Comparison of partially and fully chemically-modified siRNA in conjugate-mediated delivery in vivo

Abstract

Small interfering RNA (siRNA)-based drugs require chemical modifications or formulation to promote stability, minimize innate immunity, and enable delivery to target tissues. Partially modified siRNAs (up to 70% of the nucleotides) provide significant stabilization in vitro and are commercially available; thus are commonly used to evaluate efficacy of bio-conjugates for in vivo delivery. In contrast, most clinically-advanced non-formulated compounds, using conjugation as a delivery strategy, are fully chemically modified (100% of nucleotides). Here, we compare partially and fully chemically modified siRNAs in conjugate mediated delivery. We show that fully modified siRNAs are retained at 100x greater levels in various tissues, independently of the nature of the conjugate or siRNA sequence, and support productive mRNA silencing. Thus, fully chemically stabilized siRNAs may provide a better platform to identify novel moieties (peptides, aptamers, small molecules) for targeted RNAi delivery.

Diverse lipid conjugates for functional extra-hepatic siRNA deliveryin vivo

“…The siRNAs used in these studies were fully chemically modified for maximal stability and minimal innate immune activation as described (see Figure 1a and Methods). We and others have recently shown that full chemical stabilization is essential for conjugated-mediated delivery [19][20][21] . The chemical compositions of lipids had a profound effect on the hydrophobicity of lipid-siRNA, based on retention time in reversed-phase HPLC 36 , which ranged from 1 to 12.2 minutes (Figure 1b).…”

“…Indeed, unmodified siRNAs are rapidly degraded and cleared from circulation by kidney filtration, leading to minimal bioavailability in tissues 12 . Thus, full chemical stabilization of siRNAs is essential for conjugate-mediated delivery 19 . The clinical success of GalNAc conjugated siRNAs was only achieved when the siRNAs are extensively modified 20,21 .…”

Gene Silencing With siRNA (RNA Interference): A New Therapeutic Option During Ex Vivo Machine Liver Perfusion Preservation

“…GalNAc‐conjugated siRNA, which drives receptor‐mediated uptake by hepatocytes, is another promising candidate for delivery with machine perfusion to the liver. GalNAc‐conjugated siRNAs are fully modified using an alternating 2’‐fluoro, 2’‐O‐methylation pattern to improve stability . Other 3’ lipid modifications to siRNA can drastically change distribution patterns in liver uptake, with the most lipophilic capable of uniformly penetrating mouse liver tissue without interrupting the siRNA’s ability to interact with RISC machinery (Fig.…”

“…GalNAc-conjugated siR-NAs are fully modified using an alternating 2'-fluoro, 2'-O-methylation pattern to improve stability. (47,57,58) Other 3' lipid modifications to siRNA can drastically change distribution patterns in liver uptake, with the most lipophilic capable of uniformly penetrating mouse REVIEW ARtIclE | 149 liver tissue without interrupting the siRNA's ability to interact with RISC machinery (Fig. 2).…”

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

“…In order to define design parameters for optimizing therapeutic oligonucleotide delivery, we investigated the influence of structurally diverse lipids on conjugate-mediated siRNA biodistribution, efficacy, and safety in vivo. These studies were enabled by the use of a clinicallyvalidated, chemically-modified siRNA scaffold that is nuclease resistant and metabolically stable 8 . Here, we demonstrate that lipid conjugation modulates the hydrophobicity of hsiRNA.…”