A novel system for the detection and quantification of anti-AAV neutralizing antibodies.

Abstract

The present invention relates to a novel system for the detection and quantification of neutralizing antibodies to either adeno-associated virus (AAV) or recombinant AAV vectors, with improved sensitivity and specificity.

Description

[Technical field]

[0001] The present invention relates inter alia to a novel two-component system for the detection and quantification of neutralizing antibodies against adeno-associated virus (AAV) or recombinant AAV vectors. The invention comprises (i) a packaging cell line for the production of an AAV serotype or recombinant AAV vector against which antibodies are to be detected and which contains a tag sequence within the viral genome or within the transgene construct, and (ii) reporter gene cell lines incorporating reporter gene promoter constructs specifically responsive to the tag sequence and methods of using same to detect and optimally quantitate anti-AAV neutralizing antibodies against capsid, viral genome, and / or transgene or transgene product in a test sample. The invention further relates to high throughput screening methods for detecting and optimally monitoring immune responses to AAV with optimal sensitivity, signal strength, and biological fidelity. In a broader sense, the invention also relates to the use of packaging cell lines in combination with reporter gene cell lines in diagnostics. [Background technology]

[0002] Recombinant adeno-associated virus (AAV) vectors are used to treat an increasing number of genetic disorders, and although AAV-based gene therapy is well tolerated in the majority of cases, immune responses against either the AAV capsid, AAV genome, transgene, or transgene product are associated with reduced levels or complete loss of transgene expression and thus loss of efficacy (1). The development of neutralizing antibodies against recombinant AAV vectors is often associated with previous exposure to one or more AAV serotypes, usually during early childhood, and the high degree of cross-reactivity of anti-AAV antibodies across different AAV serotypes (1,2).

[0003] AAV is a single-stranded DNA virus whose genome consists of three genes, rep, cap, and aap, flanked by 145 bp inverted terminal repeats (ITRs), the first 125 nucleotides of which are palindromic sequences that fold back on themselves to form a T-shaped hairpin loop secondary structure. The ITRs act as primers for second strand synthesis and are required in cis for viral replication or transgene expression (3). The rep genes encode four nonstructural proteins, Rep78, Rep68, Rep52, and Rep40, that are encoded by a single ORF using two different promoters, p5 for Rep78 / 68 and p19 for Rep52 / 40, and are produced by alternative splicing. The Rep78 and Rep68 proteins bind to the ITRs and are required for genome replication, while Rep52 and Rep40 are responsible for packaging the viral genome into capsids during viral replication. The Rep proteins also play a role in site-specific genome integration. The cap gene encodes three structural proteins, Vp1, Vp2, and Vp3, transcribed from the same ORF under the control of the p40 promoter using alternative start codons and alternative splicing, which assemble in a 1:1:10 ratio to form the viral capsid (4). The Aap gene encodes an assembly activating protein (AAP) from an alternative reading frame within the Cap ORF. AAP localizes AAV capsid proteins to the nucleolus and assembles the three Cap proteins into a 60-mer icosahedral viral capsid (3).

[0004] Recombinant AAV vectors, lacking the rep, cap and aap genes, are limited in size to approximately 4.8 kb and consist of a suitable promoter, a transgene, and a polyA tail contained within the ITRs, which may be constitutive or tissue-specific depending on the intended use. AAV is replication-deficient and requires a helper virus, usually adenovirus,5 to supply the early helper proteins required for viral replication or transgene expression. Herpes simplex virus (HSV), and to some extent vaccinia virus, can substitute for the presence of adenovirus for the supply of early helper proteins (5,6). However, the early genes required for efficient AAV vector production can also be supplied by transfecting appropriate packaging cell lines with plasmids expressing the helper proteins, in addition to the transgene promoter construct and plasmids encoding the rep, cap and aap genes (7).

[0005] The capsid plays a key role in serotype-specific virus-cell interactions by first binding to carbohydrates on the cell surface. Thus, AAV serotypes 2, 3, and 6 bind heparin sulfate proteoglycans, while AAV1, 4, 5, and 6 bind sialic acid, and AAV9 binds galactose (8). Most AAV serotypes then interact with distinct parts of the novel AAV receptor (AAVR) that are required for cellular internalization (9). An additional highly conserved G protein-coupled receptor-like protein, GPR108, localized in the trans-Golgi, was recently identified as required for entry of all AAV serotypes, except for the highly divergent AAV5 serotype, which is AAVR-dependent but GPR108-independent (10). Thus, all AAV serotypes identified so far require either AAVR or GPR108, or both AAVR and GPR108, for cell transduction (9,10).

[0006] Although AAV infection is not associated with any disease in humans or other mammals, the efficacy of AAV-mediated gene therapy is often limited by the development of anti-AAV antibodies, which are often associated with previous exposure to AAV and a high degree of cross-reactivity between different serotypes, resulting in reduced or complete loss of expression levels of the transgene and thus loss of efficacy. Cell-based assays based on the use of reporter AAV vectors that are incubated with the test sample before in vitro transduction of cell lines, so-called transduction inhibition assays, often require a high multiplicity of infection (MOI), result in low sensitivity, suffer from non-specific effects and are at best a surrogate for the immune response to the actual AAV serotype or recombinant AAV vector against which the antibody response is determined. Therefore, there is a need to develop improved methods with increased sensitivity and specificity for the detection and quantification of neutralizing antibody responses to both AAV infection and treatment with recombinant AAV vectors. Summary of the Invention

[0007] The present invention has been made in light of the above-mentioned prior art, and it is an object of the present invention to provide a novel system for the detection and quantification of neutralizing antibodies against either adeno-associated virus (AAV) or recombinant AAV vectors, with improved sensitivity and specificity.

[0008] In order to obtain the above technical effects and provide a much improved method and cell line in this regard, the present invention relates to, inter alia, a cell line, which may be referred to as a packaging cell line, and may also be referred to as the first component or component 1, all of which are used interchangeably herein. The packaging cell line may comprise one or more different tag sequences.

[0009] In yet another aspect, the present invention relates to a reporter gene cell line incorporating a reporter gene promoter construct that is specifically responsive to one or more tag sequences of the packaging cell line. The reporter gene cell line may be considered as the second component or may be referred to as component 2, all used interchangeably herein.

[0010] In one embodiment of the present invention, component 1 and component 2 may function in concert or may be used in combination with each other.

[0011] In a further aspect, the present invention relates to a method of using components 1 and 2 for detecting and optimally quantifying anti-AAV neutralizing antibodies in a test sample. In particular, the present invention relates to the use of components 1 and 2 for improving the sensitivity and specificity for the detection and quantification of neutralizing antibody responses to both AAV infection and treatment with recombinant AAV vectors.

[0012] In another aspect, the invention relates to a two-component system comprising a packaging cell line (component 1) for producing an AAV serotype or recombinant AAV vector to which an antibody response is to be determined, comprising a tag sequence in the viral genome or transgene construct, and a reporter gene cell line (component 2) incorporating a reporter gene promoter construct that specifically responds to the tag sequence of component 1, and a method of using the same to detect and optimally quantitate anti-AAV neutralizing antibodies in a test sample. The invention further relates to a method of high throughput screening to detect and monitor immune responses to AAV with optimal sensitivity, signal strength and biological fidelity.

[0013] With respect to the tag sequence of the present invention, the cell line disclosed herein may comprise one or more tag sequences.When several tag sequences are present, the tag sequences may be identical or different.In another embodiment, when several tag sequences are present, a group of tag sequences may be identical, but the remaining tag sequences may be different.

[0014] In one embodiment, the tag sequence can be, for example, a tag sequence contained within the AAV genome, or, for example, a transgene promoter construct encoding a Gal4 DNA binding domain, or a tag sequence contained within the AAV genome, or a transgene promoter construct encoding a cGMP-specific receptor protein (CRP), or, for example, a tag sequence contained within the AAV genome, or a transgene construct encoding the trans-silencer VanR fused to the transactivator VP16.

[0015] As is apparent from the above, in embodiments where component 1 is used in combination with component 2, the reporter gene cell line incorporating the reporter gene promoter construct will specifically respond to one or more tag sequences of the packaging cell line, such that the reporter gene promoter construct will respond to each of the same or different tag sequences in component 1.

[0016] Component 1: Packaging cell line. In one aspect, the invention relates to a packaging cell line in which the components required for expression of an AAV serotype or recombinant AAV vector are provided by transient transfection or, preferably, are partially or fully integrated into the genome of the selected cell line, which may be referred to as the host cell line.

[0017] The choice of host cell line is determined by its safety profile, either in terms of analysis of the biological sample and the ability of the cells to withstand the toxicity of the AAV Rep protein following regulated expression of the rep gene and E4orf6.(11) Such cell lines include, but are not limited to, the human lung adenocarcinoma cell line A549 (ATCC catalog number CCL-185) or the human embryonic kidney cell line HEK293T (ATCC catalog number ACS-4500).

[0018] The selected cell line (host cell line) is transfected with the selected transgene under the control of a suitable promoter. The promoter can in principle be any suitable promoter known in the art. In one embodiment, the promoter can be an inducible promoter. In another embodiment, the promoter can be either a constitutive promoter, such as the cytomegalovirus (CMV) immediate early promoter, or a suitable tissue-specific promoter that regulates the expression of the transgene selected according to the desired tissue-specific expression of the transgene, and a suitable polyadenylation site, such as that from SV40, or an alternative polyadenylation site, contained within the ITR of AAV2 (as shown in Figure 1). Importantly, the cell line of component 1 contains a tag sequence within the ITR to monitor the specific recombinant AAV vector selected.

[0019] The host cells can further be cotransfected with the cap gene under the control of a constitutive promoter, such as the CMV minimal promoter (as shown in Figure 1). Because expression of the cap and rep genes on separate plasmids has been reported to increase AAV vector production (12), the host cells can be cotransfected with the cap and rep genes on two independent plasmids.

[0020] In contrast to HEK293T cells, the genes encoding E1 and E1A and E1B ORFs are not expressed in A549 cells. Therefore, A549 cells were transfected with the E1A gene and expressed under the control of a suitable inducible promoter to prevent the E1a protein from activating the Rep, E2 and E4 genes, resulting in cytotoxicity. Thus, in example 1, the rep and E1 genes are expressed under the control of a rapamycin-inducible promoter consisting of a 12-fold tandem repeat of the DNA-binding domain ZFHD1 fused to the FK506-binding protein (FKBP). The FKBP-rapamycin-related protein (FRAP) is fused to the VP16 transactivator from HSV-1, such that the addition of rapamycin induces the formation of a heterodimer between FKBP and FRAP, resulting in tightly regulated expression of the genes encoding the E1 early proteins.

[0021] Regulated expression of Rep78 / 68 has also been shown to increase the level of AAV vector production (12). It has been reported that transfection of HEK293 cells with E4 orf6 alone is sufficient for the production of recombinant AAV vectors without adenovirus (13), whereas the early proteins E2A, E4, and VA RNA are required for efficient production of recombinant AAV (14). Thus, in one embodiment, the packaging cell line can be co-transfected with expression vectors expressing E2A, E4, and VA RNA.

