Raman-based method for distinguishing AAV particle serotypes and AAV particle load status

Abstract

Herein is reported a method for determining viral particles having capsid nucleic acids in an aqueous sample using Raman spectroscopy, the method comprising the steps of: providing a sample and irradiating the sample with a light source; measuring a total intensity of Raman scattered light for each of a first plurality of preselected wavenumbers and / or ranges of wavenumbers to obtain a first data set for the sample; a first group of mathematical data processing step is carried out on the first data set; and determining the viral particles with capsified nucleic acids in the sample based on outputs of the first set of mathematical data processing steps, wherein the first set of mathematical data processing steps comprises principal component analysis and the determination is based on a first principal component.

Description

[0001] The present invention belongs to the field of analytical methods. In more detail, a method for determining the serotype of AAV particles and the loading status of AAV particles using Raman spectroscopy in combination with a classification or regression method such as principal component analysis is reported herein. Using the method according to the present invention, different serotypes of AAV particles as well as their loading status can be distinguished. Background Art

[0002] Adeno-associated virus (AAV) particles have good safety characteristics, high therapeutic efficacy and the possibility of target-specific engineering, and are commonly used as gene transfer vectors for research and clinical methods. For recombinant production, accurate and robust analytical methods for viral vector characterization are required. Typically, vector genome titration is performed by droplet digital PCR (ddPCR), which is a method for absolute quantification of nucleic acids. These methods are sensitive and specific, but laborious, expensive and time-consuming.

[0003] Raman spectroscopy is a powerful analytical method for determining the vibrational patterns of molecular bonds. Raman spectroscopy is often used in chemistry to provide structural fingerprints by which molecules can be identified. Given the technical nature of the method, Raman spectroscopy can be applied rapidly and non-invasively to a wide range of Raman-active samples, not only for identification purposes, but also for quantitative purposes. Furthermore, state-of-the-art chemometric and statistical data analysis methods allow for multi-attribute Raman analysis of different compounds present in one common sample.

[0004] Raman spectroscopy, especially surface enhanced Raman spectroscopy (SERS) and tip enhanced Raman spectroscopy (TERS), has been applied to clinical diagnosis and food quality insurance applications for a variety of viral diagnostic methods in recent years (Hermann, P., et al., The Analyst 136 (2011) 1148). Most methods focus on the detection and quantification of specific viral contaminants and the identification of virus-infected cells (Gogone, ICVP, et al. Spectrochim. Acta A 249 (2021) 119336; Moor, K., et al., J. Biomed. Optics 23 (2018) 097001). However, these sensitive Raman methods usually require nanoparticles conjugated with substances that can specifically bind to viral antigens for analysis, which raises challenges in test comparability and robustness.

[0005] WO 2020 / 136376 discloses a method for determining viral titer using Raman spectroscopy, wherein the method is based on a 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 data set for a sample, wherein the plurality of wavenumber ranges are preselected and are characteristic of the viruses in the sample.

[0006] WO 2022 / 003359 discloses a method for analyzing viruses using Raman spectroscopy, wherein the viral nucleic acid content of a sample is first determined 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 wavenumber ranges, the first plurality of wavenumber ranges being preselected and characteristic of viral nucleic acids in the sample, the first plurality of wavenumber ranges being combined with a second determination of the viral content in the sample, the second determination being 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 wavenumber ranges, the second plurality of wavenumber ranges being preselected and characteristic of one or more viral structural molecules of the virus in the sample. Thus, the document discloses determining the full / empty ratio based on two subsequent measurements combined with respective data analysis workflows, i.e., calculating the ratio based on the results of both (two) measurements.

[0007] US 6 040 191 discloses a Raman spectroscopic method for determining the ligand binding capacity of a biological product. The method is a non-destructive process for determining the ability of a test biological product to bind to at least one ligand. Therefore, this document focuses on determining the ligand binding capacity of a biological product.

[0008] US2009 / 086201 discloses a surface enhanced Raman spectroscopy (SERS) system for detecting viruses and a method for using the same. The method is a method for detecting at least one biomolecule in a sample, comprising attaching at least one first biomolecule to a nanorod array on a substrate and measuring a surface enhanced Raman spectroscopy (SERS) spectrum.

[0009] FR 3 109 819 discloses a method for detecting the presence of pathogens in biological fluids based on surface enhanced Raman spectroscopy (SERS), by bringing the sample into contact with non-magnetic metal nanoparticles and depositing the solution or the suspension on a support.

[0010] Hermann et al. disclose an evaluation of tip-enhanced Raman spectroscopy for characterizing different virus strains (Analyst 136 (2011) 1148-1152). 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, and can also be used to study single bacterial cells in vivo. As a disadvantage, they conclude that detecting small biological structures such as single virus particles additionally requires improved spatial resolution and significantly higher sensitivity. Their solution is disclosed to be 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. disclosed Raman spectroscopy for virus detection and unorthodox food safety implementation (Trends Food Sci. Technol. 116 (2021) 525-532). In it, Thomas (Appl. Spectrosc. 30 (1976) 483-494) was referenced. He investigated the components of viruses, RNA, DNA, and proteins from the collected Raman spectra, and found that the Raman signals of viruses were mostly similar, and it was difficult to highlight the differences, and applied statistical analysis of the spectra to solve these problems. Summary of the invention

[0012] Here we report a method for the non-invasive, rapid, and efficient analysis of biological process-related AAV samples using Raman spectroscopy (i.e., micro-Raman spectroscopy).

[0013] The present invention is based at least in part on the discovery that Raman spectroscopy combined with statistical and machine learning / deep learning techniques can be used to i) detect full and empty particles of different AAV serotype capsids at low concentrations in aqueous buffer solutions, ii) distinguish full from empty particles in a capsid sample, and iii) distinguish particles with capsids of different serotypes.

[0014] In further detail, the present invention is based, at least in part, on the discovery that conventional micro-Raman spectroscopy combined with principal component data analysis (PCA) can be used to i) detect full particles of AAV2 and AAV8 serotypes at low concentrations in aqueous buffer solutions, ii) distinguish full particles from empty particles in a sample, and iii) distinguish particles of different serotypes.

[0015] It has been found that using the method according to the invention, it is possible to distinguish between AAV particles of different serotypes present in a sample, such as, for example, AAV2, AAV5, AAV8 and AAV9. Furthermore, the method according to the invention allows the direct determination of the loading state of the AAV particles, i.e., whether the particles contain encapsidated DNA (i.e., are full particles) or do not contain encapsidated DNA (i.e., are empty particles).

[0016] The method according to the invention is suitable for the rapid, non-invasive analysis of samples comprising AAV particles for use, for example, in process control, quality assurance and control and (real-time) release analysis.

[0017] The present invention relates to a method for determining viral particles with encapsidated nucleic acid in an aqueous sample using Raman spectroscopy, the method comprising the following steps:

[0018] (a) providing a sample and illuminating the sample with a light source;

[0019] (b)(i) measuring the total intensity of Raman scattered light at each of a first plurality of preselected wavenumbers and / or wavenumber ranges to obtain a first data set for the sample;

[0020] (ii) performing a first set of mathematical data processing steps on the first data set; and

[0021] (c) determining viral particles having encapsidated nucleic acid in the sample based on the output of the first set of mathematical data processing steps.

[0022] The determination in the method according to the invention can be performed qualitatively as well as quantitatively. In the case of a quantitative determination, the number of viral particles with encapsidated nucleic acid is determined. The total number of viral particles can be determined by any method known in the art. The difference between the total number of particles and the number of particles with encapsidated nucleic acid is the number of particles without encapsidated nucleic acid. Therefore, this number can also be determined based on the method according to the invention.

[0023] It must be pointed out that in the method according to the invention, no ligands are required to bind to the matrix. That is, the method according to the invention does not include binary classification (presence / absence). The method according to the invention uses micro-Raman spectroscopy

[0024] It has to be further pointed out that in the method according to the invention a single measurement is used for the determination. In particular and surprisingly it has been found that this allows to distinguish full AAV particles from empty AAV particles.

[0025] It has to be further pointed out that in the method according to the invention no tagging or labeling of the AAV particles is required.

[0026] The present invention encompasses the following embodiments:

[0027] 1. A method for determining viral particles with encapsidated nucleic acids in an aqueous sample using Raman spectroscopy, the method comprising the following steps:

[0028] (a) providing a sample and illuminating the sample with a light source;

[0029] (b)(i) measuring the total intensity of Raman scattered light of the sample or of at least each of a first plurality of preselected wavenumbers and / or wavenumber ranges to obtain a first data set for the sample;

[0030] (ii) performing a first set of mathematical data processing steps on the first data set; and

[0031] (c) determining viral particles having encapsidated nucleic acid in the sample based on the output of the first set of mathematical data processing steps.

[0032] 2. A method for determining viral particles without encapsidated nucleic acid in an aqueous sample using Raman spectroscopy, the method comprising the following steps:

[0033] (a) providing a sample and illuminating the sample with a light source;

[0034] (b)(i) measuring the total intensity of Raman scattered light of the sample or of at least each of a first plurality of preselected wavenumbers and / or wavenumber ranges to obtain a first data set for the sample;

[0035] (ii) performing a first set of mathematical data processing steps on the first data set; and

[0036] (c) determining, based on the output of the first set of mathematical data processing steps, that viral particles in the sample do not have encapsidated nucleic acid.

[0037] 3. A method according to any one of embodiments 1 to 2, wherein the first set of mathematical data processing steps on the first data set is a classification or regression method.

[0038] 4. A 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.

[0039] 5. A method according to any one of embodiments 1 to 4, wherein the first set of mathematical data processing steps for the first data set 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), autoencoder, T-distributed stochastic neighbor embedding (t-SNE), unified manifold approximation and projection (UMAP), K-nearest neighbor algorithm (k-NN), kernel or graph-based kernel PCA, and low-dimensional embedding using PCA, LDA, CCA or NMF technology as a preprocessing step, followed by clustering by K-NN.

[0040] 6. A method according to any one of embodiments 1 to 5, wherein the first set of mathematical data processing steps on the first data set is principal component analysis, and viral particles with or without encapsidated nucleic acid are determined based on the first principal component.

[0041] 7. The method according to any one of embodiments 1 to 6, wherein step (i) comprises:

[0042] (α) measures the total intensity of the Raman scattered light of the sample,

[0043] (β) eliminating wavenumbers outside the first plurality of preselected wavenumbers and / or wavenumber ranges,

[0044] (γ) the first deviation of the function generating the data, and

[0045] (δ) Normalize the spectrum,

[0046] To obtain a first data set for the sample.

[0047] 8. A method according to embodiment 7, wherein the first deviation of the generated data function is achieved by applying a Savitzky-Golay (SG) filter.

[0048] 9. The method according to any one of embodiments 1 to 8, wherein the Raman scattered light is determined using a confocal Raman microscope or a micro-Raman spectroscopy device.

[0049] 10. The method of any one of embodiments 1 to 9, wherein the sample has a volume of 5 μL to 1000 μL.

[0050] 11. The method of any one of embodiments 1 to 10, wherein the sample has a volume of 20 μL to 250 μL.

[0051] 12. The method of any one of embodiments 1 to 11, wherein the sample has a volume of about 200 μL.

[0052] 13. The method of any one of embodiments 1 to 11, wherein the sample has a volume of about 30 to 50 μL.

[0053] 14. The method according to any one of embodiments 1 to 13, wherein the sample is a crude sample / without pre-treatment.

[0054] 15. The method according to any one of embodiments 1 to 14, wherein the viral particles are parvoviral particles.

[0055] 16. The method according to any one of embodiments 1 to 15, wherein the encapsidated nucleic acid is single-stranded DNA.

[0056] 17. The method of any one of embodiments 1 to 16, wherein the viral particle is an adeno-associated viral particle.

[0057] 18. A method according to any one of embodiments 1 to 17, wherein the viral particle is an adeno-associated virus particle of serotype 2 or 5 or 8 or 9.

[0058] 19. The method of any one of embodiments 1 to 18, wherein the light source has a wavelength of about 532 nm or about 785 nm.

[0059] 20. The method of any one of embodiments 1 to 19, wherein the viral particles are AAV particles of serotype 2 and the light source has a wavelength of about 532 nm or about 785 nm.

[0060] 21. The method of any one of embodiments 1 to 19, wherein the viral particles are AAV particles of serotype 8 and the light source has a wavelength of about 785 nm.

[0061] 22. The method according to any one of embodiments 1 to 21, wherein the first plurality of preselected wavenumbers and / or wavenumber ranges consist of DNA / RNA / protein-specific nucleotide bond deformations and amide I bond stretching.

[0062] 23. The method of any one of embodiments 1 to 22, wherein the first plurality of preselected wavenumbers and / or wavenumber ranges consists of 489 to 728 cm-1 or / and 1645 to 1680 cm-1.

[0063] 24. The method of any one of embodiments 1 to 23, wherein the viral particle is an AAV2 viral particle.

[0064] 25. The method of any one of embodiments 1 to 21, wherein the first plurality of preselected wavenumbers and / or wavenumber ranges consist of protein-specific vibrations of SS Cys bridges, Tyr-specific vibrations, CC stretching in β-sheets, Phe-specific vibrations, or / and amide I bond stretching.

[0065] 26. The method of any one of embodiments 1 to 21 and 25, wherein the first plurality of preselected wavenumbers and / or wavenumber 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, or / and 1645 to 1680 cm-1.

[0066] 27. The method of any one of embodiments 1 to 21 and 25 to 26, wherein the viral particle is an AAV8 viral particle.

[0067] 28. The method of any one of embodiments 1 to 21, wherein the first plurality of preselected wavenumbers and / or wavenumber ranges consist of protein- and nucleic acid-specific vibrations of the SS Cys bridge, Tyr-specific vibrations, nucleotide ring deformation and stretching, A / G and U / C ring vibrations, Phe-specific vibrations, or / and amide I bond stretching.

[0068] 29. The method of any one of embodiments 1 to 21 and 28, wherein the first plurality of preselected wavenumbers and / or wavenumber 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, or / and 1645 to 1680 cm-1.

[0069] 30. The method of 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 encapsidated nucleic acid.

[0070] 31. The method of any one of embodiments 1 to 21, wherein the first plurality of preselected wavenumbers and / or wavenumber ranges consist of protein-specific vibrations of SS Cys bridges, Tyr-specific vibrations, protein CC stretching in helical structures, CC stretching in β-sheets, Phe-specific vibrations, or / and amide I bond stretching.

[0071] 32. The method of any one of embodiments 1 to 21 and 31, wherein the first plurality of preselected wavenumbers and / or wavenumber ranges consists 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, or / and 1645 to 1680 cm-1.

[0072] 33. The method of 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 having encapsidated nucleic acid.

[0073] 34. The method of 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 having encapsidated nucleic acid.

[0074] 35. The method of any one of embodiments 1 to 21 and 31 to 32, wherein in step (c), the calculation is not conclusive because the sample comprises a mixture of AAV particles of different serotypes.

[0075] 36. A method according to any one of embodiments 1 to 35, wherein the first plurality of preselected wavenumbers and / or wavenumber ranges consists of one or more or all or at least all of 551 cm-1, 645 cm-1, 631-728 cm-1 or / and 1645-1680 cm-1.

[0076] 37. A method according to any one of embodiments 1 to 21, wherein the first plurality of preselected wavenumbers and / or wavenumber ranges consist of V(SS) disulfides in proteins, polysaccharides, DNA, protein vC-C α-helices, amide III, RNA, and amide I vibrations / stretches.

[0077] 38. A method according to any one of embodiments 1 to 21 and 37, wherein the first plurality of preselected wavenumbers 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, 850 cm-1, 936 cm-1, 1230 cm-1, 1244 cm-1, 1265 cm-1 or / and 1671 cm-1.

[0078] 39. The method of any one of embodiments 1 to 21 and 37 to 38, wherein the viral particle is an AAV2 viral particle.

[0079] 40. The method of any one of embodiments 1 to 21, wherein the first plurality of preselected wavenumbers and / or wavenumber ranges consists of polysaccharides, DNA, protein vC-C β-sheets, phenylalanine, nucleic acids, RNA, tyrosine, and amide I stretches.

[0080] 41. A method according to any one of embodiments 1 to 21 and 40, wherein the first plurality of preselected wavenumbers consists of one or more or all of the 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 or / and 1671 cm-1.

[0081] 42. The method of any one of embodiments 1 to 21 and 40 to 41, wherein the viral particle is an AAV8 viral particle.

[0082] 43. The method of any one of embodiments 1 to 21, wherein the first plurality of preselected wavenumbers and / or wavenumber ranges consist of V(SS) disulfides in proteins, polysaccharides, DNA, protein vC-Cβ-sheets, phenylalanine, amide III, and amide I stretches.

[0083] 44. A method according to any one of embodiments 1 to 21 and 43, wherein the first plurality of preselected wavenumbers 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 or / and 1671 cm-1.

[0084] 45. The method of any one of embodiments 1 to 21 and 43 to 44, wherein the viral particles are AAV2 particles and the sample further comprises AAV2 viral particles without encapsidated nucleic acid.

[0085] 46. ​​A method according to any one of embodiments 1 to 21, wherein the first plurality of preselected wavenumbers and / or wavenumber ranges consist of polysaccharides, DNA, protein vC-C α-helix, protein vC-C β-fold, phenylalanine, nucleic acids, RNA, CH vibrations (proteins), phenylalanine, proteins, tyrosine, and amide I stretching.

[0086] 47. A method according to any one of embodiments 1 to 21 and 46, wherein the first plurality of preselected wavenumbers 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 or / and 1671 cm-1.

[0087] 48. The method of 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 encapsidated nucleic acid.

[0088] 49. The method of 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 encapsidated nucleic acid.

