Treating mitochondrial disease caused by pathogenic mtDNA mutations

Periodic Reporting for period 4 - mtDNA-CURE (Treating mitochondrial disease caused by pathogenic mtDNA mutations)

Período documentado: 2022-07-01 hasta 2023-06-30

Mutations of mtDNA are important causes of mitochondrial disease and are also thought to have important roles in age-associated diseases and ageing. Human pathogenic mutations of mtDNA frequently affect tRNA genes and are typically heteroplasmic, i.e. present only in a fraction of all mtDNA copies in a cell. If the levels of mutated mtDNA exceed a certain threshold, the oxidative phosphorylation will be impaired and cause dysfunction of affected organs. Remarkably, maternal relatives to mitochondrial disease patients can be perfectly healthy despite having very high, but subthreshold, level of mutated mtDNA. The corollary to these observations is that a small increase of wild-type mtDNA may prevent mitochondrial dysfunction and disease. It is currently unknown whether an increase of the absolute amount of wild-type mtDNA will prevent disease manifestations even if the proportion of mutated mtDNA remains the same. Pathogenic mtDNA mutations typically act in a “recessive” (loss of function) way and it is therefore reasonable to assume that increase of the absolute amount of wild-type mtDNA may restore respiratory chain function. Based on this hypothesis, we propose that treatments aiming to increase overall mtDNA copy number are a reasonable strategy to treat or even cure a large group of human patients with mitochondrial diseases.

The specific aims of the proposal are:
1. To investigate how mtDNA copy number manipulation affects the phenotypic expression of disease causing mtDNA mutations in flies and mice.
We were able to show that an increased gene dosage of WT mtDNA has a major impact on ameliorating OXPHOS function despite that mutated mtDNA is the most abundant genome. Thus, the absolute levels of WT mtDNA are an important determinant of the pathological manifestations, suggesting that pharmacological or gene therapy approaches to selectively increase mtDNA copy number provide a potential treatment strategy for human mtDNA mutation disease. (PMID: 30949583)

2. To identify the regulatory mechanism at the end of the displacement (D)-loop region that controls the switch between abortive and genome-length mammalian mtDNA replication.
We were able to show that by extensive phenotyping of ageing Mgme1 knockout animals and show that they develop a dramatic phenotype as they age and display progressive weight loss, cataract and retinopathy. The findings link the faulty mtDNA synthesis to severe inflammatory disease and thus show that defective mtDNA replication can trigger an immune response that causes age-associated progressive pathology in the kidney.(PMID: 35533204)

3. To develop novel small molecule chemical stimulators that increase mtDNA copy number.
We have worked together with the Lead Discovery Center, Dortmund, Germany and have set up a robust drug screening strategy to identify small molecular stimulators of mtDNA transcription and were able to identify 2 compounds undergoing further optimization.

Aim 1: To investigate how mtDNA copy number manipulation affects the phenotypic expression of disease-causing mtDNA mutations in flies and mice.
Mouse model with a single mutation in the tRNA gene of mtDNA (C5024T in tRNAAla), recaptitulates a pathogenic human mtDNA mutation (G5650A in tRNAAla). We have investigated to what extent the absolute levels of wild-type (WT) mtDNA influence disease manifestations by manipulating TFAM levels in C5024T mice. We found that increased total mtDNA levels ameliorated pathology in multiple tissues. A reduction in mtDNA levels worsened the phenotype in postmitotic tissues, such as heart, whereas there was an unexpected beneficial effect in rapidly proliferating tissues, such as colon, by enhancing the selective elimination of mutated mtDNA. The absolute levels of WT mtDNA are an important determinant of the pathological manifestations, suggesting that pharmacological or gene therapy approaches to selectively increase mtDNA copy number provide a potential treatment strategy for human mtDNA mutation disease. (Filograna, R., et al Science advances 2019)

Aim 2:To identify the regulatory mechanism at the end of the displacement (D)-loop region that controls the switch between abortive and genome-length mammalian mtDNA replication.
We found that Mgme1 knockout mice display tissue specific replication stalling patterns and sequence coverage patterns of mtDNA. We found that Mgme1 knockout mice develop a dramatic phenotype as they age and display progressive weight loss, cataract and retinopathy. Surprisingly, aged animals also develop kidney inflammation, glomerular changes and severe chronic progressive nephropathy, causing nephrotic syndrome. The findings link faulty mtDNA synthesis to severe inflammatory disease and show that defective mtDNA replication can trigger an immune response that causes age-associated progressive pathology in the kidney. (Milenkovic, D., et al PLoS Genetics 2022)

To establish a screen to detect physiologically relevant protein interactors of MGME1, we established HEK-293 cell lines stably expressing wild-type and mutant MGME1-biotin ligase fusion proteins using the Flp-In TREx system. As expected, we found constituents of the mitochondrial replisome, such as POLg and TWINKLE, enriched against both the wild-type and mutant MGME1 baits. Interestingly, we found the EXD2 nuclease (Exonuclease 3'-5' domain-containing protein 2) of unknown function in mitochondria among enriched preys and we will follow up on the putative role of this protein in mtDNA maintenance.

Aim 3: To develop novel small molecule chemical stimulators that increase mtDNA copy number.
After applying a drug-likeness filter and repeated analysis, we identified a list of 528 active compounds out from 201 000 with EC30 lower than 10 μM. Following characterization for compound specificity on mitochondrial transcription, we are optimizing 2 compounds.

Our published work (Bonekamp et al. 2020) identified novel first-in-class specific inhibitors of mitochondrial transcription (IMTs) that target the mammalian mitochondrial RNA polymerase (POLRMT), which is essential for biogenesis of the oxidative phosphorylation (OXPHOS) system. The results showed that enzymes inside mammalian mitochondria can be efficiently drugged and has opened up the field to develop chemical compounds that specifically target the mtDNA gene expression machinery.

Additionally, the project has shown that upregulation of total mtDNA copy number have beneficial effects and counteract deleterious effects caused by heteroplasmic mtDNA mutations. This finding is unexpected and argues that mtDNA mutations typically have no dominant effect but rather that it is instead the lack of normal gene products that causes disease.

We have developed a database available via web interface to systematize our proteomic data and to facilitate its accessibility to the research division members and other researchers.

The discoveries in this proposal have identified novel principles for treating mitochondrial disease and drugging mitochondria. I co-founded Pretzel Therapeutics Inc. (www.pretzeltx.com) which has received a very substantial series A funding. Pretzel has very active screening projects for small-molecule compounds that can manipulate mitochondrial function. These compounds will be valuable tools for chemical biology and will also undergo the drug development process with the aim of initiating clinical trials in 2025.

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Periodic Reporting for period 4 - mtDNA-CURE (Treating mitochondrial disease caused by pathogenic mtDNA mutations)

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