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FDA approves 3rd siRNA therapeutic Oxlumo to treat the metabolic disease primary hyperoxaluria type 1 and the structural significance of 2 nucleotide 3' overhang

 For the oligonucleotide-based genetic medicine, the recent approval of multiple siRNA drugs by Food and Drug Administration marks an important pharmacological shift to molecular biological therapeutics.  These include Onpattro approved in 2018 for treating hereditary transthyretin-mediated amyloidosis, and Givlaari approved in 2019 for treating acute hepatic porphyria.  The approval is also a recognition of the seminal contributions that preceded their development, including the discovery of RNA interference by A. Fire (Johns Hopkins University, USA; Nobel prize, 2006), assembly of siRNA from synthetic oligonucleotides for targeted mRNA degradation, production of highly pure chemically modified siRNAs by the biopharmaceutical industries, and participation of numerous patients in clinical trials to assess their efficacy.  Of 195 ongoing clinical trials examining 60 oligonucleotide drugs (for various disorders including cancer), 17 trials are currently evaluating the siRNA drugs.

 For therapy, the attractiveness of siRNAs lies in their ability for a repeated utilization, thus enabling the degradation of multiple mRNA copies by a given siRNA molecule.  Briefly, the mechanism of RNA interference entails (1) processing of long double stranded RNA by DICER and (2) the cleavage of targeted mRNA by RISC (RNA-induced silencing complex).  Human DICER is a multi-domain protein comprised of helicase, double stranded RNA binding domain, RNAse III, and PAZ (Piwi/Ago/Zwille) (Paturi et al., 2021), which functions in gene regulation, antiviral defense and development.  Human DICER belongs to a RNase III (class IV) family and its substrate selection/cleavage activity is modulated by 2 double strand RNA binding proteins: PACT and TRBP (HIV-1 TAR RNA-binding protein) that interact with its helicase domain.  (Chendrimada et al, 2005; Hasse et al, 2005;  Taylor et al., 2013).

 To generate microRNA (miRNA), nascent transcript (i.e. primary RNA, 'pri-miRNA') is initially processed by DROSHA (nucleus) into 70 bp 'pre-miRNA' with a stem-loop structure and 2 nucleotide 3' overhang, which is then cleaved by DICER (cytoplasm) to generate mature microRNA.  Wheareas dsRNA with 2 nucleotide 3' overhang (pre-miRNA) is recognized by its PAZ domain, dsRNAs with blunt terminii are recognized by its helicase domain (requires ATP to unwind) for the subsequent cleavage by its RNase III domain.  For human DICER, the distance between the PAZ and RNase III domains may determine the length (21 bp) of siRNA or miRNA generated (Lau et al., 2012; MacRae et al., 2006).

In Drosophila, the characteristics of 21-23 bp dsRNA products generated (i.e. 5'-monophosphates and 3' hydroxyls at 5' ends; 2 nucleotides overhang at 3' ends; staggered cuts) indicated that DICER belongs to the RNase III family (as determined by Tuschl and colleagues, Max Planck Institute, Germany)  (Elbashir et al., 2000).

The RISC complex is comprised of DICER, TRBP, and Argonaut 2 (RNase that cleaves mRNA).    In vivo, dsRNA generated by DICER appears to be fed directionally to RISC for the cleavage of target mRNA (hence may select the guide strand) due to proximity; however, synthetic siRNAs may engage RISC in either orientation (Elbashir et al., 2000). 


Previously it was thought that the 5' phosphate may not be essential for silencing; however, a recent report suggests that its presence may be necessary as it contacts a domain in Argonaut 2 of RISC (Roberts et al., 2020).

For 3' overhang, 2-nucleotide was most potent (in silencing) while 4-6 nucleotide was inactive.  Blunt ends or 1-nucleotide 5' overhangs were inconsistent in activity (Elbashir et al., 2000).   Replacing 2 nucleotide 3' overhang with deoxy-form had no effect though complete replacement of all bases with 2'-deoxy or 2'-O-methyl group blocked silencing (nevertheless, for Givlaari, every nucleotide is chemically modified with 2'-F or 2'-O-methyl group).   A commonr industry practice is to use 2 dT residues for 3' overhang (for ease of synthesis, cost, nuclease stability)' however, it may reduce maximum silencing potential (if does not match target sequence or hybridize with a strand containing non-dT overhang) in Drosophila (Boutla et al., 2003) and some suggested that the ribose form may be more potent in humans (Hohjoh, 2002).  Further, the deoxy form of 3' overhang (as well as the sequence of siRNA) may affect the "duration" of silencing negatively whereas 2'-O-methyl group has no effect (Strapp et al., 2010).  Structurally, Patel and colleagues (Sloan Kettering Cancer Center, USA) determined that the PAZ domain (of human Argonaut protein eIF2c1) makes extensive contacts with the guide strand (anchors 2-nucleotide 3' overhang by turning it from the duplex into a protein pocket) and a minimal contact wth the complementary strand (5'-terminal residue) of a siRNA-like molecule (Ma et al., 2004).

