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UCSD Team Publishes Data on Charge-neutralized RNAi Molecules

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NEW YORK (GenomeWeb) – Researchers from the University of California, San Diego have published new data on an RNAi drug delivery approach, licensed to startup Solstice Biologics, showing that problems associated with the negative charge of siRNAs can be overcome through the use of reversible charge-neutralizing phosphotriester backbone modifications.

Molecules synthesized in the fashion, known as short interfering ribonucleic neutrals (siRNNs), can be easily delivered into cells where they are converted into siRNAs that induce RNAi responses, according to the report, which appeared in Nature Biotechnology.

The paper represents a formal unveiling of the siRNN technology, whose development was led by Solstice Co-founder and UCSD investigator Steve Dowdy. In early 2014, Solstice closed an $18 million Series A round, giving the firm an estimated three years of cash.

Standard siRNAs have long proved challenging to deliver into cells due to their large size and negative charge, and current approaches to address this problem largely involve delivery nanoparticles and conjugates.

But because efficient RNAi responses require TAR element binding protein, or TRBP, enzymatic loading of negatively charged double-stranded A-form siRNAs into Argonaute 2, the negatively charged phosphodiester siRNA backbone "has remained recalcitrant to chemical manipulation and presents a significant unsolved problem for siRNA delivery," Dowdy and his colleagues wrote in Nature Biotechnology.

To overcome this problem, the scientists created siRNNs, which feature neutralizing bioreversible phosphotriester groups that permit intracellular entry. Once inside cells, siRNNs are converted by cytoplasmic thioesterases into native, charged phosphodiester-backbone siRNAs.

To test the in vivo efficacy of siRNNs, the researchers designed siRNNs against apolipoprotein B and conjugated them with GalNAc ligands, which have a high affinity for the asialoglycoprotein receptors expressed on the surface of hepatocytes, using conjugatable A-SATE phosphotriester groups.

When the siRNNs were delivered subcutaneously in mice, a large dose — 25 mg/kg — was required to achieve significant apoB knockdown with the siRNNs at 72 hours, something that the scientists chalked up to the "biophysical attributes of the target ApoB mRNA." A similar dose was required to achieve target knockdown with GalNAc-conjugated siRNAs against apoB, according to the paper.

Unlike siRNAs, siRNNs avidly bind to serum albumin, preventing their clearance from circulation by the kidneys. In line with this, the team found that the same 25 mg/kg dose of the apoB GalNAc-siRNNs could also produce a strong RNAi effect in mice after intravenous administration. In contrast, IV delivered GalNAc-siRNAs induced a poor RNAi response.

Additionally, kinetic analyses of the intravenous dose of GalNAc-siRNN into apoB-treated mice showed partial apoB RNAi responses at 24 and 48 hours, which reached a near maximum at 72 hours and were maintained for more than 12 days.

Based on their study results, Dowdy and his team wrote that siRNNs "represent a technology that will open new avenues for development of RNAi therapeutics," pointing to their high solubility and serum stability, their ability to be delivered systemically, and because they can be produced using standard solid-phase oligonucleotide synthesis conditions.

"The collective attributes of siRNNs combined with distinct phosphotriester terminal groups and conjugation handles opens up entirely new avenues for molecularly sculpting the siRNN surface to optimize pharmacokinetics, cellular delivery, and endosomal escape into a given tissue," they added.

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