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 present invention provides (i) a packaging cell line for the production of an AAV serotype or recombinant AAV vector, wherein an antibody is to be detected and the viral genome or the transgene construct contains a tag sequence therein, and (ii) a reporter gene cell line incorporating a reporter gene promoter construct that specifically responds to the tag sequence, and a method of using it to detect and optimally quantify anti-AAV neutralizing antibodies against the capsid, viral genome, and / or transgene or transgene product in a test sample. The present invention further relates to a high-throughput screening method for detecting and optimally monitoring an immune response against AAV with optimal sensitivity, signal intensity, and biological fidelity. In a broader sense, the present invention also relates to the use of a packaging cell line in combination with a reporter gene cell line in diagnosis.

Background Art

[0002] Recombinant adeno-associated virus (AAV) vectors are used to treat an increasing number of genetic disorders, and in most cases AAV-based gene therapy is well tolerated, but an immune response against any of the AAV capsid, AAV genome, transgene, or transgene product is associated with a decrease or complete loss of the level 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 in infancy and a 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 a 145 bp reverse terminal repeat (ITR), the first 125 nucleotides of which are palindromic sequences that fold over themselves to form a T-shaped hairpin loop secondary structure. The ITR acts as a primer for second-strand synthesis and is required in cis for viral replication or transgene expression (3). The rep gene encodes four non-structural proteins, Rep78, Rep68, Rep52, and Rep40, which are encoded by a single ORF using two different promoters, p5 for Rep78 / 68 and p19 for Rep52 / 40, and produced by alternative splicing. The Rep78 and Rep68 proteins bind to the ITR and are required for genome replication, while Rep52 and Rep40 play a role in packaging the viral genome into the capsid during viral replication. The Rep proteins also play a role in site-directed 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, and assembled in a 1:1:10 ratio to form the viral capsid (4). The Aap gene encodes an assembly activation protein (AAP) from an alternative reading frame within the Cap ORF. AAP localizes the AAV capsid protein 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 to a size of approximately 4.8 kb and consist of a suitable promoter, transgene, and poly-A tail contained within the ITR, 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 initial helper proteins necessary 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 initial helper proteins (5,6). However, the initial genes necessary for efficient AAV vector production can also be supplied by transfecting a suitable packaging cell line with a plasmid expressing the helper proteins, in addition to the transgene promoter construct and plasmids encoding the rep, cap, and aap genes (7).

[0005] Capsids play a crucial role in serotype-specific virus-cell interactions by initially binding to carbohydrates on the cell surface. Thus, 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). Most AAV serotypes then interact with different parts of the novel AAV receptor (AAVR) required for intracellular initiation (9). It has recently been identified that another highly conserved G protein-coupled receptor-like protein localized in the trans-Golgi is required for entry of all AAV serotypes except for the highly branched AAV5 serotype, which is AAVR-dependent but GPR108-independent (10). Therefore, all AAV serotypes identified to date require either AAVR or GPR108, or both, for cytotransduction (9,10).

[0006] Although AAV infection is not associated with any disease in humans or other mammals, the effectiveness of AAV-mediated gene therapy is often limited by the development of anti-AAV antibodies, which are associated with prior exposure to AAV and high levels of cross-reactivity between different serotypes, leading to decreased or complete loss of transgene expression levels and, consequently, loss of effectiveness. Cell-based assays, so-called transduction inhibition assays, which use reporter AAV vectors incubated with test samples before in vitro transduction of cell lines, often require a high degree of infection multiplicity (MOI), resulting in low sensitivity, nonspecific effects, and at best, being a surrogate for the immune response to the actual AAV serotype or recombinant AAV vector, which determines the antibody response. 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. [Overview of the project]

[0007] The present invention is made in light of the above-mentioned prior art, and the object of the present invention is 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] To obtain the above technical effects and to provide a much improved method and cell line in connection therewith, the present invention relates, in particular, to a cell line. This cell line 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 contain one or more different tag sequences.

[0009] Furthermore, in another embodiment, the present invention relates to a reporter gene cell line incorporating a reporter gene promoter construct that specifically responds to one or more tag sequences of a packaging cell line. The reporter gene cell line may be considered a second component or referred to as component 2, all of which are used interchangeably herein.

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

[0011] In a further embodiment, the present invention relates to a method for using components 1 and 2 to detect and optimally quantify anti-AAV neutralizing antibodies in a test sample. Specifically, 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 embodiment, the present invention relates to a two-component system comprising a packing cell line (component 1) for producing an AAV serotype or recombinant AAV vector that determines the antibody response, which includes 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 in component 1, and a method for using this system to detect and optimally quantify anti-AAV neutralizing antibodies in a test sample. The present invention further relates to a high-throughput screening method for detecting and monitoring the immune response to AAV with optimal sensitivity, signal intensity, and biological fidelity.

[0013] With respect to the tag sequences of the present invention, the cell lines disclosed herein may comprise one or more tag sequences. If several tag sequences are present, they may be identical or different. In another embodiment, if several tag sequences are present, the group of tag sequences may be identical, while the remaining tag sequences may be different.

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

[0015] As is clear from the above, in the embodiment in which component 1 is used in combination with component 2, the reporter gene cell line incorporating the reporter gene promoter construct responds specifically to one or more tag sequences of the packaging cell line, and as a result, the reporter gene promoter construct responds to each of the same or different tag sequences in component 1.

[0016] Component 1: Packaging cell line. In one embodiment, the present invention relates to a packing cell line in which components necessary for the expression of an AAV serotype or recombinant AAV vector are supplied by transient transfection, or preferably partially or completely integrated into the genome of a selected cell line. The selected cell line may be referred to as a host cell line.

[0017] The selection of host cell lines is determined by analysis of the biological sample and by their safety profile regarding either the cell's ability to tolerate the toxicity of the AAV Rep protein after controlled 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 no. CCL-185) or the human embryonic kidney cell line HEK293T (ATCC catalog no. ACS-4500).

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

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

[0020] The genes encoding E1, E1A, and E1B ORFs are not expressed in A549 cells, in contrast to HEK293T 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 and causing 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 FK506-binding protein (FKBP). The FKBP-rapamycin-related protein (FRAP) is fused to an HSV-1-derived VP16 transactivator such that the addition of rapamycin induces the formation of a heterodimer between FKBP and FRAP, resulting in the strictly controlled expression of the gene encoding the E1 initial protein.

[0021] Regulated expression of Rep78 / 68 has also been shown to increase the level of AAV vector production (12). Transfection of HEK293 cells with E4 orf6 alone has been reported to be sufficient for adenovirus-free recombinant AAV vector production (13), but initial proteins E2A, E4, and VA RNA have been reported to be necessary for efficient recombinant AAV production (14). Therefore, in one embodiment, packaging cell lines can be co-transfected with expression vectors expressing E2A, E4, and VA RNA.

[0022] Component 2: Reporter cell line. In one embodiment, the present invention also relates to a developed reporter gene cell line that specifically responds to a tag sequence contained within the AAV genome or transgene construct (a tag sequence in the ITR for monitoring a selected specific recombinant AAV vector of component 1) such that contact between a reporting cell and a packaging cell line that produces a virus expressing a tag sequence within the genome of an AAV serotype or recombinant AAV vector for which a neutralizing antibody is quantified results in activation of a reporter gene expressed by the reporting cell. To enhance sensitivity, it is understood that the reporter gene cell line can be stably transfected with either the cell surface AAV receptor alone (9) or the endosomal AAV receptor alone (10), or both the cell surface receptor and the endosomal AAV receptor. In Example 1, the packaging cell line expresses a transgene promoter construct containing a tag sequence encoding 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 in the reporting cell. Specifically, in Example 1, the AAV-2 responsive reporter cell line contains a 5-fold tandem repeat sequence of Gal4 UAS that regulates the 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 include a second reporter gene, such as the sea urchin 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 enable the normalization of AAV-inducible firefly luciferase activity. The sea urchin luciferase (RL) normalization gene makes it possible to define different types of human serum based on their ability to activate AAV-responsive firefly luciferase and / or sea urchin luciferase normalization gene (Table 1).Therefore, the apparent neutralizing effect of two human serum HS0 and HS4 from normal individuals can be distinguished from the actual neutralizing activity of the human IV-IgG pool based on the activation of both the AAV-responsive FL reporter gene and the sea urchin luciferase normalization gene by the two serum HS0 and HS4, in contrast to the activation of FL activity by human IV-IgG alone, which reflects nonspecific inhibition of AAV activity due to cytotoxicity (Figures 18-20).

