SUPERACTIVE MUTANT THYMIDINE KINASE FOR USE IN CANCER THERAPY
20210085756 · 2021-03-25
Assignee
Inventors
Cpc classification
C12N15/86
CHEMISTRY; METALLURGY
International classification
A61P35/00
HUMAN NECESSITIES
C12N15/86
CHEMISTRY; METALLURGY
Abstract
The present invention relates to a mutant thymidine kinase, specifically a mutant human thymidine kinase 1 (Tkl1). The activity of the mutant thymidine kinase is increased compared to the activity of wildtype thymidine kinase. Provided herein are uses of the mutant thymidine kinase in therapy, such as in cancer therapy. Also methods of treating cancer comprising administering the mutant thymidine kinase are disclosed herein. The present invention relates, inter alia, to a nucleic acid for use hi treating cancer, wherein said nucleic acid comprises a nucleotide sequence, wherein said nucleotide sequence encodes a mutant thymidine kinase.
Claims
1. A nucleic acid for use in treating cancer, wherein said nucleic acid comprises a nucleotide sequence, wherein said nucleotide sequence encodes a mutant human thymidine kinase 1, wherein said mutant human thymidine kinase 1 comprises one or more amino acid substitutions at positions corresponding to positions 70 to 100 of wild-type human thymidine kinase 1.
2. A method of treating cancer, comprising administering a nucleic acid to a subject, wherein said nucleic acid comprises a nucleotide sequence, wherein said nucleotide sequence encodes a mutant human thymidine kinase 1, wherein said mutant human thymidine kinase 1 comprises one or more amino acid substitutions at positions corresponding to positions 70 to 100 of wild-type human thymidine kinase 1.
3. The nucleic acid for use of claim 1, or the method of claim 2, wherein the activity of said mutant human thymidine kinase 1 is increased compared to the activity of wildtype human thymidine kinase1.
4. The nucleic acid for use of claim 3, or the method of claim 3, wherein said activity is specific activity.
5. The nucleic acid for use of claim 3 or 4, or the method of claim 3 or 4, wherein said activity of said mutant human thymidine kinase 1 is at least 9-fold increased compared to the activity of wildtype human thymidine kinase 1.
6. The nucleic acid for use of claim 5, or the method of claim 5, wherein said mutant human thymidine kinase 1 comprises one or more amino acid substitutions at positions corresponding to positions 71 to 95 of wild-type human thymidine kinase 1.
7. The nucleic acid for use of claim 6, or the method of claim 6, wherein said mutant human thymidine kinase 1 comprises one or more amino acid substitutions at positions corresponding to one or more positions 73, 75, 83, 84, 90 and 95 of wild-type human thymidine kinase 1.
8. The nucleic acid for use of claim 7, or the method of claim 7, wherein said mutant human thymidine kinase 1 comprises one or two amino acid substitutions at positions corresponding to positions 84 and/or 90 of wild-type human thymidine kinase 1.
9. The nucleic acid for use of any one of claims 1 and 3 to 8, or the method of any one of claims 2 to 8, wherein said mutant human thymidine kinase 1 comprises one or more aliphatic, nonpolar, and neutral amino acid, one or more acid/amide, acidic polar and negatively charged amino acid and/or one or more acid/amide, polar and neutral amino acid at one or more positions corresponding to positions 70 to 100 of wild-type human thymidine kinase 1.
10. The nucleic acid for use of any one of claims 1 and 3 to 9, or the method of any one of claims 2 to 9, wherein said mutant human thymidine kinase 1 comprises one or more aliphatic, nonpolar, and neutral amino acid at one or more positions corresponding to positions 73, 75, 83, 90, and 95 of wild-type human thymidine kinase 1.
11. The nucleic acid for use of any one of claims 1 and 3 to 9, or the method of any one of claims 2 to 9, wherein said mutant human thymidine kinase 1 comprises one or more aliphatic, nonpolar, and neutral amino acid at one or more positions corresponding to positions 73, 83, 90, and 95 of wild-type human thymidine kinase 1.
12. The nucleic acid for use of any one of claims 1 and 3 to 9, or the method of any one of claims 2 to 9, wherein said mutant human thymidine kinase 1 comprises one or more aliphatic, nonpolar, and neutral amino acid at one or more positions corresponding to positions 75 of wild-type human thymidine kinase 1.
13. The nucleic acid for use of any one of claims 1 and 3 to 9, or the method of any one of claims 2 to 9, wherein said mutant human thymidine kinase 1 comprises one or more aliphatic, nonpolar, and neutral amino acid at one or more positions corresponding to position 90 of wild-type human thymidine kinase 1.
14. The nucleic acid for use of any one of claims 1 and 3 to 9, or the method of any one of claims 2 to 9, wherein said mutant human thymidine kinase 1 comprises one or more acid/amide, acidic polar and negatively charged amino acid at one or more positions corresponding to positions 75, 84 and 90 of wild-type human thymidine kinase 1.
15. The nucleic acid for use of any one of claims 1 and 3 to 9, or the method of any one of claims 2 to 9, wherein said mutant human thymidine kinase 1 comprises one or two acid/amide, acidic polar and negatively charged amino acid at one or two positions corresponding to positions 84 and 90 of wild-type human thymidine kinase 1.
16. The nucleic acid for use of any one of claims 1 and 3 to 9, or the method of any one of claims 2 to 9, wherein said mutant human thymidine kinase 1 comprises a acid/amide, polar and neutral amino acid at a position corresponding to position 75 of wild-type human thymidine kinase 1.
17. The nucleic acid for use of any one of claims 1 and 3 to 9, or the method of any one of claims 2 to 9, a) wherein said mutant human thymidine kinase 1 comprises an aliphatic, nonpolar, neutral amino acid at a position corresponding to position 73 of wild-type human thymidine kinase 1; b) wherein said mutant human thymidine kinase 1 comprises an aliphatic, nonpolar, neutral amino acid or an acid/amide, polar, neutral amino acid or an acid/amide, acidic polar, negatively charged amino acid at a position corresponding to position 75 of wild-type human thymidine kinase 1; c) wherein said mutant human thymidine kinase 1 comprises an aliphatic, nonpolar, neutral amino acid at a position corresponding to position 83 of wild-type human thymidine kinase 1; d) wherein said mutant human thymidine kinase 1 comprises an acid/amide, acidic polar, negatively charged amino acid at a position corresponding to position 84 of human wild-type thymidine kinase 1; e) wherein said mutant human thymidine kinase 1 comprises an aliphatic, nonpolar, neutral amino acid or an acid/amide, acidic polar, negatively charged amino acid at a position corresponding to position 90 of wild-type human thymidine kinase 1; and/or f) wherein said mutant human thymidine kinase 1 comprises an aliphatic, nonpolar, neutral amino acid at a position corresponding to position 95 of wild-type human thymidine kinase 1.
18. The nucleic acid for use of any one of claims 1 and 3 to 17, or the method of any one of claims 2 to 17, wherein said mutant human thymidine kinase 1 comprises a) aliphatic, nonpolar, and neutral amino acids at positions corresponding to positions 73, 83, 90 and 95 of wild-type human thymidine kinase 1 and an acid/amide, acidic polar and negatively charged amino acid at a position corresponding to position 75 of wild-type human thymidine kinase 1; b) aliphatic, nonpolar, and neutral amino acids at positions corresponding to positions 73, 83, and 90 of wild-type human thymidine kinase 1 and an acid/amide, acidic polar and negatively charged amino acid at a position corresponding to position 75 of wild-type human thymidine kinase 1; c) an acid/amide, acidic polar and negatively charged amino acid at a position corresponding to position 84 of wild-type human thymidine kinase 1 and an aliphatic, nonpolar, and neutral amino acid at a position corresponding to position 90 of wild-type human thymidine kinase 1; d) aliphatic, nonpolar, and neutral amino acids at positions corresponding to positions 73, 90 and 95 of wild-type human thymidine kinase 1 and an acid/amide, acidic polar and negatively charged amino acid at a position corresponding to position 75 of wild-type human thymidine kinase 1; e) aliphatic, nonpolar, and neutral amino acids at positions corresponding to positions 73 and 90 of wild-type human thymidine kinase 1 and an acid/amide, acidic polar and negatively charged amino acid at a position corresponding to position 75 of wild-type human thymidine kinase 1; f) an aliphatic, nonpolar, and neutral amino acid at a position corresponding to position 90 of wild-type human thymidine kinase 1; g) acid/amide, acidic polar and negatively charged amino acids at positions corresponding to positions 75 and 90 of wild-type human thymidine kinase 1; h) an acid/amide, acidic polar and negatively charged amino acid at a position corresponding to position 84 of wild-type human thymidine kinase 1 and an aliphatic, nonpolar, and neutral amino acid at position corresponding to position 90 of wild-type human thymidine kinase 1; i) aliphatic, nonpolar, and neutral amino acids at positions corresponding to positions 75 and 90 of wild-type human thymidine kinase 1 and an acid/amide, acidic polar and negatively charged amino acid at a position corresponding to position 84 of wild-type human thymidine kinase 1; j) an acid/amide, acidic polar and negatively charged amino acid at a position corresponding to position 90 of wild-type human thymidine kinase 1; k) an aliphatic, nonpolar, and neutral amino acid at a position corresponding to position 90 of wild-type human thymidine kinase 1; or l) an acid/amide, polar and neutral amino acid at a position corresponding to position 75 of wild-type human thymidine kinase 1 and an acid/amide, acidic polar and negatively charged amino acid at a position corresponding to position 84 of wild-type human thymidine kinase 1.
19. The nucleic acid for use of any one of claims 1 and 3 to 17, or the method of any one of claims 2 to 17, wherein said mutant thymidine kinase comprises a) an aliphatic, nonpolar, and neutral amino acid at a position corresponding to position 90 of wild-type human thymidine kinase 1; or b) an acid/amide, acidic polar and negatively charged amino acid at a position corresponding to position 84 of wild-type human thymidine kinase 1 and an aliphatic, nonpolar, and neutral amino acid at a position corresponding to position 90 of wild-type human thymidine kinase 1.
20. The nucleic acid for use of any one of claims 9 to 13 and 17 to 19, or the method of any one of claims 9 to 13 and 17 to 19, wherein said aliphatic, nonpolar, and neutral amino acid is one or more of alanine, glycine, valine, isoleucine, leucine and methionine.
21. The nucleic acid for use of any one of claims 9 to 11, 17a), 17c), 17e) and 17f), or the method of any one of claims 9 to 11, 17a), 17c), 17e) and 17f), wherein said aliphatic, nonpolar, and neutral amino acid is alanine.
22. The nucleic acid for use of any one of claims 9, 10, 12 and 17b), or the method of any one of claims 9, 10, 12 and 17b), wherein said aliphatic, nonpolar, and neutral amino acid is glycine.
23. The nucleic acid for use of any one of claims 9, 10, 13 and 17e), or the method of any one of claims 9, 10, 13 and 17e), wherein said aliphatic, nonpolar, and neutral amino acid is valine.
24. The nucleic acid for use of any one of claims 9, 14, 15, and 17 to 19, or the method of any one of claims 9, 14, 15, and 17 to 19, wherein said acid/amide, acidic polar and negatively charged amino acid is one or more of aspartic acid and glutamic acid.
25. The nucleic acid for use of any one of claims 9, 14, 17b), 17d), 17e), 18a), 18b), 18d), 18e), 18g), 18h) and 18j), or the method of any one of claims 9, 14, 17b), 17d), 17e), 18a), 18b), 18d), 18e), 18g), 18h) and 18j), wherein said acid/amide, acidic polar and negatively charged amino acid is aspartic acid.
26. The nucleic acid for use of any one of claims 9, 15, 17d), 17e), 18c), 18g), 18i) and 18l), or the method of any one of claims 9, 15, 17d), 17e), 18c), 18g), 18i) and 18l), wherein said acid/amide, acidic polar and negatively charged amino acid is glutamic acid.
