RU2771622C2 - Methods for aav detection - Google Patents

Abstract

FIELD: biotechnology; virology.

SUBSTANCE: methods for serotyping and/or determination of heterogeneity of a viral particle are described, using the determination of weight, in particular, using liquid chromatography/mass-spectrometry (LC/MS) or liquid chromatography/mass-spectrometry-mass-spectrometry (LC/MS/MS). Methods for the improvement of rAAV particle stability, improvement of rAAV particle assembly in a cell or improvement of rAAV particle transduction in a cell are also described, using an increase in a degree of acetylation and/or deamidation of capsid proteins. The group of inventions also relates to capsid AAV protein for the production of AAV particle and a recombinant AAV particle, a pharmaceutical composition, a kit and a product for the delivery of nucleic acid to a host cell.

EFFECT: group of inventions allows for improvement of methods for characterizing viral particles.

64 cl, 30 dwg, 11 tbl, 5 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority in US Provisional Application No. 62/375314, filed August 15, 2016, which is incorporated herein by reference in its entirety.

SEQUENCE LIST

[0002] The contents of the following presented ASCII text file are incorporated herein by reference in their entirety: Computer Readable Form (CRF) of the Sequence Listing (File Name: 159792014140SEQLIST.txt, Compiled Date: August 14, 2017, Size: 51 kB).

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

[0003] The present invention relates to methods for serotyping and/or determining the heterogeneity of a viral particle (e.g., an adeno-associated virus (AAV) particle) by mass determination, for example, by using liquid chromatography/mass spectrometry (LC/MS) or liquid chromatography /mass spectrometry-mass spectrometry (LC/MS/MS). In some aspects, the present invention relates to methods for improving the stability of AAV particles.

BACKGROUND OF THE INVENTION

[0004] A complete characterization of the viral capsid proteins of viral vectors (e.g., AAV-based vectors), including their sequence and post-translational modifications, is desirable in gene therapy research and development because viral capsid proteins (VPs) are essential for infectivity viruses.

[0005] Viral vector products, such as recombinant adeno-associated virus (rAAV) products, are typically identified by molecular means targeting the nucleic acid transgene. These methods may include polymerase chain reaction (PCR) techniques targeting transgene-specific sequences and restriction fragment length polymorphism (RFLP). As rAAV-based technology advances, many institutions are beginning to study the multiple AAV capsid serotypes encoding their therapeutic transgene in order to increase targeted tissue tropism.

[0006] Conventional molecular identification techniques identify products containing unique transgenes, but it is not possible to distinguish between those that have distinct AAV capsid serotypes. Currently, most tests for identifying AAV serotype are based on SDS-PAGE striation patterns, antibody-based ELISA, or Western blotting. However, striation patterns and antibodies are not specific enough to differentiate between different AAV serotypes. Gel-LC/MS/MS has been described as a method for identifying capsid serotype. However, this method involves several steps, including SDS-PAGE, gel digestion, and LC/MS/MS, and thus requires several days for analysis while providing limited sequence coverage. Methods for identifying vectors, such as rAAV-based vectors, are of interest in relation to gene therapy vectors (see, for example, US PG Publication No. US20110275529). Thus, it would be useful to improve methods for characterizing viral particles.

[0007] All literature references cited herein, including patent applications and publications, are incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE INVENTION

[0008] Using rAAV as an example, this document describes the use of LC/MS as an analytical tool to specifically identify different viral capsid serotypes (eg, rAAV capsid serotypes). As part of virus characterization, LC/MS can be used to complement molecular identification methods. This assay combination can satisfy regulatory requirements by delimiting both the features of the therapeutic transgene product and the features of the capsid serotype. This method can be used, for example, as a test for AAV serotype traits or to control for viral capsid protein heterogeneity in the development of recombinant AAV gene therapy. It can also be used to confirm VP sequences in capsid development studies. In addition, this technique can be used to study the effect of post-translational modifications, such as N-terminal acetylation of viral capsid proteins, on transfection efficiency and intracellular protein migration.

[0009] The methods described herein can also be used to design AAV particles for higher stability and/or increased transduction efficiency; for example, by altering the amino acid residue at position 2 of VP1 and/or VP3 of the AAV capsid such that the amino acid at position 2 is more acetylated than the wild-type AAV capsid. In some embodiments, the methods can be used to design AAV particles with reduced transduction efficiency; for example, by altering the amino acid residue at position 2 of VP1 and/or VP3 of the AAV capsid such that the amino acid at position 2 is more or less deacetylated compared to the wild-type AAV capsid.

[0010] In some aspects, the present invention provides a method for determining the serotype of a viral particle, comprising a) denaturing the viral particle, b) subjecting the denatured viral particle to liquid chromatography/mass spectrometry (LC/MS), and c) determining the masses of one or more capsid proteins viral particle; while a specific combination of masses of one or more capsid proteins characterizes the serotype of the virus. In some embodiments, the calculated masses of one or more capsid proteins are compared to theoretical masses of one or more capsid proteins of one or more virus serotypes.

[0011] In some aspects, the present invention provides a method for determining the heterogeneity of a viral particle, comprising a) denaturing the viral particle, b) subjecting the denatured viral particle to liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS), c) determining the masses of one or more capsid proteins of the viral particle and d) comparing the masses from step c) with the theoretical masses of one or more capsid proteins of the virus serotype; while the deviation of one or more masses of one or more capsid proteins characterizes the heterogeneity of the virus capsids. In some embodiments, heterogeneity includes one or more of mixed serotypes, variant capsids, capsid amino acid substitutions, truncated capsids, or modified capsids.

[0012] In some embodiments of the above aspects, liquid chromatography is reverse phase liquid chromatography, size exclusion chromatography, hydrophilic interaction liquid chromatography, or cation exchange chromatography. In some embodiments, the implementation of the viral particle contains a viral vector encoding a heterologous transgene.

[0013] In some aspects, the present invention provides a method for determining the serotype of a viral particle, comprising a) denaturing the viral particle, b) subjecting the denatured viral particle to reduction and/or alkylation, c) subjecting the denatured viral particle to cleavage to form fragments of one or more capsid proteins of the viral particle, d) subjecting the fragments of one or more capsid proteins to liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS), and e) determining the masses of the fragments of one or more capsid proteins of the viral particle; while a specific combination of masses of fragments of one or more capsid proteins characterizes the serotype of the virus. In some embodiments, the calculated masses of fragments of one or more capsid proteins are compared with the theoretical masses of fragments of one or more capsid proteins of one or more virus serotypes.

[0014] In some aspects, the present invention provides a method for determining the heterogeneity of the serotype of a viral particle, comprising a) denaturing the viral particle, b) subjecting the denatured viral particle to reduction and/or alkylation, c) subjecting the denatured viral particle to cleavage to form fragments of one or more capsid of viral particle proteins, d) subjecting fragments of one or more capsid proteins to liquid chromatography/mass spectrometry-mass spectrometry (LC/MS/MS), e) determining the masses of fragments of one or more viral particle capsid proteins, and f) comparing the masses from step e) with the theoretical masses of fragments of one or more capsid proteins of the virus serotype; while the deviation of one or more masses of one or more capsid proteins characterizes the heterogeneity of the virus capsid. In some embodiments, heterogeneity includes one or more of mixed serotypes, variant capsids, capsid amino acid substitutions, truncated capsids, or modified capsids. In some embodiments, the liquid chromatography is reverse phase liquid chromatography, size exclusion chromatography, hydrophilic interaction liquid chromatography, or cation exchange chromatography.

[0015] As shown herein, methods can be performed in the absence of a gel separation step (eg, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)).

[0016] In some embodiments of the above aspects and embodiments, the viral particle comprises a viral vector encoding a heterologous transgene. In some embodiments, the viral particle belongs to a family of viruses selected from the group consisting of Adenoviridae, Parvoviridae, Retroviridae, Baculoviridae, and Herpesviridae. In some embodiments, the viral particle belongs to a genus of viruses selected from the group consisting of Atadenovirus, Aviadenovirus, Ichtadenovirus, Mastadenovirus, Siadenovirus, Ambidensovirus, Brevidensovirus, Hepandensovirus, Iteradensovirus, Penstyldensovirus, Amdoparvovirus, Aveparvovirus, Bocaparvovirus, Copiparvovirus, Dependoparvovirus, Erythroparvovirus, Protoparvovirus .

[0017] In some aspects, the present invention provides a method for determining the serotype of an adeno-associated virus (AAV) particle, comprising a) denaturing the AAV particle, b) subjecting the denatured AAV particle to liquid chromatography/mass spectrometry (LC/MS), and c) determining VP1 masses , VP2 and VP3 AAV particles; while the specific combination of masses VP1, VP2 and VP3 characterizes the AAV serotype. In some embodiments, the calculated masses of VP1, VP2, and VP3 are compared to the theoretical masses of VP1, VP2, and VP3 of one or more of the AAV serotypes.

[0018] In some aspects, the present invention provides a method for determining the heterogeneity of an AAV particle, comprising a) denaturing the AAV particle, b) subjecting the denatured AAV particle to liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS), c) determining the masses VP1, VP2 and VP3 of the AAV particle and d) comparing the masses from step c) with the theoretical masses VP1, VP2 and VP3 of the AAV particle; while the deviation of one or more masses of VP1, VP2 and VP3 characterizes the heterogeneity of the AAV capsid. In some embodiments, heterogeneity includes one or more of mixed serotypes, variant capsids, capsid amino acid substitutions, truncated capsids, or modified capsids.

[0019] In some embodiments of the above aspects and embodiments, the AAV particle is denatured with acetic acid, guanidine hydrochloride, and/or an organic solvent. In some embodiments, the liquid chromatography is reverse phase liquid chromatography, size exclusion chromatography, hydrophilic interaction liquid chromatography, or cation exchange chromatography. In some embodiments, the liquid chromatography is reverse phase liquid chromatography. In some embodiments, reversed phase chromatography is C4 or C8 reversed phase chromatography. In some embodiments, chromatography uses mobile phase A containing formic acid in water. In some embodiments, mobile phase A contains approximately 0.1% formic acid. In some embodiments, the chromatography uses mobile phase B containing formic acid in acetonitrile. In some embodiments, mobile phase B contains approximately 0.1% formic acid. In some embodiments, the proportion of mobile phase B in the chromatography is increased over time. In some embodiments, the proportion of mobile phase B in the chromatography is increased in steps. In some embodiments, mobile phase B is increased from about 10% to about 20%, from about 20% to about 30%, and from about 30% to about 38%. In some embodiments, mobile phase B is increased from about 10% to about 20% over about 6 minutes, from about 20% to about 30% over about 10 minutes, and from about 30% to about 38% over about 40 minutes. In some embodiments, the liquid chromatography is ultra performance chromatography (UPLC).

[0020] In some embodiments of the above aspects and embodiments, mass spectrometry provides for a capillary voltage of approximately 3.5 kV. In some embodiments, the mass spectrometry involves a sampling cone voltage of approximately 45 V. In some embodiments, the mass spectrometry involves a calibration performed by an additional tool. In some embodiments, sodium iodide is used as a calibrant.

[0021] In some embodiments of the above aspects and embodiments, the N-terminus of VP1 and/or VP3 is acetylated. In some embodiments, the AAV particle is a recombinant AAV (rAAV) particle. In some embodiments, the AAV particle comprises an AAV1 capsid, AAV2 capsid, AAV3 capsid, AAV4 capsid, AAV5 capsid, AAV6 capsid, AAV7 capsid, AAV8 capsid, AAVrh8 capsid, AAV9 capsid, AAV10 capsid, AAVrh10 capsid, AAV11 capsid, AAV12 capsid, AAV LK03, capsid AAV2R471A, capsid AAV2/2-7m8, capsid AAV DJ, capsid AAV DJ8, capsid AAV2 N587A, capsid AAV2 E548A, capsid AAV2 N708A, capsid AAV V708K, capsid AAV goat, chimeric capsid AAV1/AAV2, capsid AAV large bovine or mouse AAV capsid rAAV2/HBoV1 (chimeric AAV/human boca virus 1). In some embodiments, the AAV capsid contains a tyrosine mutation or a mutation affecting heparin binding. In some embodiments, the masses of VP1, VP2, and VP3 are compared to the theoretical masses of one or more of AAV1 capsid, AAV2 capsid, AAV3 capsid, AAV4 capsid, AAV5 capsid, AAV6 capsid, AAV7 capsid, AAV8 capsid, AAVrh8 capsid, AAV9 capsid, AAV10 capsid , capsid AAVrh10, capsid AAV11, capsid AAV12, capsid AAV LK03, capsid AAV2R471A, capsid AAV2/2-7m8, capsid AAV DJ, capsid AAV DJ8, capsid AAV2 N587A, capsid AAV2 E548A, capsid AAV2 N708A, capsid AAV V708K, capsid goat, AAV1/AAV2 chimeric capsid, bovine AAV capsid, or rAAV2/HBoV1 mouse AAV capsid (human bocavirus chimeric AAV/virus 1), AAV2HBKO capsid, AAVPHP.B capsid, or AAVPHP.eB capsid.

[0022] In some embodiments of the above aspects and embodiments, the viral particle comprises ITR AAV1, ITR AAV2, ITR AAV3, ITR AAV4, ITR AAV5, ITR AAV6, ITR AAV7, ITR AAV8, ITR AAVrh8, ITR AAV9, ITR AAV10, ITR AAVrh10 , ITR AAV11 or ITR AAV12. In some embodiments, the AAV particle contains an AAV-based vector encoding a heterologous transgene.

[0023] In some aspects, the present invention provides a method for determining the serotype of an adeno-associated virus (AAV) particle, comprising a) denaturing the AAV particle, b) subjecting the denatured AAV particle to reduction and/or alkylation, c) subjecting the denatured AAV particle to cleavage to form VP1 fragments , VP2 and/or VP3 AAV particles, d) subjecting the VP1, VP2 and/or VP3 fragments to liquid chromatography/mass spectrometry-mass spectrometry (LC/MS/MS) and e) determining the masses of the VP1, VP2 and/or VP3 fragments AAV particles; wherein the specific combination of VP1, VP2 and/or VP3 fragment masses characterizes the AAV serotype. In some embodiments, the calculated masses of VP1, VP2, and VP3 fragments are compared to the theoretical masses of VP1, VP2, and/or VP3 fragments of one or more AAV serotypes.

[0024] In some aspects, the present invention provides a method for determining serotype heterogeneity of an AAV particle, comprising a) denaturing the AAV particle, b) subjecting the denatured AAV particle to reduction and/or alkylation, c) subjecting the denatured AAV particle to cleavage to form VP1, VP2 fragments, and /or VP3 of the AAV particle, d) subjecting the VP1, VP2 and/or VP3 fragments to liquid chromatography/mass spectrometry-mass spectrometry (LC/MS/MS), and e) determining the masses of the VP1, VP2 and VP3 fragments of the AAV particle; and f) comparing the masses from step e) with the theoretical masses of the AAV serotype VP1, VP2 and VP3 fragments; while the deviation of one or more masses of VP1, VP2 or VP3 characterizes the heterogeneity of the AAV capsid. In some embodiments, heterogeneity includes one or more of mixed serotypes, variant capsids, capsid amino acid substitutions, truncated capsids, or modified capsids. In some embodiments, recovery is accomplished by exposing the AAV particle to dithiothreitol, beta-mercaptoethanol, or tris(2-carboxyethyl)phosphine (TCEP). In some embodiments, alkylation is accomplished by exposing the AAV particle to iodoacetic acid, iodoacetamide, or 4-vinylpyridine. In some embodiments, the cleavage is enzymatic cleavage or chemical cleavage. In some embodiments, the enzymatic cleavage is an endopeptidase cleavage. In some embodiments, the enzymatic digestion is trypsin digestion, LysC digestion, Asp-N digestion, or Glu-C digestion. In some embodiments, the chemical cleavage is cyanogen bromide cleavage or acid cleavage. In some embodiments, the AAV particle is denatured with acetic acid, guanidine hydrochloride, and/or an organic solvent.

[0025] In some embodiments of the above aspects and embodiments, the liquid chromatography is reverse phase liquid chromatography, size exclusion chromatography, hydrophilic interaction liquid chromatography, or cation exchange chromatography. In some embodiments, the liquid chromatography is reverse phase liquid chromatography. In some embodiments, the reversed phase chromatography is C18 reversed phase chromatography. In some embodiments, chromatography uses mobile phase A containing formic acid in water. In some embodiments, mobile phase A contains approximately 0.1% formic acid. In some embodiments, the chromatography uses mobile phase B containing formic acid in acetonitrile. In some embodiments, mobile phase B contains approximately 0.1% formic acid. In some embodiments, the proportion of mobile phase B in the chromatography is increased over time. In some embodiments, mobile phase B is increased from about 2% to about 60%. In some embodiments, mobile phase B is increased from about 2% to about 60% over about 121 minutes. In some embodiments, the liquid chromatography is high performance liquid chromatography (HPLC).

[0026] In some embodiments of the above aspects and embodiments, mass spectrometry provides for a capillary voltage of approximately 3.5 kV. In some embodiments, the mass spectrometry involves a sampling cone voltage of approximately 45 V. In some embodiments, the mass spectrometry involves a calibration performed by an additional tool. In some embodiments, sodium iodide is used as a calibrant.

[0027] In some embodiments of the above aspects and embodiments, the N-terminus of VP1 and/or VP3 is acetylated. In some embodiments, the AAV particle is a recombinant AAV (rAAV) particle. In some embodiments, the AAV particle comprises an AAV1 capsid, AAV2 capsid, AAV3 capsid, AAV4 capsid, AAV5 capsid, AAV6 capsid, AAV7 capsid, AAV8 capsid, AAVrh8 capsid, AAV9 capsid, AAV10 capsid, AAVrh10 capsid, AAV11 capsid, AAV12 capsid, AAV LK03, capsid AAV2R471A, capsid AAV2/2-7m8, capsid AAV DJ, capsid AAV DJ8, capsid AAV2 N587A, capsid AAV2 E548A, capsid AAV2 N708A, capsid AAV V708K, capsid AAV goat, chimeric capsid AAV1/AAV2, capsid AAV large bovine or mouse AAV capsid rAAV2/HBoV1 (chimeric AAV/human boca virus 1). In some embodiments, the AAV capsid contains a tyrosine mutation or a mutation affecting heparin binding. In some embodiments, the masses of VP1, VP2, and VP3 are compared to the theoretical masses of one or more of AAV1 capsid, AAV2 capsid, AAV3 capsid, AAV4 capsid, AAV5 capsid, AAV6 capsid, AAV7 capsid, AAV8 capsid, AAVrh8 capsid, AAV9 capsid, AAV10 capsid , capsid AAVrh10, capsid AAV11, capsid AAV12, capsid AAV LK03, capsid AAV2R471A, capsid AAV2/2-7m8, capsid AAV DJ, capsid AAV DJ8, capsid AAV2 N587A, capsid AAV2 E548A, capsid AAV2 N708A, capsid AAV V708K, capsid goat, AAV1/AAV2 chimeric capsid, bovine AAV capsid, or rAAV2/HBoV1 mouse AAV capsid (human bocavirus chimeric AAV/virus 1).

[0028] In some embodiments of the above aspects and embodiments, the viral particle comprises ITR AAV1, ITR AAV2, ITR AAV3, ITR AAV4, ITR AAV5, ITR AAV6, ITR AAV7, ITR AAV8, ITR AAVrh8, ITR AAV9, ITR AAV10, ITR AAVrh10 , ITR AAV11 or ITR AAV12. In some embodiments, the AAV particle contains an AAV-based vector encoding a heterologous transgene.

[0029] In some embodiments, the present invention provides a recombinant AAV (rAAV) particle comprising an amino acid substitution at amino acid residue 2 of VP1 and/or VP3; wherein the amino acid substitution at amino acid residue 2 of VP1 and/or VP3 provides a change in N-terminal acetylation compared to N-terminal acetylation at amino acid residue 2 of VP1 and/or VP3 of the original AAV particle. In some embodiments, the substitution results in a higher frequency of N-terminal acetylation or a lower frequency of N-terminal acetylation. In some embodiments, the rAAV particle contains an amino acid substitution at amino acid residue 2 of VP1; wherein the amino acid substitution at amino acid residue 2 of VP1 provides a change in N-terminal acetylation compared to N-terminal acetylation at amino acid residue 2 of VP1 of the original AAV particle. In some embodiments, the rAAV particle contains an amino acid substitution at amino acid residue 2 of VP3; wherein the amino acid substitution at amino acid residue 2 of VP3 provides a change in N-terminal acetylation compared to N-terminal acetylation at amino acid residue 2 of VP3 of the original AAV particle. In some embodiments, amino acid residue 2 is replaced with Cys, Ser, Thr, Val, Gly, Asn, Asp, Glu, Ile, Leu, Phe, Gln, Lys, Met, Pro, or Tyr. In some embodiments, amino acid residue 2 is replaced by Ser, Asp, or Glu.

[0030] In some embodiments of the above aspects and embodiments, the AAV particle comprises a capsid of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV LK03, AAV2R471A , AAV2/2-7m8, AAV DJ, AAV DJ8, serotype capsid AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV V708K, AAV goat, chimeric AAV1/AAV2, AAV bovine, AAV mouse, rAAV2/HBoV1, AAV2HBKO, AAVPHP .B or AAVPHP.eB. In some embodiments, the AAV capsid further comprises a tyrosine mutation or a mutation affecting heparin binding. In some embodiments, the rAAV particle contains an rAAV vector. In some embodiments, the rAAV-based vector contains one or more AAV ITRs. In some embodiments, the rAAV-based vector comprises ITR AAV1, ITR AAV2, ITR AAV3, ITR AAV4, ITR AAV5, ITR AAV6, ITR AAV7, ITR AAV8, ITR AAVrh8, ITR AAV9, ITR AAV10, ITR AAVrh10, ITR AAV11, or ITR AAV12 . In some embodiments, the AAV capsid and AAV ITR are derived from the same serotype. In some embodiments, the AAV capsid and AAV ITR are from different serotypes. In some embodiments, the AAV particle contains an AAV-based vector encoding a heterologous transgene flanked by one or more AAV ITRs.

[0031] In some embodiments of the above aspects and embodiments, the rAAV-based vector is a self-complementary vector. In some embodiments, the rAAV-based vector comprises a first nucleic acid sequence encoding a transgene and a second nucleic acid sequence encoding a sequence complementary to the transgene, wherein the first nucleic acid sequence can form intrastrand base pairs with the second nucleic acid sequence for most or all of its length. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are joined by a mutant AAV ITR, wherein the mutant AAV ITR is characterized by a D-region deletion and is characterized by a terminal resolution sequence mutation.

[0032] In some embodiments of the above aspects and embodiments, an rAAV particle is obtained by transfecting a host cell with a nucleic acid encoding a rAAV vector and a nucleic acid encoding the rep and cap AAV functional elements, and obtaining a nucleic acid encoding the functional elements assistant AAV. In some embodiments, AAV helper functional elements are obtained by transfecting a host cell with a nucleic acid encoding AAV helper functional elements. In some embodiments, AAV helper functionality is obtained by infecting a host cell with an AAV helper virus that provides AAV helper functionality. In some embodiments, the AAV helper virus is an adenovirus, herpes simplex virus, or baculovirus. In some embodiments, an rAAV particle is generated by an AAV producer cell containing a nucleic acid encoding an rAAV vector and a nucleic acid encoding AAV rep and cap functional elements and producing a nucleic acid encoding AAV helper functional elements. In some embodiments, the AAV producer cell contains a nucleic acid encoding the functional elements of an AAV helper. In some embodiments, the AAV helper functional elements are obtained by infecting AAV producing cells with an AAV helper virus that provides the AAV helper functional elements. In some embodiments, the AAV helper virus is an adenovirus, herpes simplex virus, or baculovirus. In some embodiments, the AAV cap functional elements provide an amino acid substitution at amino acid residue 2 of VP1 and/or VP3; wherein the amino acid substitution at amino acid residue 2 of VP1 and/or VP3 provides a change in N-terminal acetylation compared to N-terminal acetylation at amino acid residue 2 of VP1 and/or VP3 of the original AAV particle.

[0033] In some aspects, the present invention provides a pharmaceutical composition containing the particle rAAV described in this document. In some aspects, the present invention provides a kit containing the particle rAAV or pharmaceutical composition described in this document. In some aspects, the present invention provides an article of manufacture containing the rAAV particle or pharmaceutical composition described herein.

[0034] In some aspects, the present invention provides an AAV capsid protein comprising an amino acid substitution at amino acid residue 2 of the parent AAV capsid protein; wherein the amino acid substitution at amino acid residue 2 provides a change in N-terminal acetylation compared to N-terminal acetylation at amino acid residue 2 of the parent AAV capsid protein. In some embodiments, the substitution results in a higher frequency of N-terminal acetylation or a lower frequency of N-terminal acetylation. In some embodiments, the AAV capsid protein is VP1 or VP3. In some embodiments, amino acid residue 2 of the AAV capsid protein is replaced by Cys, Ser, Thr, Val, Gly, Asn, Asp, Glu, Ile, Leu, Phe, Gln, Lys, Met, Pro, or Tyr. In some embodiments, amino acid residue 2 of the AAV capsid protein is replaced by Ser, Asp, or Glu. In some embodiments, the amino acid substitution results in less deamidation of the AAV capsid.

[0035] In some embodiments of the above aspects and embodiments, the AAV particle comprises a capsid of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV LK03, AAV2R471A , AAV2/2-7m8, AAV DJ, AAV DJ8, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV V708K, Goat AAV, Chimeric AAV1/AAV2, Bovine AAV, Mouse AAV, rAAV2/HBoV1, AAV2HBKO serotype capsid, AAVPHP .B or AAVPHP.eB. In some embodiments, the AAV capsid further comprises a tyrosine mutation or a mutation affecting heparin binding.

[0036] In some aspects, the present invention provides a method for improving the stability of an rAAV particle, comprising replacing amino acid residue 2 of VP1 and/or VP3 of the original VP1 and/or VP3; while replacing amino acid residue 2 provides a change in the N-terminal acetylation of VP1 and/or VP3 compared to amino acid residue 2 of the original VP1 and/or VP3. In some aspects, the present invention provides a method for improving the assembly of rAAV particles in a cell, comprising replacing amino acid residue 2 of VP1 and/or VP3 of the original VP1 and/or VP3; while replacing the amino acid at position 2 provides a change in the N-terminal acetylation of VP1 and/or VP3 compared to amino acid residue 2 of the original VP1 and/or VP3. In some aspects, the present invention provides a method for improving the transduction of rAAV particles in a cell, comprising replacing amino acid residue 2 of VP1 and/or VP3 of the original VP1 and/or VP3; while replacing amino acid residue 2 provides a change in the N-terminal acetylation of VP1 and/or VP3 compared to amino acid residue 2 of the original VP1 and/or VP3. In some embodiments, the amino acid substitution results in a higher frequency of N-terminal acetylation or a lower frequency of N-terminal acetylation. In some embodiments, the amino acid substitution is at amino acid residue 2 of VP1. In some embodiments, the amino acid substitution is at amino acid residue 2 of VP3. In some embodiments, amino acid residue 2 is replaced with Cys, Ser, Thr, Val, Gly, Asn, Asp, Glu, Ile, Leu, Phe, Gln, Lys, Met, Pro, or Tyr. In some embodiments, amino acid residue 2 is replaced by Ser, Asp, or Glu. In some aspects, the present invention provides a method for reducing transduction of rAAV particles in a cell, comprising replacing amino acid residue 2 of VP1 and/or VP3; wherein the changed amino acid at position 2 provides for a change in the N-terminal acetylation of VP1 and/or VP3 compared to amino acid residue 2 of the original VP1 and/or VP3.

[0037] In some embodiments of the above aspects and embodiments, the AAV particle comprises a capsid of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV LK03, AAV2R471A , AAV2/2-7m8, AAV DJ, AAV DJ8, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV V708K, Goat AAV, Chimeric AAV1/AAV2, Bovine AAV, Mouse AAV, rAAV2/HBoV1, AAV2HBKO serotype capsid, AAVPHP .B or AAVPHP.eB. In some embodiments, the AAV capsid further comprises a tyrosine mutation or a mutation affecting heparin binding. In some embodiments, the rAAV particle contains an rAAV vector. In some embodiments, the rAAV-based vector contains one or more AAV ITRs. In some embodiments, the rAAV-based vector comprises ITR AAV1, ITR AAV2, ITR AAV3, ITR AAV4, ITR AAV5, ITR AAV6, ITR AAV7, ITR AAV8, ITR AAVrh8, ITR AAV9, ITR AAV10, ITR AAVrh10, ITR AAV11, or ITR AAV12 . In some embodiments, the AAV capsid and AAV ITR are derived from the same serotype. In some embodiments, the AAV capsid and AAV ITR are from different serotypes. In some embodiments, the AAV particle contains an AAV-based vector encoding a heterologous transgene flanked by one or more AAV ITRs.

[0038] In some embodiments of the above aspects and embodiments, the rAAV-based vector is a self-complementary vector. In some embodiments, the rAAV-based vector comprises a first nucleic acid sequence encoding a transgene and a second nucleic acid sequence encoding a sequence complementary to the transgene, wherein the first nucleic acid sequence can form intrastrand base pairs with the second nucleic acid sequence for most or all of its length. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are joined by a mutant AAV ITR, wherein the mutant AAV ITR is characterized by a D-region deletion and is characterized by a terminal resolution sequence mutation.

[0039] In some embodiments of the above aspects and embodiments, the rAAV particle is obtained by transfecting a host cell with a nucleic acid encoding a vector based on rAAV and a nucleic acid encoding the rep and cap functional elements of AAV, and obtaining a nucleic acid encoding functional elements assistant AAV. In some embodiments, AAV helper functional elements are obtained by transfecting a host cell with a nucleic acid encoding AAV helper functional elements. In some embodiments, AAV helper functionality is obtained by infecting a host cell with an AAV helper virus that provides AAV helper functionality. In some embodiments, the AAV helper virus is an adenovirus, herpes simplex virus, or baculovirus. In some embodiments, an rAAV particle is generated by an AAV producer cell containing a nucleic acid encoding an rAAV vector and a nucleic acid encoding AAV rep and cap functional elements and producing a nucleic acid encoding AAV helper functional elements. In some embodiments, the AAV producer cell contains a nucleic acid encoding the functional elements of an AAV helper. In some embodiments, the AAV helper functional elements are obtained by infecting AAV producing cells with an AAV helper virus that provides the AAV helper functional elements. In some embodiments, the AAV helper virus is an adenovirus, herpes simplex virus, or baculovirus. In some embodiments, the AAV cap functional elements provide an amino acid substitution at amino acid residue 2 of VP1 and/or VP3; wherein the amino acid substitution at amino acid residue 2 of VP1 and/or VP3 provides a change in N-terminal acetylation compared to N-terminal acetylation at amino acid residue 2 of VP1 and/or VP3 of the original AAV particle.

[0040] In some aspects, the present invention provides a recombinant AAV (rAAV) particle containing one or more amino acid substitutions for the amino acid residue A35, N57, G58, N382, G383, N511, G512, N715, or G716 VP1 or VP3 of the original particle, where Residue numbering is based on VP1 AAV2; wherein one or more amino acid substitutions provide a change in deamidation compared to deamidation of the VP1 and/or VP3 particles of the original AAV. In some embodiments, one or more amino acid substitutions are at amino acid residue A35, N57 VP1, G58 VP1, N382 VP3, G383 VP3, N511 VP3, G512 VP3, N715 VP3, or G716 VP3 and provide a change in deamidation from that of VP1 and/or VP3 particles of the original AAV. In some embodiments, one or more amino acid substitutions are an Asp substitution at N57 VP1, N382 VP3, N511 VP3, or N715 VP3 and result in a higher rate of deamidation compared to deamidation of VP1 and/or VP3 of the parent AAV particle. In some embodiments, one or more amino acid substitutions is a substitution in N57K or N57Q and results in a lower frequency of deamidation compared to deamidation of the VP1 and/or VP3 particles of the original AAV. In some embodiments, the one or more amino acid substitutions are an Asp substitution at A35 of VP1 and result in a higher rate of deamidation compared to that of the VP1 deamidation of the parent AAV particle. In some embodiments, one or more amino acid substitutions are in G58 VP1, G383 VP3, G512 VP3, or G716 VP3 and result in a lower frequency of deamidation compared to deamidation of the VP1 and/or VP3 of the parent AAV particle. In some embodiments, G58 VP1 is replaced by Asp. In some embodiments, the rAAV particle is an AAV1 particle or an AAV2 particle.