[0023] In a preferred embodiment, a reporter cell line (component 2) is transiently or stably transfected with the earliest 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 between packaging cell lines and reporter cells In one embodiment, the present invention relates to components 1 and 2 combined so that two cell lines interact with each other (Figures 4 to 6). Therefore, in one embodiment, the present invention is (a) A step of quantifying FL reporter gene activity using a suitable substrate such as the commercially available substrate Bright-Glo (Promega, Madison, Wisconsin), or any other suitable substrate that enables detection by any means that allows sequential quantification of FL activity and RL activity in a single well of a microtiter plate; and a suitable substrate such as the commercially available substrate Dual-Glo (Promega, Madison, Wisconsin). (b) After bringing the virus-producing packaging cells into contact with the sample to be tested at various temperatures including or encompassing any one of the following temperatures: +4, 20, or 37°C, or any temperature within the range of approximately +4°C to approximately 37°C, for various durations, the packaging cell line and the sample are incubated with reporter cells at 37°C for various durations, preferably approximately 6 to approximately 18 hours or more, and then the FL reporter gene activity alone is quantified using a suitable substrate, such as the commercially available Bright-Glo (Promega, Madison, Wisconsin), or the FL&RL activity is quantified using Dual-Glo or any other suitable substrate that enables detection by any means. (c) A virus-producing packaging cell line is brought into contact with the sample to be tested and reporter cells, and directly incubated at approximately 37°C for various times, preferably approximately 18 hours or more. The FL reporter gene activity is then quantified using a suitable substrate, such as the commercially available Bright-Glo (Promega, Madison, Wisconsin), or any other suitable substrate that enables detection by any means, or FL&RL activity is quantified using Dual-Glo or any other suitable substrate that enables detection by any means. (d) Freeze the virus-producing packaging cell line together with reporter cells at an appropriate cell concentration in a suitable cryogenic protective medium, thaw the cells, contact the packaging cell / reporter gene cell with the sample to be tested, incubate the sample, packaging cell-reporter cell at approximately 37°C for a variety of times, preferably about 18 hours or more, and then quantify the FL reporter gene activity using a suitable substrate such as the commercially available substrate Bright-Glo (Promega, Madison, Wisconsin) or any other suitable substrate that enables detection by any means, or quantify the FL&RL activity using Dual-Glo or any other suitable substrate that enables detection by any means. (e) In a preferred embodiment, the method comprises the steps of separately freezing a virus-producing packaging cell line and reporter cells at appropriate cell concentrations in a suitable cryoprotective medium, 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 reporter cells at approximately 37°C for various times, preferably about 6 to 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) or any other suitable substrate that enables detection by any means, or Dual-Glo or any other suitable substrate that enables detection by any means. The use of freeze-thawed virus-producing packaging cells eliminates the need for the end user to produce a specific AAV serotype or recombinant AAV vector for which a neutralizing antibody should be detected, and contacting the virus-producing packaging cell line with the sample to be tested eliminates the need to extract and purify the challenge virus.

[0025] Cell-free AAV challenge virus / recombinant AAV vector or AAV virus / AAV recombinant vector-producing packaging cells interact with reporter gene cell lines, resulting in virus uptake, internalization, and AAV serotype-specific activation of the FL reporter gene. The initial interaction between the cell-free challenge virus or AAV-producing packaging cells and 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 to heparan sulfate proteoglycans, while AAV serotypes 1, 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 cell internalization (9) and / or a 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 luciferase, such as firefly luciferase, Renilla luciferase, Metridia luciferase, or a novel luciferase such as those described in European Patent Application No. 21170068.7, which is incorporated herein by reference in its entirety, or a green fluorescent protein (EGFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), blue fluorescent protein (BFP) and variants thereof, horseradish peroxidase (HRP) or various conjugates thereof, secreted human placental alkaline phosphatase (SEAP) or various conjugates thereof, chloramphenicol acetyltransferase (CAT), or a luciferase or fluorescent protein, including a soluble active monomer such as various linker proteins, or a fusion protein with another protein containing luciferase, present in solution or a novel luciferase attached to a solid surface such as particles, beads, assay plates or tubes.

[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 a further embodiment, the reporter cells are under the control of a chimeric promoter that specifically responds to a tag sequence present within the AAV genome or a tag sequence present within a recombinant AAV coding transgene promoter construct contained within the AAV ITR, such as a first reporter gene like the firefly luciferase (FL) reporter gene, and are co-transfected with a second reporter gene such as RL under the control of a constitutive promoter that enables normalization of the tag sequence-activated FL activity with respect to the constitutive expression level of Renilla luciferase (RL) activity, whereby the assay results are independent of the cell number and a means is provided to compensate for inter-sample variation in cell density due to cell loss or variation in the number of seeded cells. The ability to normalize AAV-activated FL activity relative 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) immediate enhancer / promoter, SV40 promoter, UBC promoter, PGK promoter, human β-actin (hACTB), human elongation factor-1α (hEF-1α), and cytomegalovirus immediate enhancer / chicken β-actin (CAG) promoter.

[0028] As referred to herein, the present invention also provides a method for detecting and operatively quantifying the activity of anti-AAV neutralizing antibodies in a test sample, said method comprising the following steps: (i) providing a test sample suspected of containing an anti-AAV antibody, together with a control sample that does not contain an anti-AAV antibody and a positive control sample known to contain an anti-AAV neutralizing antibody; (ii) A step of contacting the test sample with either a cell-free AAV challenge virus / recombinant AAV vector or a virus-producing packaging cell according to the present invention, wherein the AAV challenge virus / recombinant AAV vector expresses an AAV serotype or a transgene promoter construct, for example, a sequence tag in the genome of the Gal4 DNA-binding domain, (iii) A step of contacting the test sample together with a reporter cell with either a cell-free AAV challenge virus / recombinant AAV vector or a virus-producing packaging cell according to the present invention, wherein the cell expresses a reporter gene promoter construct under the control of a tandem repeat sequence of Gal4 UAS, such as firefly luciferase, which specifically responds to a tag sequence in the genome of an AAV serotype or transgene construct produced by the packaging cell. (iv) Optionally, in a preferred embodiment, to provide assay normalization, the reporter cell line according to the present invention further comprises a construct for a constitutive product of a reporter gene different from that used in the AAV-responsive reporter gene construct. For example, the constitutive product may be a constitutive product of a second luciferase, e.g., sea urchin luciferase, metridial luciferase, or a novel luciferase, such as that described in European Patent Application No. 21170068.7, which is incorporated herein in whole by reference. In a preferred embodiment, the reporter gene cell line according to the present invention is seeded in a 96, 384, or 1536-well assay plate. (v) Measuring the activity of the first reporter protein in the cell line using a suitable substrate, for example, the commercially available substrate Dual-Glo or any commercially available luciferase substrate, (vi) The step of measuring the activity of a second reporter protein in the cell line after the activity of a tag-responsive reporter gene luciferase has been measured in the same sample using, for example, a Stop&Glo reagent from a Dual-Glo system or any commercially available luciferase substrate that efficiently inhibits the activity of the first reporter protein, such as firefly luciferase (Lallemand C. et al J Immunol Res. 390:1-19, 2017, which is incorporated herein by reference in its entirety). (vii) The activity of the first luciferase normalized to the activity of the second luciferase is described in U.S. Patent Application Publication 2011 / 0189658, which is incorporated in its entirety herein by reference. (viii) The step of measuring the activity of a first tag-responsive reporter protein in reporter cells of a first cell sample, normalized or unnormalized to the activity of a second luciferase, is described in U.S. Patent Application Publication 2011 / 0189658, which is incorporated herein by reference in its entirety. (ix) The step of measuring the activity of the first tag-responsive reporter protein in cells of a second control cell sample, which is normalized or unnormalized to the activity of the second luciferase, is described in U.S. Patent Application Publication 2011 / 0189658, which is incorporated herein by reference in its entirety. (x) A step of providing a reporter activity ratio of a first control cell sample to a second control cell sample, wherein a ratio less than 1 (1st / 2nd) indicates the presence of an antibody against an AAV serotype or recombinant AAV vector in the sample.

