CRISPR/Cas is a revolutionary gene editing technology with wide-ranging utility.[1] The safe, non-viral delivery of CRISPR/Cas components would greatly improve future therapeutic utility.[1e] We report the synthesis and development of zwitterionic amino lipids (ZALs) that are uniquely able to (co)deliver long RNAs including Cas9 mRNA and sgRNAs. ZAL nanoparticle (ZNP) delivery of low sgRNA doses (15 nM) reduces protein expression by >90% in cells. In contrast to transient therapies (e.g. RNAi), we show that ZNP delivery of sgRNA enables permanent DNA editing with an indefinitely sustained 95% decrease in protein expression. ZNP delivery of mRNA results in high protein expression at low doses in vitro (<600 pM) and in vivo (1 mg/kg). Intravenous co-delivery of Cas9 mRNA and sgLoxP induced expression of floxed tdTomato in the liver, kidneys, and lungs of engineered mice. ZNPs provide a chemical guide for rational design of long RNA carriers, and represent a promising step towards improving the safety and utility of gene editing.
Cholesterol 25-hydroxylase (CH25H) is an interferon-stimulated gene that converts cholesterol to the oxysterol 25-hydroxycholesterol (25HC). Circulating 25HC modulates essential immunological processes including antiviral immunity, inflammasome activation and antibody class switching; and dysregulation of CH25H may contribute to chronic inflammatory disease and cancer. Although 25HC is a potent regulator of cholesterol storage, uptake, efflux and biosynthesis, how these metabolic activities reprogram the immunological state of target cells remains poorly understood. Here, we used recently designed toxin-based biosensors that discriminate between distinct pools of plasma membrane cholesterol to elucidate how 25HC prevents Listeria monocytogenes from traversing the plasma membrane of infected host cells. The 25HC-mediated activation of acyl-CoA:cholesterol acyltransferase (ACAT) triggered rapid internalization of a biochemically defined fraction of cholesterol, termed 'accessible' cholesterol, from the plasma membrane while having little effect on cholesterol in complexes with Reprints and permissions information is available at www.nature.com/reprints.
CRISPR/Cas is a revolutionary gene editing technology with wide-ranging utility. [1] The safe, non-viral delivery of CRISPR/Cas components would greatly improve future therapeutic utility. [1e] We report the synthesis and development of zwitterionic amino lipids (ZALs) that are uniquely able to (co)deliver long RNAs including Cas9 mRNA and sgRNAs. ZAL nanoparticle (ZNP) delivery of low sgRNA doses (15 nM) reduces protein expression by >90% in cells. In contrast to transient therapies (e.g. RNAi), we show that ZNP delivery of sgRNA enables permanent DNA editing with an indefinitely sustained 95% decrease in protein expression. ZNP delivery of mRNA results in high protein expression at low doses in vitro (<600 pM) and in vivo (1 mg/kg).Correspondence to: Daniel J. Siegwart. + These authors contributed equally.Supporting information for this article can be found under: http://dx.doi.org/10.1002/anie.20X. HHS Public Access Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptIntravenous co-delivery of Cas9 mRNA and sgLoxP induced expression of floxed tdTomato in the liver, kidneys, and lungs of engineered mice. ZNPs provide a chemical guide for rational design of long RNA carriers, and represent a promising step towards improving the safety and utility of gene editing. Graphical abstractWe report the synthesis and development of zwitterionic amino lipids (ZALs) that are uniquely able to deliver long RNAs (Cas9 mRNA and targeted sgRNA) from a single ZAL nanoparticle (ZNP) to enable CRISPR/Cas gene editing.
SUMMARY The non-canonical NF-κB signaling cascade is essential for lymphoid organogenesis, B-cell maturation, osteoclast differentiation, and inflammation in mammals1,2, whereas dysfunction of this system is associated with human diseases, including immunological disorders and cancer3–6. While controlled expression of NF-κB Inducing Kinase (NIK) is the rate-limiting step in non-canonical NF-κB activation2,7, mechanisms of inhibition remain largely unknown. Here, we report the identification of the sine oculis homeobox homolog family transcription factors SIX1 and SIX2 as essential inhibitory components of the non-canonical NF-κB signaling pathway. The developmentally silenced SIX-proteins are reactivated in differentiated macrophages by NIK-mediated suppression of the ubiquitin proteasome pathway. Consequently, SIX1 and SIX2 target a subset of inflammatory gene promoters and directly inhibit RelA and RelB trans-activation function in a negative feedback circuit. In support of a physiologically pivotal role for SIX-proteins in host immunity, human SIX1 transgene suppressed inflammation and promoted the recovery of mice from endotoxic shock. In addition, SIX1 and SIX2 protected RAS/p53-driven lung adenocarcinoma cells from inflammatory cell death induced by SMAC-mimetic chemotherapeutic agents, small-molecule activators of the non-canonical NF-κB pathway. Collectively, our study reveals a NIK-SIX signaling axis that fine-tunes inflammatory gene expression programs under both physiological and pathological conditions.
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