Thymidylate kinase fusions and uses thereof
The application relates to a composition comprising: a stably integrating delivery vector; a modified mammalian thymidylate kinase (tmpk) wherein the modified mammalian tmpk increases phosphorylation of a prodrug relative to phosophorylation of the prodrug by wild-type human tmpk; and a detection cassette fused to tmpk. The application also relates to use of these compositions in methods of treatment of diseases such as graft versus host disease and cancer.
1. A composition comprising:
a stably integrating delivery vector;
a modified human thymidylate kinase (tmpk) polynucleotide that encodes a modified human tmpk polypeptide that increases phosphorylation of AZT relative to phosphorylation of AZT by wild type human tmpk polypeptide;
a detection cassette polynucleotide that encodes a detection cassette polypeptide that is cell surface expressed, wherein the detection cassette polynucleotide is fused to the modified human tmpk polynucleotide and the detection cassette polypeptide is fused to the modified human tmpk polypeptide; and
a therapeutic polynucleotide cassette,
wherein the modified human tmpk polypeptide comprises a modification selected from (a) a F to Y mutation at amino acid position 105 of SEQ ID NO: 11, (b) a R to G point mutation at amino acid position 16 of SEQ ID NO: 12, or (c) a R to A mutation at amino acid position 200 of SEQ ID NO: 16.
2. The composition of claim 1 wherein the modified human tmpk polynucleotide comprises a polynucleotide with at least 90% sequence identity to the tmpk polynucleotide of any one of SEQ ID NOS: 15, 21, and 22.
3. The composition of claim 1 wherein the modified human tmpk polypeptide further comprises SEQ ID NO: 17 or the amino acid sequence RARGEL.
4. The composition of claim 1 wherein the therapeutic polynucleotide cassette is selected from the group consisting of adenosine deaminase, γc interleukin receptor subunit, α-galactosidase A, codon optimized α-galactosidase A, acid ceramidase, galactocerebrosidase, and transmembrane conductance regulator (CFTR) molecules.
5. A composition of claim 1 wherein the delivery vector is selected from the group consisting of a retroviral vector, an adenoviral vector, an adeno-associated viral vector, a spumaviral vector and a plasmid.
6. The composition of claim 5 wherein the delivery vector is a lentiviral vector that has a pHR′ backbone, a pDY backbone or a pCCL backbone and comprises 5′-Long terminal repeat (LTR), human immunodeficiency virus (HIV) signal sequence, HIV Psi signal 5′-splice site (SD), delta-GAG element, Rev Responsive Element (RRE), 3′-splice site (SA), Elongation factor (EF) 1-alpha promoter and 3′-Self inactivating LTR (SIN-LTR).
7. The composition of claim 1 wherein the detection cassette is fused to the tmpk polynucleotide by a linker.
8. The composition of claim 1 wherein the detection cassette polypeptide fused to the modified human tmpk polypeptide comprises the sequence of SEQ ID NO:38.
9. The composition of claim 1 , wherein the composition comprises a promoter functional in a mammalian cell and the coding sequence of the tmpk/detection cassette fusion polynucleotide is operably connected to the promoter.
10. A cell fate control system comprising:
a) the composition of claim 1 ;
a) AZT.
11. The composition of claim 1 , wherein the detection cassette polypeptide is CD19, truncated CD19, low affinity nerve growth receptor (LNGFR) or truncated LNGFR.
12. A cell destruction component of an expression vector comprising:
a modified human tmpk polynucleotide encoding a modified human tmpk polypeptide wherein the modified human tmpk polypeptide increases phosphorylation of AZT relative to phosphorylation of AZT by wild type human tmpk of claim 1 ;
a detection cassette polynucleotide that encodes a detection cassette polypeptide that is cell surface expressed, wherein the detection cassette polynucleotide is fused to the modified human tmpk polynucleotide and the detection cassette polypeptide is fused to the modified human tmpk polypeptide; and
a therapeutic polynucleotide for expression,
wherein the modified human tmpk polypeptide comprises a modification selected from (a) a F to Y mutation at amino acid position 105 of SEQ ID NO: 11, (b) a R to G point mutation at amino acid position 16 of SEQ ID NO: 12, or (c) a R to A mutation at amino acid position 200 of SEQ ID NO: 16.
13. The cell destruction component of claim 12 wherein the therapeutic polynucleotide is selected from the group consisting of adenosine deaminase, γc interleukin receptor subunit, acid ceramidase, codon optimized α-galactosidase A, acid ceramidase, galactocerebrosidase, and transmembrane conductance regulator (CFTR) molecules.
14. The cell destruction component of claim 12 , wherein the detection cassette polypeptide is CD19, truncated CD19, low affinity nerve growth receptor (LNGFR) or truncated LNGFR.
15. An isolated polynucleotide encoding an isolated polypeptide comprising:
a modified human thymidylate kinase (tmpk) polypeptide that increases phosphorylation of AZT relative to phosphorylation of AZT by wild type human tmpk polypeptide; and
a detection cassette polypeptide fused to the modified human tmpk polypeptide,
wherein the detection cassette polypeptide is a cell surface expressed detection cassette polypeptide,
the modified human tmpk polypeptide comprises a modification encoding selected from (a) a F to Y mutation at amino acid position 105 of SEQ ID NO: 11, (b) a R to G point mutation at amino acid position 16 of SEQ ID NO: 12, or (c) a R to A mutation at amino acid position 200 of SEQ ID NO: 16 and the detection cassette polypeptide fused to the modified human tmpk polypeptide is a tmpk/detection cassette fusion polypeptide.
16. The isolated polynucleotide of claim 15 , wherein the detection cassette polypeptide is CD19, truncated CD19, low affinity nerve growth receptor (LNGFR) or truncated LNGFR.
17. An isolated cell that expresses a modified tmpk/detection cassette fusion polynucleotide comprising:
a stably integrating delivery vector;
a modified human thymidylate kinase (tmpk) polynucleotide that encodes a modified human tmpk polypeptide that increases phosphorylation of AZT relative to phosphorylation of AZT by wild type human tmpk polypeptide;
a detection cassette polynucleotide that encodes a detection cassette polypeptide that is cell surface expressed, wherein the detection cassette polynucleotide is fused to the modified human tmpk polynucleotide and the detection cassette polypeptide is fused to the modified human tmpk polypeptide; and
a therapeutic polynucleotide cassette,
wherein the modified human tmpk polypeptide comprises a modification selected from (a) a F to Y mutation at amino acid position 105 of SEQ ID NO: 11, (b) a R to G point mutation at amino acid position 16 of SEQ ID NO: 12, or (c) a R to A mutation at amino acid position 200 of SEQ ID NO: 16.