Raman-based method for the discrimination of AAV particle serotype and AAV particle loading status

Abstract

In this specification, a method for determining virus particles having encapsulated nucleic acid in an aqueous sample using Raman spectroscopy is reported, which includes preparing a sample and irradiating the sample with a light source, measuring the total intensity of Raman scattered light for each of a first plurality of preselected wavenumbers and / or wavenumber ranges to obtain a first data set regarding the sample, performing a first set of mathematical data processing steps on the first data set, and determining virus particles having encapsulated nucleic acid in the sample based on the output of the first set of mathematical data processing steps, wherein the first set of mathematical data processing steps includes principal component analysis and the determining step is based on a first principal component.

Description

Technical Field

[0001] The present invention belongs to the field of analytical methods. More specifically, the present specification reports a method for determining AAV particle serotype and AAV particle load state using Raman spectroscopy in combination with classification or regression methods such as principal component analysis. The method according to the present invention can distinguish different AAV particle serotypes and their load states.

Background Art

[0002] Adeno-associated virus (AAV) particles are commonly used as gene delivery vehicles for research and clinical procedures due to their favorable safety profile, high therapeutic efficacy, and potential for target-specific manipulation. For recombinant production, an accurate and robust analytical method for characterization of viral vectors is required. Generally, vector genome titration is performed by droplet digital PCR (ddPCR), a method for absolute quantification of nucleic acids. These methods are sensitive and specific, but are laborious, costly, and time-consuming.

[0003] Raman spectroscopy is a powerful analytical technique for determining the vibrational modes of molecular bonds. Raman spectroscopy is commonly used in chemistry to provide a structural fingerprint that can identify molecules. Given the technical nature of this method, Raman spectroscopy can be rapidly and non-invasively applied to a variety of Raman-active samples for both identification and quantitative purposes. Furthermore, state-of-the-art chemical and statistical data analysis techniques enable multi-attribute Raman analysis of different compounds present in a single common sample.

[0004] Raman spectroscopy, especially surface-enhanced Raman spectroscopy (SERS) and tip-enhanced Raman spectroscopy (TERS), has recently been applied to various virus diagnostic methods for clinical diagnosis and food quality assurance purposes (Hermann, P., et al., The Analyst 136 (2011) 1148 (Non-Patent Document 1)). Most methods have focused on the detection and quantification of specific virus contaminants and the identification of virus-infected cells (Gogone, I.C.V.P., et al. Spectrochim. Acta A 249 (2021) 119336 (Non-Patent Document 2); Moor, K., et al., J. Biomed. Optics 23 (2018) 097001 (Non-Patent Document 3)). However, these high-sensitivity Raman methods often require nanoparticles conjugated to substances that can specifically bind to virus antigens for analysis, which poses challenges to the comparability and robustness of the tests.

[0005] International Publication No. 2020 / 136376 (Patent Document 1) discloses a method for determining virus titer using Raman spectroscopy based on partial least squares analysis of the total intensity of Raman scattered light within each of a plurality of wavenumber ranges to obtain a wavenumber intensity dataset of a sample, where the plurality of wavenumber ranges are preselected and are characteristic of the virus in the sample.

[0006] International Publication No. WO 2022 / 003359 (Patent Document 2) discloses a method for analyzing viruses using Raman spectroscopy, where a first determination of the viral nucleic acid content of a sample, based on the output of a mathematical data processing step performed on the total intensity of Raman scattered light within each of a first plurality of preselected wavenumber ranges that are characteristic of the viral nucleic acids in the sample, is combined with a second determination of the viral content in the sample, based on the output of a mathematical data processing step performed on the total intensity of Raman scattered light within each of a second plurality of preselected wavenumber ranges that are characteristic of one or more viral structural molecules of the virus in the sample. Thus, this document discloses the determination of a full / empty ratio based on two subsequent measurements combined with respective data analysis workflows, i.e., this ratio is calculated based on the results of both (two) measurements.

[0007] U.S. Patent No. 6,040,191 (Patent Document 3) discloses a Raman spectroscopy method for determining the ligand binding ability of biological substances. The method is a non-destructive process for determining the ability of a test biological substance to bind to at least one ligand. Thus, this document focuses on the determination of the ligand binding ability of biological substances.

[0008] U.S. Patent Application Publication No. 2009 / 086201 (Patent Document 4) discloses a surface-enhanced Raman spectroscopy (SERS) system for detecting viruses and a method of using the same. The method is a method for detecting at least one biomolecule in a sample, the method comprising binding at least one first biomolecule to an array of nanorods on a substrate and measuring a surface-enhanced Raman spectroscopy (SERS) spectrum.

[0009] French Patent No. 3,109,819 (Patent Document 5) discloses a method for detecting the presence of pathogens in biological fluids based on surface-enhanced Raman spectroscopy (SERS) by contacting the sample with non-magnetic metal nanoparticles and depositing the solution or suspension on a support.

[0010] Hermann et al. disclose the evaluation of tip-enhanced Raman spectroscopy for characterizing various virus strains (Analyst 136 (2011) 1148-1152 (Non-Patent Document 1)). They outline that optical techniques such as confocal Raman spectroscopy have proven to be rapid, non-destructive, and highly sensitive for chemical and biological analysis, enabling in vivo investigations of single bacterial cells. As a drawback, they conclude that the detection of small biological structures such as single virus particles requires further improved spatial resolution and significantly higher sensitivity. The disclosed solution is tip-enhanced Raman spectroscopy, an analytical technique that combines the advantages of atomic force microscopy (AFM) or scanning tunneling microscopy (STM) with SERS.

[0011] Huang et al. disclose Raman spectroscopy for virus detection and the realization of non-traditional food safety (Trends Food Sci. Technol. 116 (2021) 525-532 (Non-Patent Document 4)). Here, the viral constituent RNA, DNA, and proteins are investigated from the collected Raman spectra, and it is found that the Raman signals of viruses are mostly similar, making it difficult to highlight differences, and it is mentioned that spectral statistical analysis should be applied to overcome these problems (Thomas (Appl. Spectrosc. 30 (1976) 483-494) (Non-Patent Document 5)).

Prior Art Documents

Patent Documents

[0012]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Non-Patent Documents

[0013]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Summary of the Invention

[0014] This specification reports a method for the non-invasive, rapid, and efficient analysis of AAV samples related to bioprocesses using Raman spectroscopy, i.e., micro-Raman spectroscopy.

[0015] The present invention is at least partially based on the finding that by using Raman spectroscopy combined with statistical and machine learning / deep learning techniques, i) low-concentration complete particles and empty particles of various AAV serotype capsids in aqueous buffer solutions can be detected, ii) complete particles and empty particles in the capsid sample can be discriminated, and iii) particles having capsids of different serotypes can be discriminated.

[0016] More specifically, the present invention is at least partially based on the finding that by using conventional micro-Raman spectroscopy in combination with principal component analysis (PCA), i) low concentrations of AAV2 and AAV8 serotype complete particles in an aqueous buffer solution can be detected, ii) complete particles and empty particles in a sample can be discriminated, and iii) particles of different serotypes can be discriminated.

[0017] It has been found that different serotype AAV particles present in a sample, such as AAV2, AAV5, AAV8 and AAV9, can be distinguished using the method according to the present invention. Furthermore, the method according to the present invention enables a direct determination of the loading state of AAV particles, i.e., whether the particles contain DNA encapsulated (i.e., are complete particles) or do not contain encapsulated DNA (i.e., are empty particles).

[0018] The method according to the present invention is suitable for rapid and non-invasive analysis of AAV particle-containing samples, for example, for in-process control, quality assurance and control, and (real-time) release analysis.

[0019] The present invention is a method for determining virus particles having encapsulated nucleic acid in an aqueous sample using Raman spectroscopy, comprising: (a) preparing a sample and irradiating the sample with a light source; (b) (i) measuring the total intensity of one Raman scattered light for each of a first plurality of preselected wavenumbers and / or wavenumber ranges to obtain a first data set regarding the sample; (ii) performing a first set of mathematical data processing steps on the first data set; (c) determining virus particles having encapsulated nucleic acid in the sample based on the output of the first set of mathematical data processing steps. The present invention is directed to a method comprising the above steps.

[0020] The determination in the method according to the invention can be made both qualitatively and quantitatively. In the case of a quantitative determination, the number of virus particles having encapsulated nucleic acid is determined. The total number of virus particles can be determined by any method known in the art. The difference between the total number of particles and the number of particles having encapsulated nucleic acid is the number of particles without encapsulated nucleic acid. Thus, this number can likewise be determined based on the method according to the invention.

[0021] It must be pointed out that in the method according to the invention, ligand binding to the matrix is not required. That is, a binary classification (present / absent) is not included in the method according to the invention. The method according to the invention uses micro-Raman spectroscopy.

[0022] It must further be pointed out that in the method according to the invention, a single measured value is used for the determination. In particular, surprisingly, it has been found that this enables discrimination between complete AAV particles and empty AAV particles.

[0023] It must further be pointed out that in the method according to the invention, neither tagging nor labeling of AAV particles is required.

[0024] The present invention encompasses the following embodiments.

[0025] 1. A method for determining virus particles having encapsulated nucleic acid in an aqueous sample using Raman spectroscopy, comprising: (a) preparing a sample and irradiating the sample with a light source; (b) (i) measuring the total intensity of Raman scattered light of the sample, or at least each one of a first plurality of preselected wavenumbers and / or wavenumber ranges, to obtain a first data set regarding the sample; (ii) performing a first set of mathematical data processing steps on the first data set; (c) Based on the output of the first set of mathematical data processing steps, determining virus particles having encapsulated nucleic acid in the sample, and A method comprising.

[0026] 2. A method for determining virus particles without encapsulated nucleic acid in an aqueous sample using Raman spectroscopy, comprising: (a) Preparing a sample and irradiating the sample with a light source; and (b) (i) Measuring the total intensity of Raman scattered light of the sample or at least each one of a first plurality of preselected frequencies and / or frequency ranges to obtain a first data set regarding the sample; and (ii) Executing a first set of mathematical data processing steps on the first data set; and (c) Based on the output of the first set of mathematical data processing steps, determining virus particles without encapsulated nucleic acid in the sample, and A method comprising.

[0027] 3. The method according to embodiment 1 or 2, wherein the first set of mathematical data processing steps for the first data set is a classification method or a regression method.

[0028] 4. The method according to any one of embodiments 1 to 3, wherein the first set of mathematical data processing steps for the first data set is an artificial neural network or a decision tree-based model or principal component analysis, preferably principal component analysis.

[0029] 5. The first set of mathematical data processing steps for the first dataset is selected from the group consisting of principal component analysis (PCA), non-negative matrix factorization (NMF), linear discriminant analysis (LDA), generalized discriminant analysis (GDA), canonical correlation analysis (CCA), autoencoders, t-distributed stochastic neighbor embedding (t-SNE), uniform manifold approximation and projection (UMAP), k-nearest neighbors (k-NN), kernel or graph-based kernel PCA, and dimensionality reduction embedding that uses PCA, LDA, CCA, or NMF techniques as preprocessing steps followed by clustering by K-NN, the method according to any one of Embodiments 1 to 4.

[0030] 6. The first set of mathematical data processing steps for the first dataset is principal component analysis, and virus particles with or without encapsulated nucleic acids are determined based on the first principal component, the method according to any one of Embodiments 1 to 5.

[0031] 7. Step (i) is to (alpha) measure the total intensity of Raman scattered light of the sample, (beta) remove wavenumbers outside a first plurality of preselected wavenumbers and / or wavenumber ranges, (gamma) create a first deviation of the data function, and (delta) normalize the spectrum including, the method according to any one of Embodiments 1 to 6.

[0032] 8. Creating a first deviation of the data function is by applying a Savitzky-Golay filter, the method according to Embodiment 7.

[0033] 9. The Raman scattered light is determined using a confocal Raman microscope or a micro-Raman spectroscopy device, the method according to any one of Embodiments 1 to 8.

[0034] 10. The sample has a volume of 5 μL to 1000 μL, the method according to any one of Embodiments 1 to 9.

[0035] 11. The method according to any one of Embodiments 1 to 10, wherein the sample has a volume of 20 μL to 250 μL.

[0036] 12. The method according to any one of Embodiments 1 to 11, wherein the sample has a volume of about 200 μL.

[0037] 13. The method according to any one of Embodiments 1 to 11, wherein the sample has a volume of about 30 to 50 μL.

[0038] 14. The method according to any one of Embodiments 1 to 13, wherein the sample is a crude sample / not pretreated.

[0039] 15. The method according to any one of Embodiments 1 to 14, wherein the virus particles are parvovirus particles.

[0040] 16. The method according to any one of Embodiments 1 to 15, wherein the encapsulated nucleic acid is single-stranded DNA.

[0041] 17. The method according to any one of claims 1 to 16, wherein the virus particles are adeno-associated virus particles.

[0042] 18. The method according to any one of claims 1 to 17, wherein the virus particles are adeno-associated virus particles of serotype 2 or 5 or 8 or 9.

[0043] 19. The method according to any one of Embodiments 1 to 18, wherein the light source has a wavelength of about 532 nm or about 785 nm.

[0044] 20. The method according to any one of Embodiments 1 to 19, wherein the virus particles are AAV particles of serotype 2, and the light source has a wavelength of about 532 nm or about 785 nm.

[0045] 21. The method according to any one of claims 1 to 19, wherein the virus particles are AAV particles of serotype 8, and the light source has a wavelength of about 785 nm.

[0046] 22. The method according to any one of Embodiments 1 to 21, wherein the first plurality of preselected frequencies and / or frequency ranges consist of DNA / RNA / protein-specific nucleotide binding deformations and amide I bond elongations.

[0047] 23. The method according to any one of Embodiments 1 to 22, wherein the first plurality of preselected frequencies and / or frequency ranges consist of 489 - 728 cm-1 and / or 1645 - 1680 cm-1.

[0048] 24. The method according to any one of Embodiments 1 to 23, wherein the viral particles are AAV2 viral particles.

[0049] 25. The method according to any one of Embodiments 1 to 21, wherein the first plurality of preselected frequencies and / or frequency ranges consist of protein-specific vibrations of S-S Cys crosslinks, Tyr-specific vibrations, C-C elongations in beta sheets, Phe-specific vibrations, and / or amide I bond elongations.

[0050] 26. The method according to any one of Embodiments 1 to 21 and 25, wherein the first plurality of preselected frequencies and / or frequency ranges consist of one or more or all of 551 cm-1, 645 cm-1, 1000 cm-1, 1003 cm-1, 1530 - 1630 cm-1, and / or 1645 - 1680 cm-1.

[0051] 27. The method according to any one of Embodiments 1 to 21 and 25 to 26, wherein the viral particles are AAV8 viral particles.

[0052] 28. The method according to any one of Embodiments 1 to 21, wherein the first plurality of preselected frequencies and / or frequency ranges consist of protein- and nucleic acid-specific vibrations of S-S Cys crosslinks, Tyr-specific vibrations, nucleotide ring deformations and elongations, A / G and U / C ring vibrations, Phe-specific vibrations, and / or amide I bond elongations.

[0053] 29. The method according to any one of embodiments 1 to 21 and 28, wherein the first plurality of preselected frequencies and / or frequency ranges consist of one or more or all of 551 cm-1, 645 cm-1, 631 to 787 cm-1, 1425 to 1485 cm-1, 1003 cm-1, and / or 1645 to 1680 cm-1.

[0054] 30. The method according to any one of embodiments 1 to 21 and 28 to 29, wherein the viral particles are AAV2 particles and the sample further comprises AAV2 viral particles without encapsulated nucleic acid.

[0055] 31. The method according to any one of embodiments 1 to 21, wherein the first plurality of preselected frequencies and / or frequency ranges consist of protein-specific vibrations of S-S Cys crosslinks, Tyr-specific vibrations, protein C-C stretches in helical structures, C-C stretches in beta sheets, Phe-specific vibrations, and / or stretches of amide I bonds.

[0056] 32. The method according to any one of embodiments 1 to 21 and 31, wherein the first plurality of preselected frequencies and / or frequency ranges consist of one or more or all of 551 cm-1, 645 cm-1, 920 to 950 cm-1, 1000 cm-1, 1003 cm-1, 1530 to 1630 cm-1, and / or 1645 to 1680 cm-1.

[0057] 33. The method according to any one of embodiments 1 to 21 and 31 to 32, wherein the viral particles are AAV2 viral particles and the sample further comprises AAV8 viral particles with encapsulated nucleic acid.

[0058] 34. The method according to any one of embodiments 1 to 21 and 31 to 32, wherein the viral particles are AAV8 viral particles and the sample further comprises AAV2 viral particles with encapsulated nucleic acid.

[0059] 35. In step (c), since the sample contains a mixture of AAV particles of different serotypes, the method according to any one of embodiments 1 to 21 and 31 to 32, where the calculation is not conclusive.

[0060] 36. The method according to any one of embodiments 1 to 35, wherein the first plurality of pre-selected frequencies and / or frequency ranges consists of one or more, all, or at least all of 551 cm-1, 645 cm-1, 631 - 728 cm-1, and / or 1645 - 1680 cm-1.

[0061] 37. The method according to any one of embodiments 1 to 21, wherein the first plurality of pre-selected frequencies and / or frequency ranges consists of V(S-S) disulfide in protein, polysaccharide, DNA, protein vC-C alpha helix, amide III, RNA, and amide I vibration / extension.

[0062] 38. The method according to any one of embodiments 1 to 21 and 37, wherein the first plurality of pre-selected frequencies and / or frequency ranges consists of one or more, or all of the Raman shifts at 530 cm-1, 565 cm-1, 665 cm-1, 725 cm-1, 785 cm-1, 850 cm-1, 936 cm-1, 1230 cm-1, 1244 cm-1, 1265 cm-1, and / or 1671 cm-1.