[0041] In some aspects, the present invention provides pharmaceutical compositions that contain AAV particles containing one or more amino acid substitutions for the amino acid residue A35, N57, G58, N382, G383, N511, G512, N715, or G716 VP1 or VP3, while Residue numbering is based on VP1 AAV2; where the amino acid change provides a change in deamidation compared to the deamidation of VP1 and/or VP3 particles of the original AAV. In some aspects, the present invention provides kits containing AAV particles or compositions containing AAV particles, wherein the AAV particles contain one or more amino acid substitutions for the amino acid residue A35, N57, G58, N382, G383, N511, G512, N715, or G716 VP1 or VP3, with residue numbering based on VP1 AAV2; where the amino acid change provides a change in deamidation compared to the deamidation of VP1 and/or VP3 particles of the original AAV. In some aspects, the present invention provides articles containing AAV particles or compositions containing AAV particles, wherein the AAV particles contain one or more amino acid substitutions for the amino acid residue A35, N57, G58, N382, G383, N511, G512, N715, or G716 VP1 or VP3, with residue numbering based on VP1 AAV2; where the amino acid change provides a change in deamidation compared to the deamidation of VP1 and/or VP3 particles of the original AAV. In some aspects, the present invention provides an AAV capsid protein comprising an amino acid substitution of the parent AAV capsid protein; where the amino acid change provides a change in capsid deamidation compared to the capsid protein of the original AAV.

[0042] In some aspects, the present invention provides a method for improving the stability of an rAAV particle, comprising replacing one or more amino acid residues, wherein one or more amino acid residues are residue A35, N57, G58, N382, G383, N511, G512, N715, or G716, with residue numbering based on VP1 AAV2; where the amino acid change provides a change in deamidation compared to the deamidation of VP1 and/or VP3 particles of the original AAV. In some aspects, the present invention provides a method for improving the assembly of rAAV particles in a cell, comprising replacing one or more amino acid residues, wherein one or more amino acid residues is an A35, N57, G58, N382, G383, N511, G512, N715 residue, or G716, with residue numbering based on VP1 AAV2; where the amino acid change provides a change in deamidation compared to the deamidation of VP1 and/or VP3 particles of the original AAV. In some aspects, the present invention provides a method for improving the transduction of rAAV particles in a cell, comprising replacing one or more amino acid residues, wherein one or more amino acid residues is residue A35, N57, G58, N382, G383, N511, G512, N715, or G716, with residue numbering based on VP1 AAV2; where the amino acid change provides a change in deamidation compared to the deamidation of VP1 and/or VP3 particles of the original AAV. In some embodiments, one or more amino acid substitutions are in A35 VP1, N57 VP1, G58 VP1, N382 VP3, G383 VP3, N511 VP3, G512 VP3, N715 VP3, or G716 VP3; where the replacement of the amino acid residue provides a change in deamidation compared to the deamidation of VP1 and/or VP3 particles of the original AAV. In some embodiments, the original Ala residue at position 35 of VP1 is replaced by Asn. In some embodiments, the original Gly residue at position 58 of VP1 is replaced by Asp. In some embodiments, the rAAV particle is an AAV1 particle or an AAV2 particle.

[0043] In some embodiments, the present invention provides a method for improving the stability, assembly, and/or transduction efficiency of an rAAV particle, comprising replacing one or more amino acid residues, wherein one or more amino acid residues are A35, N57, G58, N382, G383 , N511, G512, N715, or G716, with residue numbering based on VP1 AAV2; where the amino acid replacement provides a change in deamidation compared to the deamidation of VP1 and/or VP3 of the original AAV particle described above, while the AAV particle contains the capsid of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV LK03, AAV2R471A, AAV2/2-7m8, AAV DJ, AAV DJ8, AAV2 N587A capsid, AAV2 E548A, AAV2 N708A, AAV V708K, AAV goat, chimeric AAV1/AAV2, AAV bovine cattle, mouse AAV, or rAAV2/HBoV1. In some embodiments, the AAV capsid further comprises a tyrosine mutation or a mutation affecting heparin binding. In some embodiments, the rAAV particle contains an rAAV vector. In some embodiments, the rAAV-based vector contains one or more AAV ITRs. In some embodiments, the rAAV-based vector comprises ITR AAV1, ITR AAV2, ITR AAV3, ITR AAV4, ITR AAV5, ITR AAV6, ITR AAV7, ITR AAV8, ITR AAVrh8, ITR AAV9, ITR AAV10, ITR AAVrh10, ITR AAV11, or ITR AAV12 . In some embodiments, the AAV capsid and AAV ITR are derived from the same serotype. In some embodiments, the AAV capsid and AAV ITR are from different serotypes. In some embodiments, the AAV particle contains an AAV-based vector encoding a heterologous transgene flanked by one or more AAV ITRs.

[0044] In some embodiments of the above aspects and embodiments, the rAAV-based vector is a self-complementary vector. In some embodiments, the rAAV-based vector comprises a first nucleic acid sequence encoding a transgene and a second nucleic acid sequence encoding a sequence complementary to the transgene, wherein the first nucleic acid sequence can form intrastrand base pairs with the second nucleic acid sequence for most or all of its length. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are joined by a mutant AAV ITR, wherein the mutant AAV ITR is characterized by a D-region deletion and is characterized by a terminal resolution sequence mutation.

[0045] In some embodiments of the above aspects and embodiments, an rAAV particle is obtained by transfecting a host cell with a nucleic acid encoding a vector based on rAAV and a nucleic acid encoding the rep and cap functional elements of AAV, and obtaining a nucleic acid encoding functional elements assistant AAV. In some embodiments, AAV helper functional elements are obtained by transfecting a host cell with a nucleic acid encoding AAV helper functional elements. In some embodiments, AAV helper functional elements are obtained by infecting a host cell with an AAV helper virus that provides AAV helper functional elements. In some embodiments, the AAV helper virus is an adenovirus, herpes simplex virus, or baculovirus. In some embodiments, an rAAV particle is generated by an AAV producer cell containing a nucleic acid encoding an rAAV vector and a nucleic acid encoding AAV rep and cap functional elements and producing a nucleic acid encoding AAV helper functional elements. In some embodiments, the AAV producer cell contains a nucleic acid encoding functional elements of an AAV helper. In some embodiments, the AAV helper functional elements are obtained by infecting AAV producing cells with an AAV helper virus that provides the AAV helper functional elements. In some embodiments, the AAV helper virus is an adenovirus, herpes simplex virus, or baculovirus. In some embodiments, the AAV cap functional elements provide an amino acid substitution for VP1 and/or VP3, wherein the amino acid substitution provides for modulation of capsid deamidation compared to the parent AAV particle.

BRIEF DESCRIPTION OF GRAPHICS

[0046] FIG. 1A-D are total ion chromatograms of AAV2 VP obtained by LC/MS. Fig. 1A: 10 cm BEH C4 column with 1.7%/min gradient; fig. 1B: 10 cm BEH C4 column with 0.5%/min gradient; fig. 1C: 15 cm BEH C4 column with 0.5%/min gradient; fig. 1D: 15 cm BEH C8 column with 0.5%/min gradient.

[0047] FIG. 2A and B show the mass spectra after deconvolution based on peak 1 of FIG. 1D (FIG. 2A) and peak 2 of FIG. 1D (FIG. 2B).

[0048] FIG. 3 shows sequence coverage of AAV2 VP1 (SEQ ID NO:3): green for tryptic peptides, blue for Lys-C peptides, pink for Asp-N peptides.

[0049] FIG. 4A-4C are MS/MS spectra of N-terminal AAV2 VP peptides. Fig. 4A: N-terminal tryptic peptide VP1 A(Ac)ADGYLPDWLEDTLSEGIR (SEQ ID NO: 4), FIG. 4B: N-terminal Asp-N-peptide VP2 APGKKRPVEHSPVEP (SEQ ID NO: 15). Fig. 4C: Derived from N-terminal Asp-N-peptide VP-3 A(Ac)TGSGAPM (SEQ ID NO: 5).

[0050] FIG. 5 shows the sequence alignment of 13 AAV serotypes in black letters on a white background - dissimilar; in blue letters on a blue background - conservative; in black letters on a green background - a block of similar ones; in red letters on a yellow background - identical; in green letters on a white background - similar to a low degree. AAVRh10 (SEQ ID NO: 17); AAV10 (SEQ ID NO: 18); AAV8 (SEQ ID NO: 19); AAV7 (SEQ ID NO: 20); AAV1 (SEQ ID NO: 21); AAV6 (SEQ ID NO: 22); AAV2 (SEQ ID NO: 23); AAV3 (SEQ ID NO: 24); AAV11 (SEQ ID NO: 25); AAV12 (SEQ ID NO: 26); AAV4 (SEQ ID NO: 27); AAV5 (SEQ ID NO: 28); AAV9 (SEQ ID NO: 29); consensus (SEQ ID NO: 30).

[0051] FIG. 6A and 6B show the results of LC/MS/MS analysis comparing the percentage deamidation of AAV1 and AAV2 particles obtained using the TTx and PCL methods. T9 YLGPF NG LDK peptide (SEQ ID NO: 9) was used to control the potential N57 deamidation site in both AAV1 and AAV2.

[0052] In FIG. 7A and 7B show the results of LC/MS/MS analysis comparing the percentage deamidation of AAV1 and AAV2 particles obtained using the TTx and PCL methods. Peptides T49 YNL NG R (SEQ ID NO: 11) and YHL NG R (SEQ ID NO: 12) were used to control the potential N511 deamidation site in AAV1 and AAV2, respectively.

[0053] FIG. 8A and 8B show the results of LC/MS/MS analysis comparing the percentage deamidation of AAV1 and AAV2 particles obtained using the TTx and PCL methods. Peptides T67 SANVDFTVDN NG LYTEPR (SEQ ID NO: 13) and SVNVDFTVDT NG VYSEPR (SEQ ID NO: 14) were used to control the potential N715 deamidation site in AAV1 and AAV2, respectively.

[0054] FIG. 9 shows the results of the SYPRO protein gel analysis for the production and VP1:VP2:VP3 ratio of deacetylated AAV5 mutant variants.

[0055] FIG. 10 shows an in vitro transduction assay to investigate the transduction efficiency of deacetylated AAV5 variants.

[0056] FIG. 11 shows the cell entry efficiency of these deacetylated AAV5 variants or the original unmodified AAV5 as measured by vector genome copy number/µg protein. Three cell lines were used: 293, HeLa and HuH7.

[0057] FIG. 12 shows eGFP expression (measured by ELISA) in cells transduced with the indicated deacetylated AAV5 variants compared to transduction of the original unmodified AAV5. Three cell lines were used: 293, HeLa and HuH7.

[0058] FIG. 13 shows a sequence alignment of 13 AAV serotypes, highlighting the conserved N57G58 deamidation site and the A35 residue in AAV2. AAVRh10 (SEQ ID NO: 31); AAV10 (SEQ ID NO: 31); AAV8 (SEQ ID NO: 32); AAV7 (SEQ ID NO: 33); AAV1 (SEQ ID NO: 31); AAV6 (SEQ ID NO: 31); AAV2 (SEQ ID NO: 34); AAV3 (SEQ ID NO: 35); AAV11 (SEQ ID NO: 31); AAV12 (SEQ ID NO: 36); AAV4 (SEQ ID NO: 37); AAV5 (SEQ ID NO: 38); AAV9 (SEQ ID NO: 39); consensus (SEQ ID NO: 40).

[0059] FIG. 14 shows the VP1, VP2 and VP3 capsid proteins from AAV1 or AAV2 particles obtained by the PCL or TTx method in protein gel. *truncated VP1 protein (tVP1) isolated.

[0060] FIG. 15 shows the results of LC/MS analysis of deamidation of these AAV2 mutants compared to control AAV2 capsids.

[0061] In FIG. 16 shows the results of SYPRO protein gel analysis for the production and ratio of VP1:VP2:VP3 deamidation mutant AAV2 variants.

[0062] In FIG. 17 shows an in vitro transduction assay to investigate the transduction efficiency of deamidated AAV2 variants.

[0063] FIG. 18 shows the cell entry efficiency of the indicated deamidated AAV2 variants or the original unmodified AAV2 as measured by vector genome copy number/µg protein. Three cell lines were used: 293, HeLa and HuH7.

[0064] FIG. 19 shows eGFP expression (as measured by ELISA) in cells transduced with the indicated deamidated AAV2 variants compared to transduction of the original unmodified AAV2. Three cell lines were used: 293, HeLa and HuH7.

DETAILED DESCRIPTION

[0065] In some aspects, the present invention provides a method for determining the serotype of adeno-associated virus (AAV) particle(s), comprising a) denaturing the AAV particle, b) subjecting the denatured AAV particle to liquid chromatography/mass spectrometry (LC/MS), and c) determination of the masses VP1, VP2 and VP3 of the AAV particle; while the specific combination of masses VP1, VP2 and VP3 characterizes the AAV serotype.

[0066] In other aspects, the present invention provides a method for determining the heterogeneity of an AAV particle, comprising a) denaturing the AAV particle, b) subjecting the denatured AAV particle to liquid chromatography/mass spectrometry (LC/MS), and c) determining the masses of VP1, VP2 and VP3 of the AAV particle and comparing the masses from step c) with the theoretical masses of VP1, VP2 and VP3 of the AAV particle; while the deviation of one or more masses of VP1, VP2 or VP3 characterizes the heterogeneity of the AAV capsid.

[0067] In other aspects, the present invention provides a method for determining the serotype of an adeno-associated virus (AAV) particle, comprising a) denaturing the AAV particle, b) subjecting the denatured AAV particle to reduction and/or alkylation, c) subjecting the denatured AAV particle to cleavage to form VP1 fragments , VP2 and/or VP3 AAV particles, d) subjecting the VP1, VP2 and/or VP3 fragments to liquid chromatography/mass spectrometry-mass spectrometry (LC/MS/MS) and e) determining the masses of the VP1, VP2 and/or VP3 fragments AAV particles; wherein the specific combination of VP1, VP2 and/or VP3 fragment masses characterizes the AAV serotype.

[0068] In other aspects, the present invention provides a method for determining serotype heterogeneity of an AAV particle, comprising a) denaturing the AAV particle, b) subjecting the denatured AAV particle to reduction and/or alkylation, c) subjecting the denatured AAV particle to cleavage to form VP1, VP2 fragments, and /or VP3 of the AAV particle, d) subjecting the VP1, VP2 and/or VP3 fragments to liquid chromatography/mass spectrometry-mass spectrometry (LC/MS/MS), e) determining the masses of the VP1, VP2 and VP3 fragments of the AAV particle and f) comparison of the masses from step e) with the theoretical masses of the VP1, VP2 and VP3 fragments of the AAV serotype; while the deviation of one or more masses of VP1, VP2 or VP3 characterizes the heterogeneity of the AAV capsid.

[0069] In some aspects, the present invention provides a recombinant AAV (rAAV) particle containing an amino acid substitution at amino acid residue 2 of VP1 and/or VP3; wherein the amino acid substitution at amino acid residue 2 of VP1 and/or VP3 provides a change in N-terminal acetylation compared to N-terminal acetylation at amino acid residue 2 of VP1 and/or VP3 of the original AAV particle.

[0070] In some aspects, the present invention provides a method for improving the assembly of rAAV particles in a cell, comprising replacing amino acid residue 2 of VP1 and/or VP3; wherein the replaced amino acid at position 2 is N-acetylated at a higher frequency than amino acid residue 2 of the original VP1 and/or VP3. In some aspects, the present invention provides a method for improving the transduction of rAAV particles in a cell, comprising replacing amino acid residue 2 of VP1 and/or VP3; wherein the replaced amino acid at position 2 is N-acetylated at a higher frequency than amino acid residue 2 of the original VP1 and/or VP3.

I. General methods

[0071] The techniques and procedures described or referred to herein are generally widely accepted and often used by those skilled in the art using conventional methodology, such as the widely used techniques described in Molecular Cloning: A Laboratory Manual (Sambrook et al., 4th ^ed ., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2012); Current Protocols in Molecular Biology (FM Ausubel, et al. eds., 2003); Methods in Enzymology series (Academic Press, Inc.); PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor eds., 1995); Antibodies, A Laboratory Manual (Harlow and Lane, eds., 1988); Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications (RI ^Freshney , 6th ed., J. Wiley and Sons, 2010); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JE Cellis, ed., Academic Press, 1998); Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, Plenum Press, 1998); Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JB Griffiths, and DG Newell, eds., J. Wiley and Sons, 1993-8); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds., 1996); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (JE Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Ausubel et al., eds., J. Wiley and Sons, 2002); Immunobiology (CA Janeway et al., 2004); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995) and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., JB Lippincott Company, 2011).

II. Definitions

[0072] The term "vector" as used herein refers to a recombinant plasmid or virus that contains a nucleic acid to be delivered to a host cell either in vitro or in vivo.

[0073] The terms "polynucleotide" or "nucleic acid" as used herein refer to the polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the term includes, without limitation, single-, double- or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer containing purine and pyrimidine bases or other natural, chemically or biochemically modified, other than natural, or derivatized nucleotide bases. The backbone of a nucleic acid may contain sugars and phosphate groups (which can typically be found in RNA or DNA) or modified or substituted sugar or phosphate groups. Alternatively, the nucleic acid backbone may comprise a polymer of synthetic subunits such as phosphoramidates and thus may be an oligodeoxynucleoside phosphoramidate (P-NH ₂ ) or a mixed phosphoramidate-phosphodiester oligomer. In addition, a double-stranded nucleic acid can be obtained from a single-stranded polynucleotide chemical synthesis either by synthesizing the complementary strand and hybridizing the strands under appropriate conditions, or by synthesizing the complementary strand de novo using a DNA polymerase with an appropriate primer.

[0074] The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length. Such polymers of amino acid residues may contain natural or non-natural amino acid residues, and include, without limitation, peptides, oligopeptides, dimers, trimers and multimers of amino acid residues. This definition covers both full-length proteins and their fragments. The terms also include post-translational modifications of a polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. In addition, for the purposes of the present invention, the expression "polypeptide" refers to a protein whose native sequence contains modifications such as deletions, additions and substitutions (usually conservative in nature), provided that the protein retains the desired activity. These modifications may be intentional, such as by site-directed mutagenesis, or may be accidental, such as due to mutations in hosts that produce proteins, or errors due to PCR amplification.

[0075] The expression "recombinant viral vector" refers to a recombinant polynucleotide vector containing one or more heterologous sequences (ie, a nucleic acid sequence not derived from a virus). In the case of recombinant AAV-based vectors, the recombinant nucleic acid is flanked with at least one, eg two, inverted terminal repeat (ITR) sequences.

[0076] The expression "recombinant AAV-based vector (rAAV-based vector)" refers to a polynucleotide vector containing one or more heterologous sequences (i.e., a nucleic acid sequence not derived from AAV) that are flanked by at least one, for example, two sequences of inverted terminal repeats (ITR). Such rAAV-based vectors can replicate and package into infectious viral particles when they are in a host cell that has been infected with an appropriate helper virus (or that expresses appropriate helper functional elements) and that expresses AAV rep and cap gene products (i.e. e. Rep and Cap AAV proteins). If the rAAV-based vector is inserted into a larger polynucleotide (e.g., into a chromosome or into another vector such as a plasmid used for cloning or transfection), then the rAAV-based vector may be referred to as a "provector" which can be "rescued" by replication and capsid entrapment in the presence of packaging functionalities and suitable AAV helper functionalities. The rAAV vector can be in any of a variety of forms, including but not limited to plasmids, linear artificial chromosomes complexed with lipids, encapsulated in liposomes and, in embodiments, encapsulated in a viral particle, in particular an AAV particle. The rAAV vector can be packaged into an AAV capsid to form a "recombinant adeno-associated virus particle (rAAV particle)".

[0077] "rAAV virus" or "rAAV virus particle" refers to a viral particle consisting of at least one AAV capsid protein and an encapsulated rAAV vector genome.

[0078] "Original AAV particle" and "original AAV capsid protein" as used herein in the context of comparing N-acetylation and/or deamidation refers to an AAV particle or capsid protein that has been introduced with amino acid modifications to modulate N-acetylation and /or deamidation (for example, an AAV particle/capsid protein that is similar to or similar to the AAV particle/capsid of the present invention, but does not contain mutations that provide for the modulation/change of N-acetylation and/or deamidation, as described herein ). In some embodiments, the original AAV particle is a recombinant AAV particle containing the recombinant AAV genome. In some embodiments, the parent AAV capsid particle or parent AAV capsid protein contains amino acid substitutions that affect other aspects of the AAV particle. For example, the original AAV particle may contain amino acid substitutions that affect the binding of the AAV to its receptor, eg, affect the binding of AAV2 to heparin sulfate proteoglycan (eg, the AAV2 HBKO particle). The HBKO AAV2 particle can be mutated with amino acid substitutions that provide modulation of N-acetylation and/or deamidation. This mutated AAV particle can then be compared to the original AAV2 HBKO particle in the aspects of the present invention described herein. The parent AAV capsid protein may include the parent VP1 capsid protein, the parent VP2 capsid protein, or the parent VP3 capsid protein.

[0079] Used in this document, the term "modulate" or "change" in relation to the original molecule means to change the property of the original molecule. For example, an N-acetylation altered AAV particle may have increased or decreased N-acetylation relative to the parent AAV particle, and a deamidated modified AAV particle may have increased or decreased deamidation than the parent AAV particle.

[0080] "Heterologous" means derived from an entity that is genotypically different from the rest of the entity to which it is compared or into which it is introduced or inserted. For example, a nucleic acid introduced by genetic engineering techniques into another type of cell is a heterologous nucleic acid (and may encode a heterologous polypeptide when expressed). Similarly, a cellular sequence (eg, a gene or part thereof) inserted into a viral vector is a heterologous nucleotide sequence with respect to the vector.

[0081] The term "transgene" refers to a nucleic acid introduced into a cell and capable of being transcribed into RNA and optionally translated and/or expressed under appropriate conditions. In some aspects, it imparts the desired property to the cell into which it has been introduced, or otherwise results in the desired therapeutic or diagnostic effect. In another aspect, it can be transcribed into a molecule that mediates RNA interference, such as siRNA.

[0082] The terms "genomic particles (gp)", "genomic equivalents", or "genome copies", as used in relation to viral titer, refer to the number of virions containing the recombinant AAV DNA genome, regardless of infectivity or functionality. The number of genomic particles in a particular vector preparation can be measured using procedures such as those described in the examples herein or, for example, in Clark et al. (1999) Hum. Gene Ther., 10:1031-1039; Veldwijk et al. (2002) Mol. Ther. 6:272-278.

[0083] The terms "infectious unit (iu)", "infectious particle", or "replication unit", as used in relation to viral titer, refer to the number of infectious and replication competent particles representing a recombinant AAV vector, as measured by an assay infection centers, also known as replication site assay, described for example in McLaughlin et al. (1988) J. Virol., 62:1963-1973.

[0084] The term "transducing unit (tu)", as used in relation to viral titer, refers to the number of infectious particles representing a vector based on recombinant AAV, which lead to the production of a functional transgenic product, measured in functional assays, such as those described in the examples. in this document or, for example, in Xiao et al. (1997) Exp. Neurobiol., 144:113-124; or in Fisher et al. (1996) J. Virol., 70:520-532 (LFU analysis).

[0085] The sequence "inverted terminal repeats" or "ITR" is a term commonly used in the art and refers to the relatively short sequences found at the ends of viral genomes that have the opposite orientation.

[0086] The "AAV inverted terminal repeat (ITR)" sequence, a term well known in the art, refers to a sequence of about 145 nucleotides that is present at both ends of the native single-stranded AAV genome. The last 125 nucleotides of the ITR can be present in one of two alternative orientations, resulting in heterogeneity between different AAV genomes and between two ends of the same AAV genome. The outermost 125 nucleotides also contain several shorter self-complementary regions (referred to as A-, A'-, B-, B'-, C-, C'- and D-regions) which allow intrastrand base pairing to form within this portion. ITR.

[0087] "End resolution sequence" or "trs" is a sequence in the D region of AAV ITR that is cleaved off by AAV rep proteins during viral DNA replication. The mutant end resolution sequence is resistant to cleavage by AAV rep proteins. "Functional elements of the AAV helper" refers to the functional elements that allow the replication and packaging of AAV in the host cell. The functional elements of an AAV helper can be in any of a variety of forms, including but not limited to helper virus or helper virus genes that promote AAV replication and packaging. Other functional elements of the AAV helper are known in the art, such as genotoxic agents.

[0088] "Functional elements of the AAV helper" refers to the functional elements that allow the replication and packaging of AAV in the host cell. The functional elements of an AAV helper can be in any of a variety of forms, including but not limited to helper virus or helper virus genes that facilitate AAV replication and packaging. Other functional elements of the AAV helper are known in the art, such as genotoxic agents.

[0089] The expression "helper virus" for AAV refers to a virus that allows AAV (which is a defective parvovirus) to replicate and package in a host cell. Many such helper viruses have been identified, including adenoviruses, herpesviruses, poxviruses such as vaccinia and baculovirus. Adenoviruses cover many different subgroups, but adenovirus type 5 subgroup C (Ad5) is the most widely used. Numerous adenoviruses affecting humans, non-human mammals and birds are known and available from depositories such as ATCC. Viruses of the herpesvirus family, which are also available from depositories such as ATCC, include, for example, herpes simplex viruses (HSV), Epstein-Barr viruses (EBV), cytomegaloviruses (CMV) and pseudorabies viruses (PRV). Baculoviruses available from depositories include Autographa californica nuclear polyhedrosis virus.

[0090] The expression "percent (%) sequence identity" relative to a reference polypeptide or nucleic acid sequence is defined as the percentage of amino acid residues or nucleotides in a candidate sequence that are identical to amino acid residues or nucleotides in a reference polypeptide or nucleic acid sequence after sequence alignment and, if necessary, introducing gaps to achieve maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for the purposes of determining percent identity of amino acid sequences or nucleic acid sequences can be achieved in various ways that are within the skill of the art, for example, using publicly available computer programs, such as those described in Current Protocols in Molecular Biology (Ausubel et al., eds., 1987), app. 30, section 7.7.18, table 7.7.1, and including BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. A potential alignment program is ALIGN Plus (Scientific and Educational Software, Pennsylvania). Those skilled in the art are able to determine the appropriate parameters for evaluating the alignment, including any algorithms necessary to achieve maximum alignment over the full length of the compared sequences. For the purposes of this document, the % amino acid sequence identity attributable to a given amino acid sequence A in relation to, with, or in comparison to, a given amino acid sequence B (which can alternatively be rephrased as a given amino acid sequence A that is characterized by or has a certain % amino acid identity sequence in relation to, with or in comparison with a given amino acid sequence B) is calculated as follows: 100 times the quotient of X/Y, where X is the number of amino acid residues counted as identical matches by the sequence alignment software in that software alignment A and B, and where Y is the total number of amino acid residues in B. Note that if the length of the amino acid sequence of A is not equal to the length of the amino acid sequence of B, then the % identity of the amino acid sequence value of A against B will not be equal to the % amino acid sequence identity of B against A. For the purposes of this document, the % nucleic acid sequence identity attributable to a given nucleic acid sequence C in relation to, with or in comparison with a given nucleic acid sequence D ( which can alternatively be paraphrased as a given nucleic acid sequence C that is characterized or has a certain % nucleic acid sequence identity with respect to, with or in comparison with a given nucleic acid sequence D) is calculated as follows: 100 times the quotient of W/ Z, where W is the number of nucleotides counted as identical matches by the program to align sequences in such software alignment of C and D, and where Z is the total number of nucleotides in D. Note that if the length of the sequence Since the nucleic acid C is not equal to the length of the D nucleic acid sequence, then the % nucleic acid sequence identity C to D will not be equal to the % nucleic acid sequence identity D to C.

[0091] The expression "isolated" molecule (eg, nucleic acid or protein) or cell means that it has been identified and separated and/or extracted from a component of its natural environment.

[0092] "Mass spectrometry" refers to an analytical chemistry technique associated with the identification of the amount and/or type of a compound (eg, polypeptide) by measuring the mass-to-charge ratio and excess of gas phase ions. The term "mass spectrometry" can be used interchangeably herein.

[0093] "Heterogeneity" when used in relation to an AAV capsid refers to an AAV capsid characterized by one or more capsid polypeptides that are observed to deviate from a reference mass of a VP1, VP2 and/or VP3 polypeptide or fragment thereof. The reference mass may include, without limitation, the theoretical, predicted, or expected mass of a VP1, VP2, and/or VP3 polypeptide, such as a known AAV serotype. For example, an AAV capsid is considered to exhibit heterogeneity if it has one or more of the following (but not limited to): mixed serotype, variant capsid, capsid amino acid substitution, truncated capsid, or modified capsid.

[0094] Reference to "about" in relation to a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, a description referring to "about X" includes a description of "X".

[0095] The singular forms used herein include references to the plural unless otherwise noted.

[0096] It is understood that the aspects and embodiments of the present invention described herein include "comprising", "consisting of" and/or "consisting essentially of" aspects and embodiments.

III. Ways

[0097] Certain aspects of the present invention relate to methods for determining the serotype of a viral particle. Other aspects of the present invention relate to methods for determining the heterogeneity of a viral particle. As described below, the exact masses of VP1, VP2, and VP3 of each AAV serotype are unique and can be used to identify or differentiate AAV capsid serotypes. These methods are based in part on the discovery described herein that direct LC/MS of various AAV types after denaturation can be used to monitor protein sequencing and post-translational modifications with accurate mass measurements at the intact protein level. In addition, VP1 and VP3 N-terminal acetylation events can also be identified and/or monitored in various AAV serotypes. Based on these AAV results and the guidance provided herein, it is believed that such methods can be readily applied to profiling a number of viruses, eg families, subfamilies and genera of the viruses of the present invention. These methods of the present invention may find use, for example, in obtaining VP profiles to control expression processes, post-translational modifications and truncations of VP and to ensure product homogeneity during VLP preparation, to confirm site-directed mutagenesis or structural characterization of capsid engineering applications. proteins, and/or to control or detect the heterogeneity of the viral particle or preparation.

[0098] In some embodiments, the methods include denaturing the viral particle. In some embodiments, a viral particle, such as an AAV particle, can be denatured with a detergent, heat, high salt concentration, or low or high pH buffering. In certain embodiments, the AAV particle can be denatured using acetic acid or guanidine hydrochloride. One skilled in the art will appreciate that a number of methods are available in the art suitable for promoting and/or controlling protein denaturation and will be able to appropriately select a denaturation method that is compatible with liquid chromatography/mass spectrometry. For example, if heat denaturation is used, care must be taken to avoid protein precipitation and plugging of the reverse phase column. Similarly, high salt denaturation can be combined with a desalting step prior to LC/MS or LC/MS/MS. In other embodiments, high pH denaturation, low pH denaturation, or organic solvent denaturation is used.

[0099] In some embodiments, the methods involve subjecting a denatured viral particle of the present invention to liquid chromatography/mass spectrometry (LC/MS). As is known in the art, LC/MS uses liquid chromatography to physically separate ions and mass spectrometry to obtain mass spectral data from the ions. Such mass spectral data can be used, for example, to determine molecular weight or structure, identify particles by weight, quantity, purity, and the like. This data may represent properties of the ions being detected, such as signal strength (eg, intensity) over time (eg, hold time) or relative intensity versus mass-to-charge ratio.