27. The nucleic acid for use of any one of claims 9 and 16 to 18, or the method of any one of claims 9 and 16 to 18, wherein said acid/amide, polar and neutral amino acid is one or more of glutamine, asparagine.
28. The nucleic acid for use of any one of claims 9, 16, 17b), and 18l), or the method of any one of claims 9, 16, 17b), and 18l), wherein said acid/amide, polar and neutral amino acid is glutamine.
29. The nucleic acid for use of any one of claims 1 and 3 to 28, or the method of any one of claims 2 to 28, wherein said mutant human thymidine kinase 1 comprises a) alanine at positions corresponding to positions 73, 83, 90 and 95 of wild-type human thymidine kinase 1 and aspartic acid at a position corresponding to position 75 of wild-type human thymidine kinase 1; b) alanine at positions corresponding to positions 73, 83, and 90 of wild-type human thymidine kinase 1 and aspartic acid at a position corresponding to position 75 of wild-type human thymidine kinase 1; c) glutamic acid at a position corresponding to position 84 of wild-type human thymidine kinase 1 and alanine at a position corresponding to position 90 of wild-type human thymidine kinase 1; d) alanine at positions corresponding to positions 73, 90 and 95 of wild-type human thymidine kinase 1 and aspartic acid at a position corresponding to position 75 of wild-type human thymidine kinase 1; e) alanine at positions corresponding to positions 73, and 90 of wild-type human thymidine kinase 1 and aspartic acid at a position corresponding to position 75 of wild-type human thymidine kinase 1; f) alanine at a position corresponding to position 90 of wild-type human thymidine kinase 1; g) aspartic acid at a position corresponding to position 75 of wild-type human thymidine kinase 1 and glutamic acid at a position corresponding to position 90 of wild-type human thymidine kinase 1; h) aspartic acid at a position corresponding to position 84 of wild-type human thymidine kinase 1 and alanine at position corresponding to position 90 of wild-type human thymidine kinase 1; i) glycine at a position corresponding to position 75 of wild-type human thymidine kinase 1, glutamic acid at a position corresponding to position 84 of wild-type human thymidine kinase 1 and alanine at a position corresponding to position 90 of wild-type human thymidine kinase 1; j) aspartic acid at a position corresponding to position 90 of wild-type human thymidine kinase 1; k) valine at a position corresponding to position 90 of wild-type human thymidine kinase 1; or l) glutamine at a position corresponding to position 75 of wild-type human thymidine kinase 1 and glutamic acid at a position corresponding to position 84 of wild-type human thymidine kinase 1.
30. The nucleic acid for use of any one of claims 1 and 3 to 29, or the method of any one of claims 2 to 29, wherein said mutant human thymidine kinase 1 comprises a) alanine at a position corresponding to position 90 of wild-type human thymidine kinase 1; or b) aspartic acid amino acid at a position corresponding to position 84 of wild-type human thymidine kinase 1 and an alanine at a position corresponding to position 90 of wild-type human thymidine kinase 1.
31. The nucleic acid for use of any one of claims 1 and 3 to 30, or the method of any one of claims 2 to 30, wherein said human wild-type thymidine kinase 1 has an amino acid sequence shown in SEQ ID NO. 28.
32. The nucleic acid for use of any one of claims 1 and 3 to 31, or the method of any one of claims 2 to 31, wherein said nucleic acid is selected from the group consisting of: a) a nucleic acid comprising a nucleotide sequence, wherein said nucleotide sequence encodes a mutant thymidine kinase, wherein said mutant human thymidine kinase 1 comprises an amino acid sequence as depicted in any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24; b) a nucleic acid comprising a nucleotide sequence, wherein said nucleotide sequence encodes a mutant human thymidine kinase 1, and wherein said nucleotide sequence is depicted in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 and 23; c) a nucleic acid hybridizing under stringent conditions to the complementary strand of the nucleic acid as defined in (a) or (b) and wherein said nucleic acid comprises a nucleotide sequence wherein said nucleotide sequence encodes a mutant human thymidine kinase 1; d) a nucleic acid comprising a nucleotide sequence with at least 70% identity to the nucleotide sequence of the nucleic acids of any one of (a) to (c) and wherein said nucleotide sequence encodes a mutant human thymidine kinase 1; and e) a nucleic acid comprising a nucleotide sequence which is degenerate as a result of the genetic code to the nucleotide sequence of a nucleic acid of any one of (a) to (d) and wherein said nucleotide sequence encodes a mutant human thymidine kinase 1.
33. The nucleic acid for use of any one of claims 1 and 3 to 31, or the method of any one of claims 2 to 31, wherein said nucleic acid is selected from the group consisting of: a) a nucleic acid comprising a nucleotide sequence, wherein said nucleotide sequence encodes a mutant human thymidine kinase 1, wherein said mutant thymidine kinase comprises an amino acid sequence as depicted in any one of SEQ ID NOs: 10 and 12; b) a nucleic acid comprising a nucleotide sequence, wherein said nucleotide sequence encodes a mutant human thymidine kinase 1, and wherein nucleotide sequence is depicted in SEQ ID NO: 9 and 11; c) a nucleic acid hybridizing under stringent conditions to the complementary strand of the nucleic acid as defined in (a) or (b) and wherein said nucleic acid comprises a nucleotide sequence wherein said nucleotide sequence encodes a mutant human thymidine kinase 1; d) a nucleic acid comprising a nucleotide sequence with at least 70% identity to the nucleotide sequence of the nucleic acids of any one of (a) to (c) and wherein said nucleotide sequence encodes a mutant human thymidine kinase 1; and e) a nucleic acid comprising a nucleotide sequence which is degenerate as a result of the genetic code to the nucleotide sequence of a nucleic acid of any one of (a) to (d) and wherein said nucleotide sequence encodes a mutant human thymidine kinase 1.
34. Nucleic acid as defined in any one of claims 1 to 33.
35. Vector comprising the nucleic acid of claim 34.
36. The vector of claim 35, wherein said vector is a gene therapy vector.
37. The vector of claim 35 or 36, wherein said vector is an AAV vector, adenovirus vector, or a lentivirus vector.
38. Protein encoded by the nucleic acid of claim 34.
39. Composition comprising the nucleic acid of claim 34, a vector of any one of claims 35 to 37, or the protein of claim 38.
40. The composition of claim 39, wherein said composition is a pharmaceutical composition.
41. The nucleic acid of claim 34, the vector of any one of claims 35 to 37, the protein of claim 38, or the composition of claim 39 or 40, wherein said nucleic acid, said vector, said protein, or said composition is for use as a medicament.
42. The vector of any one of claims 35 to 37, the protein of claim 38, or the composition of claim 39 or 40, wherein said vector, said protein or said composition is for use in treating cancer.
43. The nucleic acid for use of any one of claims 1 and 3 to 33, or the method of any one of claims 2 to 33, the vector for use of claim 42, the protein for use of claim 42, or the composition for use of claim 42, wherein said treatment of cancer comprises administration of deoxythymidine.
44. The nucleic acid for use of any one of claims 1, 3 to 33 and 43, or the method of any one of claims 2 to 33 and 43, the vector for use of claim 42 or 43, the protein for use of claim 42 or 43, or the composition for use of claim 42 or 43, wherein said treatment of cancer comprises administration of additional chemotherapeutic agents, surgery and/or radiotherapy.
45. The nucleic acid for use of claim 44, or the method of claim 44, the vector for use of claim 44, the protein for use of claim 44, or the composition for use of claim 44, wherein said chemotherapeutic agents are Cytarabin (araC) and/or 5-Fluoruracil (5-FU).
46. The nucleic acid for use of any one of claims 1, 3 to 33, 43, 44 and 45, or the method of any one of claims 2 to 33, 43, 44 and 45, the vector for use of any one of claims 42 to 45, the protein for use of any one of claims 42 to 45, or the composition for use of any one of claims 42 to 45, wherein said cancer is a solid cancer.
47. The nucleic acid for use of any one of claims 1 and 3 to 33, 43, 44 and 45, or the method of any one of claims 2 to 33, 43, 44 and 45, the vector for use of any one of claims 42 to 45, the protein for use of any one of claims 42 to 45, or the composition for use of any one of claims 42 to 45, wherein said cancer is selected from the group consisting of cervical cancer, prostate carcinoma, ductal mammary carcinoma, melanoma, colon cancer, lung cancer, liver cancer, glioblastoma, and nerve cell cancer.
Description
[0616] The following examples illustrate the invention:
[0617] The Figures show:
[0618]
[0619] The human superTk1 nt sequence is depicted as SEQ ID NO: 30.
[0620]
[0621] The differences to the wt Tk1 sequences (NP_003249) are underlined and printed in bold; especially the point mutation at aa 90 that causes a transition from G>A is relevant for the increase in specific activity of the superTk1. All the other changes are consequences of the origin of the cDNA and/or due to subcloning conditions. The human superTk1 aa sequence is depicted as SEQ ID NO: 12.
[0622]
TABLE-US-00010 Loading scheme of the 1.2% agarose gel: Lane Sample Description 1 1 kb ladder (10 l) 2 PC-3 pUHDsupTK1 clone 1 A very faint superTK1 band was detectable 3 PG-3 pUHDsupTK1 clone 2 A strong superTK1 band was detectable 4 PC-3 pUHDsupTK1 clone 7 All detectable bands are very strong due to overloading of too much DNA 5 PC-3 pUHDsupTK1 clone 10 A faint superTK1 band was detectable 6 PC-3 pUHDsupTK1 clone 11 A faint superTK1 band was detectable 7 PC-3 pUHDsupTK1 clone 12 A faint superTK1 band was detectable 8 PC-3 pUHDsupTK1 clone 13 A strong superTK1 band was detectable 9 Empty hygromycin vector Only the hygromycin band was detectable at 149 bp 10 Plasmid pUHDsuperTK1 #117 A superTK1 band at 665 bp and a hygromycin band at 149 bp was detectable
[0623]
[0624] The histogram shows the relative cell proliferation of PC-3 prostate carcinoma cells with stably integrated (into the genome) superTK1 gene (pUDHHyg driven), verified by a genomic PCR. The cytostatic treatment with 0.5 mM deoxythymidine (dTh) lasted for seven days, replenished every day. The bars show the mean values of cell numbers of sextuplicates, each sample measured twice; the error bars display the standard error of the means. Cell batches that expressed superTk1 received 5 g/ml doxycyclin replenished on a daily basis. The control samples received all other constituents (dTh, antibiotics) but no doxycyclin. In addition, the cell culture medium was renewed completely every 72 hrs in all wells in order to avoid unwanted starvation or deprivation effects.
[0625]
[0626] The PC-3 proliferation data recorded during inhibition by 0.5 mM dTh were subjected to a statistical analysis; Box and Whiskers plots are presented
[0627]
[0628] The histogram shows the relative cell proliferation of PC-3 prostate carcinoma cells with stably integrated wtTK1 gene (pUDHHyg driven), verified by a genomic PCR. The cytostatic treatment with 0.5 mM deoxythymidine (dTh) lasted for five days, replenished every day. The bars show the mean values of cell numbers of triplicates, each sample measured twice; the error bars display the standard error of the means. Cell batches that expressed wtTk1 received 5 g/ml doxycyclin replenished on a daily basis. The control samples received all other constituents (dTh, antibiotics) but no doxycyclin. In addition, the cell culture medium was renewed completely after 72 hrs in all wells in order to avoid unwanted starvation or deprivation effects.
[0629]
[0630] a) and b) show curves acquired by the FACS Calibur a) untreated cells and b) depicts cells supplied with 0.5 mM dTh and 5 g/ml doxycycline for three days. c) The bar graph shows which percentage of untreated cells (control) or those supplied with either substrate only (0.5 mM dTh) or doxycycline 5 g/ml only (doxy), or both (0.5 dTh+doxy), is undergoing which cell cycle phase at the time point of ethanol fixation on day 3.
