Replication-defective viral vectors and methods for measuring viral infectivity

Abstract

To provide an improved method for measuring infectivity of a replication-deficient virus and a virus vector.SOLUTION: A method for determining infectivity of a test composition including virus particles to infectivity of a reference composition including virus particles includes: a. a step of inoculating the test composition and a reference composition separately into a target cell; b. a step of cleaning the inoculated cell to remove virus particles outside the cell; c. a step of severally isolating a test nucleic acid sample and a reference nucleic acid sample from the target cell to which the test composition and reference composition have been inoculated; and d. a step of determining a ratio of virus genome copy (VGC):target cell genome copy (TCGC) in the test nucleic acid sample and the reference nucleic acid sample.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 745,859, filed October 15, 2018, which is incorporated herein by reference in its entirety. [Background technology]

[0002] background Recombinant adeno-associated virus (AAV)-based vectors are currently the most widely used gene therapy products under development. The use of rAAV vector systems is favored, in part, due to the absence of disease associated with wild-type viruses, the ability of AAV to transduce both non-dividing and dividing cells, and the resulting long-term, robust transgene expression observed in clinical trials, indicating great potential for delivery in gene therapy indications. In addition, various natural and recombinant rAAV vector serotypes can specifically target various tissues, organs, and cells, helping to circumvent any pre-existing immunity to the vector, thereby expanding the therapeutic application of AAV-based gene therapy.

[0003] Before replication-deficient virus (e.g., AAV)-based gene therapy can be more widely adopted for late-stage clinical and commercial use, new methods for large-scale production of recombinant viral particles must be developed. Absolute quantification of infectious titer by limiting dilution endpoint analysis (also known as the TCID50 infectious titer assay) has become the standard method for measuring the infectivity of recombinant viral (e.g., AAV) preparations in vitro. While the TCID50 infectious titer assay is useful for confirming the infectivity of AAV vector preparations, high assay variability (geometric coefficient of variation up to 200%) limits its applicability for supporting product suitability, comparability, and stability. Therefore, a more accurate method for measuring the infectivity of compositions containing replication-deficient viral particles (e.g., rAAV particles) is needed. Summary of the Invention

[0004] overview The present disclosure provides methods for determining the infectivity of a test composition comprising viral particles relative to the infectivity of a reference composition comprising viral particles, the methods comprising the steps of contacting target cells with the test composition and the reference composition under conditions that allow inoculation of the viral particles into the target cells; removing extracellular viral particles; isolating a test nucleic acid sample and a reference nucleic acid sample from the target cells inoculated with the test composition and the reference composition, respectively; and determining the ratio of viral genome copies (VGC):target cell genome copies (TCGC) in the test nucleic acid sample and the reference nucleic acid sample. In some embodiments, the target cells are contacted with serial dilutions of the test composition and the reference composition. In some embodiments, the serial dilutions of the test composition and the reference composition are less than 10-fold dilutions. In some embodiments, the serial dilutions of the test composition and the reference composition are 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 8-fold dilutions. In some embodiments, the serial dilutions of the test sample and the reference sample are 2-fold dilutions. In some embodiments, the method further comprises calculating the infectivity of the test sample relative to the reference sample using a parallel line model. In some embodiments, the VGC and TCGC in the nucleic acid sample are determined by polymerase chain reaction. In some embodiments, the polymerase chain reaction is quantitative polymerase chain reaction. In some embodiments, the polymerase chain reaction is digital polymerase chain reaction. In some embodiments, the viral particles are replication-deficient viral particles. In some embodiments, the replication-deficient viral particles are AAV, adenovirus, vaccinia, or lentivirus particles. In some embodiments, the replication-deficient viral particles are retrovirus particles. In some embodiments, the replication-deficient viral particles are AAV particles, e.g., recombinant AAV particles. In some embodiments, the AAV is recombinant AAV (rAAV).In some embodiments, the rAAV is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP. The capsid proteins of the rAAV include those of the AAV.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16 serotypes. In some embodiments, the rAAV comprises capsid proteins of the AAV8 or AAV9 serotype. In some embodiments, the target cells are BHK21, HEK293, BEAS-2BS, HeLaS3, Huh-7, Hepa1-6, or A549 cells. In some embodiments, the target cells are Huh-7 cells.

[0005] The present disclosure provides an isolated polynucleotide having about 15 to about 40 nucleotides comprising the nucleotide sequence of one of SEQ ID NOs: 1-6. In some embodiments, the polynucleotide is detectably labeled, wherein the detectable label is covalently attached to the polynucleotide. In some embodiments, the detectable label is a fluorescent label. In some embodiments, the detectable label comprises one or more of FAM, JOE, TAMRA, and ROX.

[0006] The present disclosure provides a method for generating a polynucleotide of interest, the method comprising subjecting DNA from a biological sample to polymerase chain reaction using one or more polynucleotides described herein.

[0007] The present disclosure provides kits for detecting rAAV in a sample containing one or more polynucleotides described herein.

[0008] The present disclosure provides kits for determining the infectivity of a test sample containing viral particles relative to the infectivity of a reference sample containing viral particles, the kit comprising one or more of: (a) forward and reverse primers capable of amplifying a viral sequence, optionally with a probe; (b) forward and reverse primers capable of amplifying a target cell genomic sequence, optionally with a probe; and (c) a viral reference sample. In some embodiments, the viral particles are rAAV particles. In some embodiments, the forward and reverse primers capable of amplifying a viral sequence, optionally with a probe, comprise a polynucleotide disclosed herein. In some embodiments, the forward and reverse primers capable of amplifying a target cell genomic sequence, optionally with a probe, comprise a polynucleotide disclosed herein.

[0009] The present disclosure further provides a method for determining the relative infectivity of a composition of viral particles under different conditions, comprising the steps of inoculating target cells with the composition comprising viral particles under first and second sets of conditions, washing the inoculated cells to remove extracellular viral particles, isolating first and second nucleic acid samples from the target cells inoculated under the first and second sets of conditions, respectively, and determining the ratio of viral genome copies (VGC):target cell genome copies (TCGC) in the first and second nucleic acid samples.

[0010] In some embodiments, the present disclosure provides: [1] A method for determining the infectivity of a test composition comprising viral particles relative to the infectivity of a reference composition comprising viral particles, comprising: [a] inoculating target cells separately with the test composition and the reference composition; [b] washing the inoculated cells to remove extracellular viral particles; [c] isolating a test nucleic acid sample and a reference nucleic acid sample from the target cells inoculated with the test composition and the reference composition, respectively; [d] determining the ratio of viral genome copies (VGC):target cell genome copies (TCGC) in the test nucleic acid sample and the reference nucleic acid sample. [2] A method for determining the infectivity of a test composition comprising viral particles relative to the infectivity of a reference composition comprising viral particles, comprising: [a] preparing serial dilutions of the test and reference compositions; [b] separately inoculating target cells with the serial dilutions of the test composition and the reference composition; [c] washing the inoculated cells to remove extracellular viral particles; [d] isolating a test nucleic acid sample and a reference nucleic acid sample from the target cells inoculated with the test composition and the reference composition, respectively; [e] determining the ratio of viral genome copies (VGC):target cell genome copies (TCGC) in the test nucleic acid sample and the reference nucleic acid sample. [3] The method according to [2], wherein the serial dilutions are dilutions of less than 10 times. [4] The method according to [2], wherein the serial dilutions are 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 8-fold dilutions. [5] The method according to [2], wherein the serial dilutions are two-fold dilutions. [6] The method according to any one of [2] to [5], wherein the serial dilutions include at least two types of dilutions, at least three types of dilutions, at least five types of dilutions, or at least ten types of dilutions. [7] The method according to any one of [2] to [5], wherein the serial dilutions include 2 to 20 types of dilutions. [8] The method according to any one of [2] to [7], further comprising the step of calculating the infectivity of the test composition relative to the reference composition using a parallel line model. [9] The step of calculating the infectivity of the test composition relative to the reference composition comprises: [a] calculating the VGC:TCGC ratio for each dilution of the test composition and the reference composition; [b] plotting the log VGC:TCGC ratio versus log dilution for the test composition and the reference composition; [c] fitting the test and reference composition data points to a test and reference composition line using a common slope; [d] determining the infectivity of the test composition relative to the reference composition; Calculate as TIFF0007822436000001.tif8128 The method according to [8], comprising:

[10] The method according to any one of [1] to [9], wherein the coefficient of variation (cv) is less than about 100%, less than about 50%, or less than about 25%.

[11] The step of inoculating target cells comprises inoculating the target cells in the presence of viral particles. [a] For about 5 minutes to about 3 days, [b] Between approximately 12 hours and approximately 36 hours, [c] Between approximately 18 and 30 hours [d] Approximately 1 hour, approximately 2 hours, approximately 6 hours, approximately 12 hours, approximately 18 hours, approximately 24 hours, approximately 30 hours, or approximately 36 hours, "e" about 1 day, or about 1.5 days, or about 2 days, or [f] Approximately 24 hours The method according to any one of [1] to

[10] , comprising incubating the mixture for a period of time.

[12] The method according to any one of [1] to

[11] , wherein the VGC and TCGC in the nucleic acid composition are determined by polymerase chain reaction.

[13] The method according to

[12] , wherein the polymerase chain reaction is a quantitative polymerase chain reaction.

[14] The method according to

[12] , wherein the polymerase chain reaction is a digital polymerase chain reaction.

[15] The method according to any one of [1] to

[14] , wherein the virus particles are replication-defective viruses.

[16] The method according to

[15] , wherein the replication-deficient virus is AAV, adenovirus, vaccinia, or lentivirus.

[17] The method according to

[15] , wherein the replication-deficient virus is a retrovirus.

[18] The method according to

[15] , wherein the replication-deficient virus is AAV.

[19] The method according to

[18] , wherein the AAV is recombinant AAV (rAAV).

[20] The rAAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh 8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP. 19. The method of claim 19, wherein the capsid protein of the AAV.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 serotype is present.

[21] The method according to

[20] , wherein the rAAV comprises a capsid protein of the AAV8 serotype or the AAV9 serotype.

[22] The method according to any one of [1] to

[21] , wherein the target cells are BHK21, HEK293, BEAS-2BS, HeLaS3, Huh-7, Hepa1-6, or A549 cells.

[23] The method according to

[22] , wherein the target cells are Huh-7 cells.

[24] The method according to any one of [1] to

[23] , wherein the test composition and the reference composition have the same titer, and the titer is measured as genome copies (GC) per milliliter.

[25] The method according to any one of [1] to

[23] , wherein the test composition and the reference composition have different titers, and the titers are measured as genome copies (GC) per milliliter.

[26] The method of

[24] or

[25] , wherein the titer of the test composition is about 1×10e+10 GC / ml to about 1×10e+13 GC / ml of rAAV particles.

[27] The method according to any one of

[24] to

[26] , wherein the titer of the reference composition is between about 1×10e+10 GC / ml and about 1×10e+13 GC / ml of rAAV particles.

[28] The following nucleotide sequence containing 0, 1, 2, 3, 4, or 5 substitutions: An isolated polynucleotide having about 15 to about 40 nucleotides comprising TIFF0007822436000002.tif55129.

[29] An isolated polynucleotide having about 15 to about 40 nucleotides and comprising the nucleotide sequence of one of SEQ ID NOs: 1 to 6.

[30] An isolated polynucleotide consisting of one of the nucleotide sequences of SEQ ID NO:1 to 6.

[31] A composition comprising: (i) the polynucleotide according to any one of

[28] to

[30] ; and (ii) a detectable label covalently attached to the polynucleotide.

[32] The composition according to

[31] , wherein the detectable label is a fluorescent label.

[33] The composition of

[32] , wherein the detectable label comprises one or more of FAM, JOE, TAMRA, and ROX.

[34] A pair of forward and reverse primers, wherein the forward and reverse primers comprise the polynucleotide sequences of SEQ ID NOs: 1 and 2, respectively.

[35] A pair of forward and reverse primers, wherein the forward and reverse primers comprise the polynucleotide sequences of SEQ ID NOs: 4 and 5, respectively.

[36] A combination of a probe, a forward primer, and a reverse primer, wherein the forward primer, the reverse primer, and the probe comprise polynucleotides consisting of the nucleotide sequences of SEQ ID NOs: 1, 2, and 3, respectively.

[37] A combination of a probe, a forward primer, and a reverse primer, wherein the forward primer, the reverse primer, and the probe comprise polynucleotides consisting of the nucleotide sequences of SEQ ID NOs: 4, 5, and 6, respectively.

[38] A method for generating a target polynucleotide, the method comprising the step of subjecting DNA from a biological sample to polymerase chain reaction using a pair of forward and reverse primers described in

[34] or

[35] .

[39] A method for generating a target polynucleotide, the method comprising the step of subjecting DNA from a biological sample to a polymerase chain reaction using a combination of the probe, forward primer, and reverse primer described in

[36] or

[37] .

[40] A kit for detecting rAAV in a sample, the kit comprising one or more polynucleotides selected from the group consisting of SEQ ID NOs: 1 to 3.

[41] A kit for detecting rAAV in a sample, comprising the pair of forward and reverse primers described in

[34] .

[42] A kit for detecting rAAV in a sample, comprising a combination of the probe, forward primer, and reverse primer described in

[36] .

[43] A kit for determining the infectivity of an rAAV test sample relative to the infectivity of a reference sample, the kit comprising the pair of forward and reverse primers described in

[34] . The kit according to

[43] , further comprising a pair of forward and reverse primers according to

[44] and

[35] .

[45] A kit for determining the infectivity of an rAAV test composition relative to the infectivity of a reference composition, the kit comprising a combination of the probe, forward primer, and reverse primer described in

[36] .

[46] The kit according to

[45] , further comprising a combination of the probe, forward primer, and reverse primer according to

[37] .

[47] The kit according to any one of

[40] to

[46] , further comprising an rAAV reference composition.

[48] ​​A method for determining the relative infectivity of a composition of viral particles under different conditions, comprising the steps of: [a] inoculating target cells with the composition comprising viral particles under a first and a second set of conditions; [b] washing the inoculated cells to remove extracellular viral particles; [c] isolating first and second nucleic acid samples from the target cells inoculated under the first and second sets of conditions, respectively; [d] determining the ratio of viral genome copies (VGC):target cell genome copies (TCGC) in the first and second nucleic acid samples.

[49] The method of

[48] , wherein the first and second sets of conditions use the same target cells.

[50] The method of

[48] , wherein the first and second sets of conditions use different target cells.

[51] The method of

[50] , wherein the different target cells comprise different genetic modifications.

[52] The method of

[50] , wherein the different target cells are identical except for the presence of a genetic modification in one of the target cells.

[53] The method according to any one of

[49] to

[52] , wherein the step of inoculating target cells comprises inoculating target cells with serial dilutions of the composition.

[54] The method according to

[53] , wherein the serial dilutions are two-fold dilutions.

[55] The method of

[53] or

[54] , wherein the step of inoculating target cells comprises inoculating target cells with serial dilutions of the composition.

[56] The method according to any one of

[53] to

[55] , further comprising the step of calculating the relative infectivity of the composition under the first and second sets of conditions using a parallel line model.

[57] The step of calculating the relative infectivity of the composition under the first and second sets of conditions comprises: [a] calculating the VGC:TCGC ratio for each dilution of the first and second sets of conditions; [b] plotting the log VGC:TCGC ratio against the log dilution for the first and second sets of conditions; [c] fitting the data points of the first and second sets of conditions to lines of first and second conditions using a common slope; [d] the infectivity under said first condition relative to said second condition; The method described in

[56] , comprising calculating the image as TIFF0007822436000003.tif8128.

[58] The method according to any one of

[53] to

[57] , wherein the coefficient of variation (cv) is less than about 100%, less than about 50%, or less than about 25%.

[59] The method according to any one of

[53] to

[58] , wherein the VGC and TCGC in the nucleic acid composition are determined by polymerase chain reaction.

[60] The method according to

[59] , wherein the polymerase chain reaction is a quantitative polymerase chain reaction.

[61] The method according to

[59] , wherein the polymerase chain reaction is a digital polymerase chain reaction.

[62] The method according to any one of

[48] to

[61] , wherein the virus particles are replication-defective viruses.

[63] The method according to

[62] , wherein the replication-deficient virus is AAV, adenovirus, vaccinia, or lentivirus.

[64] The method according to

[62] , wherein the replication-deficient virus is a retrovirus.

[65] The method according to

[62] , wherein the replication-deficient virus is AAV.

[66] The method according to

[65] , wherein the AAV is recombinant AAV (rAAV).

[67] The rAAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh 8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP. 66]. The method of claim 66, comprising a capsid protein of an AAV.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 serotype.

[68] The method according to

[67] , wherein the rAAV comprises a capsid protein of the AAV8 serotype or the AAV9 serotype.

[69] The method according to any one of

[48] to

[68] , wherein the target cells under at least one set of conditions comprise BHK21, HEK293, BEAS-2BS, HeLaS3, Huh-7, Hepa1-6, or A549 cells.

[70] The method of

[22] or

[69] , wherein the target cells under at least one set of conditions comprise Huh-7 cells.

[0011] In some embodiments, the methods disclosed herein include determining the infectivity of a composition comprising isolated rAAV particles, the composition being produced by isolating the rAAV particles from an impure feed (e.g., an rAAV production culture), wherein the method for isolating the rAAV particles includes one or more processing steps. In some embodiments, the processing is at least one of harvesting the cell culture, clarification of the harvested cell culture (e.g., by centrifugation or depth filtration), tangential flow filtration, affinity chromatography, anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, and sterile filtration. In further embodiments, the processing includes at least two, at least three, at least four, at least five, or at least six of harvesting the cell culture, clarification of the harvested cell culture (e.g., by centrifugation or depth filtration), tangential flow filtration, affinity chromatography, anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, and sterile filtration. In some embodiments, the processing does not include centrifugation of the harvested cell culture.

