with a pH value of about pH 7.4 in the presence of a salt,

[0030] (b) Applying a solution comprising full, empty and partially filled AAV particles in a solution / buffer with a pH value of about pH 7.4 (i.e. about the same as the solution / buffer of step a)) to the column obtained in step a) in the presence of a salt, thereby binding the AAV particles to the AAV affinity column (material),

[0031] (c) Optionally washing the column obtained in step b) with a solution / buffer with a pH value of about 7.4 in the presence of a salt,

[0032] (d) Washing the column of step b) or step c) with a solution / buffer with a pH value in the range of pH 5.5 to pH 4 in the presence of a salt,

[0033] (e) Eluting full AAV particles from the column applying a combined pH and salt gradient, wherein the pH gradient is from the pH of the solution / buffer used in step d) to a pH in the range of 4 to 2.5 wherein the pH of step d) (washing step) and the pH of step e) (eluting step) differ in a least 0.5 pH units, wherein the salt gradient is from the concentration of the salt in step e) to a salt concentration in the range of 0 mM to 1000 mM,

[0034] (f) recovering full AAV particles from the eluate of step e), and thereby separating full AAV particles from empty (and partially filled) AAV particles.

[0035] 10. The method according to any one of embodiments 1 to 9, wherein the pH gradient is to a pH in the range of 3.45 to 2.5. 11. The method according to any one of embodiments 1 to 10, wherein all buffers of steps a) to c) are the same.

[0036] 12. The method according to any one of embodiment 8 to 11, wherein the solution of step a) or step b) (applying step) comprises a salt at a concentration in the range of 0 to 2000 mM.

[0037] 13. The method according to any one of embodiments 8 to 12, wherein the solution of step a) or step b) (applying step) comprises a salt at a concentration in the range of 1000 mM to 2000 mM and the gradient of step b) or step e) (eluting step) is to a salt concentration of 0 mM to 200 mM.

[0038] 14. The method according to any one of embodiment 8 or 12, wherein the solution of step a) comprises a salt at a concentration in the range of 0 mM to 200 mM and the solution of step b) comprises a salt at a concentration of about 1000 mM.

[0039] 15. The method according to any one of embodiments 1 to 14, wherein the steps of the method are performed in the presence of NaCl or MgC12 or Na2SO4 as a salt.

[0040] 16. The method according to any one of embodiments 1 to 15, wherein the AAV particle is a recombinant AAV particle (rAAVp).

[0041] 17. The method according to any one of embodiments 1 to 16, wherein the AAV particle is an AAV particle of serotype 2, serotype 8 or serotype 9.

[0042] 18. The method according to any one of embodiments 1 to 17, wherein the elution buffer used in step b) or e) (eluting step) comprises formic acid or acetic acid or citric acid.

[0043] 19. The method according to any one of embodiments 1 to 18, wherein the AAV particle is an AAV particle of serotype 2 or serotype 8.

[0044] 20. The method according to any one of embodiments 1 to 19, wherein the elution buffer used in step b) or e) (elution step) comprises formic acid or citric acid.

[0045] 21. The method according to any one of embodiments 1 to 20, wherein the pH gradient in step b) or step e) is from pH 4 to pH 2.5.

[0046] 22. The method according to any one of embodiments 1 to 20, wherein the pH gradient in step b) or step e) is from pH 4 to pH 3.0. 23. The method according to any one of embodiments 1 to 20, wherein the pH gradient in step b) or step e) is from pH 3.45 to pH 2.5.

[0047] 24. The method according to any one of embodiments 1 to 20, wherein the pH gradient in step b) or step e) is from pH 3.45 to pH 3.0.

[0048] 25. The method according to any one of embodiments 1 to 18, wherein the AAV particle is an AAV particle of serotype 9.

[0049] 26. The method according to embodiment 25, wherein the pH gradient in step b) or step e) (eluting step) is from pH 5.5 to pH 3.5.

[0050] 27. The method according to any one of embodiments 1 to 26, wherein the solution / buffer used in step b) or e) (eluting step) are free of sorbitol.

[0051] 28. The method according to any one of embodiments 1 to 26, wherein the solution / buffer used in step b) or e) (eluting step) comprises sorbitol.

[0052] 29. The method according to any one of embodiments 1 to 28, wherein the solution / buffer used in step b) or e) (eluting step) comprises NaCl.

[0053] 30. The method according to any one of embodiments 1 to 29, wherein the solution / buffer used in step b) or e) (eluting step) comprises MgC12.

[0054] 31. The method according to any one of embodiments 1 to 30, wherein the solution / buffer used in step b) or e) (eluting step) comprises Na2SO4.

[0055] 32. The method according to any one of embodiments 1 to 31, wherein the solution / buffer used in step b) or e) (eluting step) comprises Poloxamer 188.

[0056] 33. The method according to any one of embodiments 1 to 32, wherein the AAV affinity column (material) comprises a camelid single-domain antibody fragment as ligand.

[0057] 34. The method according to any one of embodiments 1 to 33, wherein the AAV affinity column (material) comprises a camelid single-domain antibody fragment capable of binding an AAV capsid as ligand. 35. The method according to any one of embodiments 1 to 34, wherein the AAV affinity column (material) comprises a camelid single-domain antibody fragment capable of binding an AAV capsid as ligand and a matrix of crosslinked poly [styrene divinylbenzene],

[0058] 36. The method according to any one of embodiments 1 to 35, wherein the AAV affinity column (material) comprises a camelid single-domain variable heavy chain (V(H)H) antibody fragment capable of binding an AAV capsid as ligand and a matrix of crosslinked poly [styrene divinylbenzene].

[0059] 37. The method according to any one of embodiments 1 to 36, wherein the AAV affinity column (material) comprises a camelid single-domain antibody fragment capable of binding an AAV capsid as ligand and a matrix of crosslinked poly [styrene divinylbenzene], and wherein the single-domain antibody fragment hast a size of about 5 to about 15 kDa.

[0060] 38. The method according to any one of embodiments 1 to 37, wherein the AAV affinity column (material) comprises a camelid single-domain antibody fragment capable of binding an AAV capsid as ligand and a matrix of crosslinked poly [styrene divinylbenzene], and wherein the single-domain antibody fragment has a size of about 14 kDa.

[0061] 39. The method according to any one of embodiments 1 to 38, wherein the AAV affinity column (material) comprises a camelid single-domain antibody fragment capable of binding an AAV capsid as ligand and a matrix of crosslinked poly [styrene divinylbenzene], and wherein the single-domain antibody fragment is serotype-agnostic.

[0062] 40. The method according to any one of embodiments 1 to 39, wherein the AAV affinity column (material) comprises a camelid single-domain antibody fragment capable of binding an AAV capsid as ligand and a matrix of crosslinked poly [styrene divinylbenzene], and wherein the single-domain antibody fragment is serotype-agnostic towards a panel of at least 15 AAVs, including AAV2, AAV8, AAV9, PHP.B and Anc80.

[0063] 41. The method according to any one of embodiments 1 to 40, wherein the AAV affinity column (material) comprises a camelid single-domain antibody fragment capable of binding an AAV capsid as ligand and a matrix of crosslinked poly [styrene divinylbenzene], and wherein the matrix of crosslinked poly [styrene divinylbenzene] is covalently linked to the camelid single-domain antibody fragment.

[0064] 42. The method according to any one of embodiments 1 to 41, wherein the AAV affinity column (material) is POROS™ Captures elect™ AAVX column (material).

[0065] DESCRIPTION OF THE FIGURES

[0066] Figure 1: Experiment 1, full view: data is plotted in CV (column volumes, 1 mL / CV) on the x-axis vs. mAU (absorbance units) measured by a 10 mm UV flow cell on the primary y-axis and pH on the secondary y-axis. It is shown: the UV-signal at 280 nm (long / short-dashed line) and 260 nm (solid line) and pH (dashed line).

[0067] Figure 2: Experiment 1, zoom on elution peak: data is plotted in CV (column volumes, 1 mL / CV) on the x-axis vs. mAU (absorbance units) measured by a 10 mm UV flow cell on the primary y-axis. It is shown: the UV-signal at 280 nm (long / short-dashed line) and 260 nm (solid line).

[0068] Figure 3: Experiment 1: Mass Photometry Histograms of fraction 1,

[0069] Figure 4: Experiment 1: Mass Photometry Histograms of fraction 2.

[0070] Figure 5: Experiment 4, full view: data is plotted in CV (column volumes, 1 mL / CV) on the x-axis vs. mAU (absorbance units) measured by a 10 mm UV flow cell on the primary y-axis and pH on the secondary y-axis. It is shown: the UV-signal at 280 nm (long / short-dashed line) and 260 nm (solid line) and pH (dashed line).

[0071] Figure 6: Experiment 4, zoom on elution peak: data is plotted in CV (column volumes, 1 mL / CV) on the x-axis vs. mAU (absorbance units) measured by a 10 mm UV flow cell on the primary y-axis. It is shown: the UV-signal at 280 nm (long / short-dashed line) and 260 nm (solid line).

[0072] Figure 7: Experiment 6, zoom on elution peak: data is plotted in CV (column volumes, 1 mL / CV) on the x-axis vs. mAU (absorbance units) measured by a 10 mm UV flow cell on the primary y-axis. It is shown: the UV-signal at 280 nm (long / short-dashed line) and 260 nm (solid line). Figure 8: Experiment 10, Run 1, zoom on elution peak: data is plotted in CV (column volumes, 1 mL / CV) on the x-axis vs. mAU (absorbance units) measured by a 2 mm UV flow cell on the primary y-axis. It is shown: the UV-signal at 280 nm (long / short-dashed line) and 260 nm (solid line).

[0073] Figure 9: Experiment 18, full view: data is plotted in CV (column volumes, 1 mL / CV) on the x-axis vs. mAU (absorbance units) measured by a 10 mm UV flow cell on the primary y-axis and pH on the secondary y-axis. It is shown: the UV-signal at 280 nm (long / short-dashed line) and 260 nm (solid line) and pH (dashed line).

[0074] Figure 10: Experiment 18, zoom on elution peak: data is plotted in CV (column volumes, 1 mL / CV) on the x-axis vs. mAU (absorbance units) measured by a 10 mm UV flow cell on the primary y-axis. It is shown: the UV-signal at 280 nm (long / short-dashed line) and 260 nm (solid line).

[0075] Figure 11: Experiment 21, full view: data is plotted in CV (column volumes, 1 mL / CV) on the x- axis vs. mAU (absorbance units) measured by a 10 mm UV flow cell on the primary y-axis and pH on the secondary y-axis. It is shown: the UV-signal at 280 nm (long / short-dashed line) and 260 nm (solid line) and pH (dashed line). The pH shows a sharp drop first, because Citrate does not buffer at pH 7.4.

[0076] Figure 12: Experiment 21, zoom on elution peak: data is plotted in CV (column volumes, 1 mL / CV) on the x-axis vs. mAU (absorbance units) measured by a 10 mm UV flow cell on the primary y-axis. It is shown: the UV-signal at 280 nm (long / short-dashed line) and 260 nm (solid line).

[0077] DEFINITIONS

[0078] Unless otherwise defined herein, scientific and technical terms used in connection with the current invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0079] General information regarding the nucleotide sequences of human immunoglobulins light and heavy chains is given in: Kabat, E.A., etal., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0080] Useful methods and techniques for carrying out the current invention are described in e.g. Ausubel, F.M. (ed.), Current Protocols in Molecular Biology, Volumes I to III (1997); Glover, N.D., and Hames, B.D., ed., 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 (1986); Watson, J.D., et al., Recombinant DNA, Second Edition, CHSL Press (1992); Winnacker, E.L., From Genes to Clones; N.Y., VCH Publishers (1987); Celis, J., ed., Cell Biology, Second Edition, Academic Press (1998); Freshney, R.I., Culture of Animal Cells: A Manual of Basic Technique, second edition, Alan R. Liss, Inc., N.Y. (1987).

[0081] The use of recombinant DNA technology enables the generation of derivatives of a nucleic acid. Such derivatives can, for example, be modified in individual or several nucleotide positions by substitution, alteration, exchange, deletion, or insertion. The modification or derivatization can, for example, be carried out by means of site directed mutagenesis. Such modifications can easily be carried out by a person skilled in the art (see e.g., Sambrook, J., et al., Molecular Cloning: A laboratory manual (1999) Cold Spring Harbor Laboratory Press, New York, USA; Hames, B.D., and Higgins, S.G., Nucleic acid hybridization - a practical approach (1985) IRL Press, Oxford, England).

