A macrophage-specific lncRNA regulates apoptosis and atherosclerosis by tethering HuR in the nucleus.

Long non-coding RNAs (lncRNAs) are emerging regulators of pathophysiological processes including atherosclerosis. Using RNA-seq profiling of the intima of lesions, here we identify a macrophage-specific lncRNA MAARS (Macrophage-Associated Atherosclerosis lncRNA Sequence). Aortic intima expression of MAARS increases by 270-fold with atherosclerotic progression and decreases with regression by 60%. MAARS knockdown reduces atherosclerotic lesion formation by 52% in LDLR-/- mice, largely independent of effects on lipid profile and inflammation, but rather by decreasing macrophage apoptosis and increasing efferocytosis in the vessel wall. MAARS interacts with HuR/ELAVL1, an RNA-binding protein and important regulator of apoptosis. Overexpression and knockdown studies verified MAARS as a critical regulator of macrophage apoptosis and efferocytosis in vitro, in an HuR-dependent manner. Mechanistically, MAARS knockdown alters HuR cytosolic shuttling, regulating HuR targets such as p53, p27, Caspase-9, and BCL2. These findings establish a mechanism by which a macrophage-specific lncRNA interacting with HuR regulates apoptosis, with implications for a broad range of vascular disease states.

Poly(Beta-Amino Ester) Nanoparticles Enable Nonviral Delivery of CRISPR-Cas9 Plasmids for Gene Knockout and Gene Deletion.

The CRISPR-Cas9 system is a powerful gene-editing tool with wide-ranging applications, but the safe and efficient intracellular delivery of CRISPR components remains a challenge. In this study, we utilized biodegradable poly(beta-amino ester) nanoparticles to codeliver plasmid DNA encoding Cas9 and short guide RNA (sgRNA), respectively, to enable gene knockout following a CRISPR-mediated cleavage at one genomic site (1-cut edit), as well as gene deletion following DNA cleavage at two sites flanking a region of interest (2-cut edits). We designed a reporter system that allows for easy evaluation of both types of edits: gene knockout can be assessed by a decrease in near-infrared fluorescent protein (iRFP) fluorescence, whereas deletion of an expression stop cassette turns on a red-enhanced nanolantern fluorescence/luminescence dual reporter. Nanoparticles enabled up to 70% gene knockout due to small indels, as well as 45% gain-of-function expression after a 600-bp deletion edit. The efficiency of 2-cut edits is more sensitive than 1-cut edits to Cas9 and the sgRNA expression level. We demonstrate promising biodegradable nanoparticle formulations for gene editing. Our findings also provide new insights into the screening and transfection requirements for different types of gene edits, which are applicable for designing nonviral delivery systems for the CRISPR-Cas9 platform.