[0012] The present disclosure provides methods for producing a pharmaceutical composition comprising isolated recombinant adeno-associated virus (rAAV) particles, the method comprising: (a) isolating rAAV particles from an impure feed by one or more of centrifugation, depth filtration, tangential flow filtration, ultrafiltration, affinity chromatography, size exclusion chromatography, ion exchange chromatography, and hydrophobic interaction chromatography; determining the infectivity of the rAAV particles using a method disclosed herein; and formulating the isolated rAAV particles to produce the pharmaceutical composition.

[0013] The present disclosure provides methods for producing a pharmaceutical unit dose comprising isolated recombinant adeno-associated virus (rAAV) particles, the method comprising: (a) isolating rAAV particles from an impure feed by one or more of centrifugation, depth filtration, tangential flow filtration, ultrafiltration, affinity chromatography, size exclusion chromatography, ion exchange chromatography, and hydrophobic interaction chromatography; determining the infectivity of the rAAV particles using a method disclosed herein; and formulating the isolated rAAV particles.

[0014] [The present invention 1001] 1. A method for determining the infectivity of a test composition comprising viral particles relative to the infectivity of a reference composition comprising viral particles, comprising: a. inoculating target cells separately with the test composition and the reference composition; b. washing the inoculated cells to remove extracellular viral particles; c. isolating a test nucleic acid sample and a reference nucleic acid sample from the target cells inoculated with the test composition and the reference composition, respectively; d. determining the ratio of viral genome copies (VGC): target cell genome copies (TCGC) in the test nucleic acid sample and the reference nucleic acid sample; The method comprising: [The present invention 1002] 1. A method for determining the infectivity of a test composition comprising viral particles relative to the infectivity of a reference composition comprising viral particles, comprising: a. preparing serial dilutions of the test and reference compositions; b. separately inoculating target cells with the serial dilutions of the test composition and the reference composition; c. washing the inoculated cells to remove extracellular viral particles; d. isolating a test nucleic acid sample and a reference nucleic acid sample from the target cells inoculated with the test composition and the reference composition, respectively; e. determining the ratio of viral genome copies (VGC): target cell genome copies (TCGC) in the test nucleic acid sample and the reference nucleic acid sample; The method comprising: [The present invention 1003] 1002. The method of claim 10, wherein the serial dilutions are less than 10-fold dilutions. [The present invention 1004] 1002. The method of claim 1002, wherein said serial dilutions are 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 8-fold dilutions. [The present invention 1005] 1002. The method of claim 1002, wherein said serial dilutions are two-fold dilutions. [The present invention 1006] 1006. The method of any of claims 1002 to 1005, wherein said serial dilutions comprise at least two dilutions, at least three dilutions, at least five dilutions, or at least ten dilutions. [The present invention 1007] 1006. The method of any one of claims 1002 to 1005, wherein the serial dilutions comprise 2 to 20 different dilutions. [The present invention 1008] The method of any of claims 1002 to 1007, further comprising the step of calculating the infectivity of said test composition relative to said reference composition using a parallel line model. [The present invention 1009] calculating the infectivity of the test composition relative to the reference composition, a. calculating the VGC:TCGC ratio for each dilution of the test composition and the reference composition; b. plotting the log VGC:TCGC ratio against the log dilution for the test composition and the reference composition; c. fitting the test and reference composition data points to a test and reference composition line using a common slope; d. determining the infectivity of the test composition relative to the reference composition. Calculate as TIFF0007822436000004.tif8128 The method of the present invention 1008, comprising: [The present invention 1010] 1009. The method of any of claims 1001 to 1009, wherein the coefficient of variation (cv) is less than about 100%, less than about 50%, or less than about 25%. [The present invention 1011] The step of inoculating target cells comprises inoculating the target cells in the presence of viral particles. a.From about 5 minutes to about 3 days b. Between approximately 12 hours and approximately 36 hours c. Between approximately 18 and 30 hours d. Approximately 1 hour, approximately 2 hours, approximately 6 hours, approximately 12 hours, approximately 18 hours, approximately 24 hours, approximately 30 hours, or approximately 36 hours; e. about 1 day, or about 1.5 days, or about 2 days, or f. Approximately 24 hours The method of any one of 1001 to 1010, comprising incubating for a period of time. [The present invention 1012] 1012. The method of any one of claims 1001 to 1011, wherein said VGC and TCGC in the nucleic acid composition are determined by polymerase chain reaction. [The present invention 1013] 1012. The method of claim 1012, wherein said polymerase chain reaction is a quantitative polymerase chain reaction. [The present invention 1014] 1012. The method of claim 1012, wherein the polymerase chain reaction is a digital polymerase chain reaction. [The present invention 1015] The method of any one of claims 1001 to 1014, wherein the virus particles are replication-defective viruses. [The present invention 1016] 1015. The method of claim 1015, wherein said replication-deficient virus is AAV, adenovirus, vaccinia, or lentivirus. [The present invention 1017] 1015. The method of claim 10, wherein said replication-deficient virus is a retrovirus. [The present invention 1018] 1015. The method of claim 10, wherein said replication-deficient virus is AAV. [The present invention 1019] The method of claim 1018, wherein the AAV is a recombinant AAV (rAAV). [The present invention 1020] The rAAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, A AV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB , AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 serotype capsid protein. [The present invention 1021] The method of claim 1020, wherein the rAAV comprises capsid proteins of the AAV8 serotype or the AAV9 serotype. [The present invention 1022] 1022. The method of any one of claims 1001 to 1021, wherein said target cells are BHK21, HEK293, BEAS-2BS, HeLaS3, Huh-7, Hepa1-6, or A549 cells. [The present invention 1023] 1023. The method of claim 1022, wherein said target cells are Huh-7 cells. [The present invention 1024] 1024. The method of any of claims 1001 to 1023, wherein said test composition and said reference composition have the same titer, said titer being measured as genome copies (GC) per milliliter. [The present invention 1025] 1024. The method of any of claims 1001 to 1023, wherein said test composition and said reference composition have different titers, said titers being measured in genome copies (GC) per milliliter. [The present invention 1026] The method of claim 1024 or claim 1025, wherein the titer of the test composition is between about 1×10e+10 GC / ml and about 1×10e+13 GC / ml of rAAV particles. [The present invention 1027] 1027. The method of any of claims 1024 to 1026, wherein the titer of the reference composition is between about 1 x 10e+10 GC / ml and about 1 x 10e+13 GC / ml of rAAV particles. [The present invention 1028] The following nucleotide sequence containing 0, 1, 2, 3, 4, or 5 substitutions: An isolated polynucleotide having about 15 to about 40 nucleotides comprising TIFF0007822436000005.tif41150. [The present invention 1029] An isolated polynucleotide having about 15 to about 40 nucleotides and comprising the nucleotide sequence of one of SEQ ID NOs: 1-6. [The present invention 1030] An isolated polynucleotide consisting of the nucleotide sequence of one of SEQ ID NOs: 1 to 6. [The present invention 1031] A composition comprising (i) a polynucleotide of any one of claims 1028 to 1030, and (ii) a detectable label covalently attached to the polynucleotide. [The present invention 1032] 1031. The composition of claim 1031, wherein said detectable label is a fluorescent label. [The present invention 1033] The composition of claim 1032, wherein the detectable label comprises one or more of FAM, JOE, TAMRA, and ROX. [The present invention 1034] A pair of forward and reverse primers, wherein the forward and reverse primers comprise the polynucleotide sequences of SEQ ID NOs: 1 and 2, respectively. [This invention 1035] A pair of forward and reverse primers, wherein the forward and reverse primers comprise the polynucleotide sequences of SEQ ID NOs: 4 and 5, respectively. [The present invention 1036] A combination of a probe, a forward primer, and a reverse primer, wherein the forward primer, the reverse primer, and the probe comprise polynucleotides consisting of the nucleotide sequences of SEQ ID NOs: 1, 2, and 3, respectively. [This invention 1037] A combination of a probe, a forward primer, and a reverse primer, wherein the forward primer, the reverse primer, and the probe comprise polynucleotides consisting of the nucleotide sequences of SEQ ID NOs: 4, 5, and 6, respectively. [The present invention 1038] A method for producing a target polynucleotide, comprising the step of subjecting DNA from a biological sample to polymerase chain reaction using a pair of forward and reverse primers of the present invention 1034 or 1035. [This invention 1039] A method for generating a target polynucleotide, comprising the step of subjecting DNA from a biological sample to polymerase chain reaction using a combination of a probe, a forward primer, and a reverse primer of the present invention 1036 or 1037. [The present invention 1040] A kit for detecting rAAV in a sample, comprising one or more polynucleotides selected from the group consisting of SEQ ID NOs: 1 to 3. [This invention 1041] A kit for detecting rAAV in a sample, comprising a pair of forward and reverse primers of the present invention 1034. [The present invention 1042] A kit for detecting rAAV in a sample, comprising a combination of a probe, a forward primer, and a reverse primer of the present invention 1036. [This invention 1043] A kit for determining the infectivity of an rAAV test sample relative to the infectivity of a reference sample, comprising a pair of forward and reverse primers of the present invention 1034. [This invention 1044] The kit of claim 1043, further comprising the pair of forward and reverse primers of claim 1035. [This invention 1045] A kit for determining the infectivity of an rAAV test composition relative to the infectivity of a reference composition, said kit comprising a combination of a probe, a forward primer, and a reverse primer of the present invention 1036. [The present invention 1046] The kit of claim 1045, further comprising a combination of the probe, forward primer, and reverse primer of claim 1037. [This invention 1047] The kit of any of claims 1040 to 1046, further comprising an rAAV reference composition. [This invention 1048] 1. A method for determining the relative infectivity of a composition of viral particles under different conditions, comprising the steps of: a. inoculating target cells with said composition comprising viral particles under a first and a second set of conditions; b. washing the inoculated cells to remove extracellular viral particles; c. isolating first and second nucleic acid samples from target cells inoculated under said first and second sets of conditions, respectively; d. Determining the ratio of viral genome copies (VGC): target cell genome copies (TCGC) in said first and second nucleic acid samples. [This invention 1049] 1049. The method of claim 88, wherein said first and second sets of conditions use the same target cells. [The present invention 1050] 1049. The method of claim 88, wherein said first and second sets of conditions use different target cells. [This invention 1051] The method of claim 1050, wherein said different target cells comprise different genetic modifications. [This invention 1052] 1050. The method of claim 1050, wherein said different target cells are identical except for the presence of a genetic modification in one of said target cells. [This invention 1053] 1053. The method of any of claims 1049 to 1052, wherein said step of inoculating target cells comprises inoculating target cells with serial dilutions of said composition. [This invention 1054] 1053. The method of claim 1053, wherein said serial dilutions are two-fold dilutions. [This invention 1055] 105. The method of claim 1053 or claim 1054, wherein said step of inoculating target cells comprises inoculating target cells with serial dilutions of said composition. [This invention 1056] The method of any of claims 1053 to 1055, further comprising the step of calculating the relative infectivity of said composition under said first and second sets of conditions using a parallel line model. [This invention 1057] said step of calculating the relative infectivity of said composition under said first and second sets of conditions comprising: a. calculating the VGC:TCGC ratio for each dilution of said first and second sets of conditions; b. plotting the log VGC:TCGC ratio against the log dilution for said first and second sets of conditions; c. fitting the data points of the first and second sets of conditions to lines of first and second conditions using a common slope; d. Infectivity under the first condition relative to the second condition Calculate as TIFF0007822436000006.tif8128 The method of the present invention 1056, comprising: [This invention 1058] The method of any of claims 1053 to 1057, wherein the coefficient of variation (cv) is less than about 100%, less than about 50%, or less than about 25%. [This invention 1059] 1059. The method of any of claims 1053 to 1058, wherein said VGC and TCGC in the nucleic acid composition are determined by polymerase chain reaction. [The present invention 1060] 1059. The method of claim 1059, wherein said polymerase chain reaction is a quantitative polymerase chain reaction. [This invention 1061] 1059. The method of claim 1059, wherein said polymerase chain reaction is a digital polymerase chain reaction. [This invention 1062] The method of any one of claims 1048 to 1061, wherein the virus particle is a replication-defective virus. [This invention 1063] 1063. The method of claim 1062, wherein said replication-deficient virus is an AAV, adenovirus, vaccinia, or lentivirus. [This invention 1064] 1063. The method of claim 1062, wherein said replication-deficient virus is a retrovirus. [This invention 1065] 1063. The method of claim 1062, wherein the replication-deficient virus is AAV. [The present invention 1066] 1065. The method of claim 1065, wherein said AAV is recombinant AAV (rAAV). [This invention 1067] The rAAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, A AV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB , AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 serotype capsid protein. [The present invention 1068] 1067. The method of claim 1067, wherein the rAAV comprises capsid proteins of the AAV8 serotype or the AAV9 serotype. [The present invention 1069] 1068. The method of any of claims 1048 to 1068, wherein said target cells under at least one set of conditions comprise BHK21, HEK293, BEAS-2BS, HeLaS3, Huh-7, Hepa1-6, or A549 cells. [The present invention 1070] The method of any one of claims 1022 to 1069, wherein said target cells under at least one set of conditions comprise Huh-7 cells. Still other features and advantages of the compositions and methods described herein will become more apparent from the following detailed description when read in conjunction with the accompanying drawings. [Brief explanation of the drawings]

[0015] [Figure 1] Relative infectivity method workflow. [Figure 2] Calculation of relative infectivity using the parallel line model. DETAILED DESCRIPTION OF THE INVENTION

[0016] Detailed Description Provided herein are methods for determining the infectivity of compositions containing replication-deficient viruses, e.g., AAV vectors. The inventors have surprisingly found that the methods disclosed herein significantly improve accuracy. Specifically, the methods disclosed herein offer significant advantages over the current standard for measuring infectivity, the TCID50 assay. These advantages include improved accuracy, improved reproducibility, and faster results with less sample processing. The improved accuracy and speed of the methods disclosed herein make them suitable for a variety of applications in the development and production of pharmaceutical compositions containing replication-deficient viruses (e.g., AAV). For example, the methods disclosed herein are highly suitable for use in formulation development, as they enable rapid and highly accurate comparison of the infectivity of multiple samples containing different excipients and / or stored under different conditions for different lengths of time. The methods disclosed herein are also highly suitable for determining the biological activity of pharmaceutical doses, e.g., in lot release assays.

[0017] In some embodiments, the methods described herein are capable of detecting and quantifying small differences in the infectivity of compositions comprising replication-deficient viruses (e.g., AAV vectors). In some embodiments, the methods involve infecting adherent cells with a dilution series of a replication-deficient virus test sample in parallel with a dilution series of a reference standard. In some embodiments, the dilution series is a 2-fold, 3-fold, or 5-fold dilution series. After the infection period, the cells are washed, harvested, and subjected to PCR (e.g., ddPCR) to quantify the viral vector DNA present in the cells. The amount of viral vector DNA recovered in each dilution is used to calculate the infectivity of the sample relative to the reference standard. The methods described herein can be used to compare the infectivity of different batches of compositions comprising replication-deficient viruses and to quantify changes in infectivity due to degradation. The methods described herein can also be used as a platform method to support process and formulation development across different projects, to compare the ability of replication-deficient viral vectors to infect different human cells, to evaluate improved infectivity in recombinantly engineered variants, to explore viral infection kinetics, or to evaluate the activity of variants (e.g., the activity of variants containing different capsids). In some embodiments of the methods described herein, the replication-deficient viral vector is AAV. Those skilled in the art will understand that the methods described herein can be used to screen and identify conditions for improved infectivity by viral compositions, for example, to screen and identify cells permissive for infection by replication-deficient viral compositions. In one embodiment, the methods described herein can be used to determine the relative infectivity of viral preparations in different cell lines. In one embodiment, the methods described herein can be used to determine the relative infectivity of viral preparations for variants of cell lines containing genetic modifications (e.g., containing transgenes).

[0018] In some embodiments, the methods described herein are directed to a recombinant vector comprising a recombinant vector selected from, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV. The method is suitable for any rAAV serotype, including AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16, as well as derivatives, modifications, or pseudotypes thereof. In some embodiments, the method is used to measure the infectivity of rAAV8 particles. In some embodiments, the method is used to measure the infectivity of rAAV8 derivative particles, rAAV8 modified particles, or rAAV8 pseudotype particles. In some embodiments, the method is used to measure the infectivity of rAAV9 particles. In some embodiments, the methods are used to measure the infectivity of rAAV9 derivative particles, rAAV9 modified particles, or rAAV9 pseudotyped particles.

[0019] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. To facilitate understanding of the methods of this disclosure, several terms and phrases are defined below.

[0020] For example, "about," as used in the methods provided herein to modify the amount of a component in a composition, the concentration of a component in a composition, flow rate, rAAV particle yield, feed volume, salt concentration, and similar values ​​and ranges thereof, refers to variations in a numerical quantity that may occur, for example, due to typical measuring and handling procedures used to make concentrates or use solutions; due to inadvertent errors in these procedures; due to differences in the manufacture, source, or purity of components used in making the composition or performing the method; and similar considerations. The term "about" also encompasses amounts that vary with aging of a composition or mixture having a particular initial concentration. The term "about" also encompasses amounts that vary with mixing or processing of a composition or mixture having a particular initial concentration. Whether modified by the term "about," the claims include the equivalent of that quantity. In some embodiments, the term "about" refers to a range of about 10-20% more or less than the stated number or range. In further embodiments, "about" refers to plus or minus 10% of the stated number or range. For example, "about 10%" indicates a range of 9% to 11%.

