In vitro assay for detecting enhancers and inhibitors of adeno associated virus (AAV) vector transduction and / or detecting or quantitating Anti-AAV binding antibodies

Abstract

To provide methods for analyzing for or detecting the presence of non-antibody inhibitors and / or enhancers of adeno-associated virus (AAV) vector cell transduction in a biological sample from a subject, and also provide methods for analyzing for, or detecting the presence of, AAV binding antibodies that inhibit, reduce or decrease AAV vector cell transduction in a biological sample from a subject.SOLUTION: A method comprises steps of: providing infectious recombinant AAV particles comprising a recombinant AAV vector, wherein the vector comprises a reporter transgene; providing cells that can be infected with the infectious recombinant AAV particles; contacting the cells with the infectious recombinant AAV particles; and measuring the expression level of the reporter transgene.SELECTED DRAWING: None

Description

[Background technology]

[0001]

[0002] Adeno-associated virus (AAV) vector gene transfer has demonstrated clinical efficacy in the treatment of Leber's congenital amaurosis and in human clinical trials for the bleeding disorders hemophilia A and B. Exposure to wild-type AAV results in a variable percentage of humans with antibodies that bind to the capsid, which can inhibit or prevent AAV vector cell transduction. Such antibodies binding to AAV are a major obstacle for AAV-based gene therapy vectors, preventing some patients from accessing potentially life-saving treatments. As a result, subjects who test positive for neutralizing antibodies (NAbs) to AAV are often excluded from enrollment in gene therapy trials and are suboptimal candidates for gene therapy treatment.

[0002]

[0003] Anti-AAV antibodies can be measured using binding assays that detect IgG binding to the virus, or cell transduction inhibition assays that measure the efficiency of cell transduction of reporter vectors in vitro. Antibody binding assays are easy to initiate, but do not identify which bound antibodies affect AAV vector transduction. Conversely, cell-based NAb assays do measure the degree of inhibition of vector transduction mediated by anti-AAV circulating factors, but are time-consuming and pose challenges in terms of both sensitivity and accuracy. Furthermore, the lack of standardization of procedures for NAb assays poses a barrier to the interpretation of results from gene therapy trials. Summary of the Invention

[0003]

[0004] As disclosed herein, there are other factors distinct from AAV binding antibodies that can inhibit, reduce or decrease AAV vector cell transduction. As also disclosed herein, there are factors that can enhance AAV vector cell transduction. These enhancing and inhibiting factors may be present in certain subjects who may be treated with AAV-based gene therapy or participate in AAV-based gene therapy clinical trials. Typically, subjects are evaluated for the presence of AAV binding antibodies to determine suitability / eligibility for gene therapy treatment. Subjects may also be evaluated for the presence of AAV binding antibodies after undergoing gene therapy treatment or subsequent re-administration of gene therapy treatment to monitor antibody development. However, if the subject in which it is desired to measure AAV binding antibodies has an enhancer or inhibitor of AAV vector cell transduction, typical cell-based antibody assays will give inaccurate quantitative results in terms of AAV binding antibody titers.

[0004]

[0005] For example, if a factor that enhances AAV vector cell transduction is present in a subject with AAV-binding antibodies, the amount of AAV-binding antibodies will be underestimated and, if low enough, may result in a false negative for AAV-binding antibodies in the subject, where such a false negative subject is actually positive for AAV-binding antibodies.

[0005]

[0006] If a factor that inhibits AAV vector cell transduction is present in a subject, even in the absence of detectable AAV binding antibodies, if an inhibitor is present that inhibits or reduces AAV vector cell transduction, the test result will be falsely positive for AAV binding antibodies in the subject. Such false positive subjects are actually negative for AAV binding antibodies or have relatively low AAV antibody titers.

[0006]

[0007] The invention therefore provides, inter alia, methods for analyzing a sample, such as a biological sample from a subject, for and methods for detecting the presence of such enhancer or inhibitory factors.

[0008] In certain embodiments, a method for analyzing for or detecting the presence of an enhancer of adeno-associated viral (AAV) vector cell transduction in a biological sample from a subject includes: (a) providing a. infectious recombinant AAV particles comprising a recombinant AAV vector, the recombinant AAV vector comprising a reporter transgene operably linked to one or more expression control elements, b. empty capsid AAV particles, c. cells permissive for AAV infection, and d. a biological sample from a subject; (b) performing a first assay by: a. contacting a cell with an infectious recombinant AAV particle under conditions such that the cell is transduced with the infectious recombinant AAV particle and a reporter transgene is expressed by the cell; and b. measuring expression of the reporter transgene and assigning a value labeled MAX; (c) performing a second assay by: a. mixing the biological sample with empty capsid AAV particles to form a mixture (M) and incubating M under conditions that allow the empty capsid AAV particles to bind to any AAV-binding antibodies present in the biological sample; b. contacting a cell with M and the infectious recombinant AAV particles under conditions in which the cell is transduced with the infectious recombinant AAV particles and a reporter transgene is expressed by the cell; and c. measuring expression of the reporter transgene and assigning a value denoted S.EV. (d) comparing S.EV to MAX, where if S.EV is greater than MAX, the biological sample from the subject contains an enhancer of AAV vector cell transduction. Includes.

[0007]

[0009] In certain embodiments, a method for analyzing for or detecting the presence of an inhibitor of adeno-associated viral (AAV) vector cell transduction in a biological sample from a subject comprises: (a) providing a. infectious recombinant AAV particles comprising a recombinant AAV vector, the recombinant AAV vector comprising a reporter transgene operably linked to one or more expression control elements, b. empty capsid AAV particles, c. cells permissive for AAV infection, and d. a biological sample from a subject; (b) performing a first assay by: a. contacting a cell with an infectious recombinant AAV particle under conditions such that the cell is transduced with the infectious recombinant AAV particle and a reporter transgene is expressed by the cell; and b. measuring expression of the reporter transgene and assigning a value labeled MAX; (c) performing a second assay by: a. mixing the biological sample with empty capsid AAV particles to form a mixture (M) and incubating M under conditions that allow the empty capsid AAV particles to bind to any AAV-binding antibodies present in the biological sample; b. contacting a cell with M and the infectious recombinant AAV particles under conditions in which the cell is transduced with the infectious recombinant AAV particles and a reporter transgene is expressed by the cell; and c. measuring expression of the reporter transgene and assigning a value denoted S.EV. (d) comparing S.EV to MAX, and if S.EV is less than MAX, the biological sample from the subject contains an inhibitor of AAV vector cell transduction. Includes.

[0008]

[0010] In certain embodiments, a method for analyzing for or detecting the presence of an enhancer of adeno-associated viral (AAV) vector cell transduction in a biological sample from a subject includes: (a) providing infectious recombinant AAV particles comprising a recombinant AAV vector, wherein (i) the vector comprises a reporter transgene, (ii) the reporter transgene comprises a single-stranded or self-complementary genome, (iii) the reporter transgene is operably linked to one or more expression control elements, and (iv) the reporter transgene is adjacent to one or more flanking elements; (b) providing a cell capable of being infected with an infectious recombinant AAV particle; (c) contacting the cells of (b) with the infectious recombinant AAV particles of (a) under conditions such that the cells of (b) are transduced by the infectious recombinant AAV particles of (a) and a reporter transgene is expressed by the cells of (b); (d) measuring expression of the reporter transgene and assigning a value labeled MAX that reflects the amount of reporter transgene expression in (c); (e) providing an infectious recombinant AAV particle (a); (f) providing a biological sample from a subject; (g) providing a cell capable of being infected with an infectious recombinant AAV particle; (h) combining the biological sample of (f) with empty capsid AAV particles to produce a mixture (M) and incubating M under conditions that allow the empty capsid AAV particles to bind to any AAV-binding antibodies present in the biological sample; (i) contacting the cells of (g) with M and the infectious recombinant AAV particles of (e) under conditions such that the infectious recombinant AAV particles of (e) are capable of transducing the cells of (g) and expressing a reporter transgene in the cells of (g); (j) measuring expression of the reporter transgene and assigning a value, denoted S.EV, that reflects the amount of reporter transgene expression in (i); (k) comparing S.EV to MAX, where if S.EV is greater than MAX, the biological sample from the subject contains an enhancer of AAV vector cell transduction. Includes.

[0009]

[0011] In certain embodiments, a method for assaying for or detecting the presence of an inhibitor of adeno-associated virus (AAV) vector cell transduction in a biological sample from a subject includes the steps of: (a) providing infectious recombinant AAV particles comprising a recombinant AAV vector, wherein (i) the vector comprises a reporter transgene, (ii) the reporter transgene comprises a single-stranded or self-complementary genome, (iii) the reporter transgene is operably linked to one or more expression control elements, and (iv) the reporter transgene is flanked by one or more flanking elements; (b) providing a cell capable of being infected with an infectious recombinant AAV particle; (c) contacting the cells of (b) with the infectious recombinant AAV particles of (a) under conditions such that the cells of (b) are transduced by the infectious recombinant AAV particles of (a) and a reporter transgene is expressed by the cells of (b); (d) measuring expression of the reporter transgene and assigning a value labeled MAX that reflects the amount of reporter transgene expression in (c); (e) providing an infectious recombinant AAV particle (a); (f) providing a biological sample from a subject; (g) providing a cell capable of being infected with an infectious recombinant AAV particle; (h) combining the biological sample of (f) with empty capsid AAV particles to produce a mixture (M) and incubating M under conditions that allow the empty capsid AAV particles to bind to any AAV-binding antibodies present in the biological sample; (i) contacting the cells of (g) with M and the infectious recombinant AAV particles of (e) under conditions that allow the infectious recombinant AAV particles of (e) to transduce the cells of (g) and express a reporter transgene in the cells of (g); (j) measuring expression of the reporter transgene and assigning a value, denoted S.EV, that reflects the amount of reporter transgene expression in (i); (k) comparing S.EV to MAX, where if S.EV is less than MAX, the biological sample from the subject contains an inhibitor of AAV vector cell transduction, expression or secretion of a protein encoded by the vector. Includes.

[0010]

[0012] In certain embodiments, a method for analyzing for or detecting the presence of an enhancer of adeno-associated viral (AAV) vector cell transduction in a biological sample from a subject includes: (a) providing infectious recombinant AAV particles comprising a recombinant AAV vector, wherein (i) the vector comprises a reporter transgene, (ii) the reporter transgene comprises a single-stranded or self-complementary genome, (iii) the reporter transgene is operably linked to one or more expression control elements, and (iv) the reporter transgene is adjacent to one or more flanking elements; (b) providing a cell capable of being infected with an infectious recombinant AAV particle; (c) providing a biological sample from a subject; (d) combining the biological sample of (c) with empty capsid AAV particles to produce a mixture (M) and incubating M under conditions that allow the empty capsid AAV particles to bind to any AAV-binding antibodies present in the biological sample; (e) contacting the cells of (b) with M and the infectious recombinant AAV particles of (a) under conditions that allow the infectious recombinant AAV particles of (a) to transduce the cells of (b) and express a reporter transgene in the cells of (b); (f) measuring expression of the reporter transgene; (g) comparing the expression of (f) to a positive (+) control, the + control being expression of the reporter transgene without the sample from the subject and without the addition of empty capsid AAV particles, where if the expression of (f) is greater than the + control, then the biological sample from the subject contains an AAV vector cell transduction enhancer. Includes.

[0011]

[0013] In certain embodiments, a method for analyzing for or detecting the presence of an inhibitor of adeno-associated viral (AAV) vector cell transduction in a biological sample from a subject comprises: (a) providing infectious recombinant AAV particles comprising a recombinant AAV vector, wherein (i) the vector comprises a reporter transgene, (ii) the reporter transgene comprises a single-stranded or self-complementary genome, (iii) the reporter transgene is operably linked to one or more expression control elements, and (iv) the reporter transgene is adjacent to one or more flanking elements; (b) providing a cell capable of being infected with an infectious recombinant AAV particle; (c) providing a biological sample from a subject; (d) combining the biological sample of (c) with empty capsid AAV particles to produce a mixture (M) and incubating M under conditions that allow the empty capsid AAV particles to bind to any AAV-binding antibodies present in the biological sample; (e) contacting the cells of (b) with M and the infectious recombinant AAV particles of (a) under conditions that allow the infectious recombinant AAV particles of (a) to transduce the cells of (b) and express a reporter transgene in the cells of (b); (f) measuring expression of the reporter transgene; (g) comparing the expression of (f) to a positive (+) control, the + control being expression of the reporter transgene without the sample from the subject and without the addition of empty capsid AAV particles, and if the expression of (f) is less than the + control, then the biological sample from the subject contains an inhibitor of AAV vector cell transduction. Includes.

[0012]

[0014] The present invention also provides, inter alia, methods for assaying for, detecting or quantifying AAV-binding antibodies that inhibit, reduce or decrease AAV vector cell transduction in a sample, such as a biological sample, from a subject.

[0013]

[0015] In certain embodiments, a method for detecting or quantifying AAV binding antibodies that inhibit AAV vector cell transduction in a biological sample from a subject comprises: (a) providing infectious recombinant AAV particles comprising a recombinant AAV vector, wherein (i) the vector comprises a reporter transgene, (ii) the reporter transgene comprises a single-stranded or self-complementary genome, (iii) the reporter transgene is operably linked to one or more expression control elements, and (iv) the reporter transgene is adjacent to one or more flanking elements; (b) providing a cell capable of being infected with an infectious recombinant AAV particle; (c) contacting the cells of (b) with the infectious recombinant AAV particles of (a) under conditions such that the cells of (b) are transduced by the infectious recombinant AAV particles of (a) and a reporter transgene is expressed by the cells of (b); (d) measuring expression of the reporter transgene and assigning a value labeled MAX that reflects the amount of reporter transgene expression in (c); (e) providing an infectious recombinant AAV particle (a); (f) providing a diluted biological sample from a subject; (g) providing a cell capable of being infected with an infectious recombinant AAV particle; (h) mixing the diluted biological sample of (f) with empty capsid AAV particles to produce a mixture (M) and incubating M under conditions that allow the empty capsid AAV particles to bind to any AAV-binding antibodies present in the biological sample; (i) contacting the cells of (g) with M and the infectious recombinant AAV particles of (e) under conditions that allow the infectious recombinant AAV particles of (e) to transduce the cells of (g) and express a reporter transgene in the cells of (g); (j) measuring expression of the reporter transgene and assigning a value, denoted S.EV, that reflects the amount of reporter transgene expression in (i); (k) providing an infectious recombinant AAV particle (a); (l) providing a diluted biological sample from the same subject that provided the sample of (f); (m) providing a cell capable of being infected with an infectious recombinant AAV particle; (n) mixing the diluted biological sample of (l) with the infectious recombinant AAV particles of (k) to produce a mixture (M); (o) contacting the cells of (m) with M under conditions that allow the infectious recombinant AAV particles of (k) to transduce the cells of (m) and express a reporter transgene in the cells of (m); (p) measuring expression of the reporter transgene and assigning a value labeled S that reflects the amount of reporter transgene expression of (o); (q) performing steps (h)-(j) and (n)-(p) at least twice with different sample dilutions; (r) if S is less than MAX and S.EV is equal to or greater than MAX, then an AAV-binding antibody is present in the diluted sample that inhibits AAV vector cell transduction; Optionally, (s) measuring expression of a negative control in cells capable of being infected with, but not infected by, the infectious recombinant AAV particles (a) to provide a background value, denoted MIN, where MIN may be subtracted from any one of S, MAX and / or S.EV; Includes.

[0014]

[0016] In certain embodiments, the method steps may be performed in any suitable order, unless otherwise indicated herein.

[0015]

[0017] In certain embodiments, the method further comprises calculating, after step (r) or after step (s), a titer of the AAV-binding antibody in the biological sample corresponding to the lowest dilution of the biological sample that inhibits reporter transgene expression by about 50% or more, where if the lowest dilution that inhibits reporter transgene expression by about 50% or more is greater than or equal to about 1 to 5, the titer is the dilution that results in about 50% or more inhibition as determined by the formula S / MAX, or if the lowest dilution that inhibits reporter transgene expression by about 50% or more is less than about 1 to 5, the titer is the dilution that results in about 50% or more inhibition as determined by the formula S / S.EV.

[0016]

[0018] In certain embodiments, the method further comprises a step (t) of calculating a titer of the AAV-binding antibody in the biological sample, a titer corresponding to the lowest dilution of the biological sample that inhibits reporter transgene expression by about 50% or more, where if the lowest dilution that inhibits reporter transgene expression by about 50% or more is greater than or equal to about 1 to 5, the titer is the dilution that results in about 50% or more inhibition as determined by the formula 100-[[(S-MIN) / (MAX-MIN)]×100], or if the lowest dilution that inhibits reporter transgene expression by about 50% or more is less than about 1 to 5, the titer is the dilution that results in about 50% or more inhibition as determined by the formula 100-[[(S-MIN) / (S.EV-MIN)]×100].

[0017]

[0019] In certain embodiments, the method further comprises the steps of: (a) providing infectious recombinant AAV particles; providing cells capable of being infected with the infectious recombinant AAV particles; providing empty capsid AAV particles and contacting the cells with the provided empty capsid AAV particles; (b) transducing the cells with the infectious recombinant AAV particles of (a), and contacting the cells contacted with the empty capsid AAV particles with the infectious recombinant AAV particles of (a) under conditions such that a reporter transgene is expressed by the cells; and (c) measuring expression of the reporter transgene and assigning a value denoted MAX.EV that reflects the amount of reporter gene expression.

[0018]

[0020] In certain embodiments, the method further comprises calculating a signal to noise ratio, denoted as S / N, where S / N is equal to MAX / MIN.

[0019]

[0021] In certain embodiments, the method further comprises calculating the percent coefficient of variation (%CV), where %CV=(standard deviation / mean)×100%.

[0020]

[0022] In one particular embodiment, the method further includes calculating EV interference, where EV interference=MAX / MAX.EV.

