Self-containing
The invention relates to a recombinant Lactococcus strain, with environmentally limited growth and viability. More particularly, it relates to a recombinant Lactococcus that can only survive in a medium, where well-defined medium compounds are present. A preferred embodiment is a Lactococcus that may only survive in a host organism, where such medium compounds are present, but cannot survive outside the host organism in the absence of such medium compounds.
1. An isolated thymidylate synthase (thyA) mutant of a parent strain of Lactococcus species, said thyA mutant comprising:
an inactive Lactococcus thymidylate synthase gene; and
a gene encoding a heterologous therapeutic molecule;
wherein said parent strain comprises SEQ ID NO: 3 and or SEQ ID NO: 5,
wherein the thyA mutant expresses the heterologous therapeutic molecule.
2. The isolated thymidylate synthase (thyA) mutant of a parent strain of Lactococcus species of claim 1 , wherein said (thyA) mutant is transformed with a transforming plasmid, wherein said transforming plasmid does not encode an active thymidylate synthase.
3. The isolated thymidylate synthase (thyA) mutant of a parent strain of Lactococcus species of claim 2 , wherein the transforming plasmid comprises the gene encoding a heterologous therapeutic molecule.
4. The isolated thymidylate synthase (thyA) mutant of a parent strain of Lactococcus species of any one of claims 2 , 3 , or 1 , wherein the Lactococcus species is Lactococcus lactis.
5. The isolated thymidylate synthase (thyA) mutant of a parent strain of Lactococcus species of any one of claim 2 , 3 , or 1 , wherein the gene encoding a heterologous therapeutic molecule encodes Interleukin-10.
6. The isolated thymidylate synthase (thyA) mutant of a parent strain of Lactococcus species of any one of claims 2 , 3 , or 1 , wherein the gene encoding a heterologous therapeutic molecule encodes Interleukin-10 and wherein the Lactococcus species is Lactococcus lactis.
7. An isolated thymidylate synthase (thyA) mutant of a parent strain of Lactococcus species, the thyA mutant comprising:
an inactive Lactococcus thymidylate synthase gene and a gene encoding a heterologous therapeutic molecule, wherein the inactive Lactococcus thymidylate synthase gene has been inactivated by gene disruption;
wherein the parent strain of Lactococcus species comprises:
SEQ ID NO: 3 or SEQ ID NO: 5, and wherein the thyA mutant expresses the heterologous therapeutic molecule.
8. An isolated thymidylate synthase (thyA) mutant of a parent strain of Lactococcus species, wherein the thyA mutant lacks active Lactococcus thymidylate synthase gene and comprises a gene encoding a heterologous therapeutic molecule, wherein the thyA mutant expresses the heterologous therapeutic molecule, and wherein the thyA mutant is produced by a process comprising:
providing a parent strain of Lactococcus species comprising a thymidylate synthase gene comprising wherein said Lactococcus thymidylate synthase gene comprises SEQ ID NO: 3 or SEQ ID NO: 5; and
altering by gene disruption the Lactococcus thymidylate synthase gene of the parent strain so as to inactivate the thymidylate synthase encoded thereby.
9. The isolated thymidylate synthase (thyA) mutant of a parent strain of Lactococcus species according to claim 8 , wherein the gene encoding the heterologous therapeutic molecule is integrated within, or replaces at least a part of the thymidylate synthase gene of said parent strain.
10. The isolated thymidylate synthase (thyA) mutant of a parent strain of Lactococcus species according to claim 8 , wherein said (thyA) mutant is transformed with a transforming plasmid,
wherein said transforming plasmid does not encode an active thymidylate synthase.
11. The isolated thymidylate synthase (thyA) mutant of a parent strain of Lactococcus species according to claim 10 , wherein the transforming plasmid comprises the gene encoding a heterologous therapeutic molecule.
12. The isolated thymidylate synthase (thyA) mutant of a parent strain of Lactococcus species according to any of claims 8 , 9 , 10 or 11 , wherein the Lactococcus species is Lactococcus lactis.
13. The isolated thymidylate synthase (thyA) mutant of a parent strain of Lactococcus species according to any of claims 8 , 9 , 10 , or 11 , wherein the gene encoding a heterologous therapeutic molecule encodes Interleukin-10.
14. The isolated thymidylate synthase (thyA) mutant of a parent strain of Lactococcus species according to any of claims 8 , 9 , 10 , or 11 , wherein the gene encoding a heterologous therapeutic molecule encodes Interleukin-10 and wherein the Lactococcus species is Lactococcus lactis.
15. A composition comprising:
the isolated thymidylate synthase (thyA) mutant of claim 1 .
16. The composition of claim 15 , wherein said (thyA) mutant is transformed with a transforming plasmid, wherein said transforming plasmid does not encode an active thymidylate synthase.
17. The composition of claim 16 , wherein the transforming plasmid comprises the gene encoding a heterologous therapeutic molecule.
18. The composition of any of claim 15 , 17 , or 16 , wherein the gene encoding a heterologous therapeutic molecule encodes Interleukin-10.
19. The composition of any of claim 15 , 17 , or 16 , wherein said Lactococcus species is Lactococcus lactis.
20. The composition of any of claim 15 , 17 , or 16 , wherein the gene encoding a heterologous therapeutic molecule encodes Interleukin-10 and wherein said Lactococcus species is Lactococcus lactis.
21. A method for delivering a heterologous therapeutic molecule to a subject, said method comprising administering the transformed strain of Lactococcus species of any of claims 1 , 7 , and 8 to the subject.
22. A method for delivering a heterologous molecule to a human subject, the method comprising administering the Lactococcus species of any of claims 1 , 7 , and 8 to the human subject.
23. A method of treating inflammatory bowel disease in a subject, said method comprising:
administering to the subject a transformed strain of Lactococcus species of any of claims 1 , 7 , and 8 .
24. The method of claim 23 , wherein the gene encoding a heterologous therapeutic molecule encodes Interleukin-10.