Aav vector and assay for anti-aav (adeno-associated virus) neutralizing antibodies

Abstract

Virus vectors, virus particles, and methods and uses of screening for, detecting, analyzing and determining amounts of virus antibody, or neutralizing antibody activity of samples are provided. Such virus vectors, virus particles, and methods and uses are applicable to a broad range of virus types, such as lentiviruses, adenovirus, and adeno-associated virus (AAV) serotypes. Methods and uses include virus antibody screening, such as anti-virus immunoglobulins screened for, detected, analyzed and amounts determined.

What is claimed is: 
     
         1 . A recombinant AAV vector sequence, wherein the vector sequence comprises a reporter transgene, wherein the reporter transgene comprises (a) a single-stranded or a self-complementary genome, (b) is operably linked to one or more expression regulatory elements, and (c) is flanked by one or more flanking elements. 
     
     
         2 . A recombinant AAV vector, wherein the vector comprises a reporter transgene, wherein the reporter transgene comprises (a) a single-stranded or a self-complementary genome, (b) is operably linked to one or more expression regulatory elements, and (c) is flanked by one or more flanking elements, wherein the recombinant AAV vector comprises an infectious recombinant AAV particle. 
     
     
         3 . A method for analyzing for or detecting or measuring antibodies that bind to AAV, comprising:
 (a) providing infectious recombinant AAV particles that encapsidate a recombinant vector, wherein (i) the vector comprises a reporter transgene, (ii) the reporter transgene comprises a single-stranded or a self-complementary genome, and (iii) the reporter transgene is operably linked to one or more expression regulatory elements and flanked by one or more flanking elements;   (b) providing a biological sample from a subject for analyzing or detecting antibodies that bind to AAV;   (c) providing cells that can be infected with said infectious recombinant AAV particles;   (d) contacting or incubating the infectious recombinant AAV particles of (a) with the biological sample of (b) thereby producing a resulting mixture (M);   (e) contacting the cells of (c) with resulting mixture (M) under conditions in which the infectious recombinant AAV particles of (a) can infect and express the reporter transgene in said cells;   (f) measuring expression of the reporter transgene; and   (g) comparing said reporter transgene expression of (f) to reporter transgene of a control, said control either a negative (−) control (i) lacking antibodies that bind to AAV, or (ii) having a predetermined amount of antibodies that bind to AAV, thereby analyzing for or detecting or measuring antibodies that bind to AAV.   
     
     
         4 . The method of  claim 3 , wherein the cells comprise mammalian cells. 
     
     
         5 . The method of  claim 3 , wherein the cells provide nucleic acid sequences encoding helper functions for AAV replication and/or genomic integration. 
     
     
         6 . The method of  claim 3 , wherein the cells can be infected with AAV particles comprising a VP1, VP2 or VP3 sequence 90% or more identical to a VP1, VP2 or VP3 sequence of AAV serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh74, or Rh10, or a hybrid or chimera of any of the foregoing AAV serotypes. 
     
     
         7 . The method of  claim 3 , wherein the cells can be infected with AAV serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh74, or Rh10, or a hybrid or chimera of any of the foregoing AAV serotypes. 
     
     
         8 . The method of  claim 3 , wherein the cells comprise mammalian cells. 
     
     
         9 . The method of  claim 3 , wherein the cells comprise HEK-293, CHO, BHK, MDCK, 10T1/2, WEHI cells, COS, BSC 1, BSC 40, BMT 10, VERO, WI38, MRCS, A549, HT1080, 293, B-50, 3T3, NIH3T3, HepG2, Saos-2, Huh7, HER, HEK, HEL, or HeLa cells. 
     
     
         10 . The method of  claim 3 , wherein the cells express AAV E4 gene. 
     
     
         11 . The method of  claim 3 , wherein the cells comprise 2V6.11 cells. 
     
     
         12 . The AAV vector of  claim 1  or  2 , or method of  claim 2 , wherein the reporter transgene encodes a protein that provides an enzymatic, colorimetric, fluorescent, luminescent, chemiluminescent, or electrochemical signal. 
     
     
         13 . The AAV vector of  claim 1  or  2 , or method of  claim 2 , wherein the reporter transgene comprises a luciferase gene. 
     
     
         14 . The method of  claim 13 , wherein the luciferase gene comprises a renilla luciferase or a firefly luciferase gene. 
     
     
         15 . The AAV vector of  claim 1  or  2 , or method of  claim 2 , wherein the reporter transgene comprises a β-galactosidase gene, a β-glucoronidase gene, or a chloramphenicol transferase gene. 
     
     
         16 . The AAV vector of  claim 1  or  2 , or method of  claim 2 , wherein the reporter transgene encodes a green fluorescent protein (GFP), a red fluorescent protein (RFP) or an alkaline phosphatase. 
     
     
         17 . The AAV vector of  claim 1  or  2 , or method of  claim 2 , wherein the recombinant vector comprises an AAV vector. 
     