[0021]

[0023] In certain embodiments, the method further comprises the steps of: (a) measuring expression of the reporter transgene under control conditions comprising one or more dilutions of an AAV binding antibody that binds to the AAV vector and assigning a value denoted HQC to the expression measurement of the dilution that provides a preselected amount of reporter transgene expression relative to MAX or MAX-MIN; and / or (b) measuring expression of the reporter transgene under control conditions comprising a dilution of step (a) that provides a preselected amount of reporter transgene expression relative to MAX or MAX-MIN in the presence of empty capsid AAV particles and assigning a value denoted HQC.EV.

[0022]

[0024] The preselected amount of reporter transgene expression in step (a) can be equal to or less than about 75% of MAX or MAX-MIN, for example, without limitation, equal to or less than about 60% of MAX or MAX-MIN, equal to or less than about 50% of MAX or MAX-MIN, equal to or less than about 40% of MAX or MAX-MIN, equal to or less than about 30% of MAX or MAX-MIN. In certain embodiments, the preselected amount of reporter transgene expression in step (a) is equal to or less than about 25% of MAX or MAX-MIN. In certain embodiments, the preselected amount of reporter transgene expression in step (a) is equal to or less than about 20% of MAX or MAX-MIN.

[0023]

[0025] In certain embodiments, the method further comprises calculating EV validity, where EV validity=HQC.EV / HQC.

[0024]

[0026] In certain embodiments, steps (h)-(j) and (n)-(p) are performed at least 3, 4, 5, or 6 times, with 3, 4, 5, or 6 different dilutions of the biological sample.

[0025]

[0027] In certain embodiments, the sample is diluted from about 1:1 to about 1:5000 prior to contacting or incubating with infectious recombinant AAV particles of (a), (e), or (k).

[0026]

[0028] In certain embodiments, the sample is diluted from about 1:1 to about 1:1000 prior to contacting or incubating with infectious recombinant AAV particles of (a), (e), or (k).

[0027]

[0029] In certain embodiments, the sample is diluted from about 1:1 to about 1:500 prior to contacting or incubating with infectious recombinant AAV particles of (a), (e), or (k).

[0028]

[0030] In certain embodiments, the sample is diluted from about 1:1 to about 1:100 prior to contacting or incubating with infectious recombinant AAV particles of (a), (e), or (k).

[0029]

[0031] In certain embodiments, steps (h)-(j) and (n)-(p) are performed at a dilution of the biological sample of about 1:1, about 1:2.5, about 1:5, about 1:10, about 1:100, and / or about 1:1000.

[0030]

[0032] In certain embodiments, the method is completed within about 48 hours from the time step (c) or (d) begins or from the time step (c) or (d) is completed.

[0031]

[0033] In certain embodiments, the cells capable of being infected with the infectious recombinant AAV particles of any of steps (c), (e), (i), or (o) are contacted within about 2 hours after the cells capable of being infected with the infectious recombinant AAV particles are thawed from freezing.

[0032]

[0034] In certain embodiments, the cells capable of being infected with the infectious recombinant AAV particles of any of steps (c), (e), (i), or (o) are contacted within about 1 hour after the cells capable of being infected with the infectious recombinant AAV particles are thawed from freezing.

[0033]

[0035] In certain embodiments, the cells that can be infected with the infectious recombinant AAV particles of any of steps (c), (e), (i), or (o) are at least about 60% confluent.

[0034]

[0036] In certain embodiments, the cells that can be infected with the infectious recombinant AAV particles of any of steps (c), (e), (i), or (o) are at least about 70% confluent.

[0035]

[0037] In certain embodiments, the cells that can be infected with the infectious recombinant AAV particles of any of steps (c), (e), (i), or (o) are at least about 80% confluent.

[0036]

[0038] In certain embodiments, the cells that can be infected with the infectious recombinant AAV particles of any of steps (c), (e), (i), or (o) are at least about 90% confluent.

[0037]

[0039] The invention also provides, inter alia, carriers and plates having one or more components for use in the methods of the invention. The carriers and plates can include, for example and without limitation, cells that can be infected with infectious recombinant AAV particles, a biological sample from a subject, such as, but not limited to, a diluted biological sample from a subject, and / or empty capsid AAV particles.

[0038]

[0040] In certain embodiments, the carrier or plate includes: (a) a cell capable of being infected with an infectious recombinant AAV particle comprising a reporter transgene, and the infectious recombinant AAV particle comprising a reporter transgene; (b) a diluted biological sample from a subject, cells capable of being infected with infectious recombinant AAV particles and empty capsid AAV particles, and / or (c) a diluted biological sample from the same subject that provided the sample in (b), a cell capable of being infected with an infectious recombinant AAV particle containing a reporter transgene, and an infectious recombinant AAV particle containing a reporter transgene. are arranged separately.

[0039]

[0041] In certain embodiments, each of the components on the carrier or plate can be in a separate well, hi certain embodiments, each of the components on the carrier or plate can be in the amounts disclosed for the methods herein.

[0040]

[0042] In certain embodiments, the carrier or plate has each of the (a), (b) and (c) components disposed within separate tubes or separate single wells of a multi-well carrier or plate.

[0041]

[0043] In certain embodiments, the carrier or plate comprises plastic or glass.

[0042]

[0044] In certain embodiments, the carrier or plate is a multi-well plate or dish.

[0043]

[0045] As described herein, in certain embodiments, a method for analyzing for or detecting the presence of an enhancer of adeno-associated viral (AAV) vector cell transduction in a biological sample from a subject includes: (a) providing infectious recombinant AAV particles comprising a recombinant AAV vector, wherein (i) the vector comprises a reporter transgene, (ii) the reporter transgene comprises a single-stranded or self-complementary genome, (iii) the reporter transgene is operably linked to one or more expression control elements, and (iv) the reporter transgene is adjacent to one or more flanking elements; (b) providing a cell that can be infected with the infectious recombinant AAV particle; (c) contacting cells of (b) with infectious recombinant AAV particles of (a) under conditions such that cells of (b) are transduced with the infectious recombinant AAV particles of (a) and a reporter transgene is expressed by said cells of (b); (d) measuring expression of the reporter transgene and assigning a value labeled MAX that reflects the amount of reporter transgene expression in (c); (e) providing an infectious recombinant AAV particle (a); (f) providing a biological sample from a subject; (g) providing a cell capable of being infected with the infectious recombinant AAV particle; (h) combining the biological sample of (f) with empty capsid AAV particles to produce a mixture (M) and incubating said M under conditions that allow the empty capsid AAV particles to bind to any AAV-binding antibodies present in the biological sample; (i) contacting the cell of (g) with the M and the infectious recombinant AAV particles of (e) under conditions such that the infectious recombinant AAV particles of (e) can transduce the cell of (g) and express a reporter transgene in the cell of (g); (j) measuring expression of the reporter transgene and assigning a value, denoted S.EV, that reflects the amount of reporter transgene expression in (i); (k) comparing the S.EV to the MAX, where if the S.EV is greater than the MAX, the biological sample from the subject contains an AAV vector cell transduction enhancer. Includes.

[0044]

[0046] In certain embodiments, one or more of steps (c), (d), (h), (i), (j), or (k) may be performed using an automated system.

[0045]

[0047] In certain embodiments, the automated system includes a contacting component, a measuring component, a mixing component, an incubating component, a processor, and a non-transitory electronic storage device. The non-transitory electronic storage device is designed to allow the processor to control the contacting component, the measuring component, the mixing component, and the incubating component. The method includes: (c) contacting cells of (b) with infectious recombinant AAV particles of (a) using a contact component under conditions such that the cells of (b) are transduced with the infectious recombinant AAV particles of (a) and a reporter transgene is expressed by said cells of (b); (d) measuring expression of the reporter transgene using a measurement component, assigning using the processor a value designated MAX reflecting the amount of reporter transgene expression of (c), and storing using the processor the value designated MAX in a non-transitory electronic storage device; (h) mixing the biological sample of (f) with empty capsid AAV particles using a mixing component to produce a mixture (M) and incubating said M with an incubating component under conditions that allow the empty capsid AAV particles to bind to any AAV-binding antibodies present in the biological sample that inhibit, reduce or decrease AAV vector cell transduction; (i) contacting the cell of (g) with the M and the infectious recombinant AAV particles of (e) using a contact component under conditions such that the infectious recombinant AAV particles of (e) can transduce the cell of (g) and express a reporter transgene in the cell of (g); (j) measuring expression of the reporter transgene using a measurement component and assigning, using a processor, a value designated S.EV that reflects the amount of reporter transgene expression of (i); and storing, using the processor, the value designated S.EV in a non-transitory electronic storage device; (k) comparing, with a processor, the S.EV to the MAX, where if the S.EV is greater than the MAX, the processor determines that the biological sample from the subject contains an enhancer for AAV vector cell transduction, and optionally outputting, with the processor, an indication that the biological sample from the subject contains the enhancer for display. Further includes:

[0046]

[0048] As described herein, in certain embodiments, a method for analyzing for or detecting the presence of an inhibitor of adeno-associated viral (AAV) vector cell transduction in a biological sample from a subject includes: (a) providing infectious recombinant AAV particles comprising a recombinant AAV vector, wherein (i) the vector comprises a reporter transgene, (ii) the reporter transgene comprises a single-stranded or self-complementary genome, (iii) the reporter transgene is operably linked to one or more expression control elements, and (iv) the reporter transgene is adjacent to one or more flanking elements; (b) providing a cell that can be infected with the infectious recombinant AAV particle; (c) contacting cells of (b) with infectious recombinant AAV particles of (a) under conditions such that cells of (b) are transduced with the infectious recombinant AAV particles of (a) and a reporter transgene is expressed by said cells of (b); (d) measuring expression of the reporter transgene and assigning a value labeled MAX that reflects the amount of reporter transgene expression in (c); (e) providing an infectious recombinant AAV particle (a); (f) providing a biological sample from a subject; (g) providing a cell capable of being infected with the infectious recombinant AAV particle; (h) combining the biological sample of (f) with empty capsid AAV particles to produce a mixture (M) and incubating said M under conditions that allow the empty capsid AAV particles to bind to any AAV-binding antibodies present in the biological sample; (i) contacting the cell of (g) with the M and the infectious recombinant AAV particles of (e) under conditions that allow the infectious recombinant AAV particles of (e) to transduce the cell of (g) and express a reporter transgene in the cell of (g); (j) measuring expression of the reporter transgene and assigning a value, denoted S.EV, that reflects the amount of reporter transgene expression in (i); (k) comparing said S.EV to said MAX, wherein if said S.EV is less than said MAX, then the biological sample from the subject contains an inhibitor of AAV vector cell transduction, expression or secretion of a protein encoded by the vector. Includes.

[0047]

[0049] In certain embodiments, one or more of steps (c), (d), (h), (i), (j), or (k) may be performed using an automated system.

[0048]

[0050] In certain embodiments, the automated system includes a contacting component, a measuring component, a mixing component, an incubating component, a processor, and a non-transitory electronic storage device. The non-transitory electronic storage device is designed to allow the processor to control the contacting component, the measuring component, the mixing component, and the incubating component. The method includes: (c) contacting cells of (b) with infectious recombinant AAV particles of (a) using a contact component under conditions such that the cells of (b) are transduced with the infectious recombinant AAV particles of (a) and a reporter transgene is expressed by said cells of (b); (d) measuring expression of the reporter transgene using a measurement component, assigning using the processor a value designated MAX reflecting the amount of reporter transgene expression of (c), and storing using the processor the value designated MAX in a non-transitory electronic storage device; (h) mixing the biological sample of (f) with empty capsid AAV particles using a mixing component to produce a mixture (M) and incubating said M with an incubating component under conditions that allow the empty capsid AAV particles to bind to any AAV-binding antibodies present in the biological sample that inhibit, reduce or decrease AAV vector cell transduction; (i) contacting the cell of (g) with the M and the infectious recombinant AAV particles of (e) with a contact component under conditions that allow the infectious recombinant AAV particles of (e) to transduce the cell of (g) and express a reporter transgene in the cell of (g); (j) measuring expression of the reporter transgene using a measurement component and assigning, using a processor, a value designated S.EV that reflects the amount of reporter transgene expression of (i); and storing, using the processor, the value designated S.EV in a non-transitory electronic storage device; (k) comparing, with a processor, the S.EV to the MAX, where if the S.EV is less than the MAX, the processor determines that the biological sample from the subject contains an inhibitor of AAV vector cell transduction, expression or secretion of a protein encoded by the vector, and optionally outputting, with the processor, an indication that the biological sample from the subject contains the inhibitor. Further includes:

[0049]

[0051] As described herein, in certain embodiments, a method for analyzing for or detecting the presence of an enhancer of adeno-associated viral (AAV) vector cell transduction in a biological sample from a subject includes: (a) providing infectious recombinant AAV particles comprising a recombinant AAV vector, wherein (i) the vector comprises a reporter transgene, (ii) the reporter transgene comprises a single-stranded or self-complementary genome, (iii) the reporter transgene is operably linked to one or more expression control elements, and (iv) the reporter transgene is adjacent to one or more flanking elements; (b) providing a cell that can be infected with the infectious recombinant AAV particle; (c) providing a biological sample from a subject; (d) combining the biological sample of (c) with empty capsid AAV particles to produce a mixture (M) and incubating said M under conditions that allow the empty capsid AAV particles to bind to any AAV-binding antibodies present in the biological sample; (e) contacting the cells of (b) with the M and the infectious recombinant AAV particles of (a) under conditions that allow the infectious recombinant AAV particles of (a) to transduce the cells of (b) and express a reporter transgene in the cells of (b); (f) measuring expression of the reporter transgene; (g) comparing said expression of (f) to a positive (+) control, which is expression of a reporter transgene without the sample from the subject and without the addition of empty capsid AAV particles, and if expression of (f) is greater than said + control, then the biological sample from the subject contains an enhancer of AAV vector cell transduction. Includes.

[0050]

[0052] In certain embodiments, one or more of steps (d), (e), (f), or (g) may be performed using an automated system.

[0051]

[0053] In certain embodiments, the automated system includes a contacting component, a measuring component, a mixing component, an incubating component, a processor, and a non-transitory electronic storage device. The non-transitory electronic storage device is designed to allow the processor to control the contacting component, the measuring component, the mixing component, and the incubating component. The method includes: (d) mixing the biological sample of (c) with empty capsid AAV particles using a mixing component to produce a mixture (M) and incubating said M with an incubating component under conditions that allow the empty capsid AAV particles to bind to any AAV-binding antibodies present in the biological sample that inhibit, reduce or decrease AAV vector cell transduction; (e) contacting the cells of (b) with the M and the infectious recombinant AAV particles of (a) with a contact component under conditions that allow the infectious recombinant AAV particles of (a) to transduce the cells of (b) and express a reporter transgene in the cells of (b); (f) measuring expression of the reporter transgene using a measurement component; (g) comparing said expression of (f) with a positive (+) control using a processor, where if said expression of (f) is greater than said + control, then the processor determines that the biological sample from the subject contains an enhancer of AAV vector cell transduction, expression or secretion of a protein encoded by the vector, and optionally outputting for display, using the processor, an indication that the biological sample from the subject contains the enhancer. Further includes:

[0052]

[0054] As described herein, in certain embodiments, a method for analyzing for or detecting the presence of an inhibitor of adeno-associated viral (AAV) vector cell transduction in a biological sample from a subject includes: (a) providing infectious recombinant AAV particles comprising a recombinant AAV vector, wherein (i) the vector comprises a reporter transgene, (ii) the reporter transgene comprises a single-stranded or self-complementary genome, (iii) the reporter transgene is operably linked to one or more expression control elements, and (iv) the reporter transgene is adjacent to one or more flanking elements; (b) providing a cell that can be infected with the infectious recombinant AAV particle; (c) providing a biological sample from a subject; (d) mixing the biological sample of (c) with empty capsid AAV particles to produce a mixture (M) and incubating said M under conditions that allow the empty capsid AAV particles to bind to any AAV-binding antibodies present in the biological sample; (e) contacting the cells of (b) with the M and the infectious recombinant AAV particles of (a) under conditions that allow the infectious recombinant AAV particles of (a) to transduce the cells of (b) and express a reporter transgene in the cells of (b); (f) measuring expression of the reporter transgene; (g) comparing said expression of (f) to a positive (+) control, which is expression of a reporter transgene without the sample from the subject and without the addition of empty capsid AAV particles, and if expression of (f) is less than said + control, then the biological sample from the subject contains an inhibitor of AAV vector cell transduction. Includes.

[0053]

[0055] In certain embodiments, one or more of steps (d), (e), (f), or (g) may be performed using an automated system.

