Patent
The present invention relates to a mutant Toxoplasma gondii which exhibits enhanced homologous recombination. The mutant of the present invention is a knockout in the KU80-dependent nonhomologous end-joining pathway which finds application in generating T. gondii gene knockouts and gene replacements for use in vaccine and drug development.
1. An isolated mutant Toxoplasma gondii that exhibits enhanced homologous recombination resulting from knockout mutation of the locus encoding KU80 protein of SEQ ID NO:2.
2. The mutant of claim 1 , wherein the mutation comprises replacing the coding region or promoter of the locus with a nucleic acid molecule encoding a selectable marker.
3. The mutant of claim 2 , wherein the selectable marker is hypoxanthine-xanthine-guanine phosphoribosyltransferase, thymidine kinase, hygromycin resistance, cytosine deaminase, dihydrofolate reductase, bleomycin, chloramphenicol acetyl transferase, or a combination thereof.
4. The mutant of claim 1 , wherein said mutant is attenuated.
5. The mutant of claim 4 , wherein the mutant is attenuated by pyrimidine auxotrophy.
6. The mutant of claim 5 , wherein pyrimidine auxotrophy comprises mutation of one or more proteins involved in pyrimidine synthesis or pyrimidine salvage.
7. The mutant of claim 6 , wherein the proteins are selected from carbamoyl phosphate synthetase II, aspartate transcarbamylase, dihydroorotase, dihydroorotase dehydrogenase, orotate phosphoribosyltransferase, orotidine 5′-monophosphate decarboxylase, uridine phosphorylase, uracil phosphoribosyltransferase, a nucleobase/nucleoside transporter of pyrimidine bases or nucleosides, or combinations thereof.
8. The mutant of claim 1 , wherein the mutant further comprises in its genome one or more nucleic acid molecules encoding exogenous proteins.
9. The mutant of claim 8 , wherein the nucleic acid molecule replaces the coding region or promoter of the locus encoding KU80 protein of SEQ ID NO:2.
10. The mutant of claim 8 , wherein the exogenous protein is a therapeutic antibody, protein, enzyme or peptide.
11. The mutant of claim 8 , wherein the exogenous protein is a non- Toxoplasma gondii antigen.
12. The mutant of claim 11 , wherein the antigen is a bacterial, viral, fungal, parasitic or tumor antigen.
13. The mutant of claim 8 , wherein the exogenous protein produces a non- Toxoplasma gondii antigen.
14. The mutant of claim 13 , wherein the antigen is a lipid or polysaccharide.
15. The mutant of claim 8 , wherein the exogenous protein is a human therapeutic target protein or enzyme.