Adenovirus vectors, packaging cell lines, compositions, and methods for preparation and use

Abstract

The present invention relates to methods for gene therapy, especially to adenovirus-based gene therapy, and related cell lines and compositions. In particular, novel nucleic acid constructs and packaging cell lines are disclosed, for use in facilitating the development of high-capacity and targeted vectors. The invention also discloses a variety of high-capacity adenovirus vectors and related compositions and kits including the disclosed cell lines and vectors. Finally, the invention discloses methods of preparing and using the disclosed vectors, cell lines and kits.

1 - 30 . (canceled) 
     
     
         31 . A helper-independent fiberless recombinant adenovirus vector genome comprising genes which:
 (a) encodes all adenovirus structural gene products but do not express sufficient adenovirus fiber protein to package a fiber-containing adenovirus particle without complementation of the fiber gene or the genome lacks at least the fiber gene, and   (b) encodes an exogenous protein.   
     
     
         32 . The adenovirus vector genome of  claim 31  wherein the adenovirus vector genome does not encode one or more functional proteins selected from the group consisting of E1A, E1B, E2A, E2B, E3 and E4 protein. 
     
     
         33 . The adenovirus vector genome of  claim 31  wherein the adenovirus vector genome is Ad5.βgal.ΔF. 
     
     
         34 . The adenovirus vector genome of  claim 33  wherein the adenovirus vector genome has a nucleotide sequence shown in SEQ ED NO:27 and corresponds to Ad5.βgal.ΔF. 
     
     
         35 . The adenovirus vector genome of  claim 31  wherein the adenovirus vector genome is contained in the adenovirus particle deposited under ATCC accession VR-2636 corresponding to Ad5.βgal.ΔF. 
     
     
         36 . The adenovirus vector genome of  claim 31  wherein the exogenous protein is a therapeutic gene product. 
     
     
         37 . An isolated nucleic acid that comprises the adenovirus vector genome of  claim 31 . 
     
     
         38 - 59 . (canceled) 
     
     
         60 . A method for specifically targeting an adenovirus vector to a cell of choice comprising introducing a helper-independent or helper-dependent fiberless recombinant adenovirus vector genome into a packaging cell line for producing a fiber gene-deleted adenovirus vector, wherein a gene for a missing fiber protein is complemented with a gene for a desired modification for targeting the vector to a cell of choice. 
     
     
         61 - 70 . (canceled) 
     
     
         71 . A composition for preparing a therapeutic vector, the composition comprising a plasmid comprising an adenovirus genome lacking a nucleotide sequence encoding a fiber protein or a genome that is incapable of expressing sufficient fiber to result in packaging. 
     
     
         72 - 73 . (canceled) 
     
     
         74 . The recombinant adenovirus vector genome of  claim 31 , wherein no fiber protein is expressed. 
     
     
         75 . (canceled) 
     
     
         76 . The recombinant adenovirus genome of  claim 31 , wherein the genome expresses insufficient fiber to allow incorporation of the protein into a particle such that the particle cannot use the fiber pathway for infection. 
     
     
         77 . A method for producing a gutless adenoviral vector particle comprising:
 a) delivering a helper adenovirus vector genome to an adenovirus vector packaging cell, wherein the helper adenovirus vector genome lacks any gene encoding adenovirus fiber protein or lacks the ability to encode sufficient adenovirus fiber protein to produce an adenoviral vector comprising fiber protein in the absence of complementation by the packing cell and wherein the packaging cell comprises the nucleic acid molecule of claim  2  operably linked to a promoter and to an adenoviral fiber protein or to a chimeric protein that includes an adenovirus fiber protein tail domain;   (b) delivering a gutless adenovirus vector genome to the packaging cell; and   (c) recovering the gutless adenoviral vector particle produced by the cell.   
     
     
         78 . The method of  claim 77 , wherein the helper adenovirus vector genome is delivered by viral infection. 
     
     
         79 . The method of  claim 78 , wherein the gutless adenovirus vector genome is delivered by transfection. 
     
     
         80 . The method of  claim 77 , wherein the gutless adenovirus vector genome comprises an operable packaging sequence. 
     
     
         81 . The method of  claim 80 , wherein the helper adenovirus vector genome has a mutation in its packaging sequence that renders the genome substantially incapable of being packaged as an adenoviral vector particle by the packaging cell. 
     
     
         82 . The method of  claim 80 , wherein the helper adenovirus vector genome comprises recombinase sites flanking its packaging sequence and the packaging cell further comprises a nucleotide sequence encoding a recombinase. 
     
     
         83 . The method of  claim 82 , wherein the recombinase site is a lox site and the recombinase is Cre. 
     
     
         84 . A helper adenovirus particle comprising an adenovirus vector genome that does not encode or does not express sufficient adenovirus fiber protein to support packaging of a fiber-containing adenovirus particle without complementation of the fiber gene, wherein said genome has a mutation in its packaging sequence that renders the genome substantially incapable of being packaged. 
     
     
         85 . The helper adenovirus particle of  claim 84 , wherein the mutation comprises a deletion of at least one nucleotide in the packaging sequence. 
     
     
         86 . The helper adenovirus particle of  claim 85 , wherein the adenovirus vector genome does not encode functional proteins selected from the group consisting of E1A, E1B, E2A, E2B, E3, and E4 proteins. 
     
     
         87 - 94 . (canceled)