Can siRNA inclisiran compete with the established PCSK9 antibodies in hypercholesterolemia?
It is nearly 20 years since Andrew Fire and Craig Mello first described the mechanism of RNA interference (RNAi) in C. elegans (1) and just over 15 years after Elbashir et al. demonstrated that short synthetic small interfering RNAs (siRNAs) could induce RNAi in mammalian cells (2). As with many new technologies, an era of inflated expectation followed these initial discoveries. RNAi therapeutics were expected to largely replace antibody therapeutics within a few years, as everyone thought that they could be pushed through discovery and development much faster than antibodies. Also, unlike therapeutic antibodies which bind to and inhibit their target protein but do not directly eliminate it, siRNAs result in the destruction of their target mRNA and thus reduce production of the protein. Additionally, the synthetic nature of siRNAs significantly simplifies large-scale production and thus makes siRNAs more economical than antibodies.
Another advantage for siRNAs is the amplification effect inherent to the mechanism of action. SiRNAs are approximately 19 bp long double-stranded RNA molecules with additional 3′ overhangs of 2 nucleotides on each strand. Once introduced into mammalian cells, the double-stranded siRNAs are loaded onto the RNA-induced silencing complex (RISC) containing Argonaute 2 (Ago-2). Ago-2 removes the “passenger strand” and leaves the “guide strand”– the strand with the lower thermodynamic stability on the 5′ end – free to bind mRNAs with sequence complementarity to at least the seed region of the guide strand (bases 2-8). If there is complete sequence complementarity between the full length of the guide strand and the bound mRNA, Ago-2 cleaves the mRNA between bases 10 and 11 as counted from the 5′ end of the guide strand. The cleaved mRNA dissociates, making the guide-strand loaded RISC complex available to bind and cleave further mRNAs. Thus, a single siRNA molecule mediates the degradation of multiple mRNA molecules containing the target sequence (3) and the overall process is catalytic.
However, incomplete sequence complementarity beyond the seed region can cause off-target silencing via inhibition of mRNA translation in a micro-RNA like mechanism if the seed sequence is complementary to the 3′ UTR of off-target mRNAs. Alternatively, the seed region of the guide strand can directly bind to endogenous miRNAs and thus interfere with their normal function (4). Another problem with siRNA soon became apparent: double-stranded RNAs with less than 30 bp were not guaranteed to evade innate immune recognition as initially thought. Furthermore, as experienced with other oligonucleotide therapeutics, issues with nucleic acid stability, delivery to the target tissue and cell type were identified. By 2010 most big pharma companies had given up on the field because they believed RNAi therapeutics would be unable to overcome these challenges and live up to their potential to rival the success of monoclonal antibody therapeutics.
However, in the few years since then substantial progress in oligonucleotide chemistry led to vastly improved stability and significantly reduced immune recognition of siRNAs. In addition, it was discovered that tri-antennary N-acetylgalactosamine (GalNAc) could mediate highly efficient targeted delivery of siRNAs to hepatocytes via binding to the asialoglycoprotein receptor (read more about GalNAc here).
Having overcome the stability, immune response and delivery challenges, the question now arises if siRNAs really can compete with monoclonal antibodies in the therapeutic field. Currently, the most advanced RNAi therapeutic in clinical trials that combines the increased stability from chemical modifications with GalNAc delivery is inclisiran (ALN-PCSsc; Alnylam Pharmaceuticals and The Medicines Company) for the treatment of hypercholesterolemia.
In most cases, hypercholesterolemia is an acquired consequence of a high-fat diet and sedentary lifestyle in combination with genetic risk factors. High levels of cholesterol and in particular high levels of low-density lipoprotein (LDL; > 4.1 mmol/L or 160 mg/dL), are directly linked with an increased risk of cardiovascular disease. In therapeutic intervention trials with statins, the most widely applied treatment for hypercholesterolemia, each 1 mmol/L (or 40 mg/dL) reduction in LDL cholesterol reduced the incidence of major vascular events by ~20% (5). More severe hypercholesterolemia that cannot be sufficiently controlled by statins is caused by mutations in the hepatic low-density lipoprotein receptor (LDLR) that decrease or abolish LDLR-mediated removal of LDL particles from the bloodstream (6). This genetic disease is called familial hypercholesterolemia (FH) and can also be caused by mutations of apolipoprotein B, the main protein component of LDL particles which facilitates binding of LDL particles to LDLR. A third, if much rarer, cause of FH are gain-of-function mutations in the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene. When PCSK9 protein is bound to LDL-loaded LDLR during endocytosis, the complex is directed to the lysosome for degradation, while LDL-loaded LDLR without PSCK9 offloads the LDL particle and is then returned to the cell surface (7). Thus, gain-of-function PCSK9 mutations result in increased degradation of LDLR and reduced uptake of LDL particles from the bloodstream. Accumulation of LDL cholesterol in the bloodstream accelerates the progress of atherosclerosis and leads to premature death from cardiovascular events due to atherosclerotic lesion rupture. Conversely, loss-of-function mutations in PCSK9 result in significantly lower LDL cholesterol levels (8) due to decreased LDLR degradation and increased recirculation of the receptor to the cell surface. This facilitates increased LDL clearance and reduced LDL levels in the bloodstream, thus ameliorating atherosclerosis progression.