[0022] Component 2: Reporter cell line. In one aspect, the present invention also relates to a reporter gene cell line developed that responds specifically to a tag sequence contained within the AAV genome or transgene construct (a tag sequence within the ITR for monitoring the selected specific recombinant AAV vector of component 1) such that contact of the reporter cell with a packaging cell line producing a virus preparation or a virus expressing a tag sequence within the genome of the AAV serotype or recombinant AAV vector for which neutralizing antibodies are quantified results in activation of the reporter gene expressed by the reporter cell. It is understood that to increase sensitivity, the reporter gene cell line can be stably transfected with either a cell surface AAV receptor alone (9) or an endosomal AAV receptor alone (10), or with both a cell surface receptor and an endosomal AAV receptor. In example 1, the packaging cell line expresses a transgene promoter construct that includes a tag sequence encoding a Gal4 DNA binding domain that specifically binds to a Gal4 upstream activating sequence (UAS) that regulates the expression of the firefly luciferase (FL) gene in the reporter cell. Specifically, in Example 1, the AAV-2-responsive reporter cell line contains a 5-fold tandem repeat of Gal4 UAS that regulates expression of the FL reporter gene construct in HEK293 host cells (Figure 2). In a preferred embodiment, the AAV-responsive reporter cell line may also contain a second reporter gene, such as the Renilla luciferase reporter gene shown in Figure 3, under the control of a constitutive promoter, such as the SV40 minimal promoter shown in Figure 3, to allow normalization of AAV-induced firefly luciferase activity. The Renilla luciferase (RL) normalization gene allows for the definition of different types of human sera based on their ability to activate the AAV-responsive firefly luciferase and / or Renilla luciferase normalization gene (Table 1).Thus, the apparent neutralizing effect of the two human sera HS0 and HS4 from normal individuals can be distinguished from the actual neutralizing activity of the pool of human IV-IgG based on the activation of both the AAV-responsive FL reporter gene and the Renilla luciferase normalization gene by the two sera HS0 and HS4, in contrast to the activation of FL activity alone by human IV-IgG, which reflects nonspecific inhibition of AAV activity due to cytotoxicity (Figures 18-20).

[0023] In a preferred embodiment, the reporter cell line (component 2) is transiently or stably transfected with the immediate early enhancer protein E4orf6 (Figures 7 and 8), which significantly increases GAL4-UAS-regulated firefly luciferase expression when incubated with packaging cells (component 1) containing a cap gene encoding either the AAV2 cap protein or the AAV5 cap protein.

[0024] Interaction of packaging cell lines with reporter cells In one aspect, the present invention relates to a combination of component 1 and component 2 such that the two cell lines interact with each other (FIGS. 4 to 6). Thus, in one aspect, the present invention provides (a) contacting the virus produced by the packaging cells according to the invention with the sample to be tested for various times at various temperatures including or encompassing any one of temperatures +4, 20 or 37°C, or any temperature within the range of about +4°C to about 37°C, followed by incubation of the virus preparation and the sample with reporter cells at about 37°C for various times, preferably about 18 hours or more, and then quantifying the FL reporter gene activity using a suitable substrate such as the commercially available substrate Bright-Glo (Promega, Madison, WI) or any other suitable substrate allowing detection by any means that allows sequential quantification of FL activity and RL activity in a single well of a microtiter plate; (b) contacting the virus producing packaging cells with the sample to be tested for various times at various temperatures including or including any one of +4, 20 or 37°C, or any one of any temperature in the range of, for example, about +4°C to about 37°C, followed by incubating the packaging cell line and the sample with the reporter cells at 37°C for various times, preferably from about 6 hours to about 18 hours or more, and then quantifying either FL reporter gene activity alone using a suitable substrate, such as, for example, the commercially available substrate Bright-Glo (Promega, Madison, Wis.), or FL&RL activity using Dual-Glo or any other suitable substrate allowing detection by any means; (c) contacting the virus-producing packaging cell line with the sample to be tested and the reporter cells and directly incubating at about 37° C. for various times, preferably about 18 hours or more, and then quantifying FL reporter gene activity using a suitable substrate, such as, for example, the commercially available substrate Bright-Glo (Promega, Madison, Wis.), or any other suitable substrate allowing detection by any means, or FL&RL activity using Dual-Glo or any other suitable substrate allowing detection by any means; (d) freezing the virus-producing packaging cell line together with reporter cells at an appropriate cell concentration in a suitable cryoprotectant medium, thawing the cells, contacting the packaging cells / reporter gene cells with the sample to be tested, and incubating the sample, packaging cells-reporter cells at about 37° C. for various times, preferably about 18 hours or more, and then quantifying FL reporter gene activity using a suitable substrate such as the commercially available substrate Bright-Glo (Promega, Madison, Wis.) or any other suitable substrate allowing detection by any means, or FL&RL activity using Dual-Glo or any other suitable substrate allowing detection by any means; (e) In a preferred embodiment, the method comprises the steps of freezing the virus-producing packaging cell line and the reporter cells separately in a suitable cryoprotectant medium at an appropriate cell concentration, thawing the cells, contacting the virus-producing packaging cell line with the sample to be tested, incubating the virus-producing packaging cells and the sample with the reporter cells at about 37° C. for various times, preferably from about 6 hours to about 18 hours or more, and then quantifying the FL reporter gene activity using a suitable substrate such as the commercially available substrate Bright-Glo (Promega, Madison, Wis.) or any other suitable substrate allowing detection by any means, or the FL&RL activity using Dual-Glo or any other suitable substrate allowing detection by any means. The use of freeze-thawed virus-producing packaging cells obviates the need for the end user to generate the specific AAV serotype or recombinant AAV vector against which neutralizing antibodies are to be detected, and contacting the virus-producing packaging cell line with the sample to be tested obviates the need to extract and purify the challenge virus.

[0025] The cell-free AAV challenge virus / recombinant AAV vector or AAV virus / AAV recombinant vector producing packaging cells interact with the reporter gene cell line, resulting in virus uptake, internalization, and AAV serotype-specific activation of the FL reporter gene. The initial interaction of the cell-free challenge virus or AAV producing packaging cells with the reporter cells is likely mediated by the interaction of the capsid with carbohydrates on the cell surface in virus release and serotype-specific interactions. Thus, AAV serotypes 2, 3, and 6 are known to bind heparin sulfate proteoglycans, while AAV serotypes 1, 4, 5, and 6 bind sialic acid, and AAV9 binds galactose (8). The AAV serotypes then interact with either the AAV receptor required for cellular internalization (9) and / or the novel G protein-coupled receptor-like protein GPR108 localized in the trans-Golgi (10). In one embodiment, the reporter gene encodes an enzyme. In a preferred embodiment, the reporter is a luciferase, e.g. firefly luciferase, Renilla luciferase, Metridia luciferase, or novel luciferases such as those described in European Patent Application No. 21170068.7, which is incorporated herein by reference in its entirety, or novel luciferases present in solution as soluble active monomers comprising luciferases or fluorescent proteins such as green fluorescent protein (EGFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), blue fluorescent protein (BFP) and variants thereof exhibiting different excitation / emission spectra, horseradish peroxidase (HRP) or its various conjugates, secreted human placental alkaline phosphatase (SEAP) or its various conjugates, chloramphenicol acetyltransferase (CAT), or various linker proteins, or as fusion proteins with other proteins comprising luciferases, or attached to a solid surface such as a particle, bead, assay plate or tube.

[0026] In another embodiment, the reporter gene encodes a fluorescent protein. Useful fluorescent proteins include green fluorescent protein (GFP) and related fluorescent proteins, such as enhanced green fluorescent protein (EGFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), blue fluorescent protein (BFP), and variants thereof that exhibit different excitation / emission spectra.

[0027] In further embodiments, reporter cells may be co-transfected with a first reporter gene, such as a firefly luciferase (FL) reporter gene under the control of a chimeric promoter that responds specifically to a tag sequence present in the AAV genome or a tag sequence present in a recombinant AAV coding transgene promoter construct contained within the AAV ITRs, and a second reporter gene, such as Renilla luciferase (RL), under the control of a constitutive promoter that allows for normalization of tag sequence-activated FL activity with respect to the constitutive expression level of RL activity, making the assay results independent of cell number and providing a means to compensate for sample-to-sample variations in cell density due to cell loss or variations in the number of cells plated. The ability to normalize AAV-activated FL activity to the constitutive expression of RL activity also provides a means to compensate for serum matrix effects (15). Useful constitutively active promoters include, but are not limited to, the cytomegalovirus (CMV) early enhancer / promoter, the SV40 promoter, the UBC promoter, the PGK promoter, human β-actin (hACTB), human elongation factor-1 alpha (hEF-1α) and the cytomegalovirus early enhancer / chicken β-actin (CAG) promoter.

[0028] As mentioned herein, the present invention also provides a method for detecting and operationally quantifying the activity of anti-AAV neutralizing antibodies in a test sample, the method comprising the steps of: (i) providing a test sample suspected of containing anti-AAV antibodies, together with a control sample containing no anti-AAV antibodies and a positive control sample known to contain anti-AAV neutralizing antibodies; (ii) contacting the test sample with either a cell-free AAV challenge virus / recombinant AAV vector or a virus-producing packaging cell according to the invention, wherein the AAV challenge virus / recombinant AAV vector expresses a sequence tag within the genome of an AAV serotype or a transgene promoter construct, e.g., a Gal4 DNA binding domain; (iii) contacting the test sample with either a cell-free AAV challenge virus / recombinant AAV vector or a virus-producing packaging cell according to the invention together with a reporter cell, said cell expressing a reporter gene promoter construct, e.g. firefly luciferase, under the control of a tandem repeat of Gal4 UAS that specifically responds to a tag sequence within the genome of the AAV serotype or transgene construct produced by the packaging cell. (iv) Optionally, in a preferred embodiment, in order to provide assay normalization, the reporter cell line according to the present invention further comprises a construct for constitutive production of a reporter gene different from that used in the AAV-responsive reporter gene construct. For example, the constitutive production can be the constitutive production of a second luciferase, such as Renilla luciferase, Metridia luciferase, or a novel luciferase, such as those described in European Patent Application No. 21170068.7, the entirety of which is incorporated herein by reference. In a preferred embodiment, the reporter gene cell line according to the present invention is seeded into a 96, 384 or 1536 well assay plate. (v) measuring the activity of the first reporter protein in said cell line using a suitable substrate, such as the commercially available substrate Dual-Glo or any commercially available luciferase substrate; (vi) Measuring the activity of a second reporter protein in the cell line after a tag-responsive reporter gene luciferase is measured in the same sample using, for example, Stop&Glo reagent from the Dual-Glo system or any commercially available luciferase substrate that efficiently inhibits the activity of the first reporter protein, e.g., firefly luciferase (Lallemand C. et al J Immunol Res. 390:1-19, 2017, incorporated herein by reference in its entirety). (vii) The activity of a first luciferase normalized to the activity of a second luciferase is described in U.S. Patent Application Publication No. 2011 / 0189658, which is incorporated by reference in its entirety. (viii) Measuring the activity of the first tag-responsive reporter protein in reporter cells of the first cell sample, with or without normalization to the activity of the second luciferase, is described in U.S. Patent Application Publication No. 2011 / 0189658, which is incorporated by reference in its entirety. (ix) Measuring the activity of a first tag-responsive reporter protein in cells of a second control cell sample, with or without normalization to the activity of a second luciferase, is also described in U.S. Patent Application Publication No. 2011 / 0189658, the entire contents of which are incorporated herein by reference. (x) providing a ratio of reporter activity between a first control cell sample and a second control cell sample, wherein a ratio (first / second) lower than 1 indicates the presence of antibodies against an AAV serotype or recombinant AAV vector in the sample.