[0021] As used herein, the term "replication-deficient" refers to a viral vector incapable of complete and efficient replication. A replication-deficient virus is a mutant or is defective in one or more functions essential for viral genome replication or viral particle synthesis and assembly. A replication-deficient virus can be propagated in a complementing cell line that expresses the defective gene product. However, in normal target cells, a replication-deficient virus can express viral gene products but does not replicate to form infectious progeny viral particles. In some embodiments, a replication-deficient virus or viral vector is a virus or vector mutant or is defective in one or more functions essential for viral genome replication. In some embodiments, a replication-deficient virus or viral vector is a virus or vector mutant or is defective in one or more functions essential for viral particle synthesis and assembly. In some embodiments, a replication-deficient virus or viral vector is a retrovirus or retroviral vector, such as a lentivirus or lentiviral vector. In some embodiments, a replication-deficient virus or viral vector is an adenovirus or adenoviral vector, an HSV or HSV vector, or an influenza virus or viral vector. In some embodiments, the replication-deficient virus or viral vector is AAV virus or viral vector.Replication-deficient viral vectors are known to those skilled in the art, and are disclosed in, for example, United States Patent No. 7,198,784, United States Patent No. 9,408,905, United States Patent No. 9,862,931, United States Patent No. 8,067,156, United States Patent Application Publication No. 20150291935, United States Patent No. 20120220492, United States Patent No. 20180291351 and United States Patent Application Publication No. 20170175137 (each of which is incorporated herein by reference in its entirety).

[0022] "AAV" is an abbreviation for adeno-associated virus and can be used to refer to the virus itself or any modification, derivative, or pseudotype thereof. The term encompasses all subtypes and both native and recombinant forms, unless otherwise required. The abbreviation "rAAV" refers to recombinant adeno-associated virus. The term "AAV" includes AAV type 1 (AAV1), AAV type 2 (AAV2), AAV type 3 (AAV3), AAV type 4 (AAV4), AAV type 5 (AAV5), AAV type 6 (AAV6), AAV type 7 (AAV7), AAV type 8 (AAV8), AAV type 9 (AAV9), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV, as well as modifications, derivatives, or pseudotypes thereof. "Primate AAV" refers to AAV that infects primates, "non-primate AAV" refers to AAV that infects non-primate mammals, "bovine AAV" refers to AAV that infects bovine mammals, etc. In some embodiments, the AAV particles are selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.Anc80L65 ... AV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6 , AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16.In some embodiments, the rAAV particles are selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV. AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV. is a derivative, modification, or pseudotype of HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16.

[0023] "Recombinant" as applied to an AAV particle means that the AAV particle is the product of one or more procedures that result in an AAV particle construct that is distinct from the AAV particle itself.

[0024] A recombinant adeno-associated viral particle, or "rAAV particle," refers to a viral particle composed of at least one AAV capsid protein and an encapsidated polynucleotide rAAV vector containing a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, e.g., a transgene to be delivered to a mammalian cell). The rAAV particle can be any AAV serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10, or a derivative / modification / pseudotype thereof), including any modification, derivative, or pseudotype. Such AAV serotypes and derivatives / modifications / pseudotypes, as well as methods for generating such serotypes / derivatives / modifications / pseudotypes, are known in the art (see, e.g., Asokan et al., Mol. Ther. 20(4):699-708 (2012)). In some embodiments, the rAAV particles are selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.P The capsid protein comprises an AAV capsid serotype selected from HP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16.In some embodiments, the rAAV particles are selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, including capsid proteins that are derivatives, modifications, or pseudotypes of the AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 capsid proteins.

[0025] The rAAV particles of the present disclosure can be of any serotype or any combination of serotypes (e.g., an rAAV particle population comprising two or more serotypes (e.g., comprising two or more of rAAV2, rAAV8, and rAAV9 particles)). In some embodiments, the rAAV particles are rAAV1, rAAV2, rAAV3, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAV10, or other rAAV particles, or a combination of two or more thereof. In some embodiments, the rAAV particles are rAAV8 or rAAV9 particles. In some embodiments, the rAAV particles are selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHPB, AAV. and comprising capsid proteins from two or more serotypes selected from PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16.In some embodiments, the rAAV particles are selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV. and capsid proteins that are derivatives, modifications, or pseudotypes of two or more serotypes selected from AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16 capsid proteins.

[0026] In some embodiments, the rAAV particles have AAV capsid proteins of a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, or derivatives, modifications, or pseudotypes thereof. In some embodiments, the rAAV particles have AAV capsid proteins of a serotype selected from the group consisting of AAV8, AAV9, or derivatives, modifications, or pseudotypes thereof. In some embodiments, the rAAV particles have AAV capsid proteins of a serotype selected from the group consisting of AAV7, AAV8, AAV9, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHB, AAV.PHP.eB, and AAV.7m8. In some embodiments, the rAAV particles have AAV capsid proteins with high sequence homology to AAV8 or AAV9, e.g., AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37.

[0027] As used herein, the terms "digital PCR" or "dPCR" refer to any PCR method in which a sample is divided into multiple small subsamples, each of which is then subjected to a PCR amplification reaction. After PCR amplification, the ratio of subsamples containing target-specific PCR end products (positive reactions) to subsamples not containing target-specific PCR end products (negative reactions) is determined by detecting the presence or absence of target-specific PCR end products in each subsample. The copy number and concentration of the target sequence in the starting sample are calculated from the ratio of positive and negative subsample reactions, taking into account a Poisson distribution. Unlike conventional PCR, digital PCR does not depend on the number of amplification cycles performed to determine the target concentration of the initial sample, eliminating the reliance on uncertain exponential data to quantify target nucleic acids and providing absolute quantification. As used herein, "digital droplet PCR" refers to a digital PCR method in which the initial sample is subdivided into several droplets. In some embodiments, the digital PCR reaction is a multiplex PCR, which allows for the quantification of multiple target sequences in a single dPCR reaction. In some embodiments, the dPCR reaction is a Digital Droplet PCR™ or ddPCR™ reaction in which an initial sample is subdivided into several droplets that constitute sub-samples.

[0028] The terms "reference standard," "reference standard," or "reference composition" refer to a well-characterized vector sample utilized to produce viral particles, and these terms are used interchangeably throughout. A reference standard may be representative of clinical material or otherwise certified, or characterized to produce viral particles in a consistent manner. For example, a reference standard may be selected from an internal or external trusted source.

[0029] As used herein, the terms "purifying," "purifying," "separating," "separating," "separating," "isolating," "isolating," or "isolation" refer to increasing the degree of purity for rAAV particles from a sample containing a target product and one or more impurities. Typically, the degree of purity of the target product is increased by removing (completely or incompletely) at least one impurity from the sample. In some embodiments, the degree of purity of rAAV in a sample is increased by removing (completely or incompletely) one or more impurities from the sample using the methods described herein.

[0030] As used in this disclosure and claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise.

[0031] Whenever embodiments are described herein using the term "comprising," it should be understood that other similar embodiments described in terms of "consisting of" and / or "consisting essentially of" are also provided. Also, whenever embodiments are described herein using the term "consisting essentially of," it should be understood that other similar embodiments described in terms of "consisting of" are also provided.

[0032] The term "and / or," when used herein in phrases such as "A and / or B," is intended to include both A and B, A or B, A alone, and B alone. Similarly, the term "and / or," when used in phrases such as "A, B, and / or C," is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A alone; B alone; and C alone.

[0033] When embodiments of the present disclosure are described in terms of Markush groups or other alternative groups, the methods of the present disclosure include not only the entire group listed as a whole, but also each member of the group individually, all possible subgroups of the main group, and the main group in the absence of one or more of the group members. The methods of the present disclosure also contemplate the explicit exclusion of any one or more of the group members in the methods of the present disclosure.

[0034] Method for determining the infectivity of a test composition containing viral particles - Patent Application 20070122997 In some embodiments, the present disclosure provides a method for determining the infectivity of a test composition containing viral particles relative to the infectivity of a reference composition containing viral particles, the method comprising the steps of contacting target cells with the test composition and the reference composition under conditions that allow inoculation of the viral particles into the target cells; removing extracellular viral particles; isolating a test nucleic acid sample and a reference nucleic acid sample from the target cells inoculated with the test composition and the reference composition, respectively; and determining the ratio of viral genome copies (VGC):target cell genome copies (TCGC) in the test nucleic acid sample and the reference nucleic acid sample. Without being bound by any theory, it is believed that infection of the target cells with viral particles in the test or reference composition results in introduction of viral genomes into the target cells. Removal of extracellular viral particles after inoculation removes viral genomes not introduced into the target cells by infection. In some embodiments, the target cells are contacted with serial dilutions of the test composition and the reference composition. In some embodiments, the serial dilutions of the test composition and the reference composition are 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 8-fold dilutions. In some embodiments, the serial dilutions of the test sample and reference sample are 2-fold dilutions. In some embodiments, the serial dilutions of the test sample and reference sample are less than 2-fold dilutions. In some embodiments, the serial dilutions of the test sample and reference sample are 2-fold to 10-fold dilutions. In some embodiments, the step of inoculating the target cells comprises incubating the target cells in the presence of viral particles for between about 5 minutes and about 3 days. In some embodiments, the VGC and TCGC in the nucleic acid sample are determined by polymerase chain reaction, optionally by digital polymerase chain reaction. In some embodiments, the method further comprises calculating the infectivity of the test sample relative to the reference sample using a parallel line model.The parallel line method is a robust biostatistical analysis method for comparing one or more test substances to a reference substance based on their relative potency (Finney, DJ, Statistical methods in biological assay (Charles Griffin & Co., Ltd. 1952); Wardlaw, AC, Practical Statistics for Experimental Biologists 210 (Wiley 1986) (2000)). In some embodiments, the viral particle is a replication-deficient viral particle. In some embodiments, the replication-deficient viral particle is an AAV particle, e.g., a recombinant AAV particle. In some embodiments, the rAAV comprises a capsid protein of a serotype selected from the group consisting of AAV7, AAV8, AAV9, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHB.eB, AAV.PHP.eB, and AAV.7m8. In some embodiments, the rAAV comprises a capsid protein with high sequence homology to AAV8 or AAV9, e.g., AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37. In some embodiments, the rAAV comprises a capsid protein of the AAV8 or AAV9 serotype. In some embodiments, the target cell is a BHK21, HEK293, BEAS-2BS, HeLaS3, Huh-7, Hepa1-6, or A549 cell. In some embodiments, the target cell is a Huh-7 cell.

[0035] In some embodiments, the present disclosure provides a method for determining the infectivity of a test composition comprising viral particles relative to the infectivity of a reference composition comprising viral particles, the method comprising the steps of preparing serial dilutions of the test composition and the reference composition, contacting target cells with the test composition and the reference composition under conditions that allow inoculation of the viral particles into the target cells, removing extracellular viral particles, isolating a test nucleic acid sample and a reference nucleic acid sample from the target cells inoculated with the test composition and the reference composition, respectively, and determining the ratio of viral genome copies (VGC):target cell genome copies (TCGC) in the test nucleic acid sample and the reference nucleic acid sample. In some embodiments, the serial dilutions of the test composition and the reference composition are 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 8-fold dilutions. In some embodiments, the serial dilutions of the test sample and the reference sample are 2-fold dilutions. In some embodiments, the serial dilutions of the test sample and the reference sample are less than 2-fold dilutions. In some embodiments, the serial dilutions of the test sample and the reference sample are 2-fold to 10-fold dilutions. In some embodiments, the step of inoculating the target cells comprises incubating the target cells in the presence of the viral particles for between about 5 minutes and about 3 days. In some embodiments, the VGC and TCGC in the nucleic acid sample are determined by polymerase chain reaction, optionally by digital polymerase chain reaction. In some embodiments, the method further comprises calculating the infectivity of the test sample relative to a reference sample using a parallel line model. In some embodiments, the viral particles are replication-deficient viral particles. In some embodiments, the replication-deficient viral particles are AAV particles, e.g., recombinant AAV particles. In some embodiments, the rAAV comprises a capsid protein of a serotype selected from the group consisting of AAV7, AAV8, AAV9, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHB.B, AAV.PHP.eB, and AAV.7m8.In some embodiments, the rAAV comprises a capsid protein with high sequence homology to AAV8 or AAV9, e.g., AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37. In some embodiments, the rAAV comprises a capsid protein of the AAV8 or AAV9 serotype. In some embodiments, the target cell is a BHK21, HEK293, BEAS-2BS, HeLaS3, Huh-7, Hepa1-6, or A549 cell. In some embodiments, the target cell is a Huh-7 cell.

[0036] In some embodiments, a method disclosed herein for determining the infectivity of a test composition containing viral particles relative to the infectivity of a reference composition includes the steps of inoculating target cells separately with the test composition and the reference composition, washing the inoculated cells to remove extracellular viral particles, isolating a test nucleic acid sample and a reference nucleic acid sample from the target cells inoculated with the test composition and the reference composition, respectively, and determining the ratio of viral genome copies (VGC):target cell genome copies (TCGC) in the test nucleic acid sample and the reference nucleic acid sample. In some embodiments, the target cells are contacted with serial dilutions of the test composition and the reference composition. In some embodiments, the serial dilutions of the test composition and the reference composition are 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 8-fold dilutions. In some embodiments, the serial dilutions of the test sample and the reference sample are 2-fold dilutions. In some embodiments, the serial dilutions of the test sample and the reference sample are less than 2-fold dilutions. In some embodiments, the serial dilutions of the test sample and the reference sample are 2-fold to 10-fold dilutions. In some embodiments, the step of inoculating the target cells comprises incubating the target cells in the presence of the viral particles for between about 5 minutes and about 3 days. In some embodiments, the VGC and TCGC in the nucleic acid sample are determined by polymerase chain reaction, optionally by digital polymerase chain reaction. In some embodiments, the method further comprises calculating the infectivity of the test sample relative to a reference sample using a parallel line model. In some embodiments, the viral particles are replication-deficient viral particles. In some embodiments, the replication-deficient viral particles are AAV particles, e.g., recombinant AAV particles. In some embodiments, the rAAV comprises a capsid protein of a serotype selected from the group consisting of AAV7, AAV8, AAV9, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHB.B, AAV.PHP.eB, and AAV.7m8.In some embodiments, the rAAV comprises a capsid protein with high sequence homology to AAV8 or AAV9, e.g., AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37. In some embodiments, the rAAV comprises a capsid protein of the AAV8 or AAV9 serotype. In some embodiments, the target cell is a BHK21, HEK293, BEAS-2BS, HeLaS3, Huh-7, Hepa1-6, or A549 cell. In some embodiments, the target cell is a Huh-7 cell.

[0037] In some embodiments, a method disclosed herein for determining the infectivity of a test composition containing viral particles relative to the infectivity of a reference composition includes preparing serial dilutions of the test composition and the reference composition, inoculating target cells with the serial dilutions of the test composition and the reference composition, washing the inoculated cells to remove extracellular viral particles, isolating a test nucleic acid sample and a reference nucleic acid sample from the target cells inoculated with the test composition and the reference composition, respectively, and determining the ratio of viral genome copies (VGC):target cell genome copies (TCGC) in the test nucleic acid sample and the reference nucleic acid sample. In some embodiments, the serial dilutions of the test composition and the reference composition are 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 8-fold dilutions. In some embodiments, the serial dilutions of the test sample and the reference sample are 2-fold dilutions. In some embodiments, the serial dilutions of the test sample and the reference sample are less than 2-fold dilutions. In some embodiments, the serial dilutions of the test sample and the reference sample are 2-fold to 10-fold dilutions. In some embodiments, the step of inoculating the target cells comprises incubating the target cells in the presence of the viral particles for between about 5 minutes and about 3 days. In some embodiments, the VGC and TCGC in the nucleic acid sample are determined by polymerase chain reaction, optionally by digital polymerase chain reaction. In some embodiments, the method further comprises calculating the infectivity of the test sample relative to a reference sample using a parallel line model. In some embodiments, the viral particles are replication-deficient viral particles. In some embodiments, the replication-deficient viral particles are AAV particles, e.g., recombinant AAV particles. In some embodiments, the rAAV comprises a capsid protein of a serotype selected from the group consisting of AAV7, AAV8, AAV9, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHB.B, AAV.PHP.eB, and AAV.7m8.In some embodiments, the rAAV comprises a capsid protein with high sequence homology to AAV8 or AAV9, e.g., AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37. In some embodiments, the rAAV comprises a capsid protein of the AAV8 or AAV9 serotype. In some embodiments, the target cell is a BHK21, HEK293, BEAS-2BS, HeLaS3, Huh-7, Hepa1-6, or A549 cell. In some embodiments, the target cell is a Huh-7 cell.

[0038] The method disclosed herein can be used to determine the relative infectivity of test samples containing replication-deficient viral particles.In some embodiments, the replication-deficient viral particles are AAV, adenovirus, vaccinia, or lentivirus particles.In some embodiments, the replication-deficient viral particles are retrovirus particles.In some embodiments, the replication-deficient viral particles are AAV particles.In some embodiments, the replication-deficient viral particles are recombinant AAV particles. In some embodiments, the replication-deficient viral particles are selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B In some embodiments, the replication-deficient viral particle is a rAAV particle comprising capsid proteins of the AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 serotype. In some embodiments, the replication-deficient viral particle is a rAAV particle comprising capsid proteins of the AAV8 or AAV9 serotype.

[0039] In some embodiments, the test composition and the reference composition contain genetically identical isolated viral particles. It is understood that genetically identical isolated viral particles contain identical or substantially identical genomes and identical or substantially identical viral polypeptides. In some embodiments, the test composition and the reference composition contain genetically identical viral particles that have been separately isolated or separately processed after isolation. Thus, one skilled in the art will understand that the methods of the present disclosure can be used to compare the infectivity of genetically identical isolated viral particles produced in different batches. In some embodiments, different batches of genetically identical isolated viral particles are produced using the same process. In some embodiments, different batches of genetically identical isolated viral particles are produced using different upstream and / or downstream processes. In some embodiments, the different upstream processes use one or more of different host cells, different culture media, different tissue culture processes, and different harvest processes. In some embodiments, the different downstream processes use one or more of different purification steps, different buffers, different processing temperatures, different formulation buffers, and different storage temperatures. In some embodiments, the different batches of genetically identical isolated viral particles are stored for different time periods.

