Amgen publishing on 'emerging modality', RNAi:Plasma and liver protein binding of GalNAc conjugated | ARWR Message Board Posts

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Msg  10004 of 10065  at  6/17/2019 12:44:04 PM  by

Aspector

The following message was updated on 6/17/2019 12:47:14 PM.

Amgen publishing on 'emerging modality', RNAi:Plasma and liver protein binding of GalNAc conjugated siRNA

http://dmd.aspetjournals.org/content/dmd/early/2019/05/16/dmd.119.086967.full.pdf

Abstract
Understanding siRNA fraction unbound (fu) in relevant physiological compartments is critical for establishing pharmacokinetic-pharmacodynamic relationships for this emerging modality. In our attempts to isolate the equilibrium free fraction of GalNAc-conjugated siRNA using classical small molecule in vitro techniques, we observed that hydrodynamic radius was critical in determining the size exclusion limit requirements for fu isolation; largely validating the siRNA 'rigid rod' hypothesis and providing insight into size-based kidney and lymphatic filtration of these molecules. With this knowledge, we developed an orthogonally-validated 50 kDa MWCO ultrafiltration assay to quantify fu in biological matrices including human, non-human primate, rat, mouse plasma, and human liver homogenate. To enhance understanding of the siRNA-plasma interaction landscape, we examined the effects of various common oligonucleotide therapeutic modifications to the ribose and helix backbone on siRNA fu,plasma and found that chemical modifications can modulate plasma protein binding by at least 20%. Finally, to gain insight into which specific plasma proteins bind to siRNA, we developed a qualitative screen to identify binding "hits" across a panel of select purified human plasma proteins.

A sample excerpt:

"Given that GalNAc-siRNA is delivered to the liver via rapid ASGPR uptake, understanding siRNAprotein interactions at the site of action may aid understanding of the long duration of response. Our
findings indicate siRNA is highly bound in the liver at equilibrium. If the binding turns out to be highaffinity, this could confirm the existence of a protein-bound ‘depot’ – with gradual release of siRNA to
RISC. Other prevailing theories suggest that the ‘depot’ is a sub-cellular organelle like the endosome
(Juliano et al. 2015, Dominska et al. 2010), or a consequence of RISC-mediated RNAi being a catalytic
process with a long-lived Ago2-siRNA or Ago2-antisense complex (Wang et al. 2009, Okamura et al.
2004, Nakanishi 2016). Consequently, the contribution of liver protein binding to GalNAc-siRNA
remains an open question."


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Replies
Msg # Subject Author Recs Date Posted
10005 Re: Amgen publishing on 'emerging modality', RNAi:Plasma and liver protein binding of GalNAc conjugated siRNA biochemyst 9 6/17/2019 2:10:58 PM


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