[0029] A further aspect of the present invention relates to a method for high-throughput screening of anti-AAV antibodies, the method comprising the following steps: (i) A step of providing a test sample that is thought to contain 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) A step of contacting the test sample and the control sample with a challenge virus preparation or virus-producing packaging cell according to the present invention, wherein the cell is for the production of an AAV serotype or recombinant AAV vector from which an antibody response is determined, and the challenge virus / recombinant AAV vector contains a tag sequence within the viral genome or transgene construct. (iii) A step of contacting the test sample, control sample and challenge virus / virus-producing packaging cells with a reporter gene cell line according to the present invention, wherein the reporter cell line contains a first heterologous polynucleotide comprising a heterologous cis-action regulatory sequence that responds to treatment of the cell line with a tag sequence present in the genome of an AAV serotype or a recombinant AAV vector transgene promoter construct in which an antibody present in the test sample is detected or detected and quantified. (iv) Here, the promoter of the reporter cell line is operably linked to an open reading frame encoding a first reporter protein, including, for example, firefly luciferase, sea urchin luciferase, metridial luciferase, or one described in European Patent Application No. 21170068.7, which is incorporated herein in whole by reference. In a preferred embodiment, the cell line according to the present invention is seeded in a 96, 384, or 1536 well assay plate. (v) In a preferred embodiment, to provide assay normalization, the reporter cell line according to the present invention further has a construct for a constitutive product of a luciferase different from that used in the AAV-responsive reporter gene construct. For example, the constitutive product may be a second luciferase, such as sea urchin luciferase, firefly luciferase, metridial luciferase, or a constitutive product of a novel luciferase, such as that described in European Patent Application No. 21170068.7, which is incorporated herein in whole by reference. (vi) The step of measuring the activity of a second reporter protein in the cell line after the activity of a tag-responsive reporter gene luciferase has been measured in the same sample using, for example, a Stop&Glo reagent from the Dual-Glo system (Promega, Madison, Wisconsin) that efficiently inhibits the activity of a 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 2011 / 0189658, which is incorporated in whole herein by reference. (vii) The step of measuring the activity of a first tag-responsive reporter protein in reporter cells of a first cell sample, normalized or unnormalized to the activity of a second luciferase, is described in U.S. Patent Application Publication 2011 / 0189658, which is incorporated herein by reference in its entirety. (viii) The step of measuring the activity of the first tag-responsive reporter protein in cells of a second control cell sample, which is normalized or unnormalized to the activity of the second luciferase, is described in U.S. Patent Application Publication 2011 / 0189658, which is incorporated herein in its entirety by reference. (ix) A step of providing a reporter activity ratio of a first control cell sample to a second control cell sample, wherein a ratio less than 1 (1st / 2nd) indicates the presence of an antibody against an AAV serotype or recombinant AAV vector in the sample.

[0030] Accordingly, the present invention provides a packaging cell line that expresses the genome or naturally occurring capsid, hybrid capsid, chimeric capsid, or rationally designed capsid of a specific AAV serotype and is transfected with a gene encoding a specific recombinant AAV vector that encodes a specific transgene / promoter construct labeled with a specific tag sequence, and a reporter cell line that specifically responds to the tag sequence encoded by a specific AAV serotype or specific recombinant AAV vector. When 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, when different tag sequences are used to label individual AAV serotypes or individual recombinant AAV vectors, either different reporter cell lines that specifically respond to each individual specific tag sequence or reporter cell lines containing each individual tag are required. Its use in various situations enables detection of neutralizing antibody activity with higher sensitivity, such that the EC50 of the dose-response curve of a sample containing a neutralizing antibody against a specific AAV serotype or recombinant AAV vector determined according to the present invention is at least reduced compared to methods of the prior art. The reduction may be about 2 times, for example about 3 times, for example about 4 times, for example about 5 times, for example about 6 times, for example 7 times, for example about 8 times, for example 9 times, for example 10 times, for example 50 times, for example about 100 times, or for example about 1000 times.

[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 present 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 a neutralizing antibody. In one embodiment, the neutralizing antibody may be an antibody against a specific AAV serotype or recombinant AAV vector.

[0032] In one embodiment, the present invention relates to a cell or cell line and method relating thereto that enables higher specificity. The specificity may be at least about 75%, for example at least about 80%, for example at least about 85%, for example at least about 90%, for example at least about 95%, for example at least about 97.5%, for example at least about 98%, for example at least about 99%, for example at least about 99.5%.

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

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

[0035] In a further embodiment, the present invention provides both increased sensitivity and increased specificity. [Brief explanation of the drawing]

[0036] [Figure 1]Component 1: This figure shows the molecular constructs used to establish the packaging cell line of Example 1. Human lung adenocarcinoma cell line A549 was stably transfected with the following constructs: a preferred promoter, the CMV constitutive promoter in the shown example, and a 2A autocleavage peptide contained within the ITR of a given AAV serotype AAV2 in the shown example, isolated by the sequence encoding F2A in the shown example, under the control of a suitable promoter, the CMV constitutive promoter in the shown example, and the gal4-VP16 tag sequence. The rep and cap genes are expressed on different plasmids under the control of the constitutive promoter. The E1 gene is expressed under the control of an inductive promoter, which in the shown example consists of a 12-fold tandem repeat sequence of the ZFHD1 DNA-binding domain fused to FL506-binding protein (KFBP). Cells are also stably transfected with genes encoding E2A, E4, and VA RNA under the control of the constitutive promoter. [Figure 2] Component 2: This figure shows the molecular construct used to establish the reporter cell line of Example 1. Human embryonic kidney cell line HEK293 was stably transfected with a chimeric promoter consisting of a 5x tandem repeat sequence of the Gal4 upstream activating sequence (UAS) and a minimal promoter that modulates the expression of the firefly luciferase (FL) gene, along with a polyadenylation site from SV40 as shown in the example. [Figure 3] Component 2: This figure shows the molecular constructs used to establish the reporter cell lines of the examples. The human embryonic kidney cell line HEK293 was stably co-transfected with a chimeric promoter consisting of a 5x tandem repeat sequence of the Gal4 upstream activating sequence (UAS), shown as the first reporter gene, and a minimal promoter that regulates the expression of the firefly luciferase (FL) gene along with a polyadenylation site from SV40, shown as an example. The cells were also stably co-transfected with sea urchin luciferase under the control of the minimal constitutive promoter of SV40, shown as the second reporter gene. [Figure 4]This figure 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. [Figure 5] This figure shows the expression of the recombinant AAV vector genome, as well as the binding of the Gal4 DNA-binding domain-VP6 hybrid protein to a 5x tandem repeat sequence of the Gal4 upstream activation sequence (UAS), and potent activation of the firefly luciferase reporter gene, along with minimal activation of the sea urchin luciferase reporter gene under the control of a constitutive promoter, reflecting the nonspecific cytotoxicity of the virus. [Figure 6] The study demonstrates neutralization of AAV virus particles by anti-AAV antibodies (component 1) present in the sample upon entry 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 sea urchin luciferase receptor gene under the control of a constitutive promoter remains unchanged, reflecting the nonspecific cytotoxicity of the virus. [Figure 7] This figure shows the enhancement of Gal4-UAS regulated FL expression after transiently transfecting a reporter cell line (component 2) with a gene encoding the initial enhancer protein E4orf6, and then incubation the reporter cells with packaging cells (component 1) containing the cap gene encoding the AAV2 cap protein. [Figure 8] This figure shows the enhancement of Gal4-UAS regulated FL expression after transiently transfecting a reporter cell line (component 2) with a gene encoding the initial enhancer protein E4orf6, and then incubation the reporter cells with packaging cells (component 1) containing the cap gene encoding the AAV5 cap protein. [Figure 9]This figure 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. It was found that a total of 15,000 reporter cells / well in the presence of 7,500 packaging cells yielded optimal FL expression. [Figure 10] This figure shows the quantification of the neutralizing activity of a serum sample after a packaging cell line expressing a recombinant AAV2 vector was brought into contact with the sample under test at 20°C for 30 minutes, or without contact, and the packaging cell line and sample were incubated with reporter cells at 37°C for 18 hours. Subsequently, FL reporter gene activity was quantified using a commercially available substrate (Bright-Glo, Promega, Madison, Wisconsin), and luminescence was quantified using a luminometer (Glo-Max, Promega, Madison, Wisconsin). [Figure 11] This figure shows the effect of pre-incubating a sample to be tested for the presence of anti-AAV2 neutralizing antibodies with packaging cells expressing the gene encoding the AAV2 Cap protein (component 1) at room temperature for 30 minutes, or incubating it with reporter cells (component 2) at 37°C for 18 hours without pre-incubation. [Figure 12] This figure shows a comparison of the procedures used to test a sample for the presence of anti-AAV2 neutralizing antibodies using either the present invention or the transduction inhibition reference method described herein. [Figure 13] This figure shows a comparison of titrations of samples tested for the presence of anti-AAV2 neutralizing antibodies using either the present invention or the transduction inhibition reference method described herein. [Figure 14] This figure shows the titration results of human IVIG samples tested for the presence of neutralizing antibodies that cross-react with various animal AAVs using the invention described herein. [Figure 15]This figure 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] This figure shows the titration results of human IVIG samples 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] This figure shows a comparison of titrations of samples tested for the presence of anti-AAV2 neutralizing antibodies using either of the inventions described herein, which involves incubating virus-producing packaging cells with reporter cells overnight, or incubating free virus with an equivalent MOI with reporter cells overnight. [Figure 18] This figure shows the apparent neutralizing effect of a pool of two human serums and human IgG (IV-IgG) from normal individuals on the activation of a reporter cell line (component 2 of the present invention) containing both a firefly luciferase reporter gene regulated by a 5x tandem repeat sequence of Gal4 UAS and a sea urchin luciferase normalization gene regulated by a thymidine kinase constitutive promoter, in the presence of AAV8 packaging cells (component 1 of the present invention). [Figure 19] This figure shows the expression of sea urciferase (RL) in a reporter cell line (component 2 of the present invention) that contains both a firefly luciferase (FL) reporter gene regulated by a 5x tandem repeat sequence of UAS and a sea urciferase (RL) normalization gene regulated by a thymidine kinase constitutive promoter. Sea urciferase expression is stable when using IV-IgG, but decreases when using two human serums (HS0 and HS4) from normal individuals, demonstrating that the apparent neutralizing effect of serum HS0 and HS4 in the presence of AAV8 packaging cells (component 1 of the present invention) is due to nonspecific cytotoxicity. [Figure 20]This figure shows the ratio of FL to RL activity in a pool of two human serum HS0 and HS4 from normal individuals, as well as human IgG (IV-IgG), in the presence of AAV8 packaging cells (component 1 of the present invention) to activation of a reporter cell line (component 2 of the present invention) containing both an FL reporter gene regulated by a 5x tandem repeat sequence of Gal4 UAS and an RL normalization gene regulated by a thymidine kinase constitutive promoter. It indicates that the apparent neutralizing effect of serum HS0 and HS4 was actually due to nonspecific cytotoxicity. [Modes for carrying out the invention]