[0029] A further aspect of the invention relates to a method for high throughput screening of anti-AAV antibodies, said method comprising the steps of: (i) providing a test sample suspected of containing anti-AAV antibodies together with a control sample that does not contain anti-AAV antibodies and, in a preferred embodiment, an anti-AAV standard sample; (ii) contacting the test and control samples with a challenge virus preparation or virus-producing packaging cells according to the invention, wherein the cells are for the production of an AAV serotype or recombinant AAV vector against which an antibody response is determined, and wherein the challenge virus / recombinant AAV vector comprises a tag sequence within the viral genome or transgene construct. (iii) contacting the test sample, control sample and challenge virus / virus-producing packaging cells with a reporter gene cell line according to the invention, wherein the reporter cell line contains a first heterologous polynucleotide comprising a heterologous cis-acting regulatory sequence that is responsive to treatment of the cell line with a tag sequence present in the genome of the AAV serotype or a transgene promoter construct of a recombinant AAV vector, against which antibodies present in the test sample are detected or detected and quantified. (iv) wherein said promoter of the reporter cell line is operably linked to an open reading frame encoding a first reporter protein comprising, for example, firefly luciferase, Renilla luciferase, Metridia luciferase, or a novel luciferase such as those described in European Patent Application No. 21170068.7, the entirety of which is incorporated herein by reference. In a preferred embodiment, the cell line according to the invention is seeded into 96, 384 or 1536 well assay plates. (v) In a preferred embodiment, in order to provide assay normalization, the reporter cell line according to the invention further comprises a construct for the constitutive production of a luciferase different from that used in the AAV-responsive reporter gene construct. For example, the constitutive production may be the constitutive production of a second luciferase, such as Renilla luciferase, firefly luciferase, Metridia luciferase, or a novel luciferase as described in European Patent Application No. 21170068.7, the entire contents of which are incorporated herein by reference, using a suitable substrate, such as the commercially available Dual-Glo luciferase reagent from Dual-Glo Systems, or any commercially suitable luciferase substrate, to measure the activity of the first reporter protein in said cell line. (vi) measuring the activity of a second reporter protein in said cell line after a tag-responsive reporter gene luciferase is measured in the same sample using, for example, Stop&Glo reagent from the Dual-Glo system (Promega, Madison, Wis.), which effectively inhibits the activity of the first reporter protein. The activity of the first luciferase normalized to the activity of the second luciferase is described in U.S. Patent Application Publication No. 2011 / 0189658, which is incorporated herein by reference in its entirety. (vii) Measuring the activity of a first tag-responsive reporter protein in reporter cells of a first cell sample, with or without normalization to the activity of a second luciferase, as described in U.S. Patent Application Publication No. 2011 / 0189658, which is incorporated by reference in its entirety. (viii) Measuring the activity of the first tag-responsive reporter protein in cells of a second control cell sample, with or without normalization to the activity of the second luciferase, as described in U.S. Patent Application Publication No. 2011 / 0189658, which is incorporated by reference in its entirety. (ix) providing a ratio of reporter activity between a first control cell sample and a second control cell sample, wherein a ratio (first / second) lower than 1 indicates the presence of antibodies against an AAV serotype or recombinant AAV vector in the sample.

[0030] Thus, the present invention provides packaging cell lines transfected with genes encoding specific recombinant AAV vectors that express the genome or naturally occurring capsid, hybrid capsid, chimeric capsid or rationally designed capsid of a specific AAV serotype and encode specific transgene / promoter constructs labeled with specific tag sequences, and reporter cell lines that respond specifically to the tag sequences encoded by specific AAV serotypes or specific recombinant AAV vectors. If a common tag sequence is used to label each specific AAV serotype or each specific recombinant AAV vector, a single reporter cell line can be used to detect all different AAV serotypes or recombinant AAV vectors labeled with the same tag sequence. However, if different tag sequences are used to label each individual AAV serotype or each individual recombinant AAV vector, either different reporter cell lines that respond specifically to each individual specific tag sequence or reporter cell lines that contain each individual tag are required. Its use in various contexts allows for detection of neutralizing antibody activity with greater sensitivity, such that the EC50 of a dose-response curve of a sample containing neutralizing antibodies against a particular AAV serotype or recombinant AAV vector determined according to the present invention is at least reduced compared to prior art methods. The reduction can be about a 2-fold, such as about a 3-fold, such as about a 4-fold, such as about a 5-fold, such as about a 6-fold, such as about a 7-fold, such as about a 8-fold, such as about a 9-fold, such as a 10-fold, such as a 50-fold, such as about a 100-fold, or such as about a 1000-fold reduction.

[0031] In one embodiment, the use of components 1 and 2 in a diagnostic method, or any method comprising the use of components 1 and 2 according to the invention, may involve co-incubation of cells of component 1 and cells of component 2 with a biological sample taken from a subject to be tested for the presence of neutralizing antibodies. In one embodiment, the neutralizing antibodies may be antibodies against a particular AAV serotype or recombinant AAV vector.

[0032] In one aspect, the present invention relates to cells or cell lines and methods related thereto, which allow for higher specificity. The specificity may be at least about 75%, such as at least about 80%, such as at least about 85%, such as at least about 90%, such as at least about 95%, such as at least about 97.5%, such as at least about 98%, such as at least about 99%, such as at least about 99.5%.

[0033] In one aspect, the present invention provides increased sensitivity.

[0034] In another aspect, the present invention provides increased specificity.

[0035] In a further aspect, the present invention provides both increased sensitivity and increased specificity. [Brief description of the drawings]

[0036] [Figure 1]Component 1: Molecular constructs used to establish packaging cell lines of Example 1. Human lung adenocarcinoma cell line A549 was stably transfected with the following constructs: a selected transgene and a gal4-VP16 tag sequence separated by a sequence encoding 2A self-cleaving peptide contained within the ITR of a given AAV serotype AAV2 in the examples shown, F2A in the examples shown, under the control of a suitable promoter, a CMV constitutive promoter in the examples shown, and a polyadenylation site of SV40 in the examples shown. The rep and cap genes are expressed on different plasmids under the control of a constitutive promoter. The E1 gene is expressed under the control of an inducible promoter, which in the examples shown consists of a 12-fold tandem repeat of the ZFHD1 DNA binding domain fused to the FL506 binding protein (KFBP). The cells are also stably transfected with genes encoding E2A, E4 and VA RNA under the control of a constitutive promoter. [Diagram 2] Component 2: Molecular constructs used to establish the reporter cell line of Example 1. The human embryonic kidney cell line HEK293 was stably transfected with a chimeric promoter consisting of a minimal promoter controlling the expression of the firefly luciferase (FL) gene together with a 5-fold tandem repeat of the Gal4 upstream activating sequence (UAS) and a polyadenylation site from SV40 as shown in the example. [Diagram 3] Component 2: Molecular constructs used to establish reporter cell lines of the examples. Human embryonic kidney cell line HEK293 was stably co-transfected with a chimeric promoter consisting of a 5-fold tandem repeat of Gal4 upstream activating sequence (UAS) and a minimal promoter regulating the expression of the firefly luciferase (FL) gene with a polyadenylation site from SV40 shown in the examples, shown as the first reporter gene. Cells were also stably co-transfected with Renilla luciferase under the control of the SV40 minimal constitutive promoter, shown as the second reporter gene. [Figure 4]FIG. 1 shows component 1: production of recombinant AAV virions by the packaging cell line of Example 1, and component 2: their uptake into the reporter cell line of Example 1. [Diagram 5] FIG. 1 shows expression of the genome of the recombinant AAV vector and binding of the Gal4 DNA binding domain-VP6 hybrid protein to a 5-fold tandem repeat of the Gal4 upstream activating sequence (UAS), and strong activation of the firefly luciferase reporter gene, along with minimal activation of the Renilla luciferase reporter gene under the control of a constitutive promoter, reflecting the nonspecific cytotoxicity of the virus. [Figure 6] It shows neutralization of AAV viral particles by anti-AAV antibodies present in the sample (component 1) as they enter from the packaging cell line, thereby preventing them from entering the reporter cell line (component 2) and activating the Gal-4-UAS-regulated firefly luciferase reporter gene. This results in a lower firefly luciferase signal in the presence of neutralizing anti-AAV antibodies. Expression of the Renilla luciferase reporter gene under the control of a constitutive promoter remains unchanged, reflecting the nonspecific cytotoxicity of the virus. [Figure 7] FIG. 1 shows enhanced Gal4-UAS regulated FL expression after transient transfection of a reporter cell line (component 2) with a gene encoding the immediate early enhancer protein E4orf6 and incubation of the reporter cells with packaging cells (component 1) containing the cap gene encoding the AAV2 cap protein. [Figure 8] FIG. 1 shows enhanced Gal4-UAS regulated FL expression after transient transfection of a reporter cell line (component 2) with a gene encoding the immediate early enhancer protein E4orf6 and incubation of the reporter cells with packaging cells (component 1) containing the cap gene encoding the AAV5 cap protein. [Figure 9]Figure 1 shows the relationship between the number of reporter cells (component 2) seeded per well of a microtiter plate and Gal4-UAS regulated FL expression in the presence of packaging cells (component 1) containing the cap gene encoding the AAV2 cap protein. A total of 15,000 reporter cells / well in the presence of 7,500 packaging cells was found to give optimal FL expression. [Figure 10] FIG. 1 shows quantification of neutralizing activity of serum samples after contacting a packaging cell line expressing a recombinant AAV2 vector with the sample to be tested for 30 minutes at 20° C., followed by incubation of the packaging cell line and sample with reporter cells for 18 hours at 37° C. with or without contact, and then quantifying FL reporter gene activity using a commercially available substrate (Bright-Glo, Promega, Madison, WI) and quantifying luminescence with a luminometer (Glo-Max, Promega, Madison, WI). [Figure 11] FIG. 1 shows the effect of preincubating samples tested for the presence of anti-AAV2 neutralizing antibodies with packaging cells expressing the gene encoding the AAV2 Cap protein (component 1) for 30 minutes at room temperature, or without preincubation, followed by incubation with reporter cells (component 2) for 18 hours at 37° C. [Figure 12] FIG. 1 shows a comparison of procedures used to test samples for the presence of anti-AAV2 neutralizing antibodies using either the present invention or the transduction inhibition reference method described herein. [Figure 13] FIG. 1 shows a comparison of titrations of samples tested for the presence of anti-AAV2 neutralizing antibodies using either the invention or the transduction inhibition reference method described herein. [Figure 14] FIG. 1 shows the results of titration of samples of human IVIG tested for the presence of neutralizing antibodies that cross-react with various animal AAVs using the invention described herein. [Figure 15]FIG. 1 shows a comparison of titrations of serum samples from individual normal human donors tested for the presence of anti-AAV2 neutralizing antibodies using the invention described herein before and after ultrafiltration at a protein cutoff of 100,000 kDa. [Figure 16] FIG. 1 shows the results of titration of samples of human IVIG tested for the presence of neutralizing antibodies against various human wild-type AAV serotypes and AAV expressing chimeric CJ capsids using the invention described herein. [Figure 17] FIG. 1 shows a comparison of titrations of samples tested for the presence of anti-AAV2 neutralizing antibodies using any of the methods described herein in which virus-producing packaging cells are incubated with reporter cells overnight, or free virus at an equivalent multiplicity of infection (MOI) is incubated with reporter cells overnight. [Figure 18] FIG. 1 shows the apparent neutralizing effect of two human sera from normal individuals and a pool of human IgG (IV-IgG) on the activation of a reporter cell line (component 2 of the invention) containing both a firefly luciferase reporter gene regulated by a 5-fold tandem repeat of the Gal4 UAS and a Renilla luciferase normalization gene regulated by a thymidine kinase constitutive promoter in the presence of AAV8 packaging cells (component 1 of the invention). [Figure 19] Figure 1 shows the expression of Renilla luciferase (RL) in a reporter cell line (component 2 of the invention) containing both a firefly luciferase (FL) reporter gene regulated by a UAS 5-fold tandem repeat and a Renilla luciferase (RL) normalizer gene regulated by a thymidine kinase constitutive promoter. Renilla expression is stable when IV-IgG is used, but is reduced when two human sera from normal individuals (HS0 and HS4) are used, demonstrating that the apparent neutralizing effect of sera HS0 and HS4 in the presence of AAV8 packaging cells (component 1 of the invention) is due to non-specific cytotoxicity. [Figure 20]Figure 1 shows the ratio of FL to RL activity of two human sera HS0 and HS4 from normal individuals, and a pool of human IgG (IV-IgG), upon activation of a reporter cell line (component 2 of the invention) containing both a FL reporter gene regulated by a Gal4 UAS 5-fold tandem repeat and a RL normalization gene regulated by a thymidine kinase constitutive promoter in the presence of AAV8 packaging cells (component 1 of the invention), indicating that the apparent neutralizing effect of sera HS0 and HS4 was in fact due to non-specific cytotoxicity. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] In describing embodiments of the present invention, specific terminology is used for the sake of clarity, however, the invention is not intended to be limited to the specific terminology so selected, and it is understood that each specific term includes all technical equivalents that operate in a similar manner to accomplish a similar purpose.

