Di-valent siRNA-mediated silencing of MSH3 blocks somatic repeat expansion in mouse models of Huntington's disease.

Huntington's disease (HD) is a severe neurodegenerative disorder caused by the expansion of the CAG trinucleotide repeat tract in the huntingtin gene. Inheritance of expanded CAG repeats is needed for HD manifestation, but further somatic expansion of the repeat tract in non-dividing cells, particularly striatal neurons, hastens disease onset. Called somatic repeat expansion, this process is mediated by the mismatch repair (MMR) pathway. Among MMR components identified as modifiers of HD onset, MutS homolog 3 (MSH3) has emerged as a potentially safe and effective target for therapeutic intervention. Here, we identify a fully chemically modified short interfering RNA (siRNA) that robustly silences Msh3 in vitro and in vivo. When synthesized in a di-valent scaffold, siRNA-mediated silencing of Msh3 effectively blocked CAG-repeat expansion in the striatum of two HD mouse models without affecting tumor-associated microsatellite instability or mRNA expression of other MMR genes. Our findings establish a promising treatment approach for patients with HD and other repeat expansion diseases.

Divalent siRNAs are bioavailable in the lung and efficiently block SARS-CoV-2 infection.

The continuous evolution of SARS-CoV-2 variants complicates efforts to combat the ongoing pandemic, underscoring the need for a dynamic platform for the rapid development of pan-viral variant therapeutics. Oligonucleotide therapeutics are enhancing the treatment of numerous diseases with unprecedented potency, duration of effect, and safety. Through the systematic screening of hundreds of oligonucleotide sequences, we identified fully chemically stabilized siRNAs and ASOs that target regions of the SARS-CoV-2 genome conserved in all variants of concern, including delta and omicron. We successively evaluated candidates in cellular reporter assays, followed by viral inhibition in cell culture, with eventual testing of leads for in vivo antiviral activity in the lung. Previous attempts to deliver therapeutic oligonucleotides to the lung have met with only modest success. Here, we report the development of a platform for identifying and generating potent, chemically modified multimeric siRNAs bioavailable in the lung after local intranasal and intratracheal delivery. The optimized divalent siRNAs showed robust antiviral activity in human cells and mouse models of SARS-CoV-2 infection and represent a new paradigm for antiviral therapeutic development for current and future pandemics.

Mutant huntingtin messenger RNA forms neuronal nuclear clusters in rodent and human brains.

Mutant messenger RNA (mRNA) and protein contribute to the clinical manifestation of many repeat-associated neurological disorders, with the presence of nuclear RNA clusters being a common pathological feature. Yet, investigations into Huntington's disease-caused by a CAG repeat expansion in exon 1 of the huntingtin (HTT) gene-have primarily focused on toxic protein gain-of-function as the primary disease-causing feature. To date, mutant HTT mRNA has not been identified as an in vivo hallmark of Huntington's disease. Here, we report that, in two Huntington's disease mouse models (YAC128 and BACHD-97Q-ΔN17), mutant HTT mRNA is retained in the nucleus. Widespread formation of large mRNA clusters (∼0.6-5 µm3) occurred in 50-75% of striatal and cortical neurons. Cluster formation was independent of age and driven by expanded repeats. Clusters associate with chromosomal transcriptional sites and quantitatively co-localize with the aberrantly processed N-terminal exon 1-intron 1 mRNA isoform, HTT1a. HTT1a mRNA clusters are observed in a subset of neurons from human Huntington's disease post-mortem brain and are likely caused by somatic expansion of repeats. In YAC128 mice, clusters, but not individual HTT mRNA, are resistant to antisense oligonucleotide treatment. Our findings identify mutant HTT/HTT1a mRNA clustering as an early, robust molecular signature of Huntington's disease, providing in vivo evidence that Huntington's disease is a repeat expansion disease with mRNA involvement.

Engineered ionizable lipid siRNA conjugates enhance endosomal escape but induce toxicity in vivo.

