Recombinant virus replicon systems and uses thereof
The present disclosure generally relates to viral-based expression systems suitable for the production of molecules of interest. The disclosure relates to nucleic acid constructs, such as expression vectors, containing a modified replicon RNA which includes a modified 5′-unstranslated region (5′-UTR) and, optionally, at least some of its original viral sequence encoding structural proteins having been deleted. Also disclosed are methods for producing polypeptides of interest.
1. A nucleic acid molecule comprising a modified alphavirus replicon RNA, wherein the modified replicon RNA comprises a modified 5′-UTR of an alphavirus and sequences coding for biologically active alphavirus nonstructural proteins, and is devoid of at least a portion of a nucleic acid sequence encoding viral structural proteins, wherein the modified 5′UTR comprises a U to G nucleotide substitution at position 2.
2. The nucleic acid molecule of claim 1 , wherein the modified alphavirus replicon further comprises a subgenomic promoter RNA, 3′ viral sequences required in cis for replication, and a polyadenylate tract.
3. The nucleic acid molecule of claim 1 , wherein the modified alphavirus replicon RNA is devoid of a substantial portion of the nucleic acid sequence encoding viral structural proteins.
4. The nucleic acid molecule of claim 1 , further comprising one or more expression cassettes, wherein each of the one or more expression cassettes comprises a promoter operably linked to a heterologous nucleic acid sequence.
5. A composition comprising the nucleic acid molecule of claim 4 , and a pharmaceutically acceptable carrier.
6. The nucleic acid molecule of claim 1 , wherein the modified alphavirus replicon RNA is modified from an alphavirus belonging to the Alphavirus genus of the Togaviridae family.
7. The nucleic acid molecule of claim 6 , wherein the alphavirus belongs to the VEEV/EEEV (Venezuelan equine encephalitis virus/Eastern equine encephalitis virus) group, or the SF (Semliki Forest) group, or the SIN (Sindbis) group.
8. The nucleic acid molecule of claim 7 , wherein the alphavirus is VEEV.
9. The nucleic acid molecule of claim 1 , wherein the modified replicon RNA is operably linked to a heterologous regulatory element.
10. A recombinant cell comprising the nucleic acid molecule of claim 1 , wherein the recombinant cell is a prokaryotic or a eukaryotic cell.
11. A composition comprising the recombinant cell of claim 10 , and a pharmaceutically acceptable carrier.
12. A composition comprising the nucleic acid molecule of claim 1 , and a pharmaceutically acceptable carrier.
13. A nucleic acid molecule encoding the nucleic acid molecule of claim 1 .
14. A recombinant cell comprising the nucleic acid molecule of claim 13 , wherein the recombinant cell is a prokaryotic or a eukaryotic cell.
15. A method for producing a polypeptide of interest, comprising culturing a host cell comprising a nucleic acid molecule which comprises a modified alphavirus replicon RNA encoding the polypeptide of interest, wherein the modified alphavirus replicon RNA comprises a modified 5-′UTR of an alphavirus and sequences coding for biologically active alphavirus nonstructural proteins, and is devoid of at least a portion of a nucleic acid sequence encoding viral structural proteins, wherein the modified 5′-UTR comprises a U to G nucleotide substitution at position 2.
16. The method of claim 15 , wherein the modified alphavirus replicon RNA is modified from an alphavirus belonging to the Alphavirus genus of the Togaviridae family.
17. A method for producing a polypeptide of interest in a subject, comprising administering to the subject a nucleic acid molecule comprising a modified alphavirus replicon RNA encoding the polypeptide of interest, wherein the modified alphavirus replicon RNA comprises a modified 5-′UTR of an alphavirus and sequences coding for biologically active alphavirus nonstructural proteins, and is devoid of at least a portion of a nucleic acid sequence encoding viral structural proteins, wherein the modified 5′-UTR comprises a U to G nucleotide substitution at position 2.
18. The method of claim 17 , wherein the subject is a human, horse, pig, primate, mouse, cattle, swine, sheep, rabbit, cat, dog, bird, fish, goat, donkey, hamster, or buffalo.