# Triggering the Selective Removal of Deleterious mtDNA Mutations from Mammalian Cells

> **NIH NIH F31** · UNIVERSITY OF SOUTHERN CALIFORNIA · 2024 · $48,974

## Abstract

ABSTRACT
As we age, mutations arise in the mitochondrial genome (mtDNA) of stem cells, neurons, cardiomyocytes and
muscle fibers. When these mutations expand, they compromise mitochondrial energy production and enhance
the generation of reactive oxygen species. Together, these molecular changes accelerate the natural aging
process and contribute to various age-related diseases, including cancer, sarcopenia, Alzheimer’s disease and
Parkinson’s disease. In addition, mtDNA mutations are responsible for a wide array of pediatric diseases that
are characterized by epilepsy, muscle weakness and neuronal dysfunction. To this day though, no child has
been cured of an mtDNA disease, nor is there a treatment for the mtDNA component of age-related diseases.
The long-term goal of this proposal is to solve this problem. To identify treatments, we will use a variety of genetic
and molecular biology tools, combined with state-of-the-art sequencing techniques, in an attempt to purge
mammalian cells of mtDNA molecules that carry deleterious mutations. To generate the most appropriate cell
line for these experiments, we have created a cell line that carries an error-prone version of DNA polymerase
gamma (PolgA), the enzyme that replicates the mitochondrial genome. This cell line also carries a genetic switch
that allows the error-prone allele of PolgA to be replaced by a WT allele through Cre-recombination
(PolgAD257A→WT). Accordingly, we can control the mutation rate of the mitochondrial genome at will. In this
application, we propose to use the PolgAD257A→WT allele to create a population of cells that contain a broad and
highly diverse spectrum of mtDNA mutations, similar to aging humans. We will then replace the error-prone allele
with the WT allele to prevent additional mutations from arising and track the existing mutations over time with
MADD-seq, a novel mutation detection assay that can detect millions of mutations at once. This assay will allow
us to determine whether these mutations can be removed by manipulating mitochondrial dynamics. Finally, we
will interrogate the mechanistic basis of this intervention by either enhancing or preventing mitophagy and
monitoring mutation clearance under those conditions as well. Together, these experiments have the potential
to transform our understanding of mitochondrial genetics in aging organisms and set the stage for the
development of treatments aimed at preventing or ameliorating age-related and pediatric diseases associated
with mtDNA mutations.

## Key facts

- **NIH application ID:** 10826583
- **Project number:** 1F31AG084238-01A1
- **Recipient organization:** UNIVERSITY OF SOUTHERN CALIFORNIA
- **Principal Investigator:** Sarah Jean Shemtov
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $48,974
- **Award type:** 1
- **Project period:** 2024-08-15 → 2025-08-14

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10826583

## Citation

> US National Institutes of Health, RePORTER application 10826583, Triggering the Selective Removal of Deleterious mtDNA Mutations from Mammalian Cells (1F31AG084238-01A1). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10826583. Licensed CC0.

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