[0100] In some embodiments, liquid chromatography (e.g., as used in LC/MS described herein) is ultra performance liquid chromatography (UPLC; the terms "ultra performance liquid chromatography" or UHPLC may be used interchangeably herein). UPLC is known in the art as an LC technique that relies on the use of a column with a reduced particle size (e.g., less than 2 µm) and an increased flow rate to improve chromatographic resolution, efficiency, peak capacity, and sensitivity (see, for example, Plumb, R. et al (2004) Rapid Commun Mass Spectrom 18:2331-2337). In some embodiments, UPLC refers to the use of a column with a particle size of less than 2 microns in liquid chromatography. In some embodiments, UPLC refers to the use of high solvent linear velocity (eg, as seen when operating at 6000 psi or higher) in liquid chromatography. Exemplary UPLC devices are commercially available (eg, ACQUITY UPLC® from Waters; Milford, MA).

[0101] In some embodiments, mass spectrometry (eg, as used in LC/MS described herein) may refer to electrospray ionization mass spectrometry (ESI-MS). ESI-MS is known in the art as a technique that uses electrical energy to analyze ions obtained from a solution using mass spectrometry (see, for example, Yamashita, M. and Fenn, J.B. (1984) J. Phys. Chem. 88:4451-4459). Ionic particles (or neutral particles that are ionized in solution or in the gaseous phase) are transferred from solution to the gaseous phase by spraying charged droplets into an aerosol followed by evaporation of the solvent, which reduces the size of the charged particles and the release of sample ions from the charged droplets as how the solution passes through a small capillary with a voltage with respect to ground (for example, the wall of the surrounding chamber). In some embodiments, the capillary voltage is from about 2 kV to about 10 kV, or from about 2.5 kV to about 6.0 kV. In certain embodiments, liquid chromatography (eg, as used in LC/MS described herein) uses a capillary voltage of approximately 3.5 kV. In some embodiments, the implementation of the capillary voltage is in the range from about 1 kV to about 10 kV. In other embodiments, mass spectrometry (eg, as used in LC/MS described herein) may refer to matrix assisted laser desorption ionization (MALDI).

[0102] In some embodiments, mass spectrometry (eg, as used in LC/MS described herein) uses a sampling cone and/or a skimmer cone through which ions can pass before entering the analyzer. In some embodiments, for example, when applying voltage to a capillary as described above, the sampling cone is held at a lower voltage than the capillary voltage. In certain embodiments, liquid chromatography (e.g., as used in LC/MS described herein) uses a sample cone voltage of approximately 45 V. In some embodiments, the sample cone voltage ranges from approximately 0 V to approximately 200 V.

[0103] In some embodiments, mass spectrometry (eg, LC/MS as used herein) uses a calibration performed by an additional tool. Calibration, when applied to mass spectrometry, may involve the introduction of one or more compounds having a known mass (eg, standard) in order to calibrate the device with respect to mass detection (eg, m/z measurements). In some embodiments, calibration performed by an additional tool may refer to the use of a computer program to correlate the peak and/or position of a known standard (eg, calibrant) with a specific mass-to-charge (m/z) ratio. Once calibrated, the user can then perform mass spectrometry on a sample having one or more known compounds or compounds present at an unknown concentration, within a certain degree of accuracy or error and/or a desired level of reproducibility, e.g. compared to a previous or known experimental condition. Various calibrants are known in the art, including, but not limited to, sodium iodide, sodium-activated cesium iodide, polyethylene glycol, and perfluorotributylamine. In certain embodiments, sodium iodide is used as a calibrant. In some embodiments, the calibrants are Glu-1-fibrinopeptide B and leucine-enkephalin peptide for mass fixation during LC/MS.

[0104] In some embodiments, the methods include subjecting a denatured viral particle of the present invention or subjecting cleaved fragments of a denatured viral particle of the present invention to liquid chromatography/mass spectrometry-mass spectrometry (LC/MS/MS). As known in the art, LC/MS/MS (the term "liquid chromatography-tandem mass spectrometry" can be used interchangeably herein) uses liquid chromatography to physically separate ions and mass spectrometry to obtain mass spectral data based on ions, while mass spectrometry uses several stages of mass separation (for example, m/z), usually separated by a fragmentation step. For example, ions of interest in the m/z range can be separated in the first MS cycle, fragmented, and then further separated based on the individual m/z in the second MS cycle. Ion fragmentation may include, without limitation, a technique such as collision-induced dissociation (CID), high energy collision-induced dissociation (HCD), electron capture dissociation (ECD), or electron transfer dissociation (ETD).

[0105] In some embodiments, the methods involve subjecting a denatured viral particle of the present invention to reduction and/or alkylation. Means for recovering a viral particle include, but are not limited to, treatment with dithiothreitol, β-mercaptoethanol, or tris(2-carboxyethyl)phosphine (TCEP). Means for alkylating a viral particle include, without limitation, treating the AAV particle with iodoacetic acid, iodoacetamide, or 4-vinylpyridine.

[0106] In some embodiments, methods include subjecting a denatured viral particle of the present invention to cleavage, for example, to form VP1, VP2, and/or VP3 fragments of an AAV particle. For example, a denatured AAV particle can be cleaved to form peptide fragments that can be analyzed, for example, by LC for separation and MS/MS for analysis (see below for more details). In some embodiments, the cleavage is an enzymatic cleavage. In some embodiments, the cleavage employs chemical cleavage, such as instrumental fragmentation treatment with CNBr (eg, top to bottom). In some embodiments, the cleavage employs chemical cleavage, such as acid cleavage.

[0107] In some embodiments, the enzymatic cleavage is an endopeptidase cleavage. An endopeptidase may include any peptidase that catalyzes the proteolysis of peptide bonds of non-terminal amino acids of a polypeptide. Known peptidases may include, without limitation, trypsin, chymotrypsin, AspN, Glu-C, LysC, pepsin, thermolysin, glutamyl endopeptidase, elastase, and neprilysin. In certain embodiments, the enzymatic cleavage is a trypsin cleavage or a LysC cleavage.

[0108] In some embodiments, liquid chromatography (e.g., as used in LC/MS or LC/MS/MS described herein) is reverse phase liquid chromatography (the terms "reverse phase liquid chromatography" or RPLC can be used in this document interchangeably with reverse phase liquid chromatography). As known in the art, reversed phase liquid chromatography can refer to a chromatographic separation using a hydrophobic stationary phase (eg, a support or substrate such as a column) adsorbing the hydrophobic molecules into the polar mobile phase. By reducing the polarity of the mobile phase (for example, by adding an organic solvent), a gradient separation of molecules depending on hydrophobicity can be achieved, since more hydrophobic molecules will remain on the column at higher concentrations of organic solvent due to stronger hydrophobic interactions with the column. In some embodiments, separation is by capillary electrophoresis (CE), size exclusion chromatography (SEC), ion exchange chromatography (IEC) such as cation exchange chromatography, hydrophobic interaction chromatography (HIC), hydrophilic interaction liquid chromatography (HILIC), but not limited to LC /MS on-line, such as off-line division to MS; e.g. tips, columns; tablets or cartridges.

[0109] Typically, a stationary phase suitable for reverse phase liquid chromatography (e.g., a hydrophobic moiety) can be coupled to a support, including but not limited to a column or resin packed with particles or granules (e.g., particles of porous silica or polystyrene) . A number of hydrophobic stationary phases are known in the art, including, without limitation, hydrophobic alkyl chains, octyl or octadecylsilyl moieties, cyano moieties, and amino moieties. In some embodiments, the implementation of the stationary phase may include a hydrophobic alkyl chain of a certain length, such as C4, C8 or C18. In certain embodiments, reverse phase chromatography is C4 or C8 reverse phase chromatography (eg, reverse phase chromatography using a C4 or C8 stationary phase). The person skilled in the art can appropriately select the stationary phase based on the molecule of interest (eg, a denatured AAV particle or fragment thereof).

[0110] A number of mobile phases suitable for reverse phase liquid chromatography are known in the art. As described above, the reverse phase liquid chromatography mobile phase may include a mixture of organic (eg, hydrophobic) and aqueous (eg, polar) solvents. Increasing the proportion of the organic solvent increases its power to elute hydrophobic compounds from the stationary phase. The retention and/or selectivity of the compounds can be altered, for example, by changing the type or effect of the stationary phase, adding polar reagents such as end capping agents, changing the temperature and/or changing the characteristics of the mobile phase, such as organic solvent content, pH, buffers and the type of organic solvent used. In some embodiments, the implementation of the polar component of the mobile phase may include, without limitation, water or an aqueous buffer. In some embodiments, the implementation of the polar component of the mobile phase may include, without limitation, acetonitrile, methanol, ethanol, isopropyl alcohol, tetrahydrofuran (THF) and acetic acid.

[0111] In some embodiments, two or more mobile phases (eg, mobile phase A, mobile phase B, etc.) can be used in a gradient or proportion of interest. In certain embodiments, chromatography uses mobile phase A containing formic acid in water. In certain embodiments, mobile phase A contains approximately 0.1% formic acid. In certain embodiments, mobile phase A contains from about 0.1% to about 5% formic acid. In certain embodiments, the chromatography uses mobile phase B containing formic acid in acetonitrile. In certain embodiments, mobile phase B contains approximately 0.1% formic acid.

[0112] In some embodiments, the proportion of mobile phase B in the chromatography is increased over time. For example, the proportion of mobile phase B in the chromatography can be increased in steps. In certain embodiments, mobile phase B is increased from about 10% to about 20%, from about 20% to about 30%, and from about 30% to about 38%. In other embodiments, mobile phase B is increased from about 2% to about 60%. In other embodiments, mobile phase B is increased from about 2% to about 100% over a period of about 1 minute. up to approx. 200 min. In some embodiments, the remainder of the mobile phase is a second mobile phase of the present invention, such as mobile phase A. In certain embodiments, mobile phase B is increased from about 10% to about 20% over about 6 minutes, from about 20% to about 30% over about 10 minutes and from about 30% to about 38% over about 40 minutes. In other embodiments, mobile phase B is increased from about 2% to about 60% over about 121 minutes. One skilled in the art can appropriately adjust the mobile phase of interest and the gradient time based on the desired chromatographic separation and/or analyte of interest.

[0113] In some embodiments, the liquid chromatography is high performance liquid chromatography (HPLC). HPLC is known in the art as a form of liquid chromatography in which a liquid solvent containing a sample is compressed as it passes through a column containing a solid phase. While conventional or low pressure LC can use gravity to force the mobile phase through the solid phase, HPLC uses pumps to apply pressure to the mobile phase and typically uses finer solids to improve resolution. In some embodiments, HPLC uses a pressure of from about 50 bar to about 350 bar. In some embodiments, reverse phase HPLC can be used to concentrate and/or desalt proteins (eg, AAV capsid proteins) for MS analysis.

[0114] In some embodiments, one or more parameters, including but not limited to source voltage, capillary temperature, ESI voltage (when using ESI-MS), CID energy, and number of MS/MS events, can be adjusted, for example, at LC/ MS/MS described in this document, based on the results described in this document. In some embodiments, mass spectrometry (eg, as used in LC/MS/MS described herein) uses a source voltage (eg, capillary voltage) of approximately 2.5 kV. In some embodiments, mass spectrometry (eg, as used in LC/MS/MS described herein) uses a capillary temperature of approximately 275°C. In some embodiments, the temperature of the capillary is in the range from about 20°C to about 400°C.

[0115] A number of mass analyzers suitable for LC/MS and/or LC/MS/MS are known in the art, including, without limitation, time-of-flight (TOF) analyzers, quadrupole mass filters, quadrupole TOF (QTOF), and ion traps (eg Fourier transform mass spectrometer or Orbitrap). In the case of the Orbitrap, a barrel-shaped outer electrode at ground potential and a fusiform center electrode are used to capture ions in trajectories rotating elliptically about the center electrode, oscillating along the center axis, limited by the balance of centrifugal and electrostatic forces. With such devices, a Fourier transform operation is performed to convert a time signal (eg, frequency) based on shielding current detection into a high resolution mass measurement (eg, nano-LC/MS/MS). Additional descriptions and details can be found, for example, in Scheltema, R.A. et al. (2014) Mol. Cell Proteomics 13:3698-3708; Perry, R.H. et al. (2008) Mass. Spectrom. Rev. 27:661-699; and Scigelova, M. et al. (2011) Mol. Cell Proteomics 10:M111.009431.

[0116] As described above, in some embodiments, MS includes nano-LC/MS/MS, such as using an Orbitrap mass analyzer. In some embodiments, the ion source may include a ring-stacked ion guide or S-lens. As is known in the art, the S-lens can be used to focus an ion beam using radio frequency (RF), thereby increasing the transmission of ions to the device. This may increase sensitivity (for example, in the case of low intensity ions) and/or increase scanning speed. In certain embodiments, the RF level of the S lens on mass spectrometry is approximately 55%. In certain embodiments, the RF level of the S lens on mass spectrometry is from about 20% to about 100%.

[0117] In some embodiments, viral capsid protein masses can be determined, for example, based on LC/MS and/or LC/MS/MS data. In some embodiments, the VP1, VP2, and VP3 masses of the AAV particle or VP1, VP2, and VP3 fragments of the AAV particle can be determined, for example, based on LC/MS and/or LC/MS/MS data. Various methods for determining the mass and/or identity of proteins based on MS data are known in the art. For example, peptide mass fingerprinting can be used to determine a protein sequence based on MS data, or proteins can be identified based on MS/MS data related to one or more of their constituent peptides. When using tandem MS, product ion scanning can be used to analyze m/z data related to one or more peptides of a protein of interest. Computer programs known in the art can be used, for example, to match identified peaks with reference or known peaks, to group peaks into isotopomer zones, and so on. Peptide mass values can be compared to a database of known peptide sequences. For example, Mascot can be used to match observed peptides to theoretical database peptides, for example by applying a specific cleavage pattern to an in silico protein database. Other suitable computer programs include, without limitation, Proteome Discoverer, ProteinProspector, X!Tandem, Pepfinder, Bonics, or MassLynx™ (Waters). Other computer programs suitable for the various steps of MS data analysis can be found, for example, at www.ms-utils.org/wiki/pmwiki.php/Main/SoftwareList.

[0118] In some embodiments, the determined or calculated mass of the present invention (e.g., the determined or calculated mass VP1, VP2, and/or VP3 of an AAV particle) can be compared to a reference, e.g., the theoretical mass VP1, VP2, and/or VP3 of one or several AAV serotypes. The reference of the present invention may include the theoretical weight of VP1, VP2 and/or VP3 of one or more of the AAV serotypes described herein. For example, in some embodiments, the masses of VP1, VP2, and/or VP3 are compared to the theoretical masses of one or more of AAV1 capsid, AAV2 capsid, AAV3 capsid, AAV4 capsid, AAV5 capsid, AAV6 capsid, AAV7 capsid, AAV8 capsid, AAVrh8 capsid, AAVrh8 capsid, AAV9, AAV10 capsid, AAVrh10 capsid, AAV11 capsid, AAV12 capsid, AAV LK03 capsid (See US Pat. No. 9,169,299), AAV2R471A capsid, AAV2/2-7m8 capsid, AAV DJ capsid (See US Pat. No. 7,588,772), AAV capsid DJ8, AAV2 capsid N587A, AAV2 capsid E548A, AAV2 capsid N708A, AAV capsid V708K, goat AAV capsid, AAV1/AAV2 chimeric capsid, bovine AAV capsid, or rAAV2/HBoV1 mouse AAV capsid (bocavirus chimeric AAV/virus 1) human), AAV2HBKO capsid, AAVPHP.B capsid, or AAVPHP.eB capsid. In some embodiments, the determined or calculated weight of the present invention (e.g., the determined or calculated VP1, VP2, and/or VP3 weight of an AAV particle) can be compared to the theoretical VP1, VP2, and/or VP3 weight of the corresponding AAV serotype.

[0119] In some embodiments, the methods of the present invention may include determining the heterogeneity of the AAV particle. In some embodiments, a deviation of one or more VP1, VP2, and/or VP3 masses (eg, from a reference mass, such as theoretical, predicted, or expected mass) is indicative of AAV capsid heterogeneity. In some embodiments, heterogeneity may include, without limitation, one or more of the following: mixed serotypes, variant capsids, capsid amino acid substitutions, truncated capsids, or modified capsids.

[0120] In some embodiments, the use of LC/MS and LC/MS/MS described herein can be combined. In some embodiments, a method for determining the serotype of an AAV particle may include subjecting a denatured AAV particle to LC/MS (eg, as described herein) and determining the masses VP1, VP2, and VP3 of the AAV particle; and exposure of the VP1, VP2 and/or VP3 fragments to LC/MS/MS and determination of the masses of the VP1, VP2 and VP3 fragments of the AAV particle (the specific combination of masses of the VP1, VP2 and VP3 fragments characterizes the AAV serotype). In some embodiments, a method for determining the heterogeneity of an AAV particle may include subjecting a denatured AAV particle to LC/MS (e.g., as described herein), determining the VP1, VP2, and VP3 masses of the AAV particle, and comparing these masses to theoretical AAV serotype VP1, VP2, and VP3 masses. ; as well as exposure of the VP1, VP2 and/or VP3 fragments to LC/MS/MS, determination of the masses of the VP1, VP2 and VP3 fragments of the AAV particle and comparison of these masses with the theoretical masses of VP1, VP2 and VP3 of the AAV serotype (deviation of one or more masses of VP1, VP2 or VP3 characterizes the heterogeneity of the AAV capsid).

[0121] In some embodiments, the AAV particle of the present invention may be acetylated. For example, in some embodiments, the N-terminus of VP1 and/or VP3 is acetylated. As described in more detail below, the amino acid at position 2 to the initiating methionine (iMet X) of the AAV capsid protein can be mutated to determine its effect on N-terminal (Nt-) acetylation as well as the functional consequences of the effect of Nt-acetylation. on migration, transduction and/or post-translational modification of the AAV particle (eg, glycosylation, ubiquitination, etc.). In some embodiments, the N-terminus of the AAV capsid protein (eg, VP1 or VP3) may refer to the first amino acid after the initiator methionine, which in some cases may be removed, for example, by Met-aminopeptidase.

[0122] In some embodiments, an AAV particle of the present invention (eg, a recombinant AAV or rAAV particle) contains an amino acid substitution at amino acid residue 2 of VP1 and/or VP3. In some embodiments, an amino acid substitution at amino acid residue 2 of VP1 and/or VP3 results in VP1 and/or VP3 with a different frequency or proportion of N-terminal acetylation compared to a reference (e.g., the original AAV particle before the amino acid change, or the AAV particle with a different amino acid residue 2 of VP1 and/or VP3). In some embodiments, the amino acid substitution at amino acid residue 2 of VP1 and/or VP3 provides a change in N-terminal acetylation compared to N-terminal acetylation at amino acid residue 2 of VP1 and/or VP3 of the parent AAV particle. For example, in certain embodiments, the amino acid substitution at VP1 amino acid residue position 2 provides for a change in N-terminal acetylation compared to N-terminal acetylation at VP1 amino acid residue 2 of the parent AAV particle. In certain embodiments, the amino acid substitution at VP3 amino acid residue 2 provides for a change in N-terminal acetylation compared to N-terminal acetylation at VP3 amino acid residue 2 of the parent AAV particle. In some embodiments, an amino acid change (e.g., an amino acid change at amino acid residue 2 of VP1 or VP3) that "changes" N-terminal acetylation results in a higher frequency of N-terminal acetylation or a lower frequency of N-terminal acetylation, e.g. compared to VP1 or VP3 without replacement, such as the original VP1 or VP3. VP1 and/or VP3 may belong to any of the exemplary AAV serotypes described herein, including variants or hybrids thereof (eg, carrying a tyrosine mutation or mutations affecting heparin binding). Exemplary N-terminal acetylation assays include, but are not limited to, mass spectrometry, isotopic labeling (e.g., with an isotopically labeled acetyl group or its precursor), Western blotting with an acetylation-specific antibody, etc. In some embodiments, an amino acid residue 2 of the AAV capsid protein (eg, VP1 or VP3) is replaced by Cys, Ser, Thr, Val, Gly, Asn, Asp, Glu, Ile, Leu, Phe, Gln, Lys, Met, Pro, or Tyr. In some embodiments, the amino acid substitution results in less deamidation of the AAV capsid.

[0123] In some embodiments, the AAV particle of the present invention may be deamidated. For example, in some embodiments, N57 VP1 and/or N382, N511 and/or N715 VP3 are deamidated. As described in more detail below, an amino acid selected from A35 VP1, N57 VP1, G58 VP1, N382 VP3, G383 VP3, N511 VP3, G512 VP3, N715 VP3, or G716 VP3 of an AAV capsid protein (e.g., VP1 or VP3) can be mutated in order to determine its effect on deamidation, as well as the functional consequences of the effect of deamidation on migration, transduction and/or post-translational modification of the AAV particle (eg, glycosylation, ubiquitination, etc.).

[0124] In some embodiments, an AAV particle of the present invention (e.g., a recombinant AAV or rAAV particle) contains an amino acid substitution at one or more amino acid residues selected from A35 VP1, N57 VP1, G58 VP1, N382 VP3, G383 VP3, N511 VP3 , G512 VP3, N715 VP3 and G716 VP3. In some embodiments, the amino acid substitution A35 VP1, N57 VP1, G58 VP1, N382 VP3, G383 VP3, N511 VP3, G512 VP3, N715 VP3, and/or G716 VP3 results in the formation of VP1/or VP3 with a different frequency or proportion of deamidation compared to a reference (eg, a particle of the original AAV before the introduction of an amino acid substitution or an AAV particle with a different corresponding amino acid residue of 2). In some embodiments, an amino acid substitution (e.g., an amino acid substitution in A35 VP1, N57 VP1, G58 VP1, N382 VP3, G383 VP3, N511 VP3, G512 VP3, N715 VP3, or G716 VP3) that provides a "change" in deamidation results in more a high rate of deamidation; or a lower rate of deamidation, eg compared to VP1 or VP3 without replacement, such as the original VP1 or VP3. VP1 and/or VP3 may belong to any of the exemplary AAV serotypes described herein, including variants or hybrids thereof (eg, carrying a tyrosine mutation or mutations affecting heparin binding). Exemplary deamidation assays include, but are not limited to, mass spectrometry, HPLC (see, for example, Promega's ISOQUANT® Isoaspartate Detection Kit), etc. In some embodiments, N57 VP1, N382 VP3, N511 VP3, and/or N715 VP3 are replaced with Asp, and the amino acid change results in a higher frequency of deamidation compared to deamidation of the VP1 and/or VP3 of the parent AAV particle. In other embodiments, the amino acid substitution is N57K or N57Q. and the amino acid substitution results in a lower frequency of deamidation compared to deamidation of the VP1 and/or VP3 particles of the original AAV. In still other embodiments, G58 VP1, G383 VP3, G512 VP3, and/or G716 VP3 are replaced with an amino acid that is not Gly (e.g., Ala, Arg, Asn, Asp, Cys, Glu, Gln, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val), and the amino acid substitution results in a lower frequency of deamidation compared to deamidation of the VP1 and/or VP3 particles of the original AAV. In still other embodiments, A35 VP1 is replaced by Asn and results in a higher deamidation rate compared to the VP1 of the original particle.

[0125] As used herein, "N-acetylation" refers to a process in which an acetyl group is covalently added to the amino group of the N-terminal amino acid of a protein. Typically, N-terminal acetyltransferases (NATs) transfer an acetyl group from acetyl coenzyme A (Ac-CoA) to the α-amino group of the first amino acid residue of the protein.

[0126] As used herein, "deamidation" refers to a chemical reaction in which an amide functional group in the side chain of asparagine or glutamine is removed or converted to another functional group. For example, asparagine can be converted to aspartic acid or isoaspartic acid. In other examples, glutamine is converted to glutamic acid or pyroglutamic acid (5-oxoproline).

[0127] In some embodiments, the AAV particle is N-acetylated to a higher degree than the parent AAV capsid protein. In some embodiments, the AAV particle contains more than about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% more N-acetyl groups. , 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to the original AAV particle. In some embodiments, the AAV particle contains more N-acetyl groups by any value in the ranges of 5%-10%, 10%-15%, 15%-20%, 20%-25%, 25%-30%, 30%- 35%, 35%-40%, 40%-55%, 45%-50%, 50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75% , 75%-80%, 80%-85%, 85%-90%, 90%-95%, 95%-100%, 5-25%, 25-50%, 50-75%, 75%-100 %, 5-50% or 50%-100% compared to the original AAV particle. In some embodiments, the AAV particle contains more N-acetyl groups in any of more than about 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 500 -fold or 1000 times the amount compared to the particle of the original AAV. In some embodiments, the AAV particle contains more N-acetyl groups in an amount in any approximate range of 2-fold to 3-fold, 3-fold to 4-fold, 4-fold to 5-fold, 5-fold to 10x, 10x to 25x, 25x to 50x, 50x to 100x, 100x to 500x, 500x to 1000x, 2-fold to 10-fold, 10-fold to 100-fold, or 100-fold to 1000-fold as compared to the original AAV particle.

[0128] In some embodiments, the implementation of the particle AAV is N-acetylated to a lower extent compared to the capsid protein of the original AAV. In some embodiments, the AAV particle contains any number of N-acetyl groups less than about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% , 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to the original AAV particle. In some embodiments, the AAV particle contains less N-acetyl groups by any value in the ranges of 5%-10%, 10%-15%, 15%-20%, 20%-25%, 25%-30%, 30%- 35%, 35%-40%, 40%-55%, 45%-50%, 50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75% , 75%-80%, 80%-85%, 85%-90%, 90%-95%, 95%-100%, 5-25%, 25-50%, 50-75%, 75%-100 %, 5-50% or 50%-100% compared to the original AAV particle. In some embodiments, the AAV particle contains fewer N-acetyl groups in any of more than about 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 500 -fold or 1000 times the amount compared to the particle of the original AAV. In some embodiments, the AAV particle contains fewer N-acetyl groups in any approximate range of 2-fold to 3-fold, 3-fold to 4-fold, 4-fold to 5-fold, 5-fold to 10x, 10x to 25x, 25x to 50x, 50x to 100x, 100x to 500x, 500x to 1000x, 2-fold to 10-fold, 10-fold to 100-fold, or 100-fold to 1000-fold as compared to the original AAV particle.

[0129] In some embodiments, the AAV particle is deamidated to a higher degree than the original AAV particle. In some embodiments, the AAV particle is deamidated to a greater extent than about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to the original AAV particle. In some embodiments, the AAV particle is more deamidated by any value in the approximate range of 5%-10%, 10%-15%, 15%-20%, 20%-25%, 25%-30%, 30% -35%, 35%-40%, 40%-55%, 45%-50%, 50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75 %, 75%-80%, 80%-85%, 85%-90%, 90%-95%, 95%-100%, 5-25%, 25-50%, 50-75%, 75%- 100%, 5-50% or 50%-100% compared to the original AAV particle. In some embodiments, the AAV particle is more deamidated to any of more than about 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 500- multiple or 1000 times the power compared to the particle of the original AAV. In some embodiments, the AAV particle is more deamidated in any of the approximate ranges of 2-fold to 3-fold, 3-fold to 4-fold, 4-fold to 5-fold, 5-fold to 10-fold multiple, 10x to 25x, 25x to 50x, 50x to 100x, 100x to 500x, 500x to 1000x, 2- fold to 10 fold, 10 fold to 100 fold, or 100 fold to 1000 fold of the original AAV particle.

[0130] In some embodiments, the AAV capsid protein is deamidated to a lower degree than the parent AAV capsid protein. In some embodiments, the AAV particle is less than about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, more than about 5%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to the original AAV particle. In some embodiments, the AAV particle is less deamidated by any value in the approximate range of 5%-10%, 10%-15%, 15%-20%, 20%-25%, 25%-30%, 30% -35%, 35%-40%, 40%-55%, 45%-50%, 50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75 %, 75%-80%, 80%-85%, 85%-90%, 90%-95%, 95%-100%, 5-25%, 25-50%, 50-75%, 75%- 100%, 5-50% or 50%-100% compared to the original AAV particle. In some embodiments, the AAV particle is deamidated to a lesser extent in any of more than about 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 500- multiple or 1000 times the power compared to the particle of the original AAV. In some embodiments, the AAV particle is deamidated to a lesser extent in any of the approximate ranges of 2-fold to 3-fold, 3-fold to 4-fold, 4-fold to 5-fold, 5-fold to 10-fold multiple, 10x to 25x, 25x to 50x, 50x to 100x, 100x to 500x, 500x to 1000x, 2- fold to 10 fold, 10 fold to 100 fold, or 100 fold to 1000 fold of the original AAV particle.

[0131] The present invention contemplates any combination of N-acetylation and deamidation. For example, the AAV capsid protein may be N-acetylated to a higher degree than the parent AAV capsid protein and deamidated to a higher degree than the parent AAV capsid protein, the AAV capsid protein may be N-acetylated to a higher degree than the parent AAV capsid protein. parent AAV, and deamidated to the same extent as the parent AAV capsid protein, the AAV capsid protein may be N-acetylated to a higher degree than the parent AAV capsid protein and deamidated to a lower degree than the parent AAV capsid protein, the AAV capsid protein may be N-acetylated to the same extent as the parent AAV capsid protein and deamidated to a higher degree than the parent AAV capsid protein, the AAV capsid protein may be N-acetylated to the same extent as parent AAV capsid protein, and deamidated to the same extent as the parent AAV capsid protein, the AAV capsid protein may be N-acet ylated to the same extent as the parent AAV capsid protein and deamidated to a lower degree than the parent AAV capsid protein, the AAV capsid protein may be N-acetylated to a lower degree than the parent AAV capsid protein and deamidated to more to a higher degree than the parent AAV capsid protein, the AAV capsid protein may be N-acetylated to a lower degree than the parent AAV capsid protein and deamidated to the same degree as the parent AAV capsid protein, or the AAV capsid protein may be N α-acetylated to a lower degree than the parent AAV capsid protein and deamidated to a lower degree than the parent AAV capsid protein.

IV. Vectors

[0132] In certain aspects, the present invention relates to viral particles suitable for use in any of the methods described herein, which may contain AAV-based vectors (for example, rAAV-based vectors) or vectors derived from another virus. In some embodiments, the implementation of the viral particle contains a vector encoding a heterologous nucleic acid, for example, a heterologous transgene. In some embodiments, the AAV particle comprises an AAV vector genome encoding a heterologous nucleic acid, eg, a heterologous transgene.

[0133] The present invention provides for the use of a recombinant viral genome to introduce one or more nucleic acid sequences encoding a therapeutic polypeptide and/or nucleic acid for packaging into a rAAV viral particle. The recombinant viral genome may contain any element to allow expression of a therapeutic polypeptide and/or nucleic acid, such as a promoter, an ITR of the present invention, a ribosome binding element, a terminator, an enhancer, a selectable marker, an intron, a poly(A) signal, and/or an origin. replication.

[0134] In some embodiments, the heterologous nucleic acid encodes a therapeutic polypeptide. A therapeutic polypeptide may, for example, replace a polypeptide and/or enzymatic activity that is absent or present at a reduced level in a cell or organism. Alternatively, the therapeutic polypeptide may replace the polypeptide and/or enzymatic activity that indirectly counteract the imbalance in the cell or organism. For example, a therapeutic polypeptide used against a metabolite accumulation disorder caused by a deficiency of a metabolic enzyme or activity may replace the missing metabolic enzyme or activity, or it may provide another metabolic enzyme or activity resulting in a decrease in the level of the metabolite. The therapeutic polypeptide can also be used to reduce the activity of a polypeptide (e.g., a polypeptide that is overexpressed, activated by an acquisition mutation, or a polypeptide whose activity is otherwise dysregulated) by acting, for example, as a dominant negative effect polypeptide.

[0135] The nucleic acids of the present invention may encode polypeptides that are intracellular proteins anchored in the cell membrane, stored in the cell, or secreted by the cell transduced with the vectors of the present invention. In the case of polypeptides secreted by the cell containing the vector, the polypeptide may be soluble (ie not attached to the cell). For example, soluble polypeptides lack a transmembrane region and are secreted from the cell. Techniques for identifying and removing nucleic acid sequences that encode transmembrane domains are known in the art.

