Virus vectors and methods of making and administering the same

Abstract

The present invention provides genetically-engineered parvovirus capsids and viruses designed to introduce a heterologous gene into a target cell. The parvoviruses of the invention provide a repertoire of vectors with altered antigenic properties, packaging capabilities, and/or cellular tropisms as compared with current AAV vectors.

1 . A hybrid virus particle comprising: 
 a  parvovirus  capsid; and    a nucleic acid comprising at least one adeno-associated virus (AAV) serotype 5 inverted terminal repeat packaged within said  parvovirus  capsid, subject to the proviso that said  parvovirus  capsid is not an AAV serotype 5 capsid.    
     
     
         2 . The hybrid virus particle of  claim 1 , wherein said nucleic acid comprises at least one heterologous nucleotide sequence.  
     
     
         3 . The hybrid virus particle of  claim 1 , wherein said  parvovirus  capsid is an autonomous  parvovirus  capsid.  
     
     
         4 . The hybrid virus particle of  claim 1 , wherein said  parvovirus  capsid is a B19 capsid.  
     
     
         5 . The hybrid virus particle of  claim 1 , wherein said  parvovirus  capsid is an AAV capsid.  
     
     
         6 . The hybrid virus particle of  claim 5 , wherein said AAV capsid is of a serotype selected from the group consisting of AAV serotypes 1, 2, 3, 4 and 6.  
     
     
         7 . The hybrid virus particle of  claim 6  selected from the group consisting of: 
 (a) a hybrid virus particle comprising an AAV serotype-1 capsid and at least one AAV serotype-5 inverted terminal repeat,    (b) a hybrid virus particle comprising an AAV serotype-2 capsid and at least one AAV serotype-5 inverted terminal repeat, and    (c) a hybrid virus particle comprising an AAV serotype-6 capsid and at least one AAV serotype-5 inverted terminal repeat.    
     
     
         8 . The hybrid virus particle of  claim 1 , wherein said nucleic acid does not comprise the AAV cap genes or the AAV rep genes.  
     
     
         9 . The hybrid virus particle of  claim 2  comprising two AAV inverted terminal repeats that flank said at least one heterologous nucleotide sequence.  
     
     
         10 . The hybrid virus particle of  claim 2 , wherein said at least one heterologous nucleotide sequence encodes a protein or peptide.  
     
     
         11 . The hybrid virus-particle of  claim 10 , wherein said protein or peptide is a therapeutic protein or peptide.  
     
     
         12 . The hybrid virus particle of  claim 10 , wherein said protein or peptide is an immunogenic protein or peptide.  
     
     
         13 . The hybrid virus particle of  claim 10 , wherein said at least one heterologous nucleotide sequence encodes dystrophin, a mini-dystrophin, a clotting factor, β-glucocerebrosidase, erythropoietin, cystic fibrosis transmembrane regulator protein, a cytokine, β-globin, a hormone, α-globin or a growth factor.  
     
     
         14 . The hybrid virus particle of  claim 2 , wherein said at least one heterologous nucleotide sequence encodes an untranslated RNA.  
     
     
         15 . A pharmaceutical formulation comprising the hybrid virus particle of  claim 1  in a pharmaceutically-acceptable carrier.  
     
     
         16 . An isolated nucleic acid for producing the hybrid virus particle of  claim 1 , wherein said isolated nucleic acid comprises  parvovirus  cap genes and adeno-associated virus (AAV) rep genes, subject to the proviso that said  parvovirus  cap genes are not AAV serotype 5 cap genes.  
     
     
         17 . The isolated nucleic acid of  claim 16 , wherein said  parvovirus  cap genes are operably associated with an authentic  parvovirus  promoter.  
     
     
         18 . The isolated nucleic acid of  claim 16 , wherein said  parvovirus  cap genes are B19 cap genes.  
     
     
         19 . The isolated nucleic acid of  claim 18 , wherein said AAV rep genes are AAV serotype-5 rep genes.  
     
     
         20 . The isolated nucleic acid of  claim 19 , wherein said AAV cap genes are of a serotype selected from the group consisting of AAV serotypes 1, 2, 3, 4 and 6.  
     
