Oncolytic adenoviruses with increased proportion of the 156R splicing isoform of the E1B protein
The invention relates to a recombinant adenovirus that has an oncolytic effect in a cancer cell. By modulating the level and type of splice isoforms of the E1B gene product, expressed from the E1B gene, the oncolytic activity of such viruses can be enhanced. The invention provides a recombinant adenovirus in which the proportion of the E1B-156R isoform is increased relative to wild-type levels. Such a recombinant adenovirus may selectivity replicate in cancer cells, thereby killing cancer cells while sparing normal cells.
1. A recombinant adenovirus for which the proportion of the E1B-156R isoform is increased relative to wild-type levels, wherein the adenovirus has an oncolytic effect in a cancer cell, and wherein the recombinant adenovirus carries a mutation in the sequence of the E1B gene of the adenovirus and the mutation in the E1B gene is A3216G wherein the numbering is relative to the adenovirus Ad5 genome (accession number AC_000008.1) (SEQ ID NO: 41) (position 1503 in the E1B gene (SEQ ID NO: 1)) or a guanine point mutation at an equivalent position to 3216 in any other adenovirus serotype.
2. The recombinant adenovirus according to claim 1 , where the adenovirus is adenovirus serotype Ad5, or adenovirus serotype Ad5 strain pTG3602.
3. The recombinant adenovirus according to claim 1 , where the wild-type E1B gene has the polynucleotide sequence according to SEQ ID NO: 1.
4. The recombinant adenovirus according to claim 1 , where:
(a) the proportion of the E1B-156R isoform is increased relative to an E1B-496R isoform;
(b) the proportion of an E1B-496R isoform is decreased relative to wild-type levels;
(c) the proportion of the E1B-156R isoform is increased relative to an E1B-93R isoform;
(d) the proportion of the E1B-156R isoform is increased relative to an E1B-84R isoform; and/or
(e) the proportion of an E1B-93R isoform is decreased relative to wild-type levels.
5. The recombinant adenovirus according to claim 1 , where the proportion of the E1B-156R isoform is increased at least 2-fold, 4-fold, 10-fold, 100-fold, 1,000-fold or 10,000-fold, and/or the proportion of an E1B-496R isoform is decreased at least 2-fold, 4-fold, 10-fold, 100-fold, 1,000-fold or 10,000-fold.
6. The recombinant adenovirus according to claim 4 , where the proportion of the E1B-156R, E1B-496R, E1B-93R, or E1B-84R isoforms refers to:
a) the level of the isoform protein that is expressed; and/or
b) the level of the isoform mRNA that is transcribed.
7. The recombinant adenovirus according to claim 4 , where:
(a) the E1B-156R isoform has a polynucleotide sequence that has at least 80% sequence identity to SEQ ID NO: 2;
(b) the E1B-156R isoform has a polypeptide sequence that has at least 80% sequence identity to SEQ ID NO: 3;
(c) the E1B-496R isoform has a polynucleotide sequence that has at least 80% sequence identity to SEQ ID NO: 4;
(d) the E1B-496R isoform has a polypeptide sequence that has at least 80% sequence identity to SEQ ID NO: 5;
(e) the E1B-93R isoform has a polynucleotide sequence that has at least 80% sequence identity to SEQ ID NO: 6;
(f) the E1B-93R isoform has a polypeptide sequence that has at least 80% sequence identity to SEQ ID NO: 7;
(g) the E1B-84R isoform has a polynucleotide sequence that has at least 80% sequence identity to SEQ ID NO: 8; and/or
(h) the E1B-84R isoform has a polypeptide sequence that has at least 80% sequence identity to SEQ ID NO: 9.
8. The recombinant adenovirus according to claim 1 , where the cancer cell is a neoplastic cell.
9. The recombinant adenovirus according to claim 1 , where the oncolytic effect is determined by measuring:
a) viral infection of cells;
b) selective replication of the viral genome in cancer cells leading to preferential virus-mediated cell lysis in cancer cells, and the release of viral particles for further infection events.
10. A polynucleotide encoding the recombinant adenovirus according to claim 1 , which is optionally a vector suitable for adenovirus production in a host cell.
11. An isolated host cell comprising a polynucleotide encoding the recombinant adenovirus according to claim 1 .
12. A pharmaceutical composition comprising a recombinant adenovirus, wherein the recombinant adenovirus is characterised in that the proportion of the E1B-156R isoform is increased relative to wild-type levels, and wherein the adenovirus has an oncolytic effect in a cancer cell, and wherein the recombinant adenovirus carries a mutation in the sequence of the E1B gene of the adenovirus and the mutation in the E1B gene is A3216G wherein the numbering is relative to the adenovirus Ad5 genome (accession number AC_000008.1) (SEQ ID NO: 41) (position 1503 in the E1B gene (SEQ ID NO: 1) or a guanine point mutation at an equivalent position to 3216 in any other adenovirus serotype.