Lipid conjugation supports delivery of small interfering RNAs (siRNAs) to extrahepatic tissues, expanding the therapeutic potential of siRNAs beyond liver indications. However, siRNA silencing efficacy in extrahepatic tissues remains inferior to that routinely achieved in liver, partially due to the low rate of endosomal escape following siRNA internalization. Improving siRNA endosomal release into cytoplasm is crucial to improving efficacy of lipid-conjugated siRNAs. Given the ability of ionizable lipids to enhance endosomal escape in a context of lipid nanoparticles (LNP), here, we provide the first report on the effect of an ionizable lipid conjugate on siRNA endosomal escape, tissue distribution, efficacy, and toxicity in vivo. After developing a synthetic route to covalently attach the ionizable lipid, DLin-MC3-DMA, to siRNAs, we demonstrate that DLin-MC3-DMA enhances endosomal escape in cell culture without compromising siRNA efficacy. In mice, DLin-MC3-DMA conjugated siRNAs exhibit a similar overall tissue distribution profile to the similarly hydrophobic cholesterol-conjugated siRNA. However, only DLin-MC3-DMA conjugated siRNAs accumulated in vascular compartments, suggesting an effect of conjugate structure on intratissue distribution. Interestingly, we observed non-specific modulation of gene expression in tissues with high accumulation of DLin-MC3-DMA siRNAs (>20 pmol/mg of tissue) while limited non-specific gene modulation has been observed in tissues with lower siRNA accumulation. These findings suggest modulating the nature of the conjugate is a promising strategy to alter siRNA intratissue and intracellular trafficking. Fine-tuning the nature of the conjugate to optimize endosomal escape while minimizing toxicity will be critical for the progression of therapeutic siRNA applications beyond the liver.

Single-Stranded Phosphorothioated Regions Enhance Cellular Uptake of Cholesterol-Conjugated siRNA but Not Silencing Efficacy.

Small interfering RNAs (siRNAs) have potential to silence virtually any disease-causing gene but require chemical modifications for delivery to the tissue and cell of interest. Previously, we demonstrated that asymmetric, phosphorothioate (PS)-modified, chemically stabilized, cholesterol-conjugated siRNAs, called hsiRNAs, support rapid cellular uptake and efficient mRNA silencing both in cultured cells and in vivo. Here, we systematically evaluated the impact of number, structure, and sequence context of PS-modified backbones on cellular uptake and RNAi-mediated silencing efficacy. We find that PS enhances cellular internalization in a sequence-dependent manner but only when present in a single-stranded but not double-stranded region. Furthermore, the observed increase in cellular internalization did not correlate with functional silencing improvement, indicating that PS-mediated uptake may drive compounds to non-productive sinks. Thus, the primary contributing factor of PS modifications to functional efficacy is likely stabilization rather than enhanced cellular uptake. A better understanding of the relative impact of different chemistries on productive versus non-productive uptake will assist in improved design of therapeutic RNAs.

Cell Type Impacts Accessibility of mRNA to Silencing by RNA Interference.

RNA interference (RNAi) is a potent mechanism that silences mRNA and protein expression in all cells and tissue types. RNAi is known to exert many of its functional effects in the cytoplasm, and thus, the cellular localization of target mRNA may impact observed potency. Here, we demonstrate that cell identity has a profound impact on accessibility of apolipoprotein E (ApoE) mRNA to RNAi. We show that, whereas both neuronal and glial cell lines express detectable ApoE mRNA, in neuronal cells, ApoE mRNA is not targetable by RNAi. Screening of a panel of thirty-five chemically modified small interfering RNAs (siRNAs) did not produce a single hit in a neuronal cell line, whereas up to fifteen compounds showed strong efficacy in glial cells. Further investigation of the cellular localization of ApoE mRNA demonstrates that ApoE mRNA is partially spliced and preferentially localized to the nucleus (∼80%) in neuronal cells, whereas more than 90% of ApoE mRNA is cytoplasmic in glial cells. Such an inconsistency in intracellular localization and splicing might provide an explanation for functional differences in RNAi compounds. Thus, cellular origin might have an impact on accessibility of mRNA to RNAi and should be taken into account during the screening process.