[0089] 50. A method according to any one of embodiments 1 to 21 and 46 to 49, wherein the first plurality of preselected wavenumbers consists of one or more or all of the Raman shift of 1671 cm-1 and one or more of 565 cm-1, 665 cm-1, 785 cm-1, 985 cm-1, 1003 cm-1 or / and 1244 cm-1.

[0090] 51. A method for determining viral particles without encapsidated nucleic acid in an aqueous sample using Raman spectroscopy, the method comprising the steps of:

[0091] a) determining viral particles with encapsidated nucleic acid in an aqueous sample using Raman spectroscopy using a method according to any one of embodiments 1 to 50,

[0092] b) determine the total number of viral particles in an aqueous sample,

[0093] c) The number of viral particles without encapsidated nucleic acid is obtained by subtracting the number obtained in a) from the number obtained in b).

[0094] 52. A method according to embodiment 51, wherein the total number of viral particles in the aqueous sample is determined by enzyme-linked immunosorbent assay.

[0095] 53. A method according to any one of embodiments 1 to 52, wherein the method is used for quantification.

[0096] 54. A method according to embodiment 53, wherein quantification is accomplished 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 attack regression, Bayesian ridge regression, orthogonal matching pursuit, (artificial) neural network (ANN), (nu) support vector regression, random forest regression, decision tree, XGBoost regression, gradient boosting regression, adaboost regression, autogluon and autokeras on the Raman data.

[0097] 55. Use of Raman spectroscopy in combination with mathematical analysis or mathematical processing of the total intensity of Raman scattered light of a sample or of at least each of a first plurality of preselected wavenumbers and / or wavenumber ranges for determining viral particles with encapsidated nucleic acid in a sample.

[0098] 56. Use of Raman spectroscopy in combination with mathematical analysis or mathematical processing of the total intensity of Raman scattered light of a sample or of at least each of a first plurality of preselected wavenumbers and / or wavenumber ranges for determining viral particles without encapsidated nucleic acid in a sample.

[0099] 57. The use according to any one of embodiments 55 to 56, wherein the mathematical analysis or mathematical processing is a classification or regression method.

[0100] 58. The use according to any one of embodiments 55 to 57, wherein the mathematical analysis or mathematical processing is by artificial neural network or decision tree based model or principal component analysis.

[0101] 59. The 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), unified manifold approximation and projection (UMAP), K-nearest neighbor algorithm (k-NN), kernel or graph-based kernel PCA, and low-dimensional embedding using PCA, LDA, CCA or NMF techniques as a pre-processing step followed by clustering by K-NN.

[0102] 60. The use according to any one of embodiments 55 to 59, wherein the mathematical analysis or mathematical processing is by principal component analysis, and the viral particles with or without encapsidated nucleic acid are determined based on the first principal component.

[0103] 61. The use according to any one of embodiments 55 to 60, wherein the use is for real-time determination.

[0104] 62. The use according to any one of embodiments 55 to 60, wherein the use is for non-invasive determination.

[0105] 63. The use according to any one of embodiments 55 to 60, wherein the use is for process control determinations.

[0106] 64. The use according to any one of embodiments 55 to 60, wherein the use is for quality assurance determination.

[0107] 65. The use according to any one of embodiments 55 to 60, wherein the use is for analytical determination.

[0108] 66. The use according to any one of embodiments 55 to 60, wherein the use is for quality control determination.

[0109] 67. The use according to any one of embodiments 55 to 60, wherein the use is for release assay determination.

[0110] 68. The use according to any one of embodiments 55 to 67, wherein the principal component analysis encompasses

[0111] (α) measures the total intensity of the Raman scattered light of the sample,

[0112] (β) eliminating wavenumbers outside the first plurality of preselected wavenumbers and / or wavenumber ranges,

[0113] (γ) the first deviation of the function generating the data, and

[0114] (δ) Normalize the spectrum.

[0115] 69. The use according to embodiment 68, wherein the first deviation of the data function is generated by applying a Savitzky-Golay filter.

[0116] 70. Use according to any one of embodiments 55 to 69, wherein in the Raman spectroscopy, the Raman scattered light is determined using a confocal Raman microscope or micro-Raman.

[0117] 71. The use according to any one of embodiments 55 to 10, wherein the sample is a crude sample / without pre-treatment.

[0118] 72. The use according to any one of embodiments 55 to 71, wherein the viral particle is a parvoviral particle.

[0119] 73. The use according to any one of embodiments 55 to 72, wherein the encapsidated nucleic acid is single-stranded DNA.

[0120] 74. The use according to any one of embodiments 55 to 73, wherein the viral particle is an adeno-associated viral particle.

[0121] 75. The use according to any one of embodiments 55 to 74, wherein the viral particle is an adeno-associated virus particle of serotype 2 or 5 or 8 or 9.

[0122] 76. The 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.

[0123] 77. The use according to any one of embodiments 55 to 76, wherein the viral particle is an AAV particle of serotype 2 and the Raman spectroscopy uses a wavelength of about 532 nm or about 785 nm.

[0124] 78. The use according to any one of embodiments 55 to 76, wherein the viral particle is an AAV particle of serotype 8 and the Raman spectroscopy uses a wavelength of about 785 nm.

[0125] 79. The use according to any one of embodiments 55 to 78, wherein the principal component analysis first plurality of preselected wavenumbers and / or wavenumber ranges consist of DNA / RNA / protein specific nucleotide bond deformations and / or amide I bond stretching.

[0126] 80. The use according to any one of embodiments 55 to 79, wherein the first plurality of preselected wavenumbers and / or wavenumber ranges consists of 489 to 728 cm-1 or / and 1645 to 1680 cm-1.

[0127] 81. The use according to any one of embodiments 55 to 80, wherein the viral particle is an AAV2 viral particle.

[0128] 82. The use according to any one of embodiments 55 to 78, wherein the first plurality of preselected wavenumbers and / or wavenumber ranges consists of protein-specific vibrations of SS Cys bridges, Tyr-specific vibrations, CC stretching in β-sheets, Phe-specific vibrations, Tyr-specific vibrations or / and amide I bond stretching.

[0129] 83. The use according to any one of embodiments 55 to 78 and 82, wherein the first plurality of preselected wavenumbers and / or wavenumber 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, or / and 1645 to 1680 cm-1.

[0130] 84. The use according to any one of embodiments 55 to 78 and 82 to 83, wherein the viral particle is an AAV8 viral particle.

[0131] 85. The use according to any one of embodiments 55 to 78, wherein the first plurality of preselected wavenumbers and / or wavenumber ranges consist of protein- and nucleic acid-specific vibrations of the SS Cys bridge, Tyr-specific vibrations, nucleotide ring deformation and stretching, A / G and U / C ring vibrations, Phe-specific vibrations, or / and amide I bond stretching.

[0132] 86. The use according to any one of embodiments 55 to 78 and 85, wherein the first plurality of preselected wavenumbers and / or wavenumber 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 to 1680 cm-1.

[0133] 87. The method of 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 without encapsidated nucleic acid.

[0134] 88. The use according to any one of embodiments 55 to 78, wherein the first plurality of preselected wavenumbers and / or wavenumber ranges consists of protein-specific vibrations of SS Cys bridges, Tyr-specific vibrations, protein CC stretching in helical structures, CC stretching in β-sheets, Phe-specific vibrations, Tyr-specific vibrations or / and amide I bond stretching.

[0135] 89. The use according to any one of embodiments 55 to 78 and 88, wherein the first plurality of preselected wavenumbers and / or wavenumber ranges consists 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, or / and 1645 to 1680 cm-1.

[0136] 90. The use according to any one of embodiments 55 to 78 and 88 to 89, wherein the viral particles are AAV2 viral particles and the sample further comprises AAV8 viral particles having encapsidated nucleic acid.

[0137] 91. The use according to any one of embodiments 55 to 78 and 88 to 89, wherein the viral particles are AAV8 viral particles and the sample further comprises AAV2 viral particles having encapsidated nucleic acid.

[0138] 92. The use according to any one of embodiments 55 to 78 and 88 to 89, wherein in step (c), the calculation is not conclusive because the sample comprises a mixture of AAV particles of different serotypes.

[0139] 93. The use according to any one of embodiments 55 to 78, wherein the first plurality of preselected wavenumbers and / or wavenumber ranges consists of at least 551 cm-1, 645 cm-1, 631-728 cm-1, and 1645-1680 cm-1.

[0140] 94. The use according to any one of embodiments 55 to 78, wherein the first plurality of preselected wavenumbers and / or wavenumber ranges consist of V(SS) disulfides in proteins, polysaccharides, DNA, protein vC-C α-helices, amide III, RNA or / and amide I vibrations / stretches.

[0141] 95. The use according to any one of embodiments 55 to 78 and 94, wherein the first plurality of preselected wavenumbers consists of Raman shifts of one or more or all 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, or / and 1671 cm-1.

[0142] 96. The use according to any one of embodiments 55 to 78 and 94 to 95, wherein the viral particle is an AAV2 viral particle.

[0143] 97. The use according to any one of embodiments 55 to 78, wherein the first plurality of preselected wavenumbers and / or wavenumber ranges consist of polysaccharides, DNA, protein vC-C β-sheets, phenylalanine, nucleic acids, RNA, tyrosine or / and amide I stretches.

[0144] 98. The use according to any one of embodiments 55 to 78 and 97, wherein the first plurality of preselected wavenumbers consists of Raman shifts of one or more or all of 460 cm-1, 670-785 cm-1, 985 cm-1, 1003 cm-1, 1080 cm-1, 1244 cm-1, 1613 cm-1, and 1671 cm-1.

[0145] 99. The use according to any one of embodiments 55 to 78 and 97 to 98, wherein the viral particle is an AAV8 viral particle.

[0146] 100. The use according to any one of embodiments 55 to 78, wherein the first plurality of preselected wavenumbers and / or wavenumber ranges consists of V(SS) disulfides in proteins, polysaccharides, DNA, protein vC-C β-sheets, phenylalanine, amide III, and amide I stretches.

[0147] 101. The use according to any one of embodiments 55 to 78 and 100, wherein the first plurality of preselected wavenumbers consists of Raman shifts of one or more or all 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, or / and 1671 cm-1.

[0148] 102. The method of 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 without encapsidated nucleic acid.

[0149] 103. The use according to any one of embodiments 55 to 78, wherein the first plurality of preselected wavenumbers and / or wavenumber ranges consists of polysaccharides, DNA, protein vC-C α-helix, protein vC-C β-sheet, phenylalanine, nucleic acids, RNA, CH vibrations (proteins), phenylalanine, proteins, tyrosine or / and amide I stretching.

[0150] 104. The use according to any one of embodiments 55 to 78 and 103, wherein the first plurality of preselected wavenumbers consists of Raman shifts of one or more or all 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, or / and 1671 cm-1.

[0151] 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 encapsidated nucleic acid.

[0152] 106. The use according to any one of embodiments 55 to 78 and 103 to 105, wherein the viral particles are AAV8 viral particles and the sample further comprises AAV2 viral particles having encapsidated nucleic acid.

[0153] 107. The use according to any one of embodiments 55 to 78 and 103 to 106, wherein the first plurality of preselected wavenumbers consists of Raman shifts of one or more of 1671 cm-1 and one or more of 565 cm-1, 665 cm-1, 785 cm-1, 985 cm-1, 1003 cm-1, or / and 1244 cm-1.

[0154] 108. The method of any one of claims 1 to 54, wherein the determining is in solution.

[0155] 108. The method of any one of claims 1 to 54, wherein the viral particles are in solution.

[0156] 110. The method of any one of claims 1 to 54, wherein the viral particles are not immobilized or attached to a solid surface.

[0157] 111. A method according to any one of claims 1 to 54 and 108 to 110, wherein the measurement performed in step b(i) is the only Raman measurement in the method.

[0158] 112. The method of any one of claims 1 to 54 and 108 to 110, wherein the measurement performed in step b(i) is the only measurement in the method.

[0159] 113. The method of any one of claims 1 to 54 and 108 to 112, wherein the viral particles are neither derivatized, nor tagged or labeled.

[0160] 114. The method of any one of claims 1 to 54 and 108 to 112, wherein the viral particle is an underivatized viral particle or an untagged viral particle or an unlabeled viral particle.

[0161] 115. The use according to any one of claims 55 to 107, wherein the determination is in solution.

[0162] 116. The use according to any one of claims 55 to 107, wherein the viral particles are in solution.

[0163] 117. The use according to any one of claims 55 to 107, wherein the viral particles are not immobilized or attached to a solid surface.

[0164] 118. The use according to any one of claims 55 to 107 and 115 to 117, wherein Raman spectroscopy is the only Raman measurement.

[0165] 119. The use according to any one of claims 55 to 107 and 115 to 117, wherein Raman spectroscopy is the only measurement.

[0166] 120. The use according to any one of claims 55 to 107 and 115 to 119, wherein the viral particles are neither derivatized, nor tagged or labeled.

[0167] 121. The use according to any one of claims 55 to 107 and 115 to 119, wherein the viral particle is an underivatized viral particle or an untagged viral particle or an unlabeled viral particle. DETAILED DESCRIPTION

[0168] The present invention is based, at least in part, on the discovery that conventional micro-Raman spectroscopy combined with principal component data analysis can be used to i) detect low concentrations of fully filled AAV2 and AAV8 particles in aqueous buffer solutions, ii) distinguish fully filled AAV particles from empty AAV particles in a sample, and iii) distinguish particles of different serotypes.

[0169] definition

[0170] Methods and techniques that can be used to practice the present invention are described in, for example, Ausubel, FM (ed.), Current Protocols in Molecular Biology, Volumes I to III (1997); Glover, ND and Hames, BD, eds., DNA Cloning: A Practical Approach, Volumes I and II (1985), Oxford University Press; Freshney, RI (ed.), Animal Cell Culture—a practical approach, IRL Press Limited (1986); Watson, JD et al., Recombinant DNA, Second Edition, CHSL Press (1992); Winnacker, EL, From Genes to Clones; NY, VCH Publishers (1987); Celis, J., ed., Cell Biology, Second Edition, Academic Press (1998); Freshney, RI, Culture of Animal Cells: A Manual of Basic Technique, Second Edition, Alan R. Liss, Inc., NY (1987).

[0171] It must be noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth. Likewise, the terms "a", "one or more", and "at least one" may be used interchangeably herein. It should also be noted that the terms "comprising", "including", and "having" may be used interchangeably.

[0172] The term "AAV helper function" refers to AAV-derived coding sequences (proteins) that can be expressed to provide AAV gene products and AAV particles, which in turn function in trans for productive AAV replication and packaging. Thus, AAV helper functions include AAV open reading frames (ORFs), including rep and cap, and other functions for certain AAV serotypes, such as AAP. The rep gene expression product has been shown to have many functions, including: recognition, binding and cleavage of AAV origins of DNA replication; DNA helicase activity; and transcriptional regulation from AAV (or other heterologous) promoters. The cap gene expression product (capsid protein) provides the necessary packaging function. AAV helper functions are used to supplement trans AAV functions that are missing from the AAV vector genome.

[0173] The term "comprising" also encompasses the term "including".

[0174] The terms "empty capsid" and "empty particle" refer to an AAV particle that has an AAV protein capsid but lacks in whole or in part a nucleic acid encoding a protein or transcribed into a target transcript flanking the AAV ITRs, i.e., a vector. Thus, an empty capsid is an AAV particle that does not have an encapsidated nucleic acid (payload). Thus, an empty capsid cannot function to transfer a nucleic acid encoding a protein or transcribed into a target transcript into a host cell.

[0175] The term "mammalian cell comprising an exogenous nucleotide sequence" encompasses cells into which one or more exogenous nucleic acids have been introduced, including progeny of such cells. These can be starting points for further genetic modification. Therefore, the term "mammalian cell comprising an exogenous nucleotide sequence" encompasses cells comprising an exogenous nucleotide sequence at a single site integrated into the locus of the genome of the mammalian cell, wherein the exogenous nucleotide sequence at least comprises a first recombination recognition site and a second recombination recognition site (these recombination recognition sites are different) flanking at least one first selection marker. In certain embodiments, the mammalian cell comprising an exogenous nucleotide sequence is a cell comprising an exogenous nucleotide sequence at a single site integrated into the locus of the genome of the cell, wherein the exogenous nucleotide sequence comprises a first recombination recognition sequence and a second recombination recognition sequence flanking at least one first selection marker, and a third recombination recognition sequence between the first recombination recognition sequence and the second recombination recognition sequence, and all recombination recognition sequences are different.

[0176] Both "mammalian cells comprising exogenous nucleotide sequences" and "recombinant cells" are "transfected cells". The term includes the primary transfected cell and progeny derived therefrom, without regard to the number of passages. The progeny may, for example, not be completely identical to the nucleic acid content of the parent cell, but may contain mutations. Mutant progeny having the same function or biological activity as in the original transfected cell are encompassed.

[0177] "Nucleic acid encoding AAV packaging proteins" generally refers to one or more nucleic acid molecules that include nucleotide sequences that provide AAV functions that are deleted from AAV vectors and are used to produce transduction-active recombinant AAV particles. Nucleic acids encoding AAV packaging proteins are generally used to provide expression of AAV rep and / or cap genes to supplement the missing AAV functions required for AAV replication; however, the nucleic acid construct lacks AAV ITR and can neither replicate nor package itself. Nucleic acids encoding AAV packaging proteins can be in the form of plasmids, phages, transposons, cosmids, viruses or particles. Many nucleic acid constructs have been described, such as the commonly used plasmids pAAV / Ad and pIM29+45, which encode both rep and cap gene expression products. See, for example, Samulski et al. (1989) J. Virol. 63: 3822-3828; and McCarty et al. (1991) J. Virol. 65: 2936-2945. Different plasmids encoding the expression products of the rep and / or cap genes have been described (eg, US 5,139,941 and US 6,376,237). Any of these nucleic acids encoding AAV packaging proteins may comprise a DNA element or nucleic acid according to the invention.

