Abstract
State-of-the-art small interfering RNA (siRNA) therapeutics such as givosiran and fitusiran are constructed from three variable components: a fully-modified RNA core that conveys metabolic stability, a targeting moiety that mediates target-cell uptake, and a linker. This structural complexity poses challenges for metabolite characterization and risk assessment after long-term patient exposure. In this study, we show that basic phosphorothioate modification of a siRNA targeting the oncoprotein Lin28B provides a useful increase in metabolic stability, without greatly compromising potency. We found that its stability in vitro matched that of nanoparticle-free patisiran in serum and surpassed it in liver tritosome extracts, although it did not reach the stability of the fitusiran siRNA core structure. Liver and kidney were the main sites of accumulation after its subcutaneous administration in mice. Despite the lack of a delivery agent-free antitumor effect, we anticipate our study to be a starting point to develop alternative siRNA scaffolds that can be degraded into naturally-occurring metabolites and help alleviate the aforementioned challenges. Furthermore, Lin28B is a promising target for cancers, and the development of such simplified siRNA analogs, possibly together with novel targeting units, holds potential.
Highlights
Therapeutic oligonucleotides have emerged as a third pillar of modern pharmacotherapy, expanding the portfolio of traditional small molecule drugs and biologics [1,2]
We found that its stability in vitro matched that of nanoparticle-free patisiran in serum and surpassed it in liver tritosome extracts, it did not reach the stability of the fitusiran small interfering RNA (siRNA) core structure
To be efficient in patients, state-of-the-art siRNAs are constructed from three essential components: a fully modified RNA core that conveys metabolic stability, a targeting moiety which mediates uptake into the desired cell population, and a linker which provides optimal spacing for interaction with the designated cell surface receptors
Summary
Therapeutic oligonucleotides have emerged as a third pillar of modern pharmacotherapy, expanding the portfolio of traditional small molecule drugs and biologics [1,2]. A key advantage of this new drug class is its inherent potential to regulate the expression of any gene of interest through the rational design of a complementary oligonucleotide, including disease-causing genes that have been previously considered undruggable. Givosiran is administered as a nanoparticle-free solution. For this purpose, this siRNA relies on complex chemical modification of the natural oligoribonucleotide in three main structural ways: (1) modification of every nucleoside with 2¢-OMe or 2¢-F moieties and terminal phosphorothioate (PS) linkages, (2) conjugation through a binary linker to (3), a trivalent Nacetylgalactosamine ligand for targeted delivery to hepatocytes [15]. The structural complexity of such molecules poses an additional challenge in terms of metabolite characterization and risk assessment with respect to long-term exposure
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