[0136] The nucleic acids of the present invention (eg, the genome of an AAV-based vector) may contain, as a transgene, a nucleic acid encoding a protein or functional RNA that modulates or treats a CNS-associated disorder. The following is a non-limiting list of genes associated with CNS-associated disorders: neuronal apoptosis inhibitory protein (NAIP) gene, nerve growth factor (NGF) gene, glial growth factor (GDNF) gene, brain growth factor (BDNF) gene, ciliary neurotrophic factor (CNTF), tyrosine hydroxylase (TM) gene, GTP-cyclohydrolase (GTPCH) gene, aspartoacylase (ASPA) gene, superoxide dismutase (SOD1) gene, antioxidant gene, anti-angiogenic polypeptide gene, anti-inflammatory polypeptide gene, and amino acid decarboxylase (AADC) gene. For example, a transgene useful in the treatment of Parkinson's disease encodes TH, which is an enzyme that limits the rate of dopamine synthesis. A transgene encoding a GTPCH that generates the TH cofactor, tetrahydrobiopterin, can also be used in the treatment of Parkinson's disease. A transgene encoding GDNF, or BDNF, or AADC, which promotes the conversion of L-Dopa to DA, can also be used to treat Parkinson's disease. A transgene useful in the treatment of ALS may encode for GDNF, BDNF, or CNTF. A transgene useful in the treatment of ALS may also encode a functional RNA, eg shRNA, miRNA, which inhibits SOD1 expression. A transgene useful in the treatment of ischemia may encode for NAIP or NGF. A transgene encoding beta-glucuronidase (GUS) may be useful in the treatment of certain lysosomal storage diseases (eg, mucopolysaccharidosis type VII (MPS VII)). A transgene encoding a gene product that activates a prodrug, such as HSV thymidine kinase, which converts ganciclovir to a toxic nucleotide that interferes with DNA synthesis and results in cell death, may be useful in the treatment of certain forms of cancer, for example when administered in combination with a prodrug. A transgene encoding an endogenous opioid such as β-endorphin may be useful in the treatment of pain. Examples of antioxidants include, without limitation SOD1; SOD2; catalase; sirtuins 1, 3, 4 or 5; NRF2; PGC1a; GCL (catalytic subunit); GCL (modifying subunit); adiponectin; glutathione peroxidase 1 and neuroglobin. Examples of antiangiogenic polypeptides include, but are not limited to, angiostatin, endostatin, PEDF, soluble VEGF receptor, and soluble PDGF receptor. Examples of anti-inflammatory polypeptides include, without limitation, IL-10, soluble IL17R, soluble TNF-R, TNF-R-Ig, an IL-1 inhibitor, and an IL18 inhibitor. Other examples of transgenes that can be used in the rAAV vectors of the present invention will be apparent to those skilled in the art (see, for example, Costantini LC, et al., Gene Therapy (2000) 7, 93-109).

[0137] In some embodiments, the heterologous nucleic acid encodes a therapeutic nucleic acid. In some embodiments, the therapeutic nucleic acid may include, without limitation, a siRNA, shRNA, RNAi agent, miRNA, antisense RNA, ribozyme, or DNA enzyme. Thus, a therapeutic nucleic acid can encode an RNA that, when transcribed from the vector nucleic acids, can provide treatment for the disorder by interfering with translation or transcription of the abnormal or excess protein associated with the disorder of the present invention. For example, the nucleic acids of the present invention may encode an RNA that provides treatment for a disorder by highly specific elimination or reduction of mRNA encoding abnormal and/or redundant proteins. Therapeutic RNA sequences include RNAi agents, small inhibitory RNAs (siRNAs), microRNAs (miRNAs), and/or ribozymes (such as hammerhead ribozymes and hairpin ribozymes) that can provide treatment for disorders by highly specific elimination or reduction of mRNA encoding abnormal and/or redundant proteins.

[0138] In some embodiments, a therapeutic polypeptide or therapeutic nucleic acid is used to treat a CNS disorder. Without wishing to be bound by any theory, it is believed that a therapeutic polypeptide or therapeutic nucleic acid can be used to reduce or eliminate the expression and/or activity of a polypeptide whose gain-of-function mutation has been associated with the disorder, or to enhance the expression and/or activity of a replenishing polypeptide. a deficiency that has been associated with a disorder (eg, mutation of a gene whose expression exhibits a similar or related activity). Non-limiting examples of disorders of the present invention that can be treated with a therapeutic polypeptide or therapeutic nucleic acid of the present invention (illustrative genes that can be targeted or repaired are given in brackets for each disorder) include stroke (e.g., caspase- 3, Beclin1, Ask1, PAR1, HIF1α, PUMA and/or any of the genes described in Fukuda, A.M. and Badaut, J. (2013) Genes (Basel) 4:435-456), Huntington's disease (HTT mutant), epilepsy (e.g., SCN1A, NMDAR, ADK, and/or any of the genes described in Boison, D. (2010) Epilepsia 51:1659-1668), Parkinson's disease (alpha-synuclein gene), Lou Gehrig's disease (also known as amyotrophic lateral multiple sclerosis; SOD1), Alzheimer's disease (tau, amyloid precursor protein genes), corticobasal degeneration or CBD (tau protein gene), corticobasal ganglion degeneration or CBGD (tau protein gene), frontotemporal dementia or FTD ( tau- protein), progressive supranuclear palsy or PSP (tau protein gene), multiple system atrophy or MSA (alpha synuclein gene), brain cancer (eg, a mutated or overexpressed oncogene involved in brain cancer), and lysosomal storage diseases (LSD ). Disorders of the present invention may include disorders involving large areas of the cerebral cortex, for example, several functional areas of the cerebral cortex, several lobes of the cerebral cortex and/or the entire cerebral cortex. Other non-limiting examples of disorders of the present invention that can be treated with a therapeutic polypeptide or therapeutic nucleic acid of the present invention include traumatic brain injury, disorders involving enzymatic dysfunction, mental disorders (including post-traumatic stress syndrome), neurodegenerative diseases, and cognitive impairment (including forms of dementia, autism and depression). Disorders involving enzymatic dysfunction include, without limitation, forms of leukodystrophy (including Canavan disease) and any of the lysosomal storage diseases described below.

[0139] In some embodiments, the therapeutic polypeptide or therapeutic nucleic acid is used to treat lysosomal storage disease. As is well known in the art, lysosomal storage diseases are rare hereditary metabolic disorders characterized by defects in lysosome function. Such disorders are often caused by a deficiency of an enzyme necessary for the proper metabolism of mucopolysaccharides, glycoproteins and/or lipids, which leads to an abnormal accumulation of cellular materials stored in lysosomes. Non-limiting examples of lysosomal storage diseases of the present invention that can be treated with a therapeutic polypeptide or therapeutic nucleic acid of the present invention (illustrative genes that can be targeted or repaired are given in brackets for each disorder) include type 2 Gaucher disease. or 3 types (beta-glucosidase gene, GBA), GM1 gangliosidosis (beta-galactosidase-1 gene, GLB1), Hunter disease (iduronate-2-sulfatase gene, IDS), Krabbe disease (galactosylceramidase gene, GALC), α-mannosidosis (mannosidase gene such as alpha-D-mannosidase, MAN2B1), β-mannosidosis disease (beta-mannosidase gene, MANBA), metachromatic leukodystrophy disease (pseudoarylsulfatase A gene, ARSA) , a disease that is type II/III mucolipidosis (N-acetylglucosamine-1-phosphotransferase gene, GNPTAB), Niemann-Pick type A disease ( acid sphingomyelinase gene, ASM), Niemann-Pick disease type C (gene for the protein associated with Niemann-Pick disease type C, NPC1), Pompe disease (gene for acid alpha-1,4-glucosidase, GAA), Sandhoff disease (beta gene -subunits of hexosaminidase, HEXB), Sanfilippo disease type A (N-sulfoglucosamine sulfohydrolase gene, MPS3A), Sanfilippo type B disease (N-alpha-acetylglucosaminidase gene, NAGLU), Sanfilippo type C disease (heparin-acetyl-CoA-alpha-glucosaminide gene -N-acetyltransferase, MPS3C), Sanfilippo type D disease (N-acetylglucosamine-6-sulfatase gene, GNS), Schindler's disease (alpha-N-acetylgalactosaminidase gene, NAGA), Sly disease (beta-glucuronidase gene, GUSB), Tay-Sachs (hexosaminidase alpha subunit gene, HEXA) and Wolman's disease (lysosomal acid lipase gene, LIPA).

[0140] Additional lysosomal storage diseases, as well as defective enzymes associated with each disease, are listed in Table 1 below. In some embodiments, the disease identified in the table below is treated with a therapeutic polypeptide or therapeutic nucleic acid of the present invention that repairs or otherwise compensates for the corresponding enzyme defect.

Table 1. Lysosomal storage disorders and defective enzymes associated with them

Lysosomal storage disease Defective enzyme Aspartylglucosaminuria Aspartylglucosaminidase Fabry disease Alpha-galactosidase A Infantile form of Batten disease (CNL1) Palmitoylprotein thioesterase Classic late infantile form of Batten disease (CNL2) tripeptidyl peptidase Juvenile Batten disease (CNL3) Lysosomal transmembrane protein Batten disease, other forms (CNL4-CNL8) Several gene products cystinosis Cysteine transporter Farber's disease Acid ceramidase Fucosidosis Acidic alpha-L-fucosidase Galactosialidosis Protective protein/cathepsin A Gaucher disease types 1, 2 and 3 Acid beta-glucosidase GM1 gangliosidosis Acid beta-galactosidase Hunter disease Iduronate-2-sulfatase Hurler-Scheie disease Alpha-L-iduronidase Krabbe disease Galactocerebrosidase alpha-mannosidosis Acid alpha-mannosidase Beta-mannosidosis Acid beta-mannosidase Maroto-Lami disease Arylsulfatase B Metachromatic leukodystrophy Arylsulfatase A Morquio disease type A N-acetylgalactosamine-6-sulfatase Morquio disease type B Acid beta-galactosidase Mucolipidosis type II/III N-acetylglucosamine-1-phosphotransferase Niemann-Pick disease type A, B Acid sphingomyelinase Niemann-Pick disease type C NPC-1 Pompe disease Alpha glucosidase Sandhoff disease Beta-hexosaminidase B Sanfilippo disease type A Heparan-N-sulfatase Sanfilippo disease type B Alpha-N-acetylglucosaminidase Sanfilippo disease type C Acetyl-CoA-alpha-glucosaminide-N-acetyltransferase Sanfilippo disease type D N-acetylglucosamine-6-sulfatase Schindler's disease Alpha-N-acetylgalactosaminidase Kanzaki-Schindler disease Alpha-N-acetylgalactosaminidase Sialidosis Alpha neuraminidase Sly disease Beta-glucuronidase Tay-Sachs disease Beta-hexosaminidase A Wolman's disease acid lipase

[0141] In some embodiments, the heterologous nucleic acid is operably linked to a promoter. Exemplary promoters include, without limitation, cytomegalovirus (CMV) immediate-early promoter, RSV LTR, MoMLV LTR, phosphoglycerate kinase-1 (PGK) gene promoter, simian virus 40 (SV40) and CK6 promoter, transthyretin (TTR) gene promoter, TK promoter, tetracycline sensitive promoter (TRE), HBV promoter, hAAT promoter, LSP promoter, chimeric liver specific promoters (LSP), E2F promoter, telomerase gene promoter (hTERT); a composite cytomegalovirus enhancer promoter/chicken beta-actin gene promoter/rabbit β-globin gene intron (CAG promoter; Niwa et al., Gene, 1991, 108(2):193-9) and elongation factor 1-alpha (EF1- alpha) (Kim et al., Gene, 1990, 91(2):217-23 and Guo et al., Gene Ther., 1996, 3(9):802-10). In some embodiments, the promoter comprises a human β-glucuronidase promoter or a promoter from a cytomegalovirus enhancer coupled to a chicken β-actin (CBA) promoter. The promoter may be a constitutive, inducible or repressible promoter. In some embodiments, the present invention provides a recombinant vector containing a nucleic acid encoding a heterologous transgene of the present invention operably linked to a CBA promoter. Illustrative promoters and descriptions thereof can be found, for example, in US Patent Application No. 20140335054 published prior to the grant of the patent.

[0142] Examples of constitutive promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with an RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with a CMV enhancer) [see, e.g., Boshart et al., Cell, 41:521-530 (1985)], SV40 promoter, dihydrofolate reductase gene promoter, 13-actin gene promoter, phosphoglycerol kinase (PGK) gene promoter, and EF1a promoter [Invitrogen].

[0143] Inducible promoters allow the regulation of gene expression, and they can be regulated by exogenously administered compounds, environmental factors such as temperature, or the presence of a particular physiological state, such as an acute phase, a certain state of cell differentiation, or function only in proliferating cells. Inducible promoters and inducible systems are available from a number of commercial sources including, but not limited to, Invitrogen, Clontech, and Ariad. Many other systems have been described and can be easily selected by a person skilled in the art. Examples of inducible promoters regulated by exogenously provided compounds include the zinc inducible ovine metallothionein (MT) gene promoter, the dexamethasone (Dex) inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (WO 98/10088); ecdysone-inducible insect gene promoter (No et al., Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)), tetracycline-dependent repression system (Gossen et al., Proc. Natl. Acad. Sci USA, 89:5547-5551 (1992)), a tetracycline-dependent induction system (Gossen et al., Science, 268:1766-1769 (1995), see also Harvey et al., Curr. Opin. Chem. Biol., 2:512-518 (1998)), RU486 dependent induction system (Wang et al., Nat. Biotech., 15:239-243 (1997) and Wang et al., Gene Ther., 4: 432-441 (1997)) and a rapamycin-dependent induction system (Magari et al., J. Clin. Invest., 100:2865-2872 (1997)). Several other types of inducible promoters that may be applicable in this context are promoters that are regulated by a specific physiological state, such as temperature, acute phase, a certain state of cell differentiation, or function only in proliferating cells.

[0144] In another embodiment, a native promoter or fragment thereof will be used for the transgene. A native promoter can be used if the expression of the transgene is required to mimic the expression of the native gene. A native promoter can be used in cases where the expression of the transgene is to be regulated in time, or depending on the stage of development, or in a tissue-specific manner, or in response to specific transcriptional stimuli. In a further embodiment, other native expression control elements, such as enhancer elements, polyadenylation sites, or Kozak consensus sequences, can also be used to mimic native gene expression.

[0145] In some embodiments, the regulatory sequences confer the ability for tissue-specific gene expression. In some cases, tissue-specific regulatory sequences bind tissue-specific transcription factors that induce transcription in a tissue-specific manner. Such tissue-specific regulatory sequences (eg, promoters, enhancers, etc.) are well known in the art.

[0146] In some embodiments, the implementation of the vector contains an intron. For example, in some embodiments, the intron is a chimeric intron derived from the chicken beta actin and rabbit beta globin gene. In some embodiments, the intron is a mouse small virus (MVM) intron.

[0147] In some embodiments, the vector contains a polyadenylation sequence (poly(A)). Numerous examples of polyadenylation sequences are known in the art, such as the bovine growth hormone (BGH) poly(A) sequence (see, for example, accession number EF592533), the SV40 polyadenylation sequence, and the pA TK HSV polyadenylation sequence.

V. Virus Particles and Methods for Producing Virus Particles

[0148] Certain aspects of the present invention relate to recombinant viral particles (eg, rAAV particles).

[0149] Based on the guidance provided herein, the techniques of the present invention may be appropriately adapted by one of skill in the art for use with a variety of different viruses.

[0150] In some embodiments, the virus belongs to the Adenoviridae family, which includes non-enveloped viruses commonly known as adenoviruses. In some embodiments, the virus is genus Atadenovirus, Aviadenovirus, Ichtadenovirus, Mastadenovirus, or Siadenovirus.

[0151] In some embodiments, the virus belongs to the Parvoviridae family, which includes nonenveloped viruses such as AAV and bocaparvovirus. In some embodiments, the implementation of the virus belongs to the subfamily Densovirinae. In some embodiments, the virus is of the genus Ambidensovirus, Brevidensovirus, Hepandensovirus, Iteradensovirus, or Penstyldensovirus. In some embodiments, the implementation of the virus belongs to the subfamily Parvovirinae. In some embodiments, the virus is of the genus Amdoparvovirus, Aveparvovirus, Bocaparvovirus, Copiparvovirus, Dependoparvovirus, Erythroparvovirus, Protoparvovirus, or Tetraparvovirus.

[0152] In some embodiments, the implementation of the virus belongs to the family Retroviridae, which includes enveloped viruses, including lentivirus. In some embodiments, the implementation of the virus belongs to the subfamily Orthoretrovirinae. In some embodiments, the virus is of the genus Alpharetrovirus, Betaretrovirus, Deltaretrovirus, Epsilonretrovirus, Gammaretrovirus, or Lentivirus. In some embodiments, the implementation of the virus belongs to the subfamily Spumaretrovirinae. In some embodiments, the implementation of the virus belongs to the genus Spumavirus.

[0153] In some embodiments, the virus belongs to the Baculoviridae family, which includes enveloped viruses, including alpha baculovirus. In some embodiments, the virus is of the genus Alphabaculovirus, Betabaculovirus, Deltabaculovirus, or Gammabaculovirus.

[0154] In some embodiments, the virus belongs to the Herpesviridae family, which includes enveloped viruses such as herpes simplex viruses HSV-1 and HSV-2. In some embodiments, the implementation of the virus belongs to the subfamily Alphaherpesvirinae. In some embodiments, the virus is of the genus Iltovirus, Mardivirus, Simplexvirus, or Varicellovirus. In some embodiments, the implementation of the virus belongs to the subfamily Betaherpesvirinae. In some embodiments, the virus is of the genus Cytomegalovirus, Muromegalovirus, Proboscivirus, or Roseolovirus. In some embodiments, the implementation of the virus belongs to the subfamily Gammaherpesvirinae. In some embodiments, the virus is of the genus Lymphocryptovirus, Macavirus, Percavirus, or Rhadinovirus.

[0155] In some embodiments, the virus is an AAV virus. In the case of the AAV particle, the nucleic acid is enclosed in the capsid of the AAV particle. The AAV particle also contains capsid proteins. In some embodiments, the nucleic acid comprises a heterologous nucleic acid and/or one or more of the following: components operably linked in the direction of transcription, control sequences, including initiation and termination sequences, thereby allowing the formation of an expression cassette.

[0156] In some embodiments, the viral particle contains an AAV ITR sequence. For example, an expression cassette can be flanked at the 5' and 3' ends with at least one functional AAV ITR sequence. By "functional AAV ITR sequences" is meant that the ITR sequences function as putative for rescue, replication and packaging of the AAV virion. See Davidson et al., PNAS, 2000, 97(7)3428-32; Passini et al., J. Virol., 2003, 77(12):7034-40; and Pechan et al., Gene Ther., 2009, 16:10-16, all of which are incorporated herein by reference in their entirety. To practice some aspects of the present invention, the recombinant vectors contain at least all of the AAV sequences required to be enclosed in a capsid and the physical structures for infection with rAAV. AAV ITRs for use in the vectors of the present invention must not have a wild-type nucleotide sequence (e.g., as described in Kotin, Hum. Gene Ther., 1994, 5:793-801), and they can be altered by insertion, deletion, or substitution of nucleotides , or ITR AAV can be derived from any of several AAV serotypes. To date, over 40 AAV serotypes are known, and new serotypes and variants of existing serotypes continue to be identified. See Gao et al., PNAS, 2002, 99(18): 11854-6; Gao et al., PNAS, 2003, 100(10):6081-6; and Bossis et al., J. Virol., 2003, 77(12):6799-810. The use of any AAV serotype is considered to be within the scope of the present invention. In some embodiments, the rAAV-based vector is a vector derived from an AAV serotype, including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV LK03, AAV2R471A, AAV DJ, AAV DJ8, goat AAV, bovine AAV, or mouse AAV ITR, or the like. , AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV LK03, AAV2R471A, AAV DJ, AAV DJ8, AAV goat, AAV bovine or ITR AAV mouse or etc. In some embodiments, the AAV particle comprises an AAV-based vector encoding a heterologous transgene flanked by one or more AAV ITRs.

[0157] In some embodiments, the rAAV particle comprises an encapsulated protein selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6 (e.g., a wild-type AAV6 capsid or a variant AAV6 capsid, such as ShH10 as described in the US patent application 2012/0164106, pre-grant), AAV7, AAV8, AAVrh8, AAVrh8R, AAV9 (e.g., wild-type AAV9 capsid or modified AAV9 capsid as described in U.S. Patent Application Publication 2013/0323226, published pre-grant), AAV10, AAVrh10, AAV11, AAV12, tyrosine-mutant capsid, heparin-binding mutant capsid, AAV2R471A capsid, AAVAAV2/2-7m8 capsid, AAV LK03 capsid, AAV DJ capsid (e.g. AAV-DJ/8 capsid, AAV-DJ/ capsid 9 or any other of the capsids described in U.S. Patent Application 2012/0066783 published prior to the grant of the patent), AAV2 capsid N587A, AAV2 capsid E548A, AAV2 capsid N708A, AAV capsid V708K, goat AAV capsid, AAV1/AAV2 chimeric capsid, capsid AAV large horn bovine, mouse AAV capsid, rAAV2/HBoV1 capsid, AAV2HBKO capsid, AAVPHP.B capsid or AAVPHP.eB capsid or AAV capsid as described in US Pat. No. 8,283,151 or International Publication No. WO/2003/042397. In further embodiments, the rAAV particle comprises AAV serotype capsid proteins from clades A-F.

[0158] Certain aspects of the present invention relate to an AAV capsid protein (e.g., rAAV) containing an amino acid substitution at amino acid residue 2. In some embodiments, the amino acid substitution at amino acid residue 2 provides a change in N-terminal acetylation compared to N-terminal acetylation at amino acid residue 2 of the original AAV capsid protein. As described herein, the amino acid at position 2 to the initiating methionine (iMet X) of the AAV capsid protein can be investigated for effects on N-terminal acetylation migration, transduction and/or other post-translational modification(s). (modifications) (eg, glycosylation, ubiquitation, etc.). Any assay described herein to investigate acetylation or its implications for functions associated with AAV particles can be used to evaluate N-terminal acetylation. In some embodiments, amino acid residue 2 of the AAV capsid protein (e.g., VP1 or VP3) is replaced by Cys, Ser, Thr, Val, Gly, Asn, Asp, Glu, Ile, Leu, Phe, Gln, Lys, Met, Pro, or Tyr. In some embodiments, the amino acid substitution results in less deamidation of the AAV capsid.

[0159] Other aspects of the present disclosure relate to an AAV capsid protein (eg, rAAV) containing an amino acid substitution that provides a change in deamidation. In some embodiments, an amino acid substitution (e.g., an amino acid substitution in A35 VP1, N57 VP1, G58 VP1, N382 VP3, G383 VP3, N511 VP3, G512 VP3, N715 VP3, or G716 VP3) that provides a "change" in deamidation results in more a high rate of deamidation; or a lower rate of deamidation, eg compared to VP1 or VP3 without replacement, such as the original VP1 or VP3. As described herein, a potential deamidation site for an AAV capsid protein (e.g., VP1 or VP3) can be investigated for effects on deamidation, migration, transduction, and/or other post-translational modification(s) (e.g., glycosylation, ubiquitation, etc.). Any assay described herein to investigate deamidation or its implications for functions associated with AAV particles can be used to evaluate deamidation.

[0160] Several potential deamidation sites are described in this document. In some embodiments, the amino acid substitution that provides the change in deamidation is selected from A35 VP1, N57 VP1, G58 VP1, N382 VP3, G383 VP3, N511 VP3, G512 VP3, N715 VP3, or G716 VP3. For example, in some embodiments, N57 VP1, N382 VP3, N511 VP3, and/or N715 VP3 are replaced with Asp and the amino acid substitution results in a higher frequency of deamidation compared to deamidation of VP1 and/or VP3 of the original AAV particle. In other embodiments, the amino acid substitution is N57K or N57Q and the amino acid substitution results in a lower frequency of deamidation compared to deamidation of VP1 and/or VP3 of the original AAV particle. In still other embodiments, G58 VP1, G383 VP3, G512 VP3, and/or G716 VP3 are replaced with an amino acid that is not Gly (e.g., Ala, Arg, Asn, Asp, Cys, Glu, Gln, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val), and the amino acid substitution results in a lower frequency of deamidation compared to deamidation of VP1 and/or VP3 of the original AAV particle.

[0161] In some embodiments, the AAV capsid protein is VP1, VP2, or VP3. The AAV particle may comprise any of the exemplary AAV capsid serotypes described herein, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV LK03 . The AAV capsid protein may further comprise any of the capsid protein mutations described herein, such as tyrosine mutations and/or mutations affecting heparin binding.

[0162] Other aspects of the present invention relate to methods for improving the stability of the rAAV particle. In some embodiments, the implementation of the methods include the implementation of the replacement of amino acid residue 2 VP1 and/or VP3, for example, as described in this document. For example, in some embodiments, amino acid residue 2 of VP1 is substituted. In other embodiments, VP3 amino acid residue 2 is substituted. In some embodiments, the substituted amino acid at position 2 undergoes N-acetylation at a higher frequency than amino acid residue 2 of the original VP1 and/or VP3, for example, as described herein. In some embodiments, the replacement of amino acid residue 2 of VP1 and/or VP3 provides an improvement in the stability of the rAAV particle by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95 % or at least about 100%. In some embodiments, the stability of an rAAV particle with a changed amino acid at position 2 can be compared to a wild-type AAV capsid or a native AAV capsid, for example, of the same serotype. For example, in some embodiments, the replacement of amino acid residue 2 of VP1 and/or VP3 provides an improvement in the stability of the rAAV particle by any amount from about 10% to about 100%, from about 20% to about 100%, from about 30% to about 100%, from about 40% to about 100%, from about 50% to about 100%, from about 60% to about 100%, from about 70% to about 100%, from about 80% to about 100%, from about 90% to about 100%, from about 10% to about 90%, from about 20% to about 90%, from about 30% to about 90%, from about 40% to about 90%, from about 50% to about 90%, from about 60% to about 90%, about 70% to about 90%, about 80% to about 90%, about 10% to about 80%, about 20% up to about 80%, from about 30% to about 80%, from about 40% to about 80%, from about 50% to about 80%, from about 60% to about 80%, from about 70% to about 80%, from about 10% to about 70%, from about 20% to about 70%, from about 30% to about 70%, from about 40% to about 70%, from about 50% to about 70%, from about 60% to about 70%, about 10% to about 60%, about 20% to about 60%, about 30% to about 60%, about 40% to about 60%, about 50% to about 60%, from about 10% to about 50%, about 20% to about 50%, about 30% to about 50%, about 40% to about 50%, about 10% to about 40%, about 20% to about positively 40%, from about 30% to about 40%, from about 10% to about 30%, from about 20% to about 30%, or from about 10% to about 20%, for example, compared with the stability of an rAAV particle containing wild-type capsid. The stability of the AAV particle can be measured using a variety of assays known in the art, including, but not limited to, differential scanning fluorimetry (DSF), differential scanning calorimetry (DSC), other thermal denaturation assays, proteolysis susceptibility, imaging, or structural analysis with denaturation observation (e.g., using an electron microscope), transduction efficiency, or other functional assay for compositions based on AAV particles stored for a certain period of time at a certain temperature (e.g., room temperature or 4°C in the case of thermal stability) or processed at a certain value pH (e.g. stability depending on pH values), etc.

[0163] Other aspects of the present invention relate to methods for improving the assembly of a rAAV particle. In some embodiments, the implementation of the methods include the implementation of the replacement of amino acid residue 2 VP1 and/or VP3, for example, as described in this document. For example, in some embodiments, amino acid residue 2 of VP1 is substituted. In other embodiments, VP3 amino acid residue 2 is substituted. In some embodiments, the substituted amino acid at position 2 undergoes N-acetylation at a higher frequency than amino acid residue 2 of the original VP1 and/or VP3, for example, as described herein. In some embodiments, the replacement of amino acid residue 2 of VP1 and/or VP3 provides an improvement in assembly of the rAAV particle by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95 % or at least about 100%. In some embodiments, the assembly of an rAAV particle with an amino acid substituted at position 2 can be compared to a wild-type AAV capsid or a parental AAV capsid, for example, of the same serotype. For example, in some embodiments, the replacement of amino acid residue 2 of VP1 and/or VP3 provides an improvement in assembly of the rAAV particle by any amount from about 10% to about 100%, from about 20% to about 100%, from about 30% to about 100%, from about 40% to about 100%, from about 50% to about 100%, from about 60% to about 100%, from about 70% to about 100%, from about 80% to about 100%, from about 90% to about 100%, from about 10% to about 90%, from about 20% to about 90%, from about 30% to about 90%, from about 40% to about 90%, from about 50% to about 90%, from about 60% to about 90%, about 70% to about 90%, about 80% to about 90%, about 10% to about 80%, about 20% to about about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 60% to about 80%, about 70% to about 80%, from about 10% to about 70%, about 20% to about 70%, about 30% to about 70%, about 40% to about 70%, about 50% to about 70%, about 60% to about 70%, about 10% to about 60%, about 20% to about 60%, about 30% to about 60%, about 40% to about 60%, about 50% to about 60%, about 10% to about 50%, about 20% to about 50%, about 30% to about 50%, about 40% to about 50%, about 10% to about 40%, about 20% to approx. but 40%, about 30% to about 40%, about 10% to about 30%, about 20% to about 30%, or about 10% to about 20%, for example, compared to an rAAV particle assembly containing wild-type capsid. Assembly of an AAV particle can be measured using a variety of assays known in the art, including, but not limited to, measuring the number and/or rate of particle formation, quantifying capsid formation (e.g., after purification using any of the methods described herein), assaying full capsid formation with vectors versus empty capsids, measuring transduction efficiency, visualization or structural analysis to observe particle formation (e.g. using an electron microscope), formation of AAV capsid proteins (e.g. analyzed by Western blotting), etc. .P.

[0164] Other aspects of the present invention relate to methods for improving the transduction of the rAAV particle. In some embodiments, the implementation of the methods include the implementation of the replacement of amino acid residue 2 VP1 and/or VP3, for example, as described in this document. For example, in some embodiments, amino acid residue 2 of VP1 is substituted. In other embodiments, VP3 amino acid residue 2 is substituted. In some embodiments, the substituted amino acid at position 2 undergoes N-acetylation at a higher frequency than amino acid residue 2 of the original VP1 and/or VP3, for example, as described herein. In some embodiments, the replacement of amino acid residue 2 of VP1 and/or VP3 provides an improvement in transduction of the rAAV particle by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95 % or at least about 100%. In some embodiments, transduction of an rAAV particle with an amino acid substituted at position 2 can be compared to a wild-type AAV capsid or a native AAV capsid, eg, of the same serotype. For example, in some embodiments, the replacement of amino acid residue 2 of VP1 and/or VP3 provides an improvement in the transduction of the rAAV particle by any amount from about 10% to about 100%, from about 20% to about 100%, from about 30% to about 100%, from about 40% to about 100%, from about 50% to about 100%, from about 60% to about 100%, from about 70% to about 100%, from about 80% to about 100%, from about 90% to about 100%, from about 10% to about 90%, from about 20% to about 90%, from about 30% to about 90%, from about 40% to about 90%, from about 50% to about 90%, from about 60% to about 90%, about 70% to about 90%, about 80% to about 90%, about 10% to about 80%, about 20% up to about 80%, from about 30% to about 80%, from about 40% to about 80%, from about 50% to about 80%, from about 60% to about 80%, from about 70% to about 80%, from about 10% to about 70%, from about 20% to about 70%, from about 30% to about 70%, from about 40% to about 70%, from about 50% to about 70%, from about 60% to about 70%, about 10% to about 60%, about 20% to about 60%, about 30% to about 60%, about 40% to about 60%, about 50% to about 60%, from about 10% to about 50%, about 20% to about 50%, about 30% to about 50%, about 40% to about 50%, about 10% to about 40%, about 20% to about 40%, about 30% to about 40%, about 10% to about 30%, about 20% to about 30%, or about 10% to about 20%, for example, compared to transduction of an rAAV particle containing wild-type capsid. Transduction of the AAV particle can be measured using a variety of assays known in the art, including, but not limited to, the transduction efficiency assays described herein. In some embodiments, the present invention provides methods for reducing transduction of an rAAV particle; for example, by replacing amino acid residue 2 of VP1 and/or VP3.