Hydrophobicity drives the systemic distribution of lipid-conjugated siRNAs via lipid transport pathways.

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.

Nuclear Localization of Huntingtin mRNA Is Specific to Cells of Neuronal Origin.

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 subcellular localization might play a role in important neuronal regulatory mechanisms.

Novel Cluster and Monomer-Based GalNAc Structures Induce Effective Uptake of siRNAs in Vitro and in Vivo.

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.

CDKN2A/B T2D Genome-Wide Association Study Risk SNPs Impact Locus Gene Expression and Proliferation in Human Islets.

Genome-wide association studies link the CDKN2A/B locus with type 2 diabetes (T2D) risk, but mechanisms increasing risk remain unknown. The CDKN2A/B locus encodes cell cycle inhibitors p14, p15, and p16; MTAP; and ANRIL, a long noncoding RNA. The goal of this study was to determine whether CDKN2A/B T2D risk SNPs impact locus gene expression, insulin secretion, or ß-cell proliferation in human islets. Islets from donors without diabetes (n = 95) were tested for SNP genotype (rs10811661, rs2383208, rs564398, and rs10757283), gene expression (p14, p15, p16, MTAP, ANRIL, PCNA, KI67, and CCND2), insulin secretion (n = 61), and ß-cell proliferation (n = 47). Intriguingly, locus genes were coregulated in islets in two physically overlapping cassettes: p14-p16-ANRIL, which increased with age, and MTAP-p15, which did not. Risk alleles at rs10811661 and rs2383208 were differentially associated with expression of ANRIL, but not p14, p15, p16, or MTAP, in age-dependent fashion, such that younger homozygous risk donors had higher ANRIL 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 ANRIL coding SNP rs564398 had reduced ß-cell proliferation index. In conclusion, CDKN2A/B locus SNPs may impact T2D risk by modulating islet gene expression and ß-cell proliferation.

Visualization of self-delivering hydrophobically modified siRNA cellular internalization.

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.

Exosome-mediated Delivery of Hydrophobically Modified siRNA for Huntingtin mRNA Silencing.

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.

Long-range effects of familial hypertrophic cardiomyopathy mutations E180G and D175N on the properties of tropomyosin.

Cardiac α-tropomyosin (Tm) single-site mutations D175N and E180G cause familial hypertrophic cardiomyopathy (FHC). Previous studies have shown that these mutations increase both Ca(2+) sensitivity and residual contractile activity at low Ca(2+) concentrations, which causes incomplete relaxation during diastole resulting in hypertrophy and sarcomeric disarray. However, the molecular basis for the cause and the difference in the severity of the manifested phenotypes of disease are not known. In this work we have (1) used ATPase studies using reconstituted thin filaments in solution to show that these FHC mutants result in an increase in Ca(2+) sensitivity and an increased residual level of ATPase, (2) shown that both FHC mutants increase the rate of cleavage at R133, ~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 that for D175N, and (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 suggest increases in flexibility that may weaken the ability of Tm to inhibit activity at low Ca(2+) concentrations for D175N and to a greater degree for E180G, which may contribute to differences in the severity of FHC.

Tropomyosin is in a reduced state in rabbit psoas muscle.

Tropomyosin (Tm) purified from skeletal and cardiac muscle often contains disulfide bonds due to oxidation of cysteine groups that are in close proximity in the coiled-coil structure. Are these disulfide crosslinks present in the muscle or produced by oxidation during preparation? To answer this question we reacted one part of freshly dissected rabbit psoas muscle fibers, which was permeabilized with Triton X-100, with N-ethyl maleimide (NEM) to block cysteine groups and another part with 5,5'-dithiobis(2-nitro benzoate) (DTNB) to facilitate disulfide bond formation by interchain sulfhydryl-disulfide exchange. We found, by high resolution gradient SDS polyacrylamide gels, that the NEM-treated muscle was only composed of uncrosslinked Tm and the DTNB treated muscle was composed of disulfide-crosslinked Tm. This work indicates that Tm exists in a reduced state in rabbit psoas muscle.