[0063] 39. The method according to any one of embodiments 1 to 21 and 37 to 38, wherein the viral particles are AAV2 viral particles.

[0064] 40. The method according to any one of embodiments 1 to 21, wherein the first plurality of pre-selected frequencies and / or frequency ranges consists of polysaccharide, DNA, protein vC-C beta sheet, phenylalanine, nucleic acid, RNA, tyrosine, and amide I extension.

[0065] 41. The method according to any one of Embodiments 1 to 21 and 40, wherein the first plurality of preselected frequencies and / or frequency ranges consists of one or more or all of Raman shifts of 460 cm-1, 670 - 785 cm-1, 985 cm-1, 1003 cm-1, 1080 cm-1, 1244 cm-1, 1613 cm-1, and / or 1671 cm-1.

[0066] 42. The method according to any one of Embodiments 1 to 21 and 40 - 41, wherein the viral particles are AAV8 viral particles.

[0067] 43. The method according to any one of Embodiments 1 to 21, wherein the first plurality of preselected frequencies and / or frequency ranges consists of V(S - S) disulfide in proteins, polysaccharides, DNA, protein vC - C beta - sheets, phenylalanine, amide III, and amide I stretching.

[0068] 44. The method according to any one of Embodiments 1 to 21 and 43, wherein the first plurality of preselected frequencies and / or frequency ranges consists of one or more or all of Raman shifts of 530 cm-1, 565 cm-1, 665 cm-1, 725 cm-1, 785 cm-1, 985 cm-1, 1003 cm-1, 1265 cm-1, and / or 1671 cm-1.

[0069] 45. The method according to any one of Embodiments 1 to 21 and 43 - 44, wherein the viral particles are AAV2 particles, and the sample further comprises AAV2 viral particles that do not have encapsulated nucleic acid.

[0070] 46. The method according to any one of Embodiments 1 to 21, wherein the first plurality of preselected frequencies and / or frequency ranges consists of polysaccharides, DNA, protein vC - C alpha - helices, protein vC - C beta - sheets, phenylalanine, nucleic acids, RNA, C - H vibrations (proteins), phenylalanine, proteins, tyrosine, and amide I stretching.

[0071] 47. The method according to any one of embodiments 1 to 21 and 46, wherein the first plurality of preselected frequencies and / or frequency ranges consists of one or more or all of the Raman shifts of 460 cm-1, 565 cm-1, 665 cm-1, 750 cm-1, 825 cm-1, 855 cm-1, 936 cm-1, 985 cm-1, 1003 cm-1, 1080 cm-1, 1244 cm-1, 1450 cm-1, 1585 cm-1, 1613 cm-1, and / or 1671 cm-1.

[0072] 48. The method according to any one of embodiments 1 to 21 and 46 to 47, wherein the viral particles are AAV2 viral particles and the sample further comprises AAV8 viral particles having encapsulated nucleic acid.

[0073] 49. The method according to any one of embodiments 1 to 21 and 46 to 47, wherein the viral particles are AAV8 viral particles and the sample further comprises AAV2 viral particles having encapsulated nucleic acid.

[0074] 50. The method according to any one of embodiments 1 to 21 and 46 to 49, wherein the first plurality of preselected frequencies and / or frequency ranges consists of one or more or all of the Raman shifts of 1671 cm-1 and 565 cm-1, 665 cm-1, 785 cm-1, 985 cm-1, 1003 cm-1, and / or 1244 cm-1.

[0075] 51. A method for determining virus particles without encapsulated nucleic acid in an aqueous sample using Raman spectroscopy, comprising: a) determining virus particles having encapsulated nucleic acid in the aqueous sample using Raman spectroscopy by the method according to any one of embodiments 1 to 50; b) determining the total number of virus particles in the aqueous sample; c) obtaining the number of virus particles without encapsulated nucleic acid by subtracting the number obtained in b) from the number obtained in a). The method comprising the above steps.

[0076] 52. The method according to embodiment 51, wherein the total number of virus particles in the aqueous sample is determined by an enzyme-linked immunosorbent assay.

[0077] 53. The method according to any one of embodiments 1 to 52, which is for quantification.

[0078] 54. Quantification is performed by applying a statistical or machine learning / deep learning method from the group of methods consisting of partial least squares (PLS), Lasso, Lasso-Lars, ridge regression, elastic net, Huber regression, passive aggressive regression, Bayesian ridge regression, orthogonal matching pursuit, (artificial) neural network (ANN), (nu) support vector regression, random forest regression, decision tree, XGBoost regression, gradient boost regression, Adaboost regression, autogluon, and AutoKeras to the Raman data. The method according to embodiment 53.

[0079] 55. Use of Raman spectroscopy in combination with a mathematical analysis or mathematical processing of the total intensity of Raman scattered light of at least each of the sample or a first plurality of preselected frequencies and / or frequency ranges for the determination of virus particles having encapsulated nucleic acid in a sample.

[0080] 56. Use of Raman spectroscopy in combination with a mathematical analysis or mathematical processing of the total intensity of Raman scattered light of at least each of the sample or a first plurality of preselected frequencies and / or frequency ranges for the determination of virus particles not having encapsulated nucleic acid in a sample.

[0081] 57. The use according to any one of embodiments 55 to 56, wherein the mathematical analysis or mathematical processing is a classification method or a regression method.

[0082] 58. Use according to any one of embodiments 55 to 57, wherein the mathematical analysis or mathematical processing is by an artificial neural network or a decision tree-based model or principal component analysis.

[0083] 59. Use according to any one of embodiments 55 to 58, wherein the mathematical analysis or mathematical processing is selected from the group consisting of principal component analysis (PCA), non-negative matrix factorization (NMF), linear discriminant analysis (LDA), generalized discriminant analysis (GDA), canonical correlation analysis (CCA), autoencoders, t-distributed stochastic neighbor embedding (t-SNE), uniform manifold approximation and projection (UMAP), k-nearest neighbors (k-NN), kernel or graph-based kernel PCA, and low-dimensional embedding using PCA, LDA, CCA, or NMF techniques as a preprocessing step followed by clustering by K-NN.

[0084] 60. Use according to any one of embodiments 55 to 59, wherein the mathematical analysis or mathematical processing is principal component analysis, and virus particles with or without encapsulated nucleic acids are determined based on the first principal component.

[0085] 61. Use according to any one of embodiments 55 to 60, which is for real time.

[0086] 62. Use according to any one of embodiments 55 to 60, which is for non-invasive determination.

[0087] 63. Use according to any one of embodiments 55 to 60, which is for in-process management decisions.

[0088] 64. Use according to any one of embodiments 55 to 60, which is for quality assurance decisions.

[0089] 65. Use according to any one of embodiments 55 to 60, which is for analysis decisions.

[0090] 66. Use according to any one of embodiments 55 to 60, which is for quality control decisions.

[0091] Use according to any one of Embodiments 55 to 60, which is for release analysis determination.

[0092] 68. The principal component analysis (alpha) measuring the total intensity of Raman scattered light of the sample; (beta) removing wavenumbers outside a first plurality of preselected wavenumbers and / or wavenumber ranges; (gamma) creating a first deviation of a data function; and (delta) normalizing the spectrum Use according to any one of Embodiments 55 to 67, including the above.

[0093] 69. The use according to Embodiment 68, wherein creating a first deviation of a data function is by applying a Savitzky-Golay filter.

[0094] 70. Use according to any one of Embodiments 55 to 69, wherein in Raman spectroscopy, Raman scattered light is determined using a confocal Raman microscope or micro-Raman.

[0095] 71. Use according to any one of Embodiments 55 to 70, wherein the sample is a crude sample / not pretreated.

[0096] 72. Use according to any one of Embodiments 55 to 71, wherein the virus particles are parvovirus particles.

[0097] 73. Use according to any one of Embodiments 55 to 72, wherein the encapsulated nucleic acid is single-stranded DNA.

[0098] 74. Use according to any one of Embodiments 55 to 73, wherein the virus particles are adeno-associated virus particles.

[0099] 75. Use according to any one of embodiments 55 to 74, wherein the viral particles are adeno-associated viral particles of serotype 2 or 5 or 8 or 9.

[0100] 76. Use according to any one of embodiments 55 to 75, wherein the Raman spectroscopy uses a wavelength of about 532 nm or about 785 nm.

[0101] 77. Use according to any one of embodiments 55 to 76, wherein the viral particles are AAV particles of serotype 2 and the Raman spectroscopy uses a wavelength of about 532 nm or about 785 nm.

[0102] 78. Use according to any one of embodiments 55 to 76, wherein the viral particles are AAV particles of serotype 8 and the Raman spectroscopy uses a wavelength of about 785 nm.

[0103] 79. Use according to any one of embodiments 55 to 78, wherein the first plurality of preselected wavenumbers and / or wavenumber ranges consist of DNA / RNA / protein-specific nucleotide binding deformations and / or amide I bond stretches.

[0104] 80. Use according to any one of embodiments 55 to 79, wherein the first plurality of preselected wavenumbers and / or wavenumber ranges consist of 489 - 728 cm-1 and / or 1645 - 1680 cm-1.

[0105] 81. Use according to any one of embodiments 55 to 80, wherein the viral particles are AAV2 viral particles.

[0106] 82. Use according to any one of embodiments 55 to 78, wherein the first plurality of preselected wavenumbers and / or wavenumber ranges consist of protein-specific vibrations of S-S Cys bridges, Tyr-specific vibrations, C-C stretches in beta sheets, Phe-specific vibrations, Tyr-specific vibrations, and / or amide I bond stretches.

[0107] 83. Use according to any one of embodiments 55 to 78 and 82, wherein the first plurality of preselected frequencies and / or frequency ranges consists of one or more or all of 551 cm-1, 645 cm-1, 1000 cm-1, 1003 cm-1, 1530 - 1630 cm-1, and / or 1645 - 1680 cm-1.

[0108] 84. Use according to any one of embodiments 55 to 78 and 82 to 83, wherein the viral particles are AAV8 viral particles.

[0109] 85. Use according to any one of embodiments 55 to 78, wherein the first plurality of preselected frequencies and / or frequency ranges consists of protein and nucleic acid specific vibrations of S-S Cys crosslinks, Tyr specific vibrations, nucleotide ring deformations and stretches, A / G and U / C ring vibrations, Phe specific vibrations, and / or amide I bond stretches.

[0110] 86. Use according to any one of embodiments 55 to 78 and 85, wherein the first plurality of preselected frequencies and / or frequency ranges consists of one or more or all of 551 cm-1, 645 cm-1, 631 - 787 cm-1, 1425 - 1485 cm-1, 1003 cm-1, and 1645 - 1680 cm-1.

[0111] 87. Use according to any one of embodiments 55 to 78 and 85 to 86, wherein the viral particles are AAV2 particles and the sample further comprises AAV2 viral particles that do not have encapsulated nucleic acid.

[0112] 88. Use according to any one of embodiments 55 to 78, wherein the first plurality of preselected frequencies and / or frequency ranges consists of protein specific vibrations of S-S Cys crosslinks, Tyr specific vibrations, protein C-C stretches in helical structures, C-C stretches in beta sheets, Phe specific vibrations, Tyr specific vibrations, and / or stretches of amide I bonds.

[0113] 89. Use according to any one of embodiments 55 - 78 and 88, wherein the first plurality of pre - selected frequencies and / or frequency ranges consist of one or more or all of 551 cm-1, 645 cm-1, 920 - 950 cm-1, 1000 cm-1, 1003 cm-1, 1530 - 1630 cm-1, and / or 1645 - 1680 cm-1.

[0114] 90. Use according to any one of embodiments 55 - 78 and 88 - 89, wherein the viral particles are AAV2 viral particles and the sample further comprises AAV8 viral particles having encapsulated nucleic acid.

[0115] 91. Use according to any one of embodiments 55 - 78 and 88 - 89, wherein the viral particles are AAV8 viral particles and the sample further comprises AAV2 viral particles having encapsulated nucleic acid.

[0116] 92. Use according to any one of embodiments 55 - 78 and 88 - 89, wherein in step (c), since the sample contains a mixture of AAV particles of different serotypes, the calculation is not decisive.

[0117] 93. Use according to any one of embodiments 55 - 78, wherein the first plurality of pre - selected frequencies and / or frequency ranges consist of at least 551 cm-1, 645 cm-1, 631 - 728 cm-1 and 1645 - 1680 cm-1.

[0118] 94. Use according to any one of embodiments 55 - 78, wherein the first plurality of pre - selected frequencies and / or frequency ranges consist of V(S - S) disulfide in proteins, polysaccharides, DNA, protein vC - C alpha - helix, amide III, RNA and / or amide I vibrations / extensions.

[0119] 95. Use according to any one of embodiments 55 to 78 and 94, wherein the first plurality of preselected frequencies and / or frequency ranges consist of one or more or all of the Raman shifts of 530 cm-1, 565 cm-1, 665 cm-1, 725 cm-1, 785 cm-1, 850 cm-1, 936 cm-1, 1230 cm-1, 1244 cm-1, 1265 cm-1, and / or 1671 cm-1.

[0120] 96. Use according to any one of embodiments 55 to 78 and 94 to 95, wherein the viral particles are AAV2 viral particles.

[0121] 97. Use according to any one of embodiments 55 to 78, wherein the first plurality of preselected frequencies and / or frequency ranges consist of polysaccharides, DNA, protein vC-C beta sheets, phenylalanine, nucleic acids, RNA, tyrosine, and / or amide I stretches.

[0122] 98. Use according to any one of embodiments 55 to 78 and 97, wherein the first plurality of preselected frequencies and / or frequency ranges consist of one or more or all of the Raman shifts of 460 cm-1, 670 to 785 cm-1, 985 cm-1, 1003 cm-1, 1080 cm-1, 1244 cm-1, 1613 cm-1, and / or 1671 cm-1.

[0123] 99. Use according to any one of embodiments 55 to 78 and 97 to 98, wherein the viral particles are AAV8 viral particles.

[0124] 100. Use according to any one of embodiments 55 to 78, wherein the first plurality of preselected frequencies and / or frequency ranges consist of V(S-S) disulfides in proteins, polysaccharides, DNA, protein vC-C beta sheets, phenylalanine, amide III, and amide I stretches.

[0125] 101. The use according to any one of embodiments 55 to 78 and 100, wherein the first plurality of preselected frequencies consists of one or more or all of the Raman shifts of 530 cm-1, 565 cm-1, 665 cm-1, 725 cm-1, 785 cm-1, 985 cm-1, 1003 cm-1, 1265 cm-1, and / or 1671 cm-1.

[0126] 102. The use according to any one of embodiments 55 to 78 and 100 to 101, wherein the viral particles are AAV2 particles and the sample further comprises AAV2 viral particles having no encapsulated nucleic acid.

[0127] 103. The use according to any one of embodiments 55 to 78, wherein the first plurality of preselected frequencies and / or frequency ranges consists of polysaccharides, DNA, protein vC-C alpha helix, protein vC-C beta sheet, phenylalanine, nucleic acid, RNA, C-H vibration (protein), phenylalanine, protein, tyrosine, and / or amide I stretch.

[0128] 104. The use according to embodiments 55 to 78 and 103, wherein the first plurality of preselected frequencies consists of one or more or all of the Raman shifts of 460 cm-1, 565 cm-1, 665 cm-1, 750 cm-1, 825 cm-1, 855 cm-1, 936 cm-1, 985 cm-1, 1003 cm-1, 1080 cm-1, 1244 cm-1, 1450 cm-1, 1585 cm-1, 1613 cm-1, and / or 1671 cm-1.

[0129] 105. The use according to any one of embodiments 55 to 78 and 103 to 104, wherein the viral particles are AAV2 viral particles and the sample further comprises AAV8 viral particles having encapsulated nucleic acid.

[0130] 106. Use according to any one of embodiments 55 - 78 and 103 - 105, wherein the viral particle is an AAV8 viral particle and the sample further comprises AAV2 viral particles having encapsulated nucleic acid.

[0131] 107. Use according to any one of embodiments 55 - 78 and 103 - 106, wherein the first plurality of pre - selected wave numbers consists of Raman shifts at 1671 cm-1 and one or more of 565 cm-1, 665 cm-1, 785 cm-1, 985 cm-1, 1003 cm-1, and / or 1244 cm-1.

[0132] 108. The method according to any one of claims 1 - 54, wherein said determining is performed in solution.

[0133] 108. The method according to any one of claims 1 - 54, wherein the viral particle is in solution.

[0134] 110. The method according to any one of claims 1 - 54, wherein the viral particle is neither immobilized nor attached to a solid surface.

[0135] 111. The method according to any one of claims 1 - 54 and 108 - 110, wherein the measurement performed in step b(i) is the only Raman measurement in this method.

[0136] 112. The method according to any one of claims 1 - 54 and 108 - 110, wherein the measurement performed in step b(i) is the only measurement in this method.

[0137] 113. The method according to any one of claims 1 - 54 and 108 - 112, wherein the viral particle is not derivatized, tagged, or labeled.

[0138] 114. The method according to any one of claims 1 - 54 and 108 - 112, wherein the viral particle is a non - derivatized viral particle or a non - tagged viral particle or a non - labeled viral particle.

[0139] 115. The use according to any one of claims 55 to 107, wherein said determining is performed in solution.

[0140] 116. The use according to any one of claims 55 to 107, wherein the viral particles are in solution.

[0141] 117. The use according to any one of claims 55 to 107, wherein the viral particles are neither immobilized nor attached to a solid surface.

[0142] 118. The use according to any one of claims 55 to 107 and 115 to 117, wherein Raman spectroscopy is the only Raman measurement.

[0143] 119. The use according to any one of claims 55 to 107 and 115 to 117, wherein Raman spectroscopy is the only measurement.