[0054]

[0056] In certain embodiments, the automated system includes a contacting component, a measuring component, a mixing component, an incubating component, a processor, and a non-transitory electronic storage device. The non-transitory electronic storage device is designed to allow the processor to control the contacting component, the measuring component, the mixing component, and the incubating component. The method includes: (d) mixing the biological sample of (c) with empty capsid AAV particles using a mixing component to produce a mixture (M) and incubating said M with an incubating component under conditions that allow the empty capsid AAV particles to bind to any AAV-binding antibodies present in the biological sample that inhibit, reduce or decrease AAV vector cell transduction; (e) contacting the cells of (b) with the M and the infectious recombinant AAV particles of (a) with a contact component under conditions that allow the infectious recombinant AAV particles of (a) to transduce the cells of (b) and express a reporter transgene in the cells of (b); (f) measuring expression of the reporter transgene using a measurement component; (g) comparing said expression of (f) with a positive (+) control using a processor, where if said expression of (f) is less than said + control, then the processor determines that the biological sample from the subject contains an inhibitor of AAV vector cell transduction, expression or secretion of a protein encoded by the vector, and optionally outputting for display, using the processor, an indication that the biological sample from the subject contains the inhibitor. Further includes:

[0055]

[0057] As described herein, in certain embodiments, a method for assaying for and detecting or quantifying AAV binding antibodies that inhibit AAV vector cell transduction in a biological sample from a subject comprises: (a) providing infectious recombinant AAV particles comprising a recombinant AAV vector, wherein (i) the vector comprises a reporter transgene, (ii) the reporter transgene comprises a single-stranded or self-complementary genome, (iii) the reporter transgene is operably linked to one or more expression control elements, and (iv) the reporter transgene is adjacent to one or more flanking elements; (b) providing a cell that can be infected with the infectious recombinant AAV particle; (c) contacting the cells of (b) with the infectious recombinant AAV particles of (a) under conditions such that the cells of (b) are transduced by the infectious recombinant AAV particles of (a) and a reporter transgene is expressed by the cells of (b); (d) measuring expression of the reporter transgene and assigning a value labeled MAX that reflects the amount of reporter transgene expression in (c); (e) providing an infectious recombinant AAV particle (a); (f) providing a diluted biological sample from a subject; (g) providing a cell capable of being infected with the infectious recombinant AAV particle; (h) mixing the diluted biological sample of (f) with empty capsid AAV particles to produce a mixture (M) and incubating said M under conditions that allow the empty capsid AAV particles to bind to any AAV-binding antibodies present in the biological sample; (i) contacting the cell of (g) with the M and the infectious recombinant AAV particles of (e) under conditions that allow the infectious recombinant AAV particles of (e) to transduce the cell of (g) and express a reporter transgene in the cell of (g); (j) measuring expression of the reporter transgene and assigning a value, denoted S.EV, that reflects the amount of reporter transgene expression in (i); (k) providing an infectious recombinant AAV particle (a); (l) providing a diluted biological sample from the same subject that provided the sample of (f); (m) providing a cell capable of being infected with the infectious recombinant AAV particle; (n) mixing the diluted biological sample of (l) with the infectious recombinant AAV particles of (k) to produce a mixture (M); (o) contacting the cells of (m) with M under conditions that allow the infectious recombinant AAV particles of (k) to transduce the cells of (m) and express the reporter transgene in the cells of (m); (p) measuring expression of the reporter transgene and assigning a value denoted S that reflects the amount of reporter transgene expression of (o); (q) performing steps (h)-(j) and (n)-(p) at least twice with different sample dilutions; (r) if S is less than MAX and S.EV is equal to or greater than MAX, then an AAV-binding antibody is present in the diluted sample that inhibits AAV vector cell transduction; and optionally, (s) measuring expression of a negative control in cells capable of being infected by, but not infected with, said infectious recombinant AAV particles (a) to provide a background value, denoted MIN, where MIN may be subtracted from any one of S, MAX and / or S.EV. Includes.

[0056]

[0058] In certain embodiments, one or more of steps (c), (d), (h), (i), (j), (n), (o), (p), or (s) may be performed using an automated system.

[0057]

[0059] In certain embodiments, the automated system includes a contacting component, a measuring component, a mixing component, an incubating component, a processor, and a non-transitory electronic storage device. The non-transitory electronic storage device is designed to allow the processor to control the contacting component, the measuring component, the mixing component, and the incubating component. The method includes: (c) contacting cells of (b) with infectious recombinant AAV particles of (a) using a contact component under conditions such that the cells of (b) are transduced with the infectious recombinant AAV particles of (a) and a reporter transgene is expressed by said cells of (b); (d) measuring expression of the reporter transgene using a measurement component, assigning using the processor a value designated MAX reflecting the amount of reporter transgene expression of (c), and storing using the processor the value designated MAX in a non-transitory electronic storage device; (h) mixing the diluted biological sample of (f) with empty capsid AAV particles using a mixing component to produce a mixture (M) and incubating said M with an incubating component under conditions that allow the empty capsid AAV particles to bind to any AAV-binding antibodies present in the biological sample that inhibit, reduce or decrease AAV vector cell transduction; (i) contacting the cell of (g) with the M and the infectious recombinant AAV particles of (e) with a contact component under conditions that allow the infectious recombinant AAV particles of (e) to transduce the cell of (g) and express a reporter transgene in the cell of (g); (j) measuring expression of the reporter transgene using a measurement component and assigning, using a processor, a value designated S.EV that reflects the amount of reporter transgene expression of (i); and storing, using the processor, the value designated S.EV in a non-transitory electronic storage device; (n) mixing the diluted biological sample of (l) with the infectious recombinant AAV particles of (e) using a mixing component to produce a mixture M; (o) contacting the cells of (m) with M using a contact component under conditions that allow the infectious recombinant AAV particles of (k) to transduce the cells of (m) and express a reporter transgene in the cells of (m); (p) measuring expression of the reporter transgene using a measurement component and assigning using a processor a value designated S reflective of the amount of reporter transgene expression of (o); and storing using the processor the value designated S in a non-transitory electronic storage device; (s) optionally measuring expression of a negative control of cells capable of being infected with said infectious recombinant AAV particles (a), but not infected with said infectious recombinant AAV particles (a), using a measurement component to provide a background value, denoted MIN, where MIN may be subtracted by the processor from any one of S, MAX, and / or S.EV. Further includes:

[0058]

[0060] In certain embodiments, the method may further include a step (s) or (t) of calculating, using a processor, a titer of the AAV-binding antibody, said titer corresponding to the minimum dilution of the biological sample that results in about 50% or more inhibition of reporter transgene expression, the processor being configured such that if the minimum dilution that results in about 50% or more inhibition of reporter transgene expression is greater than or equal to about 1 to 5, the titer is determined by the formula S / MAX, or if the minimum dilution that results in about 50% or more inhibition of reporter transgene expression titer is less than about 1 to 5, the titer is determined by the formula S / S.EV, and optionally outputting, using the processor, an indication of the titer for display.

[0059]

[0061] In certain embodiments, the method may further include a step (t) of calculating, using a processor, a titer of the AAV binding antibody, said titer corresponding to the minimum dilution of the biological sample that results in about 50% or more inhibition of reporter transgene expression, the processor being configured such that if the minimum dilution that results in about 50% or more inhibition of reporter transgene expression is greater than or equal to about 1 to 5, the titer is determined by the formula 100-[[(S-MIN) / (MAX-MIN)]×100], or if the minimum dilution that results in about 50% or more inhibition of reporter transgene expression titer is less than about 1 to 5, the titer is determined by the formula 100-[[(S-MIN) / (S.EV-MIN)]×100], and optionally outputting, using the processor, an indication of the titer for display.

[0060]

[0062] In certain embodiments, the method further comprises the steps of providing infectious recombinant AAV particles (a); providing a cell capable of being infected with the infectious recombinant AAV particles; providing empty capsid AAV particles; contacting the cell with the provided empty capsid AAV particles using a contacting component; contacting the cell that has been contacted with the empty capsid AAV particles with the infectious recombinant AAV particles of (a) using a contacting component under conditions such that the cell of (b) is transduced by the infectious recombinant AAV particles of (a) and a reporter transgene is expressed by the cell; measuring expression of the reporter transgene using a measuring component, assigning a value denoted MAX.EV reflecting the amount of reporter transgene expression using a processor, and storing the value denoted MAX.EV in a non-transitory electronic storage device using a processor.

[0061]

[0063] In certain embodiments, the method further includes the steps of calculating, with a processor, a signal-to-noise ratio, denoted as S / N, where S / N is equal to MAX / MIN; storing, with the processor, the S / N in non-transitory electronic storage; and optionally outputting, with the processor, an indication of the S / N for display.

[0062]

[0064] In certain embodiments, the method further includes calculating, with a processor, a percent coefficient of variation (%CV), %CV=(standard deviation / mean)×100%, storing, with the processor, the %CV in non-transitory electronic storage, and optionally outputting, with the processor, an indication of the %CV for display.

[0063]

[0065] In a particular embodiment, the method further includes calculating, with a processor, the EV interference, EV interference=MAX / MAX.EV; storing, with the processor, the EV interference in non-transitory electronic storage; and optionally outputting, with the processor, an indication of the EV interference for display.

[0064]

[0066] In certain embodiments, the method further includes calculating, using a processor, an HQC based on expression measurements of a dilution that provides a preselected amount of reporter transgene expression relative to MAX or MAX-MIN, and / or an HQC EV and / or HQC EV / HQC based on expression of the reporter transgene under controlled conditions including the dilution of step (a) that provides a preselected amount of reporter transgene expression relative to MAX or MAX-MIN in the presence of empty capsid AAV particles, storing, using a processor, the HQC and / or HQC EV and / or HQC EV / HQC in non-transitory electronic storage, and optionally outputting, using a processor, an indication of the HQC and / or HQC EV and / or HQC EV / HQC for display. [Brief description of the drawings]

[0065] [Figure 1A] Figure 1A shows that 27% of healthy donor (HD; normal individuals without any known disease) samples had enhancers (E) that resulted in inaccurate estimates of AAV NAb titers in these samples. 49% of HD had AAV NAb titers >1:40, 14% of HD had AAV NAb titers between 1:1 and 1:20, and 10% of HD were negative for AAV NAb. [Figure 1B] FIG 1B shows that in the presence of enhancers, calculation of AAV NAb titers is inaccurate. "BAb" = AAV binding antibody [Diagram 2] FIG. 2 shows detection of AAV NAbs in serum using empty capsid AAV particles (EV). [Diagram 3] Figures 3A-3E show the results of a 2-day assay using preincubation with empty capsid AAV particles (EVs). X-axis titers are 1:1, 1:2.5, 1:5, 1:10, 1:100, and 1:1000. A) NAb+ (1:10); B) NAb- (<1:1); C) NAb- (<1:1), enhancer; D) NAb- (<1:1), inhibitor, false positive; E) NAb+ (1:1), enhancer, false negative. [Figure 4] FIG. 4 shows a decision tree for AAV NAb titer calculation. [Figure 5A] Figure 5A shows a representative formula for calculating % inhibition (MAX). MIN = background [Figure 5B] FIG. 5B shows a representative formula for calculating % inhibition (SEV). MIN=background [Figure 6A] Figure 6A shows an example of AAV NAb titer calculation. Decision tree for assigning AAV NAb titers according to test sample dilution. [Figure 6B] FIG. 6B shows an example of an AAV NAb titer calculation. [Figure 6C] Figure 6C shows an example of AAV NAb titer calculation. Low NAb titer [Figure 6D] FIG. 6D shows an example of AAV NAb titer calculation. [Figure 6E] Figure 6D shows an example of AAV NAb titer calculation. High NAb titers [Figure 7] FIG. 7 shows an exemplary assay plate layout. [Figure 8A] Figure 8A shows the assay criteria (1). Signal to noise ratio (S / N) [Figure 8B] Figure 8B shows assay criteria (1). EV potency. [Figure 8C] Figure 8C shows assay criteria (1). EV interference. [Figure 9A] 9A-9C show assay standard (2). [Figure 9B] 9A-9C show assay standard (2). [Figure 9C] 9A-9C show assay standard (2). [Figure 10A] FIG. 10A shows an exemplary FACT dilution scheme (wells 3B-3D). [Figure 10B] FIG. 10B shows an exemplary sample dilution scheme (wells 4B-4G). [Figure 11] FIG. 11 shows an exemplary transfer of diluted samples to a neutralization plate. [Figure 12] FIG. 12 shows an exemplary addition of FBS to a neutralization plate (wells: MIN=2B; S=4B-4G; S.EV=5B-5G; FACT=3B-3D; FACT.EV=3E, 3F; MAX=2G; and MAX.EV=3G). [Figure 13A] Figure 13A shows an exemplary addition of empty capsid AAV particles (EV) to a neutralization plate (wells: MIN=2B; S=4B-4G; S.EV=5B-5G; FACT=3B-3D; FACT.EV=3E, 3F; MAX=2G; and MAX.EV=3G). [Figure 13B] FIG. 13B shows an exemplary addition of DMEM media to a neutralization plate (Wells: MIN=2B; S=4B-4G; S.EV=5B-5G; FACT=3B-3D; FACT.EV=3E, 3F; MAX=2G; and MAX.EV=3G). [Figure 14] FIG. 14 shows exemplary addition of AAV reporter vectors to a neutralization plate (wells: MIN=2B; S=4B-4G; S.EV=5B-5G; FACT=3B-3D; FACT.EV=3E, 3F; MAX=2G; and MAX.EV=3G). [Figure 15] FIG. 15 shows the transfer of controls and samples from the neutralization plate to the transduction plate (wells: MIN=2B-2D; S=2E-2G; S.EV=9B-9G, 10B-10G, and 11B-11G; FACT=3B-3D, 4B-4D, and 5B-5D; FACT.EV=3E, 3F, 4E, 4F, 5E, and 5F; MAX=2E-2G; and MAX.EV=3G, 4G, and 5G). [Figure 16]FIG. 16 shows a schematic diagram of the CAG promoter sequence (SEQ ID NO:3), which is composed of the cytomegalovirus (CMV) early enhancer element ("C"), the first exon and first intron ("A") of the chicken beta-actin gene, and the splice acceptor ("G") of the rabbit beta-globin gene. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0066]

[0086] Disclosed herein are methods for the detection of enhancers and inhibitors of AAV vector transduction, as well as methods for detecting and / or quantifying AAV binding antibodies that inhibit, reduce or diminish AAV vector cell transduction, otherwise known as neutralizing antibodies (NAbs). The methods according to the invention rely in part on the transduction of AAV permissive cell lines with a reporter vector (AAV particles carrying a reporter transgene). The methods according to the invention also rely in part on the use of empty capsid AAV particles that absorb most or all of the AAV binding antibodies in a sample from a subject, thereby revealing the presence, if any, of enhancers or inhibitors of AAV vector cell transduction in the sample analyzed for AAV NAbs. The methods according to the invention may be used, inter alia, to more accurately determine AAV NAb titers in a sample from a subject, where the presence of enhancers or inhibitors can result in false negatives and false positives, respectively.

[0067]

[0087] In certain embodiments, certain steps of the assay method according to the invention are optimized. In certain embodiments, the assay completion time is reduced. In certain embodiments, the assay variability is reduced and / or matrix interference is reduced. In particular, compared to, for example, conventional NAb assays (Meliani et al., 2015, Human Gene Therapy Methods, 26:45-53, doi:10.1089 / hgtb.2015.037), the assay method according to the invention can be performed in about 2 days instead of 3 days, the intra-assay variability is reduced from 30.5% to 8.7%, as assessed by the coefficient of variation (%CV) of triplicate treatments, and the intra-assay precision evaluation of the assay revealed a %CV of 12.5% ​​for quality control samples.

[0068]

[0088] The methods according to the invention provide an efficient, reliable and more accurate assay that can be used to detect or quantify AAV binding antibodies (AAV neutralizing antibodies (NAb)) that inhibit, reduce or decrease AAV vector cell transduction in a variety of situations. For example, in certain embodiments, the assay can be used to analyze, detect or quantify AAV NAb to assist in selecting subjects for or excluding subjects from gene therapy treatment. In certain embodiments, the assay can be used to analyze, detect or quantitate AAV NAb to select subjects for or exclude subjects from inclusion in a gene therapy trial. In certain embodiments, the assay can be used to analyze, detect or quantitate AAV NAb to monitor subjects for the development of anti-AAV antibodies after undergoing gene therapy treatment. In certain embodiments, the assay can be used to analyze, detect or quantitate AAV NAb to monitor AAV NAb in subjects who may need to reduce the amount of AAV NAb or may be undergoing treatment to reduce it.

[0069]

[0089] In certain embodiments, a method for analyzing for, detecting or quantitating AAV-binding antibodies that inhibit, reduce or decrease AAV vector cell transduction in a biological sample from a subject.

[0070]

[0090] In certain embodiments, a method for analyzing for, detecting or quantitating AAV neutralizing antibodies that inhibit, reduce or decrease AAV vector cell transduction in a biological sample from a subject.

[0071]

[0091] Adeno-associated viral (AAV) vectors are viral vectors that infect, inter alia, primates, such as humans.

[0072]

[0092] As used herein, the term "recombinant" as a modifier of viral vectors, such as, but not limited to, recombinant AAV (rAAV) vectors, and sequences, such as recombinant polynucleotides and polypeptides, means that the composition has been manipulated (i.e., engineered) in a manner that does not generally occur in nature. A particular example of a recombinant AAV vector would be when a nucleic acid (heterologous polynucleotide) that is not normally present in the wild-type AAV genome is inserted into the viral genome. An example would be when a nucleic acid (e.g., gene) encoding a reporter transgene is cloned into the vector. The term "recombinant" is not necessarily used herein with respect to AAV vectors, as well as sequences, such as transgenes, but recombinant forms, including AAV vectors, polynucleotides, etc., are expressly included despite any such omission.

[0073]

[0093] A "rAAV vector" is derived from the wild-type genome of AAV, for example, by using molecular methods to remove all or part of the wild-type AAV genome and replace it with a non-native (heterologous) nucleic acid, such as a reporter transgene encoding a reporter protein. Typically, in a rAAV vector, one or both of the inverted terminal repeat (ITR) sequences of the AAV genome are retained. rAAV is distinct from the AAV genome because all or part of the AAV genome is replaced with a non-native sequence with respect to the AAV genomic nucleic acid, such as a reporter transgene encoding a reporter protein. Thus, the incorporation of a non-native (heterologous) sequence defines the AAV as a "recombinant" AAV vector, which can be referred to as a "rAAV vector."

[0074]

[0094] Recombinant AAV vector sequences can be packaged for subsequent infection (transduction) of cells ex vivo, in vitro or in vivo, and are referred to herein as "particles." When recombinant vector sequences are encapsidated or packaged in an AAV particle, the particle can also be referred to as a "rAAV," "rAAV particle," and / or "rAAV virion." Such rAAV, rAAV particles, and rAAV virions include proteins that encapsidate or package the vector genome. Particular examples include, in the case of AAV, capsid proteins.

