ONCOLYTIC ADENOVIRUS COMPOSITIONS WITH ENHANCED REPLICATION PROPERTIES
Tumor-selective recombinant adenoviruses that possess deletions or modifications in the E3 region are described. Recombinant adenoviruses that express adenovirus death protein (ADP) but have a deletion of at least three of the remaining six E3 genes exhibit enhanced virus replication. The recombinant adenoviruses further include additional modifications to allow selective replication in tumor cells and to detarget viruses from the liver. Use of the recombinant adenoviruses for cancer treatment is described.
1 - 40 . (canceled)
41 . A method of inhibiting tumor cell viability, comprising contacting the tumor cell with a recombinant adenovirus comprising a genome comprising:
an E1A region encoding a modified E1a protein;
an E3 region encoding an adenovirus death protein (ADP) and comprising a modification or deletion of the coding sequences of each of E3 genes 12.5 k, 6.7 k, 19 k, RIDα, RIDβ and 14.7 k, wherein the modification prevents expression of each of the encoded proteins; and
an E4 region comprising a modification or deletion of the E4orf6/7 coding sequence that prevents expression of the encoded protein, abolishes or impairs its E2F binding site, and/or deletes or impairs the nuclear localization signal.
42 . The method of claim 41 , wherein the method is an in vitro method.
43 . The method of claim 41 , wherein the method is an in vivo method and contacting the tumor cell comprises administering a therapeutically effective amount of the recombinant adenovirus, to a subject with a tumor comprising the tumor cell.
44 . The method of claim 43 , wherein the therapeutically effective amount of the recombinant adenovirus inhibits tumor progression or reduces tumor volume in the subject.
45 . A method of treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of a recombinant adenovirus comprising a genome comprising:
an E1A region encoding a modified E1a protein;
an E3 region encoding an adenovirus death protein (ADP) and comprising a modification or deletion of the coding sequences of each of E3 genes 12.5 k, 6.7 k, 19 k, RIDα, RIDβ and 14.7 k, wherein the modification prevents expression of each of the encoded proteins; and
an E4 region comprising a modification or deletion of the E4orf6/7 coding sequence that prevents expression of the encoded protein, abolishes or impairs its E2F binding site, and/or deletes or impairs the nuclear localization signal,
thereby treating cancer in the subject.
46 . (canceled)
47 . (canceled)
48 . (canceled)
49 . (canceled)
50 . The method of claim 45 , wherein treating the cancer comprises inhibiting tumor progression or reducing tumor volume in the subject.
51 . The method of claim 45 , wherein the modified Ela protein comprises:
a deletion of the LXCXE motif;
a deletion of residues 2-11;
a C124G substitution;
a Y47H substitution;
a Y47H substitution and a C124G substitution; or
a Y47H substitution, a C124G substitution and a deletion of residues 2-11,
wherein the numbering of the preceding modifications is relative to SEQ ID NO: 23.
52 . The method of claim 45 , wherein the modification of the coding sequences of each of E3 genes 12.5 k, 6.7 k, 19 k, RIDα, RIDβ and 14.7 k comprises a mutation of a start codon, a mutation that introduces a premature stop codon, or both.
53 . The method of claim 45 , wherein the genome further comprises a modification or deletion of E4orf3, wherein the modification prevents expression of the encoded protein.
54 . The method of claim 45 , wherein the recombinant adenovirus comprises at least one modification to detarget the recombinant adenovirus from the liver.
55 . The method of claim 54 , wherein the modification comprises a mutation in the hexon protein.
56 . The method of claim 55 , wherein the hexon mutation is an E451Q mutation, wherein the numbering is relative to SEQ ID NO: 34.
57 . The method of claim 45 , wherein the genome encodes a chimeric fiber protein.
58 . The method of claim 57 , wherein the chimeric fiber protein comprises a fiber shaft from a first adenovirus serotype and a fiber knob from a second adenovirus serotype.
59 . The method of claim 58 , wherein the first adenovirus serotype is Ad5 and the second adenovirus serotype is Ad3, Ad9, Ad11, Ad12, Ad34 or Ad37.
60 . The method of claim 58 , wherein the first adenovirus serotype is Ad5 and the second adenovirus serotype is Ad34.
61 . The method of claim 45 , wherein the genome further comprises a heterologous open reading frame (ORF).
62 . The method of claim 61 , wherein the heterologous ORF is operably linked to and in the same reading frame as a self-cleaving peptide coding sequence and the ADP coding sequence, wherein the self-cleaving peptide coding sequence is disposed between the heterologous ORF and the ADP coding sequence.
63 . The method of claim 62 , wherein the self-cleaving peptide is a 2A peptide, or variant thereof.
64 . The method of claim 63 , wherein the 2A peptide comprises a porcine teschovirus-1 (PTV1) 2A (P2A) peptide, a foot and mouth disease virus (FMDV) 2A (F2A) peptide, an equine rhinitis A virus (ERAV) 2A (E2A) peptide or a Thosea asigna virus (TaV) 2A (T2A) peptide, or a variant thereof.
65 . The method of claim 45 , wherein the nucleotide sequence of the genome is at least 95% identical to SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, or SEQ ID NO: 59.
66 . The method of claim 45 , wherein:
the modified Ela protein comprises a deletion of the LXCXE motif;
the genome comprises at least one modification in the hexon protein to detarget an adenovirus from the liver;
the genome comprises a heterologous ORF operably linked to and in the same reading frame as a self-cleaving peptide coding sequence and the ADP coding sequence; and
the genome encodes a chimeric fiber protein comprising a fiber shaft from a first adenovirus serotype and a fiber knob from a second adenovirus serotype.
67 . The method of claim 66 , wherein:
the modification or deletion of the coding sequences of each of the E3 genes 12.5 k, 6.7 k, 19 k, RIDα, RIDβ and 14.7 k comprises deletion of each of the E3 genes 12.5 k, 6.7 k, 19 k, RIDα, RIDβ and 14.7 k;
the hexon modification is E451Q, wherein the numbering is relative to SEQ ID NO: 34;
the self-cleaving peptide is a 2A peptide;
the first adenovirus serotype is Ad5 and the second adenovirus serotype is Ad3, Ad9, Ad11, Ad12, Ad34 or Ad37.
68 . The method of claim 67 , wherein the first adenovirus serotype is Ad5 and the second adenovirus serotype is Ad34.
69 . The method of claim 67 , wherein the recombinant adenovirus is Ad5.
70 . The method of claim 67 , wherein the heterologous ORF is operably linked to and in the same reading frame as the self-cleaving peptide and the ADP coding sequence encodes YPet-P2A-ADP.
71 . The method of claim 70 , wherein the first adenovirus serotype is Ad5 and the second adenovirus serotype is Ad34, and wherein the adenovirus is Ad5.
72 . The method of claim 63 , wherein the variant comprises a Gly-Ser-Gly at the N-terminus.
73 . The method of claim 72 , wherein the heterologous ORF encodes a fluorescent protein.
74 . The method of claim 73 , wherein the fluorescent protein is YPet.
75 . The method of claim 45 , wherein the nucleotide sequence of the genome comprises SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, or SEQ ID NO: 59.
76 . The method of claim 45 , wherein the genome comprises SEQ ID NO: 13.
77 . The method of claim 45 , wherein the genome consists of SEQ ID NO: 13.
78 . The method of claim 45 , wherein the genome encodes a capsid-swapped adenovirus.
79 . The method of claim 71 , wherein the genome encodes a capsid-swapped adenovirus.