[0037] Certain terms are used for clarity when describing embodiments of the present invention. However, the present invention is not intended to be limited to the specific terms thus selected, and it is understood that each specific term includes all technical equivalents that operate in a similar manner to achieve a similar purpose.

[0038] The present invention is made in light of the above-described prior art, and the object of the present invention is 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 achieve this object, the present invention provides, in particular, a packing cell line (component 1) for producing AAV serotypes or recombinant AAV vectors in which an antibody response can be determined, comprising a tag sequence in the viral genome or an transgene promoter construct contained within the AAV ITR.

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

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

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

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

[0043] Therefore, the present invention relates to a cell or cell line (component 1), wherein the cell is (i) An AAV genome or transgene promoter construct containing one or more tag sequences within the AAV ITR, (ii) Cap gene of an AAV serotype or recombinant AAV vector encoding any of the following: a naturally occurring Cap, hybrid Cap, chimeric Cap, or rationally designed Cap, operably linked to a constitutive or inductive promoter; (iii) AAV rep gene operably linked to an inductive promoter, (iv) AAV E2A, E4 and VA genes operably linked to different individual innate or inducible promoters, (v) AAV E1A gene operably linked to an inductive promoter It includes at least one or more of the following.

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

[0045] In a particular embodiment, the cell includes all of (i), (ii), (iii), (iv), and (v).

[0046] In one embodiment, the inductive promoters (iii), (iv), and (v) are different from each other, and in a Tet-on / Tet-off system, cumate The following can be selected: the inductive repressor CymR system, the flavonoid phloretin-modulated TtgR repressor system, and the vanillic acid-modulated VanR repressor / KRAP system.

[0047] In a further embodiment, the AAV E1A gene may be regulated by an inductive promoter that eliminates the need to control the rep gene or any of the E2A, E4, and VA genes by using one or more inductive promoters. In other words, in one embodiment, it is only necessary that the E1A gene be regulated by an inductive promoter. Therefore, in one embodiment, the inductive promoter of (ii) may be an inductive promoter that eliminates the need to control the rep gene or any of 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 VanO 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] To enhance sensitivity, it is understood that host cell lines can be stably transfected with either the cell surface AAV receptor alone (9) or the endosomal AAV receptor alone (10), or with both the cell surface receptor and the endosomal AAV receptor.

[0051] The selection of host cell lines is determined by analysis of the biological sample and its safety profile regarding the cell's ability to tolerate the toxicity of the AAV Rep protein after controlled expression of the rep gene and E4orf6 (11). In one embodiment, the host cell is a metazoan cell. In another embodiment, the host cell line may include, but is not limited to, the human lung adenocarcinoma cell line A549 (ATCC catalog no. CCL-185) or the human embryonic kidney cell line HEK293T (ATCC catalog no. 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 an appropriate tissue-specific promoter that modulates the expression of the transgene selected according to the desired tissue-specific expression of the transgene, and a suitable polyadenylation site contained within the ITR of AAV2, such as one derived from SV40, or an alternative polyadenylation site, for example (Figure 1). In another embodiment, the constitutive promoter may be, for example, the SV40, UBC, EF1A, PGK, and CAGG promoters. In a further embodiment, the tissue-specific promoter may be, for example, human retina-specific promoters 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, and the vitiligo macular dystrophy (VMD2) promoter. Common muscle-specific promoters include neuron-specific promoters such as the creatine kinase (CK) and myosin heavy chain (MyHC) promoters, or the synapsin promoter.

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

[0054] Since the genes encoding E1, 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 sequence of the DNA-binding domain ZFHD1 fused to FK506-binding protein (FKBP). The FKBP-rapamycin-related protein (FRAP) is fused to an HSV-1-derived VP16 transactivator such that the addition of rapamycin induces the formation of a heterodimer between FKBP and FRAP, resulting in the strictly controlled expression of the gene encoding the E1 initial protein.

[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 adenovirus-free recombinant AAV vector production (14), but early helper proteins E2A, E4, and VA RNA have been reported to be necessary for efficient recombinant AAV production (15). Therefore, packaging cell lines can be co-transfected with expression vectors expressing E2A, E4, and VA RNA in a single case.

[0056] Component 2: Reporter cell line. In one embodiment, 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 contact between reporter cells and packaging cells producing an AAV challenge virus / recombinant AAV vector, or a required AAV serotype or recombinant AAV vector for which a neutralizing antibody is quantified, results in activation of a reporter gene expressed by the reporter cell line. In Example 1, the packaging cell line expresses a transgene promoter construct containing 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. Specifically, in Example 1, the reporter cell contains a 5-fold tandem repeat sequence of the Gal4 UAS that regulates the expression of the FL reporter gene in a HEK293 cell background (Figure 1).

[0057] Interaction between packaging cell lines and reporter cells In various embodiments, the present invention relates to a method comprising at least one of the following steps: (a) The virus produced by the packaging cells (component 1) according to the present invention is brought into contact with the sample to be tested for various times and at various temperatures including approximately +4°C, approximately 20°C, or approximately 37°C. The virus preparation and the sample are then incubated with reporter cells (component 2) at approximately 37°C for various times, preferably approximately 15 to approximately 18 hours or more, and the FL reporter gene activity is then quantified using a suitable substrate, for example, the commercially available substrate Bright-Glo (Promega, Madison, Wisconsin). (b) A step in which the virus-producing packaging cells (component 1) are brought into contact with the sample to be tested at various temperatures including approximately +4°C, approximately 20°C, or approximately 37°C for various durations, the virus-producing packaging cells and the sample are incubated with reporter cells (component 2) at approximately 37°C for various durations, preferably approximately 15 to approximately 18 hours or more, and then the FL reporter gene activity is quantified. (c) In a preferred embodiment, virus-producing packaging cells (component 1) are brought into contact with the sample to be tested and reporter cells (component 2), and after direct incubation at approximately 37°C for various times, preferably about 6 to about 18 hours or more, the FL reporter gene activity is quantified using a suitable substrate, for example, the commercially available substrate Bright-Glo (Promega, Madison, Wisconsin). (c) Freeze virus-producing packaging cells (component 1) together with reporter cells (component 2) at an appropriate cell concentration in a suitable cryogenic protective medium, thaw the cells, contact the packaging cells / reporter gene cells with the sample to be tested, incubate the sample, packaging cells-reporter cells at approximately 37°C for various times, preferably about 18 hours or more, and then quantify the FL reporter gene activity using a suitable substrate such as the commercially available substrate Bright-Glo (Promega, Madison, Wisconsin). (d) In a preferred embodiment, virus-producing packaging cells (component 1) and reporter cells (component 2) are separately frozen in a suitable cryogenic protective medium at an appropriate cell concentration, the cells are thawed, the packaging cell line is brought into contact with the sample to be tested for various times and at various temperatures including approximately +4°C, approximately 20°C, or approximately 37°C, the packaging cells and the sample are incubated with reporter cells at approximately 37°C for various times, preferably approximately 18 hours or more, and then the FL reporter gene activity is quantified using a suitable substrate such as the commercially available substrate Bright-Glo (Promega, Madison, Wisconsin).