[0165] Other aspects of the present invention relate to methods for improving the stability of the rAAV particle. In some embodiments, the methods involve performing a VP1 and/or VP3 amino acid substitution that provides a change in deamidation, for example, as described herein. For example, in some embodiments, A35 VP1, N57 VP1, G58 VP1, N382 VP3, G383 VP3, N511 VP3, G512 VP3, N715 VP3, or G716 VP3 are substituted. In some embodiments, the substituted amino acid undergoes deamidation at a higher frequency than the amino acid residue of the original VP1 and/or VP3, for example, as described herein. In some embodiments, substitution of A35 VP1, N57 VP1, G58 VP1, N382 VP3, G383 VP3, N511 VP3, G512 VP3, N715 VP3, and/or G716 VP3 provides an improvement in the stability of the rAAV particle by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45 %, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80% , at least about 85%, at least about 90%, at least about 95%, or at least about 100%. In some embodiments, the stability of an rAAV particle with A35 VP1, N57 VP1, G58 VP1, N382 VP3, G383 VP3, N511 VP3, G512 VP3, N715 VP3, or G716 VP3 substituted can be compared to a wild-type AAV capsid or a parent AAV capsid, e.g., one and the same serotype. For example, in some embodiments, substitution of A35 VP1, N57 VP1, G58 VP1, N382 VP3, G383 VP3, N511 VP3, G512 VP3, N715 VP3, and/or G716 VP3 provides an improvement in the stability of the rAAV particle by any amount from about 10% to about 100 %, from about 20% to about 100%, from about 30% to about 100%, from about 40% to about 100%, from about 50% to about 100%, from about 60% to about 100%, from about 70 % to about 100%, about 80% to about 100%, about 90% to about 100%, about 10% to about 90%, about 20% to about 90%, about 30% to about 90% , from about 40% to about 90%, from about 50% to about 90%, from about 60% to about 90%, from about 70% to about 90%, from about 80% to about 90%, from approx. but 10% to about 80%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 60% to about 80%, about 70% to about 80%, about 10% to about 70%, about 20% to about 70%, about 30% to about 70%, about 40% to about 70%, from about 50% to about 70%, about 60% to about 70%, about 10% to about 60%, about 20% to about 60%, about 30% to about 60%, about 40% to about 60 %, from about 50% to about 60%, from about 10% to about 50%, from about 20% to about 50%, from about 30% to about 50%, from about 40% to about 50%, from about 10 % before about 40%, about 20% to about 40%, about 30% to about 40%, about 10% to about 30%, about 20% to about 30%, or about 10% to about 20%, e.g. compared to the stability of the rAAV particle containing the wild-type capsid. The stability of the AAV particle can be measured using a variety of assays known in the art, including, but not limited to, differential scanning fluorimetry (DSF), differential scanning calorimetry (DSC), other thermal denaturation assays, proteolysis susceptibility, imaging, or structural analysis with denaturation observation (e.g., using an electron microscope), transduction efficiency, or other functional assay for compositions based on AAV particles stored for a certain period of time at a certain temperature (e.g., room temperature or 4°C in the case of thermal stability) or processed at a certain value pH (e.g. stability depending on pH values), etc.

[0166] Other aspects of the present invention relate to methods for improving the assembly of a rAAV particle. In some embodiments, the methods involve performing a VP1 and/or VP3 amino acid substitution that provides a change in deamidation, for example, as described herein. For example, in some embodiments, A35 VP1, N57 VP1, G58 VP1, N382 VP3, G383 VP3, N511 VP3, G512 VP3, N715 VP3, or G716 VP3 are substituted. In some embodiments, the substituted amino acid undergoes deamidation at a higher frequency than the amino acid residue of the original VP1 and/or VP3, for example, as described herein. In some embodiments, substitution of A35 VP1, N57 VP1, G58 VP1, N382 VP3, G383 VP3, N511 VP3, G512 VP3, N715 VP3, and/or G716 VP3 provides an improvement in assembly of the rAAV particle by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45 %, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80% , at least about 85%, at least about 90%, at least about 95%, or at least about 100%. In some embodiments, the stability of an rAAV particle with A35 VP1, N57 VP1, G58 VP1, N382 VP3, G383 VP3, N511 VP3, G512 VP3, N715 VP3, or G716 VP3 substituted can be compared to a wild-type AAV capsid or a parent AAV capsid, e.g., one and the same serotype. For example, in some embodiments, substitution of A35 VP1, N57 VP1, G58 VP1, N382 VP3, G383 VP3, N511 VP3, G512 VP3, N715 VP3, and/or G716 VP3 provides an improvement in assembly of the rAAV particle by any amount from about 10% to about 100 %, from about 20% to about 100%, from about 30% to about 100%, from about 40% to about 100%, from about 50% to about 100%, from about 60% to about 100%, from about 70 % to about 100%, about 80% to about 100%, about 90% to about 100%, about 10% to about 90%, about 20% to about 90%, about 30% to about 90% , about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, about 80% to about 90%, about 10% up to about 80%, from about 20% to about 80%, from about 30% to about 80%, from about 40% to about 80%, from about 50% to about 80%, from about 60% to about 80%, from about 70% to about 80%, from about 10% to about 70%, from about 20% to about 70%, from about 30% to about 70%, from about 40% to about 70%, from about 50% to about 70%, about 60% to about 70%, about 10% to about 60%, about 20% to about 60%, about 30% to about 60%, about 40% to about 60%, from about 50% to about 60%, about 10% to about 50%, about 20% to about 50%, about 30% to about 50%, about 40% to about 50%, about 10% to about significantly 40%, about 20% to about 40%, about 30% to about 40%, about 10% to about 30%, about 20% to about 30%, or about 10% to about 20%, e.g. compared to an rAAV particle assembly containing a wild-type capsid. Assembly of an AAV particle can be measured using a variety of assays known in the art, including, but not limited to, measuring the number and/or rate of particle formation, quantifying capsid formation (e.g., after purification using any of the methods described herein), assaying full capsid formation with vectors versus empty capsids, measuring transduction efficiency, visualization or structural analysis to observe particle formation (e.g. using an electron microscope), formation of AAV capsid proteins (e.g. analyzed by Western blotting), etc. .P.

[0167] Other aspects of the present invention relate to methods for improving the transduction of the rAAV particle. In some embodiments, the methods involve performing a VP1 and/or VP3 amino acid substitution that provides a change in deamidation, for example, as described herein. For example, in some embodiments, A35 VP1, N57 VP1, G58 VP1, N382 VP3, G383 VP3, N511 VP3, G512 VP3, N715 VP3, or G716 VP3 are substituted. In some embodiments, the substituted amino acid undergoes deamidation at a higher frequency than the amino acid residue of the original VP1 and/or VP3, for example, as described herein. In some embodiments, substitution of A35 VP1, N57 VP1, G58 VP1, N382 VP3, G383 VP3, N511 VP3, G512 VP3, N715 VP3, and/or G716 VP3 provides an improvement in transduction of the rAAV particle by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45 %, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80% , at least about 85%, at least about 90%, at least about 95%, or at least about 100%. In some embodiments, the stability of an rAAV particle with A35 VP1, N57 VP1, G58 VP1, N382 VP3, G383 VP3, N511 VP3, G512 VP3, N715 VP3, or G716 VP3 substituted can be compared to a wild-type AAV capsid or a parent AAV capsid, e.g., one and the same serotype. For example, in some embodiments, substitution of A35 VP1, N57 VP1, G58 VP1, N382 VP3, G383 VP3, N511 VP3, G512 VP3, N715 VP3, and/or G716 VP3 provides an improvement in rAAV particle transduction by any amount from about 10% to about 100 %, from about 20% to about 100%, from about 30% to about 100%, from about 40% to about 100%, from about 50% to about 100%, from about 60% to about 100%, from about 70 % to about 100%, about 80% to about 100%, about 90% to about 100%, about 10% to about 90%, about 20% to about 90%, about 30% to about 90% , from about 40% to about 90%, from about 50% to about 90%, from about 60% to about 90%, from about 70% to about 90%, from about 80% to about 90%, from about 10% to about 80%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 60% to about 80%, about 70% to about 80%, about 10% to about 70%, about 20% to about 70%, about 30% to about 70%, about 40% to about 70%, from about 50% to about 70%, about 60% to about 70%, about 10% to about 60%, about 20% to about 60%, about 30% to about 60%, about 40% to about 60 %, from about 50% to about 60%, from about 10% to about 50%, from about 20% to about 50%, from about 30% to about 50%, from about 40% to about 50%, from about 10 % to p about 40%, about 20% to about 40%, about 30% to about 40%, about 10% to about 30%, about 20% to about 30%, or about 10% to about 20%, for example compared to transduction of an rAAV particle containing a wild-type capsid. Transduction of the AAV particle can be measured using a variety of assays known in the art, including, but not limited to, the transduction efficiency assays described herein.

[0168] In some aspects, the present invention provides viral particles containing a recombinant self-complementary genome (eg, a self-complementary or self-complementing rAAV-based vector). AAV virus particles with self-complementary vector genomes and methods for using self-complementary AAV genomes are described in US Patent Nos. 6,596,535; 7125717; 7 465 583; 7785888; 7790154; 7846729; 8093054 and 8361457; and Wang Z., et al., (2003) Gene Ther 10:2105-2111, each of which is incorporated herein by reference in its entirety. An rAAV containing a self-complementary genome will quickly form a double-stranded DNA molecule due to its partially complementary sequences (eg, complementary coding and non-coding heterologous nucleic acid strands). In some embodiments, the vector comprises a first nucleic acid sequence encoding a heterologous nucleic acid and a second nucleic acid sequence encoding a complementary nucleic acid sequence, wherein the first nucleic acid sequence can form intrastrand base pairs with the second nucleic acid sequence along most or all of its length. .

[0169] In some embodiments, a first heterologous nucleic acid sequence and a second heterologous nucleic acid sequence are joined by a mutant ITR (eg, a right ITR). In some embodiments, the ITR comprises the polynucleotide sequence 5'-CACTCCCTCTGCGCGCTCGCTCGCTCACTGAGGCC GGGCGACCAAAGGTCGCCCACGCCCGGGCTTTGCCCGGGCG-3' (SEQ ID NO: 8). The mutant ITR is characterized by a deletion of the D region containing the end resolution sequence. As a result, upon replication of the AAV viral genome, the rep proteins will not cleave the viral genome at the site of the mutated ITR, and therefore the recombinant viral genome containing the following, in order from 5' to 3', will be packaged in the viral capsid: AAV ITR , a first heterologous polynucleotide sequence, including regulatory sequences, a mutant AAV ITR, a second heterologous polynucleotide in reverse orientation to the first heterologous polynucleotide, and a third AAV ITR.

[0170] Different AAV serotypes are used to optimize transduction of specific target cells or to target specific cell types in a specific target tissue (eg, diseased tissue). The rAAV particle may contain viral proteins and viral nucleic acids of the same serotype or mixed serotype. For example, a rAAV particle may contain one or more ITRs and a capsid derived from the same AAV serotype, or an rAAV particle may contain one or more ITRs derived from an AAV serotype different from the capsid serotype of the rAAV particle.

[0171] In some embodiments, the AAV capsid contains a mutation, for example, the capsid contains a mutated capsid protein. In some embodiments, the mutation is a tyrosine mutation or a mutation affecting heparin binding. In some embodiments, the mutant capsid protein retains the ability to form an AAV capsid. In some embodiments, the rAAV particle contains an AAV2 or AAV5 tyrosine-mutant capsid (see, e.g., Zhong L. et al., (2008) Proc Natl Acad Sci U S A 105(22):7827-7832), e.g. in Y444 or Y730 (numbering according to AAV2). In additional embodiments, the rAAV particle comprises AAV serotype capsid proteins from clades A-F (Gao, et al., J. Virol. 2004, 78(12):6381).

[0172] In some embodiments, the implementation of the capsid protein contains one or more amino acid substitutions at one or more positions that are involved in interaction with heparin sulfate proteoglycan, or at one or more positions corresponding to amino acids 484, 487, 527, 532, 585 or 588, when In this case, the numbering is given according to the VP1 AAV2 numbering. It is known in the art that heparan sulfate proteoglycan (HSPG) acts as a cellular receptor for AAV2 particles (Summerford, C. and Samulski, R.J. (1998) J. Virol. 72(2):1438-45). Binding between the AAV2 particle and HSPG on the cell membrane is necessary for the attachment of the particle to the cell. Other cell surface proteins, such as the fibroblast growth factor receptor and αvβ5 integrin, may also facilitate cell infection. Once bound, the AAV2 particle can enter the cell through mechanisms including receptor-mediated endocytosis via clathrin-coated pits. The AAV2 particle can be released from the endocytic vesicle upon endosomal acidification. This promotes the migration of the AAV2 particle to the perinuclear region and then to the cell nucleus. AAV3 particles are also known to bind to heparin (Rabinowitz, J. E., et al. (2002) J. Virol. 76(2):791-801).

[0173] Binding between AAV2 and HSPG capsid proteins is known to occur through electrostatic interactions between basic residues of AAV2 capsid proteins and negatively contaminated glycosaminoglycan residues (Opie, SR et al., (2003) J. Virol. 77:6995-7006; Kern, A et al., (2003) J. Virol 77:11072-11081). Specific capsid residues involved in these interactions include R484, R487, K527, K532, R585 and R588. Mutations at these residues have been shown to reduce AAV2 binding to Hela cells and heparin itself (Opie, SR et al., (2003) J. Virol. 77:6995-7006; Kern, A et al., (2003) J Virol 77:11072-11081; WO 2004/027019 A2 US Patent No. 7629322). In addition, without being bound by any theory, it is believed that by amino acid(s) substitution(s) at one or more residues corresponding to amino acids 484, 487, 527, 532, 585, or 588, which are numbered according to AAV2 VP1 numbering, one can modulate the transduction properties of AAV capsid types that do not bind to HSPG, or one can modulate the transduction properties of AAV capsid types regardless of their ability to bind to HSPG. In some embodiments, one or more amino acid substitutions include a substitution at position R484, R487, K527, K532, R585, and/or R588 of VP1, VP2, and/or VP3, which are numbered according to the AAV2 VP1 numbering.

[0174] In some embodiments, one or more amino acid substitutions reduce the binding of the rAAV particle to heparin sulfate proteoglycan by at least about 10%, at least about 25%, at least about 50%, at least about 75%, or about at least 100%. In some embodiments, the one or more amino acid substitutions reduce the binding of the rAAV particle to heparin sulfate proteoglycan by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%. %, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65% , at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% ( compared to binding to an rAAV particle containing a wild-type capsid). In some embodiments, one or more amino acid substitutions reduce the binding of the rAAV particle to heparin sulfate proteoglycan by any amount from about 10% to about 100%, from about 20% to about 100%, from about 30% to about 100%, from about 40% to about 100%, from about 50% to about 100%, from about 60% to about 100%, from about 70% to about 100%, from about 80% to about 100%, from about 90% to about 100%, from about 10% to about 90%, from about 20% to about 90%, from about 30% to about 90%, from about 40% to about 90%, from about 50% to about 90%, from about 60% to 90%, 70% to 90%, 80% to 90%, 10% to 80%, approx. about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 60% to about 80%, about 70% to about 80%, about 10% to about 70%, about 20% to about 70%, about 30% to about 70%, about 40% to about 70%, about 50% to about 70%, from about 60% to about 70%, about 10% to about 60%, about 20% to about 60%, about 30% to about 60%, about 40% to about 60%, about 50% to about 60 %, from about 10% to about 50%, from about 20% to about 50%, from about 30% to about 50%, from about 40% to about 50%, from about 10% to about 40%, from about 20% to about 40%, about 30% to about 40%, about 10% to about 30%, about 20% to about 30%, or about 10% to about 20% (versus rAAV particle binding, containing a wild-type capsid). In some embodiments, one or more amino acid substitutions result in no detectable binding of the rAAV particle to heparin sulfate proteoglycan compared to binding of the wild-type rAAV particle. Methods for measuring the binding of AAV particles to HSPG are known in the art; for example, binding to a heparin sulfate-based chromatographic medium, or binding to a cell known to express HSPG on its surface. For example, see Opie, SR et al., (2003) J. Virol. 77:6995-7006, and Kern, A et al., (2003) J. Virol. 77:11072-11081. In some embodiments, the one or more amino acid substitutions provide an improvement in the efficiency of transduction of the rAAV particle into a cell (e.g., an ocular or CNS cell) by at least about 10%, at least about 15%, at least about 20%, at least about at least 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95 % or at least approximately 100% (compared to the transduction efficiency of an rAAV particle containing a wild-type capsid). In some embodiments, one or more amino acid substitutions provide an improvement in the efficiency of transduction of the rAAV particle into a cell (e.g., an ocular or CNS cell) by any amount from about 10% to about 100%, from about 20% to about 100%, from about 30% to about 100%, from about 40% to about 100%, from about 50% to about 100%, from about 60% to about 100%, from about 70% to about 100%, from about 80% to about 100%, from about 90% to about 100%, from about 10% to about 90%, from about 20% to about 90%, from about 30% to about 90%, from about 40% to about 90%, from about 50% to about 90%, from about 60% to about 90%, from about 70% to about 90%, from about 80% to about 90%, from about 10% to about 80%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 60% to about 80%, from about 70% to about 80%, about 10% to about 70%, about 20% to about 70%, about 30% to about 70%, about 40% to about 70%, about 50% to about 70%, about 60% to about 70%, about 10% to about 60%, about 20% to about 60%, about 30% to about 60%, about 40% to about 60%, about 50% to about 60%, about 10% to about 50%, about 20% to about 50%, about 30% to about 50%, about 40% to about 50%, about 10% to about about 40%, about 20% to about 40%, about 30% to about 40%, about 10% to about 30%, about 20% to about 30%, or about 10% to about 20% (according to compared with the transduction efficiency of the rAAV particle containing the wild-type capsid). Methods for measuring the efficiency of transduction of AAV particles into a cell (eg, a cell in culture or a piece of tissue) are known in the art. For example, a population of cells (eg, in a culture or piece of tissue) can be infected with a collection of rAAV particles containing a vector whose expression in cells produces a reporter that can be analyzed (eg, GFP fluorescence, sFLT production, etc.).

AAV capsid proteins

[0175] In some aspects, the present invention provides an AAV capsid protein comprising an amino acid substitution at amino acid residue 2; wherein the amino acid substitution at amino acid residue 2 provides a change in N-terminal acetylation compared to N-terminal acetylation at amino acid residue 2 of the original AAV capsid protein. In some embodiments, the AAV capsid protein is VP1 or VP3. In some embodiments, amino acid residue 2 of the AAV capsid protein (e.g., VP1 or VP3) is replaced with Cys, Ser, Thr, Val, Gly, Asn, Asp, Glu, Ile, Leu, Phe, Gln, Lys, Met, Pro, or Tyr. In some embodiments, the amino acid substitution results in less deamidation of the AAV capsid protein. Non-limiting examples of AAV capsid proteins of the present invention include VP1 and/or VP3 of any of the following AAV serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV LK03, AAV2R471A, AAV2/2-7m8, AAV DJ, AAV DJ8, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV V708K, Goat AAV, Chimeric AAV1/AAV2, Bovine AAV, Mouse AAV or rAAV2/HBoV1 . In some embodiments, the AAV capsid further comprises a tyrosine mutation or a mutation affecting heparin binding.

Preparation of AAV particles

[0176] Numerous methods for producing rAAV-based vectors are known in the art, including transfection, the establishment of stable cell lines, and the use of production systems based on infectious hybrid viruses, which include adenovirus-AAV hybrids, herpes virus-AAV hybrids (Conway, JE et al., (1997) J. Virology 71(11):8780-8789) and baculovirus-AAV hybrids (Urabe, M. et al., (2002) Human Gene Therapy 13(16):1935-1943; Kotin, R. (2011) Hum Mol Genet 20(R1): R2-R6). To produce rAAV viral particles, all cultures for rAAV production require: 1) suitable host cells, 2) suitable helper virus functional element, 3) AAV rep and cap genes and gene products; 4) a nucleic acid (such as a therapeutic nucleic acid) flanked by at least one AAV ITR sequence (eg, the AAV genome encoding GNPTAB); and 5) suitable media and media components to support rAAV production. In some embodiments, a suitable host cell is a host cell derived from a primate. In some embodiments, a suitable host cell is a human-derived cell line, such as HeLa, A549, 293, or Perc.6 cells. In some embodiments, a suitable helper virus functional element is provided by a wild-type or mutant adenovirus (such as heat sensitive adenovirus), herpesvirus (HSV), baculovirus, or plasmid construct providing helper functional elements. In some embodiments, the AAV rep and cap gene products may be from any AAV serotype. In general, but not necessarily, the AAV rep gene product is of the same serotype as the ITR from the rAAV-based vector genome, provided that the rep gene products can allow the rAAV genome to replicate and package. Suitable media known in the art can be used to generate rAAV-based vectors. These media include, without limitation, those manufactured by Hyclone Laboratories and JRH, including Modified Eagle's Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), custom formulations such as those described in US Pat. No. 6,566,118, and Sf medium. -900 II SFM described in US Pat. No. 6,723,551, each of which is incorporated herein by reference in its entirety, especially with respect to custom media formulations for use in the production of recombinant AAV-based vectors. In some embodiments, the functional elements of the AAV helper are adenovirus or HSV. In some embodiments, the functional elements of the AAV helper are baculovirus and the host cell is an insect cell (eg, Spodoptera frugiperda (Sf9) cells). In some embodiments, the AAV cap functional elements provide an amino acid substitution at amino acid residue 2 of VP1 and/or VP3; wherein the amino acid substitution at amino acid residue 2 of VP1 and/or VP3 provides a change in N-terminal acetylation compared to N-terminal acetylation at amino acid residue 2 of VP1 and/or VP3 of the original AAV particle. In some embodiments, amino acid residue 2 of the AAV capsid protein (e.g., VP1 or VP3) is replaced with Cys, Ser, Thr, Val, Gly, Asn, Asp, Glu, Ile, Leu, Phe, Gln, Lys, Met, Pro, or Tyr. In some embodiments, the amino acid substitution results in less deamidation of the AAV capsid.

[0177] One method for producing rAAV particles is a triple transfection method. Briefly, a plasmid containing a rep gene and a capsid gene together with an adenoviral helper plasmid can be introduced by transfection (e.g., using a calcium phosphate method) into a cell line (e.g., HEK-293 cells), and the virus can be harvested and optionally cleanse. Thus, in some embodiments, an rAAV particle has been generated by triple transfection of a nucleic acid encoding an rAAV vector, a nucleic acid encoding AAV rep and cap, and a nucleic acid encoding functional elements of an AAV helper virus into a cell host, wherein the introduction by transfection of nucleic acids into host cells results in a host cell capable of producing rAAV particles.

[0178] In some embodiments, rAAV particles can be obtained using a method using a producer cell line (see Martin et al., (2013) Human Gene Therapy Methods 24:253-269, US Patent Application No. US 2004/0224411 published prior to the grant of the patent, and Liu, X. L. et al (1999) Gene Ther 6:293-299). Briefly, a cell line (eg, HeLa, 293, A549, or Perc.6 cell line) can be stably transfected with a plasmid containing a rep gene, a capsid gene, and a vector genome containing a nucleic acid sequence with a heterologous promoter (eg, GNPTAB). Cell lines can be screened to select a lead clone for rAAV production, which can then be expanded in a production reactor and infected with a helper virus (eg, adenovirus or HSV) to initiate rAAV production. The virus can then be harvested, the adenovirus can be inactivated (eg, by heat) and/or removed, and the rAAV particles can be purified. In this regard, in some embodiments, the rAAV particle was obtained using a producer cell line containing one or more of a nucleic acid encoding a vector based on rAAV, a nucleic acid encoding rep and cap AAV, and a nucleic acid encoding functional elements AAV helper virus. As described herein, the production cell line method may be advantageous for producing gene-enlarged rAAV particles over the triple transfection method.

[0179] In some embodiments, the nucleic acid encoding the AAV rep and cap genes and/or the rAAV genome is stably maintained in a producer cell line. In some embodiments, a nucleic acid encoding the AAV rep and cap genes and/or the rAAV genome is introduced into a cell line using one or more plasmids to produce a producer cell line. In some embodiments, rep AAV, cap AAV, and the rAAV genome are introduced into the cell using the same plasmid. In other embodiments, rep AAV, cap AAV, and the rAAV genome are introduced into the cell using different plasmids. In some embodiments, in a cell line stably transformed with a plasmid, the plasmid is maintained for several passages of the cell line (eg, 5, 10, 20, 30, 40, 50, or more than 50 cell passages). For example, the plasmid(s) may be replicated during cell division, or the plasmid(s) may be integrated into the genome of the cell. Many sequences have been identified that allow autonomous replication of a plasmid in a cell (eg, human cell) (see, for example, Krysan, P.J. et al. (1989) Mol. Cell Biol. 9:1026-1033). In some embodiments, the plasmid(s) may contain a selectable marker (eg, an antibiotic resistance marker) that allows selection of cells containing the plasmid. Selectable markers commonly used in mammalian cells include, but are not limited to, blasticidin, G418, hygromycin B, zeocin, puromycin, and derivatives thereof. Methods for introducing nucleic acids into a cell are known in the art and include, but are not limited to, viral transduction, cationic transfection (for example, using a cationic polymer such as DEAE-dextran or a cationic lipid such as lipofectamine), transfection using calcium phosphate, microinjection, particle bombardment, electroporation and transfection using nanoparticles (for more details see, for example, Kim, T.K. and Eberwine, J.H. (2010) Anal. Bioanal. Chem. 397:3173-3178).

[0180] In some embodiments, the nucleic acid encoding the AAV rep and cap genes and/or the rAAV genome is stably integrated into the genome of the producer cell line. In some embodiments, a nucleic acid encoding the AAV rep and cap genes and/or the rAAV genome is introduced into a cell line using one or more plasmids to produce a producer cell line. In some embodiments, rep AAV, cap AAV, and the rAAV genome are introduced into the cell using the same plasmid. In other embodiments, rep AAV, cap AAV, and the rAAV genome are introduced into the cell using different plasmids. In some embodiments, the plasmid(s) may contain a selectable marker (eg, an antibiotic resistance marker) that allows selection of cells containing the plasmid. Methods for stably integrating nucleic acids into multiple host cell lines are known in the art. For example, reselection (eg, through the use of a selectable marker) can be used to select cells containing an integrated nucleic acid containing a selectable marker (and the AAV cap and rep genes and/or the rAAV gene). In other embodiments, nucleic acids can be integrated into a cell line in a site-specific manner to produce a producer cell line. Several site-specific recombination systems are known in the art such as FLP/FRT (see e.g. O'Gorman, S. et al. (1991) Science 251:1351-1355), Cre/loxP (see e.g. , Sauer, B. and Henderson, N. (1988) Proc. Natl. Acad. Sci. 85:5166-5170) and phi C31-att (see, for example, Groth, A. C. et al. (2000) Proc. Natl Acad. Sci. 97:5995-6000).

[0181] In some embodiments, the producer cell line is derived from a primate cell line (eg, a non-human primate cell line, such as a Vero or FRhL-2 cell line). In some embodiments, the cell line is derived from a human cell line. In some embodiments, the producer cell line is derived from HeLa, 293, A549, or PERC.6® (Crucell) cells. For example, prior to the introduction and/or stable maintenance/integration of the nucleic acid encoding the AAV rep and cap genes and/or the expanded rAAV genome into a cell line to produce a producer cell line, the cell line is a HeLa, 293, A549 or PERC.6® (Crucell) or derivative.

[0182] In some embodiments, the producer cell line is adapted to grow in suspension. As is known in the art, substrate-dependent cells are generally unable to grow in suspension without a substrate, such as microcarrier beads. Adaptation of the cell line to growth in suspension may involve, for example, growing the cell line in cultures with constant agitation using a stirring paddle, using a culture medium that does not contain calcium and magnesium ions to prevent aggregation (and optionally an antifoam agent), using a culture vessel coated with a siliconizing compound, and selecting cells in culture (rather than in large clusters or on the walls of the vessel) at each passage. For further description, see, for example, the ATCC FAQ document (available on the World Wide Web at atcc.org/Global/FAQs/9/1/Adapting%20a%20monolayer%20cell%20line%

20to%20suspension-40.aspx) and references mentioned in this document.

[0183] Suitable culture media for AAV production of the present invention can be supplemented with blood serum or recombinant proteins derived from blood serum at the level of 0.5% - 20% (v/v or w/v). Alternatively, as is known in the art, AAV-based vectors can be produced under serum-free conditions, which may also mean the use of an animal product-free medium. One of skill in the art will appreciate that commercial or custom media designed to allow production of AAV vectors can also be supplemented with one or more cell culture components known in the art, including but not limited to glucose, vitamins , amino acids and/or growth factors, in order to increase the titer of AAV in cultures for production.

[0158] AAV production cultures can be grown under various conditions (over a wide temperature range, for various times, etc.) suitable for the particular host cell used. As known in the art, cultures for AAV production include attachment-dependent cultures that can be grown in suitable attachment-dependent culture vessels such as, for example, roller bottles, hollow fiber filters, microcarriers, and packed-bed or fluidized-bed bioreactors. . Cultures for the production of AAV-based vectors may also include suspension-adapted host cells such as HeLa, 293 and SF-9 cells, which can be cultured in a variety of ways including, for example, shaking flasks, bioreactors with mechanical agitation and disposable systems such as the Wave bag system.

[0159] The AAV vector-based particles of the present invention can be harvested from AAV production cultures by lysing host cells from the production culture or by collecting spent media from the production culture, provided that the cells are cultured under conditions which are known in the art to cause the release of AAV particles from intact cells into the medium, as described in more detail in US Pat. No. 6,566,118. , microfluidization and treatment with chemicals such as detergents and/or proteases.

[0160] In additional embodiments, the AAV particles are purified. The term "purified" as used herein means a formulation of AAV particles that is free from at least some other components that may also be present in the environment where the AAV particles are found naturally or from which they were originally derived. Thus, for example, isolated AAV particles can be obtained using a purification technique to enrich a feed mixture, such as a culture lysate or a production culture supernatant. Enrichment can be measured in a variety of ways, such as, for example, by the proportion of DNase-resistant particles (DRP) or genome copies (gc) present in solution, or by infectivity, or it can be measured in relation to a second potentially interfering substance present in the initial mixture, such as contaminants, including contaminants from the production culture or contaminants formed during production, including helper virus, media components, etc.

[0161] In some embodiments, the AAV production culture harvest is decontaminated to remove host cell debris. In some embodiments, the production culture harvest is decontaminated by filtration through a series of porous filters including, for example, a Millipore Millistak+ HC brand DOHC modular filter, a Millipore Millistak+ HC brand A1HC modular filter, and a 0.2 µm Opticap XL1O hydrophilic filter. membrane filter Millipore Express SHC. Purification of impurities can also be carried out using a number of other standard techniques known in the art, such as centrifugation or filtration through any cellulose acetate filter with a pore size of 0.2 μm or larger known in the art.

[0162] In some embodiments, the harvest of the AAV production culture is further processed with ^Benzonase® to cleave any high molecular weight DNA present in the production culture. In some embodiments, ^Benzonase® digestion is carried out under standard conditions known in the art, including, for example, at a final concentration of 1-2.5 ^Benzonase® units/mL, at a temperature ranging from ambient temperature to 37°C, for a period of time from 30 minutes to several hours.

[0163] AAV particles can be isolated or purified using one or more of the following purification steps: equilibrium centrifugation; flow anion exchange filtration; tangential flow filtration (TFF) for concentrating AAV particles; capturing AAV by apatite chromatography; thermal inactivation of the helper virus; capturing AAV by hydrophobic interaction chromatography; buffer exchange by size exclusion chromatography (SEC); nanofiltration and capture of AAV by anion exchange chromatography, cation exchange chromatography or affinity chromatography. These steps can be used individually, in various combinations, or in any order. In some embodiments, the implementation of the method includes all stages in the order described below. Methods for purifying AAV particles are found, for example, in Xiao et al., (1998) Journal of Virology 72:2224-2232; U.S. Patent Nos. 6,989,264 and 8,137,948 and WO 2010/148143.