[0178] The term "nucleic acid encoding an auxiliary protein" generally refers to one or more nucleic acid molecules comprising nucleotide sequences encoding proteins and / or RNA molecules that provide adenoviral auxiliary functions. Plasmids with nucleic acids encoding auxiliary proteins can be transfected into suitable cells, wherein the plasmid is then able to support the production of AAV particles in the cells. Any of these nucleic acids encoding auxiliary proteins may comprise a DNA element or nucleic acid according to the present invention. The term explicitly excludes infectious viral particles, as they occur in nature, such as adenovirus, herpes virus or vaccinia virus particles.

[0179] The term "packaging protein" refers to non-AAV derived viral and / or cellular functions on which AAV replication depends. Thus, the term encompasses proteins and RNAs required for AAV replication, including those involved in AAV gene transcriptional activation, stage-specific AAV mRNA splicing, AAV DNA replication, Cap expression product synthesis, and AAV capsid assembly. Virus-based helper functions can be derived from any known helper virus, such as adenovirus, herpes virus (except herpes simplex virus type I) and vaccinia virus.

[0180] As used herein, "AAV packaging proteins" refer to AAV-derived sequences that function in trans for efficient AAV replication. Therefore, AAV packaging proteins are encoded by the main AAV open reading frames (ORFs), rep and cap. The rep protein has been shown to have many functions, including: recognition, binding and cleavage of AAV origins of DNA replication; DNA helicase activity; and transcriptional regulation from AAV (or other heterologous) promoters. Cap (capsid) proteins provide the necessary packaging functions. AAV packaging proteins are used herein to supplement trans AAV functions missing from AAV vectors.

[0181] "Plasmid" is a form of nucleic acid or polynucleotide, which usually has additional elements for expression (e.g., transcription, replication, etc.) or proliferation (replication) of the plasmid. Plasmid as used herein can also be used to refer to such nucleic acid or polynucleotide sequences. Therefore, in all aspects, the compositions and methods of the present invention are applicable to nucleic acids, polynucleotides and plasmids, such as for the production of cells (which produce viral (e.g., AAV) vectors), for the production of viral (e.g., AAV) particles, for the production of cell culture media containing viral (e.g., AAV) particles, etc.

[0182] As used herein, the term "recombinant cell" refers to a cell that has been genetically modified, such as, for example, a cell that expresses a polypeptide of interest or produces rAAV particles of interest and can be used to produce the polypeptide of interest or rAAV particles of interest on any scale. For example, a "mammalian cell comprising an exogenous nucleotide sequence" that has undergone recombinase-mediated cassette exchange (RMCE) whereby the coding sequence of the polypeptide of interest has been introduced into the genome of the host cell is a "recombinant cell". Although the cell is still capable of further RMCE reactions, it is not desirable to do so.

[0183] A "recombinant AAV vector" is derived from the wild-type genome of a virus (such as AAV) by using molecular biological methods to remove the wild-type genome from a virus (such as AAV) and replace it with a non-natural nucleic acid (such as a nucleic acid transcribed into a transcript or encoding a protein). Typically, for AAV, one or two 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 relative to the viral genomic nucleic acid. Therefore, the incorporation of non-natural sequences defines a viral vector (e.g., AAV) as a "recombinant" vector, which in the case of AAV can be referred to as a "rAAV vector."

[0184] Recombinant vector (e.g., AAV) sequences can be packaged - referred to herein as "particles" - for subsequent cell infection (transduction) ex vivo, in vitro or in vivo. Wherein when the recombinant vector sequence is encapsulated or packaged into an AAV particle, the particle may also be referred to as "rAAV". Such particles include proteins that encapsulate or package the vector genome. Specific examples include viral envelope proteins, and in the case of AAV, capsid proteins such as AAV VP1, VP2 and VP3.

[0185] As used herein, the term "serotype" is a distinction based on serologically different AAV capsid proteins. Serological uniqueness is determined based on the lack of cross-reactivity between antibodies to one AAV compared to antibodies to another AAV. Such cross-reactivity differences are usually due to differences in capsid protein sequences / antigenic determinants (e.g., due to differences in VP1, VP2 and / or VP3 sequences of AAV serotypes). Although AAV variants, including capsid variants, may be serologically indistinguishable from reference AAV or other AAV serotypes, they differ in at least one nucleotide or amino acid residue compared to the reference AAV serotype or other AAV serotypes.

[0186] Under the traditional definition, serotype refers to the target virus that has been tested for serum, which has specificity for neutralizing activity to all existing and characterized serotypes, and no antibodies to neutralize the target virus are found. With the discovery of more naturally occurring virus isolates and / or the generation of capsid mutants, there may be or may not be serological differences with any currently existing serotype. Therefore, in the case where a new virus (such as AAV) does not have serological differences, the new virus (such as AAV) will be a subgroup or variant of the corresponding serotype. In many cases, serological tests for neutralizing activity have not yet been performed on mutant viruses with modified capsid sequences to determine whether they have another serotype according to the traditional definition of serotype. Therefore, for convenience and to avoid repetition, the term "serotype" refers broadly to serologically different viruses (such as AAV) and serologically no different viruses (such as AAV), which may be within a subgroup or variant of a given serotype.

[0187] The term "transgene" is used herein for convenience to refer to a nucleic acid that is intended to be or has been introduced into a cell or organism. A transgene includes any nucleic acid, such as a gene, that is transcribed into a transcript or encodes a polypeptide or protein.

[0188] "Vector" refers to a portion of a recombinant plasmid sequence that is ultimately packaged or encapsulated directly or in the form of a single strand or RNA to form a virus (e.g., AAV) particle. In the case where a recombinant plasmid is used to construct or manufacture a recombinant virus particle, the virus particle does not include a "plasmid" portion that does not correspond to the vector sequence of the recombinant plasmid. This non-vector portion of the recombinant plasmid is called a "plasmid backbone," which is important for cloning and amplification of the plasmid, a process required for propagation and recombinant virus production, but it itself is not packaged or encapsulated into a virus (e.g., AAV) particle. Therefore, "vector" refers to a nucleic acid packaged or encapsulated by a virus particle (e.g., AAV).

[0189] Recombinant cells

[0190] Generally, for efficient and large-scale production of a target protein compound, such as, for example, rAAV particles or therapeutic polypeptides, cells that express and, if possible, also secrete the protein compound are necessary. Such cells are referred to as "recombinant cells" or "recombinant production cells".

[0191] To generate a "recombinant production cell", suitable mammalian cells are transfected with the desired nucleic acid sequence encoding the protein compound of interest. It may be necessary to transfect additional auxiliary polypeptides.

[0192] In order to generate stable recombinant production cells, a second step follows in which single cells that stably express the protein compound of interest are selected. This can be done, for example, based on the co-expression of a selection marker that has been co-transfected with the nucleic acid sequence encoding the protein compound of interest, or on the expression of the protein compound itself.

[0193] In order to express the coding sequence, i.e., to express the open reading frame, other regulatory elements, such as promoter and polyadenylation signal (sequence) are necessary. Therefore, the open reading frame is operably connected to the other transcriptional regulatory elements. This can be achieved by integrating it into the so-called expression cassette. The minimum regulatory element required for the expression cassette to work in mammalian cells is the promoter that works in the mammalian cell, which is located at the upstream of the open reading frame, i.e. 5', and the polyadenylation signal (sequence) that works in the mammalian cell, which is located at the downstream of the open reading frame, i.e. 3'. In addition, the terminator sequence may be present in the 3' of the polyadenylation signal (sequence). In order to express, promoter, open reading frame / coding region and polyadenylation signal sequence must be arranged in an operably connected form.

[0194] Similarly, nucleic acids that are transcribed into non-protein coding RNAs are referred to as "RNA genes". Also for the expression of RNA genes, additional regulatory elements, such as promoters and transcription termination signals or polyadenylation signals (sequences) are also necessary. The nature and positioning of such elements depend on the RNA polymerase intended to drive the expression of the RNA gene. Therefore, RNA genes are also typically integrated into expression cassettes.

[0195] If the target protein compound is an AAV particle, which is composed of different (monomeric) capsid polypeptides and a single-stranded DNA molecule, and which additionally requires other adenoviral helper functions for production and packaging, multiple expression cassettes different from the included open reading frames / coding sequences are required. In this case, at least an expression cassette is required for each transgene, the different polypeptides forming the capsid of the AAV vector, the required helper functions and the VA RNA. Thus, a separate expression cassette is required for each helper E1A, E1B, E2A, E4orf6, VA RNA, rep and cap gene.

[0196] As outlined in the previous paragraph, the more complex the target protein compound or the higher the number of auxiliary polypeptides and / or RNA required, the higher the number of different expression cassettes required. The total size of the nucleic acid is also inherently related to the number of expression cassettes. However, there is a practical upper limit to the transferable nucleic acid size, which ranges from about 15kbps (kilobase pairs). Exceeding this limit, processing and processing efficiency will drop significantly. This problem can be solved by using two or more separate plasmids. Therefore, different expression cassettes are assigned to different plasmids, so that each plasmid only contains some expression cassettes.

[0197] For the development of stable cell lines, random integration (RI) of one or more nucleic acids carrying an expression cassette for a protein compound of interest may be used. Generally, by using RI, the nucleic acid or fragment thereof is randomly integrated into the genome of the host cell.

[0198] Alternatively to RI, targeted integration (TI) can also be used for CLD. In TI CLD, one or more nucleic acids comprising different expression cassettes are introduced into a predetermined locus in the host cell genome.

[0199] In TI, homologous recombination or recombinase-mediated cassette exchange reaction (RMCE) can be used to integrate the nucleic acid containing the corresponding expression cassette into a specific locus in the genome of the TI host cell.

[0200] Adeno-associated virus (AAV)

[0201] For a general review of AAV and adenovirus or herpes helper functions, see Berns and Bohensky, Advances in Virus Research, Academic Press., 32 (1987) 243-306. The genome of AAV is described in Srivastava et al., J. Virol., 45 (1983) 555-564. Design considerations for constructing recombinant AAV vectors are described in US 4,797,368 (see also WO 93 / 24641). Additional references describing AAV vectors are West et al., Virol. 160 (1987) 38-47; Kotin, Hum. Gene Ther. 5 (1994) 793-801; and Muzyczka J. Clin. Invest. 94 (1994) 1351. The construction of recombinant AAV vectors is described in the following literature: US 5,173,414; Lebkowski et al., Mol. Cell. Biol. 8 (1988) 3988-3996; Tratschin et al., Mol. Cell. Biol. 5 (1985) 3251-3260; Tratschin et al., Mol. Cell. Biol., 4 (1994) 2072-2081; Hermonat and Muzyczka Proc. Natl. Acad. Sci. USA 81 (1984) 6466-6470; Samulski et al. J. Virol. 63 (1989) 3822-3828.

[0202] Adeno-associated virus (AAV) is a replication-defective parvovirus. It can replicate only in cells where certain viral functions are provided by co-infecting helper viruses such as adenovirus, herpes virus, and in some cases poxviruses such as vaccinia. However, if the appropriate helper virus functions are present, AAV can replicate in almost any cell line of human, simian or rodent origin.

[0203] In the absence of helper viral genes, AAV establishes latency in its host cells. Its genome integrates into a specific site on chromosome 19 [(Chr)19(q13.4)], which is called adeno-associated virus integration site 1 (AAVS1). For specific serotypes, such as AAV-2, other integration sites have been found, such as, for example, on chromosome 5 [(Chr)5(p13.3)], called AAVS2, and on chromosome 3 [(Chr)3(p24.3)], called AAVS3.

[0204] AAV is divided into different serotypes. These are assigned based on parameters such as hemagglutination, tumorigenicity, and DNA sequence homology. To date, more than 10 different serotypes and more than 100 sequences corresponding to different branches of AAV have been identified.

[0205] The capsid protein type and symmetry determine the tissue tropism of the corresponding AAV. For example, AAV-2, AAV-4, and AAV-5 are retina-specific, AAV-2, AAV-5, AAV-8, AAV-9, and AAVrh-10 are brain-specific, AAV-1, AAV-2, AAV-6, AAV-8, and AAV-9 are heart tissue-specific, AAV-1, AAV-2, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, and AAV-10 are liver-specific, and AAV-1, AAV-2, AAV-5, and AAV-9 are lung-specific.

[0206] Pseudotyping refers to the process that involves cross-packaging of the AAV genome between different serotypes, i.e., the genome is packaged with capsid proteins from different sources.

[0207] The wild-type AAV genome has a size of about 4.7 kb. The AAV genome further comprises two overlapping genes, called rep and cap, which contain multiple open reading frames (see, e.g., Srivastava et al., J. Viral., 45 (1983) 555-564; Hermonat et al., J. Viral. 51 (1984) 329-339; Tratschin et al., J. Virol., 51 (1984) 611-619). The Rep protein encoding open reading frame provides four proteins of different sizes, which are called Rep78, Rep68, Rep52 and Rep40. These are related to the replication, rescue and integration of AAV. The Cap protein encoding open reading frame provides four proteins, which are called VP1, VP2, VP3 and AAP. VP1, VP2 and VP3 are part of the protein capsid of AAV particles. The combined rep and cap open reading frames are flanked at their 5' and 3' ends by so-called inverted terminal repeats (ITRs). For replication, AAV requires, in addition to the Rep and Cap proteins, the products of the adenoviral genes E1A, E1B, E4orf6, E2A and VA or the corresponding factors of another helper virus.

[0208] In the case of AAV of serotype 2 (AAV-2), for example, each ITR has a length of 145 nucleotides and is flanked by a coding sequence region of about 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 functions as a primer during viral replication. The remaining 20 non-paired nucleotides are represented as D sequences.

[0209] The AAV genome contains three transcription promoters P5, P19 and P40 (Laughlin et al., Proc. Natl. Acad. Sci. USA 76 (1979) 5567-5571) for the expression of rep and cap genes.

[0210] ITR sequences must be present in the coding region in cis. ITR provides a functional origin of replication (ori), is integrated into the target cell genome required signal, and is efficiently excised and rescued from the host cell chromosome or recombinant plasmid. ITR further comprises an origin of replication-like element, such as a Rep protein binding site (RBS) and a terminal resolution site (TRS). It has been found that 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).

[0211] For replication and encapsidation of the viral single-stranded DNA genome, the trans organization of the rep and cap gene products, respectively, is required.

[0212] The rep locus comprises two internal promoters, called P5 and P19. It comprises open reading frames of four proteins. Promoter P5 is operably linked to a nucleic acid sequence that provides a non-spliced ​​4.2 kb mRNA encoding Rep protein Rep78 (chromatin nickase that blocks the cell cycle) and a spliced ​​3.9 kb mRNA encoding Rep protein Rep68 (site-specific endonuclease). Promoter P19 is operably linked to a nucleic acid sequence that provides a non-spliced ​​mRNA encoding Rep protein Rep52 and a spliced ​​3.3 kb mRNA encoding Rep protein Rep40 (DNA helicase for accumulation and packaging).

[0213] The two larger Rep proteins, Rep78 and Rep68, are essential for AAV duplex DNA replication, whereas the smaller Rep proteins, Rep52 and Rep40, appear to be essential for progeny single-stranded DNA accumulation (Chejanovsky and Carter, Virology 173 (1989) 120-128).

[0214] The larger Rep proteins Rep68 and Rep78 can bind specifically to the hairpin conformation of AAV ITR. They exhibit defined enzymatic activity, which is required for resolving AAV terminal replication. Expression of Rep78 or Rep68 can be sufficient for the formation of infectious particles (Holscher, C., et al. J. Virol. 68 (1994) 7169-7177 and 69 (1995) 6880-6885).

[0215] All Rep proteins, primarily Rep78 and Rep68, are believed to exhibit regulatory activities such as induction and repression of AAV genes and inhibition of cell growth (Tratschin et al., Mol. Cell. Biol. 6 (1986) 2884-2894; Labow et al., Mol. Cell. Biol., 7 (1987) 1320-1325; Khleif et al., Virology, 181 (1991) 738-741).

[0216] Recombinant overexpression of Rep78 results in a phenotype of reduced cell growth due to induction of DNA damage, thereby arresting host cells in the S phase, thereby promoting latent infection of the virus (Berthet, C., et al., Proc. Natl. Acad. Sci. USA 102 (2005) 13634-13639).

[0217] Tratschin et al. reported that the P5 promoter is negatively autoregulated by Rep78 or Rep68 (Tratschin et al., Mol. Cell. Biol. 6 (1986) 2884-2894). Due to the toxic effects of Rep protein expression, it is reported that after stable integration of AAV, some cell lines have only very low expression (see, for example, Mendelson et al., Virol. 166 (1988) 154-165).

[0218] The cap locus contains a promoter, called P40. The promoter P40 is operably linked to a nucleic acid sequence that provides 2.6 kb mRNA, which encodes the Cap proteins VP1 (87 kDa, non-spliced ​​mRNA transcripts), VP2 (72 kDa, from spliced ​​mRNA transcripts) and VP3 (61 kDa, from alternative start codons) by alternative splicing and using alternative start codons. VP1 to VP3 constitute the structural unit of the viral capsid. The capsid has the function of binding to cell surface receptors and allowing intracellular transport of the virus. VP3 accounts for about 90% of the total viral particle protein. However, all three proteins are essential for effective capsid production.

[0219] Inactivation of all three capsid proteins VP1 to VP3 reportedly prevents the accumulation of single-stranded progeny AAV DNA. Mutations at the amino terminus of VP1 ("Lip negative" or "Inf negative") still allow single-stranded DNA to assemble into viral particles, thereby greatly reducing infectious titers.

[0220] The AAP open reading frame encodes the assembly activation protein (AAP). It has a size of about 22 kDa and transports the native VP protein to the nucleolar region for capsid assembly. This open reading frame is located upstream of the VP3 protein coding sequence.

