Investigating the pharmacodynamic durability of GalNAc–siRNA conjugates

Abstract One hallmark of trivalent N-acetylgalactosamine (GalNAc)-conjugated siRNAs is the remarkable durability of silencing that can persist for months in preclinical species and humans. Here, we investigated the underlying biology supporting this extended duration of pharmacological activity. We found that siRNA accumulation and stability in acidic intracellular compartments is critical for long-term activity. We show that functional siRNA can be liberated from these compartments and loaded into newly generated Argonaute 2 protein complexes weeks after dosing, enabling continuous RNAi activity over time. Identical siRNAs delivered in lipid nanoparticles or as GalNAc conjugates were dose-adjusted to achieve similar knockdown, but only GalNAc–siRNAs supported an extended duration of activity, illustrating the importance of receptor-mediated siRNA trafficking in the process. Taken together, we provide several lines of evidence that acidic intracellular compartments serve as a long-term depot for GalNAc–siRNA conjugates and are the major contributor to the extended duration of activity observed in vivo.

Due to unequal variance, Welch's independent t-test was utilized to compare the mean value between the control and peptide-treated group at each timepoint.
Statistics for Figure 7A -Comparison of serum TTR levels between control and treatment group where peptide was administered at day 7 following ESC siRNA (1.5mg/kg, siTTR-1). Statistics for Figure 7B -Comparison of serum TTR levels between control and treatment group where peptide was administered at day 7 following Advanced ESC siRNA (0.5mg/kg, siTTR-3). Statistics for Figure 7C -Comparison of serum TTR levels between control and treatment group where peptide was administered 15 minutes following ESC siRNA (1.5mg/kg, siTTR-1). Statistics for Figure 7D -Comparison of serum TTR levels between control and treatment group where peptide was administered 15 minutes following ESC siRNA (0.5mg/kg, siTTR-1). Method of analysis: Due to unequal variance, Welch's independent t-test was utilized to compare the mean value between the control and peptide-treated group at each timepoint.
Statistics for Figure 8A -Comparison of Serum TTR levels between control and treatment group where peptide was administered at 8 hours following Advanced ESC GalNAc-siRNA (0.5 mg/kg, siTTR-2). Statistics for Figure 8B -Comparison of serum TTR levels between control and treatment group where peptide was administered at day 11 following Advanced ESC GalNAc-siRNA (0.5 mg/kg, siTTR-2). Statistics for Figure 8C -Comparison of serum TTR levels between control and treatment group where peptide was administered at day 14 following Advanced ESC GalNAc-siRNA (0.5 mg/kg, siTTR-2). Statistics for Figure 8D -Comparison of serum TTR levels between control and treatment group where peptide was administered at day 21 following Advanced ESC GalNAc-siRNA (0.5 mg/kg, siTTR-2).

Method of analysis:
Due to unequal variance, Welch's independent t-test was utilized to compare the mean value between the control and peptide-treated group at each timepoint.
Statistics for Figure 9C -Comparison of sense strand siRNA liver levels between control and treatment group where peptide was administered 15 minutes following ESC GalNAc-siRNA (0.5 mg/kg, siTTR-1). Statistics for Figure 9D -Comparison of antisense strand siRNA liver levels between control and treatment group where peptide was administered 15 minutes following ESC GalNAc-siRNA (0.5 mg/kg, siTTR-1). Statistics for Figure 9E -Comparison of sense strand RISC loading between control and treatment group where peptide was administered 15 minutes following ESC GalNAc-siRNA (0.5 mg/kg, siTTR-1). Statistics for Figure 9F -Comparison of antisense strand RISC loading between control and treatment group where peptide was administered 15 minutes following ESC GalNAc-siRNA (0.5 mg/kg, siTTR-1).

Method of analysis:
Due to unequal variance, Welch's independent t-test was utilized to compare the mean value of siRNA liver levels and RISC loading between the control and +AAV groups at the day 14, 21 and 28 timepoints.
Statistics for Figure 10C -Comparison of antisense strand siRNA liver levels between control (no AAV) and +AAV groups following Advanced ESC GalNAc-siRNA (3 mg/kg, siF12-1). Statistics for Figure 10D -Ago-APP Comparison (data bars 2 through 7) -Comparison of antisense strand siRNA loaded into RISC between control (no AAV) and +AAV groups using the Ago-APP pulldown approach. Statistics for Figure 10D -FLAG-mAgo2 Comparison (data bars 9 through 14) -Comparison of antisense strand siRNA loaded into RISC between control (no AAV) and +AAV groups using the FLAG pulldown approach.   strand per gram of liver) were measured from total liver (A) and plotted on a log10 scale. RISC-loaded antisense siRNA levels were also measured (ng/g, ng of antisense strand per gram of liver) and plotted for ESC (B) and Advanced ESC (C) siRNAs. F7 mRNA knockdown was quantified and normalized to Gapdh for all samples (B-C).
Data is represented as mean ± SD, for N = 3.