Mitochondrial diseases caused by harmful changes in mitochondrial DNA (mtDNA) significantly impact children, often leading to severe health issues and dramatically reduced life expectancy. These diseases, such as Pearson Syndrome and Kearns-Sayre syndrome, frequently become apparent in infancy and can result in serious complications early in life. Unfortunately, effective treatments are currently lacking for these conditions. This research proposal focuses on developing innovative treatment options for these mitochondrial diseases, particularly those caused by large-scale deletions in mtDNA. We are exploring a cutting-edge approach using γ- peptide nucleic acids (γPNAs), which can precisely target and interact with mitochondrial DNA. Our primary hypothesis is that by designing specific γPNAs, we can promote the growth of healthy mtDNA and help restore the vital energy functions disrupted by these pathogenic mutations. Building on our prior work, we have improved our methods for delivering these γPNAs and have identified new target sequences in mtDNA that can be effectively addressed. By focusing on these areas, we hope to create solutions that directly target the problems posed by the disease. If successful, this project could lead to groundbreaking changes in the treatment of mitochondrial diseases. Our goal is to enhance the quality of life and extend the lifespan of affected children, bringing hope to families dealing with these challenging conditions and contributing to advancements in mitochondrial medicine.
Mitochondria—often described as the “powerhouses” of the cell—contain their own small genome (mtDNA), which is essential for generating the energy that powers our bodies. When large pieces of mtDNA are lost, a condition known as an mtDNA deletion, cells can no longer produce enough energy. This has devastating consequences for energy-demanding tissues such as muscle and brain, leading to progressive and often fatal diseases. Currently, there are no treatments available that can slow, stop, or reverse mtDNA deletion disorders. To address this urgent need, we have developed a new research tool that enables us to precisely recreate the same mtDNA deletions found in patients and investigate how human cells respond. Using this system, we conducted a large-scale genetic test of every human gene to identify which ones make deletion-bearing cells weaker and which ones unexpectedly help them survive. Remarkably, we discovered a handful of “protective” genes that appear to buffer the harmful effects of mtDNA deletions, allowing cells to remain alive even when their mitochondria are severely damaged. In this project, we will carefully confirm which protective genes are most effective and investigate how they help cells adapt. We will also explore whether turning these genes on or off—or targeting them with drugs—can restore energy production. By extending our studies to muscle and nerve cells, the tissues most affected in patients, we aim to uncover protective pathways that could be translated into new treatments. Our long-term goal is to find strategies that not only buffer the harmful effects of mtDNA deletions but also potentially reduce or eliminate them altogether. This work has the potential to deliver entirely new therapeutic approaches for patients and families affected by mtDNA deletion disorders, bringing hope where currently no options exist.
Mitochondrial disorders can be caused by pathogenic mitochondrial DNA (mtDNA) variation (i.e., mutation), generally in a heteroplasmic state. In heteroplasmy, some mtDNA are healthy, “good,” and some are unhealthy, “bad.” A subset of sporadic mtDNA mutations lacks a substantial portion of the normal sequence, characterized as single large-scale deletions in mtDNA, which causes progressive external ophthalmoplegia (PEO), Kearns-Sayre syndrome (KSS), and Pearson syndrome (PS). There are no effective therapies or cures for patients affected by a mtDNA deletion.
The inability to remove disease-causing mtDNA sequences remains a blockade in treating mtDNA-borne disorders. Evidence suggests that eliminating bad mtDNA or adding good mtDNA can cause positive effects. While other experimental approaches are being developed, effective treatment will likely require multiple approaches. We seek to develop a specific strategy to remove bad mtDNA using molecules similar to DNA called peptide nucleic acids (PNA).
In the past, PNAs have been utilized to attempt to remove bad mtDNA; however, this approach failed because the PNAs could not reach the mtDNA. One additional issue with that approach is that PNAs have limited ability to penetrate genomic DNA. Recently, new chemistry and concepts involving gamma-substituted PNAs (γPNAs) have greatly improved this and have enabled whole animal DNA editing and somatic cell therapies, evidence of DNA penetration, and block of DNA replication. However, the current challenge is establishing efficient two-step delivery into the cell and then the mitochondria.
In this study, we will use next-generation approaches to solve the mtDNA targeting problem for a designer γPNA sequence specific to the mtDNA “common” deletion found in patients (γ-P3). Our preliminary data shows the successful adaptation of a recently described polymer strategy that yields improved delivery of γ-PNAs into the cell. Objective 1 will develop strategies to optimize polymer-mediated delivery of a fluorescent γ-P3 and establish optimal formulations for cellular delivery. Preliminary data shows no toxicity in animal models with polymer delivery. Objective 2 will synthesize and test γ-P3 with a mitochondrial import sequence on isolated mitochondria from animals and cells to establish evidence of interaction with the “bad” mtDNA. Objective 3 will determine whether the optimized delivery of γ-P3 can reduce mtDNA common deletion from a heteroplasmic cell to improve mitochondrial function. We can directly test for γ-P3 interaction with the correct template and block of replication.
Successful completion of this work will enable future in vitro studies in more relevant cell types directly applicable to patient therapy but challenging to work with for rapid γ-PNA development.
Mitochondria are the parts of the cell that produce most of the energy we generate from food, and this process depends on the many small circles of DNA they contain - mitochondrial DNAs. Alterations (mutations) of mitochondrial DNA are among the most frequent causes of genetic diseases, which can manifest at any stage of life and can affect any part of the body. One type of these mutations, the single large-scale mitochondrial DNA deletions (SLSMDs), involves the loss of a large portion of the mitochondrial DNA and they can cause Pearson’s and Kearns-Sayre syndromes. Importantly, the deleted molecules coexist with normal copies, and mitochondrial malfunction and disease manifest when the damaged DNAs reach high levels. Consequently, finding a way to decrease the number of deleted mitochondrial DNAs and increase the good copies could radically improve the length and quality of life of patients with SLSMDs.
Recently, we discovered that we can cripple mitochondria that contain one type of mutant mitochondrial DNA, while permitting those with good copies of mitochondrial DNA to thrive, using compounds that change nutrient usage inside the cell. This represents an important breakthrough in mitochondrial medicine, as the compounds could benefit patients with SLSMDs.
With this project we aim to i) test the small molecules against deleted mitochondrial DNAs, in cultured cells; ii) assess the effects of one of them in a mouse that shows mitochondrial dysfunction owing to a similar kind of mutant mtDNA, and iii) discover the key changes inside the cell that select the ‘good’ mitochondrial DNAs. Achieving these goals will bring us closer to the goal of finding a cure for SLSMD Syndromes.