[0038] The present invention has been made in light of the above-mentioned prior art, and it is an object of the present invention to provide a novel system for the detection and quantification of neutralizing antibodies against either adeno-associated virus (AAV) or recombinant AAV vectors with improved sensitivity and specificity. To this end, the present invention provides, inter alia, a packaging cell line (component 1) for producing AAV serotypes or recombinant AAV vectors against which antibody responses can be determined, comprising a tag sequence within the viral genome or a transgene promoter construct contained within the AAV ITRs.

[0039] The present invention also relates to a reporter gene cell line (component 2) incorporating a reporter gene promoter construct that specifically responds to the tag sequence of component 1, and a method of using same to detect and optimally quantitate anti-AAV neutralizing antibodies in a test sample.

[0040] The present invention further relates to high throughput screening methods for detecting and optimally monitoring immune responses to AAV with optimal sensitivity, signal strength, and biological fidelity.

[0041] Furthermore, the present invention also relates to the use of component 1 and / or component 2 in diagnosis or diagnostic methods, for example for the detection and quantification of neutralizing antibodies (NAb) against either adeno-associated virus (AAV) or recombinant AAV vectors.

[0042] Component 1: Packaging cell line As mentioned herein, the present invention relates to AAV packaging cell lines that may contain the components necessary for expression of an AAV serotype or recombinant AAV vector, either provided by transient transfection or preferably partially or fully integrated into the genome of the selected cell line, which may be referred to as a host cell or host cell line.

[0043] The present invention therefore relates to a cell or cell line (component 1), the cell comprising: (i) an AAV genome or transgene promoter construct comprising one or more tag sequences contained within the AAV ITRs; (ii) a cap gene of an AAV serotype or recombinant AAV vector encoding a naturally occurring Cap, a hybrid Cap, a chimeric Cap, or a rationally designed Cap operably linked to a constitutive or inducible promoter; (iii) an AAV rep gene operably linked to an inducible promoter; (iv) the AAV E2A, E4, and VA genes, each operably linked to a different respective native or inducible promoter; (v) an AAV E1A gene operably linked to an inducible promoter; The present invention includes at least one or more of the following:

[0044] In one embodiment, the cell comprises some or all of (i), (ii), (iii), (iv) and (v).

[0045] In certain embodiments, the cell comprises all of (i), (ii), (iii), (iv) and (v).

[0046] In one embodiment, the inducible promoters (iii), (iv), and (v) are different from each other and may be selected from the Tet-on / Tet-off system, the cumulative inducible repressor CymR system, the flavonoid phloretin-regulated TtgR repressor system, and the vanillic acid-regulated VanR repressor / KRAP system.

[0047] In a further embodiment, the AAV E1A gene can be regulated by an inducible promoter, which eliminates the need to regulate either the rep gene or the E2A, E4 and VA genes, by using one or more promoters that are inducible. In other words, in one embodiment, it is only necessary that the E1A gene be regulated by an inducible promoter. Thus, in one embodiment, the inducible promoter in (ii) can be an inducible promoter, which eliminates the need to regulate either the rep gene or the E2A, E4 and VA genes.

[0048] In one embodiment, the inducible promoter in (ii) may be selected from vanillic acid inducible promoters, for example using the octameric Van O operator VanO8.

[0049] In another embodiment, the inducible promoter in (ii) may be selected from, for example, the Tet-On, Tet-off, and Ponasterone A / Ecdysone systems.

[0050] It will be appreciated that to enhance sensitivity, host cell lines can be stably transfected with either cell surface AAV receptors alone (9) or endosomal AAV receptors alone (10), or with both cell surface and endosomal AAV receptors.

[0051] The choice of host cell line is determined by the analysis of biological samples and its safety profile for the ability of the cells to withstand the toxicity of AAV Rep protein after regulated expression of rep genes and E4orf6 (11). In one embodiment, the host cell is a metazoan cell. In another embodiment, the host cell line can include, but is not limited to, the human lung adenocarcinoma cell line A549 (ATCC catalog number CCL-185) or the human embryonic kidney cell line HEK293T (ATCC catalog number ACS-4500).

[0052] The selected host cell line can be transfected with the selected transgene under the control of either a constitutive promoter, such as the CMV promoter, or a suitable tissue-specific promoter that regulates the expression of the selected transgene depending on the desired tissue-specific expression of the transgene, and a suitable polyadenylation site, such as that from SV40, or an alternative polyadenylation site, such as that contained within the ITRs of AAV2 (Figure 1). In another embodiment, the constitutive promoter can be, for example, the SV40, UBC, EF1A, PGK, and CAGG promoters. In a further embodiment, the tissue-specific promoter can be, for example, a human retina-specific promoter, including the red opsin 2.1 and 1.7 promoters, the green opsin 2.1 and 1.7 promoters, the rhodopsin kinase 2 (hGPK1) promoter, the cone arrestin hCAR promoter, the vitelliform macular dystrophy (VMD2) promoter. Common muscle-specific promoters include the creatine kinase (CK) and myosin heavy chain (MyHC) promoters, or neuron-specific promoters such as the synapsin promoter.

[0053] Host cells can further be cotransfected with the cap gene under the control of a constitutive promoter, such as the CMV immediate early promoter (Figure 1). Because expression of the cap and rep genes on separate plasmids has been reported to increase AAV vector production (12), cells can be cotransfected with the cap and rep genes on two independent, regulated plasmids.

[0054] Since the genes encoding E1 and E1A and E1B ORFs are not expressed in A549 cells in contrast to HEK293T cells, A549 cells were transfected with the E1 gene under the control of a suitable inducible promoter to prevent the E1a protein from activating the Rep, E2 and E4 genes. Thus, as shown in Example 1 and in one embodiment of the present invention, the E1 gene is expressed under the control of a rapamycin-inducible promoter consisting of a 12-fold tandem repeat of the DNA-binding domain ZFHD1 fused to FK506-binding protein (FKBP). The FKBP-rapamycin-related protein (FRAP) is fused to the VP16 transactivator from HSV-1, such that the addition of rapamycin induces the formation of a heterodimer between FKBP and FRAP, resulting in tightly regulated expression of the genes encoding the E1 early proteins.

[0055] Regulated expression of Rep78 / 68 has also been shown to increase the level of AAV vector production (9). Transfection of HEK293 cells with E4 orf6 alone has been reported to be sufficient for the production of recombinant AAV vectors without adenovirus (14), while the early helper proteins E2A, E4, and VA RNA have been reported to be required for efficient production of recombinant AAV (15). Thus, packaging cell lines can be cotransfected with expression vectors expressing E2A, E4, and VA RNA in one case.

[0056] Component 2: Reporter cell line. In one aspect, the present invention relates to a developed reporter gene cell line (also referred to herein as component 2) that can specifically respond to a tag sequence contained within the AAV genome or transgene construct of component 1 such that contacting the reporter cell with an AAV challenge virus / recombinant AAV vector or a packaging cell producing the required AAV serotype or recombinant AAV vector for which neutralizing antibodies are quantified results in activation of the reporter gene expressed by the reporter cell line. In example 1, the packaging cell line expresses a transgene promoter construct that includes a tag sequence that encodes a Gal4 DNA binding domain that specifically binds to the Gal4 upstream activating sequence (UAS) that regulates the expression of the firefly luciferase (FL) gene. Specifically, in example 1, the reporter cell contains a 5-fold tandem repeat of the Gal4 UAS that regulates the expression of the FL reporter gene in a HEK293 cell background (Figure 1).

[0057] Interaction of packaging cell lines with reporter cells In various embodiments, the present invention relates to a method comprising at least one of the following steps: (a) contacting the virus produced by the packaging cells according to the present invention (component 1) with the sample to be tested for various times and at various temperatures including about +4°C, about 20°C or about 37°C, followed by incubating the virus preparation and the sample with reporter cells (component 2) at about 37°C for various times, preferably about 15 to about 18 hours or more, and then quantifying the FL reporter gene activity using a suitable substrate, for example the commercially available substrate Bright-Glo (Promega, Madison, Wisconsin). (b) contacting virus-producing packaging cells (component 1) with the sample to be tested for various periods of time at various temperatures including about +4°C, about 20°C, or about 37°C, followed by incubating the virus-producing packaging cells and the sample with reporter cells (component 2) at about 37°C for various periods of time, preferably about 15 to about 18 hours or more, and then quantifying FL reporter gene activity. (c) In a preferred embodiment, the step of contacting the virus-producing packaging cells (component 1) with the sample to be tested and the reporter cells (component 2) and directly incubating at about 37°C for various times, preferably from about 6 to about 18 hours or more, followed by quantifying the FL reporter gene activity using a suitable substrate, for example the commercially available substrate Bright-Glo (Promega, Madison, Wis.). (c) freezing the virus-producing packaging cells (component 1) together with the reporter cells (component 2) at an appropriate cell concentration in a suitable cryoprotectant medium, thawing the cells, contacting the packaging cells / reporter gene cells with the sample to be tested, and incubating the sample, packaging cells-reporter cells at about 37° C. for various times, preferably about 18 hours or more, followed by quantification of FL reporter gene activity using a suitable substrate, such as the commercially available substrate Bright-Glo (Promega, Madison, Wis.); (d) In a preferred embodiment, the virus-producing packaging cells (component 1) and the reporter cells (component 2) are frozen separately in a suitable cryoprotectant medium at an appropriate cell concentration, the cells are thawed and the packaging cell line is contacted with a sample to be tested for various times and at various temperatures including either about +4°C, about 20°C, or about 37°C, followed by incubating the packaging cells and sample with the reporter cells at about 37°C for various times, preferably about 18 hours or more, and then quantifying FL reporter gene activity using a suitable substrate such as the commercially available substrate Bright-Glo (Promega, Madison, Wisconsin).

[0058] Thus, in one aspect of the present invention, the incubation period between component 1, component 2 and the biological sample can be any time range, such as about 1 hour to about 48 hours, for example, about 6 hours to about 36 hours, for example, about 9 hours to about 32 hours, for example, about 12 hours to about 24 hours, or about 1 hour, about 4 hours, about 6 hours, about 9 hours, about 12 hours, about 15 hours, about 18 hours, about 20 hours, about 24 hours, about 28 hours, about 30 hours, about 32 hours. In one aspect, the incubation period can be, for example, overnight, which can be any time range between about 6 hours to about 10 hours, or any time range between about 6 hours to about 18 hours. In another aspect, the incubation period can be, for example, about 15 hours to about 30 hours.

[0059] In further embodiments, the incubation period between component 1, component 2 and the biological sample can be any period from about 15 hours to about 18 hours.

