METHODS: Using biochemical assays and confocal microscopy, we provide evidence that accumulated lysosomal SM and cholesterol can be released by different treatments.
RESULTS: Defective SMase activity in these fibroblasts results in a 2.5-fold increased cellular mass of SM and cholesterol, increased de novo endogenous cholesterol synthesis, and decreased cholesterol esterification, demonstrating impaired intracellular cholesterol
homeostasis. Depletion of exogenous addition of cholesterol for 24 hours or addition of the cholesterol acceptor apolipoprotein LY2835219 manufacturer A-I are sufficient to restore normal homeostatic responses. In an effort to correct the Blebbistatin nmr lysosomal storage phenotype of NPB, we infected the fibroblasts with a lentivirus expressing the phosphotyrosine binding domain of the adapter protein GULP (PTB-GULP). We have previously shown that expression of PTB-GULP in Chinese hamster ovary cells promotes intracellular cholesterol trafficking and ABCA1-mediated cholesterol efflux. We find that expression of PTB-GULP in NPB fibroblasts results in increased ABCA1 expression, increased cellular cholesterol efflux and lysosomal cholesterol redistribution, independent of the impaired SMase and cholesterol presence.
CONCLUSION:
We provide extensive functional characterization of a novel compound heterozygote mutation and provide a novel functional mechanism to overcome lysosomal storage disease defects. (C) 2013 National Lipid Association. All rights reserved.”
“Purpose of review
Primary immunodeficiencies (PIDs) are
an often-devastating class find more of genetic disorders that can be effectively treated by hematopoietic stem cell transplantation, but the lack of a suitable donor precludes this option for many patients. Gene therapy overcomes this obstacle by restoring gene expression in autologous hematopoietic stem cells and has proven effective in clinical trials, but widespread use of this approach has been impeded by the occurrence of serious complications. In this review, we discuss recent advances in gene therapy with an emphasis on strategies to improve safety, including the emergence of gene targeting technologies for the treatment of PIDs.
Recent findings
New viral vectors, including lentiviral vectors with self-inactivating long terminal repeats, have been shown to have improved safety profiles in preclinical studies, and clinical trials using these vectors are now underway. Preclinical studies using engineered nucleases to stimulate precise gene targeting have also demonstrated correction of disease phenotypes for X-linked severe combined immunodeficiency, chronic granulomatous disease, and other diseases.