[0082] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth. As well, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein. It is also to be noted that the terms "comprising", "including", and "having" can be used interchangeably.

[0083] The term “about” denotes a range of + / - 20 % of the thereafter following numerical value. In one embodiment the term about denotes a range of + / - 10 % of the thereafter following numerical value. In one embodiment the term about denotes a range of + / - 5 % of the thereafter following numerical value.

[0084] The term “affinity” refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to the intrinsic binding affinity that reflects a 1 : 1 interaction between the members of a binding pair (e.g., antibody and antigen). Affinity can be measured by common methods known in the art.

[0085] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s)” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms or words that do not preclude the possibility of additional acts or structures. The term “comprising” also encompasses the term “consisting of’. The present disclosure also contemplates other embodiments “comprising”, “consisting of’ and “consisting essentially of’ the embodiments or elements presented herein, whether explicitly set forth or not.

[0086] An "effective amount" of an agent, e.g., a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0087] The term “isolated” refers to a protein, peptide or nucleic acid that is not found in nature and is free or substantially free from other macromolecular species found in a cellular environment

[0088] The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning the use of such therapeutic products.

[0089] The term "pharmaceutical formulation" refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein, e.g., of a therapeutic antibody, to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.

[0090] “Substantially free”, as used herein, means the protein, peptide or nucleic acid of interest comprises more than 80% (on a molar basis) of the macromolecular species present, preferably more than 90% and more preferably more than 95.

[0091] The terms “empty recombinant AAV particle” and “empty rAAVp”, which can be used interchangeably, denote a protein shell composed of adeno-associated capsid polypeptides without a therein encapsidated / packaged functional nucleic acid (rAAVp = recombinant adeno-associated virus particle). That is, an empty rAAVp either may be free of encapsidated nucleic acid or comprises a nucleic acid or part thereof that is not transcribed at all or not transcribed into a functional transcript. Accordingly, an empty rAAVp does not function to transfer a nucleic acid that encodes a functional protein or is transcribed into a functional transcript of interest into a target cell. In certain embodiments of all aspects and embodiments, the functional protein or the functional transcript of interest has a therapeutic effect. The terms “full recombinant AAV particle” or “full rAAVp”, which can be used interchangeably, denote a non-covalent complex formed of a protein shell composed of adeno-associated capsid polypeptides and a therein encapsidated / packaged functional nucleic acid sequence. That is, a full rAAVp comprises a nucleic acid that is transcribed into a functional transcript. Accordingly, the full rAAVp functions to transfer a nucleic acid that encodes a protein or is transcribed into a transcript of interest into a target cell. In certain embodiments, a functional nucleic acid comprises at least one coding nucleic acid sequence interspaced between two adeno-associated viral inverted terminal repeats (ITRs).

[0092] The term “full to empty ratio” denotes the mathematical ratio of the number of full recombinant AAV particles (full rAAVp) to the total number of recombinant AAV particles (sum of full rAAVp and empty rAAVp) in a sample or in a recombinant AAV particle preparation. As the number of full rAAVp can be at most the same as the total number of rAAVp, the ratio can be at most 1. Generally, the ratio is less than 1 and is expressed as a percentage. The number of full rAAVp can be determined by determining the number of nucleic acid sequences interspaced between two AAV ITRs in the sample or preparation. This can be done by PCR, especially digital droplet PCR (ddPCR) or quantitative PCR (qPCR). The total number of rAAVp can be determined by determining the number of protein shells formed of adeno-associated capsid polypeptides in the sample or preparation. This can be done by ELISA, especially by a capsid polypeptide specific ELISA.

[0093] The term “transgene” denotes a nucleic acid derived from a wild-type genome of an adeno-associated virus, wherein except for the ITRs (adeno-associated virus Inverted Terminal Repeats) all other endogenous parts of the AAV genome are replaced by one or more exogenous nucleic acid(s). For example, such an exogenous nucleic acid can be a nucleic acid transcribed into a transcript of interest or that encodes a therapeutic protein or a therapeutic nucleic acid. Typically, for a transgene one or both ITRs of the wild-type AAV genome are retained. Thus, a transgene, which is a synthetic construct, can be distinguished from a wild-type AAV genome, since all or at least a part of the viral genome has been replaced with a non-native (i.e. exogenous) nucleic acid(s) with respect to the virus. Incorporation of a non-native nucleic acid therefore defines the transgene as a "recombinant" nucleic acid. It has to be pointed out that the serotype of the ITRs in the transgene does not need to be the same as the serotype of the adeno-associated capsid polypeptides forming the shell of the rAAVp comprising said transgene. In principle, any non- AAV nucleic acid can be packaged into a shell composed of adeno-associated capsid polypeptides resulting in a rAAVp, e.g. for subsequent infection (transduction) of a cell, ex vivo, in vitro or in vivo, i.e. a rAAVp can contain any nucleic acid as long as the size requirements for packaging are fulfilled.

[0094] As used herein, the term "serotype" is used to classify different wild- type and recombinant AAV particles (rAAVps) based on the amino acid sequence of the polypeptides forming the protein shell (capsid) of the respective AAV particle. Originally, serologic distinctiveness was determined based on the lack of cross-reactivity between antibodies to one AAV particle as compared to another AAV particle. Such cross-reactivity differences are usually due to differences in capsid polypeptide sequences and the respective antigenic determinants (e.g., due to VP1, VP2, and / or VP3 sequence differences of AAV serotypes). Despite the possibility that AAV variants including capsid variants may not be serologically distinct from a reference or wild-type AAV or other AAV serotype, they differ by at least one amino acid residue compared to the reference or wild-type or other AAV serotype.

[0095] Under the traditional definition, a serotype means that the virus of interest has been tested against serum specific for all existing and characterized serotypes for neutralizing activity and no antibodies have been found that neutralize the virus of interest. As more naturally occurring virus isolates are discovered and / or capsid mutants generated, there may or may not be serological differences with any of the currently existing serotypes. Thus, in cases where the new AAV particle has no serological difference, this new AAV particle would be a subgroup or variant of the corresponding wild-type serotype. In many cases, serology testing for neutralizing activity has yet to be performed on mutant viruses with capsid sequence modifications to determine if they are of another serotype according to the traditional definition of serotype.

[0096] DETAILED DESCRIPTION OF THE INVENTION

[0097] GENERAL METHODS FOR PRODUCING RAAVP

[0098] WO 1999 / 11764 reported methods for generating high titer helper-free preparations of recombinant AAV vectors. Not further defined AAV producer cells grown in suspension in bioreactors were infected with Adenovirus Type 5 (Ad5) at a multiplicity of infection (MOI) of 10 in low serum media at 1.5 L scale at different pH values. At a culture pH of 7.2, 4.7 E+12 total particles were obtained, at a culture pH of 7.4 1.95 E+13 total particles were obtained, at a culture pH of 7.6 1.84 E+13 total particles were obtained and at a culture pH of 8.0 1.63 E+13 total particles were obtained. The cultivation was performed in a 1.5 L bioreactor and, thus, the cultivation volume can be calculated (75 % of the nominal value) to have been about 1.125 L. Therefore the total particle number correspond to 4.2 E+09 vp / mL (pH 7.2), 1.7 E+10 vp / mL (pH 7.4), 1.6 E+10 vp / mL (pH 7.6) and 1.5 E+10 vp / mL (pH 8), respectively.

[0099] WO 2000 / 14205 reported the production of AAV particles in a non-defined cell type denoted as JL- 14 cells by co-infection with adenoviral helper virus, whereby at a pH value of 7.4 the highest number of AAV particles (sum of intracellular and secreted AAV particles), at a pH value of 8 AAV particles with the highest infectivity and at a pH of 7.6 the highest ratio of number of AAV particles to infectivity was obtained. The cultivation was performed in a volume of 1.5 L medium and, thus, the total particle number correspond to 3.0 E+09 vp / mL (pH 7.2), 1.3 E+10 vp / mL (pH 7.4), 1.2 E+10 vp / mL (pH 7.6), 3.3 E+09 vp / mL (pH 7.8) and 1.1 E+10 vp / mL (pH 8), respectively. Based on the provided infectivity data it can be assumed that the full / empty ratio of the thereby produced rAAV particles is below 1 %.

[0100] Piras, B.A., et al. (Mol. Ther. Meth. Clin. Dev. 3 (2016) 16015) compared distribution of AAV8 in cell culture media and lysates on days 3, 5, 6 and 7 post-transfection and found increasing viral production through day 6, with the proportion of viral particles in the media increasing from 76% at day 3 to 94% by day 7. Larger-scale productions showed that the ratio of full-to-empty AAV particles is similar in media and lysate, and that AAV harvested on day 6 post-transfection provides equivalent function in mice compared to AAV harvested on day 3. AAV-

[0101] Piras et al. employed adherent HEK293T / 17 cells cultured in Dulbecco’s Modified Eagle’s Medium with 10% fetal bovine serum supplemented with 2 mmol / 1 GlutaMAX (Life Technologies, Grand Island, NY). AAV was produced by two-plasmid transfection using PEIpro(TM) (Polyplustransfection SA, Illkirch, France) 1 day after seeding cells at a density of 7.26x1 E+04 cells / cm2

[0102] Powers, A.D., et al. (Hum. Gene Ther. Meth. 27 (2016) 112-121) reported the development and optimization of AAV hFIX particle production by transient transfection in an iCELLis(R) fixed-bed bioreactor. A yield to as high as 9 E+14 viral particles per square meter of fixed bed were obtained. On day 3 after inoculation with HEK293T / 17 cells, the vessel was transfected with plasmid scAAV- LPl-hFIXco-helpv3 and plasmid CR21+LTAAV help 2-8 at a plasmid mass ratio of 3: 1, respectively, using polyethylenimine (PEIpro(TM) Transfection Reagent Cat #115-375; Polyplus) in IMDM (Lonza) or DMEM supplemented with 10% FBS and 6 mM GlutaMAX(TM). The PEI and DNA solutions were combined at a 2: 1 ratio.

[0103] Poulain, A., et al. (J. Biotechnol. 255 (2017) 16-27) reported rapid protein production from stable CHO cell pools using plasmid vector and the cumate gene-switch. Cells were transfected using linear polyethylenimine (PEIpro(TM)) from Polyplus-Transfection (Illkirch, France). On the day of transfection, cells were suspended at a density of 2 x 1 E+06 cells / mL in CD DG44 medium (Life Technologies Inc., Burlington, ON, Canada), supplemented with 4 mM glutamine and 0,1% Kolliphor® P 188. The cell suspension was distributed in 6-well plates (1.8 mL / well). The DNA:PEIpro(TM) complexes were prepared at a ratio of 1 :5 (w:w), with a total of 2 pg DNA per well to transfect in 100 pL of complete culture medium.

[0104] WO 2017 / 096039 reported scalable methods for producing recombinant AAV vectors in serum-free suspension cell culture systems suitable for clinical use. Production of rAAV vectors was performed in bioreactors with HEK293F cells using triple transfection at a cell density of 1 E+06 cells / mL (1.000.000 cells / mL) with a plasmid ratio of 1: 1: 1 and a PELbased transfection reagent (PEI / DNA weight ratio of 2: 1 with ’ / 2of PEI as free PEI) at a temperature of 37 °C and a pH value of 7.2.

[0105] Nyamay’antu, A., et al. (Cell Gen. Ther. Ins. 4 (2018) 71-79) reported that PEI is widely used due to its affordability and high DNA delivery efficiency, in both adherent and suspension cells grown in serum-free medium. PEIpro(TM) is suited for small- to large-scale production of various viruses, notably AAV particles. In stirred-tank bioreactors using HEK293 or HEK293T cells titers in the range of 0.8- 1.5 E+09-E+10 vg / mL can be obtained.

