Drug Discovery, Development & Delivery
Returning to Basics of siRNA Design to Fulfil Therapeutic Potential The recent FDA approval of siRNA therapeutics has re-energised the RNA interference (RNAi) field. The RNAi mechanism was first proposed with the discovery of microRNA in 19931,2 and soon after it was demonstrated that microRNAs regulate gene expression in normal cellular biology and disease. In a Nobel prize-winning discovery in 1998, RNAi was described in C. elegans, where small interfering RNA (siRNA) caused sequence-specific gene knockdown3. The discovery of RNAi-mediated silencing in mammalian cells4 and parallel availability of the whole human genome sequence allowed for creation of research tools to study gene function using siRNA. Manufacturing siRNAs using chemical synthesis was straightforward and the prevailing thought was that siRNA could be used to silence any target gene in any cell. In fact, siRNAs targeting the whole human genome were synthesised in 2006 and whole genome screens have shown them to be a powerful genomics research tool5. However, due to challenges in siRNA delivery and pharmacokinetics, its potential as a therapeutic was not realised until the FDA approval of the siRNA therapeutics ONPATTRO® (patisiran) and GIVLAARI® (givosiran) in 2018 and 2019, respectively. The success of these therapeutics builds on almost two decades of siRNA design and encourages the field to pursue additional gene targets for therapeutic intervention. Mechanism To design a functional and specific synthetic siRNA therapeutic, the RNAi mechanism must be considered. The endogenous RNAi mechanism uses transcribed microRNA to regulate gene expression (Figure 1). Synthetic RNA can enter the RNAi mechanism at different steps to effect gene knockdown. Longer double-stranded RNAs [>19 base pairs (bp)] can enter the pathway as Dicer substrates which are cleaved into siRNAs by Dicer, or synthetic siRNAs (<19 bp) can enter the cell and be loaded directly 56 INTERNATIONAL PHARMACEUTICAL INDUSTRY
Figure 1: Synthetic siRNA can enter the endogenous RNAi mechanism to specifically downregulate gene expression. Primary microRNAs are transcribed in the nucleus and processed by the Drosha-containing complex (microprocessor) to yield the precursor microRNA, which is then shuttled out of the nucleus into the cytoplasm by Exportin-5. The Dicer complex cleaves the pre-microRNA hairpin into a double-stranded microRNA duplex. The double-stranded microRNA is unwound, and one strand is selected for incorporation into the RISC. RNA-programmed RISC binds to target mRNA with imperfect (seedmediated) or perfect complementarity, respectively. Seed-mediated complementarity causes transcript destabilisation and subsequent downregulation of protein expression while perfect complementarity causes catalytic mRNA cleavage and robust gene silencing.
into the RNA-induced silencing complex (RISC). These synthetic siRNAs must be able to enter the cell and interact with RNAi machinery including: Dicer to cleave the molecule into siRNA, RISC components for unwinding, loading, and then finally WatsonCrick base pairing one strand to the mRNA target to result in gene silencing. RNA Synthesis and Chemical Modifications This discovery that siRNAs can cause robust gene knockdown excited the field because now it is possible to easily knock down the expression of a gene by delivering a complementary RNA sequence. siRNAs are comprised of two strands (active and passenger) that form a helical duplex (Figure 2). Compared to traditional organic molecule therapeutics, siRNAs do not have drug characteristics. They are relatively large in size, the backbone contains many negative charges, and the molecule is hydrophilic. Fortunately, the short length of
siRNA allows almost any sequence to easily be synthesised by various chemistries: TBDMS (tert-butyldimethylsilyl), ACE (5'-silyl-2'-acetoxyethylorthoester), and TOM (2'-O-[(triisopropylsilyl)oxy]methyl) chemistries have all been used6. In addition, chemical modifications can be introduced to the siRNAs at multiple positions (nucleoside base, phosphate backbone, 5’ or 3’ ends, demonstrated by the coloured ovals in Figure 2) to alter the therapeutically relevant properties of the molecule. The advantage of siRNA is that its pharmacokinetic properties are primarily defined by the molecular structure, while the targeting properties are defined by the RNA sequence. This means that a successful molecular structure can be applied to a different target sequence and the development time to therapeutic can be substantially shortened. In theory, it should be possible to target any gene, making previously undruggable proteins amenable to therapeutic approaches. Autumn 2020 Volume 12 Issue 3