[0040] In some embodiments, the viral particles contained in the test composition and the reference composition are not genetically identical. In some embodiments, the test viral particles and the reference viral particles comprise different genomes. In some embodiments, the test viral particles and the reference viral particles comprise different viral polypeptides. In some embodiments, the test viral particles and the reference viral particles comprise one or more capsid polypeptides with different amino acid sequences.

[0041] In some embodiments, the test composition and the reference composition comprise genetically identical rAAV particles. In some embodiments, the test composition and the reference composition comprise genetically identical rAAV particles. In some embodiments, the test composition and the reference composition comprise genetically identical rAAV particles. In some embodiments, the test composition and the reference composition comprise genetically identical rAAV particles. In some embodiments, the test and reference compositions comprise genetically identical rAAV particles comprising AAV capsid proteins from an AAV capsid serotype selected from AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC 16. In some embodiments, the test and reference compositions comprise genetically identical rAAV particles comprising AAV8 or AAV9 capsid proteins.

[0042] In some embodiments of the methods described herein, the test composition and reference composition are diluted before contacting the target cells with the virus particles. In some embodiments, the test composition and reference composition are serially diluted before contacting the target cells with the virus particles. In some embodiments, the test composition and reference composition are serially diluted using the same dilution factor. In some embodiments, the test composition and reference composition are serially diluted by a dilution factor of less than 10. In some embodiments, the test composition and reference composition are serially diluted by a dilution factor of 1.5, 2, 3, 4, 5, 6, 7, 8, or 9. In some embodiments, the test composition and reference composition are serially diluted by a dilution factor of 2. In some embodiments, the serial dilutions of the test composition and reference composition are 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, or 9-fold dilutions. In some embodiments, the serial dilutions of the test composition and reference composition are 2-fold.

[0043] In some embodiments of the methods disclosed herein, the serial dilutions of the test composition and the reference composition include at least two dilutions, at least three dilutions, at least five dilutions, or at least 10 dilutions. In some embodiments of the methods disclosed herein, the serial dilutions of the test composition and the reference composition include 2 to 20 dilutions. In some embodiments of the methods disclosed herein, the serial dilutions of the test composition and the reference composition include 2 to 30 dilutions. In some embodiments of the methods disclosed herein, the serial dilutions of the test composition and the reference composition include 3 dilutions, 4 dilutions, 5 dilutions, 6 dilutions, 7 dilutions, 8 dilutions, 9 dilutions, 10 dilutions, 15 dilutions, or 20 dilutions.

[0044] Those skilled in the art will understand that the methods disclosed herein are also suitable for determining how variables other than virus composition affect the efficacy of the infection process. Accordingly, provided herein are methods for determining the relative infectivity of a composition of virus particles under different conditions. In one embodiment, the method disclosed herein includes inoculating target cells with a composition containing virus particles under first and second sets of conditions; washing the inoculated cells to remove extracellular virus particles; isolating first and second nucleic acid samples from the target cells inoculated under the first and second sets of conditions, respectively; and determining the ratio of viral genome copies (VGC):target cell genome copies (TCGC) in the first and second nucleic acid samples. In some embodiments, the first and second sets of conditions use different target cells. In some embodiments, the first and second sets of conditions use different target cells containing different genetic modifications. In some embodiments, the first and second sets of conditions use different target cells that are identical except for the presence of a genetic modification in one of the target cells. In some embodiments, the genetic modification is the presence of a transgene. In some embodiments, the first and second sets of conditions use target cells representing different tissue types. In some embodiments, the first and second sets of conditions use target cells that are different lineages derived from a parent cell line. In some embodiments, the target cells are contacted with serial dilutions of the virus composition. In some embodiments, the serial dilutions of the virus composition are 1.5x, 2x, 3x, 4x, 5x, or 8x dilutions. In some embodiments, the serial dilutions of the test sample and reference sample are 2x dilutions. In some embodiments, the serial dilutions of the test sample and reference sample are less than 2x dilutions. In some embodiments, the serial dilutions of the test sample and reference sample are 2x to 10x dilutions. In some embodiments, the step of inoculating the target cells comprises incubating the target cells in the presence of the virus particles for about 5 minutes to about 3 days.In some embodiments, the VGC and TCGC in the nucleic acid sample are determined by polymerase chain reaction, optionally by digital polymerase chain reaction. In some embodiments, the method further comprises calculating the infectivity of the test sample relative to a reference sample using a parallel line model. In some embodiments, the viral particles are replication-deficient viral particles. In some embodiments, the replication-deficient viral particles are AAV particles, e.g., recombinant AAV particles. In some embodiments, the rAAV comprises a capsid protein of a serotype selected from the group consisting of AAV7, AAV8, AAV9, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHB.B, AAV.PHP.eB, and AAV.7m8. In some embodiments, the rAAV comprises a capsid protein with high sequence homology to AAV8 or AAV9, e.g., AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37. In some embodiments, the rAAV comprises a capsid protein of the AAV8 or AAV9 serotype. In some embodiments, at least one of the different conditions comprises the use of target cells selected from the group consisting of BHK21, HEK293, BEAS-2BS, HeLaS3, Huh-7, Hepa1-6, and A549 cells. In some embodiments, at least one of the different conditions comprises the use of Huh-7 cells as target cells.

[0045] Any cells or cell lines known in the art to be susceptible to infection by viral particles in the test and reference samples can serve as target cells for use in the methods described herein. In some embodiments, the target cells are adherent cells. In some embodiments, the target cells are suspension cells. In some embodiments, the methods disclosed herein use mammalian cells. In some embodiments, the target cells are human cells. In some embodiments, the target cells are BHK21, HEK293, HEK293-derived cells (e.g., HEK293T cells, HEK293F cells), BEAS-2BS, HeLaS3, Huh-7, Hepa1-6, or A549 cells. In some embodiments, the target cells are Huh-7 cells. In some embodiments, the target cells are BHK cells, COS cells, PerC6 cells, or Vero cells. In some embodiments, the methods disclosed herein use insect cells, such as SF-9 cells. In some embodiments, the methods disclosed herein use HEK293 cells.

[0046] Target cells can be maintained in any suitable medium known to those of skill in the art. Such media include, but are not limited to, media produced by Hyclone Laboratories and JRH, including Modified Eagle's Medium (MEM) and Dulbecco's Modified Eagle's Medium (DMEM). In some embodiments, the medium comprises Dynamis™ medium, FreeStyle™ 293 Expression Medium, or Expi293™ Expression Medium from Invitrogen / ThermoFisher. In some embodiments, the medium comprises Dynamis™ medium. In some embodiments, the methods disclosed herein use cell cultures comprising serum-free medium, animal component-free medium, or chemically defined medium. In some embodiments, the medium is animal component-free medium. In some embodiments, the medium comprises serum. In some embodiments, the medium comprises fetal bovine serum. In some embodiments, the medium is glutamine-free medium. In some embodiments, the medium comprises glutamine. In some embodiments, the medium is supplemented with one or more of nutrients, salts, buffers, and additives (e.g., antifoaming agents). In some embodiments, the medium is supplemented with glutamine. In some embodiments, the medium is supplemented with serum. In some embodiments, the medium is supplemented with fetal bovine serum.

[0047] In some embodiments of the methods described herein, the target cells are serum-starved. In some embodiments, the target cells are serum-starved for 24 hours before contacting them with the viral particles.

[0048] In some embodiments of the methods described herein, target cells are contacted with virus particles in a multi-well plate, for example, a 96-well or 384-well plate. In some embodiments, the methods disclosed herein are carried out in a 96-well plate. It should be understood that the use of a multi-well plate allows for duplicate, triplicate, or even multiple replicates of the same inoculation reaction in the same assay.

[0049] In some embodiments of the methods described herein, the step of inoculating the target cells comprises incubating the target cells in the presence of the viral particles under conditions suitable for the viral particles to enter the target cells. In some embodiments, the step of inoculating the target cells comprises incubating the target cells in the presence of the viral particles for between about 5 minutes and about 3 days. In some embodiments, the step of inoculating the target cells comprises incubating the target cells in the presence of the viral particles for between about 12 hours and about 36 hours. In some embodiments, the step of inoculating the target cells comprises incubating the target cells in the presence of the viral particles for between about 18 hours and about 30 hours. In some embodiments, the step of inoculating the target cells comprises incubating the target cells in the presence of the viral particles for about 1 hour, about 2 hours, about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 30 hours, or about 36 hours. In some embodiments, the step of inoculating the target cells comprises incubating the target cells in the presence of the viral particles for about 1 day, about 1.5 days, or about 2 days. In some embodiments, the step of inoculating the target cells comprises incubating the target cells in the presence of the viral particles for about 24 hours.

[0050] In some embodiments of the methods described herein, the step of washing the inoculated cells to remove extracellular viral particles comprises contacting the cells with any buffer suitable for removing extracellular viral particles without lysing the inoculated target cells, hi some embodiments, the cells are washed with phosphate buffered saline or Dulbecco's phosphate buffered saline.

[0051] Any method known to those skilled in the art can be used to isolate nucleic acid samples from the inoculated cells. In some embodiments, nucleic acid samples are isolated using commercially available systems and reagents suitable for processing multi-well plates (e.g., 96-well plates). Suitable systems and reagents include Extracta™ DNA Prep Extraction Reagent, Wizard® SV 96 Genomic DNA Purification System, and GenElute 96-Well Tissue Genomic DNA Purification Kit.

[0052] In some embodiments, the nucleic acid sample is a DNA sample. In some embodiments, the nucleic acid sample is an RNA sample.

[0053] Any method known to those skilled in the art can be used to determine the ratio of viral genome copies (VGC):target cell genome copies (TCGC) in a nucleic acid sample. In some embodiments, the VGC:TGCG ratio is determined by next-generation sequencing, quantitative PCR (qPCR), or digital PCR (dPCR). In some embodiments, the VGC:TGCG ratio is determined by qPCR. In some embodiments, the VGC:TGCG ratio is determined by dPCR, for example, droplet digital PCR (ddPCR). In some embodiments, the PCR (e.g., dPCR) reaction used to determine the VGC:TGCG ratio is a multiplex PCR reaction. In some embodiments, the multiplex PCR (e.g., dPCR) reaction includes a viral genome-specific reaction and a target cell genome-specific reaction.

[0054] Those skilled in the art will understand that any viral genome-specific sequence can be targeted for amplification by PCR to determine the number or concentration of viral genome copies (VGCs) in a sample using qPCR or dPCR. Similarly, any target cell genome-specific sequence can be targeted for amplification by PCR to determine the number or concentration of target cell genome copies (TCGCs) in a sample using qPCR or dPCR. Software tools for designing combinations of forward primers, reverse primers, and probes to determine the copy number or concentration of viral genomes or target cell genome-specific sequences in a sample are well known to those skilled in the art and are available online, for example, on the websites of Takara, New England Biolabs, Integrated DNA Technologies, and BioRad. Any of these software tools can be used to design primers and probes for determining the number or concentration of viral genome copies (VGCs) and target cell genome copies in a sample according to the methods disclosed herein.

[0055] In some embodiments, the viral genome-specific sequence target is a rabbit beta-globin polyA element. In some embodiments, the forward primer and reverse primer capable of amplifying a target sequence within the rabbit beta-globin polyA element comprise the nucleotide sequences of SEQ ID NOs: 1 and 2, respectively. In some embodiments, the forward primer, reverse primer, and probe capable of detecting a target sequence within the rabbit beta-globin polyA element comprise polynucleotides consisting of the nucleotide sequences of SEQ ID NOs: 1, 2, and 3, respectively. In some embodiments, the probe further comprises a first fluorescent label covalently attached to the 5' end and a second fluorescent label covalently attached to the 3' end of the oligonucleotide. In some embodiments, the first fluorescent label is FAM and the second fluorescent label is TAMRA.

[0056] In some embodiments, the target cell genome-specific sequence target is the human albumin gene. In some embodiments, the forward primer and reverse primer capable of amplifying a target sequence within the human albumin gene consist of the nucleotide sequences of SEQ ID NOs: 4 and 5, respectively. In some embodiments, the forward primer, reverse primer, and probe capable of detecting a target sequence within the human albumin gene comprise polynucleotides consisting of the nucleotide sequences of SEQ ID NOs: 4, 5, and 6, respectively. In some embodiments, the probe further comprises a first fluorescent label covalently attached to the 5' end and a second fluorescent label covalently attached to the 3' end of the oligonucleotide. In some embodiments, the first fluorescent label is FAM and the second fluorescent label is TAMRA.

[0057] In some embodiments, the present disclosure provides methods for determining the infectivity of a test composition relative to the infectivity of a reference composition, the methods comprising calculating the infectivity of the test composition relative to the reference composition from the ratio of viral genome copies (VGC):target cell genome copies (TCGC) determined in a test nucleic acid sample and a reference nucleic acid sample. In some embodiments, the methods described herein comprise calculating the infectivity of the test composition relative to the reference composition using a parallel line model. In some embodiments, calculating the infectivity of the test composition relative to the reference composition comprises calculating the VGC:TCGC ratio for each dilution of the test composition and the reference composition, plotting the log VGC:TCGC ratio against the log dilution for the test composition and the reference composition, fitting the data points of the test composition and the reference composition to a line for the test composition and the reference composition using a common slope, and calculating the infectivity of the test composition relative to the reference composition as the antilogarithm ((intercept(test sample)-intercept(reference sample) / common slope)).

[0058] In some embodiments, the reproducibility and precision of the methods for determining relative infectivity disclosed herein are greater than the reproducibility and precision of a 50% tissue culture infectious dose (TCID50) assay. In some embodiments, the coefficient of variation (cv) of relative infectivity measurements by the methods described herein is less than about 100%, less than about 50%, or less than about 25%. In some embodiments, the coefficient of variation (cv) of relative infectivity measurements by the methods described herein is less than about 25%.

[0059] In some embodiments, the present disclosure provides methods for determining the infectivity of a test composition relative to the infectivity of a reference composition, wherein the test composition and the reference composition have the same titer. In some embodiments, the present disclosure provides methods for determining the infectivity of a test composition relative to the infectivity of a reference composition, wherein the test composition and the reference composition have different titers. In some embodiments, the titer is measured as viral genome copies (GC) per milliliter. In some embodiments, the test composition and the reference composition comprise rAAV particles.

[0060] In some embodiments of the methods described herein, the titer of the test composition is about 1×10e+10 GC / ml to about 1×10e+13 GC / ml of virus particles. In some embodiments, the titer of the test composition is about 1×10e+10 GC / ml to about 1×10e+11 GC / ml of virus particles. In some embodiments, the titer of the test composition is about 5×10e+10 GC / ml to about 1×10e+12 GC / ml of virus particles. In some embodiments, the titer of the test composition is about 5×10e+10 GC / ml to about 1×10e+13 GC / ml of virus particles. In some embodiments, the titer of the test composition is about 1×10e+11 GC / ml to about 1×10e+13 GC / ml of virus particles. In some embodiments, the titer of the test composition is about 5×10e+10 GC / ml to about 5×10e+12 GC / ml of viral particles. In some embodiments, the titer of the test composition is about 1×10e+11 GC / ml to about 5×10e+12 GC / ml of viral particles. In some embodiments, the viral particles are rAAV particles. In some embodiments, the rAAV particles comprise capsid proteins of a serotype selected from the group consisting of AAV7, AAV8, AAV9, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHB.B, AAV.PHP.eB, and AAV.7m8. In some embodiments, the rAAV particles comprise capsid proteins with high sequence homology to AAV8 or AAV9, e.g., AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37. In some embodiments, the rAAV particles are of the AAV8 or AAV9 serotype.

[0061] In some embodiments of the methods described herein, the titer of the test composition is at least about 5×10e+10 GC / ml of virus particles. In some embodiments, the titer of the test composition is at least about 1×10e+11 GC / ml of virus particles. In some embodiments, the titer of the test composition is at least about 5×10e+11 GC / ml of virus particles. In some embodiments, the titer of the test composition is at least about 1×10e+12 GC / ml of virus particles. In some embodiments, the titer of the test composition is at least about 5×10e+12 GC / ml of virus particles. In some embodiments, the titer of the test composition is at least about 1×10e+13 GC / ml of virus particles. In some embodiments, the titer of the test composition is at least about 5×10e+13 GC / ml of virus particles. In some embodiments, the virus particles are rAAV particles. In some embodiments, the rAAV particles comprise capsid proteins of a serotype selected from the group consisting of AAV7, AAV8, AAV9, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHB.B, AAV.PHP.eB, and AAV.7m8. In some embodiments, the rAAV particles comprise capsid proteins with high sequence homology to AAV8 or AAV9, e.g., AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37. In some embodiments, the rAAV particles are of the AAV8 or AAV9 serotype.

[0062] In some embodiments of the methods described herein, the titer of the reference composition is about 1×10e+10 GC / ml to about 1×10e+13 GC / ml of virus particles. In some embodiments, the titer of the reference composition is about 1×10e+10 GC / ml to about 1×10e+11 GC / ml of virus particles. In some embodiments, the titer of the reference composition is about 5×10e+10 GC / ml to about 1×10e+12 GC / ml of virus particles. In some embodiments, the titer of the reference composition is about 5×10e+10 GC / ml to about 1×10e+13 GC / ml of virus particles. In some embodiments, the titer of the reference composition is about 1×10e+11 GC / ml to about 1×10e+13 GC / ml of virus particles. In some embodiments, the titer of the reference composition is about 5×10e+10 GC / ml to about 5×10e+12 GC / ml of viral particles. In some embodiments, the titer of the reference composition is about 1×10e+11 GC / ml to about 5×10e+12 GC / ml of viral particles. In some embodiments, the viral particles are rAAV particles. In some embodiments, the rAAV particles comprise capsid proteins of a serotype selected from the group consisting of AAV7, AAV8, AAV9, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHB.B, AAV.PHP.eB, and AAV.7m8. In some embodiments, the rAAV particles comprise capsid proteins with high sequence homology to AAV8 or AAV9, e.g., AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37. In some embodiments, the rAAV particles are of the AAV8 or AAV9 serotype.

