Fragile-X Syndrome (FXS) is a multi-organ disease leading to mental retardation, macro-orchidism in males, and premature ovarian insufficiency in female carriers. FXS is also a prominent monogenic disease associated with autism spectrum disorders (ASD). FXS is typically caused by the loss of FRAGILE X-MENTAL RETARDATION 1 (FMR1) expression, which encodes for the RNA-binding protein (RBP), FMRP. We report the discovery of distinct RNA recognition elements (RREs) that correspond to the two independent RNA binding domains of FMRP, and the binding sites within the mRNA targets for wild-type and I304N mutant FMRP isoforms and its paralogs, FXR1 and FXR2. RRE frequency, ratio, and distribution determine target mRNA association with FMRP. Among highly-enriched targets, we identified many genes involved in ASD and demonstrate that FMRP affects their protein levels in cell culture, mice, and human brain. Unexpectedly, we discovered that these targets are also dysregulated in Fmr1-/- mouse ovaries, showing signs of premature follicular overdevelopment. These results indicate that FMRP targets shared signaling pathways across different cellular contexts. As it is become increasingly appreciated that signaling pathways are important to FXS and ASD, our results here provide a molecular guide towards the pursuit of novel therapeutic targets for these neurological disorders.

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.

RNA-based cancer therapies are hindered by the lack of delivery vehicles that avoid cancer-induced organ dysfunction, which exacerbates carrier toxicity. We address this issue by reporting modular degradable dendrimers that achieve the required combination of high potency to tumors and low hepatotoxicity to provide a pronounced survival benefit in an aggressive genetic cancer model. More than 1,500 dendrimers were synthesized using sequential, orthogonal reactions where ester degradability was systematically integrated with chemically diversified cores, peripheries, and generations. A lead dendrimer, 5A2-SC8, provided a broad therapeutic window: identified as potent [EC 50 < 0.02 mg/kg siRNA against FVII (siFVII)] in doseresponse experiments, and well tolerated in separate toxicity studies in chronically ill mice bearing MYC-driven tumors (>75 mg/kg dendrimer repeated dosing). Delivery of let-7g microRNA (miRNA) mimic inhibited tumor growth and dramatically extended survival. Efficacy stemmed from a combination of a small RNA with the dendrimer's own negligible toxicity, therefore illuminating an underappreciated complication in treating cancer with RNA-based drugs.dendrimers | miRNA | cancer

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.