[0106] Koo, T., et al. (Nat. Commun. 9 (2018) 1855) reported that CRISPR-LbCpfl prevents choroidal neovascularization in a mouse model of age-related macular degeneration. To produce AAV vectors, they were pseudotyped in AAV9 capsids. HEK293T cells (ATCC, CRL-3216) were transfected with pAAV-ITR-LbCpfl-crRNA, pAAV2 / 9 encoding for AAV2rep and AAV9cap, and helper plasmid. HEK293T cells were cultured in DMEM with 2% FBS. Recombinant pseudotyped AAV vector stocks were generated using PEI coprecipitation with PEIpro(TM) (Polyplus-transfection) and tripletransfection with plasmids at a molar ratio of 1 : 1 : 1 in HEK293T cells. After 72 h of incubation, cells were lysed and particles were purified by iodixanol step-gradient ultracentrifugation. The Rep proteins from AAV2 are commonly and nearly exclusively used in the production of rAAVps derived from the serotypes AAV1 to AAV13 (Daya, S., and Berns, K.I., Clin. Microbiol. Rev. 21 (2008) 583-593; Zincarelli, C., et al., Mol. Ther. 16 (2008) 1073-1080).

[0107] WO 2019 / 094253 reported means and methods for preparing viral vectors and uses thereof. Adherent HEK293 cells were cultivated in bioreactors at a pH value of 7.23 and triple transfected (plasmid ratio 1:1 :1) with PEI / DNA at a PEI-plasmid ratio of about 1 : 1 by weight.

[0108] Collaud, F. et al. (Mol. Ther. Meth. Clin. Dev. 12 (2019) 157-174) reported titers for recombinant AAV8 particles of 6.0 ± 1.89 E+04 vg / cell and 1.77 ± 1.37 E+04 vg / cell for (single stranded) and (self-complementary) AAV, respectively, for adherent HEK293 cells. A fully scalable method based on triple transfection of HEK293 cells cultured in suspension was also reported. Triple transfection of HEK293 cells was performed with polyethylenimine (PEIpro(TM), Polyplus) directly in 10 L bioreactors. AAV vectors were recovered from both supernatant and cells by mild detergent lysis followed by AVB Sepharose affinity column purification. Purified vectors were then concentrated and tested for quality and potency. No information about the pH value and obtained titers are provided.

[0109] Nyamay’antu, A., et al. (Cell Gen. Ther. Ins. 6 (2020) 655-661) reported that the efficiency of the delivery process is essential to obtain a high number of producing cells. Of the existing transfection methods, the use of PEI-based transfection reagent is predominant in gene therapy as it combines affordability and compatibility for transfection of adherent and suspension cells. In comparison to the gold standard PEIpro(TM) used for viral vector manufacturing, FectoVIR(TM)-AAV has been found to improve significantly recombinant AAV2 particle production yield of both viral genome production and packaging efficiency in suspension cells of an rAAV2-GFP of up to 10-fold compared to PEIMax(TM) and up to 2-fold compared to PEIpro(TM), respectively, when each transfection reagent is used under the recommended conditions. In more detail, suspension HEK293T cells were transfected using the respective transfection reagent under the recommended conditions. rAAV2-GFP were harvested 72 hours post transfection. The obtained titer with VectoVIR(TM) is in the range of 1 E+04 to 4.5 E+04 vg / cell depending on the used volume of complexation (1%-10%) corresponding to 1 E+12 vg / mL. The respective functional titers are about 2-8 E+08 TU / mL. The results are almost independent of the employed cultivation medium.

[0110] In a blog article entitled “Optimization of AAV production for high-yielding and scalable GMP processes with Catalent” (www.polyplus-transfection.com) different transfection reagent to DNA ratios were tested with the two serotypes AAV9 (1 : 1 and 2: 1) and AAV2 (3:1.5 and 5:2.5). The AAV2 vector yield was not affected as notably, with a 4-5-fold increase in the vector genome titer and a 3- 6-fold increase in the viral particle titer with FectoVIR(TM)- AAV as compared to PEIpro(TM). These results show that improvement in yield may vary with the AAV serotype. In a further study comparing additional AAV2 and AAV5 vectors (different from the previous AAV2 and AAV5 vectors) and using a DoE approach to optimization, experiments were conducted varying transfection reagent to DNA ratios (3:2, 3:1.5) and plasmid DNA molar ratios (1: 1: 1, 2:1 :2, 1:2: 1) were performed. A 3-5-fold increase for AAV2 and a 1.1-1.6-fold increase for AAV5 in the vector genome titer with F ecto VIR(TM)- AAV compared to PEIpro(TM) was observed. The viral particle titer increased 3.5- 4.5-fold for AAV2 and 2.5-3.75-fold for AAV5. Reagent-to-DNA ratios of 2:1 and 1.5:1 and plasmid ratios of 1 : 1 : 1 to 2: 1 :2 to 1 :2: 1 were used. The obtained titer with VectoVIR(TM) was in the range of 4 E+l 1 to 1 E+12 vg / mL.

[0111] Rossi, A. and Peigne, C-M. (Cell Culture Dish Article May 17, 2021) outlined that, typically, AAV production titers are around 1 E+l 1 to 1 E+12 in vg / mL and 1 E+08 to 1 E+09 TU / mL.

[0112] Wosnitzka, K., et al. (Cell Gen. Ther. Ins. 7 (2021) 1-7) reported that analysis of physical titers revealed a 3 -fold increase in both viral particles (VP) and viral genome (VG) per ml of cell culture when using FectoVIR(TM)-AAV transfection reagent compared to PEIpro(TM).

[0113] Porte, M., et al. (poster entitled “Next-Generation Transfection Reagent for Large Scale AAV Manufacturing”, Polyplus, Illkirch, France) reported the transfection of suspension-HEK293T cells with the optimal conditions for the other PEI- based reagent (1.5 pg / million cells, ratio DNA : PEI of 1 pg : 4 pL) and FectoVIR(TM)-AAV (1 pg / million cells, ratio DNA : reagent of 1 pg : 1 pL) following the recommended protocol for each reagent. A titer of about 5 E+l 1 vg / mL versus 1.5 E+l 1 vg / mL using FectoVIR(TM) and PEI-based transfection reagent, respectively, with a packaging efficiency of 20 % vs. about 13.5 %, respectively, was obtained.

[0114] Nakamura et al. (Eur. J. Haematol. 73 (2004) 285-294) reported about the development of packaging cell lines for generation of adeno-associated virus vectors by lentiviral gene transfer of trans- complementary components. It is outlined that adeno-associated virus (AAV) vector systems have several useful advantages with regard to in vitro and in vivo gene transfer. However, their usages have been limited by cumbersome and labor-intensive vector production in the traditional method. To overcome limitations in AAV production, Nakamura et al. explored the possibility of generating AAV packaging cell line, 293T R / C.VA.E2A.E4. cells, by using lentivirus-mediated transduction of Rep / Cap gene of AAV-2, VA RNA, E2A, and E4 genes of Ad5 into 293T cells. In packaging cell lines, it is important that supply of the AAV vector can be stably performed for long time. They showed that the 293T R / C. VA.E2A.E4. cells have stably maintained the transduced components after more than 10 passages and yielded high-titer AAV vectors, and the titer of AAV vectors did not decline even if culture of the packaging cells was continued for long time. The Rep / Cap and E4 gene products caused no remarkable cytotoxicity. The 293T R / C.VA.E2A.E4. cells might be able to tolerate the Rep / Cap and E4 gene products, or have less copy numbers of the Rep / Cap and E4 genes than the traditional method. Moreover, they showed that the AAV vectors derived from 293T R / C. VA.E2A.E4. cells infected the primary human CD34+ haematopoietic progenitor cells with high efficiency (50-70%). In the 293T R / C. VA.E2A.E4. cells, the AAV vectors can be generated by the transfection of one AAV vector plasmid, and large-scale AAV production can be easily achieved. It is important that cumbersome, variable, and costly transfection is avoided.

[0115] WO 2018 / 192983 reported an adeno-associated virus (AAV) producer cell comprising nucleic acid sequences encoding rep / cap gene; helper virus genes; and the DNA genome of the AAV vector particle, wherein said nucleic acid sequences are all integrated together at a single locus within the AAV producer cell genome. Reported are also nucleic acid vectors comprising a non-mammalian origin of replication and the ability to hold at least 25 kilo bases (kb) of DNA, characterized in that said nucleic acid vector comprises nucleic acid sequences encoding: rep / cap gene, and helper virus genes as well as uses and methods using said nucleic acid vector in order to produce stable AAV packaging and producer cell lines.

[0116] WO 2018 / 194438 reported a cell line for producing a non-replicating adenovirus, and a preparation method therefor and, more specifically, to: a cell line for producing a replication-deficient adenovirus by expressing any one or more selected from an El protein, and an El A protein or an E1B protein of an adenovirus; and a method for preparing the same. In addition, it is reported the use of the cell line, for expressing any one or more selected from an El protein, and an El A protein or an E1B protein of an adenovirus.

[0117] WO 2020 / 078953 reported adeno-associated virus (AAV) vector producer cell comprising nucleic acid sequences encoding AAV rep and cap genes, helper virus genes, and a DNA genome of the AAV vector; the AAV rep gene comprising an intron, the intron comprising a transcription termination sequence with a first recombination site located upstream and a second recombination site located downstream of the transcription termination sequence; and the nucleic acid sequences all integrated together at a single locus within the AAV vector producer cell genome.

[0118] WO 2020 / 132059 reported a mammalian cell line for producing adeno-associated virus (AAV), suitably including nucleic acids encoding helper genes and AAV genes, under the control of derepressible promoters. The disclosure also relates to isolated nucleic acid molecules that encode such genes, as well as methods of using the mammalian cells for producing AAVs. Especially is reported a mammalian cell for producing an adeno-associated virus (AAV), comprising (a) a nucleic acid molecule encoding a viral helper gene under control of a first derepressible promoter; (b) a nucleic acid molecule encoding an AAV gene under control of a second derepressible promoter; and (c) a nucleic acid molecule encoding a repressor element of the first and the second derepressible promoters.

[0119] EP 3 822 346 reported the use of an engineered mammalian packaging cell line for producing recombinant virus particles, wherein the cell line is engineered to lack cell surface expression of heparan sulfate. Further disclosed are a method for producing recombinant virus particles and a recombinant virus particle obtainable by the method. Further disclosed is a mammalian packaging cell line deposited under number DSM ACC3355 or DSM ACC3356.

[0120] WO 2022 / 112218 reported methods for the production of Adeno-associated vims (AAV), comprising steps of providing a stable AAV producer cell line in which at least some or all genes encoding the components necessary for the production of AAV are stably integrated into the cell genome, and culturing said cells in perfusion culture during the AAV production step (i.e., during the N step), wherein said perfusion culture encompasses continuous replacement of spent media with fresh media, and wherein said continuous replacement of spent media with fresh media continues after the induction of AAV production. In the cell at least (a) a gene encoding the AAV Rep protein Rep78 or Rep68, (b) a gene encoding the AAV Rep protein Rep52 or Rep40; (c) the genes encoding the adenoviral helper functions E4orf6 and E2A stably integrated into the host cell genome. Further at least the following genes are stably integrated into the host cell genome (a) the genes encoding the AAV Cap proteins VP1, VP2, VP3; (b) a gene encoding the AAV Rep protein Rep78 or Rep68; (c) a gene encoding the AAV Rep protein Rep52 or Rep40; (d) the genes encoding the adenoviral helper functions E4orf6 E2A; (e) the gene of interest flanked by AAV ITRs. WO 2022 / 173944 reported methods for producing an adeno-associated virus (AAV) in an El complementary producer cell. Especially is reported a method of producing an adeno-associated virus (AAV) in an El complementary producer cell, comprising (a) transfecting the El complementary producer cell with one or more vectors comprising (1) an El A adenovirus helper gene; (2) an adenovirus helper gene selected from E2A, E4, or both; (3) a viral-associated, non-coding RNA (VA RNA); and (4) an AA V gene selected from Rep, Cap, or both; (b) culturing the transfected El complementary producer cell under conditions suitable for producing the AAV; and (c) purifying the AAV from the cultured El complementary producer cell, thereby obtaining the AAV.

[0121] WO 2022 / 192261 reported compositions and methods for producing and characterizing stable viral vector producer cell lines that enable industrial scale production of viral vectors. Novel viral vector genome constructs, in which the constructs can be precisely mapped and viral vector genome constructs precisely quantified, are also disclosed for efficient production and characterization of viral vectors in mammalian cells.