[0631]
[0632] The histogram shows the relative cell proliferation of PC-3 prostate carcinoma cell clone 13 with stable integrated superTK1 gene (pUDHHygr driven), verified by genomic PCR. The cytostatic treatment with 0.1 mM deoxythymidine (dTh) lasted for four days, replenished every day. The bars show the mean values of cell numbers of triplicates, each sample measured twice; the error bars display the standard error of the means. Cell batches that expressed superTk1 received 5 g/ml doxycyclin replenished on a daily basis. The control samples received all other constituents (dTh, antibiotics) but no doxycyclin. In addition, the cell culture medium was renewed completely every 72 hrs in all wells in order to avoid unwanted starvation or deprivation effects.
[0633]
[0634] The PC-3 proliferation data recorded during inhibition by 0.1 mM dTh were subjected to a statistical analysis; Box and Whiskers Plots are presented. The Kruskal-Wallis test shows a P-value, which is less than 0.05 from day 3 on; this means that there is a statistically significant difference amongst the medians of the treated sample and its control at the 95.0% confidence level. The method used to discriminate among the means is Fisher's least significant difference (LSD) procedure
[0635]
[0636] The MFM-223 pUHDsuperTK1 clone 8 was chosen for the growth experiments because it was the fastest growing clone of this slowly growing cell line despite having only a weaker superTK1 band compared to others (like as clone 16 in lane 7) at the correct length of approx. 665 bp on a 1.2% arose gel (see red box).
TABLE-US-00011 Loading scheme of the 1.2% agarose gel: Lane Sample Description 1 1 kb ladder (10 l) 2 MFM-223 pUHDsuperTK1 A very strong superTK1 band clone 1 was detectable 3 MFM-223 pUHDsuperTK1 A very strong superTK1 band clone 7 was detectable 4 MFM 223 pUHDsuperTK1 A very strong superTK1 band clone 5 was detectable 5 MFM-223 pUHDsuperTK1 A very faint superTK1 band clone 15 was detectable 6 MFM-223 pUHDsuperTK1 A faint superTK1 band clone 8 was detectable 7 MFM-223 pUHDsuperTK1 A very strong superTK1 band clone 16 was detectable 8 pUHDhygr_empty vector Only the hygromycin band was detectable at 149 bp 9 Plasmid pUHDsuperTK1 #117 A superTK1 band at 665 bp and a hygromycin band at 149 bp was detectable
[0637]
[0638] The histogram presented in
[0639]
[0640] The MFM-223 breast carcinoma cell proliferation data recorded during inhibition by 0.5 mM dTh were subjected a statistical analysis; Box and Whiskers plots are presented
[0641]
[0642] The bar graph presented in
[0643]
[0644] The PC-3 proliferation data recorded during inhibition by 0.5 mM dTh and pAA driven recombinant superTK1 (recombinant Adeno-associated viral vector) were subjected a statistical analysis; Box and Whiskers plots are presented
[0645]
[0646] The histogram shows the growth curve of HeLa117 clone 3 (superTk1 stable transfectant) cultivated in the presence of 0.1 mM dTh (replenished daily) and induced with 5 g/ml doxycyclin (renewed daily, black bars) or without (white bars). Each sample was measured twice and setup as triplicates.
[0647]
[0648] The Box and Whiskers Plots (
[0649]
[0650]
[0651] The Box and Whisker Plots show minimum and maximum (top and bottom of the grey boxes) No whiskers are visible due to the low amount of data in the exploratory experiment.
[0652]
[0653] The histogram shows the growth curve of HeLa117 clone 3 (superTk11 stable transfectant) cultivated in the presence of 0.1 mM dTh and 5 M 5-fluorouracil (both replenished daily), induced with 5 g/ml doxycyclin (renewed daily, black bars) or without (white bars). Each sample was set up as triplets and measured twice.
[0654]
[0655] The Box and Whiskers Plots show the minimum and maximum (vertical whiskers) as well as the data ranges from 25-75% (grey boxes). Horizontal lines indicate the median and pluses the mean value. Data which are not included in the whiskers are plotted as outliers (small squares).
[0656]
[0657] The histogram shows the growth curve of HeLa117 clone 3 (superTk1 stable transfectant) cultivated in the presence of 0.1 mM dTh and 5 M cytarabin (both replenished daily), induced with 5 g/ml doxycyclin (renewed daily, black bars) or without (white bars). Each sample was set up as triplets and measured twice.
[0658]
[0659] The Box and Whiskers Plots show the minimum and maximum (vertical whiskers) as well as the data ranges from 25-75% (grey boxes). Horizontal lines indicate the median and pluses the mean value. Data which are not included in the whiskers are plotted as outliers (small squares).
[0660]
[0661] Legend to FIG. 12ABC: The FACS analysis presented is mainly determined by 2 main values: [0662] Forward scatter (FSC): gives an idea of the size of the cell [0663] Side scatter (SSC): gives an idea of the surface of the cell
[0664]
[0665]
[0666]
[0667]
[0668]
[0669]
[0670] The bar graph shows the percentage of the control absorbance of the converted MTT reagent in PC-3 stable transfectants. The bars show the means of triplicates of the absorbance and the error bars display the standard error of the means.
[0671] The mitochondrial activity in untreated PC-3 cells in the control bar on the left defines the control absorbance of the converted dye. Its value was set at 100%. When PC3 cells received 0.5 mM dTh daily for 3 days, their ability to convert the MTT dye was reduced to 55% (second bar from left). The addition of doxycycline instead of dTh to the cell culture medium (third bar) lessened the value to 30%. The combination of both drugs (0.5 dTh+doxy) reduced the mitochondrial activity down to 20% of normal control.
[0672] The Examples illustrate the invention.
EXAMPLE 1: MATERIAL AND METHODS
[0673] Cell Lines
[0674] Cancer cell lines and cultivation conditions, received from the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrae 7B 38124 Braunschweig, Germany
TABLE-US-00012 Cell line: PC-3 DSMZ no.: ACC 465 Species: Human (homo sapiens) Cell type: Primary prostate carcinoma Origin: Established from the bone marrow metastasis isolated post-mortem from a 62-year-old Caucasian man with grade IV prostate cancer (poorly differentiated adenocarcinoma) after androgen suppression therapy; described to form tumors in nude mice, to grow in soft agar, and to be unresponsive to androgen treatment Morphology: Epithelial-like cells growing adherently in monolayers or in multilayer foci Medium: 45% Ham's F12 45% RPMI 1640 10% FCS h.i 1 Qmax 1 P/S Subculture: Split confluent culture 1:3 to 1:10 every 3-5 days using trypsin/EDTA; seed out at ca. 1 10.sup.4 cells/cm.sup.2 Incubation: At 37 C. Doubling time: Ca. 50 hours
TABLE-US-00013 Cell line: MFM-223 DSMZ no.: ACC 57 Species: Human (homo sapiens) Cell type: Primary breast carcinoma Origin: Established from the pleural effusion of a post- menopausal breast cancer patient without any prior treatment (tumor: widespread ductal mammary carcinoma T4 N2 M1 with histological grade III); cells were described to express androgen, estrogen and progesterone receptors Morphology: Adherent cells growing as monolayer with reticulate growth pattern Medium: 85% MEM (with Earle's salts) 15% FCS h.i. 1x insulin-transferrin-sodium selenite 1x Qmax 1x P/S Subculture: Cells grow very slowly, slit confluent culture 1:2 every 3-4 days using trypsin/EDTA; seed out at ca. 1 10.sup.6 cells/25 cm.sup.2 in 10 ml medium Incubation: At 37 C. Doubling Ca. 5-7 days time:
TABLE-US-00014 Cell line: HeLa DSMZ no.: ACC 422 Species: Human (homo sapiens) Cell type: Cervix carcinoma Origin: established from the epitheloid cervix carcinoma of a 31-year-old black woman in 1951; later diagnosis changed to adenocarcinoma; first aneuploid, continuously cultured human cell line Morphology: epithelial-like cells growing in monolayers Medium; 90% MEM (with Earle's salts + 10% h.i. FBS + 2 mM L-glutamine + non-essential amino acids (cells also grow well in 90-95% RPMI 1640 + 5-10% h.i. FBS) 1x Qmax 1x P/S Subculture: split confluent culture 1:4 to 1:6 every 3-5 days using trypsin/EDTA; cells reach confluence quickly, seed out at ca. 1-2 10.sup.6 cells/80 cm.sup.2 Incubation: At 37 C. Doubling ca. 48 hours time:
[0675] SuperTK1
[0676] For experiments performed in examples 2-5, human Tk mutG90A sequence (SEQ ID No. 12), here called superTk1, was used. The corresponding nucleotide sequence is shown in
[0677] Transfection and Isolation of Stable Transfectants of PC-3 and MFM-223 Cells with the Plasmid pUHD superTK1
[0678] The Effectene Transfection Reagent (Qiagen) that is a nonliposomal lipid reagent for DNA transfection into a broad range of cell types was used according to the protocol provided by the vendor. For transfection cancer cells were grown to reach 40-80% confluence, then 1.8 g plasmid DNA were added to buffer EC to reach a final volume of 270 l. 14.4 l of the Enhancer were added, the sample was vortexed for 1 second and incubated at room temperature for 5 minutes to allow complex formation. Subsequently the mixture was centrifuged briefly and 45 l of Effectene Trafo Reagent were added, the solution was mixed on a vortex shaker for 10 seconds and incubated at room temperature for another 5 minutes. Meanwhile, the cells were washed three times with PBS and a new cell culture medium was added. 1.8 ml of cell culture medium were added to the plasmid-DNA mix and mixed by pipetting up and down twice. Then the whole transfection mix was added to the cells. After 48 hours the cells were trypsinized and split 1:3 and to each plate Hygromycin B was added at a final concentration of 100 g/ml. The transfected cells were cultivated under this selection pressure for at least one month and the growth medium was renewed regularly, always containing HygromycinB, until distinct clones could be detected and isolated.
[0679] Isolating Single Clones from Stable Transfectants
[0680] The tissue culture plate was carefully washed twice with PBS, a sterilized cloning cylinder was placed around each clone with a tweezer that was sterilized by flaming, and one drop of trypsin was put onto the cells inside the cylinder. Onto the plate some drops of medium were added, so smaller clones stayed alive for harvesting them at a later timepoint. After some minutes detached cells were suspended with growth medium and recovered from the cloning cylinder. Single clones were first propagated in separate wells starting with a 96 well plate and carefully transferred to 24, 12 and finally to 6 well plates as soon as the cells reached confluence.
[0681] Isolation of Genomic DNA from Cancer Cells and Transfectants (Modified Version of Following Protocol: [73]
[0682] Cells were grown to 50-100% confluence and the cells still attached to the tissue culture plate were washed three times with PBS. Afterwards, 300 l of digestion buffer (5 mM EDTA, pH 8.0, 200 mM NaCl, 100 mMTris, pH 8.0, 0.2% sodium dodecyl sulfate (SDS)) were added to the cells before harvesting them with a sterile silicone rubber cell scraper. The lysate was transferred into a microfuge tube, 0.4 mg proteinase K per ml digestion buffer were added, and the sample was vortexed and incubated over night at 55 C.
[0683] After the proteinase K digestion 1 ml 96% ethanol was added, the sample was vortex and incubated on ice for 1 hour before centrifugation (25000 g, 10 min, 0 C.). Afterwards the ethanol supernatant was discarded, 750 l of 70% ethanol were added to rinse the pellet which was vortexed and centrifuged again (25 000 g, 10 min., 0 C.). Again, the ethanol supernatant was discarded and the cap of the microfuge tube was left open for the evaporation of remaining alcohol. Subsequently the DNA was resuspended in 100 l 1TE buffer (10: 100 mM Tris, 10 mM EDA) and incubated for approx. 30 min. at 55 C. with the cap opened to allow the ethanol to evaporate. The DNA sample was vortexed repeatedly in between. Then the DNA content was measured using the Nanodrop device at 260 nm.
