Assays for the detection of aav neutralizing antibodies

Abstract

The present invention relates to improved methods and assays for the detection of AAV neutralizing antibodies in sera. The present invention provides safe, sensitive and high throughput neutralization assays for antibody detection.

We claim: 
     
         1 . A kit for determining the titer of adeno-associated virus (AAV) neutralizing antibodies in a human serum sample, said kit comprising: (i) at least one recombinant AAV vector comprising a heterologous sequence, (ii) a population of AAV permissive cells; and (iii) instructions, wherein the instructions include the following steps:
 (a) incubating the recombinant AAV vector comprising the heterologous sequence with the human serum sample to form a serum/vector mixture;   (b) exposing the AAV permissive cells to the serum/vector mixture to produce AAV transduced cells which have been transduced with the recombinant AAV vectors from the serum/vector mixture;   (c) measuring the quantity of the AAV transduced cells; and   (d) determining the AAV neutralizing antibody titer in the human serum sample based on the measured quantity of the AAV transduced cells;   wherein the human serum sample is collected from a patient in need of an AAV mediated gene therapy, a patient after a dose of AAV gene therapy, or a patient suspected of exposure to an AAV vector.   
     
     
         2 . The kit of  claim 1 , wherein the recombinant AAV vector comprises a GFP tag. 
     
     
         3 . The kit of  claim 2 , wherein the AAV permissive cells are selected from Huh7 cells, human cancer cell line HeLa cells, HeLa S3 cells, murine hepatoma Hepa1-6 cells, HEK293, human hepatoma cells HepG2 and IMY-N9. 
     
     
         4 . The kit of  claim 2 , wherein the AAV permissive cells are HeLa S3 cells. 
     
     
         5 . The kit of  claim 2 , wherein the AAV transduced cells are measured for GFP signal using flow cytometry to identify GFP-positive AAV transduced cells. 
     
     
         6 . The kit of  claim 5 , wherein the GFP-positive AAV transduced cells are evaluated to determine the AAV neutralizing antibody titers in the serum sample. 
     
     
         7 . The kit of  claim 1 , wherein the step of exposing the AAV permissive cells to the serum/vector mixture does not include an adenovirus or another helper virus to facilitate transduction. 
     
     
         8 . A kit for determining the titer of adeno-associated virus (AAV) neutralizing antibodies in a human serum sample, said kit comprising: (i) at least one recombinant AAV vector comprising a heterologous sequence, (ii) a population of AAV permissive cells; and (iii) instructions, wherein the instructions include the following steps
 (a) incubating the AAV vector with the human serum sample to form a serum/vector mixture;   (b) exposing the AAV permissive whole cells to the serum/vector mixture to produce AAV transduced cells which have been transduced with the AAV vectors from the serum/vector mixture;   (c) measuring the quantity of whole cells transduced by AAV; and   (d) determining the AAV neutralizing antibody titer in the serum sample based on the measured quantity of the AAV transduced whole cells.   
     
     
         9 . The method of  claim 8 , wherein the AAV vector comprises a GFP tag. 
     
     
         10 . The method of  claim 9 , wherein the AAV permissive whole cells are selected from Huh7 cells, human cancer cell line HeLa cells, HeLa S3 cells, murine hepatoma Hepa1-6 cells, HEK293, human hepatoma cells HepG2 and IMY-N9. 
     
     
         11 . The method of  claim 9 , wherein the AAV permissive whole cells are HeLa S3 cells. 
     
     
         12 . The method of  claim 9 , wherein the AAV transduced cells are measured for GFP signal using flow cytometry to identify GFP-positive AAV transduced cells. 
     
     
         13 . The method of  claim 12 , wherein the GFP-positive AAV transduced cells are evaluated to determine the AAV neutralizing antibody titers in the serum sample. 
     
     
         14 . The method of  claim 12 , wherein the GFP positive AAV transduced cells are evaluated as whole cells to determine the AAV neutralizing antibody titers in the serum sample. 
     
     
         15 . The method of  claim 13 , wherein the serum sample is collected from a patient in need of an AAV mediated gene therapy, a patient after a dose of AAV gene therapy, or a subject suspected of exposure to AAV. 
     
     
         16 . A kit for determining the titer of adeno-associated virus (AAV) neutralizing antibodies in a human cerebrospinal fluid (CSF) sample, said kit comprising: (i) at least one recombinant AAV vector comprising a heterologous sequence, (ii) a population of AAV permissive cells; and (iii) instructions, wherein the instructions include the following steps:
 (a) incubating the recombinant AAV vector comprising the heterologous sequence with the human CSF sample to form a CSF/vector mixture;   (b) exposing the AAV permissive cells to the CSF/vector mixture to produce AAV transduced cells which have been transduced with the recombinant AAV vectors from the CSF/vector mixture;   (c) measuring the quantity of the AAV transduced cells; and   (d) determining the AAV neutralizing antibody titer in the human CSF sample based on the measured quantity of the AAV transduced cells.   
     
     
         17 . The method of  claim 16 , wherein the AAV vector comprises a GFP tag. 
     
     
         18 . The method of  claim 17 , wherein the AAV permissive whole cells are HeLa S3 cells. 
     
     
         19 . The method of  claim 18 , wherein the AAV transduced cells are measured for GFP signal using flow cytometry to identify GFP-positive AAV transduced cells. 
     
     
         20 . The method of  claim 19 , wherein the CSF sample is collected from a patient in need of an AAV mediated gene therapy, a patient after a dose of AAV gene therapy, or a subject suspected of exposure to AAV.