[0144] 120. The use according to any one of claims 55 to 107 and 115 to 119, wherein the viral particles are not derivatized, tagged or labeled.

[0145] 121. The use according to any one of claims 55 to 107 and 115 to 119, wherein the viral particles are non-derivatized viral particles or non-tagged viral particles or non-labeled viral particles.

BEST MODE FOR CARRYING OUT THE INVENTION

[0146] Detailed Description of Embodiments of the Invention The present invention is at least partially based on the finding that by using a conventional micro-Raman spectroscopy combined with principal component analysis, i) complete particles of low concentrations of AAV2 and AAV8 in an aqueous buffer solution can be detected, ii) complete particles and empty particles in a sample can be discriminated, and iii) particles of different serotypes can be discriminated.

[0147] Definitions Useful methods and techniques for carrying out the present invention are described, for example, in 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).

[0148] It should be noted that, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents 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 the same applies to others. Similarly, the terms "a" (or "an"), "one or more", and "at least one" may also be used synonymously herein. It should also be noted that the terms "comprising", "including", and "having" may be used synonymously.

[0149] The term "AAV helper function" refers to AAV gene products that function in trans to provide AAV replication and packaging for production, and AAV-derived coding sequences (proteins) that can be expressed to provide AAV particles. Thus, the AAV helper function includes AAV open reading frames (ORFs) including rep and cap, as well as others such as AAP for specific AAV serotypes. The rep gene expression products have been shown to have many functions including, among other things, recognition, binding and nicking of the AAV DNA replication origin; DNA helicase activity; and regulation of transcription from AAV (or other heterologous) promoters. The cap gene expression products (capsid proteins) supply the necessary packaging functions. The AAV helper function is used to complement the trans AAV functions missing from the AAV vector genome.

[0150] The term "comprising" also encompasses the term "consisting of".

[0151] The terms "empty capsid" and "empty particle" refer to AAV particles that have an AAV protein shell but lack all or part of the nucleic acid that encodes a protein or is transcribed into the transcript of interest adjacent to the vector, i.e., the AAV ITR. Thus, an empty capsid is an AAV particle that does not have encapsidated nucleic acid (payload). Therefore, an empty capsid does not function to transfer nucleic acid that encodes a protein or is transcribed into the transcript of interest into a host cell.

[0152] The term "mammalian cell comprising an exogenous nucleotide sequence" encompasses cells into which one or more exogenous nucleic acids have been introduced, including the progeny of such cells. These can serve as a starting point for further genetic modification. Thus, the term "mammalian cell comprising an exogenous nucleotide sequence" includes cells comprising an exogenous nucleotide sequence integrated at a single site within a locus of the genome of said mammalian cell, said exogenous nucleotide sequence comprising at least a first and a second recombination recognition site (which are different) adjacent to at least one first selectable marker. In certain embodiments, a mammalian cell comprising an exogenous nucleotide sequence is a cell comprising an exogenous nucleotide sequence integrated at a single site within a locus of the genome of said cell, said exogenous nucleotide sequence comprising a first recombination recognition sequence and a second recombination recognition sequence adjacent to at least one first selectable marker, and a third recombination recognition sequence located between the first recombination recognition sequence and the second recombination recognition sequence, and all of the recombination recognition sequences are different.

[0153] Both "mammalian cell comprising an exogenous nucleotide sequence" and "recombinant cell" are "transfected cells". This term includes primary transfected cells and their progeny, regardless of the number of passages. The progeny may, for example, contain mutations even if their nucleic acid content is not identical to that of the parental cells. Mutant progeny having the same function or biological activity as the initially transfected cells are included.

[0154] The term "nucleic acid encoding AAV packaging protein" generally refers to one or more nucleic acid molecules containing nucleotide sequences that provide AAV functions deleted from an AAV vector and are used to produce transduction-competent recombinant AAV particles. Nucleic acids encoding AAV packaging proteins are commonly used to provide expression of the AAV rep and / or cap genes to complement the defective AAV functions required for AAV replication. However, the nucleic acid constructs lack AAV ITRs and cannot replicate or package themselves. Nucleic acids encoding AAV packaging proteins can be in the form of plasmids, phages, transposons, cosmids, viruses or particles. Some nucleic acid constructs, such as the commonly used plasmids pAAV / Ad and pIM 29+45 that encode both the rep gene expression product and the cap gene expression product, have been described. See, for example, Samulski et al. (1989) J. Virol. 63:3822-3828; and McCarty et al. (1991) J. Virol. 65:2936-2945. Various plasmids encoding the rep and / or cap gene expression products have been described (e.g., U.S. Patent No. 5,139,941 and U.S. Patent No. 6,376,237). Any one of these nucleic acids encoding AAV packaging proteins can contain a DNA element or nucleic acid according to the invention.

[0155] The term "nucleic acid encoding helper protein" generally refers to one or more nucleic acid molecules containing nucleotide sequences that encode proteins and / or RNA molecules that provide adenovirus helper functions. A plasmid having a nucleic acid encoding a helper protein can be transfected into a suitable cell, where the plasmid can assist in the production of AAV particles in the cell. Any one of these nucleic acids encoding helper proteins can contain a DNA element or nucleic acid according to the invention. Infectious virus particles existing in nature, such as adenovirus, herpesvirus or vaccinia virus particles, are specifically excluded from this term.

[0156] The term "packaging protein" refers to non-AAV-derived viral and / or cellular functions on which AAV depends for its replication. Thus, this term encompasses the proteins and RNAs necessary for AAV replication, including those involved in AAV gene transcription, stage-specific AAV mRNA splicing, AAV DNA replication, synthesis of the Cap expression products, and activation of AAV capsid assembly. Virus-based accessory functions can be derived from any of the known helper viruses such as adenovirus, herpesvirus (other than herpes simplex virus type I), and vaccinia virus.

[0157] As used herein, "AAV packaging protein" refers to AAV-derived sequences that function in trans to produce AAV replication. Thus, AAV packaging proteins are encoded by the major AAV open reading frames (ORFs), rep and cap. The Rep proteins have been shown to have many functions, including, among other things, recognition, binding, and nicking of the AAV origin of DNA replication; DNA helicase activity; and regulation of transcription from AAV (or other heterologous) promoters. The Cap (capsid) proteins provide the necessary packaging functions. AAV packaging proteins are used herein to complement the trans AAV functions missing from AAV vectors.

[0158] A "plasmid" is typically in the form of a nucleic acid or polynucleotide having additional elements for expression (e.g., transcription, replication, etc.) or propagation (replication) of the plasmid. A plasmid can also be used herein to refer to such nucleic acid or polynucleotide sequences. Thus, in all aspects, the compositions and methods of the invention are applicable to nucleic acids, polynucleotides, and plasmids, for example, to generate cells that produce viral (e.g., AAV) vectors, to produce viral (e.g., AAV) particles, to generate cell culture media containing viral (e.g., AAV) particles, and the like.

[0159] As used herein, the term "recombinant cell" means a cell after final genetic modification, e.g., a cell that expresses a polypeptide of interest or produces rAAV particles of interest and can be used for the production of the polypeptide of interest or rAAV particles of interest at any scale. For example, a "mammalian cell comprising an exogenous nucleotide sequence" that has been subjected to recombinase-mediated cassette exchange (RMCE) whereby the coding sequence of a polypeptide of interest has been introduced into the genome of the host cell is a "recombinant cell". This cell can still perform an RMCE reaction, but that is not the purpose.

[0160] A "recombinant AAV vector" is obtained from the wild-type genome of a virus such as AAV by using molecular biological methods to remove the wild-type genome from the virus (e.g., AAV) and replace it with a non-natural nucleic acid, e.g., a nucleic acid that is transcribed into a transcript or encodes a protein. Typically, for AAV, one or both inverted terminal repeat (ITR) sequences of the wild-type AAV genome are retained in the recombinant AAV vector. A "recombinant" AAV vector is distinguished from the wild-type viral AAV genome because all or part of the viral genome has been replaced with a non-natural (i.e., heterologous) sequence with respect to the viral genomic nucleic acid. Thus, the incorporation of a non-natural sequence defines a viral vector (e.g., AAV) as a "recombinant" vector, which in the case of AAV can be referred to as an "rAAV vector".

[0161] Recombinant vectors (e.g., AAV) are packaged for subsequent infection (transduction) of cells ex vivo, in vitro, or in vivo and may be referred to herein as "particles." When a recombinant vector sequence is encapsulated or packaged into AAV particles, the particles may also be referred to as "rAAV." Such particles contain proteins that encapsulate or package the vector genome. Specific examples include viral envelope proteins, and in the case of AAV, capsid proteins such as AAV VP1, VP2, and VP3.

[0162] As used herein, the term "serotype" is a distinction based on serologically distinct AAV capsid proteins. Serological specificity is determined based on the lack of cross-reactivity between antibodies to one AAV compared to other AAVs. Such differences in cross-reactivity are typically due to differences in the capsid protein sequence / epitope (e.g., differences in the VP1, VP2, and / or VP3 sequences of AAV serotypes). AAV variants, including capsid variants, may have at least one nucleotide or amino acid residue different compared to a reference AAV or other AAV serotype, despite being serologically indistinguishable from the reference AAV or other AAV serotypes.

[0163] Under the conventional definition, a serotype means that the virus of interest is tested against sera specific to all existing and characterized serotypes for neutralizing activity and no antibodies that neutralize the virus of interest have been found. As more naturally occurring virus isolates are discovered and / or capsid variants are generated, there may or may not be serological differences from any of the currently existing serotypes. Thus, if a new virus (e.g., AAV) has no serological differences, this new virus (e.g., AAV) is a subgroup or variant of the corresponding serotype. In many cases, serological testing for neutralizing activity has not yet been done on mutant viruses with capsid sequence modifications to determine whether they are other serotypes according to the conventional definition of serotypes. Thus, for convenience and to avoid repetition, the term "serotype" is used herein in a broad sense to refer to both serologically distinct viruses (e.g., AAV) and serologically distinct viruses (e.g., AAV) that can be within a subgroup or variant of a given serotype.

[0164] The term "transgene" is used herein for convenience to refer to a nucleic acid that is intended or has been introduced into a cell or organism. A transgene includes any nucleic acid, e.g., a gene that is transcribed into a transcript or encodes a polypeptide or protein.

[0165] "Vector" refers to a portion of a recombinant plasmid sequence that is ultimately packaged or encapsulated, either directly or in the form of single-stranded or RNA, to form viral (e.g., AAV) particles. When using a recombinant plasmid to construct or produce recombinant viral particles, the viral particles do not contain the portion of the "plasmid" that does not correspond to the vector sequence of the recombinant plasmid. This non-vector portion of the recombinant plasmid is called the "plasmid backbone", which is important for plasmid cloning and amplification, processes necessary for growth and recombinant virus production, but is not itself packaged or encapsulated in viral (e.g., AAV) particles. Thus, "vector" refers to nucleic acids packaged or encapsulated by viral particles (e.g., AAV).

[0166] Recombinant cell Generally, for the efficient and large-scale production of a proteinaceous compound of interest, such as rAAV particles or a therapeutic polypeptide, cells that express and, if possible, secrete the proteinaceous compound are required. Such cells are referred to as "recombinant cells" or "recombinant production cells".

[0167] To generate "recombinant production cells", appropriate mammalian cells are transfected with the nucleic acid sequences necessary to encode the proteinaceous compound of interest. Transfection of additional helper polypeptides may be required.

[0168] To generate stable recombinant production cells, a second step follows, where a single cell that stably expresses the proteinaceous compound of interest is selected. This can be done, for example, based on co-expression of a selectable marker co-transfected with the nucleic acid sequence encoding the proteinaceous compound of interest, or it can be the expression of the proteinaceous compound itself.

[0169] The expression of a coding sequence, i.e., an open reading frame, requires additional regulatory elements such as a promoter and a polyadenylation signal (sequence). Thus, the open reading frame is operably linked to the above-mentioned additional regulatory elements for transcription. This can be achieved by incorporating it into a so-called expression cassette. The minimal control elements required for an expression cassette to function in mammalian cells are a promoter functional in the mammalian cells located upstream, i.e., on the 5' side, of the open reading frame, and a polyadenylation signal (sequence) functional in the mammalian cells located downstream, i.e., on the 3' side, of the open reading frame. Further, a terminator sequence may be present on the 3' side of the polyadenylation signal (sequence). For expression, the promoter, open reading frame / coding region, and polyadenylation signal sequence must be arranged in an operably linked form.

[0170] Similarly, nucleic acids transcribed into non-protein-coding RNAs are called "RNA genes". For the expression of RNA genes, additional regulatory elements such as a promoter and a transcription termination signal or a polyadenylation signal (sequence) are also required. The nature and localization of such elements depend on the RNA polymerase intended to drive the expression of the RNA gene. Thus, RNA genes are usually also incorporated into an expression cassette.

[0171] If the proteinaceous compound of interest is composed of different (monomeric) capsid polypeptides and single-stranded DNA molecules and further requires the helper functions of other adenoviruses for production and encapsidation, a number of expression cassettes with different open reading frames / coding sequences contained therein are required. In this case, at least one expression cassette is required for each of the transgene, the different polypeptides forming the capsid of the AAV vector, and VA RNA for the required helper functions. Thus, individual expression cassettes for each of the helper E1A, E1B, E2A, E4orf6, VA RNA, rep, and cap genes are required.

[0172] As outlined in the previous paragraph, the more complex the proteinaceous compound of interest or the greater the number of additional helper polypeptides and / or RNAs required, the greater the number of different expression cassettes required, respectively. In essence, along with the number of expression cassettes, so is the total size of the nucleic acid. However, there is a practical upper limit to the size of transferable nucleic acids, which is in the range of about 15 kbp (kilobase pairs). Beyond this limit, handling and processing efficiency significantly decreases. This problem can be addressed by using two or more separate plasmids. Thereby, different expression cassettes are assigned to different plasmids, and each plasmid contains only a part of the expression cassette.

[0173] For the generation of stable cell lines, random integration (RI) of nucleic acids having an expression cassette for the proteinaceous compound of interest can be used. Generally, by using RI, the nucleic acid or a fragment thereof is randomly integrated into the genome of the host cell.

[0174] Alternatively, for RI, targeted integration (TI) into the CLD can be used. In TI CLD, one or more nucleic acids containing different expression cassettes are introduced into a predetermined locus within the genome of the host cell.

[0175] At TI, either homologous recombination or recombinase-mediated cassette exchange reaction (RMCE) can be used to integrate nucleic acids containing respective expression cassettes into specific loci in the genome of a TI host cell.

[0176] Adeno-associated virus (AAV) For a general review of the helper functions of AAV and adenovirus or herpesvirus, see Berns and Bohensky, Advances in Virus Research, Academic Press., 32 (1987) 243-306. The AAV genome is described in Srivastava et al., J. Virol., 45 (1983) 555-564. U.S. Patent No. 4,797,368 describes design considerations for constructing recombinant AAV vectors (see also International Publication No. 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 described in U.S. Patent No. 5,173,414; Lebkowski et al., Mol. Cell. Biol. 8 (1988) 3988-3996; Tratschin et al., Mol. Cell. Biol. 5 (1985) 3251-3260; Tratschin et al., Mol. Cell. Biol., 4 (1994) 2072-2081; Hermonat and Muzyczka Proc. Natl. Acad. Sci. USA 81 (1984) 6466-6470; Samulski et al. J. Virol. 63 (1989) 3822-3828.

[0177] Adeno-associated virus (AAV) is a replication-defective parvovirus. It can replicate only in cells in which specific viral functions are provided by co-infecting helper viruses such as adenovirus, herpesvirus, and in some cases poxviruses such as vaccinia. Nevertheless, AAV can replicate in substantially any cell line of human, simian, or rodent origin if appropriate helper virus functions are present.

[0178] In the absence of helper virus genes, AAV establishes a latent period in its host cell. Its genome integrates into a specific site on chromosome 19 [(Chr)19(q13.4)], called the adeno-associated virus integration site 1 (AAVS1). For certain serotypes such as AAV-2, other integration sites have been found, such as on chromosome 5 [(Chr)5(p13.3)], called AAVS2, and on chromosome 3 [(Chr)3(p24.3)], called AAVS3.

[0179] AAV is classified into different serotypes. These are assigned based on parameters such as hemagglutination, tumorigenicity, and DNA sequence homology. To date, more than 10 different serotypes and more than 100 sequences corresponding to different clades of AAV have been identified.

[0180] The type and symmetry of the capsid protein determine the tissue tropism of each AAV. For example, AAV-2, AAV-4, and AAV-5 are specific for the retina, AAV-2, AAV-5, AAV-8, AAV-9, and AAVrh-10 are specific for the brain, AAV-1, AAV-2, AAV-6, AAV-8, and AAV-9 are specific for heart tissue, AAV-1, AAV-2, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, and AAV-10 are specific for the liver, and AAV-1, AAV-2, AAV-5, and AAV-9 are specific for the lung.

[0181] Pseudotyping refers to a process that involves the cross-packaging of AAV genomes between various serotypes, i.e., the genome is packaged with capsid proteins of different origins.

[0182] The wild-type AAV genome has a size of approximately 4.7 kb. The AAV genome further contains two overlapping genes called rep and cap, which include multiple open reading frames (see, for example, Srivastava et al., J. Viral., 45 (1983) 555-564; Hermonat et al., J. Viral. 51 (1984) 329-339; Tratschin et al., J. Virol., 51 (1984) 611-619). The Rep proteins encoded by the open reading frames provide four proteins of different sizes called Rep78, Rep68, Rep52, and Rep40. These are involved in the replication, rescue, and integration of AAV. The open reading frame encoding the Cap proteins provides four proteins called VP1, VP2, VP3, and AAP. VP1, VP2, and VP3 are part of the proteinaceous capsid of the AAV particle. The combined rep and cap open reading frames are flanked at their 5' and 3' ends by the so-called inverted terminal repeats (ITRs). For replication, AAV requires, in addition to the Rep and Cap proteins, the products of the adenovirus genes E1A, E1B, E4orf6, E2A, and VA or the corresponding factors of another helper virus.

