Comparison of DLin-MC3-DMA and ALC-0315 for siRNA Delivery to Hepatocytes and Hepatic Stellate Cells

Abstract

Ionizable cationic lipids are essential for efficient in vivo delivery of RNA by lipid nanoparticles (LNPs). DLin-MC3-DMA (MC3), ALC-0315, and SM-102 are the only ionizable cationic lipids currently clinically approved for RNA therapies. ALC-0315 and SM-102 are structurally similar lipids used in SARS-CoV-2 mRNA vaccines, while MC3 is used in siRNA therapy to knock down transthyretin in hepatocytes. Hepatocytes and hepatic stellate cells (HSCs) are particularly attractive targets for RNA therapy because they synthesize many plasma proteins, including those that influence blood coagulation. While LNPs preferentially accumulate in the liver, evaluating the ability of different ionizable cationic lipids to deliver RNA cargo into distinct cell populations is important for designing RNA-LNP therapies with minimal hepatotoxicity. Here, we directly compared LNPs containing either ALC-0315 or MC3 to knock-down coagulation factor VII (FVII) in hepatocytes and ADAMTS13 in HSCs. At a dose of 1 mg/kg siRNA in mice, LNPs with ALC-0315 achieved a 2- and 10-fold greater knockdown of FVII and ADAMTS13, respectively, compared to LNPs with MC3. At a high dose (5 mg/kg), ALC-0315 LNPs increased markers of liver toxicity (ALT and bile acids), while the same dose of MC3 LNPs did not. These results demonstrate that ALC-0315 LNPs achieves potent siRNA-mediated knockdown of target proteins in hepatocytes and HSCs, in mice, though markers of liver toxicity can be observed after a high dose. This study provides an initial comparison that may inform the development of ionizable cationic LNP therapeutics with maximal efficacy and limited toxicity.

Keywords: RNA therapy; gene therapy; hemostasis; nanomedicine; thrombotic thrombocytopenic purpura; von Willebrand factor.

Figure 1.. ALC-0315 induces greater knockdown of FVII in hepatocytes than MC3.

Mice were injected with a single dose of siRNA FVII (siFVII) or control siLuc, encapsulated in ALC-0315 or MC3 LNPs. Livers and blood were collected one-week post-injection to quantify FVII mRNA and plasma protein levels. A) Hepatic FVII mRNA levels, relative to siLuc-treated. B) Representative western blot of FVII in blood plasma from mice. N = 3. **P < 0.01, ***P < 0.001. Error bars represent mean±SEM.

Figure 2.. ALC-0315 enables siRNA-mediated knockdown of ADAMTS13 in HSCs.

Mice were injected with a single dose of siADAMTS13 (siA13) or control siLuc, encapsulated in ALC-0315 or MC3 LNPs. Livers and blood were collected one-week post-injection to quantify ADAMTS13 mRNA, protein, and activity levels. A) Hepatic Adamts13 mRNA levels, relative to siLuc-treated. B) Representative western blot of ADAMTS13 in blood plasma from mice. C) Plasma ADAMTS13 activity measured by cleavage of a fluorescent substrate. Representative ADAMTS13 activity in blood plasma from the same mice used in the western blot. D) Mice were injected with a single dose of siADAMTS13 or siLuc encapsulated in ALC-0315 LNPs. One-week post-injection, HSCs were isolated from the liver, and qPCR was performed to quantify ADAMTS13 mRNA. PPIA was used as an internal control. Grey dashed line represents a maximum amplification cycle of 55. N = 4. *P < 0.05; ns indicates no significant difference. Error bars represent mean±SEM.

Figure 3.. ALC-0315 causes increased markers of liver toxicity after a 5 mg/kg dose.

Serum was collected five hours after administration of LNPs with MC3 or ALC-0315 at a dose of 5 mg siRNA/kg body weight, or control PBS. Markers of liver toxicity were measured, including ALT (A), AST (B), and bile acids (C). N = 4. *P < 0.05, ***P < 0.001, ns indicates no significant difference. Error bars represent mean±SEM.