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

Abstract

In one embodiment, the invention provides a herpes simplex virus (HSV) vector that does not express toxic HSV genes in non-complementing cells and which comprises a genome comprising one or more transgenes, wherein the vector is capable of expression of a transgene for at least 28 days in non-complementing cells. The invention also relates to viral stocks of the inventive vectors, compositions thereof suitable for use therapeutically or for in vitro applications, and methods relating thereto. In another aspect, the invention provides a complementing cell engineered to express ICP4 and ICP27 when the cell is infected with HSV for the production of the inventive vector.

1 - 59 . (canceled) 
     
     
         60 . A herpes simplex virus (HSV) vector comprising a polynucleotide encoding one or more transgenes, wherein the HSV vector does not express ICP0, ICP4, ICP22, and ICP27 as immediate early genes, and wherein the HSV vector is capable of expressing the one or more transgenes for at least 28 days post infection of non-complementing cells with the HSV vector. 
     
     
         61 . The HSV vector of  claim 60 , wherein the HSV vector further does not express ICP47 as an immediate early gene. 
     
     
         62 . The HSV vector of  claim 60 , wherein the non-complementing cells are cultured human dermal fibroblast (HDF) cells. 
     
     
         63 . The HSV vector of  claim 60 , comprising an inactivating deletion in one or more of the ICP0, ICP4, ICP22, and ICP27 loci. 
     
     
         64 . The HSV vector of  claim 63 , wherein the inactivating deletion in one or more of the ICP0, ICP4, ICP22, and ICP27 loci is a complete deletion of the coding sequence within said locus. 
     
     
         65 . The HSV vector of  claim 60 , comprising an inactivating deletion in each of the ICP0, ICP4, ICP22, and ICP27 loci. 
     
     
         66 . The HSV vector of  claim 60 , wherein one or more of the ICP0, ICP4, ICP22, and ICP27 genes is expressed as an early or late gene. 
     
     
         67 . The HSV vector of  claim 66 , wherein ICP22 is expressed as an early gene. 
     
     
         68 . The HSV vector of  claim 67 , wherein ICP22 within said genome is under the control of an early promoter. 
     
     
         69 . The HSV vector of  claim 60 , further comprising an inactivating deletion in one or more non-essential genes. 
     
     
         70 . The HSV vector of  claim 69 , wherein the one or more non-essential genes comprises UL41. 
     
     
         71 . The HSV vector of  claim 60 , further comprising a deletion of the internal repeat (joint) region. 
     
     
         72 . The HSV vector of  claim 60 , further comprising a gene encoding a mutant glycoprotein that enhances infectivity of the HSV vector relative to an HSV vector comprising the wild-type glycoprotein or that directs entry of the HSV vector into cells through non-canonical receptors. 
     
     
         73 . The vector of  claim 72 , wherein said glycoprotein is selected from gB, gC, gD, gH, and gK. 
     
     
         74 . The HSV vector of  claim 60 , wherein the polynucleotide encoding one or more transgenes is operably linked to a constitutive mammalian promoter. 
     
     
         75 . The HSV vector of  claim 74 , wherein the promoter is a cell- or tissue-specific promoter. 
     
     
         76 . The HSV vector of  claim 60 , wherein the polynucleotide encoding one or more transgenes is polycistronic. 
     
     
         77 . The HSV vector of  claim 60 , wherein the polynucleotide encoding one or more transgenes comprises one or more binding sites for a microRNA. 
     
     
         78 . The HSV vector of  claim 60 , wherein the one or more transgenes are selected from Oct4, Klf4, Sox2, c-Myc, L-myc, dominant-negative p53, Nanog, Glisl, Lin28, TFIID, GATA4, Nkx2.5, Tbx5, Mef2C, Myocd, Hand2, SRF, Mespl, SMARCD3, SERCA2a, Pax3, MyoD, Lhx2, FoxGl, FoxP2, Isll, Ctip2, Tbrl, Ebfl, Gsx2, Srebp2, Factor VIII, Factor IX, Dystrophin, CFTR, GlyRal, enkephalin, a GAD isoform, a neurotrophic factor, Ascll, Nurrl, Lmxl A, Brn2, Mytll, NeuroDl, FoxA2, Hnf4α, Foxal, Foxa2 or Foxa3, a microRNA, combination of miRNAs, or one or more non-coding RNA(s) (ncRNA(s)). 
     
     
         79 . The HSV vector of  claim 60 , wherein the HSV genome further comprises one or two consensus sequences for a recombinase enzyme. 
     
     
         80 . The HSV vector of  claim 79 , wherein the consensus sequence for the site-specific recombinase enzyme is inserted into an intergenic region. 
     
     
         81 . A herpes simplex virus (HSV) vector comprising a polynucleotide encoding one or more transgenes, wherein the HSV vector does not express ICP0, ICP4, ICP22, and ICP27 as immediate early genes, and wherein the HSV vector is capable of expressing the one or more transgenes for at least 28 days post infection of non-complementing cells with the HSV vector. 
     
     
         82 . A viral stock comprising the HSV vector of  claim 60 . 
     
     
         83 . A pharmaceutical composition comprising the HSV vector of  claim 60  and a pharmaceutically acceptable carrier. 
     
     
         84 . A method of expressing a transgene in a cell, comprising infecting a non-complementing cell with the HSV vector of  claim 60 , such that one or more of said transgenes is expressed within the cell. 
     
     
         85 . The method of  claim 84 , wherein the cell is in vitro or in vivo.