[0063] In some embodiments of the methods described herein, the titer of the reference composition is at least about 5×10e+10 GC / ml of virus particles. In some embodiments, the titer of the reference composition is at least about 1×10e+11 GC / ml of virus particles. In some embodiments, the titer of the reference composition is at least about 5×10e+11 GC / ml of virus particles. In some embodiments, the titer of the reference composition is at least about 1×10e+12 GC / ml of virus particles. In some embodiments, the titer of the reference composition is at least about 5×10e+12 GC / ml of virus particles. In some embodiments, the titer of the reference composition is at least about 1×10e+13 GC / ml of virus particles. In some embodiments, the titer of the reference composition is at least about 5×10e+13 GC / ml of virus particles. In some embodiments, the virus particles are rAAV particles. In some embodiments, the rAAV particles comprise capsid proteins of a serotype selected from the group consisting of AAV7, AAV8, AAV9, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHB.B, AAV.PHP.eB, and AAV.7m8. In some embodiments, the rAAV particles comprise capsid proteins with high sequence homology to AAV8 or AAV9, e.g., AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37. In some embodiments, the rAAV particles are of the AAV8 or AAV9 serotype.

[0064] In some embodiments of the methods described herein, the titer of the test composition and the titer of the reference composition are from about 1×10e+10 GC / ml to about 1×10e+13 GC / ml of virus particles. In some embodiments, the titer of the test composition and the titer of the reference composition are from about 1×10e+10 GC / ml to about 1×10e+11 GC / ml of virus particles. In some embodiments, the titer of the test composition and the titer of the reference composition are from about 5×10e+10 GC / ml to about 1×10e+12 GC / ml of virus particles. In some embodiments, the titer of the test composition and the titer of the reference composition are from about 5×10e+10 GC / ml to about 1×10e+13 GC / ml of virus particles. In some embodiments, the titer of the test composition and the titer of the reference composition are from about 1×10e+11 GC / ml to about 1×10e+13 GC / ml of virus particles. In some embodiments, the titer of the test composition and the titer of the reference composition are between about 5x10e+10 GC / ml and about 5x10e+12 GC / ml of virus particles. In some embodiments, the titer of the test composition and the titer of the reference composition are between about 1x10e+11 GC / ml and about 5x10e+12 GC / ml of virus particles. In some embodiments, the virus particles are rAAV particles. In some embodiments, the rAAV particles comprise capsid proteins of a serotype selected from the group consisting of AAV7, AAV8, AAV9, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHB.B, AAV.PHP.eB, and AAV.7m8. In some embodiments, the rAAV particles comprise capsid proteins with high sequence homology to AAV8 or AAV9, e.g., AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37. In some embodiments, the rAAV particles are of the AAV8 or AAV9 serotype.

[0065] In some embodiments of the methods described herein, the titer of the test composition and the titer of the reference composition are at least about 5×10e+10 GC / ml of virus particles. In some embodiments, the titer of the test composition and the titer of the reference composition are at least about 1×10e+11 GC / ml of virus particles. In some embodiments, the titer of the test composition and the titer of the reference composition are at least about 5×10e+11 GC / ml of virus particles. In some embodiments, the titer of the test composition and the titer of the reference composition are at least about 1×10e+12 GC / ml of virus particles. In some embodiments, the titer of the test composition and the titer of the reference composition are at least about 5×10e+12 GC / ml of virus particles. In some embodiments, the titer of the test composition and the titer of the reference composition are at least about 1×10e+13 GC / ml of virus particles. In some embodiments, the titer of the test composition and the titer of the reference composition are at least about 5×10e+13 GC / ml of virus particles. In some embodiments, the viral particle is a rAAV particle. In some embodiments, the rAAV particle comprises a capsid protein of a serotype selected from the group consisting of AAV7, AAV8, AAV9, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHB.B, AAV.PHP.eB, and AAV.7m8. In some embodiments, the rAAV particle comprises a capsid protein with high sequence homology to AAV8 or AAV9, e.g., AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37. In some embodiments, the rAAV particle is of the AAV8 or AAV9 serotype.

[0066] The methods disclosed herein can be used to assess the infectivity of rAAV particles containing capsid proteins from any AAV capsid serotype. In some embodiments, the rAAV particles include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.P The capsid protein comprises an AAV capsid serotype selected from HP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16. In some embodiments, the rAAV particles are selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, including capsid proteins that are derivatives, modifications, or pseudotypes of the AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 capsid proteins.

[0067] In some embodiments, the rAAV particles comprise capsid proteins from an AAV capsid serotype selected from AAV8 and AAV9. In some embodiments, the rAAV particles have an AAV capsid serotype of AAV8. In some embodiments, the rAAV particles have an AAV capsid serotype of AAV9.

[0068] In some embodiments, the rAAV particles comprise capsid proteins from an AAV capsid serotype selected from the group consisting of AAV7, AAV8, AAV9, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHB.B, AAV.PHP.eB, and AAV.7m8. In some embodiments, the rAAV particles comprise capsid proteins with high sequence homology to AAV8 or AAV9, e.g., AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37.

[0069] In some embodiments, the rAAV particles comprise capsid proteins that are derivatives, modifications, or pseudotypes of the AAV8 or AAV9 capsid proteins. In some embodiments, the rAAV particles comprise capsid proteins that are at least 80% identical to the VP1, VP2, and / or VP3 sequences of the AAV8 capsid protein, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical to the AAV8 capsid protein.

[0070] In some embodiments, the rAAV particles comprise capsid proteins that are derivatives, modifications, or pseudotypes of the AAV9 capsid protein. In some embodiments, the rAAV particles comprise capsid proteins that are at least 80% identical to the VP1, VP2, and / or VP3 sequences of the AAV9 capsid protein, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical to the AAV9 capsid protein.

[0071] In some embodiments, the rAAV particles comprise capsid proteins that have at least 80% or more identity, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identity, to the VP1, VP2, and / or VP3 sequences of AAV7, AAV8, AAV9, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHP.B, AAV.PHP.eB, or AAV.7m8 capsid proteins. In some embodiments, the rAAV particles comprise AAV capsid proteins with high sequence homology to AAV8 or AAV9, e.g., capsid proteins with at least 80% identity, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identity, to the VP1, VP2, and / or VP3 sequences of AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37.

[0072] In additional embodiments, the rAAV particles comprise mosaic capsids. In additional embodiments, the rAAV particles comprise pseudotyped rAAV particles. In additional embodiments, the rAAV particles comprise capsids that include capsid protein chimeras of two or more AAV capsid serotypes.

[0073] rAAV particles The provided methods are suitable for use in producing any of the isolated recombinant AAV particles, in producing compositions comprising any of the isolated recombinant AAV particles, or in methods for treating a disease or disorder in a subject in need thereof, the method comprising administration of any of the isolated recombinant AAV particles. As such, rAAV can be any serotype, modification, or derivative known in the art, or any combination thereof (e.g., a population of rAAV particles comprising two or more serotypes, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65 ... AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, or other rAAV particles, or a combination of two or more thereof).

[0074] In some embodiments, the rAAV particles are selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV. The capsid protein is from an AAV serotype selected from AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, or a derivative, modification, or pseudotype thereof.In some embodiments, the rAAV particles may be any of a variety of vectors, including, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, rAAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16, AAV.HSC17, AAV.HSC18, AAV.HSC19 ... and / or AAV.HSC16, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, that is, capsid proteins that are at least 80% identical or greater, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical, to the VP1, VP2, and / or VP3 sequences of an AAV capsid serotype selected from AAV.HSC16, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16.

[0075] In some embodiments, the rAAV particles are selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV. The capsid protein comprises an AAV capsid serotype selected from AV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, or a derivative, modification, or pseudotype thereof.In some embodiments, the rAAV particles may be any of a variety of vectors, including, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16, AAV.HSC17, AAV.HSC18, AAV.HSC19 ... and / or AAV.HSC16, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, that is, capsid proteins that are at least 80% identical or greater, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical, to the VP1, VP2, and / or VP3 sequences of an AAV capsid serotype selected from AAV.HSC16, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16.

[0076] In some embodiments, the rAAV particles comprise capsids of Anc80 or Anc80L65, as described in Zinn et al., 2015, Cell Rep. 12(6):1056-1068 (incorporated by reference in their entireties). In certain embodiments, the rAAV particles comprise capsids with one of the amino acid insertions: LGETTRP or LALGETTRP, as described in U.S. Patent Nos. 9,193,956, 9,458,517, and 9,587,282, and U.S. Patent Application Publication No. 2016 / 0376323 (each of which is incorporated by reference in its entirety). In some embodiments, the rAAV particles comprise an AAV.7m8 capsid, as described in U.S. Patent Nos. 9,193,956, 9,458,517, and 9,587,282, and U.S. Patent Application Publication No. 2016 / 0376323 (each of which is incorporated by reference in its entirety). In some embodiments, the rAAV particles comprise any AAV capsid disclosed in U.S. Patent No. 9,585,971, such as AAVPHP.B. In some embodiments, the rAAV particles comprise any AAV capsid disclosed in U.S. Patent No. 9,840,719 and WO 2015 / 013313 (each of which is incorporated by reference in its entirety), such as AAV.Rh74 and RHM4-1. In some embodiments, the rAAV particles comprise any of the AAV capsids disclosed in WO2014 / 172669 (incorporated herein by reference in its entirety), e.g., AAV rh.74. In some embodiments, the rAAV particles comprise an AAV2 / 5 capsid, as described in Georgiadis et al., 2016, Gene Therapy 23:857-862 and Georgiadis et al., 2018, Gene Therapy 25:450 (each of which is incorporated herein by reference in its entirety). In some embodiments, the rAAV particles comprise any of the AAV capsids disclosed in WO2017 / 070491 (incorporated herein by reference in its entirety), e.g., AAV2tYF.In some embodiments, the rAAV particles comprise an AAVLK03 or AAV3B capsid as described in Puzzo et al., 2017, Sci. Transl. Med. 29(9):418 (incorporated by reference in its entirety). In some embodiments, the rAAV particles comprise any AAV capsid disclosed in U.S. Patent Nos. 8,628,966, 8,927,514, 9,923,120, and WO2016 / 049230, e.g., HSC1, HSC2, HSC3, HSC4, HSC5, HSC6, HSC7, HSC8, HSC9, HSC10, HSC11, HSC12, HSC13, HSC14, HSC15, or HSC16 (each of which is incorporated by reference in its entirety).

[0077] In some embodiments, the rAAV particles are manufactured using the methods disclosed in the following patents and patent applications (each of which is incorporated by reference in its entirety): U.S. Patent Nos. 7,282,199, 7,906,111, 8,524,446, 8,999,678, 8,628,966, 8,927,514, 8,734,809, 9,284,357, 9,409,953, 9,169,299, 9,193, 956, 9458517, and 9,587,282, U.S. Patent Application Publication Nos. 2015 / 0374803, 2015 / 0126588, 2017 / 0067908, 2013 / 0224836, 2016 / 0215024, and 2017 / 0051257, and International Patent Application Nos. PCT / US2015 / 034799 and PCT / EP2015 / 053335. In some embodiments, the rAAV particles have capsid proteins that are at least 80% identical or greater, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical, to the VP1, VP2, and / or VP3 sequences of the AAV capsid disclosed in any of the following patents and patent applications (each of which is incorporated herein by reference in its entirety): U.S. Patent Nos. 7,282,199, 7,906,111, 8,524,446, and 8,906,111. Nos. 99,678, 8,628,966, 8,927,514, 8,734,809, US 9,284,357, 9,409,953, 9,169,299, 9,193,956, 9,458,517, and 9,587,282, U.S. Patent Application Publication No. US2015 / 0374803, 2015 / 0126588, 2017 / 0067908, 2013 / 0224836, 2016 / 0215024, 2017 / 0051257, and International Patent Applications Nos. PCT / US2015 / 034799 and PCT / EP2015 / 053335.

[0078] In some embodiments, the rAAV particles are prepared using the methods described in International Patent Application Publication Nos. WO2003 / 052051 (see, e.g., SEQ ID NO:2), WO2005 / 033321 (see, e.g., SEQ ID NOs:123 and 88), WO03 / 042397 (see, e.g., SEQ ID NOs:2, 81, 85, and 97), WO2006 / 068888 (see, e.g., SEQ ID NOs:1 and 3-6), WO2006 / 110689 (see, e.g., SEQ ID NOs:5-38), WO2009 / 104964 (see, e.g., SEQ ID NOs:1-5, 7, 9, 20, 22, 24, and 31), WO2010 / 127097 (see, e.g., SEQ ID NOs:1-3, 3-4, 5-6, 7-8, 9-10, 10-12, 10-14, 10-16, 10-18, 11-19, 12-20, 13-21, 13-22, 14-24, and 15-26), WO2010 / 127097 (see, e.g., SEQ ID NOs:1-3, 3-4, 5-6, 7-8, 9-10, 11-12, 13 ... Nos. 5-38), and WO2015 / 191508 (see, e.g., SEQ ID NOs:80-294), and U.S. Patent Application Publication No. 20150023924 (see, e.g., SEQ ID NOs:1, 5-10), the contents of each of which are incorporated herein by reference in their entireties.In some embodiments, the rAAV particles have capsid proteins that are at least 80% identical or greater, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical, to the VP1, VP2, and / or VP3 sequences of the AAV capsids disclosed in International Patent Application Publication Nos. WO2003 / 052051 (see, e.g., SEQ ID NO:2), WO2005 / 033321 (see, e.g., SEQ ID NOs:123 and 88), WO03 / 042397 (see, e.g., SEQ ID NOs:2, 81, 85, and 97), WO2006 / 068888 (see, e.g., SEQ ID NOs:3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 59, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 69, 69, 70, Nos. 1 and 3-6), WO2006 / 110689 (see, e.g., SEQ ID NOs: 5-38), WO2009 / 104964 (see, e.g., SEQ ID NOs: 1-5, 7, 9, 20, 22, 24, and 31), WO2010 / 127097 (see, e.g., SEQ ID NOs: 5-38), and WO2015 / 191508 (see, e.g., SEQ ID NOs: 80-294), and U.S. Patent Application Publication No. 20150023924 (see, e.g., SEQ ID NOs: 1, 5-10).

[0079] Nucleic acid sequences of AAV-based viral vectors, as well as methods for producing recombinant AAV and AAV capsids, are taught, for example, in U.S. Pat. Nos. 7,282,199, 7,906,111, 8,524,446, 8,999,678, 8,628,966, 8,927,514, 8,734,809, US Pat. Nos. 9,284,357, 9,409,953, 9,169,299, 9,193,956, 9458517, and 9,587,282; U.S. Patent Application Publication Nos. 2015 / 0374803 and 2015 / 0126588. No. 2017 / 0067908, No. 2013 / 0224836, No. 2016 / 0215024, No. 2017 / 0051257, International Patent Application No. PCT / US2015 / 034799, No. PCT / EP2015 / 053335, No. WO2003 / 052051, No. WO2005 / 033321, WO03 / 042397, WO2006 / 068888, WO2006 / 110689, WO2009 / 104964, WO2010 / 127097, and WO2015 / 191508, and U.S. Patent Application Publication No. 20150023924.

