Delivery of packaged RNA to mammalian cells
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.
1. A recombinant alphavirus particle, comprising:
a. an alphavirus replicon vector construct generated from a Sindbis virus or Venezuelan equine encephalitis virus which directs the expression of a heterologous nucleic acid molecule, wherein said alphavirus replicon vector construct comprises (i) a 5′ sequence which initiates transcription of alphavirus RNA, (ii) a Rous sarcoma virus packaging signal, (iii) a nucleotide sequence encoding Sindbis virus or Venezuelan equine encephalitis virus nonstructural proteins NSP1, NSP2 NSP3 and NSP4, (iv) a viral junction region promoter which directs the expression of the heterologous nucleic acid sequence, and (v) an RNA polymerase recognition sequence, wherein said heterologous nucleic acid sequence replaces an alphavirus structural protein gene;
b. a Rous sarcoma virus capsid protein, and
c. an envelope glycoprotein or fusogenic protein heterologous to the alphavirus of said alphavirus construct and capable of binding to a host eukaryotic cell.
2. The recombinant alphavirus particle according to claim 1 , wherein said envelope glycoprotein or fusogenic protein is 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 recombinant alphavirus particle according to claim 1 , wherein said alphavirus particle expresses the heterologous nucleic acid molecule in the host eukaryotic cell.
4. The recombinant alphavirus particle according to claim 3 , wherein said alphavirus particle is not cytopathic to the eukaryotic cell.
5. The recombinant alphavirus particle according to claim 3 , wherein the heterologous nucleic acid sequence encodes an antisense RNA, wherein the antisense RNA knocks down expression of a gene in the eukaryotic cell.
6. The recombinant alphavirus particle according to claim 3 , wherein the heterologous nucleic acid sequence 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 the recombinant alphavirus particle according to claim 1 comprising:
(1) cotranfecting a eukaryotic cell with:
a. a first alphavirus replicon vector construct generated from a Sindbis virus or Venezuelan equine encephalitis virus which directs the expression of a heterologous nucleic acid molecule, wherein said alphavirus replicon vector construct comprises (i) a 5′ sequence which initiates transcription of alphavirus RNA, (ii) a Rous sarcoma virus packaging signal, (iii) a nucleotide sequence encoding Sindbis virus or Venezuelan equine encephalitis virus nonstructural proteins NSP1, NSP2 NSP3 and NSP4, (iv) a viral junction region promoter which directs the expression of the heterologous nucleic acid sequence, and (v) an RNA polymerase recognition sequence, wherein said heterologous nucleic acid sequence replaces an alphavirus structural protein gene,
b. a second vector construct comprising a polynucleotide sequence encoding the retroviral Rous sarcoma virus gag protein, and
c. a third vector construct comprising a polynucleotide sequence encoding an envelope glycoprotein or fusogenic protein heterologous to the alphavirus of said first alphavirus construct and capable of binding to a host eukaryotic cell;
(2) culturing the cotranfected eukaryotic cell under conditions suitable to cause each vector to produce its encoded product, thereby producing the recombinant alphavirus particle; and
(3) isolating the recombinant alphavirus particle from the cell supernatant;
wherein neither the vectors nor the cell contain any alphavirus structural protein genes.
8. A method of expressing a heterologous nucleic acid sequence in a eukaryotic cell comprising contacting said eukaryotic cell with the recombinant alphavirus particle according to claim 1 .