[0060] The temperature during incubation can be any range of temperatures from about 4° C. to about 37° C., alternatively about 4° C., about 10° C., about 15° C., about 20° C., about 25° C., about 30° C., about 35° C., or about 37° C. Alternatively, the temperature can be in the range of about 30° C. to about 39° C. Thus, the temperature can relate to the incubation of component 1 and / or component 2 cells and / or the biological sample to be analyzed.

[0061] In a preferred embodiment, as shown in FIG. 9, about 7,500 packaging cells (component 1) are mixed with about 15,000 reporter cells (component 2) without pre-incubation of the sample with the packaging cells (component 1) prior to contact with the reporter cells (component 2) and incubation at 37° C. for about 18 hours (FIG. 10).

[0062] In one aspect, the AAV preparation or virus-producing packaging cells interact with the reporter gene cells, resulting in virus uptake, internalization, and AAV serotype-specific activation of the FL reporter gene. The initial interaction of the virus preparation or virus-producing packaging cells with the reporter cells is likely mediated by the interaction of the capsid with carbohydrates on the cell surface in the release of the virus and serotype-specific interaction. Thus, it is known that AAV serotypes 2, 3, and 6 bind to heparin sulfate proteoglycans, while AAV1, 4, 5, and 6 bind to sialic acid, and AAV9 binds to galactose (8). The AAV serotypes then interact with either the AAV receptor required for cellular internalization (9) and / or the novel G protein-coupled receptor-like protein GPR108 localized in the trans-Golgi (10). In one embodiment, the reporter gene encodes an enzyme or any other means that allows detection in a suitable manner. In a preferred embodiment, the reporter is a luciferase, e.g. firefly luciferase, or Renilla luciferase, Metridia luciferase, novel luciferases such as those described in European Patent Application No. 21170068.7, which is incorporated herein by reference in its entirety, or novel luciferases present in solution as soluble active monomers or as fusion proteins with other proteins comprising luciferases, such as green fluorescent protein (EGFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), blue fluorescent protein (BFP) and variants thereof exhibiting different excitation / emission spectra, horseradish peroxidase (HRP) or its various conjugates, secreted human placental alkaline phosphatase (SEAP) or its various conjugates, chloramphenicol acetyltransferase (CAT), or various linker proteins, or novel luciferases attached to solid surfaces such as particles, beads, assay plates or tubes.

[0063] In another embodiment, the reporter gene encodes a fluorescent protein. Useful fluorescent proteins include green fluorescent protein (GFP) and related fluorescent proteins, such as enhanced green fluorescent protein (EGFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), blue fluorescent protein (BFP), and variants thereof that exhibit different excitation / emission spectra.

[0064] In a further embodiment, the reporter cells are co-transfected with a first reporter gene, such as a firefly luciferase (FL) reporter gene under the control of a chimeric promoter that responds specifically to a tag sequence present in the AAV genome or a tag sequence present in a recombinant AAV-encoding transgene construct (obtained from component 1), and a second reporter gene, such as Renilla luciferase (RL), under the control of a constitutive promoter that allows for normalization of tag sequence-activated FL activity with respect to the constitutive expression level of RL activity, making the assay independent of cell number and providing a means to compensate for sample-to-sample variations in cell density due to cell loss or variations in the number of cells plated. The ability to normalize AAV-activated FL activity to the constitutive expression of RL activity also provides a means to compensate for serum matrix effects (16). Useful constitutively active promoters include, but are not limited to, the cytomegalovirus (CMV) early enhancer / promoter, the SV40 promoter, the UBC promoter, the PGK promoter, human β-actin (hACTB), human elongation factor-1 alpha (hEF-1α), the thymidine kinase (TK) promoter, and the cytomegalovirus early enhancer / chicken β-actin (CAG) promoter.

[0065] The present invention provides a novel system for the detection and quantification of neutralizing antibodies against wild-type AAV serotypes or recombinant AAV vectors with improved sensitivity, accuracy and specificity.

[0066] To solve the technical problems outlined above, a two-component system is described that comprises a packaging cell line (component 1) for producing the AAV serotype or recombinant AAV vector to which the antibody response is to be determined, comprising a tag sequence in the viral genome or transgene construct, and a reporter gene cell line (component 2) incorporating a reporter gene promoter construct that responds specifically to the tag sequence of component 1, and a method of using the same to detect and optimally quantify anti-AAV neutralizing antibodies in a test sample. The invention further relates to a method of high throughput screening to detect and optimally monitor the immune response to AAV infection or treatment with recombinant AAV vectors with optimal sensitivity, signal strength and biological fidelity.

[0067] Component 1: Packaging cell lines. Stable AAV packaging cell lines can be established in which the components required for expression of an AAV serotype or recombinant AAV vector are supplied by transient transfection or, in preferred embodiments of the invention, are integrated into the genome of the selected cell line.

[0068] As a non-limiting example, stable cell lines for the production of a particular AAV serotype or recombinant AAV vector can be established by transfecting the human lung adenocarcinoma cell line A549 (ATCC catalog number CCL-185) with a transgene of choice under the control of either a constitutive promoter or an appropriate tissue-specific promoter, selected depending on the intended use, and a polyadenylation site contained within the ITRs of AAV2 (as shown in FIG. 1).

[0069] In order to construct an expression vector for a transgene promoter construct that confers the highest possible level of expression, various types of mutants of the promoter are designed in silico to reduce the size of the promoter and increase the transcription of the transgene. Codon-optimized transgene promoter constructs containing tag sequences, such as the Gal4 DNA binding domain of Example 1, are then synthesized in vitro and used to construct a series of chimeric transgene promoter sequences and either a synthetic polyadenylation site contained within the ITR of AAV2, an SV40 polyadenylation site, or a human growth hormone polyadenylation site, selected according to their effectiveness.

[0070] In one embodiment, the cells in component 1 may express a transgene promoter construct comprising a tag sequence encoding a Gal4 DNA binding domain that specifically binds to a Gal4 upstream activating sequence (UAS) that regulates expression of the firefly luciferase (FL) gene, or express a VanR sequence within the AAV genome or transgene construct, or an AAV genome or transgene promoter construct encoding a cGMP-specific receptor protein (CRP).

[0071] In a further embodiment of the invention, the tag sequence contained within the AAV genome or transgene construct may encode the trans-silencer VanR fused to the transactivator VP16.

[0072] In a further embodiment, the cells in component 1 may contain one or more tag sequences.

[0073] In further embodiments, in instances where more than one tag sequence is present, such tag sequences may be different from one another.

[0074] In another embodiment, the tag sequences may be the same / identical.

[0075] In one embodiment, the cells of component 1 may contain a recombinant AAV vector.

[0076] Transgene expression can be increased by modifying the ITRs to generate self-complementary intermediates so that the transgene is expressed without the need for second-strand DNA synthesis (13). These constructs are then tested for their ability to drive transgene transcription in a series of transient transfection experiments in packaging cell lines established in, for example, A549 cells, which contain all the early helper proteins necessary to replicate the transgene encoded by the AAV vector, but lack the transgene promoter construct with the tag sequence contained within the ITR of AAV2. Expression levels are monitored by Western blot using an appropriate detection antibody and by the level of activation of the firefly luciferase (FL) reporter gene under the control of the chimeric transgene-responsive promoter. Results are normalized with respect to the expression of Renilla luciferase (RL) after co-transfection of cells with the RL reporter gene under the control of a constitutive promoter.

[0077] If the normalized level of transgene expression monitored by Western blot is estimated to be optimal relative to expression of the standard ITRs and the normalized increase in FL expression is estimated to be sufficient using a reporter cell line such as that of Example 1 expressing tandem repeats of the Gal4 UAS that regulate the expression of a firefly luciferase reporter gene that responds specifically to the Gal4 DNA binding domain tag contained within the AAV genome or recombinant AAV vector, the construct is used to establish a packaging cell line by transient transfection or, in a preferred embodiment, by establishment of a stable packaging cell line in which the necessary components have been stably integrated into the genome of a host cell line such as A549 cells.

[0078] Packaging cell lines can be transfected with an expression vector of the cap gene under the control of a constitutive promoter. Thus, in example 1, the cap gene is expressed under the control of the CMV immediate early promoter and used to establish packaging cell lines after transient transfection with the constituents, or in a preferred embodiment by establishing stable packaging cell lines in which the necessary components are stably integrated into the genome of the host cell line, e.g., A549 cells.

[0079] To ensure optimal regulated expression of the rep and early genes required to express the transgene in Example 1, the gene encoding E1 was expressed under the control of a rapamycin-inducible promoter consisting of a tandem repeat of a mutant of the DNA-binding domain ZFHD1 fused to an in silico designed FK506-binding protein (FKBP). A mutant of the FKBP-rapamycin-related protein (FRAP) was fused to the VP16 transactivator from HSV-1, also designed in silico, such that addition of rapamycin induces the formation of a heterodimer between FKBP and FRAP, resulting in tightly regulated expression of the gene encoding the E1 early helper protein.

[0080] Constructs were then synthesized in vitro and tested for their ability to regulate the expression of E1 proteins in a series of transient transfection experiments in the A549 packaging cell line. The protein encoded by the E1A gene initiates AAV replication by increasing rep expression by transcriptionally activating the p5 and p19 promoters. To ensure controlled expression of the Rep78 / 68 protein (14), which has been shown to increase AAV vector production levels, the rep gene was also placed under the control of the ZFHD1 promoter. Tandem repeats of the DNA-binding domain ZFHD1 fused to FK506-binding protein (FKBP) and a mutant of FKBP-rapamycin-related protein (FRAP) fused to the VP16 transcriptional activator were designed in silico, synthesized in vitro, and tested in transient transfection experiments in A549 cells, such that the addition of rapamycin induces the formation of a heterodimer between FKBP and FRAP, which results in tightly regulated expression of the gene encoding the E1 early enhancer protein.

[0081] In Example 1, in A549 cells expressing E1 under the control of the ZFHD1 promoter, the early enhancer proteins E2A, E4, and VA RNA required for efficient production of recombinant AAV were expressed as a single polycistronic sequence incorporating the self-cleaving 2A peptide to ensure that the three individual proteins were expressed at the required levels and in constant stoichiometry under the control of E1a. The functionality of the constructs was monitored by the production efficiency of the normalized levels of expression of the encapsulated transgene determined using the reporter cells of the invention.

[0082] In a preferred embodiment of the present invention, the components of the packaging cell line are integrated into the genome of the selected cell line as described in Example 1. Human A549 cells were co-transfected with the developed optimal transgene promoter construct. The cells were also co-transfected with both the rep and E1A genes under the control of the rapamycin-regulated ZFHD1 promoter and the E2A, E4 and VA genes, whose expression is regulated by E1A expression. Human A549 cells were transfected with the above constructs using an integrase / transposon system that ensures the integration of the transgene in the transcriptionally active region of chromatin. Individual stable clones were isolated and characterized by Western blot for rapamycin inducibility after transient co-transfection with a vector expressing the firefly luciferase (FL) gene under the control of a 12-fold tandem repeat of the ZFHD1 promoter, as well as for the expression levels of E1A, Rep and E2 / E4. Individual clones were also tested for the expression levels of the total encapsulation-competent recombinant AAV vectors as determined by qPCR. Quantification of AAV vector genomes was based on the use of AAV2 ITR-specific target sequences, which allows for direct comparison of results between laboratories, but relies on the use of linear or circular ITA standards and extended denaturation cycles due to extensive secondary hairpin structures in the AAV2 ITR ( 15 ).

[0083] The stability of the selected clones was tested at regular intervals over 20 passages to determine the stability of the transgene in human A549 cells. The selected clones did not show any loss of levels of total encapsulated competent recombinant AAV vector as monitored by the activation level of the transgene-responsive reporter cell line. The clones also showed stable growth characteristics, including both doubling time and maximum cell density achieved under standardized growth conditions in medium containing the required selection agent.

