Modified nodavirus RNA for gene delivery
View Patent ↗The invention provides a nodavirus RNA1 molecule modified to include a heterologous insertion which is downstream of its replicase ORF and, preferably, its B2 ORF. The insertion preferably comprises one or more protein-coding regions. The modified RNA1 may be packaged in a VLP, such as a papillomavirus VLP. The small size of nodavirus RNA1 makes it ideal for HPV packaging.
1. A modified nodavirus RNA1 molecule which includes a heterologous insertion downstream of the replicase ORF of said RNA1.
2. The RNA1 molecule of claim 1 , wherein the heterologous insertion is also downstream of the B2 ORF of said RNA1.
3. The RNA1 molecule of claim 1 , wherein the heterologous insertion is more than 5 nucleotides upstream of the 3′ end of said RNA1.
4. The RNA1 molecule of claim 1 , wherein the heterologous insertion comprises a protein-coding region.
5. The RNA1 molecule of claim 1 , wherein said RNA1 comprises a sequence that can specifically interact with a protein sequence.
6. A VLP containing the RNA1 molecule of claim 1 .
7. A single-stranded nucleic acid complementary to the RNA1 of claim 1 .
8. A single-stranded nucleic acid comprising a sequence complementary to the RNA1 of claim 1 .
9. A process for producing the RNA1 of claim 1 , comprising the steps of: (a) obtaining nucleic acid comprising or encoding a nodavirus RNA1 sequence, and (b) inserting a heterologous sequence downstream of the replicase ORF within said RNA1 sequence.
10. The RNA1 molecule of claim 2 , wherein the heterologous insertion is more than 5 nucleotides downstream of the replicase and B2 ORFs.
11. The RNA1 molecule of claim 4 , wherein the heterologous insertion comprises an IRES upstream of the protein-coding region.
12. The RNA1 molecule of claim 5 , wherein the sequence that can specifically interact with a protein sequence is a TAR sequence.
13. The RNA1 molecule of claim 12 , wherein the TAR sequence is within the heterologous insertion.
14. The VLP of claim 6 , wherein the VLP is a papillomavirus VLP.
15. A process for producing the VLP of claim 6 , comprising the steps of: transfecting a cell with a single-stranded nucleic acid complementary to a modified nodavirus RNA1 molecule which comprises a heterologous insertion downstream of the replicase ORF of said RNA1; transfecting a cell with nucleic acid encoding a VLP capsid protein, optionally modified to include a motif specific for modified RNA1; and purifying VLPs from the cell.
16. A process for delivering a nucleic acid sequence of interest to a cell, comprising the step of introducing the VLP of claim 6 into said cell, wherein said nucleic acid sequence of interest is the modified RNA1 molecule contained in said VLP.
17. A process for delivering a nucleic acid sequence to a cell, comprising the step of introducing the VLP of claim 6 into said cell.
18. The VLP of claim 14 , wherein the VLP comprises an L1 capsid protein comprising an insertion of a tat sequence, and the heterologous insertion of the modified nodavirus RNA1 molecule comprises an IRES upstream of a protein-coding region.
19. The VLP of claim 14 , wherein the VLP comprises an L1 capsid protein comprising an insertion of a tat sequence, and the heterologous insertion of the modified nodavirus RNA1 molecule comprises a TAR sequence.
20. The VLP of claim 19 , wherein the tat and TAR are from HIV or BIV.
21. A single-stranded nucleic acid complementary to the nucleic acid of claim 7 .
22. A double-stranded nucleic acid, in which one of the strands is the single-stranded nucleic acid of claim 7 .
23. A double-stranded nucleic acid, in which one of the strands is the single-stranded nucleic acid of claim 8 .
24. The process of claim 9 , wherein step (b) comprises inserting a heterologous sequence downstream of the replicase and B2 ORFs within said RNA1 sequence.
25. An RNA comprising a RNA1 sequence from a nodavirus, wherein the RNA1 sequence contains a heterologous insertion downstream of its replicase ORF.
