Expression and processing of mature human frataxin after gene therapy in mice.

Friedreich's ataxia is a degenerative and progressive multisystem disorder caused by mutations in the highly conserved frataxin (FXN) gene that results in FXN protein deficiency and mitochondrial dysfunction. While gene therapy approaches are promising, consistent induction of therapeutic FXN protein expression that is sub-toxic has proven challenging, and numerous therapeutic approaches are being tested in animal models. FXN (hFXN in humans, mFXN in mice) is proteolytically modified in mitochondria to produce mature FXN. However, unlike endogenous hFXN, endogenous mFXN is further processed into N-terminally truncated, extra-mitochondrial mFXN forms of unknown function. This study assessed mature exogenous hFXN expression levels in the heart and liver of C57Bl/6 mice 7-10 months after intravenous administration of a recombinant adeno-associated virus encoding hFXN (AAVrh.10hFXN) and examined the potential for hFXN truncation in mice. AAVrh.10hFXN induced dose-dependent expression of hFXN in the heart and liver. Interestingly, hFXN was processed into truncated forms, but found at lower levels than mature hFXN. However, the truncations were at different positions than mFXN. AAVrh.10hFXN induced mature hFXN expression in mouse heart and liver at levels that approximated endogenous mFXN levels. These results suggest that AAVrh.10hFXN can likely induce expression of therapeutic levels of mature hFXN in mice.

Bile Acid Metabolism Mediates Cholesterol Homeostasis and Promotes Tumorigenesis in Clear Cell Renal Cell Carcinoma.

Clear cell renal cell carcinoma (ccRCC) incidence has risen steadily over the last decade. Elevated lipid uptake and storage is required for ccRCC cell viability. As stored cholesterol is the most abundant component in ccRCC intracellular lipid droplets, it may also play an important role in ccRCC cellular homeostasis. In support of this hypothesis, ccRCC cells acquire exogenous cholesterol through the high-density lipoprotein receptor SCARB1, inhibition or suppression of which induces apoptosis. Here, we showed that elevated expression of 3 beta-hydroxy steroid dehydrogenase type 7 (HSD3B7), which metabolizes cholesterol-derived oxysterols in the bile acid biosynthetic pathway, is also essential for ccRCC cell survival. Development of an HSD3B7 enzymatic assay and screening for small-molecule inhibitors uncovered the compound celastrol as a potent HSD3B7 inhibitor with low micromolar activity. Repressing HSD3B7 expression genetically or treating ccRCC cells with celastrol resulted in toxic oxysterol accumulation, impaired proliferation, and increased apoptosis in vitro and in vivo. These data demonstrate that bile acid synthesis regulates cholesterol homeostasis in ccRCC and identifies HSD3B7 as a plausible therapeutic target. SIGNIFICANCE: The bile acid biosynthetic enzyme HSD3B7 is essential for ccRCC cell survival and can be targeted to induce accumulation of cholesterol-derived oxysterols and apoptotic cell death.

Expression and processing of mature human frataxin after gene therapy in mice.

Friedreich's ataxia is a degenerative and progressive multisystem disorder caused by mutations in the highly conserved frataxin (FXN) gene that results in FXN protein deficiency and mitochondrial dysfunction. While gene therapy approaches are promising, consistent induction of therapeutic FXN protein expression that is sub-toxic has proven challenging, and numerous therapeutic approaches are being tested in animal models. FXN (hFXN in humans, mFXN in mice) is proteolytically modified in mitochondria to produce mature FXN. However, unlike endogenous hFXN, endogenous mFXN is further processed into N-terminally truncated, extra-mitochondrial mFXN forms of unknown function. This study assessed mature exogenous hFXN expression levels in the heart and liver of C57Bl/6 mice 7-10 months after intravenous administration of a recombinant adeno-associated virus encoding hFXN (AAVrh.10hFXN) and examined the potential for hFXN truncation in mice. AAVrh.10hFXN induced dose-dependent expression of hFXN in the heart and liver. Interestingly, hFXN was processed into truncated forms, but found at lower levels than mature hFXN. However, the truncations were at different positions than mFXN. AAVrh.10hFXN induced mature hFXN expression in mouse heart and liver at levels that approximated endogenous mFXN levels. These results demonstrate that AAVrh.10hFXN may induce expression of therapeutic levels of mature hFXN in mice.