Pharmaceutical compositions

[0164] In some embodiments, the AAV particle of the present invention (eg, rAAV particle) is in a pharmaceutical composition. Pharmaceutical compositions may be suitable for any route of administration described herein or known in the art. In some embodiments, the pharmaceutical composition comprises rAAV particles modified to improve stability and/or improve the efficiency of transduction of rAAV particles; for example, for use in making amino acid substitutions at position 2 of VP1 and/or VP3 to improve acetylation of rAAV capsid proteins. In some embodiments, the pharmaceutical composition comprises rAAV particles modified to modulate the stability and/or efficiency of transduction of rAAV particles (e.g., increase the stability and/or efficiency of transduction, or decrease the stability and/or efficiency of transduction), for example, for use in performing amino acid residue substitutions. , which provide modulation of deamidation (for example, increasing the degree of deamidation or reducing the degree of deamidation).

[0165] In some embodiments, the rAAV particle is in a pharmaceutical composition containing a pharmaceutically acceptable excipient. As is well known in the art, pharmaceutically acceptable excipients are relatively inert substances that facilitate the administration of a pharmacologically effective substance and may be provided as liquid solutions or suspensions, as emulsions, or as solid forms suitable for dissolution or suspension in a liquid before application. For example, the excipient may give shape or texture or act as a diluent. Suitable excipients include, but are not limited to, stabilizing agents, wetting and emulsifying agents, osmolarity salts, encapsulating agents, pH buffering agents, and buffers. Such excipients include any pharmaceutical suitable for direct delivery to the eye that can be administered without undue toxicity. Pharmaceutically acceptable excipients include, without limitation, sorbitol, any of the various TWEEN compounds, and liquids such as water, saline, glycerin, and ethanol. They may include pharmaceutically acceptable salts, for example mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and organic acid salts such as acetates, propionates, malonates, benzoates, and the like. A comprehensive discussion of pharmaceutically acceptable excipients is available in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., NJ 1991). In some embodiments, a pharmaceutical composition comprising the rAAV particle described herein and a pharmaceutically acceptable carrier is suitable for administration to a human. Such carriers are well known in the art (see, for example, Remington's Pharmaceutical Sciences, 15th ^Edition , pp. 1035-1038 and 1570-1580).

[0166] Such pharmaceutically acceptable carriers can be sterile liquids such as water and oil, including oils of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, and the like. Saline and aqueous solutions of dextrose, polyethylene glycol (PEG) and glycerin can also be used as liquid carriers, in particular for injection solutions. The pharmaceutical composition may further contain additional ingredients such as preservatives, buffers, tonicity agents, antioxidants and stabilizers, non-ionic wetting or clarifying agents, thickeners, and the like. The pharmaceutical compositions described herein may be packaged in unit dosage forms. or in multi-dose forms. Typically, the compositions are formulated as a sterile or substantially isotonic solution.

Kits and products

[0167] The present invention also provides kits or articles containing any of the rAAV particles and/or pharmaceutical compositions of the present invention. Kits or articles may contain any of the rAAV particles or rAAV particle compositions of the present invention. In some embodiments, the kits are used to improve the stability and/or improve the efficiency of transduction of rAAV particles; for example, for use in making amino acid substitutions at position 2 of VP1 and/or VP3 to improve acetylation of rAAV capsid proteins. In some embodiments, the kits are used to modulate the stability and/or efficiency of transduction of rAAV particles (e.g., increase the stability and/or efficiency of transduction, or decrease the stability and/or efficiency of transduction), for example, for use in making substitutions of amino acid residues that provide modulation of deamidation (for example, an increase in the degree of deamidation or a decrease in the degree of deamidation).

[0168] In some embodiments, the kits or articles further comprise instructions for administering the rAAV particle composition. The kits or articles described herein may additionally contain other materials necessary from a commercial and consumer point of view, including other buffers, diluents, filters, needles, syringes, and inserts with instructions for performing any of the methods described in this document. Suitable packaging materials may also be included and may be any packaging materials known in the art, including, for example, flasks (such as sealed flasks), vials, ampoules, bottles, jars, flexible packaging (for example, sealed Mylar bags or plastic bags), etc. Such articles may be further sterilized and/or sealed.

[0169] In some embodiments, kits or articles further comprise one or more of the buffers and/or pharmaceutically acceptable excipients described herein (e.g., those described in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991). In some embodiments, The kits or articles of manufacture contain one or more of the pharmaceutically acceptable excipients, carriers, solutions and/or additional ingredients described herein.The kits or articles described herein may be packaged in unit dosage forms or multi-dose forms. or articles, as a rule, formulated in a sterile form, and it can be lyophilized or provided in the form of a practically isotonic solution.

EXAMPLES

[0170] The present invention will be better understood with reference to the following examples. However, they should not be construed as limiting the scope of the present invention. It is understood that the examples and embodiments described herein are illustrative only, and that various modifications or changes to take them into account will be understood by those skilled in the art and should be within the spirit and scope of this application, as well as the scope of the attached invention formulas.

Example 1 Direct LC/MS and LC/MS/MS for Complete Characterization of Recombinant AAV Viral Capsid Protein

[0171] Recombinant adeno-associated viruses (rAAVs) have become common vectors for gene therapy due to their non-pathogenic nature, ability to infect both dividing and non-dividing cells, and long period of gene expression. AAV-based gene therapies are currently being used in clinical trials for numerous targeted diseases such as muscular dystrophy, hemophilia, Parkinson's disease, Leber's congenital amaurosis, and macular degeneration.

[0172] AAV is a small and unenveloped parvovirus with a single-stranded DNA genome encapsulated in an icosahedral envelope. Each capsid contains sixty copies of the three viral capsid proteins VP1 (87 kDa), VP2 (73 kDa), and VP3 (62 kDa) in an approximate ratio of 1:1:10. All three viral capsid proteins are expressed from the same open reading frame through alternative splicing and an atypical start codon and thus have overlapping sequences. VP1 has ~137 additional N-terminal amino acid residues compared to VP3, while VP2 has ~65 additional N-terminal amino acid residues compared to VP3. At least 13 AAV serotypes and ~150 gene sequences have been isolated from human and non-human primate tissues; AAV serotypes differ in the amino acid sequence of the viral capsid proteins and their respective cellular receptors and co-receptors for targeting.

[0173] The AAV capsid, in addition to protecting the genome from the inside, plays an important role in mediating receptor binding, virus escape from the endosome, and transport of viral DNA into the nucleus in the viral infection cycle, thereby directly affecting the infectivity of the virus. It has been shown that the N-terminus of VP1 contains the PLA2 phospholipase domain (amino acids 52–97), which is essential for endosomal escape of the virus [1–3]. The N-termini of VP1 and VP2 also contain three major amino acid clusters as intranuclear localization signals. These sequences are highly conserved among different AAV serotypes. Mutations of these amino acids have been shown to reduce or completely eliminate infectivity [4]. In addition, each AAV serotype is characterized by corresponding sequence-specific receptors and co-receptors. For example, the proteoglycan heparin sulfate has been identified as the main AAV2 receptor and several other co-receptors, including αVβ5 integrin, fibroblast growth factor receptor 1 and hepatocyte growth factor receptor have been identified [5–8]. As a result of mutational analysis of AAV2 capsid proteins, a group of basic amino acids (arginine 484, 487, 585, and lysine 532) was identified as a heparin-binding motif that promotes binding of heparin and HeLa cells [9]. The NGR domain of AAV2 was identified as a domain that binds to the α5β1 integrin, which is necessary for the entry of the virus into the cell [10]. Thus, the sequences of viral capsid proteins are important for cell targeting and migration in the viral infection cycle. Since different production conditions can cause different levels of expression of viral capsid proteins, post-translational modifications and truncations, viral capsid proteins must be characterized and controlled in order to ensure product homogeneity in gene therapy development programs.

[0174] Traditionally, SDS-PAGE has been used to characterize AAV viral capsid proteins, providing approximate molecular weight information such as 87 kDa, 73 kDa, and 62 kDa. Based on Edman sequencing, no sequence information was obtained, possibly due to blocked N-termini of viral capsid proteins other than VP2. Although the X-ray structures of numerous AAVs have been determined, only the sequence of the VP3 region has been observed in the crystal structures. Fifteen N-terminal amino acid residues of VP3 were still absent in the X-ray structure, apparently due to its internal disturbance [11-13]. Apparently, the lack of information about the N-terminal regions of VP1 and VP2 in the atomic structure could be due to the low stoichiometry of VP1 and VP2 in the capsid. In addition, the N-terminals of VP1 and VP2 are immersed inside the capsid and are inaccessible to antibodies in the native state, as described in some literature sources [3, 14, 15]. Typically, the Gel-LC/MS method (SDS-PAGE, tryptic gel digestion, and LC/MS/MS) was used to characterize VP [16–18]. However, the N-terminus of VP1, VP2, and VP3 was not determined using this approach because 100% coverage of VP sequences could not be obtained using this method due to limited peptide recovery from the gel.

[0175] Direct analysis using MALDI-TOF MS has been described for several viral capsid proteins, including tobacco mosaic virus U2 after dissociation with organic acid [19]. After amide-hydrogen exchange and mass spectrometry, direct peptide mapping was used to study pH-induced structural changes in the brome mosaic virus (BMV) capsid [20]. Since AAVs are non-enveloped viruses containing only capsid proteins and genome, AAV capsids can be directly analyzed using protein RP-LC/MS and LC/MS/MS peptide mapping to achieve 100% sequence coverage after capsid dissociation without separation by SDS-PAGE. DNA fragments can be eluted in "dead volume" and thus avoid interference in the detection of proteins/peptides using LC/MS. To study these methods, direct LC/MS of various AAV types after denaturation was used to monitor protein sequence and post-translational modifications of AAV capsid proteins. As described herein, the N-terminus of VP1, VP2 and VP3 AAV was determined using mass spectrometry. N-terminal acetylations of VP1 and VP3 were identified in another AAV serotype. A direct peptide mapping of AAV by LC/MS/MS was also developed to provide coverage of the VP1, VP2 and VP3 sequences and confirm N-terminal acetylation of VP1 and VP3.

Ways

Materials and reagents

[0176] Dithiothreitol (DTT), 4-vinylpyridine, ultrapure formic acid, acetic acid, guanidine-HCl, Tris-HCl, and Tris base were purchased from Sigma Chemicals (St. Louis, MO). Amicon ultra-4 filters were purchased from Millipore (Billerica, MA). Sequencing grade porcine trypsin was purchased from Promega (Milwaukee, WI). Endoproteinase Lys-C and Asp-N were purchased from Roche (Germany). Slide-A-Lyzer cassettes with 10,000 MWCO were purchased from Pierce (Rockford, IL).

Getting and clearing vectors

[0177] AAV vectors were obtained using the triple transient transfection method described previously (Xiao, 1998 #123). Briefly, HEK293 cells were transfected with polyethyleneimine, PEI and three plasmids in a 1:1:1 ratio (ITR based vector, AAV rep/cap and helper plasmid Ad). The vector plasmid contains the CBA-EGFP vector genome and ITR sequences from AAV2. EGFP expression was driven by a hybrid promoter (CBA) based on the chicken beta actin promoter and the CMV enhancer as described (Miyazaki, 1989 #124) (Niwa, 1991 #125). The rep/cap AAV helper elements contained rep sequences from AAV2 and serotype-specific capsid sequences designated rep2/cap2, rep2/cap5, rep2/cap7, etc. The pAd helper element used was pHelper (Stratagene/Agilent Technologies, Santa Clara, California). AAV purification was performed as described by Qu et al. (2007, J. Virol. Methods 140:183-192).

Analysis of Intact Proteins by LC/MS

[0178] AAV virions were concentrated using an Amicon ultra-4 filter (10 kDa MWCO) and denatured with 10% acetic acid followed by direct analysis in an Acquity UPLC-Xevo® QTOF MS device (Waters, Milford, MA). Separations were performed using a C4 or C8 UPLC BEH column (1.7 µm, 2.1 mm i.d.) at a flow rate of 0.25 ml/min. Mobile phase A was 0.1% formic acid in water, while mobile phase B was 0.1% formic acid in acetonitrile. The final gradient was 10% B to 20% B over 6 minutes, 20% B to 30% B over 10 minutes, then 30% to 38% B over 40 minutes. In the case of MS, capillary voltage and sampling cone voltage were set to 3.5 kV and 45 V, respectively. Mass spectra were obtained in sensitivity mode with a m/z range of 500-4000. Calibration performed with an additional tool using sodium iodide as a calibrating agent was carried out in the case of mass calibration. MaxEnt1 in the Masslynx computer program was used to deconvolve proteins.

Enzymatic digests of VP AAV2

[0179] Concentrated AAV2 virions were denatured with 6 M guanidine-HCl, 0.1 M Tris at pH 8.5. Proteins were reduced with 30 mM DTT at 55°C for 1 hour in the dark and alkylated with 0.07% 4-vinylpyridine at room temperature for 2 hours. Reactions were quenched by adding 1 M DTT. Samples were dialyzed with Slide-A-Lyzer cassettes (10,000 MWCO) against 25 mM Tris buffer at pH 8.5 for ~18 hours. After dialysis, the samples were divided into three aliquots. Each aliquot was digested with trypsin at an enzyme:protein (w/w) ratio of 1:25 or Lys-C at 1:50 or Asp-N at 1:100 for 18 hours at 37°C, respectively.

Peptide mapping with LC/MS/MS

[0180] Nano-LC/MS/MS was performed with a NanoAcquity HPLC system (Waters, Milford, MA) coupled with an Orbitrap Velos mass spectrometer (Thermo-Fisher Scientific, Woltham, MA) using a factory-packaged nanocolumn. -LC (75 µm x 10 mm) with Magic C18 with packaging material (5 µm, Bruker, Billerica, MA) at 300 NL/min. Mobile phases A and B were 0.1% formic acid in water and acetonitrile, respectively. The gradient was from 2% B to 60% B over 121 min.

[0181] The source parameters in the case of Velos were as follows: source voltage: 2.5 kV, capillary temperature 275°C; S-lens RF level: 55%. Data were obtained using the data-driven top ten method with MS accuracy at 60,000 resolution and 10 MS/MS in an ion trap. Mascot against AAV2 viral capsid protein sequences was used for database searches. Stability MS 10 ppm and stability ms/ms 0.8 Da was used for searching the database.

Peptide mapping with UPLC/MS/MS

[0182] Digested proteins were also analyzed by UPLC/MS/MS in Acquity UPLC-Xevo qTOF MS. A C18 BEH300 column (2.1×150 mm) was used in mobile phases with 0.1% formic acid in a water/acetonitrile gradient at a flow rate of 0.25 ml/min. Mass spectra were obtained in positive mode MSe in the mass range of 200-2000.

results

AAV denaturation method

[0183] AAV can be denatured in a number of ways using a detergent, heat, high salt, or low or high pH buffer. Heat denaturation can lead to protein precipitation and as a result, reverse phase columns easily clog and overcompress. High salt denaturation requires an additional desalting step prior to LC/MS analysis. Denaturation with 10% acetic acid was used to analyze intact proteins by LC/MS as it provided a clean mass spectrum. In the case of peptide mapping, 0.1% RapiGest or 6 M guanidine HCl can be used as the denaturing reagent.

Development of a method for analyzing intact proteins

[0184] Initial analysis of intact AAV2 proteins was performed using a C4 UPLC BEH column with a fast gradient. Under this condition, only one peak was observed in the total ion chromatogram with a mass corresponding to VP3 (FIG. 1A). Without wishing to be bound by any theory, it is believed that the absence of VP1 and VP2 may be due to the low stoichiometry of VP1 and VP2 or suppression of VP1 and VP2 signals by VP3 if all VPs co-elute. An attempt was made to increase sample input or column length using a gentle gradient and using alternative columns in order to detect VP1 and VP2. Higher load (1.7 µg) using a gentle gradient at 0.5% B/min. led to a shoulder peak on the left (Fig. 1B). Increasing the column length from 10 cm to 15 cm did not increase the separation of the shoulder peak (Fig. 1C). However, the shoulder peak was further separated from the main peak using a C8 BEH column, and increased signal intensities were observed (FIG. 1D).

[0185] As a result, VP1 and VP2 masses were obtained in this shoulder peak at the signal intensities shown in FIG. 2A. The masses of VP1 and VP3 correspond to amino acids 2-735 (acetylation) and amino acids 204-735 (acetylation), respectively (FIGS. 2A and 2B). In the case of VP2 (amino acids 139-735), no acetylation was observed. In addition, a small peak was observed with a mass less than VP3, with the mass corresponding to the amino acid sequence 212-735 with one acetylation (Fig. 2B). These data are consistent with DNA sequences because VP3 contains two ATG initiation codons in AAV2: ATG GCTACAGGCAGTGGCGCACCA ATG GCAGAC (SEQ ID NO:1), resulting in two possible N-terminals (underlined): M ATGSGAP M AD (SEQ ID NO: 2). N-terminal methionine residues were not present in both VP1 and VP3 as measured by intact protein analysis. Acetylation of VP1 and VP3 is not an artifact caused by the method (denaturation of AAV with 10% acetic acid), as acetylation of VP1 and VP3 was also observed in the preparation of AAV using an alternative method of denaturation without the use of acetic acid. Intact protein data also confirmed that glycosylation was not present in viral capsid proteins, even though several N-linked consensus sequences were present [16].

Peptide mapping with LC/MS/MS

[0186] To further confirm the N-terminus and acetylation observed in the analysis of intact proteins, peptide mapping was performed using several enzymes and analyzed using several devices. Various sample preparation methods were evaluated, including denaturation methods and desalting steps. The final digestion method, involving denaturation with 6 M guanidine-HCl, reduction and alkylation with 4-vinylpyridine, and dialysis with a Slide-A-lyzer followed by enzymatic digestion, resulted in a clean peptide mapping with low artificial modifications during the digestion process. Only 5 μg of starting material was examined, resulting in complete sequence coverage by nano-LC/MS/MS and UPLC/MS/MS.

[0187] Mascot's search for tryptic hydrolysates by nano-LC/MS/MS alone resulted in a 78% coverage of sequences with an ion cutoff of 13 shown in FIG. 3. Two large missing tryptic peptides, T27 and T38 (boxed) based on nano-LC/MS/MS, were detected in LC/MS in Xevo TOF MS using a C18 BEH column (FIG. 3). In addition, most of the T27 and T38 peptide sequences were further confirmed by nano-LC/MS/MS Asp-N hydrolysates shown in italics in FIG. 3. The complete N-terminal and C-terminal peptides were encompassed by the Lys-C hydrolysates underlined in FIG. 3. Thus, 100% coverage of the VP1 sequence was achieved by several enzymatic digests and by two LC/MS/MS methods.

[0188] LC/MS/MS confirmed the N- and C-termini of VP1, VP2 and VP3 and the N-terminal acetylation of VP1 and VP3 observed when analyzing intact proteins. In FIG. 4A-4C show the MS/MS spectrum of the N-terminal VP1 tryptic peptide A(Ac)ADGYLPDWLEDTLSEGIR (SEQ ID NO:4) (FIG. 4A) derived from the N-terminal Asp-N VP2 peptide (APGKKRPVEHSPVEP) (SEQ ID NO: 15) (FIG. 4B) and the N-terminal Asp-N peptide of VP3 A(Ac)TGSGAPM (SEQ ID NO:5) (FIG. 4C). MS/MS confirmed the position of acetylation at the N-terminal alanine residues in both VP1 and VP3 peptides. The presence of unmodified y18 and y17 ions, as well as all detectable b ions with a mass shift of 42 Da in FIG. 4A indicates that the 42 Da modification is located at the N-terminus of VP1. Similarly, the presence of unmodified y3-y8 ions in FIG. 4C confirmed the position of the acetylation at the N-terminal alanine residue.

N-terminal comparison of VP AAV

[0189] In addition to AAV2, AAV1, AAV5, AAV7, AAV9 and AAV Rh10 were also analyzed using intact protein analysis. Theoretical and predicted VP AAV masses are shown in Table 1. 2.

Table 2. Theoretical mass and experimental mass of VP AAV

Serotype Isoform Predicted amino acid sequence
validity Actual amino acid sequence
validity theoretical
Ms (Yes) Experiment
Ms (Yeah) AAV1 VP1 1-736 2(ac)-736 81286 81291 VP2 138-736 139-736 66093 66098 VP3 203-736 204(ac)-736 59517 59520 AAV2 VP1 1-735 2(ac)-735 81856 81856 VP2 138-735 139-735 66488 66488 VP3 203-735 204(ac)-735 59974 59974 AAV5 VP1 1-724 2(ac)-724 80336 80336 VP2 137-724 138-724 65283 65284 VP3 193-724 194(ac)-724 59463 59463 AAV7 VP1 1-737 2(ac)-737 81564 81567 VP2 138-737 139-737 66372 66374 VP3 204-737 213(ac)-737 59101 59103 AAV9 VP1 1-736 2(ac)-736 81291 81288 VP2 138-736 139-736 66210 66209 VP3 203-736 204(ac)-736 59733 59733 AAVRh10 VP1 1-738 2(ac)-738 81455 81455 VP2 138-738 139-738 66253 66252 VP3 204-738 205(ac)-738 59634 59634

[0190] The N-termini, as well as their post-translational modifications, are highly conserved among the analyzed AAV serotypes, even though AAV5 is described as the most diverse sequence of the AAV serotype, as shown by the sequence alignments in FIG. 5. In 11 of the 13 AAV serotypes, the N-terminals of VP1 have an identical 13 amino acid sequence (MAADGYLPDWLED) (SEQ ID NO:6), while in all 13 AAV serotypes TAP... N-terminal sequences were identified in VP2 (Fig. 5). LC/MS AAV2 indicated that T was absent from VP2 at the protein level. The N-terminus of VP3 is the most diverse among all three viral capsid proteins, with 8 out of 13 AAV serotypes having the MA… N-terminal sequence. Similarly, AAV2, AAV1 and AAV Rh10 also have two ATG initiation codons, with the former as the predominant N-terminus based on intact protein analysis by LC/MS. Interestingly, although AAV7 has two potential initiation codons ( GTG GCTGCAGGCGGTGGCGCACCA ATG GCAGACAATAAC…) (SEQ ID NO:7), the second initiation codon (ATG) is preferred based on analysis of intact proteins: VP3` c 213(ac )-737 was the dominant peak, while VP3 with 203(ac)-737 was a minor peak.

findings

Applications of LC/MS Intact Protein Analysis and LC/MS/MS VP AAV Peptide Mapping in Gene Therapy Research and Development

[0191] These results show that direct LC/MS of various AAV types after denaturation has proven to be a simple and effective way to monitor protein sequence and post-translational modifications with accurate mass measurements at the intact protein level. The N-terminus of VP1, VP2 and VP3 AAV was determined using mass spectrometry. N-terminal acetylations of VP1 and VP3 have been identified in other AAV serotypes. A direct peptide mapping of AAV by LC/MS/MS was also developed to ensure 100% coverage of VP1, VP2 and VP3 sequences and confirm N-terminal VP acetylation. Theoretical masses of predicted sequences of 13 AAV serotypes based on sequence alignment and analysis of intact proteins of several AAV serotypes are shown in Table 1. 3.

Table 3. Predicted sequences and masses

Forecasts
VP1 sequence Weight (Yes) Forecasts
VP2 sequence Weight (Yes) Forecasts
VP3 sequence Weight (Yes) AAV1 2(ac)-736 81286 139-736 66093 204(ac)-736 59517 AAV2 2(ac)-735 81856 139-735 66488 204(ac)-735 59974 AAV3 2(ac)-736 81571 139-736 66319 204(ac)-736 59849 AAV4 2(ac)-734 80550 138-734 65626 198(ac)-734 59529 AAV5 2(ac)-724 80336 138-724 65283 194(ac)-724 59463 AAV6 2(ac)-736 81322 139-736 66096 204(ac)-736 59519 AAV7 2(ac)-737 81564 139-737 66372 213(ac)-737 59101 AAV8 2(ac)-738 81667 139-738 66519 205(ac)-738 59805 AAV9 2(ac)-736 81291 139-736 66210 204(ac)-736 59733 AAV10 2(ac)-738 81477 139-738 66271 205(ac)-738 59638 AAV11 2(ac)-733 80987 139-733 65794 198(ac)-733 59696 AAV12 2(ac)-742 82106 139-742 66905 207(ac)-742 59846 AAVRh10 2(ac)-738 81455 139-738 66253 205(ac)-738 59634

[0192] The exact masses of VP1, VP2 and VP3 of each serotype are unique and thus analysis of intact proteins can be used as an identity test to differentiate AAV capsid serotypes. In table. 4-6 show the differences in VP masses among the 13 common AAV serotypes. Delta masses greater than 10 are shown in normal font, while delta masses less than 10 are shown in bold.

Table 4. Differences in VP1 masses among 13 AAV isotypes

Table 5. Differences in VP2 masses among 13 AAV isotypes

Table 6. Differences in VP3 masses among 13 AAV isotypes

[0193] Mass differences within 10 Da of all three VPs between the two isotypes were not observed. Even though VP2 and VP3 have only a 2 Da difference between AAV1 and AAV6, the VP1 mass difference between AAV1 and AAV6 is 36, which is significant enough to be distinguished by accurate mass measurements. Thus, the measurement of intact VP1, VP2 and VP3 proteins is highly specific as an identity test.

[0194] These results show that intact protein analysis and LC/MS/MS can be used to profile VP to control expressions, post-translational modifications and truncations of VP and to ensure product homogeneity during VLP production. These two assays can also be used to confirm site-directed mutagenesis or structural characterization in case of capsid protein engineering applications.

Example 2 Role of N-Terminal Acetylation of AAV Capsid Proteins

[0195] Chemical modification of cellular proteins is a common means of controlling their functions (Arnesen, T. (2006) Virology 353(2): 283-293). N-terminal acetylation (Nt-acetylation), which involves the transfer of an acetyl group from acetyl coenzyme A to the α-amino group of the first amino acid residue of the protein (Brown, J. L. and Roberts, W. K. (1976) J Biol Chem 251: 1009-1014; Arnesen, T. et al (2009) Proc Natl Acad Sci USA 106: 8157-8162) is the most common of the protein modifications. Unlike most other protein modifications, Nt-acetylation is irreversible; it occurs primarily during protein synthesis catalyzed by ribosome-associated N-terminal acetyltransferases (NATs) (Gautschi, M. et al. (2003) Mol Cell Biol 23: 7403-7414; Pestana, A. and Pitot, H.C. ( 1975) Biochemistry 14: 1404-1412 Polevoda, B. et al (2003) J Biol Chem 278: 30686-97). There are several different NATs in eukaryotes -NatA-NatF- each consisting of one or more subunits and each of them acetylates a specific subgroup of the N-terminus depending on the amino acid sequence of the first few amino acids (Jornvall, H. (1975) J Theor Biol 55 : 1-12 Persson, B. et al (1985) Eur J Biochem 152: 523-527).

[0196] Experimental data indicate that acetylated N-terminal proteins are more stable in vivo than non-acetylated proteins; i.e., Nt-acetylation protects proteins from degradation (Hershko, A. et al. (1984) Proc Natl Acad Sci U S A 81: 7021-7025). One explanation for this may be the discovery in 2004 that another N-terminal modification, ubiquitination, which involves direct attachment of the small protein ubiquitin to the N-terminal amino acid residue, promotes subsequent protein degradation (Ben Saadon, R. et al. (2004) J Biol Chem 279: 41414-41421). In contrast, Nt-acetylation signals may also be part of a quality control mechanism to degrade unfolded or misfolded proteins to regulate protein stoichiometry in vivo (Hwang, C.S. et al. (2010) Science 327: 973-977).

[0197] A systematic analysis of the predicted N-terminal processing of cytosolic proteins compared to those intended for sorting into the secretory pathway showed that cytosolic proteins were significantly biased towards processing, however, there is an equal and opposite bias against such modification in the case of secretory proteins (Forte, G.M.A. et al. (2011) PLoS Biology, 4 May 2011, Volume 9). Mutations of secretory signal sequences that promote their acetylation lead to disruption of sorting in the cytosol in a manner dependent on the mechanism of N-terminal processing. In this regard, N-terminal acetylation represents an early step in cell sorting of resulting polypeptides that present an increased level of rigidity in order to ensure that proteins that are intended to remain in the cytosol are actually in the cytosol. A eukaryotic cell contains several different compartments, called organelles, necessary for specific functions. The proteins in these compartments are synthesized in the cytoplasm and thus require complex sorting mechanisms to ensure their delivery to the appropriate organelle. Proteins are modified by acetylation of their amino terminus at a very early stage in their synthesis. There is a marked difference between the likelihood of such modification in cytoplasmic proteins and those destined for one of the major organelles, the endoplasmic reticulum (ER): while cytoplasmic proteins are usually acetylated, those bound to the ER are mostly unmodified. Moreover, by designing specific ER proteins to induce their acetylation, their targeting to the ER was inhibited (Forte, G.M.A. et al. (2011) PLoS Biology, 4 May 2011, Volume 9).

[0198] The contractile proteins actin and tropomyosin have been shown to require NatB-mediated Nt-acetylation for their respective function, particularly involving actin and tropomyosin binding and regulation of actomyosin (Coulton, A.T. et al. (2010) J Cell Sci 123: 3235 -3243 Polevoda, B. et al (2003) J Biol Chem 278: 30686-97). Thus, Nt-acetylation of the AAV capsid proteins may be important in the transduction potential of rAAV-based vectors. If AAV vectors cannot access the nucleus, they cannot subsequently transduce cells. The role of actin filaments and FKBP52 (FK506 binding protein p52) in translocation of AAV capsids from endosome to nucleus is well defined (Zhao, W. et al. (2006) Virology 353(2): 283-293). Importantly, Nt-acetylation is required for actin filament function by modulating protein-protein interactions (Coulton, A.T. et al. (2010) J Cell Sci 123: 3235-3243; Polevoda, B. et al. (2003) J Biol Chem 278: 30686-97).

[0199] Although N-terminal acetylation of proteins is a well-known phenomenon, the biological significance of Nt-acetylation of AAV capsid proteins is not well understood. Both the predicted N-terminus of VP1 and VP3 based on DNA sequencing are methionine followed by alanine. It has been described that removal of the N-terminal methionine by Met-aminopeptidases often results in Nt-acetylation of the resulting N-terminal alanine, valine, serine, threonine, and cysteine residues, and that N-terminal acetylation acts as a potential degradation signal [21]. Ubiquitination of viral capsid proteins has been suggested as a potential signal for capsid processing during virion disassembly [22]. The relationship between N-acetylation of VP1 and VP3 and disruption and shedding of the viral capsid envelope prior to entry into the nucleus is further investigated.

[0200] To understand the functional implications of N-terminal acetylation on AAV capsid proteins, site-directed mutagenesis of the N-terminal VP3 initiation codons is used to generate AAV mutants.

Ways

[0201] AAV capsid proteins are prepared with different amino acids at position 2 relative to the initiator methionine (iMet X) to determine whether Nt-acetylation is inhibited or attenuated, and then the functional consequences are determined. The ability of capsid proteins to migrate intracellularly and/or acquire post-translational modifications such as glycosylation is determined, and then it is determined whether this ability affects the infectivity of the assembled AAV particle. In addition, the effect of acetylation on ubiquitination/degradation and targeting to the lysosome, ER, Golgi apparatus or inner nuclear membrane is determined.

[0202] For example, for migration or targeting assays, AAV particles with capsid proteins having mutant position 2 (eg, iMet X) are fluorescently labeled and used to infect cells (eg, HeLa cells). Such AAV particles are analyzed for one or more of: viral particle uptake time, colocalization of AAV particles with particular compartment markers (e.g. Golgi apparatus, ER or lysosomal proteins or other markers), nuclear accumulation (e.g. when assayed by colocalization with nuclear marker or dye) and/or migration sensitivity to specific inhibitors of early endosomal escape (such as bafilomycin A or ammonium chloride), compared to fluorescently labeled wild-type AAV particles used to infect the same cell line (see, e.g., Bartlett, J. S. et al (2000) J. Virol 74:2777-2785 for a description of such assays).