[0684] Genomic PCR (Protocol: Promea Product Information: GoTaqR G2 Colorless Master Mix)
[0685] The following primers were used:
TABLE-US-00015 superTK1antisense(pUHDhTk1as): (SEQIDNO46) 5ATTAACCTGCCCACTGTGCT3 superTK1sense(pUHDhumTK1s): (SEQIDNO47) 5CTGTGGGGCAAAGAGCTTC3 hygromycinsense: (SEQIDNO49) 5GATGTAGGAGGGCGTGGATA3 hygromycinantisense: (SEQIDNO50) 5ATAGGTCAGGCTCTCGCTGA3
[0686] The following primer pair can also be used:
TABLE-US-00016 superTK1antisense(pUHDhTk1as): (SEQIDNO46) 5ATTAACCTGCCCACTGTGCT3 superTKIsense(pUHDhumTK1s): (SEQIDNO48) 5CTGTGGGCCAAAGAGCTTC3
[0687] Procedure: The GoTaqR G2 Colorless Master Mix was thawed at room temperature and kept on ice for further use. The whole reaction was prepared on ice, then homogenized and spun down before it was transferred to the PCR machine.
TABLE-US-00017 GoTaq.sup.R G2 Colorless Master Mix, 2x 25 l Primer 1: pUHDhTK1as 10 M 5 l final conc.: 1 M Primer 2: pUHDhumTK1s 10 M 5 l final conc.: 1 M Primer 3: hygromycin sense 10 M 5 l final conc.: 1 M Primer 4: hygromycin antisense 10 M 5 l final conc.: 1 M DNA template 3 g Nuclease free water to 50 l
[0688] DNA templates for the controls: Negative control: empty pUHDhygromycin vector: 1 pg positive control: Plasmid pUHDsuperTK1 #117: 1 g
[0689] PCR Protocol:
TABLE-US-00018 lid 99 C. beginning 94 C. for 120 seconds 35 cycles: 94 C. for 45 seconds 60 C. for 60 seconds 72 C. for 60 seconds lid off 4 C. per second end 8 C. forever
[0690] Realization of the Growth Experiments:
[0691] a) Stable Transfected Clones with pUDHsuperTk1 Treated with Different Amounts of dTh
[0692] On day zero 510.sup.4 cells per well of a 6 well plate were plated in 4 ml medium containing hygromycin (0.1 mg/ml): to the upper three wells dTh and doxycycline were added, the lower wells served as controls, only dTh was added. Depending on the number of duplicates and on the incubation time, 5 up to 14 independent 6 well plates were needed. From day one on, the cells were counted at different time points (days) using the Casy Cell Counter (OLS). Triplicates or up to sextuplicates were counted, each measurement was done twice. For a longer time periods independent six well plates had to be set up to generate the necessary cell number of later timepoints, up to eleven days. dTh and doxycycline were added freshly each day at the appropriate concentration and the medium was completely renewed after 72 hours in order to avoid starvation effects. Depending on the growth rate of the cells, the cell number measurements were done daily or every second day. Experiments: [0693] PC-3 clone 2: 0.5 mM dThsextuplicates+/doxycycline (5 g/ml)days 1 to 7, measured daily [0694] PC-3 clone 2: 0.1 mM dThtriplicates+/doxycycline (5 g/ml)days 1 to 4, measured daily [0695] MFM-223 clone 8: 0.5 mM dThsextuplicates+/doxycycline (5 g/ml) days 1 to 11, measured every second day
[0696] b) Human Carcinoma Cells Infected with superTK1 Recombinant Adeno-Associated Viral Vectors
[0697] The recombinant Adeno-associated Virus (AAV) expression is a very attractive alternative to the Adenovirus based one because it causes lesser immunological problems, especially after repeated administration in test animals. The helper virus free recombinant adeno-associated virus expression systems were developed to serve as important tools for gene delivery [61]. We used the AAV Helper-Free System distributed by Agilent Technologies [62], which is also the expression system of choice for animal experiments. The components are: [0698] pAAV-MCS vector [0699] pAAV-LacZ vector [0700] pAAV-RC plasmid [0701] pMCS-MCS-vector [0702] pHelper plasmid
[0703] The cloning strategy, propagation, and the use of the AAV-system was very straight forward, taking advantage of the BamHI restriction site in the pAAV-MCS vector. By a triple transfection together with the pAAV-RC and pHelper plasmids into HEK-293 cells, high titer AAV virussuperTK1 particles could be produced and isolated by centrifugation. The big advantage of the AAV system is, in addition to causing fewer immunological problems, that all target cells can be infected independent of their state of growth (G0, G1, S, G2 or M-phase). Establishment of the pAAVsuperTK1 carrying constructs in detail:
[0704] On day zero 510.sup.4 cells were plated in 2 ml medium per well of a 6 well plate and incubated over night at 37 C. The available viral supernatant was mixed with a 2% heat inactivated FCS containing medium and added to the cells, which were then incubated for 2 hours at 37 C., swirled gently several times in between. To the controls only 1 ml of the medium containing 2% heat inactivated FCS was added (without virus). After the incubation time, 1 ml of medium containing 18% heat inactivated FCS and the desired amount of dTh was added to each well, then the cells were put back to the incubator. Depending on the number of repetitions and on the incubation time, 5 up to 14 independent 6 well plates were set up. From day one on, the cells were counted at different time points (days) using the Casy Cell Counter (OLS). Triplicates or even sextuplicates were counted, each measurement was done twice. The remaining six well plates were incubated for a longer time period to receive the cell number of later timepoints, up to eleven days. dTh was added each day to all wells at the appropriate concentration and the medium was changed after 72 hours. Depending on the growth rate of the cells, the cell number measurements were done daily or every second day. Experiments: [0705] PC-3+virus: 0.5 mM dThsextuplicates+/Virusdays 1 to 7, measured daily (
[0706] Procedure:
[0707] The cells of a well were trypsinized and resuspended in 1 ml medium, 50 l were taken and diluted in 5 ml of Casy solution and the cells were counted using the Casy device. The remaining cells were put to special tubes for the FACS measurements.
[0708] Isolation and Purification of Recombinant AAV Particles:
[0709] AAV recombinant particles were purified by a CsCl purification as described with minor modifications [74].
[0710] In general recombinant Adeno-associated viral vectors are the system of choice, because they can be easily purified, used in vitro and in vivo, and they do not cause any immunological problems a priori. The CsCl purification protocol has more than one purification step, it contains the lysis of the cells, precipitation of DNA and proteins, ultracentrifugation in a CsCl gradient, followed by dialysis and finally a filtration step is done. In comparison, iodixanol purified AAV vectors, are purer, but CsCl purified vectors contain less empty particles (not even 1%) and are therefore more bioactive.
[0711] Growing Adeno Associated Viruses in an Adherent Cell Line: AAV293 [0712] AAV293 cells were grown in Dulbecco's modified Eagle's medium, supplemented with 10% FCS, Qmax and P/S (100 U/mL penicillin and 100 g/mL streptomycin) [0713] Ten 15 cm tissue culture plates were prepared 3 days before transfection: 210.sup.6 cells were seeded per plate [0714] On the day of transfection cells were 70 to 80% confluent [0715] 25,384 g of each plasmid (pHelper, pAAV-RC, recombinant pAAV expression plasmiddissolved in sterile ddH.sub.2O) were added to 2 ml of 300 mM CaCl2 [0716] The DNA-CaCl2 mixture was added dropwise to 2 ml 2HBS buffer (2 Hepes-buffered saline, 50 mM HEPES, 280 mM NaCl, 1.5 mM Na.sub.2HPO.sub.4) [0717] The mixture was incubated for approx. 2 min at room temperature and then added dropwise to the tissue culture plate while it was swirled gently [0718] After 5 to 6 hours the medium was replaced with a fresh one [0719] The AAV293 cells were then grown for 72 hours at 37 C.
[0720] Harvesting of the Adeno Associated Viruses from the Cells: [0721] For harvesting the cells, EDTA was diluted in PBS to get a final concentration of 6.25 mM [0722] The medium of the cells was collected in conical tubes, the culture plates were then washed two times with PBS, afterwards the 6.25 mM EDTA were added to the plates which were then left for approx. 2 min at room temperature. Then, the cells were harvested and put to the conical tubes as well. [0723] The collected cells were centrifuged for 10 min at 1000 g, room temperature. [0724] The supernatant was discarded and the pellets were resuspended in lysis buffer (50 mM Tris, 150 mM NaCl, 2 mM MgCl.sub.2, pH 8.5). For all 10 plates together, 30 ml of lysis buffer were used. [0725] The cells were lysed by three freeze-thaw cycles using a dried iceethanol bath and a 37 C. water bath. [0726] For the 10 initially transfected plates, 1000 units of Benzonase were added, the sample was incubated for one hour at 37 C. [0727] Centrifugation 15 min., 2500 g, room temperature: the pellet was discarded and the supernatant was stored at 80 C. for further purification.
[0728] CsCl Purification: [0729] 300 l of 2.5M CaCl.sub.2 were added to get a final concentration of 25 mM for pelleting residual DNA [0730] the sample was incubated on ice for one hour [0731] centrifugation: 2500 g, 15 min, 4 C. [0732] The supernatant was transferred to a new tube and the pellet was discarded [0733] 40% PEG-8000 was added to get a final concentration of 8% for precipitating proteins including AAVs [0734] incubation on ice for 3 hours [0735] centrifugation 2500 g, 30 min, 4 C. [0736] the supernatant was discarded and the pellet was resuspended in 30 ml resuspension buffer (50 mM HEPES, 150 mM NaCl, 25 mM EDTA, pH 7.4) over night on a tube rotator at 4 C. [0737] if the pellet was not dissolved completely on the next day, a 5 ml Gilson pipette was used for pipetting up and down to resuspend the pellet, then it was left for one more hour on a tube rotator at 4 C.. [0738] the sample was centrifuged for 30 min at 2500 g at 4 C. [0739] CsCl was added to the supernatant to reach a 3,149M solution with an RI of 1,3710 (15.8g were added to the viral supernatant) [0740] Ultracentrifuge tubes were filled, weighed and closed and an ultracentrifugation was started: 23 h, 63000 rpm, 21 C. (90Ti rotor) [0741] 1 ml fractions were taken from the top of the gradient and the RI was measured [0742] the fractions with an RI between 1,3703 and 1,3758 were collected and dialysed over night against 1PBS, the buffer was changed altogether 4 times [0743] (the viral supernatants were concentrated using Amicon Ultra 15 centrifugal filter units) [0744] 60% glycerol was added to get a final concentration of 10% [0745] the samples were sterile filtered by using Ultra free-CL filter tubes [0746] samples were quick-frozen and stored at 80 C. for further usage
[0747] DMEM growth medium: DMEM (4.5 g/L glucose, 110 mg/L sodium pyruvate, 2 mM L-glutamine) 10% (v/v) fetal bovine serum 2 mM L-glutamine
[0748]
[0749] Construction of the Vector pAAV-supTK1-IRES-hrGFP
[0750] Digestion of the Empty Vector a AV-IRES-hrGFP with BamH: Reaction Mixture:
TABLE-US-00019 H.sub.2O 5 l NEB 3.1 buffer (conc. 10x) from Biotabs 3 l Vector DNA (pAAV-IRES-hrGFP, probe T) 20 l for 5 g Enzyme BamHI (20 U/l) 2 l (=max!) Total amount: 30 l
[0751] This reaction was left in the thermo block at 37 C. for one hour.
[0752] Treating the Vector AAV-IRES-hrGFP with Alkaline Phosphatase:
[0753] Reaction Mixture:
TABLE-US-00020 Digested vector pAAV-IRES-hrGFP probe T mixture from All above 30 l Antartic reaction buffer 10x 5 l ddH.sub.2O 14 l Antarctic phosphatase (5 U/l) 1 l Total amount 50 l
[0754] The microfuge tube was mixed by slightly snipping against its bottom with one finger, then the sample was left for 15 min at 37 C. in the thermo mixer. Afterwards the enzyme was inactivated by incubation for 5 min. at 70 C. before it was frozen at 20 C.
