Anti-cancer therapeutic strategy to overcome cancer resistance and to enable tailoring treatment to patients
View Patent ↗A gene construct comprising a programmed-cell-death executioner gene having a nuclear localization signal, a deleted signal peptide, an inhibitor-resistant binding site, a promoter, and an activator. A method of making a gene construct, by modifying a programmed-cell-death executioner gene by adding a nuclear localization signal, deleting a signal peptide, mutating a binding site for an inhibitor to make it inhibitor-resistant, adding a promoter for exclusive expression in selected cells, and adding an activator. A method of eliminating undesired cells from a patient. A method of treating cancer. An array comprising at least two gene constructs wherein all of the gene constructs differ with respect to the programmed-cell-death executioner gene and the nuclear localization signal. A method of personalizing anti-cancer treatment. A method of increasing DNase 1 resistance to actin binding. A method of increasing catalytic activity of DNase 1 binding.
1. A gene construct comprising a nucleotide sequence encoding a DNAse 1 protein operatively linked to a nucleotide sequence encoding a nuclear localization signal (NLS), wherein said nucleotide sequence encoding said DNAse 1 protein includes at least one mutation rendering an actin binding site of said DNAse 1 protein resistant to the binding of actin, said gene construct lacking the nucleotide sequence encoding a secretion signal peptide, said gene construct additionally including at least one promoter operatively linked to said nucleotide sequence encoding said DNAse 1 protein and to said nucleotide sequence encoding said NLS.
2. The gene construct of claim 1 , wherein said at least one promoter is chosen from the group consisting of the promoter from the prostate-specific antigen (PSA) gene; the promoter from the prostate-specific membrane antigen (PSMA) gene; the promoter from the prostatic acid phosphatase (PAP) gene; the probasin promoter ARR2PB; the promoter from the human secretoglobin family 2A member 2 (hSCGB2A2) gene; the promoter from the DF3/MUC1 gene; the promoter from the thyroglobulin gene; the promoter from the calcitonin (CT) gene; a nucleotide encoding the TTF1 gene operatively linked to a promoter of the human telomerase reverse transcriptase (hTERT) gene and to 5 tandem copies of at least a portion of a nucleotide encoding the human signal-induced proliferation-associated gene 1 (hSPA1); the promoter from the hexokinase II gene; the promoter from the phosphoenolpyrovate carboxykinase gene; the promoter from the albumin gene; the promoter RIP from the rat insulin gene; the promoter from the GFAP gene; the promoter from the gametophytic factor 2 (gfa2) gene; the promoter from the neuron specific enolase (NSE) gene; the 2nd intron of the Nestin gene operatively linked upstream to the 5′ upstream region (2iNP) of the Nestin gene; the promoter from the HLA-Dr alpha gene; the promoter from the CD4 gene; the promoter from the CD19 gene; the promoter from the Ig kappa gene; the long terminal repeat (LTR) promoter of HIV-1; and the promoter from cytomegalovirus (CMV).
3. The gene construct of claim 1 , wherein said nuclear localization signal is chosen from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5.
4. The gene construct of claim 1 , wherein said at least one promoter is a melanocyte-specific promoter.
5. The gene construct of claim 4 , wherein said melanocyte-specific promoter is chosen from the group consisting of the promoter from the tyrosinase gene, the promoter from the melanoma inhibitory activity (MIA) gene, the promoter from the SILV/PMEL17/GP100 gene, the promoter from the Melan-A/MART-1 gene, the promoter from the melanocortin-1 receptor (MC1R) gene, and the promoter from the microphthalmia-associated transcription factor (MITF) gene.
6. The gene construct of claim 1 or claim 4 , additionally including an enhancer operatively linked to said promoter, said enhancer being chosen from the group consisting of the enhancer of the PSA gene, operatively linked to a rat probasin promoter; the 1455 bp PSA enhancer; the prostate-specific chimeric enhancer PSES; and the enhancer of the DF3/MUC1 gene, said enhancer of said DF3/MUC1 gene operatively linked to the promoter from said DF3/MUC1 gene.
7. The gene construct of claim 1 , additionally including, operatively linked to said nucleotide sequence encoding said DNAse 1 protein and to said nucleotide sequence encoding said NLS, an activator binding sequence, and said gene construct including a nucleotide sequence encoding an inducible transcriptional activator system chosen from the group consisting of a transcriptional activator system inducible by an antibiotic, and a transcriptional activator system inducible by radiation.
8. The gene construct of claim 7 additionally including a promoter operatively linked to said gene construct encoding an inducible transcriptional activator system.
9. The gene construct of claim 1 , wherein said actin-resistant binding site is an actin binding site including a mutation chosen from the group consisting of Glu-13, His-44, Asp-53, Tyr-65, Val-66, Val-67, Glu-69, Ala-114, and combinations thereof.
10. The gene construct of claim 1 , additionally including, operatively linked to said nucleotide sequence encoding said DNAse 1 protein and to said nucleotide sequence encoding said NLS, a nucleotide sequence encoding the translation regulator Musashi.