Stimulating mitochondrial biosynthesis in patient-derived cell models of genetic POLG-related disease

• Applicant: Dr. Hans Zempel
• Subject Area: Human Genetics
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 556027894

POLG-spectrum disorders (POLG-SDs), which lead i.a. to multi-organ failure and premature death often within infancy/childhood, are detrimental genetic diseases/mitochondriopathies. Polymerase-γ (Polγ), encoded by the gene POLG, replicates mitochondrial DNA (mtDNA), essential for mitochondria as important cellular organelles. Mitochondrial biosynthesis (MB) is the process of mitochondrial replication, which also results in replication of mtDNA. MB can be chemically and genetically stimulated. In the majority of POLG-SDs, there is significant residual function of Polγ. Still, disease mechanisms are understudied, therapeutic strategies are lacking, functional tests for POLG unclear genetic variants are lacking. There is no cure nor an effective treatment available for POLG-SDs. As we assume mtDNA loss in most POLG-SDs to be slow (happening over the time course of several months-years) and gradual, we hypothesize that, due to significant residual function of Polγ in most forms of POLG-SDs, stimulation of MB can elicit sufficient mtDNA synthesis to counteract mtDNA loss in POLG-SD disease settings. We here aim to establish and characterize human cell lines, and test the effect of pharmacologic and genetic stimulation of MB in human primary fibroblasts and iPSC-derived neurons mimicking human disease. We have obtained several fibroblasts lines from POLG-SD-patients, have initially characterized them, and show here that these cells show mtDNA reduction and loss of mitochondrial mass. Here, we aim to establish a patient-derived cell study by collecting i.a. patient skin samples (used to generate fibroblasts), characterise them for mtDNA amount, mitochondrial mass, distribution and function, as well as general cell parameters, and reprogram selected fibroblasts to iPSCs, for targeted differentiation. Further, we will create iPSCs mimicking POLG-SDs via i) knockin (KI; CRISPR/Prime editing-based or commercial) to obtain an iPSC line carrying the most prevalent A467T mutation, and ii) iPSCs with lentivirus-based shRNA-mediated knockdown of POLG. Reintroduction of human disase-associated POLG will shed light on the cellular effect and pathomechanisms of different disease-associated mutations. We will assay mitochondrial and neuronal function, with the aim to establish a well-characterized human neuronal cell model of POLG-SDs. We will increase Polγ-activity via expression of i.a. the MB regulators TFAM and PPARGC1A, and substances known to increase mitochondrial biosynthesis, e.g. AICAR, fluoxetine, PDE inhibitors, and formoterol. We will characterize benefits for mitochondrial mass and function, cellular health, and effects on neuronal function. Establishing human POLG-SD models and stimulation of MB in the context of human disease may provide a therapeutic strategy also for related human genetic disorders/mitochondriopathies.

• DFG Programme: Research Grants