Delivery represents a significant barrier to the clinical advancement of oligonucleotide therapeutics for the treatment of neurological disorders, such as Huntington's disease. Small, endogenous vesicles known as exosomes have the potential to act as oligonucleotide delivery vehicles, but robust and scalable methods for loading RNA therapeutic cargo into exosomes are lacking. Here, we show that hydrophobically modified small interfering RNAs (hsiRNAs) efficiently load into exosomes upon co-incubation, without altering vesicle size distribution or integrity. Exosomes loaded with hsiRNAs targeting Huntingtin mRNA were efficiently internalized by mouse primary cortical neurons and promoted dose-dependent silencing of Huntingtin mRNA and protein. Unilateral infusion of hsiRNA-loaded exosomes, but not hsiRNAs alone, into mouse striatum resulted in bilateral oligonucleotide distribution and statistically significant bilateral silencing of up to 35% of Huntingtin mRNA. The broad distribution and efficacy of hsiRNA-loaded exosomes delivered to brain is expected to advance the development of therapies for the treatment of Huntington's disease and other neurodegenerative disorders.

siRNAs are a new class of therapeutic modalities with promising clinical efficacy that requires modification or formulation for delivery to the tissue and cell of interest. Conjugation of siRNAs to lipophilic groups supports efficient cellular uptake by a mechanism that is not well characterized. Here we study the mechanism of internalization of asymmetric, chemically stabilized, cholesterol-modified siRNAs (sd-rxRNAs®) that efficiently enter cells and tissues without the need for formulation. We demonstrate that uptake is rapid with significant membrane association within minutes of exposure followed by the formation of vesicular structures and internalization. Furthermore, sd-rxRNAs are internalized by a specific class of early endosomes and show preferential association with epidermal growth factor (EGF) but not transferrin (Tf) trafficking pathways as shown by live cell TIRF and structured illumination microscopy (SIM). In fixed cells, we observe ∼25% of sd-rxRNA co-localizing with EGF and <5% with Tf, which is indicative of selective endosomal sorting. Likewise, preferential sd-rxRNA co-localization was demonstrated with EEA1 but not RBSN-containing endosomes, consistent with preferential EGF-like trafficking through EEA1-containing endosomes. sd-rxRNA cellular uptake is a two-step process, with rapid membrane association followed by internalization through a selective, saturable subset of the endocytic process. However, the mechanistic role of EEA1 is not yet known. This method of visualization can be used to better understand the kinetics and mechanisms of hydrophobic siRNA cellular uptake and will assist in further optimization of these types of compounds for therapeutic intervention.

Genome-wide association studies link the locus with type 2 diabetes (T2D) risk, but mechanisms increasing risk remain unknown. The locus encodes cell cycle inhibitors ,, and ;; and , a long noncoding RNA. The goal of this study was to determine whether T2D risk SNPs impact locus gene expression, insulin secretion, or β-cell proliferation in human islets. Islets from donors without diabetes ( = 95) were tested for SNP genotype (rs10811661, rs2383208, rs564398, and rs10757283), gene expression (, ,, ,, ,, and ), insulin secretion ( = 61), and β-cell proliferation ( = 47). Intriguingly, locus genes were coregulated in islets in two physically overlapping cassettes: -, which increased with age, and -, which did not. Risk alleles at rs10811661 and rs2383208 were differentially associated with expression of , but not, , or, in age-dependent fashion, such that younger homozygous risk donors had higher expression, equivalent to older donor levels. We identified several risk SNP combinations that may impact locus gene expression, suggesting possible mechanisms by which SNPs impact locus biology. Risk allele carriers at coding SNP rs564398 had reduced β-cell proliferation index. In conclusion, locus SNPs may impact T2D risk by modulating islet gene expression and β-cell proliferation.

Efficient delivery of therapeutic RNA beyond the liver is the fundamental obstacle preventing its clinical utility. Lipid conjugation increases plasma half-life and enhances tissue accumulation and cellular uptake of small interfering RNAs (siRNAs). However, the mechanism relating lipid hydrophobicity, structure, and siRNA pharmacokinetics is unclear. Here, using a diverse panel of biologically occurring lipids, we show that lipid conjugation directly modulates siRNA hydrophobicity. When administered in vivo, highly hydrophobic lipid-siRNAs preferentially and spontaneously associate with circulating low-density lipoprotein (LDL), while less lipophilic lipid-siRNAs bind to high-density lipoprotein (HDL). Lipid-siRNAs are targeted to lipoprotein receptor-enriched tissues, eliciting significant mRNA silencing in liver (65%), adrenal gland (37%), ovary (35%), and kidney (78%). Interestingly, siRNA internalization may not be completely driven by lipoprotein endocytosis, but the extent of siRNA phosphorothioate modifications may also be a factor. Although biomimetic lipoprotein nanoparticles have been explored for the enhancement of siRNA delivery, our findings suggest that hydrophobic modifications can be leveraged to incorporate therapeutic siRNA into endogenous lipid transport pathways without the requirement for synthetic formulation.