[0183] For example, in the case of AAV of serotype 2 (AAV-2), the ITRs each have a length of 145 nucleotides and are adjacent to a coding sequence region of approximately 4470 nucleotides. Of the 145 nucleotides of the ITR, 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 unpaired nucleotides are shown as the D sequence.

[0184] The AAV genome carries three transcription promoters, P5, P19, and P40, for the expression of the rep and cap genes (Laughlin et al., Proc. Natl. Acad. Sci. USA 76 (1979) 5567-5571).

[0185] The ITR sequences must be present in cis with respect to the coding region. The ITRs provide a functional origin of replication (ori), signals necessary for integration into the genome of the target cell, and efficient excision and rescue from the host cell chromosome or recombinant plasmid. The ITRs further contain origin-of-replication-like elements such as the Rep protein binding site (RBS) and the terminal resolution site (TRS). It has been found that the ITR itself can have the function of a transcription promoter in an AAV vector (Flotte et al., J. Biol. Chem. 268 (1993) 3781-3790; Flotte et al., Proc. Natl. Acad. Sci. USA 93 (1993) 10163-10167).

[0186] Trans-complementation of the rep gene product and the cap gene product is required for replication and encapsidation of the viral single-stranded DNA genome, respectively.

[0187] The rep locus contains two internal promoters called P5 and P19. It contains open reading frames for four proteins. Promoter P5 is operably linked to a nucleic acid sequence that provides a non-spliced 4.2 kb mRNA encoding the Rep protein Rep78 (a chromatin nickase for arresting the cell cycle) and a spliced 3.9 kb mRNA encoding the Rep protein Rep68 (a site-specific endonuclease). Promoter P19 is operably linked to a nucleic acid sequence that yields a non-spliced mRNA encoding the Rep protein Rep52 and a spliced 3.3 kb mRNA encoding the Rep protein Rep40 (a DNA helicase for accumulation and packaging).

[0188] The two larger Rep proteins, Rep78 and Rep68, are essential for AAV double-stranded DNA replication, while the smaller Rep proteins, Rep52 and Rep40, appear to be essential for progeny single-stranded DNA accumulation (Chejanovsky & Carter, Virology 173 (1989) 120-128).

[0189] The larger Rep proteins, Rep68 and Rep78, can specifically bind to the hairpin conformation of the AAV ITR. They exhibit certain enzymatic activities required to resolve replication at the AAV termini. Expression of Rep78 or Rep68 may be sufficient for infectious particle formation (Holscher, C., et al. J. Virol. 68 (1994) 7169-7177 and 69 (1995) 6880-6885).

[0190] All Rep proteins, mainly Rep78 and Rep68, are thought to exhibit regulatory activities such as induction and repression of AAV genes and inhibitory effects on cell 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).

[0191] Overexpression of recombinant Rep78 results in a phenotype associated with a decrease in cell growth due to induction of DNA damage. This causes the host cell to arrest in the S phase, thereby promoting latent infection by the virus (Berthet, C., et al., Proc. Natl. Acad. Sci. USA 102 (2005) 13634-13639).

[0192] Tratschin et al. reported that the P5 promoter is negatively autoregulated by Rep78 or Rep68 (Tratschin et al., Mol. Cell. Biol. 6 (1986) 2884-2894). Due to the toxic effects of Rep protein expression, very low expression has been reported for certain cell lines after stable integration of AAV (see, for example, Mendelson et al., Virol. 166 (1988) 154-165).

[0193] The cap locus contains one promoter called P40. Promoter P40 is operably linked to a nucleic acid sequence that provides a 2.6 kb mRNA encoding the Cap proteins VP1 (87 kDa, unspliced mRNA transcript), VP2 (72 kDa from a spliced mRNA transcript), and VP3 (61 kDa from an alternative start codon) by alternative splicing and alternative start codon usage. VP1-VP3 constitute the components of the viral capsid. The capsid has a function of binding to cell surface receptors and enabling intracellular transport of the virus. VP3 accounts for approximately 90% of all viral particle proteins. Nevertheless, all three proteins are essential for efficient capsid production.

[0194] Inactivation of all three capsid proteins VP1-VP3 has been reported to prevent the accumulation of single-stranded progeny AAV DNA. Mutations at the VP1 amino terminus ("lipid negative" or "Inf negative") still allow the assembly of single-stranded DNA into viral particles, thereby significantly reducing the infectivity titer.

[0195] The AAP open reading frame encodes the assembly activation protein (AAP). This has a size of approximately 22 kDa and transports the native VP proteins to the nucleolar region for capsid assembly. This open reading frame is located upstream of the VP3 protein coding sequence.

[0196] Individual AAV particles contain only single-stranded DNA molecules, which can be either the "plus" or "minus" strand. AAV viral particles containing the DNA molecule are infectious. Inside the infected cell, the parental infecting single strand is converted to double-stranded and then amplified. Amplification results in a large pool of double-stranded DNA molecules from which single strands are displaced and packaged into capsids.

[0197] Adeno-associated virus (AAV) vectors can transduce both dividing and quiescent cells. Transgenes introduced into target cells using AAV vectors are thought to be expressed for a long time. One drawback of using AAV vectors is the size limitation of the transgenes that can be introduced into cells.

[0198] Viral vectors such as parvovirus particles including AAV serotypes and their variants provide a means for delivering nucleic acids to cells ex vivo, in vitro, and in vivo that encode proteins such that the cells express the encoded proteins. AAV is a virus useful as a gene therapy vector because it can penetrate cells and introduce nucleic acid / genetic material so that the nucleic acid / genetic material can be stably maintained within the cells. Furthermore, these viruses can introduce nucleic acid / genetic material into specific sites, for example. Since AAV is not associated with pathogenic diseases in humans, AAV vectors can deliver heterologous polynucleotide sequences (e.g., therapeutic proteins and agents) to human patients without causing substantial AAV etiology or disease.

[0199] Viral vectors that can be used include, but are not limited to, adeno-associated virus (AAV) particles of multiple serotypes (e.g., AAV-1 to AAV-12, etc.) and hybrid / chimeric AAV particles.

[0200] AAV particles can be advantageously used as vehicles for effective gene delivery. Such particles have several desirable features for such uses, including tropism for both dividing and non-dividing cells. Even in the early clinical experience with these vectors, no persistent toxicity was shown and the immune response was minimal or undetectable. AAV is known to infect a wide variety of cell types in vivo and in vitro by receptor-mediated endocytosis or transcytosis. These vector systems have been tested in humans targeting retinal epithelium, liver, skeletal muscle, airway, brain, joints, and hematopoietic stem cells.

[0201] Recombinant AAV particles typically do not contain virus genes related to the etiology. Such vectors typically have one or more of the wild-type AAV genes that are wholly or partially deleted, such as, for example, the rep and / or cap genes, but retain at least one functional adjacent ITR sequence as required for rescue, replication, and packaging of the recombinant vector into AAV particles. For example, only the essential parts of the vector, such as the ITR element and the LTR element respectively, are included. Thus, the AAV vector genome will contain the sequences necessary in cis for replication and packaging (e.g., functional ITR sequences).

[0202] Recombinant AAV vectors, and methods and uses thereof, include any viral strain or serotype. By way of non-limiting example, recombinant AAV vectors can be based on any AAV genome, such as AAV-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, 2i8, AAV rh74 or AAV 7m8. Such vectors can be based on the same strain or serotype (or subgroup or variant), or can differ from one another. By way of non-limiting example, a recombinant AAV vector based on one serotype genome can be identical to one or more of the capsid proteins that package the vector. Further, a recombinant AAV vector genome can be based on an AAV (e.g., AAV2) serotype genome that is distinct from one or more of the AAV capsid proteins that package the vector. For example, the AAV vector genome can be based on AAV2, but at least one of the three capsid proteins can be, for example, AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV rh74, AAV 7m8 or a variant thereof. AAV variants include variants and chimeras of the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV rh74 and AAV 7m8 capsids.

[0203] In certain embodiments of all aspects and embodiments, adeno-associated virus (AAV) vectors or particles include, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV rh74 and AAV 7m8 and variants thereof (e.g., capsid variants such as amino acid insertions, additions, substitutions and deletions) as described in International Publication No. WO 2013 / 158879, International Publication No. WO 2015 / 013313 and U.S. Patent Application Publication No. 2013 / 0059732 (disclosing LK01, LK02, LK03, etc.).

[0204] AAV and AAV variant (e.g., capsid variant) serotypes (e.g., VP1, VP2, and / or VP3 sequences) may or may not be distinguished from other AAV serotypes including, for example, AAV1 to AAV12 (e.g., different from the VP1, VP2, and / or VP3 sequences of any of the AAV1 to AAV12 serotypes).

[0205] In certain embodiments of all aspects and embodiments, the AAV particles related to the reference serotype have a polynucleotide, polypeptide, or a subsequence thereof that contains or consists of a sequence that is at least 80% or more identical (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc.) to one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV rh74, or AAV 7m8 (e.g., ITR sequence, or VP1 sequence, VP2 sequence, and / or VP3 sequence, etc.).

[0206] The methods and uses of the present invention include AAV sequences (polypeptides and nucleotides) and subsequences thereof that exhibit less than 100% sequence identity to reference AAV serotypes such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV rh74, or AAV 7m8, but are different from and not identical to known AAV genes or proteins such as the genes or proteins of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV rh74, or AAV 7m8. In certain embodiments of all aspects and embodiments, the AAV polypeptide or subsequence thereof has a sequence that is at least 75% identical to any reference AAV sequence or subsequence thereof, such as 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, up to 100% identical, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV rh74, or AAV 7m8 (e.g., VP1, VP2 and / or VP3 capsid or ITR). In certain embodiments, the AAV variant has 1, 2, 3, 4, 5, 5-10, 10-15, 15-20 or more amino acid substitutions.

[0207] Recombinant AAV particles, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV rh74 or AAV7m8, as well as variant, related, hybrid and chimeric sequences, can be constructed using recombinant techniques known to those skilled in the art to contain one or more nucleic acid sequences (transgenes) adjacent to one or more functional AAV ITR sequences.

[0208] Recombinant particles (e.g., rAAV particles) can be incorporated into pharmaceutical compositions. Such pharmaceutical compositions are useful, inter alia, for in vivo or ex vivo administration and delivery to a subject. In certain embodiments, the pharmaceutical composition contains a pharmaceutically acceptable carrier or excipient. Such excipients include any pharmaceutical that can be administered without inducing an immune response harmful to the individual receiving the composition itself and without undue toxicity.

[0209] Protocols for the generation of adenoviral vectors are described in U.S. Patent No. 5,998,205; U.S. Patent No. 6,228,646; U.S. Patent No. 6,093,699; U.S. Patent No. 6,100,242; International Publication No. 94 / 17810 and International Publication No. 94 / 23744.

[0210] Viral Genome Quantification To enable accurate viral genome quantification, the sample must not contain plasmid DNA nor unpackaged vector genomes, both of which should not contain at least a portion of the viral genome. Further, the packaged AAV genome must be available from the first PCR cycle.

[0211] To remove unpackaged DNA that may interfere with the ddPCR process, DNAseI digestions are commonly used.

[0212] To make the encapsulated DNA accessible to ddPCR, capsid opening is required. This can be done either by incubation at high temperature or by proteinase K digestion. The need for proteinase K digestion is highly debated in the art.

[0213] Droplet Digital Polymerase Chain Reaction Droplet digital PCR (ddPCR) enables the absolute quantification of viral genomes without the need to generate a standard curve.

[0214] More specifically, droplet digital polymerase chain reaction (ddPCR) enables absolute quantification of nucleic acids by randomly partitioning the PCR reaction mixture into separate compartments, some of which have no nucleic acid target sequences while others contain one or more template copies (Hindson, B., et al. 2011). The partitioning results in thousands of independent PCR reactions being carried out during thermal cycling. At the endpoint, the proportion of target-positive compartments is read out and used to calculate the initial template DNA concentration (Pinheiro, L., et al. 2011).

[0215] First, up to 20,000 droplets having a volume of approximately 1 nL are formed in a water-oil emulsion. Thereby, a PCR reaction mixture containing a ddPCR supermix that includes a nucleic acid template, forward (fwd) and reverse (rev) primers, a TaqMan probe, as well as Thermus aquaticus (Taq) DNA polymerase, dNTPs, and a PCR buffer is partitioned (see, e.g., Hindson, B., et al. 2011; Taylor, S, et al. 2017). In each droplet, individual PCR reactions are carried out during thermal cycling depending on the presence or absence of the DNA target.

[0216] In droplets containing template DNA, the target sequence is amplified. During amplification, the 5'-to-3' exonuclease activity of Taq polymerase hydrolyzes the TaqMan probe bound to the template strand. Due to the degradation of the probe into smaller fragments, the intact fluorophore located at the 5' end is no longer in proximity to the quencher located at its 3' end. This relieves signal quenching and generates a fluorescent signal. Compartments lacking the template sequence do not show amplification as the fluorescence of the intact fluorophore located at the 5' end remains quenched and thus do not show hydrolysis of the TaqMan probe and fluorescence generation respectively (see for example, Holland, P., et al. 1991). Since probes with distinct intact fluorophores having different excitation and emission wavelengths are available, the ddPCR reaction can be performed as a multiplex reaction within a single droplet. The intact fluorophores commonly used in two-dimensional ddPCR are 6-carboxyfluorescein (FAM) and hexachloro-6-carboxyfluorescein (HEX), both of which are quenched by Black Hole Quencher 1 (BHQ1) (see for example, Furuta-Hanawa, B., et al. 2019).

[0217] As an endpoint analysis, the fluorescence signal of each droplet after thermal cycling is read out. Using Poisson statistics, the copy number (λ) of the target sequence can be calculated from the ratio (p) of positive reads to total reads according to Equation 1 (see for example, Hindson, B., et al. 2011). λ = -ln(1 - p) (1)

[0218] Since ddPCR relies on endpoint measurement, the quantification of the target sequence is to some extent independent of the PCR reaction efficiency. This is in contrast to real-time PCR (qPCR) commonly used for viral genome titration (see for example, Taylor, S, et al. 2017). Furthermore, there is no need to use a standard or calibration sample (see for example, Dorange, F., Bec, C. 2018).

[0219] Recombinant AAV particles Various methods known in the art for producing rAAV particles. For example, transfection using an AAV plasmid and an AAV helper sequence in combination with co-infection with one AAV helper virus (e.g., adenovirus, herpesvirus, or vaccinia virus), or transfection with a recombinant AAV plasmid, an AAV helper plasmid, and a helper function plasmid. Non-limiting methods for producing rAAV particles are described, for example, in U.S. Patent No. 6,001,650, U.S. Patent No. 6,004,797, International Publication No. 2017 / 096039, and International Publication No. 2018 / 226887. After recombinant rAAV particle production (i.e., particle production in a cell culture system), the rAAV particles can be obtained and purified from host cells and cell culture supernatants.

[0220] Production of recombinant AAV particles requires the expression of Rep protein and Cap protein, helper proteins E1A, E1B, E2A, and E4orf6, and adenovirus VA RNA in a single mammalian cell. The helper proteins E1A, E1B, E2A, and E4orf6 can be expressed using any promoter shown by Matsushita et al. (Gene Ther. 5 (1998) 938-945), particularly the CMV IE promoter. Thus, any promoter can be used.

[0221] Generally, to produce recombinant AAV particles, different complementary plasmids are co-transfected into host cells. One of the plasmids contains a transgene flanked by two cis-acting AAV ITRs. The defective AAV elements required for replication of the progeny recombinant genome and subsequent packaging, namely the open reading frames of the Rep and Cap proteins, are contained in trans on a second plasmid. Overexpression of the Rep protein results in an inhibitory effect on cell growth (Li, J., et al., J. Virol. 71 (1997) 5236 - 5243). Furthermore, a third plasmid containing helper virus genes, namely E1, E4orf6, E2A and VA from adenovirus, is required for AAV replication.

[0222] To reduce the number of plasmids required, the Rep, Cap and adenovirus helper genes may be combined on a single plasmid.

[0223] Alternatively, the host cell may already stably express the E1 gene product. Such cells are HEK293 cells. The human embryonic kidney clone designated 293 was generated in 1977 by integrating adenovirus DNA into human embryonic kidney cells (HEK cells) (Graham, F. L., et al., J. Gen. Virol. 36 (1977) 59 - 74). The HEK293 cell line contains base pairs 1 - 4344 of the adenovirus serotype 5 genome. This includes the E1A and E1B genes as well as the adenovirus packaging signal (Louis, N., et al., Virology 233 (1997) 423 - 429).

[0224] When using HEK293 cells, the missing E2A, E4orf6, and VA genes can be introduced by co-infection with adenovirus or by co-transfection with E2A, E4orf6, and VA expression plasmids (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; Chiorini, J.A., et al., Hum. Gene Ther. 6 (1995) 1531-1541; Ferrari, F.K., et al., J. Virol. 70 (1996) 3227-3234; Salvetti, A., et al., Hum. Gene Ther. 9 (1998) 695-706; Xiao, X., et al., J. Virol. 72 (1998) 2224-2232; Grimm, D., et al., Hum. Gene Ther. 9 (1998) 2745-2760; Zhang, X., et al., Hum. Gene Ther. 10 (1999) 2527-2537). Alternatively, adenovirus / AAV or herpes simplex virus / AAV hybrid vectors 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).

[0225] Therefore, cell lines in which the rep gene is integrated and expressed tend to grow slowly or express the Rep protein at very low levels.

