Viral-mediated gene transfer system

Abstract

A recombinant neurotropic HSV-1 vector carrying the gene for human HPRT under the direction of the viral TK promoter expresses a high level of human enzyme in host neuronal cells, yet has reduced cytopathicity. The virulence of the HSV-1 vector is further reduced by rendering it replication-defective through UV-irradiation or the use of a replication-defective HSV-1 deletion mutant in its construction. The recombinant vector is also provided with a non-viral site of initiation of DNA synthesis, which permits the HPRT gene to replicate independently of the viral genome, thus providing means for amplification of the gene in the host. The recombinant HSV-1 vector's high level of expression of HPRT coupled with decreased virulence allows treatment of HPRT deficieny by direct infection of the host organism.

What is claimed is:  
     
         1 . A recombinant viral vector capable of expressing a gene in a selected host cell or tissue comprising: 
 a) at least a portion of the genome of a DNA virus which exhibits tropicity for said selected host cell or tissue, which portion is capable of infection of said selected host cell or tissue; said genome having been modified to have a level of virulence less than the level of virulence present in a wild type virulent strain of the virus from which said vector is derived; and    b) a gene operatively linked thereto wherein said gene is capable of expression in said selected host cell or tissue after infection of said selected host cell or tissue.    
     
     
         2 . The recombinant vector of    claim 1   , wherein said vector further comprises a DNA sequence containing a nonviral initiation site of DNA synthesis operatively linked to said gene for amplification of said gene.  
     
     
         3 . The recombinant vector of    claim 1   , wherein said DNA virus is a double-stranded neurotropic virus, said human gene is the gene for hypoxanthine-guanine phosphoribosyltransferase, and said vector further comprises a promoter sequence of DNA for promoting expression of said gene.  
     
     
         4 . The recombinant vector of    claim 3   , wherein said DNA virus is herpes simplex virus and said vector is synthesized by a process comprising: 
 a) cloning a fragment containing a herpes virus thymidine kinase promoter into a suitable plasmid;    b) linearizing said plasmid with Bg1 II to obtain plasmid DNA;    c) obtaining a fragment containing human HPRT cDNA, and blunt-ending and ligated said fragment to BamH1 linkers to form a ligatable fragment;    ligating said ligatable fragment to said linear plasmid DNA to form a recombinant plasmid containing a TK promoter-HPRT minigene;    linearizing and recombining said recombinant plasmid with an intact wild-type herpes virus genome to form recombinant viral progeny containing the TK promoter-HPRT minigene.    
     
     
         5 . The recombinant vector of    claim 3   , wherein said portion of said viral genome contains a deletion in a viral gene necessary for replication of said genome.  
     
     
         6 . The recombinant vector of    claim 3   , wherein said DNA sequence containing said nonviral initiation site is of murine origin.  
     
     
         7 . A method of viral-mediated gene therapy for the treatment of a selected host organism comprising the steps of: 
 a) providing at least a portion of a genome of a DNA virus, said portion being capable of infecting cells of said selected host organism;    b) operatively linking a human gene to said portion to form a recombinant viral vector, said gene being capable of expression in said host cell after infection of said cell by said vector;    c) modifying said recombinant viral vector to have a level of virulence less than the level of virulence present in a wild type virulent strain from which said portion of said genome is derived; and    d) directly contacting said selected host organism with sufficient quantity of said viral vector and for a time effective for said viral vector to infect said organism.    
     
     
         8 . The method of    claim 7   , further comprising the step of providing said recombinant vector with a nonviral site of initation of DNA synthesis.  
     
     
         9 . The method of    claim 8   , wherein said portion of said genome of said virus is replication-defective.  
     
     
         10 . The method of    claim 9   , wherein said virus is a double-stranded neurotropic virus and said site of nonviral initiation is contained within as autonomous replicating sequence of murine orgin.  
     
     
         11 . The method of    claim 10   , wherein said virus is a herpes simplex virus, said gene is the gene for hypoxanthine-guanine phosphoribosyltransferase, and said recombinant vector further comprises a viral promoter sequence for promoting expression of said gene.  
     
     
         12 . A method for providing means for amplification of a gene carried on a recombinant viral vector comprising the steps of: 
 a) providing at least a portion of the genome of a virus, said portion being capable of infecting a host organism;    b) operatively linking a human gene to said portion to form a recombinant viral vector, wherein said gene is capable of expression in said host organism after infection of said organism by said vector; and    c) providing a nonviral site of initiation of neuclic acid synthesis for amplification of said gene.    
     
     
         13 . The method of    claim 12   , wherein said method further comprises the step of modifying said portion of said genome of said virus replication-defective.  
     
     
         14 . The method of    claim 12   , wherein said nonviral site of initiation resides on an autonomous replicating sequence of nucleic acids.  
     
     
         15 . The method of    claim 14   , wherein said virus is a double stranded DNA virus which exhibits tissue-tropism, said human gene codes for an enzyme which is selectable in culture, and said autonomous replicating sequence is of murine orgin.  
     
     
         16 . The method of    claim 15   , wherein said virus is herpes simplex virus, said gene is the gene for hypoxanthine-guanine phosphoribosyltransferase and said recombinant vector further comprises a DNA sequence for the herpes simplex thymidine kinase promoter operatively linked thereto.