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Permanent Alteration of PCSK9 With In Vivo CRISPR-Cas9 Genome Editing
Rationale: Individuals with naturally occurring loss-of-function proprotein convertase subtilisin/kexin type 9 (PCSK9) mutations experience reduced low-density lipoprotein cholesterol levels and protection against cardiovascular disease.
Objective: The goal of this study was to assess whether genome editing using a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system can efficiently introduce loss-of-function mutations into the endogenous PCSK9 gene in vivo.
Methods and Results: We used adenovirus to express CRISPR-associated 9 and a CRISPR guide RNA targeting Pcsk9 in mouse liver, where the gene is specifically expressed. We found that 50%. This resulted in decreased plasma PCSK9 levels, increased hepatic low-density lipoprotein receptor levels, and decreased plasma cholesterol levels (by 35–40%). No off-target mutagenesis was detected in 10 selected sites.
Conclusions: Genome editing with the CRISPR–CRISPR-associated 9 system disrupts the Pcsk9 gene in vivo with high efficiency and reduces blood cholesterol levels in mice. This approach may have therapeutic potential for the prevention of cardiovascular disease in humans.Stem Cell and Regenerative Biolog
The Role Of Folliculin In Hepatic Lipid Metabolism And The Pathogenesis Of Nafld And Nash
Nonalcoholic fatty liver disease (NAFLD) is characterized by abnormal lipid accumulation in the liver. Steatosis can induce lipotoxicity and subsequent development of nonalcoholic steatohepatitis (NASH), characterized not only by steatosis but also inflammation and fibrosis. The pathophysiology of NAFLD and NASH are complex, with the dysregulation of several hepatic lipid processes, and there is currently no FDA-approved pharmacotherapy. Given the central role of the mechanistic target of rapamycin complex 1 (mTORC1) in lipid homeostasis, mTORC1 has the potential to be a therapeutic target. However, how mTORC1 controls hepatic lipids remains incompletely understood, and multiple studies have yielded seemingly conflicting conclusions. Here, we reconcile these studies by showing that selective inhibition of one arm of mTORC1 signaling, via genetic deletion of the RagC/D GTPase-activating protein FLCN in the mouse liver, promotes activation of the transcription factor TFE3 without affecting other mTORC1 targets. Flcn deletion profoundly protects against NAFLD and NASH in mice, induced by multiple diets, and reverses these processes after they have been established. Mechanistically, disease protection is mediated via TFE3, which simultaneously induces lipid-clearance pathways (such as lysosomal biogenesis), promotes mitochondrial respiratory capacity, suppresses anabolic de novo lipogenesis (DNL). The latter is accomplished by suppressing proteolytic processing of SREBP-1c, the central driver of DNL, and by functional interaction with SREBP-1c on chromatin at DNL genes. Initial data show that Flcn deletion also activates VLDL-TAG secretion, suggesting the contribution of another lipid process for NAFLD/NASH protection. All together, our data illuminate previously conflicting studies, and highlight selective inhibition of mTORC1 via suppression of the FLCN:mTORC1:TFE3 axis as a potentially specific and coordinated therapeutic approach to treat NASH/NAFLD