[0226] To restrict transgene activity to specific tissues, i.e., to restrict the site of integration, the transgene can be operably linked to an inducible or tissue-specific promoter (see, for example, Yang, Y., et al. Hum. Gene. Ther. 6 (1995) 1203-1213).

[0227] To date, the main difficulty in the production of rAAV particles has been the inefficient packaging of the rAAV vector, which results in low titers. Packaging has been difficult for several reasons, including the following. - The preferential capsid formation when wild-type AAV genomes are present; - The difficulty in generating sufficient complementing functions as provided by the wild-type rep and cap genes due to the inhibitory effects associated with the rep gene products; - The limited efficiency of co-transfection of plasmid constructs.

[0228] These are all based on the biological properties of the Rep protein. In particular, the inhibitory (cytostatic and cytotoxic) properties of the Rep protein, as well as the ability to reverse the immortalized phenotype of cultured cells, are problematic. Furthermore, the Rep protein down-regulates its own expression when the widely used AAV P5 promoter is used (see, for example, Tratschin et al., Mol. Cell. Biol. 6 (1986) 2884-2894).

[0229] In certain embodiments of all aspects and embodiments, the rAAV particles are derived from an AAV selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, Rh10, Rh74, and 7m8.

[0230] In certain embodiments of all aspects and embodiments, the rAAV particles comprise a capsid sequence having at least 70% sequence identity with the capsid sequence of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, Rh10, Rh74 or 7m8.

[0231] In certain embodiments of all aspects and embodiments, the rAAV particles comprise an ITR sequence having at least 70% sequence identity with the ITR sequence of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10.

[0232] E1A, E1B, E2 and E4 The coding sequences of E1A and E1B (open reading frames) can be derived from human adenoviruses, such as, for example, human adenovirus serotype 2 or serotype 5 in particular. Exemplary sequences of human Ad5 (adenovirus serotype 5) are found in GenBank entry X02996, AC_000008, and exemplary sequences of human Ad2 are found in GenBank entry AC_000007. Nucleotides 505 to 3522 comprise the nucleic acid sequences encoding E1A and E1B of human adenovirus serotype 5. Plasmid pSTK146 reported in European Patent No. 1230354, as well as plasmids pGS119 and pGS122 reported in International Publication No. 2007 / 056994, can also be used as sources of the E1A and E1B open reading frames.

[0233] E1A is the first viral helper gene expressed after adenovirus DNA enters the cell nucleus. The E1A gene encodes 12S and 13S proteins based on the same E1A mRNA by alternative splicing. The expression of 12S and 13S proteins leads to the activation of other viral functions E1B, E2, E3, and E4. Furthermore, the expression of 12S and 13S proteins pushes the cell into the S phase of the cell cycle. When only E1A-derived proteins are expressed, the cells die (apoptosis).

[0234] E1B is the second viral helper gene expressed. It is activated by the E1A-derived proteins 12S and 13S. The mRNA derived from the E1B gene can be spliced in two different ways, resulting in the first 55 kDa transcript and the second 19 kDa transcript. The E1B 55 kDa protein is involved in the regulation of the cell cycle, the prevention of the transport of cellular mRNA in the late stages of infection, and the prevention of E1A-induced apoptosis. The E1B 19 kDa protein is involved in the prevention of E1A-induced apoptosis in cells.

[0235] The E2 gene encodes different proteins. The E2A transcript encodes a single-stranded binding protein (SSBP) that is essential for AAV replication.

[0236] Also, the E4 gene encodes several proteins. The 34 kDa protein (E4orf6) derived from the E4 gene, together with the E1B 55 kDa protein, prevents the accumulation of cellular mRNA in the cytoplasm but also promotes the transport of viral RNA from the nucleus to the cytoplasm.

[0237] Adenovirus VA RNA gene Virus-associated RNA (VA RNA) is a non-coding RNA of adenovirus (Ad) that regulates translation. The adenovirus genome contains two independent copies: VAI (VA RNAI) and VAII (VA RNAII). Both are transcribed by RNA polymerase III from type 2 polymerase III promoters (see, for example, Machitani, M., et al., J. Contr. Rel. 154 (2011) 285-289). For recombinant production, the adenovirus VA RNA gene can be driven by any promoter.

[0238] The structure, function, and evolution of adenovirus-associated RNA using phylogenetic methods were investigated by Ma, Y. and Mathews, M.B. (J. Virol. 70 (1996) 5083-5099). They provided alignment and consensus VA RNA sequences based on 47 known human adenovirus serotypes.

[0239] VA RNA, VAI, and VAII consist of 157-160 nucleotides (nt).

[0240] Depending on the serotype, adenoviruses contain one or two VA RNA genes. VA RNAI is thought to play a dominant proviral 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).

[0241] VA RNA is not essential but plays an important role in efficient virus growth by overcoming the antiviral mechanisms of cells. That is, although VA RNA is not essential for virus growth, VA RNA-deleted adenoviruses may not be able to grow during the initial stages of vector production where there are only a few copies of the viral genome per cell, probably because viral genes other than VA RNA that block the antiviral mechanisms of cells are not sufficiently expressed (see Maekawa, A., et al. Nature Sci. Rep. 3 (2013) 1136).

[0242] Maekawa, A., et al. (Nature Sci. Rep. 3 (2013) 1136) reported the efficient production of adenovirus vectors lacking viral-related RNA genes that disrupt the cellular RNAi mechanism. Here, HEK293 cells constitutively and highly expressing flippase recombinase were infected to obtain VA RNA-deleted adenoviruses by FLP recombinase-mediated excision of the VA RNA locus.

[0243] Human adenovirus 2 VA RNAI corresponds to nucleotides 10586 - 10810 of the GenBank entry AC_000007 sequence. Human adenovirus 5 VA RNAI corresponds to nucleotides 10579 - 10820 of the GenBank entry AC_000008 sequence.

[0244] Methods for producing rAAV particles Carter et al. showed that the entire rep and cap open reading frames in the wild-type AAV genome can be deleted and replaced with a transgene (Carter, B. J., in ‘‘Handbook of Parvoviruses’’, ed. by P. Tijssen, CRC Press, pp. 155 - 168 (1990)). Furthermore, it has been reported that the ITRs must be maintained to retain the functions of replication, rescue, packaging, and integration of the transgene into the genome of target cells.

[0245] When cells containing each viral helper gene are transduced by an AAV vector, or vice versa, or when cells containing the integrated AAV provirus are transduced by an appropriate helper virus, the AAV provirus is activated and enters the 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).

[0246] Aspects of the invention are methods of transducing cells with a nucleic acid (e.g., a plasmid) that contains all of the elements necessary for the production of recombinant AAV particles, and the methods according to the invention are used for the determination of complete AAV particles. Thus, since the plasmid encodes viral packaging proteins and / or helper proteins, the cells can produce recombinant virus particles that contain a nucleic acid encoding the protein of interest or contain sequences that are transcribed into the transcript of interest.

[0247] The invention provides a virus (e.g., AAV) particle production platform that includes features that distinguish it from current "industry standard" virus (e.g., AAV) particle production processes by using the methods according to the invention.

[0248] More generally, cells that are transfected or transduced with DNA for the recombinant production of AAV particles can be referred to as "recombinant cells". Such cells can be, for example, yeast cells, insect cells, or mammalian cells, and are used as recipients of nucleic acids (plasmids) encoding packaging proteins such as AAV packaging proteins, nucleic acids (plasmids) encoding helper proteins, nucleic acids (plasmids) encoding proteins or transcribed into transcripts of interest, i.e., transgenes placed between two AAV ITRs, or other introduced nucleic acids (plasmids). This term includes the progeny of the originally transfected or transduced cells. It is understood that the progeny of a single parental cell may not necessarily be identical in form or genomic or total nucleic acid complement to the original parent due to natural, accidental, or intentional mutations.

[0249] A number of suitable cell growth media are commercially available to maintain cell viability or to bring about cell growth and / or proliferation. Examples of such media include serum-free eukaryotic growth media such as media for maintaining viability or providing 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.

[0250] Helper protein plasmids can be in the form of plasmids, phages, transposons or cosmids. In particular, it has been demonstrated that a perfect complement of the adenovirus gene is not required for helper function. For example, adenovirus mutants unable to perform DNA replication and late gene synthesis have been shown to permit AAV replication. Ito et al., J. Gen. Virol. 9(1970)243; Ishibashi et al., Virology 45(1971)317.

[0251] Variants within the E2B and E3 regions have been shown to support AAV replication, indicating that the E2B and E3 regions are probably not involved in providing helper functions. Carter et al., Virology 126(1983)505. However, adenoviruses that are defective in the E1 region or deleted for the E4 region are unable to assist AAV replication. Thus, in the case of adenovirus helper proteins, the E1A and E4 regions are likely required for AAV replication either directly or indirectly (see, for example, Laughlin et al., J. Virol. 41(1982)868; Janik et al., Proc. Natl. Acad. Sci. USA 78(1981)1925; Carter et al., Virology 126(1983)505). Other characteristic adenovirus variants include the following: E1B (Laughlin et al. (1982), supra; Janik et al. (1981), supra; Ostrove et al., Virology 104(1980)502); E2A (Handa et al., J. Gen. Virol. 29(1975)239; Strauss et al., J. Virol. 17(1976)140; Myers et al., J. Virol. 35(1980)665; Jay et al., Proc. Natl. Acad. Sci. USA 78(1981)2927; Myers et al., J. Biol. Chem. 256(1981)567); E2B (Carter, Adeno-Associated Virus Helper Functions, in I CRC Handbook of Parvoviruses (P. Tijssen ed., 1990)); E3 (Carter et al. (1983), supra); and E4 (Carter et al. (1983), supra; Carter(1995)).

[0252] Studies of helper proteins provided by adenoviruses having mutations in E1B have reported that the E1B 55 kDa protein is required for AAV particle production, but the E1B 19 kDa protein is not. Further, WO 97 / 17458 and Matshushita et al. (Gene Therapy 5 (1998) 938-945) described helper function plasmids encoding various adenovirus genes. Examples of helper plasmids include the adenovirus VA RNA coding region, the adenovirus E4 or f6 coding region, the adenovirus E2A 72 kDa coding region, the adenovirus E1A coding region, and the adenovirus E1B region lacking the intact 55 kDa coding region of E1B (see, e.g., WO 01 / 83797).

[0253] Accordingly, provided herein is a method for producing the recombinant AAV vector or AAV particles comprising the recombinant AAV vector, for use in complete capsid determination, using the method according to the invention, the recombinant AAV vector comprising a nucleic acid encoding a protein or a nucleic acid transcribed into a transcript of interest.

[0254] One aspect of the invention is a method for producing a recombinant AAV vector comprising a nucleic acid encoding a protein or a nucleic acid transcribed into a transcript of interest, or an AAV particle comprising the recombinant AAV vector, comprising: (i) providing one or more plasmids comprising a nucleic acid encoding an AAV packaging protein and / or a nucleic acid encoding a helper protein; (ii) providing a plasmid comprising a nucleic acid encoding a protein of interest or a nucleic acid transcribed into a transcript of interest; (iii) contacting one or more mammalian or insect cells with the provided plasmids; (iv) further adding a transfection reagent and optionally incubating the plasmid / transfection reagent / cell mixture or providing physical means such as an electric current to introduce the nucleic acids into the cells; (v) culturing the transfected cells; (vi) recovering the cultured cells and / or the culture medium from the cultured cells to produce a recovery of the cells and / or the culture medium; (vii) lysing the cells and optionally isolating a recombinant AAV vector or AAV particles from the cell and / or culture medium recovery lysate; (viii) determining a complete AAV particle fraction during and / or after step (vi) and / or step (vii) using the method according to the invention; comprising thereby producing a recombinant AAV vector or AAV particles encoding a protein of interest or comprising a nucleic acid transcribed into a transcript of interest.

[0255] One aspect of the invention is a method for producing a recombinant AAV vector comprising a nucleic acid encoding a protein or a nucleic acid transcribed into a transcript of interest or AAV particles comprising said recombinant AAV vector, comprising: (i) preparing one or more plasmids comprising a nucleic acid encoding an AAV packaging protein and / or a nucleic acid encoding a helper protein; (ii) preparing a plasmid comprising a nucleic acid encoding a protein of interest or a nucleic acid transcribed into a transcript of interest; (iii) (a) contacting a stable transfected cell with - one or more mammalian cells or insect cells with the plasmid provided in (i); - further adding a transfection reagent and optionally incubating the plasmid / transfection reagent / cell mixture or providing physical means such as an electric current to introduce the nucleic acid into the cell; - selecting a first stably transfected cell; - contacting the selected first stably transfected cell with the plasmid provided in (ii); - Further add a transfection reagent and optionally incubate the plasmid / transfection reagent / cell mixture or provide physical means such as an electric current to introduce the nucleic acid into the cell; or (b) Transiently transfect the cells with - Contact one or more mammalian or insect cells with the provided plasmid in (i) and (ii); - Further add a transfection reagent and optionally incubate the plasmid / transfection reagent / cell mixture or provide physical means such as an electric current to introduce the nucleic acid into the cell; (iv) Culture the transfected cells in (iii); (v) Recover the cultured cells and / or the culture medium from the cultured cells to produce a recovery of the cells and / or the culture medium; (vi) Lyse the cells and optionally isolate the recombinant AAV vector or AAV particles from the cell and / or culture medium recovery lysate; (vii) Determine the complete capsid during or after step (v) or / and step (vi) using the method according to the present invention and thereby produce a recombinant AAV vector or AAV particles comprising a nucleic acid encoding a protein of interest or a nucleic acid transcribed into a transcript of interest.

[0256] One aspect of the present invention is a method for producing a recombinant AAV vector comprising a nucleic acid encoding a protein or a nucleic acid transcribed into a transcript of interest or AAV particles comprising said recombinant AAV vector, comprising: (i) Preparing a mammalian or insect cell comprising a nucleic acid encoding an AAV packaging protein and / or a nucleic acid encoding a helper protein; (ii) Preparing a plasmid comprising a nucleic acid encoding a protein of interest or a nucleic acid transcribed into a transcript of interest; (iii) (a) Stable transfected cells are generated by: - contacting one or more mammalian or insect cells with the provided plasmid(s) of (i); - further adding a transfection reagent and optionally incubating the plasmid / transfection reagent / cell mixture or providing physical means such as an electric current to introduce the nucleic acid into the cell; - selecting the first stably transfected cells; - contacting the selected first stably transfected cells with the provided plasmid(s) of (ii); - further adding a transfection reagent and optionally incubating the plasmid / transfection reagent / cell mixture or providing physical means such as an electric current to introduce the nucleic acid into the cell; or alternatively (b) Transiently transfected cells are generated by: - contacting one or more mammalian or insect cells with the provided plasmids of (i) and (ii); - further adding a transfection reagent and optionally incubating the plasmid / transfection reagent / cell mixture or providing physical means such as an electric current to introduce the nucleic acid into the cell; (iv) culturing the transfected cells of (iii); (v) recovering the cultured cells and / or the culture medium from the cultured cells to generate a recovery of the cells and / or the culture medium; (vi) lysing the cells and optionally isolating and / or purifying recombinant AAV vectors or AAV particles from the cell and / or culture medium recovery; (vii) determining intact AAV particles during and / or after step (v) or / and step (vi) using the method according to the invention; A method for producing a recombinant AAV vector or AAV particle that contains a nucleic acid encoding a protein of interest or that is transcribed into a transcript of interest thereby.

[0257] The introduction of nucleic acid (plasmid) into cells can be carried out by a plurality of methods.

[0258] A variety of methods for DNA transfer into mammalian cells have been reported in the art. All of these are useful in the method according to the present 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, inorganic substances (such as calcium phosphate / DNA co-precipitates, etc.), cationic polymers (such as polyethyleneimine, DEAE-dextran, etc.) or cationic lipids (lipofection) are used for nucleic acid transfer / transfection. Calcium phosphate and polyethyleneimine are the most commonly used reagents for transfection for nucleic acid transfer on a larger scale (see, for example, Baldi et al., Biotechnol. Lett. 29 (2007) 677-684), and polyethyleneimine is preferred.

[0259] In certain embodiments of all aspects and embodiments, the nucleic acid (plasmid) is provided in a composition in combination with polyethyleneimine (PEI), optionally in combination with cells. In certain embodiments, the composition comprises a plasmid / PEI mixture having the following components: (a) one or more plasmids comprising a nucleic acid encoding an AAV packaging protein and / or a nucleic acid encoding a helper protein; (b) a plasmid comprising a nucleic acid encoding a protein or transcribed into a transcript of interest; (c) a polyethyleneimine (PEI) solution. In certain embodiments, the plasmids are in a molar ratio range of about 1:0.01 to about 1:100, or in a molar ratio range of about 100:1 to about 1:0.01, and the mixture of components (a), (b), and (c) is optionally incubated for a period of about 10 seconds to about 4 hours.

[0260] In certain embodiments of all aspects and embodiments, the composition further comprises cells. In certain embodiments, the cells are in contact with the plasmid / PEI mixture of components (a), (b), and / or (c).

[0261] In certain embodiments of all aspects and embodiments, the composition further comprises free PEI, optionally in combination with cells. In certain embodiments, the cells are in contact with free PEI.

[0262] In certain embodiments of all aspects and embodiments, the cells are in contact with the mixture of components (a), (b), and / or (c) for at least about 4 hours, or about 4 hours to about 140 hours, or about 4 hours to about 96 hours. In a preferred embodiment, the cells are in contact with the mixture of components (a), (b), and / or (c), and optionally free PEI, for at least about 4 hours.

[0263] The composition may further comprise additional plasmid(s) and / or cells. Such plasmids and cells may be in contact with free PEI. In certain embodiments, the plasmid(s) and / or cells are in contact with free PEI for at least about 4 hours, or from about 4 hours to about 140 hours, or from about 4 hours to about 96 hours.