[0221] In a single AAV particle, only one single-stranded DNA molecule is contained. This can be either the "plus" strand or the "minus" strand. AAV viral particles containing DNA molecules are infectious. Inside infected cells, the parental infectious single strand is converted to a double strand, which is then amplified. Amplification produces a large number of double-stranded DNA molecules in which the single strand is displaced and packaged into capsids.

[0222] Adeno-associated virus (AAV) vectors can transduce dividing cells as well as dormant cells. It can be assumed that transgenes introduced into target cells using AAV vectors will be expressed long term. One disadvantage of using AAV vectors is that the size of the transgene that can be introduced into the cell is limited.

[0223] Viral vectors such as parvovirus particles, including AAV serotypes and variants thereof, provide a means of delivering protein-encoding nucleic acids to cells in vitro, in vitro, and in vivo, so that the cells express the encoded proteins. AAV is a virus that can be used as a gene therapy vector because they can penetrate cells and introduce nucleic acids / genetic material so that the nucleic acids / genetic material can be stably maintained in cells. In addition, for example, these viruses can introduce nucleic acids / genetic material into specific sites. Since AAV is not related to human pathogenic diseases, AAV vectors are able to deliver heterologous polynucleotide sequences (e.g., therapeutic proteins and agents) to human patients without causing substantial AAV pathogenesis or disease.

[0224] Viral vectors that can be used include, but are not limited to, adeno-associated virus (AAV) particles of various serotypes (eg, AAV-1 to AAV-12, etc.) and hybrid / chimeric AAV particles.

[0225] AAV particles can be used to advantage as vehicles for efficient gene delivery. Such particles have many of the characteristics desired for such applications, including tropism for both dividing and non-dividing cells. Early clinical experience with these vectors also suggests no persistent toxicity and minimal or undetectable immune responses. AAV is known to infect a variety of cell types in vivo and in vitro, either by receptor-mediated endocytosis or by transcytosis. These vector systems have been tested in humans against the retinal epithelium, liver, skeletal muscle, airways, brain, joints, and hematopoietic stem cells.

[0226] Recombinant AAV particles generally do not contain viral genes associated with pathogenesis. Such vectors generally have one or more wild-type AAV genes that are completely or partially deleted, such as rep and / or cap genes, but retain at least one functional flanking ITR sequence, which is necessary for rescue, replication, and packaging of the recombinant vector into AAV particles. For example, only the essential parts of the vector are included, such as ITR and LTR elements, respectively. Therefore, the AAV vector genome will include cis sequences (e.g., functional ITR sequences) required for replication and packaging.

[0227] Recombinant AAV vectors and methods and uses thereof include any strain or serotype. As a non-limiting example, recombinant AAV vectors can be based on any AAV genome, such as, for example, AAV-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, 2i8, AAV rh74 or AAV 7m8. Such vectors can be based on the same strain or serotype (or subgroup or variant), or different from each other. As a non-limiting example, a recombinant AAV vector based on a serotype genome can be the same as one or more capsid proteins of the packaging vector. In addition, the recombinant AAV vector genome can be based on an AAV (e.g., AAV2) serotype genome different from one or more AAV capsid proteins of the packaging vector. For example, the AAV vector genome can be based on AAV2, while at least one of the three capsid proteins can be, for example, AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV rh74, AAV 7m8, or a variant thereof. AAV variants include variants and chimeras of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV rh74, and AAV 7m8 capsids.

[0228] 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, AAVrh74 and AAV 7m8, and variants thereof (e.g., capsid variants, such as amino acid insertions, additions, substitutions and deletions), as described in WO2013 / 158879, WO 2015 / 013313 and US2013 / 0059732 (disclosing LK01, LK02, LK03, etc.).

[0229] AAV and AAV variant (e.g., capsid variant) serotypes (e.g., VP1, VP2 and / or VP3 sequences) may or may not be different from other AAV serotypes, including, for example, AAV1-AAV12 (e.g., different from the VP1, VP2 and / or VP3 sequences of any of the AAV1-AAV12 serotypes).

[0230] In certain embodiments of all aspects and embodiments, the AAV particles associated with a reference serotype have a polynucleotide, polypeptide, or subsequence thereof that comprises or consists of a sequence that is at least 80% or more (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc.) identical to one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV rh74, or AAV 7m8 (e.g., such as ITRs, or VP1, VP2, and / or VP3 sequences).

[0231] 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 a reference AAV serotype (such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV rh74 or AAV 7m8), but are different and not identical to known AAV genes or proteins (such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAVrh74 or AAV 7m8 genes or proteins, etc.). In certain embodiments of all aspects and embodiments, the AAV polypeptide or a subsequence thereof comprises or consists of a sequence that is at least 75% or more identical, e.g., 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identical to any reference AAV sequence or a subsequence thereof, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAVrh74 or AAV 7m8 (e.g., VP1, VP2 and / or VP3 capsid or ITRs). In certain embodiments, the AAV variant has 1, 2, 3, 4, 5, 5-10, 10-15, 15-20, or more amino acid substitutions.

[0232] Recombinant AAV particles, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV rh74 or AAV 7m8, as well as variants, related, hybrid and chimeric sequences, can be constructed using recombinant techniques known to those of skill in the art to include one or more nucleic acid sequences (transgenes) flanked by one or more functional AAV ITR sequences.

[0233] Recombinant particles (e.g., rAAV particles) can be incorporated into pharmaceutical compositions. Such pharmaceutical compositions can be used, among other things, 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 agent that does not itself induce an immune response that is harmful to the individual receiving the composition and can be administered without excessive toxicity.

[0234] Protocols for generating adenoviral vectors have been described in US 5,998,205; US 6,228,646; US 6,093,699; US 6,100,242; WO 94 / 17810 and WO 94 / 23744.

[0235] Viral genome quantification

[0236] For correct viral genome quantification, the sample must be free of plasmid DNA as well as unpackaged vector genomes, both of which contain at least partial viral genomes. In addition, packaged AAV genomes must be available in the first PCR cycle.

[0237] To remove unpackaged DNA that could interfere with the ddPCR process, DNAseI digestion is often used.

[0238] In order to make the encapsulated DNA available for ddPCR, the capsid needs to be opened. This can be done by high temperature incubation or proteinase K digestion. The need for proteinase K digestion has been much discussed in the art.

[0239] Droplet digital polymerase chain reaction

[0240] Droplet digital PCR (ddPCR) allows for absolute quantification of viral genomes without the need to generate a standard curve.

[0241] In more detail, droplet digital polymerase chain reaction (ddPCR) achieves absolute quantification of nucleic acids by randomly partitioning the PCR reaction mixture into discrete partitions, some of which are devoid of nucleic acid target sequences and others in which one or more copies of the template are present (Hindson, B., et al. 2011). Due to the partitioning, thousands of independent PCR reactions are performed during thermal cycling. At the endpoint, the fraction of target-positive partitions is read out and used to calculate the initial template DNA concentration (Pinheiro, L., et al. 2011)

[0242] First, up to 20,000 droplets of approximately 1 nL in volume are formed in a water-oil emulsion. Thus, a PCR reaction mixture consisting of a nucleic acid template, forward (fwd) and reverse (rev) primers, TaqMan probes, and a ddPCR supermix containing Thermus aquaticus (Taq) DNA polymerase, dNTPs, and PCR buffer is partitioned. (See, e.g., Hindson, B., et al. 2011; Taylor, S, et al. 2017). During thermal cycling, a separate PCR reaction is performed in each droplet depending on the presence or absence of the DNA target.

[0243] In the droplet containing template DNA, the target sequence is amplified. During amplification, the 5' to 3' exonuclease activity of Taq polymerase will hydrolyze the TaqMan probe bound to the template chain. Because the probe is degraded into smaller fragments, the fluorophore at the 5' position is no longer close to the quencher at its 3' position. Thereby eliminating signal quenching and generating fluorescent signals. The partition lacking the template sequence does not show amplification, and therefore the TaqMan probe will not be hydrolyzed and will not generate fluorescence, because the fluorescence of the fluorophore at the 5' position remains quenched (see, for example, Holland, P., et al. 1991). Since probes with different fluorophores can be used, these probes have different excitation and emission wavelengths, so ddPCR reactions can be performed as multiple reactions in one droplet. The 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).

[0244] As an endpoint analysis, the fluorescence signal of each droplet after thermal cycling is read. Using Poisson statistics, the copy number (λ) of the target sequence can be calculated according to the ratio of positive reads to total reads (p) according to Equation 1 (see, e.g., Hindson, B., et al. 2011).

[0245] λ = - ln (1 – p) (1)

[0246] Since ddPCR relies on endpoint measurement, target sequence quantification is somewhat independent of PCR reaction efficiency. This is in contrast to real-time PCR (qPCR), which is commonly used for viral genome titration (see, e.g., Taylor, S, et al. 2017). In addition, there is no need to use standards or calibration samples (see, e.g., Dorange, F., Bec, C. 2018).

[0247] Recombinant AAV particles

[0248] Different methods for generating rAAV particles are known in the art. For example, transfection using AAV plasmids and AAV auxiliary sequences and combined with a co-infection of an AAV helper virus (e.g., adenovirus, herpes virus or vaccinia virus), or transfection using recombinant AAV plasmids, AAV helper plasmids and auxiliary function plasmids. Non-limiting methods for generating rAAV particles are described in, for example, US 6,001,650, US 6,004,797, WO 2017 / 096039 and WO 2018 / 226887. After recombinant rAAV particle production (i.e., generating particles in a cell culture system), rAAV particles can be obtained from host cells and cell culture supernatants and purified.

[0249] In order to generate recombinant AAV particles, it is necessary to express Rep and Cap proteins, accessory proteins E1A, E1B, E2A and E4orf6, and adenovirus VA RNA in a single mammalian cell. Accessory proteins E1A, E1B, E2A and E4orf6 can be expressed using any promoter, as shown by Matsushita et al. (Gene Ther. 5 (1998) 938-945), in particular the CMV IE promoter. Therefore, any promoter can be used.

[0250] In general, to produce recombinant AAV particles, different complementary plasmids are co-transfected into host cells. One of the plasmids contains a transgene sandwiched between two cis-acting AAV ITRs. The deletion AAV elements (i.e., the open reading frames of Rep and Cap proteins) required for the replication and subsequent packaging of the progeny recombinant genome are included in the second plasmid in trans. Overexpression of Rep protein leads to an inhibitory effect on cell growth (Li, J., et al., J. Virol. 71 (1997) 5236-5243). In addition, AAV replication requires a third plasmid containing helper virus genes (i.e., E1, E4orf6, E2A and VA from adenovirus).

[0251] To reduce the number of plasmids required, Rep, Cap and adenoviral helper genes can be combined on a single plasmid.

[0252] Alternatively, the host cell may have stably expressed the E1 gene product. Such cells are HEK293 cells. The human embryonic kidney clone, denoted 293, was generated as early as 1977 by integrating adenovirus DNA into human embryonic kidney cells (HEK cells) (Graham, FL, et al., J. Gen. Virol. 36 (1977) 59-74). The HEK293 cell line contains base pairs 1 to 4344 of the adenovirus serotype 5 genome. This covers E1A and E1B genes and adenovirus packaging signals (Louis, N., et al., Virology 233 (1997) 423-429).

[0253] When HEK293 cells are used, the deleted E2A, E4orf6 and VA genes can be introduced by co-infection with adenovirus or by co-transfection with plasmids expressing E2A, E4orf6 and VA (see, e.g., Samulski, RJ, et al., J. Virol. 63 (1989) 3822-3828; Allen, JM, et al., J. Virol. 71 (1997) 6816-6822; Tamayose, K., et al., Hum. Gene Ther. 7 (1996) 507-513; Flotte, TR, et al., Gene Ther. 2 (1995) 29-37; Conway, JE, et al., J. Virol. 71 (1997) 8780-8789; Chiorini, JA, et al., Hum. Gene Ther. 6 (1995) 1531-1541; Ferrari, FK, et al., J. Virol. 70 (1996) 3227-3234; Salvetti, A., et al., Hum. Gene Ther. 9 (1998) 695-706; Xiao, X., et al., J. Virol. 72 (1998) 2224-2232; Grimm, D., et al., Hum. Gene Ther. 9 (1998) 2745-2760; Zhang, X., et al., Hum. Gene Ther. 10 (1999) 2527-2537). Alternatively, adenovirus / AAV or herpes simplex virus / AAV hybrid vectors may be used (see, e.g., Conway, JE, et al., J. Virol. 71 (1997) 8780-8789; Johnston, KM, et al., Hum. Gene Ther. 8 (1997) 359-370; Thrasher, AJ, et al., Gene Ther. 2 (1995) 481-485; Fisher, JK, et al., Hum. Gene Ther. 7 (1996) 2079-2087; Johnston, KM, et al., Hum. Gene Ther. 8 (1997) 359-370).

[0254] Therefore, cell lines in which the rep gene is integrated and expressed tend to grow slowly or express Rep protein at very low levels.

[0255] To restrict transgene activity to specific tissues, ie, to limit integration sites, the transgene can be operably linked to an inducible or tissue-specific promoter (see, eg, Yang, Y., et al. Hum. Gene. Ther. 6 (1995) 1203-1213).

[0256] A major difficulty in rAAV particle production is the inefficiency of rAAV vector packaging, resulting in low titers. Packaging has been difficult for a number of reasons, including

[0257] - Encapsidation of the wild-type AAV genome is preferred (if present);

[0258] - Difficulty in generating adequate complementary functions, such as those provided by wild-type rep and cap genes, due to repression associated with the rep gene product;

[0259] - The efficiency of co-transfection of plasmid constructs is limited.

[0260] All of this is based on the biological properties of the Rep protein. In particular, the inhibitory (cytostatic and cytotoxic) properties of the Rep protein and its ability to reverse the immortalized phenotype of cultured cells are problematic. In addition, when the widely used AAV P5 promoter is adopted, the Rep protein downregulates its own expression (see, e.g., Tratschin et al., Mol. Cell. Biol. 6 (1986) 2884-2894).

[0261] 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.

[0262] In certain embodiments of all aspects and embodiments, the rAAV particle comprises a capsid sequence having 70% or more sequence identity to an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, Rh10, Rh74, or 7m8 capsid sequence.

[0263] In certain embodiments of all aspects and embodiments, the rAAV particle comprises an ITR sequence having 70% or more sequence identity to an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10 ITR sequence.

[0264] E1A, E1B, E2 and E4

[0265] The coding sequences (open reading frames) of E1A and E1B may be derived from human adenoviruses, such as, for example, in particular human adenovirus serotype 2 or serotype 5. Exemplary sequences of human Ad5 (adenovirus serotype 5) are found in GenBank entries X02996, AC_000008, and the sequence of an exemplary human Ad2 is found in GenBank entry AC_000007. Nucleotides 505 to 3522 comprise the nucleic acid sequence encoding E1A and E1B of human adenovirus serotype 5. Plasmid pSTK146 as reported in EP 1 230 354 and plasmids pGS119 and pGS122 as reported in WO2007 / 056994 may also be used as sources of E1A and E1B open reading frames.

[0266] E1A is the first viral helper gene expressed after adenoviral DNA enters the cell nucleus. The E1A gene encodes 12S and 13S proteins, which are 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. In addition, the expression of 12S and 13S proteins forces cells to enter the S phase of the cell cycle. If only E1A-derived proteins are expressed, cells will stain (apoptosis).

[0267] E1B is the second viral helper gene expressed. It is activated by E1A-derived proteins 12S and 13S. The mRNA derived from the E1B gene can be spliced ​​in two different ways, producing a first 55kDa transcript and a second 19kDa transcript. The E1B 55kDa protein is involved in the regulation of the cell cycle, the prevention of mRNA trafficking in cells late in infection, and the prevention of E1A-induced apoptosis. The E1B 19kDa protein is involved in the prevention of E1A-induced apoptosis.

[0268] The E2 genes encode different proteins. The E2A transcript encodes the single-stranded binding protein (SSBP), which is essential for AAV replication.

[0269] In addition, the E4 gene encodes multiple proteins. The 34kDa protein (E4orf6) derived from the E4 gene, together with the E1B 55kDa protein, prevents the accumulation of cellular mRNA in the cytoplasm, but also promotes the transport of viral RNA from the nucleus to the cytoplasm.

[0270] Adenovirus VARNA gene

[0271] 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 a type 2 polymerase III promoter (see, e.g., 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.

[0272] The structure, function and evolution of adenovirus-associated RNAs were investigated using a phylogenetic approach by Ma, Y. and Mathews, MB (J. Virol. 70 (1996) 5083-5099). They provided an alignment based on 47 known human adenovirus serotypes and a consensus VA RNA sequence.

[0273] VA RNA, VAI and VAII consist of 157-160 nucleotides (nt).

[0274] Depending on the serotype, adenovirus contains one or two VA RNA genes. VA RNAI is thought to play a major proviral role, while VA RNAII can partially compensate for the absence of VA RNAI (Vachon, VK and Conn, GL, Virus Res. 212 (2016) 39-52).

[0275] VA RNA is not essential, but plays an important role in efficient viral growth by overcoming cellular antiviral mechanisms. That is, although VA RNA is not essential for viral growth, adenoviruses lacking VA RNA cannot grow during the initial steps of vector generation, where only a few copies of the viral genome are present per cell, probably because viral genes other than VA RNA that block cellular antiviral mechanisms may not be fully expressed (see Maekawa, A., et al. Nature Sci. Rep. 3 (2013) 1136).

[0276] Maekawa, A., et al. (Nature Sci. Rep. 3 (2013) 1136) reported the efficient production of adenoviral vectors lacking virus-associated RNA genes that disrupt the cellular RNAi machinery, in which HEK293 cells constitutively and highly expressing flippase recombinase were infected to obtain VA RNA-deficient adenovirus through FLP recombinase-mediated excision of the VA RNA locus.