[0080] The provided methods are suitable for use in generating a recombinant AAV encoding a transgene. In certain embodiments, the transgene is from Tables 1A-1C. In some embodiments, the rAAV genome comprises a vector comprising the following components: (1) AAV inverted terminal repeats flanking an expression cassette, (2) regulatory control elements, e.g., a) promoter / enhancer, b) polyA signal, and c) optionally, an intron, and (3) a nucleic acid sequence encoding the transgene. In other embodiments for expressing an intact or substantially intact monoclonal antibody (mAb), the rAAV genome comprises a vector comprising the following components: (1) AAV inverted terminal repeats flanking an expression cassette, (2) regulatory control elements, e.g., a) promoter / enhancer, b) polyA signal, and c) optionally, an intron, and (3) a nucleic acid sequence encoding the light chain Fab and heavy chain Fab of the antibody, or at least the heavy chain or light chain Fab, and optionally, the heavy chain Fc region. In yet other embodiments for expressing intact or substantially intact mAbs, the rAAV genome comprises a vector containing the following components: (1) AAV inverted terminal repeats flanking the expression cassette; (2) regulatory control elements, e.g., a) promoter / enhancer, b) polyA signal, and c) optionally introns;and (3) nucleic acid sequences encoding the heavy chain Fab of the following: anti-VEGF (e.g., sevacizumab, ranibizumab, bevacizumab, and brolucizumab), anti-EpoR (e.g., LKA-651), anti-ALK1 (e.g., ascribacumab), anti-C5 (e.g., tesidolumab and eculizumab), anti-CD105 (e.g., carotuximab), anti-CC1Q (e.g., ANX-007), anti-TNFα (e.g., adalimumab, infliximab, and golimumab). anti-IL-5 (e.g., mepolizumab), anti-IL12 / IL23 (e.g., ustekinumab), anti-CD19 (e.g., inebilizumab), anti-ITGF7 (e.g., pamrevlumab), anti-IL6R (e.g., satralizumab and sarilumab), anti-IL4R (e.g., dupilumab), anti-IL17A (e.g., ixekizumab and secukinumab), anti-IL-5 (e.g., mepolizumab), anti-IL12 / IL23 (e.g., ustekinumab), anti-CD19 (e.g., inebilizumab), anti-ITGF7 mAb (e.g., etrolizumab), anti-SOST mAb (e.g., romosozumab), anti-pKal mAb (e.g., lanadelumab), anti-ITGA4 (e.g., natalizumab), anti-ITGA4B7 (e.g., vedolizumab), anti-BLyS (e.g., belimumab), anti-PD-1 (e.g., nivolumab and pembrolizumab), anti-RANKL (e.g., densomab), anti-PCSK9 (e.g., alirocumab and evolocumab), anti-ANGPTL3 (e.g., evinacumab*), anti-OxPL (e.g., E06), anti-fD (e.g., lampalizumab), or anti-MMP9 (e.g., andecaliximab); optionally, an Fc polypeptide of the same isotype as the native form of the therapeutic antibody, e.g., an IgG isotype amino acid sequence IgG1, IgG2, or IgG4, or a modified Fc thereof;and nucleic acid sequences encoding the light chains of: anti-VEGF (e.g., sevacizumab, ranibizumab, bevacizumab, and brolucizumab), anti-EpoR (e.g., LKA-651), anti-ALK1 (e.g., ascribacumab), anti-C5 (e.g., tesidolumab and eculizumab), anti-CD105 or anti-ENG (e.g., carotuximab), anti-CC1Q (e.g., ANX-007), anti-TNFα (e.g., adalimumab, infliximab, and goli anti-IL-5 (e.g., mepolizumab), anti-IL12 / IL23 (e.g., ustekinumab), anti-CD19 (e.g., inebilizumab), anti-ITGF7 (e.g., pamrevlumab), anti-IL6R (e.g., satralizumab and sarilumab), anti-IL4R (e.g., dupilumab), anti-IL17A (e.g., ixekizumab and secukinumab), anti-IL-5 (e.g., mepolizumab), anti-IL12 / IL23 (e.g., ustekinumab), anti-CD19 (e.g., inebilizumab), anti-ITGF7 mAb (e.g., etrolizumab), anti-SOST mAb (e.g., romosozumab), anti-pKal mAb (e.g., lanadelumab), anti-ITGA4 (e.g., natalizumab), anti-ITGA4B7 (e.g., vedolizumab), anti-BLyS (e.g., belimumab), anti-PD-1 (e.g., nivolumab and pembrolizumab), anti-RANKL (e.g., densomab), anti-PCSK9 (e.g., alirocumab and evolocumab), anti-ANGPTL3 (e.g., evinacumab), anti-OxPL (e.g., E06), anti-fD (e.g., lampalizumab), or anti-MMP9 (e.g., andecaliximab). Here, the heavy chain (Fab and optionally the Fc region) and the light chain are separated by a self-cleaving furin (F) / F2A or a flexible linker, ensuring that the heavy and light chain polypeptides are expressed in equal amounts;

[0081] (Table 1A) TIFF0007822436000007.tif249165TIFF0007822436000008.tif245164TIFF0007822436000009.tif249164TIFF0007822436000010.tif145164

[0082] (Table 1B) TIFF0007822436000011.tif102159TIFF0007822436000012.tif249161TIFF0007822436000013.tif63158

[0083] (Table 1C) TIFF0007822436000014.tif180159TIFF0007822436000015.tif245159

[0084] In some embodiments, provided herein are rAAV viral vectors encoding anti-VEGF Fabs. In specific embodiments, provided herein are rAAV8-based viral vectors encoding anti-VEGF Fabs. In more specific embodiments, provided herein are rAAV8-based viral vectors encoding ranibizumab. In some embodiments, provided herein are rAAV viral vectors encoding iduronidase (IDUA). In specific embodiments, provided herein are rAAV9-based viral vectors encoding IDUA. In some embodiments, provided herein are rAAV viral vectors encoding iduronate 2-sulfatase (IDS). In specific embodiments, provided herein are rAAV9-based viral vectors encoding IDS. In some embodiments, provided herein are rAAV viral vectors encoding low-density lipoprotein receptor (LDLR). In specific embodiments, provided herein are rAAV8-based viral vectors encoding LDLR. In some embodiments, provided herein are rAAV viral vectors encoding tripeptidyl peptidase 1 (TPP1) protein. In specific embodiments, provided herein are rAAV9-based viral vectors encoding TPP1. In some embodiments, provided herein are rAAV viral vectors encoding a non-membrane-bound splice variant of VEGF receptor 1 (sFlt-1).In some embodiments, the proteins described herein include gamma-sarcoglycan, Rab escort protein 1 (REP1 / CHM), retinoid isomerohydrolase (RPE65), cyclic nucleotide-gated channel alpha 3 (CNGA3), cyclic nucleotide-gated channel beta 3 (CNGB3), aromatic L-amino acid decarboxylase (AADC), lysosome-associated membrane protein 2 isoform B (LAMP2B), factor VIII, factor IX, retinitis pigmentosa GTPase regulator (RPGR), retinoschisin (R S1), sarcoplasmic reticulum calcium ATPase (SERCA2a), aflibercept, battenin (CLN3), transmembrane ER protein (CLN6), glutamic acid decarboxylase (GAD), glial cell line-derived neurotrophic factor (GDNF), aquaporin 1 (AQP1), dystrophin, myotubularin 1 (MTM1), follistatin (FST), glucose-6-phosphatase (G6Pase), apolipoprotein A2 (APOA2), uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) , arylsulfatase B (ARSB), N-acetyl-alpha-glucosaminidase (NAGLU), alpha-glucosidase (GAA), alpha-galactosidase (GLA), beta-galactosidase (GLB1), lipoprotein lipase (LPL), alpha 1-antitrypsin (AAT), phosphodiesterase 6B (PDE6B), ornithine carbamoyltransferase 9 (OTC), survival motor neuron (SMN1), survival motor neuron (SMN2), neurturin (NRTN), neuroto Provided are rAAV viral vectors encoding lophin-3 (NT-3 / NTF3), porphobilinogen deaminase (PBGD), nerve growth factor (NGF), mitochondrial-encoded NADH:ubiquinone oxidoreductase core subunit 4 (MT-ND4), protective protein cathepsin A (PPCA), dysferlin, MER proto-oncogene tyrosine kinase (MERTK), cystic fibrosis transmembrane conductance regulator (CFTR), or tumor necrosis factor receptor (TNFR)-immunoglobulin (IgG1) Fc fusions.

[0085] In additional embodiments, the rAAV particles comprise pseudotyped AAV capsids. In some embodiments, the pseudotyped AAV capsids are rAAV2 / 8 or rAAV2 / 9 pseudotyped AAV capsids. Methods for producing and using pseudotyped rAAV particles are known in the art (see, e.g., Duan et al., J. Virol., 75:7662-7671 (2001); Halbert et al., J. Virol., 74:1524-1532 (2000); Zolotukhin et al., Methods 28:158-167 (2002); and Auricchio et al., Hum. Molec. Genet. 10:3075-3081, (2001)).

[0086] In additional embodiments, the rAAV particles comprise capsids that include capsid proteins that are chimeric of two or more AAV capsid serotypes. In some embodiments, the capsid proteins are selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP. and a chimera of two or more AAV capsid proteins from an AAV serotype selected from AAV.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16.

[0087] In certain embodiments, single-stranded AAV (ssAAV) can be used. In certain embodiments, self-complementary vectors, such as scAAV, can be used (see, e.g., Wu, 2007, Human Gene Therapy, 18(2):171-82; McCarty et al, 2001, Gene Therapy, Vol. 8, Number 16:1248-1254; and U.S. Patent Nos. 6,596,535, 7,125,717, and 7,456,683, each of which is incorporated herein by reference in its entirety).

[0088] In some embodiments, the rAAV particles comprise capsid proteins from an AAV capsid serotype selected from AAV8 or AAV9. In some embodiments, the rAAV particles comprise capsid proteins from an AAV capsid serotype selected from the group consisting of AAV7, AAV8, AAV9, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHB.B, AAV.PHP.eB, and AAV.7m8. In some embodiments, the rAAV particles comprise capsid proteins with high sequence homology to AAV8 or AAV9, e.g., AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37. In some embodiments, the rAAV particles have an AAV1, or a derivative, modified, or pseudotyped AAV capsid serotype thereof. In some embodiments, the rAAV particles have an AAV4, or a derivative, modified, or pseudotyped AAV capsid serotype thereof. In some embodiments, the rAAV particles have an AAV5, or a derivative, modified, or pseudotyped AAV capsid serotype thereof. In some embodiments, the rAAV particles have an AAV8, or a derivative, modified, or pseudotyped AAV capsid serotype thereof. In some embodiments, the rAAV particles have an AAV9, or a derivative, modified, or pseudotyped AAV capsid serotype thereof.

[0089] In some embodiments, the rAAV particles comprise capsid proteins that are derivatives, modifications, or pseudotypes of the AAV8 or AAV9 capsid proteins. In some embodiments, the rAAV particles comprise capsid proteins that are at least 80% identical to the VP1, VP2, and / or VP3 sequences of the AAV8 capsid protein, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical to the AAV8 capsid protein.

[0090] In some embodiments, the rAAV particles comprise capsid proteins that are derivatives, modifications, or pseudotypes of the AAV9 capsid protein. In some embodiments, the rAAV particles comprise capsid proteins that are at least 80% identical to the VP1, VP2, and / or VP3 sequences of the AAV9 capsid protein, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical to the AAV8 capsid protein.

[0091] In some embodiments, the rAAV particles comprise capsid proteins that have at least 80% or more identity, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identity, to the VP1, VP2, and / or VP3 sequences of AAV7, AAV8, AAV9, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHP.B, AAV.PHP.eB, or AAV.7m8 capsid proteins. In some embodiments, the rAAV particles comprise AAV capsid proteins with high sequence homology to AAV8 or AAV9, e.g., capsid proteins with at least 80% identity, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identity, to the VP1, VP2, and / or VP3 sequences of AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37.

[0092] In additional embodiments, the rAAV particles comprise a mosaic capsid. Mosaic AAV particles are composed of a mixture of viral capsid proteins from different AAV serotypes. In some embodiments, the rAAV particles are selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP and a mosaic capsid comprising capsid proteins of a serotype selected from AAV.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16.

[0093] In some embodiments, the rAAV particles comprise a mosaic capsid comprising capsid proteins of a serotype selected from AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.8, and AAVrh.10.

[0094] In additional embodiments, the rAAV particles comprise pseudotyped rAAV particles. In some embodiments, the pseudotyped rAAV particles comprise (a) a nucleic acid vector comprising AAV ITRs and (b) a capsid composed of capsid proteins derived from an AAVx (e.g., AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16). In additional embodiments, the rAAV particles are selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AA In additional embodiments, the rAAV particles comprise pseudotyped rAAV particles comprised of capsid proteins of an AAV serotype selected from AAV.V2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16. In additional embodiments, the rAAV particles comprise pseudotyped rAAV particles comprised of AAV8 capsid proteins. In additional embodiments, the rAAV particles comprise pseudotyped rAAV particles comprised of AAV9 capsid proteins. In some embodiments, the pseudotyped rAAV8 or rAAV9 particles are rAAV2 / 8 or rAAV2 / 9 pseudotyped particles.Methods for producing and using pseudotyped rAAV particles are known in the art (see, e.g., Duan et al., J. Virol., 75:7662-7671 (2001); Halbert et al., J. Virol., 74:1524-1532 (2000); Zolotukhin et al., Methods 28:158-167 (2002); and Auricchio et al., Hum. Molec. Genet. 10:3075-3081, (2001)).

[0095] In additional embodiments, the rAAV particles comprise capsids that include capsid proteins that are chimeric of two or more AAV capsid serotypes, hi further embodiments, the capsid proteins are selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP. and a chimera of two or more AAV capsid proteins from an AAV serotype selected from: eB, AAV2.5, AAV2tYF, AAV3B, rAAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16. In a further embodiment, the capsid protein is a chimera of two or more AAV capsid proteins from an AAV serotype selected from AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.8, and AAVrh.10.

[0096] In some embodiments, the rAAV particles comprise an AAV8 capsid protein and one of the following: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB. , AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16. In some embodiments, the rAAV particles comprise an AAV capsid protein that is a chimera of an AAV8 capsid protein and one or more AAV capsid proteins from an AAV serotype selected from AAV1, AAV2, AAV5, AAV6, AAV7, AAV9, AAV10, AAVrh.8, and AAVrh.10.

[0097] In some embodiments, the rAAV particles comprise an AAV9 capsid protein and one of the following: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.e and AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16.

[0098] In some embodiments, the rAAV particles comprise an AAV capsid protein that is a chimera of an AAV9 capsid protein and a capsid protein of one or more AAV capsid serotypes selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.8, and AAVrh.10.

[0099] Methods for isolating rAAV particles In some embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising isolated recombinant adeno-associated virus (rAAV) particles, the method comprising: isolating rAAV particles from an impure feed (e.g., an rAAV production culture), determining the genomic titer of the isolated rAAV particles, determining the infectivity of the isolated rAAV particles using a method disclosed herein, and formulating the isolated rAAV particles to produce a pharmaceutical composition. In some embodiments, the method for producing a pharmaceutical composition comprising isolated recombinant adeno-associated virus (rAAV) particles comprises: isolating rAAV particles from an impure feed (e.g., an rAAV production culture), determining the infectivity of the isolated rAAV particles using a method disclosed herein, and formulating the isolated rAAV particles to produce a pharmaceutical composition.

[0100] In some embodiments, the disclosure further provides a method for producing a pharmaceutical unit dose comprising isolated recombinant adeno-associated virus (rAAV) particles, the method comprising: isolating rAAV particles from an impure feed (e.g., an rAAV production culture), determining the genomic titer of the isolated rAAV particles, determining the infectivity of the isolated rAAV particles using a method disclosed herein, and formulating the isolated rAAV particles. In some embodiments, the method for producing a pharmaceutical unit dose comprising isolated recombinant adeno-associated virus (rAAV) particles comprises: isolating rAAV particles from an impure feed (e.g., an rAAV production culture), determining the infectivity of the isolated rAAV particles using a method disclosed herein, and formulating the isolated rAAV particles.

[0101] Isolated rAAV particles can be isolated using methods known in the art. In some embodiments, the method of isolating rAAV particles includes downstream processing, such as harvesting the cell culture, clarification of the harvested cell culture (e.g., by centrifugation or depth filtration), tangential flow filtration, affinity chromatography, anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, sterile filtration, or any combination(s) thereof. In some embodiments, the downstream processing includes at least two, at least three, at least four, at least five, or at least six of harvesting the cell culture, clarification of the harvested cell culture (e.g., by centrifugation or depth filtration), tangential flow filtration, affinity chromatography, anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, and sterile filtration. In some embodiments, downstream processing comprises harvesting the cell culture, clarification of the harvested cell culture (e.g., by depth filtration), sterile filtration, tangential flow filtration, affinity chromatography, and anion exchange chromatography. In some embodiments, downstream processing comprises clarification of the harvested cell culture, sterile filtration, tangential flow filtration, affinity chromatography, and anion exchange chromatography. In some embodiments, downstream processing comprises clarification of the harvested cell culture by depth filtration, sterile filtration, tangential flow filtration, affinity chromatography, and anion exchange chromatography. In some embodiments, clarification of the harvested cell culture comprises sterile filtration. In some embodiments, downstream processing does not comprise centrifugation. In some embodiments, the rAAV particles comprise capsid proteins of the AAV8 serotype. In some embodiments, the rAAV particles comprise capsid proteins of the AAV9 serotype.

[0102] In some embodiments, methods of isolating rAAV particles comprise harvesting a cell culture, clarification of the harvested cell culture (e.g., by depth filtration), a first sterile filtration, a first tangential flow filtration, affinity chromatography, anion exchange chromatography (e.g., monolith anion exchange chromatography or AEX chromatography with a quaternary amine ligand), a second tangential flow filtration, and a second sterile filtration. In some embodiments, methods of isolating rAAV particles disclosed herein comprise harvesting a cell culture, clarification of the harvested cell culture (e.g., by depth filtration), a first sterile filtration, affinity chromatography, anion exchange chromatography (e.g., monolith anion exchange chromatography or AEX chromatography with a quaternary amine ligand), tangential flow filtration, and a second sterile filtration. In some embodiments, methods for isolating rAAV particles produced according to the methods disclosed herein comprise clarification of the harvested cell culture, a first sterile filtration, a first tangential flow filtration, affinity chromatography, anion exchange chromatography (e.g., monolith anion exchange chromatography or AEX chromatography with a quaternary amine ligand), a second tangential flow filtration, and a second sterile filtration. In some embodiments, methods for isolating rAAV particles disclosed herein comprise clarification of the harvested cell culture, a first sterile filtration, affinity chromatography, anion exchange chromatography (e.g., monolith anion exchange chromatography or AEX chromatography with a quaternary amine ligand), tangential flow filtration, and a second sterile filtration. In some embodiments, methods for isolating rAAV particles produced according to the methods disclosed herein include clarification of the harvested cell culture by depth filtration, a first sterile filtration, a first tangential flow filtration, affinity chromatography, anion exchange chromatography (e.g., monolith anion exchange chromatography or AEX chromatography using a quaternary amine ligand), a second tangential flow filtration, and a second sterile filtration.In some embodiments, the methods for isolating rAAV particles disclosed herein comprise clarification of the harvested cell culture by depth filtration, a first sterile filtration, affinity chromatography, anion exchange chromatography (e.g., monolith anion exchange chromatography or AEX chromatography using a quaternary amine ligand), tangential flow filtration, and a second sterile filtration. In some embodiments, the method does not involve centrifugation. In some embodiments, clarification of the harvested cell culture comprises sterile filtration. In some embodiments, the rAAV particles comprise capsid proteins of the AAV8 serotype. In some embodiments, the rAAV particles comprise capsid proteins of the AAV9 serotype.

[0103] Numerous methods for producing rAAV particles are known in the art, including transfection, stable cell line generation, and infectious hybrid virus production systems (including adenovirus-AAV hybrids, herpesvirus-AAV hybrids, and baculovirus-AAV hybrids). Any rAAV production culture for generating rAAV viral particles requires (1) suitable host cells (including, in the case of baculovirus systems, for example, human-derived cell lines (e.g., HeLa, A549, or HEK293 cells and their derivatives (HEK293T cells, HEK293F cells)), mammalian cell lines (e.g., Vero), or insect-derived cell lines (e.g., SF-9)); (2) suitable helper virus functions provided by wild-type or mutant adenovirus (e.g., temperature-sensitive adenovirus), herpesvirus, baculovirus, or a plasmid construct providing helper functions; (3) AAV rep and cap genes and gene products; (4) a transgene (e.g., a therapeutic transgene) flanked by AAV ITR sequences; and (5) suitable media and media components to support rAAV production. Suitable media known in the art can be used for the production of rAAV vectors. Such media include, but are not limited to, Modified Eagle's Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), and media produced by Hyclone Laboratories and JRH, including Sf-900 II SFM medium as described in U.S. Pat. No. 6,723,551, which is incorporated herein by reference in its entirety.

