Non-toxic hsv vectors for efficient gene delivery applications and complementing cells for their production

Abstract

Disclosed is a method for administering a transgene into a fibroblast in a subject comprising: a) providing a herpes simplex virus (HSV) comprising a recombinant herpes simplex virus genome, wherein said recombinant herpes simplex virus genome comprises one or more transgenes encoding a polypeptide to be expressed in said fibroblast; and b) providing a pharmaceutically acceptable carrier; wherein said HSV has reduced cytotoxicity as compared to a wild-type herpes simplex virus.

1 . A method for administering a transgene into a fibroblast in a subject comprising: a) providing a herpes simplex virus (HSV) comprising a recombinant herpes simplex virus genome, wherein said recombinant herpes simplex virus genome comprises one or more transgenes encoding a polypeptide to be expressed in said fibroblast; and b) providing a pharmaceutically acceptable carrier; wherein said HSV has reduced cytotoxicity as compared to a wild-type herpes simplex virus. 
     
     
         2 . The method of  claim 1 , wherein said one or more transgenes is in operable connection with one or more insulator sequences in said recombinant herpes simplex virus genome. 
     
     
         3 . The method of  claim 1 , wherein said HSV further comprises an inactivating deletion in a UL55 locus of said recombinant herpes simplex virus genome. 
     
     
         4 . The method of  claim 1 , wherein said HSV is capable of expression of said one or more transgenes for a plurality of days post infection in said fibroblast. 
     
     
         5 . The method of  claim 4 , wherein said plurality of days is at least 14 days. 
     
     
         6 . The method of  claim 5 , wherein said plurality of days is at least 28 days. 
     
     
         7 . The method of  claim 1 , further comprising inactivation of at least one immediate-early protein. 
     
     
         8 . The method of  claim 7 , further comprising complete deletion of a respective coding sequence(s) of said at least one immediate-early protein. 
     
     
         9 . The method of  claim 1 , wherein said polynucleotide encoding one or more transgenes is operably linked to a constitutive promoter. 
     
     
         10 . The method of  claim 9 , wherein said constitutive promoter is a cell- or tissue-specific promoter. 
     
     
         11 . The method of  claim 1 , wherein said one or more transgenes is polycistronic. 
     
     
         12 . The method of  claim 1 , wherein said one or more transgenes comprises one or more binding sites for a microRNA. 
     
     
         13 . The method of  claim 1 , wherein said one or more transgenes is operably connected with one or more insulator sequences. 
     
     
         14 . The method of  claim 1 , wherein said HSV further comprises one or two consensus sequences for a recombinase enzyme. 
     
     
         15 . The method of  claim 14 , wherein said one or two consensus sequences for said recombinase enzyme is inserted into an intergenic region. 
     
     
         16 . The method of  claim 1 , wherein said one or more transgenes further comprises a transcription-terminating region. 
     
     
         17 . The method of  claim 16 , wherein said transcription-terminating region is a polyadenylation sequence located 3′ of said one or more transgenes. 
     
     
         18 . The method of  claim 1 , wherein said HSV further does not express ICP47 as an immediate early gene. 
     
     
         19 . The method of  claim 1 , wherein said HSV further does not express ICP22 as an immediate early gene. 
     
     
         20 . The method of  claim 1 , wherein said HSV further comprises an inactivating deletion in one or more of the ICP4, ICP27, ICP22, and ICP47 loci. 
     
     
         21 . The method of  claim 20 , wherein said inactivating deletion in one or more of said ICP4, ICP27, ICP22, and ICP47 loci is a complete deletion of the coding sequence within said locus. 
     
     
         22 . The method of  claim 1 , wherein one or more of ICP4, ICP22, ICP47 and ICP27 genes is expressed as an early or late gene. 
     
     
         23 . The method of  claim 22 , wherein said one or more of ICP4, ICP22, ICP47 and ICP27 is operably linked to an HSV thymidine kinase promoter. 
     
     
         24 . The method of  claim 23 , wherein said ICP22 is under the control of an early promoter. 
     
     
         25 . The method of  claim 1 , wherein said HSV vector does not express UL41. 
     
     
         26 . The method of  claim 1 , wherein said HSV vector further comprises at least one additional nucleic acid sequence. 
     
     
         27 . The method of claim of  claim 26 , wherein said at least one additional nucleic acid sequence encodes a chimeric protein for combination therapy. 
     
     
         28 . The method of  claim 26 , wherein said at least one additional nucleic acid sequence is controlled by the same control sequence as said one or more transgenes. 
     
     
         29 . The method of  claim 26 , wherein said at least one additional nucleic acid sequence encodes a mutant glycoprotein that enhances infectivity of said HSV vector relative to an HSV vector comprising a wild-type glycoprotein or that directs entry of said HSV vector into cells through non-canonical receptors. 
     
     
         30 . The method of  claim 29 , wherein said mutant glycoprotein is selected from the group consisting of gB, gC, gD, gH, and gK.