[0755] Digesting the Insert supTK1 from the Vector pAAV supTK1 with BamHI: [0756] DNA plasmid pAAV without IRES, containing the wanted insert supTK1, clone number 3: [0757] clone 3 with the correct sequence and correct orientation of the gene was picked: vector length approx. 6 kb, the insert length was about 700 nucleotides [proportion of about one to eight] [0758] for digestion 0.5 to 1 g insert would have been preferred 8 g of plasmid were needed to get the wanted amount of insert digestion reaction: The reaction mixture was put to the thermo block to 37 C. for one hour.
[0759] Checking Vector and Insert on an Agarose Gel:
[0760] Vector and insert were loaded onto a 0.9% Agarose gel to check their length and to purify them:
TABLE-US-00021 Slot sample 1 1 kb DNA ladder 2 Vector pAAV-IRES-hrGFP, sample T, not digested 3 + 4 Vector pAAV-IRES-hrGFP BamHI digested and phosphatase treated 5 Free 6 pAAV-supTK1 without digestion 7 pAAV-supTK1 BamHI digested 8 pGSX TK1 90GA: old probe which was eluted 9 pAAV-IRES-OLD: old probe from a vector prep 10 100 bp ladder
[0761] Purification of the Vector and Insert from the Gel:
[0762] The BamHI cut vector pAAV-IRES-hrGFP and the BamHI cut insert supTK1 were purified from the gel lanes 3, 4 and 7.
[0763] Ligation of the Vector and the Insert:
[0764] The reaction mixture should be as concentrated as possible, 15-20 l of volume should be used
TABLE-US-00022 pAAV-IRES-hrGFP cut with BamHI, phosphatase 5 l (0.5 g) treated Insert supTK1, cut with BamHi 12 l 10x T4 DNA ligase buffer 2 l T4 DNA ligase (400 U/l) 1 l Total volume 20 l
[0765] Also, a negative control was done: 12 l ddH.sub.2O instead of the insert were used, the other components stayed exactly the same
[0766] The reaction mixtures where put to the 4 C. cold room into a water bath which had 16 C. overnight.
[0767] Transformation of the New AAV-supTK1-IRES-hrGFP into Competent E. coli Cells and Checking the Direction of the Insert on an Agarose Gel:
[0768] The vector map showing the superTK1 in addition to the hrGFP reporter gene is presented in
[0769] In order to determine the proper insertion of the superTK1 into the pAAV-IRES-hrGFP a restriction digest was performed with EcoRI and StuI. Both enzymes cut only once in the whole new plasmid. EcoRI has a recognition site on the vector backbone close to the superTK1 insert and StuI at the 3end of it. Dependent on the orientation of the inserted gene, the products of the digestion with both enzymes generated different sizes. The correct orientation of the insert leads to one really large and one really small fragment, the wrong orientation of the insert leads to a smaller big band and to a bigger small band.
TABLE-US-00023 Plasmid DNA 1 g CutSmart 10x buffer 2 l EcoR1-HF (20 U/l) 1 l StuI (10 U/l) 1 l ddH.sub.2O up to 20 l
[0770] The digested inserts were analyzed on a 1% agarose gel.
[0771] Sequence Analysis: [0772] Sequencing of pAAV_supTK1_IRES_hrGFP, where the gene was inserted in the correct direction: clones 3 and 4 were sent out to Microsynth: twice, 1.2 g DNA were diluted to get a final volume of 15 l 2 primersone forward and one reversedwere used for sequence analysis. The sequences were checked using the software MegAlign (DNASTAR-Lasergene): both sequences were correct without any point mutations!
[0773] Transfections of AAV-293 Cells with Different Plasmids for Generating Viruses [75]
[0774] 3 plasmids were used per transfection: The pHelper and the pAAV-RC plasmid were the same all the time, the third plasmid contained the gene of interest in a recombinant vector containing ITR regions and/or GFP as a reporter gene, in addition to superTK1, and, as a control, LacZ alone. These plasmids were used for independent transfections: the recombinant constructs pAAVsuperTK1, pAAV-supTK1-IRES-hrGFP and the plasmid pAAV-LacZ. The pHelper plasmid contains most of the adenovirus genes like E2A, E4 and VA RNA genes, which were needed for producing infective AAV particles (for more details of the recombinant pAAV expression system see [62]).
[0775]
[0776]
[0777]
EXAMPLE 2: CELL GROWTH AND INHIBITION EXPERIMENTS IN STABLY TRANSFECTED IN HUMAN PRIMARY PROSTATE CARCINOMA (PC-3) AND MAMMARY CARCINOMA (MFM-223) CELLS
[0778] In the case of the stable transfectants, superTk1 (Tk1 mutG90A) carried on a plasmid pUDHHyg10.3 was transfected into human prostate PC-3 or human ductal mammary carcinoma cells (MFM-223). Clones of stable transfectants were selected and analyzed for full length superTk1 DNA by specific PCR. Subsequently, growth curves under inhibitory conditions were performed to analyze the cytostatic exertion of superTk1 expression combined with various, very low levels of dTh (0.1 mM) in the growth medium.
[0779] First, superTk1 cDNA was integrated into the pUHDhygr expression vector, transfected it into PC-3 cells and selected for stable transfectant clones that present a strong band at 665 nt in a genomic PCR. The proper insert was verified by sequence analysis to make sure that no unwanted point mutation got was introduced during the subcloning procedure (see
[0780] Human Primary Prostate Carcinoma (PC-3) Treated with dTh
[0781]
[0782] The Kruskal-Wallis test determines a p-value, which is less than 0.05 from day 2 on; this means that there is a statistically significant difference amongst the medians of the treated (superTk1 expressing sample) sample and its control at the 95.0% confidence level. In this test the method used to discriminate among the means was Fisher's least significant difference (LSD) procedure (cf.
TABLE-US-00024 day1 day2 day3 day4 day5 day6 day7 p value (KrusKal 0.11847 0.01932 0.00003 0.00003 0.00003 0.00003 0.00003 Wallis, comparison of the medians)
[0783]
[0784] The administration of both drugs simultaneously, which induced the expression of superTK and supplied the substrate, had the strongest effect onto the cell cycle distribution of the cells. No cells were detectable in the G2/M phase, approx. one fourth of the cells was present in the G1 phase. Over 75% of the cells were measured while undergoing the S phase. Due to the addition of dTh and doxycycline in combination, the cells stopped dividing, they rested in the S phase and did not proliferate anymore.
[0785]
[0786] The growth curves of PC-3 cells treated with 0.1 mM dTh showed a very similar growth inhibition as seen with 0.5 mM dTh presented in
TABLE-US-00025 day1 day2 day3 day4 p value (Kruskall Wallis, 0.12753 0.07764 0.00648 0.00395 comparison of the medians)
[0787] Human Mammary Carcinoma (MFM223) Cells Treated with dTh
[0788] The MFM-223 pUHDsuperTK1 clone 8 was chosen for the growth experiments because it was the fastest growing clone of this slowly growing cell line despite having only a weaker superTK1 band compared to others (such as clone 16 in lane 7) at the correct length of approx. 665 bp on a 1.2% Agarose gel (see black box); see
[0789]
[0790] Comparable to the PC-3 prostate carcinoma cells in
TABLE-US-00026 day1 day3 day5 day7 day9 day11 p value 0.00556 0.00009 0.00003 0.00003 0.00003 0.00003 (Kruskall Wallis, comparison of the medians)
[0791]
[0792] After the infection with the rAAVs the cell proliferation was slowed down during the treatment period of 5 days. Similar to the transfected and genomically integrated superTK1 in
[0793] The Kruskal-Wallis test determines a p-value, which is less than 0.05 from day 1 on; this means that there is a statistically significant difference amongst the medians of the treated sample and its control at the 95.0% confidence level; cf.
TABLE-US-00027 day1 day2 day3 day4 day5 p value (Kruskall 0.01631 0.00648 0.00395 0.00388 0.00388 Wallis, comparison of the medians)
EXAMPLE 3: HUMAN HELA CELLS (CERVIX CARCINOMA) TREATED WITH DTH AND WITH ARAC OR 5-FU IN COMBINATION
[0794] One of the major aims was to find out the lowest dTh concentration still able to cause a growth halt in tumor cells stable transfected with superTK1. For this task this gene was integrated in pUHDhygr and stable transfected into HeLa cervix carcinoma cells. One of the clones showing the highest amount of superTk1 cDNA was clone 117[76] These transfected HeLa cells were grown under permanent hygromycin and puromycin selection pressure for 4 to 7 days, starting with 50.000 cells/well (12.500 cells/ml). In order to establish a perfect growth curve cell counts on a daily basis with a Casy cell counting system were mandatory. Tumor cell cultures expressing the super TK1 and cultivated under 0.1 mM dTh and doxycyclin induction still showed a clear growth repression.
[0795] That means even 1/50 of standard dTh concentration was sufficient for the superTk1 to cause a S-phase block in HeLa cells (
[0796] The Kruskal-Wallis test determines a p-value which is less than 0.05 except for days 1 and 2; this means that there is a statistically significant difference amongst the medians of the treated samples versus its controls at the 95.0% confidence level.; cf.
[0797] In addition to growth inhibition with dTh alone we wanted to explore the synergistic effects on tumor cell growth in HeLa clone 117 superTk1 transfectants when one of the nucleoside analogues, cytarabine and 5-fluorouracil in very low concentrations was applied. At first, concentration series of 5, 10, 20 and 50 M as well as 1, 2, 5 and 5 M AraC and 5-FU, respectively, were set up in order to investigate the lowest concentration still able to cause cell growth arrest when combined with dTh treatment. In both cases, the extremely low concentrations of 5 M, seemed to be the most sufficient ones for AraC or 5-FU (see
[0798] In this case now it is of interest to extrapolate the cell culture conditions with serum levels currently given during a clinical treatment. For the case of 5-FU, a regular therapeutic cytostatic dosis in cancer treatment is 400 mg/m2/day or a serum level, which corresponds to 55.44 g/ml c.sub.max in the serum (according to: [77]). Our working concentration 5 M results in a calculated serum concentration of 0.65 g/ml.
[0799] This means that there still is a statistically significant inhibition with a 1/85 of the amount of 5-FU used in the literature. For AraC the values are comparable. (see
[0800] The biggest impact appeared to be evident on day 4, where the cell number of the doxycyclin induced superTk1 affected cells was reduced almost to half of the amount of the control without doxycyclin induction in both the AraC and 5-FU triplets. In this experiment, cytarabin appeared to be the drug with the higher cell growth arrest potential. In a direct comparison of the control groups without doxycycline treatment, the relative cell proliferation on day 4 reached only 2.4 times the number of day 1 in the cytarabin group. Whereas in the case of the 5-FU-group, a 3.3-fold higher cell number was reached on day 4 compared to day 1. Another concentration series was set up, with 5, 2.5 and 1 M AraC or 5-FU, respectively. The concentrations lower than 5 m of the chemotherapeutics obviously were too low to exert any inhibitory effects.
[0801] One Way Anova results were acquired with Stat Graphics: Since the experiment was thought of as an exploratory one, each sample was set up only once (one 6-well plate) and measured twice-thus it was not possible to determine meaningful p-values. The Multiple Range Test identifies 2 different groups and shows a statistically significant difference level from day 3-4 (5 M 5-FU), day 2 (10 M 5-FU), none (20 M 5-FU) and day 4 (50 M 5-FU). In this test the method used to determinate among the means was Fisher's least significant difference (LSD) procedure; see
[0802] Since the experiment was planned as an exploratory one, each sample was set up only once (one 6-well plate) and measured twicethus it was not possible to determine meaningful p-values. The Multiple Range Test identifies 2 different groups and shows a statistically significant difference level on day 4 (5 M AraC), day 1-2 (10 M AraC), day 2 (20 M AraC) and none (50 M AraC). In this test the method used to determinate among the means was Fisher's least significant difference (LSD) procedure; (data not shown)
[0803]
[0804] The Kruskal-Wallis test determines a p-value which is less than 0.05 on days 3 and 4; this means that there is a statistically significant difference amongst the medians of the treated samples versus its controls at the 95.0% confidence level; cf.