[0058] Accordingly, in one embodiment of the present invention, the incubation period between component 1, component 2 and the biological sample may 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, or about 32 hours. In one embodiment, the incubation period may be, for example, overnight, which may be any period between about 6 hours and about 10 hours, or any period between about 6 hours and about 18 hours. In another embodiment, the incubation period may be, for example, about 15 hours to about 30 hours.

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

[0060] The incubation temperature may be any range of approximately 4°C to approximately 37°C, or approximately 4°C, approximately 10°C, approximately 15°C, approximately 20°C, approximately 25°C, approximately 30°C, approximately 35°C, or approximately 37°C. Alternatively, the temperature may be in the range of approximately 30°C to approximately 39°C. Therefore, the temperature may be relevant to the incubation of the cells of component 1 and / or component 2 and / or the biological sample to be analyzed.

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

[0062] In one embodiment, an AAV preparation or virus-producing packaging cell interacts with a reporter gene cell, resulting in viral uptake, internalization, and AAV serotype-specific activation of the FL reporter gene. The initial interaction between the virus preparation or virus-producing packaging cell and the reporter cell is likely mediated by the interaction of the capsid with a carbohydrate on the cell surface in the release of the virus and serotype-specific interactions. 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 serotype then interacts with either the AAV receptor (9) and / or a novel G protein-coupled receptor-like protein GPR108 localized in the trans-Golgi, which are necessary for intracellular internalization (10). In one embodiment, the reporter gene encodes an enzyme or any other means that enables detection in a preferred manner. In a preferred embodiment, the reporter is a novel luciferase, such as firefly luciferase, or sea urchin luciferase, metridial luciferase, as described in European Patent Application No. 21170068.7, which is incorporated in whole herein by reference, or a novel luciferase present in solution as a soluble active monomer containing a luciferase or fluorescent protein, such as green fluorescent protein (EGFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), blue fluorescent protein (BFP) and their variants, 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 a fusion protein with other proteins containing luciferase, or attached to a solid surface 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 their variants exhibiting different excitation / emission spectra.

[0064] In a further embodiment, 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, which specifically responds to a tag sequence present in the AAV genome or in a recombinant AAV coding transgene construct (obtained from component 1), and a second reporter gene, such as RL, under the control of a constitutive promoter, which allows for the normalization of tag sequence-activated FL activity with respect to the constitutive expression level of sea urchin luciferase (RL) activity, thereby making the assay cell number independent and providing a means to compensate for inter-sample variability in cell density due to cell loss or variations in the number of seeded cells. The ability to normalize AAV-activated FL activity with respect 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, 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.

[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 address the technical problems outlined above, a two-component system is described, comprising a packing cell line (component 1) for producing an AAV serotype or recombinant AAV vector that determines the antibody response, containing a tag sequence within 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 in component 1, and a method for using this system to detect and optimally quantify anti-AAV neutralizing antibodies in a test sample. The present invention further relates to a high-throughput screening method for detecting and optimally monitoring immune responses to AAV infection or treatment with recombinant AAV vectors with optimal sensitivity, signal intensity, and biological fidelity.

[0067] Component 1: Packaging cell line. A stable AAV packaging cell line can be established in which the components necessary for the expression of an AAV serotype or recombinant AAV vector are supplied by transient transfection or integrated into the genome of a cell line selected in a preferred embodiment of the present invention.

[0068] As a non-limiting example, a stable cell line for the production of a specific 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 selected under the control of either a constitutive promoter or an appropriate tissue-specific promoter, depending on the intended use, and a polyadenylation site contained within the ITR of AAV2 (as shown in Figure 1).

[0069] To construct expression vectors for transgene promoter constructs that confer the highest possible level of expression, various types of promoter variants are designed in silico to reduce promoter size and increase transgene transcription. Then, codon-optimized transgene promoter constructs containing tag sequences, such as the Gal4 DNA-binding domain in Example 1, are synthesized in vitro and used to construct a series of chimeric transgene promoter sequences and, depending on their effectiveness, one of the synthetic polyadenylation sites contained within the ITR of AAV2, an SV40 polyadenylation site, or a human growth hormone polyadenylation site.

[0070] In one embodiment, cells in component 1 may express an transgene promoter construct containing 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, or they may express an AAV genome or a VanR sequence within the transgene construct, or an AAV genome or a transgene promoter construct encoding a cGMP-specific receptor protein (CRP).

[0071] In a further aspect of the present invention, a tag sequence contained within the AAV genome or transgene construct may encode the transsilencer VanR fused to the transactivator VP16.

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

[0073] In a further embodiment, in cases where there are two or more tag sequences, 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 ITR to generate a self-complementary intermediate so that the transgene is expressed without requiring 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 a packaging cell line established in, for example, A549 cells, which contains all the initial helper proteins necessary for replicating the transgene encoded by the AAV vector but lacks a transgene promoter construct with a tag sequence contained within the ITR of AAV2. Expression levels are monitored by Western blotting using appropriate detection antibodies and by the level of activation of the firefly luciferase (FL) reporter gene under the control of a chimeric transgene-responsive promoter. Results are normalized with respect to the expression of sea urchin 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 blotting is estimated to be optimal for standard ITR expression, and the normalized increase in FL expression is estimated to be sufficient using a reporter cell line such as Example 1 which expresses a tandem repeat of Gal4 UAS that modulates the expression of a firefly luciferase reporter gene that responds specifically to a Gal4 DNA-binding domain tag contained in the AAV genome or recombinant AAV vector, then the construct is used to establish a packaging cell line by transient transfection, or, in a preferred embodiment, by establishing a stable packaging cell line in which the necessary components of the genome of a host cell line, such as A549 cells, are stably incorporated.

[0078] Packaging cell lines can be transfected with an expression vector for the cap gene under the control of a constitutive promoter. Therefore, in Example 1, the cap gene is expressed under the control of the CMV early promoter and used to establish a packaging cell line after transient transfection with the components, or, in a preferred embodiment, to establish a stable packaging cell line in which the necessary components are stably incorporated into the genome of a host cell line, such as A549 cells.

[0079] In Example 1, to ensure optimally regulated expression of the rep gene and initial gene necessary for expressing the transgene, the gene encoding E1 was expressed under the control of a rapamycin-inducible promoter consisting of a tandem repeat sequence of a DNA-binding domain ZFHD1 mutant fused to an in silico-designed FK506-binding protein (FKBP). A variant of FKBP-rapamycin-related protein (FRAP) was fused to an in silico-designed HSV-1-derived VP16 transactivator such that the addition of rapamycin induced the formation of a heterodimer between FKBP and FRAP, resulting in strictly controlled expression of the gene encoding the E1 initial helper protein.

[0080] Next, constructs were synthesized in vitro and tested for their ability to regulate E1 protein expression 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 through transcriptional activation of the p5 and p19 promoters. To ensure the 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. A tandem repeat sequence of DNA-binding domain ZFHD1 fused to FK506-binding protein (FKBP) and a variant of FKBP-rapamycin-associated protein (FRAP) fused to VP16 transcription activator was designed in silico, synthesized in vitro, and tested in transient transfection experiments in A549 cells, so that the addition of rapamycin induces the formation of a heterodimer between FKBP and FRAP, resulting in tightly controlled 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 initial enhancer proteins E2A, E4, and VA RNA, necessary for the efficient production of recombinant AAV, were expressed as a single polycistronic sequence incorporating a self-cleaving 2A peptide, to ensure that the three individual proteins were expressed at the required levels and in constant stoichiometric amounts under the control of E1a. The functionality of the construct was monitored by the production efficiency of the encapsulated transgene at normalized levels of expression determined using the reporter cells of the present invention.