[0277] Human adenovirus 2VA RNAI corresponds to nucleotides 10586-10810 of the sequence of GenBank entry AC_000007. Human adenovirus 5VA RNAI corresponds to nucleotides 10579-10820 of the sequence of GenBank entry AC_000008.

[0278] Methods for producing rAAV particles

[0279] Carter et al. have shown that the entire rep and cap open reading frames in the wild-type AAV genome can be deleted and replaced with transgenes (Carter, BJ, in "Handbook of Parvoviruses", edited by P. Tijssen, CRC Press, pp. 155-168 (1990)). In addition, it is reported that ITRs must be maintained to retain the functions of replication, rescue, packaging and integration of transgenes into the target cell genome.

[0280] When cells containing the corresponding viral helper genes are transduced with AAV vectors, or vice versa, when cells containing integrated AAV provirus are transduced with appropriate helper virus, the AAV provirus is activated and enters the lytic infection cycle again (Clark, KR, et al., Hum. Gene Ther. 6 (1995) 1329-1341; Samulski, RJ, Curr. Opin. Genet. Dev. 3 (1993) 74-80).

[0281] Aspects of the invention are methods of transducing cells with nucleic acids (e.g., plasmids) comprising all the elements required to produce recombinant AAV particles, wherein the methods according to the invention are used to determine full AAV particles. Thus, when the plasmid encodes viral packaging proteins and / or accessory proteins, the cells can produce recombinant viral particles that include nucleic acids encoding a protein of interest or that include sequences that are transcribed into a transcript of interest.

[0282] The present invention provides a viral (e.g., AAV) particle production platform that includes features that distinguish it from current "industry standard" viral (e.g., AAV) particle production processes through the use of methods according to the present invention.

[0283] More generally, cells transfected or transduced with DNA for recombinant production of AAV particles may be referred to as "recombinant cells". Such cells may be, for example, yeast cells, insect cells or mammalian cells, and have been used as receptors for nucleic acids (plasmids) encoding packaging proteins such as AAV packaging proteins, nucleic acids (plasmids) encoding auxiliary proteins, and nucleic acids (plasmids) encoding proteins or transcribed into target transcripts (i.e., transgenes placed between two AAV ITRs). The term includes progeny of the original cells that have been transduced or transfected. It should be understood that the progeny of a single parental cell may not be exactly the same as the original parent in morphology or genome or total nucleic acid complement due to natural, accidental or deliberate mutations.

[0284] Many cell growth media suitable for maintaining cell viability or providing cell growth and / or proliferation are commercially available. The example of such culture medium includes serum-free eukaryotic growth medium, such as for maintaining the viability of mammalian (e.g., human) cells or providing the culture medium for the growth of mammalian (e.g., human) cells. Non-limiting examples include Ham's F12 or F12K culture medium (Sigma-Aldrich), FreeStyle (FS) F17 culture medium (Thermo-Fisher Scientific), MEM, DMEM, RPMI-1640 (Thermo-Fisher Scientific) and mixtures thereof. Such culture medium can be supplemented with vitamins and / or trace minerals and / or salts and / or amino acids, such as the essential amino acids of mammalian (e.g., human) cells.

[0285] The helper protein plasmid can be in the form of a plasmid, phage, transposon or cosmid. In particular, it has been demonstrated that helper function does not require a full complement of adenoviral genes. For example, adenoviral mutants that are unable to carry out DNA replication and late gene synthesis have been shown to allow AAV replication. Ito et al., J. Gen. Virol. 9 (1970) 243; Ishibashi et al., Virology 45 (1971) 317.

[0286] Mutants within the E2B and E3 regions have been shown to support AAV replication, indicating that the E2B and E3 regions may not be involved in providing auxiliary functions. Carter et al., Virology 126 (1983) 505. However, adenoviruses with defects in the E1 region or deletions in the E4 region cannot support AAV replication. Therefore, for adenovirus auxiliary proteins, the E1A and E4 regions may be directly or indirectly required for AAV replication (see, e.g., Laughlin et al., J. Virol. 41 (1982) 868; Janik et al., Proc. Natl. Acad. Sci. USA 78 (1981) 1925; Carter et al., Virology 126 (1983) 505). Other characterized adenovirus mutants include: E1B (Laughlin et al. (1982), supra; Janik et al. (1981), supra; Ostrove et al., Virology 104 (1980) 502); E2A (Handa et al., J. Gen. Virol. 29 (1975) 239; Strauss et al., J. Virol. 17 (1976) 140; Myers et al., J. Virol. 35 (1980) 665; Jay et al., Proc. Natl. Acad. Sci. USA 78 (1981) 2927; Myers et al., J. Biol. Chem. 256 (1981) 567); E2B (Carter, Adeno-Associated Virus Helper Functions, in I CRC Handbook of Parvoviruses (P. Tijssen, ed., 1990)); E3 (Carter et al. (1983), supra); and E4 (Carter et al. (1983), supra; Carter (1995)).

[0287] Studies of helper proteins provided by adenoviruses with mutations in E1B report that the E1B 55 kDa protein is essential for AAV particle production, whereas the E1B 19 kDa is not. In addition, WO 97 / 17458 and Matshushita et al. (Gene Therapy 5 (1998) 938-945) describe helper plasmids encoding various adenovirus genes. Examples of helper plasmids include the adenovirus VA RNA coding region, the adenovirus E4orf6 coding region, the adenovirus E2A 72 kDa coding region, the adenovirus E1A coding region, and the adenovirus E1B region lacking the complete E1B 55 kDa coding region (see, e.g., WO 01 / 83797).

[0288] Therefore, the present invention provides a method for producing a recombinant AAV vector or an AAV particle comprising the recombinant AAV vector using the method determined according to the present invention for full capsid, wherein the recombinant AAV vector comprises a nucleic acid encoding a protein or transcribed into a target transcript.

[0289] One aspect of the present invention is a method for producing a recombinant AAV vector or an AAV particle comprising the recombinant AAV vector, wherein the recombinant AAV vector comprises a nucleic acid encoding a protein or transcribed into a target transcript, the method comprising the following steps:

[0290] (i) providing one or more plasmids comprising nucleic acids encoding AAV packaging proteins and / or nucleic acids encoding accessory proteins;

[0291] (ii) providing a plasmid comprising a nucleic acid encoding a target protein or transcribed into a target transcript;

[0292] (iii) contacting one or more mammalian or insect cells with a provided plasmid;

[0293] (iv) further adding a transfection reagent and optionally incubating the plasmid / transfection reagent / cell mixture; or providing a physical means, such as an electric current, to introduce the nucleic acid into the cells;

[0294] (v) culturing the transfected cells;

[0295] (vi) harvesting the cultured cells and / or medium from the cultured cells to produce a cell and / or medium harvest; and

[0296] (vii) lysing the cells and optionally harvesting the lysate from the cells and / or culture medium to isolate the recombinant AAV vector or AAV particles;

[0297] (viii) determining the fraction of full AAV particles using a method according to the present invention in or after step (vi) or / and step (vii);

[0298] Thereby, a recombinant AAV vector or AAV particle comprising a nucleic acid encoding a protein of interest or transcribed into a transcript of interest is produced.

[0299] One aspect of the present invention is a method for producing a recombinant AAV vector or an AAV particle comprising the recombinant AAV vector, wherein the recombinant AAV vector comprises a nucleic acid encoding a protein or transcribed into a target transcript, the method comprising the following steps:

[0300] (i) providing one or more plasmids comprising nucleic acids encoding AAV packaging proteins and / or nucleic acids encoding accessory proteins;

[0301] (ii) providing a plasmid comprising a nucleic acid encoding a target protein or transcribed into a target transcript;

[0302] (iii)

[0303] (a) Generate stably transfected cells by

[0304] - contacting one or more mammalian or insect cells with the plasmid provided by (i);

[0305] - further adding a transfection reagent and optionally incubating the plasmid / transfection reagent / cell mixture; or providing physical means, such as an electric current, to introduce the nucleic acid into the cells;

[0306] - selecting a first stably transfected cell;

[0307] - contacting the selected first stably transfected cell with the plasmid provided by (ii); and

[0308] - further adding a transfection reagent and optionally incubating the plasmid / transfection reagent / cell mixture; or providing physical means, such as an electric current, to introduce the nucleic acid into the cells;

[0309] or

[0310] (b) Transiently transfected cells were generated by

[0311] - contacting one or more mammalian or insect cells with the plasmids provided by (i) and (ii); and

[0312] - further adding a transfection reagent and optionally incubating the plasmid / transfection reagent / cell mixture; or providing physical means, such as an electric current, to introduce the nucleic acid into the cells;

[0313] (iv) culturing the transfected cells of (iii);

[0314] (v) harvesting cultured cells and / or culture medium from the cultured cells to produce a cell and / or culture medium harvest;

[0315] (vi) lysing the cells and optionally harvesting the lysate from the cells and / or culture medium to isolate the recombinant AAV vector or AAV particles;

[0316] (vii) determining full capsids using a method according to the present invention during or after step (v) and / or step (vi);

[0317] Thereby, a recombinant AAV vector or AAV particle comprising a nucleic acid encoding a protein of interest or transcribed into a transcript of interest is produced.

[0318] One aspect of the present invention is a method for producing a recombinant AAV vector or an AAV particle comprising the recombinant AAV vector, wherein the recombinant AAV vector comprises a nucleic acid encoding a protein or transcribed into a target transcript, the method comprising the following steps:

[0319] (i) providing a mammalian or insect cell comprising a nucleic acid encoding an AAV packaging protein and / or a nucleic acid encoding an accessory protein;

[0320] (ii) providing a plasmid comprising a nucleic acid encoding a target protein or transcribed into a target transcript;

[0321] (iii)

[0322] (a) Generate stably transfected cells by

[0323] - contacting one or more mammalian or insect cells with the plasmid provided by (i);

[0324] - further adding a transfection reagent and optionally incubating the plasmid / transfection reagent / cell mixture; or providing physical means, such as an electric current, to introduce the nucleic acid into the cells;

[0325] - selecting a first stably transfected cell;

[0326] - contacting the selected first stably transfected cell with the plasmid provided by (ii); and

[0327] - further adding a transfection reagent and optionally incubating the plasmid / transfection reagent / cell mixture; or providing physical means, such as an electric current, to introduce the nucleic acid into the cells;

[0328] or

[0329] (b) Transiently transfected cells were generated by

[0330] - contacting one or more mammalian or insect cells with the plasmids provided by (i) and (ii); and

[0331] - further adding a transfection reagent and optionally incubating the plasmid / transfection reagent / cell mixture; or providing physical means, such as an electric current, to introduce the nucleic acid into the cells;

[0332] (iv) culturing the transfected cells of (iii);

[0333] (v) harvesting cultured cells and / or culture medium from the cultured cells to produce a cell and / or culture medium harvest;

[0334] (vi) lysing the cells and optionally isolating and / or purifying the recombinant AAV vector or AAV particles from the cells and / or culture medium harvest; and

[0335] (vii) determining full AAV particles in or after step (v) or / and step (vi) using a method according to the present invention;

[0336] Thereby, a recombinant AAV vector or AAV particle comprising a nucleic acid encoding a protein of interest or transcribed into a transcript of interest is produced.

[0337] Introduction of nucleic acids (plasmids) into cells can be performed in a variety of ways.

[0338] The various methods of transferring DNA to mammalian cells have been reported in the art. These can be used according to the method of the present invention. In some embodiments of all aspects and embodiments, electroporation, nuclear transfection or microinjection are used for nucleic acid transfer / transfection. In some embodiments of all aspects and embodiments, inorganic substances (such as, for example, calcium phosphate / DNA coprecipitation), cationic polymers (such as, for example, polyethyleneimine, DEAE-dextran) or cationic lipids (lipofection) are used for nucleic acid transfer / transfection. Calcium phosphate and polyethyleneimine are the most commonly used reagents in the transfection for larger-scale nucleic acid transfer (see, for example, Baldi et al., Biotechnol.Lett.29 (2007) 677-684), wherein polyethyleneimine is preferred.

[0339] In certain embodiments of all aspects and embodiments, nucleic acid (plasmid) is provided in the form of a composition in combination with polyethyleneimine (PEI), optionally in combination with cells. In certain embodiments, the composition includes a plasmid / PEI mixture having multiple components: (a) one or more plasmids comprising nucleic acids encoding AAV packaging proteins and / or nucleic acids encoding auxiliary proteins; (b) a plasmid comprising a nucleic acid encoding a protein or transcribed into a target transcript; and (c) a polyethyleneimine (PEI) solution. In certain embodiments, the molar ratio of the plasmid ranges from about 1:0.01 to about 1:100, or a molar ratio ranges from 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.

[0340] In certain embodiments of all aspects and embodiments, the composition further comprises a cell. In certain embodiments, the cell is contacted with the plasmid / PEI mixture of components (a), (b) and / or (c).

[0341] In certain embodiments of all aspects and embodiments, the composition optionally combined with the cells further comprises free PEI. In certain embodiments, the cells are contacted with free PEI.

[0342] In certain embodiments of all aspects and embodiments, the cell has been in contact with a mixture of components (a), (b) and / or (c) for at least about 4 hours, or from about 4 hours to about 140 hours, or from about 4 hours to about 96 hours. In a preferred embodiment, the cell has been in contact with a mixture of components (a), (b) and / or (c) and optionally free PEI for at least about 4 hours.

[0343] Said composition may further comprise plasmid or / and cell.Such plasmid and cell may be contacted with free PEI.In certain embodiments, plasmid and / or cell have been contacted with free PEI for at least about 4 hours, or about 4 hours to about 140 hours, or about 4 hours to about 96 hours.

[0344] Also provided is a method for producing a recombinant AAV vector or AAV particle comprising a nucleic acid encoding a protein or transcribed into a target transcript, the method comprising: providing one or more plasmids comprising a nucleic acid encoding an AAV packaging protein and / or a nucleic acid encoding an auxiliary protein; providing a plasmid comprising a nucleic acid encoding a target protein or transcribed into a target transcript; providing a solution comprising polyethyleneimine (PEI); mixing the above plasmids with the PEI solution, wherein the molar ratio of the plasmids ranges from about 1:0.01 to about 1:100, or the molar ratio ranges from about 100:1 to about 1:0.01, to produce a plasmid / PEI mixture (and optionally incubating the plasmid / PEI mixture for a period of time ranging from about 10 seconds to about 4 hours); and incubating cells with the plasmid / PEI solution produced as described above. I mixture to produce a plasmid / PEI cell culture; adding free PEI to the plasmid / PEI cell culture produced as described above to produce a free PEI / plasmid / PEI cell culture; incubating the produced plasmid / PEI cell culture or free PEI / plasmid / PEI cell culture for at least about 4 hours to produce transfected cells; harvesting the produced transfected cells and / or culture medium from the produced transfected cells to produce a cell and / or culture medium harvest; lysing the cells and optionally harvesting the lysate from the cells and / or culture medium to isolate the recombinant AAV vector or particle, thereby determining the full capsid in the lysate or isolated AAV particle using the method according to the present invention; and thereby producing a recombinant AAV vector or particle comprising a nucleic acid encoding a protein or transcribed into a target transcript.

[0345] Methods for producing recombinant AAV vectors or AAV particles using methods according to the invention may include one or more additional steps or features. Exemplary steps or features include, but are not limited to, steps of harvesting the produced cultured cells and / or harvesting culture medium from the produced cultured cells to produce a cell and / or culture medium harvest. Additional exemplary steps or features include, but are not limited to, lysing the harvested cells and optionally isolating the recombinant AAV vector or AAV particle from the cell and / or culture medium harvested lysate; thereby determining the viral genome copy number using methods according to the invention; and thereby producing a recombinant AAV vector or AAV particle comprising a nucleic acid encoding a protein or being transcribed into a target transcript.

[0346] 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.

[0347] In certain embodiments of all aspects and embodiments, the encoded accessory proteins include adenoviral E1A and E1B, adenoviral E2 and / or E4, VA RNA and / or non-AAV accessory proteins.

[0348] In certain embodiments of all aspects and embodiments, the first plasmid comprises a nucleic acid encoding an AAV packaging protein and the second plasmid comprises a nucleic acid encoding an accessory protein.

[0349] In certain embodiments of all aspects and embodiments, in the co-transfection, the molar ratio of the plasmid comprising the nucleic acid encoding the protein or being transcribed into the target transcript to the first plasmid comprising the nucleic acid encoding the AAV packaging protein to the second plasmid comprising the nucleic acid encoding the auxiliary protein is in the range of about 1-5:1:1, or 1:1-5:1, or 1:1:1-5.

[0350] In certain embodiments of all aspects and embodiments, the cell is a eukaryotic cell. In certain embodiments, the eukaryotic cell is a mammalian cell. In a preferred embodiment, the cell is a HEK293 cell or a CHO cell.

[0351] The usual conditions for culturing eukaryotic cells at about 37° C., 95% humidity and 8 vol.-% CO2 can be used for culturing. Cultivation can be carried out in serum-containing or serum-free medium, adherent culture or suspension culture. Suspension culture can be carried out in any fermentation vessel, such as, for example, in a stirred tank reactor, a wave reactor, a rocking bioreactor, a shaking container or a rotating container or a so-called roller bottle. Transfection can be carried out in a high-throughput format and in screening, for example in a 96 or 384-well format, respectively.

[0352] The methods according to the invention include AAV particles of any serotype or variants thereof. In certain embodiments of all aspects and embodiments, the recombinant AAV particles comprise: any one of AAV serotypes 1 to 12; AAV VP1, VP2 and / or VP3 capsid proteins; or modified or variant AAV VP1, VP2 and / or VP3 capsid proteins; or wild-type AAV VP1, VP2 and / or VP3 capsid proteins. In certain embodiments of all aspects and embodiments, the AAV particles comprise an AAV serotype or an AAV pseudotype, wherein the AAV pseudotype comprises an AAV capsid serotype different from the ITR serotype.