26. The RNA of claim 25 , wherein the heterologous insertion is downstream of the B2 ORF.
27. A modified nodavirus RNA1 molecule that comprises a heterologous insertion that comprises a protein-coding region, wherein the heterologous insertion is more than 5 nucleotides downstream of the replicase ORF and the B2 ORF of said RNA1 and is more than 5 nucleotides upstream of the 3′ end of said RNA1.
28. The RNA1 molecule of claim 27 , wherein the heterologous insertion comprises an IRES upstream of the protein-coding region.
29. The RNA1 molecule of claim 27 , wherein said RNA1 comprises a sequence that can specifically interact with a protein sequence.
30. A VLP containing the RNA1 molecule of claim 27 .
31. A single-stranded nucleic acid complementary to the RNA1 of claim 27 .
32. A single-stranded nucleic acid comprising a sequence complementary to the RNA1 of claim 27 .
33. A process for producing the RNA1 of claim 27 , comprising the steps of: (a) obtaining nucleic acid comprising or encoding a nodavirus RNA1 sequence, and (b) inserting a heterologous sequence downstream of the replicase ORF within said RNA1 sequence.
34. The RNA1 molecule of claim 29 , wherein the sequence that can specifically interact with the protein sequence is a TAR sequence.
35. The RNA1 molecule of claim 34 , wherein the TAR sequence is within the heterologous insertion.
36. The VLP of claim 30 , wherein the VLP is a papillomavirus VLP.
37. A process for producing the VLP of claim 30 , comprising the steps of: transfecting a cell with a single-stranded nucleic acid complementary to a modified nodavirus RNA1 molecule that comprises a heterologous insertion that comprises a protein-coding region, wherein the heterologous insertion is more than 5 nucleotides downstream of the replicase ORF and the B2 ORF of said RNA1 and is more than 5 nucleotides upstream of the 3′ end of said RNA1; transfecting a cell with nucleic acid encoding a VLP capsid protein, optionally modified to include a motif specific for modified RNA1; and purifying VLPs from the cell.
38. A process for delivering a nucleic acid sequence of interest to a cell, comprising the step of introducing the VLP of claim 30 into said cell, wherein said nucleic acid sequence of interest is the modified RNA1 molecule contained in said VLP.
39. A process for delivering a nucleic acid sequence to a cell, comprising the step of introducing the VLP of claim 30 into said cell.
40. The VLP of claim 36 , wherein the VLP comprises an L1 capsid protein comprising an insertion of a tat sequence, and the heterologous insertion of the modified nodavirus RNA1 molecule comprises an IRES upstream of a protein-coding region.
41. The VLP of claim 36 , wherein the VLP comprises an L1 capsid protein comprising an insertion of a tat sequence, and the heterologous insertion of the modified nodavirus RNA1 molecule comprises a TAR sequence.
42. The VLP of claim 41 , wherein the tat and TAR are from HIV or BIV.
43. A double-stranded nucleic acid, in which one of the strands is the single-stranded nucleic acid of claim 31 .
44. A double nucleic acid, in which one of the strands is the single-stranded nucleic acid of claim 32 .
45. The process of claim 33 , wherein step (b) comprises inserting a heterologous sequence downstream of the replicase and B2 ORFs within said RNA1 sequence.
46. The process of claim 39 , performed in vitro.
47. The process of claim 39 , performed in vivo.
48. A modified nodavirus RNA1 molecule comprising a heterologous insertion downstream of the replicase ORF of said RNA1, wherein said heterologous insertion is situated between the last nucleotide of the replicase ORF of said RNA1 and the last nucleotide of the RNA1 molecule.
49. An RNA comprising a RNA1 sequence from a nodavirus, wherein the RNA1 sequence contains a heterologous insertion downstream of its replicase ORF, wherein the heterologous insertion is situated between the last nucleotide of the replicase ORF of said RNA1 and the last nucleotide of the RNA1 sequence.