     
         18 . The AAV vector or method of  claim 17 , wherein the AAV vector comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh74, or Rh10 vector, or a hybrid or chimera of any of the foregoing AAV vectors. 
     
     
         19 . The AAV vector of  claim 1  or  2 , or method of  claim 2 , wherein the expression regulatory elements comprise a promoter and/or enhancer nucleic acid sequence operable in mammalian cells. 
     
     
         20 . The AAV vector of  claim 1  or  2 , or method of  claim 2 , wherein the expression regulatory element comprises a cytomegalovirus (CMV) immediate early promoter/enhancer, the Rous sarcoma virus (RSV) promoter/enhancer, SV40 promoter, dihydrofolate reductase (DHFR) promoter, chicken β-actin (CBA) promoter, phosphoglycerol kinase (PGK) promoter, or elongation factor-1 alpha (EF1-alpha) promoter. 
     
     
         21 . The AAV vector of  claim 1  or  2 , or method of  claim 2 , wherein the reporter transgene is a single stranded genome. 
     
     
         22 . The AAV vector of  claim 1  or  2 , or method of  claim 2 , wherein the reporter transgene is a self-complementary genome. 
     
     
         23 . The AAV vector of  claim 1  or  2 , or method of  claim 2 , wherein the flanking element(s) comprises one or more AAV inverted terminal repeat sequences (ITRs). 
     
     
         24 . The method of  claim 23 , wherein the reporter transgene is positioned between the one or more 5′ and/or 3′ AAV ITRs. 
     
     
         25 . The AAV vector of  claim 1  or  2 , or method of  claim 2 , wherein the self-complementary reporter transgene genome comprises a double strand inverted repeat sequence structure. 
     
     
         26 . The AAV vector of  claim 1  or method of  claim 2 , wherein the flanking element(s) comprises a mutated or variant AAV ITR that is not processed by AAV Rep protein. 
     
     
         27 . The AAV vector of  claim 1  or method of  claim 2 , wherein the flanking element(s) comprises a mutated or variant AAV ITR that allows or facilitates formation of the self-complementary reporter transgene genome into a double strand inverted repeat sequence structure in the infectious recombinant AAV particles. 
     
     
         28 . The AAV vector or method of  claim 27  or  28 , wherein the mutated, modified or variant AAV ITR has a deleted D sequence, and/or a mutated, modified or variant terminal resolution site (TRS) sequence. 
     
     
         29 . The AAV vector or method of  claim 28 , wherein the mutated, modified or variant terminal resolution site (TRS) sequence comprises: CGGTTG. 
     
     
         30 . The AAV vector or method of  claim 27  or  28 , wherein the mutated or variant AAV ITR is positioned between the self-complementary sequences of the reporter transgene. 
     
     
         31 . The AAV vector or method of  claim 27  or  28 , wherein the mutated or variant AAV ITR comprises an AAV2 ITR having nucleotides corresponding to nucleotides 122-144 of AAV2 genome sequence deleted. 
     
     
         32 . The AAV vector of  claim 1  or  2 , or method of  claim 2 , wherein the recombinant vector comprises a first inverted terminal repeat (ITR) of an AAV; a promoter operable in mammalian cells; the reporter transgene; a polyadenylation signal; and optionally a second ITR of an AAV. 
     
     
         33 . The AAV vector of  claim 1  or  2 , or method of  claim 2 , wherein the recombinant vector comprises a restriction site to allow insertion of the reporter transgene downstream of a promoter operable in mammalian cells, and a posttranscriptional regulatory element downstream of the restriction site, wherein the promoter, the restriction site and the posttranscription regulatory element are located downstream of a 5′ AAV ITR and upstream of an optional 3′ AAV ITR. 
     
     
         34 . The AAV vector of  claim 1  or  2 , or method of  claim 2 , wherein the infectious recombinant AAV particles comprise an AAV serotype that infects primates. 
     
     
         35 . The AAV vector of  claim 1  or  2 , or method of  claim 2 , wherein the infectious recombinant AAV particles comprise an AAV serotype that infects humans or rhesus macaques. 
     
     
         36 . The AAV vector of  claim 1  or  2 , or method of  claim 2 , wherein the infectious recombinant AAV particles comprise a VP1, VP2 or VP3 sequence 90% or more identical to a VP1, VP2 or VP3 sequence of AAV serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh74, or Rh10, or a hybrid or chimera of any of the foregoing AAV serotypes. 
     
     
         37 . The AAV vector of  claim 1  or  2 , or method of  claim 2 , wherein the infectious recombinant AAV particles comprise AAV serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh74, or Rh10, or a hybrid or chimera of any of the foregoing AAV serotypes. 
     
     
         38 . The method of  claim 2  or  3 , wherein the biological sample comprises a primate sample. 
     
     
         39 . The method of  claim 2  or  3 , wherein the biological sample comprises serum. 
     
     
         40 . The method of  claim 2  or  3 , wherein the biological sample comprises plasma. 
     
     
         41 . The method of  claim 2  or  3 , wherein the biological sample comprises human serum or human plasma. 
     