[0353] The methods according to the invention that provide or include AAV vectors or particles may also include other elements. Examples of such elements include, but are not limited to, introns, expression control elements, one or more adeno-associated virus (AAV) inverted terminal repeats (ITRs) and / or filler / stuffing polynucleotide sequences. Such elements may be within or flanking a nucleic acid encoding a protein or transcribed into a target transcript, or an expression control element may be operably linked to a nucleic acid encoding a protein or transcribed into a target transcript, or an AAV ITR may flank the 5' or 3' end of a nucleic acid encoding a protein or transcribed into a target transcript, or a stuffer polynucleotide sequence may flank the 5' or 3' end of a nucleic acid encoding a protein or transcribed into a target transcript.

[0354] Expression control elements include constitutive or regulatable control elements, such as tissue-specific expression control elements or promoters.

[0355] The ITR may be any of AAV2 or AAV6 or AAV8 or AAV9 serotypes, or a combination thereof. The AAV particles may include any VP1, VP2 and / or VP3 capsid proteins having 75% or more sequence identity with any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV10, AAV11, AAV-2i8, AAV rh74 or AAV 7m8 VP1, VP2 and / or VP3 capsid proteins, or 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.

[0356] After producing recombinant virus (e.g., AAV) particles as described herein, if desired, a variety of conventional methods can be used to purify and / or isolate virus (e.g., rAAV) particles from host cells. Such methods include column chromatography, CsCl gradients, iodixanol gradients, etc.

[0357] For example, multiple column purification steps can be used, such as purification by anion exchange column, affinity column and / or cation exchange column. (See, for example, WO 02 / 12455 and US2003 / 0207439). Alternatively or additionally, iodixanol or CsCl gradient steps can be used (see, for example, US 2012 / 0135515; and US2013 / 0072548). In addition, if infectious viruses are used to express packaging proteins and / or auxiliary proteins, various methods can be used to inactivate residual viruses. For example, adenovirus can be inactivated by heating to a temperature of about 60° C. for, for example, 20 minutes or more. This treatment effectively inactivates the helper virus because AAV is heat stable and the helper adenovirus is heat unstable.

[0358] The goal in rAAV vector production and purification systems is to implement strategies to minimize / control the generation of production-related impurities (such as protein, nucleic acid, and vector-related impurities, including wild-type / pseudo-wild-type AAV species (wtAAV) and residual DNA impurities encapsulated by AAV).

[0359] Considering that rAAV particles account for only a small part of the biomass, rAAV particles need to be purified to a purity level that can be used as a clinical human gene therapy product (see, for example, Smith PH, et al., Mo. Therapy 7 (2003) 8348; Chadeuf G., et al., Mo. Therapy 12 (2005) 744; report from the CHMP gene therapy expert group meeting, European Medicines Agency EMEA / CHMP 2005, 183989 / 2004).

[0360] As an initial step, cultured cells producing rAAV particles are typically harvested, optionally combined with harvested cell culture supernatant (culture medium), in which cells producing rAAV particles (suspension or adherent cells) have been cultured. The harvested cells and optional cell culture supernatant can be used as is, as appropriate, lysed or concentrated. In addition, if infection is used to express auxiliary functions, residual helper viruses may be inactivated. For example, adenoviruses can be inactivated by heating to a temperature of about 60° C. for, for example, 20 minutes or more, which only inactivates helper viruses because AAV is heat stable, while helper adenoviruses are heat unstable.

[0361] The supernatant of the cell and / or harvest is lysed by destroying the cells, for example, by chemical or physical means, such as detergent, microfluidization and / or homogenization, to release rAAV particles. During cell lysis or subsequently after cell lysis, a nuclease such as Benzonase is added to degrade contaminated DNA. Typically, the resulting lysate is clarified to remove cell debris, for example, by filtering or centrifugation, to obtain a clarified cell lysate. In a particular example, the lysate is filtered with a micron diameter pore size filter (such as a 0.1-10.0 μm pore size filter, for example a 0.45 μm and / or pore size 0.2 μm filter) to produce a clarified lysate.

[0362] The lysate (optionally clarified) contains AAV particles (including rAAV vectors with encapsidated nucleic acids and empty particles) and production / process-related impurities, such as soluble cellular components from host cells, which may include cell proteins, lipids, and / or nucleic acids, and cell culture medium components, among others. The optionally clarified lysate is then subjected to a purification step to purify the AAV particles (including rAAV vectors) from impurities using chromatography. Prior to the first chromatography step, the clarified lysate may be diluted or concentrated with an appropriate buffer.

[0363] Following cell lysis, optional clarification, and optional dilution or concentration, multiple subsequent and sequential chromatography steps may be used to purify the rAAV particles.

[0364] The first chromatography step can be a cation exchange chromatography or anion exchange chromatography. If the first chromatography step is a cation exchange chromatography, the second chromatography step can be anion exchange chromatography or size exclusion chromatography (SEC). Thus, in certain embodiments of all aspects and embodiments, rAAV particle purification is performed via cation exchange chromatography, followed by purification via anion exchange chromatography.

[0365] Alternatively, if the first chromatography step is cation exchange chromatography, the second chromatography step can be size exclusion chromatography (SEC). Thus, in certain embodiments of all aspects and embodiments, rAAV particle purification is performed via cation exchange chromatography followed by purification via size exclusion chromatography (SEC).

[0366] Still alternatively, the first chromatography step can be affinity chromatography. If the first chromatography step is affinity chromatography, the second chromatography step can be anion exchange chromatography. Thus, in certain embodiments of all aspects and embodiments, rAAV particle purification is performed via affinity chromatography followed by purification via anion exchange chromatography.

[0367] Optionally, a third chromatography step may be added to the preceding chromatography steps. Typically, the optional third chromatography step follows cation exchange, anion exchange, size exclusion or affinity chromatography.

[0368] Thus, in certain embodiments of all aspects and embodiments, rAAV particle purification is performed via cation exchange chromatography, followed by purification via anion exchange chromatography, followed by purification via size exclusion chromatography (SEC).

[0369] Furthermore, in certain embodiments of all aspects and embodiments, further purification of the rAAV particles is performed via cation exchange chromatography, followed by purification via size exclusion chromatography (SEC), followed by purification via anion exchange chromatography.

[0370] In yet further embodiments of all aspects and embodiments, rAAV particle purification is performed via affinity chromatography, followed by purification via anion exchange chromatography, followed by purification via size exclusion chromatography (SEC).

[0371] In yet further embodiments of all aspects and embodiments, rAAV particle purification is performed via affinity chromatography, followed by purification via size exclusion chromatography (SEC), followed by purification via anion exchange chromatography.

[0372] The role of cation exchange chromatography is to separate AAV particles from cells and other components present in the clarified lysate and / or column eluate from affinity chromatography or size exclusion chromatography. Examples of strong cation exchange resins capable of binding rAAV particles over a wide pH range include, but are not limited to, any sulfonic acid-based 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, POROSXS, POROS SP, and POROS S (strong cation exchangers available from Thermo Fisher Scientific, Inc., Waltham, MA, USA). Additional examples include Capto S, Capto S ImpAct, Capto SImpRes (strong cation exchangers available from GE Healthcare, Marlborough, MA, USA), and commercially available from Aldrich Chemical Company (Milliwaukee, WI, USA). and Series of resins. Weak cation exchange resins include, but are not limited to, any carboxylic acid-based resins. Exemplary cation exchange resins include carboxymethyl (CM) resins, phosphoric acid resins (based on phosphate functional groups), methyl sulfonate (S) resins, and sulfopropyl (SP) resins.

[0373] The effect of anion exchange chromatography separates AAV particles from proteins, cells and other components present in the lysate and / or column eluent of the clarification from affinity or cation exchange or size exclusion chromatography. Anion exchange chromatography can also be used to reduce and therefore control the amount of empty particles in the eluent. For example, the anion exchange column with the rAAV particles bound thereto can be washed with a solution of NaCl comprising a moderate concentration (e.g., about 100-125mM, such as 110-115mM), and a portion of empty particles can be eluted during the flow-through process without a large amount of eluted rAAV particles. Subsequently, the rAAV particles bonded to the anion exchange column can be eluted using a solution comprising a higher concentration of NaCl (e.g., about 130-300mM NaCl), thereby producing a column eluent of rAAV particles comprising rAAV vectors with an empty capsid with a reduced or depleted amount and a proportionally increased amount.

[0374] Exemplary anion exchange resins include, but are not limited to, those based on polyamine resins and other resins.Examples of strong anion exchange resins include those generally based on quaternized nitrogen atoms, including but not limited to quaternary ammonium salt resins such as trialkylbenzylammonium resins. Suitable exchange chromatography materials include, but are not limited to, MACRO PREP Q (strong anion exchanger available from BioRad, Hercules, CA, USA); UNOSPHERE Q (strong anion exchanger available from BioRad, Hercules, CA, USA); POROS 50HQ (strong anion exchanger available from Applied Biosystems, Foster City, CA, USA); POROS XQ (strong anion exchanger available from Applied Biosystems, Foster City, CA, USA); POROS SOD (weak anion exchanger available from Applied Biosystems, Foster City, CA, USA); POROS 50PI (weak anion exchanger available from Applied Biosystems, Foster City, CA, USA); CaptoQ, Capto XQ, Capto Q ImpRes and SOURCE 30Q (strong 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 strong anion exchanger available from Amersham Biosciences, Piscataway, NJ, USA). Additional exemplary anion exchange resins include aminoethyl (AE), diethylaminoethyl (DEAE), diethylaminopropyl (DEPE), and quaternary aminoethyl (QAE).

[0375] WO 2019 / 006390 reports an exemplary process for purification of recombinant AAV particles.

[0376] The recombinant adeno-associated virus particle (rAAV particle) purification and production methods outlined below are scalable on a large scale. For example, to 5, 10, 10-20, 20-50, 50-100, 100-200 or more liters of suspension culture volume. The recombinant adeno-associated virus particle purification and production methods are applicable to multiple AAV serotypes / capsid variants.

[0377] In certain embodiments of all aspects and embodiments, purification of rAAV particles comprises the following steps:

[0378] (a) harvesting cells and / or cell culture supernatant containing rAAV particles to produce a harvest;

[0379] (b) optionally concentrating the harvest produced in step (a) to produce a concentrated harvest;

[0380] (c) lysing the harvest produced in step (a) or the concentrated harvest produced in step (b) to produce a lysate;

[0381] (d) treating the lysate produced in step (c) to reduce contaminating nucleic acids in the lysate, thereby producing a nucleic acid-reduced lysate;

[0382] (e) optionally filtering the nucleic acid-reduced lysate produced in step (d) to produce a clarified lysate, and optionally diluting the clarified lysate to produce a diluted clarified lysate;

[0383] (f) subjecting the nucleic acid-reduced lysate of step (d), the clarified lysate of step (e), or the diluted clarified lysate produced in step (e) to cation exchange column chromatography to produce a column eluate comprising rAAV particles, thereby separating the rAAV particles from protein impurities or other production / process related impurities, and optionally diluting the column eluate to produce a diluted column eluate;

[0384] (g) subjecting the column eluate produced in step (f) or the diluted column eluate to anion exchange chromatography to produce a second column eluate comprising rAAV particles, thereby separating the rAAV particles from protein impurities or production / process-related impurities, and optionally concentrating the second column eluate to produce a concentrated second column eluate;

[0385] (h) subjecting the second column eluate produced in step (g) or the concentrated second column eluate to size exclusion column chromatography (SEC) to produce a third column eluate comprising rAAV particles, thereby separating the rAAV particles from protein impurities or production / process-related impurities, and optionally concentrating the third column eluate to produce a concentrated third column eluate; and

[0386] (i) filtering the third column eluate or the concentrated third column eluate produced in step (h) to produce purified rAAV particles;

[0387] Full particles are thus determined with the method according to the invention during or after one or more of steps (a) to (i).

[0388] In certain embodiments of all aspects and embodiments, the AAV affinity column comprises a protein or ligand that binds to an AAV capsid protein. Non-limiting examples of proteins include antibodies that bind to an AAV capsid protein. More specific non-limiting examples include single-chain llama antibodies that bind to an AAV capsid protein.

[0389] In certain embodiments of all aspects and embodiments, the cells are cells growing in suspension or adherently.

[0390] In certain embodiments of all aspects and embodiments, the cell is a mammalian cell. Non-limiting examples include HEK cells, such as HEK-293 cells, and CHO cells, such as CHO-K1 cells.

[0391] Methods for determining the infectious titer of rAAV particles containing a transgene are known in the art (see, e.g., Zhen et al., Hum. Gene Ther. 15 (2004) 709). Methods for determining the infectious titer of empty particles and rAAV particles with packaged transgenes are known (see, e.g., Grimm et al., Gene Therapy 6 (1999) 1322-1330; Sommer et al., Malec. Ther. 7 (2003) 122-128).

[0392] To determine the presence or amount of degraded / denatured capsids, purified rAAV particles can be subjected to SDS-polyacrylamide gel electrophoresis consisting of any gel (e.g., gradient gel) capable of separating the three capsid proteins, then running the gel until the sample is separated, and blotting the gel onto a nylon or nitrocellulose membrane. Anti-AAV capsid antibodies are then used as primary antibodies that bind to denatured capsid proteins (see, e.g., Wobus et al., J. Viral. 74 (2000) 9281-9293). The secondary antibody bound to the primary antibody contains a means for detecting the primary antibody. Semi-quantitative detection of the binding between the primary antibody and the secondary antibody determines the amount of the capsid. Another method is to use an analytical HPLC or analytical ultracentrifuge using a SEC column.

[0393] Data Dimensionality Reduction Technology

[0394] Fast spectroscopic "fingerprinting" techniques, such as near-infrared, mid-infrared, Raman or two-dimensional fluorescence spectroscopy, are relatively inexpensive and well suited for analyzing complex mixtures. These methods generate large amounts of high-dimensional data that can only be processed by chemometric methods such as principal component analysis (PCA) or partial least squares (PLS) modeling. The combination of sophisticated spectroscopic methods and chemometrics is often used for identity testing of raw materials or as a tool for raw material classification.

[0395] For classification methods, such as distinguishing "full" and "empty" capsids in the method according to the present invention, any suitable statistical and machine learning / deep learning techniques can be applied. Without being limited thereto, further, the following but not limited to these alternative methods can be used for classification:

[0396] -Principal Component Analysis (PCA)

[0397] -Non-negative matrix factorization (NMF)

[0398] -Linear Discriminant Analysis (LDA)

[0399] -Generalized Discriminant Analysis (GDA)

[0400] -Canonical Correlation Analysis (CCA)

[0401] -Autoencoder

[0402] -T-Distributed Stochastic Neighbor Embedding (t-SNE)

[0403] - Unified Manifold Approximation and Projection (UMAP)

[0404] -K-Nearest Neighbor Algorithm (k-NN)

[0405] - Kernel or graph-based kernel PCA

[0406] - Low-dimensional embedding: Use PCA, LDA, CCA or NMF techniques as a preprocessing step, followed by clustering via K-NN.

[0407] In more detail, T., et al. have reported the use of principal component analysis (PCA) and partial least squares (PLS) to process and model complex data ( T., et al., NIR Publications, (2002)). In WO 2009 / 086083, a method for hierarchical organization of data using PLS is reported. In WO 2008 / 146059, an analyzer and method for determining the relative importance of fractions of a biological mixture are reported. In WO 2009 / 061326, an evaluation of chromatographic materials is reported.

[0408] Maitra, S., and Yan, J., reviewed principal component analysis and partial least squares, two dimensionality reduction techniques for regression (Casualty Actuarial Society, 2008 Discussion Paper Program).

[0409] 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 highly correlated independent variables. PCA does not reflect the correlation between the independent variables in the dataset and the dependent variable. On the contrary, PLS takes into account the said correlation of the dependent and independent variables. Therefore, PCA is an unsupervised methodology, whereas PLS is a supervised methodology.

[0410] PCA is often used to reduce the number of predictor variables and solve the problem of multicollinearity (Bair, E., et al., J. Am. Stat. Assoc. 101 (2006) 119-137). The advantage of PCA is that it retains most of the information contained in the data set. The data of the variables of the data set are combined in a limited number of linear combinations.

[0411] PCA can be done with any statistical software package. The statistical software generates the eigenvectors of linear coefficients and the mean and standard deviation of each predictor variable. This is the basis for calculating the principal components through regression. That is, the principal components are obtained through the eigenvalue decomposition of the covariance or correlation matrix of the predictor variables under consideration.

[0412] PCA depends on the X vector or the predictor variables. In particular, the relationship of the predictor variables with the dependent variable or the target variable is not important. Hence, PCA is an unsupervised technique. Therefore, most of the information contained in the original predictor variables and the relationship between the predictor variables and the target variable is retained.

[0413] Compared with PCA, partial least squares takes into account the correlation of dependent and independent variables and is therefore a supervised methodology. PLS is particularly suitable for situations where the predictor variables consist of many different measurements in the experiment and the relationship between these variables is difficult to understand (Kleinbaum, DG, et al., in "Applied Regression Analysis and Multivariable Methods", 3rd edition (Pacific Grove, Ca, USA, Brooks / Cole Publishing Company, 1998).

[0414] In certain embodiments of the method according to the invention, physical information has to be removed by spectral preprocessing prior to PCA analysis.

[0415] Additionally, baseline shifts can be removed by applying a multiplicative scatter correction (MSC).

[0416] In order 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 spectrum can be converted to its first derivative.

[0417] In certain embodiments of the invention, PCA analysis is performed on pre-processed spectra.