[0122] WO 2023 / 077078 reported recombinant adeno-associated virus (rAAV) packaging and / or producer cell lines which have been engineered to reduce expression and / or activity of one or more genes and / or proteins to increase rAAV titers. Especially is reported a recombinant adeno-associated virus (rAAV) packaging and / or producer cell line comprising cells in which the expression of a gene selected from the group consisting of RIG-1 (DDX58), IFIT3, MDA5 (IFIH1), CGAS (cGAS), CHUK (IKK-a), DDX41, DHX58 (LGP2), IFI6, IKBKB (IKK-a), IRF3, IRF7, MAVS, MYD88, NFKB1, NFKB2, TBK1, TRIP, and TRIM25, and any combination thereof is reduced compared to that in control parental cells.

[0123] WO 2023 / 102549 reported systems for increasing AAV particle production. These systems comprise producer cell lines adapted for the production of AAV particles, as well as methods of producing AAV particles using said producer cell lines. Also provided are AAV particles produced by said production systems, producer cell lines and methods. Especially it is reported a genetically engineered producer cell line in which the expression of at least one of TMED10, M0N2, TMED2, HS2ST1, C3orfS8, SPPL3, SURF4, LSMS, ARF1, and PI4KB is reduced as compared to a control cell line, and / or in which the expression of at least one of B4GALT7, B3GAT3, OAF, EXT2, C0MMD3, SLC3SD1, B3GALT6, SDC1, NDST1, RAC1, CSK, GLCE, PDCL, FAM20B, TM4SFS, DGAT2, POMT1, YY1, and DPF2 is increased as compared to a control cell line. WO 2023 / 114897 reported methods for the production of recombinant adeno-associated virus (rAAV) particles. These methods are particularly useful for the large-scale production of AAV particles. Especially it is reported A method for producing recombinant AAV (rAAV) particles, comprising (a) introducing into a mammalian cell a first polynucleotide comprising an rAAV genome, to generate an AA V producer cell; (b) culturing the AA V producer cell in a first culture medium at a first temperature for a first period of time; (c) culturing the AAV producer cell in a second culture medium at a second temperature for a second period of time, wherein the second temperature is about 38°C to about 42°C, such that rAAV particles are produced by the AAV producer cell, wherein the rAAV particles comprise an rAAV genome comprising a transgene, and an AAV capsid comprising an AAV capsid protein.

[0124] WO 2023 / 166026 reported cell lines in which DNA fragmentation is inhibited, uses of cell lines in which DNA fragmentation is inhibited for the production of adeno-associated virus (AAV), related methods of producing AAV, and methods of producing AAV, comprising the step of exposing the cells in which AAV is produced to an inhibitor of DNA fragmentation during the AAV production phase.

[0125] WO 2023 / 171698 reported a producer cell for the production of an adeno-associated virus wherein cell damage is avoided or suppressed at the establishment of a cell line, a method for producing the producer cell, and a method for producing an AAV using the producer cell. In the producer cell a Cap gene under the control of a foreign promoter and a Rep gene under the control of a foreign promoter is integrated in the chromosome, that is free from a VA-RNA gene and / or an E4 gene, and that is a mammalian cell.

[0126] The content of all documents outlined in this section are expressly incorporated by reference herein.

[0127] RECOMBINANT CELL

[0128] Generally, for efficient as well as large-scale production of a rAAVp a cell expressing and, if possible, also secreting said rAAVp is used. Such a cell is termed “recombinant producer cell” or short “producer cell”.

[0129] For the generation of a recombinant producer cell a suitable mammalian cell is transfected with the nucleic acids required for producing said rAAVp, including the required AAV helper functions. Generally, for expression of a coding sequence, i.e. of an open reading frame, additional regulatory elements, such as a promoter and a polyadenylation signal (sequence), are necessary. Thus, for functional transcription an open reading frame has to be and is operably linked to said additional regulatory elements. This can be achieved by combining these parts into a so-called expression cassette. The minimal regulatory elements required for an expression cassette to be functional in a mammalian cell are a promoter functional in said mammalian cell, which is located upstream, i.e. 5’, to the open reading frame, and a polyadenylation signal (sequence) functional in said mammalian cell, which is located downstream, i.e. 3’, to the open reading frame. Additionally, a terminator sequence may be present 3’ to the polyadenylation signal (sequence). For expression, the promoter, the open reading frame / coding region and the polyadenylation signal sequence have to be arranged in an operably linked form.

[0130] Likewise, a nucleic acid that is transcribed into a non-protein coding RNA is called “RNA gene”. Also, for expression of an RNA gene, additional regulatory elements, such as a promoter and a transcription termination signal or polyadenylation signal (sequence), are necessary. The nature and localization of such elements depends on the RNA polymerase that is intended to drive the expression of the RNA gene. Thus, an RNA gene is normally also integrated into an expression cassette.

[0131] In case of an rAAVp, which is composed of different (monomeric) capsid polypeptides and a therein encapsidated single stranded DNA molecule and which in addition requires other viral helper functions for production and encapsidation, a multitude of expression cassettes differing in the contained open reading frames / coding sequences are required. In this case, at least an expression cassette for each of the transgene, for the polypeptides forming the capsid of the rAAVp, for the required viral helper functions are required. Thus, individual expression cassettes at least for each of the helper functions E1A, E1B, E2A, E4orf6, the rep and cap genes are required. HEK293 cells express the El A and E1B helper functions constitutively.

[0132] ADENO-ASSOCIATED VIRUS (AAV)

[0133] For a general review of AAVs and of the adenovirus or herpes helper functions see, Berns and Bohensky, Advances in Virus Research, Academic Press., 32 (1987) 243-306. The genome of AAV is described in Srivastava et al., J. Virol., 45 (1983) 555-564. In US 4,797,368 design considerations for constructing recombinant AAV vectors are described (see also WO 93 / 24641). Additional references describing AAV vectors are West et al., Virol. 160 (1987) 38-47; Kotin, Hum. Gene Ther. 5 (1994) 793-801; and Muzyczka J. Clin. Invest. 94 (1994) 1351. Construction of recombinant AAV vectors is described in US 5,173,414; Lebkowski etal., 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.

[0134] An AAV is a replication-deficient parvovirus. It can replicate only in cells, in which certain viral functions are provided by a co-infecting helper virus, such as adenoviruses, herpesviruses and, in some cases, poxviruses such as vaccinia. Nevertheless, an AAV can replicate in virtually any cell line of human, simian or rodent origin provided that the appropriate helper viral functions are present.

[0135] Without helper viral genes being present, an AAV establishes latency in its host cell. Its genome integrates into a specific site in chromosome 19 [(Chr) 19 (ql3.4)], which is termed the adeno- associated virus integration site 1 (AAVS1). For specific serotypes, such as AAV2 other integration sites have been found, such as, e.g., on chromosome 5 [(Chr) 5 (pl 3.3)], termed AAVS2, and on chromosome 3 [(Chr) 3 (p24.3)], termed AAVS3.

[0136] AAVs are categorized into different serotypes. These have been allocated based on parameters, such as hemagglutination, tumorigenicity and DNA sequence homology. Up to now, more than 12 different serotypes and more than a hundred sequences corresponding to different clades of AAV have been identified.

[0137] The capsid protein type and symmetry determines the tissue tropism of the respective AAV. For example, AAV2, AAV4 and AAV5 are specific to retina, AAV2, AAV5, AAV8, AAV9 and AAV- rh.10 are specific for brain, AAV1, AAV2, AAV6, AAV8 and AAV9 are specific for cardiac tissue, AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9 and AAV10 are specific for liver, AAV1, AAV2, AAV5 and AAV9 are specific for lung.

[0138] Pseudotyping denotes a process comprising the cross packaging of the AAV genome between various serotypes, i.e. the genome is packaged with differently originating capsid proteins.

[0139] The wild- type AAV genome has a size of about 4.7 kb. The AAV genome further comprises two overlapping genes named rep and cap, which comprise multiple open reading frames (see, e.g., Srivastava et al., J. Viral., 45 (1983) 555-564; Hermonat et al., J. Viral. 51 (1984) 329-339; Tratschin et al., J. Virol., 51 (1984) 611-619). The Rep protein encoding open reading frame provides for four proteins of different size, which are termed Rep78, Rep68, Rep52 and Rep40. These are involved in replication, rescue and integration of the AAV. The Cap protein encoding open reading frame provides four proteins, which are termed VP1, VP2, VP3, and AAP. VP1, VP2 and VP3 are part of the proteinaceous capsid of the AAV particles. 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, an AAV requires in addition to the Rep and Cap proteins the products of the genes El A, El B, E4orf6, E2A and VA of an adenovirus or corresponding factors of another helper virus.

[0140] In the case of an AAV of the serotype 2 (AAV2), for example, the ITRs each have a length of 145 nucleotides and flank a coding sequence region of about 4470 nucleotides. Of the ITR’s 145 nucleotides 125 nucleotides have a palindromic sequence and can form a T-shaped hairpin structure. This structure has the function of a primer during viral replication. The remaining 20, non-paired, nucleotides are denoted as D-sequence.

[0141] The wild-type AAV genome harbors three transcription promoters P5, Pl 9, and P40 (Laughlin et al., Proc. Natl. Acad. Sci. USA 76 (1979) 5567-5571) for the expression of the rep and cap genes.

[0142] The ITR sequences have to be present in cis to the coding region. The ITRs provide a functional origin of replication (ori), signals required for integration into the target cell’s genome, and efficient excision and rescue from host cell chromosomes or recombinant plasmids. The ITRs further comprise origin of replication like-elements, such as a Rep-protein binding site (RBS) and a terminal resolution site (TRS). It has been found that the ITRs themselves can have the function of a transcription promoter (Flotte et al., J. Biol. Chem. 268 (1993) 3781-3790; Flotte et al., Proc. Natl. Acad. Sci. USA 93 (1993) 10163-10167).

[0143] For replication and encapsidation, respectively, of the viral single-stranded DNA genome an in trans organization of the rep and cap gene products is required.

[0144] The rep gene locus comprises two internal promoters, termed P5 and Pl 9. It comprises open reading frames for four proteins. Promoter P5 is operably linked to a nucleic acid sequence providing for nonspliced 4.2 kb mRNA encoding the Rep protein Rep78 (chromatin nickase to arrest cell cycle), and a spliced 3.9 kb mRNA encoding the Rep protein Rep68 (site-specific endonuclease). Promoter P19 is operably linked to a nucleic acid sequence providing for a non-spliced mRNA encoding the Rep protein Rep52 and a spliced 3.3 kb mRNA encoding the Rep protein Rep40 (DNA helicases for accumulation and packaging). The two larger Rep proteins, Rep78 and Rep68, are essential for AAV duplex DNA replication, whereas the smaller Rep proteins, Rep52 and Rep40, seem to be essential for progeny and singlestrand DNA accumulation (Chejanovsky & Carter, Virology 173 (1989) 120-128).

[0145] The larger Rep proteins, Rep68 and Rep78, can specifically bind to the hairpin conformation of the AAV ITR. They exhibit defined enzyme activities, which are required for resolving replication at the AAV termini. Expression of Rep78 or Rep68 could be sufficient for infectious particle formation (Holscher, C., et al. J. Virol. 68 (1994) 7169-7177 and 69 (1995) 6880-6885).

[0146] It is deemed that all Rep proteins, primarily Rep78 and Rep68, exhibit regulatory activities, such as induction and suppression of AAV genes as well as inhibitory effects on 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).

[0147] Recombinant overexpression of Rep78 results in phenotype with reduced cell growth due to the induction of DNA damage. Thereby the host cell is arrested in the S phase, whereby latent infection by the virus is facilitated (Berthet, C., et al., Proc. Natl. Acad. Sci. USA 102 (2005) 13634-13639).

[0148] Tratschin et al. reported that the P5 promoter is negatively auto-regulated by Rep78 or Rep68 (Tratschin et al., Mol. Cell. Biol. 6 (1986) 2884-2894). Due to the toxic effects of expression of the Rep protein, only very low expression has been reported for certain cell lines after stable integration of AAV (see, e.g., Mendelson et al., Virol. 166 (1988) 154-165).