SUMMARY Huntington’s disease (HD) is a monogenic neurodegenerative disorder representing an ideal candidate for gene silencing with oligonucleotide therapeutics (i.e., antisense oligonucleotides [ASOs] and small interfering RNAs [siRNAs]). Using an ultra-sensitive branched fluorescence in situ hybridization (FISH) method, we show that ~50% of wild-type HTT mRNA localizes to the nucleus and that its nuclear localization is observed only in neuronal cells. In mouse brain sections, we detect Htt mRNA predominantly in neurons, with a wide range of Htt foci observed per cell. We further show that siRNAs and ASOs efficiently eliminate cytoplasmic HTT mRNA and HTT protein, but only ASOs induce a partial but significant reduction of nuclear HTT mRNA. We speculate that, like other mRNAs, HTT mRNA sub-cellular localization might play a role in important neuronal regulatory mechanisms.

Cardiac α-tropomyosin (Tm) single site mutations, D175N and E180G, cause familial hypertrophic cardiomyopathy (FHC). Previous studies have shown that these mutations increase both Ca2+-sensitivity and residual contractile activity at low Ca2+, which causes incomplete relaxation during diastole resulting in hypertrophy and sarcomeric disarray. However, the molecular basis for the cause and the difference in severity of the manifested phenotypes of disease is not known. In this work we have: 1) used ATPase studies on reconstituted thin filaments in solution to show that these FHC mutants result in an increase in Ca2+-sensitivity and increased residual ATPase; 2) shown that both FHC mutants increase the rate of cleavage at R133, about 45 residues N-terminal to the mutations, when free and bound to actin; 3) shown that for Tm-E180G, the increase in the rate of cleavage is greater than for D175N; 4) shown that for E180G, cleavage also occurs at a new site 53 residues C-terminal to E180G, in parallel with cleavage at R133. The long-range decreases in dynamic stability due to these two single site mutations, suggests increases in flexibility that may decrease the ability of Tm to inhibit activity at low Ca2+ for D175N and to a greater degree for E180G, which may contribute to differences in the severity of FHC.

Efficient delivery of therapeutic RNA is the fundamental obstacle preventing its clinical utility. Lipid conjugation improves plasma half-life, tissue accumulation, and cellular uptake of small interfering RNAs (siRNAs). However, the impact of conjugate structure and hydrophobicity on siRNA pharmacokinetics is unclear, impeding the design of clinically relevant lipid-siRNAs. Using a panel of biologically-occurring lipids, we show that lipid conjugation modulates siRNA hydrophobicity and governs spontaneous partitioning into distinct plasma lipoprotein classes in vivo. Lipoprotein binding influences siRNA distribution by delaying renal excretion and promoting uptake into lipoprotein receptor-enriched tissues. Lipid-siRNAs elicit mRNA silencing without causing toxicity in a tissue-specific manner. Lipid-siRNA internalization occurs independently of lipoprotein endocytosis, and is mediated by siRNA phosphorothioate modifications. Although biomimetic lipoprotein nanoparticles have been considered for the enhancement of siRNA delivery, our findings suggest that hydrophobic modifications can be leveraged to incorporate therapeutic siRNA into endogenous lipid transport pathways without the requirement for synthetic formulation. IntroductionFor over a decade, the underlying obstacle preventing the widespread clinical use of small interfering RNA (siRNA)-based therapies has been efficient and safe in vivo delivery. siRNAs are large (~14 kDa), polyanionic macromolecules that require extensive modifications to improve their inherently-poor pharmacological properties (e.g. plasma half-life of <5 min before renal excretion) 1,2 . Lipid-and polymer-based nanoparticles, which are effective transfection agents in vitro, can prolong circulation time and improve stability and bioavailability in vivo. However, nanoparticle delivery is typically limited to clearance organs with fenestrated and/or discontinuous endothelium (e.g. liver, spleen, and certain tumors). Moreover, nanoparticles can interact with opsonin proteins that enhance clearance by macrophage phagocytosis.

GalNAc conjugation is emerging as a dominant strategy for delivery of therapeutic oligonucleotides to hepatocytes. The structure and valency of the GalNAc ligand contributes to the potency of the conjugates. Here we present a panel of multivalent GalNAc variants using two different synthetic strategies. Specifically, we present a novel conjugate based on a support-bound trivalent GalNAc cluster, and four others using a GalNAc phosphoramidite monomer that was readily assembled into tri- or tetravalent designs during solid phase oligonucleotide synthesis. We compared these compounds to a clinically used trivalent GalNAc cluster both in vitro and in vivo. In vitro, cluster-based and phosphoramidite-based scaffolds show a similar rate of internalization in primary hepatocytes, with membrane binding observed as early as 5 min. All tested compounds provided potent, dose-dependent silencing, with 2-4% of injected dose recoverable from liver after 1 week. The two preassembled trivalent GalNAc clusters showed higher tissue accumulation and gene silencing relative to di-, tri-, or tetravalent GalNAc conjugates assembled via phosphoramidite chemistry.