Cell specific replication-competent viral vectors comprising a self processing peptide cleavage site
Cell specific replication-competent viral vectors comprising a self processing peptide cleavage sequence are provided. The targeted replication-competent viral vectors include two or more co-transcribed genes under transcriptional control of the same heterologous transcriptional regulatory element (TRE), wherein at least a second gene is under translational control of a self processing cleavage sequence or 2A sequence. Exemplary vector constructs may further include an additional proteolytic cleavage site which provides a means to remove the self processing peptide sequence from the viral vector.
1 . A cytolytic replication competent adenovirus vector comprising in sequential order: a left ITR, a heterologous transcriptional regulatory element (TRE) operably linked to the coding sequence for a first adenoviral gene, a sequence encoding a self-processing cleavage site, the coding sequence for a second adenoviral gene and a right ITR.
2 . The adenovirus vector according to claim 1 , wherein said first adenoviral gene is an adenoviral gene essential for replication.
3 . The adenovirus vector according to claim 2 , wherein said adenoviral gene essential for replication is an early gene.
4 . The adenovirus vector according to claim 3 , wherein said adenoviral gene essential for replication is selected from the group consisting of E1A, E1B, E2 and E4.
5 . The adenovirus vector according to claim 4 , wherein said adenoviral gene essential for replication is E1A or E1B.
6 . The adenovirus vector according to claim 5 , wherein E1A or E1B has a mutation in, or deletion of its endogenous promoter.
7 . The adenovirus vector according to claim 2 , wherein said adenoviral gene essential for replication is a late gene.
8 . An adenovirus vector according to claim 1 , wherein said second adenoviral gene is an adenoviral gene essential for replication.
9 . The adenovirus vector according to claim 8 , wherein said adenoviral gene essential for replication is an early gene.
10 . The adenovirus vector according to claim 9 , wherein said adenoviral gene essential for replication is selected from the group consisting of E1A, E1B, E2 and E4.
11 . The adenovirus vector according to claim 10 , wherein said adenoviral gene essential for replication is E1A or E1B.
12 . The adenovirus vector according to claim 11 , wherein E1A or E1B has a mutation in or deletion of its endogenous promoter.
13 . The adenovirus vector according to claim 7 , wherein said adenoviral gene essential for replication is a late gene.
14 . An adenovirus vector according to claim 4 , wherein the sequence encoding said self-processing cleavage site comprises a 2A sequence.
15 . An adenovirus vector according to claim 14 , wherein said 2A sequence is a Foot and Mouth Disease Virus (FMDV) sequence.
16 . An adenovirus vector according to claim 15 , wherein said 2A sequence encodes an oligopeptide comprising amino acid residues LLNFDLLKLAGDVESNPGP (SEQ ID NO:1) or TLNFDLLKLAGDVESNPGP (SEQ ID NO:2).
17 . An adenovirus vector according to claim 15 , further comprising an additional proteolytic cleavage site wherein said cleavage site is a furin cleavage site with the consensus sequence RXK(R)R (SEQ ID NO:10).
18 . An adenovirus vector according to claim 4 , wherein said heterologous TRE comprises a promoter selected from the group consisting of a tissue-specific promoter, a tumor-specific promoter, a developmental stage-specific promoter and a cell status specific promoter.
19 . An adenovirus vector according to claim 18 , wherein said heterologous TRE further comprises an enhancer.
20 . An adenovirus vector according to claim 4 , wherein said heterologous TRE is a selected from the group consisting of an E2F responsive promoter, a TERT promoter, a prostate-specific antigen (PSA) transcriptional regulatory element (PSA-TRE), a probasin transcriptional regulatory element (PB-TRE), a human glandular kallikrein transcriptional regulatory element (HKLK2-TRE), a carcinoembryonic antigen transcriptional regulatory element (CEA-TRE), an alpha-fetoprotein transcriptional regulatory element (AFP-TRE), a uroplakin II transcriptional regulatory element (UPII-TRE); a PRL-3 transcriptional regulatory element TRE (PRL-3 TRE); a melanocyte cell-specific transcriptional response element (melanocyte TRE) and a CRG-L2 transcriptional regulatory element (CRG-L2 TRE).
21 . An adenovirus vector according to claim 20 , wherein said heterologous TRE is an E2F responsive promoter.
22 . An adenovirus vector according to claim 21 , wherein said E2F responsive promoter comprises SEQ ID NO:15.
23 . An adenovirus vector according to claim 20 , wherein said heterologous TRE is a TERT promoter.
24 . An adenovirus vector according to claim 23 , wherein said TERT promoter is a human TERT promoter.
25 . An adenovirus vector according to claim 24 , wherein said TERT promoter comprises SEQ ID NO:16 or SEQ ID NO:17.
26 . An adenovirus vector according to claim 5 , wherein the EIB gene has a deletion of the 19-kDa region.
27 . An adenovirus vector according to claim 4 , further comprising a mutation or deletion in an E3 coding region.
28 . An adenovirus vector according to claim 27 , wherein at least one E3 coding region has been deleted from said backbone.
29 . An adenovirus vector according to claim 4 , wherein the E3 region in said backbone codes for at least one of the native E3 proteins.
30 . An adenovirus vector according to claim 29 , wherein said E3 coding region is selected from the group consisting of E3-6.7, KDa, gp19 KDa, 11.6 KDa (ADP), 10.4 KDa (RIDα), 14.5 KDa (RIDβ), and E3-14.7 Kda.
31 . An adenovirus vector according to claim 29 wherein the E3 region in said backbone codes for all of the native E3 proteins.
32 . An isolated host cell comprising the adenovirus vector of claim 15 .
