Methods to produce chimeric adeno-associated virus/bocavirus parvovirus

Abstract

A method of preparing a chimeric virus comprising bocavirus capsid protein (VP) and a recombinant adeno-associated (AAV) viral genome, and isolated mutant bocavirus genomes, are provided.

1 . (canceled) 
     
     
         2 . A method of preparing a chimeric virus comprising a bocavirus VP1 capsid polypeptide and a recombinant adeno-associated (AAV) viral genome, comprising:
 a) isolating the chimeric virus from a host cell that:
 (1) expresses the bocavirus VP1 capsid polypeptide, and 
 (2) does not express one or more of the bocavirus NS1, NS2, NS4 polypeptides, or any combination thereof. 
   
     
     
         3 . The method of  claim 2 , wherein the host cell expresses a bocavirus NS3 polypeptide. 
     
     
         4 . The method of  claim 2 , wherein the host cell does not express a functional bocavirus NP1 polypeptide. 
     
     
         5 . The method of  claim 2 , wherein the host cell is a mammalian cell. 
     
     
         6 . The method of  claim 2 , wherein the host cell is an insect cell. 
     
     
         7 . The method of  claim 2 , wherein the host cell comprises a rAAV genome. 
     
     
         8 . The method of  claim 7 , wherein the rAAV genome does not express the functional bocavirus NP1 polypeptide in trans. 
     
     
         9 . The method of  claim 2 , wherein the host cell does not express one or more of NS1, NS2, NS4 polypeptides, or any combination thereof. 
     
     
         10 . The method of  claim 2 , wherein the host cell expresses a bocavirus VP2 capsid polypeptide, a bocavirus VP3 capsid polypeptide, or a combination thereof. 
     
     
         11 . The method of  claim 2 , wherein the rAAV vector comprises an expression cassette encoding a heterologous gene product. 
     
     
         12 . The method of  claim 11 , wherein the gene product encodes a therapeutic protein. 
     
     
         13 . The method of  claim 2 , wherein the rAAV genome is a rAAV-2 genome. 
     
     
         14 . The method of  claim 2 , wherein the gene product is cystic fibrosis transmembrane conductance regulator, b-globin, g-globin, tyrosine hydroxylase, glucocerebrosidase, aryl sulfatase A, factor VIII, dystrophin, alpha 1-antitrypsin, surfactant protein SP-D, SP-A or SP-C, C1 inhibitor gene, C1-INH gene, SERPING gene erythropoietin, HBoV protein, influenza virus protein, RSV protein, a neutralizing antibody or an antigen binding fragment thereof, SARS virus protein, or a cytokine. 
     
     
         15 . The method of  claim 2 , wherein the bocavirus is a human bocavirus. 
     
     
         16 . The method of  claim 2 , wherein the method is independent of bocavirus NP1. 
     
     
         17 . The method of  claim 16 , wherein the bocavirus VP1 capsid polypeptide is encoded by a nucleic acid sequence comprising a silent mutation in one or more polyadenylation signals, relative to a wild type polyadenylation signal. 
     
     
         18 . The method of  claim 2 , wherein one or more of the following is stably integrated into a genome of the host cell: (1) a coding sequence encoding the bocavirus VP1 capsid polypeptide, (2) a coding sequence encoding a bocavirus NS3 polypeptide, and (3) the recombinant adeno-associated (AAV) viral genome. 
     
     
         19 . The method of  claim 2 , wherein one or more of the following is not stably integrated into a genome of the host cell: (1) a coding sequence encoding the bocavirus VP1 capsid polypeptide, (2) a coding sequence encoding a bocavirus NS3 polypeptide, and (3) the recombinant adeno-associated (AAV) viral genome. 
     
     
         20 . The method of  claim 2 , wherein none of the following are stably integrated into a genome of the host cell: (1) a coding sequence encoding the bocavirus VP1 capsid polypeptide, (2) a coding sequence encoding a bocavirus NS3 polypeptide, and (3) the recombinant adeno-associated (AAV) viral genome. 
     
     
         21 . A host cell comprising a chimeric virus, the chimeric virus comprising a bocavirus VP1 capsid polypeptide and a recombinant adeno-associated (AAV) viral genome,
 wherein the host cell (1) expresses the bocavirus VP1 capsid polypeptide; and (2) does not express one or more of the bocavirus NS1, NS2, NS4 polypeptides, or any combination thereof.   
     
     
         22 . The host cell of  claim 21 , wherein the host cell expresses a bocavirus NS3 polypeptide. 
     
     
         23 . The host cell of  claim 21 , wherein the host cell is a mammalian cell. 
     
     
         24 . The host cell of  claim 21 , wherein the host cell is an insect cell. 
     