[0082] In a preferred embodiment of the present invention, the components of the packaging cell line are integrated into the genome of a 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 gene and the E1A gene under the control of the rapamycin-regulated ZFHD1 promoter, whose expression is regulated by E1A expression, as well as the E2A, E4, and VA genes. Human A549 cells were transfected with the above construct 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 blotting for rapamycin-inducible ability after transient co-transfection with a vector expressing the firefly luciferase (FL) gene under the control of a 12-fold tandem repeat sequence 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 level of the fully inclusion competent recombinant AAV vector, as determined by qPCR. The quantification of AAV vector genomes was based on the use of AAV2 ITR-specific target sequences, which allowed for direct comparison of interlaboratory results, but relied on the use of linear or circular ITA standards and extended denaturation cycles due to the extensive secondary hairpin structure of AAV2 ITRs (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 the total inclusion competent recombinant AAV vector, monitored by the activation level of a transgene-responsive reporter cell line. The clones also exhibited stable growth characteristics, including both doubling time and maximum cell density achieved under standardized growth conditions in a medium containing the required selector.

[0084] definition vector The term "vector" refers to a DNA molecule used as a vehicle for transferring recombinant genetic material into host cells. The four main types of vectors are plasmids, bacteriophages and other viruses, cosmids, and artificial chromosomes. A vector itself is generally a DNA sequence consisting of an insert (a heterologous nucleic acid sequence, or transgene) and a larger sequence that functions as the vector's "skeleton." The purpose of a vector for transmitting genetic information to a host is typically to isolate, grow, or express the insert in target cells. Vectors called expression vectors (or expression constructs) are specifically adapted for the expression of a heterologous sequence in target cells and generally have a promoter sequence that drives the expression of the heterologous sequence. The choice of vector used in embodiments of the present invention depends on the specific application of the polypeptide or polynucleotide-encoding vector.

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

[0086] Operablely connected The term "operatably linked" refers to the linking of elements that are part of a functional unit, such as a gene or an open reading frame. Therefore, by operatably 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 enables the transcription of the nucleic acid sequence directed by the promoter. By operatably linking two heterologous nucleic acid sequences encoding polypeptides, the sequences become part of an open reading frame that encodes a functional unit, i.e., a fusion protein containing the amino acid sequence encoded by the heterologous nucleic acid sequences. By operatably linking two amino acid sequences, the sequences become part of the same functional unit, i.e., a polypeptide. By operatably linking two heterologous amino acid sequences, a hybrid (fusion) polypeptide is produced.

[0087] Minimal promoter Promoters that direct the expression of reporter genes are typically minimally constitutively active in mammalian host cells used to establish the reporter cell lines of the present invention and, on their own, do not respond to cell treatment with pharmacologically active molecules. Useful constitutively active promoters include, but are not limited to, the 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.

[0088] Inducible promoter An inducible promoter is a DNA sequence in which mRNA transcription occurs following a reversible change in the presence, abundance, or conformation of a regulatory factor or protein in a cell, enabling an activated 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 lead to promoter induction.

[0089] Chemically modified promoters are one of the most common inducible promoters. The positive 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-regulated transcription activator) is normally inactive and cannot bind to tetracycline responsive factors (TREs) in the promoter. Tetracyclines and their derivatives act as inducers that enable promoter activation.

[0090] One of the most commonly used prokaryotic promoters is the negative inducible pLac promoter. This promoter requires the removal of the lac repressor (lacI protein) for transcription activation. In the presence of lactose or the lactose analog IPTG, the lac repressor undergoes a conformational change that removes it from the lacO site within the promoter, halting the repression of the target gene. Simplified lac-inducible systems are found in many bacterial expression vectors.

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

[0092] Temperature-sensitive expression systems typically exhibit less leakage than chemically induced promoters and exhibit near-zero expression at normal temperatures but can be induced by exposure to heat or cold. Examples, though not limited to these, include heat-shock-inducible Hsp70 or Hsp90-derived promoters, where selected genes are expressed only after brief exposure to heat shock. In the case of Hsp70, heat shock releases heat shock factor 1 (HSF-1), which subsequently binds to the heat shock element within the promoter, thereby activating transcription. Other non-limiting examples include heat-shock-inducible Cre and Cas9 for genome engineering in species such as the nematode (C. elegans) and the fruit fly (Drosophila).

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

[0094] In describing embodiments of the present invention, not all possible combinations and permutations of embodiments are explicitly described. Nevertheless, the mere fact that certain means are listed in different dependent items or described in different embodiments does not indicate that combinations of these means cannot be used advantageously. The present invention assumes all possible combinations and substitutions of the described embodiments.

[0095] The terms “comprising,” “comprise,” and “comprises” as used herein are intended to be optionally substituted in all cases with the terms “consisting of,” “consist of,” and “consist of,” respectively. The present invention will be more fully understood by referring to the detailed description of the invention. However, this should not be construed as limiting the scope of the invention. All references to the literature are incorporated herein by reference in their entirety. [Examples]

