Search

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 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 to 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.

Huntington’s Disease (HD) is a severe neurodegenerative disorder caused by 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 fully chemically modified short interfering RNA (siRNA) that robustly silence MSH3in vitroandin vivo. When synthesized in a di-valent scaffold, siRNA-mediated silencing of MSH3 effectively blocked CAG repeat expansion in striatum of two HD mouse models without impacting tumor-associated microsatellite instability. Our findings establish a novel paradigm for treating patients with HD and other repeat expansion diseases.One Sentence SummarySilencing MSH3 in the CNS of two models of Huntington’s disease using di-valent siRNA blocks disease-accelerating somatic expansion of CAG repeats.

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.

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., Graphical Abstract, Ferguson et al. show that neuronal and glial cell lines express detectable ApoE mRNA, but ApoE mRNA is not targetable by RNAi in neuronal cells. Thus, cell identity has a profound impact on accessibility of ApoE mRNA to RNAi and should be taken into account during the screening process.

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 subcellular localization might play a role in important neuronal regulatory mechanisms. : Huntington’s disease (HD) is a monogenic neurodegenerative disorder representing an ideal candidate for gene silencing with oligonucleotide therapeutics. Didiot et al. examine the subcellular localization of HTT mRNA in non-neuronal and neuronal cells and the efficiency of oligonucleotide therapeutics on HTT mRNA subcellular fractions. Keywords: Huntington’s disease, HTT mRNA, CAG repeat RNA foci, RNA fluorescence in situ hybridization, confocal microscopy, siRNAs, ASOs

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 β-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 β-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 β-cell proliferation index. In conclusion, CDKN2A/B locus SNPs may impact T2D risk by modulating islet gene expression and β-cell proliferation.

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

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.

Familial hypertrophic cardiomyopathy (FHC) is a genetically transmitted disease that causes progressive remodeling of the myocardium and may result in sudden death. FHC is caused by mutations in sarcomeric proteins including the thin filament regulatory proteins troponin (Tn) and tropomyosin (Tm) (1-3). The single site mutations, D175N and E180G, in cardiac α-tropomyosin have been the subject of several early studies (4, 5), including force measurements on fibers (6, 7), in vitro motility assays (8), ATPase measurements on Tm mutants exchanged into myofibrils (9), physiological measurements on mouse model hearts (10) and myosin subfragment 1 (S1) binding kinetics using reconstituted thin filaments (11). These studies suggest that incomplete relaxation of the heart at the low Ca2+ concentrations present during diastole coupled with the increased Ca2+-sensitivity can cause some of the phenotypes observed in the disease. In most cases the Ca+2 -sensitivity increase was greater for E180G than D175N. However, the molecular basis for the in vivo abnormal functional properties of these Tm mutants is not known. Our early study on the properties of the D175N and E180G Tm mutants showed that: 1) the binding of these mutants to actin in the absence and presence of Tn is weakened (12), (note label mixup, (13)); 2) the thermal stability of E180G is decreased; 3) the region of the molecule near position 190 is destabilized. The greatest effects occur for the E180G FHC mutant. More recent biochemical studies have verified the weakened binding to actin (14). These mutations are located outside of the hydrophobic region of the coiled-coil but may be involved in ionic interactions across the chains (15). Thus, the single site mutations D175N and E180G dramatically affect the global properties of Tm, but the underlying mechanisms are not clear. It is also unknown how these effects cause the FHC phenotype. Tm is a coiled-coil molecule stabilized primarily by hydrophobic side-chain interactions between the two parallel α-helical polypeptide chains. In the actinTmTn thin filament, Tm acts in two ways: 1) by sterically inhibiting the interaction of myosin heads with actin at low Ca2+ and 2) by inhibiting the transition of myosin heads on actin to the force-generating state. This is the basis of the 3-state model consisting of Blocked (− Ca2+), Closed (Ca+2-indiuced) and Open (myosin-induced) (16) and is correlated with 3 positions of Tm on actin (17). The 3-state model has provided a useful framework for understanding normal muscle regulation. Recently, we proposed the inclusion of a 4th state, an active state present in the absence of Ca2+ produced by strong-binding myosin heads (18) in order to help understand how mutations, involving most of the sarcomeric muscle proteins, can cause cardiac diseases such as familial hypertrophic cardiomyopathy (FHC) and dilated cardiomyopathy (DCM). The FHC effects are mimicked by NEM-S1 (19), a strong binding analogue of S1 which does not dissociate from actin in the presence of ATP, indicating that the increase in Ca2+-sensitivity involves myosin. In this work, we showed that substitution of Gly and Gln neutral amino acids for negatively charged Glu and Asp, respectively, on the outside of the Tm coiled coil affects the protein local stability about 50 residues away, a phenomenon of long range information transfer. The decreased stability of the FHC mutants suggests a greater flexibility of Tm on the thin filament which may be important in their malfunction. The results are discussed within the framework of the 4-state model recently published whereby the contribution of an active state in the absence of Ca2+ can explain several effects produced by strong binding myosin heads (18).

