In vivo CRISPR base editing of PCSK9 durably lowers cholesterol in primates.

Gene-editing technologies, which include the CRISPR-Cas nucleases1-3 and CRISPR base editors4,5, have the potential to permanently modify disease-causing genes in patients6. The demonstration of durable editing in target organs of nonhuman primates is a key step before in vivo administration of gene editors to patients in clinical trials. Here we demonstrate that CRISPR base editors that are delivered in vivo using lipid nanoparticles can efficiently and precisely modify disease-related genes in living cynomolgus monkeys (Macaca fascicularis). We observed a near-complete knockdown of PCSK9 in the liver after a single infusion of lipid nanoparticles, with concomitant reductions in blood levels of PCSK9 and low-density lipoprotein cholesterol of approximately 90% and about 60%, respectively; all of these changes remained stable for at least 8 months after a single-dose treatment. In addition to supporting a 'once-and-done' approach to the reduction of low-density lipoprotein cholesterol and the treatment of atherosclerotic cardiovascular disease (the leading cause of death worldwide7), our results provide a proof-of-concept for how CRISPR base editors can be productively applied to make precise single-nucleotide changes in therapeutic target genes in the liver, and potentially in other organs.

Histone Deacetylase 3 Inhibition Decreases Cerebral Edema and Protects the Blood-Brain Barrier After Stroke.

We have previously shown that selective inhibition of histone deacetylase 3 (HDAC3) decreases infarct volume and improves long-term functional outcomes after stroke. In this study, we examined the effects of HDAC3 inhibition on cerebral edema and blood-brain barrier (BBB) leakage and explored its underlying mechanisms. Adult male Wistar rats were subjected to 2-h middle cerebral artery occlusion (MCAO) and randomly treated i.p. with either vehicle or a selective HDAC3 inhibitor (RGFP966) at 2 and 24 h after stroke. Modified neurological severity scores (mNSS) were calculated at 2 h, 1 day, and 3 days. H&E, Evans blue dye (EBD) assay, and fluorescein isothiocyanate (FITC)-dextran were employed to assess cerebral edema and BBB leakage. Western blot for matrix metalloproteinase-9 (MMP9), MMP-9 zymography, and immunostaining for HDAC3, GFAP, Iba-1, albumin, aquaporin-4, claudin-5, ZO-1, and NF-kB were performed. Early RGFP966 administration decreased cerebral edema (p = 0.002) and BBB leakage, as measured by EBD assay, FITC-dextran, and albumin extravasation (p < 0.01). RGFP966 significantly increased tight junction proteins (claudin-5 and ZO-1) in the peri-infarct area. RGFP966 also significantly decreased HDAC3 in GFAP + astrocytes, which correlated with better mNSS (r = 0.67, p = 0.03) and decreased cerebral edema (r = 0.64, p = 0.04). RGFP966 decreased aquaporin-4 in GFAP + astrocytes (p = 0.002), as well as, the inflammatory markers Iba-1, NF-kB, and MMP9 in the ischemic brain (p < 0.05). Early HDAC3 inhibition decreases cerebral edema and BBB leakage. BBB protection by RGFP966 is mediated in part by the upregulation of tight junction proteins, downregulation of aquaporin-4 and HDAC3 in astrocytes, and decreased neuroinflammation.

Neuroprotective Effects of Selective Inhibition of Histone Deacetylase 3 in Experimental Stroke.

Histone deacetylase 3 (HDAC3) has been implicated as neurotoxic in several neurodegenerative conditions. However, the role of HDAC3 in ischemic stroke has not been thoroughly explored. We tested the hypothesis that selective inhibition of HDAC3 after stroke affords neuroprotection. Adult male Wistar rats (n = 8/group) were subjected to 2 h of middle cerebral artery occlusion (MCAO), and randomly selected animals were treated intraperitoneally twice with either vehicle (1% Tween 80) or a selective HDAC3 inhibitor (RGFP966, 10 mg/kg) at 2 and 24 h after MCAO. Long-term behavioral tests were performed up to 28 days after MCAO. Another set of rats (n = 7/group) were sacrificed at 3 days for histological analysis. Immunostaining for HDAC3, acetyl-Histone 3 (AcH3), NeuN, TNF-alpha, toll-like receptor 4 (TLR4), cleaved caspase-3, cleaved poly (ADP-ribose) polymerase (PARP), Akt, and TUNEL were performed. Selective HDAC3 inhibition improved long-term functional outcome (p < 0.05) and reduced infarct volume (p < 0.0001). HDAC3 inhibition increased levels of AcH3 in the ischemic brain (p = 0.016). Higher levels of AcH3 were significantly correlated with better neurological scores and smaller infarct volumes (r = 0.74, p = 0.002; r = 0.6, p = 0.02, respectively). The RGFP966 treatment reduced apoptosis-TUNEL+, cleaved caspase-3+, and cleaved PARP+ cells-and neuroinflammation-TNF-alpha+ and TLR4+ cells-in the ischemic border compared to vehicle control (p < 0.05). The RGFP966 treatment also increased Akt expression in the ipsilateral cortex (p < 0.001). Selective HDAC3 inhibition after stroke improves long-term neurological outcome and decreases infarct volume. The neuroprotective effects of HDAC3 inhibition are associated with a reduction in apoptosis and inflammation and upregulation of the Akt pathway.