Oncolytic HSV vector
The present invention provides a recombinant oncolytic Herpes Simplex Virus (oHSV) comprising a non-HSV ligand specific for a molecule (protein, lipid, or carbohydrate determinant) present on the surface of a cell (such as a cancer cell) and one or more copies of one or more microRNA target sequences inserted into one or more HSV gene loci, preferably one or more HSV gene(s) required for replication of HSV in normal (i.e., non-cancerous) cells. The invention further provides stocks and pharmaceutical compositions comprising the inventive oHSV and methods for killing tumor cells employing the inventive oHSV.
1. A recombinant herpes simplex virus (HSV), comprising:
(a) a mutation of glycoprotein B (gB) at position 285 or 549; and
(b) a plurality of copies of one or more microRNA target sequences inserted into a locus of one or more HSV genes required for HSV replication,
wherein expression of one or more genes in the internal repeat (joint) region in the HSV genome is silenced, and
wherein the internal repeat (joint) region comprises one copy of the ICP0, ICP34.5, LAT, and ICP4 genes and the promoter of the ICP47 gene.
2. The HSV of claim 1 , wherein the one or more genes in the internal repeat (joint) region that have silenced expression comprises the ICP0 gene or the ICP34.5 gene.
3. The HSV of claim 1 , wherein the expression is silenced by deletion or limited mutagenesis of the one or more genes in the internal repeat (joint) region.
4. The HSV of claim 1 , wherein the one or more microRNA target sequences are inserted in the 3′ untranslated region (3′ UTR) of the one or more HSV genes.
5. The HSV of claim 1 , comprising two or more copies of the microRNA target sequences inserted into the locus of the one or more HSV genes.
6. The HSV of claim 5 , comprising 4 copies of the microRNA target sequences inserted into the locus of the one or more HSV genes.
7. The HSV of claim 1 , wherein at least one of the one or more microRNA target sequences is a target sequence for miR-124.
8. The HSV of claim 7 , wherein the target sequence for miR-124 is present in an ICP4 gene.
9. The HSV of claim 1 , comprising a plurality of copies of at least two microRNA target sequences selected from the group consisting of a miR-122 target sequence, a miR-124 target sequence, a miR-128 target sequence, a miR-137 target sequence, and a miR-199 target sequence.
10. The HSV of claim 1 , wherein the mutation of the gB at position 285 or 549 is a missense mutation selected from the group consisting of D285N and A549T.
11. The HSV of claim 1 , wherein the HSV comprises a mutation of the gB at both positions 285 and 549.
12. The HSV of claim 1 , further comprising a non-HSV ligand displayed on the surface of the HSV envelope, which is specific for a molecule present on the surface of a cancer cell.
13. The HSV of claim 1 , further comprising a transgene that encodes an oncolytic factor, a protein or polypeptide that induces patient immune response against cancer, or a protein or polypeptide that enhances the lateral spread of the HSV.
14. A nucleic acid encoding the HSV of claim 1 .
15. A viral stock comprising the HSV of claim 1 , wherein the viral stock has a viral titer of at least 10 5 plaque-forming units (pfu).
16. A composition comprising:
(a) the HSV of claim 1 , a nucleic acid encoding the HSV, or a viral stock comprising the HSV; and
(b) a pharmaceutically acceptable carrier.
17. A method of killing a cancerous cell, comprising exposing the cancerous cell to the HSV of claim 1 , a nucleic acid encoding the HSV, or a viral stock comprising the HSV under conditions sufficient for the HSV, the nucleic acid, or the viral stock to infect the cancerous cell, whereby replication of the HSV within the cancerous cell results in death of the cancerous cell.
18. The method of claim 17 , wherein the cell is in vivo.
19. The method of claim 17 , wherein the cell is a human cell.
20. The method of claim 17 , wherein the cell is within a tumor.
21. The method of claim 20 , wherein the tumor is glioblastoma multiforme.
22. The method of claim 17 , wherein the cell is within the brain of an animal.
23. The method of claim 22 , wherein the HSV, the nucleic acid, or the viral stock is exposed to the cell by intracranially injecting the HSV, the nucleic acid, or the viral stock to the animal.