[0075]

[0095] As used herein, "infectious recombinant AAV particle(s)" refers to a packaged recombinant AAV vector sequence capable of infecting or transducing a cell ex vivo, in vitro or in vivo. As used herein, "cell(s) capable of being infected" refers to a cell that is receptive to infection or transduction by an infectious recombinant AAV particle. In certain embodiments, the methods of the invention use cells that can be infected in vitro by infectious rAAV particles.

[0076]

[0096] "Vector genome", sometimes abbreviated as "vg", refers to the portion of the recombinant plasmid sequence that is ultimately packaged or encapsidated to form the rAAV particle. In cases where a recombinant plasmid is used to construct or produce a recombinant AAV vector, the AAV vector genome does not include the portion of the "plasmid" that does not correspond to the vector genome sequence of the recombinant plasmid. This non-vector genome portion of the recombinant plasmid is called the "plasmid backbone", which is important for plasmid cloning and amplification, steps necessary for propagation and recombinant AAV vector production, but is not itself packaged or encapsidated into the rAAV particle. Thus, "vector genome" refers to the nucleic acid that is packaged or encapsidated by the rAAV.

[0077]

[0097] Empty capsid AAV particles (EV) or empty vector (EV) are AAV particles that lack a vector genome. Empty capsid AAV particles are useful for absorbing (binding to) AAV-binding antibodies to assay for or detect the presence of enhancers of AAV vector cell transduction or inhibitors of AAV vector cell transduction. Empty capsid AAV particles are also useful for quantifying AAV NAb titers at relatively low titers (e.g., less than about 1 to 5).

[0078]

[0098] As used herein, the term "serotype" in reference to an AAV vector refers to a capsid that is serologically distinct from other AAV serotypes. Serological distinctiveness is determined based on the lack of cross-reactivity between antibodies against one AAV compared to another AAV. Differences in cross-reactivity are usually due to differences in capsid protein sequences / antigenic determinants (e.g., due to differences in VP1, VP2, and / or VP3 sequences of AAV serotypes). Antibodies against one AAV capsid serotype may cross-react with one or more other AAV capsid serotypes due to homology of capsid protein sequences or similar or identical conformational epitopes.

[0079]

[0099] Under the traditional definition, a serotype means that the virus of interest has been tested against all existing serotype-specific sera characterized for neutralizing activity, and no antibodies have been found that neutralize the virus of interest. As more naturally occurring virus isolates are discovered and / or capsid mutants are created, there may or may not be serological differences from any of the extant serotypes. Thus, if a new virus (e.g., AAV) does not have serological differences, it is considered to be a subgroup or variant of the corresponding serotype. In many cases, serological testing for neutralizing activity has not yet been performed on mutant viruses with capsid sequence modifications to determine whether they are a different serotype according to the traditional definition of a serotype. Therefore, for convenience and to avoid repetition, the term "serotype" is broadly used to refer to both serologically distinct viruses (e.g., AAV) and viruses (e.g., AAV) that are not serologically distinct and may be within a subgroup or variant of a given serotype.

[0080]

[0100] The rAAV vector and empty capsid AAV particles include any virus strain or serotype. For example, without limitation, the AAV vector genome or particle (capsid such as VP1, VP2 and / or VP3) can be based on any AAV serotype, such as AAV-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, -rh74, -rh10, AA3B or AAV-2i8. Such vectors and empty capsid AAV particles can be based on the same strain or serotype (or subgroup or variant) or different from each other. For example, without limitation, the rAAV vector genome or particle (capsid) based on one serotype genome can be identical to one or more of the capsid proteins that package the vector. Additionally, the rAAV vector genome can be based on an AAV serotype genome that is distinct from one or more of the capsid proteins that package the vector genome, where at least one of the three capsid proteins can be a different AAV serotype, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, -rh74, -rh10, AAV3B, AAV-2i8, SPK1 (SEQ ID NO:1), SPK2 (SEQ ID NO:2), or, for example, a variant thereof. More specifically, a rAAV vector genome can contain AAV2 ITRs, but capsids from different serotypes, such as AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, -rh74, -rh10, AAV3B, AAV-2i8, SPK1 (SEQ ID NO:1), SPK2 (SEQ ID NO:2), or variants thereof. Thus, a rAAV vector can contain gene / protein sequences identical to those characteristic of a particular serotype, as well as "mixed" serotypes, which can also be referred to as "pseudotypes."

[0081]

[0101] In certain embodiments, the reporter-containing AAV vector has the same capsid serotype as the empty capsid AAV particle. However, the capsid serotype of the empty capsid AAV particle does not have to be the same serotype as the capsid serotype of the reporter-containing AAV vector, as long as the empty capsid AAV particle can absorb (bind to) an antibody that binds to the reporter-containing AAV vector, e.g., due to cross-reactivity.

[0082]

[0102] In certain embodiments, the rAAV vector or empty capsid AAV particle comprises or consists of a capsid sequence that is at least 70% or more (e.g., 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc.) identical to one or more AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, -rh74, -rh10, AAV3B, AAV-2i8, SPK1 (SEQ ID NO:1), SPK2 (SEQ ID NO:2) capsid proteins (VP1, VP2, and / or VP3 sequences). In certain embodiments, the rAAV vector comprises or consists of a sequence that is at least 70% or more (e.g., 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc.) identical to one or more AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, -rh74, -rh10 ITR(s).

[0083]

[0103] In certain embodiments, the rAAV vector or empty capsid AAV particle can be prepared using methods described, for example, in International Publication No. WO 2013 / 158879 (International Application No. PCT / US2013 / 037170), International Publication No. WO 2015 / 013313 (International Application No. PCT / US2014 / 047670), and U.S. Patent Application Publication No. 2013 / 0059732 (U.S. Provisional Patent Application No. 13 / 594,773). AAV vectors that contain AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, AAV3B, and AAV-2i8 variants thereof (e.g., capsid variants such as amino acid insertions, additions, substitutions, and deletions, and ITR nucleotide insertions, additions, substitutions, and deletions in the context of a rAAV vector).

[0084]

[0104] rAAV and empty capsid AAV particles, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, -rh74, -rh10, AAV3B, AAV-2i8, SPK1 (SEQ ID NO: 1), SPK2 (SEQ ID NO: 2), and variants, hybrids, and chimeric sequences, can be constructed using recombinant techniques known to those skilled in the art to contain one or more heterologous polynucleotide sequences (transgenes) flanked at the 5' and / or 3' ends by one or more functional AAV ITR sequences. rAAV vectors typically retain at least one functional flanking ITR sequence(s) necessary for rescue, replication, and packaging of the recombinant vector into rAAV vector particles. Thus, it is believed that the rAAV vector genome contains sequences (e.g., functional ITR sequences) necessary in cis for replication and packaging.

[0085]

[0105] In certain embodiments, AAV vectors are used to transduce target cells with a reporter transgene that is subsequently transcribed and, optionally, translated, thereby generating a detectable signal for detecting or quantifying transgene expression. The amount of signal is proportional to the efficiency of cell transduction and subsequent expression. Antibodies or inhibitor agents that bind to vector proteins that package or encapsidate the reporter transgene will inhibit, reduce or decrease vector cell transduction, subsequent reporter expression and, therefore, the amount of detectable signal.

[0086]

[0106] In the assays described herein for analyzing, detecting, and quantifying antibodies, the selection of the particular capsid protein(s) serotype that packages or encapsidates the reporter transgene can be used to identify the serotype(s) to which the NAb binds. For example, if it is desired to detect AAV-2 antibodies, the reporter transgene can be encapsidated with AAV-2 capsid protein(s). If it is desired to detect AAV-8 antibodies, the reporter transgene can be encapsidated with AAV-8 capsid protein(s). If it is desired to detect AAV-9 antibodies, the reporter transgene can be encapsidated with AAV-9 capsid protein(s). If an antibody is present, the antibody binds to the capsid protein(s) that encapsidates the reporter transgene and inhibits, reduces, or decreases vector cell transduction and the resulting reporter transgene expression. The greater the amount or titer of antibody that binds to the envelope or capsid protein(s), the less the vector transduction of cells and the resulting reporter transgene expression and signal. Thus, the methods herein for assaying for, detecting, and quantifying antibodies that bind to vector proteins, such as viral (e.g., AAV) capsid proteins, can also be used to confirm the presence or absence of antibodies that bind to any particular AAV capsid serotype.

[0087]

[0107] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein and refer to any form of nucleic acid, oligonucleotide, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleic acids include genomic DNA, cDNA and antisense DNA, as well as spliced ​​or unspliced ​​mRNA, rRNA, tRNA and inhibitory DNA or RNA (RNAi, e.g., small or short hairpin (sh)RNA, microRNA (miRNA), small or short interfering (si)RNA, trans-splicing RNA, or antisense RNA).

[0088]

[0108] Nucleic acids include naturally occurring, synthetic, and intentionally modified or altered polynucleotides. Nucleic acids can be single-stranded, double-stranded, or triple-stranded, linear or circular, and can be of any length. When discussing nucleic acids, the sequence or structure of a particular polynucleotide may be described herein according to the convention of providing the sequence in the 5' to 3' direction.

[0089]

[0109] A "heterologous" transgene or nucleic acid is a polynucleotide inserted into a vector (e.g., AAV) for vector-mediated transfer / delivery of the polynucleotide into a cell. A heterologous transgene is distinct from the vector (e.g., AAV) nucleic acid, i.e., non-native with respect to the viral (e.g., AAV) nucleic acid sequence. Once transferred / delivered into a cell, a heterologous transgene, although contained within the virion, can be expressed (e.g., transcribed and, if necessary, translated). The term "heterologous" is not necessarily used herein in reference to a transgene or nucleic acid, but reference to a transgene or nucleic acid in the absence of the modifier "heterologous" is intended to include heterologous transgenes and nucleic acids despite the omission.

[0090]

[0110] As used herein, a "reporter" transgene is a polynucleotide that produces a detectable signal. The detectable signal may be produced by the reporter transgene itself, a transcript of the transgene, or a protein encoded by the reporter transgene.

[0091]

[0111] Expressly included are all mammalian and non-mammalian types of transgenes, both known and unknown, including the non-limiting examples of reporter transgenes and encoded proteins disclosed herein. Thus, methods according to the invention include reporter transgenes and proteins from microorganisms and other organisms, which reporter transgenes and proteins are detectable in cells following transduction or transfection as described herein.

[0092]

[0112] In certain embodiments, the reporter transgene encodes a secreted or secretable protein, hi certain embodiments, the reporter transgene encodes a protein that provides an enzymatic, colorimetric, fluorescent, luminescent, chemiluminescent, or electrochemical signal.

[0093]

[0113] For example, and without limitation, reporter transgenes include luciferase genes that encode luciferase proteins, such as, but not limited to, Renilla luciferase, firefly luciferase, and Gaussia luciferase genes.

[0094]

[0114] For example, and without limitation, the reporter transgene may encode β-galactosidase, β-glucuronidase, chloramphenicol transferase, green fluorescent protein (GFP), red fluorescent protein (RFP), and alkaline phosphatase.

[0095]

[0115] "Polypeptides," "proteins," and "peptides" encoded by a "transgene" include, as in naturally occurring proteins, the full-length native sequence, as well as functional subsequence modifications or sequence variants, so long as the subsequence modifications or variants retain some degree of functionality of the native full-length protein. In the present invention, such polypeptides, proteins, and peptides encoded by a transgene can be, but are not required to be, identical to the wild-type protein.

[0096]

[0116] In certain embodiments, the reporter transgene (the transgene provides a detectable signal) can comprise a single-stranded or self-complementary genome that becomes double-stranded or a double-stranded dimer upon packaging into a viral particle (e.g., an AAV vector) or upon viral vector cell transduction and viral unthreshing within the transduced cell.

[0097]

[0117] The term "complementary" or "complement" when used in relation to nucleic acids such as transgenes refers to a number of chemical bases that "specifically hybridize" or bind (anneal) or are capable of doing so with another single-stranded sequence through base pairing to form a double-stranded or duplex molecule. The ability of two single-stranded sequences to specifically hybridize or bind (anneal) to each other to form a double-stranded (or duplex) molecule is due to the functional group of a base on one strand (e.g., sense) that hydrogen bonds with another base on the opposite nucleic acid strand (e.g., antisense). Complementary bases that can bind to each other are typically A and T and C and G in DNA, and C and G, and U and A in RNA. Thus, an example of a self-complementary sequence could be ATCGXXXCGAT, where X represents a non-complementary base, and the structure of such a double-stranded or duplex molecule with a non-hybridizing X base would be as follows:

[0098] [ka]

[0099]

[0118] The term "complementary" or "complement" when used in reference to a polynucleotide or nucleic acid, such as a transgene, is therefore intended to describe the physical state in which a double-stranded or duplex polynucleotide or nucleic acid molecule is formed, or simply describes the sequence relationship between two polynucleotides or nucleic acid molecules such that each single-stranded molecule is believed to be capable of forming a duplex with the other. Thus, "complementary" or "complement" refers to the relationship of the bases in each polynucleotide or nucleic acid molecule strand, and does not mean that the two strands must exist in a double-stranded (or duplex) configuration or in the physical state of each other in a duplex.

[0100]

[0119] Typically, in viral vectors that package single-stranded nucleic acids, such as AAV, inverted terminal repeat (ITR) sequences participate in replication to form hairpin loops that contribute to self-priming to allow the initiation and synthesis of a second DNA strand. After synthesis of the second DNA strand, the AAV ITRs have so-called terminal separation sites (TRS), so that the hairpin loop is cleaved into two single strands, each with a 5' and 3' terminal repeat for viral packaging.

[0101]

[0120] At least one ITR has a deleted, mutated, modified or non-functional TRS, which allows the formation of a double-stranded duplex that is not cleaved by the TRS. In a self-complementary reporter transgene double-stranded duplex structure, an ITR with a deleted, mutated or variant TRS is typically located between two complementary strands. With a non-cleavable or non-degradable TRS, the double-stranded form is not cleaved, allowing the formation of a self-complementary reporter transgene double-stranded duplex structure. Either a non-degradable ITR with a deleted, mutated or variant TRS or a separable ITR may be suitable for virus packaging. The separable AAV ITR does not need to be a wild-type ITR sequence, as long as the ITR mediates the desired function, such as packaging, self-priming, replication, etc.

[0102]

[0121] The ITR and TRS sequences of various AAV serotypes, which may be deleted, mutated, modified, or changed, include any AAV serotypes shown herein or known to those skilled in the art. For example, without limitation, the ITR and TRS sequences of various AAV serotypes include AAV-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -rh74, -rh10. Another example is a modified or variant AAV ITR that is not processed by AAV Rep protein. Another example is a mutated, modified, or variant AAV ITR with a deleted D sequence and / or a mutated, modified, or variant terminal separation site (TRS) sequence. In AAV2, a representative mutated TRS sequence is "CGGTTG".

[0103]

[0122] For self-complementary reporter transgene sequences located outside one or more ITRs, expression control sequences, downstream sequences, etc., such sequences of vector sequences outside the reporter transgene can, but need not, be self-complementary. Thus, self-complementary can be used in certain contexts, e.g., in relation to a transgene, such as a reporter transgene, where only the transgene, such as the reporter transgene, is self-complementary and other non-transgene sequences can, but need not, be self-complementary.

[0104]

[0123] To be self-complementary, not all bases in one strand must be complementary to all bases in each of the opposite complementary strands. There only need to be a sufficient number of complementary nucleotide or nucleoside bases to allow two polynucleotides or nucleic acid molecules to specifically hybridize or bind (anneal) to each other. Thus, there may be short sequence segments or regions of non-complementary bases between self-complementary polynucleotides or nucleic acid molecules. For example, without limitation, there may be 1-5, 5-10, 10-20, 20-30, 30-40, 40-50, 50-75, 75-100, or 100-150 or more consecutive or non-consecutive non-complementary bases, but there may be sufficient complementary bases over the length of the two sequences so that the two polynucleotides or nucleic acid molecules can specifically hybridize or bind (anneal) to each other to form a double-stranded (or duplex) sequence. Thus, the sequences of the two single-stranded regions may be less than 100% complementary to each other and still be able to form a double-stranded duplex molecule. In certain embodiments, the two single-stranded sequences have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or more complementary to each other.

[0105]

[0124] Such segments or regions of non-complementary bases between self-complementary polynucleotide or nucleic acid molecules can be internal sequences, such that when the complementary portions of the two single-stranded molecules form a double strand or duplex, the non-complementary bases form a loop or bulge configuration and the overall structure resembles a hairpin. Such segments or regions of non-complementary bases between self-complementary polynucleotide or nucleic acid molecules can also be adjacent to complementary regions, in which case either or both of the 5' or 3' flanking regions need not form a double-stranded duplex.

[0106]

[0125] In a cell carrying a transgene, the transgene has been introduced / transferred via a vector, such as a viral vector (e.g., AAV). This process is called "transduction" or "transfection" of the cell. The terms "transduce" and "transfect" refer to the introduction of a molecule, such as a transgene, into a cell.

[0107]

[0126] A cell into which a transgene has been introduced is referred to as a "transduced" or "transfected" cell. Thus, a "transduced" or "transfected" cell refers to the change in a cell following the uptake of an exogenous molecule, such as a polynucleotide (e.g., an AAV vector containing a transgene), into the cell. Thus, a "transduced" or "transfected" cell is, for example, a cell into which an exogenous molecule has been introduced, or a progeny thereof. The cell(s) can grow and the introduced transgene can be transcribed and / or protein expressed.