EXAMPLE 4: CALCULATION OF HELICITY DETERMINATION OF ACTIVITY OF WILD-TYPE AND MUTANT HUMAN THYMIDIN KINASES
[0805] The helicity calculations of the protein domain aa 71-95 of the wt human TK1 and the mutations were done using GOR IV. [78] Results are presented in
[0806] As a result of this procedure the mutant G90A is characterized by a 1.16 fold increase of helicity in relation to the wtTK1 (
[0807] Other methods for secondary structure prediction can be used, e.g.: SOPM [79], SOPMA [80], HNN [81], MLRC [81], DPM [82], DSC [83], GOR I [84], GOR III [85], PHD [86], PREDATOR [87], or SIMPA96 [88].
[0808] All GOR IV helicity calculations were performed by Network Protein Sequence Analysis, provided from the PBIL-IBCP Institute of Biology and Protein Chemistry 7, passage of Vercors 69367 Lyon Cedex 07, FRANCE TIBS [41] https://npsa-prabi.ibcp.fr/cgibin/npsa_automat.pl?page=/NPSA/npsa_seccons.html
[0809]
[0810] Calculation of TK Activity
[0811] For the determination of a specific activity a quantification of the protein solution was done first, according the method of Bradford. [43] The dye stock solution (Biorad) containing methanol and acetic acid was diluted 1:4 with ddH2O. An aliquot of the protein solution was then filled up to 1 ml with the diluted Bradford solution. (Usually 5l of protein solution are diluted in 995 ml Bradford solution. The protein concentration is calculated by multiplying the absorbance at =595 nm with 17 and dividing this by 5, resulting in the concentration in g/l.) Depending on the concentration of the kinases 5-20 l of the sample solution were used.
[0812] The following components were mixed for the enzyme assay:
[0813] 10 fold mix: [0814] 0.5 M Tris-HCl, pH 7.5 [0815] 0.1 M DTT [0816] 25 mM ATP [0817] 25 mM MgCl.sub.2
[0818] Set up for the activity test: 5.0 l 10-fold mix [0819] 2.5 l BSA (60 mg/ml) [0820] 1.0 l Chaps (25 mM) [0821] 1.0 l NaF (0.15 M) [0822] 1.0 l .sup.3H-dTh or 3H-dCyd (500 M, 2 Ci/mmol) [0823] 5.0-20 l of the protein solution [0824] Filled up with ddH.sub.2O to 50 l
[0825] The set-up was incubated at 37 C. At distinct times 10 l aliquots were removed from the reaction mix and pipetted onto small pieces of DE81-filters. After removal of every aliquot, the filter papers were washed in a big volume of ammonium formiat (5 mM for TK assay), transferred to water and rinsed, and finally dried after shortly immersing them in ethanol. Then, after the dried filters were transferred into the scintillation tubes, the bound radioactive nucleotides were eluted by addition of 500 l elution buffer (0.1 M HCl, 0.2 M KCl). 2.5 ml of scintillation solution were added before measuring the radioactivity. A standard was created in order to convert the cpm-values into pmol: 5 l from some of the reactions were removed and pipetted on DE81-filters, which were not washed, but directly transferred to the scintillation tubes, treated like described above, and measured. 5 l of the standard contained 50 pMol of the substrate or the product. From the cpm-values of these samples one got the converting factor UF (is equal to the specific activity of the used substrate): UF=cpm standard/50 pMol
[0826] For the calculation of the whole enzymatic activity of one fraction (U) the following formula was used:
[0827] 10 l were pipetted at each time point onto the filter, and 20 l of the protein solution was initially used. cpm=(cpmt2cpm t1)/t2t1 (cpm/min); t1=start of the reaction; t2=end of the reaction, e.g. 5 min; Vol=total volume of the fraction (l); UF is the specific activity (see above) (cpm/pMol); U is the whole activity of the enzyme per fraction (pMol/minl=Mol/minml).
TABLE-US-00028 TABLE 2 Correlation of the calculated protein secondary structure parameter (degree of helicity) with the measured specific enzyme activity of human cytosolic Tk1 mutants with elevated and one with decreased specific activity humTK1- Helicity measured specific Tk1 Relative in-/decrease variant (AUC) enzyme activity of helicity to wt Tk1 wt 332924 37 pmol/mg min 1.00 L8081F 255025 1.4 pmol/mg min 0.77 G90A 385426 375 pmol/mg min* 1.16 V84DG90A 366809 393 pmol/mg min 1.10 *data retrieved from a different experiment
[0828] Legend to table 2: Helicity scores given in the table were calculated by NPS@: Network Protein Sequence Analysis [42]; the AUC was obtained by integration of the area under the helicity curve between aa 71-95 of the TK1 variants and equals the degree of helicity of the protein domain
EXAMPLE 5: CELL VIABILITYCYTOTOXICITY MTT ASSAY OF STABLE PUHDSUPERTK1 TRANSFECTANTS OF PC-3 CELLS TREATED +/DTH, +/DOXY
[0829] The MTT assay is a colorimetric assay for assessing cell metabolic activity; see J Immunol Methods. 1983 Dec. 16; 65(1-2):55-63; Sigma Aldrich. (2016). MTT Cell Viability Applications. The control shows the mitochondrial activity in untreated PC-3 cells with endogenous wild-type TK1 only, without addition of dTh or induction of superTK1 (
EXAMPLE 6: VALIDATION IN ANIMAL MODELS
[0830] For this experiment human Tk1 mutG90A sequence (SEQ ID No. 12), here called superTk1, is used. The corresponding nucleotide sequence is shown in
[0831] Experimental Approach 1
[0832] SuperTK1 carrying glioblastoma cells (310.sup.6/50 l; stable transfected) are subcutaneously implanted as xenografts in SCID mice. Due to the fact that all cells carry the superTK1 integrated into the genomic DNA, every single tumor cell is targeted by the induction of the recombinant superTK1 by doxycyclin and thus harmed by gene therapy. After 2 weeks of tumor growth in the breast region of the animal, the therapy is started by adding doxycyclin to the drinking water. In parallel, osmotic mini pumps are implanted in the neck of the SCID mice, prefilled with the necessary amount of dTh and, for some test animals with the cytostatics AraC or 5-FU as well, if the test animal is to be treated by a combination therapy.
[0833] After 3 weeks of therapy regimen, the successful treatment is analyzed by physical, histological, and molecular analyses. In the same manner, untransfected and previously untreated glioblastoma cells are implanted and function as controls.
[0834] Number of test animals for this milestone: [0835] 1) 3 mice for the glioblastoma cell line without any treatment (control) [0836] 2) pUDHsuperTK1 transfected glioblastoma cells treated with 0.1 mM dTh alone (3 mice) [0837] 3) pUDHsuperTK1 transfected glioblastoma cells treated with 0.1 mM dTh plus 0.5 mM AraC (3 mice) [0838] 4) pUDHsuperTK1 transfected glioblastoma cells treated with 0.1 mM dTh plus 0.5 mM 5-FU (3 mice) [0839] 5) pUDH-0 (empty vector control) transfected glioblastoma cells treated with 0.1 mM dTh alone (3 mice)
[0840] In total 15 test animals are needed for this experimental approach
[0841] Experimental Approach 2:
[0842] Based on the results of the first experiment, in this experiment previously untreated glioblastoma cells are implanted in SCID mice (310.sup.6/100 l). After development of about 2 weeks, the solid tumor is analyzed by MRT to get a most precise 3D-information about the size and structure of the tumor. Usually, the solid tumor has grown to a size of 6-10 mm. The infection with superTK1-AAV recombinant particles is exactly planned and based on the stereoscopic data. The infection is accomplished by infiltration of the solid tumor at several recombinant virus infusion areas. The necessary infiltration spots as well as the total amounts in MOI/l are optimized by pretests with 8-10 SCID mice. For this purpose, rAAVsuperTK1-GFP constructs are used that already have been generated and characterized by FACS analyses. They allow the visual tracing of successful infection by green fluorescence protein expression. The aim is to generate highly concentrated recombinant AAVsuperTK1 particles with a high MOI in order to be able to reduce the necessary liquid amounts to a minimum and avoid generating bulbs. On the other hand, it is essential to infect not only the tumor cells but also the stroma. The artificial expression is induced up to 3 weeks/therapy cycle. The tumor cell survival rate is evaluated by physical, cell- and molecular biological analyses. The histo-immuno-cytological examinations are performed in a similar way as described for the first approach in the histolab of MFPL.
[0843] Necessary test animals for the second milestone: [0844] 1) 10 mice for the pretests to optimize the rAAVsuperTK1 infection/infiltration process 2) the 3 untreated control mice have already been analyzed in approach 1 [0845] 3) pAAVsuperK1 infected glioblastoma cells treated with 0.1 mM dTh treatment (3 mice) [0846] 4) pAAVsuperK1 infected glioblastoma cells treated with 0.1 mM dTh alone (3 mice) [0847] 5) pAAVsuperK1 infected glioblastoma cells treated with 0.1 mM dTh plus 0.5 mM AraC (3 mice) [0848] 6) pAAVsuperK1 infected glioblastoma cells treated with 0.1 mMdTh plus 0.5 mM 5-FU (3 mice) In total 22 test animals are needed for this experimental approach.
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[0937] All references cited herein are fully incorporated by reference. Having now fully described the invention, it will be understood by a person skilled in the art that the invention may be practiced within a wide and equivalent range of conditions, parameters and the like, without affecting the spirit or scope of the invention or any embodiment thereof.
[0938] The present invention refers to the following nucleotide and amino acid sequences: The wild-type sequences provided herein are available in the NCBI database and can be retrieved from www.ncbi.nlm.nih.gov/sites/entrez?db=gene; Theses sequences also relate to annotated and modified sequences. The present invention also provides techniques and methods wherein homologous sequences, and variants of the concise sequences provided herein are used.
[0939] Preferably, such variants are genetic variants.