[0264] A method for producing a recombinant AAV vector or AAV particle comprising a nucleic acid encoding a protein or a nucleic acid transcribed into a transcript of interest, the method comprising: providing one or more plasmids comprising a nucleic acid encoding an AAV packaging protein and / or a nucleic acid encoding a helper protein; providing a plasmid comprising a nucleic acid encoding a protein of interest or a nucleic acid transcribed into a transcript of interest; providing a solution comprising polyethyleneimine (PEI); mixing the aforementioned plasmids with the PEI solution, wherein the plasmids are in a molar ratio range of about 1:0.01 to about 1:100, or in a molar ratio range of about 100:1 to about 1:0.01, to produce a plasmid / PEI mixture (and optionally incubating the plasmid / PEI mixture for a period ranging from about 10 seconds to about 4 hours); contacting cells with the plasmid / PEI mixture produced as described to produce a plasmid / PEI cell culture; adding free PEI to the plasmid / PEI cell culture produced as described to produce a free PEI / plasmid / PEI cell culture; incubating the plasmid / PEI cell culture or the produced free PEI / plasmid / PEI cell culture for at least about 4 hours to produce transfected cells; recovering the produced transfected cells and / or the culture medium from the produced transfected cells to produce a cell and / or culture medium recovery; lysing the cells and optionally isolating the recombinant AAV vector or particle from the cell and / or culture medium recovery lysate, whereby the capsid is determined in the lysate or the isolated AAV particles using the method of the present invention; and thereby further providing a method comprising producing a recombinant AAV vector or particle comprising a nucleic acid encoding a protein or a nucleic acid transcribed into a transcript of interest.

[0265] Methods for producing recombinant AAV vectors or AAV particles using the methods according to the invention may include one or more additional steps or features. Exemplary steps or features include, but are not limited to, the step of harvesting the generated cultured cells and / or harvesting the culture medium from the generated cultured cells to produce a cell and / or culture medium harvest. Further exemplary steps or features include, but are not limited to, lysing the harvested cells and optionally isolating the recombinant AAV vector or AAV particles from the cell and / or culture medium harvest lysate, whereby the viral genome copy number is determined using the methods according to the invention; whereby a recombinant AAV vector or AAV particle is produced that contains a nucleic acid encoding a protein or transcribed into a transcript of interest.

[0266] In certain embodiments of all aspects and embodiments, the encoded AAV packaging proteins include AAV rep and / or AAV cap. In certain embodiments of all aspects and embodiments, such AAV packaging proteins include AAV rep and / or AAV cap proteins of any AAV serotype.

[0267] In certain embodiments of all aspects and embodiments, the encoded helper proteins include adenovirus E1A and / or E1B, adenovirus E2 and / or E4, VA RNA, and / or non-AAV helper proteins.

[0268] In certain embodiments of all aspects and embodiments, the first plasmid contains a nucleic acid encoding an AAV packaging protein, and the second plasmid contains a nucleic acid encoding a helper protein.

[0269] In certain embodiments of all aspects and embodiments, the molar ratio of a plasmid comprising a nucleic acid encoding a protein or a nucleic acid transcribed into a transcript of interest, a first plasmid comprising a nucleic acid encoding an AAV packaging protein, and a second plasmid comprising a nucleic acid encoding a helper protein is in the range of about 1-5:1:1, or 1:1-5:1, or 1:1:1-5 in co-transfection.

[0270] In certain embodiments of all aspects and embodiments, the cell is a eukaryotic cell. In certain embodiments, the eukaryotic cell is a mammalian cell. In one preferred embodiment, the cell is a HEK293 cell or a CHO cell.

[0271] The culture can be carried out using conditions of about 37 °C, 95% humidity and 8% by volume CO2, which are commonly used for culturing eukaryotic cells. The culture can be carried out in a serum-containing medium or a serum-free medium, and in adherent culture or suspension culture. Suspension culture can be carried out in any fermentation vessel such as a stirred tank reactor, a wave reactor, a shaker vessel or a spinner vessel, or a so-called roller bottle. Transfection can be carried out in high-throughput format and screening, for example, in 96 or 384 well format.

[0272] The method according to the present invention includes AAV particles of any serotype or variants thereof. In certain embodiments of all aspects and embodiments, the recombinant AAV particles include AAV serotypes 1-12, AAV VP1, VP2 and / or VP3 capsid proteins, or modified or variant AAV VP1, VP2 and / or VP3 capsid proteins, or wild-type AAV VP1, VP2 and / or VP3 capsid proteins. In certain embodiments of all aspects and embodiments, the AAV particles include an AAV serotype or an AAV pseudotype, and the AAV pseudotype includes an AAV capsid serotype different from the ITR serotype.

[0273] The methods according to the invention that provide or comprise AAV vectors or particles can also comprise other elements. Examples of such elements include, but are not limited to, introns, expression control elements, one or more adeno-associated virus (AAV) inverted terminal repeat sequences (ITRs) and / or filler / stuffer polynucleotide sequences. Such elements may be present in or adjacent to a nucleic acid encoding a protein or transcribed into a transcript of interest, or an expression control element may be operably linked to a nucleic acid encoding a protein or transcribed into a transcript of interest, or an AAV ITR may be adjacent to the 5' or 3' end of a nucleic acid encoding a protein or transcribed into a transcript of interest, or a filler polynucleotide sequence may be adjacent to the 5' or 3' end of a nucleic acid encoding a protein or transcribed into a transcript of interest.

[0274] Expression control elements include constitutive or regulatable control elements such as tissue-specific expression control elements or promoters.

[0275] The ITR can be of any of the AAV2, AAV6, AAV8, or AAV9 serotypes, or a combination thereof. The AAV particles can comprise any VP1, VP2, and / or VP3 capsid protein having at least 75% sequence identity to any of the VP1, VP2, and / or VP3 capsid proteins of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV10, AAV11, AAV-2i8, AAV rh74, or AAV 7m8, or can comprise a modified or variant VP1, VP2, and / or VP3 capsid protein selected from any of the following: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV10, AAV11, AAV-2i8, AAV rh74, and AAV 7m8 AAV serotypes.

[0276] After the production of the recombinant virus (e.g., AAV) particles described herein, if desired, the virus (e.g., rAAV) particles can be purified and / or isolated from host cells using various conventional methods. Such methods include column chromatography, CsCl gradient, iodixanol gradient, and the like.

[0277] For example, multiple column purification steps such as purification by anion exchange columns, affinity columns, and / or cation exchange columns can be used. (See, for example, WO 02 / 12455 and US 2003 / 0207439). Alternatively or additionally, iodixanol gradient or CsCl gradient steps can be used (see, for example, US 2012 / 0135515; and US 2013 / 0072548). Further, when using infectious virus to express packaging and / or helper proteins, various methods can be used to inactivate residual virus. For example, adenovirus can be inactivated by heating to a temperature of approximately 60°C for, for example, 20 minutes or more. Since AAV is heat stable while helper adenovirus is heat labile, this treatment effectively inactivates the helper virus.

[0278] The goal of the rAAV vector production and purification system is to implement strategies to minimize / control the production of production-related impurities such as wild-type / pseudo-wild-type AAV species (wtAAV) and protein, nucleic acid, and vector-related impurities including AAV encapsidated residual DNA impurities.

[0279] Considering that rAAV particles represent a very small fraction of the biomass, rAAV particles need to be purified to a level of purity that can be used as a clinical human gene therapy product (see, for example, Smith P.H., et al., Mo. Therapy 7(2003)8348; Chadeuf G., et al, Mo. Therapy 12(2005)744; CHMP gene therapy expert group meeting, European Medicines Agency EMEA / CHMP 2005, 183989 / 2004 for reports).

[0280] As a first step, typically, the cultured cells that produce rAAV particles are recovered, optionally in combination with the harvested cell culture supernatant (medium) in which the cells (suspension or adherent) that produce rAAV particles were cultured. The recovered cells and optionally the cell culture supernatant can be used as is, lysed or concentrated as needed. Further, if infection is used to express helper functions, residual helper virus can be inactivated. For example, adenovirus can be inactivated by heating to a temperature of approximately 60°C, for example, for 20 minutes or more, which inactivates only the helper virus since AAV is thermostable while the helper adenovirus is thermolabile.

[0281] The cells and / or the supernatant of the harvest are lysed by disrupting the cells, for example, by chemical or physical means such as detergents, microfluidization and / or homogenization to release the rAAV particles. During or after cell lysis, a nuclease, for example, benzonase, is added to degrade contaminating DNA. Typically, the resulting lysate is clarified, for example, by filtration or centrifugation to remove cell debris, giving a clarified cell lysate. In certain examples, the lysate is filtered through a filter with a pore diameter in microns (for example, a filter with a pore diameter of 0.1 - 10.0 μm, for example, a filter with a pore diameter of 0.45 μm and / or 0.2 μm) to produce a clarified lysate.

[0282] The lysate (optionally clarified) contains AAV particles (including nucleic acids encapsulated in the rAAV vector and empty particles) as well as soluble cell components from the host cells that may contain production / process-related impurities, such as, among others, cellular proteins, lipids and / or nucleic acids, and cell culture medium components. The optionally clarified lysate is then subjected to a purification process to purify the AAV particles (including the rAAV vector) from the impurities using chromatography. The clarified lysate can be diluted or concentrated with a suitable buffer prior to the first chromatography step.

[0283] After cell lysis, any clarification, and any dilution or concentration, the rAAV particles can be purified using a plurality of subsequent consecutive chromatography steps.

[0284] The first chromatography step can be cation exchange chromatography or anion exchange chromatography. If the first chromatography step is cation exchange chromatography, the second chromatography step can be anion exchange chromatography or size exclusion chromatography (SEC). Thus, in certain embodiments of all aspects and embodiments, the rAAV particle purification is by cation exchange chromatography, followed by purification by anion exchange chromatography.

[0285] Alternatively, if the first chromatography step is cation exchange chromatography, the second chromatography step can be size exclusion chromatography (SEC). Thus, in certain embodiments of all aspects and embodiments, the rAAV particle purification is by cation exchange chromatography, followed by purification by size exclusion chromatography (SEC).

[0286] Alternatively, the first chromatographic step may be affinity chromatography. If the first chromatographic step is affinity chromatography, the second chromatographic step can be anion exchange chromatography. Thus, in certain embodiments of all aspects and embodiments, rAAV particle purification is by affinity chromatography, followed by purification by anion exchange chromatography.

[0287] Optionally, a third chromatography can be added to the aforementioned chromatographic steps. Typically, any third chromatographic step follows cation exchange, anion exchange, size exclusion or affinity chromatography.

[0288] Thus, in certain embodiments of all aspects and embodiments, rAAV particle purification is by cation exchange chromatography, followed by purification by anion exchange chromatography, followed by purification by size exclusion chromatography (SEC).

[0289] Furthermore, in certain embodiments of all aspects and embodiments, further rAAV particle purification is by cation exchange chromatography, followed by purification by size exclusion chromatography (SEC), followed by purification by anion exchange chromatography.

[0290] In further embodiments of all aspects and embodiments, rAAV particle purification is by affinity chromatography, followed by purification by anion exchange chromatography, followed by purification by size exclusion chromatography (SEC).

[0291] In further embodiments of all aspects and embodiments, rAAV particle purification is by affinity chromatography, followed by purification by size exclusion chromatography (SEC), followed by purification by anion exchange chromatography.

[0292] Cation exchange chromatography functions to separate AAV particles from cell components and other components present in the lysate and / or column eluate clarified from affinity chromatography or size exclusion chromatography. Examples of strong cation exchange resins that can bind rAAV particles over a wide pH range include, but are not limited to, any sulfonic acid-based resin indicated by the presence of a 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). Further examples include Capto S, Capto S ImpAct, Capto S ImpRes (strong cation exchangers available from GE Healthcare, Marlborough, MA, USA), as well as the commercially available DOWEX®, AMBERLITE®, and AMBERLYST® resin families available from Aldrich Chemical Company (Milliwaukee, WI, USA). Examples of weak cation exchange resins include, but are not limited to, any carboxylic acid-based resin. Exemplary cation exchange resins include carboxymethyl (CM), phospho (based on a phosphate functional group), sulfomethyl (S) and sulfopropyl (SP) resins.

[0293] Anion exchange chromatography functions to separate AAV particles from proteins, cellular components, and other components present in the clarified lysate and / or column eluate from affinity chromatography or cation exchange chromatography or size exclusion chromatography. The amount of empty capsids in the eluate can also be reduced and thereby controlled using anion exchange chromatography. For example, an anion exchange column to which rAAV particles are bound can be washed with a solution containing a moderate concentration (e.g., about 100-125 mM, e.g., 110-115 mM) of NaCl, and a portion of the empty capsids can be eluted in the flow-through without substantially eluting the rAAV particles. Subsequently, the rAAV particles bound to the anion exchange column can be eluted using a solution containing a higher concentration (e.g., about 130-300 mM NaCl) of NaCl to produce a column eluate having a reduced or depleted amount of empty capsids and a proportionally increased amount of rAAV vector-containing rAAV particles.

[0294] Exemplary anion exchange resins include, but are not limited to, those based on polyamine resins and other resins. Examples of strong anion exchange resins include, but are not limited to, those generally based on quaternized nitrogen atoms, including quaternary ammonium salt resins such as trialkylbenzylammonium resins. Suitable exchange chromatography materials include, but are not limited to, MACRO PREP Q (a strong anion exchanger available from BioRad, Hercules, CA, USA); UNOSPHERE Q (a strong anion exchanger available from BioRad, Hercules, CA, USA); POROS 50HQ (a strong anion exchanger available from Applied Biosystems, Foster City, CA, USA); POROS XQ (a strong anion exchanger available from Applied Biosystems, Foster City, CA, USA); POROS SOD (a weak anion exchanger available from Applied Biosystems, Foster City, CA, USA); POROS 50PI (a weak anion exchanger available from Applied Biosystems, Foster City, CA, USA); Capto Q, Capto XQ, Capto Q ImpRes, and SOURCE 30Q (powerful anion exchangers available from GE healthcare, Marlborough, MA, USA); DEAE Sepharose (a weak anion exchanger available from Amersham Biosciences, Piscataway, NJ, USA); Q Sepharose (a powerful anion exchanger available from Amersham Biosciences, Piscataway, NJ, USA). Further exemplary anion exchange resins include aminoethyl (AE), diethylaminoethyl (DEAE), diethylaminopropyl (DEPE), and quaternary aminoethyl (QAE).

[0295] An exemplary process for the purification of recombinant AAV particles is reported in International Publication No. WO 2019 / 006390.

[0296] The method for purifying and manufacturing recombinant adeno-associated virus particles (rAAV particles) outlined below can be scaled up to large scales. For example, up to suspension cultures with volumes of 5, 10, 10-20, 20-50, 50-100, 100-200 liters or more. The method for purifying and producing recombinant adeno-associated virus particles is applicable to a wide variety of AAV serotypes / capsid variants.

[0297] In certain embodiments of all aspects and embodiments, the purification of rAAV particles comprises the following steps: (a) Recovering a cell culture supernatant containing cells and / or rAAV particles to generate a recoverate; (b) Optionally, concentrating the recoverate generated in step (a) to generate a concentrated recoverate; (c) Lysing the recoverate generated in step (a) or the concentrated recoverate generated in step (b) to generate a lysate; (d) Treating the lysate generated in step (c) to reduce contaminating nucleic acids in the lysate, thereby generating a nucleic acid-reduced lysate; (e) Optionally, filtering the nucleic acid-reduced lysate generated in step (d) to generate a clarified lysate, and optionally diluting the clarified lysate to generate a diluted clarified lysate; (f) Subjecting the nucleic acid-reduced lysate of step (d), the clarified lysate of step (e), or the diluted clarified lysate generated in step (e) to cation exchange column chromatography to generate a column eluate containing rAAV particles, thereby separating the rAAV particles from protein impurities or other manufacturing / process-related impurities, and optionally diluting the column eluate to generate a diluted column eluate; (g) Subjecting the column eluate or diluted column eluate generated in step (f) to anion exchange chromatography to generate a second column eluate containing rAAV particles, thereby separating the rAAV particles from protein impurities or manufacturing / process-related impurities, and optionally concentrating the second column eluate to generate a concentrated second column eluate; (h) Subjecting the second column eluate or concentrated second column eluate produced in step (g) to size exclusion column chromatography (SEC) to produce a third column eluate containing rAAV particles, thereby separating the rAAV particles from protein impurities or manufacturing / process-related impurities, and optionally concentrating the third column eluate to produce a concentrated third column eluate; and (i) Filtering the third column eluate or concentrated third column eluate produced in step (h) to thereby produce purified rAAV particles comprising, whereby, during or after one or more of steps (a)-(i), full particles are determined using the method according to the invention.

[0298] In certain embodiments of all aspects and embodiments, the AAV affinity column comprises a protein or ligand that binds to the AAV capsid protein. Non-limiting examples of proteins include antibodies that bind to the AAV capsid protein. More specific non-limiting examples include single-chain camelid antibodies (Camelid) that bind to the AAV capsid protein.

[0299] In certain embodiments of all aspects and embodiments, the cells are suspension-growing cells or adherent-growing cells.

[0300] In certain embodiments of all aspects and embodiments, the cells are mammalian cells. Non-limiting examples include HEK cells such as HEK-293 cells, and CHO cells such as CHO-K1 cells.

[0301] Methods for determining the infectivity titer of rAAV particles containing a transgene are known in the art (see, e.g., Zhen et al., Hum. Gene Ther. 15 (2004) 709). Methods for assaying empty particles and rAAV particles packaged with a transgene are known (see, e.g., Grimm et al., Gene Therapy 6 (1999) 1322-1330; Sommer et al., Malec. Ther. 7 (2003) 122-128).

