Fragile X syndrome (FXS), the most common monogenic cause of inherited intellectual disability and autism, is caused by the silencing of the FMR1 gene, leading to the loss of fragile X mental retardation protein (FMRP), a synaptically expressed RNA-binding protein regulating translation. The Fmr1 knockout model recapitulates the main traits of the disease. Uncontrolled activity of metabotropic glutamate receptor 5 (mGluR5) and mammalian target of rapamycin (mTOR) signaling seem crucial in the pathology of this disease. The endocannabinoid system (ECS) is a key modulator of synaptic plasticity, cognitive performance, anxiety, nociception and seizure susceptibility, all of which are affected in FXS. The cannabinoid receptors CB1 (CB1R) and CB2 (CB2R) are activated by phospholipid-derived endocannabinoids, and CB1R-driven long-term regulation of synaptic strength, as a consequence of mGluR5 activation, is altered in several brain areas of Fmr1 knockout mice. We found that CB1R blockade in male Fmr1 knockout (Fmr1(-/y)) mice through pharmacological and genetic approaches normalized cognitive impairment, nociceptive desensitization, susceptibility to audiogenic seizures, overactivated mTOR signaling and altered spine morphology, whereas pharmacological blockade of CB2R normalized anxiolytic-like behavior. Some of these traits were also reversed by pharmacological inhibition of mTOR or mGluR5. Thus, blockade of ECS is a potential therapeutic approach to normalize specific alterations in FXS.

Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder caused by expanded CAG repeats in the huntingtin (HTT) gene. Although several palliative treatments are available, there is currently no cure and patients generally die 10-15 y after diagnosis. Several promising approaches for HD therapy are currently in development, including RNAi and antisense analogs. We developed a complementary strategy to test repression of mutant HTT with zinc finger proteins (ZFPs) in an HD model. We tested a "molecular tape measure" approach, using long artificial ZFP chains, designed to bind longer CAG repeats more strongly than shorter repeats. After optimization, stable ZFP expression in a model HD cell line reduced chromosomal expression of the mutant gene at both the protein and mRNA levels (95% and 78% reduction, respectively). This was achieved chromosomally in the context of endogenous mouse HTT genes, with variable CAG-repeat lengths. Shorter wild-type alleles, other genomic CAG-repeat genes, and neighboring genes were unaffected. In vivo, striatal adeno-associated virus viral delivery in R6/2 mice was efficient and revealed dose-dependent repression of mutant HTT in the brain (up to 60%). Furthermore, zinc finger repression was tested at several levels, resulting in protein aggregate reduction, reduced decline in rotarod performance, and alleviation of clasping in R6/2 mice, establishing a proof-of-principle for synthetic transcription factor repressors in the brain.

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