Barth syndrome (BTHS) is a rare mitochondrial disease that affects heart and skeletal muscle and has no curative treatment. It is caused by recessive mutations in the X-linked gene TAZ, which encodes tafazzin. To develop a clinically relevant gene therapy to restore tafazzin function and treat BTHS, three different adeno-associated virus serotype 9 vectors were tested and compared to identify the optimal promoter-cytomegalovirus (CMV), desmin (Des), or a native tafazzin promoter (Taz)-for TAZ expression following intravenous administration of 1 × 10 vector genomes/kilogram to a mouse model of BTHS as either neonates (1-2 days of age) or adults (3 months of age). At 5 months of age, evaluations of biodistribution and TAZ expression levels, mouse activity assessments, fatigue in response to exercise, muscle strength, cardiac function, mitochondrial structure, oxygen consumption, and electron transport chain complex activity assays were performed to measure the extent of improvement in treated mice. Each promoter was scored for significant improvement over untreated control mice and significant improvement compared with the other two promoters for every measurement and within each age of administration. All three of the promoters resulted in significant improvements in a majority of the assessments compared with untreated BTHS controls. When scored for overall effectiveness as a gene therapy, the Des promoter was found to provide improvement in the most assessments, followed by the CMV promoter, and finally Taz regardless of injection age. This study provides substantial support for translation of an adeno-associated virus serotype 9-mediated TAZ gene replacement strategy using a Des promoter for human BTHS patients in the clinic.
Background Transgender women (TW) are disproportionately affected by both HIV and cardiovascular disease (CVD). Objectives We aim to quantify prevalence of elevated predicted CVD risk for TW compared to cisgender women (CW) and cisgender men (CM) in HIV care and describe the impact of multiple operationalizations of CVD risk score calculations for TW. Design We conducted a cross-sectional analysis of patients engaged in HIV care between October 2014 and February 2018.
regions for treated and untreated mutants compared to wild type mice, using X-ray fluorescence microscopy (XFM). XFM enables sensitive, quantitative measurement of the spatial distribution of biometals at image resolutions approaching the subcellular level. Ten days after rAAV9-ATP7A administration, copper levels in cerebral cortex, caudate and choroid plexus in the lateral ventricle in combination treated mice were all normal or slightly higher than normal compared to wild type mice and untreated mutants. Immunohistochemistry demonstrated robust ATP7A expression in choroid plexus epithelia as well as in neurons throughout the brain in the treated animals. These preliminary data support the hypothesis that choroid plexus is the major mediator of brain copper delivery by pumping copper into the CSF via ATP7A. Our findings provide further support for CSFdirected viral gene therapy in human subjects with Menkes disease.
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.
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