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

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

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

[0099] Example 3 Packaging cell lines (component 1), established in HEK293 cells, possessing a Gal-4 tag sequence between ITRs and expressing the cap gene encoding the AAV2 capsid, as shown in Figure 1, were co-incubated overnight with a certain number of reporter cell lines containing the firefly luciferase reporter gene, under the control of a 5x tandem repeat sequence of GAL4 UAS (component 2), as shown in Figure 2. In parallel, in the same experiment, purified cell-free AAV2 virions with increasing MOI were co-incubated overnight with a certain number of reporter cell lines containing the firefly luciferase reporter gene, under the control of a 5x tandem repeat sequence of GAL4 UAS (component 2), as shown in Figure 2. Unexpectedly, direct contact between an AAV2-producing packaging cell line (component 1) and a reporter cell line containing the firefly luciferase receptor gene, under the control of a 5x tandem repeat sequence of GAL4 UAS (component 2) as shown in Figure 2, resulted in greater activation of FL receptor gene activity than the addition of purified cell-free AAV2 virions with an equivalent MOI and overnight incubation with the reporter cell line containing the firefly luciferase receptor gene under the control of a 5x tandem repeat sequence of GAL4 UAS (component 2). This is shown in Figure 17, where a given amount of AAV2-producing packaging cell line (component 1), incubated overnight with a reporter cell line containing the firefly luciferase reporter gene under the control of a 5x tandem repeat sequence of GAL4 UAS (component 2) shown in Figure 2, resulted in greater activation of the FL reporter gene than the addition of an equivalent MOI of free AAV2 virions incubated overnight with the reporter cell line (component 2). This suggests that co-incubation of the two-component system of the present invention over a period of time, 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, in particular, direct contact between the reporter cells and purified cell-free viral virions with an equivalent MOI.Therefore, the effective antigen loading provided by contact between virus-producing packaging cells (component 1) and reporter cells (component 2) over a given period of time using a sample believed to contain neutralizing antibodies against the wild-type AAV serotype or recombinant AAV vector of the present invention is effectively lower than that of conventional neutralization assays in which cell-free viral virions are contacted with a sample believed to contain neutralizing antibodies against the wild-type AAV serotype or recombinant AAV vector at 37°C for a period of often less than one hour, resulting in increased sensitivity to the present 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 recombinant AAV vector, in which the antibody response is determined, (i) AAV genome or transgene promoter construct containing a tag sequence within the AAV ITR, (ii) Cap gene of an AAV serotype or recombinant AAV vector encoding any of a naturally occurring Cap, hybrid Cap, or chimeric Cap, which is operablely linked to a constitutive promoter. (iii) AAV rep gene operably linked to an inductive promoter, (iv) AAV E2A, E4 and VA genes operably linked to their respective intrinsic or inducible promoters, (v) AAV E1A gene operably linked to an inductive promoter The first metazoan cell (component 1), which includes [the specified element]. 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. Furthermore, a method for using the same to detect and optimally quantify anti-AAV neutralizing antibodies in a test sample. 2. The cells described in item 1, which are baculovirus-infected insect cells, e.g., SF9 cells; avian cells, e.g., DT-40, MSB1, or LMH; mouse cells, e.g., L929 cells or LS variants; Chinese hamster ovary (CHO) cells, e.g., CHO-K1, CHO-DXB11, CHO-DG44, CHOK1SV (including all variants), CHOK1SV-GSKO (glutamine synthase knockout) (including all variants); human cells, e.g., HEK293 cells (including all variants and all suspension or adherent variants); HeLa cells, HT1080 cells, ARPE-19, U937, Jurkat, HuH-7, HepG2, K562, A431, or A549 cells. 3. Component 1 cells in which the tag sequence contained within the AAV genome or transgene promoter construct is a Gal4 DNA-binding domain. 4. Component 1 cells in which a tag sequence contained within the AAV genome or transgene promoter construct encodes cGMP-specific receptor protein (CRP). 5. Component 1 cells in which a tag sequence contained within the AAV genome or transgene construct encodes the transsilencer VanR fused to the transactivator VP16. 6. A chimeric promoter comprising a Gal4 upstream activation sequence (UAS) and a minimal promoter comprising the transcription start site TATAA or a variant thereof, or 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 the transgene construct of component 1, which is operably ligated to a chimeric promoter comprising a tangent repeat sequence of a Gal4 UAS, preferably five times the tangent repeat sequence thereof. 7. A chimeric promoter comprising a GTA operator sequence and a minimal promoter, or a chimeric promoter comprising a tandem repeat sequence, preferably four times the tandem repeat sequence, comprising 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 the transgene promoter construct of component 1, which is operably ligated to the chimeric promoter. 8. Cells 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 the transgene construct of component 1, which is operably linked to a chimeric promoter including a VanO operator module, preferably an octameric VanO operator module (Van08), and a minimal promoter such as the CMV initial promoter. 9. Cells of component 1 as described in any one of the above items, comprising at least five recombinant target sites. 10. Cells of component 2 as described in any one of items 1 to 3, wherein the reporter is a luciferase such as firefly luciferase, sea urchin luciferase, metridial luciferase, or a novel luciferase described in European Patent Application No. 21170068.7, or a novel luciferase or other protein including a fluorescent protein such as 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, to provide assay normalization, the cell of component 2 according to the present invention further has a construct for constitutive expression of a luciferase different from that used in a reporter gene construct that responds to a tag sequence contained in the AAV genome or a transgene promoter construct. For example, the constitutive product may be a constitutive product of a second luciferase, such as sea urchin luciferase, firefly luciferase, metridial luciferase, or a novel luciferase such as that described in European Patent Application No. 21170068.7. 11. A method for detecting and optionally quantifying the activity of anti-AAV neutralizing antibodies in a test sample, (i) A method comprising the step of providing a test sample that is thought to contain an anti-AAV antibody together with a control sample that does not contain an anti-AAV antibody, and a positive control sample that is known to contain an anti-AAV neutralizing antibody, in a preferred embodiment. (ii) The step of bringing the test sample into contact with either a virus produced by a cell of component 1 described in any one of the above items, or a virus-producing cell of component 1. (iii) Before measuring the activity of the AAV tag-responsive first reporter protein in the cell line, the test sample is incubated with a virus produced by the cells of component 1 described in any one of the above items, or with the virus-producing cells of component 1, and with the cells of component 2 at various temperatures, preferably 37°C, for various durations of 6 to 18 hours or more. The method according to any of the above items, wherein the reporter cell line expresses a second reporter protein, and the method further comprises the following: (iv) A step of measuring the activity of the first reporter protein in the cell line, (v) A step of providing a ratio of the activity of the first reporter protein to the activity of the second reporter protein. (vi) The step of measuring the activity of a first tag-responsive reporter protein in reporter cells of a first cell sample, normalized or unnormalized to the activity of a second luciferase, is described in U.S. Patent Application Publication No. 2011 / 0189658. (vii) The step of measuring the activity of the first tag-responsive reporter protein in cells of a second control cell sample, which is normalized or unnormalized to the activity of the second luciferase, is described in U.S. Patent Application Publication No. 2011 / 0189658. (viii) A step of providing a reporter activity ratio of a first control cell sample to a second control cell sample, wherein a ratio less than 1 (1st / 2nd) indicates the presence of an antibody 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 an immune response to an AAV capsid, AAV genome, transgene, or transgene product, (i) A step of providing a test sample consisting of patient samples to be screened, (ii) The step of bringing the test sample into contact with the challenge virus or virus-producing packaging cell of component 1 according to the present invention and any one of the above items, (iii) Before measuring the activity of the AAV tag-responsive reporter protein 1 in the cell line, incubate the test sample with the challenge virus or virus-producing cells of component 1 and the cells of component 2, at various temperatures, preferably about 37°C, for various times, preferably about 6 to about 18 hours or more. A method that includes this. The method according to any of the above items, wherein the cell line expresses a second reporter protein, and the method further comprises the following: (iv) A step of measuring the activity of the first tag-responsive reporter protein in the cell line, (v) A step of providing a ratio of the activities of the cell line component 2, wherein component 2 comprises a first heterologous polynucleotide comprising a heterologous cis-acting regulatory sequence that responds 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, the promoter being operably linked to an open reading frame encoding a first reporter protein such as a luciferase, e.g., sea urchin luciferase, firefly luciferase, or metridial luciferase. In a preferred embodiment, to provide assay normalization, the cell of component 2 according to the present invention further has a construct for constitutive expression of a luciferase different from that used in a reporter gene construct that responds to a tag sequence contained in the AAV genome or transgene construct. For example, the constitutive product may be a constitutive product of a second luciferase, such as sea urchin luciferase, firefly luciferase, metridial luciferase, or a novel luciferase such as that described in European Patent Application No. 21170068.7. (vi) In a preferred embodiment, the cells according to the present invention are seeded in a 96, 384, or 1536 well plate assay plate. 13. A first metazoan cell (component 1) for the production of an AAV serotype or recombinant AAV vector in which an antibody response is determined, (i) Transgene promoter constructs containing tag sequences within the AAV genome or AAV ITR, (ii) Cap gene of an AAV serotype or recombinant AAV vector encoding any of a naturally occurring Cap, hybrid Cap, or chimeric Cap, which is operablely linked to a constitutive promoter. (iii) AAV rep gene operably linked to an inductive promoter, (iv) AAV E2A, E4 and VA genes, each operably linked to an individual native or inducible promoter, (v) A first metazoan cell (component 1) containing the AAV E1A gene operably linked to an inductive promoter. A second metazoan cell (component 2) incorporating a reporter gene that specifically responds to a tag sequence in the AAV genome or transgene promoter construct of component 1, and a method for detecting and optimally quantifying anti-AAV neutralizing antibodies in a test sample. 14. The cells described in item 1, which are baculovirus-infected insect cells, e.g., SF9 cells; avian cells, e.g., DT-40, MSB1, or LMH; mouse cells, e.g., L929 cells or LS variants; Chinese hamster ovary (CHO) cells, e.g., CHO-K1, CHO-DXB11, CHO-DG44, CHOK1SV (including all variants), CHOK1SV-GSKO (glutamine synthase knockout) (including all variants); human cells, e.g., HEK293 cells (including all variants and all suspension or adherent variants); HeLa cells, HT1080 cells, ARPE-19, U937, Jurkat, HuH-7, HepG2, K562, A431, or A549 cells. 