[0149] The cap gene locus comprises one promoter, termed P40. Promoter P40 is operably linked to a nucleic acid sequence providing for 2.6 kb mRNA, which, by alternative splicing and use of alternative start codons, encodes the Cap proteins VP1 (87 kDa, non-spliced mRNA transcript), VP2 (72 kDa, from the spliced mRNA transcript), and VP3 (61 kDa, from alternative start codon). VP1 to VP3 constitute the building blocks of the viral capsid. The capsid has the function to bind to a cell surface receptor and allow for intracellular trafficking of the virus. VP3 accounts for about 90 % of total viral particle protein. Nevertheless, all three proteins are essential for effective capsid production.

[0150] It has been reported that inactivation of all three capsid proteins VP1 to VP3 prevents accumulation of single-strand progeny AAV DNA. Mutations in the VP1 amino-terminus ("Lip-negative" or "Inf- negative") still allows for assembly of single-stranded DNA into viral particles whereby the infectious titer is greatly reduced. The AAP open reading frame is encoding the assembly activating protein (AAP). It has a size of about 22 kDa and transports the native VP proteins into the nucleolar region for capsid assembly. This open reading frame is located upstream of the VP3 protein encoding sequence.

[0151] In individual AAV particles, only one single-stranded DNA molecule is contained. This may be either the "plus" or "minus" strand. AAV particles containing a DNA molecule are infectious. Inside the infected cell, the parental infecting single stranded DNA is converted into a double stranded DNA, which is subsequently amplified. The amplification results in a large pool of double stranded DNA molecules from which single strands are displaced and packaged into capsids.

[0152] Adeno-associated viral (AAV) vectors can transduce dividing cells as well as resting cells. It can be assumed that a transgene introduced using an AAV vector into a target cell will be expressed for a long period. One drawback of using an AAV vector is the limitation of the size of the transgene that can be introduced into cells.

[0153] Parvovirus particles, including AAV serotypes and variants thereof, provide a means for ex vivo, in vitro and in vivo delivery of nucleic acid, which encode proteins, into cells such that the infected cells express the encoded protein. AAVs are viruses useful as gene therapy vectors as they can penetrate cells and introduce nucleic acid / genetic material so that the nucleic acid / genetic material may be stably maintained in the infected cells. Because AAV are not associated with pathogenic disease in humans, AAVs are able to deliver heterologous polynucleotide sequences (e.g., therapeutic proteins and agents) to human patients without causing substantial AAV-related pathogenesis or disease.

[0154] AAV particles used as vehicles for effective gene delivery possess a number of desirable features for such applications, including tropism for dividing and non-dividing cells. Early clinical experience with these vectors also demonstrated no sustained toxicity and immune responses were minimal or undetectable. AAV are known to infect a wide variety of cell types in vivo and in vitro by receptor- mediated endocytosis or by transcytosis. These vector systems have been tested in humans targeting retinal epithelium, liver, skeletal muscle, airways, brainjoints and hematopoietic stem cells.

[0155] Recombinant AAV particles do not typically include viral genes associated with pathogenesis. Such particles typically comprise a genome, wherein one or more of the wild-type AAV genes have been deleted in whole or in part, for example, rep and / or cap genes, but retain at least one functional flanking ITR sequence, as necessary for the rescue, replication, and packaging of the recombinant vector into an rAAV. Thus, an AAV vector includes sequences required in cis for replication and packaging (i.e. functional ITR sequences).

[0156] Recombinant AAV particles, as well as methods and uses thereof, can be based on any wild-type AAV genome or serotype or combination thereof. As a non-limiting example, a rAAV can be based upon any wild-type AAV genome, i.e. comprise the respective ITR sequences, such as AAV1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, 2i8, rh.74, rh.10 or 7m8 for example. Such particles can be based on the same strain or serotype (or subgroup or variant), or be different from each other. As a nonlimiting example, a rAAV based upon one wild-type genome can be identical or different to one or more of the capsid proteins that package the vector. In addition, a recombinant AAV vector can be based upon an AAV (e.g., AAV2) wild-type serotype genome distinct from one or more of the AAV capsid proteins that package the vector. For example, the AAV vector can be based upon AAV2, whereas at least one of the three capsid proteins could be an AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-218, AAV-rh.74, AAV-rh.10 or AAV-7m8 or a variant thereof, for example. AAV variants include variants and chimeras of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-218, AAV- rh.74, AAV-rh.10 and AAV-7m8 capsids.

[0157] In certain embodiments of all aspects and embodiments of the invention, the rAAVp is derived from a wild-type AAV particle selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-218, AAV-rh.74, AAV-rh.10 and AAV-7m8, as well as variants (e.g., capsid variants, such as amino acid insertions, additions, substitutions and deletions) thereof, for example, as set forth in WO 2013 / 158879, WO 2015 / 013313 and US 2013 / 0059732 (disclosing LK01, LK02, LK03, etc.).

[0158] In certain embodiments of all aspects and embodiments of the invention, the rAAVp comprises a capsid polypeptides with an amino acid sequence having 70 % or more sequence identity to an wildtype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV-rh.10, AAV-rh.74, or AAV-7m8 capsid sequence.

[0159] In certain embodiments of all aspects and embodiments of the invention, the rAAVp comprises one or two ITR sequence having 70 % or more sequence identity to a wild-type AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 or AAV12 ITR sequence. Recombinant AAV particles can be incorporated into pharmaceutical compositions. Such pharmaceutical compositions are useful for, among other things, administration and delivery to a subject in vivo or ex vivo. In certain embodiments, the pharmaceutical composition contains a pharmaceutically acceptable carrier or excipient. Such excipients include any pharmaceutical agent that does not itself induce an immune response harmful to the individual receiving the composition, and which may be administered without undue toxicity.

[0160] Protocols for the generation of adenoviral 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, which are incorporated herein by reference in their entirety.

[0161] RECOMBINANT ADENO-ASSOCIATED VIRAL PARTICLES (RAAVPS)

[0162] Different methods are known in the art for generating recombinant AAV particles. For example, transfection with an AAV vector comprising plasmid and a plasmid comprising AAV helper sequences (rep and cap) in conjunction with co-infection with one AAV helper virus (e.g., adenovirus, herpesvirus, or vaccinia virus) or transfection with a recombinant AAV vector comprising plasmid, an AAV helper plasmid (comprising rep and cap), and an helper function plasmid. Non-limiting methods for generating rAAV are described, for example, in US 6,001,650, US 6,004,797, WO 2017 / 096039, and WO 2018 / 226887. Following rAAV production (i.e. particle generation in cell culture systems), rAAV can be obtained from the host cells and / or cell culture supernatant and purified.

[0163] For the generation of recombinant AAV particles, expression of the Rep and Cap proteins, the helper proteins E1A, E1B, E2A and E4orf6 as well as optionally the adenoviral VA RNA in a single mammalian cell is required. The helper 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. Thus, any promoter can be operably linked to said genes for functional expression.

[0164] Generally, to produce rAAV, different, complementing plasmids are co-transfected into a host cell. One of the plasmids comprises the transgene sandwiched between the two cis acting AAV ITRs. The missing AAV elements required for replication and subsequent packaging of progeny recombinant genomes, i.e. the open reading frames for the Rep and Cap proteins, are contained in trans on a second plasmid. The over expression of the Rep proteins results in inhibitory effects on cell growth (Li, J., et al., J. Virol. 71 (1997) 5236-5243). Additionally, a third plasmid comprising the genes of a helper virus, i.e. El, E4orf6, E2A and VA from adenovirus, is required for rAAV production.

[0165] To reduce the number of required plasmids, rep, cap and the adenovirus helper genes may be combined on a single plasmid.

[0166] Alternatively, the host cell may already stably express the El gene products. Such a cell is a HEK293 cell. The human embryonic kidney clone denoted as 293 was generated back in 1977 by integrating adenoviral DNA into human embryonic kidney cells (HEK cells) (Graham, F.L., et al., J. Gen. Virol. 36 (1977) 59-74). The HEK293 cell line comprises base pair 1 to 4344 of the adenovirus serotype 5 genome. This encompasses the El A and E1B genes as well as the adenoviral packaging signals (Louis, N., et al., Virology 233 (1997) 423-429).

[0167] When using HEK293 cells the missing E2A, E4orf6 and VA genes can be introduced either by coinfection with an adenovirus or by co-transfection with an E2A-, E4orf6- and VA-expressing plasmid (see, e.g., 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 (1997) 8780-8789; Chionm, J.A., et al., Hum. Gene Ther. 6 (1995) 1531-1541; Ferrari, F.K., et al., J. Virol. 70 (1996) 3227-3234; Salvetti, A., etal., Hum. Gene Ther. 9 (1998) 695-706; Xiao, X., et al., J. Virol. 72 (1998) 2224-2232; Grimm, D., et al., Hum. Gene Ther. 9 (1998) 2745-2760; Zhang, X., et al., Hum. Gene Ther. 10 (1999) 2527-2537). Alternatively, adenovirus / AAV or herpes simplex virus / AAV hybrid vectors can be used (see, e.g., Conway, 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).

[0168] In order to limit the transgene activity to specific tissues, i.e. to limit the site of action, the transgene can be operably linked to an inducible or tissue specific promoter (see, e.g., Yang, Y., et al. Hum. Gene. Ther. 6 (1995) 1203-1213).

[0169] The coding sequences of El A and E1B (open reading frames) can be derived from a human adenovirus, such as, e.g., in particular of human adenovirus serotype 2 or serotype 5. An exemplary sequence of human Ad5 (adenovirus serotype 5) is found in GenBank entries X02996, AC 000008 and that of an exemplary human Ad2 in GenBank entry AC_000007. Nucleotides 505 to 3522 comprise the nucleic acid sequences encoding El A and E1B of human adenovirus serotype 5. Plasmid pSTK146 as reported in EP 1 230 354, as well as plasmids pGSl 19 and pGS122 as reported in WO 2007 / 056994, can also be used as a source for the El A and E1B open reading frames.

[0170] El A is the first viral helper gene that is expressed after adenoviral DNA enters the cell nucleus. The El A gene encodes the 12S and 13S proteins, which are based on the same El A mRNA by alternative splicing. Expression of the 12S and 13S proteins results in the activation of the other viral functions E1B, E2, E3 and E4. Additionally, expression of the 12S and 13S proteins force the cell into the S phase of the cell cycle. If only the ElA-derived proteins are expressed, the cell will die (apoptosis).

[0171] E1B is the second viral helper gene that is expressed. It is activated by the ElA-derived proteins 12S and 13S. The E1B gene derived mRNA can be spliced in two different ways resulting in a first 55 kDa transcript and a second 19 kDa transcript. The E1B 55 kDa protein is involved in the modulation of the cell cycle, the prevention of the transport of cellular mRNA in the late phase of the infection, and the prevention of El A-induced apoptosis. The E1B 19 kDa protein is involved in the prevention of ElA-induced apoptosis of cells.

[0172] The E2 gene encodes different proteins. The E2A transcript codes for the single strand-binding protein (SSBP), which is essential for AAV replication

[0173] In addition, the E4 gene encodes several proteins. The E4 gene derived 34 kDa protein (E4orf6) prevents the accumulation of cellular mRNAs in the cytoplasm together with the E1B 55 kDa protein, but also promotes the transport of viral RNAs from the cell nucleus into the cytoplasm.

[0174] The viral associated RNA (VA RNA) is a non-coding RNA of adenovirus (Ad), regulating translation. The adenoviral genome comprises two independent copies: VAI (VA RNAI) and VAII (VA RNAII). Both are transcribed by RNA polymerase III (see, e.g., Machitani, M., et al., J. Contr. Rel. 154 (2011) 285-289) from a type 2 polymerases III promoter. For recombinant AAV particle production, the adenoviral VA RNA gene can be driven by any promoter.

[0175] The structure, function, and evolution of adenovirus-associated RNA using a phylogenetic approach was investigated by Ma, Y. and Mathews, M.B. (J. Virol. 70 (1996) 5083-5099). They provided alignments as well as consensus VA RNA sequences based on 47 known human adenovirus serotypes. Said disclosure is herewith incorporated by reference in its entirety into the current application.

[0176] VA RNAs, VAI and VAII, are consisting of 157-160 nucleotides (nt). Depending on the serotype, adenoviruses contain one or two VA RNA genes. VA RNAI is believed to play the dominant pro-viral role, while VA RNAII can partially compensate for the absence of VA RNAI (Vachon, V.K. and Conn, G.L., Virus Res. 212 (2016) 39-52).