[0084] definition vector The term "vector" refers to a DNA molecule used as a vehicle to transfer recombinant genetic material to a host cell. The four main types of vectors are plasmids, bacteriophages and other viruses, cosmids, and artificial chromosomes. The vector itself is generally a DNA sequence consisting of an insert (heterologous nucleic acid sequence, transgene) and a larger sequence that serves as the "backbone" of the vector. The purpose of a vector to transfer genetic information to a host is typically to isolate, propagate, or express the insert in a target cell. Vectors called expression vectors (expression constructs) are specifically adapted for the expression of heterologous sequences in target cells and generally have a promoter sequence that drives the expression of the heterologous sequence. The choice of vector to be used in embodiments of the invention depends on the particular application of the vector to encode a polypeptide or polynucleotide.

[0085] Transgene A transgene is a segment of DNA that encodes an open reading frame (ORF), initiation and termination codons, and is frequently transferred from one organism to another using a suitable vector.

[0086] operably linked The term "operably linked" refers to the connection of elements that are part of a functional unit, such as a gene or an open reading frame. Thus, by operably linking a promoter to a nucleic acid sequence encoding a polypeptide (open reading frame, ORF), the two elements become part of a functional unit, i.e., a gene. Linking an expression control sequence (promoter) to a nucleic acid sequence allows transcription of the nucleic acid sequence as directed by the promoter. By operably linking two heterologous nucleic acid sequences encoding a polypeptide, the sequences become part of a functional unit, i.e., an open reading frame that encodes a fusion protein that includes the amino acid sequences encoded by the heterologous nucleic acid sequences. By operably linking two amino acid sequences, the sequences become part of the same functional unit, i.e., a polypeptide. By operably linking two heterologous amino acid sequences, a hybrid (fusion) polypeptide is generated.

[0087] Minimal constitutive promoter The promoter directing the expression of the reporter gene is typically minimally constitutively active in the mammalian host cells used to establish the reporter cell line of the present invention, and alone is not responsive to treatment of the cells with pharmacologically active molecules. Useful constitutively active promoters include, but are not limited to, the cytomegalovirus (CMV) early enhancer / promoter, the SV40 promoter, the UBC promoter, the PGK promoter, the human β-actin (hACTB), the human elongation factor-1α (hEF-1α), the thymidine kinase (TK) promoter, and the cytomegalovirus early enhancer / chicken β-actin (CAG) promoter.

[0088] Inducible promoters An inducible promoter is a DNA sequence from which transcription of mRNA occurs following a reversible change in the presence or abundance or conformation of a regulatory factor or protein in a cell that allows an activating transcription factor to recruit RNA polymerase II. According to the present invention, an inducible promoter can be any promoter that responds to or is activated (induced) by chemical agents, temperature and light, all of which are examples of factors that can result in the induction of a promoter.

[0089] Chemically regulated promoters are one of the most common inducible promoters. The positively inducible tetracycline-ON (Tet-On) system is a versatile tool developed for use in prokaryotes and eukaryotes, and acts via direct activation. In this system, the activator rtTA (reverse tetracycline-controlled transcription activator) is normally inactive and cannot bind to the tetracycline response element (TRE) in the promoter. Tetracycline and its derivatives act as inducers that allow promoter activation.

[0090] One of the most commonly used prokaryotic promoters is the negatively inducible pLac promoter. This promoter requires the removal of the lac repressor (lacI protein) for transcription to be activated. In the presence of lactose or the lactose analog IPTG, the lac repressor undergoes a conformational change that removes it from the lacO site in the promoter, terminating repression of the target gene. A simplified lac inducible system is found in many bacterial expression vectors.

[0091] The negatively inducible promoter pBad is another common prokaryotic promoter that is often used in bacterial protein purification. In the absence of arabinose, the regulatory protein AraC binds to the O and I1 sites upstream of pBad and blocks transcription. The addition of arabinose causes AraC to bind the I1 and I2 sites and initiate transcription. In addition to arabinose, cAMP complexed with cAMP-activating protein (CAP) can also stimulate AraC binding to the I1 and I2 sites. Supplementing the cell growth medium with glucose reduces cAMP, represses pBad, and reduces promoter leakage.

[0092] Temperature-sensitive expression systems are typically less leaky than chemically induced promoters, which show near-zero expression at normal temperatures but can be induced by exposure to heat or low temperatures. Examples include, but are not limited to, heat shock-inducible Hsp70 or Hsp90-derived promoters, where the selected gene is expressed only after exposure to a brief heat shock. In the case of Hsp70, heat shock releases heat shock factor 1 (HSF-1), which then binds to heat shock elements in the promoter, thereby activating transcription. Other non-limiting examples are heat shock-inducible Cre and Cas9 for genome engineering in species such as C. elegans and Drosophila.

[0093] Light is another technique to activate gene expression, and two-component systems used in synthetic biology use light to regulate transcription. The red flame plasmid pDawn contains the blue light-sensing protein YFI. In the absence of light, YFI phosphorylates FixJ, which binds to the FixK2 promoter and induces transcription of the phage repressor cI. Repressor cI inhibits transcription from the phage promoter pR, preventing reporter gene expression. In the presence of light, YFI is inactive, preventing repressor cI synthesis and allowing reporter gene transcription to occur.

[0094] When describing the embodiments of the present invention, all possible combinations and permutations of the embodiments are not explicitly described. Nevertheless, the mere fact that certain measures are recited in mutually different subitems or described in different embodiments does not indicate that a combination of these measures cannot be used to advantage. The present invention contemplates all possible combinations and permutations of the described embodiments.

[0095] The terms "comprising," "comprise," and "comprises" herein are intended in all instances to be optionally interchangeable with the terms "consisting of," "consist of," and "consist of," respectively. The present invention will be more fully understood by reference to the detailed description of the invention. However, this should not be construed as limiting the scope of the invention. All literature citations are incorporated herein by reference in their entirety. EXAMPLES

[0096] The invention is further illustrated in the following non-limiting examples.

[0097] Example 1 A packaging cell line (component 1) as shown in Figure 1, established in HEK293 cells and expressing the cap gene encoding the capsid of AAV2 with a Gal-4 tag sequence between the ITRs, was co-incubated overnight or for any period of time between about 15 hours and about 30 hours at about 37°C with a reporter cell line containing a firefly luciferase reporter gene under the control of a 5-fold tandem repeat of the GAL4 UAS (component 2) as shown in Figure 2, in the presence of a sample of human serum to be tested for the presence of neutralizing antibodies to AAV2. Firefly luciferase expression was then quantified in a luminometer using Bright-Glo substrate (Figure 10).

[0098] Example 2 A packaging cell line (component 1) as shown in FIG. 1, established in HEK293 cells and expressing the cap gene encoding the capsid of AAV2 with a Gal-4 tag sequence between the ITRs, was co-incubated overnight with a reporter cell line containing a firefly luciferase reporter gene under the control of a 5-fold tandem repeat of the GAL4 UAS (component 2) as shown in FIG. 2, using the invention described herein, in the presence of a sample of serum from a normal human donor to be tested for the presence of neutralizing antibodies against AAV2, either without pretreatment or after ultrafiltration with a cutoff for proteins of 100,000 kDa. Firefly luciferase expression was then quantified in a luminometer using Bright-Glo substrate. As shown in FIG. 15, both the untreated serum and the material retained by the filter showed similarly reduced RLU levels (RLU values), reflecting the presence of neutralizing antibodies against AAV2. In contrast, the filtrate showed unreduced RLU levels, indicating the absence of neutralizing antibodies against AAV2. Sample 58 is a sample of control serum from a normal human donor known to not exhibit neutralizing antibodies to AAV2. Sample 58 was found to exhibit similar RLU values ​​both before and after ultrafiltration.

[0099] Example 3 Increasing numbers of packaging cell lines as shown in Figure 1 (component 1), established in HEK293 cells and expressing the cap gene encoding the AAV2 capsid with a Gal-4 tag sequence between the ITRs, were co-incubated overnight with a number of reporter cell lines containing a firefly luciferase reporter gene under the control of a 5-fold tandem repeat of the GAL4 UAS (component 2) as shown in Figure 2. In parallel, in the same experiment, increasing multiplicities of infection (MOI) of purified cell-free AAV2 virions were co-incubated overnight with a number of reporter cell lines containing a firefly luciferase reporter gene under the control of a 5-fold tandem repeat of the GAL4 UAS (component 2) as shown in Figure 2. Unexpectedly, direct contact of the AAV2 producing packaging cell line (component 1) with a reporter cell line containing the firefly luciferase reporter gene under the control of a 5-fold tandem repeat of the GAL4 UAS (component 2) shown in Figure 2 resulted in greater activation of FL receptor gene activity than addition of a comparable MOI of purified cell-free AAV2 virions and overnight incubation with the reporter cell line containing the firefly luciferase reporter gene under the control of a 5-fold tandem repeat of the GAL4 UAS (component 2). This is shown in FIG. 17, which shows that a given amount of an AAV2-producing packaging cell line (component 1) incubated overnight with a reporter cell line containing a firefly luciferase reporter gene under the control of a 5-fold tandem repeat of the GAL4 UAS (component 2) shown in FIG. 2 resulted in greater activation of FL reporter gene activity than the addition of an equivalent MOI of free AAV2 virions incubated overnight with the reporter cell line (component 2), suggesting that co-incubation over a period of time of the two-component system of the present invention, and thus direct contact between the virus-producing packaging cells (component 1) and the reporter cells (component 2), is inherently more efficient in activating the reporter gene than, inter alia, direct contact between an equivalent MOI of purified cell-free virus virions and the reporter cells.Thus, the effective antigen load provided by contacting virus-producing packaging cells (component 1) and reporter cells (component 2) for a given period of time with a sample suspected of containing neutralizing antibodies to a wild-type AAV serotype or recombinant AAV vector of the invention is effectively lower than in conventional neutralization assays in which cell-free viral virions are contacted with a sample suspected of containing neutralizing antibodies to a wild-type AAV serotype or recombinant AAV vector at 37°C for periods often up to one hour, resulting in increased sensitivity of the invention.