[0302] To determine the presence or amount of degraded / denatured capsid, purified rAAV particles are subjected to SDS-polyacrylamide gel electrophoresis on any gel capable of separating the three capsid proteins, e.g., a gradient gel, and then the gel is run until the samples are separated and the gel can be blotted onto a nylon or nitrocellulose membrane. An anti-AAV capsid antibody is then used as the primary antibody that binds to the denatured capsid protein (see, e.g., Wobus et al., J. Viral. 74 (2000) 9281-9293). The secondary antibody that binds to the primary antibody includes means for detecting the primary antibody. The binding between the primary antibody and the secondary antibody is detected semi-quantitatively to determine the amount of capsid. Another method is analytical HPLC using an SEC column or an analytical ultracentrifuge.

[0303] Data dimensionality reduction techniques Fast spectroscopic "fingerprinting" techniques such as near-infrared, mid-infrared, Raman, or 2D fluorescence spectroscopy are relatively inexpensive and suitable for analyzing complex mixtures. These methods generate very large amounts of high-dimensional data that can only be handled by chemometric methods such as principal component analysis (PCA) or partial least squares (PLS) modeling. The combination of complex spectroscopy and chemometrics is generally used in the identity testing of raw materials or as a tool for the classification of raw materials.

[0304] In classification techniques such as the identification of "complete" and "empty" capsids in the method according to the present invention, any suitable statistical and machine learning / deep learning techniques can be applied. Without limitation, but not limited to, the following further alternative methods can be used for classification: - Principal Component Analysis (PCA) - Non-Negative Matrix Factorization (NMF) - Linear Discriminant Analysis (LDA) - Generalized Discriminant Analysis (GDA) - Canonical Correlation Analysis (CCA) - Autoencoder - t-Distributed Stochastic Neighbor Embedding (t-SNE) - Uniform Manifold Approximation and Projection (UMAP) - k-Nearest Neighbors (k-NN) - Kernel or graph-based Kernel PCA - Low-dimensional embedding that uses PCA, LDA, CCA, or NMF techniques as a preprocessing step, followed by clustering with K-NN.

[0305] More specifically, the use of Principal Component Analysis (PCA) and Partial Least Squares (PLS) for processing and modeling complex data has been reported by Naes, T. et al. (Naes, T., et al., NIR Publications, (2002)). International Publication No. 2009 / 086083 reports a method for hierarchically organizing data using PLS. An analyzer and method for determining the relative importance of fractions of biological mixtures are reported in International Publication No. 2008 / 146059. International Publication No. 2009 / 061326 reports the evaluation of chromatographic materials.

[0306] Maitra, S. and Yan, J. examined Principal Component Analysis and Partial Least Squares, two-dimensional reduction techniques for regression (Casualty Actuarial Society, 2008 Discussion Paper Program).

[0307] Principal component analysis (PCA) and partial least squares (PLS) are two established methods for dimensionality reduction of complex datasets. These techniques are applied when the underlying dataset contains independent variables that are highly correlated. PCA does not reflect the correlation between the independent and dependent variables within the dataset. In contrast, PLS takes into account the said correlation between the dependent and independent variables. Thus, PCA is an unsupervised methodology, whereas PLS is a supervised methodology.

[0308] PCA is commonly used to reduce the number of predictors and solve the multicollinearity problem (Bair, E., et al., J. Am. Stat. Assoc. 101 (2006) 119 - 137). The advantage of PCA is that most of the information contained in the dataset is retained. The data of the variables in the dataset are combined by a limited number of linear combinations.

[0309] PCA can be performed by any statistical software package. The statistical software generates the Eigen vectors of the linear coefficients along with the mean and standard deviation of each predictor variable. This is the basis for the calculation of the principal components by regression. That is, the principal components are obtained by the eigenvalue decomposition of the covariance or correlation matrix of the predictor variables under consideration.

[0310] PCA depends on the X vector or predictor variables. In particular, the relationship between the predictor variables and the dependent or target variables is not important. Thus, PCA is an unsupervised technique. As a result, most of the information contained in the raw predictor variables and the relationship between the predictor variables and the target variables is retained.

[0311] In contrast to PCA, partial least squares takes into account the correlation between the dependent and independent variables and is thus a supervised methodology. PLS is particularly suitable when the predictor variables consist of many different measurements in an experiment and the relationships between these variables are not well understood (Kleinbaum, D.G., et al., in ‘‘Applied Regression Analysis and Multivariable Methods’’, 3rd Edition (Pacific Grove, Ca, USA, Brooks / Cole Publishing Company, 1998)).

[0312] In certain embodiments of the method according to the invention, physical information must be removed by spectral preprocessing before PCA analysis.

[0313] Furthermore, baseline shifts can be removed by applying multiplicative scatter correction (MSC).

[0314] To enhance the variance between samples, in certain embodiments of the method according to the invention, Savitzky-Golay filtering and smoothing methods are applied. Optionally, the spectra can be converted to their first derivatives.

[0315] PCA analysis is performed on the preprocessed spectra in certain embodiments of the invention.

[0316] The method according to the invention The present invention is based at least in part on the finding that Raman spectroscopy, combined with statistical and machine learning / deep learning techniques, can be used to i) detect low concentrations of full and empty particles of various AAV serotype capsids in aqueous buffer solutions, ii) distinguish full particles from empty particles in a capsid sample, and iii) distinguish particles of capsids of different serotypes.

[0317] More specifically, the present invention is based at least in part on the finding that by using conventional micro-Raman spectroscopy in combination with principal component analysis (PCA), i) low concentrations of AAV2 and AAV8 serotype complete particles in an aqueous buffer solution can be detected, ii) complete particles and empty particles in a sample can be discriminated, and iii) particles of different serotypes can be discriminated.

[0318] Generally, recombinant AAV particles being evaluated and developed for clinical use are small non-enveloped virus vectors having a diameter of 20-25 nm and a recombinant single-stranded DNA genome of about 4.7 kb (see FIG. 1). Two wild-type AAV genes encode different rep and cap gene products by splicing required for virus replication and capsid structure formation, respectively.

[0319] AAV particles are on average composed of 74% protein and 26% DNA by molecular weight. The maximum DNA payload capacity is about 4.7 kb. Naturally occurring AAV capsids, as well as genetically engineered AAV capsids, have been intensively evaluated in different studies and compatibility tests for clinical use. The AAV capsid amino acid sequences are highly conserved among different serotypes, in the range of 53% to >99% (see Table 2A of Vance, M., et al. in ‘‘Gene Therapy’’, ed. by Doaa Hashad, InTechOpen, 2014, reproduced below as Table 1).

[0320] (Table 1) BLAST alignments of double combinations of AAV serotypes to determine percentage of homology TIFF2025517987000001.tif61145

[0321] Studies using particles formed from naturally occurring AAV serotypes and engineered capsid proteins have revealed that even minor differences in the amino acid sequence of the capsid protein can affect the tissue tropism of AAV particles and, most favorably, can be used to improve the intended clinical applications. This has created a need for rapid, non-invasive, and sensitive analytical methods for the discrimination of particles of different AAV serotypes and for the quality control of recombinant AAV particles.

[0322] Raman spectroscopy is generally used to identify individual chemical components by detecting the compound-specific vibrational responses of the chemical bonds involved. The intensity of such responses correlates linearly with the amount of each bond in the analyte / sample.

[0323] Results from various conventional Raman spectroscopic studies (using visible or NIR excitation) in the literature indicate that for certain bioprocess-related analytes, the detection limit is between 0.50 mM and 2.0 mM. This would lead those skilled in the art to conclude that conventional Raman spectroscopy is not applicable to the analysis of AAV particles contained in relevant bioprocess samples due to the (very) low concentrations and high molecular similarity of AAV particles of different serotypes.

[0324] Raman spectroscopic techniques for quantifying the intended compounds and analytes based on concentration-correlated spectral characteristics and relative peak intensities are known (Pelletier, M. J., Appl. Spectrosc., 57 (2003) 20A - 42A; Ma, X., et al., Front. Chem., 6 (2018) Article 400). A major advantage of this technique is the potential to quantify specific analytes in complex matrices. In general, and without limitation to these, statistical and machine learning / deep learning methods for quantifying compounds based on Raman spectral data can be used, including: - Without limitation, linear algorithms such as - PLS; - Lasso; - Lasso - Lars; - Ridge regression; - Elastic net; - Huber regression; - Passive aggressive regression; - Bayesian ridge regression; - Orthogonal matching pursuit - Although not limited to, non-linear algorithms such as the following - (Artificial) neural network (ANN); - (Nu) support vector regression; - Tree-based algorithms: Random forest regression, decision tree; - Boosting regression: - XGBoost regression, gradient boosting regression, Adaboost regression; - AutoML: autogluon, AutoKeras.

[0325] Partial least squares regression (PLS): Partial least squares regression is a linear regression technique that finds a linear regression model by projecting the predictor and observation variables into a new space. Lasso, on the other hand, is a form of regularized linear regression where the number of components controls the strength of regularization.

[0326] Lasso: Lasso (Least Absolute Shrinkage and Selection Operator) is a linear regression technique for estimating sparse coefficients and consists of a linear model with an added regularization term. It is known to effectively reduce the number of features on which a given solution depends.

[0327] Lasso Lars: Lasso Lars is a linear regression technique that uses the Least Angle Regression (LARS) algorithm. LARS is a regression algorithm for high-dimensional data similar to forward stepwise regression. It finds the feature most correlated with the target. This does not include all features at each step. The estimated coefficients increase in a direction equiangular to their respective correlation with the residual.

[0328] Ridge Regression: Ridge regression is a linear regression technique. It solves a regression model where the loss function is the linear least squares function. Regularization is given by the l2-norm. Similar to elastic net regression, ridge regression introduces a penalty coefficient. However, ridge regression squares and does not consider the magnitude of the coefficients as in elastic net regression.

[0329] Elastic Net: Elastic net is a linear regression technique. It combines L1 and L2 priors as regularizers. This combination allows learning a sparse model where some of the weights are non-zero while still maintaining the regularization properties of Ridge.

[0330] Huber Regression: Huber regression is a linear regression technique that is robust to outliers. It uses a loss function different from the conventional least squares method. When the residuals are small, it is identical to the least squares penalty. However, for large residuals, the penalty is lower and increases linearly rather than quadratically.

[0331] Passive Aggressive Regression: Passive aggressive regression is a linear regression technique for one of large scale learning and few online learning algorithms. This means that the algorithm is updated incrementally. It is similar to the perceptron and does not require a learning rate. In contrast to the perceptron, it includes a regularization parameter.

[0332] Bayesian Ridge Regression: Bayesian ridge regression is a linear regression technique. Statistical analysis is performed within the context of Bayesian inference. This assumes that the errors are normally distributed and the prior distribution has a specific form. The explicit result is available as the posterior probability distribution of the model's parameters. The algorithm is similar to classical ridge regression.

[0333] Neural Network: A neural network is a multi-layer perceptron. The output of an artificial neuron is calculated by some non-linear function of the sum of its inputs and can be the input to another neuron. Here, a maximum of five layers and a maximum of five neurons per layer were used.

[0334] Nu Support Vector Regression: Nu support vector regression is a support vector regression with a new parameter that determines the ratio of the number of support vectors that it is desirable to hold in the solution to the total number of samples in the dataset.

[0335] Support Vector Regression: A support vector machine constructs a hyperplane or a set of hyperplanes in a high-dimensional space.

[0336] Decision Tree Regression: Decision tree regression is a tree-based approach. The model predicts the value of the target variable by learning simple decision rules inferred from data features. The tree can be viewed as a piecewise constant approximation.

[0337] Random Forest: Random forest is a tree-based approach. The number of classification decision trees is fitted to various subsamples of the dataset. Averaging is used to improve the accuracy of the prediction and control overfitting.

[0338] Adaboost Regression: Adaboost is a tree-based approach. A series of weak prediction models (i.e., models that are slightly better than random guesses such as small decision trees) are fitted to repeatedly modified versions of the data. The final prediction is then made by a weighted majority vote of all predictions.

[0339] Gradient Boosting Regression: Gradient boosting is a tree-based approach. As with Adaboost, the model is constructed step by step, but generalizes Adaboost and other boosting methods by enabling the optimization of any differentiable loss function.

[0340] Autogluron: Autogluron is AutoML. The focus is on tree-based models, model ensembles, neural networks, and linear models.

[0341] Autokeras: Autokeras is an efficient neural architecture search system. It focuses on neural networks.

[0342] Generally, specific wavelength shifts can be assigned to nucleic acids and proteins in Raman spectroscopy. Some are summarized in Tables 2 to 4 below.

[0343] (Table 2) Nucleic acid (DNA / RNA) analysis by Raman spectroscopy - Assignment of common bands in ribose / phosphate / backbone (adopted from Hobro, A.J., et al. Nucl. Acids Res. 35 (2007) 1169 - 1177) TIFF2025517987000002.tif82143

[0344] (Table 3) DNA / RNA analysis by Raman spectroscopy - Assignment of common bands in bases (adopted from Hobro et al. above) TIFF2025517987000003.tif161143

[0345] (Table 4) Protein analysis by Raman spectroscopy - Common band assignment in proteins (adopted from Beattie, J.R., et al., J. Raman Spec. 48 (2017) 813 - 821) TIFF2025517987000004.tif211143

[0346] The method according to the present invention uses micro-Raman spectroscopy. Thereby, most of the problems of AAV particle analysis are overcome. The method according to the present invention is · Non-invasive (natural sample, no preparation required); · Fast (seconds to minutes); ·Multi-attribute approach with one sample / analysis (enabling detection, identification, characterization, discrimination, and quantification simultaneously); ·Required small sample volume (e.g., 200 μL); ·Automatable (currently up to 96 samples in one sequence, 384 possible); ·Standalone solution with small laboratory footprint.

[0347] In an exemplary description of the method according to the present invention, ·The time for performing the analysis can be reduced (approximately 100 times faster); ·The required landscape of analytical instruments can be reduced (approximately 1 / 2 to 1 / 4 reduction); ·The throughput can be improved (enabling an "unlimited" number of samples); ·The representativeness can be enhanced (because it is non-invasive and sample preparation is not required).

[0348] The method according to the present invention is exemplified below using commercially available AAV particle standards of AAV2 and AAV8 serotypes, for particles with nucleic acid and particles without encapsulated nucleic acid, respectively. Samples were processed by a well-plate-based micro-Raman system XPloRa Plus manufactured by Horiba. This is presented merely to exemplify the present invention. It should not be construed as a limitation. The true scope of the present invention is set forth in the appended claims.

[0349] Samples containing AAV virus particles with encapsulated nucleic acid payload are shown herein as "complete" (AAV2 or AAV8 is complete), and samples containing AAV virus particles without encapsulated nucleic acid payload are shown herein as "empty" (AAV2 or AAV8 is empty).

[0350] The Raman spectra of the AAV2 complete samples and empty samples and their corresponding buffers were each acquired 50 times (50 repetitions) using an XPloRa Plus confocal Raman microscope. A laser with a wavelength of 532 nm was used. An exemplary overlay of the spectra is shown in Figure 2.

[0351] The Raman spectra of the AAV8 complete samples and empty samples and their corresponding buffers were each acquired 5 times (5 repetitions) using an XPloRa Plus confocal Raman microscope. A laser with a wavelength of 785 nm was used. An exemplary overlay of the spectra is shown in Figure 3.

[0352] To enable a direct comparison between particles of the AAV2 and AAV8 serotypes, the Raman spectra of the AAV2 complete samples and empty samples were further acquired 5 times using a laser wavelength of 785 nm.

[0353] In the first experiment, the feasibility of the method according to the invention for distinguishing between the AAV2 complete sample and the AAV8 complete sample, respectively, and the buffer blank sample was demonstrated. Virus particles were used at a concentration of 2×10^13 vg / ml.

[0354] The results obtained for the AAV2 complete sample are shown in Figure 4. Surprisingly, it can be seen that the AAV2 complete sample can be clearly distinguished from the blank buffer sample using the method according to the invention. Furthermore, the replicated complete sample and blank buffer measurements were clustered in distinct populations separated mainly by the first principal component (PC-1), explaining 55% of the variance of the dataset. The other low-rank PCA components, i.e., PC-2 and below, contribute substantially nothing, i.e., the second PC-2 contributes substantially nothing to the separation of AAV2 from the buffer control, as can be seen in the PCA plot of Figure 4. PC-2 accounts for only 2% of the variance of the dataset. All samples were measured by laser wavelength excitation at 532 nm.

[0355] These results indicate that the method according to the present invention enables the determination of virus particles having encapsulated nucleic acids by PC load analysis. Thereby, it is possible to identify important spectral features at specific Raman wavelength shifts that contribute to population separation by each PC. To determine AAV2 particles having encapsulated nucleic acids, it was found that shifts in the wavelength ranges of 489 to 728 cm-1 and 1645 to 1680 cm-1 can be used (see Figure 5). Without being bound by this theory, it is assumed that the first shift region corresponds to deformations and stretching vibrations of DNA / RNA-specific nucleotide bonds and nucleic acids such as proteins, while the second shift region corresponds to stretching of the amide I bond.

