Title: Specific targeting of tumour mitochondrial DNA for cancer gene therapy
Funder: Cancer Research UK

Abstract: Background: In a significant fraction of tumours, almost all mitochondrial DNA (mtDNA) has a specific, often synonymous, mutation, that is distinct from surrounding and normal tissue. These mutations have been so far ignored since they have no effect on tumour biology. However, in 10-30% of tumours depending on the tissue of origin, the mutations occur sufficiently early in tumourigenesis to be present in near homoplasmy, i.e. almost all copies of mtDNA carry the mutation in almost all tumour cells. Recent studies showed that mutant mtDNA can be eliminated selectively in vivo by gene targeting methods such as zinc-finger nucleases and TALENs, leading to near complete depletion of mutant mtDNAs. In tumours with homoplasmic mtDNA mutations this approach would lead to near complete depletion of mtDNA, resulting in collapse of cellular energy homeostasis, block of proliferation and cell death. Vitally, cells which lack the mutant mtDNA are unaffected by this therapy. The mtDNA sequence is thus both a tumour-specific biomarker and potential therapeutic target. Aims: We propose that tumour-specific elimination of mtDNA, carrying unique homoplasmic mutations could be exploited to inhibit tumour growth and/or increase the therapeutic efficacy of appropriate chemotherapies without needing to be directly targeted at tumour cells. In the proposal we aim to provide proof-of-principle for: (i) efficient delivery and mtDNA depletion by mitochondria targeted TALENs (mitoTALENs) in mouse in vivo tumour models bearing tumour-specific homoplasmic mtDNA mutations, (ii) mtDNA depletion-mediated inhibition of tumour growth or induction of tumour cell death. Methods: In order to provide proof-of-principle of in vivo mitoTALEN delivery and detrimental effects of complete mtDNA depletion on the tumours, we will use human breast cancer patient derived xenografts (PDXs). We will apply viral delivery of fluorescently labelled mitoTALENs against tumour/human-specific mtDNA sequences to deplete all mtDNA in the PDX, leaving all mouse wild type mtDNA intact. We will assess: (i) delivery efficiency by cell sorting and imaging of fluorescent tags, (ii) mtDNA depletion by quantitative PCR, (iii) the effect of the full depletion of mtDNA by measuring tumour growth and apoptosis. How the results of this research will be used: We will provide proof-of-principle for targeting mtDNA mutations in cancer as the basis of development of refined strategies to achieve therapeutic effect. Moreover, we envisage that the PDX models and mtDNA targeting approach will be a valuable generic platform technology for testing combinational therapies synergising with mtDNA depletion, such as chemotherapies or nutrient deprivation-based approaches.

Lay abstract: Background: In a significant fraction of tumours, almost all mitochondrial DNA (mtDNA) has a specific, often synonymous, mutation, that is distinct from surrounding and normal tissue. These mutations have been so far ignored since they have no effect on tumour biology. However, in 10-30% of tumours depending on the tissue of origin, the mutations occur sufficiently early in tumourigenesis to be present in near homoplasmy, i.e. almost all copies of mtDNA carry the mutation in almost all tumour cells. Recent studies showed that mutant mtDNA can be eliminated selectively in vivo by gene targeting methods such as zinc-finger nucleases and TALENs, leading to near complete depletion of mutant mtDNAs. In tumours with homoplasmic mtDNA mutations this approach would lead to near complete depletion of mtDNA, resulting in collapse of cellular energy homeostasis, block of proliferation and cell death. Vitally, cells which lack the mutant mtDNA are unaffected by this therapy. The mtDNA sequence is thus both a tumour-specific biomarker and potential therapeutic target. Aims: We propose that tumour-specific elimination of mtDNA, carrying unique homoplasmic mutations could be exploited to inhibit tumour growth and/or increase the therapeutic efficacy of appropriate chemotherapies without needing to be directly targeted at tumour cells. In the proposal we aim to provide proof-of-principle for: (i) efficient delivery and mtDNA depletion by mitochondria targeted TALENs (mitoTALENs) in mouse in vivo tumour models bearing tumour-specific homoplasmic mtDNA mutations, (ii) mtDNA depletion-mediated inhibition of tumour growth or induction of tumour cell death. Methods: In order to provide proof-of-principle of in vivo mitoTALEN delivery and detrimental effects of complete mtDNA depletion on the tumours, we will use human breast cancer patient derived xenografts (PDXs). We will apply viral delivery of fluorescently labelled mitoTALENs against tumour/human-specific mtDNA sequences to deplete all mtDNA in the PDX, leaving all mouse wild type mtDNA intact. We will assess: (i) delivery efficiency by cell sorting and imaging of fluorescent tags, (ii) mtDNA depletion by quantitative PCR, (iii) the effect of the full depletion of mtDNA by measuring tumour growth and apoptosis. How the results of this research will be used: We will provide proof-of-principle for targeting mtDNA mutations in cancer as the basis of development of refined strategies to achieve therapeutic effect. Moreover, we envisage that the PDX models and mtDNA targeting approach will be a valuable generic platform technology for testing combinational therapies synergising with mtDNA depletion, such as chemotherapies or nutrient deprivation-based approaches.

Research activity codes: 5.1 PHARMACEUTICALS
Health categories: CANCER AND NEOPLASMS