[0177] The VA RNAs are not essential, but play an important role in efficient viral growth by overcoming cellular antiviral machinery. That is, although VA RNAs are not essential for viral growth, VA RNA- deleted adenovirus cannot grow during the initial step of vector generation, where only a few copies of the viral genome are present per cell, possibly because viral genes other than VA RNAs that block the cellular antiviral machinery may not be sufficiently expressed (see Maekawa, A., et al. Nature Sci. Rep. 3 (2013) 1136).

[0178] Maekawa, A., et al. (Nature Sci. Rep. 3 (2013) 1136) reported efficient production of adenovirus vector lacking genes of virus-associated RNAs that disturb cellular RNAi machinery, wherein HEK293 cells that constitutively and highly express flippase recombinase were infected to obtain VA RNA-deleted adenovirus by FLP recombinase-mediated excision of the VA RNA locus.

[0179] The human adenovirus 2 VA RNAI corresponds to nucleotides 10586-10810 of GenBank entry AC_000007 sequence. The human adenovirus 5 VA RNAI corresponds to nucleotides 10579-10820 of GenBank entry AC 000008 sequence.

[0180] GENERAL DESCRIPTION OF RECOMBINANT AAV PARTICLE PRODUCTION

[0181] After entry into the host cell nucleus, AAV can follow either one of two distinct and interchangeable pathways of its life cycle: the lytic or the lysogenic. The former develops in cells infected with a helper virus such as Ad or herpes simplex virus (HSV) whereas the latter is established in host cells in the absence of a helper virus.

[0182] When a latently infected cell is super-infected with a helper virus, the AAV gene expression program is activated leading to the AAV Rep-mediated rescue (i.e., excision) of the provirus DNA from the host cell chromosome followed by replication and packaging of the viral genome. Finally, upon helper virus-induced cell lysis, the newly assembled virions (particles) are released. Thus, the lytic phase of the AAV life cycle is induced.

[0183] Therefore, in the presence of Ad helper functions, the rAAV vector is subjected to the wild-type AAV lytic processes by being rescued from the plasmid backbone, replicated and packaged into preformed AAV capsids as single-stranded molecules (Goncalves, M.A.F.V., Virol. I., 2 (2005) 43). Generation of a recombinant AAV particle involves replacing a majority of the AAV's wild-type genome with a desired transgene and providing the viral genes that are essential for virus packaging in-trans on a separate plasmid. Once all components are transfected together into a packaging cell line, recombinant AAV particles are assembled using the cell’s cellular machineries. The process of viral assembly and encapsulation takes roughly two days, after which the cells are lysed to release the rAAV for further purification and concentration

[0184] (https: / / old.abmgood.com / marketing / knowledge_base / Adeno_Associated_Virus_Production_and_ Modification of AAV. php) .

[0185] AAV is not released very efficiently from the cells, although major differences have been observed between serotypes (see, e.g., Strobel, B., et al., Lamia T. Comparative Analysis of Cesium Chloride- and lodixanol-Based Purification of Recombinant Adeno-Associated Viral Vectors for Preclinical Applications. Hum. Gene Ther. Methods 26 (2015) 147-157). When harvesting the culture, a cell disruption method is usually applied to recover the vectors entrapped in the cells.

[0186] Historically, manufacturing of rAAVps was performed by double transfection of a plasmid containing the rep and the cap ORFs and a plasmid with the gene of interest flanked by ITRs. Then, a helper virus, typically Adenovirus, was co-infected (see, e.g., Aponte-Ubillus, J.J., et al., Appl. Microbiol. Biotechnol. 102 (2018) 1045-1054; Muzyczka, N., Curr. Top. Microbiol. Immunol. 158 (1992) 97- 129). In this setting, the separation of the helper virus from the final product was difficult, but a critical element to avoid induction of inflammatory responses after injection into patients (see, e.g., Schnell, M.A., et al., Mol. Ther. 3 (2001) 708-722.). Therefore, production of rAAVps nowadays moved towards an adenovirus-free approach by utilizing triple transfection (see, e.g., Large, E.E., et al., Viruses 13 (2021) 1336). To this end, three components are needed: one plasmid encoding the genes for Rep and Cap without the ITRs, a second plasmid with the transgene of interest flanked by ITRs, and a helper plasmid to provide the helper genes of the helper virus (see, e.g., Aponte-Ubillus, J. J., et al., Appl. Microbiol. Biotechnol. 102 (2018) 1045-1054; Farris, K.D. and Pintel, D.J., Hum. Gene Ther. 19 (2008) 1421-1427; Grimm, D., et al., Hum. Gene Ther. 9 (1998) 2745-2760; Ferrari, F.K., et al., Nat. Med. 3 (1997) 1295-1297). For example, the Adenovirus helper bears the minimal required adenoviral genes E2A, E4 and VA. It is important to note, that the Human Embryonic Kidney cells 293 (HEK293) constitutively express the adenoviral genes E1A / B, which are also required for production of rAAVps. Therefore, HEK293 cells are classic producer cells for rAAVps and for manufacturing. Other cell types require a supplementation of E1A / B. 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 a transgene (Carter, B. J., in "Handbook of Parvoviruses", ed. by P. Tijssen, CRC Press, pp. 155-168 (1990)). Further, it has been reported that the ITRs have to be maintained to retain the function of replication, rescue, packaging, and integration of the transgene into the genome of the target cell.

[0187] When cells comprising the respective viral helper genes are transduced by an AAV vector, or, vice versa, when cells comprising an integrated AAV provirus are transduced by a suitable helper virus, then the AAV provirus is activated and enters a lytic infection cycle again (Clark, K.R., et al., Hum. Gene Ther. 6 (1995) 1329-1341; Samulski, R.J., Curr. Opin. Genet. Dev. 3 (1993) 74-80).

[0188] Producer cells contain the rep and cap gene sequences, as well as the transgene cassette flanked by ITR sequences on one or more plasmids that are retained, e.g., via drug selection. Production of rAAV in these cell lines generally occurs after their infection with the required helper functions. Therefore, cells are infected either with replication-competent adenovirus (usually wild type Ad5) or a plasmid comprising the respective helper genes to supply helper virus proteins and initiate rAAV production. A packaging cell line differs from a producer cell line as it only contains the rep and cap genes.

[0189] More generally, cells transfected or transduced with DNA for the recombinant production of AAV particles can be referred to as a "recombinant cell". Such a cell can be any mammalian cell that has been used as recipient of a nucleic acid (plasmid) encoding packaging proteins, such as AAV packaging proteins, a nucleic acid (plasmid) encoding helper proteins, and a nucleic acid (plasmid) that encodes a protein or is transcribed into a transcript of interest, i.e. a transgene placed between two AAV ITRs. The term includes the progeny of the original cell, which has been transduced or transfected. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total nucleic acid complement as the original parent, due to natural, accidental, or deliberate mutation.

[0190] Numerous cell growth media appropriate for sustaining cell viability or providing cell growth and / or proliferation are commercially available. Examples of such medium include serum free eukaryotic growth mediums, such as medium for sustaining viability or providing for the growth of mammalian (e.g., human) cells. Non-limiting examples include Ham's F12 or F12K medium (Sigma-Aldrich), FreeStyle (FS) F17 medium (Thermo-Fisher Scientific), MEM, DMEM, RPMI-1640 (Thermo-Fisher Scientific) and mixtures thereof. Such media can be supplemented with vitamins and / or trace minerals and / or salts and / or amino acids, such as essential amino acids for mammalian (e.g., human) cells.

[0191] For producing rAAV, three plasmids are co-transfected into a mammalian cell. The transgene plasmid encodes the expression cassette, which is cloned between the AAV ITRs, whereas rep and cap genes are provided in trans by co-transfecting a second, packaging plasmid (rep / cap plasmid) to ensure AAV replication and packaging. The third plasmid, also referred to as helper plasmid, contains the minimal helper virus factors, commonly adenoviral E2A, E4orf6 and VA genes, but lacking AAV ITRs.

[0192] Diverse methods for the DNA transfer into mammalian cells have been reported in the art. These are all useful in the methods according to the current invention. In certain embodiments of all aspects and embodiments, electroporation, nucleofection, or microinjection for nucleic acid transfer / transfection is used. In certain embodiments of all aspects and embodiments, an inorganic substance (such as, e.g., calcium phosphate / DNA co-precipitation), a cationic polymer (such as, e.g., polyethylenimine, DEAE-dextran), or a cationic lipid (lipofection) is used for nucleic acid transfer / transfection is used. Calcium phosphate and polyethylenimine are the most commonly used reagents for transfection for nucleic acid transfer in larger scales (see, e.g., Baldi et al., Biotechnol. Lett. 29 (2007) 677-684), whereof polyethylenimine is preferred.

[0193] The growth in serum-free suspension culture and improvement of efficiency and reproducibility of transfection conditions using PEI as a transfection reagent permits ready scale-up the AAV production using shake-flasks, wave, or stirred-tank bioreactors.

[0194] The composition may comprise further plasmids or / and cells. Such plasmids and cells may be in contact with free PEI.

[0195] In addition to PEI, valproic acid (VP A) can be used to improve transfection efficiency. VP A, a branched short-chain fatty acid and inhibits histone deacetylase activity. Due to this reason, it is commonly added to mammalian cell culture as an enhancer of recombinant protein production.

[0196] Encoded AAV packaging proteins include, in certain embodiments of all aspects and embodiments, AAV rep and / or AAV cap. Such AAV packaging proteins include, in certain embodiments of all aspects and embodiments, AAV rep and / or AAV cap proteins of any AAV serotype.

[0197] Encoded helper proteins include, in certain embodiments of all aspects and embodiments, adenovirus El A and E1B, adenovirus E2 and / or E4, VA RNA, and / or non- AAV helper proteins. The cultivation can be performed using the generally used conditions for the cultivation of eukaryotic cells of about 37 °C, 95 % humidity and 8 vol.-% CO2. The cultivation can be performed in serum containing or serum free medium, in adherent culture or in suspension culture. The suspension cultivation can be performed in any fermentation vessel, such as, e.g., in stirred tank reactors, wave reactors, rocking bioreactors, shaker vessels or spinner vessels or so called roller bottles. Transfection can be performed in high throughput format and screening, respectively, e.g. in a 96 or 384 well format.

[0198] Methods according to the current invention can include AAV particles of any serotype, or a variant thereof. In certain embodiments of all aspects and embodiments, a recombinant AAV particle comprises any of AAV serotypes 1-12, an AAV VP1, VP2 and / or VP3 capsid protein, or a modified or variant AAV VP1 , VP2 and / or VP3 capsid protein, or wild-type AAV VP1 , VP2 and / or VP3 capsid protein. In certain embodiments of all aspects and embodiments, an AAV particle comprises an AAV serotype or an AAV pseudotype, where the AAV pseudotype comprises an AAV capsid serotype different from an ITR serotype.

[0199] Expression control elements include constitutive or regulatable control elements, such as a tissuespecific expression control element or promoter.

[0200] ITRs can be any of AAV2 or AAV6 or AAV8 or AAV9 serotypes, or a combination thereof. AAV particles can include any VP1, VP2 and / or VP3 capsid protein having 75 % or more sequence identity to any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV10, AAV11, AAV12, AAV 218, AAV rh.10, AAV rh.74 or AAV 7m8 VP1, VP2 and / or VP3 capsid proteins, or comprises a modified or variant VP1, VP2 and / or VP3 capsid protein selected from any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAV9, AAV-218, AAV-rh.10, AAV-rh.74 and AAV-7m8 AAV serotypes.

[0201] Following production of recombinant viral (e.g., AAV) particles, if desired, the viral (e.g., rAAV) particles can be purified and / or isolated from host cells using a variety of conventional methods. Such methods include column chromatography, CsCl gradients, iodixanol gradient and the like.

[0202] For example, a plurality of column purification steps such as purification over an anion exchange column, an affinity column and / or a cation exchange column can be used. (See, e.g., WO 02 / 12455 and US 2003 / 0207439). Alternatively, or in addition, an iodixanol or CsCl gradient steps can be used (see, e.g., US 2012 / 0135515; and US 2013 / 0072548). Further, if the use of infectious virus is employed to express the packaging and / or helper proteins, residual virus can be inactivated, using various methods. For example, adenovirus can be inactivated by heating to temperatures of approximately 60 °C for, e.g., 20 minutes or more. This treatment effectively inactivates the helper virus since AAV is heat stable while the helper adenovirus is heat labile.