[0100] In certain embodiments, the present invention relates to the following items: 1. A first metazoan cell (component 1) for the production of an AAV serotype or a recombinant AAV vector, against which an antibody response is determined, (i) an AAV genome or transgene promoter construct, comprising a tag sequence contained within the AAV ITRs; (ii) a cap gene of an AAV serotype or recombinant AAV vector encoding either a naturally occurring Cap, a hybrid Cap, or a chimeric Cap operably linked to a constitutive promoter; (iii) an AAV rep gene operably linked to an inducible promoter; (iv) the AAV E2A, E4, and VA genes, each operably linked to its respective native or inducible promoter; (v) an AAV E1A gene operably linked to an inducible promoter; A first metazoan cell (component 1), comprising: A second metazoan cell (component 2) incorporating a reporter gene that specifically responds to the tag sequence within the AAV genome or transgene promoter construct of component 1. and methods of using same to detect and optimally quantitate anti-AAV neutralizing antibodies in a test sample. 2. The cell according to item 1, which is a baculovirus-infected insect cell, such as an SF9 cell, an avian cell, such as a DT-40, MSB1 or LMH, a mouse cell, such as an L929 cell or an LS mutant, a Chinese Hamster Ovary (CHO) cell, such as a CHO-K1, CHO-DXB11, CHO-DG44, CHOK1SV (including all mutants), CHOK1SV-GSKO (glutamine synthetase knockout) (including all mutants), a human cell, such as a HEK293 cell (including all mutants and all suspension or adherent mutants), a HeLa cell, an HT1080 cell, an ARPE-19, U937, Jurkat, HuH-7, HepG2, K562, A431 or A549 cell. 3. The cell of component 1, wherein the tag sequence contained within the AAV genome or transgene promoter construct is a Gal4 DNA binding domain. 4. The cell of component 1, wherein the tag sequence contained within the AAV genome or transgene promoter construct encodes a cGMP-specific receptor protein (CRP). 5. The cells of component 1, wherein the tag sequence contained within the AAV genome or transgene construct encodes the transsilencer VanR fused to the transactivator VP16. 6. A cell of component 2 incorporating a reporter gene that specifically responds to a gal4 tag sequence in the AAV genome of component 1 or a gal4 tag sequence in an introduced gene construct of component 1, operably linked to a chimeric promoter comprising a Gal4 upstream activating sequence (UAS) and a minimal promoter comprising a transcription start site TATAA or a mutant thereof, or a tandem repeat sequence of the Gal4 UAS, in a preferred embodiment a tandem repeat sequence five times that of the Gal4 UAS. 7. A cell of component 2 incorporating a reporter gene that specifically responds to the CRP sequence in the AAV genome of component 1 or the CRP sequence in a transgene promoter construct of component 1, operably linked to a chimeric promoter comprising a GTA operator sequence and a minimal promoter, or a tandem repeat thereof, in a preferred embodiment a four-fold tandem repeat thereof. 8. A component 2 cell incorporating a reporter gene that specifically responds to a VanR sequence in the AAV genome of component 1 or a VanR sequence in a transgene construct of component 1, operably linked to a VanO operator module, in a preferred embodiment an octameric VanO operator module (Van08), and a chimeric promoter comprising a minimal promoter such as the CMV immediate early promoter. 9. The cell of component 1 according to any one of the preceding items, comprising at least five recombination target sites. 10. The cell of component 2 according to any one of items 1 to 3, wherein the reporter is a luciferase such as firefly luciferase, Renilla luciferase, Metridia luciferase or the novel luciferases described in European Patent Application No. 21170068.7, or other proteins including novel luciferases or fluorescent proteins such as, for example, green fluorescent protein, enhanced green fluorescent protein, red fluorescent protein, yellow fluorescent protein, blue fluorescent protein and variants exhibiting different excitation / emission spectra. (i) In a preferred embodiment, in order to provide for normalization of the assay, the cells of component 2 according to the invention further comprise a construct for the constitutive expression of a luciferase different from that used in the reporter gene construct responsive to a tag sequence contained within the AAV genome or transgene promoter construct. For example, the constitutive product can be the constitutive product of a second luciferase, such as Renilla luciferase, firefly luciferase, Metridia luciferase, or a novel luciferase, such as those described in European Patent Application No. 21170068.7. 11. A method for detecting and optionally quantifying anti-AAV neutralizing antibody activity in a test sample, comprising: (i) providing a test sample suspected of containing anti-AAV antibodies together with a control sample containing no anti-AAV antibodies, and in a preferred embodiment, a positive control sample known to contain anti-AAV neutralizing antibodies. (ii) contacting the test sample with either a virus produced by a cell of component 1 according to any one of the preceding paragraphs or a virus-producing cell of component 1; (iii) prior to measuring the activity of the AAV tag-responsive first reporter protein in the cell line, incubating the test sample with either a virus produced by the cells of component 1 described in any one of the above items, or the virus-producing cells of component 1, and the cells of component 2, at various temperatures, preferably 37°C, for various times, preferably 6 to 18 hours or more. The method of any of the preceding items, wherein the reporter cell line expresses a second reporter protein, and the method further comprises: (iv) measuring the activity of the first reporter protein in the cell line; (v) providing a ratio between the activity of the first reporter protein and the activity of the second reporter protein. (vi) measuring the activity of a first tag-responsive reporter protein in reporter cells of the first cell sample, with or without normalization to the activity of a second luciferase, as described in U.S. Patent Application Publication No. 2011 / 0189658. (vii) measuring the activity of the first tag-responsive reporter protein in cells of a second control cell sample, with or without normalization to the activity of the second luciferase, as described in U.S. Patent Application Publication No. 2011 / 0189658. (viii) providing a ratio of reporter activity between the first control cell sample and the second control cell sample, wherein a ratio (first / second) lower than 1 indicates the presence of antibodies against an AAV serotype or recombinant AAV vector in the sample. 12. A method for high throughput screening of patient samples to detect and optimally monitor immune responses to either an AAV capsid, an AAV genome, a transgene, or a transgene product, comprising: (i) providing a test sample comprising a patient sample to be screened; (ii) contacting said test sample with a challenge virus or virus-producing packaging cells of component 1 according to the present invention and according to any one of the preceding paragraphs; (iii) incubating the test sample with the challenge virus or virus-producing cells of component 1 and the cells of component 2 described in any one of the above items at various temperatures, preferably about 37° C., for various times, preferably about 6 to about 18 hours or more, before measuring the activity of the AAV tag-responsive first reporter protein in the cell line. The method includes: The method of any of the preceding items, wherein the cell line expresses a second reporter protein and the method further comprises: (iv) measuring the activity of a first tag-responsive reporter protein in the cell line; (v) providing a ratio between the activities of said cell line component 2, said component 2 containing a first heterologous polynucleotide comprising a heterologous cis-acting regulatory sequence responsive to treatment of the cell line with a tag sequence present in the genome of an AAV serotype or recombinant AAV vector construct operably linked to a downstream promoter sequence, said promoter being operably linked to an open reading frame encoding a first reporter protein, such as a luciferase, e.g., Renilla luciferase, firefly luciferase or Metridia luciferase. In a preferred embodiment, in order to provide for normalization of the assay, the cells of component 2 according to the invention further comprise a construct for the constitutive expression of a luciferase different from that used in the reporter gene construct responsive to the tag sequence contained in the AAV genome or transgene construct. For example, the constitutive product can be the constitutive product of a second luciferase, such as Renilla luciferase, firefly luciferase, Metridia luciferase, or a novel luciferase, such as those described in European Patent Application No. 21170068.7. (vi) In a preferred embodiment, the cells according to the invention are seeded into 96, 384 or 1536 well plate assay plates. 13. A first metazoan cell (component 1) for the production of an AAV serotype or a recombinant AAV vector against which an antibody response is determined, (i) a transgene promoter construct comprising a tag sequence contained within the AAV genome or AAV ITRs; (ii) a cap gene of an AAV serotype or recombinant AAV vector encoding either a naturally occurring Cap, a hybrid Cap, or a chimeric Cap operably linked to a constitutive promoter; (iii) an AAV rep gene operably linked to an inducible promoter; (iv) the AAV E2A, E4, and VA genes, each operably linked to its respective native or inducible promoter; (v) a first metazoan cell (component 1), comprising an AAV E1A gene operably linked to an inducible promoter. A second metazoan cell (component 2) incorporating a reporter gene that specifically responds to a tag sequence within the AAV genome or transgene promoter construct of component 1, and a method of using same to detect and optimally quantify anti-AAV neutralizing antibodies in a test sample. 14. The cell according to item 1, which is a baculovirus-infected insect cell, such as an SF9 cell, an avian cell, such as a DT-40, MSB1 or LMH, a mouse cell, such as an L929 cell or an LS mutant, a Chinese Hamster Ovary (CHO) cell, such as a CHO-K1, CHO-DXB11, CHO-DG44, CHOK1SV (including all mutants), CHOK1SV-GSKO (glutamine synthetase knockout) (including all mutants), a human cell, such as a HEK293 cell (including all mutants and all suspension or adherent mutants), a HeLa cell, an HT1080 cell, an ARPE-19, U937, Jurkat, HuH-7, HepG2, K562, A431 or A549 cell. 15. The cell of component 1, wherein the tag sequence contained within the AAV genome or the transgene promoter construct is a Gal4 DNA binding domain. 16. The cell of component 1, wherein the tag sequence contained within the AAV genome or transgene promoter construct encodes a cGMP-specific receptor protein (CRP). 17. The cell of component 1, wherein the tag sequence contained within the AAV genome or transgene construct encodes the transsilencer VanR fused to the transactivator VP16. 18. A cell of component 2 incorporating a reporter gene that specifically responds to a gal4 tag sequence in the AAV genome of component 1 or a gal4 tag sequence in an introduced gene construct of component 1, operably linked to a chimeric promoter comprising a Gal4 upstream activating sequence (UAS) and a minimal promoter comprising a transcription start site TATAA or a variant thereof, or a tandem repeat sequence of the Gal4 UAS, in a preferred embodiment a tandem repeat sequence five times that of the Gal4 UAS. 19. A cell of component 2 incorporating a reporter gene that specifically responds to a CRP sequence in the AAV genome of component 1 or a CRP sequence in a transgene promoter construct, operably linked to a chimeric promoter comprising a GTA operator sequence and a minimal promoter, or a tandem repeat thereof, in a preferred embodiment a 4-fold tandem repeat thereof. 20. A cell of component 2 incorporating a reporter gene that specifically responds to a VanR sequence in the AAV genome of component 1 or a VanR sequence in a transgene construct of component 1, operably linked to a VanO operator module, in a preferred embodiment an octameric VanO operator module (Van08), and a chimeric promoter comprising a minimal promoter such as the CMV immediate early promoter. 21. A cell of component 1 according to any one of the preceding items, comprising at least five recombination target sites. 22. The cell of component 2 according to any one of items 1 to 3, wherein the reporter is a luciferase, such as firefly luciferase, Renilla luciferase, Metridia luciferase, or the novel luciferases described in European Patent Application No. 21170068.7, or other proteins, including novel luciferases or fluorescent proteins, such as, for example, green fluorescent protein, enhanced green fluorescent protein, red fluorescent protein, yellow fluorescent protein, blue fluorescent protein and variants exhibiting different excitation / emission spectra. (i) In a preferred embodiment, in order to provide for normalization of the assay, the cells of component 2 according to the invention further comprise a construct for the constitutive expression of a luciferase different from that used in the reporter gene construct responsive to a tag sequence contained within the AAV genome or transgene promoter construct. For example, the constitutive product can be the constitutive product of a second luciferase, such as Renilla luciferase, firefly luciferase, Metridia luciferase, or a novel luciferase, such as those described in European Patent Application No. 21170068.7. 23. A method for detecting and optionally quantifying anti-AAV neutralizing antibody activity in a test sample, comprising: (i) providing a test sample suspected of containing anti-AAV antibodies together with a control sample that does not contain anti-AAV antibodies, and in a preferred embodiment, a positive control sample that is known to contain anti-AAV neutralizing antibodies. (ii) contacting the test sample with either a virus produced by a cell of component 1 according to any one of the preceding paragraphs or a virus-producing cell of component 1; (iii) prior to measuring the activity of the AAV tag-responsive first reporter protein in the cell line, incubating the test sample with either a virus produced by the cells of component 1 described in any one of the above items, or the virus-producing cells of component 1, and the cells of component 2, at various temperatures, preferably 37°C, for various times, preferably 6 to 18 hours or more. The method of any of the preceding items, wherein the reporter cell line expresses a second reporter protein, and the method further comprises: (iv) measuring the activity of the first reporter protein in the cell line; (v) providing a ratio between the activity of the first reporter protein and the activity of the second reporter protein. (vi) measuring the activity of a first tag-responsive reporter protein in reporter cells of the first cell sample, with or without normalization to the activity of a second luciferase, as described in U.S. Patent Application Publication No. 2011 / 0189658. (vii) measuring the activity of the first tag-responsive reporter protein in cells of a second control cell sample, with or without normalization to the activity of the second luciferase, as described in U.S. Patent Application Publication No. 2011 / 0189658. (viii) providing a ratio of reporter activity between the first control cell sample and the second control cell sample, wherein a ratio (first / second) lower than 1 indicates the presence of antibodies against an AAV serotype or recombinant AAV vector in the sample. 24. A method for high throughput screening of patient samples to detect and optimally monitor immune responses to either an AAV capsid, an AAV genome, a transgene, or a transgene product, comprising: (iv) providing a test sample comprising the patient sample to be screened; (v) contacting the test sample with a challenge virus or virus-producing packaging cells of component 1 according to the present invention and according to any one of the preceding paragraphs; (vi) A method comprising the step of incubating the test sample with a challenge virus or virus-producing packaging cells of component 1 and cells of component 2 described in any one of the preceding items at various temperatures, preferably 37°C, for various times, preferably 6 to 18 hours or more, prior to measuring the activity of an AAV tag-responsive first reporter protein in the cell line. The method of any of the preceding items, wherein the cell line expresses a second reporter protein and the method further comprises: (iv) measuring the activity of a first tag-responsive reporter protein in the cell line; Providing a ratio between the activities of said cell line component 2, said component 2 containing a first heterologous polynucleotide comprising a heterologous cis-acting regulatory sequence responsive to treatment of the cell line with a tag sequence present in the genome of an AAV serotype or recombinant AAV vector construct operably linked to a downstream promoter sequence, said promoter being operably linked to an open reading frame encoding a first reporter protein, such as a luciferase, e.g., Renilla luciferase, firefly luciferase, Metridia luciferase, or a novel luciferase such as those described in European Patent Application No. 21170068.7. (vii) In a preferred embodiment, in order to provide for normalization of the assay, the cells of component 2 according to the invention further comprise a construct for the constitutive expression of a luciferase different from that used in the reporter gene construct responsive to a tag sequence contained within the AAV genome or transgene construct. For example, the constitutive product can be the constitutive product of a second luciferase, such as Renilla luciferase, firefly luciferase, Metridia luciferase, or a novel luciferase, such as those described in European Patent Application No. 21170068.7. (viii) In a preferred embodiment, the cells according to the invention are seeded into 96, 384 or 1536 well plate assay plates. 25. A first metazoan cell (component 1) for the production of an AAV serotype or a recombinant AAV vector against which an antibody response is determined, (i) a transgene promoter construct comprising a tag sequence contained within the AAV genome or AAV ITRs; (ii) a cap gene of an AAV serotype or recombinant AAV vector encoding either a naturally occurring Cap, a hybrid Cap, or a chimeric Cap operably linked to a constitutive promoter; (iii) an AAV rep gene operably linked to an inducible promoter; (iv) the AAV E2A, E4, and VA genes, each operably linked to its respective native or inducible promoter; (v) a first metazoan cell (component 1), comprising an AAV E1A gene operably linked to an inducible promoter. A second metazoan cell (component 2) incorporating a reporter gene that specifically responds to the tag sequence within the AAV genome or transgene promoter construct of component 1. and methods of using same to detect and optimally quantitate anti-AAV neutralizing antibodies in a test sample. 26. The cell of component 1, wherein the tag sequence contained within the AAV genome or transgene promoter construct encodes a cGMP-specific receptor protein (CRP). 27. A cell of component 2 incorporating a FL luciferase reporter gene that specifically responds to a gal4 tag sequence in the AAV genome of component 1 or a gal4 tag sequence in an introduced gene construct of component 1, operably linked to a chimeric promoter comprising a Gal4 upstream activating sequence (UAS) and a minimal promoter comprising a transcription start site TATAA or a mutant thereof, or a tandem repeat sequence of the Gal4 UAS, in a preferred embodiment a tandem repeat sequence five times that of the Gal4 UAS. 28. A method for detecting and optionally quantifying anti-AAV neutralizing antibody activity in a test sample, comprising: (i) providing a test sample from a normal human donor suspected of containing anti-AAV antibodies together with a control sample containing no anti-AAV antibodies, and in a preferred embodiment, a positive control sample known to contain anti-AAV neutralizing antibodies. (ii) contacting the test sample with a virus-producing cell of component 1 according to any one of the preceding paragraphs; (iii) prior to measuring the activity of the AAV tag-responsive first reporter protein in the cell line, incubating the test sample with a virus-producing cell of component 1 described in any one of the above items and a cell of component 2 at various temperatures, preferably 37°C, for various times, preferably 6 to 18 hours or more. The method of any of the preceding items, wherein the reporter cell line expresses a second reporter protein, and the method further comprises: (iv) measuring the activity of the first reporter protein in the cell line; (v) providing a ratio between the activity of the first reporter protein and the activity of the second reporter protein. (vi) measuring the activity of a first tag-responsive reporter protein in reporter cells of the first cell sample, with or without normalization to the activity of a second luciferase, as described in U.S. Patent Application Publication No. 2011 / 0189658. (vii) measuring the activity of the first tag-responsive reporter protein in cells of a second control cell sample, with or without normalization to the activity of the second luciferase, as described in U.S. Patent Application Publication No. 2011 / 0189658. (viii) providing a ratio of reporter activity between the first control cell sample and the second control cell sample, wherein a ratio (first / second) lower than 1 indicates the presence of antibodies against an AAV serotype or recombinant AAV vector in the sample. 29. A method for high throughput screening of patient samples to detect and optimally monitor an immune response to either an AAV capsid, an AAV genome, a transgene, or a transgene product, comprising: (vii) providing a test sample comprising a patient sample to be screened; (viii) contacting the test sample with a challenge virus or virus-producing packaging cells of component 1 according to the present invention and according to any one of the preceding paragraphs; (ix) incubating the test sample with the challenge virus or virus-producing cells of component 1 and the cells of component 2 described in any one of the above items at various temperatures, preferably about 37° C., for various times, preferably about 6 to about 18 hours or more, before measuring the activity of the AAV tag-responsive first reporter protein in the cell line. The method includes: The method of any of the preceding items, wherein the cell line expresses a second reporter protein and the method further comprises: (iv) measuring the activity of a first tag-responsive reporter protein in the cell line; Providing a ratio between the activities of said cell line component 2, said component 2 containing a first heterologous polynucleotide comprising a heterologous cis-acting regulatory sequence responsive to treatment of the cell line with a tag sequence present in the genome of an AAV serotype or recombinant AAV vector construct operably linked to a downstream promoter sequence, said promoter being operably linked to an open reading frame encoding a first reporter protein, such as a luciferase, e.g., Renilla luciferase, firefly luciferase, Metridia luciferase, or a novel luciferase such as those described in European Patent Application No. 21170068.7. (ix) In a preferred embodiment, in order to provide for normalization of the assay, the cells of component 2 according to the invention further comprise a construct for the constitutive expression of a luciferase different from that used in the reporter gene construct responsive to a tag sequence contained within the AAV genome or transgene construct. For example, the constitutive product can be the constitutive product of a second luciferase, such as Renilla luciferase, firefly luciferase, Metridia luciferase, or a novel luciferase, such as those described in European Patent Application No. 21170068.7. (x) In a preferred embodiment, the cells according to the present invention are seeded into 96, 384 or 1536 well plate assay plates.

