Research Themes
Research in the Kaufman lab centers on understanding the fundamental biology around mitochondrial genome (mtDNA) stability and how it contributes to health. Our research is centered around detecting mtDNA alterations in disease and exploring novel approaches for genetic correction. These alterations are detected in primary mitochondrial disease, where genes involved in mitochondrial respiration are altered, or in secondary diseases, including heart, lung, and kidney diseases, which involve mitochondrial dysfunction as a key step in the pathophysiology of the disease. Our work often includes studies with novel transgenic animals, cell culture models, genome engineering methods, and experimental therapeutic approaches. Our lab has several senior members, postdocs, and graduate students to mentor new scientists to develop a passion for research.
Mechanisms of mtdna stability
We are extending our understanding of how helicases interact with specific mtDNA sequences and structures to ensure genome stability and expression. Through NIH funding, we are employing the latest genome-wide high density sequencing approaches, super-resolution microscopy, and genome editing approaches to understand how mtDNA instability contributes to both respiratory dysfunction and mtDNA damage dependent inflammation in animal models of sudden cardiac arrest, ischemia reperfusion injury, kidney injury, chronic obstruction pulmonary disease (COPD), and idiopathic pulmonary fibrosis
Involvement of mtDNA in human disorders
Our broader objective is to test the contribution of mtDNA aberrations to disease progression. We have developed a rapid, high throughput mtDNA screening capability to test for these aberrations in samples relevant to diseases with known mitochondrial involvement, including age-related disorders such as cancer, diabetes, neurodegeneration, and cardiovascular disease. Recent work included cell-free mtDNA association with stress and disease in human samples and animal models of COPD, heart failure, psychological stress, and preterm birth
mtDNA therapies
Alterations in mtDNA are the cause of many disease phenotypes in both primary and secondary mitochondrial disease. Often, these diseases are aggressive in young children causing muscle weakness, stunted growth, and delays in development. We are looking into novel ways to address the pathologies caused by these alterations. In current projects, we are investigating how the presence of novel DNA structures can be used to influence disease phenotype. Additionally, we plan on investigating known mutant proteins associated with mitochondrial disease to determine their structure and role in disease pathology.
Our Projects
G- Quadruplexes
Within the mitochondrial genome, grouped guanine bases can form planar tetrads, which can Hoogsteen base pair and stack with a center metal cation resulting in structures known as G-Quadruplexes (G4). The evolutionary conserved G4-forming sequences are capable of regulating replication and transcriptions. The misregulated and mutagenic G4s are implicated in fatal mitochondrial diseases and genomic breakpoints. There are predicted to be hundreds of potential G4-forming sequences in mitochondria, and the full scope of their impact is unknown. Here we are looking at developing tools and methodologies capable of probing and perturbing G4s in mitochondria. We are implementing single-chain variable fragments (scFv); these antibody-like fusion proteins can bind G4s. Coupling this with small molecules to stabilize the G-quadruplexes, we can extend G4's influence on the mitochondria. We are utilizing small molecules and scFvs to determine G4 forming sequences, identify interacting partners of G4s, and induce heteroplasmy shift on mutagenic G4 forming sequences in the mitochondria.
ScFV Project Summary
ScFv have a high affinity for epitopes while maintaining a small size (<30kDa) and being genetically encoded and are comprised of a variable light (VL) and Variable Heavy (VH) domain connected by a linker. Encoding phage gIII enables the display of the scFv on the surface of the phage. PCR mutagenesis has created scFv libraries with theoretical diversity greater than 10^9 by mutagenesis of the complementarity-determining regions (CDRs). Previously, we have shown that the common m.10191T>C mtDNA pathogenic variant that causes Leigh Syndrome (LS) can form a unique G-quadruplex (G4) secondary structure. This LS sequence variant can form a zero-loop G4 called S2, but the wild-type (WT) sequence cannot. Libraries were generated by screening for candidate binders for the mutant and wild-type G4 structures. Compared to previous methods of library analysis, next-generation sequencing allows for the consideration of all candidates in the library, as opposed to only those able to produce bacterial colonies. A simple workflow can cluster specific sequences with minimal computation skills using the NGS data from enriched and unenriched libraries. This workflow can identify candidate sequences for recombinant biochemical assays. Highly enriched clusters are identified as possible candidates for scFv to generate to shift heteroplasmy. Clusters identified from these libraries would be candidates that could be used to shift heteroplasmy in this condition. Additionally, the analysis of variable regions from these clusters may be used to identify amino acid sequences that preferentially interact with the mutant G4 sequences.
Gamma PNA Project
The gamma Peptide Nucleic Acid project (gPNA) is centered around developing a novel DNA based therapeutic for mitochondrial disease. Mitochondrial diseases arise from mutations in mitochondrial DNA (mtDNA). The primary mitochondrial DNA diseases include Pearson Disease/Kearns-Sayre syndrome, Leigh Syndrome and MELAS, which primarily affect children and frequently result in death before the teenage years. In many cases, mitochondrial cardiomyopathy is responsible for death in patients with mitochondrial disease. In this project, we aim to use gPNAs, which act as synthetic analogs of DNA, to target mutated mtDNA. The overall goal of this project work is to deliver gPNAs to the mtDNA to halt the replication of mutated mtDNA so that only healthy DNA is available. This approach leverages that most mitochondrial disease exists in a state of heteroplasmy, meaning there is both healthy and mutated mtDNA in the genome. The idea is to eliminate only mutated mtDNA. In our current work, we employ a novel polymeric delivery system that has been shown to deliver small molecules to the cell without being endocytosed. The gPNA will be delivered to the mitochondria via a mitochondrial targeting moiety. Successful mtDNA targeting will demonstrate a heteroplasmy shift.
Transduction of Psychological Stress into Systematic Inflammation by Mitochondrial DNA Signaling
The Kaufman Lab collaborates with Martin Picard, PhD, New York State Psychiatric Institute and Anna Marsland, PhD, University of Pittsburgh on this NIMHS-funded project.
Under conditions of psychosocial stress, mitochondria release mitochondrial DNA (mtDNA), a proinflammatory molecule. In humans, circulating cell-free (ccf)-mtDNA and markers of inflammation are elevated in blood after a non-violent suicide attempt and in major depressive disorder. Our preliminary work provides initial evidence that acute socio-evaluative laboratory stress is sufficient to induce a robust and selective elevation in serum ccf-mtDNA after 30 minutes [5]. Moreover, the stress mediator glucocorticoid promotes mtDNA release from mitochondria in cultured human cells. However, the extent to which stress-induced increases in ccf-mtDNA contribute to the delayed increase of circulating proinflammatory mediators after acute psychological stress in humans remains unknown. We hypothesize that psychosocial stress regulates ccf-mtDNA release, which, in turn, signals a pro-inflammatory response. We utilize human and cell-based studies to examine this novel pathway at the systems, cellular, and molecular levels