[0203] An objective in the rAAV 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 AAV- encapsulated residual DNA impurities.

[0204] Considering that the rAAV represents only a minor fraction of the biomass, rAAV need to be purified to a level of purity, which can be used as a clinical human gene therapy product (see, e.g., Smith P.H., et al., Mo. Therapy 7 (2003) 8348; Chadeuf G., et al, Mo. Therapy 12 (2005) 744; report from the CHMP gene therapy expert group meeting, European Medicines Agency EMEA / CHMP 2005, 183989 / 2004).

[0205] In certain embodiments of all aspects and embodiments of the method according to the current invention, as an initial step, typically the cultivated cells that produce the rAAVps are harvested, optionally in combination with harvesting cell culture supernatant (medium) in which the cells (suspension or adherent) producing recombinant AAV particles have been cultured. The harvested cells and optionally cell culture supernatant may be used as is, as appropriate, lysed or concentrated. Further, if infection is employed to express helper functions, residual helper virus can be inactivated. For example, adenovirus can be inactivated by heating to temperatures of approximately 60 °C for, e.g., 20 minutes or more, which inactivates only the helper virus since AAV is heat stable while the helper adenovirus is heat labile.

[0206] The cells in the harvested cultivation broth can be lysed using methods now in the art, such as, e.g., detergent lysis or freeze-thaw cycles, to release the rAAV particles. Concurrently during cell lysis or subsequently after cell lysis, a nuclease, such as, e.g., benzonase, is added to degrade contaminating DNA. Typically, the resulting lysate is clarified to remove cell debris, e.g. by filtering or centrifuging, to render a clarified cell lysate. In a particular example, the lysate is filtered with a micron diameter pore size filter (such as a 0.1-10.0 pm pore size filter, for example, a 0.45 pm and / or pore size 0.2 pm filter), to produce a clarified lysate.

[0207] The lysate (optionally clarified) contains recombinant AAV particles (comprising full as well as empty rAAVps) and production / process related impurities, such as soluble cellular components from the host cells that can include, inter alia, cellular proteins, lipids, and / or nucleic acids, and cell culture medium components. The optionally clarified lysate is then subjected to purification steps to purify the rAAV (comprising rAAV vectors) from impurities using chromatography. The clarified lysate may be diluted or concentrated with an appropriate buffer prior to the first chromatography step.

[0208] After cell lysis, optional clarifying, and optional dilution or concentration, a plurality of subsequent and sequential chromatography steps can be used to purify the rAAV.

[0209] The first chromatography step is preferably an affinity chromatography step using an AAV affinity chromatography ligand.

[0210] If the first chromatography step is affinity chromatography the second chromatography step can be anion exchange chromatography. Thus, in certain embodiments of all aspects and embodiments, rAAV purification is via affinity chromatography, followed by purification via anion exchange chromatography or / and cation exchange chromatography or / and size exclusion chromatography, in any order or sequence or combination.

[0211] The removal of empty capsids from full ones, for example, during downstream processing is based on their different isoelectric points (pl) in anion exchange chromatography. The average calculated pl across all serotypes is 5.9 for full capsids and 6.3 for empty capsids (Venkatakrishnan, B., et al., J. Virol. 87 (2013) 4974-4984).

[0212] Cation exchange chromatography functions to separate the AAV from cellular and other components present in the clarified lysate and / or column eluate from an affinity or size exclusion chromatography. Examples of strong cation exchange resins capable of binding rAAV over a wide pH range include, without limitation, any sulfonic acid based resin as indicated by the presence of the sulfonate functional group, 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). Additional examples include Capto S, Capto S ImpAct, Capto S ImpRes (strong cation exchangers available from GE Healthcare, Marlborough, MA, USA), and commercial DOWEX®, AMBERLITE®, and AMBERLYST® families of resins available from Aldrich Chemical Company (Milliwaukee, WI, USA). Weak cation exchange resins include, without limitation, any carboxylic acid based resin. Exemplary cation exchange resins include carboxymethyl (CM), phospho (based on the phosphate functional group), methyl sulfonate (S) and sulfopropyl (SP) resins.

[0213] Anion exchange chromatography functions to separate rAAV from proteins, cellular and other components present in the clarified lysate and / or column eluate from an affinity or cation exchange or size exclusion chromatography. Anion exchange chromatography can also be used to reduce and thereby control the amount of empty rAAV in the eluate. For example, the anion exchange column having full and empty rAAV bound thereto can be washed with a solution comprising NaCl at a modest concentration (e.g., about 100-125 mM, such as 110-115 mM) and a portion of the empty rAAV can be eluted in the flow through without substantial elution of the full rAAV. Subsequently, full rAAV bound to the anion exchange column can be eluted using a solution comprising NaCl at a higher concentration (e.g., about 130-300 mMNaCl), thereby producing a column eluate with reduced or depleted amounts of empty rAAVps and proportionally increased amounts of full rAAV comprising an rAAV vector.

[0214] Exemplary anion exchange resins include, without limitation, those based on polyamine resins and other resins. Examples of strong anion exchange resins include those based generally on the quaternized nitrogen atom including, without limitation, quaternary ammonium salt resins such as trialkylbenzyl ammonium resins. Suitable exchange chromatography materials include, without limitation, MACRO PREP Q (strong anion-exchanger available from BioRad, Hercules, CA, USA); UNOSPHERE Q (strong anion-exchanger available from BioRad, Hercules, CA, USA); POROS 50HQ (strong anion-exchanger available from Applied Biosystems, Foster City, CA, USA); POROS XQ (strong anion-exchanger available from Applied Biosystems, Foster City, CA, USA); POROS SOD (weak anion-exchanger available from Applied Biosystems, Foster City, CA, USA); POROS 50PI (weak anion-exchanger available from Applied Biosystems, Foster City, CA, USA); Capto Q, Capto XQ, Capto Q ImpRes, and SOURCE 30Q (strong anion-exchanger available from GE healthcare, Marlborough, MA, USA); DEAE SEPHAROSE (weak anion-exchanger available from Amersham Biosciences, Piscataway, NJ, USA); Q SEPHAROSE (strong anion-exchanger available from Amersham Biosciences, Piscataway, NJ, USA). Additional exemplary anion exchange resins include aminoethyl (AE), diethylaminoethyl (DEAE), diethylaminopropyl (DEPE) and quaternary amino ethyl (QAE). A commercial manufacturing process to purify recombinant AAV particles intended as a product to treat human disease should achieve the following objectives: 1) consistent particle purity, potency and safety; 2) manufacturing process scalability; and 3) acceptable cost of manufacturing.

[0215] Exemplary processes for recombinant AAV particle purification are reported in WO 2019 / 006390.

[0216] Methods to determine infectious titer of rAAV particles containing a transgene are known in the art (see, e.g., Zhen et al., Hum. Gene Ther. 15 (2004) 709). Methods for assaying for empty rAAV and full rAAV with packaged transgenes are known (see, e.g., Grimm et al., Gene Therapy 6 (1999) 1322- 1330; Sommer et al., Malec. Ther. 7 (2003) 122-128).

[0217] To determine the presence or amount of degraded / denatured capsid, purified rAAV can be subjected to SDS-polyacrylamide gel electrophoresis, consisting of any gel capable of separating the three capsid proteins, for example, a gradient gel, then running the gel until sample is separated, and blotting the gel onto nylon or nitrocellulose membranes. Anti- AAV capsid antibodies are then used as primary antibodies that bind to denatured capsid proteins (see, e.g., Wobus et al., J. Viral. 74 (2000) 9281- 9293). A secondary antibody that binds to the primary antibody contains a means for detecting the primary antibody. Binding between the primary and secondary antibodies is detected semi- quantitatively to determine the amount of capsids. Another method would be analytical HPLC with a SEC column or analytical ultracentrifuge.

[0218] DESCRIPTION OF THE SEQUENCES

[0219] SEQ ID NO: 1 Primer CMV_enh_fwd

[0220] SEQ ID NO: 2 Primer CMV_enh_rev

[0221] SEQ ID NO: 3 Primer CMV_enh_probe

[0222] EXAMPLES

[0223] Example 1

[0224] Cultivation of HEK293 cells and production of recombinant AAV preparations

[0225] Generally, the cultivation methods have been adapted from standard protocols (see, e.g., Lindl, T., “Zell- und Gewebekultur: Einfuhrung in die Grundlagen sowie ausgewahlte Methoden und Anwendungen”, Spektrum Akademischer Verlag GmbH, Heidelberg / Berlin, 2002) and operating instructions of the respective supplier.

[0226] Pre- cultivation

[0227] HEK cells were thawed and propagated in shake flasks at 37 °C, 85 % humidity, a pCO2 of 5% and a shaking frequency of 120 rpm for two to three weeks in cultivation medium. Cells were split every three to four days and expanded in medium to the volume required for inoculation of the production cultivation.

[0228] Production cultivation

[0229] For producing recombinant AAV particles, the respective pre-cultivated HEK293 cells were cultured in the respective reactor in a batch or fed-batch process under the indicated conditions. rAAVp preparation: 1) with particles comprising capsid variant derived from AAV2 serotype and therapeutic transgene; 2) with particles comprising an AAV2 wild type capsid and green fluorescent protein (GFP) transgene: reactor: Ambrl5 cell line: HEK293 cell line stably transfected with all elements required for rAAVp production; doxycycline inducible cell line cultivation medium: Gibco™ Protein Expression Medium (PEM; Thermo Fisher Scientific; Cat-No.: 10621864) + 4 mM GlutaMAX™ (Thermo Fisher Scientific; Cat-No.: 35050061) feed: HEK FS (Sartorius; Cat-No.: 871-0001) temperature: about 37 °C pH: about 7.2 inoculation VCD: ~ 30 E+05 cells / mL induction: day 3; doxycycline duration: 7 days Example 2

[0230] Lysis

[0231] In case lysis was included in the process, it has been performed as follows: To release the AAV particles into the cell culture broth, 5 % (v / v) of lysis buffer (10 % Triton CG 110, 40 mM MgCh) was added to the culture broth. Additionally, 100 U / ml Benzonase™ nuclease (Merck). The cell culture broth was then incubated for about one hour at 37 °C with stirring, without aeration and pH control. After the respective incubation, 5 M NaCl solution was added and the lysate was sterile filtered.

[0232] Example 3

[0233] AAV particle purification

[0234] General Method:

[0235] Load Preparation:

[0236] The following steps were performed sequentially:

[0237] 1.1. CCCF (concentrated cell culture fluid) containing the AAV particles was thawed in a waterbath at 25 °C and aliquoted into equal portions containing a total of 1 El 4 capsids.

[0238] 1.2. The aliquots were frozen at -70 °C

[0239] 1.3. Per experiment, one aliquot was thawed in a waterbath at 25 °C, filtered using a 0.22 pm Sartorius Minisart High Flow syringe filter.

[0240] 1.4. To collect capsids remaining in the filter, the load was chased with 1 mL equilibration buffer (25 mM Tns / Tns-HCl, 150 mM NaCl, 2 mM MgC12, 0.005% Poloxamer 188, pH 7.4)

[0241] 1.5. The load was stored on ice and immediately processed

[0242] Chromatography:

[0243] Chromatographic AAV-purification was performed using a prepacked 0.5 x 5 cm 1 mL POROS™ GoPure™ AAVX column (Thermo Scientific™) (the POROS™ Captures elect™ AAVX in this GoPure™ column has a AAVX CaptureSelect™ material) on an AEKTA Avant 25 system (GE Healthcare). All chromatographic steps were done in downflow.

[0244] Elution Buffer Preparation:

[0245] If only one buffer is required, the ingredients for the buffer were mixed in a cylinder. The pH-values of the buffer was adjusted using the respective amount of 10 mol / L NaOH The conductivity of the buffer got measured.

[0246] If the ingredients for both buffers are identical, the ingredients for both buffers were mixed in one graduated cylinder and later divided into 2 fractions of equal volume to finalize the buffers. The pH- values of both buffers were adjusted using the respective amount of 10 mol / L NaOH The conductivity of both buffers got measured. As more NaOH is needed for the “high pH”-buffer, its conductivity is higher. To ensure elution based on a pH-Gradient, the conductivity was strictly kept constant. Therefore, the conductivity of the “low pH” buffer was titrated to match the “high-pH” buffer conductivity using NaCl.