[0418] Method according to the invention

[0419] The present invention is based at least in part on the discovery that Raman spectroscopy combined with statistical and machine learning / deep learning techniques can be used to i) detect low concentrations of full particles as well as empty particles of capsids of different AAV serotypes in aqueous buffer solutions, ii) distinguish full particles from empty particles in a capsid sample, and iii) distinguish particle capsids of different serotypes.

[0420] In further detail, the present invention is based, at least in part, on the discovery that conventional micro-Raman spectroscopy combined with principal component data analysis (PCA) can be used to i) detect full particles of AAV2 and AAV8 serotypes at low concentrations in aqueous buffer solutions, ii) distinguish full particles from empty particles in a sample, and iii) distinguish particles of different serotypes.

[0421] In general, recombinant AAV particles being evaluated and developed for clinical use are small nonenveloped viral vectors with a diameter of 20 to 25 nm and a recombinant single-stranded DNA genome of approximately 4.7 kb (see Figure 1 ). The two wild-type AAV genes encode different rep and cap gene products through splicing, which are required for viral replication and capsid structure formation, respectively.

[0422] Calculated by molecular weight, AAV particles are composed of 74% protein and 26% DNA on average. The maximum DNA payload capacity is about 4.7kb. Naturally occurring AAV capsids and genetically engineered AAV capsids are being evaluated in depth in different research and clinical application suitability tests. The AAV capsid amino acid sequence is highly conserved between different serotypes, ranging from 53% to >99% (see Vance, M., et al. in "Gene Therapy", ed. by Doaa Hashad, InTechOpen, Table 2A of 2014, reproduced in Table 1 below).

[0423] Table 1: Blast alignment of double combinations of AAV serotypes to determine percent homology.

[0424] AAV1 AAV2 AAV3 AAV4 AAV5 AAV6 AAV7 AAV8 AAV9 AAV10 AAV11 AAV12 AAV1 100％ 83％ 86％ 64％ 59％ 99％ 85％ 84％ 82％ 85％ 67％ 61％ AAV2 83％ 100％ 87％ 61％ 58％ 83％ 82％ 83％ 82％ 84％ 63％ 60％ AAV3 86％ 87％ 100％ 63％ 58％ 87％ 84％ 85％ 83％ 85％ 65％ 61％ AAV4 64％ 61％ 63％ 100％ 53％ 63％ 64％ 64％ 63％ 64％ 82％ 79％ AAV5 59％ 58％ 58％ 53％ 100％ 59％ 59％ 58％ 57％ 57％ 53％ 53％ AAV6 99％ 83％ 87％ 63％ 59％ 100％ 85％ 84％ 82％ 85％ 66％ 61％ AAV7 85％ 82％ 84％ 64％ 59％ 85％ 100％ 88％ 81％ 88％ 67％ 62％ AAV8 84％ 83％ 85％ 64％ 58％ 84％ 88％ 100％ 85％ 93％ 66％ 62％ AAV9 82％ 82％ 83％ 63％ 57％ 82％ 81％ 85％ 100％ 86％ 64％ 60％ AAV10 85％ 84％ 85％ 64％ 57％ 85％ 88％ 93％ 86％ 100％ 67％ 61％ AAV11 67％ 63％ 65％ 82％ 53％ 66％ 67％ 66％ 64％ 67％ 100％ 84％ AAV12 61％ 60％ 61％ 79％ 53％ 61％ 62％ 62％ 60％ 61％ 84％ 100％

[0425] Studies on particles formed from naturally occurring AAV serotypes and engineered capsid proteins have shown that even small differences in the capsid protein amino acid sequence may affect the tissue tropism of AAV particles and, ideally, could be exploited to improve the intended clinical application. Thus, a rapid, non-invasive, and sensitive analytical method is needed to distinguish particles from different AAV serotypes and for recombinant AAV particle quality control.

[0426] Raman spectroscopy is often used to identify individual chemical components by detecting the compound-specific vibrational responses of the chemical bonds they contain. The intensity of such responses is linearly related to the number of corresponding bonds in the analyte / sample.

[0427] Results from various conventional Raman spectroscopy studies in the literature (using visible or near-infrared excitation) indicate that for certain bioprocess-related analytes, the detection limit is between 0.50 mM and 2.0 mM. Thus, one skilled in the art would consider that conventional Raman spectroscopy is not suitable for the analysis of AAV particles containing bioprocess-related samples due to the (very) low concentrations and high molecular similarity of AAV particles of different serotypes.

[0428] Raman spectroscopy methods are known for quantifying expected compounds and analytes based on concentrations and relative peak intensities associated with spectral features (Pelletier, MJ, Appl. Spectrosc., 57 (2003) 20A-42A; Ma, X., et al., Front. Chem., 6 (2018) Article 400). A significant advantage of this method is that specific analytes in complex matrices can be quantified. In general, further, the following but not limited to statistical and machine learning / deep learning methods for quantifying compounds based on Raman spectroscopy data can be used:

[0429] - Linear algorithms such as but not limited to

[0430] -PLS;

[0431] -lasso;

[0432] -lasso-lars;

[0433] - Ridge regression;

[0434] - Resilient network;

[0435] - Huber returns;

[0436] - Passive aggression returns;

[0437] - Bayesian Ridge Regression;

[0438] -Orthogonal matching pursuit;

[0439] - Non-linear algorithms, such as but not limited to

[0440] - (Artificial) Neural Networks (ANN);

[0441] -(nu) Support Vector Regression;

[0442] -Tree-based algorithms: random forest regression, decision trees;

[0443] -Boosted regression: XGBoost regression, gradient boosted regression, adaboost regression;

[0444] -autoML: autogluon, autokeras.

[0445] Partial Least Squares Regression (PLS): Partial least squares regression is a linear regression method that finds a linear regression model by projecting the predictor and observed variables into a new space. Like the Lasso, it is a form of regularized linear regression where the number of components controls the strength of the regularization.

[0446] Lasso: Lasso (Least Absolute Shrinkage and Selection Operator) is a linear regression method for estimating sparse coefficients and consists of a linear model with an additional regularization term. It is known to effectively reduce the number of features that a given solution depends on.

[0447] Lasso Lars: Lasso Lars is a linear regression method that uses the least angle regression (LARS) algorithm. LARS is a regression algorithm for high-dimensional data that is similar to forward stepwise regression. It finds the features that are most correlated with the target. It does not include all features at each step. The estimated coefficients increase in the direction equiangular to the correlation of each coefficient with the residual.

[0448] Ridge Regression: Ridge regression is a linear regression method. It solves a regression model where the loss function is a linear least squares function. Regularization is given by the l2-norm. Similar to lasso regression, ridge regression introduces a penalty factor. However, ridge regression uses the square of the coefficients instead of the magnitude of the coefficients like lasso regression.

[0449] Elastic Net: Elastic Net is a linear regression method. It combines L1 and L2 priors as regularizers. This combination allows learning sparse models where few weights are non-zero like Lasso while still maintaining the regularization properties of Ridge.

[0450] Huber Regression: Huber regression is a linear regression method that is robust to outliers. It uses a different loss function instead of the traditional least squares method. For small residuals, it has the same penalty as the least squares method. However, for large residuals, the penalty is lower and increases linearly rather than quadratically.

[0451] Passive Aggressive Regressor: The Passive Aggressive Regressor is a linear regression method for large-scale learning and is one of the 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, a regularization parameter is included.

[0452] Bayesian Ridge Regression: Bayesian Ridge Regression is a linear regression method. The statistical analysis is performed in the context of Bayesian inference. Therefore, it is assumed that the errors are normally distributed and the prior distribution has a specific form. Explicit results are obtained for the posterior probability distribution of the model parameters. The algorithm is similar to the classic ridge regression.

[0453] Neural Networks: Neural networks are multi-layer perceptrons. The output of an artificial neuron is calculated by some non-linear function of the sum of its inputs and possibly the input of another neuron. Here, up to 5 layers are used and each layer has up to 5 neurons.

[0454] Nu Support Vector Regression: Nu Support Vector Regression is a type of Support Vector Regression with a new parameter, which determines the number of support vectors we want to keep in the solution relative to the total number of samples in the dataset.

[0455] Support Vector Regression: Support vector machines construct a hyperplane or a set of hyperplanes in a high-dimensional space.

[0456] Decision Tree Regression: Decision tree regression is a tree-based method. The model predicts the value of a target variable by learning simple decision rules inferred from data features. The tree can be viewed as a piecewise constant approximation.

[0457] Random Forest: Random Forest is a tree-based method. A number of classification decision trees are fitted on various subsamples of the dataset. Averaging is used to improve the accuracy of predictions and control overfitting.

[0458] Adaboost regressor: Adaboost is a tree-based method. A series of weak prediction models (i.e., models that are only slightly better than random guessing, such as small decision trees) are fit to iteratively modified versions of the data. The final prediction is then made by a weighted majority vote of all predictions.

[0459] Gradient Boosting Regressor: Gradient boosting is a tree-based method. Like Adaboost, the model is built in a stage-wise manner, but it generalizes Adaboost and other boosting methods by allowing the optimization of arbitrary differentiable loss functions.

[0460] Autogluon: Autogluon is an AutoML. Focus is on tree-based models, model ensembles, neural networks, and linear models.

[0461] Autokeras: Autokeras is an efficient neural architecture search system. Focus is on neural networks.

[0462] In general, specific wavelength shifts can be assigned to nucleic acids and proteins in Raman spectroscopy. Some are summarized in Tables 2 to 4 below.

[0463] Table 2: Analysis of nucleic acids (DNA / RNA) by Raman spectroscopy - common band assignments in ribose / phosphate / backbone (adapted from Hobro, AJ, et al. Nucl. Acids Res. 35 (2007) 1169-1177)

[0464] <![CDATA[Raman shift (cm -1 )]]> distribute 601 Ribose 813 Symmetrical stretching of OPO, a hallmark of the A-helix 856 Ribose-phosphate 919 Ribose-phosphate, -CO-stretching 977 Ribose-phosphate, -CO-stretching 1047 PO stretching, phosphate sugar-CO-stretching 1098 <![CDATA[PO2 - Symmetrical Stretch]]> 1136 Stretching at the 2' position of the ribose

[0465] Table 3: DNA / RNA analysis by Raman spectroscopy - common band assignments among bases (adapted from Hobro et al., supra).

[0466]

[0467]

[0468] Table 4: Protein analysis by Raman spectroscopy - common band assignments in proteins (adapted from Beattie, JR, et al., J. Raman Spec. 48 (2017) 813-821).

[0469]

[0470]

[0471]

[0472] The method according to the present invention uses micro-Raman spectroscopy. Thus, most challenges of AAV particle analysis are overcome. The method according to the present invention is

[0473] Non-invasive (native samples, no preparation required);

[0474] Fast (seconds to minutes);

[0475] ·Multi-attribute approach for one sample / analysis (allowing for simultaneous detection, identification, characterization, differentiation, quantification);

[0476] The sample volume requirement is small (e.g. 200 μL);

[0477] Automatable (currently up to 96 samples in a sequence, potentially 384);

[0478] A standalone solution with a small laboratory footprint.

[0479] Improved properties of the method according to the invention

[0480] Can achieve reduced time to perform analysis (approximately 100 times faster)

[0481] The required analytical equipment environment can be reduced (approximately 2 to 4 times);

[0482] Can increase throughput (allowing an “infinite” number of samples);

[0483] • May improve representativeness (because non-invasive and no sample preparation required).

[0484] The method according to the invention is illustrated below with commercially available AAV particle standards of the AAV2 and AAV8 serotypes, each for particles with and without nucleic acid encapsidation. The samples were processed by a well plate based Raman microscope XPloRa Plus from Horiba. This is merely to illustrate the invention. It should not be construed as limiting. The true scope of the invention is set forth in the appended claims.

[0485] Samples containing AAV viral particles with an encapsidated nucleic acid payload are denoted herein as "full" (AAV2 or AAV8 full), whereas samples containing AAV viral particles without an encapsidated nucleic acid payload are denoted herein as "empty" (AAV2 or AAV8 empty).

[0486] The Raman spectra of the AAV2 full sample and empty sample and their corresponding buffer were acquired 50 times (50 repetitions) by XPloRa Plus confocal Raman microscope. The laser wavelength was 532nm. The exemplary overlay of the spectra is Figure 2 Shown in.

[0487] The Raman spectra of the AAV8 full sample and empty sample and their corresponding buffer were acquired 5 times (5 times) by XPloRa Plus confocal Raman microscope. The laser wavelength was 785nm. The exemplary overlay of the spectra is Figure 3 Shown in.

[0488] To enable direct comparison of particles of AAV2 and AAV8 serotypes, Raman spectra of AAV2 full and empty samples were additionally acquired five times using a laser wavelength of 785 nm.

[0489] In a first experiment, the feasibility of the method according to the invention to distinguish between AAV2 full samples and AAV8 full samples and buffer blank samples was shown. The concentration of the viral particles used was 2*10^13 vg / ml.

[0490] The results obtained for the AAV2 full sample were Figure 4 As can be seen, surprisingly, the AAV2 full samples can be clearly distinguished from the blank buffer samples using the method according to the present invention. It can further be seen that the replicate full sample and blank buffer measurements are clustered in different groups, which are mainly separated by the first principal component (PC-1), which explains 55% of the variance in the data set. The other lower ranked PCA components (i.e. PC-2 and lower) do not contribute substantially at all, as shown in Figure 2. Figure 4 As shown in the PCA plot of , the second PC-2 does not contribute substantially to the separation of AAV2 from the buffer control. PC-2 accounts for only 2% of the variance in the data set. All samples were measured with 532 nm laser wavelength excitation.

[0491] These results show that the method according to the invention allows the determination of viral particles with encapsidated nucleic acid by analyzing the PC load. Thus, important spectral features at specific Raman wavelength shifts can be identified, which increase the population separation of each PC. It has been found that for the determination of AAV2 particles with encapsidated nucleic acid, specific Raman 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 hypothesized that the first displacement region corresponds to nucleic acids, such as but not limited to DNA / RNA specific nucleotide bond deformation and stretching vibrations and proteins, while the second displacement region corresponds to amide I bond stretching.

[0492] Table 5: Specific Raman wavelength shifts used to identify AAV2 particles with encapsidated nucleic acid.

[0493]

[0494] The results obtained with the full AAV8 sample were Figure 6. It can be seen that, surprisingly, the AAV8 full samples can be clearly distinguished from the blank buffer samples using the method according to the invention. It can further be seen that, like the AAV2 full samples, the repeated measurements are significantly clustered in different groups, which are separated by the first principal component PC-1, which explains 49% of the variance in the data set. The other lower ranked PCA components, i.e. PC-2 and lower, do not contribute substantially at all. It has been found that in order to determine AAV8 particles with encapsidated nucleic acids, specific Raman shifts at and in the wavelength range of 551 cm-1, 645 cm-1, 1000 cm-1, 1003 cm-1, 1530–1630 cm-1 and 1645 to 1680 cm-1 can be used (see Figure 7 Without being bound by this theory, it is assumed that these correspond to protein-specific vibrations of the SS Cys bridge (551 cm-1), Tyr-specific vibrations (645 cm-1), CC stretching in the β-sheet (1000 cm-1), Phe-specific vibrations (1003 cm-1), Tyr-specific vibrations (1530–1630 cm-1), and amide I bond stretching (1645 to 1680 cm-1).

[0495] Table 6: Specific Raman wavelength shifts used to identify AAV8 particles with encapsidated nucleic acid.

[0496]

[0497] In a second experiment, the feasibility of the method according to the invention to distinguish full from empty samples was evaluated. Thus, AAV2 full and empty samples with a viral genome / particle concentration of 2*10^13 vg / ml were used.

[0498] The results are Figure 8 As can be seen, surprisingly, the full samples can be clearly distinguished from the empty samples using the method according to the present invention. It can further be seen that the repeated measurements are clustered in different groups separated mainly by the first principal component PC-1, which explains 49% of the variance in the data set. The other lower ranked PCA components (i.e. PC-2 and below) do not contribute substantially at all, as shown in Figure 2. Figure 8 As shown in the PCA plot of , the second PC-2 does not contribute substantially to the separation of full and empty samples. It has been found that in order to distinguish full and empty samples, specific Raman shifts at and in the wavelength range of 551 cm-1, 645 cm-1, 631–787 cm-1, 1425–1485 cm-1, 1003 cm-1, and 1645 to 1680 cm-1 can be used (see Fig. 9Without being bound by this theory, it is assumed that these correspond to protein- and nucleic acid-specific vibrations of the S-SCys bridge (551 cm-1), Tyr-specific vibrations (645 cm-1), nucleotide ring deformation and stretching (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 stretching (1645 to 1680 cm-1).

[0499] Table 7: Specific Raman wavelength shifts used to identify AAV2 particles with encapsidated nucleic acid in the presence of AAV2 particles without encapsidated nucleic acid.

[0500]

[0501] In the third experiment, the feasibility of distinguishing different AAV serotypes according to the method of the present invention was evaluated. Therefore, the Raman spectra of AAV2 full sample and AAV8 full sample corrected by buffer background obtained at a viral genome concentration of 2*10^13vg / ml were used. All samples were measured by excitation with a 785nm laser wavelength.

[0502] The results obtained in Fig.10 As can be seen, surprisingly, the two AAV serotypes can be clearly distinguished using the method according to the present invention. It can further be seen that the repeated measurements are clustered in different groups separated mainly by the first principal component PC-1, which explains 52% of the variance in the data set. The other lower ranked PCA components (i.e. PC-2 and below) do not contribute substantially at all, as shown in Figure 2. Fig.10 As shown in the PCA diagram of , the second PC-2 does not contribute substantially to the separation of full samples from empty samples. This is even more surprising in view of the low particle concentration and overall high amino acid sequence homology (83%) of AAV8 to AAV2 (Lochire, MA, et al., J. Virol. 80 (2006) 821-834). It has been found that in order to distinguish AAV particles of different serotypes, such as AAV2 and AAV8, specific Raman shifts at and within wavelengths of 551 cm-1, 645 cm-1, 920–950 cm-1, 1000 cm-1, 1003 cm-1, 1530–1630 cm-1 and 1645 to 1680 cm-1 can be used ( Fig.11 ).