[0108]

[0127] A cell that can be targeted for transduction with a vector (e.g., a viral vector) carrying a transgene can be any cell that is susceptible to infection or can be infected with a vector. Such cells include mammalian cells. Such cells may have a low, intermediate or high susceptibility rate to infection. Thus, target cells include cells of any tissue or organ type and of any origin (e.g., mesodermal, ectodermal or endodermal). Examples of cells that can be infected and used in the methods according to the invention include, for example and without limitation, cells of the liver (e.g., hepatocytes, sinusoidal endothelial cells), pancreas (e.g., beta pancreatic islet cells), lung, central or peripheral nervous system such as the brain (e.g., neural, glial or ependymal cells) or spine, kidney (HEK-293 cells), eye (e.g., retina, cellular components), spleen, skin, thymus, testis, lung, diaphragm, heart (cardiac), muscle or psoas, or intestine (e.g., endocrine), adipose tissue (white, brown or beige), muscle (e.g., fibroblasts), synoviocytes, chondrocytes, osteoclasts, epithelial cells, endothelial cells, salivary gland cells, inner ear neuronal cells or hematopoietic (e.g., blood or lymphatic) cells.

[0109]

[0128] In certain embodiments, cells that can be targeted for transduction with a vector (e.g., a viral vector) carrying a transgene may be seeded from a frozen cell aliquot or from a cell bank. In certain embodiments, cells that can be targeted for transduction with a vector (e.g., a viral vector) carrying a transgene may be seeded from cultured cells.

[0110]

[0129] Examples of cell lines that can be targeted for transduction with a vector (e.g., a viral vector) carrying a transgene include, for example and without limitation, 2V6.11, HEK-293, CHO, BHK, MDCK, 10T1 / 2, WEHI cells, COS, BSC 1, BSC 40, BMT 10, VERO, WI38, MRC5, A549, HT1080, B-50, 3T3, NIH3T3, HepG2, Saos-2, Huh7, HER, HEK, HEL, and HeLa cells.

[0111]

[0130] In certain embodiments, cells that can be targeted for transduction with a vector (e.g., a viral vector) carrying a transgene can transiently or stably express the E4 gene from an adenovirus. E4 gene expression ensures efficient cell transduction by the AAV vector.

[0112]

[0131] AVV vectors and vector sequences can include one or more "expression control elements" or "expression regulatory elements." Typically, an expression control or regulatory element is a nucleic acid sequence(s) that influences the expression of an operably linked polynucleotide (e.g., a transgene). The control elements present in the vector, including the expression control and regulatory elements set forth herein, such as promoters and enhancers, are included to facilitate proper transgene transcription and, where necessary, translation (e.g., promoters, enhancers, splicing signals for introns, maintenance of the correct reading frame of the polynucleotide and stop codons to allow in-frame translation of mRNA, etc.). Such elements typically act in cis, but may also act in trans.

[0113]

[0132] Expression control may be accomplished at the level of transcription, translation, splicing, message stability, etc. Typically, expression control elements that modulate transcription are juxtaposed near the 5' end (i.e., "upstream") of the transcribed polynucleotide. Expression control elements can also be located at the 3' end (i.e., "downstream") of the transcribed sequence or within the transcript (e.g., in an intron). Expression control elements can be located away from the transcribed sequence (e.g., 100-500, 500-1000, 2000-5000, 5000-10,000 or more nucleotides from the polynucleotide), even at significant distances. Nevertheless, due to polynucleotide length limitations, in AAV vectors, such expression control elements are typically within 1-1000 nucleotides from the polynucleotide.

[0114]

[0133] Functionally, expression of an operably linked transgene can be controlled, at least in part, by the element, such that the element modulates transcription of the polynucleotide and, optionally, translation of the transcript. A particular example of an expression control element is a promoter, which is usually located 5' of the transcribed sequence. Another example of an expression control element is an enhancer, which can be located 5' or 3' of the transcribed sequence, or within the transcribed sequence.

[0115]

[0134] As used herein, a "promoter" can refer to a DNA sequence that is typically located adjacent to a transgene. A promoter typically increases the amount of expression from a transgene compared to the amount expressed in the absence of the promoter.

[0116]

[0135] "Enhancer" as used herein can refer to a sequence located adjacent to a transgene. Enhancer elements are typically located upstream of promoter elements, but can also function and be located downstream or within a DNA sequence (e.g., a transgene). Thus, enhancer elements can be located 100, 200, or 300 or more base pairs upstream or downstream of a transgene. Enhancer elements typically increase expression of a transgene more than the increased expression provided by a promoter element.

[0117]

[0136] Examples of expression regulatory or expression control elements that can be used in the methods according to the invention include, for example and without limitation, CAG (SEQ ID NO:3), cytomegalovirus (CMV) immediate early promoter / enhancer, Rous sarcoma virus (RSV) promoter / enhancer, SV40 promoter, dihydrofolate reductase (DHFR) promoter, chicken beta actin (CBA) promoter, phosphoglycerol kinase (PGK) promoter, and elongation factor-1 alpha (EF1-alpha) promoter.

[0118]

[0137] Antibodies that bind to recombinant viral vectors useful for gene therapy, such as rAAV vectors, which may be referred to as "neutralizing" antibodies, can reduce, inhibit or decrease cell transduction by the viral vector, such that, without being bound by theory, cell transduction is reduced, inhibited or decreased, thereby reducing the introduction of virally packaged heterologous polynucleotides into cells and their subsequent expression and, if desired, their subsequent translation into proteins or peptides.

[0119]

[0138] An immune response, such as humoral immunity, may be generated against a wild-type virus in a subject exposed to the wild-type virus. Such exposure may result in pre-existing antibodies in the subject that bind to a viral vector based on the wild-type virus even before treatment with a gene therapy method using the viral vector. Alternatively, antibodies may be generated in a subject after treatment with a recombinant viral vector or exposure to a wild-type virus.

[0120]

[0139] Biological samples are typically obtained or produced from biological organisms. Examples of biological samples from subjects that may be analyzed using the methods according to the invention include, for example, without limitation, whole blood, serum, plasma, the like, and combinations thereof. Other biological samples from subjects that may be used in the methods according to the invention include, for example, without limitation, cerebrospinal fluid, or simply spinal fluid. Biological samples may lack cells or may include cells (e.g., red blood cells, platelets, and / or lymphocytes).

[0121]

[0140] Suitable subjects from which biological samples can be obtained for analysis by using the methods according to the present invention include mammals such as primates (e.g., humans), as well as non-human mammals. The term "subject" refers to animals, typically mammals such as humans, non-human primates (apes, gibbons, gorillas, chimpanzees, orangutans, macaques), domestic animals (dogs and cats), livestock (poultry such as chickens and ducks, horses, cows, goats, sheep, pigs), and laboratory animals (mice, rats, rabbits, guinea pigs). Suitable human subjects include fetal, neonatal, infant, juvenile, and adult subjects. Subjects also include animal disease models, e.g., other animal models such as mice and non-human primates.

[0122]

[0141] Suitable subjects (e.g., humans) from which biological samples may be analyzed using the methods according to the present invention include, for example, but are not limited to, subjects with loss-of-function and gain-of-function genetic diseases, disorders and deficiencies. Thus, subjects (e.g., humans) include subjects who are candidates for or undergoing gene replacement or supplementation therapy, such as protein / enzyme replacement therapy, as well as subjects (e.g., humans) who are candidates for or undergoing gene knockdown or knockout therapy.

[0123]

[0142] The term "loss-of-function" as used herein in relation to a gene deficiency refers to any mutation in a gene in which the protein that the gene encodes (i.e., the mutant protein) exhibits partial or complete loss of a function normally associated with the wild-type protein. This includes any disease, disorder or deficiency caused or resulting from insufficient expression or activity of a protein.

[0124]

[0143] The term "gain of function" as used herein in relation to genetic defects refers to any mutation in a gene in which the protein it encodes (i.e., mutant protein) gains a function not normally associated with the protein (i.e., wild-type protein) and causes or contributes to a disease or injury. A gain of function mutation can be a deletion, addition, or substitution of a nucleotide or nucleotides in a gene that can result in a change in the function of the encoded protein. A gain of function mutation can change the function of the mutant protein or cause interactions with other proteins. A gain of function mutation can also cause a reduction in or removal of a normal wild-type protein, for example, by interaction of the altered mutant protein with the normal wild-type protein. A gain of function mutation can result in a disorder or disease caused or resulting from abnormal, aberrant, or undesirable protein expression or activity.

[0125]

[0144] Suitable human subjects from which biological samples may be analyzed using the methods according to the invention include, for example, without limitation, subjects with a genetic disease or disorder amenable to treatment by gene therapy. Gene therapy treatment or therapy involves vector (e.g., viral vectors such as AAV vectors) mediated delivery of nucleic acids for the treatment of a disease or disorder.

[0126]

[0145] Suitable human subjects from which biological samples may be analyzed using the methods according to the invention include, for example, but are not limited to, those suffering from pulmonary diseases (e.g., cystic fibrosis), bleeding disorders (e.g., hemophilia A or hemophilia B with or without inhibitors), thalassemia, blood disorders (e.g., anemia), Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), epilepsy, lysosomal storage diseases (e.g., aspartylglucosaminuria, Batten disease, late-onset childhood neuronal ceroid lipofuscinosis type 2 (CLN2), cystinosis, Fabry disease, Gaucher disease types I, II, and III, glycogen storage disease type II, Pompe disease (caused by mutations in or loss of function or expression of acid α-glucosidase (GAA; which catalyzes the breakdown of glycogen)), GM2-gangliosidosis type I (Tay-Sachs disease ... Mucopolysaccharidosis type II (Sandhoff disease), Mucolipidosis type I (Sialidosis types I and II), II (I-cell disease), III (Pseudo-Hurler disease) and IV, Mucopolysaccharidosis (Hurler disease and variants, Hunter, Sanfilippo types A, B, C, D, Morquio types A and B, Marotouramy and Sly disease), Niemann-Pick disease types A / B, C1 and C2, and Schindler disease types I and II), Further included are subjects with hereditary angioedema (HAE), copper or iron storage disorders (e.g., Wilson's or Menkes' disease), lysosomal acid lipase deficiency, neurological or neurodegenerative disorders, cancer, type 1 or type 2 diabetes, adenosine deaminase deficiency, metabolic deficiencies (e.g., glycogen storage diseases), diseases of solid organs (e.g., brain, liver, kidney, heart), or infectious viral (e.g., Hepatitis B and C, HIV, etc.), bacterial or fungal diseases. Suitable human subjects from which biological samples may be analyzed according to the methods of the present invention further include subjects with blood clotting disorders, such as, but not limited to, hemophilia A, hemophilia B, deficiency of any of the following clotting factors: VII, VIII, IX, X, XI, V, XII, II, von Willebrand factor, or combined FV / FVIII deficiency, thalassemia, vitamin K epoxide reductase C1 deficiency, or gamma carboxylase deficiency.

[0127]

[0146] Subjects from which biological samples may be analyzed using the methods according to the invention further include, for example and without limitation, subjects who have developed inhibitory antibodies to a protein delivered to the subject for therapeutic purposes, for example and without limitation, subjects with Pompe disease, hemophilia A, or hemophilia B administered GAA, Factor VIII, and Factor IX, respectively, can develop inhibitory antibodies to GAA, Factor VIII, and Factor IX, respectively. Thus, subjects include subjects who do not have inhibitory antibodies as well as subjects who have inhibitory antibodies to the protein.

[0128]

[0147] Suitable human subjects from which biological samples may be analyzed according to the methods of the present invention further include subjects having a disease affecting or occurring in the central nervous system (CNS) or a neurodegenerative disease such as, for example, but not limited to, Alzheimer's disease, Huntington's disease, ALS, hereditary spastic hemiplegia, primary lateral sclerosis, spinal muscular atrophy, Kennedy's disease, polyglutamine repeat diseases, Parkinson's disease, and polyglutamine repeat diseases, including, for example, but not limited to, spinocerebellar ataxias (e.g., SCA1, SCA2, SCA3, SCA6, SCA7, or SCA17).

[0129]

[0148] The invention provides compositions, such as kits, that include packaging materials and one or more components. Kits typically include a label or package insert containing instructions for the components or instructions for in vitro, in vivo, or ex vivo use of the components. Kits can contain such components, e.g., recombinant viral (e.g., rAAV) vectors, empty capsid AAV particles, and optionally, one or more collections of reagents suitable for carrying out the methods of the invention.

[0130]

[0149] A kit is a physical structure that contains one or more components of the kit. The packaging material is capable of maintaining the components sterile and can be made of materials commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampoules, vials, tubes, etc.).

[0131]

[0150] The label or insert may include identification of one or more components, strength. The label or insert may include information identifying the manufacturer, lot number, manufacturing location and date, expiration date. The label or insert may include information identifying the manufacturer information, lot number, and date. The label or insert may include information on how to use the kit components. The label or insert may include instructions for using one or more of the kit components in a method or use of the invention.

[0132]

[0151] 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 invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.

[0133]

[0152] All patents, patent applications, publications, and other references cited herein, GenBank citations, and ATCC citations are incorporated by reference in their entirety. In the case of conflict, the specification, including definitions, will control.

[0134]

[0153] All features disclosed in this specification may be combined in any combination. Each feature disclosed in the specification may be replaced with an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, a disclosed feature is an example of a genus of equivalent or similar features.

[0135]

[0154] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "a nucleic acid" includes a plurality of such nucleic acids, reference to "vector" includes a plurality of such vectors, and reference to "virus" or "particle" includes a plurality of such viruses / particles.

[0136]

[0155] The term "about" as used herein refers to values ​​within 10% (i.e., plus or minus 10%) of the underlying parameter. For example, "about 1 to 10" means 1.1 to 10.1 or 0.9 to 9.9, about 5 hours means 4.5 hours or 5.5 hours, etc. The term "about" at the beginning of a series of values ​​modifies each of the values ​​by 10%.

[0137]

[0156] All numerical values ​​or numerical ranges include integers within such ranges and fractions of values ​​or integers within the range, unless the context clearly indicates otherwise. Thus, by way of example, reference to 95% or more includes 95%, 96%, 97%, 98%, 99%, 100%, etc., as well as 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, etc., 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, etc. Thus, by way of further example, reference to a numerical range such as "1 to 4" includes 1, 2, 3, as well as 1.1, 1.2, 1.3, 1.4, etc. For example, "1 to 4 weeks" includes 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days.

[0138]

[0157] Additionally, references to numerical ranges such as "0.01 to 10" include 0.011, 0.012, 0.013, etc., as well as 9.5, 9.6, 9.7, 9.8, 9.9, etc. For example, the range "from about 0.01 to about 10" includes 0.011, 0.012, 0.013, 0.014, 0.015, etc., as well as 9.5, 9.6, 9.7, 9.8, 9.9, etc.

[0139]

[0158] References to more (larger) or less integers include any numbers greater or less than the reference number, respectively. Thus, for example, reference to more than 2 includes 2.1, 2.2, 3, 3.1, 3.2, 4, 4.1, 4.2, 5, 5.1, 5.2, etc. References to "two or more" include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more times.

[0140]

[0159] Additionally, references to numerical ranges such as "1 to 90" include 1.1, 1.2, 1.3, 1.4, 1.5, etc., as well as 81, 82, 83, 84, 85, etc. For example, "between about 1 minute and about 90 days" includes 1.0 minute, 1.1 minute, 1.2 minutes, 1.3 minutes, 1.4 minutes, 1.5 minutes, etc., as well as 1 day, 2 days, 3 days, 4 days, 5 days...81 days, 82 days, 83 days, 84 days, 85 days, etc.

[0141]

[0160] The present invention is generally disclosed herein using affirmative language to describe numerous embodiments of the invention. The present invention also specifically includes embodiments in which certain subject matter, such as substances or materials, method steps and conditions, protocols, or procedures, are excluded in whole or in part. For example, in certain embodiments of the invention, materials and / or method steps are excluded. Thus, even though the present invention is not generally expressed herein in terms of what the invention does not include, aspects of the invention that are not expressly excluded are nevertheless disclosed herein.

[0142]

[0161] Several embodiments of the present invention have been described. Nevertheless, those skilled in the art can make various changes and modifications to the present invention to adapt it to various usages and conditions without departing from the spirit and scope of the present invention. Thus, the following examples are intended to illustrate but in no way limit the scope of the claimed invention. EXAMPLES

[0143] Example 1

[0162] This is a description of an exemplary cell-based in vitro assay for determining anti-AAV neutralizing antibody (NAb) titers from serum or plasma samples using an AAV vector expressing luciferase as a reporter transgene.

[0144]

[0163] This description is applicable to determining AAV NAb titers in serum and plasma and other biological samples from human clinical trial subjects, and human candidates for preclinical or nonclinical studies of gene therapy treatments, as well as subjects monitoring for AAV NAbs, such as subjects who have undergone gene therapy treatment or who are at risk or have developed AAV NAbs, and to determining the presence or amount of NAb before and / or after treatment, and optionally determining AAV NAb titers when it is desirable to reduce the amount of AAV NAb.

[0145]

[0164] The exemplary 2V6.11 cell line used (Mohammadi et al., Nucl. Acids Res. 32:2652 (2004)) is a genetically modified human embryonic kidney (HEK) 293 cell line that stably expresses the E4 gene from adenovirus. E4 gene expression ensures efficient transduction by AAV vectors. Other cells are also suitable for use.

[0146]

[0165] The serum samples serve as a source of potential AAV NAbs, which, if present, reduce AAV reporter vector transduction of 2V6.11 cells and reduce the measured luciferase activity. The serum samples are diluted, for example, but not limited to, a range of dilutions (e.g., 1:1, 1:2.5, 1:5, 1:10, 1:100, and 1:1000), and mixed with the reporter vector, such that AAV NAbs (if present) bind to the AAV reporter vector capsid surface and neutralization occurs.

[0147]

[0166] Some humans, and therefore their serum (or other biological samples), may contain other factors that do not bind AAV capsids but can affect AAV vector cell transduction positively (increase cell transduction) or negatively (decrease cell transduction), referred to herein as enhancers and inhibitors, respectively. Thus, in parallel, serially diluted serum samples are also preincubated with empty capsid AAV particles (EVs) followed by the addition of an AAV reporter vector.