[0203] For infectivity assays, AAV particles with capsid proteins having a mutated position 2 (e.g., iMet X) are used to infect cells (e.g., HeLa cells) and their transduction efficiency is compared to wild-type AAV particles (e.g., having the same same AAV serotype and infecting the same cell type).

[0204] For glycosylation assays, AAV particles with capsid proteins having a mutant position 2 (eg, iMet X) are used to infect cells (eg, HeLa cells). AAV particles from infected cells are subjected to one or more assays, including, but not limited to, chemical detection of glycosylation (e.g., use of a commercially available digooxygeninglycan (DIG) detection kit and/or fluorescent glycoprotein detection kit for denatured and electrophoretically separated capsid proteins), and mass spectrometry (e.g. FT-ICR MS), for comparison with wild-type AAV particles used to infect the same cell line (see e.g. Murray, S. et al. (2006) J. Virol. 80: 6171-6176 for a description of such assays).

[0205] For ubiquitination assay, AAV particles with capsid proteins having a mutant position 2 are used to infect cells (eg, HeLa cells). AAV particles are immunoprecipitated from infected cells with an anti-capsid antibody, then Western blotted with an anti-ubiquitin antibody and compared with wild-type AAV particles used to infect cells in the same manner. Mutant AAV particles can also be used in in vitro ubiquitination assays for comparison with wild-type AAV particles (see, for example, Yan, Z. et al. (2002) J. Virol. 76:2043-2053).

Example 3 Role of Deamidation of AAV2 Capsid Particles

[0206] Анализ последовательностей капсидного белка AAV2 показал потенциальные сайты дезамидирования, подчеркнутые в следующей аминокислотной последовательности: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPF NG LDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLG RAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFG QTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCD STWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHC HFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEY QLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLN NG SQAVGRSSFYCLEYFPSQMLRTG NNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAG ASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHL NG RDSLVNPGPAM ASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNL QRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLK HPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTS NYNKSVNVDFTVDT NG VYSEPRPIGTRYLTRNL (SEQ ID NO: 3).

[0207] In particular, a potential deamidation site was found in N57/G58 in the domain of phospholipase A2 (Ca++ binding site), shown in bold italics in the above sequence. The following experiments were aimed at investigating whether deamidation at N57 could lead to reduced activity and/or truncation of AAV2, and whether different methods of producing AAV could have different effects on deamidation. For example, using a method using a producer cell line (see Martin et al., (2013) Human Gene Therapy Methods 24:253-269; US Patent Application Publication No. US 2004/0224411, published before the grant of a patent; and Liu , X. L. et al (1999) Gene Ther 6:293-299) can induce a higher level of deamidation in N57 compared to the triple transfection method. In accordance with the crystal structure of AAV2, N57 is not shown; however, N382 and N511 are partially affected and N715 is fully affected.

Ways

Enzymatic digests of VP AAV1 and AAV2

[0208] 10 µg of each AAV1-EGFP or AAV2-EGFP material (formed by triple transfection, as well as the method using a producer cell line) was concentrated using Amicon filters (10 kDa MWCO), denatured with 6 M guanidine-HCl, 50 mM Tris at pH 8.5. Proteins were reduced with 5 mM DTT at 60° C. for 30 minutes in the dark, alkylated with 15 mM iodoacetamide at room temperature for 30 minutes, and then the buffer was changed to 25 mM Tris, pH 7.1, for digestion using Bio- Spin ^® 6 Tris. After changing the buffer, the samples were divided into two aliquots. Each aliquot was digested with trypsin at an enzyme:protein (w/w) ratio of 1:25 or Asp-N at 1:50 for 2 hours at 37°C, respectively.

Peptide mapping with UPLC/MS/MS

[0209] Digested proteins were also analyzed by UPLC/MS/MS in Acquity UPLC-Xevo qTOF MS. A C18 BEH300 column (2.1×150 mm) was used for mobile phase separation with 0.1% formic acid in a water/acetonitrile gradient at a flow rate of 0.25 ml/min. Mass spectra were obtained in the positive resolution mode MSe in the mass range of 50-2000.

Determination of Deamidation Levels in VP AAV

[0210] Extracted ion chromatograms (XIC) of peptides containing NG sites (T9, T49 and T67 in VP AA1 and AAV2) and their respective deamidated molecules were used to calculate levels of deamidation.

[0211] For comparison, AAV-based vectors obtained using triple transfection (TTx) methods and using a producer cell line (PCL), AAV1 or AAV2 labeled with EGFP, were obtained using the TTx or PCL method. Truncated VP1 (tVP1) was found to be present in PCL-derived AAV2-EGFP but not in TTx-derived AAV2-EGFP. It was found that AAV1-EGFP does not have tVP1, regardless of the method of obtaining. It has also been found that the in vitro efficacy of AAV2 produced by the PCL method is reduced compared to AAV2 produced by TTx. It was also found that the N57K and N57Q mutant AAV2 particles have reduced activity and impaired Ca++ binding.

[0212] The following table lists the tryptic peptides that were analyzed to examine each potential deamidation site as well as the corresponding residue.

Table 7. Tryptic peptides containing NG sites

Peptide (NG sequence underlined) Remainder YLGPF NG LDK (SEQ ID NO: 9) N57 EVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLN NG SQAVGRSSFYCLEYFPSQMLR (SEQ ID NO: 10) N382 YNL NG R (AAV1) (SEQ ID NO: 11) N511 YHL NG R (AAV2) (SEQ ID NO: 12) N511 SANVDFTVDN NG LYTEPR (AAV1) (SEQ ID NO: 13) N715 SVNVDFTVDT NG VYSEPR (AAV2) (SEQ ID NO: 14) N715

[0213] As shown in the table. 7, T9 YLGPF NG LDK peptide (SEQ ID NO: 9) was used to control N57, T38 EVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTL N NG SQAVGRSSFYCLEYFPSQMLR peptide (SEQ ID NO: 10) T38 EVTQNDGTTTIANNLTSTVQVFTDSEYQ (SEQ) was used to control N382, T49 YNL peptides NG R (SEQ YHL NG R (SEQ ID NO: 12) was used to control N511 in AAV1 or AAV2 (respectively) and peptides T67 SANVDFTVDN NG LYTEPR (SEQ ID NO: 13) and SVNVDFTVDT NG VYSEPR (SEQ ID NO: 14) were used to control N715 in AAV1 or AAV2 (respectively).

[0214] LC/MS/MS analysis was used to compare the percentage of deamidation in AAV1 and AAV2 particles obtained using the TTx and PCL methods. The results for the T9 peptide are shown in FIG. 6A and 6B. The results for the T49 peptide are shown in FIG. 7A and 7B. The results for the T67 peptide are shown in FIG. 8A and 8B. These results are summarized in Table. 8. The T38 peptide was not identified due to its size.

Table 8. Summary of LC/MS/MS results

% deamidation N57 N511 N715 AAV1 TTx 7.9 30.9 18.1 PCL 11.3 27.4 18.7 AAV2 TTx 6.7 39.6 27.4 PCL 18.4 42.3 28.0

[0215] In particular, PCL-derived AAV2 had an almost 3-fold increase in deamidation compared to TTx-derived AAV2. These results indicate that deamidation reduces the activity of AAV, as well as the activity of AAV2 in vitro obtained using PCL.

findings

[0216] Taken together, Examples 1-3 show methods for analyzing intact viral particle proteins (eg, AAV capsid proteins) by LC/MS. Molecular weight was measured accurately, and these techniques can also be used to determine the N-termini and/or modifications of viral capsid proteins. Moreover, these methods can be adapted as capsid serotype identity assays useful in gene therapy, for example as an analytical platform. These results further define the relationship between structure (eg, truncations, deamidation, etc.) and activity of capsid proteins, suggesting that point mutations at major sites can be used to design more efficient vectors.

Example 4 Determining the Role of N-Terminal Acetylation of AAV Capsid Proteins

[0217] As discussed above, the N-termini of AAV capsid proteins are highly conserved across serotypes (FIG. 5). The techniques described in example 1, allow you to study the expression and post-translational modifications of VP. Next, the role and biological significance of N-terminal acetylation of AAV capsid proteins was studied.

results

[0218] To determine the potential role of deacetylation of AAV capsid proteins, AAV5 deacetylation variants were investigated. The AAV5 particle expressing eGFP using the CBA promoter (AAV5-CBA-Egfp) was compared to AAV5 variants with the amino acid adjacent to the initiator methionine (iMET) mutated in the case of VP1 and VP3 (deAC-AAV5-CBA-eGFPs). Three amino acids predicted to have a low probability of NatA, NatC, or NatD acetylation were selected to generate variants: Gly, Leu, and Pro, as shown in Table 1. 9 below.

Table 9. Frequency of N-terminal acetylation

N-terminal amino acids Transferase Frequency NT-AC MET-ALA
Commonly found in VP1 and VP3 NatA High MET-SER
Usually found in VP1 and VP3 in the case of AAV5 NatA High AAV variants MET GLY NatA Low MET-LEU NatC Low MET PRO NatD/other Low

[0219] The following deacetylated AAV5 (deAC) mutants were generated:

S2GVP1 - Ser changed to Gly at position 2 in AAV5VP1

S2LVP1 - Ser changed to Leu in position 2 in AAV5VP1

S2PVP1 - Ser changed to Pro in position 2 in AAV5VP1

S2GVP3 - Ser changed to Gly at position 2 in AAV5VP3

S2LVP3 - Ser changed to Leu in position 2 in AAV5VP3

S2PVP3 - Ser changed to Pro in position 2 in AAV5VP3

S2PVP1/VP3 - Ser changed to Pro in position 2 in both VP1 and VP3 AAV5

S2GVP1/VP3 - Ser changed to Gly at position 2 in both VP1 and VP3 AAV5

S2LVP1/VP3 - Ser changed to Leu in position 2 in both VP1 and VP3 AAV5

[0220] These variants were generated using the TTX method described above. All variants of AAV5 were characterized by high productivity, while the output was more than 10 ¹³ total VG. All AAV5 variants also had a predictable ratio of VP1:VP2:VP3 proteins by SYPRO protein gel analysis (FIG. 9). Thereafter, LC/MS was used to confirm that all AAV5 variants had reduced acetylation, as shown in Table 1. ten.

Table 10. LC/MS analysis in case of AAV5 variant acetylation

Mutants Theor. VP1 Exp. VP1 ∆mass (VP1) Theor. VP2 Exp. VP2 ∆mass (VP2) Theor. VP3 Exp. VP3 ∆mass (VP3) Note one deAC-AAV5 (S2GVP1)/CBA-eGFP 80234 but. 65283 65293 ten 59463 59472 nine VP1 not detected 2 deAC-AAV5 (S2LVP1)/CBA-eGFP 80346 80501 181 65283 65292 nine 59463 59471 eight VP1 erroneous 3 deAC-AAV5 (S2GVP3)/CBA-eGFP 80234 but. 65253 65261 eight 59391 59398 7 confirmed 4 deAC-AAV5 (S2LVP3)/CBA-eGFP 80336 80363 27 65309 65309 0 59447 59620 173 VP3 erroneous 5 deAC-AAV5 (S2PVP1VP3)/CBA-eGFP 80314 80324 ten 65293 65300 7 59431 59438 7 confirmed 6 deAC-AAV5 (S2GVP1VP3)/CBA-eGFP 80234 80243 nine 65253 65261 eight 59391 59398 7 confirmed 7 deAC-AAV5 (S2PVP3)/CBA-eGFP 80336 80346 ten 65293 65292 one 59431 59430 one confirmed eight deAC-AAV5 (S2PVP1)/CBA-eGFP 80314 80313 one 65283 65291 eight 59463 59470 7 confirmed nine deAC-AAV5 (S2L VP1VP3)/CBA-eGFP 80346 but. 65309 65318 nine 59447 59629 182 VP3 erroneous

but. = not defined.

[0221] These LC/MS analyzes confirmed that the AAV5 variants were deacetylated. All variants of S2LVP1, S2LVP3, and S2LVP1/VP3 were characterized by increased mass (increased from 173 to 182) in VP1 and VP3 proteins, indicating that changing the second N-terminal amino acid to leucine in VP1 or VP3 alters the protein, leading to an increase in mass.

[0222] AAV5 variants were then analyzed in an in vitro transduction assay with eGFP as a reporter gene (FIG. 10). An assay was designed to evaluate transduction with deacetylated ^AAV5 mutant variants at a MOI of 106, comparing each variant with the original, unmodified AAV5 particle. Three cell lines were used: 293, HuH7 and HeLa cells. After infection, the cells were analyzed to determine the copy number of the vector genome (vg/μg cell protein) and eGFP expression (using ELISA). Vector genome copy number (vg/µg protein) represents the efficiency at which the AAV5 variant enters the cell, and eGFP represents the efficiency of intracellular capsid migration, since transgene expression requires capsid/vector DNA to migrate efficiently into the nucleus (Fig. 10). Vector genomes were quantified by TaqMan assay.

[0223] FIG. 11 shows that, based on vector genome analysis, the deacetylated AAV5 mutant vectors infected all three test cell lines at similar but reduced levels compared to the original unmodified AAV5 particles. In FIG. 12 shows that all deacetylated AAV5 mutant vectors resulted in reduced eGFP expression in all three cell lines compared to transduction of the original unmodified AAV5.

findings

[0224] As predicted, acetylation in mutants in which the N-terminal Ser is replaced by Pro/Leu/Gly was not observed when examined using LC/MS. The deAC AAV5 variants were characterized by sustained production of the vectors, and the deAC AAV5 variants infected cells at levels comparable to the original AAV5. However, the functional levels of proteins in cells infected with deAC variants were significantly reduced compared to the original AAV5. These data indicate that tropism is minimally impaired as a result of the absence of N-terminal deacetylation in VP1/VP3, however, subsequent processing (eg, migration and/or degradation) is significantly impaired. Since the studied variants were characterized by reduced activity in vitro, the person skilled in the art will understand that you can use options characterized by reduced or eliminated acetylation, among other things, if reduced transduction levels are desired.

Example 5 Assessment of Deamidation of AAV Capsid Proteins

[0225] Examples 1 and 3 show techniques that allow the study of post-translational modifications of AAV capsid proteins and the role of deamidation of the AAV2 capsid. In the following example, it was investigated whether deamidation reduces activity and/or induces truncation of capsid proteins, and whether different manufacturing methods can affect levels of deamidation.

Ways

AAV particles were generated and deamidation status was analyzed as described in Example 3.

results

[0226] As described in Example 3, a potential deamidation site was found at N57/G58 in the phospholipase A2 domain (Ca++ binding site) in VP1 of the AAV2 capsid. The N57/G58 motif is conserved across AAV serotypes (FIG. 13). Example 3 shows that PCL-derived AAV2 had an almost 3-fold increase in deamidation compared to TTx-derived AAV2 (see FIGS. 6A and 6B and Table 8).

[0227] When studying the production of VP1, VP2 and VP3 using protein gels, the truncated VP1 protein (tVP1) was detected only in the AAV2 capsid proteins obtained using the PCL method (Fig. 14).

[0228] After that, a number of AAV2 deamidation mutants were formed. These mutants targeted the Gly residue in the canonical NG sequence. Also generated mutations targeting residue A35 (see Fig. 13), N-terminal amino acid in the case of tVP1, as shown in table. 11. Mutants pAF277 and pAF279 carrying multiple mutations were not packaged.

Table 11. Deamidation Mutants

Name Mutation Avg. drp/cell pAF274 G58K 4.54E+03 pAF275 G58D 5.00E+03 pAF276 G58Q 5.41E+03 pAF277 G58,383,512,716K 1.2 pAF278 A35N 6.89E+03 pAF279 A35N, G58,383,512,765K 2.2 293 - 0.9 PIM45 The control 6.28E+03

K = positive charge (primary);

D = negative charge (acidic);

Q = polar.

[0229] Deamidation of the variants was then analyzed by LC/MS as described in Example 3 above. Variants AAV2A35N and AAV2G58D were characterized by altered deamidation compared to the original AAV2 (Fig. 15). In particular, the AAV2A35N mutant was characterized by increased deamidation (17.8%) compared to the original AAV2 (5.7%). The AAV2G58D variant was characterized by reduced deamidation (1.1%) compared to the original AAV2. SYPRO protein gel analysis indicated that the AAV2 deamidation mutants had an appropriate VP1:VP2:VP2 ratio (FIG. 16).

[0230] Thereafter, AAV2 deamidation variants were analyzed in an in vitro transduction assay with eGFP as a reporter gene (FIG. 17). The assay was designed to evaluate transduction of AAV2 deamidation mutant variants at a MOI of 10 ⁶ by comparing each variant with the original, unmodified AAV2 particle. Three cell lines were used: 293, HuH7 and HeLa cells. After infection, the cells were analyzed to determine the copy number of the vector genome (vg/μg cell protein) and eGFP expression (using ELISA). The copy number of the vector genome (vg/µg protein) represents the efficiency at which the AAV2 variant enters the cell, and eGFP represents the efficiency of intracellular capsid migration, since transgene expression requires capsid/vector DNA to efficiently migrate into the nucleus (Fig. 17). Vector genomes were quantified by TaqMan assay.

[0231] Analysis of the vector genomes indicated that the AAV2 deamidation mutants infected all cell lines tested at levels comparable to the original AAV2 vectors (FIG. 18). Importantly, the AAV2A35N variant was found to be more active than the original AAV2 vector for transduction into all cell lines (FIG. 19). The AAV2G58D variant was found to be more active than the original AAV2 vector in HuH7 cells (FIG. 19).

findings

[0232] In conclusion, AAV2 deamidation mutant vectors infect cells at levels comparable to the original AAV2 particles (eg, comparable to vg/μg cellular protein). However, based on the analysis of eGFP levels in the transduced cells, the AAV2A35N variant was more active than the parent AAV2 in all cell lines studied, and the AAV2G58D variant was more active than the parent AAV2 in HuH7 (liver-derived cell line). These results indicate that the A35N mutation may be effective in increasing vector activity when transduced into various cell types, and that the G58D mutation may also be effective in increasing activity in certain cell types, such as liver cells.

Literary sources

1. Girod, A., et al., The VP1 capsid protein of adeno-associated virus type 2 is carrying a phospholipase A2 domain required for virus infectivity. J Gen Virol, 2002. 83(Pt 5): p. 973-8.

2. Stahnke, S., et al., Intrinsic phospholipase A2 activity of adeno-associated virus is involved in endosomal escape of incoming particles. Virology, 2011. 409(1): p. 77-83.

3. Bleker, S., F. Sonntag, and J.A. Kleinschmidt, Mutational analysis of narrow pores at the fivefold symmetry axes of adeno-associated virus type 2 capsids reveals a dual role in genome packaging and activation of phospholipase A2 activity. J Virol, 2005. 79(4): p. 2528-40.

4. Popa-Wagner, R., et al., Impact of VP1-specific protein sequence motifs on adeno-associated virus type 2 intracellular trafficking and nuclear entry. J Virol, 2012. 86(17): p. 9163-74.

5. Kashiwakura, Y., et al., Hepatocyte growth factor receptor is a coreceptor for adeno-associated virus type 2 infection. J Virol, 2005. 79(1): p. 609-14.

6. Qing, K., et al., Human fibroblast growth factor receptor 1 is a co-receptor for infection by adeno-associated virus 2. Nat Med, 1999. 5(1): p. 71-7.

7. Sanlioglu, S., et al., Endocytosis and nuclear trafficking of adeno-associated virus type 2 are controlled by rac1 and phosphatidylinositol-3 kinase activation. J Virol, 2000. 74(19): p. 9184-96.

8. Summerford, C., J.S. Bartlett, and R.J. Samulski, AlphaVbeta5 integrin: a co-receptor for adeno-associated virus type 2 infection. Nat Med, 1999. 5(1): p. 78-82.

9. Kern, A., et al., Identification of a Heparin-Binding Motif on Adeno-Associated Virus Type 2 Capsids. J Virol, 2003. 77(20): p. 11072-11081.

10. Asokan, A., et al., Adeno-associated virus type 2 contains an integrin alpha5beta1 binding domain essential for viral cell entry. J Virol, 2006. 80(18): p. 8961-9.

11. Xie, Q., et al., The atomic structure of adeno-associated virus (AAV-2), a vector for human gene therapy. Proc Natl Acad Sci U S A, 2002. 99(16): p. 10405-10.

12. DiMattia, M.A., et al., Structural insight into the unique properties of adeno-associated virus serotype 9. J Virol, 2012. 86(12): p. 6947-58.

13. Nam, H.J., et al., Structure of adeno-associated virus serotype 8, a gene therapy vector. J Virol, 2007. 81(22): p. 12260-71.

14. Kronenberg, S., et al., A conformational change in the adeno-associated virus type 2 capsid leads to the exposure of hidden VP1 N termini. J Virol, 2005. 79(9): p. 5296-303.

15. Sonntag, F., et al., Adeno-associated virus type 2 capsids with externalized VP1/VP2 trafficking domains are generated prior to passage through the cytoplasm and are maintained until uncoating occurs in the nucleus. J Virol, 2006. 80(22): p. 11040-54.

16. Murray, S., et al., Characterization of the capsid protein glycosylation of adeno-associated virus type 2 by high-resolution mass spectrometry. J Virol, 2006. 80(12): p. 6171-6.

17. Salganik, M., et al., Evidence for pH-dependent protease activity in the adeno-associated virus capsid. J Virol, 2012. 86(21): p. 11877-85.

18. Van Vliet, K., et al., Adeno-associated virus capsid serotype identification: Analytical methods development and application. J Virol Methods, 2009. 159(2): p. 167-77.

19. Thomas, J.J., et al., Viral characterization by direct analysis of capsid proteins. Anal Chem, 1998. 70(18): p. 3863-7.

20. Wang, L., L.C. Lane, and D.L. Smith, Detecting structural changes in viral capsids by hydrogen exchange and mass spectrometry. Protein Sci, 2001. 10(6): p. 1234-43.

21. Hwang, C.S., A. Shemorry, and A. Varshavsky, N-terminal acetylation of cellular proteins creates specific degradation signals. Science, 2010. 327(5968): p. 973-7.

22. Yan, Z., et al., Ubiquitination of both Adeno-Associated Virus Type 2 and 5 Capsid Proteins Affects the Transduction Efficiency of Recombinant Vectors. J Virol, 2002. 76(5): p. 2043-2053.

SEQUENCES

All polypeptide sequences are presented in N-terminal to C-terminal direction unless otherwise indicated.

All nucleic acid sequences are presented in the 5' to 3' direction unless otherwise indicated.

Nucleotide sequences of potential VP3 AAV2 initiation codons (ATG codons are underlined)

ATG GCTACAGGCAGTGGCGCACCA ATG GCAGAC (SEQ ID NO: 1)

Polypeptide sequence corresponding to potential VP3 AAV2 initiation codons (methionines are underlined)

M ATGSGAP M AD (SEQ ID NO: 2)

AAV2 VP1 polypeptide sequence

MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYL GPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSF GGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPAR KRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSS GNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYF DFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQ VFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPS QMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRL QFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLV NPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYG SVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLM GGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWN PEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL (SEQ ID NO: 3)

N-terminal VP1 tryptic peptide (N-terminal alanine is acetylated)

AADGYLPDWLEDTLSEGIR (SEQ ID NO: 4)

N-terminal Asp-N-peptide VP3 (N-terminal alanine is acetylated)

ATGSGAPM (SEQ ID NO: 5)

Regular N-terminal sequence of VP1

MAADGYLPDWLED (SEQ ID NO: 6)

Nucleotide sequences of potential VP3 AAV7 initiation codons (start codons are underlined)

GTG GCTGCAGGCGGTGGCGCACCA ATG GCAGACAATAAC (SEQ ID NO:7)

Nucleotide sequence of the mutated ITR

CACTCCCTCTCTGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTC GCCCACGCCCGGGCTTTGCCCGGGCG (SEQ ID NO: 8)

--->

SEQUENCE LIST

<110> JIN, Xiaoying

O'RIORDAN, Catherine

LIU, Ling

Zhang, Kate

<120> METHODS FOR AAV DETECTION

<130> 159792014140

<140> Not yet assigned

<141> At the same time

<150> 62/375.314

<151> 2016-08-15

<160> 40

<170> FastSEQ for Windows version 4.0

<210> 1

<211> 33

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 1

Ala Thr Gly Gly Cys Thr Ala Cys Ala Gly Gly Cys Ala Gly Thr Gly

1 5 10 15

Gly Cys Gly Cys Ala Cys Cys Ala Ala Thr Gly Gly Cys Ala Gly Ala

20 25 30

Cys

<210> 2

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 2

Met Ala Thr Gly Ser Gly Ala Pro Met Ala Asp

1 5 10

<210> 3

<211> 735

<212> PROTEIN

<213> Adeno-associated virus 2

<400> 3

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser

1 5 10 15

Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro

20 25 30

Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Pro Val Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu His Ser Pro Val Glu Pro Asp Ser Ser Ser Gly Thr Gly

145 150 155 160

Lys Ala Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Leu Gly Gln Pro Pro

180 185 190

Ala Ala Pro Ser Gly Leu Gly Thr Asn Thr Met Ala Thr Gly Ser Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr

260 265 270

Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His

275 280 285

Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp

290 295 300

Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln Val

305 310 315 320

Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu

325 330 335

Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr

340 345 350

Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp

355 360 365

Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser

370 375 380

Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser

385 390 395 400

Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe Glu

405 410 415

Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg

420 425 430

Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr

435 440 445

Asn Thr Pro Ser Gly Thr Thr Thr Gln Ser Arg Leu Gln Phe Ser Gln

450 455 460

Ala Gly Ala Ser Asp Ile Arg Asp Gln Ser Arg Asn Trp Leu Pro Gly

465 470 475 480

Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Ser Ala Asp Asn Asn

485 490 495

Asn Ser Glu Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His Leu Asn Gly

500 505 510

Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys Asp

515 520 525

Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu Ile Phe Gly Lys

530 535 540

Gln Gly Ser Glu Lys Thr Asn Val Asp Ile Glu Lys Val Met Ile Thr

545 550 555 560

Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr

565 570 575

Gly Ser Val Ser Thr Asn Leu Gln Arg Gly Asn Arg Gln Ala Ala Thr

580 585 590

Ala Asp Val Asn Thr Gln Gly Val Leu Pro Gly Met Val Trp Gln Asp

595 600 605

Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His Thr

610 615 620

Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu Lys

625 630 635 640

His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala Asn

645 650 655

Pro Ser Thr Thr Phe Ser Ala Ala Lys Phe Ala Ser Phe Ile Thr Gln

660 665 670

Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln Lys

675 680 685

Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr

690 695 700

Asn Lys Ser Val Asn Val Asp Phe Thr Val Asp Thr Asn Gly Val Tyr

705 710 715 720

Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu

725 730 735

<210> 4

<211> 19

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<220>

<221> ACETYLING

<222> 1

<400> 4

Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser Glu

1 5 10 15

Gly Ile Arg

<210> 5

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<220>

<221> ACETYLING

<222> 1

<400> 5

Ala Thr Gly Ser Gly Ala Pro Met

fifteen

<210> 6

<211> 13

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 6

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp

1 5 10

<210> 7

<211> 39

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 7

Gly Thr Gly Gly Cys Thr Gly Cys Ala Gly Gly Cys Gly Gly Thr Gly

1 5 10 15

Gly Cys Gly Cys Ala Cys Cys Ala Ala Thr Gly Gly Cys Ala Gly Ala

20 25 30

Cys Ala Ala Thr Ala Ala Cys

35

<210> 8

<211> 78

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 8

Cys Ala Cys Thr Cys Cys Cys Thr Cys Thr Cys Thr Gly Cys Gly Cys

1 5 10 15

Gly Cys Thr Cys Gly Cys Thr Cys Gly Cys Thr Cys Ala Cys Thr Gly

20 25 30

Ala Gly Gly Cys Cys Gly Gly Gly Cys Gly Ala Cys Cys Ala Ala Ala

35 40 45

Gly Gly Thr Cys Gly Cys Cys Cys Ala Cys Gly Cys Cys Cys Gly Gly

50 55 60

Gly Cys Thr Thr Thr Gly Cys Cys Cys Gly Gly Gly Cys Gly

65 70 75

<210> 9

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 9

Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys

1 5 10

<210> 10

<211> 83

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 10

Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu Thr

1 5 10 15

Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr Val

20 25 30

Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp Val

35 40 45

Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser Gln

50 55 60

Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser Gln

65 70 75 80

Met Leu Arg

<210> 11

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 11

Tyr Asn Leu Asn Gly Arg

fifteen

<210> 12

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 12

Tyr His Leu Asn Gly Arg

fifteen

<210> 13

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 13

Ser Ala Asn Val Asp Phe Thr Val Asp Asn Asn Gly Leu Tyr Thr Glu

1 5 10 15

Pro Arg

<210> 14

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 14

Ser Val Asn Val Asp Phe Thr Val Asp Thr Asn Gly Val Tyr Ser Glu

1 5 10 15

Pro Arg

<210> 15

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 15

Ala Pro Gly Lys Lys Arg Pro Val Glu His Ser Pro Val Glu Pro

1 5 10 15

<210> 16

<211> 735

<212> PROTEIN

<213> Adeno-associated virus 2

<400> 16

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser

1 5 10 15

Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro

20 25 30

Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Pro Val Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu His Ser Pro Val Glu Pro Asp Ser Ser Ser Gly Thr Gly

145 150 155 160

Lys Ala Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Leu Gly Gln Pro Pro

180 185 190

Ala Ala Pro Ser Gly Leu Gly Thr Asn Thr Met Ala Thr Gly Ser Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr

260 265 270

Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His

275 280 285

Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp

290 295 300

Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln Val

305 310 315 320

Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu

325 330 335

Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr

340 345 350

Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp

355 360 365

Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser

370 375 380

Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser

385 390 395 400

Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe Glu

405 410 415

Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg

420 425 430

Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr

435 440 445

Asn Thr Pro Ser Gly Thr Thr Thr Gln Ser Arg Leu Gln Phe Ser Gln

450 455 460

Ala Gly Ala Ser Asp Ile Arg Asp Gln Ser Arg Asn Trp Leu Pro Gly

465 470 475 480

Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Ser Ala Asp Asn Asn

485 490 495

Asn Ser Glu Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His Leu Asn Gly

500 505 510

Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys Asp

515 520 525

Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu Ile Phe Gly Lys

530 535 540

Gln Gly Ser Glu Lys Thr Asn Val Asp Ile Glu Lys Val Met Ile Thr

545 550 555 560

Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr

565 570 575

Gly Ser Val Ser Thr Asn Leu Gln Arg Gly Asn Arg Gln Ala Ala Thr

580 585 590

Ala Asp Val Asn Thr Gln Gly Val Leu Pro Gly Met Val Trp Gln Asp

595 600 605

Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His Thr

610 615 620

Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu Lys

625 630 635 640

His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala Asn

645 650 655

Pro Ser Thr Thr Phe Ser Ala Ala Lys Phe Ala Ser Phe Ile Thr Gln

660 665 670

Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln Lys

675 680 685

Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr

690 695 700

Asn Lys Ser Val Asn Val Asp Phe Thr Val Asp Thr Asn Gly Val Tyr

705 710 715 720

Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu

725 730 735

<210> 17

<211> 239

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 17

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro

20 25 30

Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Pro Ser Pro Gln Arg Ser Pro Asp Ser Ser Thr Gly Ile

145 150 155 160

Gly Lys Lys Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln

165 170 175

Thr Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro

180 185 190

Pro Ala Gly Pro Ser Gly Leu Gly Ser Gly Thr Met Ala Ala Gly Gly

195 200 205

Gly Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser

210 215 220

Ser Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg

225 230 235

<210> 18

<211> 239

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 18

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro

20 25 30

Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Pro Ser Pro Gln Arg Ser Pro Asp Ser Ser Thr Gly Ile

145 150 155 160

Gly Lys Lys Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln

165 170 175

Thr Gly Glu Ser Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro

180 185 190

Pro Ala Gly Pro Ser Gly Leu Gly Ser Gly Thr Met Ala Ala Gly Gly

195 200 205

Gly Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser

210 215 220

Ser Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg

225 230 235

<210> 19

<211> 239

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 19

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Lys Pro

20 25 30

Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Gln Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Pro Ser Pro Gln Arg Ser Pro Asp Ser Ser Thr Gly Ile