[0503] Table 8: Specific Raman wavelength shifts used to determine AAV8 particles with encapsidated nucleic acid in the presence of AAV2 particles with encapsidated nucleic acid.

[0504]

[0505] Without being bound by this theory, it is assumed that these correspond to protein-specific vibrations of the SS Cys bridge (551 cm-1), Tyr-specific vibrations (645 cm-1), protein CC stretching in the helical structure (920–950 cm-1), CC stretching in the β-sheet (1000 cm-1), Phe-specific vibrations (1003 cm-1), Tyr-specific vibrations (1530–1630 cm-1), and amide I bond stretching (1645 to 1680 cm-1).

[0506] In a fourth experiment, the method according to the invention was used to distinguish full AAV particles of serotypes 5, 8 and 9. Samples containing full AAV particles of different serotypes were measured by micro-Raman spectroscopy. The acquired data were pre-processed (wave range: 400-1800 cm^-1; SG-filter first derivative, first order polynomial, 25pt, SNV transformation, exclusion of obvious outliers) and analyzed by PCA. The results are presented in Fig.12 As can be seen, clear differentiation between the different serotypes is possible.

[0507] In subsequent experiments, full 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 invention to distinguish AAV serotypes as well as the state of AAV payload. The acquired data were preprocessed (wave range: 400-1800 cm^-1; SG-filter first derivative, first order polynomial, 25pt, SNV transformation, exclusion of obvious outliers) and analyzed by PCA. The largest variance in the combined data set of all serotypes was explained by PC1 (71%) and PC2 (16%). The results are in Figures 13 to 15 Thus, using the method according to the present invention, full AAV particles and empty AAV particles of AAV serotypes 5, 8 and 9 can be clearly separated.

[0508] In order to determine the detection limit of the method according to the invention, full 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 acquired data were preprocessed (wave range: 400-1800 cm^-1; SG-filter first derivative, first order polynomial, 25pt, SNV transformation) and analyzed by PCA. Full and empty AAV particles were separated by PCA. The maximum variance in the combined data set of all concentrations was explained by PC1 (29%) and PC2 (13%).

[0509] exist Figures 19 to 22, showing the results of separate analyses of different concentrations. Even at a concentration of 2.5E+12 vg / mL, full and empty AAV particles clustered differently and could be identified by PCA, albeit with a lower signal-to-noise ratio compared to the other concentrations. Pretreatment resulted in an increase in the signal-to-noise ratio.

[0510] In summary and unexpectedly, the present invention is based on the unexpected discovery that conventional Raman spectroscopy combined with principal component data analysis can be used to i) detect low concentrations of AAV particles with encapsidated nucleic acid, such as AAV2 and AAV8 particles, in aqueous buffer solutions, ii) distinguish between full and empty particles comprising the sample, and iii) distinguish between particles of different serotypes. Thus, the method according to the present invention is suitable for rapid, non-invasive analysis of samples comprising AAV particles, such as for example for process control, quality assurance and control and (real-time) release analysis.

[0511] ***

[0512] In addition to the various embodiments depicted and claimed, the disclosed subject matter also relates to other embodiments having other combinations of features disclosed and claimed herein. Thus, the specific features presented herein may be combined with each other in other ways within the scope of the disclosed subject matter, such that the disclosed subject matter includes any suitable combination of features disclosed herein. For purposes of illustration and description, the foregoing description of specific embodiments of the disclosed subject matter has been presented. It is not intended to be exhaustive or to limit the disclosed subject matter to those embodiments disclosed.

[0513] All references mentioned herein are incorporated by reference in their entirety.

[0514] ***

[0515] The following examples and drawings are provided to aid the understanding of the present invention, the true scope of the invention being set forth in the appended claims.It should be understood that modifications may be made to the procedures set forth without departing from the spirit of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0516] Figure 1 Molecular characteristics of AAV. Naturally occurring and engineered AAV particles consist of a non-enveloped icosahedral 60-mer protein capsid and an approximately 4.7 kb viral single-stranded DNA genome or recombinant gene payload. This nanoscale particle is composed of 74% protein and 26% DNA by molecular weight.

[0517] Figure 2 Overlay of Raman spectra of AAV2 samples collected by XPloRa Plus confocal Raman microscope; laser wavelength is 532nm.

[0518] Figure 3 Overlay of Raman spectra of AAV8 samples collected by XPloRa Plus confocal Raman microscope; laser wavelength is 785nm.

[0519] Figure 4 AAV2 particles were detected by micro-Raman spectroscopy. The Raman signals of full AAV2 particles and blank buffer samples can be distinguished. Principal component analysis (PCA) was applied to the Raman spectra of AAV 2 full samples and buffer blank samples. Raman measurements of AAV2 full samples and buffer blank samples were repeated 50 times in the same well (n=50). The scatter plot between PC-1 and PC-2 shows the separation between the buffer blank (light gray) and AAV2 full (dark gray) sample spectra. The explained variance ratio of PC-1 is 55%, and the explained variance ratio of PC-2 is 2%.

[0520] Figure 5 AAV2-loading was detected by micro-Raman spectroscopy. The first principal component (PC-1) can distinguish the Raman signals of the AAV2-filled sample and the buffer blank sample. The loading of PC-1 shows clear differences, such as in the marked bands.

[0521] Figure 6 AAV8 particles were detected by micro-Raman spectroscopy. The Raman signals of full AAV8 particles and buffer blank samples can be distinguished. Principal component analysis (PCA) was applied to the Raman spectra of AAV8 full samples and buffer blank samples. Raman measurements of AAV8 full samples and buffer blank samples were repeated 5 times in the same well (n=5). The scatter plot between PC-1 and PC-2 shows the separation between the buffer blank (light gray) and AAV8 full (dark gray) sample spectra. The explained variance ratio of PC-1 is 49%, and the explained variance ratio of PC-2 is 10%.

[0522] Figure 7 AAV8 particle-loading was detected by micro-Raman spectroscopy. The first principal component (PC-1) could distinguish the Raman signals of the AAV8-filled sample and the buffer blank sample. The loading of PC-1 showed clear differences, such as in the marked bands.

[0523] Figure 8Discrimination of full and empty AAV2 particles. The Raman signals of AAV2 full samples and AAV2 empty samples can be distinguished. Principal component analysis (PCA) was applied to the Raman spectra of AAV2 full samples and AAV2 empty samples. Raman measurements of AAV2 full samples and AAV2 empty samples were repeated 50 times in the same well (n=50). The scatter plot between PC-1 and PC-2 shows the separation between AAV2 empty (light gray) and AAV2 full (dark gray) sample spectra. The explained variance of PC-1 is 49%, and the explained variance of PC-2 is 4%.

[0524] Fig. 9 Distinction between full and empty AAV2 particles - loading. The first principal component (PC-1) can distinguish the Raman signals of AAV2 full and AAV2 empty samples. The loading of PC-1 shows clear differences, for example in the marked bands.

[0525] Fig.10 Differentiation of AAV particle serotypes. The Raman signals of AAV2 full samples and AAV8 full samples can be distinguished. Principal component analysis (PCA) was applied to the Raman spectra of AAV2 full samples and AAV8 full samples. Raman measurements of AAV2 full samples and AAV8 full samples were repeated 5 times in the same well. The scatter plot between PC-1 and PC-2 shows the separation between the spectra of AAV8 full (dark gray) and AAV2 full (light gray) samples. The first component shows an explained variance rate of 52%, and the second component shows an explained variance rate of 8%.

[0526] Fig.11 Differentiation of AAV particle serotypes - Loading. The first principal component (PC-1) can differentiate the Raman signals of the AAV2 full sample and the AAV8 full sample. The loading of PC-1 shows clear differences, such as in the marked bands.

[0527] Fig.12 Differentiation of AAV5, AAV8 and AAV9 full capsids by PCA by micro-Raman spectroscopy. For each serotype, spectra were acquired in two different wells with five replicates per well. The loading of PC1 and PC2 for differentiation of AAV5, AAV8 and AAV9 full capsids by micro-Raman spectroscopy is also shown.

[0528] Figures 13 to 15 Differentiation of AAV5( Fig.13 )、AAV8( Fig.14 )、AAV9( Fig.15 , outlier-removed dataset) and the corresponding loadings of PC1 and PC2 analyzed using micro-Raman spectroscopy.

[0529] Figures 16 to 18 PCA used to distinguish a gradual mixture of AAV empty particles from AAV full particles by micro-Raman spectroscopy analysis. Also shown are the loadings of PC1 and PC2 used to distinguish a gradual mixture of AAV empty particles from AAV full particles by micro-Raman spectroscopy analysis. Fig.16 (AAV9), Fig.17 (AAV5, full dataset), Fig.18 (AAV5, outlier-removed dataset).

[0530] Figures 19 to 22 Micro-Raman spectroscopy was used to distinguish full and empty AAV particles using four different concentrations of PCA.

[0531] Fig.19 : 2E+13vg / mL; Fig. 20 :1E+13vg / mL; Fig.21 : 5E+12vg / mL; Fig. 22 :2.5E+12vg / mL.

[0532] Examples

[0533] Materials and methods

[0534] All measurements were performed using an XPloRa Plus confocal Raman microscope (Horiba, Longjumeau, France). A commercially available SensoPlate (Greiner Bio-One GmbH, Kremsmuenster, Austria) was used as a sample holder. The AAV-containing sample was a commercial standard from Virovek (Hayward, USA) with a virus concentration of 2E+13 vg / mL.

[0535] Data analysis

[0536] To compare AAV2 full and AAV8 full, Raman spectra of samples of AAV2 full, AAV8 full and corresponding buffer without AAV particles were recorded five times. Thereafter, all spectra were scaled by a min-max 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 comparable. In the next step, the spectra were preprocessed as follows: 1.) elimination of wavenumbers outside the fingerprint region; 2.) application of a Savitzky-Golay filter; 3.) spectral normalization. In the last step, a principal component analysis (PCA) was performed with two components and the loading diagram was plotted.

[0537] For samples of AAV2 full and buffer and for samples of AAV2 full and AAV2 empty, the Raman spectrum of each sample was determined 50 times. For samples of AAV8 full and buffer only, the Raman spectrum of each sample was determined 5 times. The spectra were pre-processed as follows: 1.) elimination of wavenumbers outside the fingerprint region; 2.) application of Savitzky-Golay filter; 3.) normalization of the spectra. In the last step, principal component analysis (PCA) was performed with two components and the loadings were plotted. After the PCs, PCs such as PC3 and below (e.g. PC4, PC5...) do not contribute significantly to explaining the observed effects. The explained variance of the following PCs is always smaller than that of PC2.

[0538] Serotype differentiation

[0539] Different "full" AAV capsids of serotypes 5, 8 and 9 were measured by micro-Raman spectroscopy in two different wells, 5 times per well. For the measurements, the settings described below were used. One measurement of AAV5 was considered an outlier, probably due to an analytical artifact, and was excluded. The data were preprocessed as outlined below and analyzed by PCA.

[0540] Measurement settings: laser wavelength 532nm, acquisition: 2x 20s, ND 100, 250-3000cm^-1, x10 objective lens, SS-MWP (stainless steel microplate)

[0541] Preprocessing: Wave range: 400-1800cm^-1; SG-filter first-order derivative, first-order polynomial, 25pt, SNV transform

[0542] The results are Fig.12 Shown in.

[0543] Full / empty distinction

[0544] Full and empty AAV particles of serotypes 5, 8, and 9 were measured by micro-Raman spectroscopy. For the measurements, the same settings as for serotype determination were used (see below). One measurement of AAV9 full was considered an outlier and excluded. The data were preprocessed as outlined below and analyzed by PCA.

[0545] The results are Fig.13 (AAV5), 14 (AAV8), and 15 (AAV9, outlier-cleaned dataset).

[0546] Full and empty AAV particles of serotypes 5, 8, and 9 could be clearly distinguished in the combined dataset by PC1 (71%) and PC2 (16%).

[0547] Measurement settings: laser wavelength 532nm, acquisition: 2x 20s, ND 100, 250-3000cm^-1, x10 objective, SS-MWP

[0548] Preprocessing: Wave range: 400-1800cm^-1; SG-filter first-order derivative, first-order polynomial, 25pt, SNV transform

[0549] Identification of a mixture of full and empty capsids

[0550] To characterize the gradual increase of full AAV particles in a matrix of empty AAV particles, the respective standards were sonicated and mixed.

[0551] The measurement is performed as follows:

[0552] - 5 replicates of 30 μL of empty standard were measured in two wells each;

[0553] - Add 5 μL of full standard to two wells, resuspend and repeat the measurement for 5 replicates (5 μL / well in total);

[0554] -Add another 5 μL of full standard to two wells, resuspend and repeat the measurement for 5 replicates (total 10 μL / well);

[0555] -Add another 5 μL of full standard to two wells, resuspend and repeat the measurement for 5 replicates (total 15 μL / well);

[0556] - Perform 5 replicates of measuring 30 μL of full standard in each of two wells.

[0557] The settings described below were used. Data were preprocessed as described below and analyzed by PCA.

[0558] The results are Fig.16 (AAV9), 17 (AAV5), and 18 (AAV5, outlier-cleaned dataset).

[0559] Full (white circles) and empty (black circles) AAV particles can be clearly separated. By adding a total of 5 μL, 10 μL and 15 μL of full AAV standards, the gradual increase of AAV full to AAV empty standards is clearly observed through the transition of the respective clusters (replicates) in PC1 and PC2 (light gray, medium gray and dark gray circles). For the combined data set, the maximum variance is explained by PC1 (51% and 30%, respectively, with and without outliers) and PC2 (8% and 13%, respectively, with and without outliers).

[0560] For one replicate of AAV5 empty and 15 μL AAV5 full, and for one replicate of AAV5 and 5 μL AAV5 full, a clear shift to PC1 or PC2 was observed. These shifts were considered likely to originate from analytical artifacts. In order to analyze the PCA separation without considering these two outliers, a new PCA was generated. Thus, by adding the total 5 μL, 10 μL and 15 μL full AAV standards, the gradual increase of AAV5 full to AAV5 empty standards was even more clearly observed by shifting the clusters (replicates) in PC1 and PC2 (light gray, medium gray and dark gray circles). Again, the largest variance was explained by PC1 (22%) and PC2 (10%).

[0561] Measurement settings: laser wavelength 532nm, acquisition: 2x 20s, ND 100, 250-3000cm^-1, x10 objective, SS-MWP

[0562] Preprocessing: Wave range: 400-1800cm^-1; SG-filter first-order derivative, first-order polynomial, 27pt, SNV transformation:

[0563] Full / empty distinction at different AAV concentrations

[0564] To determine the detection limit for DNA payload status determination, AAV2 full and empty particles were measured at four different concentrations (2E+13, 1E+13, 5E+12, and 2.5E+12 vg / mL) in two different wells with five replicates per well. The settings outlined below were used. The data were preprocessed as outlined below and all concentrations were analyzed together by PCA. The largest variance was explained by PC1 (29%) and PC2 (13%).

[0565] Concentrations were also analyzed individually by PCA. Figures 19 to 22 Individual results for different concentrations are shown. Even at a concentration of 2.5E+12 vg / mL, the clustering of full and empty particles is distinct and identifiable.

[0566] Measurement settings: laser wavelength 532nm, acquisition: 2x 20s, ND 100, 250-3000cm^-1, 600gr / mm, x10 objective, SS-MWP

[0567] Preprocessing: Wave range: 400-1800cm^-1; SG-filter first-order derivative, first-order polynomial, 27pt, SNV transform.

Claims

1. A method for determining viral particles with encapsidated nucleic acids in an aqueous sample using Raman spectroscopy, the method comprising the following steps: (a) providing a sample and illuminating the sample with a light source; (b)(i) measuring the total intensity of Raman scattered light of the sample or of at least each of a first plurality of preselected wavenumbers and / or wavenumber ranges to obtain a first data set for the sample; (ii) performing principal component analysis on the first data set; as well as (c) determining said viral particles in said sample having encapsidated nucleic acid based on the output of the first set of mathematical data processing steps, Wherein the viral particles are in solution. 2 . The method according to claim 1 , wherein the viral particles having encapsidated nucleic acid are determined based on a first principal component.

3. The method according to any one of claims 1 to 2, wherein step (i) comprises (α) measuring the total intensity of the Raman scattered light of the sample, (β) eliminating wavenumbers outside the first plurality of preselected wavenumbers and / or wavenumber ranges, (γ) the first deviation of the function generating the data, and (δ) Normalize the spectrum, to obtain a first data set for the sample.

4. The method of claim 3, wherein generating the first deviation of the data function is by applying a Savitzky-Golay filter.

5. The method according to any one of claims 1 to 4, wherein the Raman scattered light is determined using a confocal Raman microscope or a micro-Raman spectroscopy device.

6. The method according to any one of claims 1 to 5, wherein the sample is a crude sample / without pre-treatment.

7. The method according to any one of claims 1 to 6, wherein the viral particle is an adeno-associated viral particle.

8. The method according to any one of claims 1 to 7, wherein the viral particle is an adeno-associated virus particle of serotype 2 or 8.

9. The method of any one of claims 1 to 8, wherein the viral particles are AAV particles of serotype 2 and the light source has a wavelength of about 532 nm or about 785 nm.

10. The method of any one of claims 1 to 8, wherein the viral particles are AAV particles 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 consists of 489 to 728 cm-1 or / and 1645 to 1680 cm-1.

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

13. The method of any one of claims 1 to 10, wherein the first plurality of preselected wavenumbers and / or wavenumber ranges consists 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, or / and 1645 to 1680 cm-1.

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

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

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