     
         42 . The method of  claim 2  or  3 , wherein the subject is a mammal. 
     
     
         43 . The method of  claim 2  or  3 , wherein the subject is a human. 
     
     
         44 . The method of  claim 2  or  3 , wherein the subject suffers from a disorder due to insufficient expression or activity of a protein. 
     
     
         45 . The method of  claim 2  or  3 , wherein the subject suffers from a disorder due to expression or activity of an abnormal, aberrant or undesirable protein. 
     
     
         46 . The method of  claim 2  or  3 , wherein the subject suffers from a genetic disorder. 
     
     
         47 . The method of  claim 2  or  3 , wherein the subject is a candidate for gene replacement or supplement therapy. 
     
     
         48 . The method of  claim 2  or  3 , wherein the subject is a candidate for gene knockdown or knockout therapy. 
     
     
         49 . The method of  claim 2  or  3 , wherein the subject suffers from a lung disease (e.g., cystic fibrosis), a bleeding disorder (e.g., hemophilia A or hemophilia B with or without inhibitors), thalassemia, a blood disorder (e.g., anemia), Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), epilepsy, lysosomal storage diseases, a copper or iron accumulation disorders (e.g., Wilson's or Menkes disease) lysosomal acid lipase deficiency, a neurological or neurodegenerative disorder, cancer, type 1 or type 2 diabetes, Gaucher's disease, Hurler's disease, adenosine deaminase deficiency, a metabolic defect (e.g., glycogen storage diseases), a retinal degenerative disease (such as RPE65 deficiency, choroideremia, and other diseases of the eye), a disease of solid organs (e.g., brain, liver, kidney, heart), or an infectious viral (e.g., hepatitis B and C, HIV, etc.), bacterial or fungal disease. 
     
     
         50 . The method of  claim 2  or  3 , further comprising calculating the amount of antibodies that bind to AAV in the biological sample based upon the reporter transgene expression of (f) compared to (−) control. 
     
     
         51 . The method of  claim 2  or  3 , further comprising entering or including the amount of antibodies that bind to AAV in the biological sample into a report associated with said subject. 
     
     
         52 . The method of  claim 2  or  2 , further comprising entering the amount of antibodies that bind to AAV in the biological sample into a database thereby producing a database entry, the database entry associated with said subject. 
     
     
         53 . The method of  claim 2  or  3 , wherein the antibodies analyzed or detected bind to AAV capsid protein. 
     
     
         54 . The method of  claim 2  or  3 , wherein the antibodies analyzed or detected bind to AAV serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh74, or Rh10, or a hybrid or chimera of any of the foregoing AAV serotypes. 
     
     
         55 . The method of  claim 2  or  3 , wherein the predetermined amount of antibodies bind to AAV serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh74, or Rh10, or a hybrid or chimera of any of the foregoing AAV serotypes. 
     
     
         56 . The method of  claim 2  or  3 , wherein the biological sample is diluted prior to contact or incubating with the infectious recombinant AAV particles of (a). 
     
     
         57 . The method of  claim 2  or  3 , wherein a plurality of dilutions of the biological sample is analyzed. 
     
     
         58 . The method of  claim 2  or  3 , wherein a plurality of different dilution ratios of the biological sample are analyzed. 
     
     
         59 . The method of  claim 2  or  3 , wherein the biological sample is diluted between 1:1 and 1:500 prior to contacting or incubating with the infectious recombinant AAV particles of (a). 
     
     
         60 . The method of  claim 2  or  3 , wherein the biological sample is diluted between 1:500 and 1:5000 prior to contacting or incubating with the infectious recombinant AAV particles of (a). 
     
     
         61 . The method of  claim 2  or  3 , wherein at least 2, 3, 4, 5 or 6 different dilution ratios of the biological sample are analyzed. 
     
     
         62 . The method of  claim 2  or  3 , wherein the cells are contacted with the resulting mixture (d) for a period of 6-48 hours. 
     
     
         63 . The method of  claim 2  or  3 , wherein the cells are contacted with the resulting mixture (d) for a period of 12-36 hours. 
     
     
         64 . The method of  claim 2  or  3 , wherein the cells are contacted with the resulting mixture (d) for a period of 20-30 hours. 
     
     
         65 . The method of  claim 2  or  3 , wherein the cells are contacted with the resulting mixture (d) for a period of about 24 hours. 
     
     
         66 . The method of  claim 2  or  3 , wherein the cells are lysed prior to measuring expression of the reporter transgene. 
     
     
         67 . The method of  claim 2  or  3 , wherein the biological sample has been heat inactivated. 
     
     
         68 . The method of  claim 2  or  3 , wherein the biological sample has been heat inactivated at about 50-70 degrees Celsius for a period of about 15 minutes up to one hour. 
     
     
         69 . The method of  claim 2  or  3 , wherein the biological sample has been heat inactivated at about 56 degrees Celsius for a period of about 30 minutes. 
     
     
         70 . The method of  claim 2  or  3 , wherein the method is performed in vitro.