With regard to the length of the duplex, 21 bp was most potent when compared to 20, 22 or 23 bp; 24 or 25 bp lacked silencing activity (Elbashir et al., 2000).

As for the backbone, while the use phosphorothioate linkage may confer nuclease resistance, the risk of developing thrombocytopenia (bleeding disorder due to lesser clotting) has been described (Frazier et al., 2015).  The incorporation of sulfur generates chiral centers with distinct potency depending on the specific sterioisomer (Roberts et al., 2020).


For the base modification, methylated pyrimidines (ex. 5-methylcytidine, 5-methyluridine/ribothymidine) may be incorporated  to increase the Tm value.

For sugar modification, 2ʹ-O-methoxyethyl and 2ʹ-Fluoro are used to provide nuclease resistance or to enhance binding efficacy.   The use of 2ʹ-O-methoxyethyl may  help to avoid inciting innate immuity mediated by TLR (toll like receptors), RIG-1, or PKR system.

Regarding the dose, for Onpattro, ~0.3 mg per kilogram of body mass (or 30 mg for >100 kg individuals) of current GMP grade siRNA was administered intravenously per patient (once every 3 weeks for 18 months for the clinical trial).  Delivery vectors have utilized nanoparticles or N-acetylgalactosamine with the latter targeting asialoglycoprotein receptor, which is highly expressed in the liver cells.  However, for delivering to non-liver tissues, conjugation to peptides or nanoparticles modified with peptides targeting specific tissues is increasingly being sought (Roberts et al., 2020).

These advances led to the approval of the siRNA drug Oxlumo (Lumasiran; Alnylam Pharmaceuticals) to treat primary hyperoxaluria type 1 by FDA in 2020.  Its chemical modifications are patterned after Givlaari.  Hyperoxaluria type 1, which causes kidney stones, is caused by mutant alanine-glyoxylate aminotransferase in the peroxisome of liver cells.  This causes its substrate (glyoxylate) to accumulate, resulting in its production of oxalate, which is catalyzed by glycolate oxidase (it also catalyzes conversion of glycolate to glyoxylate), whose mRNA is targeted by Oxlumo for degradation.  Despite targeting liver, Oxlumo is administered subcutaneously (6 mg/kg body weight monthly for 3 doses, followed by 3 mg/kg monthly for maintenance therapy; annual list price 493,000$). 

The key to preventing epidemic is the ability to diagnose the infected early to preempt further propagation.  For this, Bio-Synthesis, Inc. provides primers and probes (as well as synthetic RNA control) for COVID-19 diagnosis via RT-PCR assay.  It specializes in oligonucleotide modification and provides an extensive array of chemically modified nucleoside analogues (over ~200) including bridged nucleic acid (BNA) in addition to mRNA synthesis.  A number of options are available to label oligonucleotides (DNA or RNA) with fluorophores either terminally or internally as well as to conjugate to peptides or antibodies.  It recently acquired a license from BNA Inc. of Osaka, Japan, for the manufacturing and distribution of BNANC, a third generation of BNA oligonucleotides.  To meet the demands of therapeutic application, its oligonucleotide products are approaching GMP grade.  Bio-Synthesis, Inc. has recently entered into collaborative agreement with Bind Therapeutics, Inc. to synthesize miR-21 blocker using BNA for triple negative breast cancer.  The BNA technology provides superior, unequalled advantages in base stacking, binding affinity, aqueous solubility and nuclease resistance.  It also improves the formation of duplexes and triplexes by reducing the repulsion between the negatively charged phosphates of the oligonucleotide backbone.  Its single-mismatch discriminating power is especially useful for diagnosis (ex. FISH using DNA probe).  For clinical application, BNA oligonucleotide exhibits lesser toxicity than other modified nucleotides.!,-an-abundant-post-transcriptional-RNA-modification.aspx



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