182. Characterization and AAV-Mediated Correction of Xeroderma Pigmentosum-Cockayne Syndrome (XP/CS)
Authors: Skylar A. Rizzo, Amy L. Donate, Bennett J. Gosiker, Katherine E. Santostefano, Naohiro Terada, Fowzan S. Alkuraya, Peter B. Kang, Christina A. Pacak
Journal: Molecular Therapy (2015)

Cockayne Syndrome (CS) is caused by mutations in several genes that encode proteins involved in DNA repair. These include traditional CS proteins (CSA/CSB), as well as several Xeroderma Pigmentosum (XP) proteins (including XPG). While traditional CS is characterized by neurodegeneration and other clinical manifestations that result in an overall phenotype of premature aging, the typical XP phenotype is photosensitivity and skin cancer susceptibility. However, XPG presents with one of two different phenotypes, depending on the individual mutations: photosensitivity alone (XP) or photosensitivity + CS (XP/CS). Although the more severe XP/CS phenotype correlates well with mutations that result in XPG truncation, a function for this protein that explains the neurological deficit has yet to be described. XPG is involved in nuclear DNA repair as part of a complex that includes CSA and CSB. As CSA and CSB have also been shown to play a role in mitochondrial DNA (mtDNA) repair, it is likely, though unproven, that XPG is also involved in mtDNA repair. Insufficient mtDNA repair in neurons would ultimately lead to mitochondrial dysfunction and cause progressive neurodegeneration. The objective of this project is two-fold: 1) to characterize the two different phenotypes observed in XPG patients through examination of patient cells representing the XP as well as the XP/CS phenotype and compare them to healthy and CSA cells in order to identify a novel role for XPG that would explain the neurological deficit that occurs when the protein is truncated, and 2) to develop adeno-associated virus (AAV) mediated gene therapy for XPG and CSA and thus, overexpress these genes in an attempt to further characterize their function in healthy and patient-derived human cells. Cellular and mitochondrial function, sensitivity to oxidative and UV-induced stress, and general cell health and morphology are all being evaluated in healthy, XPG, and CSA patient-derived fibroblasts. Preliminary studies demonstrate reduced ATP production through oxidative phosphorylation in fibroblasts from patients with an XP/CS or CS phenotype as compared to those patients displaying XP alone or healthy controls. Scratch tests revealed motility and expansion deficiencies in XP/CS and CS cells as compared to XP or healthy controls, suggesting a potential role for mitochondria in these processes. AAV-XPG and AAV-CSA constructs have been generated and are being tested for their potential to correct cellular dysfunction. We are reprogramming a subset of these cells into induced pluripotent stem cells that will then be differentiated into neurons for further mitochondrial analyses and treatment studies. These studies will determine whether XPG function correlates with mitochondrial function in an in vitro human model of disease and generate preclinical data to justify further investigations into gene therapy strategies for this devastating syndrome.