24. A recombinant Herpes Simplex Virus (HSV), comprising:
(a) a non-HSV ligand displayed on the surface of the HSV envelope, which is specific for a molecule present on the surface of a cancer cell; and
(b) a plurality of copies of one or more microRNA target sequences inserted into a locus of one or more HSV genes required for HSV replication,
wherein the expression of one or more genes in the internal repeat (joint) region in the HSV genome is silenced, and wherein the internal repeat (joint) region comprises one copy of the ICP0, ICP34.5, LAT, and ICP4 genes and the promoter of the ICP47 gene.
25. The HSV of claim 24 , wherein the one or more genes in the internal repeat (joint) region that have silenced expression comprises the ICP0 gene or the ICP34.5 gene.
26. The HSV of claim 24 , wherein the expression is silenced by deletion or limited mutagenesis of the one or more genes in the internal repeat (joint) region.
27. The HSV of claim 24 , wherein the ligand is incorporated into a viral envelope glycoprotein exposed on the HSV surface.
28. The HSV of claim 27 , wherein the viral envelope glycoprotein is gD or gC.
29. The HSV of claim 28 , wherein the ligand is incorporated between residues 1 and 25 of gD.
30. The HSV of claim 24 , wherein the non-HSV ligand is capable of specifically binding EGFR or EGFRvIII.
31. The HSV of claim 24 , wherein the ligand is a single-chain antibody (scFv), a peptide, a non-peptide hormone, or a growth factor that binds a cellular receptor.
32. The HSV of claim 24 , comprising two or more copies of the microRNA target sequences inserted into the locus of the one or more HSV genes.
33. The HSV of claim 32 , comprising 4 copies of the microRNA target sequences inserted into the locus of the one or more HSV genes.
34. The HSV of claim 32 , wherein the two or more copies of the microRNA target sequences are separated by spacers of four or more nucleotides within the HSV genome.
35. The HSV of claim 24 , wherein the one or more microRNA target sequences are inserted in the 3′ untranslated region (3′ UTR) of the one or more HSV genes.
36. The HSV of claim 24 , wherein at least one of the one or more microRNA target sequences is a target sequence for miR-124.
37. The HSV of claim 24 , comprising a plurality of copies of at least two microRNA target sequences selected from the group consisting of a miR-122 target sequence, a miR-124 target sequence, a miR-128 target sequence, a miR-137 target sequence, and a miR-199 target sequence.
38. The HSV of claim 24 , further comprising a mutant HSV gB or gH glycoprotein that facilitates vector entry through non-canonical receptors.
39. The HSV of claim 24 , further comprising a transgene that encodes an oncolytic factor, a protein or polypeptide that induces patient immune response against cancer, or a protein or polypeptide that enhances the lateral spread of the HSV.
40. A nucleic acid encoding the HSV of claim 24 .
41. A viral stock comprising the HSV of claim 24 , wherein the viral stock has a viral titer of at least 10 5 plaque-forming units (pfu).
42. A composition comprising:
(a) the HSV of claim 24 , a nucleic acid encoding the HSV, or a viral stock comprising the HSV; and
(b) a pharmaceutically acceptable carrier.
43. A method of killing a cancerous cell, comprising exposing the cancerous cell to the HSV of claim 24 , a nucleic acid encoding the HSV, or a viral stock comprising the HSV under conditions sufficient for the HSV, the nucleic acid, or the viral stock to infect the cancerous cell, whereby replication of the HSV within the cancerous cell results in death of the cancerous cell.
44. The method of claim 43 , wherein the cell is in vivo.
45. The method of claim 43 , wherein the cell is a human cell.
46. The method of claim 43 , wherein the cell is within a tumor.
47. The method of claim 46 , wherein the tumor is glioblastoma multiforme.
48. The method of claim 43 , wherein the cell is within the brain of an animal.
49. The method of claim 48 , wherein the HSV, the nucleic acid, or the viral stock is exposed to the cell by intracranially injecting the HSV, the nucleic acid, or the viral stock to the animal.