[0104] rAAV production cultures can be routinely grown under a variety of conditions (e.g., over a wide temperature range, for various lengths of time) appropriate for the particular host cells being utilized. As is known in the art, rAAV production cultures include attachment-dependent cultures that can be cultured in suitable attachment-dependent vessels (e.g., roller bottles, hollow fiber filters, microcarriers, and packed-bed or fluidized-bed bioreactors). rAAV vector production cultures can also be grown in suspension-adapted host cells, such as HeLa cells, HEK293 cells, HEK293-derived cells (e.g., HEK293T cells, HEK293F cells), Vero cells, CHO cells, CHO-K1 cells, CHO-derived cells, EB66 cells, BSC cells, HepG2 cells, LLC-MK cells, CV-1 cells, COS cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK cells, TCMK-1 cells, LLC cells, and the like. Cells may also include PK cells, PK15 cells, LLC-RK cells, MDOK cells, BHK cells, BHK-21 cells, NS-1 cells, MRC-5 cells, WI-38 cells, BHK cells, 3T3 cells, 293 cells, RK cells, Per.C6 cells, chicken embryo cells, or SF-9 cells, which can be cultured in a variety of ways, including, for example, spinner flasks, stirred tank bioreactors, and disposable systems such as the Wave bag system. In some embodiments, the cells are HEK293 cells. In some embodiments, the cells are HEK293 cells adapted to growth in suspension culture. Numerous suspension cultures for producing rAAV particles are known in the art, including, for example, those disclosed in U.S. Pat. Nos. 6,995,006, 9,783,826, and U.S. Patent Application Publication No. 20120122155 (each of which is incorporated by reference in its entirety).

[0105] In some embodiments, the rAAV production culture comprises a high-density cell culture. In some embodiments, the culture has a total cell density of about 1 x 10E+06 cells / ml to about 30 x 10E+06 cells / ml. In some embodiments, greater than about 50% of the cells are viable cells. In some embodiments, the cells are HeLa cells, HEK293 cells, HEK293-derived cells (e.g., HEK293T cells, HEK293F cells), Vero cells, or SF-9 cells. In further embodiments, the cells are HEK293 cells. In further embodiments, the cells are HEK293 cells adapted to growth in suspension culture.

[0106] In additional embodiments of the provided methods, the rAAV production culture comprises a suspension culture comprising rAAV particles. Numerous suspension cultures for producing rAAV particles are known in the art, including, for example, those disclosed in U.S. Patent Nos. 6,995,006, 9,783,826, and U.S. Patent Application Publication No. 20120122155, each of which is incorporated by reference in its entirety. In some embodiments, the suspension culture comprises a culture of mammalian cells or insect cells. In some embodiments, the suspension culture comprises a culture of HeLa cells, HEK293 cells, HEK293-derived cells (e.g., HEK293T cells, HEK293F cells), Vero cells, CHO cells, CHO-K1 cells, CHO-derived cells, EB66 cells, BSC cells, HepG2 cells, LLC-MK cells, CV-1 cells, COS cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK cells, TCMK-1 cells, LLCPK cells, PK15 cells, LLC-RK cells, MDOK cells, BHK cells, BHK-21 cells, NS-1 cells, MRC-5 cells, WI-38 cells, BHK cells, 3T3 cells, 293 cells, RK cells, Per.C6 cells, chicken embryo cells, or SF-9 cells. In some embodiments, the suspension culture comprises a culture of HEK293 cells.

[0107] In some embodiments, a method for producing rAAV particles includes providing a cell culture containing cells capable of producing rAAV, adding a histone deacetylase (HDAC) inhibitor to the cell culture to a final concentration of between about 0.1 mM and about 20 mM, and maintaining the cell culture under conditions that allow for production of the rAAV particles. In some embodiments, the HDAC inhibitor comprises a short-chain fatty acid or a salt thereof. In some embodiments, the HDAC inhibitor comprises butyric acid (e.g., sodium butyrate), valproic acid (e.g., sodium valproate), propionic acid (e.g., sodium propionate), or a combination thereof.

[0108] In some embodiments, the rAAV particles are produced as disclosed in International Patent Application No. PCT / US19 / 45926, filed August 9, 2019, entitled "SCALABLE METHOD FOR RECOMBINANT AAV PRODUCTION," which is incorporated herein by reference in its entirety.

[0109] Recombinant AAV particles can be collected from rAAV production cultures by harvesting the production culture containing the host cells or by collecting spent medium from the production culture, provided that the cells are cultured under conditions known in the art to cause rAAV particle release from intact host cells into the medium. Recombinant AAV particles can also be collected from rAAV production cultures by lysing the host cells of the production culture. Suitable methods for lysing cells are also known in the art, and include, for example, multiple freeze / thaw cycles, sonication, microfluidization, and treatment with chemicals (e.g., detergents and / or proteases).

[0110] At harvest, rAAV production cultures can contain one or more of the following: (1) host cell proteins; (2) host cell DNA; (3) plasmid DNA; (4) helper virus; (5) helper virus proteins; (6) helper virus DNA; and (7) media components (including, for example, serum proteins, amino acids, transferrin, and other low molecular weight proteins). rAAV production cultures can further contain product-related impurities, such as inactive vector forms, empty viral capsids, aggregated viral particles or capsids, misfolded viral capsids, and disassembled viral particles.

[0111] In some embodiments, the rAAV production culture harvest is clarified to remove host cell debris. In some embodiments, the production culture harvest is clarified by filtration through a series of depth filters. Clarification can also be achieved by a variety of other standard techniques known in the art, such as centrifugation or filtration through any cellulose acetate filter known in the art with a pore size of 0.2 mm or greater. In some embodiments, clarification of the harvested cell culture comprises sterile filtration. In some embodiments, the production culture harvest is clarified by centrifugation. In some embodiments, clarification of the production culture harvest does not involve centrifugation.

[0112] In some embodiments, the harvested cell culture is clarified using filtration. In some embodiments, clarification of the harvested cell culture comprises depth filtration. In some embodiments, clarification of the harvested cell culture further comprises depth filtration and sterile filtration. In some embodiments, the harvested cell culture is clarified using a filter train comprising one or more different filtration media. In some embodiments, the filter train comprises one depth filtration medium. In some embodiments, the filter train comprises one or more depth filtration media. In some embodiments, the filter train comprises two depth filtration media. In some embodiments, the filter train comprises one sterile filtration medium. In some embodiments, the filter train comprises two depth filtration media and one sterile filtration medium. In some embodiments, the depth filter medium is a porous depth filter. In some embodiments, the filter train comprises Clarisolve® 20MS, Millistak+® COHC, and sterile-grade filtration media. In some embodiments, the filter train comprises Clarisolve® 20MS, Millistak+® COHC, and Sartopore® 2 XLG 0.2 μm. In some embodiments, the harvested cell culture is pretreated prior to contacting the depth filter. In some embodiments, the pretreatment comprises adding salt to the harvested cell culture. In some embodiments, the pretreatment comprises adding a chemical flocculant to the harvested cell culture. In some embodiments, the harvested cell culture is not pretreated prior to contacting the depth filter.

[0113] In some embodiments, the production culture harvest is clarified by filtration as disclosed in PCT International Patent Application No. PCT / US2019 / 029539, filed April 27, 2019, entitled "SCALABLE CLARIFICATION PROCESS FOR RECOMBINANT AAV PRODUCTION," which is incorporated herein by reference in its entirety.

[0114] In some embodiments, rAAV production culture harvests are treated with a nuclease (e.g., Benzonase®) or endonuclease (e.g., endonuclease from Serratia marcescens) to digest high molecular weight DNA present in the production culture. Nuclease or endonuclease digestion can be routinely performed under standard conditions known in the art. For example, nuclease digestion is performed at a final concentration of 1-2.5 units / ml of Benzonase® at temperatures ranging from ambient to 37°C for a period ranging from 30 minutes to several hours.

[0115] Sterile filtration encompasses filtration using a sterile-grade filter medium. In some embodiments, the sterile-grade filtration medium is a 0.2 or 0.22 μm pore filter. In some embodiments, the sterile-grade filtration medium comprises polyethersulfone (PES). In some embodiments, the sterile-grade filtration medium comprises polyvinylidene fluoride (PVDF). In some embodiments, the sterile-grade filtration medium has a hydrophilic heterogeneous bilayer design. In some embodiments, the sterile-grade filtration medium has a hydrophilic heterogeneous bilayer design of a 0.8 μm prefilter and a 0.2 μm final filter membrane. In some embodiments, the sterile-grade filtration medium has a hydrophilic heterogeneous bilayer design of a 1.2 μm prefilter and a 0.2 μm final filter membrane. In some embodiments, the sterile-grade filtration medium is a 0.2 or 0.22 μm pore filter. In further embodiments, the sterile-grade filtration medium is a 0.2 μm pore filter. In some embodiments, the sterile-grade filtration medium is Sartopore® 2 XLG 0.2 μm, Durapore™ PVDF membrane 0.45 μm, or Sartoguard® PES 1.2 μm + 0.2 μm nominal pore size combination. In some embodiments, the sterile-grade filtration medium is Sartopore® 2 XLG 0.2 μm.

[0116] In some embodiments, the clarified feed is concentrated via tangential flow filtration ("TFF") before being applied to a chromatography medium, e.g., an affinity chromatography medium. Large-scale concentration of viruses using TFF ultrafiltration is described in Paul et al., Human Gene Therapy 4:609-615 (1993). TFF concentration of the clarified feed allows for a technically manageable amount of clarified feed to be chromatographed and also allows for more rational sizing of the column without the need for long recirculation times. In some embodiments, the clarified feed is concentrated between at least 2-fold and at least 10-fold. In some embodiments, the clarified feed is concentrated between at least 10-fold and at least 20-fold. In some embodiments, the clarified feed is concentrated between at least 20-fold and at least 50-fold. In some embodiments, the clarified feed is concentrated approximately 20-fold. Those skilled in the art will also recognize that TFF may be used to remove small molecule impurities (e.g., cell culture impurities including media components, serum albumin, or other serum proteins) from a clarified feed via diafiltration. In some embodiments, the clarified feed is subjected to diafiltration to remove small molecule impurities. In some embodiments, the diafiltration involves using between about 3 and about 10 diafiltration volumes of buffer. In some embodiments, the diafiltration involves using about 5 diafiltration volumes of buffer. Those skilled in the art will also recognize that TFF may be used at any step of the purification process where buffer exchange is desired before performing the next step in the purification process. In some embodiments, the methods for isolating rAAV from a clarified feed disclosed herein involve the use of TFF to exchange the buffer.

[0117] Affinity chromatography can be used to isolate rAAV particles from a composition. In some embodiments, affinity chromatography is used to isolate rAAV particles from a clarified feed. In some embodiments, affinity chromatography is used to isolate rAAV particles from a clarified feed that has been subjected to tangential flow filtration. Suitable affinity chromatography media are known in the art and include, but are not limited to, AVB Sepharose™, POROS™ CaptureSelect™ AAVX affinity resin, POROS™ CaptureSelect™ AAV9 affinity resin, and POROS™ CaptureSelect™ AAV8 affinity resin. In some embodiments, the affinity chromatography media is POROS™ CaptureSelect™ AAV9 affinity resin. In some embodiments, the affinity chromatography media is POROS™ CaptureSelect™ AAV8 affinity resin. In some embodiments, the affinity chromatography medium is POROS™ CaptureSelect™ AAVX affinity resin.

[0118] Anion exchange chromatography can be used to isolate rAAV particles from a composition. In some embodiments, anion exchange chromatography is used after affinity chromatography as a final concentration and polishing step. Suitable anion exchange chromatography media are known in the art and include, but are not limited to, Unosphere Q (Biorad, Hercules, Calif.), and N-charged amino or imino resins, such as POROS 50 PI, or any DEAE, TMAE, tertiary or quaternary amine, or PEI-based resins known in the art (U.S. Pat. No. 6,989,264; Brument et al., Mol. Therapy 6(5):678-686 (2002); Gao et al., Hum. Gene Therapy 11:2079-2091 (2000)). In some embodiments, the anion exchange chromatography media comprises a quaternary amine. In some embodiments, the anion exchange media is a monolithic anion exchange chromatography resin. In some embodiments, the monolithic anion exchange chromatography medium comprises a glycidyl methacrylate-ethylene dimethacrylate polymer or a styrene-divinylbenzene polymer. In some embodiments, the monolithic anion exchange chromatography medium is selected from the group consisting of CIMmultus™ QA-1 Advanced Composite Column (Quaternary Amine), CIMmultus™ DEAE-1 Advanced Composite Column (Diethylamino), CIM® QA Disk (Quaternary Amine), CIM® DEAE, and CIM® EDA Disk (Ethylenediamino). In some embodiments, the monolithic anion exchange chromatography medium is CIMmultus™ QA-1 Advanced Composite Column (Quaternary Amine). In some embodiments, the monolithic anion exchange chromatography medium is CIM® QA Disk (Quaternary Amine). In some embodiments, the anion exchange chromatography medium is CIM QA (BIA Separations, Slovenia).In some embodiments, the anion exchange chromatography medium is BIA CIM® QA-80 (80 mL column volume). One skilled in the art will appreciate that a wash buffer of suitable ionic strength can be identified so that impurities (including, but not limited to, impurities that may be introduced by upstream purification steps) are removed while the rAAV remains bound to the resin.

[0119] In some embodiments, anion exchange chromatography is performed according to the methods disclosed in International Patent Application No. PCT / US2019 / 037013, filed June 13, 2019, entitled "Anion Exchange Chromatography for Recombinant AAV production," which is incorporated herein by reference in its entirety.

[0120] In some embodiments, the method for isolating rAAV particles includes determining the vector genome titer, capsid titer, and / or the ratio of full capsids to empty capsids in a composition comprising the isolated rAAV particles. In some embodiments, the vector genome titer is determined by quantitative PCR (qPCR), digital PCR (dPCR), or droplet digital PCR (ddPCR). In some embodiments, the capsid titer is determined by serotype-specific ELISA. In some embodiments, the ratio of full capsids to empty capsids is determined by analytical ultracentrifugation (AUC) or transmission electron microscopy (TEM).

[0121] In some embodiments, the vector genome titer, capsid titer, and / or the ratio of full capsids to empty capsids is determined spectrophotometrically, for example, by measuring the absorbance of the composition at 260 nm and by measuring the absorbance of the composition at 280 nm. In some embodiments, the rAAV particles are not denatured before measuring the absorbance of the composition. In some embodiments, the rAAV particles are denatured before measuring the absorbance of the composition. In some embodiments, the absorbance of the composition at 260 nm and 280 nm is determined using a spectrophotometer. In some embodiments, the absorbance of the composition at 260 nm and 280 nm is determined using HPLC. In some embodiments, the absorbance is the peak absorbance. Several methods for measuring the absorbance of a composition at 260 nm and 280 nm are known in the art. Methods for determining vector genome titer and capsid titer of compositions containing isolated recombinant rAAV particles are disclosed in International Patent Application No. PCT / US19 / 29540, filed April 27, 2019, entitled "Systems and methods of spectrophotometry for the determination of genome copies and full / empty ratios of adeno-associated virus particles," which is incorporated herein by reference in its entirety.

[0122] In additional embodiments, the present disclosure provides a composition comprising isolated rAAV particles produced according to the methods disclosed herein. In some embodiments, the composition is a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

[0123] As used herein, the term "pharmaceutically acceptable" refers to a biologically acceptable formulation, gas, liquid, or solid, or mixture thereof, suitable for one or more routes of administration, in vivo delivery, or contact. A "pharmaceutically acceptable" composition is a material that is not biologically or otherwise undesirable; e.g., the material can be administered to a subject without causing substantial undesirable biological effects. Thus, such pharmaceutical compositions can be used, for example, when administering rAAV isolated according to the methods of the present disclosure to a subject. Such compositions include solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (e.g., oil-in-water or water-in-oil), suspensions, syrups, elixirs, dispersion and suspension media, coatings, isotonicity and absorption enhancers or delayers, that are compatible with pharmaceutical administration or in vivo contact or delivery. Aqueous and non-aqueous solvents, solutions, and suspensions can include suspending agents and thickeners. Such pharmaceutically acceptable carriers include tablets (coated or uncoated), capsules (hard or soft), microbeads, powders, granules, and crystals. Supplementary active compounds (e.g., preservatives, antimicrobial agents, antiviral agents, and antifungal agents) can also be incorporated into the compositions. Pharmaceutical compositions can be formulated to be compatible with a particular route of administration or delivery, as described herein or as known to those skilled in the art. Thus, pharmaceutical compositions include carriers, diluents, or excipients suitable for administration by various routes.Pharmaceutical compositions and delivery systems suitable for the rAAV particles and methods and uses of the invention are known in the art (e.g., Remington: The Science and Practice of Pharmacy (2003) 20th ed., Mack Publishing Co., Easton, Pa.; Remington's Pharmaceutical Sciences (1990) 18th ed., Mack Publishing Co., Easton, Pa.; The Merck Index (1996) 12th ed., Merck Publishing Group, Whitehouse, NJ; Pharmaceutical Principles of Solid Dosage Forms (1993), Technonic Publishing Co., Inc., Lancaster, Pa.; Ansel and Stoklosa, Pharmaceutical Calculations (2001) 11th ed., Lippincott Williams & Wilkins, Baltimore, Md.; and Poznansky et al., Drug Delivery Systems (1980), R.L. Juliano, ed., Oxford, NY, pp. 253-315).