Soon after the positive effects of PCSK9 loss-of-function mutations had been discovered, two fully humanized monoclonal antibodies against PCSK9 were developed: alirocumab (Praluent) by Regeneron in partnership with Sanofi and evolocumab (Repatha) by Amgen. Both can be used in addition to statin therapy and have shown LDL reductions of up to 60 % after 12 weeks of biweekly subcutaneous administration of 75-150 mg. This was true even for patients with high LDL levels that were not controlled by concurrent maximal lipid lowering therapy. Since LDL lowering is a surrogate measure of reduced cardiovascular disease risk, Praluent and Repatha were approved by the FDA in 2015. However, large-scale follow-on studies on cardiovascular outcomes were required. Such studies are ongoing for Praluent (ODYSSEY OUTCOMES; NCT01663402) or in the case of Repatha (FOURIER; NCT01764633), results have recently been announced (9). FOURIER met its primary and secondary endpoints of reducing the risk of cardiovascular events by at least 15%. In the parallel GLAGOV (NCT01813422) study, treatment with Repatha achieved a 1% reduction in percent atheroma volume compared to placebo (p < 0.001) after 76 weeks of treatment (10). It is noteworthy, that PCSK9 inhibition could not reduce LDL cholesterol in LDLR-negative homozygous FH patients (11)
These results certainly set a very high standard for any competing PCSK9 inhibitor. Yet, inclisiran more than meets the challenge. Chemical modifications with phosphorothioate backbone and 2′ ribo sugar modifications (-deoxy, -fluoro and -O-methyl) enhance metabolic stability and protect the siRNA from nuclease degradation while the tri-antennary GalNAc moiety conjugated to the 3′ end of the passenger strand delivers the siRNA effectively to hepatocytes (12). As Fitzgerald et al. now report (13), in a phase I trial (NCT02314442), a single subcutaneous injection of at least 300 mg inclisiran reduced PCSK9 levels in healthy volunteers by 75% on day 84 after treatment. Remarkably, the effect was sustained on day 180. Multiple doses of inclisiran administered on a weekly (4 x 125 mg), biweekly (2 x 250 mg) or monthly (2 x 300 mg, 2 x 500 mg) schedule further confirmed this data. Concomitant reductions in LDL cholesterol were 50% in the single dose groups (≥ 300 mg) and up to 60% in the multiple dosing groups. Again, this effect persisted on day 194. There were no serious adverse events in this phase I study, and Alnylam Pharmaceuticals, in collaboration with The Medicines Company, advanced inclisiran to phase II trials.
Data from a planned interim analysis of the ongoing ORION-1 phase II trial in patients with high cardiovascular risk and elevated LDL cholesterol (NCT02597127) were presented on 15th November 2016 at the American Heart Association conference (14). In this phase II trial, 480 subjects with atherosclerotic cardiovascular disease (ASCVD) or ASCVD-risk equivalents (Type 2 diabetes, familial hypercholesterolemia or Framingham risk score > 20%) receive either single inclisiran subcutaneous injections of 200, 300 or 500 mg or two doses of 100, 200 or 300 mg spaced 90 days apart. As reported (14, 15), the ORION-1 study met all interim analysis goals. A single injection of 300 mg inclisiran lowered LDL levels by a mean of 51% on day 60 compared to placebo in patients that were mostly already on lipid lowering therapy (~80%). The effect was sustained on day 90 (45% mean reduction). Among study participants with follow-up of >180 days, the mean LDL cholesterol reduction was 59% which persisted on day 90 (50% mean reduction) and 180 (43% mean reduction). Two injections of inclisiran (300 mg; on days 1 and 90) lowered LDL cholesterol by 57% on day 120, which was durable on day 180 (52%). All comparisons were highly significant (p < 0.0001). Incidence of treatment emergent adverse events was similar between all inclisiran treatment groups and the placebo group with a low number of injection site reactions which were all mild or moderate in the treatment groups (3.2%). Full data of ORION-1 with six- to nine-month follow-up for all patients will be presented at the American College of Cardiology’s annual scientific conference on the 17th of March 2017.
Currently, another phase II trial of inclisiran is recruiting patients with homozygous familial hypercholesterolemia (ORION-2; NCT02963311), while a third study (ORION-3; NCT03060577) will be comparing 300 mg inclisiran every 180 days with 140 mg evolocumab every 14 days is set to open in March 2017. Although results for this head-to-head comparison of RNAi vs. monoclonal antibody will not be available until 2018, preliminary data suggest that inclisiran will be at a minimum similar to evolocumab. The substantially reduced injection schedule of inclisiran (2 per year versus 26 per year for evolocumab), together with its reduced cost-of-goods (600 mg of siRNA versus 3640 mg of monoclonal antibody) could be advantages.
Another advantage of the current chemically modified siRNA-versions, that has not been as widely reported as it should, is their incredible stability under a wide variety of thermal conditions. As exemplified by fitusiran, another investigational RNAi therapy in development, lyophilized current generation siRNAs could be stored at 40 °C for 6 months or 60 °C for 28 days without loss of chemical integrity (16). Three cycles of freezing at -20 °C followed by 3 days of 50 °C also did not result in degradation. Thus, refrigeration is not actually required for these siRNAs, surely making them the ideal choice for treatments in the developing world.
Although it is early days yet with only preliminary phase II data available for inclisiran, it looks like this siRNA will beat monoclonal antibodies at their own game quite handily. We could be witnessing not just the dawning of the age of antisense, but the dawning of the age of RNAi therapeutics too.
Addendum: The full results from the ORION-1 phase II trial have now been published (17) and the slide deck from the American College of Cardiology conference is also available (18). The data confirm and extend the preliminary data with significant downregulation of LDL cholesterol 9 months after a single 300 mg dose (41%) or two doses of 300 mg at day 0 and 90 (50%).
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