TABLE-US-00029 SEQIDNo.1: HumanTK1mutM73AA75DD83AG9095Antsequence atgagctgcattaacctgcccactgtgctg 241cccggctcccccagcaagacccgggggcagatccaggtgattctcgggccgatgttctca 301ggaaaaagcacagagttgatgagacgcgtccgtcgcttccagattgctcagtacaagtgc 361ctggtgatcaagtatgccaaagacactcgctacagcagcagcttctgcacacatgaccgg 421aacaccgccgaggacctgcccgcctgcctgctccgagccgtggcccaggaggccctggct 481gtggctgtcatagctatcgacgaggggcagtttttccctgacatcgtggagttctgcgag 541gccatggccaacgccgggaagaccgtaattgtggctgcactggatgggaccttccagagg 601aagccatttggggccatcctgaacctggtgccgctggccgagagcgtggtaaagctgacg 661gcggtgtgcatggagtgcttccgggaagccgcctataccaagaggctcggcacagagaag 721gaggtcgaggtgattgggggagcagacaagtaccactccgtgtgtcggctctgctacttc 781aagaaggcctcaggacagcctgccgggccggacaacaaagagaactgcccagtgccagga 841aagccaggggaagccgtggctgccaggaagctctttgccccacagcagattctgcaatgg 901atccagacatga SEQIDNo.2: HumanTK1mutM73AA75DD83AG9095Aaasequence 1MSCINLPIVLPGSPSKTRGQIQVILGPMFSGKSTELMRRVRRFQIAQYKC 51LVIKYAKDTRYSSSFCTKDRNTAEDLPACLLRAVAQEALAVAVIAIDEGQ 101FFPDIVEFCEAMANAGKIVIVAALDGIFQRKPFGAILNLVPLAESVVKLT 151AVCMECFREAAYTKRLGTEKEVEVIGGADKYKSVCRLCYFKKASGUAGP 201DNKENCPVPGRPGEAVAARKLEAPQQILQWIQT SEQIDNo.3: HumanTK1mutM73AA75DD83AG90Antsequence atgagctgcattaacctgaccactgtgctg 241cccggctcccccagcaagacccgggggcagatccaggtgattctcgggccgatgttctca 301ggaaaaagcacagagttgatgagacgcgtccgtcgcttccagattgctcagtacaagtgc 361ctggtgatcaagtatgccaaagacactcgctacagcagcagcttctgcacacatgaccgg 421aacaccgccgaggacctgcccgcctgcctgctccgagccgtggcccaggaggccctggct 481gtggctgtcataggcatcgacgaggggcagtttttccctgacatcgtggagttctgcgag 541gccatggccaacgccgggaagaccgtaattgtggctgcactggatgggaccttccagagg 601aagccatttggggccatcctgaacctggtgccgctggccgagagcgtggtgaagctgacg 661gcggtgtgcatggagtgcttccgggaagccgcctataccaagaggctccgcacagagaag 721gaggtcgaggtgattgggggagcagacaagtaccactccgtgtgtcggctctgctacttc 781aagaaggcctcaggccagcctgccgggccggacaacaaagagaactgcccagtgccagga 841aagccaggggaagccgtggctgccaggaagctctttgccccacagcagattctgcaatgg 901atccagacatga SEQIDNo.4: HumanTK1mutM73AA75DD83AG90Aaasequence 1MSCINLPTVLPGSPSKTRGQIQVILGPMFSGKSTELMRRVRRFQIAQYKC 51LVIKYAKDTRYSSSECTHDRNTAEDLPACLLRAVAQEALAVAVIGIDEGQ 101FFPDIVEFCEAMANAGKTVIVAALDGTFQRKPFGAILNLVPLAESVVKLI 151AVCMECFREAAYTKRLGTEKEVEVIGGADKYHSVCRLCYFKKASGQPAGP 201DNKENCPVPGRPGEAVAARKLFAPGQILQWIQT SEQIDNo.5: HumanTK1mutV84EG90Antsequence atgagctgcattaacctgcccactgtgctg 241cccggctcccccagcaagacccgggggcagatccaggtgattctcgggccgatgttctca 301ggaaaaagcacagagttgatgagacgcgtccgtcgcttccagattgctcagtacaagtgc 361ctggtgatcaagtatgccaaagacactcgctacagcagcagcttctgcacacatgaccgg 421aacaccatggaggcgctgcccgcctgcctgctccgagacgaggcccaggaggccctggct 481gtggctgtcataggcatcgacgaggggcagtttttccctgacatcgtggagttctgcgag 541gccatggccaacgccgggaagaccgtaattgtggctgcactggatgggaccttccagagg 601aagccatttggggccatcctgaacctggtgccgctggccgagagcgtggtgaagctgacg 661gcggtgtgcatggagtgcttccgggaagccgcctataccaagaggctcggcacagagaag 721gaggtcgaggtgattgggggagcagacaagtaccactccgtgtgtcggctctgctacttc 781aagaaggcctcaggccagcctgccgggccggacaacaaagagaactgcccagtgccagga 841aagccaggggaagccgtggctgccaggaagctctttgccccacagcagattctgcaatgg 901atccagacatga SEQIDNo.6: HumanTK1mutV84EG90Aaasequence 1MSCINLPTVLPGSPSKTRGQIQVILCPMFSGKSTELMRRVRRFQIAQYKC 51LVIKYAKDTRYSSSFCTEDRNTMEALPACLLRDEAQEALAVAVIGIDEGQ 101FFPDIVEFCEAMANAGKTVIVAALDGTFQRKPFGAILNLVPLAESVVKLT 151AVCMECFREAAYTKKLGTEKEVEVIGGADKYHSVCRLCYFKKASGQPAGP 201DNKENCPVPGRPGEAVAARKLFAPQQILQWIQT SEDIDNo.7: HumanTK1mutMT73AA75DG9095Antsequence atgagctgcattaacctgcccactgtgctg 241cccggctcccccagcaagacccgggggcagatccaggtgattctcgggccgatgttctca 301ggaaaaagcacagagttgatgagacgcgtccgtcgcttccagattgctcagtacaagtgc 361ctggtgatcaagtatgccaaagacactcgctacagcagcagcttctgcacacatgaccgg 421aacaccgccgaggacctgcccgcctgcctgctccgagacgtggcccaggaggccctggct 481gtggctgtcatagctatcgacgaggggcagtttttccctgacatcgtggagttctgagag 541gccatggccaacgccgggaagaccgtaattgtggatgcactggatgggaccttccagagg 601aagccatttggggccatcctgaacctggtgccgctggccgagagcgtggtgaagctgacg 661gcggtgtgcatggagtgcttccgggaagccgcctataccaagaggctcggcacagagaag 721gaggtcgaggtgattgggggagcagacaagtaccactccgtgtgtcggctctgctacttc 781aagaaggcctcaggccagcctgccgggccggacaacaaagagaactgcccagtgccagga 841aagccaggggaagccgtggctgccaggaagctctttgccccacagcagattctgcaatgg 901atccagacatga SEQIDNo.8: HumanTK1mutM73AA75DG9095Aaasequence 1MSCINLPTVLPGSPSKTRGQIQVILGPMFSGKSTELMRRVRRFQIAQYKC 51LVIKYAKDTRYSSSFCTHDRNTAEDLPACLLRDVAQEALAVAVIAIDEGQ 101FFPDIVEFCEAMANAGKTVIVAALDGTFQRKPFGAILNLVPLAESVVKLT 151AVCMECFREAAYTKRLGTEKEVEVIGGADKYHSVCRLCYFKKASGQPAGP 201DNKENCPVPGRPGEAVAARKLFAPQQILQWIQT SEQIDNo.9: HumanTK1mutM73AA75DG90Antsequence atgagatgcattaacctgcccactgtgctg 241cccggctcccccagcaagacccgggggcagatccaggtgattctcgggccgatgttctca 301ggaaaaagcacagagttgatgagacgcgtccgtcgcttccagattgctcagtacaagtgc 361ctggtgatcaagtatgccaaagacactcgctacagcagcagcttctgcacacatgaccgg 421aacaccgccgaggacctgcccgcctgcctgctccgagacgtggcccaggaggccctggct 481gtggctgtcataggcatcgacgaggggcagtttttccctgacatcgtggagttctgcgag 541gccatggccaacgccgggaagaccgtaattgtggctgcactggatgggaccttccagagg 601aagccatttggggccatcctgaacctggtgccgctggccgagagcgtggtgaagctgacg 661gcggtgtgcatggagtgcttccgggaagccgcctataccaagaggctcggcacagagaag 721gaggtcgaggtgattgggggagcagacaagtaccactccgtgtgtcggctctgctacttc 781aagaaggcctcaggccagcctgccgggccggacaacaaagagaactgcccagtgccagga 841aagccaggggaagccgtggctgccaggaagctctttgccccacagcagattctgcaatgg 901atccagacatga SEQIDNo.10: HumanTK1mutM73AA75DG90Aaasequence 1MSCINLPTVLPGSPSKTRGQIQVILGPMESGKSTELMRRVRRFQIAQYKC 51LVIKYAKDTRYSSSFCTHDRNTAEDLPACLLRDVAQEALAVAVIGIDEGQ 101FFPDIVEFCEAMANAGKTVIVAALDGTFQRKPFGAILNLVPLAESVVKLT 151AVCMECFREAAYTKRLGTEKEVEVIGGADKYHSVCRLCYFKKASGQPAGP 201DNKENCPVPGRPGEAVAARKLFAPQQILQWIQT SEQIDNo.11: HumanTK1mutG90Antsequence atgagctgcattaacctgcccactgtgctg 241cccggctcccccagcaagacccgggggcagatccaggtgattctcgggccgatgttctca 301ggaaaaagcacagagttgatgagacgcgtccgtcgcttccagattgctcagtacaagtgc 361ctggtgatcaagtatgccaaagacactcgctacagcagcagcttctgcacacatgaccgg 421aacaccatggaggcgctgcccgcctgcctgctccgagacgtggcccaggaggccctggct 481gtggctgtcataggcatcgacgaggggcagtttttccctgacatcgtggagttctgcgag 541gccatggccaacgccgggaagaccgtaattgtggctgcactggatgggaccttccagagg 601aagccatttggggccatcctgaacctggtgccgctggccgagagcgtggtgaagctgacg 661gcggtgtgcatggagtgcttccgggaagccgcctataccaagaggctcggcacagagaag 721gaggtcgaggtgattgggggagcagacaagtaccactccgtgtgtcggctctgctacttc 781aagaaggcctcaggccagcctgccgggccggacaacaaagagaactgcccagtgccagga 841aagccaggggaagccgtggctgccaggaagctctttgccccacagcagattctgcaatgg 901atccagaacatga SEQIDNo.12:humansuperTK1 HumanTK1mutG90Aaasequence 1MSCINLPTVLPGSPSKTRGQIQVILGPMFSGKSTELMRRVRRFQIAQYKC 51LVIKYAKDTRYSSSFCTHDRNTMEALPACLLRDVAQEALAVAVIGIDEGQ 101FFPDIVEFCEAMANAGKTVIVAALEGTFQRKPFGAILNLVPLAESVVKLT 151AVCMECFREAAYTKRLGTEKEVEVIGGADKYHSVCRLCYFKKASGQPAGP 201DNKENCPVPGRPGEAVAARKLFAPQQILQWIQT SEQIDNo.13: HumanTK1mutA75DG90Entsequence atgagctgcattaacctgcccactgtgctg 211cccggctcccccagcaagacccgggggcagatccaggtgattctcgggccgatgttctca 301ggaaaaagcacagagttgatgagacgcgtccgtcgcttccagattgctcagtacaagtgc 361ctggtgatcaagtatgccaaagacactcgctacagcagcagcttctgcacacatgaccgg 421aacaccatggaggacctgcccgcctgcctgctccgagacgtggcccaggaggccctggag 481gtggctgtcataggcatcgacgaggggcagtttttccctgacatcgtggagtictgcgag 541gccatggccaacgccgggaagaccgtaattgtggctgcactggatgggaccttccagagg 601aagccatttggggccatcctgaacctggtgccgctggccgagagcgtggtgaagctgacg 661gcggtgtgcatggagtgcttccgggaagccgcctataccaagaggctcggcacagagaag 721gaggtcgaggtgattgggggagcagacaagtaccactccgtgtgtcggctctgctacttc 781aagaaggcctcaggccagcctgccgggccggacaacaaagagaactgcccagtgccagga 841aagccaggggaagccgtggctgccaggaagctctttgccccacagcagattctgcaatgg 901atccagacatga SEQIDNo.14: HumanTK1mutA75DG90Eaasequence 1MSCINLPTVLPGSPSKTRGQIQVILGPMFSGKSTELMRRVRRFQIAQYKC 51LVIKYAKDTRYSSSFCTHDRNTMEDLPACLLRDVAQEALEVAVIGIDEGQ 101FFPDIVEFCEAMANAGKTVIVAALDGTFQRKPFGAILNLVPLAESVVKLT 151AVCMECFREAAYTKRLGTEKEVEVIGGADKYHSVCRLCYFKKASGQPAGP 