A characteristic of trivalent N-acetylgalactosamine (GalNAc)-conjugated siRNAs is the remarkable durability of silencing, which can persist for months in both preclinical species and humans. For the sustained activity of these siRNAs, the accumulation and stability of siRNA within intracellular compartments are paramount. Several weeks after dosing, a substantial quantity of functional siRNA is released and loaded into RNA-induced silencing complex (RISC) following treatment with a GalNAc-conjugated endolytic peptide.

The GalNAc-siRNA accumulates in acidic endolysosomes. Given the technical challenges associated with directly measuring endolysosome content, a tri-GalNAc conjugated endolytic peptide (stabilized with D-amino acids) extracted from the HA2 domain of influenza virus (D-INF7) was used. When exposed to low pH (<5), the peptide undergoes a conformational change, promotes membrane destruction and thereby releasing the contents of acidic organelles (including siRNAs) into the cytoplasm. The enhanced target knockdown observed post-peptide administration indicates the release of siRNA from acidic organelles and subsequent loading into RISC.

These methods can increase functional siRNA release.  They encompass the use of osmolytic agents like nigericin and chloroquine to induce vesicle swelling and rupture, a well as cell-penetrating and fusogenic peptides such as EB1, melittin, and dfTAT to disrupt membrane integrity. Additionally, photochemical stimulation facilitates compound internalization, while inhibiting endogenous proteins involved in membrane repair nhances vesicular permeability.

GalNAc-INF7 (INF7 peptide, CAS: 185462-59-3, Sequence: GLFEAIEGFIENGWEGMIDGWYGC, NH2-Gly-Leu-Phe-Glu-Ala-Ile-Glu-Gly-Phe-Ile-Glu-Asn-Gly-Trp-Glu-Gly-Met-Ile-Asp-Gly-Trp-Tyr-Gly-Cys-COOH) increases the duration of the enhanced knockdown.Functional siRNA can be released from the acidic intracellular compartment through the endocytic GalNAc-peptide conjugate and loaded into the newly generated Argonaute 2 protein complex, thereby sustaining RNAi activity over an extended period.

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Synthesis of GalNAc-conjugated INF7 peptide



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Reference Reading:

1. Investigating the pharmacodynamic durability of GalNAc-siRNA conjugates

Brown, C.R., Gupta, S., Qin, J., Racie, T., He, G., Lentini, S., Malone, R., Yu, M., Matsuda, S., Shulga-Morskaya, S. and Nair, A.V., 2020. Nucleic acids research, 48(21), pp.11827-11844.

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.

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