A combinatorial approach for achieving CNS-selective RNAi.

RNA interference (RNAi) is an endogenous process that can be harnessed using chemically modified small interfering RNAs (siRNAs) to potently modulate gene expression in many tissues. The route of administration and chemical architecture are the primary drivers of oligonucleotide tissue distribution, including siRNAs. Independently of the nature and type, oligonucleotides are eliminated from the body through clearance tissues, where their unintended accumulation may result in undesired gene modulation. Divalent siRNAs (di-siRNAs) administered into the CSF induce robust gene silencing throughout the central nervous system (CNS). Upon clearance from the CSF, they are mainly filtered by the kidneys and liver, with the most functionally significant accumulation occurring in the liver. siRNA- and miRNA-induced silencing can be blocked through substrate inhibition using single-stranded, stabilized oligonucleotides called antagomirs or anti-siRNAs. Using APOE as a model target, we show that undesired di-siRNA-induced silencing in the liver can be mitigated through administration of liver targeting GalNAc-conjugated anti-siRNAs, without impacting CNS activity. Blocking unwanted hepatic APOE silencing achieves fully CNS-selective silencing, essential for potential clinical translation. While we focus on CNS/liver selectivity, coadministration of differentially targeting siRNA and anti-siRNAs can be adapted as a strategy to achieve tissue selectivity in different organ combinations.

Silencing Apoe with divalent-siRNAs improves amyloid burden and activates immune response pathways in Alzheimer's disease.

INTRODUCTION: The most significant genetic risk factor for late-onset Alzheimer's disease (AD) is APOE4, with evidence for gain- and loss-of-function mechanisms. A clinical need remains for therapeutically relevant tools that potently modulate APOE expression. METHODS: We optimized small interfering RNAs (di-siRNA, GalNAc) to potently silence brain or liver Apoe and evaluated the impact of each pool of Apoe on pathology. RESULTS: In adult 5xFAD mice, siRNAs targeting CNS Apoe efficiently silenced Apoe expression and reduced amyloid burden without affecting systemic cholesterol, confirming that potent silencing of brain Apoe is sufficient to slow disease progression. Mechanistically, silencing Apoe reduced APOE-rich amyloid cores and activated immune system responses. DISCUSSION: These results establish siRNA-based modulation of Apoe as a viable therapeutic approach, highlight immune activation as a key pathway affected by Apoe modulation, and provide the technology to further evaluate the impact of APOE silencing on neurodegeneration.

RNAi-based modulation of IFN-γ signaling in skin.

Aberrant activation of interferon (IFN)-γ signaling plays a key role in several autoimmune skin diseases, including lupus erythematosus, alopecia areata, vitiligo, and lichen planus. Here, we identify fully chemically modified small interfering RNAs (siRNAs) that silence the ligand binding chain of the IFN-γ receptor (IFNGR1), for the modulation of IFN-γ signaling. Conjugating these siRNAs to docosanoic acid (DCA) enables productive delivery to all major skin cell types local to the injection site, with a single dose of injection supporting effective IFNGR1 protein reduction for at least 1 month in mice. In an ex vivo model of IFN-γ signaling, DCA-siRNA efficiently inhibits the induction of IFN-γ-inducible chemokines, CXCL9 and CXCL10, in skin biopsies from the injection site. Our data demonstrate that DCA-siRNAs can be engineered for functional gene silencing in skin and establish a path toward siRNA treatment of autoimmune skin diseases.

An RNAi therapeutic targeting hepatic DGAT2 in a genetically obese mouse model of nonalcoholic steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is a severe liver disorder characterized by triglyceride accumulation, severe inflammation, and fibrosis. With the recent increase in prevalence, NASH is now the leading cause of liver transplant, with no approved therapeutics available. Although the exact molecular mechanism of NASH progression is not well understood, a widely held hypothesis is that fat accumulation is the primary driver of the disease. Therefore, diacylglycerol O-acyltransferase 2 (DGAT2), a key enzyme in triglyceride synthesis, has been explored as a NASH target. RNAi-based therapeutics is revolutionizing the treatment of liver diseases, with recent chemical advances supporting long-term gene silencing with single subcutaneous administration. Here, we identified a hyper-functional, fully chemically stabilized GalNAc-conjugated small interfering RNA (siRNA) targeting DGAT2 (Dgat2-1473) that, upon injection, elicits up to 3 months of DGAT2 silencing (>80%-90%, p < 0.0001) in wild-type and NSG-PiZ "humanized" mice. Using an obesity-driven mouse model of NASH (ob/ob-GAN), Dgat2-1473 administration prevents and reverses triglyceride accumulation (>85%, p < 0.0001) without increased accumulation of diglycerides, resulting in significant improvement of the fatty liver phenotype. However, surprisingly, the reduction in liver fat did not translate into a similar impact on inflammation and fibrosis. Thus, while Dgat2-1473 is a practical, long-lasting silencing agent for potential therapeutic attenuation of liver steatosis, combinatorial targeting of a second pathway may be necessary for therapeutic efficacy against NASH.

2'-O-Methyl at 20-mer Guide Strand 3' Termini May Negatively Affect Target Silencing Activity of Fully Chemically Modified siRNA.

Small interfering RNAs (siRNAs) have the potential to treat a broad range of diseases. siRNAs need to be extensively chemically modified to improve their bioavailability, safety, and stability in vivo. However, chemical modifications variably impact target silencing for different siRNA sequences, making the activity of chemically modified siRNA difficult to predict. Here, we systematically evaluated the impact of 3' terminal modifications (2'-O-methyl versus 2'-fluoro) on guide strands of different length and showed that 3' terminal 2'-O-methyl modification negatively impacts activity for >60% of siRNA sequences tested but only in the context of 20- and not 19- or 21-nt-long guide strands. These results indicate that sequence, modification pattern, and structure may cooperatively affect target silencing. Interestingly, the introduction of an extra 2'-fluoro modification in the seed region at guide strand position 5, but not 7, may partially compensate for the negative impact of 3' terminal 2'-O-methyl modification. Molecular modeling analysis suggests that 2'-O-methyl modification may impair guide strand interactions within the PAZ domain of argonaute-2, which may affect target recognition and cleavage, specifically when guide strands are 20-nt long. Our findings emphasize the complex nature of modified RNA-protein interactions and contribute to design principles for chemically modified siRNAs.