[0356] (Table 5) Specific Raman wavelength shifts for determining AAV2 particles having encapsulated nucleic acids TIFF2025517987000005.tif81128

[0357] The results obtained for the AAV8 full sample are shown in Figure 6. Surprisingly, it can be seen that the AAV8 full sample can be clearly distinguished from the blank buffer sample using the method according to the present invention. Furthermore, as in the case of the AAV2 full sample, it can be seen that the replicate measurements significantly cluster into distinct populations separated by the first principal component PC-1, which explains 49% of the variance of the dataset. The other lower-rank PCA components, i.e., PC-2 and below, do not substantially contribute. It was found that specific Raman shifts at wavelengths and wavelength ranges of 551 cm-1, 645 cm-1, 1000 cm-1, 1003 cm-1, 1530 - 1630 cm-1, and 1645 - 1680 cm-1 can be used to determine AAV8 particles having encapsulated nucleic acid (see Figure 7). Without being bound by this theory, these are assumed to correspond to protein-specific vibrations of S-S Cys cross-links (551 cm-1), Tyr-specific vibrations (645 cm-1), C-C stretching of beta sheets (1000 cm-1), Phe-specific vibrations (1003 cm-1), Tyr-specific vibrations (1530 - 1630 cm-1), and amide I bond stretching (1645 - 1680 cm-1).

[0358] (Table 6) Specific Raman wavelength shifts for determining AAV8 particles having encapsulated nucleic acid TIFF2025517987000006.tif96128

[0359] In a second experiment, the feasibility of the method according to the present invention for distinguishing between full and empty samples was evaluated. Therefore, AAV2 full and empty samples with a virus genome / particle concentration of 2×10^13 vg / ml were used.

[0360] The results are shown in Fig. 8. It can be seen that, surprisingly, the full samples can be clearly distinguished from the empty samples using the method according to the invention. Furthermore, it can be seen that the replicate measurements cluster into distinct populations that are mainly separated by the first principal component PC-1, which explains 49% of the variance of the data set. The other low-rank PCA components, i.e., PC-2 and below, contribute substantially nothing, i.e., the second PC-2 contributes substantially nothing to the separation of the full samples from the empty samples, as can be seen in the PCA plot of Fig. 8. It was found that specific Raman shifts in the wavelength and wavelength ranges of 551 cm-1, 645 cm-1, 631 - 787 cm-1, 1425 - 1485 cm-1, 1003 cm-1, and 1645 - 1680 cm-1, respectively, can be used to distinguish the full samples from the empty samples (see Fig. 9). Without being bound by this theory, these are assumed to correspond to protein- and nucleic acid-specific vibrations of S-S Cys cross-links (551 cm-1), Tyr-specific vibrations (645 cm-1), nucleotide ring deformations and stretches (631 - 787 cm-1), A / G and U / C ring vibrations (1425 - 1485 cm-1), Phe-specific vibrations (1003 cm-1), and amide I bond stretches (1645 - 1680 cm-1).

[0361] (Table 7) Specific Raman wavelength shifts for the determination of AAV2 particles having encapsulated nucleic acid in the presence of AAV2 particles without encapsulated nucleic acid TIFF2025517987000007.tif86128

[0362] In a third experiment, the feasibility of the method according to the invention for distinguishing different AAV serotypes was evaluated. Thus, Raman spectra of buffer background-corrected AAV2 full samples and AAV8 full samples obtained at a viral genome concentration of 2×10^13 vg / ml were used. All samples were measured by laser wavelength excitation at 785 nm.

[0363] The results obtained are shown in Figure 10. Surprisingly, it is found that both AAV serotypes can be clearly distinguished by the method according to the present invention. Furthermore, it is found that the replicate measurements cluster into distinct populations that are mainly separated by the first principal component PC-1, which explains 52% of the variance of the dataset. The other low-rank PCA components, i.e., PC-2 and below, contribute substantially nothing, i.e., the second PC-2 contributes substantially nothing to the separation of the complete samples from the empty samples, as seen in the PCA plot of Figure 10. This is even more surprising from the perspective of the low particle concentration and overall high amino acid sequence homology between AAV8 and AAV2, which was 83% (Lochire, M.A., et al., J. Virol. 80 (2006) 821-834). For the discrimination of AAV particles of different serotypes such as AAV2 from AAV8, it was found that specific Raman shifts within the wavelengths and wavelength ranges of 551 cm-1, 645 cm-1, 920 - 950 cm-1, 1000 cm-1, 1003 cm-1, 1530 - 1630 cm-1, and 1645 - 1680 cm-1 can be used (Figure 11).

[0364] (Table 8) Specific Raman wavelength shifts for the determination of AAV8 particles with encapsulated nucleic acid in the presence of AAV2 particles with encapsulated nucleic acid TIFF2025517987000008.tif124128

[0365] Without being bound by this theory, these are assumed to correspond to protein-specific vibrations of S-S Cys cross-links (551 cm-1), Tyr-specific vibrations (645 cm-1), C-C stretches of helical structures (920 - 950 cm-1), C-C stretches of beta sheets (1000 cm-1), Phe-specific vibrations (1003 cm-1), Tyr-specific vibrations (1530 - 1630 cm-1), and amide I bond stretches (1645 - 1680 cm-1).

[0366] In the fourth experiment, the method according to the present invention was used to distinguish complete AAV particles of serotypes 5, 8, and 9. Samples containing complete AAV particles of different serotypes were measured by micro-Raman spectroscopy. The obtained data was preprocessed (wavenumber range: 400 - 1800 cm^-1; SG-filter first derivative, first polynomial, 25 pt, SNV transformation, obvious outliers were excluded) and analyzed by PCA. The results are shown in Fig. 12. It can be seen that clear discrimination between different serotypes is possible.

[0367] In subsequent experiments, complete and empty AAV particles of serotypes 5, 8, and 9 were measured by micro-Raman spectroscopy to show the applicability of the method according to the present invention for discriminating AAV serotypes and AAV payload states. The obtained data was preprocessed (wavelength range: 400 - 1800 cm^-1; SG-filter first derivative, first polynomial, 25 pt, SNV transformation, obvious outliers were excluded) and analyzed by PCA. Most of the variance in the combined dataset for all serotypes is explained by PC1 (71%) and PC2 (16%). The results are shown in Figs. 13 - 15. Therefore, with the method according to the present invention, complete AAV particles and empty AAV particles of AAV serotypes 5, 8, and 9 can be clearly separated.

[0368] To determine the detection limit of the method according to the present invention, complete AAV particles and empty AAV particles of serotype 2 were analyzed at four different concentrations (2E+13, 1E+13, 5E+12, and 2.5E+12 vg / mL). The obtained data was preprocessed (wavelength range: 400 - 1800 cm^-1; SG-filter first derivative, first polynomial, 25 pt, SNV transformation) and analyzed by PCA. Complete AAV particles and empty AAV particles were separated by PCA. Most of the variance in the combined dataset for all concentrations is explained by PC1 (29%) and PC2 (13%).

[0369] Figures 19 to 22 show the results of separate analyses at different concentrations. Even at a concentration of 2.5E+12 vg / mL, the complete AAV particles and the empty AAV particles cluster separately and can be identified by PCA, despite having a lower signal-to-noise ratio compared to other concentrations. The signal-to-noise ratio increases with pretreatment.

[0370] In summary, unexpectedly, the present invention is based on the unexpected discovery that by using conventional Raman spectroscopy in combination with principal component data analysis, i) low concentrations of AAV particles such as AAV2 and AAV8 particles having nucleic acids encapsulated in an aqueous buffer solution can be detected, ii) samples containing complete particles can be distinguished from samples containing empty particles, and iii) particles of different serotypes can be distinguished. Thereby, the method according to the present invention is suitable for rapid and non-invasive analysis of AAV particle-containing samples, for example, for in-process control, quality assurance and control, and (real-time) release analysis.

[0371] In addition to the various embodiments described and claimed, the subject matter of the present disclosure also encompasses other embodiments having other combinations of the features disclosed and claimed herein. Accordingly, the specific features presented herein can be combined with each other in other ways within the scope of the disclosed subject matter so that the disclosed subject matter includes any suitable combination of the features disclosed herein. The foregoing description of specific embodiments of the present disclosure has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the present disclosure to the embodiments disclosed.

[0372] All references mentioned herein are incorporated herein by reference.

[0373] The following examples and figures are provided to assist in understanding the present invention, and the true scope of the present invention is set forth in the appended claims. It is understood that modifications can be made to the described procedures without departing from the spirit of the present invention.

Brief Description of the Drawings

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Example

[0375] Materials and Methods All measurements were performed using an XPloRa Plus confocal Raman microscope (Horiba, Longjumeau, France). As a sample holder, a commercially available SensoPlate (Greiner Bio-One GmbH, Kremsmuenster, Austria) was used. The AAV-containing samples were standard substances commercially available from Virovek (Hayward, USA) with a virus concentration of 2E+13 vg / mL.

[0376] Data Analysis To compare AAV2 full and AAV8 full, the Raman spectra of samples of AAV2 full, AAV8 full, and the corresponding buffer without AAV particles were recorded 5 times. Then, all spectra were scaled by a minimum-maximum scaler, and the average buffer spectrum was subtracted from the corresponding AAV sample spectrum. In this way, the background was removed, and the spectra of AAV2 full and AAV8 full were equivalent. In the next step, the spectra were preprocessed as follows: 1.) Removal of wavenumbers outside the fingerprint region; 2.) Application of a Savitzky-Golay filter; 3.) Normalization of the spectra. In the final step, principal component analysis (PCA) was performed using two components, and the loadings were plotted.

[0377] For the AAV2 complete sample and buffer sample, as well as the AAV2 complete sample and AAV2 empty sample, the Raman spectra of each sample were measured 50 times. For the AAV8 complete sample and the sample with only buffer, the Raman spectra of each sample were determined 5 times. The spectra were pre-processed as follows: 1.) Removal of wavenumbers outside the fingerprint region; 2.) Application of the Savitzky-Golay filter; 3.) Normalization of the spectra. In the final step, principal component analysis (PCA) was performed using two components, and the loadings were plotted. Subsequent PCs such as PC3 and subsequent PCs (e.g., PC4, PC5...) do not have a significant contribution to explaining the observed effects. The explained variance of the following PCs is always smaller than the explained variance of PC2.

[0378] Serotype discrimination Different "complete" AAV capsids of serotypes 5, 8, and 9 were measured by micro-Raman spectroscopy 5 times per well in two different wells. The following settings were used for the measurement. One measurement value of AAV5 was considered an outlier, probably due to an analytical artifact, and was excluded. The data were pre-processed and analyzed by PCA as outlined below. Measurement settings: Laser wavelength 532 nm, acquisition: 2×20 s, ND 100, 250 - 3000 cm^-1, x10 objective lens, SS-MWP (stainless steel microplate) Pre-processing: Wavenumber range: 400 - 1800 cm^-1; SG-filter first derivative, first polynomial, 25 pt, SNV transformation The results are shown in Figure 12.

[0379] Complete / empty discrimination Complete and empty AAV particles of serotypes 5, 8, and 9 were measured by micro-Raman spectroscopy. The same settings as for serotype determination were used for the measurement (see below). One measurement value of AAV9 complete was considered an outlier and was excluded. The data were pre-processed and analyzed by PCA as outlined below.

[0380] The results are shown in Figures 13 (AAV5), 14 (AAV8), and 15 (AAV9, outlier-cleared dataset).

[0381] The complete and empty AAV particles of serotypes 5, 8, and 9 can be clearly separated in the combined dataset by PC1 (71%) and PC2 (16%). Measurement settings: laser wavelength 532 nm, acquisition: 2 × 20 s, ND 100, 250 - 3000 cm^-1, x10 objective lens, SS-MWP Pretreatment: wave range: 400 - 1800 cm^-1; SG-filter first derivative, first polynomial, 25 pt, SNV conversion

[0382] Identification of a mixture of complete and empty capsids To characterize the increasing amount of complete AAV particles in the matrix of empty AAV particles, each standard was sonicated and mixed.

[0383] Measurements were performed as follows. - Five consecutive measurements of 30 μL of empty standard in two wells each; - Add 5 μL of complete standard to both wells, resuspend, and repeat five consecutive measurements (total 5 μL / well); - Add an additional 5 μL of complete standard to both wells, resuspend, and repeat five consecutive measurements (total 10 μL / well); - Add an additional 5 μL of complete standard to both wells, resuspend, and repeat five consecutive measurements (total 15 μL / well); - Five consecutive measurements of 30 μL of complete standard in two wells each.

[0384] The settings described below were used. The data was pretreated and analyzed by PCA as described below.

[0385] The results are shown in Figures 16 (AAV9), 17 (AAV5), and 18 (AAV5, outlier-cleared dataset).

[0386] Intact (white circles) and empty (black circles) AAV particles can be clearly separated. The incremental addition of intact AAV to the AAV empty standard by adding a total of 5 μL, 10 μL, and 15 μL of intact AAV standards was clearly observable by the shift of each cluster (replicate) in PC1 and PC2 (light, medium, and dark gray circles). For the combined dataset, most of the variance was explained by PC1 (51% and 30% respectively, with and without outliers) and PC2 (8% and 13% respectively, with and without outliers).

[0387] For one replicate of empty AAV5 with 15 μL of intact AAV5 and one replicate of AAV5 with 5 μL of intact AAV5, a clear shift to PC1 or PC2 was observed. The shift was thought to likely be due to an analytical artifact. A new PCA was created for the analysis of PCA separation without considering these two outliers. Thereby, the incremental addition of intact AAV5 to the AAV5 empty standard by adding a total of 5 μL, 10 μL, and 15 μL of intact AAV standards was even more clearly observable by shifting the clusters (replicates) of PC1 and PC2 (light, medium, and dark gray circles). Again, most of the variation was explained by PC1 (22%) and PC2 (10%). Measurement settings: Laser wavelength 532 nm, acquisition: 2×20 s, ND 100, 250 - 3000 cm^-1, x10 objective lens, SS-MWP Pretreatment: Wavenumber range: 400 - 1800 cm^-1; SG-filter first derivative, first polynomial, 27 pt, SNV conversion:

[0388] Discrimination of intact / empty at different AAV concentrations To determine the detection limit for determining the DNA payload state, AAV2 full and empty particles were measured in two different wells at four different concentrations (2E+13, 1E+13, 5E+12, and 2.5E+12 vg / mL) with 5 replicates for each well. The settings outlined below were used. The data was preprocessed as outlined below and all concentrations were analyzed together by PCA. Most of the variation was explained by PC1 (29%) and PC2 (13%).

[0389] The concentrations were also analyzed separately by PCA. Figures 19 - 22 show the individual results for different concentrations. Even at a concentration of 2.5E+12 vg / mL, the full and empty particles can be clustered and determined separately. Measurement settings: Laser wavelength 532 nm, acquisition: 2 x 20 s, ND 100, 250 - 3000 cm^-1, 600 gr / mm, x10 objective, SS-MWP Preprocessing: Wavenumber range: 400 - 1800 cm^-1; SG-filter first derivative, first polynomial, 27 pt, SNV transformation.

Claims

**Claim 1** A method for determining virus particles having encapsulated nucleic acid in an aqueous sample using Raman spectroscopy, comprising: (a) preparing a sample and irradiating the sample with a light source; (b) (i) measuring the total intensity of Raman scattered light of at least each one of the sample or of a first plurality of preselected wavenumbers and / or wavenumber ranges to obtain a first data set regarding the sample; (ii) performing a principal component analysis on the first data set; (c) determining the virus particles having encapsulated nucleic acid in the sample based on the output of a first set of mathematical data processing steps. The method, wherein the virus particles are in solution. **Claim 2** The method according to claim 1, wherein the virus particles having encapsulated nucleic acid are determined based on a first principal component. **Claim 3** The method according to any one of claims 1-2, wherein step (i) comprises, to obtain a first data set regarding the sample: (alpha) measuring the total intensity of the Raman scattered light of the sample; (beta) removing wavenumbers outside a first plurality of preselected wavenumbers and / or wavenumber ranges; (gamma) creating a first deviation of a data function; and (delta) normalizing the spectrum. **Claim 4** The method according to claim 3, wherein creating the first deviation of the data function is by applying a Savitzky-Golay filter. **Claim 5** The method according to any one of claims 1-4, wherein the Raman scattered light is determined using a confocal Raman microscope or a micro-Raman spectroscopy device. **Claim 6** The method according to any one of claims 1-5, wherein the sample is a crude sample / not pretreated. **Claim 7** The method according to any one of claims 1-6, wherein the virus particles are adeno-associated virus particles. **Claim 8** The method according to any one of claims 1-7, wherein the virus particles are adeno-associated virus particles of serotype 2 or 8. **Claim 9** The method according to any one of claims 1-8, wherein the virus particles are AAV particles of serotype 2 and the light source has a wavelength of about 532 nm or about 785 nm. **Claim 10** ​ ​ The method according to any one of claims 1 to 8, wherein the viral particle is an AAV particle of serotype 8 and the light source has a wavelength of about 785 nm.

11. The method according to any one of claims 1 to 10, wherein the first plurality of preselected wavenumbers and / or wavenumber ranges consist of 489 to 728 cm−1 and / or 1645 to 1680 cm−1.

12. The method according to any one of claims 1 to 10, wherein the first plurality of preselected wavenumbers and / or wavenumber ranges consist of one or more or all of 551 cm−1, 645 cm−1, 1000 cm−1, 1003 cm−1, 1530 to 1630 cm−1, and / or 1645 to 1680 cm−1.

13. The method according to any one of claims 1 to 10, wherein the first plurality of preselected wavenumbers and / or wavenumber ranges consist of one or more or all of 551 cm−1, 645 cm−1, 631 to 787 cm−1, 1425 to 1485 cm−1, 1003 cm−1, and / or 1645 to 1680 cm−1.

14. The method according to any one of claims 1 to 10, wherein the first plurality of preselected wavenumbers and / or wavenumber ranges consist of one or more or all of 551 cm−1, 645 cm−1, 920 to 950 cm−1, 1000 cm−1, 1003.

15. Use of Raman spectroscopy for the determination in an aqueous sample, in a solution of viral particles having encapsidated nucleic acid, of the sample or of at least each one of the first plurality of preselected wavenumbers and / or wavenumber ranges, in combination with a mathematical analysis or mathematical processing of the total intensity of Raman scattered light.

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