33 . An isolated host cell comprising the adenovirus vector of claim 22 .
34 . An isolated host cell comprising the adenovirus vector of claim 25 .
35 . A composition comprising a replication-competent adenovirus vector according to claim 15 and a pharmaceutically acceptable excipient.
36 . A composition comprising a replication-competent adenovirus vector according to claim 22 and a pharmaceutically acceptable excipient.
37 . A composition comprising a replication-competent adenovirus vector according to claim 25 and a pharmaceutically acceptable excipient.
38 . A cytolytic replication competent adenovirus vector comprising in sequential order: a left ITR, heterologous transcriptional regulatory element (TRE) operably linked to the coding sequence for an adenoviral gene, a sequence encoding a self-processing cleavage site, the coding sequence for a transgene and a right ITR.
39 . An adenovirus vector according to claim 38 , wherein said adenoviral gene is an adenoviral gene essential for replication.
40 . The adenovirus vector of claim 39 , wherein said adenoviral gene essential for replication is an early gene.
41 . The adenovirus vector of claim 40 , wherein said adenoviral gene essential for replication is selected from the group consisting of E1A, E1B, E2 and E4.
42 . The adenovirus vector of claim 41 , wherein said adenoviral gene essential for replication is E1A or E1B.
43 . The adenovirus vector of claim 42 , wherein E1A or E1B has a mutation in or deletion of its endogenous promoter.
44 . The adenovirus vector of claim 39 , wherein said adenoviral gene essential for replication is a late gene.
45 . An adenovirus vector according to claim 41 , wherein said transgene is a cytotoxic gene.
46 . The adenovirus vector of claim 45 , wherein said cytotoxic gene is an adenoviral death protein (ADP) gene.
47 . An adenovirus vector according to claim 41 , wherein said transgene is GM-CSF.
48 . An adenovirus vector according to claim 41 , wherein the sequence encoding said self-processing cleavage site comprises a 2A sequence.
49 . An adenovirus vector according to claim 48 , wherein said 2A sequence is a Foot and Mouth Disease Virus (FMDV) sequence.
50 . An adenovirus vector according to claim 49 , wherein said 2A sequence encodes an oligopeptide comprising amino acid residues LLNFDLLKLAGDVESNPGP (SEQ ID NO:1) or TLNFDLLKLAGDVESNPGP (SEQ ID NO:2).
51 . An adenovirus vector according to claim 41 , further comprising an additional proteolytic cleavage site is a furin cleavage site with the consensus sequence RXK(R)R (SEQ ID NO:10).
52 . An adenovirus vector according to claim 41 , wherein said heterologous TRE comprises a promoter selected from the group consisting of a tissue-specific, a tumor-specific, a developmental stage-specific and a cell status specific promoter.
53 . An adenovirus vector according to claim 52 , wherein said heterologous TRE further comprises an enhancer.
54 . An adenovirus vector according to claim 41 , wherein said heterologous TRE is a selected from the group consisting of an E2F responsive promoter, a TERT promoter, a prostate-specific antigen (PSA) transcriptional regulatory element (PSA-TRE), a probasin transcriptional regulatory element (PB-TRE), a human glandular kallikrein transcriptional regulatory element (HKLK2-TRE), a carcinoembryonic antigen transcriptional regulatory element (CEA-TRE), an alpha-fetoprotein transcriptional regulatory element (AFP-TRE), a uroplakin II transcriptional regulatory element (UPII-TRE); a PRL-3 transcriptional regulatory element TRE (PRL-3 TRE); a melanocyte cell-specific transcriptional response element (melanocyte TRE) and a CRG-L2 transcriptional regulatory element (CRG-L2 TRE).
55 . An adenovirus vector according to claim 54 , wherein said heterologous TRE is an E2F responsive promoter.
56 . An adenovirus vector according to claim 55 , wherein said E2F responsive promoter comprises SEQ ID NO:15.
57 . An adenovirus vector according to claim 41 , wherein said heterologous TRE is a TERT promoter.
58 . An adenovirus vector according to claim 57 , wherein said TERT promoter is a human TERT promoter.
59 . An adenovirus vector according to claim 58 , wherein said TERT promoter comprises SEQ ID NO:16 or SEQ ID NO:17.
60 . An adenovirus vector according to claim 43 , wherein the E1B gene has a deletion of the 19-kDa region.
61 . An adenovirus vector according to claim 41 , further comprising a mutation or deletion in an E3 coding region.
62 . An adenovirus vector according to claim 61 , wherein at least one of the E3 coding regions have been deleted from said backbone.
63 . An adenovirus vector according to claim 41 , wherein the E3 region in said backbone codes for at least one of the native E3 proteins.
64 . An adenovirus vector according to claim 63 , wherein said E3 coding region is selected from the group consisting of E3-6.7, KDa, gp19KDa, 11.6 KDa (ADP), 10.4 KDa (RIDα), 14.5 KDa (RIDβ), and E3-14.7 Kda.
65 . An adenovirus vector according to claim 63 , wherein the E3 region in said backbone codes for all of the native E3 proteins.
66 . An isolated host cell comprising the adenovirus vector of claim 49 .
67 . An isolated host cell comprising the adenovirus vector of claim 56 .
68 . An isolated host cell comprising the adenovirus vector of claim 59 .
69 . A composition comprising a replication-competent adenovirus vector according to claim 49 and a pharmaceutically acceptable excipient.
70 . A composition comprising a replication-competent adenovirus vector according to claim 56 and a pharmaceutically acceptable excipient.
71 . A composition comprising a replication-competent adenovirus vector according to claim 59 and a pharmaceutically acceptable excipient.