145 150 155 160

Gly Lys Lys Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln

165 170 175

Thr Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro

180 185 190

Pro Ala Ala Pro Ser Gly Val Gly Pro Asn Thr Met Ala Ala Gly Gly

195 200 205

Gly Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser

210 215 220

Ser Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg

225 230 235

<210> 20

<211> 239

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 20

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro

20 25 30

Lys Ala Asn Gln Gln Lys Gln Asp Asn Gly Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Ala Lys Lys Arg

130 135 140

Pro Val Glu Pro Ser Pro Gln Arg Ser Pro Asp Ser Ser Thr Gly Ile

145 150 155 160

Gly Lys Lys Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln

165 170 175

Thr Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro

180 185 190

Pro Ala Ala Pro Ser Ser Val Gly Ser Gly Thr Val Ala Ala Gly Gly

195 200 205

Gly Ala Pro Met Ala Asp Asn Glu Gly Ala Asp Gly Val Gly Asn

210 215 220

Ala Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg

225 230 235

<210> 21

<211> 238

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 21

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro

20 25 30

Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Ile Gly

145 150 155 160

Lys Thr Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro

180 185 190

Ala Thr Pro Ala Ala Val Gly Pro Thr Thr Met Ala Ser Gly Gly Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ala

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg

225 230 235

<210> 22

<211> 238

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 22

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro

20 25 30

Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Phe Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Ile Gly

145 150 155 160

Lys Thr Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro

180 185 190

Ala Thr Pro Ala Ala Val Gly Pro Thr Thr Met Ala Ser Gly Gly Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ala

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg

225 230 235

<210> 23

<211> 238

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 23

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser

1 5 10 15

Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro

20 25 30

Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Pro Val Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu His Ser Pro Val Glu Pro Asp Ser Ser Ser Gly Thr Gly

145 150 155 160

Lys Ala Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Leu Gly Gln Pro Pro

180 185 190

Ala Ala Pro Ser Gly Leu Gly Thr Asn Thr Met Ala Thr Gly Ser Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg

225 230 235

<210> 24

<211> 238

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 24

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Val Pro Gln Pro

20 25 30

Lys Ala Asn Gln Gln His Gln Asp Asn Arg Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Ile Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Gly

130 135 140

Ala Val Asp Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Val Gly

145 150 155 160

Lys Ser Gly Lys Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro

180 185 190

Ala Ala Pro Thr Ser Leu Gly Ser Asn Thr Met Ala Ser Gly Gly Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg

225 230 235

<210> 25

<211> 232

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 25

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro

20 25 30

Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Leu Glu Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Ile Gly Lys

145 150 155 160

Lys Gly Lys Gln Pro Ala Arg Lys Arg Leu Asn Phe Glu Glu Asp Thr

165 170 175

Gly Ala Gly Asp Gly Pro Pro Glu Gly Ser Asp Thr Ser Ala Met Ser

180 185 190

Ser Asp Ile Glu Met Arg Ala Ala Pro Gly Gly Asn Ala Val Asp Ala

195 200 205

Gly Gln Gly Ser Asp Gly Val Gly Asn Ala Ser Gly Asp Trp His Cys

210 215 220

Asp Ser Thr Trp Ser Glu Gly Lys

225 230

<210> 26

<211> 241

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 26

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Gln Pro

20 25 30

Lys Ala Asn Gln Gln His Gln Asp Asn Gly Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Lys Gln Leu Glu Gln Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Gln Arg Leu Ala Thr Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Ile Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Gly Val Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Leu Glu Lys Thr Pro Asn Arg Pro Thr Asn Pro Asp Ser Gly Lys

145 150 155 160

Ala Pro Ala Lys Lys Lys Gln Lys Asp Gly Glu Pro Ala Asp Ser Ala

165 170 175

Arg Arg Thr Leu Asp Phe Glu Asp Ser Gly Ala Gly Asp Gly Pro Pro

180 185 190

Glu Gly Ser Ser Ser Gly Glu Met Ser His Asp Ala Glu Met Arg Ala

195 200 205

Ala Pro Gly Gly Asn Ala Val Glu Ala Gly Gln Gly Ala Asp Gly Val

210 215 220

Gly Asn Ala Ser Gly Asp Trp His Cys Asp Ser Thr Trp Ser Glu Gly

225 230 235 240

Arg

<210> 27

<211> 232

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 27

Met Thr Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser Glu

1 5 10 15

Gly Val Arg Glu Trp Trp Ala Leu Gln Pro Gly Ala Pro Lys Pro Lys

20 25 30

Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu Pro Gly

35 40 45

Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro Val

50 55 60

Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp Gln

65 70 75 80

Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala Asp

85 90 95

Ala Glu Phe Gln Gln Arg Leu Gln Gly Asp Thr Ser Phe Gly Gly Asn

100 105 110

Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro Leu

115 120 125

Gly Leu Val Glu Gln Ala Gly Glu Thr Ala Pro Gly Lys Lys Arg Pro

130 135 140

Leu Ile Glu Ser Pro Gln Gln Pro Asp Ser Ser Thr Gly Ile Gly Lys

145 150 155 160

Lys Gly Lys Gln Pro Ala Lys Lys Lys Leu Val Phe Glu Asp Glu Thr

165 170 175

Gly Ala Gly Asp Gly Pro Pro Glu Gly Ser Thr Ser Gly Ala Met Ser

180 185 190

Asp Asp Ser Glu Met Arg Ala Ala Ala Gly Gly Ala Ala Val Glu Gly

195 200 205

Gly Gln Gly Ala Asp Gly Val Gly Asn Ala Ser Gly Asp Trp His Cys

210 215 220

Asp Ser Thr Trp Ser Glu Gly His

225 230

<210> 28

<211> 228

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 28

Met Ser Phe Val Asp His Pro Pro Asp Trp Leu Glu Glu Val Gly Glu

1 5 10 15

Gly Leu Arg Glu Phe Leu Gly Leu Glu Ala Gly Pro Pro Lys Pro Lys

20 25 30

Pro Asn Gln Gln His Gln Asp Gln Ala Arg Gly Leu Val Leu Pro Gly

35 40 45

Tyr Asn Tyr Leu Gly Pro Gly Asn Gly Leu Asp Arg Gly Glu Pro Val

50 55 60

Asn Arg Ala Asp Glu Val Ala Arg Glu His Asp Ile Ser Tyr Asn Glu

65 70 75 80

Gln Leu Glu Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala Asp

85 90 95

Ala Glu Phe Gln Glu Lys Leu Ala Asp Asp Thr Ser Phe Gly Gly Asn

100 105 110

Leu Gly Lys Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro Phe

115 120 125

Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Thr Gly Lys Arg Ile

130 135 140

Asp Asp His Phe Pro Lys Arg Lys Lys Ala Arg Thr Glu Glu Asp Ser

145 150 155 160

Lys Pro Ser Thr Ser Ser Asp Ala Glu Ala Gly Pro Ser Gly Ser Gln

165 170 175

Gln Leu Gln Ile Pro Ala Gln Pro Ala Ser Ser Leu Gly Ala Asp Thr

180 185 190

Met Ser Ala Gly Gly Gly Gly Pro Leu Gly Asp Asn Asn Gln Gly Ala

195 200 205

Asp Gly Val Gly Asn Ala Ser Gly Asp Trp His Cys Asp Ser Thr Trp

210 215 220

Met Gly Asp Arg

225

<210> 29

<211> 238

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 29

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Gln Pro

20 25 30

Lys Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ala Gly Ile Gly

145 150 155 160

Lys Ser Gly Ala Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro Pro

180 185 190

Ala Ala Pro Ser Gly Val Gly Ser Leu Thr Met Ala Ser Gly Gly Gly

195 200 205

Ala Pro Val Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg

225 230 235

<210> 30

<211> 246

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<220>

<221> OPTION

<222> 24, 148, 151, 152, 154, 155, 168, 169, 170, 171, 172, 208,

209

<223> Xaa = any amino acid

<400> 30

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Xaa Leu Lys Pro Gly Ala Pro Lys Pro

20 25 30

Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Xaa Ser Pro Xaa Xaa Gln Xaa Xaa Pro Asp Ser Ser Ser

145 150 155 160

Gly Ile Gly Lys Lys Gly Gln Xaa Xaa Xaa Xaa Xaa Gln Pro Ala Lys

165 170 175

Lys Arg Leu Asn Phe Gly Gln Thr Gly Asp Ser Glu Ser Val Pro Asp

180 185 190

Pro Gln Pro Leu Gly Glu Pro Pro Ala Ala Pro Ser Gly Leu Gly Xaa

195 200 205

Xaa Thr Met Ala Ala Gly Gly Gly Ala Pro Met Ala Asp Asn Asn Glu

210 215 220

Gly Ala Asp Gly Val Gly Asn Ala Ser Gly Asn Trp His Cys Asp Ser

225 230 235 240

Thr Trp Leu Gly Asp Arg

245

<210> 31

<211> 61

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 31

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro

20 25 30

Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys

50 55 60

<210> 32

<211> 61

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 32

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Lys Pro

20 25 30

Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys

50 55 60

<210> 33

<211> 61

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 33

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro

20 25 30

Lys Ala Asn Gln Gln Lys Gln Asp Asn Gly Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys

50 55 60

<210> 34

<211> 61

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 34

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser

1 5 10 15

Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro

20 25 30

Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys

50 55 60

<210> 35

<211> 61

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 35

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Val Pro Gln Pro

20 25 30

Lys Ala Asn Gln Gln His Gln Asp Asn Arg Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys

50 55 60

<210> 36

<211> 61

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 36

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Gln Pro

20 25 30

Lys Lys Ala Asn Gln Gln His Gln Asp Asn Gly Arg Gly Leu Val Leu

35 40 45

Pro Gly Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys

50 55 60

<210> 37

<211> 60

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 37

Met Thr Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser Glu

1 5 10 15

Gly Val Arg Glu Trp Trp Ala Leu Gln Pro Gly Ala Pro Lys Pro Lys

20 25 30

Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu Pro Gly

35 40 45

Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys

50 55 60

<210> 38

<211> 60

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 38

Met Ser Phe Val Asp His Pro Pro Asp Trp Leu Glu Glu Val Gly Glu

1 5 10 15

Gly Leu Arg Glu Phe Leu Gly Leu Glu Ala Gly Pro Pro Lys Pro Lys

20 25 30

Pro Asn Gln Gln His Gln Asp Gln Ala Arg Gly Leu Val Leu Pro Gly

35 40 45

Tyr Asn Tyr Leu Gly Pro Gly Asn Gly Leu Asp Arg

50 55 60

<210> 39

<211> 61

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 39

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Gln Pro

20 25 30

Lys Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys

50 55 60

<210> 40

<211> 61

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<220>

<221> OPTION

<222> 24

<223> Xaa = any amino acid

<400> 40

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Xaa Leu Lys Pro Gly Ala Pro Lys Pro

20 25 30

Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys

50 55 60

<---

Claims (129)

1. A method for determining the serotype of a viral particle, including: a) denaturation of the viral particle; b) direct introduction of the denatured viral particle into the intact protein assay by liquid chromatography/mass spectrometry (LC/MS); and c) determining the masses of one or more capsid proteins of the viral particle; where the specific combination of masses of one or more capsid proteins characterizes the serotype of the virus, and where the method is carried out in the absence of a separation step on the gel. 2. The method of claim 1, wherein the calculated masses of one or more capsid proteins are compared to theoretical masses of one or more capsid proteins of one or more virus serotypes. 3. A method for determining the serotype of a viral particle, including: a) denaturation of the viral particle; b) recovery and/or alkylation of the denatured viral particle; c) cleavage of the denatured viral particle to form fragments of one or more capsid proteins of the viral particle; d) liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) of fragments of one or more capsid proteins; and e) determining the masses of fragments of one or more capsid proteins of the viral particle; where the specific combination of masses of fragments of one or more capsid proteins characterizes the serotype of the virus, and where the method is carried out in the absence of a separation step on the gel. 4. The method of claim 3, wherein the calculated masses of fragments of one or more capsid proteins are compared with the theoretical masses of fragments of one or more capsid proteins of one or more virus serotypes. 5. A method for determining the heterogeneity of a viral particle, including: a) denaturation of the viral particle; b) direct introduction of the denatured viral particle into the analysis of the intact protein by liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS); c) determining the masses of one or more capsid proteins of the viral particle, and d) comparing the masses from step c) with the theoretical masses of one or more capsid proteins of the virus serotype; where the deviation of one or more masses of one or more capsid proteins characterizes the heterogeneity of the capsid of the virus, and where the method is carried out in the absence of a separation step on the gel. 6. A method for determining the heterogeneity of the serotype of a viral particle, including: a) denaturation of the viral particle; b) recovery and/or alkylation of the denatured viral particle; c) cleavage of the denatured viral particle to form fragments of one or more capsid proteins of the viral particle; d) liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) of fragments of one or more capsid proteins; e) determining the masses of fragments of one or more capsid proteins of the viral particle and f) comparing the masses from step e) with the theoretical masses of fragments of one or more capsid proteins of the virus serotype; where the deviation of one or more masses of one or more capsid proteins characterizes the heterogeneity of the capsid of the virus, and where the method is carried out in the absence of a separation step on the gel. 7. The method of claim 5 or 6, wherein the heterogeneity comprises one or more of mixed serotypes, variant capsids, capsid amino acid substitutions, truncated capsids, or modified capsids. 8. The method according to any one of paragraphs. 1-4, where (i) mass spectrometry includes: (1) a capillary voltage of 3.5 kV, and/or (2) sampling cone voltage of 45 V and/or (3) calibration performed by an additional tool; and/or (ii) the liquid chromatography is ultra high performance liquid chromatography (UPLC) and/or reverse phase liquid chromatography. 9. The method of claim 8, wherein the mass spectrometry comprises calibration performed with an additional tool, wherein sodium iodide is used as the calibrant. 10. The method according to claim 8 or 9, wherein the liquid chromatography is C4 or C8 reverse phase chromatography. 11. The method according to claim 10, in which: (1) chromatography uses mobile phase A containing formic acid in water; and/or (2) chromatography uses mobile phase B containing formic acid in acetonitrile. 12. The method according to claim 11, wherein mobile phase A containing 0.1% formic acid in water and/or mobile phase B containing 0.1% formic acid in acetonitrile is used in the chromatography. 13. The method according to claim 12, in which the proportion of mobile phase B in the chromatography is increased over time, increased in stages, increased from 10% to 20%, increased from 20% to 30%, increased from 30% to 38%, increased from 10 % to 20% within 6 minutes, increase from 20% to 30% within 10 minutes, or increase from 30% to 38% within 40 minutes. 14. The method according to any one of paragraphs. 5-7, where (i) mass spectrometry includes: (1) a source voltage of 2.5 kV, and/or (2) a capillary temperature of 275°C, and/or (3) the RF level of the S-lens being 55%; and/or (ii) the liquid chromatography is high performance liquid chromatography (HPLC) and/or reverse phase liquid chromatography. 15. The method of claim 14, wherein the liquid chromatography is reversed phase liquid chromatography, wherein the reversed phase liquid chromatography is C18 reversed phase chromatography. 16. The method according to claim 15, in which: (1) chromatography uses mobile phase A containing formic acid in water; and/or (2) chromatography uses mobile phase B containing formic acid in acetonitrile. 17. The method according to claim 16, wherein mobile phase A containing 0.1% formic acid in water and/or mobile phase B containing 0.1% formic acid in acetonitrile is used in the chromatography. 18. The method according to any one of paragraphs. 14-17, in which the proportion of mobile phase B in the chromatography is increased over time, increased from 2% to 60%, or increased from 2% to 60% within 121 minutes. 19. The method according to claim 3 or 6, where the viral particle is an AAV particle, and where (i) reduction is carried out by exposing the AAV particle to dithiothreitol, beta-mercaptoethanol or tris(2-carboxyethyl)phosphine (TCEP); and/or (ii) alkylation is carried out by exposing the AAV particle to iodoacetic acid, iodoacetamide, or 4-vinylpyridine; and/or (iii) the cleavage is enzymatic cleavage or chemical cleavage. 20. The method of claim 19 wherein the enzymatic cleavage is endopeptidase cleavage. 21. The method of claim 20 wherein cleavage with endopeptidase is trypsin digestion, LysC digestion, Asp-N digestion, or Glu-C digestion. 22. The method of claim 19 wherein the chemical cleavage is cyanogen bromide or acid cleavage. 23. The method according to any one of paragraphs. 1-22, wherein the viral particle includes a viral vector encoding a heterologous transgene. 24. The method according to any one of paragraphs. 1-23, in which (i) the viral particle is denatured with acetic acid, guanidine hydrochloride and/or an organic solvent; and/or (ii) the liquid chromatography is reverse phase liquid chromatography, size exclusion chromatography, hydrophilic interaction liquid chromatography, or cation exchange chromatography. 25. The method according to any one of paragraphs. 1-24, wherein the viral particle is an adeno-associated virus (AAV) particle and the one or more capsid proteins are VP1, VP2, and VP3. 26. The method of claim 25 wherein (A) the AAV particle is a recombinant AAV (rAAV) particle and/or (B) the N-terminus of VP1 and/or VP3 is acetylated. 27. A method for determining the serotype of an AAV particle, comprising the method of claim 1 in combination with the method of claim 3. 28. A method for determining the heterogeneity of an AAV particle, comprising the method of claim 5 in combination with the method of claim 6. 29. The method according to any one of paragraphs. 1-28, where the viral particle is an AAV particle and the AAV particle contains: (i) AAV1 capsid, AAV2 capsid, AAV3 capsid, AAV4 capsid, AAV5 capsid, AAV6 capsid, AAV7 capsid, AAV8 capsid, AAVrh8 capsid, AAV9 capsid, AAV10 capsid, AAVrh10 capsid, AAV11 capsid, AAV12 capsid, AAV LK03 capsid, capsid AAV2R471A, AAV2/2-7m8 capsid, AAV DJ capsid, AAV DJ8 capsid, AAV2 capsid N587A, AAV2 capsid E548A, AAV2 capsid N708A, AAV capsid V708K, goat AAV capsid, AAV1/AAV2 chimeric capsid, bovine AAV capsid, capsid Mouse AAV rAAV2/HBoV1, AAV2HBKO capsid, AAVPHP.B capsid, or AAVPHP.eB capsid, and/or AAV capsid contains a tyrosine mutation, a heparin binding mutation, or an HBKO mutation; and/or (ii) ITR AAV1, ITR AAV2, ITR AAV3, ITR AAV4, ITR AAV5, ITR AAV6, ITR AAV7, ITR AAV8, ITR AAVrh8, ITR AAV9, ITR AAV10, ITR AAVrh10, ITR AAV11 or ITR AAV12. 30. The method according to any one of paragraphs. 1-29, where the masses of VP1, VP2 and VP3 are compared to the theoretical masses of one or more of AAV1 capsid, AAV2 capsid, AAV3 capsid, AAV4 capsid, AAV5 capsid, AAV6 capsid, AAV7 capsid, AAV8 capsid, AAVrh8 capsid, AAV9 capsid, capsid AAV10, Capsid AAVrh10, Capsid AAV11, Capsid AAV12, Capsid AAV LK03, Capsid AAV2R471A, Capsid AAV2/2-7m8, Capsid AAV DJ, Capsid AAV DJ8, Capsid AAV2 N587A, Capsid AAV2 E548A, Capsid AAV2 N708A, Capsid AAV V708K Goat AAV, AAV1/AAV2 chimeric capsid, bovine AAV capsid, rAAV2/HBoV1 mouse AAV capsid, AAV2HBKO capsid, AAVPHP.B capsid, or AAVPHP.eB capsid. 31. The method according to any one of paragraphs. 1-18, where the virus particle belongs to a family of viruses selected from the group consisting of Adenoviridae , Parvoviridae , Retroviridae , Baculoviridae and Herpesviridae . 32. The method according to claim 31, wherein the viral particle belongs to a genus of viruses selected from the group consisting of Atadenovirus, Aviadenovirus, Ichtadenovirus, Mastadenovirus, Siadenovirus, Ambidensovirus, Brevidensovirus, Hepandensovirus, Iteradensovirus, Penstyldensovirus, Amdoparvovirus, Aveparvovirus, Bocaparvovirus, Copiparvovirus , Dependoparvovirus, Erythroparvovirus, Protoparvovirus, Tetraparvovirus, Alpharetrovirus, Betaretrovirus, Deltaretrovirus, Epsilonretrovirus, Gammaretrovirus, Lentivirus, Spumavirus, Alphabaculovirus, Betabaculovirus, Deltabaculovirus, Gammabaculovirus, Iltovirus, Mardivirus, Simplexvirus, Lycellovirus, Cytomegalovirus, Muromegalovirus, Rosemolovirus, Proboscivirus , Percavirus and Rhadinovirus . 33. A method for improving the stability of a rAAV particle, improving the assembly of rAAV particles in a cell, or improving the transduction of rAAV particles in a cell, comprising replacing the amino acid residue 2 of VP1 and/or VP3 of the original VP1 and/or VP3; where the amino acid change at position 2 provides a change in the N-terminal acetylation of VP1 and/or VP3 compared to amino acid residue 2 of the original VP1 and/or VP3. 34. The method of claim 33 wherein the substitution at amino acid residue 2 results in a higher frequency of N-terminal acetylation or a lower frequency of N-terminal acetylation. 35. The method of claim 33 or 34 wherein amino acid residue 2 is replaced with Cys, Ser, Thr, Val, Gly, Asn, Asp, Glu, Ile, Leu, Phe, Gln, Lys, Met, Pro, or Tyr. 36. The method of claim 35 wherein amino acid residue 2 is replaced by Ser, Asp, or Glu. 37. A method for improving the stability of a rAAV particle, improving the assembly of rAAV particles in a cell, or improving the transduction of rAAV particles in a cell, comprising replacing one or more VP1 and/or VP3 amino acid residues of the original VP1 and/or VP3, where one or more amino acid residues are A35, N57, G58, N382, G383, N511, G512, N715 or G716, with residue numbering based on VP1 AAV2; where the amino acid change provides a change in deamidation compared to amino acid residue 2 of the original VP1 and/or VP3. 38. The method of claim 37, wherein one or more amino acid substitutions are made at A35 VP1, N57 VP1, G58 VP1, N382 VP3, G383 VP3, N511 VP3, G512 VP3, N715 VP3, or G716 VP3; where the amino acid change provides a change in deamidation compared to the deamidation of VP1 and/or VP3 particles of the original AAV. 39. The method according to claim 37 or 38, wherein the original Ala residue at position 35 VP1 is replaced by Asn and/or the original Gly residue at position 58 VP1 is replaced by Asp. 40. The method according to any one of paragraphs. 33-39, where the rAAV particle is obtained using a) transfecting a host cell with a nucleic acid encoding an rAAV vector and a nucleic acid encoding AAV rep and cap functional elements, and obtaining a nucleic acid encoding AAV helper functional elements; or b) an AAV producing cell comprising a nucleic acid encoding an rAAV vector and a nucleic acid encoding AAV rep and cap functional elements, and producing a nucleic acid encoding AAV helper functional elements. 41. The method of claim 40, wherein the AAV cap functional elements provide an amino acid substitution for VP1 and/or VP3, wherein the VP1 and/or VP3 amino acid substitution provides a change in deamidation from that of the VP1 and/or VP3 deamidation of the parent AAV particle. 42. The method according to any one of paragraphs. 33-41, where the AAV particle contains: (i) AAV1 capsid, AAV2 capsid, AAV3 capsid, AAV4 capsid, AAV5 capsid, AAV6 capsid, AAV7 capsid, AAV8 capsid, AAVrh8 capsid, AAV9 capsid, AAV10 capsid, AAVrh10 capsid, AAV11 capsid, AAV12 capsid, AAV LK03 capsid, capsid AAV2R471A, AAV2/2-7m8 capsid, AAV DJ capsid, AAV DJ8 capsid, AAV2 capsid N587A, AAV2 capsid E548A, AAV2 capsid N708A, AAV capsid V708K, goat AAV capsid, AAV1/AAV2 chimeric capsid, bovine AAV capsid, capsid Mouse AAV rAAV2/HBoV1 (chimeric AAV/human boca virus 1), AAV2HBKO capsid, AAVPHP.B capsid, or AAVPHP.eB capsid; and/or (ii) rAAV vector. 43. The method of claim 42, wherein the AAV capsid contains a tyrosine mutation, a heparin binding mutation, or an HBKO mutation. 44. The method of claim 42 or 43, wherein the rAAV-based vector is a self-complementary vector. 45. Recombinant AAV particle (rAAV) for nucleic acid delivery to a host cell, containing an amino acid substitution at amino acid residue 2 of VP1 and/or VP3 or the original polypeptide; wherein the amino acid substitution at amino acid residue 2 of VP1 and/or VP3 provides a change in N-terminal acetylation compared to N-terminal acetylation at amino acid residue 2 of VP1 and/or VP3 of the original AAV particle. 46. The rAAV particle of claim 45, wherein a) substitution at amino acid residue 2 results in a higher frequency of N-terminal acetylation or a lower frequency of N-terminal acetylation; b) the rAAV particle contains an amino acid substitution at amino acid residue 2 of VP1, wherein the amino acid substitution at amino acid residue 2 of VP1 provides a change in N-terminal acetylation compared to N-terminal acetylation at amino acid residue 2 of VP1 of the original AAV particle; c) the rAAV particle contains an amino acid substitution at amino acid residue 2 of VP3; and wherein the amino acid substitution at amino acid residue 2 of VP3 provides for a change in N-terminal acetylation compared to N-terminal acetylation at amino acid residue 2 of VP3 of the original AAV particle; and/or d) amino acid residue 2 is replaced by Cys, Ser, Thr, Val, Gly, Asn, Asp, Glu, Ile, Leu, Phe, Gln, Lys, Met, Pro, or Tyr. 47. The rAAV particle of claim 46 wherein amino acid residue 2 is replaced by Ser, Asp or Glu. 48. Particle rAAV according to any one of paragraphs. 45-47, where the rAAV particle contains: (i) AAV1 capsid, AAV2 capsid, AAV3 capsid, AAV4 capsid, AAV5 capsid, AAV6 capsid, AAV7 capsid, AAV8 capsid, AAVrh8 capsid, AAV9 capsid, AAV10 capsid, AAVrh10 capsid, AAV11 capsid, AAV12 capsid, AAV LK03 capsid, capsid AAV2R471A, AAV2/2-7m8 capsid, AAV DJ capsid, AAV DJ8 capsid, AAV2 capsid N587A, AAV2 capsid E548A, AAV2 capsid N708A, AAV capsid V708K, goat AAV capsid, AAV1/AAV2 chimeric capsid, bovine AAV capsid, capsid Mouse AAV rAAV2/HBoV1 (chimeric AAV/human boca virus 1), AAV2HBKO capsid, AAVPHP.B capsid, or AAVPHP.eB capsid, and/or AAV capsid contains a tyrosine mutation, a mutation affecting heparin binding, or HBKO mutation; and/or (ii) rAAV vector. 49. The rAAV particle according to claim 48, wherein the rAAV particle contains the rAAV genome, wherein the rAAV genome contains: (1) one or more AAV ITRs; and/or (2) ITR AAV1, ITR AAV2, ITR AAV3, ITR AAV4, ITR AAV5, ITR AAV6, ITR AAV7, ITR AAV8, ITR AAVrh8, ITR AAV9, ITR AAV10, ITR AAVrh10, ITR AAV11 or ITR AAV12; and/or (3) an encoded heterologous transgene flanked by one or more AAV ITRs. 50. An rAAV particle according to claim 48 or 49, wherein the rAAV-based vector is a self-complementary vector. 51. The rAAV particle of claim 48, wherein the rAAV-based vector comprises a first nucleic acid sequence encoding a transgene and a second nucleic acid sequence encoding a sequence complementary to the transgene, wherein the first nucleic acid sequence forms intrastrand base pairs with the second nucleic acid sequence at most part or all of its length. 52. The rAAV particle of claim 51, wherein the first nucleic acid sequence and the second nucleic acid sequence are joined by a mutant AAV ITR, and wherein the mutant AAV ITR is characterized by a D-region deletion and is characterized by a terminal resolution sequence mutation. 53. Recombinant AAV (rAAV) particle for nucleic acid delivery to a host cell containing one or more amino acid substitutions at amino acid residue A35, N57, G58, N382, G383, N511, G512, N715 or G716 VP1 or VP3 of the original AAV particle, where the residue numbering is based on the VP1 of AAV2, and one or more amino acid substitutions provide a change in deamidation compared to the deamidation of the VP1 and/or VP3 particles of the original AAV. 54. An rAAV particle according to claim 53, wherein the rAAV particle is an AAV1 particle or an AAV2 particle. 55. The rAAV particle according to claim 53 or 54, wherein a) one or more amino acid substitutions are made at amino acid residue A35 VP1, N57 VP1, G58 VP1, N382 VP3, G383 VP3, N511 VP3, G512 VP3, N715 VP3 or G716 VP3 and provide a change in deamidation compared to deamidation of VP1 and/or VP3 original AAV particles; or b) one or more amino acid substitutions include an Asp substitution at N57 VP1, N382 VP3, N511 VP3 or N715 VP3 and result in a higher frequency of deamidation compared to deamidation of VP1 and/or VP3 of the parent AAV particle; or c) one or more amino acid substitutions include an N57K or N57Q substitution and result in a lower frequency of deamidation compared to deamidation of the VP1 and/or VP3 particles of the original AAV; or d) one or more amino acid substitutions include an Asn substitution at A35 of VP1 and result in a higher frequency of deamidation compared to deamidation of the VP1 particle of the original AAV; or e) one or more amino acid substitutions are made at G58 VP1, G383 VP3, G512 VP3, or G716 VP3 and result in a lower rate of deamidation compared to deamidation of the VP1 and/or VP3 of the parent AAV particle. 56. The rAAV particle according to claim 55, where G58 VP1 is replaced by Asp. 57. Pharmaceutical composition for delivery of a nucleic acid to a host cell containing an rAAV particle according to any one of paragraphs. 45-56. 58. Kit for delivery of nucleic acid into the host cell containing the rAAV particle according to any one of paragraphs. 45-56 or a pharmaceutical composition according to claim 57. 59. An article for delivery of a nucleic acid to a host cell, comprising an rAAV particle according to any one of paragraphs. 45-56 or a pharmaceutical composition according to claim 57. 60. An AAV capsid protein to obtain an AAV particle containing an amino acid substitution at amino acid residue 2 of the parent AAV capsid protein; wherein the amino acid substitution at amino acid residue 2 provides a change in N-terminal acetylation compared to N-terminal acetylation at amino acid residue 2 of the parent AAV capsid protein. 61. The AAV capsid protein according to claim 60, where: a) the substitution results in a higher frequency of N-terminal acetylation or a lower frequency of N-terminal acetylation; and/or b) the AAV capsid protein is VP1 or VP3; and/or c) amino acid residue 2 is replaced by Cys, Ser, Thr, Val, Gly, Asn, Asp, Glu, Ile, Leu, Phe, Gln, Lys, Met, Pro, or Tyr; and/or d) amino acid substitution results in less deamidation of the AAV capsid. 62. The AAV capsid protein of claim 61 wherein amino acid residue 2 is replaced by Ser, Asp, or Glu. 63. AAV capsid protein according to any one of paragraphs. 60-62, where the capsid protein refers to the serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV LK03, AAV2R471A, AAV2/2-7m8 , AAV DJ, AAV DJ8 capsid, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV V708K, AAV goat, chimeric AAV1/AAV2, AAV bovine, AAV mouse, rAAV2/HBoV1, AAV2HBKO, AAVPHP.B or AAVPHP.eB; and/or the AAV capsid protein further comprises a tyrosine mutation or a mutation affecting heparin binding. 64. An AAV capsid protein to obtain an AAV particle, containing an amino acid substitution of the parent AAV capsid protein; where the amino acid change provides a change in capsid deamidation compared to the capsid protein of the original AAV.

Images