[0247] Chromatographic Steps:

[0248] Here the steps and conditions common to all the examples. Compounds used in the individual steps that are varied are denoted with an “X” in this section and defined in the specific experimental table.

[0249] 2.1. Equilibration 25 mM Tris / Tris-HCl, 150 mM NaCl, 2 mM MgC12, 0.005% Poloxamer 188, pH 7.4, 30 CV (column volumes) / h, 5 CV

[0250] 2.2. Load CCCF, 25 mM Tris / Tris-HCl, 150 mM NaCl, 2 mM MgC12, 0.005% Poloxamer 188, pH 7.4, 30 CV / h

[0251] 2.3. Load Chase, 25 mM Tris / Tris-HCl, 150 mM NaCl, 2 mM MgC12, 0.005% Poloxamer 188, pH

[0252] 7.4. 30 CV / h, XI CV

[0253] 2.4. Equilibration 2, 25 mM Tris / Tris-HCl, 150 mM NaCl, 2 mM MgC12, 0.005% Poloxamer 188, pH 7.4, 30 CV / h, X2 CV

[0254] 2.5. High pH Wash, X3, X4, X5, 2 mM MgC12, 0.005% Poloxamer 188, X6, 30 CV / h, 10 CV 2.6. Elution Gradient from 100 % X3, X4, X5, 2 mM MgC12, 0.005% Poloxamer 188, X6 to 100 % X7, X8, X9, 2 mM MgC12, 0.005% Poloxamer 188, X10, 15 CV / h, XI 1, XI 2, Pooling from UV280 10 mAU to Crossover UV260 / UV280 (= fraction 1); from Crossover UV260 / UV280 to UV280 10 mAU (=fraction 2). Fractions of 1 mL each were collected by using the Akta Avant’s volume fractionation.

[0255] In the experiments (unless stated otherwise), two pools were collected. Pool 1 encompassed the fractions from UV280 = 10 mAU (2 mm UV-cell) to crossover-point UV280 / 260 (short: CO) and pool 2 encompassed the fractions from CO to 10 mAU (2 mm UV-cell). As the fractionation was decoupled from the UV-signal, the fractions could not be split exactly at the CO. In experiments 5, 6 and 14 the CO was in the middle of a 1 mL fraction. In these cases, this fraction has been collected as a separate fraction. The pooling criteria in these cases would therefore be: Pool 1 : 10 mAU to shortly before CO, Pool 2: 1 mL fraction that contains CO, Pool 3: shortly after CO to 10 mAU. For proper visualization, the fractions and pools are depicted in the respective figures.

[0256] 2.7. Low pH Wash, X7, X8, X9, 2 mM MgC12, 0.005% Poloxamer 188, XI 0, 30 CV / h, 5 CV 2.8. Regeneration 1 , 100 mM Glycine, 2 M NaCl, pH 1.5, 30 CV / h, 5 CV

[0257] 2.9. Regeneration 2, 100 mM Phosphoric Acid, 30 CV / h, 5 CV 2.10. Regeneration 3, 25 mM NaOH, 30 CV / h, 5 CV

[0258] 2.11. Equilibration 3, 25 mM Tns / Tns-HCl, 150 mM NaCl, 2 mM MgC12, 0.005% Poloxamer 188, pH 7.4, 30 CV / h, 5 CV

[0259] 2.12. Storage, 20% Ethanol, 30 CV / h, 10 CV Experimental Data: n / a = not applicable; n.d. = not determined

[0260] Results:

[0261]

[0262] Results:

[0263]

[0264] Results:

[0265]

[0266] Results:

[0267] Results:

[0268] Results:

[0269]

[0270] Results:

[0271]

[0272] Results:

[0273]

[0274] Results:

[0275]

[0276] Results:

[0277] Results: Results:

[0278] Results:

[0279]

[0280] Results:

[0281]

[0282] Results:

[0283]

[0284] Results:

[0285] Results:

[0286] Results:

[0287]

[0288] Results:

[0289]

[0290] Results:

[0291]

[0292] Results:

[0293]

[0294] Results:

[0295] Results: Example 4

[0296] Analytical methods

[0297] Titer determination

[0298] The concentration of viral capsids in samples were determined using an ElectroChemiLuminescence ImmunoAssay (ECLIA) performed with a cobas®pro e801. The assay is a sandwich assay.

[0299] Samples were individually diluted in Elecsys base buffer (Elecsys Base buffer HCG STAT II Elecsys R1 Base buffer pH 7.5; Roche Diagnostics GmbH, Mannheim, Germany) based to the expected concentration to be within 1E8 to 1E11 cp / mL.

[0300] For determination approx.. 10 pL of the concentration adjusted sample was mixed with about 40 pL capture antibody (biotinylated, anti-AAVx antibody or AAV9-specific antibody) and approx.. 40 pL of a mixture of tracer antibody (POD-labeled, anti-AAVx antibody or AAV9-specific antibody) and detection antibody (ruthenylated anti-POD antibody; Roche Diagnostics GmbH, Mannheim, Germany). The mixture was incubated for about 10 minutes to form a sandwich complex of capture antibody-AAV particle-tracer antibody-detection antibody.

[0301] Thereafter streptavidin-coated magnetic beads (SA Coated Beads E2G, Roche Diagnostics GmbH, Mannheim, Germany) were added and incubated to capture the sandwich complex.

[0302] The reaction mixture was transferred to the measuring cell. The microparticles were fixed onto the surface of the electrode by magnetic action. Subsequently, unbound substances were removed with a buffer according to the manufacturer (ProCell II M; Roche Diagnostics GmbH, Mannheim, Germany). By applying a voltage, chemiluminescent emission was induced and measured with a photomultiplier.

[0303] The results were determined based on a calibration curve pre-determined with known concentrations of AAV capsids.

[0304] Digital Polymerase Chain Reaction (dPCR)

[0305] For removal of the contaminating free nucleic acids outside the particles, rAAVv samples were treated with nuclease. To remove the rAAVv capsid and release viral genomes, samples were subjected to a Proteinase K digestion step. After Proteinase K inactivation, samples were cooled to RT.

[0306] A dPCR Master Mix was prepared using QIAcuity Probe PCR Kit (Qiagen), a FAM-labeled primer / probe stock solution and restriction enzyme Smal. Primers and probes were used in concentration recommended by Qiagen. The following primers and probes were used:

[0307] A serial dilution of the pre-processed samples was conducted. Diluted template and Master Mix were transferred to each well of dPCR 96-well plate and vortexed strongly. For partitioning, the mix was transferred to a QIAcuity Nanoplate 26k 8-well (Qiagen) according to the manufacturer’s instructions. The plate was inserted into a Qiacuity Eight (Qiagen) and samples were subjected to endpoint PCR thermal cycling with the following protocol:

[0308] After PCR amplification of the viral genome targets, results were analyzed using the QIAcuity Software Suite 2.5.0.1. During analysis, it was necessary to ensure that samples and controls are within the linear range of the method and comparable to previously gained reference values.

Claims

1. CLAIMS1. A method for separating full AAV particles from empty AAV particles, the method comprising a) Applying a solution comprising full, empty and partially filled AAV particles to an AAV affinity column and thereby binding the AAV particles to the AAV affinity column, wherein the solution has a pH in the range of 7.4 to pH 4, b) Eluting full AAV particles from the AAV affinity column by applying a pH gradient from the pH of the solution of step a) to a pH in the range of 4 to 2.5, wherein the pH of step a) (applying step) and the pH of step b) (eluting step) differ in a least 0.5 pH units, c) Recovering full AAV particles from the eluate of step b), and thereby separating full AAV particles from empty AAV particles.

2. The method according to claim 1, the method comprising after step a) and before step b) the following step al) Washing the AAV affinity column obtained in step a) with a buffer with a pH in the range of pH 5.5 to pH 4, and step b) is b) Eluting full AAV particles from the AAV affinity column by applying a pH gradient from the pH of the solution of step al) to a pH in the range of 4 to 2.5, wherein the pH of step al) (washing step) and the pH of step b) (eluting step) differ in a least 0.5 pH units.

3. The method according to claim 1 or 2, the method comprising a) Equilibrating an AAV affinity chromatography material / column with a solution / buffer with a pH value of about pH 7.4,74b) Applying a solution comprising full, empty and partially filled AAV particles in a solution / buffer with a pH value of about pH 7.4 to the column obtained in step a), thereby binding the AAV particles to the AAV affinity column, c) Optionally washing the column obtained in step b) with a solution / buffer with a pH value of about 7.4, d) Washing the column of step b) or step c) with a solution / buffer with a pH value in the range of pH 5.5 to pH 4, e) Eluting full AAV particles from the column applying a pH gradient from the pH of the solution / buffer used in step d) to a pH in the range of 4 to 2.5 wherein the pH of step d) (washing step) and the pH of step e) (eluting step) differ in a least 0.5 pH units, f) Recovering full AAV particles from the eluate of step e), and thereby separating full AAV particles from empty AAV particles.

4. The method according to any one of claims 1 to 3, wherein the pH of step a) or step b) (applying step) is in the range of 7.4 to 5.5.

5. The method according to any one of claims 1 to 4, wherein the pH of step a) or step b) (applying step) is about 7.4.

6. The method according to any one of claims 1 to 5, wherein the steps of the method are performed in the presence of a salt.

7. The method according to claim 6, wherein the salt has a concentration of 0-2000 mM, 0- 1200 mM, 0-1000 mM, preferably 100-300 mM, more preferred 100-200 mM, most preferred about 200 mM.

8. A method for separating full AAV particles from empty AAV particles, the method comprising a) Applying a solution comprising full, empty and partially filled AAV particles to an AAV affinity column comprising an AAV affinity material and thereby binding theAAV particles to the AAV affinity column material, wherein the solution has a pH in the range of pH 6 to pH 3, b) Eluting full AAV particles from the column applying a salt gradient from the salt concentration of the solution of step a) to a salt concentration in the range of 0 mM to 1000 mM, c) Recovering full AAV particles from the eluate of step b), and thereby separating full AAV particles from empty AAV particles.

9. The method according to any one of claims 1 to 7, the method comprising a) Equilibrating an AAV affinity chromatography material / column comprising an AAV affinity material with a solution / buffer with a pH value of about pH 7.4 in the presence of a salt, b) Applying a solution comprising full, empty and partially filled AAV particles in a solution / buffer with a pH value of about pH 7.4 to the column obtained in step a) in the presence of a salt, thereby binding the AAV particles to the AAV affinity column material, c) Optionally washing the column obtained in step b) with a solution / buffer with a pH value of about 7.4 in the presence of a salt, d) Washing the column of step b) or step c) with a solution / buffer with a pH value in the range of pH 5.5 to pH 4 in the presence of a salt, e) Eluting full AAV particles from the column applying a combined pH and salt gradient, wherein the pH gradient is from the pH of the solution / buffer used in step d) to a pH in the range of 4 to 2.5 wherein the pH of step d) (washing step) and the pH of step e) (eluting step) differ in a least 0.5 pH units, wherein the salt gradient is from the concentration of the salt in step e) to a salt concentration in the range of 0 mM to 1000 mM, f) recovering full AAV particles from the eluate of step e),and thereby separating full AAV particles from empty AAV particles.

10. The method according to any one of claim 8 or 9, wherein the solution of step a) or step b) (applying step) comprises a salt at a concentration in the range of 0 to 2000 mM.

11. The method according to any one of any one of claims 8 to 10, wherein the solution of step a) or step b) (applying step) comprises a salt at a concentration in the range of 1000 mM to2000 mM and the gradient of step b) or step e) (eluting step) is to a salt concentration of 0 mM to 200 mM.

12. The method according to any one of claim 8 or 10, wherein the solution of step a) comprises a salt at a concentration in the range of 0 mM to 200 mM and the solution of step b) comprises a salt at a concentration of about 1000 mM.

13. The method according to any one of claims 1 to 12, wherein the steps of the method are performed in the presence of NaCl or MgC12 or Na2SO4 as a salt.

14. The method according to any one of claims 1 to 13, wherein the AAV particle is an AAV particle of serotype 2, serotype 8 or serotype 9.

15. The method according to any one of claims 1 to 14, wherein the elution buffer used in step b) or e) (eluting step) comprises formic acid or acetic acid or citric acid.77

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