[0124] In some embodiments, the composition is a pharmaceutical unit dose. A "unit dose" refers to a physically discrete unit suitable as a unitary dosage for a subject to be treated. Each unit contains a predetermined amount, optionally with a pharmaceutical carrier (excipient, diluent, vehicle, or filler), calculated to produce a desired effect (e.g., a prophylactic or therapeutic effect) when administered in one or more doses. Unit dosage forms may be, for example, in ampoules and vials, which may contain liquid compositions or compositions in a freeze-dried or lyophilized state, to which, for example, a sterile liquid carrier may be added prior to in vivo administration or delivery. Individual unit dosage forms may be included in multi-dose kits or containers. Recombinant vector (e.g., AAV) sequences, plasmids, vector genomes, and recombinant viral particles, as well as pharmaceutical compositions thereof, may be packaged in single or multiple unit dosage forms for ease of administration and uniformity of dosage. In some embodiments, the composition comprises AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV.PHP. The present invention also includes rAAV particles comprising AAV capsid proteins from an AAV capsid serotype selected from AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16. In some embodiments, the AAV capsid serotype is AAV8. In some embodiments, the AAV capsid serotype is AAV9.

[0125] Polynucleotides In some embodiments, the present disclosure provides isolated polynucleotides. In some embodiments, the isolated polynucleotides described herein are useful for detecting or determining the genome copy number of a recombinant virus or viral vector containing a rabbit beta-globin polyA element. In some embodiments, the isolated polynucleotides described herein are useful for detecting or determining the genome copy number of a human target cell containing a human albumin gene.

[0126] In some embodiments, the isolated polynucleotides disclosed herein have the following nucleotide sequence: Contains about 15 to about 40 nucleotides, including TIFF0007822436000016.tif41141.

[0127] In some embodiments, the isolated polynucleotide disclosed herein has from about 15 to about 40 nucleotides and comprises the nucleotide sequence of one of SEQ ID NOs: 1-6. In some embodiments, the isolated polynucleotide consists of the nucleotide sequence of one of SEQ ID NOs: 1-6.

[0128] In some embodiments, the present disclosure provides a composition comprising (i) a polynucleotide described herein and (ii) a detectable label covalently attached to the polynucleotide. In some embodiments, the detectable label is a fluorescent label. In some embodiments, the detectable label comprises one or more of FAM, JOE, TAMRA, and ROX. In some embodiments, the isolated polynucleotide comprises the nucleotide sequence of one of SEQ ID NOs: 1-6.

[0129] In some embodiments, the disclosure provides a pair of a forward primer and a reverse primer, wherein the forward primer and the reverse primer comprise the polynucleotide sequences of SEQ ID NOs: 1 and 2, respectively.

[0130] In some embodiments, the disclosure provides a pair of forward and reverse primers, wherein the forward and reverse primers comprise the polynucleotide sequences of SEQ ID NOs: 4 and 5, respectively.

[0131] In some embodiments, the present disclosure provides a combination of a probe, a forward primer, and a reverse primer, wherein the forward primer, the reverse primer, and the probe comprise a polynucleotide consisting of the nucleotide sequence of SEQ ID NOs: 1, 2, and 3, respectively.

[0132] In some embodiments, the present disclosure provides a combination of a probe, a forward primer, and a reverse primer, wherein the forward primer, the reverse primer, and the probe comprise a polynucleotide consisting of the nucleotide sequence of SEQ ID NOs: 4, 5, and 6, respectively.

[0133] In some embodiments, the isolated oligonucleotide described herein is a probe, wherein the polynucleotide consists of a nucleotide sequence selected from SEQ ID NOs: 3 and 6.

[0134] In some embodiments, the probes described herein comprise a detectable label. The detectable label can be covalently attached to the polynucleotide. The detectable label can be a fluorescent label, such as FAM, JOE, TAMRA, and ROX. In some embodiments, the probes described herein comprise one or more covalently attached fluorescent labels selected from the group consisting of FAM, JOE, TAMRA, and ROX. In some embodiments, the probes described herein comprise a polynucleotide comprising a first fluorescent label covalently attached to the 5' end and a second fluorescent label covalently attached to the 3' end. In some embodiments, the probes described herein are dual-labeled probes comprising a fluorescent reporter and a quencher dye. In some embodiments, the quencher is capable of quenching fluorescence by the reporter via fluorescence resonance energy transfer (FRET). In some embodiments, the quencher is capable of quenching fluorescence by the reporter via static quenching. In some embodiments, the fluorescent reporter is selected from the group consisting of FAM, JOE, TAMRA, and ROX, and the quencher is TAMRA.

[0135] Provided herein are combinations of forward and reverse primers capable of amplifying a target sequence within a rabbit beta-globin polyA element or a human albumin gene. In some embodiments, the forward and reverse primers capable of amplifying a target sequence within a rabbit beta-globin polyA element consist of the nucleotide sequences of SEQ ID NOs: 1 and 2, respectively. In some embodiments, the forward and reverse primers capable of amplifying a target sequence within a human albumin gene consist of the nucleotide sequences of SEQ ID NOs: 4 and 5, respectively.

[0136] Provided herein are combinations of a probe, a forward primer, and a reverse primer capable of detecting a target sequence within a rabbit beta-globin polyA element or a human albumin gene in a qPCR or dPCR reaction. In some embodiments, the forward primer, the reverse primer, and the probe capable of detecting a target sequence within a rabbit beta-globin polyA element comprise polynucleotides consisting of the nucleotide sequences of SEQ ID NOs: 1, 2, and 3, respectively. In some embodiments, the forward primer, the reverse primer, and the probe capable of detecting a target sequence within a human albumin gene comprise polynucleotides consisting of the nucleotide sequences of SEQ ID NOs: 4, 5, and 6, respectively. In some embodiments, the probe further comprises a first fluorescent label covalently attached to the 5' end and a second fluorescent label covalently attached to the 3' end of the oligonucleotide. In some embodiments, the first fluorescent label is FAM and the second fluorescent label is TAMRA.

[0137] In some embodiments, the present disclosure provides a method for generating a polynucleotide of interest, the method comprising subjecting DNA from a biological sample to polymerase chain reaction using a pair of forward and reverse primers described herein.

[0138] In some embodiments, the present disclosure provides a method for generating a polynucleotide of interest, the method comprising subjecting DNA from a biological sample to polymerase chain reaction using a combination of a probe, a forward primer, and a reverse primer described herein.

[0139] kit In some embodiments, the present disclosure provides a kit for detecting rAAV in a sample, the kit comprising one or more polynucleotides selected from the group consisting of SEQ ID NOs: 1-3.

[0140] In some embodiments, the present disclosure provides a kit for detecting rAAV in a sample, the kit comprising a pair of forward and reverse primers described herein.

[0141] In some embodiments, the present disclosure provides a kit for detecting rAAV in a sample, the kit comprising a combination of a probe, a forward primer, and a reverse primer described herein.

[0142] In some embodiments, the present disclosure provides a kit for determining the infectivity of an rAAV test composition relative to the infectivity of a reference composition, the kit comprising a combination of a probe, a forward primer, and a reverse primer described herein. In some embodiments, the kit further comprises an rAAV reference composition.

[0143] In some embodiments, the present disclosure provides a kit for determining the relative infectivity of a composition of viral particles under different conditions, the kit comprising a combination of a probe, a forward primer, and a reverse primer described herein. In some embodiments, the kit further comprises an rAAV reference composition. [Example]

[0144] Example 1. Relative infectivity is a reliable method for quantifying differences in infectivity of AAV vectors in vitro. Methods for measuring in vitro infectivity are relied upon to support the product suitability, comparability, and stability of AAV gene therapy products. The TCID50 infectious titer assay is one of the most commonly used methods for measuring the in vitro infectivity of AAV viral vectors, but it suffers from significant assay variability. For example, infectivity measurements of rAAV2 and rAAV9 reference standards using the TCID50 infectious titer assay yielded geometric CVs of 191% (SD = 0.46 log10 IU / mL) and 209% (SD = 0.49 log10 IU / mL), respectively. High assay variability makes TCID50 an unreliable tool for measuring differences in infectivity between different vector preparations or changes in infectivity as a result of degradation.

[0145] A relative infectivity method has been developed that can detect and quantify small differences in the in vitro infectivity of AAV vectors. A schematic diagram of this method is shown in Figures 1 and 2. To provide accurate relative infectivity measurements, it is important to accurately quantify the vector genome concentration in the test sample and reference standard. It is also important to use well-characterized reference standards with known biological activity or infectivity.

[0146] Briefly, on day 1, HuH-7 cells were seeded at 40,000–50,000 cells / well in two 96-well Edge plates. On day 2, cells were serum-starved overnight. On day 3, serial dilutions of test samples and reference standards were prepared in duplicate in medium containing 0.001% Pluronic F-68. The diluted test samples and reference standards were transferred to the cells and incubated for 24 hours at 37°C and 5% CO2. On day 4, cells were washed with DPBS, wells were treated with Accutase™ (a cell detachment solution containing proteolytic and collagenolytic enzymes), cells were pelleted, and DNA was extracted from the cell pellets. Multiplex ddPCR reactions were set up to determine the viral genome copy number in the DNA samples and the target cell genome copy number. Viral genome copy number was determined using rabbit globin poly(A)-specific primers / probes (SEQ ID NOs: 1–3). Human albumin-specific primers / probes (SEQ ID NOs: 4-6) were used to determine target cell genome copy number. Relative infectivity was determined using a parallel line model, as shown in Figure 2.

[0147] To assess the linearity, precision, and accuracy of the assay, a starting composition containing 100% rAAV particles was used as a reference to measure the relative infectivity of test compositions containing 200%, 150%, 125%, 100%, 75%, or 50% rAAV particles. The results shown below were collected over nine separate runs (N=20). TIFF0007822436000017.tif73128

[0148] Comparison of relative infectivity after forced digestion: The relative infectivity of rAAV samples incubated at 60°C for 10 minutes was measured. The relative infectivity of untreated samples (stored at -80°C) was also measured as a control. The relative infectivity observed in samples incubated at 60°C was 375% (standard error 42%). The relative infectivity of the untreated control was 99% (standard error 6%). Therefore, the relative infectivity method described herein can detect changes in infectivity upon forced digestion. It is believed that the increase in relative infectivity is due to increased uptake of GC-containing particles due to aggregation.

[0149] Comparison of relative infectivity between different rAAV vectors. The relative infectivity of AAV9 and AAV8 vectors containing different payloads was measured. The results are shown below. Different ddPCR methods were used for the different recombinant vectors. TIFF0007822436000018.tif44128

[0150] Comparison of relative infectivity among multiple batches. The relative infectivity of multiple batches of the same recombinant AAV8 vector was measured. The results are shown below. TIFF0007822436000019.tif52128

[0151] The in vitro relative infectivity method described herein is capable of detecting small differences in AAV vector infectivity. The relative infectivity method is linear, accurate, and precise from 50-200% relative infectivity. The relative infectivity method is linear, accurate, and precise from 50-200% relative infectivity. The relative infectivity method also provides a useful tool for comparing infectivity among different preparations, products, and AAV capsid serotypes.

[0152] Example 2. Comparison of the ability of AAV vectors to infect different cells in vitro. The methods disclosed herein for measuring in vitro relative infectivity are also useful for screening and identifying cells that are permissive for viral infection, as well as for identifying factors that modulate the susceptibility of cells to viral infection. In one embodiment, the in vitro relative infectivity of a single AAV vector or reference standard with known biological activity or infectivity is measured in parallel on different cell substrates using the methods disclosed herein to screen for or identify cells and conditions that are permissive for viral infection.

[0153] Comparison of different variants of the human adherent HEK293 cell line. On day 1, 30,000 cells / well of different variants of the human adherent HEK293 cell line were seeded into 96-well Edge plates. On day 2, cells were serum-starved overnight. On day 3, serial dilutions of a single AAV8 vector reference standard were prepared in duplicate in medium containing 0.001% Pluronic F-68. The diluted reference standard was transferred to the cells and incubated for 24 hours at 37°C and 5% CO2. On day 4, cells were washed with DPBS, wells were treated with Accutase™ (a cell detachment solution containing proteolytic and collagenolytic enzymes), cells were pelleted, and DNA was extracted from the cell pellets. Multiplex ddPCR reactions were set up to determine the viral genome copy number in the DNA samples and the target cell genome copy number. Viral genome copy number was determined using rabbit globin poly(A)-specific primers / probes (SEQ ID NOs: 1-3). Human albumin-specific primers / probes (SEQ ID NOs: 4-6) were used to determine target cell genome copy numbers. Relative infectivity was determined using a parallel line model, as shown in Figure 2. The results obtained are shown below. TIFF0007822436000020.tif23153

[0154] The results demonstrate that the relative infectivity method provides a useful tool for identifying cellular modifications and factors that regulate cellular susceptibility to viral infection.

[0155] While the methods of this disclosure have been described in connection with what are presently considered to be the most practical and preferred embodiments, it is to be understood that the methods encompassed by this disclosure are not to be limited to the disclosed embodiments, but on the contrary, are intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

[0156] All publications, patents, patent applications, internet sites, and accession number / database sequences (including both polynucleotide and polypeptide sequences) cited herein are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, internet site, or accession number / database sequence was specifically and individually indicated to be incorporated by reference.

[0157] Sequence information SEQUENCE LISTING <110> REGENXBIO INC. <120> METHOD FOR MEASURING THE INFECTIVITY OF REPLICATION DEFECTIVE Viral vectors and viruses <150> US 62 / 745,859 <151> 2018-10-15 <160> 6 <170> PatentIn version 3.5 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer sequence <400> 1 ggacatcatg aagcccctt 19 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer sequence <400> 2 tccaacacac tattgcaatg aaaa 24 <210> 3 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer sequence <400> 3 agcatctgac ttctggctaa taaaggaa 28 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer sequence <400> 4 tgaaacatac gttcccaaag agttt 25 <210> 5 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer sequence <400> 5 ctctccttct cagaaagtgt gcatat 26 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer sequence <400> 6 tgctgaaaca ttcaccttcc atgca 25

Claims

1. 1. A method for producing a pharmaceutical unit dose comprising isolated recombinant adeno-associated virus (rAAV) particles, comprising: a) isolating rAAV particles from an impure feed to produce a first composition; b) determining the rAAV genome titer of said first composition; c) determining the relative infectivity of said first composition relative to the infectivity of a reference rAAV composition; d) generating a pharmaceutical unit dose using the rAAV genome titer determined in step b) and the relative infectivity determined in step c); wherein the step of determining the relative infectivity comprises: i) preparing serial dilutions of said first composition and said reference composition; ii) separately inoculating target cells with each dilution of the first composition and the reference composition, wherein the rAAV particles are unable to replicate in the target cells; iii) washing the inoculated cells to remove extracellular viral particles; iv) isolating a first nucleic acid sample and a reference nucleic acid sample from the target cells inoculated with each dilution of the first composition and the reference composition, respectively; v) determining the ratio of viral genome copies (VGC): target cell genome copies (TCGC) in each of the first nucleic acid sample and the reference nucleic acid sample; vi) plotting the log VGC:TCGC ratio against the log dilution data points for the first composition and the reference composition; vii) fitting the data points of the first composition and the reference composition to a line of the first composition and the reference composition using a common slope; viii) determining the relative infectivity of the first composition relative to the reference composition; and calculating The method comprising:

2. The step of inoculating target cells comprises inoculating the target cells in the presence of viral particles. a. Between 5 minutes and 3 days b. Between 12 and 36 hours c. Between 18 and 30 hours d. 1 hour, 2 hours, 6 hours, 12 hours, 18 hours, 24 hours, 30 hours, or 36 hours; e. 1 day, or 1.5 days, or 2 days, or f. 24 hours 2. The method of claim 1, comprising incubating for 10 minutes.

3. 3. The method of claim 1 or 2, wherein the VGC:TCGC ratio in the nucleic acid composition is determined by polymerase chain reaction.

4. 4. The method of any one of claims 1 to 3, wherein the first composition and the reference composition have the same titer, the titer being measured in genome copies (GC) per milliliter.

5. 4. The method of claim 3, wherein the polymerase chain reaction comprises using (a) primers or probes capable of detecting and amplifying viral sequences, and (b) primers or probes capable of detecting and amplifying target cell genomic sequences.

6. The polymerase chain reaction (a) a pair of forward and reverse primers comprising the polynucleotide sequences of SEQ ID NOs: 1 and 2, respectively; (b) a pair of forward and reverse primers comprising the polynucleotide sequences of SEQ ID NOs: 4 and 5, respectively; (c) a combination of a probe, a forward primer, and a reverse primer, wherein the forward primer, the reverse primer, and the probe comprise polynucleotides consisting of the nucleotide sequences of SEQ ID NOs: 1, 2, and 3, respectively; or (d) a combination of a probe, a forward primer, and a reverse primer, wherein the forward primer, the reverse primer, and the probe comprise polynucleotides consisting of the nucleotide sequences of SEQ ID NOs: 4, 5, and 6, respectively; The method of claim 3, comprising using

7. The rAAV particles are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV. rh8, AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, AAV. hu37, AAV. Anc80, AAV. Anc80L65, AAV. 7m8, AAV. PHP. B. AAV. PHP.

7. The method of any one of claims 1 to 6, wherein the capsid protein comprises a capsid protein of an AAV.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 serotype.

8. The method of any one of claims 1 to 6, wherein the rAAV particles comprise capsid proteins of AAV8 or AAV9 serotypes.

9. 9. The method of any one of claims 1 to 8, wherein the target cells under at least one set of conditions comprise BHK21, HEK293, BEAS-2BS, HeLaS3, Huh-7, Hepa1-6, or A549 cells.

10. The method of any one of claims 1 to 8, wherein the target cells under at least one set of conditions comprise Huh-7 cells.

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