[0148]

[0167] Preincubation of serum with empty capsid AAV particles (EV) provides a means for analyzing NAb samples with low titers (e.g., less than or equal to about 1:5, or ≦1:5) or negative for the presence of enhancers or inhibitors, for example, in subjects prior to AAV vector administration. This allows for the identification of false NAb positive subjects due to the presence of inhibitors and false NAb negative subjects due to the presence of enhancers. False NAb positive subjects can be treated by means of AAV vector-based gene therapy. Depending on the NAb titer, it may be decided to enroll or exclude false NAb negative subjects from the AAV vector-based gene therapy trial or method. Clinical samples taken from subjects 2 weeks after AAV vector infusion typically have high NAb titers (e.g., greater than or equal to about 1:5, or ≧1:5), and the use of empty capsid AAV particles (EV) is not necessary for NAb titer determination in these subjects.

[0149]

[0168] Quality controls (QC) can also be used to demonstrate the completeness and / or accuracy of the assay. For example, an AAV NAb control can verify that empty capsid AAV particles (EVs) are absorbing / binding AAV NAbs in order for the assay to determine the presence of any enhancers or inhibitors in the biological sample. Such quality controls are referred to herein as EV validity.

[0150]

[0169] The source of AAV NAb can be provided via one or more samples from one or more subjects expressing AAV NAb. Typically, AAV NAb is from pooled samples to ensure the presence of AAV NAb in the control. However, AAV NAb in any form, such as in a solution of PBS, can serve as a control as disclosed herein.

[0151]

[0170] 2V6.11 cells are first transduced with the prepared suspension of AAV reporter vector in 96-well plate, followed by overnight incubation. Luciferase activity is read by luminescence plate reader. After background (MIN) subtraction for the whole plate, sample results are run in triplicate wells and compared to the maximum (MAX) signal generated from positive control wells containing cells transduced with AAV reporter vector alone without serum. The dilution of serum sample at which luciferase activity is inhibited by about 50% or more is reported as the AAV NAb titer for the subject providing the sample.

[0152]

[0171] In an exemplary study, samples were collected from 89 human subjects prior to enrollment in a Phase I / II gene therapy trial for the treatment of hemophilia A. Of the 89 subjects evaluated, the assay revealed that 58 (65.2%) subjects had AAV NAb titers <1:1, which were considered negative, 16 (18%) subjects had titers ≥1:1 ​​but equal to or less than 1:10, and 15 (16.8%) subjects had titers >1:10.

[0153] Example 2

[0154] [Table 1]

[0155] [Table 2] TIFF2024174877000004.tif109149

[0156] Example 3 Materials and Equipment

[0172] The cells are the 2V6.11 cell line (Mohammadi et al., Nucl. Acids Res. 32:2652 (2004)), which is a human embryonic kidney (HEK) cell line that stably expresses the adenovirus E4 gene. Master, working, and assay ready cell banks are generated. A "cell bank" aliquot, 1e7 / mL, is stored in liquid nitrogen.

[0157] Reagents and storage conditions [Table 3]

[0158] Example 4 Reagent preparation Reagent volumes may be adjusted as needed.

[0159]

[0173] Add 50 mL of heat-inactivated FBS to 55 mL of complete DMEM (cDMEM). Add 5 mL of 100x penicillin / streptomycin / glutamine solution. Filter through a sterile bottle filter. Mark with a 1 month expiration date and store at 4°C.

[0160]

[0174] FACT QC Stock: AAV NAb titers in different lots of FACT may vary and the dilution scheme may be adapted to achieve 50% inhibition between the two intermediate dilutions. Other control AAV NAbs such as plasma or serum may be used.

[0161]

[0175] Example: For lot 3696, heat inactivated FACT sample is diluted 1:10 with heat inactivated FBS and frozen in aliquots. Store at <-60°C with an expiration date. Do not exceed 3 freeze-thaw cycles. HQC, MQC, and LQC are diluted during the assay set up using the following exemplary scheme.

[0176]

[0162] [Table 4]

[0163]

[0177] Ponasterone A: Quickly spin the virus and reconstitute in 100% ethanol at a concentration of 1 mg / mL. Vortex vigorously. Store at -20°C.

[0164]

[0178] Pluronic F68 in DPBS: Using a two-step dilution design, mix 1 part of 10% Pluronic stock solution with 10,000 parts of DPBS. Use on the day of preparation.

[0165]

[0179] AAV-CAG-luciferase vector: When the manufactured stock is first thawed, the vector is diluted with 0.001% Pluronic F68 to a stock concentration of 2 x 10 11 Obtain vg / mL. Dispense into 50 μL aliquots into sterile non-stick tubes. Store aliquots at -60°C.

[0166]

[0180] Empty capsid AAV particles (EV): After the initial thawing of the production stock, aliquot the appropriate volume into a sterile, non-stick tube and store at -60° C. After thawing the aliquot, discard the remainder.

[0167]

[0181] Test Samples: After initial thawing, serum samples are heat inactivated at 56°C for 30 minutes. 400 μL aliquots are prepared in sterile, non-stick tubes and stored at -60°C until use. After thawing, unused serum can be set aside and stored at -60°C or discarded. If set aside, label the freeze-thaw cycle on the tube. Do not exceed 3 freeze-thaw cycles.

[0168]

[0182] Dilute serum: Fetal Bovine Serum (FBS). Prepare single-use aliquots and store at -20°C.

[0169]

[0183] 1x Renilla Luciferase Assay Reagent: Thaw or bring to room temperature (ambient temperature) the assay buffer and substrate. A water bath can be used. Thawing creates a density and composition gradient, so mix well. The reagent may be thawed up to 5 times without any apparent loss of activity. To prepare the reagent, add 1 volume of 100x Renilla Luciferase Assay Substrate to 100 volumes of Renilla Luciferase Assay Buffer in a 50 mL conical tube. Mix thoroughly by vortexing the tube for 10-20 seconds. Store the reagent in the dark. Once prepared, the buffer is stable for 12 hours at ambient temperature.

[0170] Example 5 Day 1: Plating 2V6.11 cells, neutralizing samples, and transducing cells

[0184] C. Incubate the DMEM medium at 37 °C and CO 2 Place in incubator with loose cap for a minimum of 30 minutes. This allows the pH and temperature to reset. This medium will be used later for cell thawing and plating.

[0171]

[0185] Remove samples, FACT QC stocks, heat inactivated FBS and DMEM without FBS from storage and allow them to equilibrate to ambient temperature.

[0172]

[0186] Optional: If serum samples have not been heat inactivated, heat inactivate samples at 56°C for 30 minutes. Label tubes to indicate heat inactivation. Do not heat samples again if previously heat inactivated.

[0173]

[0187] Prepare dilutions from the FACT QC stock solution as shown in Figure 10A using a 96-well U-bottom tissue culture plate. This plate is called the dilution plate. The volumes indicated are sufficient for preparation of one sample and should be scaled up if necessary. Mix with a pipette until homogenous. Use the same dilution plate to prepare the sample dilutions shown in Figure 10B.

[0174]

[0188] Transfer 20 μL of each diluted FACT control and sample from the dilution plate to a new 96-well U-bottom neutralization plate, as shown in FIG.

[0175]

[0189] Add 20 μL of heat inactivated FBS to the MIN, MAX, and MAX.EV wells on the Neutralization Plate shown in FIG.

[0176]

[0190] If EVs are used only, 1.5 x 10 cells in DMEM without FBS 11 Prepare a working concentration of empty capsid AAV particles (EVs) in cp / mL. Avoid vigorous vortexing. A volume of 0.8 mL of diluted EVs is sufficient for one full assay plate.

[0177]

[0191] Only if EVs are used, add 10 μL of EVs to wells designated S.EV, FACT.EV and MAX.EV as shown in Figure 13A. Add 10 μL of DMEM without FBS to wells containing sample (S), FACT and MAX and 20 μL of DMEM without FBS to MIN control wells as shown in Figure 13B. Only if EVs are used, incubate the neutralization plate for 30±5 minutes at 37°C.

[0178]

[0192] 3.2 x 10 in DMEM without FBS 9 Prepare a working concentration of AAV reporter vector (RV) in 100 vg / mL. Avoid vigorous vortexing. A volume of 0.8 mL of diluted vector is sufficient for one full assay plate.

[0179]

[0193] Add 10 μL of RV to all wells except for wells containing the MIN control of the neutralization plate as shown in Figure 14. Incubate the neutralization plate for 30±5 minutes at 37°C.

[0180]

[0194] Remove one vial of 2V6.11 cells from cold storage and immediately place in a 37°C water bath for 4 minutes. If multiple vials are required, scale up accordingly. Remove the vial from the water bath, wash with 70% EtOH, and invert 180° twice to resuspend the cells.

[0181]

[0195] Use a 1 or 2 mL serological pipette to aspirate all the cell suspension from the cryovial and transfer it to an empty 15 mL conical tube. Cells are sensitive to shear forces at this step.

[0182]

[0196] Using a 10 mL pipette, add 9 mL of warm medium to the 15 mL conical tube. The first 3 mL should be added slowly, drop by drop. The remaining 6 mL of medium should be added more quickly from the pipette. At this point, the cells will be suspended in approximately 10 mL.

[0183]

[0197] Close the 15 mL conical tube and homogenize the cell suspension by flipping it 180° twice. Transfer 50 µL of the cell suspension to an Eppendorf tube for counting purposes and spin the remaining cell suspension at 240 x g for 10 min.

[0184]

[0198] Count live and dead cells. If viability is less than 70%, discard cells and thaw another vial.

[0185]

[0199] Cells were cultured at 4.0 x 10 in cDMEM. 5 Dilute to 100 cells / mL. Add Ponasterone A to a final concentration of 1 μg / mL. Each plate requires 10 mL of cell suspension.

[0186]

[0200] Take a flat-bottom 96-well tissue culture plate and add 100 µL of cell suspension (4.0 × 10) per well.4 Add 1000 cells (10 ... 2 Incubate in an incubator. Describe the cell bank lot number used and the viability.

[0187]

[0201] After neutralization, transfer 7.5 μL from each well on the neutralization plate to triplicate wells of the transduction plate as shown in Figure 15. Wrap the transduction plate in paraffin and store the transduction plate at 37°C, 5% CO 2 Incubate in a tissue culture incubator for 24 hours ± 30 minutes. Record the start time of incubation. Day 2: Measurement of luciferase activity

[0202] Cell culture supernatant containing secreted luciferase should be removed from the wells within approximately 24-25 hours after adding the 7.5 µL / well transferred from the neutralization plate to the transduction plate. Secreted luciferase accumulates in the culture medium over time, so altering this time will have a significant effect on the range of activity readout.

[0188]

[0203] Prepare a sufficient volume of 1x Renilla luciferase assay reagent and allow to equilibrate to room temperature prior to the assay. Remove the assay plate from the incubator. Observe the cells and note and note any signs of toxicity (low culture confluency, cells detached from the well bottom, cells that are attached but rounded, etc.).

[0189]

[0204] Turn on the GloMax Navigator System microplate reader, then turn on the tablet PC for at least 5 minutes prior to reading. After approximately 24 hours of incubation, transfer approximately 90 μL of culture supernatant to a 96-well tissue culture plate using a multichannel pipette to pipette the supernatant up and down once. Excess supernatant may be used for a second read if desired. If not used, it may be discarded or frozen at <-60°.

[0190]

[0205] Transfer 40 μL of culture supernatant into a 96-well black plate. The assay plate layout is shown in Figure 7. Optionally, if the plate is not to be read immediately, seal the plate with a sealer and store it at room temperature under dark conditions for up to 4 hours.

[0191]

[0206] Load plate into reader. Start GloMax Navigator software by tapping on the GloMax Navigator software icon. Select a protocol. Use the instrument settings detailed below. Reading mode: Illuminating Integration time: 1 second Plate type: 96 well standard Reading Area: Highlights the columns and rows being measured Injection and Delay: Injector 1: Deselect Injector 2: Select Volume: 100μL Delay: 2 seconds Speed: 230μL / sec Dark adaptation: 3 minutes Read the plate

[0192] Example 6 4. Data Analysis

[0207] The average (also called the mean), standard deviation (SD), and %CV of luminescence readings [RLU] are calculated for all control and sample dilutions.

[0193]

[0208] The average luminescence value [RLU] of the MAX3 wells and the MIN3 wells AV ] to calculate the signal-to-noise ratio (S / N): S / N = MAX[RLU AV ] / MIN[RLU AV ]

[0194]

[0209] The following calculations include the MAX, MIN, QC dilutions (e.g., HQC, MQC, LQC, HQC EV, MQC EV, and LQC EV) with and without the empty vector, respectively, and the mean luminescence values ​​[RLU] of triplicate wells of the test sample (S). AV ] to use:

[0210] % Inhibition MAX (%I.MAX)=100-[(S-MIN) / (MAX-MIN)]×100]. This is calculated for each sample (S) dilution as shown in Table 2.

[0195] [Table 5]

[0196]

[0211] % Inhibition S.EV (%I.SEV) = 100 - [(S-MIN) / (S.EV-MIN)] x 100]. This is calculated for each sample (S) dilution as shown in Table 3.

[0197] [Table 6]

[0198]

[0212] HQC% inhibition=100-[(HQC-MIN)×100) / (MAX-MIN)]

[0199]

[0213] EV interference = MAX / MAX.EV

[0200]

[0214] EV Effectiveness = HQC EV / HQC

[0201]

[0215] AAV NAb titer is defined as the lowest sample dilution with % inhibition > 50. Each sample is assigned an NAb titer as shown in Figures 6A-6E. Assay Acceptance Criteria

[0216] Assay Criterion 1: HQC (eg, FACT 1 vs. 100) must meet a predefined lot-specific %I.MAX ± 3SD.

[0202]

[0217] Assay Criterion 2: HQC (eg, FACT 1 vs. 100) must meet precision of CV%≦35% for triplicate well values ​​[RLU].

[0203]

[0218] Assay criterion 3: Positive transfection controls (MAX) must meet precision of CV%≦35% for luminescence values ​​[RLU] among triplicate wells.

[0204]

[0219] Assay criterion 4 (only applicable if EVs are used in the assay): Calculated EV interference must be in the range of 0.7-1.3.

[0205]

[0220] Assay Criterion 5 (only applicable if EV is used in the assay): The calculated EV must be ≧2.

[0206]

[0221] Assay plates that do not meet all of the above applicable criteria are considered failed plates. Results from failed plates should not be reported. The assay will be repeated.

[0207] Sample titer acceptance criteria

[0222] Criterion 1: Sample dilutions reported as final NAb titers should meet a precision of CV% ≤ 35% for luminescence values ​​[RLU] between triplicate wells. For any sample that does not meet the criteria, the result will be considered invalid and retesting will be required unless the sample is exhausted or specifically justified. Samples with valid results should not be reanalyzed unless technical errors are identified or reanalysis is desired.

[0208] Example 7

[0209] [Table 7]

[0210] Example 8

[0223] Spk1 (SEQ ID NO: 1) MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDNGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVESPVKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVP DPQPIGEPPAAPSGVGPNTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFP ADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFEFSYNFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGTAGTQQLLFSQAGPNNMSAQAKNWLPGPCYRQQRVSTTLSQNNNSNFAWTGATKYHLNGRDSLVNPGVAMATHKDDEERFFPSSGVLMFGKQGAGKD NVDYSSVMLTSEEEIKTTNPVATEQYGVVADNLQQQNAAPIVGAVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQAKLASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTEGTYSEPRPIGTRYLTRNL

[0211]

[0224] Spk2 (SEQ ID NO:2) MAADGYLPDWLEDNLSEGIREWWALQPGAPKPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGNGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVDQSPQEPDSSSGVGKSGKQPARKRLNFGQTGDSESVPD PQPLGEPPAAPTSLGSNTMASGGGAPMADNNEGADGVGNSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLSFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPAD VFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQGTTSGTTNQSRLLFSQAGPQSMSLQARNWLPGPCYRQQRLSKTANDNNNSNFPWTAASKYHLNGRDSLVNPGPAMASHKDDEEKFFPMHGNLIFGKEGTTASN AELDNVMITDEEEIRTTNPVATEQYGTVANNLQSSNTAPTTRTVNDQGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQIMIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRPL

[0212]

[0225] CAG promoter sequence (also shown diagrammatically in FIG. 16) (SEQ ID NO: 3)

[0213] [Table 8]

[0214] Related Applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 768,665, filed November 16, 2018. The entire contents of the aforementioned application are incorporated herein by reference, including all text, tables, sequence listings, and figures.