     
         25 . The host cell of  claim 21 , wherein one or more of the following is stably integrated into a genome of the host cell: (1) a coding sequence encoding the bocavirus VP1 capsid polypeptide, (2) a coding sequence encoding a bocavirus NS3 polypeptide, and (3) the recombinant adeno-associated (AAV) viral genome. 
     
     
         26 . The host cell of  claim 21 , wherein one or more of the following is not stably integrated into a genome of the host cell: (1) a coding sequence encoding the bocavirus VP1 capsid polypeptide, (2) a coding sequence encoding a bocavirus NS3 polypeptide, and (3) the recombinant adeno-associated (AAV) viral genome. 
     
     
         27 . The host cell of  claim 21 , wherein none of the following are stably integrated into a genome of the host cell: (1) a coding sequence encoding the bocavirus VP1 capsid polypeptide, (2) a coding sequence encoding a bocavirus NS3 polypeptide, and (3) the recombinant adeno-associated (AAV) viral genome. 
     
     
         28 . A population of chimeric viruses, the population comprising a bocavirus VP1 capsid polypeptide and a recombinant adeno-associated (AAV) viral genome,
 wherein the population was isolated from a host cell capable of expressing the bocavirus VP1 capsid polypeptide; and   wherein the host cell does not express one or more of bocavirus NS1, NS2, NS4 polypeptides, or any combination thereof.   
     
     
         29 . The host cell of  claim 28 , wherein the host cell expresses a bocavirus NS3 polypeptide. 
     
     
         30 . The population of  claim 28 , wherein the host cell is a mammalian cell. 
     
     
         31 . The population of  claim 28 , wherein the host cell is an insect cell. 
     
     
         32 . The population of  claim 28 , wherein one or more of the following is stably integrated into a genome of the host cell: (1) a coding sequence encoding the bocavirus VP1 capsid polypeptide, (2) a coding sequence encoding a bocavirus NS3 polypeptide, and (3) the recombinant adeno-associated (AAV) viral genome. 
     
     
         33 . The population of  claim 28 , wherein one or more of the following is not stably integrated into a genome of the host cell: (1) a coding sequence encoding the bocavirus VP1 capsid polypeptide, (2) a coding sequence encoding a bocavirus NS3 polypeptide, and (3) the recombinant adeno-associated (AAV) viral genome. 
     
     
         34 . The population of  claim 28 , wherein none of the following are stably integrated into a genome of the host cell: (1) a coding sequence encoding the bocavirus VP1 capsid polypeptide, (2) a coding sequence encoding a bocavirus NS3 polypeptide, and (3) the recombinant adeno-associated (AAV) viral genome. 
     
     
         35 . A preparation comprising a chimeric virus, the chimeric virus comprising a bocavirus VP1 capsid polypeptide and a recombinant adeno-associated (AAV) viral genome,
 wherein the chimeric virus was produced by a host cell capable of expressing the bocavirus VP1 capsid polypeptide; and   wherein the host cell does not express one or more of bocavirus NS1, NS2, NS4 polypeptides, or any combination thereof.   
     
     
         36 . The preparation of  claim 35 , wherein the host cell expresses a bocavirus NS3 polypeptide. 
     
     
         37 . The population of  claim 35 , wherein the host cell is a mammalian cell. 
     
     
         38 . The population of  claim 37 , wherein the host cell is an insect cell. 
     
     
         39 . A method of treatment comprising:
 administering to a subject a chimeric virus comprising a bocavirus VP1 capsid polypeptide and a recombinant adeno-associated (AAV) viral genome,   wherein the chimeric virus was isolated from a host cell that expresses the bocavirus VP1 capsid polypeptide; and   wherein the host cell does not express one or more of bocavirus NS1, NS2, NS4 polypeptides, or any combination thereof.   
     
     
         40 . The method of  claim 39 , wherein the host cell expresses a bocavirus NS3 polypeptide. 
     
     
         41 . A method of preparing a chimeric virus comprising bocavirus capsid protein and a recombinant adeno-associated (AAV) viral genome which method is independent of bocavirus NP1, comprising:
 a) providing a bocavirus genome vector that when introduced to mammalian cells
 i) express bocavirus NS3 and NS4 but not NS1 and NS2, 
 ii) expresses bocavirus capsid proteins VP1, VP2 and VP3, and 
 iii) does not express a functional bocavirus NP1, 
 wherein the coding region for the one or more of the VP1, VP2 or VP3 in the bocavirus genome vector is codon optimized; 
   b) introducing the bocavirus genome vector and a rAAV vector having an AAV genome into mammalian cells that do not express bocavirus NP1 in trans,   thereby producing bocavirus nonstructural proteins NS3 and NS4, bocavirus capsid proteins and a rAAV genome; and   c) collecting chimeric rAAV/bocavirus virus.