15. Component 1 cells in which the tag sequence contained within the AAV genome or transgene promoter construct is a Gal4 DNA-binding domain. 16. Component 1 cells in which a tag sequence contained within the AAV genome or transgene promoter construct encodes cGMP-specific receptor protein (CRP). 17. Component 1 cells in which a tag sequence contained within the AAV genome or transgene construct encodes the transsilencer VanR fused to the transactivator VP16. 18. A chimeric promoter comprising a Gal4 upstream activation sequence (UAS) and a minimal promoter comprising the transcription start site TATAA or a variant thereof, or 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 the transgene construct of component 1, which is operably ligated to a chimeric promoter comprising a tangent repeat sequence of a Gal4 UAS, preferably five times the tangent repeat sequence thereof. 19. A chimeric promoter comprising a GTA operator sequence and a minimal promoter, or a chimeric promoter comprising a tandem repeat sequence thereof, preferably a quadruple tandem repeat sequence thereof, incorporating a reporter gene that specifically responds to a CRP sequence in the AAV genome of component 1 or a CRP sequence in an transgene promoter construct, which is operably ligated to the chimeric promoter. 20. Cells 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 the transgene construct of component 1, which is operably linked to a chimeric promoter including a VanO operator module, preferably an octameric VanO operator module (Van08), and a minimal promoter such as the CMV initial promoter. 21. Cells of component 1 as described in any one of the above items, comprising at least five recombinant target sites. 22. Cells of component 2 as described in any one of items 1 to 3, wherein the reporter is a luciferase such as firefly luciferase, sea urchin luciferase, metridial luciferase, or a novel luciferase described in European Patent Application No. 21170068.7, or a novel luciferase or other protein including a fluorescent protein such as 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, to provide assay normalization, the cell of component 2 according to the present invention further has a construct for constitutive expression of a luciferase different from that used in a reporter gene construct that responds to a tag sequence contained in the AAV genome or a transgene promoter construct. For example, the constitutive product may be a constitutive product of a second luciferase, such as sea urchin luciferase, firefly luciferase, metridial luciferase, or a novel luciferase such as that described in European Patent Application No. 21170068.7. 23. A method for detecting and optionally quantifying the activity of anti-AAV neutralizing antibodies in a test sample, (i) A method comprising the step of providing a test sample that is thought to contain an anti-AAV antibody together with a control sample that does not contain an anti-AAV antibody, and, in a preferred embodiment, a positive control sample that is known to contain an anti-AAV neutralizing antibody. (ii) The step of bringing the test sample into contact with either a virus produced by a cell of component 1 described in any one of the above items, or a virus-producing cell of component 1. (iii) Before measuring the activity of the AAV tag-responsive first reporter protein in the cell line, the test sample is incubated with a virus produced by the cells of component 1 described in any one of the above items, or with the virus-producing cells of component 1, and with the cells of component 2 at various temperatures, preferably 37°C, for various durations of 6 to 18 hours or more. The method according to any of the above items, wherein the reporter cell line expresses a second reporter protein, and the method further comprises the following: (iv) A step of measuring the activity of the first reporter protein in the cell line, (v) A step of providing a ratio of the activity of the first reporter protein to the activity of the second reporter protein. (vi) The step of measuring the activity of a first tag-responsive reporter protein in reporter cells of a first cell sample, normalized or unnormalized to the activity of a second luciferase, is described in U.S. Patent Application Publication No. 2011 / 0189658. (vii) The step of measuring the activity of the first tag-responsive reporter protein in cells of a second control cell sample, which is normalized or unnormalized to the activity of the second luciferase, is described in U.S. Patent Application Publication No. 2011 / 0189658. (viii) A step of providing a reporter activity ratio of a first control cell sample to a second control cell sample, wherein a ratio less than 1 (1st / 2nd) indicates the presence of an antibody 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 an immune response to an AAV capsid, AAV genome, transgene, or transgene product, (iv) A step of providing a test sample consisting of patient samples to be screened, (v) The step of contacting the test sample with a challenge virus or virus-producing packaging cell of component 1 according to the present invention and any one of the above items, (vi) A method comprising the step of incubating the test sample with the challenge virus or virus-producing packaging cells of component 1 and the cells of component 2, according to any one of the above items, at various temperatures, preferably 37°C, for various times, preferably 6 to 18 hours or more. The method according to any of the above items, wherein the cell line expresses a second reporter protein, and the method further comprises the following: (iv) A step of measuring the activity of the first tag-responsive reporter protein in the cell line, A step of providing a ratio of the activities of the cell line component 2, wherein component 2 comprises a first heterologous polynucleotide comprising a heterologous cis-acting regulatory sequence that responds 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, the promoter being operably linked to an open reading frame encoding a first reporter protein such as a luciferase, e.g., a sea urchin luciferase, a firefly luciferase, a metridial luciferase, or a novel luciferase such as that described in European Patent Application No. 21170068.7. (vii) In a preferred embodiment, to provide assay normalization, the cell of component 2 according to the present invention further has a construct for constitutive expression of a luciferase different from that used in a reporter gene construct that responds to a tag sequence contained in the AAV genome or transgene construct. For example, the constitutive product may be a constitutive product of a second luciferase, such as sea urchin luciferase, firefly luciferase, metridial luciferase, or a novel luciferase such as that described in European Patent Application No. 21170068.7. (viii) In a preferred embodiment, the cells according to the present invention are seeded in a 96, 384, or 1536 well plate assay plate. 25. A first metazoan cell (component 1) for the production of an AAV serotype or recombinant AAV vector in which an antibody response is determined, (i) Transgene promoter constructs containing tag sequences within the AAV genome or AAV ITR, (ii) Cap gene of an AAV serotype or recombinant AAV vector encoding any of a naturally occurring Cap, hybrid Cap, or chimeric Cap, which is operablely linked to a constitutive promoter. (iii) AAV rep gene operably linked to an inductive promoter, (iv) AAV E2A, E4 and VA genes, each operably linked to an individual native or inducible promoter, (v) A first metazoan cell (component 1) containing the AAV E1A gene operably linked to an inductive 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. Furthermore, a method for using the same to detect and optimally quantify anti-AAV neutralizing antibodies in a test sample. 26. Component 1 cells in which a tag sequence contained within the AAV genome or transgene promoter construct encodes cGMP-specific receptor protein (CRP). 27. A chimeric promoter comprising a Gal4 upstream activation sequence (UAS) and a minimal promoter comprising the transcription start site TATAA or a variant thereof, or a cell of component 2 incorporating an 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 the transgene construct of component 1, operably linked to a chimeric promoter comprising a tangent repeat sequence of the Gal4 UAS, preferably five times the tangent repeat sequence thereof. 28. A method for detecting and optionally quantifying the activity of anti-AAV neutralizing antibodies in a test sample, (i) A method comprising the step of providing a test sample from a normal human donor that is thought to contain 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) The step of bringing the test sample into contact with a virus-producing cell of component 1 as described in any one of the above items, (iii) Before measuring the activity of the AAV tag-responsive first reporter protein in the cell line, the test sample is incubated with the virus-producing cells of component 1 and the cells of component 2 as described in any one of the above items at various temperatures, preferably 37°C, for various durations of 6 to 18 hours or more. The method according to any of the above items, wherein the reporter cell line expresses a second reporter protein, and the method further comprises the following: (iv) A step of measuring the activity of the first reporter protein in the cell line, (v) A step of providing a ratio of the activity of the first reporter protein to the activity of the second reporter protein. (vi) The step of measuring the activity of a first tag-responsive reporter protein in reporter cells of a first cell sample, normalized or unnormalized to the activity of a second luciferase, is described in U.S. Patent Application Publication No. 2011 / 0189658. (vii) The step of measuring the activity of the first tag-responsive reporter protein in cells of a second control cell sample, which is normalized or unnormalized to the activity of the second luciferase, is described in U.S. Patent Application Publication No. 2011 / 0189658. (viii) A step of providing a reporter activity ratio of a first control cell sample to a second control cell sample, wherein a ratio less than 1 (1st / 2nd) indicates the presence of an antibody 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 an AAV capsid, AAV genome, transgene, or transgene product, (vii) A step of providing a test sample consisting of patient samples to be screened, (viii) The step of contacting the test sample with a challenge virus or virus-producing packaging cell of component 1 according to the present invention and any one of the above items, (ix) Before measuring the activity of the AAV tag-responsive reporter protein 1 in the cell line, incubate the test sample with the challenge virus or virus-producing cells of component 1 and the cells of component 2, at various temperatures, preferably about 37°C, for various times, preferably about 6 to about 18 hours or more. A method that includes this. The method according to any of the above items, wherein the cell line expresses a second reporter protein, and the method further comprises the following: (iv) A step of measuring the activity of the first tag-responsive reporter protein in the cell line, A step of providing a ratio of the activities of the cell line component 2, wherein component 2 comprises a first heterologous polynucleotide comprising a heterologous cis-acting regulatory sequence that responds 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, the promoter being operably linked to an open reading frame encoding a first reporter protein such as a luciferase, e.g., a sea urchin luciferase, a firefly luciferase, a metridial luciferase, or a novel luciferase such as that described in European Patent Application No. 21170068.7. (ix) In a preferred embodiment, to provide assay normalization, the cells of component 2 according to the present invention further have a construct for constitutive expression of a luciferase different from that used in a reporter gene construct that responds to a tag sequence contained in the AAV genome or transgene construct. For example, the constitutive product may be a constitutive product of a second luciferase, such as sea urchin luciferase, firefly luciferase, metridial luciferase, or a novel luciferase such as that described in European Patent Application No. 21170068.7. (x) In a preferred embodiment, the cells according to the present invention are seeded in a 96, 384, or 1536-well plate assay plate.

[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 inducible 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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