Claims

1. 1. A method for assaying for or detecting the presence of an adeno-associated viral (AAV) vector cell transduction enhancer in a biological sample from a subject, comprising: (a) providing infectious recombinant AAV particles comprising a recombinant AAV vector, (i) the vector comprises a reporter transgene; (ii) the reporter transgene comprises a single-stranded or self-complementary genome; (iii) the reporter transgene is operably linked to one or more expression control elements; and (iv) the reporter transgene is flanked by one or more flanking elements; Steps and (b) providing a cell capable of being infected with said infectious recombinant AAV particle; (c) contacting the cells of (b) with the infectious recombinant AAV particles of (a) under conditions such that the cells of (b) are transduced by the infectious recombinant AAV particles of (a) and the reporter transgene is expressed by the cells of (b); (d) measuring expression of the reporter transgene and assigning a value designated MAX that reflects the amount of reporter transgene expression in (c); (e) providing infectious recombinant AAV particles of (a); (f) providing a biological sample from a subject; (g) providing a cell capable of being infected with said infectious recombinant AAV particles; (h) mixing the biological sample of (f) with empty capsid AAV particles to produce a mixture (M) and incubating said M under conditions that allow said empty capsid AAV particles to bind to any AAV-binding antibodies present in said biological sample; (i) contacting the cells of (g) with the M and the infectious recombinant AAV particles of (e) under conditions that allow the infectious recombinant AAV particles of (e) to transduce the cells of (g) and express the reporter transgene in the cells of (g); (j) measuring expression of the reporter transgene and assigning a value, designated S.EV, that reflects the amount of reporter transgene expression in (i); (k) comparing the S.EV to the MAX, and if the S.EV is greater than the MAX, the biological sample from the subject contains an AAV vector cell transduction enhancer. The method includes:

2. 1. A method for assaying for or detecting the presence of an inhibitor of adeno-associated viral (AAV) vector cell transduction in a biological sample from a subject, comprising: (a) providing infectious recombinant AAV particles comprising a recombinant AAV vector, (i) the vector comprises a reporter transgene; (ii) the reporter transgene comprises a single-stranded or self-complementary genome; (iii) the reporter transgene is operably linked to one or more expression control elements; and (iv) the reporter transgene is flanked by one or more flanking elements; Steps and (b) providing a cell capable of being infected with said infectious recombinant AAV particle; (c) contacting the cells of (b) with the infectious recombinant AAV particles of (a) under conditions such that the cells of (b) are transduced by the infectious recombinant AAV particles of (a) and the reporter transgene is expressed by the cells of (b); (d) measuring expression of the reporter transgene and assigning a value designated MAX that reflects the amount of reporter transgene expression in (c); (e) providing infectious recombinant AAV particles of (a); (f) providing a biological sample from a subject; (g) providing a cell capable of being infected with said infectious recombinant AAV particles; (h) mixing the biological sample of (f) with empty capsid AAV particles to produce a mixture (M) and incubating said M under conditions that allow said empty capsid AAV particles to bind to any AAV-binding antibodies present in said biological sample; (i) contacting the cells of (g) with the M and the infectious recombinant AAV particles of (e) under conditions that allow the infectious recombinant AAV particles of (e) to transduce the cells of (g) and express the reporter transgene in the cells of (g); (j) measuring expression of the reporter transgene and assigning a value, designated S.EV, that reflects the amount of reporter transgene expression in (i); (k) comparing the S.EV to the MAX, where if the S.EV is less than the MAX, the biological sample from the subject contains an inhibitor of AAV vector cell transduction, expression or secretion of a protein encoded by the vector. The method includes:

3. 1. A method for assaying for or detecting the presence of an adeno-associated viral (AAV) vector cell transduction enhancer in a biological sample from a subject, comprising: (a) providing infectious recombinant AAV particles comprising a recombinant AAV vector, (i) the vector comprises a reporter transgene; (ii) the reporter transgene comprises a single-stranded or self-complementary genome; (iii) the reporter transgene is operably linked to one or more expression control elements; and (iv) the reporter transgene is flanked by one or more flanking elements; Steps and (b) providing a cell capable of being infected with said infectious recombinant AAV particle; (c) providing a biological sample from a subject; (d) mixing the biological sample of (c) with empty capsid AAV particles to produce a mixture (M) and incubating said M under conditions that allow the empty capsid AAV particles to bind to any AAV-binding antibodies present in the biological sample; (e) contacting the cells of (b) with the M and the infectious recombinant AAV particles of (a) under conditions that allow the infectious recombinant AAV particles of (a) to transduce the cells of (b) and express the reporter transgene in the cells of (b); (f) measuring expression of the reporter transgene; (g) comparing said expression of (f) to a positive (+) control, said + control being expression of said reporter transgene without said sample from said subject and without addition of said empty capsid AAV particles, wherein if expression of (f) is greater than said + control, then said biological sample from said subject contains an enhancer of AAV vector cell transduction. The method includes:

4. 1. A method for assaying for or detecting the presence of an inhibitor of adeno-associated viral (AAV) vector cell transduction in a biological sample from a subject, comprising: (a) providing infectious recombinant AAV particles comprising a recombinant AAV vector, (i) the vector comprises a reporter transgene; (ii) the reporter transgene comprises a single-stranded or self-complementary genome; (iii) the reporter transgene is operably linked to one or more expression control elements; and (iv) the reporter transgene is flanked by one or more flanking elements; Steps and (b) providing a cell capable of being infected with said infectious recombinant AAV particle; (c) providing a biological sample from a subject; (d) mixing the biological sample of (c) with empty capsid AAV particles to produce a mixture (M) and incubating said M under conditions that allow the empty capsid AAV particles to bind to any AAV-binding antibodies present in the biological sample; (e) contacting the cells of (b) with the M and the infectious recombinant AAV particles of (a) under conditions that allow the infectious recombinant AAV particles of (a) to transduce the cells of (b) and express the reporter transgene in the cells of (b); (f) measuring expression of the reporter transgene; (g) comparing said expression of (f) to a positive (+) control, said + control being expression of said reporter transgene without said sample from said subject and without addition of said empty capsid AAV particles, wherein if expression of (f) is less than said + control, then said biological sample from said subject contains an inhibitor of AAV vector cell transduction. The method includes:

5. 1. A method for detecting or quantifying AAV binding antibodies, comprising assaying for AAV binding antibodies that inhibit AAV vector cell transduction in a biological sample from a subject, the method comprising: (a) providing infectious recombinant AAV particles comprising a recombinant AAV vector, (i) the vector comprises a reporter transgene; (ii) the reporter transgene comprises a single-stranded or self-complementary genome; (iii) the reporter transgene is operably linked to one or more expression control elements; and (iv) the reporter transgene is flanked by one or more flanking elements; Steps and (b) providing a cell capable of being infected with said infectious recombinant AAV particle; (c) contacting the cells of (b) with the infectious recombinant AAV particles of (a) under conditions such that the cells of (b) are transduced by the infectious recombinant AAV particles of (a) and the reporter transgene is expressed by the cells of (b); (d) measuring expression of the reporter transgene and assigning a value designated MAX that reflects the amount of reporter transgene expression in (c); (e) providing infectious recombinant AAV particles of (a); (f) providing a diluted biological sample from a subject; (g) providing a cell capable of being infected with said infectious recombinant AAV particles; (h) mixing the diluted biological sample of (f) with empty capsid AAV particles to produce a mixture (M) and incubating said M under conditions that allow the empty capsid AAV particles to bind to any AAV-binding antibodies present in the diluted biological sample; (i) contacting the cells of (g) with the M and the infectious recombinant AAV particles of (e) under conditions that allow the infectious recombinant AAV particles of (e) to transduce the cells of (g) and express the reporter transgene in the cells of (g); (j) measuring expression of the reporter transgene and assigning a value, designated S.EV, that reflects the amount of reporter transgene expression in (i); (k) providing infectious recombinant AAV particles of (a); (l) providing a diluted biological sample from the same subject that provided said sample of (f); (m) providing a cell capable of being infected with said infectious recombinant AAV particles; (n) mixing the diluted biological sample of (l) with the infectious recombinant AAV particles of (k) to produce a mixture (M); (o) contacting the cells of (m) with M under conditions that allow the infectious recombinant AAV particles of (k) to transduce the cells of (m) and express the reporter transgene in the cells of (m); (p) measuring expression of said reporter transgene and assigning a value denoted S that reflects the amount of reporter transgene expression in (o); (q) performing steps (h)-(j) and (n)-(p) at least two times with different sample dilutions; (r) if S is less than MAX and S.EV is equal to or greater than MAX, then an AAV-binding antibody is present in said diluted biological sample that inhibits AAV vector cell transduction; and, optionally, (s) measuring expression of a negative control in cells capable of being infected by the infectious recombinant AAV particles of (a) but not infected by the infectious recombinant AAV particles of (a) to provide a background value denoted MIN, where MIN may be subtracted from any one of S, MAX and / or S.EV. The method includes:

6. 6. The method of claim 5, further comprising calculating, after step (r) or after step (s), a titer of AAV-binding antibody in the biological sample corresponding to the lowest dilution of the biological sample that inhibits reporter transgene expression by about 50% or more, wherein if the lowest dilution that inhibits reporter transgene expression by about 50% or more is greater than or equal to about 1 to 5, the titer is the dilution that results in about 50% or more inhibition as determined by the formula S / MAX, or if the lowest dilution that inhibits reporter transgene expression by about 50% or more is less than about 1 to 5, the titer is the dilution that results in about 50% or more inhibition as determined by the formula S / S.EV.

7. 6. The method of claim 5, further comprising the step (t) of calculating a titer of AAV binding antibody in said biological sample corresponding to the lowest dilution of said biological sample that inhibits reporter transgene expression by about 50% or more, wherein if the lowest dilution that inhibits reporter transgene expression by about 50% or more is greater than or equal to about 1 to 5, said titer is the dilution that results in about 50% or more inhibition as determined by the formula 100 - [[(S-MIN) / (MAX-MIN)] x 100], or if the lowest dilution that inhibits reporter transgene expression by about 50% or more is less than about 1 to 5, said titer is the dilution that results in about 50% or more inhibition as determined by the formula 100 - [[(S-MIN) / (S.EV-MIN)] x 100].

8. Providing infectious recombinant AAV particles of (a); providing a cell capable of being infected with said infectious recombinant AAV particle; providing empty capsid AAV particles; contacting the cells with the provided empty capsid AAV particles; transducing the cells of (b) with the infectious recombinant AAV particles of (a), and contacting the cells that have been contacted with the empty capsid AAV particles with the infectious recombinant AAV particles of (a) under conditions such that the reporter transgene is expressed by the cells; and measuring expression of the reporter transgene and assigning a value designated MAX.EV that reflects the amount of reporter gene expression.

9. The method according to any one of claims 5 to 8, further comprising the step of calculating the signal to noise ratio, denoted S / N, where S / N=MAX / MIN.

10. 10. The method of any one of claims 5 to 9, further comprising the step of calculating the percent coefficient of variation (%CV), where %CV = (standard deviation / mean) x 100%.

11. The method of any one of claims 5 to 10, further comprising the step of calculating EV interference, where EV interference = MAX / MAX.EV.

12. (a) measuring expression of said reporter transgene under control conditions comprising one or more dilutions of an AAV-binding antibody that binds to an AAV vector, and assigning a value, denoted HQC, to said expression measurement of the dilution that gives a preselected amount of reporter transgene expression relative to MAX or MAX-MIN; and / or 13. The method of any one of claims 5 to 12, further comprising the step of: (b) measuring expression of said reporter transgene under control conditions comprising a dilution of step (a) that provides said preselected amount of reporter transgene expression relative to MAX or MAX-MIN in the presence of empty capsid AAV particles, and assigning a value denoted HQC.EV.

13. 13. The method of claim 12, wherein the preselected amount of reporter transgene expression in step (a) is less than or equal to about 30% of MAX or MAX-MIN.

14. 14. The method of claim 12 or 13, further comprising the step of calculating EV validity, where EV validity = HQC.EV / HQC.

15. 14. The method of any one of claims 5 to 13, wherein steps (h) to (j) and (n) to (p) are performed at least 3, 4, 5 or 6 times with 3, 4, 5 or 6 different dilutions of the biological sample.

16. 15. The method of any one of claims 5-14, wherein the biological sample is diluted from about 1:1 to about 1:1000 prior to contacting or incubating with the infectious recombinant AAV particles of (a), (e) or (k).

17. 17. The method of any one of claims 5 to 16, wherein steps (h) to (j) and (n) to (p) are performed at a dilution of the biological sample of about 1:1, about 1:2.5, about 1:5, about 1:10, about 1:100 and / or about 1:1000.

18. 17. The method of any one of claims 1 to 16, wherein the method is completed within about 48 hours of step (c) or (d).

19. The method of any one of claims 1 to 18, wherein the cells capable of being infected with the infectious recombinant AAV particles are inoculated from a frozen cell aliquot.

20. 20. The method of any one of claims 1-19, wherein the cells capable of infection with the infectious recombinant AAV particles of any of steps (c), (e), (i), or (o) are contacted within about 2 hours after the cells capable of infection with the infectious recombinant AAV particles are thawed from freezing.

21. 21. The method of any one of claims 1 to 20, wherein the cells capable of being infected with the infectious recombinant AAV particles of any of steps (c), (e), (i), or (o) are at least about 80% confluent.

22. (a) a cell capable of being infected with an infectious recombinant AAV particle comprising a reporter transgene, and said infectious recombinant AAV particle comprising said reporter transgene; (b) a diluted biological sample from a subject, cells capable of being infected with infectious recombinant AAV particles, and empty capsid AAV particles; and (c) a diluted biological sample from the same subject that provided the sample in (b), a cell capable of being infected with an infectious recombinant AAV particle comprising a reporter transgene, and the infectious recombinant AAV particle comprising the reporter transgene. A carrier or plate on which the electrodes are individually disposed.

23. 23. The carrier or plate of claim 22, wherein the carrier or plate comprises plastic or glass.

24. 24. The carrier or plate of claim 22 or 23, wherein the carrier or plate is a multi-well plate or dish.

25. 25. The carrier or plate of any one of claims 22 to 24, wherein each of (a), (b) and (c) is disposed within a separate tube or separate single well of a multi-well carrier or plate.

26. The method of any one of claims 1 to 21 or the carrier or plate of any one of claims 22 to 25, wherein the cells capable of being infected with the infectious recombinant AAV particles comprise mammalian cells.

27. 26. The method of any one of claims 1 to 21 or the carrier or plate of any one of claims 22 to 25, wherein the cells are capable of being infected with AAV particles comprising a VP1, VP2 or VP3 sequence that is 90% or more identical to the VP1, VP2 or VP3 sequence of the AAV serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV3B, AAV-2i8, Rh74, Rh10, SPK1 (SEQ ID NO: 1), SPK2 (SEQ ID NO: 2) or a hybrid or chimeric VP1, VP2 or VP3 sequence of any one of the preceding AAV serotypes.

28. 26. The method according to any one of claims 1 to 21 or the carrier or plate according to any one of claims 22 to 25, wherein the cells are capable of being infected with AAV serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV3B, AAV-2i8, Rh74, Rh10, SPK1 (SEQ ID NO: 1), SPK2 (SEQ ID NO: 2) or a hybrid or chimera of any one of the aforementioned AAV serotypes.

29. 26. The method of any one of claims 1 to 21 or the carrier or plate of any one of claims 22 to 25, wherein the cells capable of being infected with the infectious recombinant AAV particles comprise 2V6.11, HEK-293, CHO, BHK, MDCK, 10T1 / 2, WEHI cells, COS, BSC 1, BSC 40, BMT 10, VERO, WI38, MRC5, A549, HT1080, B-50, 3T3, NIH3T3, HepG2, Saos-2, Huh7, HER, HEK, HEL, or HeLa cells, optionally any of said cells expressing the adenovirus E4 gene.

30. The method of any one of claims 1 to 21 or the carrier or plate of any one of claims 22 to 25, wherein the reporter transgene encodes a secreted or secretable protein.

31. The method of any one of claims 1 to 21 or the carrier or plate of any one of claims 22 to 25, wherein the reporter transgene encodes a protein that provides an enzymatic, colorimetric, fluorescent, luminescent, chemiluminescent or electrochemical signal.

32. The method according to any one of claims 1 to 21 or the carrier or plate according to any one of claims 22 to 25, wherein said reporter transgene comprises a luciferase gene.

33. The method according to any one of claims 1 to 21 or the carrier or plate according to any one of claims 22 to 25, wherein said reporter transgene comprises Renilla luciferase, Firefly luciferase, Gaussia luciferase gene.

34. The method of any one of claims 1 to 21 or the carrier or plate of any one of claims 22 to 25, wherein the reporter transgene comprises a β-galactosidase gene, a β-glucuronidase gene, or a chloramphenicol transferase gene.

35. The method according to any one of claims 1 to 21 or the carrier or plate according to any one of claims 22 to 25, wherein the reporter transgenes encode green fluorescent protein (GFP), red fluorescent protein (RFP) and alkaline phosphatase.

36. The method according to any one of claims 1 to 21 or the carrier or plate according to any one of claims 22 to 25, wherein the expression control element comprises a promoter and / or enhancer nucleic acid sequence operable in a mammalian cell.

37. The method of any one of claims 1 to 21 or the carrier or plate of any one of claims 22 to 25, wherein the expression control element comprises CAG (SEQ ID NO: 3), a cytomegalovirus (CMV) immediate early promoter / enhancer, a Rous sarcoma virus (RSV) promoter / enhancer, an SV40 promoter, a dihydrofolate reductase (DHFR) promoter, a chicken beta actin (CBA) promoter, a phosphoglycerol kinase (PGK) promoter, or an elongation factor-1 alpha (EF1-alpha) promoter.

38. The method of any one of claims 1 to 21 or the carrier or plate of any one of claims 22 to 25, wherein the flanking elements comprise one or more AAV inverted terminal repeats (ITRs).

39. The method of any one of claims 1 to 21 or the carrier or plate of any one of claims 22 to 25, wherein the reporter transgene is located between one or more 5' and / or 3' AAV ITRs.

40. The method of any one of claims 1 to 21 or the carrier or plate of any one of claims 22 to 25, wherein the flanking elements comprise mutated, modified or variant AAV ITRs that are not processed by AAV Rep proteins.

41. The method of any one of claims 1 to 21 or the carrier or plate of any one of claims 22 to 25, wherein the flanking elements comprise mutated, modified or variant AAV ITRs that enable or promote the formation of a self-complementary reporter transgene genome into a double-stranded inverted repeat structure in an infectious recombinant AAV particle.

42. 26. The method of any one of claims 1 to 21 or the carrier or plate of any one of claims 22 to 25, wherein the mutated, modified or variant AAV ITRs have a deleted D sequence and / or a mutated, modified or variant terminal separation site (TRS) sequence.

43. The method of any one of claims 1 to 21 or the carrier or plate of any one of claims 22 to 25, wherein the recombinant vector comprises a first inverted terminal repeat (ITR) of AAV; a promoter operable in a mammalian cell; the reporter transgene; a polyadenylation signal; and optionally a second ITR of AAV.

44. 26. The method of any one of claims 1 to 21 or the carrier or plate of any one of claims 22 to 25, wherein the recombinant vector comprises a restriction site allowing insertion of the reporter transgene downstream of a promoter operable in a mammalian cell, and a post-transcriptional regulatory element downstream of said restriction site, and wherein the promoter, the restriction site and the post-transcriptional regulatory element are located downstream of the 5' AAV ITR and upstream of the 3' AAV ITR.