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.

Previously we showed that the familial hypertrophic cardiomyopathy (FHC) mutation in Tm, E180G, increased the trypsin cleavage rate at R133 and introduced another cleavage site with a similar rate at K233 (Ly & Lehrer, 2012). To understand how this mutation can produce this dynamically unstable hot spot 53 residues away, we introduced a disulfide crosslink (XL) at C190 and studied its effect on limited trypsin digestion using MALDI MS and N-terminal sequencing.Trypsin digestions at 26° of native disulfide crosslinked (XL) ααTm, FHC mutant D175N and WT, produced a XL heterodimer intermediate for all, containing one cleaved chain at R133, XL to the full length chain (134-284/1-284) (50 kDa) as well as the doubly cleaved XL homodimer (134-284/134-284) (35 kDa). For XL E180G, however, another intermediate was also present (169-284/1-284) (46 kDa). For native XL ααTm, the cleavage site at K168 was only observed at 40° where the middle of Tm is unfolded. In contrast to uncrosslinked E180G, where cleavage occurred at residue 233, no cleavage was observed at residue 233 for crosslinked E180G.These results show: 1) that in addition to the increased destabilization near R133 due to the Glu to Gly change at residue 180 in XL E180G, further destabilization occurs in the region near residue 168; 2) the long-range destabilization near residue K233 caused by the E180G FHC mutation is inhibited by the intervening XL. This suggests that interchain dynamics is involved in propagating the local perturbation at 180G due to the mutation to the K233 region.Supported by NIH HL 91162.

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.

Physiological and biochemical studies have shown that intact and reconstituted muscle thin filaments containing FHC mutants of tropomyosin Tm-D175N and Tm-E180G have a greater Ca2+-sensitivity than native systems. These mutant molecules have a lower thermal stability and bind less strongly to actin (Golitsina et al., 1997; Kremneva et al., 2004). Here we report trypsin digestion kinetics and cleavage sites using PAGE-SDS gels, MALDI MS and N-terminal sequencing. Native Tm is initially cleaved at Arg 133 to form a C-terminal 18KDa and an N-terminal 15KDa fragment due to the instability caused by a nearby Asp 137 in the hydrophobic ridge of the coiled-coil α-helix (Pato et al., 1981; Sumida et al., 2008). WT, Tm-D175N and Tm-E180G were also cleaved at R133, but Tm-D175N and Tm-E180G were cleaved 1.3X and 3.3X faster than WT, respectively. Cleavage of Tm bond to actin was slowed an order of magnitude with similar order of rate: E180G > D175N > WT.In the absence of actin, Tm-E180G, is cleaved at K233 (as well as R133), to produce intermediates of 27KDa and 6KDa. At longer times, the 18KDa fragment of all mutants gets cleaved at R167 to produce a 13.5KDa fragment. For Tm-E180G, the N-terminal 27KDa intermediate is similarly cut at R167 to produce the15KDa fragment and a new 12KDa fragment. ATPase studies of reconstituted thin filaments showed an elevated ATPase at low Ca2+ with: Tm-E180G > Tm-D175N > WT. Thus, whereas both Tm-D175N and Tm-E180G show greater instability near R133 than WT, for Tm-E180G there is an additional region of dynamic instability, near K233. The increased instability for both mutants would result in greater flexibility which appears to be involved in the increased Ca2+-sensitivity observed. Supported by NIH HL 91162.