Delivery of packaged rna to mammalian cells

Abstract

Described herein are compositions relating to alphavirus-based virus-like particles (VLPs) and methods for making and using the described VLPs. The described compositions include VLPs and vectors and cells used to produce the VLPs. Also included are related methods to produce the VLPs, to transduce cells using the VLPs, and to produce a protein or polynucleotide of interest in a target cell using the VLPs. Also described are alphavirus-based replicons that allow for expression of proteins or polynucleotides of interest in a target cell without a cytopathic effect.

What is claimed is: 
     
         1 . A virus like particle (VLP) consisting of:
 a. an alphavirus replicon isolated from Sindbis virus or Venezuelan equine encephalitis virus comprising a recombinant polynucleotide comprising a sequence encoding Sindbis virus or Venezuelan equine encephalitis virus nonstructural proteins NSP1, NSP2, NSP3, and NSP4; and a retroviral packaging signal isolated from Rous sarcoma virus,   b. a retroviral gag protein encoded by a polynucleotide comprising a genomic sequence of a Rous sarcoma virus, and   c. a fusogenic envelope protein,   
       wherein the VLP does not contain an alphavirus structural protein gene. 
     
     
         2 . The VLP of  claim 1 , wherein the fusogenic envelope protein is encoded by a polynucleotide comprising a genomic sequence encoding an envelope protein selected from the group consisting of haemagglutinin, Rous sarcoma virus fusion protein, an E protein of tick borne encephalitis virus and dengue fever virus, the E1 protein of Semliki Forest virus, baculovirus gp64, VSV-EnvA, and VSV-G protein. 
     
     
         3 . The VLP of  claim 1 , wherein the VLP is not cytopathic to a eukaryotic cell. 
     
     
         4 . The VLP of  claim 1 , wherein the alphavirus replicon comprises a promoter of a virus RNA-dependent RNA polymerase, wherein said promoter is linked to the recombinant polynucleotide. 
     
     
         5 . The VLP of  claim 1 , wherein the recombinant polynucleotide encodes an antisense RNA, that knocks down expression of a gene in the eukaryotic cell. 
     
     
         6 . The VLP of  claim 1 , wherein the recombinant polynucleotide encodes a shRNA or a miRNA, wherein the shRNA or miRNA knocks down expression of a gene in the eukaryotic cell. 
     
     
         7 . A method of producing a VLP comprising:
 a. co-transforming a eukaryotic cell with:
 i. a first vector comprising a polynucleotide sequence encoding the alphavirus replicon, wherein the alphavirus replicon includes the polynucleotide of interest, 
 ii. a second vector comprising a polynucleotide sequence encoding the retroviral Rous gag protein, and 
 iii. a third vector comprising a polynucleotide sequence encoding the fusogenic envelope protein; 
   b. culturing the co-transformed cell under conditions suitable to cause each vector to produce its encoded product, thereby producing the VLP; and   c. isolating the VLP from the cell;   
       wherein neither the vectors nor the cell contain any alphavirus structural protein genes. 
     
     
         8 . A method of treating or preventing a disease or a disorder in a subject, comprising administering to a subject the VLP of  claim 1 . 
     
     
         9 . The method of  claim 8 , wherein the vector comprising the polynucleotide sequence encoding the alphavirus replicon is pDest472-VEE or pDest472-VEE-MCS.