201DNKENCPVPGRPGEAVAARKLFAPQQILQWIQT SEQIDNo,15: HumanTK1mutV84DG90Antsequence atgagctgcattaacctgcccactgtgctg 241cccggctcccccagcaagacccgggggcagatccaggtgattctcgggccgatgttctca 301ggaaaaagcacagagttgatgagacgcgtccgtcgcttccagattgatcagtacaagtgc 361ctggtgatcaagtatgccaaagacactcgctacagcagcagcttctgcacacatgaccgg 421aacaccatggaggcgctgcccgcctgcctgctccgagacgacgcccaggaggccctggct 481gtggctgtcataggcatcgacgaggggcagtttttccctgacatcgtggagttctgcgag 541gccatggccaacgccgggaagaccgtaattgtggctgcactggatgggaccttccagagg 601aagccatttggggccatcctgaacctggtgccgctggccgagagcgtggtgaagctgacg 661gcggtgtgcatggagtgcttccgggaagccgcctataccaagaggctcggcacagagaag 721gaggtcgaggtgattgggggagcagacaagtaccactccgtgtgtcggctctgctacttc 781aagaaggcctcaggccagcctgccgggccggacaacaaagagaactgcccagtgccagga 841aagccaggggaagccgtggctgccaggaagctctttgccccacagcagattctgcaatgg 901atccagacatga SEQIDNa16: HumanTK1mutV84DG90Aaasequence 1MSCINLPTVLPGSPSKTRGQIQVILGPMFSGKSTELMRRVRRFQIAQYKC 51LVIKYAKDTRYSSSFCTHDRNTMEALPACLLRDDAQEALAVAVIGIDEGQ 101FFPDIVEFCEAMANAGKTVIVAALDGTFQRKPFGAILNLVPLAESVVKLT 151AVCMECFREAAYTKRLGTEKEVEVIGGADKYHSVCRLCYFKKASGQPAGP 201DNKENCPVPGRPGEAVAARKLFAPQQILQWIQT SEQIDNo.17: HumanTK1mutA75GA84EG90Antsequence atgagctgcattaacctgcccactgtgctg 241cccggctcccccagcaagacccgggggcagatccaggtgattctcgggccgatgttctca 301ggaaaaagcacagagttgatgagacgcgtccgtcgcttccagattgctcagtacaagtgc 361ctggtgatcaagtatgccaaagacactcgctacagcagcagcttctgcacacatgaccgg 421aacaccatggaaggcctgcccgcctgcctgctccgagacgaggcccaggaggccctggct 481gtggctgtcataggcatcgacgaggggcagtttttccctgacatcgtggagttctgcgag 541gccatggccaacgccgggaagaccgtaattgtggctgcactggatgggaccttccagagg 601aagccatttggggccatcctgaacctggtgccgctggccgagagcgtggtgaagctgacg 661gcggtgtgcatggagtgcttccgggaagccgcctataccaagaggctcggcacagagaag 721gaggtcgaggtgattgggggagcagacaagtaccactccgtgtgtcggctctgctacttc 781aagaaggcctcaggccagcctgccgggccggacaacaaagagaactgcccagtgccagga 841aagccaggggaagccgtggctgccaggaagctctttgccccacagcagattctgcaatgg 901atccagacatga SEQIDNo.18: HumanTK1mutA75GV84EG90Aaasequence 1MSCINLPTVLPGSPSKTRGQIQVILGPMFSGKSTELMRRVRRFQIAQYKC 51LVIKYAKDTRYSSSFCTHDRNTMEGLPACLLRDEAQEALAVAVIGIDEGQ 101FFPDIVEFCEAMANAGKTVIVAALDGTFQRKPFGAILNLVPLAESVVKLT 151AVCMECFREAAYTKRLGTEKEVEVIGGADKYHSVCRLCYFKKASGQPAGP 201DNKENCPVPGRPGEAVAARKLFAPQQILQWIQT SEQIDNo.19: HumanTK1mutG90Dntsequence atgagctgcattaacctgcccactgtgatg 241cccggctcccccagcaagacccgggggcagatccaggtgattctcgggccgatgttctca 301ggaaaaagcacagagttgatgagacgcgtccgtcgcttccagattgctcagtacaagtgc 361ctggtgatcaagtatgccaaagacactcgctacagcagcagcttctgcacacatgaccgg 421aacaccatggaggcgctgcccgcctgcctgctccgagacgtggcccaggaggccctggac 481gtggctgtcataggcatcgacgaggggcagtttttccctgacatcgtggagttctgcgag 541gccatggccaacgccgggaagaccgtaattgtggctgcactggatgggaccttccagagg 601aagccatttggggccatcctgaacctggtgccgctggccgagagcgtggtgaagctgacg 661gcggtgtgcatggagtgcttccgggaagccgcctataccaagaggctcggcacagagaag 721gaggtcgaggtgattgggggagcagacaagtaccactccgtgtgtcggctctgctacttc 781aagaaggcctcaggccagcctgccgggccggacaacaaagagaactgcccagtgccagga 841aagccaggggaagccgtggctgccaggaagctctttgccccacagcagattctgcaatgg 901atccagacatga SEQIDNo.20: HumanTK1mutG90Daasequence 1MSCINLPTVLPGSPSKTRGQIQVILGPMFSGKSTELMRRVRRFQIAQYKC 51LVIKYAKDTRYSSSFCTHDRNTMEALPACLLRDVAQEALDVAVIGIDEGQ 101FFPDIVEFCEAMANAGKTVIVAALDGTFQRKPFGAILNLVPLAESVVKLT 151AVCMECFREAAYTKRLGTEKEVEVIGGADKYHSVCRLCYFKKASGQPAGP 201DNKENCPVPGRPGEAVAARKLFAPQQILQWIQT SEQIDNo.21: HumanTK1mutG90Vntsequence atgagctgcattaacctgcccactgtgctg 241cccggctcccccagcaagacccgggggcagatccaggtgattctcgggccgatgttctca 301ggaaaaagcacagagttgatgagacgcgtccgtcgcttccagattgctcagtacaagtgc 361ctggtgatcaagtatgccaaagacactcgctacagcagcagcttctgcacacatgaccgg 421aacaccatggaggcgctgcccgcctgcctgctccgagacgtggcccaggaggccctggtg 481gtggctgtcataggcatcgacgaggggcagtttttccctgacatcgtggagttctgcgag 541gccatggccaacgccgggaagaccgtaattgtggctgcactggatgggaccttccagagg 601aagccatttggggccatcctgaacctggtgccgctggccgagagcgtggtgaagctgacg 661gcggtgtgcatggagtgattccgggaagccgcctataccaagaggctcggcacagagaag 721gaggtcgaggtgattgggggagcagacaagtaccactccgtgtgtcggctctgctacttc 781aagaaggcctcaggccagcctgccgggccggacaacaaagagaactgcccagtgccagga 841aagccaggggaagccgtggctgccaggaagctctttgccccacagcagattctgcaatgg 901atccagacatga SEQIDNo.22: HumanTK1mutG90Vaasequence 1MSCINLPTVLPGSPSKTRGQIQVILGPMFSGKSTELMRRVRREQIAQYKC 51LVIKYAKDTRYSSSFCTHDRNTMEALPACLLRDVAQEALVVAVIGIDEGQ 101FFPDIVEFCEAMANAGKTVIVAALDGTFQRKPFGAILNLVPLAESVVKLT 151AVCMECFREAAYTKRLGTEKEVEVIGGADKYHSVCRLCYFKKASGQPAGP 201DNKENCPVPGRPGEAVAARKLFAPQQILQWIQT SEQIDNo.23: HumanTK1mutA75QV84Entsequence atgagctgcattaacctgcccactgtgctg 241cccggctcccccagcaagacccgggggcagatccaggtgattctcgggccgatgttctca 301ggaaaaagcacagagttgatgagacgcgtccgtcgcttccagattgctcagtacaagtgc 361ctggtgatcaagtatgccaaagacactcgctacagcagcagcttctgcacacatgaccgg 421aacaccatggagcagctgcccgcctgcctgctccgagacgaggcccaggaggccctgggc 481gtggctgtcataggcatcgacgaggggcagtttttccctgacatcgtggagttctgcgag 541gccatggccaacgccgggaagaccgtaattgtggctgcactggatgggaccttccagagg 601aagccatttggggccatcctgaacctggtgccgctggccgagagcgtggtgaagctgacg 661gcggtgtgcatggagtgcttccgggaagccgcctataccaagaggctcggcacagagaag 721gaggtcgaggtgattgggggagcagacaagtaccactccgtgtgtcggctctgctacttc 781aagaaggcctcaggccagcctgccgggccggacaacaaagagaactgcccagtgccagga 841aagccaggggaagccgtggctgccaggaagctctttgccccacagcagattctgcaatgg 901atccagacatga SEQIDNo.24: HumanTK1mutA75QV84Eaasequence 1MSCINLPTVLPGSPSKTRGQIQVILGPMFSGKSTELMRRVRREQIAQYKC 51LVIKYAKDTRYSSSFCTHDRNTMEQLPACLLRDEAQEALGVAVIGIDEGQ 101FFPDIVEFCEAMANAGKTVIVAALDGTFQRKPEGAILNLVPLAESVVKLT 151AVCMECFREAAYTKRLGTEKEVEVIGGADKYHSVCRLCYFKKASGQPAGP 201DNKENCPVPGRPGEAVAARKLFAPQQILQWIQT SEQIDNo.25: HumanTK1mutL8081Fntsequence atgagctgcattaacctgcccactgtgctg 241cccggctcccccagcaagacccgggggcagatccaggtgattctcgggccgatgttctca 301ggaaaaagcacagagttgatgagacgcgtccgtcgcttccagattgctcagtacaagtgc 361ctggtgatcaagtatgccaaagacactcgctacagcagcagcttctgcacacatgaccgg 421aacaccatggaggcactgcccgcctgcttcttccgagacgaggcccaggaggccctgggc 481gtggctgtcataggcatcgacgaggggcagtttttccctgacatcgtggagttctgcgag 541gccatggccaacgccgggaagaccgtaattgtggctgcactggatgggaccttccagagg 601aagccatttggggccatcctgaacctggtgccgatggccgagagcgtggtgaagctgacg 661gcggtgtgcatggagtgcttccgggaagccgcctataccaagaggctcggcacagagaag 721gaggtcgaggtgattgggggagcagacaagtaccactcagtgtgtcggctctgctacttc 781aagaaggcctcaggccagcctgccgggccggacaacaaagagaactgcccagtgccagga 841aagccaggggaagccgtggctgccaggaagctctttgccccacagcagattctgcaatgg 901atccagacatga SEQIDNo.26: HumanTK1mutL8081Faasequence 1MSCINLPTVLPGSPSKTRGQIQVILGPMFSGKSTELMRPVRRFQIAQYKC 51LVIKYAKDTRYSSSFCTHDRNTMEALPACFFRDVAQEALGVAVIGIDEGQ 101FFPDIVEFCEAMANAGKTVIVAALDGTFQRKPFGAILNLVPLAESVVKLT 151AVCMECFREAAYTKRLGTEKEVEVIGGADKYHSVCRLCYFKKASGQPAGP 201DNKENCPVPGRPGEAVAARKLFAPQQILQWIQT SEQIDNo.27: HumanTK1wtNM_003258ntsequence(ORF) atgagctgcattaacctgcccactgtgctg 241cctggctcccccagcaagacccgggggcagatccaggtgattctcgggccgatgttctca 301ggaaaaagcacagagttgatgagacgcgtccgtcgcttccagattgctcagtacaagtgc 361ctggtgatcaagtatgccaaagacactcgctacagcagcagcttctgcacacatgaccgg 421aacaccatggaggcactgcccgcctgcctgctccgagacgtggcccaggaggccatgggc 481gtggctgtcataggcatcgacgaggggcagtttttccctgacatcgtggagttctgcgag 541gccatggccaacgccgggaagaccgtaattgtggctgcactggatgggaccttccagagg 601aagccatttggggccatcctgaacctggtgccgctggccgagagcgtggtgaagctgacg 681gcggtgtgcatggagtgcttccgggaagccgcctataccaagaggccggicacagagaag 721gaggtcgaggtgattgggggagcagacaagtaccactccgtgtgtcggctctgctacttc 781aagaaggcctcaggccagcctgccgggccggacaacaaagagaactgaccagtgccagga 841aagccaggggaagccgtggctgccaggaagctctttgccccacagcagattctgcaatgc 901agccctgccaactga SEQIDNo.28: HumanTK1wtNM_003258aasequence 1MSCINLPTVLPGSPSKTRGQIQVILGPMFSGKSTELMRRVRRFQIAQYKC 51LVIKYAKDTRYSSSFCTHDRNTMEALPACLLRDVAQEALGVAVIGIDEGQ 101FFPDIVEFCEAMANAGKTVIVAALDGTFQRKPFGAILNLVPLAESVVKLT 151AVCMECFREAAYTKRLGTEKEVEVIGGADKYHSVCRLCYFKKASGQPAGP 201DNKENCPVPGKPGEAVAARKLFAPQQILQCSPAN