     
         21 . The isolated nucleic acid of  claim 20  selected from the group consisting of: 
 (a) an isolated nucleic acid comprising AAV serotype-1 cap genes and AAV serotype-5 rep genes,    (b) an isolated nucleic acid comprising AAV serotype-2 cap genes and AAV serotype-5 rep genes,    (c) an isolated nucleic acid comprising AAV serotype-3 cap genes and AAV serotype-5 rep genes,    (d) an isolated nucleic acid comprising AAV serotype-4 cap genes and AAV serotype-5 rep genes, and    (e) an isolated nucleic acid comprising AAV serotype-6 cap genes and AAV serotype-5 rep genes.    
     
     
         22 . The isolated nucleic acid of  claim 16 , wherein said cap genes are AAV cap genes.  
     
     
         23 . The isolated nucleic acid of  claim 22 , wherein said AAV cap genes are operably associated with an authentic AAV promoter.  
     
     
         24 . The isolated nucleic acid of  claim 23 , wherein said authentic AAV promoter is an AAV p40 promoter.  
     
     
         25 . A vector comprising the isolated nucleic acid of  claim 16 .  
     
     
         26 . The vector of  claim 25 , wherein said vector is selected from the group consisting of plasmids, naked DNA vectors, bacterial artificial chromosomes, yeast artificial chromosomes, and viral vectors.  
     
     
         27 . The vector of  claim 26 , wherein said vector is a plasmid.  
     
     
         28 . A cell comprising the vector of  claim 25 .  
     
     
         29 . The cell of  claim 28 , wherein said cell is selected from the group consisting of bacterial, protozoan, yeast, fungus, plant, and animal cells.  
     
     
         30 . A cell comprising a vector, the vector comprising: 
   parvovirus  cap genes,    adeno-associated virus (AAV) rep genes, and    a nucleic acid comprising at least one AAV inverted serotype 5 terminal repeat,    subject to the proviso that said  parvovirus  cap genes are not AAV serotype 5 cap genes.    
     
     
         31 . The cell of  claim 30 , wherein said cell is a mammalian cell.  
     
     
         32 . A cell comprising  parvovirus  cap genes and adeno-associated virus (AAV) rep genes stably integrated into the genome of the cell, subject to the proviso that if said  parvovirus  cap genes are not AAV serotype 2 cap genes, the serotypes of said AAV cap genes and said AAV rep genes are different.  
     
     
         33 . The cell of  claim 32  further comprising a nucleic acid comprising at least one AAV serotype 5 inverted terminal repeat, subject to the proviso that said  parvovirus  cap genes are not AAV serotype 5 cap genes.  
     
     
         34 . A method of producing a hybrid virus particle, the method comprising: 
 providing a cell with adeno-associated virus (AAV) rep genes,  parvovirus  cap genes, a nucleic acid comprising at least one AAV serotype 5 inverted terminal repeat, and helper functions for generating a productive AAV infection; subject to the proviso that the  parvovirus  cap genes are not AAV serotype 5 cap genes, and    allowing assembly of the hybrid virus particles.    
     
     
         35 . The method of  claim 34 , further comprising collecting the hybrid virus particles.  
     
     
         36 . The method of  claim 34 , wherein the nucleic acid comprises at least one heterologous nucleotide sequence.  
     
     
         37 . The method of  claim 34 , wherein the  parvovirus  cap genes and AAV rep genes are provided by one or more transcomplementing packaging vectors.  
     
     
         38 . The method of  claim 34 , wherein the  parvovirus  cap genes and AAV rep genes are provided by a plasmid.  
     
     
         39 . The method of  claim 34 , wherein the  parvovirus  cap genes and AAV rep genes are provided by an  adenovirus  vector.  
     
     
         40 . The method of  claim 34 , wherein the  parvovirus  cap genes and AAV rep genes are stably integrated into the genome of the cell.  
     
     
         41 . The method of  claim 34 , wherein the  parvovirus  cap genes are AAV cap genes.  
     
     
         42 . A hybrid virus particle produced by the method of  claim 34 .  
     
     
         43 . A method of delivering a nucleotide sequence to a cell, comprising introducing into a cell the hybrid virus particle according to  claim 2 .  
     
     
         44 - 53 . (canceled)  
     
     
         54 . A method of administering a nucleotide sequence to a subject, comprising administering the cell of  claim 43  to a subject.  
     
     
         55 . A method of administering a nucleotide sequence to a subject, comprising administering to a subject the hybrid virus particle according to  claim 2 .  
     
     
         56 - 63 . (canceled)