45. The method of any one of claims 1 to 21 or the carrier or plate of any one of claims 22 to 25, wherein said infectious recombinant AAV particles comprise an AAV serotype that infects primates.

46. The method according to any one of claims 1 to 21 or the carrier or plate according to any one of claims 22 to 25, wherein said infectious recombinant AAV particles comprise an AAV serotype that infects humans or rhesus monkeys.

47. 26. The method of any one of claims 1 to 21 or the carrier or plate of any one of claims 22 to 25, wherein said infectious recombinant AAV particles comprise a VP1, VP2 or VP3 sequence that is 90% or more identical to the VP1, VP2 or VP3 sequence of the AAV serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV3B, AAV-2i8, Rh74, Rh10, SPK1 (SEQ ID NO: 1), SPK2 (SEQ ID NO: 2) or a hybrid or chimeric of any of the foregoing AAV serotypes.

48. 26. The method of any one of claims 1 to 21 or the carrier or plate of any one of claims 22 to 25, wherein said infectious recombinant AAV particles comprise AAV serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV3B, AAV-2i8, Rh74, Rh10, SPK1 (SEQ ID NO: 1), SPK2 (SEQ ID NO: 2) or a hybrid or chimera of any of the aforementioned AAV serotypes.

49. The method of any one of claims 1 to 21 or the carrier or plate of any one of claims 22 to 25, wherein the biological sample comprises a primate sample.

50. The method according to any one of claims 1 to 21 or the carrier or plate according to any one of claims 22 to 25, wherein the biological sample comprises serum.

51. The method according to any one of claims 1 to 21 or the carrier or plate according to any one of claims 22 to 25, wherein the biological sample comprises plasma.

52. The method according to any one of claims 1 to 21 or the carrier or plate according to any one of claims 22 to 25, wherein said biological sample comprises human serum or human plasma.

53. The method according to any one of claims 1 to 21 or the carrier or plate according to any one of claims 22 to 25, wherein the subject is a mammal.

54. The method according to any one of claims 1 to 21 or the carrier or plate according to any one of claims 22 to 25, wherein the subject is a human, optionally suffering from a genetic disease amenable to treatment by gene therapy.

55. The method according to any one of claims 1 to 21 or the carrier or plate according to any one of claims 22 to 25, wherein said subject suffers from a disorder due to insufficient expression or activity of a protein.

56. The method according to any one of claims 1 to 21 or the carrier or plate according to any one of claims 22 to 25, wherein the subject suffers from a disorder due to abnormal, aberrant or unwanted protein expression or activity.

57. The method according to any one of claims 1 to 21 or the carrier or plate according to any one of claims 22 to 25, wherein the subject suffers from a genetic disorder.

58. The method according to any one of claims 1 to 21 or the carrier or plate according to any one of claims 22 to 25, wherein the subject is a candidate for gene replacement or supplementation therapy.

59. The method according to any one of claims 1 to 21 or the carrier or plate according to any one of claims 22 to 25, wherein the subject is a candidate for gene knock-down or knock-out therapy.

60. The subject is furthermore diagnosed with a pulmonary disease (e.g., cystic fibrosis), a bleeding disorder (e.g., hemophilia A or hemophilia B with or without inhibitors), thalassemia, a blood disorder (e.g., anemia), Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), epilepsy, a lysosomal storage disease (e.g., aspartylglucosaminuria, Batten disease, cystinosis, late-onset childhood neuronal ceroid lipofuscinosis 2, type 2 (CLN2), Fabry disease, Gaucher disease types I, II, and III, glycogen storage disease type II (Pompe disease), GM2-gangliosidosis type I (Tay-Sachs disease), GM2-gangliosidosis type II (Sandhoff disease), mucolipidosis type I (sialidosis types I and II), II (I-cell disease), III (pseudo-Hurler disease) and IV, mucopolysaccharidosis (Hurler disease and variants, Hunter, San 26. The method of any one of claims 1 to 21 or the carrier or plate of any one of claims 22 to 25, wherein the patient is suffering from a genetic disorder such as rheumatoid arthritis (HLA), ...

61. The method of any one of claims 1 to 21 or the carrier or plate of any one of claims 22 to 25, further comprising the step of entering information regarding the presence or titer of AAV-binding antibodies in said biological sample into a report related to said subject.

62. The method of any one of claims 1 to 21 or the carrier or plate of any one of claims 22 to 25, further comprising the step of entering information regarding the presence or titer of AAV-binding antibodies in the biological sample into a database, thereby creating a database entry relating to said subject.

63. 26. The method of any one of claims 1 to 21 or the carrier or plate of any one of claims 22 to 25, wherein the AAV binding antibodies analysed or detected bind to AAV serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV3B, AAV-2i8, Rh74, Rh10 or a hybrid or chimera of any of the aforementioned AAV serotypes.

64. The method of any one of claims 1 to 21 or the carrier or plate of any one of claims 22 to 25, wherein the biological sample is diluted before contacting it with the infectious recombinant AAV particles of (a), (e) or (k).

65. The method according to any one of claims 1 to 21 or the carrier or plate according to any one of claims 22 to 25, wherein multiple dilutions of the biological sample are analysed.

66. The method according to any one of claims 1 to 21 or the carrier or plate according to any one of claims 22 to 25, wherein a plurality of different dilutions of the biological sample are analysed.

67. The method according to any one of claims 1 to 21 or the carrier or plate according to any one of claims 22 to 25, wherein the cells capable of being infected with the infectious recombinant AAV particles are not lysed prior to measuring the expression of the reporter transgene.

68. The method according to any one of claims 1 to 21 or the carrier or plate according to any one of claims 22 to 25, wherein the biological sample is heat inactivated.

69. 10. The method of claim 1, wherein one or more of steps (c), (d), (h), (i), (j), or (k) are performed using an automated system.

70. the automated system includes a contacting component, a measuring component, a mixing component, an incubating component, a processor, and a non-transitory electronic storage device, the non-transitory electronic storage device configured to cause the processor to control the contacting component, the measuring component, the mixing component, and the incubating component; The method further comprising: (c) contacting the cells of (b) with the infectious recombinant AAV particles of (a) with the contact component under conditions such that the cells of (b) are transduced by the infectious recombinant AAV particles of (a) and the reporter transgene is expressed by the cells of (b); (d) measuring expression of the reporter transgene using the measurement component and assigning using the processor a value designated MAX reflecting the amount of reporter transgene expression of (c); and storing using the processor the value designated MAX in the non-transitory electronic storage device; (h) mixing the biological sample of (f) with the empty capsid AAV particles using the mixing component to produce the mixture (M) and incubating the M with the incubating component under conditions that allow the empty capsid AAV particles to bind to any AAV-binding antibodies present in the biological sample that inhibit, reduce or decrease AAV vector cell transduction; (i) contacting the cell of (g) with the M and the infectious recombinant AAV particles of (e) with the contact component under conditions such that the infectious recombinant AAV particles of (e) are capable of transducing the cell of (g) and expressing the reporter transgene in the cell of (g); (j) measuring expression of the reporter transgene using the measurement component and assigning using the processor a value designated S.EV that reflects the amount of reporter transgene expression of (i); and storing using the processor the value designated S.EV in the non-transitory electronic storage device; (k) comparing the S.EV to the MAX with the processor, where if the S.EV is greater than the MAX, the processor determines that the biological sample from the subject contains an enhancer of AAV vector cell transduction, and optionally outputting for display, with the processor, an indication that the biological sample from the subject contains an enhancer.

70. The method of claim 69, further comprising:

71. 3. The method of claim 2, wherein one or more of steps (c), (d), (h), (i), (j), or (k) are performed using an automated system.

72. the automated system includes a contacting component, a measuring component, a mixing component, an incubating component, a processor, and a non-transitory electronic storage device, the non-transitory electronic storage device configured to cause the processor to control the contacting component, the measuring component, the mixing component, and the incubating component; The method further comprising: (c) contacting the cells of (b) with the infectious recombinant AAV particles of (a) with the contact component under conditions such that the cells of (b) are transduced by the infectious recombinant AAV particles of (a) and the reporter transgene is expressed by the cells of (b); (d) measuring expression of the reporter transgene using the measurement component and assigning using the processor a value designated MAX reflecting the amount of reporter transgene expression of (c); and storing using the processor the value designated MAX in the non-transitory electronic storage device; (h) mixing the biological sample of (f) with the empty capsid AAV particles using the mixing component to produce the mixture (M) and incubating the M with the incubating component under conditions that allow the empty capsid AAV particles to bind to any AAV-binding antibodies present in the biological sample that inhibit, reduce or decrease AAV vector cell transduction; (i) contacting the cell of (g) with the M and the infectious recombinant AAV particles of (e) with the contact component under conditions that allow the infectious recombinant AAV particles of (e) to transduce the cell of (g) and express the reporter transgene in the cell of (g); (j) measuring expression of the reporter transgene using the measurement component and assigning using the processor a value designated S.EV that reflects the amount of reporter transgene expression of (i); and storing using the processor the value designated S.EV in the non-transitory electronic storage device; (k) comparing the S.EV to the MAX with the processor, where if the S.EV is less than the MAX, the processor determines that the biological sample from the subject contains an inhibitor of AAV vector cell transduction, expression or secretion of a protein encoded by the vector, and optionally outputting for display, with the processor, an indication that the biological sample from the subject contains the inhibitor.

72. The method of claim 71, further comprising:

73. 4. The method of claim 3, wherein one or more of steps (d), (e), (f), or (g) are performed using an automated system.

74. the automated system includes a contacting component, a measuring component, a mixing component, an incubating component, a processor, and a non-transitory electronic storage device, the non-transitory electronic storage device configured to cause the processor to control the contacting component, the measuring component, the mixing component, and the incubating component; The method further comprising: (d) mixing the biological sample of (c) with the empty capsid AAV particles using the mixing component to produce the mixture (M) and incubating the M with the incubating component under conditions that allow the empty capsid AAV particles to bind to any AAV-binding antibodies present in the biological sample that inhibit, reduce or decrease AAV vector cell transduction; (e) contacting the cells of (b) with the M and the infectious recombinant AAV particles of (a) with the contact component under conditions that allow the infectious recombinant AAV particles of (a) to transduce the cells of (b) and express the reporter transgene in the cells of (b); (f) measuring the expression of the reporter transgene using the measurement component; (g) comparing said expression of (f) to said positive (+) control with said processor, wherein if said expression of (f) is greater than said + control, said processor determines that said biological sample from said subject contains an enhancer of AAV vector cell transduction, expression or secretion of a protein encoded by said vector, and optionally outputting for display, with said processor, an indication that said biological sample from said subject contains said enhancer.

74. The method of claim 73, further comprising:

75. 5. The method of claim 4, wherein one or more of steps (d), (e), (f), or (g) are performed using an automated system.

76. the automated system includes a contacting component, a measuring component, a mixing component, an incubating component, a processor, and a non-transitory electronic storage device, the non-transitory electronic storage device configured to cause the processor to control the contacting component, the measuring component, the mixing component, and the incubating component; The method further comprising: (d) mixing the biological sample of (c) with the empty capsid AAV particles using the mixing component to produce the mixture (M) and incubating the M with the incubating component under conditions that allow the empty capsid AAV particles to bind to any AAV-binding antibodies present in the biological sample that inhibit, reduce or decrease AAV vector cell transduction; (e) contacting the cells of (b) with the M and the infectious recombinant AAV particles of (a) with the contact component under conditions that allow the infectious recombinant AAV particles of (a) to transduce the cells of (b) and express the reporter transgene in the cells of (b); (f) measuring the expression of the reporter transgene using the measurement component; (g) comparing said expression of (f) to said positive (+) control with said processor, wherein if said expression of (f) is less than said + control, said processor determines that said biological sample from said subject contains an inhibitor of AAV vector cell transduction, expression or secretion of a protein encoded by said vector, and optionally outputting for display, with said processor, an indication that said biological sample from said subject contains said inhibitor.

76. The method of claim 75, further comprising:

77. 6. The method of claim 5, wherein one or more of steps (c), (d), (h), (i), (j), (n), (o), (p), or (s) are performed using an automated system.

78. the automated system includes a contacting component, a measuring component, a mixing component, an incubating component, a processor, and a non-transitory electronic storage device, the non-transitory electronic storage device configured to cause the processor to control the contacting component, the measuring component, the mixing component, and the incubating component; The method further comprising: (c) contacting the cells of (b) with the infectious recombinant AAV particles of (a) with the contact component under conditions such that the cells of (b) are transduced by the infectious recombinant AAV particles of (a) and the reporter transgene is expressed by the cells of (b); (d) measuring the expression of the reporter transgene using the measurement component and assigning using the processor the value designated MAX reflecting the amount of reporter transgene expression of (c); and storing using the processor the value designated MAX in the non-transitory electronic storage device; (h) mixing the diluted biological sample of (f) with the empty capsid AAV particles using the mixing component to produce the mixture (M) and incubating the M with the incubating component under conditions that allow the empty capsid AAV particles to bind to any AAV-binding antibodies present in the biological sample that inhibit, reduce or decrease AAV vector cell transduction; (i) contacting the cell of (g) with the M and the infectious recombinant AAV particles of (e) with the contact component under conditions that allow the infectious recombinant AAV particles of (e) to transduce the cell of (g) and express the reporter transgene in the cell of (g); (j) measuring the expression of the reporter transgene using the measurement component and assigning using the processor the value designated S.EV reflecting the amount of reporter transgene expression of (i); and storing using the processor the value designated S.EV in the non-transitory electronic storage device; (n) mixing the diluted biological sample of (l) with the infectious recombinant AAV particles of (e) using the mixing component to produce the mixture M; (o) contacting the cells of (m) with the M using the contact component under conditions that allow the infectious recombinant AAV particles of (k) to transduce the cells of (m) and express the reporter transgene in the cells of (m); (p) measuring expression of the reporter transgene using the measurement component and assigning using the processor the value designated S reflecting the amount of reporter transgene expression of (o); and storing using the processor the value designated S in the non-transitory electronic storage device; (s) optionally measuring expression of said negative control in cells that can be infected with said infectious recombinant AAV particles (a), but are not infected with said infectious recombinant AAV particles (a), using said measurement component to provide a background value denoted MIN, where MIN may be subtracted by said processor from any one of S, MAX and / or S.EV.

78. The method of claim 77, further comprising:

79. a titer of the AAV-binding antibody is calculated using the processor, the titer corresponding to the lowest dilution of the biological sample that results in about 50% or greater inhibition of reporter transgene expression, the processor being configured such that if the lowest dilution that results in about 50% or greater inhibition of reporter transgene expression is greater than or equal to about 1 to 5, the titer is determined by the formula S / MAX, or if the lowest dilution that results in about 50% or greater inhibition of reporter transgene expression is less than about 1 to 5, the titer is determined by the formula S / S.EV; Optionally, outputting, using said processor, an indication of said titer for display.

79. The method of claim 78, further comprising step (s) or (t).

80. a titer of the AAV binding antibody is calculated using the processor, said titer corresponding to the smallest dilution of the biological sample that results in about 50% or greater inhibition of reporter transgene expression, said processor being configured to determine said titer by the formula 100-[[(S-MIN) / (MAX-MIN)]×100] if said smallest dilution that results in about 50% or greater inhibition of reporter transgene expression is greater than or equal to about 1 to 5, or by the formula 100-[[(S-MIN) / (S.EV-MIN)]×100] if said smallest dilution that results in about 50% or greater inhibition of reporter transgene expression is less than about 1 to 5; and Optionally, outputting, using said processor, an indication of said titer for display.

80. The method of claim 78, further comprising the step (t).

81. Providing infectious recombinant AAV particles (a); providing a cell capable of being infected with said infectious recombinant AAV particle; providing empty capsid AAV particles; contacting the cells with the provided empty capsid AAV particles with the contacting component; contacting the cells contacted with the empty capsid AAV particles with the infectious recombinant AAV particles of (a) using the contact component under conditions such that the cells of (b) are transduced by the infectious recombinant AAV particles of (a) and the reporter transgene is expressed by the cells; measuring expression of the reporter transgene using the measurement component, assigning a value denoted MAX.EV reflective of the amount of reporter transgene expression using the processor, and storing the value denoted MAX.EV in the non-transitory electronic storage device using the processor. The method of any one of claims 78 to 80, further comprising:

82. 82. The method of any one of claims 78 to 81, further comprising the steps of: calculating, with the processor, a signal to noise ratio indicated by S / N, said S / N equal to MAX / MIN; storing, with the processor, the S / N in the non-transitory electronic storage; and optionally outputting, with the processor, an indication of the S / N for display.

83. 83. The method of any one of claims 78-82, further comprising the steps of calculating, with said processor, a percent coefficient of variation (%CV), %CV=(standard deviation / mean)×100%, storing, with said processor, the %CV in said non-transitory electronic storage device, and optionally outputting, with said processor, an indication of the %CV for display.

84. 84. The method of any one of claims 78 to 83, further comprising the steps of calculating, with said processor, EV interference, EV interference=MAX / MAX.EV, storing, with said processor, EV interference in said non-transitory electronic storage, and optionally outputting, with said processor, an indication of EV interference for display.

85. 85. The method of any one of claims 78-84, further comprising the steps of: calculating, with said processor, an HQC based on expression measurements of a dilution that provides a preselected amount of reporter transgene expression relative to MAX or MAX-MIN, and / or an HQC EV and / or HQC EV / HQC based on expression of the reporter transgene under control conditions comprising a dilution of step (a) that provides said preselected amount of reporter transgene expression relative to MAX or MAX-MIN in the presence of empty capsid AAV particles; storing, with said processor, the HQC and / or HQC EV and / or HQC EV / HQC in said non-transitory electronic storage; and optionally outputting, with said processor, an indication of the HQC and / or HQC EV and / or HQC EV / HQC for display.

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