[0101] References

number

Claims

1. A reagent for use in a method for detecting neutralizing antibodies against adeno-associated virus (AAV) or recombinant AAV vectors, comprising two cell lines, wherein one cell line is called component 1, and component 1 is: (i) An introduced gene promoter construct comprising one or more tag sequences contained within the AAV ITR, wherein the tag sequences are distinct from each other, Cells, (a) A tag sequence encoding the Gal4-VP16 protein that specifically binds to the Gal4 upstream activation sequence (UAS) that regulates the expression of the firefly luciferase (FL) reporter gene of component 2, (b) A tag sequence within the AAV ITR encoding the VanR-VP16 protein that binds to the octamer of the VanO operator sequence that regulates the expression of the firefly luciferase (FL) reporter gene of component 2, or (c) A transgene promoter construct within the AAV ITR expressing a tag sequence encoding cGMP-specific receptor protein (CRP) that binds to a GTA operator sequence that regulates the expression of the FL reporter gene of component 2. Express an introduced gene promoter construct that includes, Transgene promoter construct, (ii) The cap gene of a specific AAV serotype or recombinant AAV vector encoding either a naturally occurring Cap, hybrid Cap, or chimeric Cap, operably linked to a constitutive or inducible promoter. (iii) AAV rep gene operably linked to a natural or inducible promoter, (iv) AAV E2A, E4 and VA genes operably linked to their respective natural promoters, and (v) The AAV E1A gene, which is operablely linked to an inductive promoter or is endogenously present in cells including HEK293 cells. Includes, Further cell lines are referred to as component 2, and component 2 is, The component 1 comprises a reporter gene that specifically responds to one or more tag sequences described in (i)(a), (i)(b), or (i)(c) within the cell transgene promoter construct. The above reagents.

2. The reagent according to claim 1, wherein the constitutive promoter is selected from the group consisting of cytomegalovirus (CMV) early enhancer / promoter, SV40 promoter, UBC promoter, PGK promoter, human β-actin (hACTB), human elongation factor-1α (hEF-1α), thymidine kinase (TK) promoter, and cytomegalovirus early enhancer / chicken β-actin (CAG) promoter.

3. The reagent according to claim 1 or 2, wherein the inductive promoter is selected from the group consisting of the Tet-on / Tet-off system, the cumate-inducible repressor CymR system, the flavonoid phloretin-modulating TtgR repressor system, and the vanillic acid-modulating VanR repressor / KRAP system, and the inductive promoters in (iii) and (v) are different from each other.

4. The reagent according to claim 1 or 2, wherein a tag sequence contained within the transgene promoter construct encodes a transsilencer VanR fused to the transactivator VP16.

5. Component 2 is A reporter gene operably ligated to a chimeric promoter containing a Gal4 upstream activation sequence (UAS) and a minimal promoter containing the transcription start site TATAA or a variant thereof, or to a chimeric promoter containing a tandem repeat (vertical repeat sequence) of the Gal4 UAS, or a 5-fold tandem repeat (vertical repeat sequence) thereof. Includes, The reagent according to claim 1 or 2, wherein the reporter gene specifically responds to the gal4 tag sequence in the introduced gene promoter construct of component 1.

6. Component 2 is A reporter gene operably ligated to a chimeric promoter containing a GTA operator sequence and a minimal promoter, or to a chimeric promoter containing a tandem repeat (column repeat sequence) thereof, or a 4x tandem repeat (column repeat sequence) thereof. Includes, The reporter gene responds specifically to the CRP sequence within the introduced gene promoter construct of component 1, and The reagent according to claim 1 or 2, wherein the reporter protein shown as the first reporter protein is firefly luciferase.

7. Component 2 is A reporter gene operably ligated to a chimeric promoter containing a VanO operator module, or its tandem repeat (column repeat sequence), or an octameric VanO operator module, and a minimal promoter including the CMV initial promoter. Includes, The reagent according to claim 1 or 2, wherein the reporter gene specifically responds to a VanR sequence in the introduced gene construct of component 1.

8. The reagent according to claim 1 or 2, wherein component 2 further comprises a construct for constitutive expression of a luciferase different from that used in a reporter gene construct that responds to one or more tag sequences contained in the transgene promoter construct within component 1, and the construct is a constitutive product of a second luciferase selected from the group consisting of sea urchin luciferase, firefly luciferase, and metridial luciferase.

9. The reagent according to claim 1 or 2, wherein component 1 comprises at least five recombinant target sites.

10. The reagent according to claim 1 or 2, wherein the method comprises the detection and quantification of a neutralizing antibody against adeno-associated virus (AAV), AAV capsid, or recombinant AAV vector.

11. The reagent according to claim 1 or 2, wherein the cells of component 1 and component 2 are co-incubated with a biological sample that is thought to contain neutralizing antibodies against a wild-type AAV serotype or a recombinant AAV vector.

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