Mechanisms of Chemical Targeting of the Mitochondrial Genome
Abstract
The inability to remove disease-causing variant mitochondrial DNA (mtDNA) sequences remains a blockade in treating mtDNA-borne disorders. These disorders affect approximately 1/10,000 live births. Early approaches treating these afflictions focused on traditional peptide nucleic acids (PNAs), which base-pair with target DNA but use peptides as the backbone, enabling high DNA sequence selectivity and resistance to degradation. Unfortunately, traditional PNAs fail to engage the mitochondrial genome in cells. New chemistry and concepts involving PNAs have greatly enhanced their ability to target DNA, enabling genome editing in animals (i.e., gamma PNAs; gPNAs). However, establishing efficient gPNA delivery to the mitochondria is a crucial challenge. In this application, we use cutting-edge approaches to solve the mtDNA targeting problem, with the capability of mtDNA variant removal in cells and ultimately in vivo. This LEGO-style targeting strategy is flexible, modular, and increases synthetic yields over long gPNA synthesis. In Aim 1, we will engineer a modular approach to cellular uptake of gPNAs. Classically, PNAs are taken up by endocytosis and require a toxic release agent to make the PNA available to the cytoplasm to gain access to the mitochondrial protein import machinery. Chemical targeting to the mitochondria traps the PNA in the mitochondrial inner membrane. These approaches are not feasible in vivo. A recent study has identified a synthetic polymer that delivers oligoglutamate-tagged proteins to the cytoplasm that bypasses the plasma membrane without endocytosis. We have demonstrated in preliminary data that this strategy effectively delivers a tagged gPNA to the cytoplasm and nucleus. In this aim, we will develop a modular approach to polymer-mediated delivery of a fluorescent gPNA and establish optimal formulations for cytosolic delivery in vitro. This modular design can be deployed with any gPNA and is expected to be compatible with targeting nucleic acid sequences in the mitochondria, nucleus, or cytoplasm. In Aim 2, we will optimize mitochondrial delivery of gPNAs. In parallel with Aim 1, we will synthesize and test gPNAs functionalized with peptides that mediate mitochondrial delivery from the cytoplasm. We will test the uptake of fluorescent gPNAs using mitochondrial import peptides into freshly isolated mouse mitochondria. Because the mitochondria will be actively respiring and replicating, we will establish a time course of import up to 2 hours in the absence of a cell membrane barrier. We will also test the impact of mitochondrial dysfunction on import efficiency. Findings will be extended using biotinylated forms of gPNAs for chromatin IP assays to demonstrate direct and specific interaction with the target mtDNA sequence. In Aim 3, we will test cell-based heteroplasmy models to measure the ability of dual-tagged gPNAs to inhibit the replication of specific mtDNA sequences. The advantage of gPNAs over other synthetic oligonucleotides, including unmodified PNAs, is their demonstrated ability to invade specific sequences of double-stranded DNA that are the prevalent form of mtDNA in human tissue. Combining the optimal cellular and mitochondrial uptake protocols from Aims 1 and 2, we will use gPNAs targeting the single most common disease-causing variant (m.3243A>G tRNALeu) in heteroplasmic cells. We will test whether gPNA delivery achieves mitochondrial therapeutic improvement as measured by reducing variant mtDNA levels and improved mitochondrial respiration. Successful completion of this work will enable future in vitro studies in cells derived from mitochondrial disease-affected patients and in vivo mouse studies that will demonstrate the benefit and safety of gPNA therapy prior to clinical studies.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Dec 28, 2022
- Source ID
- W81XWH2210062
Entities
People
- Brett Kaufman
Organizations
- United States Army
- University of Pittsburgh